"Medicine, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Fan, Xuelai"@en . "2015-10-23T14:20:37"@en . "2015"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Rapid and reversible methods for altering the function of endogenous proteins are not only indispensable tools for probing complex biological systems, but may potentially drive the development of new therapeutics for the treatment of human diseases. Genetic approaches have provided insights into protein function, but are limited in speed, reversibility and spatiotemporal control. To overcome these limitations, we have developed a peptide-based method to degrade a given endogenous protein at the post-translational level by harnessing chaperone-mediated autophagy, a major intracellular protein degradation pathway mediated by the lysosome. This thesis presents the design and validation of SNIPER (Selective Native Protein ERadication) for use in cultured cells as well as in intact animals. \nSpecifically, we demonstrate the specificity, efficacy and generalizability of SNIPER by showing efficient knockdown of various proteins, including death-associated protein kinase 1 (160 kDa), scaffolding protein PSD-95 (95 kDa) and \u00CE\u00B1-synuclein (19 kDa), with their respective SNIPER peptides in cell lines and rat neuronal cultures. Moreover, we examine the characteristics of SNIPER-mediated protein knockdown and found that the level of protein knockdown is dose-, time- and lysosome-dependent. Furthermore, we demonstrate that SNIPER efficiently knocked down a dephosphorylated subpopulation of a given protein while sparing its phosphorylated form, attesting to the specificity of the method. Finally, using a rat model of focal ischemic stroke, we show that a single intravenous injection of a SNIPER peptide efficiently knocked down a death-promoting protein in the brains of freely moving rats and protected the rat brain from ischemic injury. \nTaken together, SNIPER is a robust and convenient research tool for manipulating the levels of endogenous proteins in situ, and may also lead to the development of novel protein knockdown\u00E2\u0080\u0093based therapeutics for treating human diseases."@en . "https://circle.library.ubc.ca/rest/handle/2429/51927?expand=metadata"@en . "HARNESSING\t \u00C2\u00A0CHAPERONE-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0AUTOPHAGY\t \u00C2\u00A0FOR\t \u00C2\u00A0THE\t \u00C2\u00A0DEGRADATION\t \u00C2\u00A0OF\t \u00C2\u00A0ENDOGENOUS\t \u00C2\u00A0PROTEINS\t \u00C2\u00A0by\t \u00C2\u00A0Xuelai\t \u00C2\u00A0Fan\t \u00C2\u00A0B.Sc.,\t \u00C2\u00A0Peking\t \u00C2\u00A0University,\t \u00C2\u00A02009\t \u00C2\u00A0A\t \u00C2\u00A0THESIS\t \u00C2\u00A0SUBMITTED\t \u00C2\u00A0IN\t \u00C2\u00A0PARTIAL\t \u00C2\u00A0FULFILLMENT\t \u00C2\u00A0OF\t \u00C2\u00A0THE\t \u00C2\u00A0REQUIREMENTS\t \u00C2\u00A0FOR\t \u00C2\u00A0THE\t \u00C2\u00A0DEGREE\t \u00C2\u00A0OF\t \u00C2\u00A0\t \u00C2\u00A0DOCTOR\t \u00C2\u00A0OF\t \u00C2\u00A0PHILOSOPHY\t \u00C2\u00A0in\t \u00C2\u00A0The\t \u00C2\u00A0Faculty\t \u00C2\u00A0of\t \u00C2\u00A0Graduate\t \u00C2\u00A0and\t \u00C2\u00A0Postdoctoral\t \u00C2\u00A0Studies\t \u00C2\u00A0(Neuroscience)\t \u00C2\u00A0\t \u00C2\u00A0THE\t \u00C2\u00A0UNIVERSITY\t \u00C2\u00A0OF\t \u00C2\u00A0BRITISH\t \u00C2\u00A0COLUMBIA\t \u00C2\u00A0(Vancouver)\t \u00C2\u00A0\t \u00C2\u00A0January\t \u00C2\u00A02015\t \u00C2\u00A0\u00C2\u00A9\t \u00C2\u00A0Xuelai\t \u00C2\u00A0Fan,\t \u00C2\u00A02015\t \u00C2\u00A0 ii ABSTRACT\t \u00C2\u00A0Rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible\t \u00C2\u00A0methods\t \u00C2\u00A0for\t \u00C2\u00A0altering\t \u00C2\u00A0the\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0only\t \u00C2\u00A0indispensable\t \u00C2\u00A0tools\t \u00C2\u00A0for\t \u00C2\u00A0probing\t \u00C2\u00A0complex\t \u00C2\u00A0biological\t \u00C2\u00A0systems,\t \u00C2\u00A0but\t \u00C2\u00A0may\t \u00C2\u00A0potentially\t \u00C2\u00A0drive\t \u00C2\u00A0the\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0new\t \u00C2\u00A0therapeutics\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0treatment\t \u00C2\u00A0of\t \u00C2\u00A0human\t \u00C2\u00A0diseases.\t \u00C2\u00A0Genetic\t \u00C2\u00A0approaches\t \u00C2\u00A0have\t \u00C2\u00A0provided\t \u00C2\u00A0insights\t \u00C2\u00A0into\t \u00C2\u00A0protein\t \u00C2\u00A0function,\t \u00C2\u00A0but\t \u00C2\u00A0are\t \u00C2\u00A0limited\t \u00C2\u00A0in\t \u00C2\u00A0speed,\t \u00C2\u00A0reversibility\t \u00C2\u00A0and\t \u00C2\u00A0spatiotemporal\t \u00C2\u00A0control.\t \u00C2\u00A0To\t \u00C2\u00A0overcome\t \u00C2\u00A0these\t \u00C2\u00A0limitations,\t \u00C2\u00A0we\t \u00C2\u00A0have\t \u00C2\u00A0developed\t \u00C2\u00A0a\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0method\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0endogenous\t \u00C2\u00A0protein\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level\t \u00C2\u00A0by\t \u00C2\u00A0harnessing\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy,\t \u00C2\u00A0a\t \u00C2\u00A0major\t \u00C2\u00A0intracellular\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0pathway\t \u00C2\u00A0mediated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0lysosome.\t \u00C2\u00A0This\t \u00C2\u00A0thesis\t \u00C2\u00A0presents\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0and\t \u00C2\u00A0validation\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0(Selective\t \u00C2\u00A0Native\t \u00C2\u00A0Protein\t \u00C2\u00A0ERadication)\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0cells\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0in\t \u00C2\u00A0intact\t \u00C2\u00A0animals.\t \u00C2\u00A0\t \u00C2\u00A0Specifically,\t \u00C2\u00A0we\t \u00C2\u00A0demonstrate\t \u00C2\u00A0the\t \u00C2\u00A0specificity,\t \u00C2\u00A0efficacy\t \u00C2\u00A0and\t \u00C2\u00A0generalizability\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0by\t \u00C2\u00A0showing\t \u00C2\u00A0efficient\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0various\t \u00C2\u00A0proteins,\t \u00C2\u00A0including\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A01\t \u00C2\u00A0(160\t \u00C2\u00A0kDa),\t \u00C2\u00A0scaffolding\t \u00C2\u00A0protein\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0(95\t \u00C2\u00A0kDa)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(19\t \u00C2\u00A0kDa),\t \u00C2\u00A0with\t \u00C2\u00A0their\t \u00C2\u00A0respective\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0in\t \u00C2\u00A0cell\t \u00C2\u00A0lines\t \u00C2\u00A0and\t \u00C2\u00A0rat\t \u00C2\u00A0neuronal\t \u00C2\u00A0cultures.\t \u00C2\u00A0Moreover,\t \u00C2\u00A0we\t \u00C2\u00A0examine\t \u00C2\u00A0the\t \u00C2\u00A0characteristics\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0and\t \u00C2\u00A0found\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090,\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0we\t \u00C2\u00A0demonstrate\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0efficiently\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0a\t \u00C2\u00A0dephosphorylated\t \u00C2\u00A0subpopulation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0while\t \u00C2\u00A0sparing\t \u00C2\u00A0its\t \u00C2\u00A0phosphorylated\t \u00C2\u00A0form,\t \u00C2\u00A0attesting\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0method.\t \u00C2\u00A0Finally,\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0rat\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0focal\t \u00C2\u00A0ischemic\t \u00C2\u00A0stroke,\t \u00C2\u00A0we\t \u00C2\u00A0show\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0intravenous\t \u00C2\u00A0injection\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0efficiently\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0a\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090promoting\t \u00C2\u00A0 iii protein\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0brains\t \u00C2\u00A0of\t \u00C2\u00A0freely\t \u00C2\u00A0moving\t \u00C2\u00A0rats\t \u00C2\u00A0and\t \u00C2\u00A0protected\t \u00C2\u00A0the\t \u00C2\u00A0rat\t \u00C2\u00A0brain\t \u00C2\u00A0from\t \u00C2\u00A0ischemic\t \u00C2\u00A0injury.\t \u00C2\u00A0\t \u00C2\u00A0Taken\t \u00C2\u00A0together,\t \u00C2\u00A0SNIPER\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0robust\t \u00C2\u00A0and\t \u00C2\u00A0convenient\t \u00C2\u00A0research\t \u00C2\u00A0tool\t \u00C2\u00A0for\t \u00C2\u00A0manipulating\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0situ,\t \u00C2\u00A0and\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0lead\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0novel\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\u00E2\u0080\u0093based\t \u00C2\u00A0therapeutics\t \u00C2\u00A0for\t \u00C2\u00A0treating\t \u00C2\u00A0human\t \u00C2\u00A0diseases.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 iv PREFACE\t \u00C2\u00A0Much\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0work\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0published,\t \u00C2\u00A0and\t \u00C2\u00A0as\t \u00C2\u00A0evidenced\t \u00C2\u00A0by\t \u00C2\u00A0multiple\t \u00C2\u00A0authorships\t \u00C2\u00A0listed\t \u00C2\u00A0below,\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0result\t \u00C2\u00A0of\t \u00C2\u00A0collaborations\t \u00C2\u00A0between\t \u00C2\u00A0my\t \u00C2\u00A0colleagues\t \u00C2\u00A0and\t \u00C2\u00A0myself.\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0interest\t \u00C2\u00A0of\t \u00C2\u00A0space,\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0only\t \u00C2\u00A0contains\t \u00C2\u00A0work\t \u00C2\u00A0that\t \u00C2\u00A0I\t \u00C2\u00A0was\t \u00C2\u00A0heavily\t \u00C2\u00A0involved\t \u00C2\u00A0in.\t \u00C2\u00A0\t \u00C2\u00A0A\t \u00C2\u00A0version\t \u00C2\u00A0of\t \u00C2\u00A0Chapter\t \u00C2\u00A04\t \u00C2\u00A0was\t \u00C2\u00A0published\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0review\t \u00C2\u00A0article:\t \u00C2\u00A0Fan\t \u00C2\u00A0X,\t \u00C2\u00A0Jin\t \u00C2\u00A0WY\t \u00C2\u00A0and\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2014a).\t \u00C2\u00A0The\t \u00C2\u00A0NMDAR\t \u00C2\u00A0multi-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0complex:\t \u00C2\u00A0a\t \u00C2\u00A0multifunctional\t \u00C2\u00A0machine\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0CNS.\t \u00C2\u00A0Front.\t \u00C2\u00A0Cell.\t \u00C2\u00A0Neurosci.\t \u00C2\u00A08:160.\t \u00C2\u00A0doi:\t \u00C2\u00A010.3389/fncel.2014.00160\t \u00C2\u00A0\t \u00C2\u00A0My\t \u00C2\u00A0colleague\t \u00C2\u00A0and\t \u00C2\u00A0I\t \u00C2\u00A0were\t \u00C2\u00A0responsible\t \u00C2\u00A0for\t \u00C2\u00A0writing\t \u00C2\u00A0the\t \u00C2\u00A0initial\t \u00C2\u00A0manuscript\t \u00C2\u00A0draft,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0modified\t \u00C2\u00A0by\t \u00C2\u00A0Dr.\t \u00C2\u00A0Yu\t \u00C2\u00A0Tian\t \u00C2\u00A0Wang\t \u00C2\u00A0and\t \u00C2\u00A0later\t \u00C2\u00A0edited\t \u00C2\u00A0for\t \u00C2\u00A0consistency\t \u00C2\u00A0by\t \u00C2\u00A0myself.\t \u00C2\u00A0\t \u00C2\u00A0Results\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0Chapters\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905\t \u00C2\u00A0were\t \u00C2\u00A0published\t \u00C2\u00A0in:\t \u00C2\u00A0Fan\t \u00C2\u00A0X*,\t \u00C2\u00A0Jin\t \u00C2\u00A0WY*,\t \u00C2\u00A0Lu\t \u00C2\u00A0J,\t \u00C2\u00A0Wang\t \u00C2\u00A0J,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2014b).\t \u00C2\u00A0Rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0by\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation.\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience.\t \u00C2\u00A017(3):471-\u00C2\u00AD\u00E2\u0080\u009080.\t \u00C2\u00A0doi:10.1038/nn.3637\t \u00C2\u00A0(*\t \u00C2\u00A0equal\t \u00C2\u00A0contribution)\t \u00C2\u00A0\t \u00C2\u00A0Fan\t \u00C2\u00A0X,\t \u00C2\u00A0Jin\t \u00C2\u00A0WY,\t \u00C2\u00A0Lu\t \u00C2\u00A0J,\t \u00C2\u00A0and\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2014).\t \u00C2\u00A0Peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090inducing\t \u00C2\u00A0kinase\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapy\t \u00C2\u00A0for\t \u00C2\u00A0stroke.\t \u00C2\u00A0Canadian\t \u00C2\u00A0Association\t \u00C2\u00A0for\t \u00C2\u00A0Neuroscience,\t \u00C2\u00A02014.\t \u00C2\u00A0\t \u00C2\u00A0Fan\t \u00C2\u00A0X,\t \u00C2\u00A0Jin\t \u00C2\u00A0WY,\t \u00C2\u00A0Lu\t \u00C2\u00A0J,\t \u00C2\u00A0Wang\t \u00C2\u00A0J\t \u00C2\u00A0and\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2012,\t \u00C2\u00A02013).\t \u00C2\u00A0Development\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0targeting\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0(TPDPD)\t \u00C2\u00A0system\t \u00C2\u00A0for\t \u00C2\u00A0rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible\t \u00C2\u00A0 v knockdown\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins.\t \u00C2\u00A0Canadian\t \u00C2\u00A0Association\t \u00C2\u00A0for\t \u00C2\u00A0Neuroscience,\t \u00C2\u00A02013;\t \u00C2\u00A0Society\t \u00C2\u00A0for\t \u00C2\u00A0Neuroscience,\t \u00C2\u00A02012.\t \u00C2\u00A0\t \u00C2\u00A0I\t \u00C2\u00A0was\t \u00C2\u00A0jointly\t \u00C2\u00A0responsible\t \u00C2\u00A0with\t \u00C2\u00A0Dr.\t \u00C2\u00A0Yu\t \u00C2\u00A0Tian\t \u00C2\u00A0Wang\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0experiments,\t \u00C2\u00A0and\t \u00C2\u00A0mainly\t \u00C2\u00A0responsible\t \u00C2\u00A0for\t \u00C2\u00A0performing\t \u00C2\u00A0the\t \u00C2\u00A0experiments\t \u00C2\u00A0and\t \u00C2\u00A0analyzing\t \u00C2\u00A0the\t \u00C2\u00A0data.\t \u00C2\u00A0The\t \u00C2\u00A0animal\t \u00C2\u00A0work\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014b\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0collaborative\t \u00C2\u00A0effort\t \u00C2\u00A0between\t \u00C2\u00A0my\t \u00C2\u00A0colleague\t \u00C2\u00A0and\t \u00C2\u00A0myself.\t \u00C2\u00A0I\t \u00C2\u00A0was\t \u00C2\u00A0chiefly\t \u00C2\u00A0responsible\t \u00C2\u00A0for\t \u00C2\u00A0writing\t \u00C2\u00A0the\t \u00C2\u00A0manuscript,\t \u00C2\u00A0with\t \u00C2\u00A0modifications\t \u00C2\u00A0from\t \u00C2\u00A0Dr.\t \u00C2\u00A0Yu\t \u00C2\u00A0Tian\t \u00C2\u00A0Wang.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0A\t \u00C2\u00A0version\t \u00C2\u00A0of\t \u00C2\u00A0Chapter\t \u00C2\u00A06\t \u00C2\u00A0was\t \u00C2\u00A0published\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0protocol\t \u00C2\u00A0article,\t \u00C2\u00A0for\t \u00C2\u00A0which\t \u00C2\u00A0I\t \u00C2\u00A0was\t \u00C2\u00A0the\t \u00C2\u00A0primary\t \u00C2\u00A0author.\t \u00C2\u00A0Fan\t \u00C2\u00A0X\t \u00C2\u00A0and\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2015).\t \u00C2\u00A0SNIPER\t \u00C2\u00A0Peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins.\t \u00C2\u00A0Current\t \u00C2\u00A0Protocols\t \u00C2\u00A0in\t \u00C2\u00A0Chemical\t \u00C2\u00A0Biology.\t \u00C2\u00A07:1-\u00C2\u00AD\u00E2\u0080\u009016.\t \u00C2\u00A0\t \u00C2\u00A0All\t \u00C2\u00A0animal\t \u00C2\u00A0work\t \u00C2\u00A0conducted\t \u00C2\u00A0was\t \u00C2\u00A0approved\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0UBC\t \u00C2\u00A0Animal\t \u00C2\u00A0Care\t \u00C2\u00A0Committee,\t \u00C2\u00A0following\t \u00C2\u00A0the\t \u00C2\u00A0MCAo\t \u00C2\u00A0protocol\t \u00C2\u00A0A07-\u00C2\u00AD\u00E2\u0080\u00900151.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 vi TABLE\t \u00C2\u00A0OF\t \u00C2\u00A0CONTENTS\t \u00C2\u00A0\t \u00C2\u00A0ABSTRACT\t \u00C2\u00A0...................................................................................................................\t \u00C2\u00A0ii\t \u00C2\u00A0PREFACE\t \u00C2\u00A0....................................................................................................................\t \u00C2\u00A0iv\t \u00C2\u00A0TABLE\t \u00C2\u00A0OF\t \u00C2\u00A0CONTENTS\t \u00C2\u00A0................................................................................................\t \u00C2\u00A0vi\t \u00C2\u00A0LIST\t \u00C2\u00A0OF\t \u00C2\u00A0TABLES\t \u00C2\u00A0........................................................................................................\t \u00C2\u00A0xii\t \u00C2\u00A0LIST\t \u00C2\u00A0OF\t \u00C2\u00A0FIGURES\t \u00C2\u00A0.....................................................................................................\t \u00C2\u00A0xiii\t \u00C2\u00A0LIST\t \u00C2\u00A0OF\t \u00C2\u00A0ABBREVIATIONS\t \u00C2\u00A0.........................................................................................\t \u00C2\u00A0xv\t \u00C2\u00A0LIST\t \u00C2\u00A0OF\t \u00C2\u00A0PLASMIDS\t \u00C2\u00A0..................................................................................................\t \u00C2\u00A0xxi\t \u00C2\u00A0ACKNOWLEDGEMENTS\t \u00C2\u00A0........................................................................................\t \u00C2\u00A0xxiii\t \u00C2\u00A0DEDICATION\t \u00C2\u00A0...........................................................................................................\t \u00C2\u00A0xxv\t \u00C2\u00A0CHAPTER\t \u00C2\u00A01:\t \u00C2\u00A0INTRODUCTION\t \u00C2\u00A0.....................................................................................\t \u00C2\u00A01\t \u00C2\u00A01.1\t \u00C2\u00A0 OVERVIEW\t \u00C2\u00A0...............................................................................................................................\t \u00C2\u00A01\t \u00C2\u00A01.2\t \u00C2\u00A0 PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0BY\t \u00C2\u00A0GENE\t \u00C2\u00A0TARGETING\t \u00C2\u00A0..............................................................\t \u00C2\u00A03\t \u00C2\u00A01.2.1\t \u00C2\u00A0 CONVENTIONAL\t \u00C2\u00A0GENETIC\t \u00C2\u00A0KNOCKOUT\t \u00C2\u00A0....................................................................................\t \u00C2\u00A03\t \u00C2\u00A01.2.2\t \u00C2\u00A0 CONDITIONAL\t \u00C2\u00A0GENETIC\t \u00C2\u00A0KNOCKOUT\t \u00C2\u00A0.....................................................................................\t \u00C2\u00A05\t \u00C2\u00A01.2.3\t \u00C2\u00A0 TARGETED\t \u00C2\u00A0GENOME\t \u00C2\u00A0EDITING\t \u00C2\u00A0...................................................................................................\t \u00C2\u00A07\t \u00C2\u00A01.3\t \u00C2\u00A0 PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0BY\t \u00C2\u00A0RNA\t \u00C2\u00A0TARGETING\t \u00C2\u00A0................................................................\t \u00C2\u00A09\t \u00C2\u00A01.3.1\t \u00C2\u00A0 ANTI-\u00C2\u00AD\u00E2\u0080\u0090SENSE\t \u00C2\u00A0OLIGONUCLEOTIDES\t \u00C2\u00A0(ASO)\t \u00C2\u00A0................................................................................\t \u00C2\u00A09\t \u00C2\u00A01.3.2\t \u00C2\u00A0 RNA\t \u00C2\u00A0INTERFERENCE\t \u00C2\u00A0(RNAi)\t \u00C2\u00A0.......................................................................................................\t \u00C2\u00A011\t \u00C2\u00A0 vii 1.4\t \u00C2\u00A0 PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0AT\t \u00C2\u00A0THE\t \u00C2\u00A0POST-\u00C2\u00AD\u00E2\u0080\u0090TRANSLATIONAL\t \u00C2\u00A0LEVEL\t \u00C2\u00A0.............................\t \u00C2\u00A013\t \u00C2\u00A01.4.1\t \u00C2\u00A0 THE\t \u00C2\u00A0UPS\t \u00C2\u00A0.........................................................................................................................................\t \u00C2\u00A015\t \u00C2\u00A01.4.2\t \u00C2\u00A0 THE\t \u00C2\u00A0ALS\t \u00C2\u00A0.........................................................................................................................................\t \u00C2\u00A016\t \u00C2\u00A01.4.2.1\t \u00C2\u00A0 MACROAUTOPHAGY\t \u00C2\u00A0...........................................................................................................\t \u00C2\u00A017\t \u00C2\u00A01.4.2.2\t \u00C2\u00A0 CMA\t \u00C2\u00A0.......................................................................................................................................\t \u00C2\u00A020\t \u00C2\u00A01.4.2.3\t \u00C2\u00A0 ENDOSOMAL\t \u00C2\u00A0MICROAUTOPHAGY\t \u00C2\u00A0(e-\u00C2\u00AD\u00E2\u0080\u0090MI)\t \u00C2\u00A0.....................................................................\t \u00C2\u00A026\t \u00C2\u00A01.4.2.4\t \u00C2\u00A0 CROSSTALK\t \u00C2\u00A0BETWEEN\t \u00C2\u00A0PROTEOLYTIC\t \u00C2\u00A0SYSTEMS\t \u00C2\u00A0.........................................................\t \u00C2\u00A026\t \u00C2\u00A01.4.3\t \u00C2\u00A0 PRIMARY\t \u00C2\u00A0AND\t \u00C2\u00A0SECONDARY\t \u00C2\u00A0DEGRONS\t \u00C2\u00A0.................................................................................\t \u00C2\u00A030\t \u00C2\u00A01.4.4\t \u00C2\u00A0 POST-\u00C2\u00AD\u00E2\u0080\u0090TRANSLATIONAL\t \u00C2\u00A0REGULATION\t \u00C2\u00A0OF\t \u00C2\u00A0DEGRONS\t \u00C2\u00A0..........................................................\t \u00C2\u00A032\t \u00C2\u00A01.4.5\t \u00C2\u00A0 TRANS-\u00C2\u00AD\u00E2\u0080\u0090TARGETING\t \u00C2\u00A0VIA\t \u00C2\u00A0PROTEIN-\u00C2\u00AD\u00E2\u0080\u0090PROTEIN\t \u00C2\u00A0INTERACTION\t \u00C2\u00A0..............................................\t \u00C2\u00A033\t \u00C2\u00A01.5\t \u00C2\u00A0 EXAMPLES\t \u00C2\u00A0OF\t \u00C2\u00A0DIRECT\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0METHODS\t \u00C2\u00A0......................................\t \u00C2\u00A034\t \u00C2\u00A01.5.1\t \u00C2\u00A0 PHYSICAL\t \u00C2\u00A0LINKAGE\t \u00C2\u00A0TO\t \u00C2\u00A0A\t \u00C2\u00A0DEGRON\t \u00C2\u00A0..........................................................................................\t \u00C2\u00A035\t \u00C2\u00A01.5.2\t \u00C2\u00A0 DEGRADATION\t \u00C2\u00A0BY\t \u00C2\u00A0TRANS-\u00C2\u00AD\u00E2\u0080\u0090TARGETING\t \u00C2\u00A0.................................................................................\t \u00C2\u00A039\t \u00C2\u00A01.5.3\t \u00C2\u00A0 LIMITATIONS\t \u00C2\u00A0OF\t \u00C2\u00A0CURRENT\t \u00C2\u00A0DEGRON-\u00C2\u00AD\u00E2\u0080\u0090BASED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0METHODS\t \u00C2\u00A0......................\t \u00C2\u00A044\t \u00C2\u00A01.6\t \u00C2\u00A0 RATIONALE\t \u00C2\u00A0AND\t \u00C2\u00A0HYPOTHESIS\t \u00C2\u00A0........................................................................................\t \u00C2\u00A046\t \u00C2\u00A01.6.1\t \u00C2\u00A0 THE\t \u00C2\u00A0CORE\t \u00C2\u00A0CONSTITUENTS\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0................................................................\t \u00C2\u00A047\t \u00C2\u00A01.6.2\t \u00C2\u00A0 OBJECTIVE\t \u00C2\u00A01:\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0PROTEINS\t \u00C2\u00A0.........\t \u00C2\u00A049\t \u00C2\u00A01.6.3\t \u00C2\u00A0 OBJECTIVE\t \u00C2\u00A02:\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0PROTEINS\t \u00C2\u00A0IN\t \u00C2\u00A0PRIMARY\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CULTURES\t \u00C2\u00A0.........................................................................................................................\t \u00C2\u00A050\t \u00C2\u00A01.6.4\t \u00C2\u00A0 OBJECTIVE\t \u00C2\u00A03:\t \u00C2\u00A0FUNCTIONAL\t \u00C2\u00A0CONSEQUENCES\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IN\t \u00C2\u00A0VITRO\t \u00C2\u00A0AND\t \u00C2\u00A0IN\t \u00C2\u00A0VIVO\t \u00C2\u00A0...............................................................................................\t \u00C2\u00A051\t \u00C2\u00A0CHAPTER\t \u00C2\u00A02:\t \u00C2\u00A0MATERIALS\t \u00C2\u00A0AND\t \u00C2\u00A0METHODS\t \u00C2\u00A0................................................................\t \u00C2\u00A059\t \u00C2\u00A02.1\t \u00C2\u00A0 CELL\t \u00C2\u00A0CULTURE\t \u00C2\u00A0.....................................................................................................................\t \u00C2\u00A059\t \u00C2\u00A02.1.1\t \u00C2\u00A0 HUMAN\t \u00C2\u00A0EMBRYONIC\t \u00C2\u00A0KIDNEY\t \u00C2\u00A0293\t \u00C2\u00A0(HEK\t \u00C2\u00A0293)\t \u00C2\u00A0CELL\t \u00C2\u00A0MAINTENANCE\t \u00C2\u00A0.................................\t \u00C2\u00A059\t \u00C2\u00A02.1.2\t \u00C2\u00A0 TRANSFECTION\t \u00C2\u00A0AND\t \u00C2\u00A0DRUG\t \u00C2\u00A0TREATMENT\t \u00C2\u00A0OF\t \u00C2\u00A0HEK\t \u00C2\u00A0CELLS\t \u00C2\u00A0.................................................\t \u00C2\u00A060\t \u00C2\u00A0 viii 2.1.3\t \u00C2\u00A0 STORAGE\t \u00C2\u00A0OF\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0CELLS\t \u00C2\u00A0.....................................................................................................\t \u00C2\u00A061\t \u00C2\u00A02.1.4\t \u00C2\u00A0 PRIMARY\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CULTURE\t \u00C2\u00A0..............................................................................................\t \u00C2\u00A061\t \u00C2\u00A02.1.5\t \u00C2\u00A0 DRUG\t \u00C2\u00A0TREATMENT\t \u00C2\u00A0OF\t \u00C2\u00A0PRIMARY\t \u00C2\u00A0CULTURED\t \u00C2\u00A0NEURONS\t \u00C2\u00A0...................................................\t \u00C2\u00A063\t \u00C2\u00A02.2\t \u00C2\u00A0 CELL\t \u00C2\u00A0AND\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0......................................................................................\t \u00C2\u00A064\t \u00C2\u00A02.2.1\t \u00C2\u00A0 HEK\t \u00C2\u00A0293\t \u00C2\u00A0CELL\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0WHOLE\t \u00C2\u00A0CELL\t \u00C2\u00A0LYSATE\t \u00C2\u00A0............................................\t \u00C2\u00A064\t \u00C2\u00A02.2.2\t \u00C2\u00A0 PRIMARY\t \u00C2\u00A0NEURON\t \u00C2\u00A0CULTURE\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0WHOLE\t \u00C2\u00A0CELL\t \u00C2\u00A0LYSATE\t \u00C2\u00A0...............\t \u00C2\u00A065\t \u00C2\u00A02.2.3\t \u00C2\u00A0 PRIMARY\t \u00C2\u00A0NEURON\t \u00C2\u00A0CULTURE\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0NUCLEAR\t \u00C2\u00A0FRACTIONATION\t \u00C2\u00A0...\t \u00C2\u00A065\t \u00C2\u00A02.2.4\t \u00C2\u00A0 PRIMARY\t \u00C2\u00A0NEURON\t \u00C2\u00A0CULTURE\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0MITOCHONDRIAL\t \u00C2\u00A0FRACTIONATION\t \u00C2\u00A0...................................................................................................................................\t \u00C2\u00A067\t \u00C2\u00A02.2.5\t \u00C2\u00A0 ANIMAL\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0LYSATE\t \u00C2\u00A0...............................................................................\t \u00C2\u00A068\t \u00C2\u00A02.2.6\t \u00C2\u00A0 ANIMAL\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0PERFUSION\t \u00C2\u00A0AND\t \u00C2\u00A0FIXATION\t \u00C2\u00A0..........................................\t \u00C2\u00A069\t \u00C2\u00A02.3\t \u00C2\u00A0 PROTEIN\t \u00C2\u00A0AND\t \u00C2\u00A0PEPTIDE\t \u00C2\u00A0ANALYSIS\t \u00C2\u00A0..................................................................................\t \u00C2\u00A070\t \u00C2\u00A02.3.1\t \u00C2\u00A0 PROTEIN\t \u00C2\u00A0CONCENTRATION\t \u00C2\u00A0....................................................................................................\t \u00C2\u00A070\t \u00C2\u00A02.3.2\t \u00C2\u00A0 ELECTROPHORESIS\t \u00C2\u00A0AND\t \u00C2\u00A0WESTERN\t \u00C2\u00A0BLOT\t \u00C2\u00A0ANALYSIS\t \u00C2\u00A0.........................................................\t \u00C2\u00A071\t \u00C2\u00A02.3.3\t \u00C2\u00A0 CO-\u00C2\u00AD\u00E2\u0080\u0090IMMUNOPRECIPITATION\t \u00C2\u00A0(CO-\u00C2\u00AD\u00E2\u0080\u0090IP)\t \u00C2\u00A0....................................................................................\t \u00C2\u00A073\t \u00C2\u00A02.3.4\t \u00C2\u00A0 HIS-\u00C2\u00AD\u00E2\u0080\u0090TAGGED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0PURIFICATION\t \u00C2\u00A0AND\t \u00C2\u00A0COOMASSIE\t \u00C2\u00A0BLUE\t \u00C2\u00A0STAINING\t \u00C2\u00A0..................\t \u00C2\u00A074\t \u00C2\u00A02.3.5\t \u00C2\u00A0 SEQUENCES\t \u00C2\u00A0OF\t \u00C2\u00A0SYNTHETIC\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0..................................................................................\t \u00C2\u00A076\t \u00C2\u00A02.4\t \u00C2\u00A0 CELL\t \u00C2\u00A0DEATH\t \u00C2\u00A0ANALYSIS\t \u00C2\u00A0.....................................................................................................\t \u00C2\u00A076\t \u00C2\u00A02.5\t \u00C2\u00A0 HISTOLOGY,\t \u00C2\u00A0IMMUNOCYTOCHEMISTRY,\t \u00C2\u00A0AND\t \u00C2\u00A0IMMUNOHISTOCHEMISTRY\t \u00C2\u00A0.......\t \u00C2\u00A077\t \u00C2\u00A02.5.1\t \u00C2\u00A0 2,3,5-\u00C2\u00AD\u00E2\u0080\u0090TRIPHENYLTETRAZOLIUM\t \u00C2\u00A0CHLORIDE\t \u00C2\u00A0(TTC)\t \u00C2\u00A0STAINING\t \u00C2\u00A0............................................\t \u00C2\u00A077\t \u00C2\u00A02.5.2\t \u00C2\u00A0 FLUORO-\u00C2\u00AD\u00E2\u0080\u0090JADE\t \u00C2\u00A0B\t \u00C2\u00A0STAINING\t \u00C2\u00A0......................................................................................................\t \u00C2\u00A078\t \u00C2\u00A02.5.3\t \u00C2\u00A0 HEMATOXYLIN\t \u00C2\u00A0(MAYER\u00E2\u0080\u0099S)\t \u00C2\u00A0AND\t \u00C2\u00A0EOSIN\t \u00C2\u00A0Y\t \u00C2\u00A0STAINING\t \u00C2\u00A0..........................................................\t \u00C2\u00A078\t \u00C2\u00A02.5.4\t \u00C2\u00A0 IMMUNOCYTOCHEMISTRY\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0....................................................................................\t \u00C2\u00A079\t \u00C2\u00A02.5.5\t \u00C2\u00A0 IMMUNOHISTOCHEMISTRY\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0DAPK1\t \u00C2\u00A0....................................................................................\t \u00C2\u00A080\t \u00C2\u00A02.6\t \u00C2\u00A0 SIRNA\t \u00C2\u00A0KNOCKOUT,\t \u00C2\u00A0RNA\t \u00C2\u00A0ISOLATION\t \u00C2\u00A0AND\t \u00C2\u00A0RT-\u00C2\u00AD\u00E2\u0080\u0090PCR\t \u00C2\u00A0.....................................................\t \u00C2\u00A081\t \u00C2\u00A0 ix 2.6.1\t \u00C2\u00A0 SIRNA\t \u00C2\u00A0KNOCKOUT\t \u00C2\u00A0AND\t \u00C2\u00A0POLY-\u00C2\u00AD\u00E2\u0080\u0090CHAIN\t \u00C2\u00A0REACTION\t \u00C2\u00A0(PCR)\t \u00C2\u00A0....................................................\t \u00C2\u00A081\t \u00C2\u00A02.6.2\t \u00C2\u00A0 TOTAL\t \u00C2\u00A0RNA\t \u00C2\u00A0ISOLATION\t \u00C2\u00A0WITH\t \u00C2\u00A0TRIZOL\t \u00C2\u00A0................................................................................\t \u00C2\u00A082\t \u00C2\u00A02.6.3\t \u00C2\u00A0 DNA\t \u00C2\u00A0ELECTROPHORESIS\t \u00C2\u00A0AND\t \u00C2\u00A0REAL-\u00C2\u00AD\u00E2\u0080\u0090TIME\t \u00C2\u00A0PCR\t \u00C2\u00A0(RT-\u00C2\u00AD\u00E2\u0080\u0090PCR)\t \u00C2\u00A0.................................................\t \u00C2\u00A083\t \u00C2\u00A02.7\t \u00C2\u00A0 GENETIC\t \u00C2\u00A0ENGINEERING\t \u00C2\u00A0....................................................................................................\t \u00C2\u00A085\t \u00C2\u00A02.7.1\t \u00C2\u00A0 PLASMID\t \u00C2\u00A0AND\t \u00C2\u00A0MINIGENE\t \u00C2\u00A0SOURCE\t \u00C2\u00A0.........................................................................................\t \u00C2\u00A085\t \u00C2\u00A02.7.2\t \u00C2\u00A0 PLASMID\t \u00C2\u00A0AMPLIFICATION,\t \u00C2\u00A0PURIFICATION\t \u00C2\u00A0AND\t \u00C2\u00A0STORAGE\t \u00C2\u00A0.............................................\t \u00C2\u00A086\t \u00C2\u00A02.7.3\t \u00C2\u00A0 MINIGENE\t \u00C2\u00A0ANNEALING\t \u00C2\u00A0............................................................................................................\t \u00C2\u00A087\t \u00C2\u00A02.7.4\t \u00C2\u00A0 SITE-\u00C2\u00AD\u00E2\u0080\u0090DIRECTED\t \u00C2\u00A0MUTAGENESIS\t \u00C2\u00A0..............................................................................................\t \u00C2\u00A087\t \u00C2\u00A02.7.5\t \u00C2\u00A0 RESTRICTION,\t \u00C2\u00A0PURIFICATION\t \u00C2\u00A0AND\t \u00C2\u00A0LIGATION\t \u00C2\u00A0...................................................................\t \u00C2\u00A088\t \u00C2\u00A02.7.6\t \u00C2\u00A0 PLASMID\t \u00C2\u00A0PRODUCTION\t \u00C2\u00A0...........................................................................................................\t \u00C2\u00A090\t \u00C2\u00A02.7.6.1\t \u00C2\u00A0 PLASMIDS\t \u00C2\u00A0FOR\t \u00C2\u00A0GFP\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0................................................................................\t \u00C2\u00A090\t \u00C2\u00A02.7.6.2\t \u00C2\u00A0 PLASMIDS\t \u00C2\u00A0FOR\t \u00C2\u00A0\u00CE\u0091-\u00C2\u00AD\u00E2\u0080\u0090SYNUCLEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0.............................................................\t \u00C2\u00A090\t \u00C2\u00A02.7.6.3\t \u00C2\u00A0 PLASMIDS\t \u00C2\u00A0FOR\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0...........................................................................\t \u00C2\u00A094\t \u00C2\u00A02.8\t \u00C2\u00A0 ANIMAL\t \u00C2\u00A0MODELS\t \u00C2\u00A0...............................................................................................................\t \u00C2\u00A097\t \u00C2\u00A02.8.1\t \u00C2\u00A0 EXPERIMENTAL\t \u00C2\u00A0ANIMALS\t \u00C2\u00A0........................................................................................................\t \u00C2\u00A097\t \u00C2\u00A02.8.2\t \u00C2\u00A0 MIDDLE\t \u00C2\u00A0CEREBRAL\t \u00C2\u00A0ARTERIAL\t \u00C2\u00A0OCCLUSION\t \u00C2\u00A0(MCAO)\t \u00C2\u00A0.........................................................\t \u00C2\u00A097\t \u00C2\u00A0CHAPTER\t \u00C2\u00A03:\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0PROTEINS\t \u00C2\u00A0..................................................................................................................................\t \u00C2\u00A0103\t \u00C2\u00A03.1\t \u00C2\u00A0 INTRODUCTION\t \u00C2\u00A0.................................................................................................................\t \u00C2\u00A0103\t \u00C2\u00A03.2\t \u00C2\u00A0 RESULTS\t \u00C2\u00A0..............................................................................................................................\t \u00C2\u00A0104\t \u00C2\u00A03.2.1\t \u00C2\u00A0 KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090LINKED\t \u00C2\u00A0GREEN\t \u00C2\u00A0FLUORESCENT\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0(GFP)\t \u00C2\u00A0IN\t \u00C2\u00A0HUMAN\t \u00C2\u00A0CELLS.\t \u00C2\u00A0......................................................................................................................................................\t \u00C2\u00A0104\t \u00C2\u00A03.2.2\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0\u00CE\u0091-\u00C2\u00AD\u00E2\u0080\u0090SYNUCLEIN\t \u00C2\u00A0.........................\t \u00C2\u00A0106\t \u00C2\u00A03.2.3\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0ACTIVATED\t \u00C2\u00A0DAPK1\t \u00C2\u00A0................\t \u00C2\u00A0107\t \u00C2\u00A0 x 3.2.4\t \u00C2\u00A0 PLACING\t \u00C2\u00A0CTM\t \u00C2\u00A0AT\t \u00C2\u00A0THE\t \u00C2\u00A0TERMINUS\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER\t \u00C2\u00A0INCREASES\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0EFFICIENCY\t \u00C2\u00A0.............................................................................................................................................\t \u00C2\u00A0109\t \u00C2\u00A0CHAPTER\t \u00C2\u00A04:\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0ENDOGENOUS\t \u00C2\u00A0PROTEINS\t \u00C2\u00A0...................................................................................................................................\t \u00C2\u00A0122\t \u00C2\u00A04.1\t \u00C2\u00A0 INTRODUCTION\t \u00C2\u00A0.................................................................................................................\t \u00C2\u00A0122\t \u00C2\u00A04.2\t \u00C2\u00A0 KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0DAPK1\t \u00C2\u00A0WITH\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0IN\t \u00C2\u00A0CULTURED\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CELLS\t \u00C2\u00A0......................................................................................................................\t \u00C2\u00A0124\t \u00C2\u00A04.3\t \u00C2\u00A0 KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0DAPK1\t \u00C2\u00A0WITH\t \u00C2\u00A0SYNTHETIC\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0IN\t \u00C2\u00A0CULTURED\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CELLS\t \u00C2\u00A0......................................................................................................................\t \u00C2\u00A0126\t \u00C2\u00A04.4\t \u00C2\u00A0 ELUCIDATING\t \u00C2\u00A0THE\t \u00C2\u00A0MECHANISM\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN.\t \u00C2\u00A0............................................................................................................................\t \u00C2\u00A0128\t \u00C2\u00A04.5\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IS\t \u00C2\u00A0GENERALIZABLE\t \u00C2\u00A0......................\t \u00C2\u00A0130\t \u00C2\u00A04.5.1\t \u00C2\u00A0 PEPTIDE-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0..............................................\t \u00C2\u00A0130\t \u00C2\u00A04.5.2\t \u00C2\u00A0 PEPTIDE-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0\u00CE\u0091-\u00C2\u00AD\u00E2\u0080\u0090SYNUCLEIN\t \u00C2\u00A0AND\t \u00C2\u00A0ITS\t \u00C2\u00A0MUTANTS\t \u00C2\u00A0.............\t \u00C2\u00A0131\t \u00C2\u00A04.6\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IS\t \u00C2\u00A0SPECIFIC\t \u00C2\u00A0....................................\t \u00C2\u00A0134\t \u00C2\u00A04.7\t \u00C2\u00A0 A\t \u00C2\u00A0SINGLE\t \u00C2\u00A0CTM\t \u00C2\u00A0REPEAT\t \u00C2\u00A0IN\t \u00C2\u00A0SNIPER\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0IS\t \u00C2\u00A0SUFFICIENT\t \u00C2\u00A0FOR\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0.............................................................................................................................\t \u00C2\u00A0137\t \u00C2\u00A0CHAPTER\t \u00C2\u00A05:\t \u00C2\u00A0NEUROPROTECTION\t \u00C2\u00A0BY\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IN\t \u00C2\u00A0VITRO\t \u00C2\u00A0AND\t \u00C2\u00A0IN\t \u00C2\u00A0VIVO\t \u00C2\u00A0..........................................................................................................................\t \u00C2\u00A0161\t \u00C2\u00A05.1\t \u00C2\u00A0 INTRODUCTION\t \u00C2\u00A0.................................................................................................................\t \u00C2\u00A0161\t \u00C2\u00A05.2\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0PROTECTS\t \u00C2\u00A0AGAINST\t \u00C2\u00A0OXIDATIVE\t \u00C2\u00A0STRESS\t \u00C2\u00A0...........................................................................................................................................\t \u00C2\u00A0163\t \u00C2\u00A0 xi 5.3\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090PEPTIDE\t \u00C2\u00A0KNOCKS\t \u00C2\u00A0DOWN\t \u00C2\u00A0DAPK1\t \u00C2\u00A0IN\t \u00C2\u00A0THE\t \u00C2\u00A0CORTEX\t \u00C2\u00A0FOLLOWING\t \u00C2\u00A0EXCITOTOXIC\t \u00C2\u00A0STIMULI\t \u00C2\u00A0............................................................................................................\t \u00C2\u00A0164\t \u00C2\u00A05.4\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IS\t \u00C2\u00A0A\t \u00C2\u00A0NEUROPROTECTIVE\t \u00C2\u00A0.....................\t \u00C2\u00A0165\t \u00C2\u00A0CHAPTER\t \u00C2\u00A06:\t \u00C2\u00A0DISCUSSION\t \u00C2\u00A0AND\t \u00C2\u00A0CONCLUSIONS\t \u00C2\u00A0.....................................................\t \u00C2\u00A0175\t \u00C2\u00A06.1\t \u00C2\u00A0 OVERALL\t \u00C2\u00A0SIGNIFICANCE\t \u00C2\u00A0..................................................................................................\t \u00C2\u00A0175\t \u00C2\u00A06.2\t \u00C2\u00A0 METHODOLOGY\t \u00C2\u00A0................................................................................................................\t \u00C2\u00A0176\t \u00C2\u00A06.2.1\t \u00C2\u00A0 CTM\t \u00C2\u00A0IS\t \u00C2\u00A0A\t \u00C2\u00A0DEGRON\t \u00C2\u00A0FOR\t \u00C2\u00A0CMA\t \u00C2\u00A0..................................................................................................\t \u00C2\u00A0176\t \u00C2\u00A06.2.2\t \u00C2\u00A0 GENERALIZABILITY\t \u00C2\u00A0AND\t \u00C2\u00A0PREDICTABILITY\t \u00C2\u00A0.........................................................................\t \u00C2\u00A0180\t \u00C2\u00A06.2.3\t \u00C2\u00A0 SPECIFICITY\t \u00C2\u00A0................................................................................................................................\t \u00C2\u00A0181\t \u00C2\u00A06.2.4\t \u00C2\u00A0 TOXICITY\t \u00C2\u00A0....................................................................................................................................\t \u00C2\u00A0183\t \u00C2\u00A06.3\t \u00C2\u00A0 CLINICAL\t \u00C2\u00A0APPLICABILITY\t \u00C2\u00A0...............................................................................................\t \u00C2\u00A0184\t \u00C2\u00A06.3.1\t \u00C2\u00A0 PEPTIDE\t \u00C2\u00A0DELIVERY\t \u00C2\u00A0...................................................................................................................\t \u00C2\u00A0184\t \u00C2\u00A06.3.2\t \u00C2\u00A0 STROKE\t \u00C2\u00A0.......................................................................................................................................\t \u00C2\u00A0185\t \u00C2\u00A06.3.3\t \u00C2\u00A0 APPLICATION\t \u00C2\u00A0AND\t \u00C2\u00A0SAFETY\t \u00C2\u00A0....................................................................................................\t \u00C2\u00A0187\t \u00C2\u00A06.4\t \u00C2\u00A0 POTENTIAL\t \u00C2\u00A0LIMITATIONS\t \u00C2\u00A0AND\t \u00C2\u00A0FUTURE\t \u00C2\u00A0DIRECTIONS\t \u00C2\u00A0............................................\t \u00C2\u00A0188\t \u00C2\u00A06.4.1\t \u00C2\u00A0 PEPTIDE\t \u00C2\u00A0STABILITY\t \u00C2\u00A0...................................................................................................................\t \u00C2\u00A0188\t \u00C2\u00A06.4.2\t \u00C2\u00A0 TARGETED\t \u00C2\u00A0DELIVERY\t \u00C2\u00A0..............................................................................................................\t \u00C2\u00A0190\t \u00C2\u00A06.5\t \u00C2\u00A0 CONCLUDING\t \u00C2\u00A0REMARKS\t \u00C2\u00A0.................................................................................................\t \u00C2\u00A0191\t \u00C2\u00A0REFERENCES\t \u00C2\u00A0.............................................................................................................\t \u00C2\u00A0192\t \u00C2\u00A0APPENDICES\t \u00C2\u00A0............................................................................................................\t \u00C2\u00A0233\t \u00C2\u00A0\t \u00C2\u00A0 xii LIST\t \u00C2\u00A0OF\t \u00C2\u00A0TABLES\t \u00C2\u00A0\t \u00C2\u00A0Table\t \u00C2\u00A01-\u00C2\u00AD\u00E2\u0080\u00901.\t \u00C2\u00A0Post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0...................................................\t \u00C2\u00A055\t \u00C2\u00A0Table\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00901.\t \u00C2\u00A0Primary\t \u00C2\u00A0antibodies\t \u00C2\u00A0.................................................................................................\t \u00C2\u00A0100\t \u00C2\u00A0Table\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00902.\t \u00C2\u00A0Synthetic\t \u00C2\u00A0peptide\t \u00C2\u00A0sequences\t \u00C2\u00A0................................................................................\t \u00C2\u00A0102\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 xiii LIST\t \u00C2\u00A0OF\t \u00C2\u00A0FIGURES\t \u00C2\u00A0\t \u00C2\u00A0Figure\t \u00C2\u00A01-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0Schematic\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090peptide\t \u00C2\u00A0mediated\t \u00C2\u00A0knockdown.\t \u00C2\u00A0.........................................\t \u00C2\u00A058\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0......................................................................\t \u00C2\u00A0113\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0Time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0GFP.\t \u00C2\u00A0..........................................\t \u00C2\u00A0115\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein.\t \u00C2\u00A0.......................\t \u00C2\u00A0116\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1.\t \u00C2\u00A0...............................\t \u00C2\u00A0117\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905\t \u00C2\u00A0Enhancing\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0CTM\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide.\t \u00C2\u00A0........\t \u00C2\u00A0119\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0Activation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0GluN2B\t \u00C2\u00A0following\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatment.\t \u00C2\u00A0.....\t \u00C2\u00A0138\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0A\t \u00C2\u00A0recombinant\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0specifically\t \u00C2\u00A0degrades\t \u00C2\u00A0activated\t \u00C2\u00A0endogenous\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0culture.\t \u00C2\u00A0..............................................................................\t \u00C2\u00A0139\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0Dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0synthetic\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM.\t \u00C2\u00A0............................................................................................................................\t \u00C2\u00A0141\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00904\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0alter\t \u00C2\u00A0DAPK1\t \u00C2\u00A0mRNA\t \u00C2\u00A0levels.\t \u00C2\u00A0....................................\t \u00C2\u00A0142\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00905\t \u00C2\u00A0Pep1-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0intracellular\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0synthetic\t \u00C2\u00A0GluN2BCTM\t \u00C2\u00A0peptide\t \u00C2\u00A0specifically\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons.\t \u00C2\u00A0.......................................\t \u00C2\u00A0143\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00906\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\u00E2\u0080\u0093induced\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0reduces\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0various\t \u00C2\u00A0subcellular\t \u00C2\u00A0compartments\t \u00C2\u00A0in\t \u00C2\u00A0neuron\t \u00C2\u00A0cultures.\t \u00C2\u00A0.......................................................\t \u00C2\u00A0145\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00907\t \u00C2\u00A0Macroautophagy\t \u00C2\u00A0inhibition\t \u00C2\u00A0cannot\t \u00C2\u00A0rescue\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090peptide\t \u00C2\u00A0mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0.............................................................................................................................\t \u00C2\u00A0147\t \u00C2\u00A0 xiv Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00908\t \u00C2\u00A0siRNA-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0reduces\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0induced DAPK1\t \u00C2\u00A0degradation.\t \u00C2\u00A0................................................................................................................\t \u00C2\u00A0149\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00909\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons.\t \u00C2\u00A0....................................................................................................................................\t \u00C2\u00A0151\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009010\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0are\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM.\t \u00C2\u00A0.\t \u00C2\u00A0153\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009011\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0are\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfected\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells.\t \u00C2\u00A0.....................................................................................\t \u00C2\u00A0155\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009012\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0is\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1.\t \u00C2\u00A0...........................................................\t \u00C2\u00A0157\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009013\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0were\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090toxic\t \u00C2\u00A024h\t \u00C2\u00A0after\t \u00C2\u00A0treatment.\t \u00C2\u00A0.....................................\t \u00C2\u00A0158\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009014\t \u00C2\u00A0A\t \u00C2\u00A0single\t \u00C2\u00A0CTM\t \u00C2\u00A0repeat\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0.......................\t \u00C2\u00A0159\t \u00C2\u00A0Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090activated\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0protecting\t \u00C2\u00A0neurons\t \u00C2\u00A0against\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neurotoxicity\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0cultures.\t \u00C2\u00A0................................................\t \u00C2\u00A0169\t \u00C2\u00A0Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cortex\t \u00C2\u00A0of\t \u00C2\u00A0animals\t \u00C2\u00A0following\t \u00C2\u00A0KA\t \u00C2\u00A0treatment.\t \u00C2\u00A0.................................................................................................................................\t \u00C2\u00A0171\t \u00C2\u00A0Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0specifically\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0ischemic\t \u00C2\u00A0brain\t \u00C2\u00A0areas\t \u00C2\u00A0and reduces\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0MCAo\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0focal\t \u00C2\u00A0ischemia\t \u00C2\u00A0in\t \u00C2\u00A0rats.\t \u00C2\u00A0..........................\t \u00C2\u00A0172\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 xv LIST\t \u00C2\u00A0OF\t \u00C2\u00A0ABBREVIATIONS\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA\t \u00C2\u00A0 3-\u00C2\u00AD\u00E2\u0080\u0090methyladenine\t \u00C2\u00A0ACC\t \u00C2\u00A0 Animal\t \u00C2\u00A0Care\t \u00C2\u00A0Committee\t \u00C2\u00A0AGO2\t \u00C2\u00A0 Argonaute\t \u00C2\u00A0protein\t \u00C2\u00A02\t \u00C2\u00A0AID\t \u00C2\u00A0 Auxin-\u00C2\u00AD\u00E2\u0080\u0090responsive\t \u00C2\u00A0degron\t \u00C2\u00A0ALS\t \u00C2\u00A0 Autophagy-\u00C2\u00AD\u00E2\u0080\u0090lysosome\t \u00C2\u00A0system\t \u00C2\u00A0APC\t \u00C2\u00A0 Anaphase-\u00C2\u00AD\u00E2\u0080\u0090promoting\t \u00C2\u00A0complex\t \u00C2\u00A0ASO\t \u00C2\u00A0 Anti-\u00C2\u00AD\u00E2\u0080\u0090sense\t \u00C2\u00A0oligonucleotides\t \u00C2\u00A0ATG\t \u00C2\u00A0 Autophagy-\u00C2\u00AD\u00E2\u0080\u0090related\t \u00C2\u00A0genes\t \u00C2\u00A0BBB\t \u00C2\u00A0 Blood\t \u00C2\u00A0brain\t \u00C2\u00A0barrier\t \u00C2\u00A0CCA\t \u00C2\u00A0 Common\t \u00C2\u00A0carotid\t \u00C2\u00A0artery\t \u00C2\u00A0CMA\t \u00C2\u00A0 Chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0CNS\t \u00C2\u00A0 Central\t \u00C2\u00A0nervous\t \u00C2\u00A0system\t \u00C2\u00A0CTM\t \u00C2\u00A0 CMA\t \u00C2\u00A0targeting\t \u00C2\u00A0motif\t \u00C2\u00A0CPP\t \u00C2\u00A0 Cell\t \u00C2\u00A0penetrating\t \u00C2\u00A0peptide\t \u00C2\u00A0 xvi CRISPR\t \u00C2\u00A0 Clustered\t \u00C2\u00A0regularly\t \u00C2\u00A0interspaced\t \u00C2\u00A0short\t \u00C2\u00A0palindromic\t \u00C2\u00A0repeat\t \u00C2\u00A0CRY2\t \u00C2\u00A0 Cryptochrome\t \u00C2\u00A02\t \u00C2\u00A0dsRNA\t \u00C2\u00A0 Double-\u00C2\u00AD\u00E2\u0080\u0090stranded\t \u00C2\u00A0RNA\t \u00C2\u00A0DAPK1\t \u00C2\u00A0 Death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A01\t \u00C2\u00A0DB\t \u00C2\u00A0 Dissection\t \u00C2\u00A0buffer\t \u00C2\u00A0DEPC\t \u00C2\u00A0 Diethylpyrocarbonate\t \u00C2\u00A0DHFR\t \u00C2\u00A0 Dihydrofolate\t \u00C2\u00A0reductase\t \u00C2\u00A0DMEM\t \u00C2\u00A0 Dulbecco\u00E2\u0080\u0099s\t \u00C2\u00A0modified\t \u00C2\u00A0eagle\t \u00C2\u00A0medium\t \u00C2\u00A0ECA\t \u00C2\u00A0 External\t \u00C2\u00A0carotid\t \u00C2\u00A0artery\t \u00C2\u00A0EF1a\t \u00C2\u00A0 Elongation\t \u00C2\u00A0factor\t \u00C2\u00A01a\t \u00C2\u00A0ESC\t \u00C2\u00A0 Embryonic\t \u00C2\u00A0stem\t \u00C2\u00A0cell\t \u00C2\u00A0FBS\t \u00C2\u00A0 Fetal\t \u00C2\u00A0bovine\t \u00C2\u00A0serum\t \u00C2\u00A0FKBP12\t \u00C2\u00A0 FK506\t \u00C2\u00A0and\t \u00C2\u00A0rapamycin-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0protein\t \u00C2\u00A012\t \u00C2\u00A0GFAP\t \u00C2\u00A0 Glial\t \u00C2\u00A0fibrillary\t \u00C2\u00A0acidic\t \u00C2\u00A0protein\t \u00C2\u00A0GSK-\u00C2\u00AD\u00E2\u0080\u00903\u00CE\u00B2\t \u00C2\u00A0 Glycogen\t \u00C2\u00A0synthase\t \u00C2\u00A0kinase\t \u00C2\u00A03\u00CE\u00B2\t \u00C2\u00A0HD\t \u00C2\u00A0 Huntington\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0 xvii HGP\t \u00C2\u00A0 Human\t \u00C2\u00A0Genome\t \u00C2\u00A0Project\t \u00C2\u00A0HPLC\t \u00C2\u00A0 High\t \u00C2\u00A0performance\t \u00C2\u00A0liquid\t \u00C2\u00A0chromatography\t \u00C2\u00A0Hsc70\t \u00C2\u00A0 Heat\t \u00C2\u00A0shock\t \u00C2\u00A0cognate\t \u00C2\u00A070\t \u00C2\u00A0Htt\t \u00C2\u00A0 Huntingtin\t \u00C2\u00A0ICA\t \u00C2\u00A0 Internal\t \u00C2\u00A0carotid\t \u00C2\u00A0artery\t \u00C2\u00A0i.c.v.\t \u00C2\u00A0 Intracerebroventricular\t \u00C2\u00A0i.n.\t \u00C2\u00A0 Intranasal\t \u00C2\u00A0i.p.\t \u00C2\u00A0 Intraperitoneal\t \u00C2\u00A0IPTG\t \u00C2\u00A0 Isopropyl\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090D-\u00C2\u00AD\u00E2\u0080\u00901-\u00C2\u00AD\u00E2\u0080\u0090thiogalactopyranoside\t \u00C2\u00A0KA\t \u00C2\u00A0 Kainic\t \u00C2\u00A0acid\t \u00C2\u00A0LAMP1\t \u00C2\u00A0 Lysosome-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0membrane\t \u00C2\u00A0protein\t \u00C2\u00A01\t \u00C2\u00A0LAMP2\t \u00C2\u00A0 Lysosome-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0membrane\t \u00C2\u00A0protein\t \u00C2\u00A02\t \u00C2\u00A0LID\t \u00C2\u00A0 Ligand-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0degradation\t \u00C2\u00A0LMP\t \u00C2\u00A0 Lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0protein\t \u00C2\u00A0LOVE\t \u00C2\u00A0 Light-\u00C2\u00AD\u00E2\u0080\u0090oxygen-\u00C2\u00AD\u00E2\u0080\u0090voltage\t \u00C2\u00A0loxP\t \u00C2\u00A0 Locus\t \u00C2\u00A0of\t \u00C2\u00A0X-\u00C2\u00AD\u00E2\u0080\u0090over\t \u00C2\u00A0P1\t \u00C2\u00A0 xviii MCAo\t \u00C2\u00A0 Middle\t \u00C2\u00A0cerebral\t \u00C2\u00A0arterial\t \u00C2\u00A0occlusion\t \u00C2\u00A0MetAP-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0 Methionine\t \u00C2\u00A0aminopeptidase-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0mHtt\t \u00C2\u00A0 Mutant\t \u00C2\u00A0huntingtin\t \u00C2\u00A0MS\t \u00C2\u00A0 Mass\t \u00C2\u00A0spectrometry\t \u00C2\u00A0MTX\t \u00C2\u00A0 Methotrexate\t \u00C2\u00A0NMDA\t \u00C2\u00A0 N-\u00C2\u00AD\u00E2\u0080\u0090methyl\t \u00C2\u00A0D-\u00C2\u00AD\u00E2\u0080\u0090aspartate\t \u00C2\u00A0NMDAR\t \u00C2\u00A0 N-\u00C2\u00AD\u00E2\u0080\u0090methyl\t \u00C2\u00A0D-\u00C2\u00AD\u00E2\u0080\u0090aspartate\t \u00C2\u00A0Receptor\t \u00C2\u00A0NP\t \u00C2\u00A0 Neurobasal\t \u00C2\u00A0plating\t \u00C2\u00A0media\t \u00C2\u00A0OCT\t \u00C2\u00A0 Optimal\t \u00C2\u00A0Cutting\t \u00C2\u00A0Temperature\t \u00C2\u00A0PBD\t \u00C2\u00A0 Protein\t \u00C2\u00A0binding\t \u00C2\u00A0domain\t \u00C2\u00A0PD\t \u00C2\u00A0 Parkinson\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0PDL\t \u00C2\u00A0 Poly-\u00C2\u00AD\u00E2\u0080\u0090D-\u00C2\u00AD\u00E2\u0080\u0090lysine\t \u00C2\u00A0PEG\t \u00C2\u00A0 Polyethylene\t \u00C2\u00A0glycol\t \u00C2\u00A0PepA\t \u00C2\u00A0 Pepstatin\t \u00C2\u00A0A\t \u00C2\u00A0PFA\t \u00C2\u00A0 Paraformaldehyde\t \u00C2\u00A0PKM2\t \u00C2\u00A0 Pyruvate\t \u00C2\u00A0kinase\t \u00C2\u00A0embryonic\t \u00C2\u00A0M2\t \u00C2\u00A0isoform\t \u00C2\u00A0 xix POI\t \u00C2\u00A0 Protein-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090interest\t \u00C2\u00A0PROTACs\t \u00C2\u00A0 Proteolysis\t \u00C2\u00A0targeting\t \u00C2\u00A0chimeric\t \u00C2\u00A0molecules\t \u00C2\u00A0PSD\t \u00C2\u00A0 Post-\u00C2\u00AD\u00E2\u0080\u0090synaptic\t \u00C2\u00A0density\t \u00C2\u00A0PVDF\t \u00C2\u00A0 Polyvinyl\t \u00C2\u00A0difluoride\t \u00C2\u00A0RISC\t \u00C2\u00A0 RNAi-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0silencing\t \u00C2\u00A0complex\t \u00C2\u00A0RNAi\t \u00C2\u00A0 RNA\t \u00C2\u00A0interference\t \u00C2\u00A0RNase\t \u00C2\u00A0A\t \u00C2\u00A0 Ribonuclease\t \u00C2\u00A0A\t \u00C2\u00A0RNase\t \u00C2\u00A0H\t \u00C2\u00A0 Ribonuclease\t \u00C2\u00A0H\t \u00C2\u00A0ROS\t \u00C2\u00A0 Reactive\t \u00C2\u00A0oxygen\t \u00C2\u00A0species\t \u00C2\u00A0rtTA\t \u00C2\u00A0 Reverse\t \u00C2\u00A0tetracycline-\u00C2\u00AD\u00E2\u0080\u0090responsive\t \u00C2\u00A0transactivator\t \u00C2\u00A0SCF\t \u00C2\u00A0 Skp1\u00E2\u0080\u0093Cullin\u00E2\u0080\u0093F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein\t \u00C2\u00A0SDS-\u00C2\u00AD\u00E2\u0080\u0090PAGE\t \u00C2\u00A0 Sodium\t \u00C2\u00A0dodecyl\t \u00C2\u00A0sulfate\t \u00C2\u00A0polyacrylamide\t \u00C2\u00A0gel\t \u00C2\u00A0electrophoresis\t \u00C2\u00A0siRNA\t \u00C2\u00A0 Short\t \u00C2\u00A0interfering\t \u00C2\u00A0RNA\t \u00C2\u00A0shRNA\t \u00C2\u00A0 Short\t \u00C2\u00A0hairpin\t \u00C2\u00A0RNA\t \u00C2\u00A0SNIPER\t \u00C2\u00A0 Selective\t \u00C2\u00A0native\t \u00C2\u00A0protein\t \u00C2\u00A0eradication\t \u00C2\u00A0TALEN\t \u00C2\u00A0 Transcription\t \u00C2\u00A0activator-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0effector\t \u00C2\u00A0nucleases\t \u00C2\u00A0 xx TTC\t \u00C2\u00A0 2,3,5-\u00C2\u00AD\u00E2\u0080\u0090Triphenyltetrazolium\t \u00C2\u00A0chloride\t \u00C2\u00A0UPS\t \u00C2\u00A0 Ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090proteasome\t \u00C2\u00A0system\t \u00C2\u00A0ZFN\t \u00C2\u00A0 Zinc-\u00C2\u00AD\u00E2\u0080\u0090finger\t \u00C2\u00A0nucleases\t \u00C2\u00A0\t \u00C2\u00A0 xxi LIST\t \u00C2\u00A0OF\t \u00C2\u00A0PLASMIDS\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0pEGFP-\u00C2\u00AD\u00E2\u0080\u0090N2\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090A1Q2-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090Q1A2-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0 \t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090A1A2-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(mCTM-\u00C2\u00AD\u00E2\u0080\u0090GFP)\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn-\u00C2\u00AD\u00E2\u0080\u0090degron\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36KFERQ\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36QKILD\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36QRFFE\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36KFERQKILD\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36QKILDQRFFE\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36KFERQQRFFE\t \u00C2\u00A0 xxii WT\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(A53T)\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm\t \u00C2\u00A06X\t \u00C2\u00A0His-\u00C2\u00AD\u00E2\u0080\u0090TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A06X\t \u00C2\u00A0His-\u00C2\u00AD\u00E2\u0080\u0090TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090DAPK1(\u00CE\u0094CaM)\t \u00C2\u00A0 xxiii ACKNOWLEDGEMENTS\t \u00C2\u00A0First,\t \u00C2\u00A0I\u00E2\u0080\u0099d\t \u00C2\u00A0like\t \u00C2\u00A0to\t \u00C2\u00A0thank\t \u00C2\u00A0my\t \u00C2\u00A0graduate\t \u00C2\u00A0supervisor\t \u00C2\u00A0Dr.\t \u00C2\u00A0Yu\t \u00C2\u00A0Tian\t \u00C2\u00A0Wang\t \u00C2\u00A0for\t \u00C2\u00A0his\t \u00C2\u00A0excellent\t \u00C2\u00A0guidance\t \u00C2\u00A0and\t \u00C2\u00A0patient\t \u00C2\u00A0supervision.\t \u00C2\u00A0His\t \u00C2\u00A0passion\t \u00C2\u00A0for\t \u00C2\u00A0scientific\t \u00C2\u00A0research\t \u00C2\u00A0and\t \u00C2\u00A0extraordinarily\t \u00C2\u00A0keen\t \u00C2\u00A0eye\t \u00C2\u00A0towards\t \u00C2\u00A0key\t \u00C2\u00A0outstanding\t \u00C2\u00A0problems\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0field\t \u00C2\u00A0is\t \u00C2\u00A0particularly\t \u00C2\u00A0inspiring.\t \u00C2\u00A0His\t \u00C2\u00A0critical\t \u00C2\u00A0thinking\t \u00C2\u00A0and\t \u00C2\u00A0rigorous\t \u00C2\u00A0logic\t \u00C2\u00A0in\t \u00C2\u00A0experimental\t \u00C2\u00A0design\t \u00C2\u00A0and\t \u00C2\u00A0data\t \u00C2\u00A0interpretation\t \u00C2\u00A0will\t \u00C2\u00A0leave\t \u00C2\u00A0long-\u00C2\u00AD\u00E2\u0080\u0090lasting\t \u00C2\u00A0impact\t \u00C2\u00A0on\t \u00C2\u00A0my\t \u00C2\u00A0future\t \u00C2\u00A0research\t \u00C2\u00A0endeavors.\t \u00C2\u00A0I\t \u00C2\u00A0would\t \u00C2\u00A0also\t \u00C2\u00A0like\t \u00C2\u00A0to\t \u00C2\u00A0thank\t \u00C2\u00A0my\t \u00C2\u00A0committee\t \u00C2\u00A0members\t \u00C2\u00A0Dr.\t \u00C2\u00A0Brian\t \u00C2\u00A0MacVicar,\t \u00C2\u00A0Dr.\t \u00C2\u00A0Terry\t \u00C2\u00A0Snutch\t \u00C2\u00A0and\t \u00C2\u00A0Dr.\t \u00C2\u00A0Ann\t \u00C2\u00A0Marie\t \u00C2\u00A0Craig,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0my\t \u00C2\u00A0program\t \u00C2\u00A0head\t \u00C2\u00A0Dr.\t \u00C2\u00A0Tim\t \u00C2\u00A0O\u00E2\u0080\u0099Conner\t \u00C2\u00A0for\t \u00C2\u00A0their\t \u00C2\u00A0invaluable\t \u00C2\u00A0guidance\t \u00C2\u00A0and\t \u00C2\u00A0constructive\t \u00C2\u00A0advice\t \u00C2\u00A0for\t \u00C2\u00A0my\t \u00C2\u00A0project.\t \u00C2\u00A0I\t \u00C2\u00A0would\t \u00C2\u00A0also\t \u00C2\u00A0like\t \u00C2\u00A0to\t \u00C2\u00A0thank\t \u00C2\u00A0Dr.\t \u00C2\u00A0Brian\t \u00C2\u00A0MacVicar\t \u00C2\u00A0and\t \u00C2\u00A0Dr.\t \u00C2\u00A0Lynn\t \u00C2\u00A0Raymond\t \u00C2\u00A0for\t \u00C2\u00A0graciously\t \u00C2\u00A0offering\t \u00C2\u00A0me\t \u00C2\u00A0the\t \u00C2\u00A0opportunity\t \u00C2\u00A0to\t \u00C2\u00A0study\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0laboratories\t \u00C2\u00A0during\t \u00C2\u00A0rotations\t \u00C2\u00A0in\t \u00C2\u00A0my\t \u00C2\u00A0first\t \u00C2\u00A0year\t \u00C2\u00A0of\t \u00C2\u00A0PhD\t \u00C2\u00A0studies.\t \u00C2\u00A0\t \u00C2\u00A0My\t \u00C2\u00A0PhD\t \u00C2\u00A0training\t \u00C2\u00A0would\t \u00C2\u00A0not\t \u00C2\u00A0be\t \u00C2\u00A0possible\t \u00C2\u00A0if\t \u00C2\u00A0not\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0generosity\t \u00C2\u00A0of\t \u00C2\u00A0multiple\t \u00C2\u00A0organizations\t \u00C2\u00A0for\t \u00C2\u00A0offering\t \u00C2\u00A0me\t \u00C2\u00A0financial\t \u00C2\u00A0support,\t \u00C2\u00A0including:\t \u00C2\u00A0the\t \u00C2\u00A0University\t \u00C2\u00A0of\t \u00C2\u00A0British\t \u00C2\u00A0Columbia\t \u00C2\u00A0Four-\u00C2\u00AD\u00E2\u0080\u0090Year\t \u00C2\u00A0Fellowship,\t \u00C2\u00A0the\t \u00C2\u00A0Izaak\t \u00C2\u00A0Walton\t \u00C2\u00A0Killam\t \u00C2\u00A0Memorial\t \u00C2\u00A0Doctoral\t \u00C2\u00A0Scholarship\t \u00C2\u00A0and\t \u00C2\u00A0Travel\t \u00C2\u00A0Award,\t \u00C2\u00A0the\t \u00C2\u00A0College\t \u00C2\u00A0for\t \u00C2\u00A0Interdisciplinary\t \u00C2\u00A0Studies\t \u00C2\u00A0Award,\t \u00C2\u00A0the\t \u00C2\u00A0CIHR\t \u00C2\u00A0Training\t \u00C2\u00A0Program\t \u00C2\u00A0in\t \u00C2\u00A0Neurobiology\t \u00C2\u00A0and\t \u00C2\u00A0Behaviour\t \u00C2\u00A0Fellowship\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0Graduate\t \u00C2\u00A0Entrance\t \u00C2\u00A0Scholarship.\t \u00C2\u00A0Various\t \u00C2\u00A0travel\t \u00C2\u00A0awards\t \u00C2\u00A0have\t \u00C2\u00A0allowed\t \u00C2\u00A0me\t \u00C2\u00A0to\t \u00C2\u00A0present\t \u00C2\u00A0my\t \u00C2\u00A0work\t \u00C2\u00A0at\t \u00C2\u00A0national\t \u00C2\u00A0and\t \u00C2\u00A0international\t \u00C2\u00A0conferences,\t \u00C2\u00A0including\t \u00C2\u00A0the\t \u00C2\u00A0Canadian\t \u00C2\u00A0Association\t \u00C2\u00A0for\t \u00C2\u00A0Neuroscience\t \u00C2\u00A0Travel\t \u00C2\u00A0Award\t \u00C2\u00A0and\t \u00C2\u00A0College\t \u00C2\u00A0for\t \u00C2\u00A0Interdisciplinary\t \u00C2\u00A0Studies\t \u00C2\u00A0Travel\t \u00C2\u00A0Award.\t \u00C2\u00A0Finally,\t \u00C2\u00A0I\u00E2\u0080\u0099d\t \u00C2\u00A0like\t \u00C2\u00A0to\t \u00C2\u00A0thank\t \u00C2\u00A0the\t \u00C2\u00A0Canadian\t \u00C2\u00A0Science\t \u00C2\u00A0Writers\u00E2\u0080\u0099\t \u00C2\u00A0Association\t \u00C2\u00A0for\t \u00C2\u00A0offering\t \u00C2\u00A0me\t \u00C2\u00A0the\t \u00C2\u00A0Banff\t \u00C2\u00A0Science\t \u00C2\u00A0Award\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0Banff\t \u00C2\u00A0 xxiv Centre\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0substantial\t \u00C2\u00A0scholarship\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0Banff\t \u00C2\u00A0Centre\u00E2\u0080\u0099s\t \u00C2\u00A0Science\t \u00C2\u00A0Communications\t \u00C2\u00A0Program,\t \u00C2\u00A0which\t \u00C2\u00A0helped\t \u00C2\u00A0broaden\t \u00C2\u00A0my\t \u00C2\u00A0scope\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0scientist\t \u00C2\u00A0and\t \u00C2\u00A0science\t \u00C2\u00A0communicator.\t \u00C2\u00A0The\t \u00C2\u00A0work\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0would\t \u00C2\u00A0not\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0possible\t \u00C2\u00A0without\t \u00C2\u00A0the\t \u00C2\u00A0help\t \u00C2\u00A0and\t \u00C2\u00A0support\t \u00C2\u00A0of\t \u00C2\u00A0my\t \u00C2\u00A0colleagues.\t \u00C2\u00A0I\t \u00C2\u00A0would\t \u00C2\u00A0particularly\t \u00C2\u00A0like\t \u00C2\u00A0to\t \u00C2\u00A0thank\t \u00C2\u00A0Ester\t \u00C2\u00A0Yu,\t \u00C2\u00A0Dr.\t \u00C2\u00A0Shu\t \u00C2\u00A0Zhang,\t \u00C2\u00A0Dr.\t \u00C2\u00A0Jie\t \u00C2\u00A0Lu,\t \u00C2\u00A0Dr.\t \u00C2\u00A0Loren\t \u00C2\u00A0Oschipok\t \u00C2\u00A0and\t \u00C2\u00A0Dr.\t \u00C2\u00A0Yuan\t \u00C2\u00A0Ge\t \u00C2\u00A0for\t \u00C2\u00A0patiently\t \u00C2\u00A0helping\t \u00C2\u00A0me\t \u00C2\u00A0orient\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0laboratory.\t \u00C2\u00A0I\t \u00C2\u00A0am\t \u00C2\u00A0especially\t \u00C2\u00A0indebted\t \u00C2\u00A0to\t \u00C2\u00A0Yuping\t \u00C2\u00A0Li\t \u00C2\u00A0for\t \u00C2\u00A0years\t \u00C2\u00A0of\t \u00C2\u00A0her\t \u00C2\u00A0technical\t \u00C2\u00A0assistance\t \u00C2\u00A0and\t \u00C2\u00A0care.\t \u00C2\u00A0I\t \u00C2\u00A0have\t \u00C2\u00A0also\t \u00C2\u00A0received\t \u00C2\u00A0invaluable\t \u00C2\u00A0advice\t \u00C2\u00A0from\t \u00C2\u00A0Dr.\t \u00C2\u00A0Lidong\t \u00C2\u00A0Liu\t \u00C2\u00A0over\t \u00C2\u00A0the\t \u00C2\u00A0years.\t \u00C2\u00A0I\t \u00C2\u00A0would\t \u00C2\u00A0also\t \u00C2\u00A0like\t \u00C2\u00A0to\t \u00C2\u00A0thank\t \u00C2\u00A0my\t \u00C2\u00A0labmates\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin,\t \u00C2\u00A0Kim\t \u00C2\u00A0Girling\t \u00C2\u00A0and\t \u00C2\u00A0Peter\t \u00C2\u00A0Axerio-\u00C2\u00AD\u00E2\u0080\u0090Cilies\t \u00C2\u00A0for\t \u00C2\u00A0excellent\t \u00C2\u00A0collaborations,\t \u00C2\u00A0discussions\t \u00C2\u00A0and\t \u00C2\u00A0troubleshooting\t \u00C2\u00A0during\t \u00C2\u00A0my\t \u00C2\u00A0PhD\t \u00C2\u00A0studies,\t \u00C2\u00A0and\t \u00C2\u00A0for\t \u00C2\u00A0keeping\t \u00C2\u00A0my\t \u00C2\u00A0spirits\t \u00C2\u00A0up\t \u00C2\u00A0when\t \u00C2\u00A0experiments\t \u00C2\u00A0fail.\t \u00C2\u00A0I\t \u00C2\u00A0was\t \u00C2\u00A0fortunate\t \u00C2\u00A0to\t \u00C2\u00A0(co)supervise\t \u00C2\u00A0many\t \u00C2\u00A0talented\t \u00C2\u00A0summer\t \u00C2\u00A0students,\t \u00C2\u00A0including\t \u00C2\u00A0Brandon\t \u00C2\u00A0Chau,\t \u00C2\u00A0Nelson\t \u00C2\u00A0Lu\t \u00C2\u00A0and\t \u00C2\u00A0Sam\t \u00C2\u00A0Sedyim.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0Last\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0least,\t \u00C2\u00A0I\t \u00C2\u00A0would\t \u00C2\u00A0like\t \u00C2\u00A0to\t \u00C2\u00A0thank\t \u00C2\u00A0my\t \u00C2\u00A0brilliant\t \u00C2\u00A0parents\t \u00C2\u00A0Dr.\t \u00C2\u00A0Ping\t \u00C2\u00A0Liang\t \u00C2\u00A0and\t \u00C2\u00A0Dr.\t \u00C2\u00A0Ming\t \u00C2\u00A0Fan\t \u00C2\u00A0for\t \u00C2\u00A0giving\t \u00C2\u00A0me\t \u00C2\u00A0the\t \u00C2\u00A0best\t \u00C2\u00A0education\t \u00C2\u00A0in\t \u00C2\u00A0my\t \u00C2\u00A0childhood\t \u00C2\u00A0and\t \u00C2\u00A0for\t \u00C2\u00A0their\t \u00C2\u00A0eternal\t \u00C2\u00A0unconditional\t \u00C2\u00A0love\t \u00C2\u00A0and\t \u00C2\u00A0support.\t \u00C2\u00A0\t \u00C2\u00A0 xxv DEDICATION\t \u00C2\u00A0\t \u00C2\u00A0For\t \u00C2\u00A0A.K.,\t \u00C2\u00A0B.A.\t \u00C2\u00A0and\t \u00C2\u00A0my\t \u00C2\u00A0parents.\t \u00C2\u00A0For\t \u00C2\u00A0always\t \u00C2\u00A0being\t \u00C2\u00A0there.\t \u00C2\u00A0 1 CHAPTER\t \u00C2\u00A01: INTRODUCTION\t \u00C2\u00A0\t \u00C2\u00A01.1 OVERVIEW\t \u00C2\u00A0In\t \u00C2\u00A01990,\t \u00C2\u00A0the\t \u00C2\u00A0Human\t \u00C2\u00A0Genome\t \u00C2\u00A0Project\t \u00C2\u00A0(HGP)\t \u00C2\u00A0launched\t \u00C2\u00A0a\t \u00C2\u00A0global\t \u00C2\u00A0effort\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0our\t \u00C2\u00A0understanding\t \u00C2\u00A0of\t \u00C2\u00A0basic\t \u00C2\u00A0human\t \u00C2\u00A0physiology\t \u00C2\u00A0and\t \u00C2\u00A0genetically\t \u00C2\u00A0inherited\t \u00C2\u00A0diseases.\t \u00C2\u00A0With\t \u00C2\u00A0the\t \u00C2\u00A0completion\t \u00C2\u00A0of\t \u00C2\u00A0its\t \u00C2\u00A0final\t \u00C2\u00A0phase\t \u00C2\u00A0in\t \u00C2\u00A02004,\t \u00C2\u00A0anticipation\t \u00C2\u00A0was\t \u00C2\u00A0high\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0genetic\t \u00C2\u00A0information\t \u00C2\u00A0gathered\t \u00C2\u00A0would\t \u00C2\u00A0propel\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0aimed\t \u00C2\u00A0at\t \u00C2\u00A0elucidating\t \u00C2\u00A0the\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0products,\t \u00C2\u00A0including\t \u00C2\u00A0RNAs\t \u00C2\u00A0and\t \u00C2\u00A0proteins.\t \u00C2\u00A0The\t \u00C2\u00A0HGP\t \u00C2\u00A0was\t \u00C2\u00A0also\t \u00C2\u00A0predicted\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0systematic\t \u00C2\u00A0identification\t \u00C2\u00A0of\t \u00C2\u00A0key\t \u00C2\u00A0heritable\t \u00C2\u00A0factors\t \u00C2\u00A0that\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0disease,\t \u00C2\u00A0thus\t \u00C2\u00A0opening\t \u00C2\u00A0new\t \u00C2\u00A0avenues\t \u00C2\u00A0for\t \u00C2\u00A0drug\t \u00C2\u00A0discovery.\t \u00C2\u00A0Indeed,\t \u00C2\u00A0as\t \u00C2\u00A0many\t \u00C2\u00A0as\t \u00C2\u00A05,000-\u00C2\u00AD\u00E2\u0080\u009010,000\t \u00C2\u00A0new\t \u00C2\u00A0drug\t \u00C2\u00A0targets\t \u00C2\u00A0may\t \u00C2\u00A0exist\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0genome\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0some\t \u00C2\u00A0estimations\t \u00C2\u00A0(Drews,\t \u00C2\u00A01998).\t \u00C2\u00A0\t \u00C2\u00A0Despite\t \u00C2\u00A0further\t \u00C2\u00A0advances\t \u00C2\u00A0in\t \u00C2\u00A0genomic\t \u00C2\u00A0and\t \u00C2\u00A0proteomic\t \u00C2\u00A0tools\t \u00C2\u00A0over\t \u00C2\u00A0the\t \u00C2\u00A0past\t \u00C2\u00A0two\t \u00C2\u00A0decades,\t \u00C2\u00A0our\t \u00C2\u00A0understanding\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0individual\t \u00C2\u00A0proteins\t \u00C2\u00A0and\t \u00C2\u00A0protein\t \u00C2\u00A0complexes\t \u00C2\u00A0remains\t \u00C2\u00A0rudimentary.\t \u00C2\u00A0A\t \u00C2\u00A0common\t \u00C2\u00A0approach\t \u00C2\u00A0in\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0to\t \u00C2\u00A0interrogate\t \u00C2\u00A0protein\t \u00C2\u00A0function\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0loss-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090function\t \u00C2\u00A0studies,\t \u00C2\u00A0that\t \u00C2\u00A0is,\t \u00C2\u00A0to\t \u00C2\u00A0examine\t \u00C2\u00A0phenotypes\t \u00C2\u00A0developed\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0absence\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090interest\t \u00C2\u00A0(POI).\t \u00C2\u00A0Current\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0seek\t \u00C2\u00A0to\t \u00C2\u00A0eliminate\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0either\t \u00C2\u00A0operate\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0genetic\t \u00C2\u00A0level\t \u00C2\u00A0or\t \u00C2\u00A0via\t \u00C2\u00A0direct\t \u00C2\u00A0degradation\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level.\t \u00C2\u00A0While\t \u00C2\u00A0immensely\t \u00C2\u00A0useful,\t \u00C2\u00A0these\t \u00C2\u00A0techniques\t \u00C2\u00A0often\t \u00C2\u00A0require\t \u00C2\u00A0manipulation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0genome\t \u00C2\u00A0or\t \u00C2\u00A0the\t \u00C2\u00A0introduction\t \u00C2\u00A0of\t \u00C2\u00A0external\t \u00C2\u00A0genetic\t \u00C2\u00A0constructs\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0organism.\t \u00C2\u00A0Thus,\t \u00C2\u00A0as\t \u00C2\u00A0explained\t \u00C2\u00A0in\t \u00C2\u00A0detail\t \u00C2\u00A0below,\t \u00C2\u00A0these\t \u00C2\u00A0methods\t \u00C2\u00A0are\t \u00C2\u00A0limited\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0speed,\t \u00C2\u00A0reversibility\t \u00C2\u00A0 2 and\t \u00C2\u00A0tunability.\t \u00C2\u00A0\t \u00C2\u00A0Unfortunately,\t \u00C2\u00A0these\t \u00C2\u00A0limitations\t \u00C2\u00A0have\t \u00C2\u00A0hindered\t \u00C2\u00A0the\t \u00C2\u00A0translation\t \u00C2\u00A0from\t \u00C2\u00A0gene\t \u00C2\u00A0sequence\t \u00C2\u00A0data\t \u00C2\u00A0to\t \u00C2\u00A0drug\t \u00C2\u00A0candidates.\t \u00C2\u00A0An\t \u00C2\u00A0emerging\t \u00C2\u00A0approach\t \u00C2\u00A0in\t \u00C2\u00A0drug\t \u00C2\u00A0development\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0destroy\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0inhibit\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090related\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0cells,\t \u00C2\u00A0particularly\t \u00C2\u00A0those\t \u00C2\u00A0that\t \u00C2\u00A0cannot\t \u00C2\u00A0be\t \u00C2\u00A0targeted\t \u00C2\u00A0by\t \u00C2\u00A0conventional\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0inhibitors\t \u00C2\u00A0(Neklesa\t \u00C2\u00A0and\t \u00C2\u00A0Crews,\t \u00C2\u00A02012).\t \u00C2\u00A0These\t \u00C2\u00A0undruggable\t \u00C2\u00A0proteins\t \u00C2\u00A0constitute\t \u00C2\u00A0roughly\t \u00C2\u00A085%\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0human\t \u00C2\u00A0proteome\t \u00C2\u00A0(Russ\t \u00C2\u00A0and\t \u00C2\u00A0Lampel,\t \u00C2\u00A02005)\t \u00C2\u00A0and\t \u00C2\u00A0include\t \u00C2\u00A0oncogene\t \u00C2\u00A0products\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0proteins\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090aggregation\t \u00C2\u00A0disorders.\t \u00C2\u00A0Compelling\t \u00C2\u00A0evidence\t \u00C2\u00A0from\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090clinical\t \u00C2\u00A0studies\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0proteins\t \u00C2\u00A0has\t \u00C2\u00A0excellent\t \u00C2\u00A0potential\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic\t \u00C2\u00A0strategy;\t \u00C2\u00A0however\t \u00C2\u00A0with\t \u00C2\u00A0current\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0approaches\t \u00C2\u00A0this\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0yet\t \u00C2\u00A0achievable\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0clinical\t \u00C2\u00A0setting,\t \u00C2\u00A0illustrating\t \u00C2\u00A0the\t \u00C2\u00A0need\t \u00C2\u00A0for\t \u00C2\u00A0novel\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0ready\t \u00C2\u00A0for\t \u00C2\u00A0clinical\t \u00C2\u00A0translation.\t \u00C2\u00A0An\t \u00C2\u00A0ideal\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0system\t \u00C2\u00A0should\t \u00C2\u00A0be\t \u00C2\u00A0efficient,\t \u00C2\u00A0highly\t \u00C2\u00A0specific,\t \u00C2\u00A0rapid,\t \u00C2\u00A0reversible\t \u00C2\u00A0and\t \u00C2\u00A0tunable,\t \u00C2\u00A0with\t \u00C2\u00A0minimal\t \u00C2\u00A0perturbation\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0endogenous\t \u00C2\u00A0cellular\t \u00C2\u00A0milieu.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0chapter,\t \u00C2\u00A0I\t \u00C2\u00A0will\t \u00C2\u00A0first\t \u00C2\u00A0review\t \u00C2\u00A0DNA-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0RNA-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0strategies\t \u00C2\u00A0and\t \u00C2\u00A0discuss\t \u00C2\u00A0their\t \u00C2\u00A0limitations.\t \u00C2\u00A0Next,\t \u00C2\u00A0I\t \u00C2\u00A0will\t \u00C2\u00A0provide\t \u00C2\u00A0an\t \u00C2\u00A0overview\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0systems\t \u00C2\u00A0and\t \u00C2\u00A0review\t \u00C2\u00A0key\t \u00C2\u00A0components\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0systems\t \u00C2\u00A0that\t \u00C2\u00A0are\t \u00C2\u00A0harnessed\t \u00C2\u00A0by\t \u00C2\u00A0existing\t \u00C2\u00A0methods\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0that\t \u00C2\u00A0operate\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level.\t \u00C2\u00A0I\t \u00C2\u00A0will\t \u00C2\u00A0then\t \u00C2\u00A0describe\t \u00C2\u00A0and\t \u00C2\u00A0evaluate\t \u00C2\u00A0a\t \u00C2\u00A0select\t \u00C2\u00A0few\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0and\t \u00C2\u00A0discuss\t \u00C2\u00A0their\t \u00C2\u00A0limitations.\t \u00C2\u00A0Finally,\t \u00C2\u00A0I\t \u00C2\u00A0will\t \u00C2\u00A0introduce\t \u00C2\u00A0the\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0system\t \u00C2\u00A0SNIPER\t \u00C2\u00A0(Selective\t \u00C2\u00A0Native\t \u00C2\u00A0Protein\t \u00C2\u00A0Eradication)\t \u00C2\u00A0developed\t \u00C2\u00A0during\t \u00C2\u00A0the\t \u00C2\u00A0course\t \u00C2\u00A0of\t \u00C2\u00A0my\t \u00C2\u00A0studies\t \u00C2\u00A0and\t \u00C2\u00A0discuss\t \u00C2\u00A0the\t \u00C2\u00A0rationale\t \u00C2\u00A0behind\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0system.\t \u00C2\u00A0 3 1.2 PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0BY\t \u00C2\u00A0GENE\t \u00C2\u00A0TARGETING\t \u00C2\u00A01.2.1 CONVENTIONAL\t \u00C2\u00A0GENETIC\t \u00C2\u00A0KNOCKOUT\t \u00C2\u00A0Targeted\t \u00C2\u00A0gene\t \u00C2\u00A0knockout\t \u00C2\u00A0through\t \u00C2\u00A0homologous\t \u00C2\u00A0recombination\t \u00C2\u00A0in\t \u00C2\u00A0mice\t \u00C2\u00A0has\t \u00C2\u00A0undoubtedly\t \u00C2\u00A0become\t \u00C2\u00A0the\t \u00C2\u00A0\u00E2\u0080\u009Cgold\t \u00C2\u00A0standard\u00E2\u0080\u009D\t \u00C2\u00A0for\t \u00C2\u00A0determining\t \u00C2\u00A0gene\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0mammals.\t \u00C2\u00A0This\t \u00C2\u00A0approach\t \u00C2\u00A0often\t \u00C2\u00A0involves\t \u00C2\u00A0mutating\t \u00C2\u00A0or\t \u00C2\u00A0deleting\t \u00C2\u00A0a\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090interest\t \u00C2\u00A0and\t \u00C2\u00A0evaluating\t \u00C2\u00A0its\t \u00C2\u00A0role\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0resulting\t \u00C2\u00A0phenotype.\t \u00C2\u00A0Gene\t \u00C2\u00A0knockout\t \u00C2\u00A0provides\t \u00C2\u00A0a\t \u00C2\u00A0means\t \u00C2\u00A0to\t \u00C2\u00A0break\t \u00C2\u00A0down\t \u00C2\u00A0complex\t \u00C2\u00A0biological\t \u00C2\u00A0phenomena\t \u00C2\u00A0into\t \u00C2\u00A0manageable\t \u00C2\u00A0parts\t \u00C2\u00A0for\t \u00C2\u00A0individual\t \u00C2\u00A0study.\t \u00C2\u00A0By\t \u00C2\u00A0synthesizing\t \u00C2\u00A0numerous\t \u00C2\u00A0such\t \u00C2\u00A0studies\t \u00C2\u00A0we\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0obtain\t \u00C2\u00A0a\t \u00C2\u00A0detailed\t \u00C2\u00A0overall\t \u00C2\u00A0picture\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0particular\t \u00C2\u00A0biological\t \u00C2\u00A0process\t \u00C2\u00A0(Austin\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004;\t \u00C2\u00A0Capecchi,\t \u00C2\u00A02005).\t \u00C2\u00A0\t \u00C2\u00A0Conventional\t \u00C2\u00A0gene\t \u00C2\u00A0knockout\t \u00C2\u00A0involves\t \u00C2\u00A0permanently\t \u00C2\u00A0inactivating\t \u00C2\u00A0a\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090interest\t \u00C2\u00A0in\t \u00C2\u00A0embryonic\t \u00C2\u00A0stems\t \u00C2\u00A0cells\t \u00C2\u00A0(ESCs)\t \u00C2\u00A0through\t \u00C2\u00A0homologous\t \u00C2\u00A0recombination.\t \u00C2\u00A0ESCs\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0desired\t \u00C2\u00A0modifications\t \u00C2\u00A0are\t \u00C2\u00A0then\t \u00C2\u00A0injected\t \u00C2\u00A0into\t \u00C2\u00A0very\t \u00C2\u00A0early\t \u00C2\u00A0embryos,\t \u00C2\u00A0which\t \u00C2\u00A0are\t \u00C2\u00A0then\t \u00C2\u00A0transplanted\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0donor\t \u00C2\u00A0mouse\t \u00C2\u00A0(Capecchi\t \u00C2\u00A01989).\t \u00C2\u00A0Mice\t \u00C2\u00A0developed\t \u00C2\u00A0from\t \u00C2\u00A0these\t \u00C2\u00A0embryos\t \u00C2\u00A0are\t \u00C2\u00A0heterozygous,\t \u00C2\u00A0carrying\t \u00C2\u00A0one\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0and\t \u00C2\u00A0one\t \u00C2\u00A0disrupted\t \u00C2\u00A0allele.\t \u00C2\u00A0Subsequent\t \u00C2\u00A0inter-\u00C2\u00AD\u00E2\u0080\u0090breeding\t \u00C2\u00A0between\t \u00C2\u00A0these\t \u00C2\u00A0chimeras\t \u00C2\u00A0produces\t \u00C2\u00A0homozygous\t \u00C2\u00A0knockout\t \u00C2\u00A0mice\t \u00C2\u00A0with\t \u00C2\u00A0both\t \u00C2\u00A0alleles\t \u00C2\u00A0disrupted.\t \u00C2\u00A0To\t \u00C2\u00A0date,\t \u00C2\u00A0these\t \u00C2\u00A0genetic\t \u00C2\u00A0studies\t \u00C2\u00A0have\t \u00C2\u00A0identified\t \u00C2\u00A0the\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0over\t \u00C2\u00A07,000\t \u00C2\u00A0mammalian\t \u00C2\u00A0genes\t \u00C2\u00A0(White\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013),\t \u00C2\u00A0attesting\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0power\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0knockout\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0research\t \u00C2\u00A0tool.\t \u00C2\u00A0Nevertheless,\t \u00C2\u00A0several\t \u00C2\u00A0limitations\t \u00C2\u00A0exist.\t \u00C2\u00A0First,\t \u00C2\u00A0disrupting\t \u00C2\u00A0essential\t \u00C2\u00A0genes\t \u00C2\u00A0for\t \u00C2\u00A0normal\t \u00C2\u00A0development\t \u00C2\u00A0can\t \u00C2\u00A0lead\t \u00C2\u00A0to\t \u00C2\u00A0embryonic\t \u00C2\u00A0lethality\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0transformed\t \u00C2\u00A0embryo.\t \u00C2\u00A0This\t \u00C2\u00A0precludes\t \u00C2\u00A0the\t \u00C2\u00A0analysis\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0functions\t \u00C2\u00A0in\t \u00C2\u00A0later\t \u00C2\u00A0stages\t \u00C2\u00A0of\t \u00C2\u00A0 4 embryonic\t \u00C2\u00A0development\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0adulthood.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0such\t \u00C2\u00A0genes\t \u00C2\u00A0may\t \u00C2\u00A0serve\t \u00C2\u00A0different\t \u00C2\u00A0functions\t \u00C2\u00A0during\t \u00C2\u00A0different\t \u00C2\u00A0periods\t \u00C2\u00A0of\t \u00C2\u00A0development,\t \u00C2\u00A0they\t \u00C2\u00A0may\t \u00C2\u00A0still\t \u00C2\u00A0be\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0pathogenesis\t \u00C2\u00A0of\t \u00C2\u00A0human\t \u00C2\u00A0diseases.\t \u00C2\u00A0The\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0genes\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0insubstantial:\t \u00C2\u00A0an\t \u00C2\u00A0estimated\t \u00C2\u00A015%\t \u00C2\u00A0of\t \u00C2\u00A0all\t \u00C2\u00A0knockout\t \u00C2\u00A0mice\t \u00C2\u00A0carry\t \u00C2\u00A0germline\t \u00C2\u00A0mutations\t \u00C2\u00A0that\t \u00C2\u00A0result\t \u00C2\u00A0in\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090term\t \u00C2\u00A0or\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090adult\t \u00C2\u00A0lethality\t \u00C2\u00A0(M\u00C3\u00BCller,\t \u00C2\u00A02011).\t \u00C2\u00A0\t \u00C2\u00A0Second,\t \u00C2\u00A0the\t \u00C2\u00A0phenotype\t \u00C2\u00A0resulting\t \u00C2\u00A0from\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0null\t \u00C2\u00A0mutation\t \u00C2\u00A0can\t \u00C2\u00A0heavily\t \u00C2\u00A0depend\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0genetic\t \u00C2\u00A0background\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0mouse\t \u00C2\u00A0strain\t \u00C2\u00A0(Wolfer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002),\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0wide\t \u00C2\u00A0variety\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0traits\t \u00C2\u00A0native\t \u00C2\u00A0to\t \u00C2\u00A0inbred\t \u00C2\u00A0strains\t \u00C2\u00A0of\t \u00C2\u00A0mice\t \u00C2\u00A0may\t \u00C2\u00A0further\t \u00C2\u00A0confound\t \u00C2\u00A0interpretation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0mutant\t \u00C2\u00A0phenotype\t \u00C2\u00A0(Crabbe\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01999;\t \u00C2\u00A0Doetschman,\t \u00C2\u00A02009).\t \u00C2\u00A0Technicalities\t \u00C2\u00A0aside,\t \u00C2\u00A0perhaps\t \u00C2\u00A0the\t \u00C2\u00A0most\t \u00C2\u00A0intractable\t \u00C2\u00A0problems\t \u00C2\u00A0arise\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0dynamic\t \u00C2\u00A0nature\t \u00C2\u00A0of\t \u00C2\u00A0biological\t \u00C2\u00A0systems.\t \u00C2\u00A0One\t \u00C2\u00A0such\t \u00C2\u00A0problem\t \u00C2\u00A0is\t \u00C2\u00A0genetic\t \u00C2\u00A0pleiotropy,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0one\t \u00C2\u00A0gene\t \u00C2\u00A0controls\t \u00C2\u00A0several\t \u00C2\u00A0distinctive\t \u00C2\u00A0and\t \u00C2\u00A0seemingly\t \u00C2\u00A0unrated\t \u00C2\u00A0phenotypic\t \u00C2\u00A0traits\t \u00C2\u00A0(Wagner\t \u00C2\u00A0and\t \u00C2\u00A0Zhang,\t \u00C2\u00A02011).\t \u00C2\u00A0Conversely,\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0gene\t \u00C2\u00A0may\t \u00C2\u00A0not\t \u00C2\u00A0produce\t \u00C2\u00A0a\t \u00C2\u00A0phenotypical\t \u00C2\u00A0outcome\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0redundancy\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0biological\t \u00C2\u00A0system\t \u00C2\u00A0(Barbaric\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02007).\t \u00C2\u00A0Similarly,\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0gene\t \u00C2\u00A0can\t \u00C2\u00A0produce\t \u00C2\u00A0multiple\t \u00C2\u00A0variants\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0alternative\t \u00C2\u00A0splicing\t \u00C2\u00A0and\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications.\t \u00C2\u00A0Moreover,\t \u00C2\u00A0proteins\t \u00C2\u00A0can\t \u00C2\u00A0participate\t \u00C2\u00A0in\t \u00C2\u00A0multiple\t \u00C2\u00A0cellular\t \u00C2\u00A0pathways\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0perform\t \u00C2\u00A0more\t \u00C2\u00A0than\t \u00C2\u00A0one\t \u00C2\u00A0biological\t \u00C2\u00A0function.\t \u00C2\u00A0Although\t \u00C2\u00A0with\t \u00C2\u00A0modern\t \u00C2\u00A0gene\t \u00C2\u00A0technology,\t \u00C2\u00A0specific\t \u00C2\u00A0splice\t \u00C2\u00A0forms\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0targeted\t \u00C2\u00A0by\t \u00C2\u00A0gene\t \u00C2\u00A0knockout,\t \u00C2\u00A0and\t \u00C2\u00A0specific\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0abolished\t \u00C2\u00A0with\t \u00C2\u00A0gene\t \u00C2\u00A0knock-\u00C2\u00AD\u00E2\u0080\u0090in,\t \u00C2\u00A0in\t \u00C2\u00A0most\t \u00C2\u00A0cases\t \u00C2\u00A0all\t \u00C2\u00A0variants\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0gene\t \u00C2\u00A0product\t \u00C2\u00A0are\t \u00C2\u00A0eliminated,\t \u00C2\u00A0thus\t \u00C2\u00A0making\t \u00C2\u00A0it\t \u00C2\u00A0difficult\t \u00C2\u00A0to\t \u00C2\u00A0tease\t \u00C2\u00A0apart\t \u00C2\u00A0the\t \u00C2\u00A0different\t \u00C2\u00A0roles\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0gene\t \u00C2\u00A0(Banaszynski\t \u00C2\u00A0and\t \u00C2\u00A0Wandless,\t \u00C2\u00A02006).\t \u00C2\u00A0Finally,\t \u00C2\u00A0cellular\t \u00C2\u00A0and\t \u00C2\u00A0 5 molecular\t \u00C2\u00A0compensation\t \u00C2\u00A0may\t \u00C2\u00A0occur\t \u00C2\u00A0during\t \u00C2\u00A0development,\t \u00C2\u00A0effectively\t \u00C2\u00A0masking\t \u00C2\u00A0the\t \u00C2\u00A0phenotype\t \u00C2\u00A0and\t \u00C2\u00A0making\t \u00C2\u00A0interpretation\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0experiments\t \u00C2\u00A0difficult.\t \u00C2\u00A01.2.2 CONDITIONAL\t \u00C2\u00A0GENETIC\t \u00C2\u00A0KNOCKOUT\t \u00C2\u00A0Conditional\t \u00C2\u00A0genetic\t \u00C2\u00A0knockout\t \u00C2\u00A0circumvents\t \u00C2\u00A0some\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0problems\t \u00C2\u00A0by\t \u00C2\u00A0restricting\t \u00C2\u00A0control\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0expression\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0temporal,\t \u00C2\u00A0spatial\t \u00C2\u00A0and\t \u00C2\u00A0tissue-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0or\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0specific\t \u00C2\u00A0manner.\t \u00C2\u00A0Perhaps\t \u00C2\u00A0the\t \u00C2\u00A0most\t \u00C2\u00A0widely\t \u00C2\u00A0used\t \u00C2\u00A0technology\t \u00C2\u00A0today\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0Cre-\u00C2\u00AD\u00E2\u0080\u0090loxP\t \u00C2\u00A0(locus\t \u00C2\u00A0of\t \u00C2\u00A0X-\u00C2\u00AD\u00E2\u0080\u0090over\t \u00C2\u00A0P1)\t \u00C2\u00A0site-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0recombination\t \u00C2\u00A0system\t \u00C2\u00A0(Sauer\t \u00C2\u00A0and\t \u00C2\u00A0Henderson,\t \u00C2\u00A01988;\t \u00C2\u00A0Nagy,\t \u00C2\u00A02000),\t \u00C2\u00A0which\t \u00C2\u00A0uses\t \u00C2\u00A0bacteriophage\t \u00C2\u00A0Cre-\u00C2\u00AD\u00E2\u0080\u0090recombinase\t \u00C2\u00A0to\t \u00C2\u00A0excise\t \u00C2\u00A0all\t \u00C2\u00A0DNA\t \u00C2\u00A0sequences\t \u00C2\u00A0located\t \u00C2\u00A0between\t \u00C2\u00A0two\t \u00C2\u00A0directly\t \u00C2\u00A0repeated\t \u00C2\u00A0loxP\t \u00C2\u00A0sites.\t \u00C2\u00A0Generally,\t \u00C2\u00A0the\t \u00C2\u00A0two\t \u00C2\u00A0loxP\t \u00C2\u00A0sites\t \u00C2\u00A0are\t \u00C2\u00A0genetically\t \u00C2\u00A0inserted\t \u00C2\u00A0around\t \u00C2\u00A0an\t \u00C2\u00A0essential\t \u00C2\u00A0exon\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090interest\t \u00C2\u00A0in\t \u00C2\u00A0one\t \u00C2\u00A0animal\t \u00C2\u00A0strain,\t \u00C2\u00A0while\t \u00C2\u00A0the\t \u00C2\u00A0Cre-\u00C2\u00AD\u00E2\u0080\u0090expressing\t \u00C2\u00A0construct\t \u00C2\u00A0is\t \u00C2\u00A0introduced\t \u00C2\u00A0into\t \u00C2\u00A0another\t \u00C2\u00A0strain;\t \u00C2\u00A0crossing\t \u00C2\u00A0the\t \u00C2\u00A0animals\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0gene\t \u00C2\u00A0excision\t \u00C2\u00A0(Gu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01994).\t \u00C2\u00A0Cre\t \u00C2\u00A0transcription\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0further\t \u00C2\u00A0rendered\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0specific\t \u00C2\u00A0and/or\t \u00C2\u00A0inducible\t \u00C2\u00A0by\t \u00C2\u00A0placing\t \u00C2\u00A0it\t \u00C2\u00A0under\t \u00C2\u00A0the\t \u00C2\u00A0control\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0specific\t \u00C2\u00A0and/or\t \u00C2\u00A0small\t \u00C2\u00A0molecule-\u00C2\u00AD\u00E2\u0080\u0090responsive\t \u00C2\u00A0promoter.\t \u00C2\u00A0A\t \u00C2\u00A0classic\t \u00C2\u00A0example\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0a\t \u00C2\u00A0system\t \u00C2\u00A0involves\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0rtTA\t \u00C2\u00A0(reverse\t \u00C2\u00A0tetracycline-\u00C2\u00AD\u00E2\u0080\u0090responsive\t \u00C2\u00A0transactivator)\t \u00C2\u00A0to\t \u00C2\u00A0control\t \u00C2\u00A0the\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0Cre.\t \u00C2\u00A0rtTA\t \u00C2\u00A0is\t \u00C2\u00A0inert\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0absence\t \u00C2\u00A0of\t \u00C2\u00A0doxycycline\t \u00C2\u00A0and\t \u00C2\u00A0activates\t \u00C2\u00A0once\t \u00C2\u00A0the\t \u00C2\u00A0drug\t \u00C2\u00A0becomes\t \u00C2\u00A0available.\t \u00C2\u00A0A\t \u00C2\u00A0cell\t \u00C2\u00A0type-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0promoter\t \u00C2\u00A0limits\t \u00C2\u00A0the\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0rtTA\t \u00C2\u00A0to\t \u00C2\u00A0defined\t \u00C2\u00A0cell\t \u00C2\u00A0lineages.\t \u00C2\u00A0Thus,\t \u00C2\u00A0an\t \u00C2\u00A0injection\t \u00C2\u00A0of\t \u00C2\u00A0doxycycline\t \u00C2\u00A0activates\t \u00C2\u00A0rtTA\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0particular\t \u00C2\u00A0population\t \u00C2\u00A0of\t \u00C2\u00A0cells,\t \u00C2\u00A0which\t \u00C2\u00A0induces\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0specific\t \u00C2\u00A0Cre\t \u00C2\u00A0expression\t \u00C2\u00A0and\t \u00C2\u00A0subsequent\t \u00C2\u00A0Cre-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0deletion\t \u00C2\u00A0of\t \u00C2\u00A0loxP-\u00C2\u00AD\u00E2\u0080\u0090flanked\t \u00C2\u00A0DNA\t \u00C2\u00A0(Utomo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01999).\t \u00C2\u00A0With\t \u00C2\u00A0 6 the\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0high-\u00C2\u00AD\u00E2\u0080\u0090throughout\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0pipeline\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0generation\t \u00C2\u00A0for\t \u00C2\u00A0conditional\t \u00C2\u00A0alleles,\t \u00C2\u00A0the\t \u00C2\u00A0International\t \u00C2\u00A0Knockout\t \u00C2\u00A0Mouse\t \u00C2\u00A0Consortium\t \u00C2\u00A0(IKMC)\t \u00C2\u00A0has\t \u00C2\u00A0thus\t \u00C2\u00A0far\t \u00C2\u00A0generated\t \u00C2\u00A0more\t \u00C2\u00A0than\t \u00C2\u00A01,700\t \u00C2\u00A0mutant\t \u00C2\u00A0mouse\t \u00C2\u00A0strains\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0biomedical\t \u00C2\u00A0research,\t \u00C2\u00A0most\t \u00C2\u00A0of\t \u00C2\u00A0them\t \u00C2\u00A0conditional\t \u00C2\u00A0(Skarnes\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Bradley\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Although\t \u00C2\u00A0sophisticated\t \u00C2\u00A0in\t \u00C2\u00A0design,\t \u00C2\u00A0the\t \u00C2\u00A0inducible\t \u00C2\u00A0Cre-\u00C2\u00AD\u00E2\u0080\u0090loxP\t \u00C2\u00A0system\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0without\t \u00C2\u00A0shortcomings.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0gene\t \u00C2\u00A0deletion\t \u00C2\u00A0often\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0loxP\t \u00C2\u00A0flanking\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0exon,\t \u00C2\u00A0unidentified\t \u00C2\u00A0alternative\t \u00C2\u00A0transcriptional\t \u00C2\u00A0start\t \u00C2\u00A0sites\t \u00C2\u00A0may\t \u00C2\u00A0allow\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0continue\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0translated\t \u00C2\u00A0even\t \u00C2\u00A0after\t \u00C2\u00A0successful\t \u00C2\u00A0recombination.\t \u00C2\u00A0Indeed,\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0cases\t \u00C2\u00A0where\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0persist\t \u00C2\u00A0despite\t \u00C2\u00A0demonstrated\t \u00C2\u00A0recombination\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0(Turlo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Once\t \u00C2\u00A0a\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090interest\t \u00C2\u00A0is\t \u00C2\u00A0excised,\t \u00C2\u00A0the\t \u00C2\u00A0deletion\t \u00C2\u00A0process\t \u00C2\u00A0is\t \u00C2\u00A0complete\t \u00C2\u00A0and\t \u00C2\u00A0irreversible,\t \u00C2\u00A0thus\t \u00C2\u00A0precluding\t \u00C2\u00A0examination\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0effects\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0dosage.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0an\t \u00C2\u00A0investigation\t \u00C2\u00A0of\t \u00C2\u00A040\t \u00C2\u00A0cre\t \u00C2\u00A0mouse\t \u00C2\u00A0strains\t \u00C2\u00A0found\t \u00C2\u00A0a\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0issues\t \u00C2\u00A0with\t \u00C2\u00A0cre\t \u00C2\u00A0function,\t \u00C2\u00A0including\t \u00C2\u00A0inconsistent\t \u00C2\u00A0recombination\t \u00C2\u00A0between\t \u00C2\u00A0littermates,\t \u00C2\u00A0differential\t \u00C2\u00A0cre\t \u00C2\u00A0activity\t \u00C2\u00A0depending\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0gene\u00E2\u0080\u0099s\t \u00C2\u00A0parental\t \u00C2\u00A0origin\t \u00C2\u00A0and\t \u00C2\u00A0unexpected\t \u00C2\u00A0activity\t \u00C2\u00A0in\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0tissues\t \u00C2\u00A0(Heffner\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0The\t \u00C2\u00A0design\t \u00C2\u00A0and\t \u00C2\u00A0generation\t \u00C2\u00A0of\t \u00C2\u00A0cre\t \u00C2\u00A0mouse\t \u00C2\u00A0lines\t \u00C2\u00A0is\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090consuming\t \u00C2\u00A0as\t \u00C2\u00A0genetic\t \u00C2\u00A0deletion\t \u00C2\u00A0may\t \u00C2\u00A0take\t \u00C2\u00A0days\t \u00C2\u00A0or\t \u00C2\u00A0even\t \u00C2\u00A0weeks\t \u00C2\u00A0(Kuhn\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01995).\t \u00C2\u00A0In\t \u00C2\u00A0higher\t \u00C2\u00A0eukaryotic\t \u00C2\u00A0cells,\t \u00C2\u00A0the\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0homologous\t \u00C2\u00A0recombination\t \u00C2\u00A0is\t \u00C2\u00A0low,\t \u00C2\u00A0ranging\t \u00C2\u00A0from\t \u00C2\u00A01\t \u00C2\u00A0in\t \u00C2\u00A0106\t \u00C2\u00A0to\t \u00C2\u00A01\t \u00C2\u00A0in\t \u00C2\u00A0107\t \u00C2\u00A0(Kim\t \u00C2\u00A0and\t \u00C2\u00A0Kim,\t \u00C2\u00A02014).\t \u00C2\u00A0Finally,\t \u00C2\u00A0as\t \u00C2\u00A0genetic\t \u00C2\u00A0deletion\t \u00C2\u00A0targets\t \u00C2\u00A0precursors\t \u00C2\u00A0(i.e.\t \u00C2\u00A0DNA)\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0the\t \u00C2\u00A0final\t \u00C2\u00A0protein\t \u00C2\u00A0product,\t \u00C2\u00A0phenotypical\t \u00C2\u00A0changes\t \u00C2\u00A0may\t \u00C2\u00A0not\t \u00C2\u00A0be\t \u00C2\u00A0measurable\t \u00C2\u00A0until\t \u00C2\u00A0the\t \u00C2\u00A0pool\t \u00C2\u00A0of\t \u00C2\u00A0already\t \u00C2\u00A0synthesized\t \u00C2\u00A0proteins\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0via\t \u00C2\u00A0normal\t \u00C2\u00A0turnover,\t \u00C2\u00A0which\t \u00C2\u00A0becomes\t \u00C2\u00A0problematic\t \u00C2\u00A0for\t \u00C2\u00A0proteins\t \u00C2\u00A0with\t \u00C2\u00A0long\t \u00C2\u00A0half-\u00C2\u00AD\u00E2\u0080\u0090lives.\t \u00C2\u00A0 7 1.2.3 TARGETED\t \u00C2\u00A0GENOME\t \u00C2\u00A0EDITING\t \u00C2\u00A0An\t \u00C2\u00A0exciting\t \u00C2\u00A0new\t \u00C2\u00A0approach\t \u00C2\u00A0to\t \u00C2\u00A0genomic\t \u00C2\u00A0engineering\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0programmable\t \u00C2\u00A0nucleases,\t \u00C2\u00A0which\t \u00C2\u00A0contain\t \u00C2\u00A0a\t \u00C2\u00A0sequence-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0DNA-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0nonspecific\t \u00C2\u00A0DNA\t \u00C2\u00A0cleavage\t \u00C2\u00A0module\t \u00C2\u00A0(Gaj\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0These\t \u00C2\u00A0nucleases\t \u00C2\u00A0induce\t \u00C2\u00A0double\t \u00C2\u00A0stranded\t \u00C2\u00A0breaks\t \u00C2\u00A0in\t \u00C2\u00A0defined\t \u00C2\u00A0DNA\t \u00C2\u00A0sites,\t \u00C2\u00A0which\t \u00C2\u00A0enhances\t \u00C2\u00A0the\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0homologous\t \u00C2\u00A0recombination\t \u00C2\u00A0by\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A0two-\u00C2\u00AD\u00E2\u0080\u0090fold\t \u00C2\u00A0(Rouet\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01994),\t \u00C2\u00A0and\t \u00C2\u00A0enables\t \u00C2\u00A0targeted\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0or\t \u00C2\u00A0gene\t \u00C2\u00A0knockout\t \u00C2\u00A0through\t \u00C2\u00A0error-\u00C2\u00AD\u00E2\u0080\u0090prone\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090homologous\t \u00C2\u00A0end\t \u00C2\u00A0joining\t \u00C2\u00A0or\t \u00C2\u00A0homology-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0repair\t \u00C2\u00A0(Bibikova\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002).\t \u00C2\u00A0There\t \u00C2\u00A0are\t \u00C2\u00A0currently\t \u00C2\u00A0three\t \u00C2\u00A0classes\t \u00C2\u00A0of\t \u00C2\u00A0genome\t \u00C2\u00A0editing\t \u00C2\u00A0nucleases:\t \u00C2\u00A0zinc-\u00C2\u00AD\u00E2\u0080\u0090finger\t \u00C2\u00A0nucleases\t \u00C2\u00A0(ZFNs),\t \u00C2\u00A0transcription\t \u00C2\u00A0activator-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0effector\t \u00C2\u00A0nucleases\t \u00C2\u00A0(TALENs)\t \u00C2\u00A0and\t \u00C2\u00A0clustered\t \u00C2\u00A0regularly\t \u00C2\u00A0interspaced\t \u00C2\u00A0short\t \u00C2\u00A0palindromic\t \u00C2\u00A0repeat\t \u00C2\u00A0(CRISPR)\u00E2\u0080\u0093CRISPR-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0system\t \u00C2\u00A0(Cas)\t \u00C2\u00A0(Urnov\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Bedell\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013;\t \u00C2\u00A0Cong\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013;\t \u00C2\u00A0Mali\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0All\t \u00C2\u00A0three\t \u00C2\u00A0types\t \u00C2\u00A0of\t \u00C2\u00A0nucleases\t \u00C2\u00A0introduce\t \u00C2\u00A0cleavages\t \u00C2\u00A0at\t \u00C2\u00A0specific\t \u00C2\u00A0chromosomal\t \u00C2\u00A0DNA\t \u00C2\u00A0sites,\t \u00C2\u00A0which\t \u00C2\u00A0activates\t \u00C2\u00A0the\t \u00C2\u00A0endogenous\t \u00C2\u00A0DNA-\u00C2\u00AD\u00E2\u0080\u0090repair\t \u00C2\u00A0system\t \u00C2\u00A0and\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0targeted\t \u00C2\u00A0genomic\t \u00C2\u00A0editing\t \u00C2\u00A0(for\t \u00C2\u00A0a\t \u00C2\u00A0review,\t \u00C2\u00A0see\t \u00C2\u00A0Kim\t \u00C2\u00A0and\t \u00C2\u00A0Kim,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0Programmable\t \u00C2\u00A0nucleases\t \u00C2\u00A0enable\t \u00C2\u00A0the\t \u00C2\u00A0manipulation\t \u00C2\u00A0of\t \u00C2\u00A0virtually\t \u00C2\u00A0any\t \u00C2\u00A0gene\t \u00C2\u00A0in\t \u00C2\u00A0multiple\t \u00C2\u00A0animal\t \u00C2\u00A0model\t \u00C2\u00A0systems\t \u00C2\u00A0(Gaj\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0These\t \u00C2\u00A0nucleases\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0successfully\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090knockout\t \u00C2\u00A0cell\t \u00C2\u00A0lines\t \u00C2\u00A0and\t \u00C2\u00A0animal\t \u00C2\u00A0models\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0analysis\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0glycoproteome\t \u00C2\u00A0(Steentoft\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011),\t \u00C2\u00A0protein\t \u00C2\u00A0methylation\t \u00C2\u00A0(Kernstock\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012)\t \u00C2\u00A0and\t \u00C2\u00A0neurodegeneration\t \u00C2\u00A0(Schmid\t \u00C2\u00A0and\t \u00C2\u00A0Haass,\t \u00C2\u00A02013).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0therapeutic\t \u00C2\u00A0potential\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0methods\t \u00C2\u00A0has\t \u00C2\u00A0also\t \u00C2\u00A0been\t \u00C2\u00A0demonstrated.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0ZFNs\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0knockdown\t \u00C2\u00A0mutant\t \u00C2\u00A0huntingtin\t \u00C2\u00A0(mHtt)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0mouse\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0Huntington\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0(HD)\t \u00C2\u00A0(Garriga-\u00C2\u00AD\u00E2\u0080\u0090Canut\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0 8 2012)\t \u00C2\u00A0and\t \u00C2\u00A0to\t \u00C2\u00A0correct\t \u00C2\u00A0a\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0mutation\t \u00C2\u00A0in\t \u00C2\u00A0human\t \u00C2\u00A0induced\t \u00C2\u00A0pluripotent\t \u00C2\u00A0cell\t \u00C2\u00A0derived\t \u00C2\u00A0from\t \u00C2\u00A0patients\t \u00C2\u00A0with\t \u00C2\u00A0Parkinson\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0(PD)\t \u00C2\u00A0(Soldner\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0The\t \u00C2\u00A0field\t \u00C2\u00A0of\t \u00C2\u00A0targeted\t \u00C2\u00A0genome\t \u00C2\u00A0editing\t \u00C2\u00A0is\t \u00C2\u00A0still\t \u00C2\u00A0in\t \u00C2\u00A0its\t \u00C2\u00A0infancy,\t \u00C2\u00A0but\t \u00C2\u00A0the\t \u00C2\u00A0technique\t \u00C2\u00A0has\t \u00C2\u00A0the\t \u00C2\u00A0potential\t \u00C2\u00A0to\t \u00C2\u00A0revolutionize\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0and\t \u00C2\u00A0enable\t \u00C2\u00A0gene\t \u00C2\u00A0therapy\t \u00C2\u00A0in\t \u00C2\u00A0humans.\t \u00C2\u00A0However,\t \u00C2\u00A0these\t \u00C2\u00A0methods\t \u00C2\u00A0have\t \u00C2\u00A0several\t \u00C2\u00A0notable\t \u00C2\u00A0limitations\t \u00C2\u00A0with\t \u00C2\u00A0regards\t \u00C2\u00A0to\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0First,\t \u00C2\u00A0the\t \u00C2\u00A0genome-\u00C2\u00AD\u00E2\u0080\u0090editing\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0engineered\t \u00C2\u00A0nuclease\t \u00C2\u00A0is\t \u00C2\u00A0widely\t \u00C2\u00A0variable,\t \u00C2\u00A0and\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0currently\t \u00C2\u00A0no\t \u00C2\u00A0reliable\t \u00C2\u00A0methods\t \u00C2\u00A0to\t \u00C2\u00A0predict\t \u00C2\u00A0nuclease\t \u00C2\u00A0activity\t \u00C2\u00A0prior\t \u00C2\u00A0experimentation\t \u00C2\u00A0(Kim\t \u00C2\u00A0and\t \u00C2\u00A0Kim,\t \u00C2\u00A02014).\t \u00C2\u00A0Second,\t \u00C2\u00A0all\t \u00C2\u00A0three\t \u00C2\u00A0types\t \u00C2\u00A0of\t \u00C2\u00A0nucleases\t \u00C2\u00A0can\t \u00C2\u00A0cause\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0mutations\t \u00C2\u00A0(Pattanayak\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Fu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0CRISPR-\u00C2\u00AD\u00E2\u0080\u0090Cas\t \u00C2\u00A0nuclease\t \u00C2\u00A0may\t \u00C2\u00A0recognize\t \u00C2\u00A0and\t \u00C2\u00A0mutate\t \u00C2\u00A0a\t \u00C2\u00A0collection\t \u00C2\u00A0of\t \u00C2\u00A0DNA\t \u00C2\u00A0sites\t \u00C2\u00A0that\t \u00C2\u00A0differ\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0site\t \u00C2\u00A0by\t \u00C2\u00A0up\t \u00C2\u00A0to\t \u00C2\u00A0five\t \u00C2\u00A0nucleotides\t \u00C2\u00A0(Fu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013),\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0there\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0thousands\t \u00C2\u00A0of\t \u00C2\u00A0potential\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0sites.\t \u00C2\u00A0Third,\t \u00C2\u00A0the\t \u00C2\u00A0optimal\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0engineered\t \u00C2\u00A0nucleases\t \u00C2\u00A0remains\t \u00C2\u00A0a\t \u00C2\u00A0problem\t \u00C2\u00A0for\t \u00C2\u00A0efficient\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0implementation.\t \u00C2\u00A0Currently,\t \u00C2\u00A0transient\t \u00C2\u00A0transfection\t \u00C2\u00A0is\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0deliver\t \u00C2\u00A0plasmid\t \u00C2\u00A0DNAs\t \u00C2\u00A0encoding\t \u00C2\u00A0the\t \u00C2\u00A0engineered\t \u00C2\u00A0nucleases\t \u00C2\u00A0into\t \u00C2\u00A0cell\t \u00C2\u00A0lines\t \u00C2\u00A0(Urnov\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005),\t \u00C2\u00A0and\t \u00C2\u00A0microinjection\t \u00C2\u00A0into\t \u00C2\u00A0single-\u00C2\u00AD\u00E2\u0080\u0090cell\t \u00C2\u00A0embryos\t \u00C2\u00A0is\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0knockout\t \u00C2\u00A0animals\t \u00C2\u00A0(Niu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Viral\t \u00C2\u00A0transfection\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0most\t \u00C2\u00A0common\t \u00C2\u00A0delivery\t \u00C2\u00A0method\t \u00C2\u00A0for\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0application,\t \u00C2\u00A0but\t \u00C2\u00A0carries\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0immunogenicity,\t \u00C2\u00A0toxicity,\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0case\t \u00C2\u00A0of\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090episomal\t \u00C2\u00A0vectors,\t \u00C2\u00A0insertional\t \u00C2\u00A0mutagenesis.\t \u00C2\u00A0More\t \u00C2\u00A0efficient\t \u00C2\u00A0delivery\t \u00C2\u00A0systems\t \u00C2\u00A0will\t \u00C2\u00A0have\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0developed\t \u00C2\u00A0before\t \u00C2\u00A0these\t \u00C2\u00A0nucleases\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0gene\t \u00C2\u00A0editing\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0treatment\t \u00C2\u00A0of\t \u00C2\u00A0human\t \u00C2\u00A0diseases.\t \u00C2\u00A0 9 1.3 PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0BY\t \u00C2\u00A0RNA\t \u00C2\u00A0TARGETING\t \u00C2\u00A01.3.1 ANTI-\u00C2\u00AD\u00E2\u0080\u0090SENSE\t \u00C2\u00A0OLIGONUCLEOTIDES\t \u00C2\u00A0(ASO)\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0late\t \u00C2\u00A01970s,\t \u00C2\u00A0Zamecnik\t \u00C2\u00A0and\t \u00C2\u00A0Stephenson\t \u00C2\u00A0made\t \u00C2\u00A0the\t \u00C2\u00A0discovery\t \u00C2\u00A0that\t \u00C2\u00A0an\t \u00C2\u00A0exogenous\t \u00C2\u00A013-\u00C2\u00AD\u00E2\u0080\u0090mer\t \u00C2\u00A0DNA\t \u00C2\u00A0sequence\t \u00C2\u00A0complementary\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0RNA\t \u00C2\u00A0sequence\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0Rous\t \u00C2\u00A0sarcoma\t \u00C2\u00A0virus\t \u00C2\u00A0blocked\t \u00C2\u00A0viral\t \u00C2\u00A0protein\t \u00C2\u00A0translation\t \u00C2\u00A0and\t \u00C2\u00A0inhibited\t \u00C2\u00A0viral\t \u00C2\u00A0replication\t \u00C2\u00A0(Zamecnik\t \u00C2\u00A0and\t \u00C2\u00A0Stephenson,\t \u00C2\u00A01978).\t \u00C2\u00A0This\t \u00C2\u00A0discovery\t \u00C2\u00A0laid\t \u00C2\u00A0the\t \u00C2\u00A0foundation\t \u00C2\u00A0of\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090sense\t \u00C2\u00A0technology.\t \u00C2\u00A0Today,\t \u00C2\u00A0ASOs\t \u00C2\u00A0have\t \u00C2\u00A0become\t \u00C2\u00A0one\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0most\t \u00C2\u00A0successful\t \u00C2\u00A0methods\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0laboratory\t \u00C2\u00A0to\t \u00C2\u00A0suppress\t \u00C2\u00A0the\t \u00C2\u00A0transmission\t \u00C2\u00A0of\t \u00C2\u00A0genetic\t \u00C2\u00A0information.\t \u00C2\u00A0The\t \u00C2\u00A0concept\t \u00C2\u00A0underlying\t \u00C2\u00A0ASOs\t \u00C2\u00A0is\t \u00C2\u00A0fairly\t \u00C2\u00A0straightforward:\t \u00C2\u00A0a\t \u00C2\u00A0short\t \u00C2\u00A0DNA\t \u00C2\u00A0sequence\t \u00C2\u00A0(13-\u00C2\u00AD\u00E2\u0080\u009025mer)\t \u00C2\u00A0designed\t \u00C2\u00A0to\t \u00C2\u00A0complement\t \u00C2\u00A0a\t \u00C2\u00A0target\t \u00C2\u00A0mRNA,\t \u00C2\u00A0once\t \u00C2\u00A0introduced\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0cell,\t \u00C2\u00A0stops\t \u00C2\u00A0mRNA\t \u00C2\u00A0translation\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0blocks\t \u00C2\u00A0the\t \u00C2\u00A0synthesis\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0POI.\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0the\t \u00C2\u00A0biological\t \u00C2\u00A0processes\t \u00C2\u00A0behind\t \u00C2\u00A0ASO-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0inhibition\t \u00C2\u00A0are\t \u00C2\u00A0numerous\t \u00C2\u00A0and\t \u00C2\u00A0complex.\t \u00C2\u00A0The\t \u00C2\u00A0most\t \u00C2\u00A0commonly\t \u00C2\u00A0studied\t \u00C2\u00A0\u00E2\u0080\u009Cclassical\u00E2\u0080\u009D\t \u00C2\u00A0ASOs\t \u00C2\u00A0(antisense\t \u00C2\u00A0gapmer\t \u00C2\u00A0oligonucleotides)\t \u00C2\u00A0recruit\t \u00C2\u00A0Ribonuclease\t \u00C2\u00A0H\t \u00C2\u00A0(RNase\t \u00C2\u00A0H),\t \u00C2\u00A0an\t \u00C2\u00A0enzyme\t \u00C2\u00A0that\t \u00C2\u00A0degrades\t \u00C2\u00A0RNA\t \u00C2\u00A0in\t \u00C2\u00A0RNA-\u00C2\u00AD\u00E2\u0080\u0090DNA\t \u00C2\u00A0duplexes,\t \u00C2\u00A0to\t \u00C2\u00A0cleave\t \u00C2\u00A0target\t \u00C2\u00A0mRNAs\t \u00C2\u00A0(Dias\t \u00C2\u00A0and\t \u00C2\u00A0Stein,\t \u00C2\u00A02002;\t \u00C2\u00A0Bennett\t \u00C2\u00A0and\t \u00C2\u00A0Swayze,\t \u00C2\u00A02010).\t \u00C2\u00A0An\t \u00C2\u00A0offshoot\t \u00C2\u00A0of\t \u00C2\u00A0antisense\t \u00C2\u00A0gapmer\t \u00C2\u00A0oligonucleotides\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0external\t \u00C2\u00A0guide\t \u00C2\u00A0sequence\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0short\t \u00C2\u00A0oligonucleotides\t \u00C2\u00A0that\t \u00C2\u00A0form\t \u00C2\u00A0tRNA-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0structures\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0that\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0targeted\t \u00C2\u00A0mRNA\t \u00C2\u00A0and\t \u00C2\u00A0induces\t \u00C2\u00A0cleavage\t \u00C2\u00A0by\t \u00C2\u00A0RNase\t \u00C2\u00A0P\t \u00C2\u00A0(Kole\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Advances\t \u00C2\u00A0in\t \u00C2\u00A0nucleotide\t \u00C2\u00A0chemistry\t \u00C2\u00A0over\t \u00C2\u00A0the\t \u00C2\u00A0years\t \u00C2\u00A0have\t \u00C2\u00A0led\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0cleavage-\u00C2\u00AD\u00E2\u0080\u0090independent\t \u00C2\u00A0ASOs.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0translation-\u00C2\u00AD\u00E2\u0080\u0090suppressing\t \u00C2\u00A0(steric-\u00C2\u00AD\u00E2\u0080\u0090blocking)\t \u00C2\u00A0oligonucleotides\t \u00C2\u00A0block\t \u00C2\u00A0mRNA\t \u00C2\u00A0sequences\t \u00C2\u00A0near\t \u00C2\u00A0the\t \u00C2\u00A0translation\t \u00C2\u00A0initiation\t \u00C2\u00A0site,\t \u00C2\u00A0hindering\t \u00C2\u00A0 10 mRNA\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0ribosome\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0inhibiting\t \u00C2\u00A0protein\t \u00C2\u00A0translation\t \u00C2\u00A0(Stirchak\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01989).\t \u00C2\u00A0Splice-\u00C2\u00AD\u00E2\u0080\u0090switching\t \u00C2\u00A0oligonucleotides\t \u00C2\u00A0restrict\t \u00C2\u00A0the\t \u00C2\u00A0splicing\t \u00C2\u00A0machinery\t \u00C2\u00A0from\t \u00C2\u00A0accessing\t \u00C2\u00A0the\t \u00C2\u00A0targeted\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090mRNA\t \u00C2\u00A0and\t \u00C2\u00A0prevent\t \u00C2\u00A0the\t \u00C2\u00A0production\t \u00C2\u00A0of\t \u00C2\u00A0undesirable\t \u00C2\u00A0splice\t \u00C2\u00A0variants\t \u00C2\u00A0(Roberts\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006;\t \u00C2\u00A0Disterer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0ASOs\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0utilized\t \u00C2\u00A0in\t \u00C2\u00A0numerous\t \u00C2\u00A0studies\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0protein\t \u00C2\u00A0accumulation\t \u00C2\u00A0disorders.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0a\t \u00C2\u00A0recent\t \u00C2\u00A0study\t \u00C2\u00A0used\t \u00C2\u00A0ASOs\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0human\t \u00C2\u00A0huntingtin\t \u00C2\u00A0(Htt)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0transgenic\t \u00C2\u00A0mouse\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0HD\t \u00C2\u00A0(Kordasiewicz\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0A\t \u00C2\u00A0single\t \u00C2\u00A0intracerebroventricular\t \u00C2\u00A0(i.c.v.)\t \u00C2\u00A0infusion\t \u00C2\u00A0of\t \u00C2\u00A0an\t \u00C2\u00A0Htt-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0ASO\t \u00C2\u00A0into\t \u00C2\u00A0symptomatic\t \u00C2\u00A0HD\t \u00C2\u00A0transgenic\t \u00C2\u00A0mice\t \u00C2\u00A0significantly\t \u00C2\u00A0decreased\t \u00C2\u00A0the\t \u00C2\u00A0total\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0Htt\t \u00C2\u00A0mRNA\t \u00C2\u00A0and\t \u00C2\u00A0protein,\t \u00C2\u00A0and\t \u00C2\u00A0delayed\t \u00C2\u00A0disease\t \u00C2\u00A0progression\t \u00C2\u00A0for\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A09\t \u00C2\u00A0months\t \u00C2\u00A0after\t \u00C2\u00A0the\t \u00C2\u00A0initial\t \u00C2\u00A0bolus\t \u00C2\u00A0treatment\t \u00C2\u00A0(Kordasiewicz\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0ASOs\t \u00C2\u00A0have\t \u00C2\u00A0also\t \u00C2\u00A0been\t \u00C2\u00A0successfully\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0specifically\t \u00C2\u00A0target\t \u00C2\u00A0the\t \u00C2\u00A0mHtt\t \u00C2\u00A0allele\t \u00C2\u00A0while\t \u00C2\u00A0leaving\t \u00C2\u00A0the\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0allele\t \u00C2\u00A0intact,\t \u00C2\u00A0thus\t \u00C2\u00A0eliminating\t \u00C2\u00A0only\t \u00C2\u00A0the\t \u00C2\u00A0pathogenic\t \u00C2\u00A0protein\t \u00C2\u00A0variant\t \u00C2\u00A0(Hu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0\t \u00C2\u00A0Despite\t \u00C2\u00A0numerous\t \u00C2\u00A0successful\t \u00C2\u00A0applications\t \u00C2\u00A0in\t \u00C2\u00A0animal\t \u00C2\u00A0models\t \u00C2\u00A0of\t \u00C2\u00A0disease,\t \u00C2\u00A0ASOs\t \u00C2\u00A0have\t \u00C2\u00A0two\t \u00C2\u00A0major\t \u00C2\u00A0problems\t \u00C2\u00A0that\t \u00C2\u00A0limit\t \u00C2\u00A0their\t \u00C2\u00A0prospects\t \u00C2\u00A0for\t \u00C2\u00A0clinical\t \u00C2\u00A0application:\t \u00C2\u00A0poor\t \u00C2\u00A0cellular\t \u00C2\u00A0uptake\t \u00C2\u00A0and\t \u00C2\u00A0chemistry-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0toxicity.\t \u00C2\u00A0Although\t \u00C2\u00A0numerous\t \u00C2\u00A0transporters,\t \u00C2\u00A0including\t \u00C2\u00A0cationic\t \u00C2\u00A0liposomes\t \u00C2\u00A0and\t \u00C2\u00A0polymers,\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0developed\t \u00C2\u00A0to\t \u00C2\u00A0improve\t \u00C2\u00A0intracellular\t \u00C2\u00A0delivery,\t \u00C2\u00A0tissue\t \u00C2\u00A0penetration\t \u00C2\u00A0remains\t \u00C2\u00A0an\t \u00C2\u00A0unresolved\t \u00C2\u00A0problem\t \u00C2\u00A0(Dias\t \u00C2\u00A0and\t \u00C2\u00A0Stein,\t \u00C2\u00A02002).\t \u00C2\u00A0ASOs\t \u00C2\u00A0have\t \u00C2\u00A0also\t \u00C2\u00A0been\t \u00C2\u00A0reported\t \u00C2\u00A0to\t \u00C2\u00A0stimulate\t \u00C2\u00A0the\t \u00C2\u00A0immune\t \u00C2\u00A0system,\t \u00C2\u00A0increasing\t \u00C2\u00A0B\t \u00C2\u00A0cell\t \u00C2\u00A0activation\t \u00C2\u00A0and\t \u00C2\u00A0secretion\t \u00C2\u00A0of\t \u00C2\u00A0inflammatory\t \u00C2\u00A0cytokines\t \u00C2\u00A0(Krieg\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01995),\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0structure-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0sequence-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0 11 manner.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0hefty\t \u00C2\u00A0cost\t \u00C2\u00A0of\t \u00C2\u00A0synthesizing\t \u00C2\u00A0ASOs\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0prohibitive\t \u00C2\u00A0for\t \u00C2\u00A0long-\u00C2\u00AD\u00E2\u0080\u0090term\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0clinical\t \u00C2\u00A0setting.\t \u00C2\u00A01.3.2 RNA\t \u00C2\u00A0INTERFERENCE\t \u00C2\u00A0(RNAi)\t \u00C2\u00A0RNAi\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0natural\t \u00C2\u00A0biological\t \u00C2\u00A0process\t \u00C2\u00A0that\t \u00C2\u00A0regulates\t \u00C2\u00A0gene\t \u00C2\u00A0expression\t \u00C2\u00A0and\t \u00C2\u00A0provides\t \u00C2\u00A0an\t \u00C2\u00A0innate\t \u00C2\u00A0defense\t \u00C2\u00A0mechanism\t \u00C2\u00A0against\t \u00C2\u00A0invading\t \u00C2\u00A0viruses\t \u00C2\u00A0and\t \u00C2\u00A0transposable\t \u00C2\u00A0elements\t \u00C2\u00A0(Davidson\t \u00C2\u00A0and\t \u00C2\u00A0McCray,\t \u00C2\u00A02011).\t \u00C2\u00A0The\t \u00C2\u00A0field\t \u00C2\u00A0of\t \u00C2\u00A0RNAi\t \u00C2\u00A0stemmed\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0Nobel\t \u00C2\u00A0Prize-\u00C2\u00AD\u00E2\u0080\u0090winning\t \u00C2\u00A0discovery\t \u00C2\u00A0that\t \u00C2\u00A0double-\u00C2\u00AD\u00E2\u0080\u0090stranded\t \u00C2\u00A0RNAs\t \u00C2\u00A0(dsRNAs)\t \u00C2\u00A0can\t \u00C2\u00A0trigger\t \u00C2\u00A0silencing\t \u00C2\u00A0of\t \u00C2\u00A0complementary\t \u00C2\u00A0mRNA\t \u00C2\u00A0sequences\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0nematode\t \u00C2\u00A0Caenorhabditis\t \u00C2\u00A0elegans\t \u00C2\u00A0(Fire\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01998).\t \u00C2\u00A0Shortly\t \u00C2\u00A0thereafter,\t \u00C2\u00A0RNAi\t \u00C2\u00A0was\t \u00C2\u00A0discovered\t \u00C2\u00A0in\t \u00C2\u00A0mice\t \u00C2\u00A0using\t \u00C2\u00A0synthesized\t \u00C2\u00A021-\u00C2\u00AD\u00E2\u0080\u009023\t \u00C2\u00A0mer\t \u00C2\u00A0dsRNAs,\t \u00C2\u00A0or\t \u00C2\u00A0small\t \u00C2\u00A0interfering\t \u00C2\u00A0RNAs\t \u00C2\u00A0(siRNAs)\t \u00C2\u00A0(McCaffrey\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002),\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0method\u00E2\u0080\u0099s\t \u00C2\u00A0therapeutic\t \u00C2\u00A0potential\t \u00C2\u00A0was\t \u00C2\u00A0demonstrated\t \u00C2\u00A0a\t \u00C2\u00A0year\t \u00C2\u00A0later\t \u00C2\u00A0(Song\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0\t \u00C2\u00A0RNAi\t \u00C2\u00A0agents\t \u00C2\u00A0include\t \u00C2\u00A0dsRNA,\t \u00C2\u00A0short\t \u00C2\u00A0hairpin\t \u00C2\u00A0RNA\t \u00C2\u00A0(shRNA)\t \u00C2\u00A0and\t \u00C2\u00A0artificial\t \u00C2\u00A0miRNA.\t \u00C2\u00A0Each\t \u00C2\u00A0agent\t \u00C2\u00A0differs\t \u00C2\u00A0in\t \u00C2\u00A0size,\t \u00C2\u00A0utility,\t \u00C2\u00A0packaging\t \u00C2\u00A0and\t \u00C2\u00A0delivery,\t \u00C2\u00A0and\t \u00C2\u00A0enters\t \u00C2\u00A0at\t \u00C2\u00A0different\t \u00C2\u00A0points\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0innate\t \u00C2\u00A0RNAi\t \u00C2\u00A0pathway.\t \u00C2\u00A0In\t \u00C2\u00A0research\t \u00C2\u00A0using\t \u00C2\u00A0cultured\t \u00C2\u00A0cells,\t \u00C2\u00A0the\t \u00C2\u00A0most\t \u00C2\u00A0common\t \u00C2\u00A0delivery\t \u00C2\u00A0method\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0directly\t \u00C2\u00A0transfect\t \u00C2\u00A021-\u00C2\u00AD\u00E2\u0080\u009022nt\t \u00C2\u00A0siRNAs\t \u00C2\u00A0into\t \u00C2\u00A0cells\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0gene\t \u00C2\u00A0silencing.\t \u00C2\u00A0These\t \u00C2\u00A0synthetic\t \u00C2\u00A0siRNAs\t \u00C2\u00A0often\t \u00C2\u00A0lack\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0triphosphates\t \u00C2\u00A0and\t \u00C2\u00A0contain\t \u00C2\u00A0appropriate\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0overhangs\t \u00C2\u00A0to\t \u00C2\u00A0minimize\t \u00C2\u00A0host\t \u00C2\u00A0immune\t \u00C2\u00A0system\t \u00C2\u00A0activation.\t \u00C2\u00A0Once\t \u00C2\u00A0inside\t \u00C2\u00A0the\t \u00C2\u00A0cell,\t \u00C2\u00A0the\t \u00C2\u00A0siRNA\t \u00C2\u00A0associates\t \u00C2\u00A0with\t \u00C2\u00A0Argonaute\t \u00C2\u00A0protein\t \u00C2\u00A02\t \u00C2\u00A0(AGO2)\t \u00C2\u00A0within\t \u00C2\u00A0the\t \u00C2\u00A0precursor\t \u00C2\u00A0RNAi-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0silencing\t \u00C2\u00A0complex\t \u00C2\u00A0(pre-\u00C2\u00AD\u00E2\u0080\u0090RISC).\t \u00C2\u00A0The\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0passenger\t \u00C2\u00A0strand\t \u00C2\u00A0is\t \u00C2\u00A0cleaved,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0mature\t \u00C2\u00A0RISC,\t \u00C2\u00A0harboring\t \u00C2\u00A0the\t \u00C2\u00A0 12 guide\t \u00C2\u00A0strand,\t \u00C2\u00A0associates\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0mRNA\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0its\t \u00C2\u00A0cleavage\t \u00C2\u00A0(Meister\t \u00C2\u00A0and\t \u00C2\u00A0Tuschl,\t \u00C2\u00A02004).\t \u00C2\u00A0In\t \u00C2\u00A0contrast\t \u00C2\u00A0to\t \u00C2\u00A0linear\t \u00C2\u00A0siRNAs,\t \u00C2\u00A0shRNA\t \u00C2\u00A0and\t \u00C2\u00A0artificial\t \u00C2\u00A0miRNAs\t \u00C2\u00A0are\t \u00C2\u00A0packaged\t \u00C2\u00A0into\t \u00C2\u00A0plasmids\t \u00C2\u00A0and\t \u00C2\u00A0delivered\t \u00C2\u00A0into\t \u00C2\u00A0isolated\t \u00C2\u00A0cells\t \u00C2\u00A0or\t \u00C2\u00A0animals\t \u00C2\u00A0via\t \u00C2\u00A0viral\t \u00C2\u00A0infection\t \u00C2\u00A0(Rubinson\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Once\t \u00C2\u00A0expressed,\t \u00C2\u00A0shRNA\t \u00C2\u00A0and\t \u00C2\u00A0miRNAs\t \u00C2\u00A0are\t \u00C2\u00A0processed\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0nucleus\t \u00C2\u00A0into\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090miRNAs,\t \u00C2\u00A0which\t \u00C2\u00A0are\t \u00C2\u00A0then\t \u00C2\u00A0transported\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cytoplasm\t \u00C2\u00A0and\t \u00C2\u00A0cut\t \u00C2\u00A0into\t \u00C2\u00A0linear\t \u00C2\u00A0miRNA-\u00C2\u00AD\u00E2\u0080\u0090miRNA*\t \u00C2\u00A0duplexes\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0complex\t \u00C2\u00A0that\t \u00C2\u00A0contains\t \u00C2\u00A0the\t \u00C2\u00A0endoribonuclease\t \u00C2\u00A0Dicer.\t \u00C2\u00A0This\t \u00C2\u00A0duplex\t \u00C2\u00A0is\t \u00C2\u00A0then\t \u00C2\u00A0processed\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0manner\t \u00C2\u00A0as\t \u00C2\u00A0siRNAs\t \u00C2\u00A0to\t \u00C2\u00A0achieve\t \u00C2\u00A0target\t \u00C2\u00A0mRNA\t \u00C2\u00A0cleavage\t \u00C2\u00A0or\t \u00C2\u00A0translational\t \u00C2\u00A0inhibition\t \u00C2\u00A0(Paddison\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002).\t \u00C2\u00A0A\t \u00C2\u00A0detailed\t \u00C2\u00A0description\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0mechanisms\t \u00C2\u00A0behind\t \u00C2\u00A0RNAi\t \u00C2\u00A0is\t \u00C2\u00A0beyond\t \u00C2\u00A0the\t \u00C2\u00A0scope\t \u00C2\u00A0of\t \u00C2\u00A0this\t \u00C2\u00A0thesis,\t \u00C2\u00A0and\t \u00C2\u00A0readers\t \u00C2\u00A0are\t \u00C2\u00A0encouraged\t \u00C2\u00A0to\t \u00C2\u00A0refer\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0recent\t \u00C2\u00A0review\t \u00C2\u00A0(Wilson\t \u00C2\u00A0and\t \u00C2\u00A0Doudna,\t \u00C2\u00A02013).\t \u00C2\u00A0Recent\t \u00C2\u00A0efforts\t \u00C2\u00A0have\t \u00C2\u00A0developed\t \u00C2\u00A0single-\u00C2\u00AD\u00E2\u0080\u0090stranded\t \u00C2\u00A0siRNAs\t \u00C2\u00A0(ss-\u00C2\u00AD\u00E2\u0080\u0090siRNAs)\t \u00C2\u00A0with\t \u00C2\u00A0increased\t \u00C2\u00A0stability\t \u00C2\u00A0and\t \u00C2\u00A0enhanced\t \u00C2\u00A0cellular\t \u00C2\u00A0uptake\t \u00C2\u00A0(Lima\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012;\t \u00C2\u00A0Nawy,\t \u00C2\u00A02012).\t \u00C2\u00A0The\t \u00C2\u00A0single-\u00C2\u00AD\u00E2\u0080\u0090strand\t \u00C2\u00A0RNAi\t \u00C2\u00A0approach\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0successfully\t \u00C2\u00A0to\t \u00C2\u00A0knockdown\t \u00C2\u00A0mHtt\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0HdhQ150/Q7\t \u00C2\u00A0heterozygous\t \u00C2\u00A0mouse\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0HD\t \u00C2\u00A0following\t \u00C2\u00A0i.c.v.\t \u00C2\u00A0infusion\t \u00C2\u00A0for\t \u00C2\u00A028\t \u00C2\u00A0days\t \u00C2\u00A0(Yu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Although\t \u00C2\u00A0RNAi\t \u00C2\u00A0has\t \u00C2\u00A0promising\t \u00C2\u00A0therapeutic\t \u00C2\u00A0potential,\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0several\t \u00C2\u00A0problems\t \u00C2\u00A0preventing\t \u00C2\u00A0its\t \u00C2\u00A0rapid\t \u00C2\u00A0clinical\t \u00C2\u00A0adoption.\t \u00C2\u00A0First,\t \u00C2\u00A0although\t \u00C2\u00A0siRNAs\t \u00C2\u00A0are\t \u00C2\u00A0designed\t \u00C2\u00A0to\t \u00C2\u00A0fully\t \u00C2\u00A0complement\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0mRNA\t \u00C2\u00A0transcript,\t \u00C2\u00A0they\t \u00C2\u00A0may\t \u00C2\u00A0inadvertently\t \u00C2\u00A0hybridize\t \u00C2\u00A0to\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0mRNAs\t \u00C2\u00A0(Jackson\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003)\t \u00C2\u00A0and\t \u00C2\u00A0confound\t \u00C2\u00A0the\t \u00C2\u00A0interpretation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0resulting\t \u00C2\u00A0phenotype.\t \u00C2\u00A0This\t \u00C2\u00A0problem\t \u00C2\u00A0is\t \u00C2\u00A0more\t \u00C2\u00A0pronounced\t \u00C2\u00A0for\t \u00C2\u00A0artificial\t \u00C2\u00A0miRNAs\t \u00C2\u00A0that\t \u00C2\u00A0complement\t \u00C2\u00A0to\t \u00C2\u00A0mRNA\t \u00C2\u00A0 13 targets\t \u00C2\u00A0via\t \u00C2\u00A0a\t \u00C2\u00A0six\t \u00C2\u00A0nucleotide\t \u00C2\u00A0\t \u00C2\u00A0\u00E2\u0080\u009Cseed\t \u00C2\u00A0region\u00E2\u0080\u009D.\t \u00C2\u00A0Second,\t \u00C2\u00A0siRNAs\t \u00C2\u00A0are\t \u00C2\u00A0prone\t \u00C2\u00A0to\t \u00C2\u00A0degradation\t \u00C2\u00A0and\t \u00C2\u00A0have\t \u00C2\u00A0a\t \u00C2\u00A0short\t \u00C2\u00A0duration\t \u00C2\u00A0of\t \u00C2\u00A0activity\t \u00C2\u00A0(Scaggiante\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0Although\t \u00C2\u00A0viral-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0shRNA\t \u00C2\u00A0can\t \u00C2\u00A0prolong\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown,\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0viral\t \u00C2\u00A0vectors\t \u00C2\u00A0carries\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0insertional\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0or\t \u00C2\u00A0triggering\t \u00C2\u00A0aberrant\t \u00C2\u00A0gene\t \u00C2\u00A0expression\t \u00C2\u00A0(Castanotto\t \u00C2\u00A0and\t \u00C2\u00A0Rossi,\t \u00C2\u00A02009).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0some\t \u00C2\u00A0viral\t \u00C2\u00A0vectors\t \u00C2\u00A0(e.g.\t \u00C2\u00A0adenoviral\t \u00C2\u00A0vectors)\t \u00C2\u00A0infect\t \u00C2\u00A0cells\t \u00C2\u00A0at\t \u00C2\u00A0multiple\t \u00C2\u00A0copies\t \u00C2\u00A0per\t \u00C2\u00A0cell.\t \u00C2\u00A0This\t \u00C2\u00A0may\t \u00C2\u00A0lead\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0higher\t \u00C2\u00A0dose\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0expressed\t \u00C2\u00A0RNAi\t \u00C2\u00A0agent\t \u00C2\u00A0and\t \u00C2\u00A0cause\t \u00C2\u00A0saturation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0RNAi\t \u00C2\u00A0machinery\t \u00C2\u00A0(Grimm\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006).\t \u00C2\u00A0Third,\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0RNAi\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0natural\t \u00C2\u00A0defense\t \u00C2\u00A0mechanism\t \u00C2\u00A0against\t \u00C2\u00A0pathogens,\t \u00C2\u00A0it\t \u00C2\u00A0inherently\t \u00C2\u00A0carries\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0inducing\t \u00C2\u00A0the\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090viral\t \u00C2\u00A0response\t \u00C2\u00A0(Judge\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0Fourth,\t \u00C2\u00A0despite\t \u00C2\u00A0significant\t \u00C2\u00A0advancements\t \u00C2\u00A0in\t \u00C2\u00A0siRNA\t \u00C2\u00A0carriers\t \u00C2\u00A0(Whitehead\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009),\t \u00C2\u00A0the\t \u00C2\u00A0systemic\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0RNAi\t \u00C2\u00A0agents\t \u00C2\u00A0remains\t \u00C2\u00A0clinically\t \u00C2\u00A0challenging.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0Finally,\t \u00C2\u00A0because\t \u00C2\u00A0RNAi\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0gene\t \u00C2\u00A0targeting\t \u00C2\u00A0and\t \u00C2\u00A0ASOs\t \u00C2\u00A0described\t \u00C2\u00A0above\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0targets\t \u00C2\u00A0nucleotide\t \u00C2\u00A0precursors\t \u00C2\u00A0and\t \u00C2\u00A0not\t \u00C2\u00A0protein\t \u00C2\u00A0end\t \u00C2\u00A0products,\t \u00C2\u00A0measurable\t \u00C2\u00A0decreases\t \u00C2\u00A0in\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0will\t \u00C2\u00A0only\t \u00C2\u00A0occur\t \u00C2\u00A0after\t \u00C2\u00A0the\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090existing\t \u00C2\u00A0pool\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0relevant\t \u00C2\u00A0protein\t \u00C2\u00A0is\t \u00C2\u00A0degraded,\t \u00C2\u00A0thus\t \u00C2\u00A0causing\t \u00C2\u00A0a\t \u00C2\u00A0significant\t \u00C2\u00A0time\t \u00C2\u00A0lag\t \u00C2\u00A0between\t \u00C2\u00A0treatment\t \u00C2\u00A0and\t \u00C2\u00A0phenotypic\t \u00C2\u00A0response.\t \u00C2\u00A0\t \u00C2\u00A01.4 PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0AT\t \u00C2\u00A0THE\t \u00C2\u00A0POST-\u00C2\u00AD\u00E2\u0080\u0090TRANSLATIONAL\t \u00C2\u00A0LEVEL\t \u00C2\u00A0Protein\t \u00C2\u00A0turnover\t \u00C2\u00A0in\t \u00C2\u00A0cells\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0highly\t \u00C2\u00A0selective\t \u00C2\u00A0and\t \u00C2\u00A0tightly\t \u00C2\u00A0regulated\t \u00C2\u00A0process\t \u00C2\u00A0that\t \u00C2\u00A0serves\t \u00C2\u00A0to\t \u00C2\u00A0maintain\t \u00C2\u00A0cellular\t \u00C2\u00A0homeostasis\t \u00C2\u00A0by\t \u00C2\u00A0eliminating\t \u00C2\u00A0misfolded\t \u00C2\u00A0or\t \u00C2\u00A0damaged\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0would\t \u00C2\u00A0otherwise\t \u00C2\u00A0interfere\t \u00C2\u00A0with\t \u00C2\u00A0cell\t \u00C2\u00A0viability\t \u00C2\u00A0and\t \u00C2\u00A0function\t \u00C2\u00A0(Goldberg,\t \u00C2\u00A02003;\t \u00C2\u00A0Mizushima\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008;\t \u00C2\u00A0Dantuma\t \u00C2\u00A0and\t \u00C2\u00A0Bott,\t \u00C2\u00A02014).\t \u00C2\u00A0Multiple\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0work\t \u00C2\u00A0in\t \u00C2\u00A0tandem\t \u00C2\u00A0to\t \u00C2\u00A0protect\t \u00C2\u00A0 14 proteomic\t \u00C2\u00A0homeostasis,\t \u00C2\u00A0ranging\t \u00C2\u00A0from\t \u00C2\u00A0simple\t \u00C2\u00A0extracellular\t \u00C2\u00A0proteases\t \u00C2\u00A0(e.g.\t \u00C2\u00A0digestive\t \u00C2\u00A0proteases)\t \u00C2\u00A0to\t \u00C2\u00A0highly\t \u00C2\u00A0elaborate\t \u00C2\u00A0proteolytic\t \u00C2\u00A0machineries\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090proteasome\t \u00C2\u00A0system\t \u00C2\u00A0(UPS)\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0autophagy-\u00C2\u00AD\u00E2\u0080\u0090lysosome\t \u00C2\u00A0system\t \u00C2\u00A0(ALS).\t \u00C2\u00A0Generally\t \u00C2\u00A0speaking,\t \u00C2\u00A0in\t \u00C2\u00A0eukaryotes\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0is\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0while\t \u00C2\u00A0the\t \u00C2\u00A0ALS\t \u00C2\u00A0degrades\t \u00C2\u00A0substrates\t \u00C2\u00A0in\t \u00C2\u00A0bulk.\t \u00C2\u00A0However,\t \u00C2\u00A0it\t \u00C2\u00A0should\t \u00C2\u00A0be\t \u00C2\u00A0noted\t \u00C2\u00A0that\t \u00C2\u00A0both\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0are\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0selective,\t \u00C2\u00A0one-\u00C2\u00AD\u00E2\u0080\u0090by-\u00C2\u00AD\u00E2\u0080\u0090one\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Wong,\t \u00C2\u00A02013).\t \u00C2\u00A0The\t \u00C2\u00A0molecular\t \u00C2\u00A0machineries\t \u00C2\u00A0underlying\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0ALS\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0extensively\t \u00C2\u00A0characterized,\t \u00C2\u00A0and\t \u00C2\u00A0this\t \u00C2\u00A0knowledge\t \u00C2\u00A0provides\t \u00C2\u00A0the\t \u00C2\u00A0basis\t \u00C2\u00A0of\t \u00C2\u00A0harnessing\t \u00C2\u00A0these\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level.\t \u00C2\u00A0\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0method\t \u00C2\u00A0developed\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0(CMA),\t \u00C2\u00A0a\t \u00C2\u00A0process\t \u00C2\u00A0involving\t \u00C2\u00A0the\t \u00C2\u00A0lysosome,\t \u00C2\u00A0I\t \u00C2\u00A0will\t \u00C2\u00A0only\t \u00C2\u00A0briefly\t \u00C2\u00A0review\t \u00C2\u00A0our\t \u00C2\u00A0current\t \u00C2\u00A0understanding\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0UPS,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0other\t \u00C2\u00A0degradation\t \u00C2\u00A0pathways\t \u00C2\u00A0mediated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0ALS.\t \u00C2\u00A0This\t \u00C2\u00A0bird\u00E2\u0080\u0099s\t \u00C2\u00A0eye\t \u00C2\u00A0view\t \u00C2\u00A0of\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0is\t \u00C2\u00A0important\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0isolation;\t \u00C2\u00A0instead,\t \u00C2\u00A0it\t \u00C2\u00A0has\t \u00C2\u00A0extensive\t \u00C2\u00A0crosstalk\t \u00C2\u00A0with\t \u00C2\u00A0other\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0that\t \u00C2\u00A0becomes\t \u00C2\u00A0especially\t \u00C2\u00A0important\t \u00C2\u00A0to\t \u00C2\u00A0consider\t \u00C2\u00A0in\t \u00C2\u00A0cases\t \u00C2\u00A0of\t \u00C2\u00A0disease\t \u00C2\u00A0(Korolchuk\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Park\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02013;\t \u00C2\u00A0Yang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0most\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0utilize\t \u00C2\u00A0the\t \u00C2\u00A0UPS,\t \u00C2\u00A0an\t \u00C2\u00A0overview\t \u00C2\u00A0of\t \u00C2\u00A0this\t \u00C2\u00A0pathway\t \u00C2\u00A0will\t \u00C2\u00A0provide\t \u00C2\u00A0the\t \u00C2\u00A0knowledge\t \u00C2\u00A0basis\t \u00C2\u00A0necessary\t \u00C2\u00A0to\t \u00C2\u00A0evaluate\t \u00C2\u00A0such\t \u00C2\u00A0methods.\t \u00C2\u00A0In\t \u00C2\u00A0sum,\t \u00C2\u00A0the\t \u00C2\u00A0focus\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0sections\t \u00C2\u00A0is\t \u00C2\u00A0on\t \u00C2\u00A0CMA;\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0comprehensive\t \u00C2\u00A0review\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0macroautophagy,\t \u00C2\u00A0see\t \u00C2\u00A0(Lecker,\t \u00C2\u00A02006;\t \u00C2\u00A0Bhattacharyya\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014)\t \u00C2\u00A0and\t \u00C2\u00A0(Feng\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013),\t \u00C2\u00A0respectively.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 15 1.4.1 THE\t \u00C2\u00A0UPS\t \u00C2\u00A0The\t \u00C2\u00A0UPS\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0major\t \u00C2\u00A0proteolytic\t \u00C2\u00A0pathway\t \u00C2\u00A0responsible\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0regulated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0short-\u00C2\u00AD\u00E2\u0080\u0090lived\t \u00C2\u00A0proteins,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0nucleus\t \u00C2\u00A0and\t \u00C2\u00A0endoplasmic\t \u00C2\u00A0reticulum\t \u00C2\u00A0(Baumeister\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01998;\t \u00C2\u00A0Vembar\t \u00C2\u00A0and\t \u00C2\u00A0Brodsky,\t \u00C2\u00A02008).\t \u00C2\u00A0Generally\t \u00C2\u00A0speaking,\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0UPS\t \u00C2\u00A0substrates\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0multi-\u00C2\u00AD\u00E2\u0080\u0090step\t \u00C2\u00A0process\t \u00C2\u00A0that\t \u00C2\u00A0centers\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0ubiquitin,\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0(8.5kDa)\t \u00C2\u00A0globular\t \u00C2\u00A0protein,\t \u00C2\u00A0to\t \u00C2\u00A0substrate\t \u00C2\u00A0proteins\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0covalent\t \u00C2\u00A0bond\t \u00C2\u00A0that\t \u00C2\u00A0involves\t \u00C2\u00A0a\t \u00C2\u00A0lysine\t \u00C2\u00A0residue\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0(Wong\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02010a).\t \u00C2\u00A0A\t \u00C2\u00A0notable\t \u00C2\u00A0exception\t \u00C2\u00A0is\t \u00C2\u00A0ornithine\t \u00C2\u00A0decarboxylase,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0directly\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A026S\t \u00C2\u00A0proteasome\t \u00C2\u00A0subunit\t \u00C2\u00A0without\t \u00C2\u00A0the\t \u00C2\u00A0need\t \u00C2\u00A0for\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0(Janse,\t \u00C2\u00A02004a;\t \u00C2\u00A0Erales\t \u00C2\u00A0and\t \u00C2\u00A0Coffino,\t \u00C2\u00A02014).\t \u00C2\u00A0In\t \u00C2\u00A0fact,\t \u00C2\u00A0this\t \u00C2\u00A0direct\t \u00C2\u00A0proteasomal\t \u00C2\u00A0degradation\t \u00C2\u00A0mechanism\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0harnessed\t \u00C2\u00A0to\t \u00C2\u00A0design\t \u00C2\u00A0a\t \u00C2\u00A0proteasome-\u00C2\u00AD\u00E2\u0080\u0090dependent,\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090independent\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0method\t \u00C2\u00A0(Matsuzawa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0conjugation\t \u00C2\u00A0of\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0protein\t \u00C2\u00A0requires\t \u00C2\u00A0the\t \u00C2\u00A0actions\t \u00C2\u00A0of\t \u00C2\u00A0three\t \u00C2\u00A0enzymes\t \u00C2\u00A0(Hershko\t \u00C2\u00A0and\t \u00C2\u00A0Ciechanover,\t \u00C2\u00A01998).\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0first\t \u00C2\u00A0step,\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090activating\t \u00C2\u00A0enzyme\t \u00C2\u00A0(E1)\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0ATP-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0process,\t \u00C2\u00A0and\t \u00C2\u00A0is\t \u00C2\u00A0subsequently\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0active\t \u00C2\u00A0site\t \u00C2\u00A0of\t \u00C2\u00A0one\t \u00C2\u00A0of\t \u00C2\u00A0many\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090conjugating\t \u00C2\u00A0enzymes\t \u00C2\u00A0(E2).\t \u00C2\u00A0Finally,\t \u00C2\u00A0a\t \u00C2\u00A0substrate-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090ligase\t \u00C2\u00A0enzyme\t \u00C2\u00A0(E3)\t \u00C2\u00A0catalyzes\t \u00C2\u00A0the\t \u00C2\u00A0transfer\t \u00C2\u00A0of\t \u00C2\u00A0E2-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0protein\t \u00C2\u00A0(Bhattacharyya\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Because\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0itself\t \u00C2\u00A0contains\t \u00C2\u00A0seven\t \u00C2\u00A0lysine\t \u00C2\u00A0residues,\t \u00C2\u00A0the\t \u00C2\u00A0process\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0repeated\t \u00C2\u00A0to\t \u00C2\u00A0form\t \u00C2\u00A0polyubiquitin\t \u00C2\u00A0chains\t \u00C2\u00A0with\t \u00C2\u00A0diverse\t \u00C2\u00A0topologies.\t \u00C2\u00A0Notably,\t \u00C2\u00A0the\t \u00C2\u00A0particular\t \u00C2\u00A0mode\t \u00C2\u00A0of\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0linkage\t \u00C2\u00A0determines\t \u00C2\u00A0the\t \u00C2\u00A0fate\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0(Komander\t \u00C2\u00A0and\t \u00C2\u00A0Rape,\t \u00C2\u00A02012).\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0case\t \u00C2\u00A0of\t \u00C2\u00A0UPS-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation,\t \u00C2\u00A0proteins\t \u00C2\u00A0 16 tagged\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0linear\t \u00C2\u00A0chain\t \u00C2\u00A0of\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A0four\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0molecules\t \u00C2\u00A0are\t \u00C2\u00A0generally\t \u00C2\u00A0recognized\t \u00C2\u00A0as\t \u00C2\u00A0UPS\t \u00C2\u00A0substrates\t \u00C2\u00A0(Jentsch\t \u00C2\u00A0and\t \u00C2\u00A0Schlenker,\t \u00C2\u00A01995).\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0selection\t \u00C2\u00A0of\t \u00C2\u00A0substrates\t \u00C2\u00A0for\t \u00C2\u00A0UPS\t \u00C2\u00A0degradation\t \u00C2\u00A0is\t \u00C2\u00A0largely\t \u00C2\u00A0dependent\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0E3\t \u00C2\u00A0enzymes,\t \u00C2\u00A0a\t \u00C2\u00A0large\t \u00C2\u00A0(>1000)\t \u00C2\u00A0family\t \u00C2\u00A0of\t \u00C2\u00A0ligases\t \u00C2\u00A0that\t \u00C2\u00A0bear\t \u00C2\u00A0little\t \u00C2\u00A0structural\t \u00C2\u00A0or\t \u00C2\u00A0chemical\t \u00C2\u00A0similarity\t \u00C2\u00A0between\t \u00C2\u00A0its\t \u00C2\u00A0members\t \u00C2\u00A0(Staub,\t \u00C2\u00A02006).\t \u00C2\u00A0E3\t \u00C2\u00A0enzymes\t \u00C2\u00A0select\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0either\t \u00C2\u00A0directly\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0through\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions\t \u00C2\u00A0or\t \u00C2\u00A0indirectly\t \u00C2\u00A0through\t \u00C2\u00A0adaptor\t \u00C2\u00A0chaperones,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0Hsp70\t \u00C2\u00A0or\t \u00C2\u00A0Hsp90\t \u00C2\u00A0(Rosser\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02007).\t \u00C2\u00A0The\t \u00C2\u00A0large\t \u00C2\u00A0diversity\t \u00C2\u00A0of\t \u00C2\u00A0E3\t \u00C2\u00A0ligases\t \u00C2\u00A0enables\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0to\t \u00C2\u00A0individually\t \u00C2\u00A0regulate\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0variety\t \u00C2\u00A0of\t \u00C2\u00A0UPS\t \u00C2\u00A0substrate\t \u00C2\u00A0proteins.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0around\t \u00C2\u00A0100\t \u00C2\u00A0types\t \u00C2\u00A0of\t \u00C2\u00A0E2\t \u00C2\u00A0enzymes,\t \u00C2\u00A0which\t \u00C2\u00A0may\t \u00C2\u00A0further\t \u00C2\u00A0confer\t \u00C2\u00A0substrate\t \u00C2\u00A0selectivity\t \u00C2\u00A0(Staub,\t \u00C2\u00A02006).\t \u00C2\u00A0As\t \u00C2\u00A0explained\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.5,\t \u00C2\u00A0many\t \u00C2\u00A0research\t \u00C2\u00A0groups\t \u00C2\u00A0have\t \u00C2\u00A0harnessed\t \u00C2\u00A0the\t \u00C2\u00A0selectivity\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0system\t \u00C2\u00A0to\t \u00C2\u00A0design\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0directly\t \u00C2\u00A0target\t \u00C2\u00A0POIs\t \u00C2\u00A0for\t \u00C2\u00A0UPS\t \u00C2\u00A0degradation.\t \u00C2\u00A01.4.2 THE\t \u00C2\u00A0ALS\t \u00C2\u00A0Lysosomes\t \u00C2\u00A0are\t \u00C2\u00A0highly\t \u00C2\u00A0acidic\t \u00C2\u00A0(pH\t \u00C2\u00A04.6-\u00C2\u00AD\u00E2\u0080\u00905.0),\t \u00C2\u00A0single\t \u00C2\u00A0membrane-\u00C2\u00AD\u00E2\u0080\u0090bound\t \u00C2\u00A0organelles\t \u00C2\u00A0that\t \u00C2\u00A0function\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0major\t \u00C2\u00A0terminal\t \u00C2\u00A0degradative\t \u00C2\u00A0compartment\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0(Luzio\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02007).\t \u00C2\u00A0The\t \u00C2\u00A0lysosome\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0fusogenic\t \u00C2\u00A0organelle,\t \u00C2\u00A0as\t \u00C2\u00A0demonstrated\t \u00C2\u00A0by\t \u00C2\u00A0its\t \u00C2\u00A0ability\t \u00C2\u00A0to\t \u00C2\u00A0fuse\t \u00C2\u00A0with\t \u00C2\u00A0late\t \u00C2\u00A0endosomes\t \u00C2\u00A0and\t \u00C2\u00A0autophagosomes\t \u00C2\u00A0to\t \u00C2\u00A0create\t \u00C2\u00A0hybrid\t \u00C2\u00A0organelles\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0bulk\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cargo\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0(Luzio\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02007).\t \u00C2\u00A0The\t \u00C2\u00A0normal\t \u00C2\u00A0functions\t \u00C2\u00A0of\t \u00C2\u00A0lysosomes\t \u00C2\u00A0depend\t \u00C2\u00A0on\t \u00C2\u00A0two\t \u00C2\u00A0main\t \u00C2\u00A0classes\t \u00C2\u00A0of\t \u00C2\u00A0proteins:\t \u00C2\u00A0acid\t \u00C2\u00A0hydrolases\t \u00C2\u00A0and\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins\t \u00C2\u00A0(LMPs).\t \u00C2\u00A0To\t \u00C2\u00A0date,\t \u00C2\u00A0over\t \u00C2\u00A050\t \u00C2\u00A0substrate-\u00C2\u00AD\u00E2\u0080\u0090selective\t \u00C2\u00A0hydrolases\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0discovered,\t \u00C2\u00A0the\t \u00C2\u00A0collective\t \u00C2\u00A0action\t \u00C2\u00A0of\t \u00C2\u00A0 17 which\t \u00C2\u00A0confer\t \u00C2\u00A0lysosomes\t \u00C2\u00A0with\t \u00C2\u00A0total\t \u00C2\u00A0catabolic\t \u00C2\u00A0ability\t \u00C2\u00A0(Saftig\t \u00C2\u00A0and\t \u00C2\u00A0Klumperman,\t \u00C2\u00A02009).\t \u00C2\u00A0LMPs\t \u00C2\u00A0are\t \u00C2\u00A0integral\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0mainly\t \u00C2\u00A0reside\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0limiting\t \u00C2\u00A0membrane\t \u00C2\u00A0and\t \u00C2\u00A0serve\t \u00C2\u00A0diverse\t \u00C2\u00A0functions,\t \u00C2\u00A0including\t \u00C2\u00A0acidification\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0lumen,\t \u00C2\u00A0internalization\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0substrates\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0cytosol\t \u00C2\u00A0and\t \u00C2\u00A0fusion\t \u00C2\u00A0of\t \u00C2\u00A0limiting\t \u00C2\u00A0membranes\t \u00C2\u00A0(Eskelinen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0The\t \u00C2\u00A0most\t \u00C2\u00A0abundant\t \u00C2\u00A0LMPs\t \u00C2\u00A0are\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins\t \u00C2\u00A01\t \u00C2\u00A0and\t \u00C2\u00A02\t \u00C2\u00A0(LAMP1\t \u00C2\u00A0and\t \u00C2\u00A0LAMP2).\t \u00C2\u00A0Although\t \u00C2\u00A0the\t \u00C2\u00A0functions\t \u00C2\u00A0of\t \u00C2\u00A0LAMP1\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0substituted\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0structurally\t \u00C2\u00A0homologous\t \u00C2\u00A0LAMP2,\t \u00C2\u00A0the\t \u00C2\u00A0converse\t \u00C2\u00A0process\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0feasible.\t \u00C2\u00A0This\t \u00C2\u00A0is\t \u00C2\u00A0because\t \u00C2\u00A0LAMP2\t \u00C2\u00A0serves\t \u00C2\u00A0several\t \u00C2\u00A0specific\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090redundant\t \u00C2\u00A0roles,\t \u00C2\u00A0including\t \u00C2\u00A0lysosomal\t \u00C2\u00A0biogenesis,\t \u00C2\u00A0lysosome\t \u00C2\u00A0enzyme\t \u00C2\u00A0targeting\t \u00C2\u00A0and\t \u00C2\u00A0CMA\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000a;\t \u00C2\u00A0Eskelinen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Autophagy\t \u00C2\u00A0refers\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0intracellular\t \u00C2\u00A0components\t \u00C2\u00A0(Mizushima\t \u00C2\u00A0and\t \u00C2\u00A0Komatsu,\t \u00C2\u00A02011).\t \u00C2\u00A0There\t \u00C2\u00A0are\t \u00C2\u00A0three\t \u00C2\u00A0major\t \u00C2\u00A0autophagic\t \u00C2\u00A0pathways\t \u00C2\u00A0that\t \u00C2\u00A0differ\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0mechanism\t \u00C2\u00A0by\t \u00C2\u00A0which\t \u00C2\u00A0substrates\t \u00C2\u00A0are\t \u00C2\u00A0delivered\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosome:\t \u00C2\u00A0macroautophagy,\t \u00C2\u00A0microautophagy\t \u00C2\u00A0and\t \u00C2\u00A0CMA.\t \u00C2\u00A0These\t \u00C2\u00A0three\t \u00C2\u00A0main\t \u00C2\u00A0types\t \u00C2\u00A0of\t \u00C2\u00A0autophagy\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0further\t \u00C2\u00A0subdivided\t \u00C2\u00A0depending\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0nature\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cargo\t \u00C2\u00A0and/or\t \u00C2\u00A0the\t \u00C2\u00A0stimuli\t \u00C2\u00A0that\t \u00C2\u00A0trigger\t \u00C2\u00A0their\t \u00C2\u00A0degradation.\t \u00C2\u00A0Microautophagy\t \u00C2\u00A0and\t \u00C2\u00A0CMA\t \u00C2\u00A0deliver\t \u00C2\u00A0substrates\t \u00C2\u00A0directly\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0lumen\t \u00C2\u00A0(Yamamoto\t \u00C2\u00A0and\t \u00C2\u00A0Yue,\t \u00C2\u00A02014),\t \u00C2\u00A0while\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0first\t \u00C2\u00A0segregates\t \u00C2\u00A0substrates\t \u00C2\u00A0into\t \u00C2\u00A0de\t \u00C2\u00A0novo\t \u00C2\u00A0vesicles\t \u00C2\u00A0prior\t \u00C2\u00A0fusing\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0lysosome.\t \u00C2\u00A0\t \u00C2\u00A01.4.2.1 MACROAUTOPHAGY\t \u00C2\u00A0Macroautophagy\t \u00C2\u00A0is\t \u00C2\u00A0by\t \u00C2\u00A0far\t \u00C2\u00A0the\t \u00C2\u00A0best-\u00C2\u00AD\u00E2\u0080\u0090characterized\t \u00C2\u00A0autophagic\t \u00C2\u00A0pathway\t \u00C2\u00A0(Yang\t \u00C2\u00A0and\t \u00C2\u00A0Klionsky,\t \u00C2\u00A02010).\t \u00C2\u00A0This\t \u00C2\u00A0pathway\t \u00C2\u00A0requires\t \u00C2\u00A0the\t \u00C2\u00A0synthesis\t \u00C2\u00A0of\t \u00C2\u00A0multilamellar\t \u00C2\u00A0vesicles\t \u00C2\u00A0(autophagosomes)\t \u00C2\u00A0 18 around\t \u00C2\u00A0to-\u00C2\u00AD\u00E2\u0080\u0090be-\u00C2\u00AD\u00E2\u0080\u0090degraded\t \u00C2\u00A0substrates\t \u00C2\u00A0and\t \u00C2\u00A0their\t \u00C2\u00A0surrounding\t \u00C2\u00A0cytosol.\t \u00C2\u00A0The\t \u00C2\u00A0autophagosome\t \u00C2\u00A0then\t \u00C2\u00A0matures\t \u00C2\u00A0and\t \u00C2\u00A0traffics\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0perinuclear\t \u00C2\u00A0area,\t \u00C2\u00A0where\t \u00C2\u00A0it\t \u00C2\u00A0fuses\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0to\t \u00C2\u00A0deliver\t \u00C2\u00A0its\t \u00C2\u00A0cargo.\t \u00C2\u00A0The\t \u00C2\u00A0core\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0machinery\t \u00C2\u00A0is\t \u00C2\u00A0composed\t \u00C2\u00A0of\t \u00C2\u00A0more\t \u00C2\u00A0than\t \u00C2\u00A030\t \u00C2\u00A0proteins\t \u00C2\u00A0encoded\t \u00C2\u00A0by\t \u00C2\u00A0autophagy-\u00C2\u00AD\u00E2\u0080\u0090related\t \u00C2\u00A0genes\t \u00C2\u00A0(ATGs),\t \u00C2\u00A0most\t \u00C2\u00A0of\t \u00C2\u00A0which\t \u00C2\u00A0are\t \u00C2\u00A0conserved\t \u00C2\u00A0through\t \u00C2\u00A0evolution\t \u00C2\u00A0(Harris\t \u00C2\u00A0and\t \u00C2\u00A0Rubinsztein,\t \u00C2\u00A02011).\t \u00C2\u00A0\t \u00C2\u00A0Macroautophagic\t \u00C2\u00A0degradation\t \u00C2\u00A0centers\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0formation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0autophagosome\t \u00C2\u00A0and\t \u00C2\u00A0consists\t \u00C2\u00A0of\t \u00C2\u00A0four\t \u00C2\u00A0phases:\t \u00C2\u00A0initiation,\t \u00C2\u00A0elongation,\t \u00C2\u00A0maturation\t \u00C2\u00A0and\t \u00C2\u00A0fusion\t \u00C2\u00A0(Feng\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013;\t \u00C2\u00A0Yamamoto\t \u00C2\u00A0and\t \u00C2\u00A0Yue,\t \u00C2\u00A02014).\t \u00C2\u00A0The\t \u00C2\u00A0process\t \u00C2\u00A0begins\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0formation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0cup-\u00C2\u00AD\u00E2\u0080\u0090shaped\t \u00C2\u00A0isolation\t \u00C2\u00A0membrane\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cytoplasm,\t \u00C2\u00A0a\t \u00C2\u00A0process\t \u00C2\u00A0dependent\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0Ulk1\t \u00C2\u00A0complex\t \u00C2\u00A0and\t \u00C2\u00A0Beclin1-\u00C2\u00AD\u00E2\u0080\u0090Vsp34\t \u00C2\u00A0kinase.\t \u00C2\u00A0The\t \u00C2\u00A0membrane\t \u00C2\u00A0then\t \u00C2\u00A0elongates\t \u00C2\u00A0and\t \u00C2\u00A0envelops\t \u00C2\u00A0the\t \u00C2\u00A0cargo,\t \u00C2\u00A0which\t \u00C2\u00A0requires\t \u00C2\u00A0Atg9\t \u00C2\u00A0and\t \u00C2\u00A0two\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0conjugation\t \u00C2\u00A0systems\t \u00C2\u00A0(Mizushima\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008)\t \u00C2\u00A0and\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0lipidation\t \u00C2\u00A0of\t \u00C2\u00A0LC3.\t \u00C2\u00A0Once\t \u00C2\u00A0formed,\t \u00C2\u00A0the\t \u00C2\u00A0autophagosome\t \u00C2\u00A0travels\t \u00C2\u00A0along\t \u00C2\u00A0microtubules\t \u00C2\u00A0towards\t \u00C2\u00A0the\t \u00C2\u00A0perinuclear\t \u00C2\u00A0area,\t \u00C2\u00A0and\t \u00C2\u00A0finally\t \u00C2\u00A0fuses\t \u00C2\u00A0with\t \u00C2\u00A0lysosomes\t \u00C2\u00A0to\t \u00C2\u00A0form\t \u00C2\u00A0the\t \u00C2\u00A0hybrid\t \u00C2\u00A0amphisomes\t \u00C2\u00A0that\t \u00C2\u00A0matures\t \u00C2\u00A0into\t \u00C2\u00A0autophagolysosomes,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0cargo\t \u00C2\u00A0is\t \u00C2\u00A0degraded.\t \u00C2\u00A0Of\t \u00C2\u00A0note,\t \u00C2\u00A0a\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090canonical\t \u00C2\u00A0form\t \u00C2\u00A0of\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0that\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0require\t \u00C2\u00A0LC3\t \u00C2\u00A0lipidation\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0discovered\t \u00C2\u00A0in\t \u00C2\u00A0embryonic\t \u00C2\u00A0tissue\t \u00C2\u00A0(Nishida\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0Although\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0was\t \u00C2\u00A0initially\t \u00C2\u00A0considered\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090specific,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0now\t \u00C2\u00A0recognized\t \u00C2\u00A0that\t \u00C2\u00A0adaptor\t \u00C2\u00A0proteins\t \u00C2\u00A0known\t \u00C2\u00A0as\t \u00C2\u00A0selectivity\t \u00C2\u00A0adaptors\t \u00C2\u00A0can\t \u00C2\u00A0promote\t \u00C2\u00A0the\t \u00C2\u00A0selective\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0aggregated\t \u00C2\u00A0proteins\t \u00C2\u00A0and\t \u00C2\u00A0organelles.\t \u00C2\u00A0Adaptor\t \u00C2\u00A0proteins\t \u00C2\u00A0identified\t \u00C2\u00A0thus\t \u00C2\u00A0far\t \u00C2\u00A0include\t \u00C2\u00A0p62,\t \u00C2\u00A0NBR1,\t \u00C2\u00A0Nix,\t \u00C2\u00A0NDP52,\t \u00C2\u00A0Alfy\t \u00C2\u00A0and\t \u00C2\u00A0OPTN\t \u00C2\u00A0(Mijaljica\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Johansen\t \u00C2\u00A0and\t \u00C2\u00A0Lamark,\t \u00C2\u00A02014).\t \u00C2\u00A0Selective\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0was\t \u00C2\u00A0originally\t \u00C2\u00A0thought\t \u00C2\u00A0to\t \u00C2\u00A0only\t \u00C2\u00A0exist\t \u00C2\u00A0in\t \u00C2\u00A0 19 mammalian\t \u00C2\u00A0cells;\t \u00C2\u00A0however,\t \u00C2\u00A0a\t \u00C2\u00A0recent\t \u00C2\u00A0study\t \u00C2\u00A0identified\t \u00C2\u00A0a\t \u00C2\u00A0similar\t \u00C2\u00A0mechanism\t \u00C2\u00A0in\t \u00C2\u00A0yeast,\t \u00C2\u00A0suggesting\t \u00C2\u00A0this\t \u00C2\u00A0pathway\t \u00C2\u00A0is\t \u00C2\u00A0evolutionarily\t \u00C2\u00A0conserved\t \u00C2\u00A0(Lu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Certain\t \u00C2\u00A0destabilizing\t \u00C2\u00A0motifs\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0surface\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cargo\t \u00C2\u00A0are\t \u00C2\u00A0either\t \u00C2\u00A0directly\t \u00C2\u00A0recognized\t \u00C2\u00A0by\t \u00C2\u00A0adaptor\t \u00C2\u00A0proteins\t \u00C2\u00A0to\t \u00C2\u00A0initiate\t \u00C2\u00A0degradation,\t \u00C2\u00A0or\t \u00C2\u00A0(in\t \u00C2\u00A0most\t \u00C2\u00A0cases)\t \u00C2\u00A0first\t \u00C2\u00A0modified\t \u00C2\u00A0by\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0actions\t \u00C2\u00A0of\t \u00C2\u00A0E3-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0ligases.\t \u00C2\u00A0Adaptor\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0bifunctional\t \u00C2\u00A0molecules\t \u00C2\u00A0that\t \u00C2\u00A0contain\t \u00C2\u00A0an\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090conjugating\t \u00C2\u00A0domain\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0binding\t \u00C2\u00A0site\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0autophagosomal\t \u00C2\u00A0protein\t \u00C2\u00A0Atg8\t \u00C2\u00A0(LC3)\t \u00C2\u00A0(Klionsky\t \u00C2\u00A0and\t \u00C2\u00A0Schulman,\t \u00C2\u00A02014).\t \u00C2\u00A0Thus\t \u00C2\u00A0adaptor\t \u00C2\u00A0proteins\t \u00C2\u00A0tether\t \u00C2\u00A0the\t \u00C2\u00A0ubiquitinated\t \u00C2\u00A0substrate\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0ALS\t \u00C2\u00A0machinery\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0Protein\t \u00C2\u00A0aggregates,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0those\t \u00C2\u00A0formed\t \u00C2\u00A0by\t \u00C2\u00A0mHtt,\t \u00C2\u00A0are\t \u00C2\u00A0often\t \u00C2\u00A0degraded\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0way\t \u00C2\u00A0(Jentsch\t \u00C2\u00A0and\t \u00C2\u00A0Schlenker,\t \u00C2\u00A01995;\t \u00C2\u00A0Lu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0While\t \u00C2\u00A0higher\t \u00C2\u00A0in\t \u00C2\u00A0specificity\t \u00C2\u00A0than\t \u00C2\u00A0classical\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0(bulk\t \u00C2\u00A0degradation\t \u00C2\u00A0after\t \u00C2\u00A0nutrient\t \u00C2\u00A0starvation),\t \u00C2\u00A0the\t \u00C2\u00A0selectivity\t \u00C2\u00A0of\t \u00C2\u00A0receptor-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0is\t \u00C2\u00A0far\t \u00C2\u00A0lower\t \u00C2\u00A0than\t \u00C2\u00A0that\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0UPS.\t \u00C2\u00A0This\t \u00C2\u00A0is\t \u00C2\u00A0in\t \u00C2\u00A0part\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0basic\t \u00C2\u00A0mechanism\t \u00C2\u00A0of\t \u00C2\u00A0substrate\t \u00C2\u00A0sequestration\t \u00C2\u00A0in\t \u00C2\u00A0macroautophagy,\t \u00C2\u00A0in\t \u00C2\u00A0that\t \u00C2\u00A0cytosolic\t \u00C2\u00A0components\t \u00C2\u00A0are\t \u00C2\u00A0unavoidably\t \u00C2\u00A0segregated\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0cargo\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0Thus\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0unlikely\t \u00C2\u00A0that\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0is\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0targeting\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0degradation,\t \u00C2\u00A0and\t \u00C2\u00A0accordingly,\t \u00C2\u00A0the\t \u00C2\u00A0pathway\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0suitable\t \u00C2\u00A0for\t \u00C2\u00A0utilization\t \u00C2\u00A0in\t \u00C2\u00A0methods\t \u00C2\u00A0aimed\t \u00C2\u00A0at\t \u00C2\u00A0selective\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Nevertheless,\t \u00C2\u00A0there\t \u00C2\u00A0is\t \u00C2\u00A0considerable\t \u00C2\u00A0interest\t \u00C2\u00A0in\t \u00C2\u00A0enhancing\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0activity\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic\t \u00C2\u00A0approach\t \u00C2\u00A0for\t \u00C2\u00A0proteinopathies\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0disorders\t \u00C2\u00A0(Rubinsztein\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0This\t \u00C2\u00A0manipulation\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0especially\t \u00C2\u00A0useful\t \u00C2\u00A0in\t \u00C2\u00A0later\t \u00C2\u00A0stages\t \u00C2\u00A0of\t \u00C2\u00A0disease\t \u00C2\u00A0whereby\t \u00C2\u00A0aberrant\t \u00C2\u00A0protein\t \u00C2\u00A0accumulation\t \u00C2\u00A0inhibits\t \u00C2\u00A0lysosomal\t \u00C2\u00A0function.\t \u00C2\u00A0An\t \u00C2\u00A0example\t \u00C2\u00A0is\t \u00C2\u00A0HD,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0a\t \u00C2\u00A0primary\t \u00C2\u00A0failure\t \u00C2\u00A0in\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0increased\t \u00C2\u00A0protein\t \u00C2\u00A0load\t \u00C2\u00A0for\t \u00C2\u00A0 20 the\t \u00C2\u00A0CMA\t \u00C2\u00A0system\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011a;\t \u00C2\u00A0Qi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0conceivable\t \u00C2\u00A0that\t \u00C2\u00A0under\t \u00C2\u00A0such\t \u00C2\u00A0pathological\t \u00C2\u00A0states,\t \u00C2\u00A0a\t \u00C2\u00A0combinatorial\t \u00C2\u00A0approach\t \u00C2\u00A0that\t \u00C2\u00A0includes\t \u00C2\u00A0targeted\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090causing\t \u00C2\u00A0proteins\t \u00C2\u00A0and\t \u00C2\u00A0reversing\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0failure\t \u00C2\u00A0may\t \u00C2\u00A0synergistically\t \u00C2\u00A0enhance\t \u00C2\u00A0the\t \u00C2\u00A0elimination\t \u00C2\u00A0of\t \u00C2\u00A0undesirable\t \u00C2\u00A0proteins.\t \u00C2\u00A0To\t \u00C2\u00A0this\t \u00C2\u00A0end,\t \u00C2\u00A0a\t \u00C2\u00A0macroautophagy-\u00C2\u00AD\u00E2\u0080\u0090enhancing\t \u00C2\u00A0peptide\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0identified\t \u00C2\u00A0(Shoji-\u00C2\u00AD\u00E2\u0080\u0090Kawata\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0\t \u00C2\u00A01.4.2.2 CMA\t \u00C2\u00A0Unlike\t \u00C2\u00A0macroautophagy,\t \u00C2\u00A0CMA\t \u00C2\u00A0has\t \u00C2\u00A0so\t \u00C2\u00A0far\t \u00C2\u00A0only\t \u00C2\u00A0been\t \u00C2\u00A0identified\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012).\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0unique\t \u00C2\u00A0amongst\t \u00C2\u00A0the\t \u00C2\u00A0autophagy\t \u00C2\u00A0pathways\t \u00C2\u00A0in\t \u00C2\u00A0that\t \u00C2\u00A0its\t \u00C2\u00A0substrate\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0identified\t \u00C2\u00A0individually\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0recognition\t \u00C2\u00A0motif\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0primary\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0sequence\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990).\t \u00C2\u00A0This\t \u00C2\u00A0motif\t \u00C2\u00A0is\t \u00C2\u00A0recognized\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0chaperone\t \u00C2\u00A0heat\t \u00C2\u00A0shock\t \u00C2\u00A0cognate\t \u00C2\u00A070\t \u00C2\u00A0(hsc70),\t \u00C2\u00A0which\t \u00C2\u00A0delivers\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0limiting\t \u00C2\u00A0membrane.\t \u00C2\u00A0The\t \u00C2\u00A0protein\t \u00C2\u00A0is\t \u00C2\u00A0then\t \u00C2\u00A0unfolded\t \u00C2\u00A0(Salvador\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02000)\t \u00C2\u00A0before\t \u00C2\u00A0being\t \u00C2\u00A0shuttled\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0lumen\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0receptor\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000a).\t \u00C2\u00A0Hence,\t \u00C2\u00A0the\t \u00C2\u00A0three\t \u00C2\u00A0major\t \u00C2\u00A0events\t \u00C2\u00A0in\t \u00C2\u00A0CMA\t \u00C2\u00A0are:\t \u00C2\u00A0(1)\t \u00C2\u00A0selected\t \u00C2\u00A0identification\t \u00C2\u00A0of\t \u00C2\u00A0individual\t \u00C2\u00A0substrates,\t \u00C2\u00A0(2)\t \u00C2\u00A0protein\t \u00C2\u00A0unfolding\t \u00C2\u00A0and\t \u00C2\u00A0(3)\t \u00C2\u00A0translocation\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0through\t \u00C2\u00A0receptor-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0binding.\t \u00C2\u00A0\t \u00C2\u00A01.4.2.2.1 The\t \u00C2\u00A0CMA\t \u00C2\u00A0targeting\t \u00C2\u00A0motif\t \u00C2\u00A0CMA\t \u00C2\u00A0was\t \u00C2\u00A0first\t \u00C2\u00A0discovered\t \u00C2\u00A0through\t \u00C2\u00A0an\t \u00C2\u00A0observation\t \u00C2\u00A0that\t \u00C2\u00A0nutrient\t \u00C2\u00A0removal\t \u00C2\u00A0degrades\t \u00C2\u00A0a\t \u00C2\u00A0select\t \u00C2\u00A0population\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0via\t \u00C2\u00A0a\t \u00C2\u00A0lysosomal-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0pathway\t \u00C2\u00A0in\t \u00C2\u00A0liver\t \u00C2\u00A0cells\t \u00C2\u00A0(Backer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01983;\t \u00C2\u00A0Dice,\t \u00C2\u00A01987).\t \u00C2\u00A0Unlike\t \u00C2\u00A0macroautophagy,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0also\t \u00C2\u00A0activated\t \u00C2\u00A0by\t \u00C2\u00A0serum\t \u00C2\u00A0deprivation,\t \u00C2\u00A0the\t \u00C2\u00A0newly\t \u00C2\u00A0identified\t \u00C2\u00A0selective\t \u00C2\u00A0proteolytic\t \u00C2\u00A0pathway\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0require\t \u00C2\u00A0the\t \u00C2\u00A0formation\t \u00C2\u00A0of\t \u00C2\u00A0 21 autophagosomes.\t \u00C2\u00A0Subsequent\t \u00C2\u00A0biochemical\t \u00C2\u00A0and\t \u00C2\u00A0genetic\t \u00C2\u00A0analysis\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0all\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0contain\t \u00C2\u00A0a\t \u00C2\u00A0pentapeptide\t \u00C2\u00A0motif\t \u00C2\u00A0that\t \u00C2\u00A0is\t \u00C2\u00A0necessary\t \u00C2\u00A0and\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0targeting\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0pathway\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990;\t \u00C2\u00A0Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012).\t \u00C2\u00A0This\t \u00C2\u00A0motif\t \u00C2\u00A0was\t \u00C2\u00A0subsequently\t \u00C2\u00A0named\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0targeting\t \u00C2\u00A0motif\t \u00C2\u00A0(CTM).\t \u00C2\u00A0Mutation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0prototypical\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0Ribonuclease\t \u00C2\u00A0A\t \u00C2\u00A0(RNase\t \u00C2\u00A0A)\t \u00C2\u00A0prevented\t \u00C2\u00A0its\t \u00C2\u00A0depletion\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0in\t \u00C2\u00A0response\t \u00C2\u00A0to\t \u00C2\u00A0serum\t \u00C2\u00A0removal\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990),\t \u00C2\u00A0and\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0motif\t \u00C2\u00A0to\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0their\t \u00C2\u00A0recognition\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0system\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011b;\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0chemical\t \u00C2\u00A0properties\t \u00C2\u00A0of\t \u00C2\u00A0its\t \u00C2\u00A0constituent\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0a\t \u00C2\u00A0specific\t \u00C2\u00A0sequence\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990).\t \u00C2\u00A0The\t \u00C2\u00A0motif\t \u00C2\u00A0is\t \u00C2\u00A0made\t \u00C2\u00A0up\t \u00C2\u00A0of\t \u00C2\u00A0five\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0including\t \u00C2\u00A0a\t \u00C2\u00A0positively\t \u00C2\u00A0charged\t \u00C2\u00A0residue\t \u00C2\u00A0(K,\t \u00C2\u00A0R),\t \u00C2\u00A0a\t \u00C2\u00A0hydrophobic\t \u00C2\u00A0residue\t \u00C2\u00A0(I,\t \u00C2\u00A0L,\t \u00C2\u00A0V,\t \u00C2\u00A0F),\t \u00C2\u00A0a\t \u00C2\u00A0negatively\t \u00C2\u00A0charged\t \u00C2\u00A0residue\t \u00C2\u00A0(D,\t \u00C2\u00A0E),\t \u00C2\u00A0another\t \u00C2\u00A0positively\t \u00C2\u00A0charged\t \u00C2\u00A0or\t \u00C2\u00A0hydrophobic\t \u00C2\u00A0residue,\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0glutamine\t \u00C2\u00A0(Q)\t \u00C2\u00A0on\t \u00C2\u00A0either\t \u00C2\u00A0side\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0pentapeptide.\t \u00C2\u00A0The\t \u00C2\u00A0Q\t \u00C2\u00A0residue\t \u00C2\u00A0is\t \u00C2\u00A0critically\t \u00C2\u00A0important\t \u00C2\u00A0as\t \u00C2\u00A0mutating\t \u00C2\u00A0it\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0alanine\t \u00C2\u00A0(A)\t \u00C2\u00A0eliminates\t \u00C2\u00A0the\t \u00C2\u00A0targeting\t \u00C2\u00A0ability\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0motif\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990;\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02004;\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Of\t \u00C2\u00A0note,\t \u00C2\u00A0an\t \u00C2\u00A0early\t \u00C2\u00A0report\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0in\t \u00C2\u00A0some\t \u00C2\u00A0cases\t \u00C2\u00A0Q\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0replaced\t \u00C2\u00A0with\t \u00C2\u00A0N\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990),\t \u00C2\u00A0although\t \u00C2\u00A0this\t \u00C2\u00A0was\t \u00C2\u00A0not\t \u00C2\u00A0further\t \u00C2\u00A0examined.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0location\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0protein\t \u00C2\u00A0is\t \u00C2\u00A0flexible,\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0only\t \u00C2\u00A0requirement\t \u00C2\u00A0being\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0exposed\t \u00C2\u00A0and\t \u00C2\u00A0available\t \u00C2\u00A0for\t \u00C2\u00A0chaperone\t \u00C2\u00A0binding\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012).\t \u00C2\u00A0Unlike\t \u00C2\u00A0other\t \u00C2\u00A0degradation\t \u00C2\u00A0signals\t \u00C2\u00A0that\t \u00C2\u00A0are\t \u00C2\u00A0only\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0prior\t \u00C2\u00A0degradation\t \u00C2\u00A0(e.g.\t \u00C2\u00A0ubiquitin),\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0always\t \u00C2\u00A0present\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0substrate.\t \u00C2\u00A0Thus\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0most\t \u00C2\u00A0likely\t \u00C2\u00A0depends\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0accessibility\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CTM.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0substrates\t \u00C2\u00A0 22 that\t \u00C2\u00A0harbor\t \u00C2\u00A0hidden\t \u00C2\u00A0CTMs\t \u00C2\u00A0may\t \u00C2\u00A0only\t \u00C2\u00A0gain\t \u00C2\u00A0the\t \u00C2\u00A0ability\t \u00C2\u00A0to\t \u00C2\u00A0attract\t \u00C2\u00A0chaperones\t \u00C2\u00A0following\t \u00C2\u00A0partial\t \u00C2\u00A0unfolding\t \u00C2\u00A0(Kiffin\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004),\t \u00C2\u00A0dissociation\t \u00C2\u00A0from\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0complex\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01995)\t \u00C2\u00A0or\t \u00C2\u00A0release\t \u00C2\u00A0from\t \u00C2\u00A0intracellular\t \u00C2\u00A0membranes\t \u00C2\u00A0or\t \u00C2\u00A0subcellular\t \u00C2\u00A0compartments\t \u00C2\u00A0(Yang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0\t \u00C2\u00A0An\t \u00C2\u00A0earlier\t \u00C2\u00A0analysis\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0~30%\t \u00C2\u00A0of\t \u00C2\u00A0cytosolic\t \u00C2\u00A0proteins\t \u00C2\u00A0contain\t \u00C2\u00A0CTMs\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0primary\t \u00C2\u00A0sequences\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012).\t \u00C2\u00A0As\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins\t \u00C2\u00A0cannot\t \u00C2\u00A0be\t \u00C2\u00A0directly\t \u00C2\u00A0translocated\t \u00C2\u00A0into\t \u00C2\u00A0lysosomes,\t \u00C2\u00A0this\t \u00C2\u00A0pool\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0is\t \u00C2\u00A0thought\t \u00C2\u00A0to\t \u00C2\u00A0lack\t \u00C2\u00A0CTMs\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo\t \u00C2\u00A02012).\t \u00C2\u00A0However,\t \u00C2\u00A0our\t \u00C2\u00A0initial\t \u00C2\u00A0in\t \u00C2\u00A0silico\t \u00C2\u00A0screen\t \u00C2\u00A0for\t \u00C2\u00A0all\t \u00C2\u00A0probable\t \u00C2\u00A0CTM\t \u00C2\u00A0combinations\t \u00C2\u00A0in\t \u00C2\u00A0various\t \u00C2\u00A0excitatory\t \u00C2\u00A0and\t \u00C2\u00A0inhibitory\t \u00C2\u00A0membrane\t \u00C2\u00A0receptor\t \u00C2\u00A0proteins\t \u00C2\u00A0revealed\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0one\t \u00C2\u00A0or\t \u00C2\u00A0more\t \u00C2\u00A0putative\t \u00C2\u00A0CTM\t \u00C2\u00A0sequences\t \u00C2\u00A0(Appendix\t \u00C2\u00A0B).\t \u00C2\u00A0Whether\t \u00C2\u00A0these\t \u00C2\u00A0motifs\t \u00C2\u00A0have\t \u00C2\u00A0biological\t \u00C2\u00A0functions\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0remains\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0seen.\t \u00C2\u00A0The\t \u00C2\u00A0functionality\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0regulated\t \u00C2\u00A0via\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0case,\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0only\t \u00C2\u00A0partially\t \u00C2\u00A0complete\t \u00C2\u00A0under\t \u00C2\u00A0basal\t \u00C2\u00A0conditions\t \u00C2\u00A0and\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications\t \u00C2\u00A0(e.g.\t \u00C2\u00A0phosphorylation)\t \u00C2\u00A0provide\t \u00C2\u00A0a\t \u00C2\u00A0missing\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0residue\t \u00C2\u00A0to\t \u00C2\u00A0make\t \u00C2\u00A0it\t \u00C2\u00A0functional.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0the\t \u00C2\u00A0glycolytic\t \u00C2\u00A0enzyme\t \u00C2\u00A0embryonic\t \u00C2\u00A0M2\t \u00C2\u00A0isoform\t \u00C2\u00A0of\t \u00C2\u00A0pyruvate\t \u00C2\u00A0kinase\t \u00C2\u00A0(PKM2)\t \u00C2\u00A0contains\t \u00C2\u00A0a\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0lysine\t \u00C2\u00A0residue\t \u00C2\u00A0in\t \u00C2\u00A0place\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0critical\t \u00C2\u00A0glutamine\t \u00C2\u00A0residue;\t \u00C2\u00A0acetylation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0lysine\t \u00C2\u00A0residue\t \u00C2\u00A0(which\t \u00C2\u00A0mimics\t \u00C2\u00A0glutamine)\t \u00C2\u00A0completes\t \u00C2\u00A0the\t \u00C2\u00A0motif\t \u00C2\u00A0and\t \u00C2\u00A0channels\t \u00C2\u00A0its\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0CMA\t \u00C2\u00A0(Lv\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0Similarly,\t \u00C2\u00A0CMA-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0region\t \u00C2\u00A0of\t \u00C2\u00A0Htt\t \u00C2\u00A0requires\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0acetylation\t \u00C2\u00A0to\t \u00C2\u00A0complete\t \u00C2\u00A0its\t \u00C2\u00A0CTM\t \u00C2\u00A0(Thompson\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0Finally,\t \u00C2\u00A0an\t \u00C2\u00A0oxidized\t \u00C2\u00A0histidine\t \u00C2\u00A0behaves\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0negative\t \u00C2\u00A0aspartic\t \u00C2\u00A0acid\t \u00C2\u00A0residue,\t \u00C2\u00A0which\t \u00C2\u00A0may\t \u00C2\u00A0complete\t \u00C2\u00A0a\t \u00C2\u00A0CTM\t \u00C2\u00A0during\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0and\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0protein\t \u00C2\u00A0quality\t \u00C2\u00A0control\t \u00C2\u00A0(Massey\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004).\t \u00C2\u00A0In\t \u00C2\u00A0sum,\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090 23 translational\t \u00C2\u00A0modification\t \u00C2\u00A0increases\t \u00C2\u00A0the\t \u00C2\u00A0pool\t \u00C2\u00A0of\t \u00C2\u00A0potential\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0and\t \u00C2\u00A0provides\t \u00C2\u00A0a\t \u00C2\u00A0way\t \u00C2\u00A0to\t \u00C2\u00A0regulate\t \u00C2\u00A0their\t \u00C2\u00A0degradation.\t \u00C2\u00A0A\t \u00C2\u00A0single\t \u00C2\u00A0copy\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0lysosomal\t \u00C2\u00A0targeting\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990).\t \u00C2\u00A0However,\t \u00C2\u00A0multiple\t \u00C2\u00A0copies\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0may\t \u00C2\u00A0confer\t \u00C2\u00A0additional\t \u00C2\u00A0advantages.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0different\t \u00C2\u00A0CTMs\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0protein\t \u00C2\u00A0may\t \u00C2\u00A0become\t \u00C2\u00A0exposed\t \u00C2\u00A0under\t \u00C2\u00A0different\t \u00C2\u00A0stimuli,\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0allow\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0conditionally\t \u00C2\u00A0degraded.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0for\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0undergo\t \u00C2\u00A0cleavage\t \u00C2\u00A0to\t \u00C2\u00A0release\t \u00C2\u00A0functional\t \u00C2\u00A0peptides,\t \u00C2\u00A0multiple\t \u00C2\u00A0CTMs\t \u00C2\u00A0may\t \u00C2\u00A0ensure\t \u00C2\u00A0the\t \u00C2\u00A0complete\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0resulting\t \u00C2\u00A0peptides\t \u00C2\u00A0by\t \u00C2\u00A0CMA.\t \u00C2\u00A0\t \u00C2\u00A01.4.2.2.2 Regulation\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0by\t \u00C2\u00A0hsc70\t \u00C2\u00A0and\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0The\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0recognized\t \u00C2\u00A0by\t \u00C2\u00A0hsc70,\t \u00C2\u00A0a\t \u00C2\u00A0multifunctional\t \u00C2\u00A0chaperone\t \u00C2\u00A0that\t \u00C2\u00A0also\t \u00C2\u00A0participates\t \u00C2\u00A0in\t \u00C2\u00A0other\t \u00C2\u00A0cellular\t \u00C2\u00A0processes\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0protein\t \u00C2\u00A0folding,\t \u00C2\u00A0protein\t \u00C2\u00A0complex\t \u00C2\u00A0disassembly\t \u00C2\u00A0and\t \u00C2\u00A0protein\t \u00C2\u00A0trafficking\t \u00C2\u00A0(Liu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Thus\t \u00C2\u00A0far\t \u00C2\u00A0hsc70\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0only\t \u00C2\u00A0chaperone\t \u00C2\u00A0identified\t \u00C2\u00A0to\t \u00C2\u00A0mediate\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0targeting\t \u00C2\u00A0(Chiang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01989),\t \u00C2\u00A0although\t \u00C2\u00A0it\t \u00C2\u00A0also\t \u00C2\u00A0plays\t \u00C2\u00A0a\t \u00C2\u00A0role\t \u00C2\u00A0in\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090assisted\t \u00C2\u00A0selective\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0(Arndt\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010)\t \u00C2\u00A0and\t \u00C2\u00A0endosomal\t \u00C2\u00A0microautophagy\t \u00C2\u00A0(Sahu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0The\t \u00C2\u00A0diverse\t \u00C2\u00A0roles\t \u00C2\u00A0of\t \u00C2\u00A0hsc70\t \u00C2\u00A0may\t \u00C2\u00A0in\t \u00C2\u00A0part\t \u00C2\u00A0depend\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0particular\t \u00C2\u00A0set\t \u00C2\u00A0of\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090chaperones\t \u00C2\u00A0it\t \u00C2\u00A0associates\t \u00C2\u00A0with,\t \u00C2\u00A0which\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0case\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0include\t \u00C2\u00A0hsp90,\t \u00C2\u00A0CHIP,\t \u00C2\u00A0BAG1\t \u00C2\u00A0and\t \u00C2\u00A0hop\t \u00C2\u00A0(Agarraberes\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02001).\t \u00C2\u00A0The\t \u00C2\u00A0chaperone\t \u00C2\u00A0complex\t \u00C2\u00A0delivers\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0and\t \u00C2\u00A0assists\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0unfolding\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0substrate\t \u00C2\u00A0before\t \u00C2\u00A0subsequent\t \u00C2\u00A0translocation\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0lumen.\t \u00C2\u00A0Unfolding\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0 24 binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0membrane,\t \u00C2\u00A0but\t \u00C2\u00A0is\t \u00C2\u00A0absolutely\t \u00C2\u00A0necessary\t \u00C2\u00A0for\t \u00C2\u00A0translocation\t \u00C2\u00A0(Salvador\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02000).\t \u00C2\u00A0Binding\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0is\t \u00C2\u00A0mediated\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0KRHH\t \u00C2\u00A0motif\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0cytosolic\t \u00C2\u00A0tail\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0receptor\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000a).\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0is\t \u00C2\u00A0one\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0three\t \u00C2\u00A0splice\t \u00C2\u00A0variants\t \u00C2\u00A0encoded\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0lamp2\t \u00C2\u00A0gene,\t \u00C2\u00A0and\t \u00C2\u00A0differs\t \u00C2\u00A0from\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902B\t \u00C2\u00A0and\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902C\t \u00C2\u00A0in\t \u00C2\u00A0its\t \u00C2\u00A0transmembrane\t \u00C2\u00A0and\t \u00C2\u00A0cytosolic\t \u00C2\u00A0domains.\t \u00C2\u00A0Only\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0is\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0CMA\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000a).\t \u00C2\u00A0Under\t \u00C2\u00A0basal\t \u00C2\u00A0conditions,\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0is\t \u00C2\u00A0present\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0monomeric\t \u00C2\u00A0form.\t \u00C2\u00A0Upon\t \u00C2\u00A0substrate\t \u00C2\u00A0binding,\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0transiently\t \u00C2\u00A0oligomerizes\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0complex\t \u00C2\u00A0of\t \u00C2\u00A0roughly\t \u00C2\u00A0700kDa,\t \u00C2\u00A0a\t \u00C2\u00A0process\t \u00C2\u00A0regulated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0coordinated\t \u00C2\u00A0activity\t \u00C2\u00A0of\t \u00C2\u00A0glial\t \u00C2\u00A0fibrillary\t \u00C2\u00A0acidic\t \u00C2\u00A0protein\t \u00C2\u00A0(GFAP),\t \u00C2\u00A0elongation\t \u00C2\u00A0factor\t \u00C2\u00A01\t \u00C2\u00A0a\t \u00C2\u00A0(EF1a),\t \u00C2\u00A0and\t \u00C2\u00A0lipids\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0(Bandyopadhyay\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008;\t \u00C2\u00A02010).\t \u00C2\u00A0Despite\t \u00C2\u00A0its\t \u00C2\u00A0name,\t \u00C2\u00A0GFAP\t \u00C2\u00A0is\t \u00C2\u00A0an\t \u00C2\u00A0intermediate\t \u00C2\u00A0filament\t \u00C2\u00A0protein\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0establishing\t \u00C2\u00A0the\t \u00C2\u00A0intracellular\t \u00C2\u00A0cytoplasmic\t \u00C2\u00A0framework,\t \u00C2\u00A0and\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0identified\t \u00C2\u00A0in\t \u00C2\u00A0multiple\t \u00C2\u00A0cell\t \u00C2\u00A0types\t \u00C2\u00A0and\t \u00C2\u00A0tissues\t \u00C2\u00A0in\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0glia\t \u00C2\u00A0(Morini\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0Upon\t \u00C2\u00A0substrate\t \u00C2\u00A0translocation\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0complex\t \u00C2\u00A0and\t \u00C2\u00A0release\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0lumen,\t \u00C2\u00A0the\t \u00C2\u00A0complex\t \u00C2\u00A0dissociates\t \u00C2\u00A0back\t \u00C2\u00A0into\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0monomers\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0help\t \u00C2\u00A0of\t \u00C2\u00A0lysosomal\t \u00C2\u00A0hsp90.\t \u00C2\u00A0Substrate\t \u00C2\u00A0interaction\t \u00C2\u00A0with\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0rate-\u00C2\u00AD\u00E2\u0080\u0090limiting\t \u00C2\u00A0step\t \u00C2\u00A0of\t \u00C2\u00A0CMA,\t \u00C2\u00A0and\t \u00C2\u00A0CMA\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0mainly\t \u00C2\u00A0regulated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0monomers\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000b),\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0efficiency\t \u00C2\u00A0of\t \u00C2\u00A0its\t \u00C2\u00A0multimerization\t \u00C2\u00A0(Bandyopadhyay\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008).\t \u00C2\u00A0 25 In\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A,\t \u00C2\u00A0CMA\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0secondarily\t \u00C2\u00A0regulated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0hsc70\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0lumen.\t \u00C2\u00A0Only\t \u00C2\u00A0a\t \u00C2\u00A0fraction\t \u00C2\u00A0of\t \u00C2\u00A0lysosomes\t \u00C2\u00A0contain\t \u00C2\u00A0lysosomal\t \u00C2\u00A0hsc70,\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0its\t \u00C2\u00A0presence\t \u00C2\u00A0lysosomes\t \u00C2\u00A0are\t \u00C2\u00A0either\t \u00C2\u00A0CMA\t \u00C2\u00A0competent\t \u00C2\u00A0or\t \u00C2\u00A0incompetent\t \u00C2\u00A0(Agarraberes\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01997).\t \u00C2\u00A0Although\t \u00C2\u00A0only\t \u00C2\u00A020%\t \u00C2\u00A0of\t \u00C2\u00A0all\t \u00C2\u00A0lysosomes\t \u00C2\u00A0are\t \u00C2\u00A0CMA-\u00C2\u00AD\u00E2\u0080\u0090competent\t \u00C2\u00A0under\t \u00C2\u00A0basal\t \u00C2\u00A0conditions,\t \u00C2\u00A0the\t \u00C2\u00A0pathway\t \u00C2\u00A0is\t \u00C2\u00A0basally\t \u00C2\u00A0operational\t \u00C2\u00A0in\t \u00C2\u00A0most\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells,\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0normal\t \u00C2\u00A0function\t \u00C2\u00A0is\t \u00C2\u00A0critical\t \u00C2\u00A0for\t \u00C2\u00A0maintaining\t \u00C2\u00A0cellular\t \u00C2\u00A0homeostasis.\t \u00C2\u00A0Indeed,\t \u00C2\u00A0blocking\t \u00C2\u00A0CMA\t \u00C2\u00A0in\t \u00C2\u00A0mouse\t \u00C2\u00A0fibroblasts\t \u00C2\u00A0increased\t \u00C2\u00A0their\t \u00C2\u00A0sensitivity\t \u00C2\u00A0to\t \u00C2\u00A0stressors\t \u00C2\u00A0(Massey\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006),\t \u00C2\u00A0and\t \u00C2\u00A0inhibition\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0with\t \u00C2\u00A0aging\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000c;\t \u00C2\u00A0Zhang\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02008)\t \u00C2\u00A0or\t \u00C2\u00A0dietary\t \u00C2\u00A0lipids\t \u00C2\u00A0(Rodriguez-\u00C2\u00AD\u00E2\u0080\u0090Navarro\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012;\t \u00C2\u00A0Rodriguez-\u00C2\u00AD\u00E2\u0080\u0090Navarro\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012)\t \u00C2\u00A0leads\t \u00C2\u00A0to\t \u00C2\u00A0alterations\t \u00C2\u00A0in\t \u00C2\u00A0lipid\t \u00C2\u00A0metabolism\t \u00C2\u00A0and\t \u00C2\u00A0protein\t \u00C2\u00A0catabolism.\t \u00C2\u00A0CMA\t \u00C2\u00A0becomes\t \u00C2\u00A0a\t \u00C2\u00A0larger\t \u00C2\u00A0player\t \u00C2\u00A0in\t \u00C2\u00A0proteolysis\t \u00C2\u00A0after\t \u00C2\u00A0exposure\t \u00C2\u00A0to\t \u00C2\u00A0cellular\t \u00C2\u00A0stressors\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0nutrient\t \u00C2\u00A0deprivation\t \u00C2\u00A0or\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0(Dohi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Under\t \u00C2\u00A0such\t \u00C2\u00A0conditions\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0upregulated\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0variety\t \u00C2\u00A0of\t \u00C2\u00A0mechanisms,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0increased\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0transcription\t \u00C2\u00A0and\t \u00C2\u00A0insertion\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane,\t \u00C2\u00A0decreased\t \u00C2\u00A0proteolysis,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0an\t \u00C2\u00A0increased\t \u00C2\u00A0number\t \u00C2\u00A0(up\t \u00C2\u00A0to\t \u00C2\u00A080%)\t \u00C2\u00A0of\t \u00C2\u00A0lysosomes\t \u00C2\u00A0with\t \u00C2\u00A0lysosomal\t \u00C2\u00A0hsc70\t \u00C2\u00A0(Kiffin\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004;\t \u00C2\u00A0Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011a).\t \u00C2\u00A0As\t \u00C2\u00A0such,\t \u00C2\u00A0CMA\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0particularly\t \u00C2\u00A0effective\t \u00C2\u00A0at\t \u00C2\u00A0degrading\t \u00C2\u00A0substrates\t \u00C2\u00A0under\t \u00C2\u00A0conditions\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0stress.\t \u00C2\u00A0Similar\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0UPS,\t \u00C2\u00A0the\t \u00C2\u00A0selectivity\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0allows\t \u00C2\u00A0the\t \u00C2\u00A0removal\t \u00C2\u00A0of\t \u00C2\u00A0specific\t \u00C2\u00A0proteins\t \u00C2\u00A0without\t \u00C2\u00A0perturbing\t \u00C2\u00A0its\t \u00C2\u00A0neighbors,\t \u00C2\u00A0thus\t \u00C2\u00A0making\t \u00C2\u00A0CMA\t \u00C2\u00A0an\t \u00C2\u00A0efficient\t \u00C2\u00A0protein\t \u00C2\u00A0quality\t \u00C2\u00A0control\t \u00C2\u00A0system\t \u00C2\u00A0for\t \u00C2\u00A0degrading\t \u00C2\u00A0damaged\t \u00C2\u00A0proteins\t \u00C2\u00A0or\t \u00C2\u00A0surplus\t \u00C2\u00A0protein\t \u00C2\u00A0subunits\t \u00C2\u00A0in\t \u00C2\u00A0protein\t \u00C2\u00A0complexes\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Wong,\t \u00C2\u00A02013).\t \u00C2\u00A0Thus\t \u00C2\u00A0far,\t \u00C2\u00A0there\t \u00C2\u00A0is\t \u00C2\u00A0no\t \u00C2\u00A0evidence\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0saturated\t \u00C2\u00A0by\t \u00C2\u00A0its\t \u00C2\u00A0normal\t \u00C2\u00A0substrates.\t \u00C2\u00A0 26 1.4.2.3 ENDOSOMAL\t \u00C2\u00A0MICROAUTOPHAGY\t \u00C2\u00A0(e-\u00C2\u00AD\u00E2\u0080\u0090MI)\t \u00C2\u00A0The\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0absolutely\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0by\t \u00C2\u00A0CMA.\t \u00C2\u00A0However,\t \u00C2\u00A0having\t \u00C2\u00A0a\t \u00C2\u00A0CTM\t \u00C2\u00A0alone\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0guarantee\t \u00C2\u00A0CMA\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0only\t \u00C2\u00A0degradation\t \u00C2\u00A0pathway.\t \u00C2\u00A0A\t \u00C2\u00A0recent\t \u00C2\u00A0study\t \u00C2\u00A0demonstrated\t \u00C2\u00A0that\t \u00C2\u00A0hsc70\t \u00C2\u00A0can\t \u00C2\u00A0also\t \u00C2\u00A0route\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0proteins\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0late\t \u00C2\u00A0endosome\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0(Sahu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0type\t \u00C2\u00A0of\t \u00C2\u00A0microautophagy\t \u00C2\u00A0that\t \u00C2\u00A0occurs\t \u00C2\u00A0in\t \u00C2\u00A0late\t \u00C2\u00A0endosomes,\t \u00C2\u00A0which\t \u00C2\u00A0can\t \u00C2\u00A0degrade\t \u00C2\u00A0proteins\t \u00C2\u00A0either\t \u00C2\u00A0in\t \u00C2\u00A0bulk\t \u00C2\u00A0or\t \u00C2\u00A0selectively\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012).\t \u00C2\u00A0Similar\t \u00C2\u00A0to\t \u00C2\u00A0CMA,\t \u00C2\u00A0selective\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0also\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0protein\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0actions\t \u00C2\u00A0of\t \u00C2\u00A0hsc70.\t \u00C2\u00A0However,\t \u00C2\u00A0the\t \u00C2\u00A0two\t \u00C2\u00A0processes\t \u00C2\u00A0differ\t \u00C2\u00A0with\t \u00C2\u00A0regards\t \u00C2\u00A0to\t \u00C2\u00A0substrate\t \u00C2\u00A0internalization,\t \u00C2\u00A0in\t \u00C2\u00A0that\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0require\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A.\t \u00C2\u00A0Instead,\t \u00C2\u00A0the\t \u00C2\u00A0substrate-\u00C2\u00AD\u00E2\u0080\u0090hsc70\t \u00C2\u00A0complex\t \u00C2\u00A0directly\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0phospholipid\t \u00C2\u00A0phosphatidylserine\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0endosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0through\t \u00C2\u00A0hydrophobic\t \u00C2\u00A0residues\t \u00C2\u00A0in\t \u00C2\u00A0hsc70.\t \u00C2\u00A0Importantly,\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0require\t \u00C2\u00A0unfolding\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0for\t \u00C2\u00A0substrate\t \u00C2\u00A0degradation\t \u00C2\u00A0(Sahu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011),\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0may\t \u00C2\u00A0potentially\t \u00C2\u00A0be\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0protein\t \u00C2\u00A0aggregates.\t \u00C2\u00A0Thus\t \u00C2\u00A0far\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0has\t \u00C2\u00A0only\t \u00C2\u00A0been\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0dendritic\t \u00C2\u00A0cells\t \u00C2\u00A0and\t \u00C2\u00A0fibroblasts,\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0contribution\t \u00C2\u00A0to\t \u00C2\u00A0protein\t \u00C2\u00A0homeostasis\t \u00C2\u00A0in\t \u00C2\u00A0other\t \u00C2\u00A0cell\t \u00C2\u00A0lineages\t \u00C2\u00A0and\t \u00C2\u00A0tissues\t \u00C2\u00A0remains\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0seen.\t \u00C2\u00A0\t \u00C2\u00A01.4.2.4 CROSSTALK\t \u00C2\u00A0BETWEEN\t \u00C2\u00A0PROTEOLYTIC\t \u00C2\u00A0SYSTEMS\t \u00C2\u00A0An\t \u00C2\u00A0important\t \u00C2\u00A0aspect\t \u00C2\u00A0when\t \u00C2\u00A0considering\t \u00C2\u00A0the\t \u00C2\u00A0physiological\t \u00C2\u00A0roles\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0isolation.\t \u00C2\u00A0CMA\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0tightly\t \u00C2\u00A0coordinated\t \u00C2\u00A0with\t \u00C2\u00A0other\t \u00C2\u00A0forms\t \u00C2\u00A0of\t \u00C2\u00A0autophagy,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0(Wong\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02010b;\t \u00C2\u00A0Park\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02013).\t \u00C2\u00A0Notably,\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0may\t \u00C2\u00A0undergo\t \u00C2\u00A0multiple\t \u00C2\u00A0degradation\t \u00C2\u00A0routes\t \u00C2\u00A0depending\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0type,\t \u00C2\u00A0the\t \u00C2\u00A0cellular\t \u00C2\u00A0 27 environment\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modification\t \u00C2\u00A0state.\t \u00C2\u00A0As\t \u00C2\u00A0such,\t \u00C2\u00A0when\t \u00C2\u00A0designing\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0system\t \u00C2\u00A0that\t \u00C2\u00A0utilizes\t \u00C2\u00A0CMA,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0important\t \u00C2\u00A0to\t \u00C2\u00A0also\t \u00C2\u00A0consider\t \u00C2\u00A0the\t \u00C2\u00A0potential\t \u00C2\u00A0contributions\t \u00C2\u00A0of\t \u00C2\u00A0other\t \u00C2\u00A0interacting\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems.\t \u00C2\u00A0Recent\t \u00C2\u00A0studies\t \u00C2\u00A0have\t \u00C2\u00A0uncovered\t \u00C2\u00A0many\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0functional\t \u00C2\u00A0interaction\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0ALS.\t \u00C2\u00A0First,\t \u00C2\u00A0changes\t \u00C2\u00A0in\t \u00C2\u00A0one\t \u00C2\u00A0pathway\t \u00C2\u00A0may\t \u00C2\u00A0affect\t \u00C2\u00A0the\t \u00C2\u00A0activity\t \u00C2\u00A0of\t \u00C2\u00A0another,\t \u00C2\u00A0an\t \u00C2\u00A0interaction\t \u00C2\u00A0particularly\t \u00C2\u00A0noticeable\t \u00C2\u00A0in\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0diseases.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0accumulation\t \u00C2\u00A0and\t \u00C2\u00A0aggregation\t \u00C2\u00A0of\t \u00C2\u00A0mHtt\t \u00C2\u00A0down\t \u00C2\u00A0regulates\t \u00C2\u00A0macroautophagy,\t \u00C2\u00A0which\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0compensatory\t \u00C2\u00A0upregulation\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011a;\t \u00C2\u00A0Qi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Although\t \u00C2\u00A0initially\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0maintaining\t \u00C2\u00A0proteome\t \u00C2\u00A0stability,\t \u00C2\u00A0CMA\t \u00C2\u00A0activity\t \u00C2\u00A0decreases\t \u00C2\u00A0with\t \u00C2\u00A0age\t \u00C2\u00A0(Rodriguez-\u00C2\u00AD\u00E2\u0080\u0090Navarro\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012),\t \u00C2\u00A0which\t \u00C2\u00A0may\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0progression\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0disease.\t \u00C2\u00A0Similarly,\t \u00C2\u00A0cells\t \u00C2\u00A0activate\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0in\t \u00C2\u00A0response\t \u00C2\u00A0to\t \u00C2\u00A0CMA\t \u00C2\u00A0blockage\t \u00C2\u00A0(Massey\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006;\t \u00C2\u00A0Kaushik\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008),\t \u00C2\u00A0which\t \u00C2\u00A0helps\t \u00C2\u00A0to\t \u00C2\u00A0temporarily\t \u00C2\u00A0maintain\t \u00C2\u00A0proteome\t \u00C2\u00A0homeostasis.\t \u00C2\u00A0Pharmacological\t \u00C2\u00A0blockage\t \u00C2\u00A0of\t \u00C2\u00A0UPS\t \u00C2\u00A0often\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0compensatory\t \u00C2\u00A0upregulation\t \u00C2\u00A0of\t \u00C2\u00A0both\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0(Pan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008)\t \u00C2\u00A0and\t \u00C2\u00A0CMA\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011a).\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0contrast\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0bi-\u00C2\u00AD\u00E2\u0080\u0090directional\t \u00C2\u00A0crosstalk\t \u00C2\u00A0between\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0and\t \u00C2\u00A0CMA,\t \u00C2\u00A0the\t \u00C2\u00A0interaction\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0seems\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0unidirectional.\t \u00C2\u00A0Here,\t \u00C2\u00A0blocking\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0reduces\t \u00C2\u00A0UPS\t \u00C2\u00A0activity,\t \u00C2\u00A0possibly\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0aberrant\t \u00C2\u00A0accumulation\t \u00C2\u00A0of\t \u00C2\u00A0p62,\t \u00C2\u00A0which\t \u00C2\u00A0competes\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A019S\t \u00C2\u00A0proteasome\t \u00C2\u00A0subunit\t \u00C2\u00A0with\t \u00C2\u00A0UPS\t \u00C2\u00A0substrates\t \u00C2\u00A0and\t \u00C2\u00A0thereby\t \u00C2\u00A0decreases\t \u00C2\u00A0the\t \u00C2\u00A0entry\t \u00C2\u00A0of\t \u00C2\u00A0other\t \u00C2\u00A0proteins\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0proteosomal\t \u00C2\u00A0core\t \u00C2\u00A0(Korolchuk\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0Under\t \u00C2\u00A0such\t \u00C2\u00A0circumstances\t \u00C2\u00A0CMA\t \u00C2\u00A0becomes\t \u00C2\u00A0crucially\t \u00C2\u00A0important\t \u00C2\u00A0for\t \u00C2\u00A0maintaining\t \u00C2\u00A0cellular\t \u00C2\u00A0homeostasis.\t \u00C2\u00A0\t \u00C2\u00A0 28 Interestingly,\t \u00C2\u00A0in\t \u00C2\u00A0contrast\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0many\t \u00C2\u00A0studies\t \u00C2\u00A0that\t \u00C2\u00A0characterize\t \u00C2\u00A0how\t \u00C2\u00A0blockage\t \u00C2\u00A0of\t \u00C2\u00A0one\t \u00C2\u00A0system\t \u00C2\u00A0influences\t \u00C2\u00A0the\t \u00C2\u00A0others,\t \u00C2\u00A0there\t \u00C2\u00A0is\t \u00C2\u00A0little\t \u00C2\u00A0research\t \u00C2\u00A0on\t \u00C2\u00A0how\t \u00C2\u00A0upregulating\t \u00C2\u00A0one\t \u00C2\u00A0system\t \u00C2\u00A0changes\t \u00C2\u00A0the\t \u00C2\u00A0activity\t \u00C2\u00A0of\t \u00C2\u00A0other\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems.\t \u00C2\u00A0There\t \u00C2\u00A0is\t \u00C2\u00A0some\t \u00C2\u00A0evidence\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0chemical\t \u00C2\u00A0upregulation\t \u00C2\u00A0of\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0modify\t \u00C2\u00A0UPS\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0activity,\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0general\t \u00C2\u00A0upregulation\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0cells\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0affect\t \u00C2\u00A0UPS\t \u00C2\u00A0or\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0efficiency\t \u00C2\u00A0(Park\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02013).\t \u00C2\u00A0Thus\t \u00C2\u00A0the\t \u00C2\u00A0crosstalk\t \u00C2\u00A0between\t \u00C2\u00A0these\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0seems\t \u00C2\u00A0to\t \u00C2\u00A0mainly\t \u00C2\u00A0have\t \u00C2\u00A0a\t \u00C2\u00A0compensatory\t \u00C2\u00A0function.\t \u00C2\u00A0A\t \u00C2\u00A0second\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0interaction\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0protein\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0used\t \u00C2\u00A0or\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0multiple\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0is\t \u00C2\u00A0normally\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0both\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0CMA\t \u00C2\u00A0in\t \u00C2\u00A0neurons,\t \u00C2\u00A0whereas\t \u00C2\u00A0its\t \u00C2\u00A0aggregated\t \u00C2\u00A0forms\t \u00C2\u00A0are\t \u00C2\u00A0preferentially\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0(Stefanis\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001;\t \u00C2\u00A0Webb\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02004).\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0ALS\t \u00C2\u00A0also\t \u00C2\u00A0require\t \u00C2\u00A0the\t \u00C2\u00A0actions\t \u00C2\u00A0of\t \u00C2\u00A0many\t \u00C2\u00A0same\t \u00C2\u00A0components\t \u00C2\u00A0for\t \u00C2\u00A0their\t \u00C2\u00A0normal\t \u00C2\u00A0functioning,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0and\t \u00C2\u00A0various\t \u00C2\u00A0chaperones\t \u00C2\u00A0(e.g.\t \u00C2\u00A0hsc70),\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0effector\t \u00C2\u00A0molecules\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0E3\t \u00C2\u00A0ligases.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0is\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0both\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0selective\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapters\t \u00C2\u00A01.4.1\t \u00C2\u00A0and\t \u00C2\u00A01.4.2.1,\t \u00C2\u00A0whereas\t \u00C2\u00A0hsc70\t \u00C2\u00A0is\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0CMA,\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0and\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090assisted\t \u00C2\u00A0selective\t \u00C2\u00A0autophagy.\t \u00C2\u00A0The\t \u00C2\u00A0factors\t \u00C2\u00A0that\t \u00C2\u00A0route\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0substrate\t \u00C2\u00A0to\t \u00C2\u00A0one\t \u00C2\u00A0system\t \u00C2\u00A0or\t \u00C2\u00A0another\t \u00C2\u00A0remain\t \u00C2\u00A0unknown.\t \u00C2\u00A0\t \u00C2\u00A0Finally,\t \u00C2\u00A0one\t \u00C2\u00A0proteolytic\t \u00C2\u00A0pathway\t \u00C2\u00A0may\t \u00C2\u00A0directly\t \u00C2\u00A0regulate\t \u00C2\u00A0the\t \u00C2\u00A0activity\t \u00C2\u00A0of\t \u00C2\u00A0another\t \u00C2\u00A0by\t \u00C2\u00A0degrading\t \u00C2\u00A0its\t \u00C2\u00A0components.\t \u00C2\u00A0As\t \u00C2\u00A0with\t \u00C2\u00A0all\t \u00C2\u00A0other\t \u00C2\u00A0cellular\t \u00C2\u00A0proteins,\t \u00C2\u00A0those\t \u00C2\u00A0that\t \u00C2\u00A0make\t \u00C2\u00A0up\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0autophagy\t \u00C2\u00A0machinery\t \u00C2\u00A0also\t \u00C2\u00A0undergo\t \u00C2\u00A0normal\t \u00C2\u00A0turnover.\t \u00C2\u00A0The\t \u00C2\u00A0catalytic\t \u00C2\u00A0core\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0during\t \u00C2\u00A0nutrient\t \u00C2\u00A0starvation,\t \u00C2\u00A0which\t \u00C2\u00A0in\t \u00C2\u00A0part\t \u00C2\u00A0 29 contributes\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0down\t \u00C2\u00A0regulation\t \u00C2\u00A0of\t \u00C2\u00A0UPS\t \u00C2\u00A0under\t \u00C2\u00A0those\t \u00C2\u00A0circumstances\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01995).\t \u00C2\u00A0A\t \u00C2\u00A0similar\t \u00C2\u00A0link\t \u00C2\u00A0exists\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0CMA,\t \u00C2\u00A0in\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A026S\t \u00C2\u00A0proteasome\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0found\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0lumen\t \u00C2\u00A0of\t \u00C2\u00A0autophagosomes\t \u00C2\u00A0and\t \u00C2\u00A0CMA-\u00C2\u00AD\u00E2\u0080\u0090capable\t \u00C2\u00A0lysosomes\t \u00C2\u00A0(Rothenberg\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010),\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0ALS\t \u00C2\u00A0may\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0turnover\t \u00C2\u00A0of\t \u00C2\u00A0ubiquitin.\t \u00C2\u00A0Similarly,\t \u00C2\u00A0the\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0adaptor\t \u00C2\u00A0protein\t \u00C2\u00A0p62\t \u00C2\u00A0undergoes\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0(Komatsu\t \u00C2\u00A0and\t \u00C2\u00A0Ichimura,\t \u00C2\u00A02010).\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0Much\t \u00C2\u00A0remains\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0understood\t \u00C2\u00A0regarding\t \u00C2\u00A0the\t \u00C2\u00A0rules\t \u00C2\u00A0that\t \u00C2\u00A0govern\t \u00C2\u00A0the\t \u00C2\u00A0interplay\t \u00C2\u00A0amongst\t \u00C2\u00A0various\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems.\t \u00C2\u00A0In\t \u00C2\u00A0particular,\t \u00C2\u00A0functional\t \u00C2\u00A0compensation\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0discovered\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0growing\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0aggregation\t \u00C2\u00A0diseases,\t \u00C2\u00A0illustrating\t \u00C2\u00A0the\t \u00C2\u00A0importance\t \u00C2\u00A0of\t \u00C2\u00A0considering\t \u00C2\u00A0such\t \u00C2\u00A0interactions\t \u00C2\u00A0when\t \u00C2\u00A0designing\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0aim\t \u00C2\u00A0to\t \u00C2\u00A0eliminate\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090causing\t \u00C2\u00A0proteins\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic\t \u00C2\u00A0approach.\t \u00C2\u00A0In\t \u00C2\u00A0particular,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0critically\t \u00C2\u00A0important\t \u00C2\u00A0to\t \u00C2\u00A0consider\t \u00C2\u00A0the\t \u00C2\u00A0stage\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0disease\t \u00C2\u00A0and\t \u00C2\u00A0select\t \u00C2\u00A0an\t \u00C2\u00A0appropriate\t \u00C2\u00A0(i.e.\t \u00C2\u00A0upregulated)\t \u00C2\u00A0proteolytic\t \u00C2\u00A0system\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0degradation,\t \u00C2\u00A0while\t \u00C2\u00A0avoiding\t \u00C2\u00A0systems\t \u00C2\u00A0that\t \u00C2\u00A0are\t \u00C2\u00A0already\t \u00C2\u00A0inhibited.\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0case\t \u00C2\u00A0of\t \u00C2\u00A0HD,\t \u00C2\u00A0routing\t \u00C2\u00A0mHtt\t \u00C2\u00A0to\t \u00C2\u00A0CMA\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0the\t \u00C2\u00A0most\t \u00C2\u00A0appropriate\t \u00C2\u00A0approach,\t \u00C2\u00A0as\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0and\t \u00C2\u00A0UPS\t \u00C2\u00A0failure\t \u00C2\u00A0occur\t \u00C2\u00A0early\t \u00C2\u00A0on\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0disorder\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011a).\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0in\t \u00C2\u00A0later\t \u00C2\u00A0stages\t \u00C2\u00A0of\t \u00C2\u00A0PD\t \u00C2\u00A0it\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0most\t \u00C2\u00A0beneficial\t \u00C2\u00A0to\t \u00C2\u00A0upregulate\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0to\t \u00C2\u00A0eradicate\t \u00C2\u00A0protein\t \u00C2\u00A0aggregates,\t \u00C2\u00A0as\t \u00C2\u00A0they\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0UPS\t \u00C2\u00A0or\t \u00C2\u00A0CMA\t \u00C2\u00A0degradation.\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0terms\t \u00C2\u00A0of\t \u00C2\u00A0developing\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0technology,\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0CMA\t \u00C2\u00A0pathways\t \u00C2\u00A0are\t \u00C2\u00A0perhaps\t \u00C2\u00A0the\t \u00C2\u00A0best\t \u00C2\u00A0routes\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0their\t \u00C2\u00A0exquisite\t \u00C2\u00A0selectivity.\t \u00C2\u00A0 30 The\t \u00C2\u00A0process\t \u00C2\u00A0of\t \u00C2\u00A0substrate\t \u00C2\u00A0selection\t \u00C2\u00A0for\t \u00C2\u00A0both\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0CMA\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0or\t \u00C2\u00A0exposure\t \u00C2\u00A0of\t \u00C2\u00A0certain\t \u00C2\u00A0motifs\t \u00C2\u00A0to\t \u00C2\u00A0various\t \u00C2\u00A0chaperones.\t \u00C2\u00A0Motifs\t \u00C2\u00A0that\t \u00C2\u00A0signal\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0are\t \u00C2\u00A0called\t \u00C2\u00A0protein\t \u00C2\u00A0destruction\t \u00C2\u00A0signals,\t \u00C2\u00A0or\t \u00C2\u00A0degrons.\t \u00C2\u00A0One\t \u00C2\u00A0characteristic\t \u00C2\u00A0of\t \u00C2\u00A0degrons\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0they\t \u00C2\u00A0confer\t \u00C2\u00A0metabolic\t \u00C2\u00A0instability\t \u00C2\u00A0when\t \u00C2\u00A0artificially\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0otherwise\t \u00C2\u00A0stable\t \u00C2\u00A0protein\t \u00C2\u00A0(Dohmen,\t \u00C2\u00A02000).\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0this\t \u00C2\u00A0transferability\t \u00C2\u00A0of\t \u00C2\u00A0degrons\t \u00C2\u00A0that\t \u00C2\u00A0forms\t \u00C2\u00A0the\t \u00C2\u00A0basis\t \u00C2\u00A0for\t \u00C2\u00A0utilizing\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A01.4.3 PRIMARY\t \u00C2\u00A0AND\t \u00C2\u00A0SECONDARY\t \u00C2\u00A0DEGRONS\t \u00C2\u00A0\t \u00C2\u00A0A\t \u00C2\u00A0fundamental\t \u00C2\u00A0question\t \u00C2\u00A0regarding\t \u00C2\u00A0intracellular\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0is\t \u00C2\u00A0how\t \u00C2\u00A0specific\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0recognized\t \u00C2\u00A0and\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0various\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0with\t \u00C2\u00A0highly\t \u00C2\u00A0characteristic\t \u00C2\u00A0half-\u00C2\u00AD\u00E2\u0080\u0090lives\t \u00C2\u00A0(Goldberg,\t \u00C2\u00A02003).\t \u00C2\u00A0Early\t \u00C2\u00A0work\t \u00C2\u00A0suggested\t \u00C2\u00A0that\t \u00C2\u00A0global\t \u00C2\u00A0structural\t \u00C2\u00A0features,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0molecular\t \u00C2\u00A0mass\t \u00C2\u00A0or\t \u00C2\u00A0isoelectric\t \u00C2\u00A0point,\t \u00C2\u00A0determine\t \u00C2\u00A0the\t \u00C2\u00A0rate\t \u00C2\u00A0of\t \u00C2\u00A0turnover\t \u00C2\u00A0(Goldberg\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A01974).\t \u00C2\u00A0However,\t \u00C2\u00A0later\t \u00C2\u00A0analyses\t \u00C2\u00A0demonstrated\t \u00C2\u00A0only\t \u00C2\u00A0a\t \u00C2\u00A0weak\t \u00C2\u00A0correlation\t \u00C2\u00A0between\t \u00C2\u00A0gross\t \u00C2\u00A0physical-\u00C2\u00AD\u00E2\u0080\u0090chemical\t \u00C2\u00A0properties\t \u00C2\u00A0and\t \u00C2\u00A0protein\t \u00C2\u00A0stability\t \u00C2\u00A0(Dice,\t \u00C2\u00A01987).\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0now\t \u00C2\u00A0widely\t \u00C2\u00A0accepted\t \u00C2\u00A0that\t \u00C2\u00A0localized\t \u00C2\u00A0features,\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0global\t \u00C2\u00A0structure,\t \u00C2\u00A0are\t \u00C2\u00A0the\t \u00C2\u00A0primary\t \u00C2\u00A0determinants\t \u00C2\u00A0that\t \u00C2\u00A0mark\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0These\t \u00C2\u00A0local\t \u00C2\u00A0destabilizing\t \u00C2\u00A0structures\t \u00C2\u00A0that\t \u00C2\u00A0render\t \u00C2\u00A0proteins\t \u00C2\u00A0metabolically\t \u00C2\u00A0unstable\t \u00C2\u00A0are\t \u00C2\u00A0commonly\t \u00C2\u00A0referred\t \u00C2\u00A0to\t \u00C2\u00A0as\t \u00C2\u00A0degrons\t \u00C2\u00A0(Varshavsky,\t \u00C2\u00A01992).\t \u00C2\u00A0Specifically,\t \u00C2\u00A0degrons\t \u00C2\u00A0are\t \u00C2\u00A0(1)\t \u00C2\u00A0any\t \u00C2\u00A0minimal\t \u00C2\u00A0element\t \u00C2\u00A0within\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0that\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0recognition\t \u00C2\u00A0and\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0proteolytic\t \u00C2\u00A0system,\t \u00C2\u00A0and\t \u00C2\u00A0(2)\t \u00C2\u00A0an\t \u00C2\u00A0element\t \u00C2\u00A0that\t \u00C2\u00A0is\t \u00C2\u00A0transferable\t \u00C2\u00A0to\t \u00C2\u00A0any\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0confer\t \u00C2\u00A0metabolically\t \u00C2\u00A0instability\t \u00C2\u00A0(Dohmen,\t \u00C2\u00A02000).\t \u00C2\u00A0Primary\t \u00C2\u00A0degrons\t \u00C2\u00A0are\t \u00C2\u00A0sequences\t \u00C2\u00A0or\t \u00C2\u00A0structures\t \u00C2\u00A0 31 within\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0protein\t \u00C2\u00A0that\t \u00C2\u00A0directly\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0components\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0proteolytic\t \u00C2\u00A0system\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0degradation.\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0secondary\t \u00C2\u00A0degrons\t \u00C2\u00A0attach\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0and\t \u00C2\u00A0function\t \u00C2\u00A0as\t \u00C2\u00A0adaptor\t \u00C2\u00A0signals\t \u00C2\u00A0to\t \u00C2\u00A0recruit\t \u00C2\u00A0components\t \u00C2\u00A0necessary\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0the\t \u00C2\u00A0polyubiquitin\t \u00C2\u00A0chain\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0secondary\t \u00C2\u00A0degron\t \u00C2\u00A0targeting\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0for\t \u00C2\u00A0UPS\t \u00C2\u00A0destruction.\t \u00C2\u00A0Primary\t \u00C2\u00A0degrons\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0are\t \u00C2\u00A0extremely\t \u00C2\u00A0diverse\t \u00C2\u00A0in\t \u00C2\u00A0terms\t \u00C2\u00A0of\t \u00C2\u00A0their\t \u00C2\u00A0chemical\t \u00C2\u00A0and\t \u00C2\u00A0structural\t \u00C2\u00A0properties,\t \u00C2\u00A0and\t \u00C2\u00A0many\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0exploited\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods.\t \u00C2\u00A0The\t \u00C2\u00A0best-\u00C2\u00AD\u00E2\u0080\u0090characterized\t \u00C2\u00A0degron\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0is\t \u00C2\u00A0arguably\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090degron,\t \u00C2\u00A0first\t \u00C2\u00A0discovered\t \u00C2\u00A0in\t \u00C2\u00A0yeast.\t \u00C2\u00A0By\t \u00C2\u00A0systematically\t \u00C2\u00A0changing\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0residue\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0series\t \u00C2\u00A0of\t \u00C2\u00A0otherwise\t \u00C2\u00A0identical\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090galactosidase\t \u00C2\u00A0substrates,\t \u00C2\u00A0it\t \u00C2\u00A0was\t \u00C2\u00A0observed\t \u00C2\u00A0that\t \u00C2\u00A0these\t \u00C2\u00A0test\t \u00C2\u00A0substrates\t \u00C2\u00A0dramatically\t \u00C2\u00A0differed\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0stability\t \u00C2\u00A0(L\u00C3\u00A9vy\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01999).\t \u00C2\u00A0This\t \u00C2\u00A0difference\t \u00C2\u00A0in\t \u00C2\u00A0half-\u00C2\u00AD\u00E2\u0080\u0090lives\t \u00C2\u00A0was\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0different\t \u00C2\u00A0binding\t \u00C2\u00A0affinities\t \u00C2\u00A0of\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0residues\t \u00C2\u00A0to\t \u00C2\u00A0Ubr1,\t \u00C2\u00A0an\t \u00C2\u00A0E3\t \u00C2\u00A0ligase\t \u00C2\u00A0that\t \u00C2\u00A0recruits\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0UPS\t \u00C2\u00A0degradation.\t \u00C2\u00A0The\t \u00C2\u00A0observation\t \u00C2\u00A0that\t \u00C2\u00A0certain\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0confer\t \u00C2\u00A0metabolic\t \u00C2\u00A0instability\t \u00C2\u00A0was\t \u00C2\u00A0subsequently\t \u00C2\u00A0named\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090end\t \u00C2\u00A0rule,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0exposed\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0region\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0UPS-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0was\t \u00C2\u00A0termed\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090degron\t \u00C2\u00A0(Hwang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Kim\t \u00C2\u00A0and\t \u00C2\u00A0Kim,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0contrast\t \u00C2\u00A0to\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090degrons\t \u00C2\u00A0that\t \u00C2\u00A0recruit\t \u00C2\u00A0simple\t \u00C2\u00A0E3\t \u00C2\u00A0ligases,\t \u00C2\u00A0PEST\t \u00C2\u00A0sequences\t \u00C2\u00A0and\t \u00C2\u00A0KEN\t \u00C2\u00A0boxes\t \u00C2\u00A0are\t \u00C2\u00A0degrons\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0E3\t \u00C2\u00A0ligase\t \u00C2\u00A0complexes,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0SCF\t \u00C2\u00A0complex\t \u00C2\u00A0(Skp1,\t \u00C2\u00A0Cullin,\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein)\t \u00C2\u00A0and\t \u00C2\u00A0APC\t \u00C2\u00A0(Anaphase-\u00C2\u00AD\u00E2\u0080\u0090Promoting\t \u00C2\u00A0Complex)\t \u00C2\u00A0(Rechsteiner\t \u00C2\u00A0and\t \u00C2\u00A0Rogers,\t \u00C2\u00A01996;\t \u00C2\u00A0Pfleger\t \u00C2\u00A0and\t \u00C2\u00A0Kirschner,\t \u00C2\u00A02000).\t \u00C2\u00A0Another\t \u00C2\u00A0interesting\t \u00C2\u00A0example\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0sequence-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0degron\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0region\t \u00C2\u00A0of\t \u00C2\u00A0ornithine\t \u00C2\u00A0decarboxylase,\t \u00C2\u00A0which\t \u00C2\u00A0recruits\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0antizyme\t \u00C2\u00A0for\t \u00C2\u00A0direct\t \u00C2\u00A0proteasomal\t \u00C2\u00A0degradation\t \u00C2\u00A0without\t \u00C2\u00A0the\t \u00C2\u00A0need\t \u00C2\u00A0for\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0(Matsuzawa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0 32 2005).\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0detail\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.5,\t \u00C2\u00A0all\t \u00C2\u00A0three\t \u00C2\u00A0primary\t \u00C2\u00A0degrons\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0methods.\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0primary\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0sequence\t \u00C2\u00A0motifs\t \u00C2\u00A0that\t \u00C2\u00A0confer\t \u00C2\u00A0instability,\t \u00C2\u00A0localized\t \u00C2\u00A0structural\t \u00C2\u00A0elements\t \u00C2\u00A0can\t \u00C2\u00A0also\t \u00C2\u00A0serve\t \u00C2\u00A0as\t \u00C2\u00A0degrons.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0exposure\t \u00C2\u00A0of\t \u00C2\u00A0hydrophobic\t \u00C2\u00A0residues\t \u00C2\u00A0is\t \u00C2\u00A0considered\t \u00C2\u00A0a\t \u00C2\u00A0hallmark\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0misfolded\t \u00C2\u00A0or\t \u00C2\u00A0unfolded\t \u00C2\u00A0protein,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0subsequently\t \u00C2\u00A0recognized\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0cellular\t \u00C2\u00A0protein\t \u00C2\u00A0quality\t \u00C2\u00A0control\t \u00C2\u00A0machinery\t \u00C2\u00A0and\t \u00C2\u00A0eliminated\t \u00C2\u00A0through\t \u00C2\u00A0UPS\t \u00C2\u00A0(Neklesa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Neklesa\t \u00C2\u00A0and\t \u00C2\u00A0Crews,\t \u00C2\u00A02012).\t \u00C2\u00A0An\t \u00C2\u00A0example\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0method\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0increasing\t \u00C2\u00A0local\t \u00C2\u00A0hydrophobicity\t \u00C2\u00A0is\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.5.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A01.4.4 POST-\u00C2\u00AD\u00E2\u0080\u0090TRANSLATIONAL\t \u00C2\u00A0REGULATION\t \u00C2\u00A0OF\t \u00C2\u00A0DEGRONS\t \u00C2\u00A0Phosphorylation\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0widely\t \u00C2\u00A0used\t \u00C2\u00A0strategy\t \u00C2\u00A0for\t \u00C2\u00A0regulating\t \u00C2\u00A0sequence-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0degrons\t \u00C2\u00A0(Dohmen,\t \u00C2\u00A02000).\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin,\t \u00C2\u00A0a\t \u00C2\u00A0multifunctional\t \u00C2\u00A0protein\t \u00C2\u00A0essential\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0Wnt\t \u00C2\u00A0signaling\t \u00C2\u00A0pathway\t \u00C2\u00A0and\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090cell\t \u00C2\u00A0adhesion\t \u00C2\u00A0(Gottardi,\t \u00C2\u00A02004),\t \u00C2\u00A0by\t \u00C2\u00A0glycogen\t \u00C2\u00A0synthase\t \u00C2\u00A0kinase\t \u00C2\u00A03\u00CE\u00B2\t \u00C2\u00A0(GSK-\u00C2\u00AD\u00E2\u0080\u00903\u00CE\u00B2)\t \u00C2\u00A0triggers\t \u00C2\u00A0its\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0SCF\t \u00C2\u00A0complex\t \u00C2\u00A0and\t \u00C2\u00A0subsequent\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0(Xu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0GSK-\u00C2\u00AD\u00E2\u0080\u00903\u00CE\u00B2\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0inhibited\t \u00C2\u00A0in\t \u00C2\u00A0many\t \u00C2\u00A0types\t \u00C2\u00A0of\t \u00C2\u00A0cancer,\t \u00C2\u00A0which\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0aberrant\t \u00C2\u00A0accumulation\t \u00C2\u00A0of\t \u00C2\u00A0dephosphorylated\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cytosol\t \u00C2\u00A0and\t \u00C2\u00A0contributes\t \u00C2\u00A0to\t \u00C2\u00A0carcinogenesis\t \u00C2\u00A0(Liu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0as\t \u00C2\u00A0mentioned\t \u00C2\u00A0above,\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0incomplete\t \u00C2\u00A0CTM\t \u00C2\u00A0may\t \u00C2\u00A0complete\t \u00C2\u00A0it\t \u00C2\u00A0and\t \u00C2\u00A0result\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0substrate.\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0can\t \u00C2\u00A0also\t \u00C2\u00A0inhibit\t \u00C2\u00A0proteolysis.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0the\t \u00C2\u00A0ectopic\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0oncogene\t \u00C2\u00A0Mos\t \u00C2\u00A0induces\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0transcriptional\t \u00C2\u00A0activator\t \u00C2\u00A0 33 c-\u00C2\u00AD\u00E2\u0080\u0090Jun\t \u00C2\u00A0in\t \u00C2\u00A0its\t \u00C2\u00A0PEST\t \u00C2\u00A0region,\t \u00C2\u00A0which\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0stabilization\t \u00C2\u00A0of\t \u00C2\u00A0c-\u00C2\u00AD\u00E2\u0080\u0090Jun\t \u00C2\u00A0(Okazaki\t \u00C2\u00A0and\t \u00C2\u00A0Sagata,\t \u00C2\u00A01995).\t \u00C2\u00A0In\t \u00C2\u00A0sum,\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications\t \u00C2\u00A0provide\t \u00C2\u00A0an\t \u00C2\u00A0additional\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0regulation\t \u00C2\u00A0in\t \u00C2\u00A0both\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0ALS.\t \u00C2\u00A0These\t \u00C2\u00A0examples\t \u00C2\u00A0from\t \u00C2\u00A0nature\t \u00C2\u00A0also\t \u00C2\u00A0suggest\t \u00C2\u00A0a\t \u00C2\u00A0means\t \u00C2\u00A0to\t \u00C2\u00A0design\t \u00C2\u00A0\u00E2\u0080\u009Ccryptic\u00E2\u0080\u009D\t \u00C2\u00A0degrons\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0that\t \u00C2\u00A0is,\t \u00C2\u00A0to\t \u00C2\u00A0introduce\t \u00C2\u00A0an\t \u00C2\u00A0inactive\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0construct\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0and\t \u00C2\u00A0conditionally\t \u00C2\u00A0trigger\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0under\t \u00C2\u00A0circumstances\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0cryptic\t \u00C2\u00A0degron\t \u00C2\u00A0is\t \u00C2\u00A0completed\t \u00C2\u00A0and/or\t \u00C2\u00A0exposed.\t \u00C2\u00A01.4.5 TRANS-\u00C2\u00AD\u00E2\u0080\u0090TARGETING\t \u00C2\u00A0VIA\t \u00C2\u00A0PROTEIN-\u00C2\u00AD\u00E2\u0080\u0090PROTEIN\t \u00C2\u00A0INTERACTION\t \u00C2\u00A0A\t \u00C2\u00A0primary\t \u00C2\u00A0destruction\t \u00C2\u00A0signal\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0a\t \u00C2\u00A0complex\t \u00C2\u00A0structural\t \u00C2\u00A0feature\t \u00C2\u00A0that\t \u00C2\u00A0involves\t \u00C2\u00A0more\t \u00C2\u00A0than\t \u00C2\u00A0one\t \u00C2\u00A0polypeptide.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0a\t \u00C2\u00A0polypeptide\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090degron\t \u00C2\u00A0can\t \u00C2\u00A0trigger\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0that\t \u00C2\u00A0contains\t \u00C2\u00A0lysine\t \u00C2\u00A0residues\t \u00C2\u00A0(for\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0acceptance)\t \u00C2\u00A0but\t \u00C2\u00A0lack\t \u00C2\u00A0a\t \u00C2\u00A0degron\t \u00C2\u00A0sequence.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0case,\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090degron\t \u00C2\u00A0acts\t \u00C2\u00A0catalytically\t \u00C2\u00A0to\t \u00C2\u00A0initiate\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0quasi-\u00C2\u00AD\u00E2\u0080\u0090UPS\t \u00C2\u00A0substrate\t \u00C2\u00A0that\t \u00C2\u00A0contains\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0acceptor\t \u00C2\u00A0sites\t \u00C2\u00A0(Johnson\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01990).\t \u00C2\u00A0In\t \u00C2\u00A0other\t \u00C2\u00A0words,\t \u00C2\u00A0a\t \u00C2\u00A0degron-\u00C2\u00AD\u00E2\u0080\u0090bearing\t \u00C2\u00A0peptide\t \u00C2\u00A0may\t \u00C2\u00A0trigger\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090substrates\t \u00C2\u00A0through\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction.\t \u00C2\u00A0Interestingly,\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0prohibit\t \u00C2\u00A0the\t \u00C2\u00A0selective\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0multi-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0complex\t \u00C2\u00A0(Schrader\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0terms\t \u00C2\u00A0of\t \u00C2\u00A0physiological\t \u00C2\u00A0function,\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0may\t \u00C2\u00A0provide\t \u00C2\u00A0a\t \u00C2\u00A0means\t \u00C2\u00A0to\t \u00C2\u00A0efficiently\t \u00C2\u00A0target\t \u00C2\u00A0a\t \u00C2\u00A0greater\t \u00C2\u00A0variety\t \u00C2\u00A0of\t \u00C2\u00A0substrates\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0actions\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0limited\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0E3\t \u00C2\u00A0ligases.\t \u00C2\u00A0An\t \u00C2\u00A0example\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0SCF\t \u00C2\u00A0complex,\t \u00C2\u00A0which\t \u00C2\u00A0contains\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0proteins,\t \u00C2\u00A0Cullin1,\t \u00C2\u00A0RBX1\t \u00C2\u00A0and\t \u00C2\u00A0Skp1.\t \u00C2\u00A0The\t \u00C2\u00A0SCF\t \u00C2\u00A0selects\t \u00C2\u00A0substrates\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0different\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0 34 complex\t \u00C2\u00A0selectively\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0proteolytic\t \u00C2\u00A0substrate\t \u00C2\u00A0and\t \u00C2\u00A0tethers\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0(Patton\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01998;\t \u00C2\u00A0Sakamoto\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001;\t \u00C2\u00A0Sakamoto,\t \u00C2\u00A02003).\t \u00C2\u00A0\t \u00C2\u00A0Trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0is\t \u00C2\u00A0widely\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0turnover\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0proteins.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0the\t \u00C2\u00A0tumour\t \u00C2\u00A0suppressor\t \u00C2\u00A0protein\t \u00C2\u00A0p53\t \u00C2\u00A0is\t \u00C2\u00A0ubiquitinated\t \u00C2\u00A0and\t \u00C2\u00A0dramatically\t \u00C2\u00A0degraded\t \u00C2\u00A0following\t \u00C2\u00A0association\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0HIV-\u00C2\u00AD\u00E2\u0080\u009016\t \u00C2\u00A0E6,\t \u00C2\u00A0the\t \u00C2\u00A0latter\t \u00C2\u00A0of\t \u00C2\u00A0which\t \u00C2\u00A0tethers\t \u00C2\u00A0p53\t \u00C2\u00A0to\t \u00C2\u00A0E6-\u00C2\u00AD\u00E2\u0080\u0090AP;\t \u00C2\u00A0E6-\u00C2\u00AD\u00E2\u0080\u0090E6AP\t \u00C2\u00A0together\t \u00C2\u00A0functions\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0E3\t \u00C2\u00A0ligase\t \u00C2\u00A0that\t \u00C2\u00A0degrades\t \u00C2\u00A0p53\t \u00C2\u00A0(Scheffner\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01993).\t \u00C2\u00A0The\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0transcriptional\t \u00C2\u00A0factor\t \u00C2\u00A0c-\u00C2\u00AD\u00E2\u0080\u0090Fos\t \u00C2\u00A0is\t \u00C2\u00A0reduced\t \u00C2\u00A0after\t \u00C2\u00A0forming\t \u00C2\u00A0a\t \u00C2\u00A0heterodimeric\t \u00C2\u00A0complex\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0phosphorylated\t \u00C2\u00A0c-\u00C2\u00AD\u00E2\u0080\u0090Jun\t \u00C2\u00A0(Tsurumi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01995).\t \u00C2\u00A0Similar\t \u00C2\u00A0mechanisms\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0discovered\t \u00C2\u00A0in\t \u00C2\u00A0Escherichia\t \u00C2\u00A0coli\t \u00C2\u00A0(Neher\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003),\t \u00C2\u00A0Xenopus\t \u00C2\u00A0(Stewart\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01994)\t \u00C2\u00A0and\t \u00C2\u00A0Saccharomyces\t \u00C2\u00A0cerevisiae\t \u00C2\u00A0(Yaglom\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01995).\t \u00C2\u00A0In\t \u00C2\u00A0terms\t \u00C2\u00A0of\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown,\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0provides\t \u00C2\u00A0a\t \u00C2\u00A0biological\t \u00C2\u00A0basis\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0idea\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0degron-\u00C2\u00AD\u00E2\u0080\u0090bearing\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0agent\t \u00C2\u00A0may\t \u00C2\u00A0degrade\t \u00C2\u00A0its\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0via\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction.\t \u00C2\u00A0As\t \u00C2\u00A0illustrated\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0above\t \u00C2\u00A0examples,\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0allows\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0specific\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0without\t \u00C2\u00A0perturbing\t \u00C2\u00A0their\t \u00C2\u00A0interacting\t \u00C2\u00A0partners.\t \u00C2\u00A0Thus\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0conceivable\t \u00C2\u00A0that\t \u00C2\u00A0by\t \u00C2\u00A0engineering\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptides\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0POI,\t \u00C2\u00A0we\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0it\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0highly\t \u00C2\u00A0specific\t \u00C2\u00A0manner.\t \u00C2\u00A01.5 EXAMPLES\t \u00C2\u00A0OF\t \u00C2\u00A0DIRECT\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0METHODS\t \u00C2\u00A0The\t \u00C2\u00A0discovery\t \u00C2\u00A0of\t \u00C2\u00A0degrons\t \u00C2\u00A0and\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0has\t \u00C2\u00A0allowed\t \u00C2\u00A0many\t \u00C2\u00A0research\t \u00C2\u00A0groups\t \u00C2\u00A0to\t \u00C2\u00A0utilize\t \u00C2\u00A0natural\t \u00C2\u00A0or\t \u00C2\u00A0synthetic\t \u00C2\u00A0degrons\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Although\t \u00C2\u00A0varied\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0specific\t \u00C2\u00A0design,\t \u00C2\u00A0the\t \u00C2\u00A0methods\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0summarized\t \u00C2\u00A0as\t \u00C2\u00A0follows,\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0their\t \u00C2\u00A0overall\t \u00C2\u00A0 35 strategy:\t \u00C2\u00A01)\t \u00C2\u00A0direct\t \u00C2\u00A0linkage\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0degron\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein,\t \u00C2\u00A0and\t \u00C2\u00A02)\t \u00C2\u00A0localization\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0various\t \u00C2\u00A0components\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0(E2,\t \u00C2\u00A0E3\t \u00C2\u00A0and\t \u00C2\u00A0proteasome)\t \u00C2\u00A0via\t \u00C2\u00A0an\t \u00C2\u00A0adaptor\t \u00C2\u00A0construct.\t \u00C2\u00A0The\t \u00C2\u00A0first\t \u00C2\u00A0strategy\t \u00C2\u00A0requires\t \u00C2\u00A0alterations\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein,\t \u00C2\u00A0whereas\t \u00C2\u00A0the\t \u00C2\u00A0second\t \u00C2\u00A0strategy\t \u00C2\u00A0in\t \u00C2\u00A0theory\t \u00C2\u00A0can\t \u00C2\u00A0target\t \u00C2\u00A0endogenous,\t \u00C2\u00A0unmodified\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0Representative\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0are\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0Table\t \u00C2\u00A01-\u00C2\u00AD\u00E2\u0080\u00901,\t \u00C2\u00A0and\t \u00C2\u00A0key\t \u00C2\u00A0examples\t \u00C2\u00A0are\t \u00C2\u00A0discussed\t \u00C2\u00A0in\t \u00C2\u00A0detail\t \u00C2\u00A0below.\t \u00C2\u00A01.5.1 PHYSICAL\t \u00C2\u00A0LINKAGE\t \u00C2\u00A0TO\t \u00C2\u00A0A\t \u00C2\u00A0DEGRON\t \u00C2\u00A0In\t \u00C2\u00A01994,\t \u00C2\u00A0Varshavsky\t \u00C2\u00A0and\t \u00C2\u00A0coworkers\t \u00C2\u00A0demonstrated\t \u00C2\u00A0the\t \u00C2\u00A0first\t \u00C2\u00A0strategy\t \u00C2\u00A0that\t \u00C2\u00A0rapidly\t \u00C2\u00A0and\t \u00C2\u00A0reversibly\t \u00C2\u00A0controls\t \u00C2\u00A0the\t \u00C2\u00A0stability\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0recombinant\t \u00C2\u00A0protein\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0engineered\t \u00C2\u00A0degron.\t \u00C2\u00A0Starting\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0dihydrofolate\t \u00C2\u00A0reductase\t \u00C2\u00A0(DHFR)\t \u00C2\u00A0mutant,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0valine\t \u00C2\u00A0(V)\t \u00C2\u00A0was\t \u00C2\u00A0replaced\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0destabilizing\t \u00C2\u00A0arginine\t \u00C2\u00A0(R)\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090end\t \u00C2\u00A0rule,\t \u00C2\u00A0the\t \u00C2\u00A0team\t \u00C2\u00A0screened\t \u00C2\u00A0for\t \u00C2\u00A0temperature-\u00C2\u00AD\u00E2\u0080\u0090sensitive\t \u00C2\u00A0mutants\t \u00C2\u00A0that\t \u00C2\u00A0were\t \u00C2\u00A0stable\t \u00C2\u00A0at\t \u00C2\u00A023\u00C2\u00B0C\t \u00C2\u00A0but\t \u00C2\u00A0rapidly\t \u00C2\u00A0degraded\t \u00C2\u00A0at\t \u00C2\u00A037\u00C2\u00B0C.\t \u00C2\u00A0The\t \u00C2\u00A0resulting\t \u00C2\u00A0mutant\t \u00C2\u00A0(R-\u00C2\u00AD\u00E2\u0080\u0090DHFRts)\t \u00C2\u00A0was\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0Cdc28,\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0maintaining\t \u00C2\u00A0cell\t \u00C2\u00A0cycle\t \u00C2\u00A0oscillations.\t \u00C2\u00A0R-\u00C2\u00AD\u00E2\u0080\u0090DHFRts-\u00C2\u00AD\u00E2\u0080\u0090Cdc28\t \u00C2\u00A0was\t \u00C2\u00A0functional\t \u00C2\u00A0at\t \u00C2\u00A023\u00C2\u00B0C,\t \u00C2\u00A0but\t \u00C2\u00A0showed\t \u00C2\u00A0no\t \u00C2\u00A0activity\t \u00C2\u00A0at\t \u00C2\u00A037\u00C2\u00B0C\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0its\t \u00C2\u00A0rapid\t \u00C2\u00A0degradation\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090end\t \u00C2\u00A0rule\t \u00C2\u00A0(Dohmen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01994).\t \u00C2\u00A0This\t \u00C2\u00A0method\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0widely\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0study\t \u00C2\u00A0the\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0multiple\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0yeast\t \u00C2\u00A0(Kanemaki\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0However,\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0requirement\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0temperature\t \u00C2\u00A0change\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0degradation,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0ideal\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells.\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0a\t \u00C2\u00A0later\t \u00C2\u00A0study\t \u00C2\u00A0the\t \u00C2\u00A0team\t \u00C2\u00A0engineered\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0R-\u00C2\u00AD\u00E2\u0080\u0090DHFR*\t \u00C2\u00A0mutant\t \u00C2\u00A0to\t \u00C2\u00A0generalize\t \u00C2\u00A0the\t \u00C2\u00A0applicability\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0to\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells.\t \u00C2\u00A0Target\t \u00C2\u00A0proteins\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0this\t \u00C2\u00A0novel\t \u00C2\u00A0 36 degron\t \u00C2\u00A0were\t \u00C2\u00A0stable\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0R-\u00C2\u00AD\u00E2\u0080\u0090DHFR*\t \u00C2\u00A0high-\u00C2\u00AD\u00E2\u0080\u0090affinity\t \u00C2\u00A0ligand\t \u00C2\u00A0methotrexate\t \u00C2\u00A0(MTX),\t \u00C2\u00A0but\t \u00C2\u00A0were\t \u00C2\u00A0rapidly\t \u00C2\u00A0degraded\t \u00C2\u00A0upon\t \u00C2\u00A0withdrawal\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0drug\t \u00C2\u00A0(Johnston\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01995).\t \u00C2\u00A0This\t \u00C2\u00A0study\t \u00C2\u00A0represents\t \u00C2\u00A0the\t \u00C2\u00A0first\t \u00C2\u00A0\u00E2\u0080\u009Cdrug-\u00C2\u00AD\u00E2\u0080\u0090off\u00E2\u0080\u009D\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0system,\t \u00C2\u00A0and\t \u00C2\u00A0paved\t \u00C2\u00A0the\t \u00C2\u00A0way\t \u00C2\u00A0for\t \u00C2\u00A0small-\u00C2\u00AD\u00E2\u0080\u0090molecule\t \u00C2\u00A0controlled\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods.\t \u00C2\u00A0The\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domains\t \u00C2\u00A0represents\t \u00C2\u00A0another\t \u00C2\u00A0key\t \u00C2\u00A0effort\t \u00C2\u00A0in\t \u00C2\u00A0engineering\t \u00C2\u00A0novel\t \u00C2\u00A0small-\u00C2\u00AD\u00E2\u0080\u0090chemical\t \u00C2\u00A0controlled\t \u00C2\u00A0degrons\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0FKBP12\t \u00C2\u00A0(FK506\t \u00C2\u00A0and\t \u00C2\u00A0rapamycin-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0protein),\t \u00C2\u00A0rapamycin\t \u00C2\u00A0and\t \u00C2\u00A0FRB\t \u00C2\u00A0associate\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0well-\u00C2\u00AD\u00E2\u0080\u0090characterized\t \u00C2\u00A0ternary\t \u00C2\u00A0complex\t \u00C2\u00A0(Banaszynski\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005);\t \u00C2\u00A0to\t \u00C2\u00A0minimize\t \u00C2\u00A0the\t \u00C2\u00A0biological\t \u00C2\u00A0effects\t \u00C2\u00A0of\t \u00C2\u00A0rapamycin\t \u00C2\u00A0on\t \u00C2\u00A0mTOR,\t \u00C2\u00A0a\t \u00C2\u00A0bump-\u00C2\u00AD\u00E2\u0080\u0090hole\t \u00C2\u00A0strategy\t \u00C2\u00A0is\t \u00C2\u00A0often\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0design\t \u00C2\u00A0rapamycin\t \u00C2\u00A0analogues\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0mutated\t \u00C2\u00A0FRB\t \u00C2\u00A0(FRB*)\t \u00C2\u00A0or\t \u00C2\u00A0FKBP\t \u00C2\u00A0(FBKP*)\t \u00C2\u00A0with\t \u00C2\u00A0higher\t \u00C2\u00A0affinity\t \u00C2\u00A0than\t \u00C2\u00A0to\t \u00C2\u00A0their\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0counterparts.\t \u00C2\u00A0While\t \u00C2\u00A0using\t \u00C2\u00A0MaRap,\t \u00C2\u00A0an\t \u00C2\u00A0engineered\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0structurally\t \u00C2\u00A0related\t \u00C2\u00A0to\t \u00C2\u00A0rapamycin,\t \u00C2\u00A0to\t \u00C2\u00A0mislocalize\t \u00C2\u00A0FRB*-\u00C2\u00AD\u00E2\u0080\u0090GSK-\u00C2\u00AD\u00E2\u0080\u00903\u00CE\u00B2\t \u00C2\u00A0via\t \u00C2\u00A0MaRap-\u00C2\u00AD\u00E2\u0080\u0090FRB*-\u00C2\u00AD\u00E2\u0080\u0090FKBP\t \u00C2\u00A0binding\t \u00C2\u00A0in\t \u00C2\u00A0mice,\t \u00C2\u00A0Stankunas\t \u00C2\u00A0and\t \u00C2\u00A0coworkers\t \u00C2\u00A0noticed\t \u00C2\u00A0decreased\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0FRB*-\u00C2\u00AD\u00E2\u0080\u0090GSK-\u00C2\u00AD\u00E2\u0080\u00903\u00CE\u00B2\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0otherwise\t \u00C2\u00A0identical\t \u00C2\u00A0fusion\t \u00C2\u00A0with\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0FRB.\t \u00C2\u00A0The\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0FRB*-\u00C2\u00AD\u00E2\u0080\u0090GSK-\u00C2\u00AD\u00E2\u0080\u00903\u00CE\u00B2\t \u00C2\u00A0were\t \u00C2\u00A0restored\t \u00C2\u00A0upon\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0MaRap\t \u00C2\u00A0(Stankunas\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0This\t \u00C2\u00A0observation\t \u00C2\u00A0led\t \u00C2\u00A0the\t \u00C2\u00A0team\t \u00C2\u00A0to\t \u00C2\u00A0further\t \u00C2\u00A0explore\t \u00C2\u00A0engineered\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domains.\t \u00C2\u00A0Error\t \u00C2\u00A0prone\t \u00C2\u00A0PCR\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0screen\t \u00C2\u00A0for\t \u00C2\u00A0FKBP\t \u00C2\u00A0mutants\t \u00C2\u00A0that\t \u00C2\u00A0conferred\t \u00C2\u00A0metabolic\t \u00C2\u00A0instability\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0absence\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0designer\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0called\t \u00C2\u00A0Shield-\u00C2\u00AD\u00E2\u0080\u00901.\t \u00C2\u00A0The\t \u00C2\u00A0resulting\t \u00C2\u00A0degron,\t \u00C2\u00A0FKBP*,\t \u00C2\u00A0was\t \u00C2\u00A0covalently\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0of\t \u00C2\u00A0various\t \u00C2\u00A0sizes,\t \u00C2\u00A0functions\t \u00C2\u00A0and\t \u00C2\u00A0cellular\t \u00C2\u00A0localizations.\t \u00C2\u00A0These\t \u00C2\u00A0fusion\t \u00C2\u00A0proteins\t \u00C2\u00A0were\t \u00C2\u00A0stable\t \u00C2\u00A0and\t \u00C2\u00A0functional\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0Shield-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0and\t \u00C2\u00A0were\t \u00C2\u00A0rapidly\t \u00C2\u00A0degraded\t \u00C2\u00A0upon\t \u00C2\u00A0withdrawal\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0ligand\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090 37 dependent\t \u00C2\u00A0manner.\t \u00C2\u00A0FKBP*\t \u00C2\u00A0was\t \u00C2\u00A0also\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0conferring\t \u00C2\u00A0instability\t \u00C2\u00A0when\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0target\t \u00C2\u00A0protein,\t \u00C2\u00A0although\t \u00C2\u00A0the\t \u00C2\u00A0efficacy\t \u00C2\u00A0was\t \u00C2\u00A0lower\t \u00C2\u00A0than\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0linkage\t \u00C2\u00A0(Banaszynski\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006).\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0FKBP*\t \u00C2\u00A0degron\t \u00C2\u00A0was\t \u00C2\u00A0later\t \u00C2\u00A0modified\t \u00C2\u00A0for\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0use.\t \u00C2\u00A0HCT116\t \u00C2\u00A0cancer\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0transfected\t \u00C2\u00A0with\t \u00C2\u00A0FKBP*-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0interleukin-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0(IL-\u00C2\u00AD\u00E2\u0080\u00902)\t \u00C2\u00A0and\t \u00C2\u00A0xenografted\t \u00C2\u00A0into\t \u00C2\u00A0nude\t \u00C2\u00A0mice.\t \u00C2\u00A0Daily\t \u00C2\u00A0intraperitoneal\t \u00C2\u00A0(i.p.)\t \u00C2\u00A0injections\t \u00C2\u00A0of\t \u00C2\u00A0Shield-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0stabilized\t \u00C2\u00A0the\t \u00C2\u00A0tumour\t \u00C2\u00A0suppressing\t \u00C2\u00A0IL-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0fusion\t \u00C2\u00A0protein\t \u00C2\u00A0and\t \u00C2\u00A0decreased\t \u00C2\u00A0tumour\t \u00C2\u00A0burden\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0saline-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0animals\t \u00C2\u00A0(Banaszynski\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008).\t \u00C2\u00A0Two\t \u00C2\u00A0additional\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domains\t \u00C2\u00A0were\t \u00C2\u00A0later\t \u00C2\u00A0engineered\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0similar\t \u00C2\u00A0approach:\t \u00C2\u00A01)\t \u00C2\u00A0a\t \u00C2\u00A0mutated\t \u00C2\u00A0E.coli\t \u00C2\u00A0DHFR,\t \u00C2\u00A0which\t \u00C2\u00A0destabilizes\t \u00C2\u00A0a\t \u00C2\u00A0linked\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0absence\t \u00C2\u00A0of\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0ligand\t \u00C2\u00A0trimethoprim\t \u00C2\u00A0(Iwamoto\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010),\t \u00C2\u00A0and\t \u00C2\u00A02)\t \u00C2\u00A0a\t \u00C2\u00A0mutated\t \u00C2\u00A0Estrogen\t \u00C2\u00A0Receptor\t \u00C2\u00A0Ligand\t \u00C2\u00A0Binding\t \u00C2\u00A0Domain,\t \u00C2\u00A0which\t \u00C2\u00A0destabilizes\t \u00C2\u00A0a\t \u00C2\u00A0linked\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0absence\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0CMP8\t \u00C2\u00A0(Miyazaki\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0\t \u00C2\u00A0Constant\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0ligand\t \u00C2\u00A0is\t \u00C2\u00A0necessary\t \u00C2\u00A0to\t \u00C2\u00A0maintain\t \u00C2\u00A0protein\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0all\t \u00C2\u00A0approaches\t \u00C2\u00A0that\t \u00C2\u00A0utilize\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domains.\t \u00C2\u00A0This\t \u00C2\u00A0becomes\t \u00C2\u00A0problematic\t \u00C2\u00A0when\t \u00C2\u00A0studying\t \u00C2\u00A0the\t \u00C2\u00A0products\t \u00C2\u00A0of\t \u00C2\u00A0essential\t \u00C2\u00A0genes,\t \u00C2\u00A0as\t \u00C2\u00A0continuous\t \u00C2\u00A0drug\t \u00C2\u00A0application\t \u00C2\u00A0may\t \u00C2\u00A0accumulate\t \u00C2\u00A0and\t \u00C2\u00A0eventually\t \u00C2\u00A0lead\t \u00C2\u00A0to\t \u00C2\u00A0toxicity.\t \u00C2\u00A0A\t \u00C2\u00A0novel\t \u00C2\u00A0approach\t \u00C2\u00A0that\t \u00C2\u00A0compliments\t \u00C2\u00A0these\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domains\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0ligand-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0degradation\t \u00C2\u00A0(LID)\t \u00C2\u00A0system.\t \u00C2\u00A0The\t \u00C2\u00A0LID\t \u00C2\u00A0domain\t \u00C2\u00A0contains\t \u00C2\u00A0an\t \u00C2\u00A0FKBP*\t \u00C2\u00A0mutant\t \u00C2\u00A0that\t \u00C2\u00A0expresses\t \u00C2\u00A0a\t \u00C2\u00A019-\u00C2\u00AD\u00E2\u0080\u0090residue\t \u00C2\u00A0peptide\t \u00C2\u00A0at\t \u00C2\u00A0its\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090terminal.\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0absence\t \u00C2\u00A0of\t \u00C2\u00A0Shield-\u00C2\u00AD\u00E2\u0080\u00901,\t \u00C2\u00A0this\t \u00C2\u00A0peptide\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0FKPB*\t \u00C2\u00A0active\t \u00C2\u00A0site\t \u00C2\u00A0and\t \u00C2\u00A0stabilizes\t \u00C2\u00A0the\t \u00C2\u00A0LID-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0protein.\t \u00C2\u00A0The\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0Shield-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0displaces\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0activate\t \u00C2\u00A0site,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0liberated\t \u00C2\u00A0peptide\t \u00C2\u00A0functions\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0UPS\t \u00C2\u00A0degron\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0actions\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0critical\t \u00C2\u00A0residues\t \u00C2\u00A0RRRG\t \u00C2\u00A0(Bonger\t \u00C2\u00A0et\t \u00C2\u00A0 38 al.,\t \u00C2\u00A02011).\t \u00C2\u00A0This\t \u00C2\u00A0represents\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0small-\u00C2\u00AD\u00E2\u0080\u0090molecule\t \u00C2\u00A0controlled\t \u00C2\u00A0\u00E2\u0080\u009Cdrug-\u00C2\u00AD\u00E2\u0080\u0090on\u00E2\u0080\u009D\t \u00C2\u00A0system,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0ligand\t \u00C2\u00A0induces\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0An\t \u00C2\u00A0alternative\t \u00C2\u00A0approach\t \u00C2\u00A0to\t \u00C2\u00A0designing\t \u00C2\u00A0degrons\t \u00C2\u00A0de\t \u00C2\u00A0novo\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0utilize\t \u00C2\u00A0the\t \u00C2\u00A0hydrophobicity\t \u00C2\u00A0protein\t \u00C2\u00A0quality\t \u00C2\u00A0control\t \u00C2\u00A0response.\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.4.3,\t \u00C2\u00A0local\t \u00C2\u00A0areas\t \u00C2\u00A0of\t \u00C2\u00A0increased\t \u00C2\u00A0hydrophobicity\t \u00C2\u00A0are\t \u00C2\u00A0efficient\t \u00C2\u00A0degrons\t \u00C2\u00A0that\t \u00C2\u00A0mark\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0UPS\t \u00C2\u00A0degradation.\t \u00C2\u00A0One\t \u00C2\u00A0such\t \u00C2\u00A0method\t \u00C2\u00A0utilizes\t \u00C2\u00A0the\t \u00C2\u00A0commercially\t \u00C2\u00A0available\t \u00C2\u00A0HaloTag-\u00C2\u00AD\u00E2\u0080\u0090dehalogenase\t \u00C2\u00A0system,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0HaloTag\t \u00C2\u00A0is\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0POI.\t \u00C2\u00A0HaloTag\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0hydrophobic\t \u00C2\u00A0compound\t \u00C2\u00A0HyT,\t \u00C2\u00A0which\t \u00C2\u00A0increases\t \u00C2\u00A0the\t \u00C2\u00A0hydrophobicity\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0fusion\t \u00C2\u00A0protein\t \u00C2\u00A0and\t \u00C2\u00A0thereby\t \u00C2\u00A0triggers\t \u00C2\u00A0its\t \u00C2\u00A0degradation\t \u00C2\u00A0via\t \u00C2\u00A0UPS\t \u00C2\u00A0(Neklesa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0As\t \u00C2\u00A0HyT\t \u00C2\u00A0is\t \u00C2\u00A0bacterial\t \u00C2\u00A0in\t \u00C2\u00A0origin,\t \u00C2\u00A0this\t \u00C2\u00A0system\t \u00C2\u00A0is\t \u00C2\u00A0unlikely\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0toxicity\t \u00C2\u00A0when\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0bio-\u00C2\u00AD\u00E2\u0080\u0090orthogonality.\t \u00C2\u00A0In\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0chemical\t \u00C2\u00A0approaches,\t \u00C2\u00A0there\t \u00C2\u00A0is\t \u00C2\u00A0now\t \u00C2\u00A0also\t \u00C2\u00A0considerable\t \u00C2\u00A0interest\t \u00C2\u00A0in\t \u00C2\u00A0combining\t \u00C2\u00A0optogenetics\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0degron\t \u00C2\u00A0system\t \u00C2\u00A0to\t \u00C2\u00A0achieve\t \u00C2\u00A0light-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0One\t \u00C2\u00A0such\t \u00C2\u00A0approach\t \u00C2\u00A0involves\t \u00C2\u00A0linking\t \u00C2\u00A0a\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0CIB1,\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0that\t \u00C2\u00A0rapidly\t \u00C2\u00A0dimerizes\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0cryptochrome\t \u00C2\u00A02\t \u00C2\u00A0(CRY2)\t \u00C2\u00A0protein\t \u00C2\u00A0from\t \u00C2\u00A0Arabidopsis\t \u00C2\u00A0upon\t \u00C2\u00A0exposure\t \u00C2\u00A0to\t \u00C2\u00A0blue\t \u00C2\u00A0light.\t \u00C2\u00A0By\t \u00C2\u00A0fusing\t \u00C2\u00A0the\t \u00C2\u00A0CRY2\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0SCF\t \u00C2\u00A0complex,\t \u00C2\u00A0light-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0dimerization\t \u00C2\u00A0helps\t \u00C2\u00A0to\t \u00C2\u00A0tether\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0SCF\t \u00C2\u00A0complex,\t \u00C2\u00A0resulting\t \u00C2\u00A0in\t \u00C2\u00A0UPS-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0(Labella\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0Although\t \u00C2\u00A0complicated\t \u00C2\u00A0in\t \u00C2\u00A0design,\t \u00C2\u00A0this\t \u00C2\u00A0approach\t \u00C2\u00A0takes\t \u00C2\u00A0full\t \u00C2\u00A0advantage\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0high\t \u00C2\u00A0spatial\t \u00C2\u00A0and\t \u00C2\u00A0temporal\t \u00C2\u00A0resolution\t \u00C2\u00A0of\t \u00C2\u00A0optogenetics\t \u00C2\u00A0in\t \u00C2\u00A0regulating\t \u00C2\u00A0protein\t \u00C2\u00A0stability.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0as\t \u00C2\u00A0CRY2\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0expressed\t \u00C2\u00A0in\t \u00C2\u00A0specific\t \u00C2\u00A0cell\t \u00C2\u00A0types\t \u00C2\u00A0and/or\t \u00C2\u00A0brain\t \u00C2\u00A0areas,\t \u00C2\u00A0this\t \u00C2\u00A0approach\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0achieve\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090lineage\t \u00C2\u00A0specific\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0In\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0CRY2,\t \u00C2\u00A0 39 light-\u00C2\u00AD\u00E2\u0080\u0090inducible\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0rely\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0light-\u00C2\u00AD\u00E2\u0080\u0090oxygen-\u00C2\u00AD\u00E2\u0080\u0090voltage\t \u00C2\u00A0(LOVE)\t \u00C2\u00A0domain\t \u00C2\u00A0of\t \u00C2\u00A0plants\t \u00C2\u00A0have\t \u00C2\u00A0also\t \u00C2\u00A0been\t \u00C2\u00A0developed\t \u00C2\u00A0(Bonger\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0In\t \u00C2\u00A0sum,\t \u00C2\u00A0the\t \u00C2\u00A0metabolic\t \u00C2\u00A0stability\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0controlled\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0ligand-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0by\t \u00C2\u00A0linking\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0molecule-\u00C2\u00AD\u00E2\u0080\u0090controlled\t \u00C2\u00A0degron.\t \u00C2\u00A0This\t \u00C2\u00A0strategy\t \u00C2\u00A0can\t \u00C2\u00A0induce\t \u00C2\u00A0the\t \u00C2\u00A0rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0cytosolic\t \u00C2\u00A0and\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0on-\u00C2\u00AD\u00E2\u0080\u0090and-\u00C2\u00AD\u00E2\u0080\u0090off\t \u00C2\u00A0manner\t \u00C2\u00A0with\t \u00C2\u00A0excellent\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependency.\t \u00C2\u00A0The\t \u00C2\u00A0positioning\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0degron\t \u00C2\u00A0is\t \u00C2\u00A0flexible\t \u00C2\u00A0in\t \u00C2\u00A0most\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0methods,\t \u00C2\u00A0in\t \u00C2\u00A0that\t \u00C2\u00A0they\t \u00C2\u00A0allow\t \u00C2\u00A0both\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0linkage.\t \u00C2\u00A0However,\t \u00C2\u00A0the\t \u00C2\u00A0above\t \u00C2\u00A0approaches\t \u00C2\u00A0are\t \u00C2\u00A0only\t \u00C2\u00A0useful\t \u00C2\u00A0for\t \u00C2\u00A0degrading\t \u00C2\u00A0genetically\t \u00C2\u00A0modified\t \u00C2\u00A0proteins\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0cannot\t \u00C2\u00A0target\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0This\t \u00C2\u00A0severely\t \u00C2\u00A0limits\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0methods\t \u00C2\u00A0in\t \u00C2\u00A0examining\t \u00C2\u00A0protein\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0situ,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0their\t \u00C2\u00A0potential\t \u00C2\u00A0as\t \u00C2\u00A0therapeutics\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0aggregation\t \u00C2\u00A0disorders.\t \u00C2\u00A01.5.2 DEGRADATION\t \u00C2\u00A0BY\t \u00C2\u00A0TRANS-\u00C2\u00AD\u00E2\u0080\u0090TARGETING\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0is\t \u00C2\u00A0often\t \u00C2\u00A0achieved\t \u00C2\u00A0by\t \u00C2\u00A0utilizing\t \u00C2\u00A0bifunctional\t \u00C2\u00A0molecules\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0and\t \u00C2\u00A0target\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0One\t \u00C2\u00A0prominent\t \u00C2\u00A0example\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0a\t \u00C2\u00A0strategy\t \u00C2\u00A0utilizes\t \u00C2\u00A0a\t \u00C2\u00A0family\t \u00C2\u00A0of\t \u00C2\u00A0adaptor\t \u00C2\u00A0molecules\t \u00C2\u00A0called\t \u00C2\u00A0proteolysis\t \u00C2\u00A0targeting\t \u00C2\u00A0chimeric\t \u00C2\u00A0molecules\t \u00C2\u00A0(PROTACs).\t \u00C2\u00A0PROTACs\t \u00C2\u00A0contain\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0chemical\t \u00C2\u00A0ligand\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0target\t \u00C2\u00A0protein,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0tethered\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0short\t \u00C2\u00A0peptide\t \u00C2\u00A0sequence\t \u00C2\u00A0that\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0E3\t \u00C2\u00A0ligase\t \u00C2\u00A0(for\t \u00C2\u00A0example,\t \u00C2\u00A0SCF\u00CE\u00B2TCRP\t \u00C2\u00A0\t \u00C2\u00A0and\t \u00C2\u00A0SCFVBL-\u00C2\u00AD\u00E2\u0080\u0090Cul2\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0utilized)\t \u00C2\u00A0via\t \u00C2\u00A0a\t \u00C2\u00A0linker\t \u00C2\u00A0(Sakamoto\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001;\t \u00C2\u00A0Jang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Cyrus\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0PROTACs\t \u00C2\u00A0catalyze\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0through\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0actions\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0particular\t \u00C2\u00A0SCF\t \u00C2\u00A0 40 E3\t \u00C2\u00A0ligase.\t \u00C2\u00A0In\t \u00C2\u00A0a\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0study,\t \u00C2\u00A0Protac-\u00C2\u00AD\u00E2\u0080\u00901,\t \u00C2\u00A0which\t \u00C2\u00A0contains\t \u00C2\u00A0the\t \u00C2\u00A0ligand\t \u00C2\u00A0OVA\t \u00C2\u00A0for\t \u00C2\u00A0methionine\t \u00C2\u00A0aminopeptidase-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0(MetAP-\u00C2\u00AD\u00E2\u0080\u00902),\t \u00C2\u00A0successfully\t \u00C2\u00A0degraded\t \u00C2\u00A0MetAP-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0in\t \u00C2\u00A0Xenopus\t \u00C2\u00A0Oocyte\t \u00C2\u00A0extracts\t \u00C2\u00A0following\t \u00C2\u00A0microinjection\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0(Sakamoto\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001).\t \u00C2\u00A0Later\t \u00C2\u00A0improvements\t \u00C2\u00A0added\t \u00C2\u00A0a\t \u00C2\u00A0poly-\u00C2\u00AD\u00E2\u0080\u0090arginine\t \u00C2\u00A0domain\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0original\t \u00C2\u00A0design\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0PROTACs\t \u00C2\u00A0to\t \u00C2\u00A0penetrate\t \u00C2\u00A0the\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane,\t \u00C2\u00A0and\t \u00C2\u00A0this\t \u00C2\u00A0new\t \u00C2\u00A0generation\t \u00C2\u00A0of\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090permeant\t \u00C2\u00A0PROTAC\t \u00C2\u00A0was\t \u00C2\u00A0shown\t \u00C2\u00A0to\t \u00C2\u00A0effectively\t \u00C2\u00A0degrade\t \u00C2\u00A0recombinant\t \u00C2\u00A0androgen\t \u00C2\u00A0receptor\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cell\t \u00C2\u00A0lines\t \u00C2\u00A0(Sakamoto\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0To\t \u00C2\u00A0further\t \u00C2\u00A0increase\t \u00C2\u00A0the\t \u00C2\u00A0efficiency\t \u00C2\u00A0of\t \u00C2\u00A0PROTACs,\t \u00C2\u00A0a\t \u00C2\u00A0two-\u00C2\u00AD\u00E2\u0080\u0090headed\t \u00C2\u00A0version\t \u00C2\u00A0was\t \u00C2\u00A0later\t \u00C2\u00A0synthesized\t \u00C2\u00A0and\t \u00C2\u00A0validated\t \u00C2\u00A0(Cyrus\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010),\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0recent\t \u00C2\u00A0study\t \u00C2\u00A0described\t \u00C2\u00A0a\t \u00C2\u00A0phosphorylation-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0PROTAC\t \u00C2\u00A0strategy\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0conditional\t \u00C2\u00A0knockout\t \u00C2\u00A0of\t \u00C2\u00A0fibroblast\t \u00C2\u00A0growth\t \u00C2\u00A0factor\t \u00C2\u00A0receptor\t \u00C2\u00A0substrate\t \u00C2\u00A02\u00CE\u00B1\t \u00C2\u00A0and\t \u00C2\u00A0phosphatidylinositol-\u00C2\u00AD\u00E2\u0080\u00903-\u00C2\u00AD\u00E2\u0080\u0090kinase\t \u00C2\u00A0(Hines\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0A\t \u00C2\u00A0major\t \u00C2\u00A0advantage\t \u00C2\u00A0of\t \u00C2\u00A0this\t \u00C2\u00A0technique\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0PROTACs\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0unmodified\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins,\t \u00C2\u00A0utilizing\t \u00C2\u00A0a\t \u00C2\u00A0variety\t \u00C2\u00A0of\t \u00C2\u00A0E3\t \u00C2\u00A0ligases.\t \u00C2\u00A0As\t \u00C2\u00A0the\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0E3\t \u00C2\u00A0enzymes\t \u00C2\u00A0is\t \u00C2\u00A0somewhat\t \u00C2\u00A0tissue\t \u00C2\u00A0specific,\t \u00C2\u00A0PROTAC\t \u00C2\u00A0may\t \u00C2\u00A0potentially\t \u00C2\u00A0achieve\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0particular\t \u00C2\u00A0tissue\t \u00C2\u00A0type.\t \u00C2\u00A0However,\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0is\t \u00C2\u00A0contingent\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0existence\t \u00C2\u00A0of\t \u00C2\u00A0small\t \u00C2\u00A0chemical\t \u00C2\u00A0ligands\t \u00C2\u00A0that\t \u00C2\u00A0selectively\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0with\t \u00C2\u00A0high\t \u00C2\u00A0affinity.\t \u00C2\u00A0Thus,\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0is\t \u00C2\u00A0limited\t \u00C2\u00A0to\t \u00C2\u00A0degrading\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0which\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0ligand\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0identified\t \u00C2\u00A0(i.e.\t \u00C2\u00A0druggable\t \u00C2\u00A0proteins).\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0circumvent\t \u00C2\u00A0the\t \u00C2\u00A0requirement\t \u00C2\u00A0for\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0ligands,\t \u00C2\u00A0Zhou\t \u00C2\u00A0and\t \u00C2\u00A0Hadley\t \u00C2\u00A0developed\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0based\t \u00C2\u00A0approach.\t \u00C2\u00A0As\t \u00C2\u00A0with\t \u00C2\u00A0first\t \u00C2\u00A0generation\t \u00C2\u00A0PROTACs,\t \u00C2\u00A0this\t \u00C2\u00A0system\t \u00C2\u00A0also\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0SCF\u00CE\u00B2TrCP\t \u00C2\u00A0E3\t \u00C2\u00A0ligase.\t \u00C2\u00A0The\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein\t \u00C2\u00A0component\t \u00C2\u00A0of\t \u00C2\u00A0SCF\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0 41 bidirectional\t \u00C2\u00A0molecule\t \u00C2\u00A0that\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0substrate\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0WD40\t \u00C2\u00A0domain\t \u00C2\u00A0and\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0Skp1/Cullin\t \u00C2\u00A0component\t \u00C2\u00A0through\t \u00C2\u00A0an\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0domain\t \u00C2\u00A0(Zhou\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02000).\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0method,\t \u00C2\u00A0a\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0domain\t \u00C2\u00A0that\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0was\t \u00C2\u00A0genetically\t \u00C2\u00A0tagged\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0natural\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein.\t \u00C2\u00A0In\t \u00C2\u00A0a\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0study,\t \u00C2\u00A0adenoviral\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0an\t \u00C2\u00A0engineered\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein\t \u00C2\u00A0containing\t \u00C2\u00A0a\t \u00C2\u00A0binding\t \u00C2\u00A0domain\t \u00C2\u00A0for\t \u00C2\u00A0p107\t \u00C2\u00A0rapidly\t \u00C2\u00A0degraded\t \u00C2\u00A0endogenous\t \u00C2\u00A0p107\t \u00C2\u00A0via\t \u00C2\u00A0UPS\t \u00C2\u00A0in\t \u00C2\u00A0cervical\t \u00C2\u00A0carcinoma\t \u00C2\u00A0C33A\t \u00C2\u00A0cells;\t \u00C2\u00A0however,\t \u00C2\u00A0the\t \u00C2\u00A0structurally\t \u00C2\u00A0similar\t \u00C2\u00A0pRB\t \u00C2\u00A0protein\t \u00C2\u00A0was\t \u00C2\u00A0also\t \u00C2\u00A0degraded,\t \u00C2\u00A0highlighting\t \u00C2\u00A0the\t \u00C2\u00A0importance\t \u00C2\u00A0of\t \u00C2\u00A0selecting\t \u00C2\u00A0target\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0binding\t \u00C2\u00A0domains\t \u00C2\u00A0in\t \u00C2\u00A0designing\t \u00C2\u00A0degradation\t \u00C2\u00A0constructs\t \u00C2\u00A0(Zhou\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02000).\t \u00C2\u00A0\t \u00C2\u00A0A\t \u00C2\u00A0similar\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0based\t \u00C2\u00A0strategy\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0used\t \u00C2\u00A0successfully\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0(Su\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003)\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0(Cong\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003)\t \u00C2\u00A0to\t \u00C2\u00A0selectively\t \u00C2\u00A0knockdown\t \u00C2\u00A0the\t \u00C2\u00A0nuclear-\u00C2\u00AD\u00E2\u0080\u0090cytosolic\t \u00C2\u00A0subpopulation\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0Wnt\t \u00C2\u00A0signaling,\t \u00C2\u00A0without\t \u00C2\u00A0perturbing\t \u00C2\u00A0the\t \u00C2\u00A0pool\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0cell\t \u00C2\u00A0adhesion.\t \u00C2\u00A0Notably,\t \u00C2\u00A0two\t \u00C2\u00A0improvements\t \u00C2\u00A0were\t \u00C2\u00A0made.\t \u00C2\u00A0First,\t \u00C2\u00A0a\t \u00C2\u00A0glycine-\u00C2\u00AD\u00E2\u0080\u0090serine\t \u00C2\u00A0linker\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0component\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0the\t \u00C2\u00A0flexibility\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0construct\t \u00C2\u00A0and\t \u00C2\u00A0enhance\t \u00C2\u00A0binding\t \u00C2\u00A0efficacy\t \u00C2\u00A0to\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin.\t \u00C2\u00A0Second,\t \u00C2\u00A0the\t \u00C2\u00A0construct\t \u00C2\u00A0incorporated\t \u00C2\u00A0multiple\t \u00C2\u00A0repeats\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0binding\t \u00C2\u00A0domain\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0specificity\t \u00C2\u00A0(Su\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0clearly\t \u00C2\u00A0demonstrates\t \u00C2\u00A0the\t \u00C2\u00A0superior\t \u00C2\u00A0selectivity\t \u00C2\u00A0of\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions,\t \u00C2\u00A0as\t \u00C2\u00A0opposed\t \u00C2\u00A0to\t \u00C2\u00A0small\t \u00C2\u00A0molecule-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0binding,\t \u00C2\u00A0in\t \u00C2\u00A0targeting\t \u00C2\u00A0a\t \u00C2\u00A0specific\t \u00C2\u00A0subpopulation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0selective\t \u00C2\u00A0degradation.\t \u00C2\u00A0\t \u00C2\u00A0However,\t \u00C2\u00A0a\t \u00C2\u00A0major\t \u00C2\u00A0problem\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0above\t \u00C2\u00A0approach\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0native\t \u00C2\u00A0WD40\t \u00C2\u00A0domain\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0chimeric\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein\t \u00C2\u00A0retains\t \u00C2\u00A0the\t \u00C2\u00A0ability\t \u00C2\u00A0target\t \u00C2\u00A0endogenous\t \u00C2\u00A0WD40-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0proteins\t \u00C2\u00A0 42 for\t \u00C2\u00A0knockdown,\t \u00C2\u00A0which\t \u00C2\u00A0may\t \u00C2\u00A0lead\t \u00C2\u00A0to\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0effects.\t \u00C2\u00A0Accordingly,\t \u00C2\u00A0the\t \u00C2\u00A0WD40\t \u00C2\u00A0domain\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0in\t \u00C2\u00A0later\t \u00C2\u00A0studies\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0specificity\t \u00C2\u00A0(Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Liu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004).\t \u00C2\u00A0\t \u00C2\u00A0An\t \u00C2\u00A0alternative\t \u00C2\u00A0strategy\t \u00C2\u00A0to\t \u00C2\u00A0decrease\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0effects\t \u00C2\u00A0while\t \u00C2\u00A0still\t \u00C2\u00A0harnessing\t \u00C2\u00A0the\t \u00C2\u00A0SCF\t \u00C2\u00A0E3\t \u00C2\u00A0ligase\t \u00C2\u00A0system\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0introduce\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090mammalian\t \u00C2\u00A0SCF\t \u00C2\u00A0E3\t \u00C2\u00A0ligases\t \u00C2\u00A0into\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0To\t \u00C2\u00A0this\t \u00C2\u00A0end,\t \u00C2\u00A0components\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0Drosophila\t \u00C2\u00A0melanogaster\t \u00C2\u00A0SCF\t \u00C2\u00A0system\t \u00C2\u00A0was\t \u00C2\u00A0expressed\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells,\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0chimeric\t \u00C2\u00A0Drosophila\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein\t \u00C2\u00A0was\t \u00C2\u00A0engineered\t \u00C2\u00A0to\t \u00C2\u00A0contain\t \u00C2\u00A0a\t \u00C2\u00A0single-\u00C2\u00AD\u00E2\u0080\u0090domain\t \u00C2\u00A0antibody\t \u00C2\u00A0fragment\t \u00C2\u00A0to\t \u00C2\u00A0GFP.\t \u00C2\u00A0The\t \u00C2\u00A0system\t \u00C2\u00A0could\t \u00C2\u00A0then\t \u00C2\u00A0be\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0any\t \u00C2\u00A0GFP-\u00C2\u00AD\u00E2\u0080\u0090fusion\t \u00C2\u00A0protein\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0SCF\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090proteasome\t \u00C2\u00A0pathway\t \u00C2\u00A0(Caussinus\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0Nishimura\t \u00C2\u00A0and\t \u00C2\u00A0coworkers\t \u00C2\u00A0developed\t \u00C2\u00A0a\t \u00C2\u00A0similar\t \u00C2\u00A0strategy\t \u00C2\u00A0utilizing\t \u00C2\u00A0a\t \u00C2\u00A0plant-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0SCF\t \u00C2\u00A0ligase\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Plants\t \u00C2\u00A0have\t \u00C2\u00A0a\t \u00C2\u00A0unique\t \u00C2\u00A0system\t \u00C2\u00A0whereby\t \u00C2\u00A0SCF-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0is\t \u00C2\u00A0induced\t \u00C2\u00A0following\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0Auxin,\t \u00C2\u00A0a\t \u00C2\u00A0plant\t \u00C2\u00A0hormone.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0strategy,\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0was\t \u00C2\u00A0genetically\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0Auxin-\u00C2\u00AD\u00E2\u0080\u0090responsive\t \u00C2\u00A0degron\t \u00C2\u00A0(AID),\t \u00C2\u00A0which\t \u00C2\u00A0couples\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0plant\t \u00C2\u00A0SCFTIR1\t \u00C2\u00A0complex\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0Auxin,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0chimeric\t \u00C2\u00A0protein\t \u00C2\u00A0was\t \u00C2\u00A0rapidly\t \u00C2\u00A0degraded\t \u00C2\u00A0(~30min)\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0pathway\t \u00C2\u00A0upon\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0Auxin\t \u00C2\u00A0(Nishimura\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Holland\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Notably,\t \u00C2\u00A0the\t \u00C2\u00A0degree\t \u00C2\u00A0of\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0was\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0and\t \u00C2\u00A0could\t \u00C2\u00A0be\t \u00C2\u00A0easily\t \u00C2\u00A0controlled\t \u00C2\u00A0by\t \u00C2\u00A0altering\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0Auxin\t \u00C2\u00A0(Nishimura\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0using\t \u00C2\u00A0mammalian\t \u00C2\u00A0SCF\t \u00C2\u00A0E3\t \u00C2\u00A0ligases\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0degradation,\t \u00C2\u00A0the\t \u00C2\u00A0methods\t \u00C2\u00A0described\t \u00C2\u00A0above\t \u00C2\u00A0are\t \u00C2\u00A0expected\t \u00C2\u00A0to\t \u00C2\u00A0have\t \u00C2\u00A0fewer\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0effects.\t \u00C2\u00A0However,\t \u00C2\u00A0both\t \u00C2\u00A0methods\t \u00C2\u00A0require\t \u00C2\u00A0the\t \u00C2\u00A0introduction\t \u00C2\u00A0of\t \u00C2\u00A0two\t \u00C2\u00A0genetic\t \u00C2\u00A0constructs\t \u00C2\u00A0(the\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090mammalian\t \u00C2\u00A0SCF\t \u00C2\u00A0system\t \u00C2\u00A0and\t \u00C2\u00A0 43 the\t \u00C2\u00A0GFP\u00E2\u0080\u0093\t \u00C2\u00A0or\t \u00C2\u00A0AID\u00E2\u0080\u0093tagged\t \u00C2\u00A0target\t \u00C2\u00A0protein),\t \u00C2\u00A0thus\t \u00C2\u00A0complicating\t \u00C2\u00A0the\t \u00C2\u00A0experiment\t \u00C2\u00A0and\t \u00C2\u00A0limiting\t \u00C2\u00A0their\t \u00C2\u00A0use\t \u00C2\u00A0as\t \u00C2\u00A0therapeutics.\t \u00C2\u00A0\t \u00C2\u00A0Post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0phosphorylation,\t \u00C2\u00A0are\t \u00C2\u00A0crucially\t \u00C2\u00A0important\t \u00C2\u00A0in\t \u00C2\u00A0regulating\t \u00C2\u00A0protein\t \u00C2\u00A0function,\t \u00C2\u00A0yet\t \u00C2\u00A0experimental\t \u00C2\u00A0tools\t \u00C2\u00A0designed\t \u00C2\u00A0to\t \u00C2\u00A0dissect\t \u00C2\u00A0the\t \u00C2\u00A0specific\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0certain\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translationally\t \u00C2\u00A0modified\t \u00C2\u00A0form\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0are\t \u00C2\u00A0limited.\t \u00C2\u00A0To\t \u00C2\u00A0explore\t \u00C2\u00A0the\t \u00C2\u00A0potential\t \u00C2\u00A0of\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0targeting\t \u00C2\u00A0a\t \u00C2\u00A0subpopulation\t \u00C2\u00A0of\t \u00C2\u00A0modified\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0degradation,\t \u00C2\u00A0Zhang\t \u00C2\u00A0and\t \u00C2\u00A0colleagues\t \u00C2\u00A0engineered\t \u00C2\u00A0a\t \u00C2\u00A0SCF\t \u00C2\u00A0complex\t \u00C2\u00A0that\t \u00C2\u00A0contains\t \u00C2\u00A0a\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0hypophosphorylated\t \u00C2\u00A0form\t \u00C2\u00A0of\t \u00C2\u00A0p107.\t \u00C2\u00A0By\t \u00C2\u00A0eliminating\t \u00C2\u00A0the\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0WD40\t \u00C2\u00A0domain\t \u00C2\u00A0and\t \u00C2\u00A0using\t \u00C2\u00A0the\t \u00C2\u00A0minimal\t \u00C2\u00A0binding\t \u00C2\u00A0sequence\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0p107,\t \u00C2\u00A0the\t \u00C2\u00A0construct\t \u00C2\u00A0specifically\t \u00C2\u00A0degraded\t \u00C2\u00A0hypo\u00E2\u0080\u0093,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0hyper-\u00C2\u00AD\u00E2\u0080\u0090phosphorylated\t \u00C2\u00A0p107\t \u00C2\u00A0(Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Interestingly,\t \u00C2\u00A0p107\t \u00C2\u00A0associates\t \u00C2\u00A0with\t \u00C2\u00A0cyclinA\t \u00C2\u00A0and\t \u00C2\u00A0cdk2\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0complex,\t \u00C2\u00A0nevertheless,\t \u00C2\u00A0the\t \u00C2\u00A0construct\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0perturb\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0either\t \u00C2\u00A0p107-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0protein,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0based\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0selective\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0component\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0multi-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0complex\t \u00C2\u00A0(Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0This\t \u00C2\u00A0seminal\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0study\t \u00C2\u00A0strongly\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0by\t \u00C2\u00A0using\t \u00C2\u00A0rational\t \u00C2\u00A0design,\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockout\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0interrogate\t \u00C2\u00A0the\t \u00C2\u00A0functions\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0certain\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translationally\t \u00C2\u00A0modified\t \u00C2\u00A0form\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090interest.\t \u00C2\u00A0\t \u00C2\u00A0Notably,\t \u00C2\u00A0all\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0rely\t \u00C2\u00A0on\t \u00C2\u00A0E3\t \u00C2\u00A0ligases\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0require\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0contains\t \u00C2\u00A0exposed\t \u00C2\u00A0lysine\t \u00C2\u00A0residues\t \u00C2\u00A0for\t \u00C2\u00A0ubiquitination,\t \u00C2\u00A0sufficient\t \u00C2\u00A0molecular\t \u00C2\u00A0mass\t \u00C2\u00A0for\t \u00C2\u00A0efficient\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0E2\t \u00C2\u00A0enzymes\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0proteasome,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0less\t \u00C2\u00A0compact\t \u00C2\u00A0structure\t \u00C2\u00A0for\t \u00C2\u00A0subsequent\t \u00C2\u00A0unfolding.\t \u00C2\u00A0 44 In\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0localization\t \u00C2\u00A0to\t \u00C2\u00A0E3\t \u00C2\u00A0ligases,\t \u00C2\u00A0methods\t \u00C2\u00A0have\t \u00C2\u00A0also\t \u00C2\u00A0been\t \u00C2\u00A0developed\t \u00C2\u00A0to\t \u00C2\u00A0tether\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0E2\t \u00C2\u00A0enzymes\t \u00C2\u00A0(Gosink\t \u00C2\u00A0and\t \u00C2\u00A0Vierstra,\t \u00C2\u00A01995)\t \u00C2\u00A0or\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0itself\t \u00C2\u00A0(Janse,\t \u00C2\u00A02004a;\t \u00C2\u00A0Matsuzawa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0The\t \u00C2\u00A0latter\t \u00C2\u00A0approach\t \u00C2\u00A0bypasses\t \u00C2\u00A0the\t \u00C2\u00A0need\t \u00C2\u00A0for\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0POI,\t \u00C2\u00A0and\t \u00C2\u00A0is\t \u00C2\u00A0particularly\t \u00C2\u00A0useful\t \u00C2\u00A0for\t \u00C2\u00A0proteins\t \u00C2\u00A0without\t \u00C2\u00A0lysine\t \u00C2\u00A0residues.\t \u00C2\u00A0An\t \u00C2\u00A0elegant\t \u00C2\u00A0demonstration\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0a\t \u00C2\u00A0method\t \u00C2\u00A0utilizes\t \u00C2\u00A0ornithine\t \u00C2\u00A0decarboxylase\t \u00C2\u00A0(molecular\t \u00C2\u00A0weight:\t \u00C2\u00A051kDa),\t \u00C2\u00A0which\t \u00C2\u00A0following\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0antizyme,\t \u00C2\u00A0directly\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A026S\t \u00C2\u00A0subunit\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0Matsuzawa\t \u00C2\u00A0and\t \u00C2\u00A0coworkers\t \u00C2\u00A0linked\t \u00C2\u00A0a\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0of\t \u00C2\u00A0ornithine\t \u00C2\u00A0decarboxylase\t \u00C2\u00A0via\t \u00C2\u00A0a\t \u00C2\u00A0linker,\t \u00C2\u00A0and\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0this\t \u00C2\u00A0construct\t \u00C2\u00A0successfully\t \u00C2\u00A0targeted\t \u00C2\u00A0POIs\t \u00C2\u00A0for\t \u00C2\u00A0proteasomal\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0cells\t \u00C2\u00A0(Matsuzawa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A01.5.3 LIMITATIONS\t \u00C2\u00A0OF\t \u00C2\u00A0CURRENT\t \u00C2\u00A0DEGRON-\u00C2\u00AD\u00E2\u0080\u0090BASED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0METHODS\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0DNA\u00E2\u0080\u0093\t \u00C2\u00A0or\t \u00C2\u00A0RNA\u00E2\u0080\u0093based\t \u00C2\u00A0techniques,\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0directly\t \u00C2\u00A0degrade\t \u00C2\u00A0proteins\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level\t \u00C2\u00A0are\t \u00C2\u00A0rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible.\t \u00C2\u00A0Most\t \u00C2\u00A0methods\t \u00C2\u00A0have\t \u00C2\u00A0excellent\t \u00C2\u00A0dose\u00E2\u0080\u0093\t \u00C2\u00A0and\t \u00C2\u00A0time\u00E2\u0080\u0093dependency,\t \u00C2\u00A0thus\t \u00C2\u00A0allowing\t \u00C2\u00A0the\t \u00C2\u00A0examination\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0graded\t \u00C2\u00A0manner\t \u00C2\u00A0and\t \u00C2\u00A0across\t \u00C2\u00A0developmental\t \u00C2\u00A0stages.\t \u00C2\u00A0Further\t \u00C2\u00A0temporal\t \u00C2\u00A0control\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0realized\t \u00C2\u00A0by\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0ligands,\t \u00C2\u00A0and\t \u00C2\u00A0both\t \u00C2\u00A0drug-\u00C2\u00AD\u00E2\u0080\u0090on\t \u00C2\u00A0and\t \u00C2\u00A0drug-\u00C2\u00AD\u00E2\u0080\u0090off\t \u00C2\u00A0systems\t \u00C2\u00A0(Banaszynski\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006;\t \u00C2\u00A0Nishimura\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Bonger\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Neklesa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013)\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0developed.\t \u00C2\u00A0Despite\t \u00C2\u00A0these\t \u00C2\u00A0advantages\t \u00C2\u00A0over\t \u00C2\u00A0genetic\t \u00C2\u00A0protein\t \u00C2\u00A0knockout\t \u00C2\u00A0methods,\t \u00C2\u00A0however,\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0three\t \u00C2\u00A0main\t \u00C2\u00A0limitations\t \u00C2\u00A0with\t \u00C2\u00A0these\t \u00C2\u00A0techniques.\t \u00C2\u00A0 45 First,\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0require\t \u00C2\u00A0direct\t \u00C2\u00A0linkage\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0degron\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0suitable\t \u00C2\u00A0for\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0unmodified\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0cells\t \u00C2\u00A0or\t \u00C2\u00A0animals\t \u00C2\u00A0in\t \u00C2\u00A0situ\t \u00C2\u00A0(Dohmen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01994;\t \u00C2\u00A0Banaszynski\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006;\t \u00C2\u00A0Bonger\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0This\t \u00C2\u00A0is\t \u00C2\u00A0problematic\t \u00C2\u00A0for\t \u00C2\u00A0both\t \u00C2\u00A0basic\t \u00C2\u00A0research\t \u00C2\u00A0and\t \u00C2\u00A0therapeutic\t \u00C2\u00A0development.\t \u00C2\u00A0The\t \u00C2\u00A0covalent\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0components\t \u00C2\u00A0necessary\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0POI\t \u00C2\u00A0degradation\t \u00C2\u00A0may\t \u00C2\u00A0interfere\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0normal\t \u00C2\u00A0localization,\t \u00C2\u00A0stability\t \u00C2\u00A0and\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0(Giepmans,\t \u00C2\u00A02006).\t \u00C2\u00A0The\t \u00C2\u00A0requirement\t \u00C2\u00A0for\t \u00C2\u00A0genetic\t \u00C2\u00A0modification\t \u00C2\u00A0also\t \u00C2\u00A0precludes\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0methods\t \u00C2\u00A0in\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0native\t \u00C2\u00A0pathogenic\t \u00C2\u00A0proteins,\t \u00C2\u00A0thus\t \u00C2\u00A0limiting\t \u00C2\u00A0their\t \u00C2\u00A0therapeutic\t \u00C2\u00A0potential.\t \u00C2\u00A0Second,\t \u00C2\u00A0although\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0induce\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0via\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0are\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0unmodified\t \u00C2\u00A0proteins,\t \u00C2\u00A0the\t \u00C2\u00A0bifunctional\t \u00C2\u00A0adaptors\t \u00C2\u00A0that\t \u00C2\u00A0tether\t \u00C2\u00A0a\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0UPS\t \u00C2\u00A0components\t \u00C2\u00A0are\t \u00C2\u00A0generally\t \u00C2\u00A0too\t \u00C2\u00A0large\t \u00C2\u00A0to\t \u00C2\u00A0penetrate\t \u00C2\u00A0the\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane.\t \u00C2\u00A0In\t \u00C2\u00A0many\t \u00C2\u00A0cases,\t \u00C2\u00A0viral\t \u00C2\u00A0infection\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0agents\t \u00C2\u00A0is\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0cells\t \u00C2\u00A0and\t \u00C2\u00A0animals\t \u00C2\u00A0(Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Matsuzawa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005),\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0substantial\t \u00C2\u00A0size\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0adapters\t \u00C2\u00A0increases\t \u00C2\u00A0the\t \u00C2\u00A0chance\t \u00C2\u00A0of\t \u00C2\u00A0immunogenicity.\t \u00C2\u00A0Third,\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0current\t \u00C2\u00A0degron-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0require\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0for\t \u00C2\u00A0degradation,\t \u00C2\u00A0they\t \u00C2\u00A0may\t \u00C2\u00A0fail\t \u00C2\u00A0in\t \u00C2\u00A0cases\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0proteasomal\t \u00C2\u00A0function\t \u00C2\u00A0is\t \u00C2\u00A0compromised.\t \u00C2\u00A0In\t \u00C2\u00A0particular,\t \u00C2\u00A0these\t \u00C2\u00A0methods\t \u00C2\u00A0may\t \u00C2\u00A0not\t \u00C2\u00A0be\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0aberrantly\t \u00C2\u00A0accumulated\t \u00C2\u00A0pathogenic\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0proteinopathies\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0disorders,\t \u00C2\u00A0as\t \u00C2\u00A0proteasomal\t \u00C2\u00A0dysfunction\t \u00C2\u00A0occurs\t \u00C2\u00A0early\t \u00C2\u00A0during\t \u00C2\u00A0pathogenesis.\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0conceivable\t \u00C2\u00A0that\t \u00C2\u00A0UPS-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0therapies\t \u00C2\u00A0would\t \u00C2\u00A0not\t \u00C2\u00A0only\t \u00C2\u00A0fail\t \u00C2\u00A0under\t \u00C2\u00A0such\t \u00C2\u00A0circumstances,\t \u00C2\u00A0 46 but\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0detrimental\t \u00C2\u00A0to\t \u00C2\u00A0cell\t \u00C2\u00A0survival\t \u00C2\u00A0as\t \u00C2\u00A0they\t \u00C2\u00A0further\t \u00C2\u00A0stress\t \u00C2\u00A0the\t \u00C2\u00A0already\t \u00C2\u00A0dysfunctional\t \u00C2\u00A0UPS\t \u00C2\u00A0system.\t \u00C2\u00A01.6 RATIONALE\t \u00C2\u00A0AND\t \u00C2\u00A0HYPOTHESIS\t \u00C2\u00A0Rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible\t \u00C2\u00A0methods\t \u00C2\u00A0for\t \u00C2\u00A0altering\t \u00C2\u00A0the\t \u00C2\u00A0expression\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0only\t \u00C2\u00A0indispensable\t \u00C2\u00A0tools\t \u00C2\u00A0for\t \u00C2\u00A0studying\t \u00C2\u00A0complex\t \u00C2\u00A0biological\t \u00C2\u00A0systems,\t \u00C2\u00A0but\t \u00C2\u00A0may\t \u00C2\u00A0potentially\t \u00C2\u00A0drive\t \u00C2\u00A0the\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0new\t \u00C2\u00A0therapeutics\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0treatment\t \u00C2\u00A0of\t \u00C2\u00A0human\t \u00C2\u00A0diseases.\t \u00C2\u00A0Techniques\t \u00C2\u00A0that\t \u00C2\u00A0regulate\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0expression\t \u00C2\u00A0by\t \u00C2\u00A0targeting\t \u00C2\u00A0DNA\t \u00C2\u00A0or\t \u00C2\u00A0mRNA\t \u00C2\u00A0have\t \u00C2\u00A0revolutionized\t \u00C2\u00A0our\t \u00C2\u00A0understanding\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0function\t \u00C2\u00A0(Lewandoski,\t \u00C2\u00A02001;\t \u00C2\u00A0Dykxhoorn\t \u00C2\u00A0and\t \u00C2\u00A0Lieberman,\t \u00C2\u00A02005),\t \u00C2\u00A0but\t \u00C2\u00A0are\t \u00C2\u00A0often\t \u00C2\u00A0plagued\t \u00C2\u00A0by\t \u00C2\u00A0problems\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0lack\t \u00C2\u00A0of\t \u00C2\u00A0specificity,\t \u00C2\u00A0speed,\t \u00C2\u00A0and\t \u00C2\u00A0tunability\t \u00C2\u00A0(Banaszynski\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0dearth\t \u00C2\u00A0of\t \u00C2\u00A0efficient\t \u00C2\u00A0systemic\t \u00C2\u00A0delivery\t \u00C2\u00A0systems\t \u00C2\u00A0stymies\t \u00C2\u00A0their\t \u00C2\u00A0use\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0treatment\t \u00C2\u00A0of\t \u00C2\u00A0human\t \u00C2\u00A0diseases.\t \u00C2\u00A0\t \u00C2\u00A0Protein\t \u00C2\u00A0degradation\t \u00C2\u00A0techniques\t \u00C2\u00A0that\t \u00C2\u00A0act\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level\t \u00C2\u00A0have\t \u00C2\u00A0the\t \u00C2\u00A0ability\t \u00C2\u00A0to\t \u00C2\u00A0overcome\t \u00C2\u00A0some\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0limitations\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0knockout,\t \u00C2\u00A0allowing\t \u00C2\u00A0rapid,\t \u00C2\u00A0reversible\t \u00C2\u00A0and\t \u00C2\u00A0graded\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0However,\t \u00C2\u00A0many\t \u00C2\u00A0techniques\t \u00C2\u00A0require\t \u00C2\u00A0the\t \u00C2\u00A0genetic\t \u00C2\u00A0manipulation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0and/or\t \u00C2\u00A0viral\t \u00C2\u00A0infection\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0targeting\t \u00C2\u00A0agents,\t \u00C2\u00A0thus\t \u00C2\u00A0severely\t \u00C2\u00A0constraining\t \u00C2\u00A0their\t \u00C2\u00A0potential\t \u00C2\u00A0for\t \u00C2\u00A0clinical\t \u00C2\u00A0adoption.\t \u00C2\u00A0Ideally,\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0should\t \u00C2\u00A0allow\t \u00C2\u00A0the\t \u00C2\u00A0specific\t \u00C2\u00A0targeting\t \u00C2\u00A0of\t \u00C2\u00A0unmodified,\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0rapid,\t \u00C2\u00A0reversible\t \u00C2\u00A0and\t \u00C2\u00A0controllable\t \u00C2\u00A0manner\t \u00C2\u00A0without\t \u00C2\u00A0increasing\t \u00C2\u00A0stress\t \u00C2\u00A0to\t \u00C2\u00A0cellular\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems.\t \u00C2\u00A0The\t \u00C2\u00A0aim\t \u00C2\u00A0of\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0develop\t \u00C2\u00A0such\t \u00C2\u00A0a\t \u00C2\u00A0method,\t \u00C2\u00A0using\t \u00C2\u00A0peptides\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0agent.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0section,\t \u00C2\u00A0I\t \u00C2\u00A0will\t \u00C2\u00A0explain\t \u00C2\u00A0the\t \u00C2\u00A0 47 rationale\t \u00C2\u00A0behind\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0and\t \u00C2\u00A0outline\t \u00C2\u00A0the\t \u00C2\u00A0steps\t \u00C2\u00A0to\t \u00C2\u00A0evaluate\t \u00C2\u00A0and\t \u00C2\u00A0improve\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0method.\t \u00C2\u00A0\t \u00C2\u00A01.6.1 THE\t \u00C2\u00A0CORE\t \u00C2\u00A0CONSTITUENTS\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0We\t \u00C2\u00A0propose\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0a\t \u00C2\u00A0multi-\u00C2\u00AD\u00E2\u0080\u0090functional\t \u00C2\u00A0peptide\t \u00C2\u00A0containing\t \u00C2\u00A0a\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0(PBD)\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0degron\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0agent.\t \u00C2\u00A0The\t \u00C2\u00A0choice\t \u00C2\u00A0of\t \u00C2\u00A0utilizing\t \u00C2\u00A0peptides\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0small\t \u00C2\u00A0molecules\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0enhance\t \u00C2\u00A0the\t \u00C2\u00A0generalizability\t \u00C2\u00A0and\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0method.\t \u00C2\u00A0Although\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0drugs\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0traditionally\t \u00C2\u00A0favoured\t \u00C2\u00A0over\t \u00C2\u00A0peptidergic\t \u00C2\u00A0agents\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0their\t \u00C2\u00A0excellent\t \u00C2\u00A0pharmacokinetics\t \u00C2\u00A0and\t \u00C2\u00A0stability,\t \u00C2\u00A0their\t \u00C2\u00A0use\t \u00C2\u00A0is\t \u00C2\u00A0limited\t \u00C2\u00A0to\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0which\t \u00C2\u00A0binding\t \u00C2\u00A0pockets\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0found.\t \u00C2\u00A0As\t \u00C2\u00A0illustrated\t \u00C2\u00A0by\t \u00C2\u00A0PROTACs,\t \u00C2\u00A0a\t \u00C2\u00A0small-\u00C2\u00AD\u00E2\u0080\u0090ligand\t \u00C2\u00A0approach\t \u00C2\u00A0greatly\t \u00C2\u00A0limits\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0targeted\t \u00C2\u00A0knockdown\t \u00C2\u00A0(Jang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Carmony\t \u00C2\u00A0and\t \u00C2\u00A0Kim,\t \u00C2\u00A02012).\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0now\t \u00C2\u00A0possible\t \u00C2\u00A0to\t \u00C2\u00A0identify\t \u00C2\u00A0binding\t \u00C2\u00A0domains\t \u00C2\u00A0for\t \u00C2\u00A0any\t \u00C2\u00A0given\t \u00C2\u00A0POI.\t \u00C2\u00A0The\t \u00C2\u00A0simplest\t \u00C2\u00A0method\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0search\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0databases\t \u00C2\u00A0(BIOGRID,\t \u00C2\u00A0Rivas)\t \u00C2\u00A0and/or\t \u00C2\u00A0the\t \u00C2\u00A0literature\t \u00C2\u00A0for\t \u00C2\u00A0well-\u00C2\u00AD\u00E2\u0080\u0090characterized\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0domains,\t \u00C2\u00A0from\t \u00C2\u00A0which\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0designed.\t \u00C2\u00A0For\t \u00C2\u00A0proteins\t \u00C2\u00A0without\t \u00C2\u00A0a\t \u00C2\u00A0known\t \u00C2\u00A0binding\t \u00C2\u00A0partner,\t \u00C2\u00A0a\t \u00C2\u00A0wide\t \u00C2\u00A0array\t \u00C2\u00A0of\t \u00C2\u00A0genetic\t \u00C2\u00A0and\t \u00C2\u00A0cell\t \u00C2\u00A0biology\t \u00C2\u00A0tools\t \u00C2\u00A0exist\t \u00C2\u00A0to\t \u00C2\u00A0identify\t \u00C2\u00A0potential\t \u00C2\u00A0binding\t \u00C2\u00A0candidates,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0yeast\t \u00C2\u00A0two-\u00C2\u00AD\u00E2\u0080\u0090hybrid\t \u00C2\u00A0assay,\t \u00C2\u00A0affinity\t \u00C2\u00A0purification\t \u00C2\u00A0and\t \u00C2\u00A0mass\t \u00C2\u00A0spectrometry,\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090immunoprecipitation\t \u00C2\u00A0and\t \u00C2\u00A0genome-\u00C2\u00AD\u00E2\u0080\u0090wide\t \u00C2\u00A0assays\t \u00C2\u00A0(Nero\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Once\t \u00C2\u00A0a\t \u00C2\u00A0potential\t \u00C2\u00A0binding\t \u00C2\u00A0partner\t \u00C2\u00A0is\t \u00C2\u00A0identified,\t \u00C2\u00A0peptide\t \u00C2\u00A0arrays\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0further\t \u00C2\u00A0identify\t \u00C2\u00A0crucial\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0stretches\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0 48 the\t \u00C2\u00A0interaction\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides.\t \u00C2\u00A0Finally,\t \u00C2\u00A0binding\t \u00C2\u00A0peptides\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0POI\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0rationally\t \u00C2\u00A0designed\t \u00C2\u00A0using\t \u00C2\u00A0available\t \u00C2\u00A0computer\t \u00C2\u00A0frameworks\t \u00C2\u00A0(Grigoryan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Das\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0Notably,\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions\t \u00C2\u00A0often\t \u00C2\u00A0depend\t \u00C2\u00A0on\t \u00C2\u00A0small\t \u00C2\u00A0clusters\t \u00C2\u00A0of\t \u00C2\u00A0key\t \u00C2\u00A0residues\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0binding\t \u00C2\u00A0interface\t \u00C2\u00A0(London\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Mason,\t \u00C2\u00A02010).\t \u00C2\u00A0Peptides\t \u00C2\u00A0designed\t \u00C2\u00A0around\t \u00C2\u00A0these\t \u00C2\u00A0hot\t \u00C2\u00A0spots\t \u00C2\u00A0are\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0highly\t \u00C2\u00A0specific\t \u00C2\u00A0high\t \u00C2\u00A0affinity\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0(Dev,\t \u00C2\u00A02004;\t \u00C2\u00A0London\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0The\t \u00C2\u00A0ability\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0short\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domains\t \u00C2\u00A0decrease\t \u00C2\u00A0the\t \u00C2\u00A0overall\t \u00C2\u00A0length\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0and\t \u00C2\u00A0facilitates\t \u00C2\u00A0its\t \u00C2\u00A0synthesis.\t \u00C2\u00A0\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0ligands,\t \u00C2\u00A0peptidergic\t \u00C2\u00A0binding\t \u00C2\u00A0domains\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0confined\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0limited\t \u00C2\u00A0amount\t \u00C2\u00A0of\t \u00C2\u00A0binding\t \u00C2\u00A0pockets,\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0they\t \u00C2\u00A0can\t \u00C2\u00A0target\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090druggable\t \u00C2\u00A0sites\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0POI.\t \u00C2\u00A0This\t \u00C2\u00A0greatly\t \u00C2\u00A0increases\t \u00C2\u00A0selectivity\t \u00C2\u00A0and\t \u00C2\u00A0is\t \u00C2\u00A0particularly\t \u00C2\u00A0useful\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0case\t \u00C2\u00A0of\t \u00C2\u00A0targeting\t \u00C2\u00A0a\t \u00C2\u00A0member\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0family\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0protein\t \u00C2\u00A0kinases\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0~60%\t \u00C2\u00A0overlap\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0primary\t \u00C2\u00A0sequences\t \u00C2\u00A0have\t \u00C2\u00A0a\t \u00C2\u00A0high\t \u00C2\u00A0probability\t \u00C2\u00A0of\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0small\t \u00C2\u00A0molecule\t \u00C2\u00A0ligand\t \u00C2\u00A0(Vieth\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005),\t \u00C2\u00A0thus\t \u00C2\u00A0prohibiting\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0small\t \u00C2\u00A0molecules\t \u00C2\u00A0in\t \u00C2\u00A0targeting\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0member\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0However,\t \u00C2\u00A0by\t \u00C2\u00A0using\t \u00C2\u00A0peptide\t \u00C2\u00A0ligands\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0sequences\t \u00C2\u00A0unique\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0kinase,\t \u00C2\u00A0it\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0possible\t \u00C2\u00A0to\t \u00C2\u00A0specifically\t \u00C2\u00A0eliminate\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0member\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0family.\t \u00C2\u00A0The\t \u00C2\u00A0second\t \u00C2\u00A0core\t \u00C2\u00A0constituent\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0degron,\t \u00C2\u00A0which\t \u00C2\u00A0recruits\t \u00C2\u00A0components\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0proteolytic\t \u00C2\u00A0system\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0degradation.\t \u00C2\u00A0Multiple\t \u00C2\u00A0degrons\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0identified\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0and\t \u00C2\u00A0ALS.\t \u00C2\u00A0However,\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0existing\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0require\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0proteosome,\t \u00C2\u00A0a\t \u00C2\u00A0CMA-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0degradation\t \u00C2\u00A0system\t \u00C2\u00A0would\t \u00C2\u00A0 49 not\t \u00C2\u00A0only\t \u00C2\u00A0complement\t \u00C2\u00A0current\t \u00C2\u00A0methods,\t \u00C2\u00A0but\t \u00C2\u00A0would\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0especially\t \u00C2\u00A0powerful\t \u00C2\u00A0under\t \u00C2\u00A0pathological\t \u00C2\u00A0conditions\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0is\t \u00C2\u00A0inhibited.\t \u00C2\u00A0Due\t \u00C2\u00A0to\t \u00C2\u00A0these\t \u00C2\u00A0reasons,\t \u00C2\u00A0we\t \u00C2\u00A0opted\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0CTM\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0degron\t \u00C2\u00A0in\t \u00C2\u00A0our\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0approach.\t \u00C2\u00A0Thus,\t \u00C2\u00A0the\t \u00C2\u00A0central\t \u00C2\u00A0mechanism\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting.\t \u00C2\u00A0Specifically,\t \u00C2\u00A0our\t \u00C2\u00A0working\t \u00C2\u00A0hypothesis\t \u00C2\u00A0is:\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide,\t \u00C2\u00A0once\t \u00C2\u00A0introduced\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0(Chapter\t \u00C2\u00A01.6.3),\t \u00C2\u00A0will\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0through\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0via\t \u00C2\u00A0its\t \u00C2\u00A0PBD,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0directs\t \u00C2\u00A0the\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0complex\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0via\t \u00C2\u00A0CMA\t \u00C2\u00A0(Figure\t \u00C2\u00A01-\u00C2\u00AD\u00E2\u0080\u00901).\t \u00C2\u00A01.6.2 OBJECTIVE\t \u00C2\u00A01:\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0PROTEINS\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.4.2.2,\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0mediated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0CTM.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0transferrable\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011b)\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0upregulated\t \u00C2\u00A0in\t \u00C2\u00A0cases\t \u00C2\u00A0whereby\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0is\t \u00C2\u00A0inhibited,\t \u00C2\u00A0we\t \u00C2\u00A0hypothesize\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0peptide\t \u00C2\u00A0containing\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0binding\t \u00C2\u00A0sequence\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0can\t \u00C2\u00A0degrade\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0through\t \u00C2\u00A0CMA,\t \u00C2\u00A0even\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0proteasomal\t \u00C2\u00A0inhibition.\t \u00C2\u00A0To\t \u00C2\u00A0potentially\t \u00C2\u00A0increase\t \u00C2\u00A0degradation\t \u00C2\u00A0efficiency,\t \u00C2\u00A0we\t \u00C2\u00A0designed\t \u00C2\u00A0a\t \u00C2\u00A0composite\t \u00C2\u00A0CTM\t \u00C2\u00A0(KFERQKILDQRFFE)\t \u00C2\u00A0from\t \u00C2\u00A0three\t \u00C2\u00A0natural\t \u00C2\u00A0CTMs\t \u00C2\u00A0identified\t \u00C2\u00A0from\t \u00C2\u00A0RNase\t \u00C2\u00A0A\t \u00C2\u00A0(KFERQ)\t \u00C2\u00A0(Backer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01983),\t \u00C2\u00A0hsc70\t \u00C2\u00A0(QKILD)\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000a)\t \u00C2\u00A0and\t \u00C2\u00A0hemoglobin\t \u00C2\u00A0(QRFFE)\t \u00C2\u00A0(Slot\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01986).\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0aim\t \u00C2\u00A0we\t \u00C2\u00A0will\t \u00C2\u00A0use\t \u00C2\u00A0a\t \u00C2\u00A0recombinant\t \u00C2\u00A0protein\t \u00C2\u00A0system\t \u00C2\u00A0to\t \u00C2\u00A0provide\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0that\t \u00C2\u00A01)\t \u00C2\u00A0direct\t \u00C2\u00A0 50 tagging\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0renders\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0metabolically\t \u00C2\u00A0unstable\t \u00C2\u00A0and\t \u00C2\u00A02)\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0can\t \u00C2\u00A0direct\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0POI\t \u00C2\u00A0for\t \u00C2\u00A0CMA\t \u00C2\u00A0degradation\t \u00C2\u00A0via\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction.\t \u00C2\u00A0\t \u00C2\u00A01.6.3 OBJECTIVE\t \u00C2\u00A02:\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0PROTEINS\t \u00C2\u00A0IN\t \u00C2\u00A0PRIMARY\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CULTURES\t \u00C2\u00A0The\t \u00C2\u00A0blood-\u00C2\u00AD\u00E2\u0080\u0090brain\t \u00C2\u00A0barrier\t \u00C2\u00A0(BBB)\t \u00C2\u00A0and\t \u00C2\u00A0cellular\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane\t \u00C2\u00A0pose\t \u00C2\u00A0a\t \u00C2\u00A0challenge\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0intracellular\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0macromolecules.\t \u00C2\u00A0Previous\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0have\t \u00C2\u00A0circumvented\t \u00C2\u00A0this\t \u00C2\u00A0problem\t \u00C2\u00A0by\t \u00C2\u00A0using\t \u00C2\u00A0viral\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0their\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0agents.\t \u00C2\u00A0While\t \u00C2\u00A0effective,\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0viral\t \u00C2\u00A0vectors\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0only\t \u00C2\u00A0technically\t \u00C2\u00A0cumbersome\t \u00C2\u00A0but\t \u00C2\u00A0also\t \u00C2\u00A0limits\t \u00C2\u00A0their\t \u00C2\u00A0use\t \u00C2\u00A0as\t \u00C2\u00A0therapeutics.\t \u00C2\u00A0Cell-\u00C2\u00AD\u00E2\u0080\u0090penetrating\t \u00C2\u00A0peptides\t \u00C2\u00A0(CPPs)\t \u00C2\u00A0are\t \u00C2\u00A0a\t \u00C2\u00A0family\t \u00C2\u00A0of\t \u00C2\u00A0membrane-\u00C2\u00AD\u00E2\u0080\u0090permeant\t \u00C2\u00A0peptides\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0delivering\t \u00C2\u00A0biologically\t \u00C2\u00A0active\t \u00C2\u00A0cargo\t \u00C2\u00A0across\t \u00C2\u00A0the\t \u00C2\u00A0BBB\t \u00C2\u00A0and\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane\t \u00C2\u00A0of\t \u00C2\u00A0cells\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0highly\t \u00C2\u00A0efficient\t \u00C2\u00A0manner\t \u00C2\u00A0(Morris\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0There\t \u00C2\u00A0are\t \u00C2\u00A0two\t \u00C2\u00A0main\t \u00C2\u00A0classes\t \u00C2\u00A0of\t \u00C2\u00A0CPPs\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0their\t \u00C2\u00A0physical\t \u00C2\u00A0chemical\t \u00C2\u00A0properties\t \u00C2\u00A0and\t \u00C2\u00A0internalization\t \u00C2\u00A0mechanisms.\t \u00C2\u00A0The\t \u00C2\u00A0first\t \u00C2\u00A0class\t \u00C2\u00A0is\t \u00C2\u00A0amphipathic\t \u00C2\u00A0CPPs,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0Pep-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0(Morris\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001),\t \u00C2\u00A0that\t \u00C2\u00A0encapsulate\t \u00C2\u00A0their\t \u00C2\u00A0cargo\t \u00C2\u00A0through\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090covalent\t \u00C2\u00A0bonds\t \u00C2\u00A0and\t \u00C2\u00A0penetrate\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0by\t \u00C2\u00A0forming\t \u00C2\u00A0a\t \u00C2\u00A0transient\t \u00C2\u00A0transmembrane\t \u00C2\u00A0pore-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0structure\t \u00C2\u00A0(Deshayes\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004).\t \u00C2\u00A0The\t \u00C2\u00A0second\t \u00C2\u00A0class\t \u00C2\u00A0is\t \u00C2\u00A0polycationic\t \u00C2\u00A0CPPs,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0TAT\t \u00C2\u00A0(Viv\u00C3\u00A8s\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01997),\t \u00C2\u00A0which\t \u00C2\u00A0are\t \u00C2\u00A0directly\t \u00C2\u00A0attached\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0cargo\t \u00C2\u00A0through\t \u00C2\u00A0covalent\t \u00C2\u00A0bounds\t \u00C2\u00A0and\t \u00C2\u00A0enter\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0via\t \u00C2\u00A0direct\t \u00C2\u00A0penetration\t \u00C2\u00A0and/or\t \u00C2\u00A0endocytosis\t \u00C2\u00A0(Gump\t \u00C2\u00A0and\t \u00C2\u00A0Dowdy,\t \u00C2\u00A02007).\t \u00C2\u00A0Both\t \u00C2\u00A0classes\t \u00C2\u00A0of\t \u00C2\u00A0CPPs\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0successfully\t \u00C2\u00A0 51 utilized\t \u00C2\u00A0to\t \u00C2\u00A0deliver\t \u00C2\u00A0a\t \u00C2\u00A0wide\t \u00C2\u00A0range\t \u00C2\u00A0of\t \u00C2\u00A0biologically\t \u00C2\u00A0active\t \u00C2\u00A0macromolecules\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0(Morris\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01999;\t \u00C2\u00A0Lim\t \u00C2\u00A0and\t \u00C2\u00A0Gleeson,\t \u00C2\u00A02011;\t \u00C2\u00A0Koren\t \u00C2\u00A0and\t \u00C2\u00A0Torchilin,\t \u00C2\u00A02012).\t \u00C2\u00A0TAT\t \u00C2\u00A0is\t \u00C2\u00A0by\t \u00C2\u00A0far\t \u00C2\u00A0the\t \u00C2\u00A0best-\u00C2\u00AD\u00E2\u0080\u0090characterized\t \u00C2\u00A0CPP.\t \u00C2\u00A0This\t \u00C2\u00A0sequence\t \u00C2\u00A0is\t \u00C2\u00A0derived\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0membrane\t \u00C2\u00A0transduction\t \u00C2\u00A0domain\t \u00C2\u00A0(47-\u00C2\u00AD\u00E2\u0080\u009057)\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0HIV\t \u00C2\u00A01\t \u00C2\u00A0Tat\t \u00C2\u00A0protein\t \u00C2\u00A0after\t \u00C2\u00A0it\t \u00C2\u00A0was\t \u00C2\u00A0found\t \u00C2\u00A0that\t \u00C2\u00A0full\t \u00C2\u00A0length\t \u00C2\u00A0Tat\t \u00C2\u00A0could\t \u00C2\u00A0be\t \u00C2\u00A0readily\t \u00C2\u00A0taken\t \u00C2\u00A0up\t \u00C2\u00A0by\t \u00C2\u00A0cells\t \u00C2\u00A0(Viv\u00C3\u00A8s\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01997).\t \u00C2\u00A0Since\t \u00C2\u00A0then,\t \u00C2\u00A0TAT\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0repeatedly\t \u00C2\u00A0demonstrated\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0safe\t \u00C2\u00A0and\t \u00C2\u00A0effective\t \u00C2\u00A0at\t \u00C2\u00A0delivering\t \u00C2\u00A0active\t \u00C2\u00A0biomolecules\t \u00C2\u00A0across\t \u00C2\u00A0the\t \u00C2\u00A0BBB\t \u00C2\u00A0and\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane\t \u00C2\u00A0in\t \u00C2\u00A0animals\t \u00C2\u00A0(Schwarze,\t \u00C2\u00A01999;\t \u00C2\u00A0Ahmadian\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004;\t \u00C2\u00A0Brebner,\t \u00C2\u00A02005;\t \u00C2\u00A0Wong\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02007;\t \u00C2\u00A0Taghibiglou\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Most\t \u00C2\u00A0importantly,\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0TAT\t \u00C2\u00A0in\t \u00C2\u00A0peptidergic\t \u00C2\u00A0therapeutics\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0validated\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0recent\t \u00C2\u00A0successful\t \u00C2\u00A0phase\t \u00C2\u00A02B\t \u00C2\u00A0clinical\t \u00C2\u00A0trial\t \u00C2\u00A0(Hill\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012),\t \u00C2\u00A0with\t \u00C2\u00A0phase\t \u00C2\u00A03\t \u00C2\u00A0in\t \u00C2\u00A0progress.\t \u00C2\u00A0\t \u00C2\u00A0Based\t \u00C2\u00A0 on\t \u00C2\u00A0 the\t \u00C2\u00A0 demonstrated\t \u00C2\u00A0 delivery\t \u00C2\u00A0 efficacy\t \u00C2\u00A0 of\t \u00C2\u00A0 CPPs,\t \u00C2\u00A0 we\t \u00C2\u00A0 hypothesize\t \u00C2\u00A0 that\t \u00C2\u00A0 SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0easily\t \u00C2\u00A0delivered\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0interior\t \u00C2\u00A0of\t \u00C2\u00A0primary\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0CPP,\t \u00C2\u00A0thus\t \u00C2\u00A0circumventing\t \u00C2\u00A0the\t \u00C2\u00A0need\t \u00C2\u00A0for\t \u00C2\u00A0viral\t \u00C2\u00A0infection.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0aim\t \u00C2\u00A0we\t \u00C2\u00A0will\t \u00C2\u00A0also\t \u00C2\u00A0examine\t \u00C2\u00A0the\t \u00C2\u00A0characteristics\t \u00C2\u00A0 of\t \u00C2\u00A0 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0 degradation\t \u00C2\u00A0 of\t \u00C2\u00A0 native\t \u00C2\u00A0 proteins,\t \u00C2\u00A0 including\t \u00C2\u00A0 time-\u00C2\u00AD\u00E2\u0080\u0090and\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependency,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0reversibility\t \u00C2\u00A0and\t \u00C2\u00A0toxicity.\t \u00C2\u00A01.6.4 OBJECTIVE\t \u00C2\u00A03:\t \u00C2\u00A0FUNCTIONAL\t \u00C2\u00A0CONSEQUENCES\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IN\t \u00C2\u00A0VITRO\t \u00C2\u00A0AND\t \u00C2\u00A0IN\t \u00C2\u00A0VIVO\t \u00C2\u00A0A\t \u00C2\u00A0major\t \u00C2\u00A0challenge\t \u00C2\u00A0in\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0technology\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0central\t \u00C2\u00A0nervous\t \u00C2\u00A0system\t \u00C2\u00A0(CNS)\t \u00C2\u00A0for\t \u00C2\u00A0rapid\t \u00C2\u00A0and\t \u00C2\u00A0controllable\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0animal\t \u00C2\u00A0models\t \u00C2\u00A0of\t \u00C2\u00A0human\t \u00C2\u00A0diseases.\t \u00C2\u00A0Such\t \u00C2\u00A0a\t \u00C2\u00A0method\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0especially\t \u00C2\u00A0useful\t \u00C2\u00A0for\t \u00C2\u00A0targeting\t \u00C2\u00A0 52 pathogenic\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0disorders\t \u00C2\u00A0or\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090inducing\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0brain\t \u00C2\u00A0injuries\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0Ischemic\t \u00C2\u00A0stroke,\t \u00C2\u00A0for\t \u00C2\u00A0example,\t \u00C2\u00A0activates\t \u00C2\u00A0multiple\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0signaling\t \u00C2\u00A0pathways,\t \u00C2\u00A0eventually\t \u00C2\u00A0leading\t \u00C2\u00A0to\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A0and\t \u00C2\u00A0loss\t \u00C2\u00A0of\t \u00C2\u00A0brain\t \u00C2\u00A0function\t \u00C2\u00A0(Lai\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0By\t \u00C2\u00A0elucidating\t \u00C2\u00A0the\t \u00C2\u00A0molecular\t \u00C2\u00A0cascades\t \u00C2\u00A0that\t \u00C2\u00A0precede\t \u00C2\u00A0irreversible\t \u00C2\u00A0cell\t \u00C2\u00A0death,\t \u00C2\u00A0multiple\t \u00C2\u00A0key\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090signaling\t \u00C2\u00A0kinases\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0identified\t \u00C2\u00A0as\t \u00C2\u00A0viable\t \u00C2\u00A0targets\t \u00C2\u00A0for\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0interventions\t \u00C2\u00A0(Osuga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02000;\t \u00C2\u00A0Bright\t \u00C2\u00A0and\t \u00C2\u00A0Mochly-\u00C2\u00AD\u00E2\u0080\u0090Rosen,\t \u00C2\u00A02005;\t \u00C2\u00A0Zhuang\t \u00C2\u00A0and\t \u00C2\u00A0Schnellmann,\t \u00C2\u00A02006;\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Coffey,\t \u00C2\u00A02014;\t \u00C2\u00A0Coultrap\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0Pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0kinases\t \u00C2\u00A0are\t \u00C2\u00A0particularly\t \u00C2\u00A0interesting\t \u00C2\u00A0drug\t \u00C2\u00A0targets\t \u00C2\u00A0as\t \u00C2\u00A0their\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0dependent\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090state;\t \u00C2\u00A0that\t \u00C2\u00A0is,\t \u00C2\u00A0they\t \u00C2\u00A0are\t \u00C2\u00A0normally\t \u00C2\u00A0inactive\t \u00C2\u00A0but\t \u00C2\u00A0activate\t \u00C2\u00A0following\t \u00C2\u00A0cytotoxic\t \u00C2\u00A0insults\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0(Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0kinases\t \u00C2\u00A0often\t \u00C2\u00A0serve\t \u00C2\u00A0as\t \u00C2\u00A0convergence\t \u00C2\u00A0points\t \u00C2\u00A0for\t \u00C2\u00A0different\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090inducing\t \u00C2\u00A0pathways\t \u00C2\u00A0(Hara\t \u00C2\u00A0and\t \u00C2\u00A0Snyder,\t \u00C2\u00A02007;\t \u00C2\u00A0Pei\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Accordingly,\t \u00C2\u00A0inhibiting\t \u00C2\u00A0the\t \u00C2\u00A0activity\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0kinase\t \u00C2\u00A0blocks\t \u00C2\u00A0multiple\t \u00C2\u00A0neurotoxic\t \u00C2\u00A0pathways\t \u00C2\u00A0and\t \u00C2\u00A0is\t \u00C2\u00A0thereby\t \u00C2\u00A0an\t \u00C2\u00A0attractive\t \u00C2\u00A0strategy\t \u00C2\u00A0for\t \u00C2\u00A0neuroprotection.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0increasing\t \u00C2\u00A0evidence\t \u00C2\u00A0shows\t \u00C2\u00A0that\t \u00C2\u00A0many\t \u00C2\u00A0kinases\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0acute\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A0also\t \u00C2\u00A0participate\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0gradual\t \u00C2\u00A0neuronal\t \u00C2\u00A0loss\t \u00C2\u00A0seen\t \u00C2\u00A0in\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0diseases\t \u00C2\u00A0(Flight,\t \u00C2\u00A02013;\t \u00C2\u00A0Hilgeroth,\t \u00C2\u00A02013;\t \u00C2\u00A0Coffey,\t \u00C2\u00A02014),\t \u00C2\u00A0making\t \u00C2\u00A0them\t \u00C2\u00A0excellent\t \u00C2\u00A0candidates\t \u00C2\u00A0for\t \u00C2\u00A0intervention.\t \u00C2\u00A0Unfortunately,\t \u00C2\u00A0most\t \u00C2\u00A0kinases\t \u00C2\u00A0are\t \u00C2\u00A0undruggable\t \u00C2\u00A0by\t \u00C2\u00A0conventional\t \u00C2\u00A0small\t \u00C2\u00A0molecules\t \u00C2\u00A0(Crews,\t \u00C2\u00A02010).\t \u00C2\u00A0However,\t \u00C2\u00A0as\t \u00C2\u00A0many\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0kinases\t \u00C2\u00A0have\t \u00C2\u00A0known\t \u00C2\u00A0binding\t \u00C2\u00A0partners\t \u00C2\u00A0with\t \u00C2\u00A0clearly\t \u00C2\u00A0defined\t \u00C2\u00A0interaction\t \u00C2\u00A0sites,\t \u00C2\u00A0it\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0possible\t \u00C2\u00A0to\t \u00C2\u00A0engineer\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0activated\t \u00C2\u00A0kinases\t \u00C2\u00A0for\t \u00C2\u00A0CMA\t \u00C2\u00A0degradation\t \u00C2\u00A0and\t \u00C2\u00A0thereby\t \u00C2\u00A0prevent\t \u00C2\u00A0cell\t \u00C2\u00A0death.\t \u00C2\u00A0\t \u00C2\u00A0 53 Recent\t \u00C2\u00A0efforts\t \u00C2\u00A0have\t \u00C2\u00A0identified\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0kinase\t \u00C2\u00A01\t \u00C2\u00A0(DAPK1)\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0key\t \u00C2\u00A0mediator\t \u00C2\u00A0of\t \u00C2\u00A0ischemic\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0(Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005;\t \u00C2\u00A0Hainsworth\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Following\t \u00C2\u00A0an\t \u00C2\u00A0ischemic\t \u00C2\u00A0insult,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activates\t \u00C2\u00A0and\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0subunit\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090Methyl\t \u00C2\u00A0D-\u00C2\u00AD\u00E2\u0080\u0090Aspartate\t \u00C2\u00A0Receptor\t \u00C2\u00A0(NMDAR)\t \u00C2\u00A0and\t \u00C2\u00A0enhances\t \u00C2\u00A0NMDAR-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0currents\t \u00C2\u00A0and\t \u00C2\u00A0excitotoxicity\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0also\t \u00C2\u00A0inhibits\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090survival\t \u00C2\u00A0pathways\t \u00C2\u00A0by\t \u00C2\u00A0sequestering\t \u00C2\u00A0ERK\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cytoplasm,\t \u00C2\u00A0thereby\t \u00C2\u00A0inhibiting\t \u00C2\u00A0the\t \u00C2\u00A0nuclear\t \u00C2\u00A0translocation\t \u00C2\u00A0of\t \u00C2\u00A0ERK\t \u00C2\u00A0and\t \u00C2\u00A0subsequent\t \u00C2\u00A0transcription\t \u00C2\u00A0of\t \u00C2\u00A0survival\t \u00C2\u00A0genes\t \u00C2\u00A0(Chen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0In\t \u00C2\u00A0addition,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0inhibit\t \u00C2\u00A0the\t \u00C2\u00A0Akt\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090survival\t \u00C2\u00A0pathway\t \u00C2\u00A0via\t \u00C2\u00A0its\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0and\t \u00C2\u00A0inactivation\t \u00C2\u00A0of\t \u00C2\u00A0CaMKK\t \u00C2\u00A0(Schumacher\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004),\t \u00C2\u00A0the\t \u00C2\u00A0latter\t \u00C2\u00A0of\t \u00C2\u00A0which\t \u00C2\u00A0activates\t \u00C2\u00A0Akt\t \u00C2\u00A0(Yano\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01998).\t \u00C2\u00A0\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0also\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0mediating\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0through\t \u00C2\u00A0non\t \u00C2\u00A0NMDAR-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0pathways,\t \u00C2\u00A0including\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0JNK\t \u00C2\u00A0pathway\t \u00C2\u00A0(Eisenberg-\u00C2\u00AD\u00E2\u0080\u0090Lerner\t \u00C2\u00A0and\t \u00C2\u00A0Kimchi,\t \u00C2\u00A02007)\t \u00C2\u00A0and\t \u00C2\u00A0inflammation\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0formation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0NLRP3\t \u00C2\u00A0inflammasome\t \u00C2\u00A0and\t \u00C2\u00A0generation\t \u00C2\u00A0of\t \u00C2\u00A0IL-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0(Turner-\u00C2\u00AD\u00E2\u0080\u0090Brannen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Yang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0aberrant\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activation\t \u00C2\u00A0has\t \u00C2\u00A0also\t \u00C2\u00A0been\t \u00C2\u00A0show\t \u00C2\u00A0to\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage\t \u00C2\u00A0in\t \u00C2\u00A0epilepsy\t \u00C2\u00A0(Henshall\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Araki\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004;\t \u00C2\u00A0Henshall\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004)\t \u00C2\u00A0and\t \u00C2\u00A0Alzheimer\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0(Kim\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014),\t \u00C2\u00A0and\t \u00C2\u00A0knocking\t \u00C2\u00A0out\t \u00C2\u00A0DAPK1\t \u00C2\u00A0confers\t \u00C2\u00A0resistance\t \u00C2\u00A0to\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0from\t \u00C2\u00A0ER\t \u00C2\u00A0stress\t \u00C2\u00A0(Gozuacik\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008)\t \u00C2\u00A0or\t \u00C2\u00A0ceramide\t \u00C2\u00A0(Pelled\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002).\t \u00C2\u00A0For\t \u00C2\u00A0a\t \u00C2\u00A0review\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0role\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0cell\t \u00C2\u00A0death,\t \u00C2\u00A0see\t \u00C2\u00A0(Fujita\t \u00C2\u00A0and\t \u00C2\u00A0Yamashita,\t \u00C2\u00A02014).\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0aim\t \u00C2\u00A0we\t \u00C2\u00A0will\t \u00C2\u00A0first\t \u00C2\u00A0determine\t \u00C2\u00A0the\t \u00C2\u00A0feasibility\t \u00C2\u00A0and\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0method\t \u00C2\u00A0in\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0vivo.\t \u00C2\u00A0We\t \u00C2\u00A0will\t \u00C2\u00A0next\t \u00C2\u00A0examine\t \u00C2\u00A0the\t \u00C2\u00A0phenotypic\t \u00C2\u00A0outcome\t \u00C2\u00A0of\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0cellular\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0and\t \u00C2\u00A0an\t \u00C2\u00A0animal\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0stroke.\t \u00C2\u00A0Given\t \u00C2\u00A0 54 the\t \u00C2\u00A0crucial\t \u00C2\u00A0role\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0mediating\t \u00C2\u00A0stroke-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neuronal\t \u00C2\u00A0death,\t \u00C2\u00A0we\t \u00C2\u00A0hypothesize\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0active\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0vivo.\t \u00C2\u00A0These\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0experiments\t \u00C2\u00A0will\t \u00C2\u00A0facilitate\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0novel\t \u00C2\u00A0therapeutics\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0pathogenic\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0vivo.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 55 Table\t \u00C2\u00A01-\u00C2\u00AD\u00E2\u0080\u00901.\t \u00C2\u00A0Post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0Strategy\t \u00C2\u00A0 Mechanism\t \u00C2\u00A0 Organism\t \u00C2\u00A0 Regulation\t \u00C2\u00A0 Reference\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0rule\t \u00C2\u00A0\t \u00C2\u00A0 Arg-\u00C2\u00AD\u00E2\u0080\u0090DHFRts\t \u00C2\u00A0 Yeast\t \u00C2\u00A0 Yes\t \u00C2\u00A0 Dohmen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01994\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0rule\t \u00C2\u00A0 Arg-\u00C2\u00AD\u00E2\u0080\u0090DHFR*\t \u00C2\u00A0 Mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0Yes,\t \u00C2\u00A0MTX\t \u00C2\u00A0 L\u00C3\u00A9vy\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01999\t \u00C2\u00A0E2\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0E2-\u00C2\u00AD\u00E2\u0080\u0090PBD\t \u00C2\u00A0tethers\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0E2\t \u00C2\u00A0Cell\t \u00C2\u00A0extracts\t \u00C2\u00A0 No\t \u00C2\u00A0 Gosink\t \u00C2\u00A0and\t \u00C2\u00A0Vierstra,\t \u00C2\u00A01995\t \u00C2\u00A0E3\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0F\t \u00C2\u00A0box-\u00C2\u00AD\u00E2\u0080\u0090PBD\t \u00C2\u00A0tethers\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0SCF\u00CE\u00B2TrCP\t \u00C2\u00A0\t \u00C2\u00A0Yeast,\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0No\t \u00C2\u00A0 Zhou\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02000;\t \u00C2\u00A0Su\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Cong\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Liu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004;\t \u00C2\u00A0Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003 E3\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0Ubiquibodies\t \u00C2\u00A0target\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0CHIP\t \u00C2\u00A0Mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0No\t \u00C2\u00A0 Portnoff\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014\t \u00C2\u00A0 56 Strategy\t \u00C2\u00A0 Mechanism\t \u00C2\u00A0 Organism\t \u00C2\u00A0 Regulation\t \u00C2\u00A0 Reference\t \u00C2\u00A0E3\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0Bi-\u00C2\u00AD\u00E2\u0080\u0090functional\t \u00C2\u00A0molecule\t \u00C2\u00A0with\t \u00C2\u00A0PBD\t \u00C2\u00A0tethers\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0cIAP\t \u00C2\u00A0\t \u00C2\u00A0Mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0No\t \u00C2\u00A0 Itoh\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A02011;\t \u00C2\u00A02012\t \u00C2\u00A0E3\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0AID\t \u00C2\u00A0tethers\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0SCFTIR1\t \u00C2\u00A0\t \u00C2\u00A0Yeast,\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0Yes,\t \u00C2\u00A0auxin\t \u00C2\u00A0\t \u00C2\u00A0 Nishimura\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009\t \u00C2\u00A0E3\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0SCFNSmlb\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090vhhGFP4\t \u00C2\u00A0targets\t \u00C2\u00A0GFP-\u00C2\u00AD\u00E2\u0080\u0090POI\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0Drosophila,\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0No\t \u00C2\u00A0 Caussinus\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011\t \u00C2\u00A0E3\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0PROTAC-\u00C2\u00AD\u00E2\u0080\u0090ligand\t \u00C2\u00A0tethers\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0E3\t \u00C2\u00A0Cell\t \u00C2\u00A0extracts,\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0No\t \u00C2\u00A0 Sakamoto\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001,\t \u00C2\u00A02003;\t \u00C2\u00A0Cyrus\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A02010;\t \u00C2\u00A0Hines\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013\t \u00C2\u00A0Proteasome\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0POI-\u00C2\u00AD\u00E2\u0080\u0090FRB\t \u00C2\u00A0and\t \u00C2\u00A0proteasome-\u00C2\u00AD\u00E2\u0080\u0090FKBP\t \u00C2\u00A0dimerization\t \u00C2\u00A0Yeast\t \u00C2\u00A0 Yes,\t \u00C2\u00A0rapamycin\t \u00C2\u00A0\t \u00C2\u00A0Janse,\t \u00C2\u00A02004b\t \u00C2\u00A0 57 Strategy\t \u00C2\u00A0 Mechanism\t \u00C2\u00A0 Organism\t \u00C2\u00A0 Regulation\t \u00C2\u00A0 Reference\t \u00C2\u00A0Proteasome\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0ODC-\u00C2\u00AD\u00E2\u0080\u0090PBD\t \u00C2\u00A0directs\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0proteasome\t \u00C2\u00A0Mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0Yes,\t \u00C2\u00A0antizyme\t \u00C2\u00A0\t \u00C2\u00A0Matsuzawa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005\t \u00C2\u00A0Destabilizing\t \u00C2\u00A0domain\t \u00C2\u00A0POI-\u00C2\u00AD\u00E2\u0080\u0090destabilizing\t \u00C2\u00A0domain\t \u00C2\u00A0Mammalian\t \u00C2\u00A0cells,\t \u00C2\u00A0animals\t \u00C2\u00A0Yes\t \u00C2\u00A0(MaRap,\t \u00C2\u00A0Shield-\u00C2\u00AD\u00E2\u0080\u00901,\t \u00C2\u00A0TMP\t \u00C2\u00A0or\t \u00C2\u00A0CMP8)\t \u00C2\u00A0\t \u00C2\u00A0Stankunas\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Banaszynski\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006;\t \u00C2\u00A02008;\t \u00C2\u00A0Iwamoto\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Miyazaki\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012\t \u00C2\u00A0Destabilizing\t \u00C2\u00A0domain\t \u00C2\u00A0POI-\u00C2\u00AD\u00E2\u0080\u0090cryptic\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domain\t \u00C2\u00A0(LID)\t \u00C2\u00A0Mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0Yes\t \u00C2\u00A0(Shield-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0or\t \u00C2\u00A0light)\t \u00C2\u00A0\t \u00C2\u00A0Bonger\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A02014\t \u00C2\u00A0Increase\t \u00C2\u00A0hydrophobicity\t \u00C2\u00A0HaloTag-\u00C2\u00AD\u00E2\u0080\u0090POI\t \u00C2\u00A0 Mammalian\t \u00C2\u00A0cells,\t \u00C2\u00A0mice\t \u00C2\u00A0Yes,\t \u00C2\u00A0HyT\t \u00C2\u00A0or\t \u00C2\u00A0HALTS1\t \u00C2\u00A0\t \u00C2\u00A0Neklesa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A02013\t \u00C2\u00A0CMA\t \u00C2\u00A0 Adaptor\t \u00C2\u00A0tethers\t \u00C2\u00A0mHtt\t \u00C2\u00A0(N-\u00C2\u00AD\u00E2\u0080\u0090term\t \u00C2\u00A090aa)\t \u00C2\u00A0to\t \u00C2\u00A0lysosome\t \u00C2\u00A0Mammalian\t \u00C2\u00A0cells,\t \u00C2\u00A0mice\t \u00C2\u00A0No\t \u00C2\u00A0 Bauer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010\t \u00C2\u00A0\t \u00C2\u00A0 58 Figure\t \u00C2\u00A01-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0Schematic\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090peptide\t \u00C2\u00A0mediated\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Schematic\t \u00C2\u00A0diagram\t \u00C2\u00A0illustrating\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090peptide\t \u00C2\u00A0mediated\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0Following\t \u00C2\u00A0administration,\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0enters\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0through\t \u00C2\u00A0its\t \u00C2\u00A0cell\t \u00C2\u00A0penetrating\t \u00C2\u00A0peptide\t \u00C2\u00A0(CPP)\t \u00C2\u00A0domain,\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0via\t \u00C2\u00A0its\t \u00C2\u00A0protein\t \u00C2\u00A0binding\t \u00C2\u00A0domain\t \u00C2\u00A0(PBD),\t \u00C2\u00A0and\t \u00C2\u00A0chaperones\t \u00C2\u00A0the\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0complex\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0via\t \u00C2\u00A0its\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0targeting\t \u00C2\u00A0motif\t \u00C2\u00A0(CTM).\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0and\t \u00C2\u00A0Wang,\t \u00C2\u00A0Current\t \u00C2\u00A0Protocols\t \u00C2\u00A0in\t \u00C2\u00A0Chemical\t \u00C2\u00A0Biology\t \u00C2\u00A02015)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 59 CHAPTER\t \u00C2\u00A02: MATERIALS\t \u00C2\u00A0AND\t \u00C2\u00A0METHODS\t \u00C2\u00A02.1 CELL\t \u00C2\u00A0CULTURE\t \u00C2\u00A02.1.1 HUMAN\t \u00C2\u00A0EMBRYONIC\t \u00C2\u00A0KIDNEY\t \u00C2\u00A0293\t \u00C2\u00A0(HEK\t \u00C2\u00A0293)\t \u00C2\u00A0CELL\t \u00C2\u00A0MAINTENANCE\t \u00C2\u00A0The\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cell\t \u00C2\u00A0line\t \u00C2\u00A0was\t \u00C2\u00A0maintained\t \u00C2\u00A0in\t \u00C2\u00A010cm\t \u00C2\u00A0Petri\t \u00C2\u00A0dishes\t \u00C2\u00A0containing\t \u00C2\u00A0Dulbecco\u00E2\u0080\u0099s\t \u00C2\u00A0Modified\t \u00C2\u00A0Eagle\t \u00C2\u00A0Medium\t \u00C2\u00A0(DMEM;\t \u00C2\u00A0Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#10566-\u00C2\u00AD\u00E2\u0080\u0090016)\t \u00C2\u00A0supplemented\t \u00C2\u00A0with\t \u00C2\u00A010%\t \u00C2\u00A0fetal\t \u00C2\u00A0bovine\t \u00C2\u00A0serum\t \u00C2\u00A0(FBS;\t \u00C2\u00A0Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#10099-\u00C2\u00AD\u00E2\u0080\u0090141)\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0incubator\t \u00C2\u00A0set\t \u00C2\u00A0at\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0with\t \u00C2\u00A05%\t \u00C2\u00A0CO2.\t \u00C2\u00A0The\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0monitored\t \u00C2\u00A0daily\t \u00C2\u00A0and\t \u00C2\u00A0medium\t \u00C2\u00A0was\t \u00C2\u00A0changed\t \u00C2\u00A0every\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0days.\t \u00C2\u00A0\t \u00C2\u00A0Once\t \u00C2\u00A0cells\t \u00C2\u00A0reached\t \u00C2\u00A080%\t \u00C2\u00A0confluence\t \u00C2\u00A0they\t \u00C2\u00A0were\t \u00C2\u00A0passaged\t \u00C2\u00A0into\t \u00C2\u00A0new\t \u00C2\u00A0dishes\t \u00C2\u00A0to\t \u00C2\u00A0sustain\t \u00C2\u00A0growth.\t \u00C2\u00A0Briefly,\t \u00C2\u00A0after\t \u00C2\u00A0removal\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0medium,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0incubated\t \u00C2\u00A0with\t \u00C2\u00A03mL\t \u00C2\u00A0Trypsin\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#25200-\u00C2\u00AD\u00E2\u0080\u0090072)\t \u00C2\u00A0in\t \u00C2\u00A0DMEM\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0incubator\t \u00C2\u00A0until\t \u00C2\u00A0they\t \u00C2\u00A0detached\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0bottom\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0dish.\t \u00C2\u00A0Trypsin\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0neutralized\t \u00C2\u00A0with\t \u00C2\u00A03mL\t \u00C2\u00A0complete\t \u00C2\u00A0growth\t \u00C2\u00A0media,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0pelleted\t \u00C2\u00A0by\t \u00C2\u00A0centrifugation\t \u00C2\u00A0at\t \u00C2\u00A01,000rpm\t \u00C2\u00A0for\t \u00C2\u00A03.5min\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0old\t \u00C2\u00A0medium\t \u00C2\u00A0removed.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090suspended\t \u00C2\u00A0in\t \u00C2\u00A07mL\t \u00C2\u00A0fresh\t \u00C2\u00A0complete\t \u00C2\u00A0growth\t \u00C2\u00A0media\t \u00C2\u00A0by\t \u00C2\u00A0pipetting\t \u00C2\u00A0up-\u00C2\u00AD\u00E2\u0080\u0090and-\u00C2\u00AD\u00E2\u0080\u0090down\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A05mL\t \u00C2\u00A0pipette.\t \u00C2\u00A0Finally,\t \u00C2\u00A01mL\t \u00C2\u00A0of\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090suspended\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0was\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A010cm\t \u00C2\u00A0dish\t \u00C2\u00A0containing\t \u00C2\u00A010mL\t \u00C2\u00A0fresh\t \u00C2\u00A0complete\t \u00C2\u00A0growth\t \u00C2\u00A0medium\t \u00C2\u00A0for\t \u00C2\u00A0further\t \u00C2\u00A0maintenance,\t \u00C2\u00A0and\t \u00C2\u00A00.5mL\t \u00C2\u00A0of\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0each\t \u00C2\u00A0well\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A06-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plate\t \u00C2\u00A0for\t \u00C2\u00A0transfection\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0day.\t \u00C2\u00A0All\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0returned\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0incubator\t \u00C2\u00A0and\t \u00C2\u00A0further\t \u00C2\u00A0monitored\t \u00C2\u00A0for\t \u00C2\u00A0confluency\t \u00C2\u00A0and\t \u00C2\u00A0health.\t \u00C2\u00A0 60 2.1.2 TRANSFECTION\t \u00C2\u00A0AND\t \u00C2\u00A0DRUG\t \u00C2\u00A0TREATMENT\t \u00C2\u00A0OF\t \u00C2\u00A0HEK\t \u00C2\u00A0CELLS\t \u00C2\u00A0Lipofectamine\t \u00C2\u00A02000(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#15338-\u00C2\u00AD\u00E2\u0080\u0090500)\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0transfection\t \u00C2\u00A0of\t \u00C2\u00A0plasmid\t \u00C2\u00A0DNA\t \u00C2\u00A0into\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0instructions\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0few\t \u00C2\u00A0modifications.\t \u00C2\u00A0Briefly,\t \u00C2\u00A0for\t \u00C2\u00A0each\t \u00C2\u00A0well\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A06-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plate,\t \u00C2\u00A02\u00CE\u00BCg\t \u00C2\u00A0of\t \u00C2\u00A0plasmid\t \u00C2\u00A0DNA\t \u00C2\u00A0was\t \u00C2\u00A0diluted\t \u00C2\u00A0in\t \u00C2\u00A0125\u00CE\u00BCL\t \u00C2\u00A0Opti-\u00C2\u00AD\u00E2\u0080\u0090MEM\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#31985-\u00C2\u00AD\u00E2\u0080\u0090070)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0sterilized\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tube.\t \u00C2\u00A02\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0Lipofectamine\t \u00C2\u00A0was\t \u00C2\u00A0diluted\t \u00C2\u00A0in\t \u00C2\u00A0another\t \u00C2\u00A0125\u00CE\u00BCL\t \u00C2\u00A0Opti-\u00C2\u00AD\u00E2\u0080\u0090MEM\t \u00C2\u00A0and\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0rest\t \u00C2\u00A0without\t \u00C2\u00A0agitation\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0for\t \u00C2\u00A05min.\t \u00C2\u00A0The\t \u00C2\u00A0two\t \u00C2\u00A0solutions\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0mixed\t \u00C2\u00A0and\t \u00C2\u00A0rested\t \u00C2\u00A0for\t \u00C2\u00A020min\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature.\t \u00C2\u00A0Across\t \u00C2\u00A0all\t \u00C2\u00A0experimental\t \u00C2\u00A0conditions\t \u00C2\u00A0the\t \u00C2\u00A0total\t \u00C2\u00A0amount\t \u00C2\u00A0of\t \u00C2\u00A0plasmid\t \u00C2\u00A0DNA\t \u00C2\u00A0was\t \u00C2\u00A0equalized\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0vector.\t \u00C2\u00A0The\t \u00C2\u00A0plasmid-\u00C2\u00AD\u00E2\u0080\u0090Lipofectamine\t \u00C2\u00A0complex\t \u00C2\u00A0solution\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0added\t \u00C2\u00A0drop-\u00C2\u00AD\u00E2\u0080\u0090by-\u00C2\u00AD\u00E2\u0080\u0090drop\t \u00C2\u00A0into\t \u00C2\u00A0each\t \u00C2\u00A0well\t \u00C2\u00A0of\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0containing\t \u00C2\u00A0750\u00CE\u00BCL\t \u00C2\u00A0fresh\t \u00C2\u00A0complete\t \u00C2\u00A0growth\t \u00C2\u00A0media.\t \u00C2\u00A0Transfected\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0returned\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0incubator\t \u00C2\u00A0for\t \u00C2\u00A0either\t \u00C2\u00A024h\t \u00C2\u00A0or\t \u00C2\u00A048h\t \u00C2\u00A0before\t \u00C2\u00A0harvesting\t \u00C2\u00A0for\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A0(see\t \u00C2\u00A0Chapter\t \u00C2\u00A02.3.2).\t \u00C2\u00A0\t \u00C2\u00A0Serum\t \u00C2\u00A0deprivation\t \u00C2\u00A0was\t \u00C2\u00A0initiated\t \u00C2\u00A0by\t \u00C2\u00A0replacing\t \u00C2\u00A0complete\t \u00C2\u00A0growth\t \u00C2\u00A0media\t \u00C2\u00A0with\t \u00C2\u00A01mL\t \u00C2\u00A0serum-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0DMEM\t \u00C2\u00A0after\t \u00C2\u00A0transfection\t \u00C2\u00A0to\t \u00C2\u00A0enhance\t \u00C2\u00A0CMA\t \u00C2\u00A0activity.\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090methyladenine\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#M9281)\t \u00C2\u00A0was\t \u00C2\u00A0dissolved\t \u00C2\u00A0in\t \u00C2\u00A0water\t \u00C2\u00A0with\t \u00C2\u00A0gentle\t \u00C2\u00A0heating\t \u00C2\u00A0and\t \u00C2\u00A0used\t \u00C2\u00A0at\t \u00C2\u00A010mM\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0macroautophagy.\t \u00C2\u00A0MG132\t \u00C2\u00A0(Biomol,\t \u00C2\u00A0cat.\t \u00C2\u00A0#PI102-\u00C2\u00AD\u00E2\u0080\u00900025)\t \u00C2\u00A0was\t \u00C2\u00A0dissolved\t \u00C2\u00A0in\t \u00C2\u00A0DMSO\t \u00C2\u00A0and\t \u00C2\u00A0used\t \u00C2\u00A0at\t \u00C2\u00A05\u00CE\u00BCM\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0proteasomal\t \u00C2\u00A0activity.\t \u00C2\u00A0Ammonium\t \u00C2\u00A0chloride\t \u00C2\u00A0(NH4Cl;\t \u00C2\u00A0Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#A0171)\t \u00C2\u00A0was\t \u00C2\u00A0dissolved\t \u00C2\u00A0in\t \u00C2\u00A0water\t \u00C2\u00A0and\t \u00C2\u00A0used\t \u00C2\u00A0at\t \u00C2\u00A020mM\t \u00C2\u00A0concentration\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0lysosomal\t \u00C2\u00A0activity.\t \u00C2\u00A0Pepstatin\t \u00C2\u00A0A\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#\t \u00C2\u00A0P5318)\t \u00C2\u00A0was\t \u00C2\u00A0dissolved\t \u00C2\u00A0in\t \u00C2\u00A0DMSO\t \u00C2\u00A0and\t \u00C2\u00A0used\t \u00C2\u00A0at\t \u00C2\u00A010\u00CE\u00BCM\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0the\t \u00C2\u00A0two\t \u00C2\u00A0primary\t \u00C2\u00A0lysosome\t \u00C2\u00A0enzymes,\t \u00C2\u00A0cathepsin\t \u00C2\u00A0D\t \u00C2\u00A0and\t \u00C2\u00A0E\t \u00C2\u00A0(Cataldo\t \u00C2\u00A0and\t \u00C2\u00A0Nixon,\t \u00C2\u00A01990;\t \u00C2\u00A0Bauer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0 61 2010).\t \u00C2\u00A0Control\t \u00C2\u00A0conditions\t \u00C2\u00A0included\t \u00C2\u00A0either\t \u00C2\u00A0DMSO\t \u00C2\u00A0or\t \u00C2\u00A0water.\t \u00C2\u00A0All\t \u00C2\u00A0treatments\t \u00C2\u00A0were\t \u00C2\u00A0initiated\t \u00C2\u00A018hrs\t \u00C2\u00A0after\t \u00C2\u00A0transfection.\t \u00C2\u00A02.1.3 STORAGE\t \u00C2\u00A0OF\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0CELLS\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0stored\t \u00C2\u00A0in\t \u00C2\u00A0liquid\t \u00C2\u00A0nitrogen.\t \u00C2\u00A0Briefly,\t \u00C2\u00A0cells\t \u00C2\u00A0grown\t \u00C2\u00A0to\t \u00C2\u00A080-\u00C2\u00AD\u00E2\u0080\u009090%\t \u00C2\u00A0confluence\t \u00C2\u00A0were\t \u00C2\u00A0trypsinized\t \u00C2\u00A0and\t \u00C2\u00A0centrifuged\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A02.1.1.\t \u00C2\u00A0Following\t \u00C2\u00A0removal\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0media,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090suspended\t \u00C2\u00A0with\t \u00C2\u00A01mL\t \u00C2\u00A0solution\t \u00C2\u00A0containing\t \u00C2\u00A090%\t \u00C2\u00A0complete\t \u00C2\u00A0growth\t \u00C2\u00A0media\t \u00C2\u00A0and\t \u00C2\u00A010%\t \u00C2\u00A0DMSO.\t \u00C2\u00A0The\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0pipetted\t \u00C2\u00A0into\t \u00C2\u00A02mL\t \u00C2\u00A0freezer\t \u00C2\u00A0tubes,\t \u00C2\u00A0wrapped\t \u00C2\u00A0in\t \u00C2\u00A0Tena\t \u00C2\u00A0underpads\t \u00C2\u00A0for\t \u00C2\u00A0extra\t \u00C2\u00A0insulation\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A02h\t \u00C2\u00A0before\t \u00C2\u00A0being\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\u00E2\u0080\u009380\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0refrigerator\t \u00C2\u00A0for\t \u00C2\u00A0overnight\t \u00C2\u00A0cooling.\t \u00C2\u00A0The\t \u00C2\u00A0freezer\t \u00C2\u00A0tubes\t \u00C2\u00A0were\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0liquid\t \u00C2\u00A0nitrogen\t \u00C2\u00A0the\t \u00C2\u00A0next\t \u00C2\u00A0day\t \u00C2\u00A0for\t \u00C2\u00A0long-\u00C2\u00AD\u00E2\u0080\u0090term\t \u00C2\u00A0storage.\t \u00C2\u00A02.1.4 PRIMARY\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CULTURE\t \u00C2\u00A0Dissociated\t \u00C2\u00A0cultures\t \u00C2\u00A0of\t \u00C2\u00A0rat\t \u00C2\u00A0cortical\t \u00C2\u00A0or\t \u00C2\u00A0hippocampal\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0prepared\t \u00C2\u00A0from\t \u00C2\u00A0Sprague-\u00C2\u00AD\u00E2\u0080\u0090Dawley\t \u00C2\u00A0rat\t \u00C2\u00A0embryos\t \u00C2\u00A0collected\t \u00C2\u00A0from\t \u00C2\u00A0timed\t \u00C2\u00A0pregnant\t \u00C2\u00A0rats\t \u00C2\u00A0E18-\u00C2\u00AD\u00E2\u0080\u0090E19\t \u00C2\u00A0after\t \u00C2\u00A0fertilization\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0day\t \u00C2\u00A0of\t \u00C2\u00A0culture.\t \u00C2\u00A0Mothers\t \u00C2\u00A0were\t \u00C2\u00A0sacrificed\t \u00C2\u00A0by\t \u00C2\u00A0overdosing\t \u00C2\u00A0with\t \u00C2\u00A03.5mL\t \u00C2\u00A025%\t \u00C2\u00A0urethane\t \u00C2\u00A0solution\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0lower\t \u00C2\u00A0abdomen\t \u00C2\u00A0was\t \u00C2\u00A0cleaned\t \u00C2\u00A0with\t \u00C2\u00A070%\t \u00C2\u00A0ethanol.\t \u00C2\u00A0An\t \u00C2\u00A0incision\t \u00C2\u00A0was\t \u00C2\u00A0made\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0skin\t \u00C2\u00A0and\t \u00C2\u00A0muscle\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0abdomen\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0uterus,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0removed\t \u00C2\u00A0in\t \u00C2\u00A0situ\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0sterile\t \u00C2\u00A010cm\t \u00C2\u00A0culture\t \u00C2\u00A0dish\t \u00C2\u00A0with\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0HBSS\t \u00C2\u00A0Dissection\t \u00C2\u00A0Buffer\t \u00C2\u00A0(DB).\t \u00C2\u00A0The\t \u00C2\u00A0recipe\t \u00C2\u00A0of\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0as\t \u00C2\u00A0follows:\t \u00C2\u00A0\t \u00C2\u00A0 62 HBSS\t \u00C2\u00A0Dissection\t \u00C2\u00A0Buffer\t \u00C2\u00A0(DB)\t \u00C2\u00A0500mL\t \u00C2\u00A01x\t \u00C2\u00A0HBSS\t \u00C2\u00A0(without\t \u00C2\u00A0Ca2+\t \u00C2\u00A0and\t \u00C2\u00A0Mg2+,\t \u00C2\u00A0Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#14170-\u00C2\u00AD\u00E2\u0080\u0090112)\t \u00C2\u00A010g\t \u00C2\u00A0glucose\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#G7528)\t \u00C2\u00A02.5g\t \u00C2\u00A0sucrose\t \u00C2\u00A0(Fisher,\t \u00C2\u00A0cat.\t \u00C2\u00A0#FL030297)\t \u00C2\u00A01.78g\t \u00C2\u00A0Hepes\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#H3784)\t \u00C2\u00A0Adjusted\t \u00C2\u00A0to\t \u00C2\u00A0pH=\t \u00C2\u00A07.37\u00E2\u0080\u00937.43\t \u00C2\u00A0and\t \u00C2\u00A0osmolarity=310-\u00C2\u00AD\u00E2\u0080\u0090320\t \u00C2\u00A0mOsm\t \u00C2\u00A0Filter\t \u00C2\u00A0to\t \u00C2\u00A0sterilize.\t \u00C2\u00A0Using\t \u00C2\u00A0sterile\t \u00C2\u00A0procedures,\t \u00C2\u00A0the\t \u00C2\u00A0uterus\t \u00C2\u00A0was\t \u00C2\u00A0opened\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0embryos\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090chilled\t \u00C2\u00A010cm\t \u00C2\u00A0Petri\t \u00C2\u00A0dish.\t \u00C2\u00A0The\t \u00C2\u00A0brain\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0embryo\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0cortex\t \u00C2\u00A0was\t \u00C2\u00A0dissected\t \u00C2\u00A0out\t \u00C2\u00A0under\t \u00C2\u00A0a\t \u00C2\u00A0microscope\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0fresh\t \u00C2\u00A010cm\t \u00C2\u00A0culture\t \u00C2\u00A0dish\t \u00C2\u00A0containing\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0DB.\t \u00C2\u00A0To\t \u00C2\u00A0digest\t \u00C2\u00A0the\t \u00C2\u00A0tissue,\t \u00C2\u00A0DB\t \u00C2\u00A0was\t \u00C2\u00A0replaced\t \u00C2\u00A0with\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00904mL\t \u00C2\u00A0of\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090warmed\t \u00C2\u00A00.25%\t \u00C2\u00A0Trypsin-\u00C2\u00AD\u00E2\u0080\u0090EDTA\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#25200-\u00C2\u00AD\u00E2\u0080\u0090072)\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0dish\t \u00C2\u00A0was\t \u00C2\u00A0placed\t \u00C2\u00A0into\t \u00C2\u00A037\u00C2\u00B0C\t \u00C2\u00A0incubator\t \u00C2\u00A0for\t \u00C2\u00A030\t \u00C2\u00A0minutes.\t \u00C2\u00A0Trypsin\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0inactivated\t \u00C2\u00A0by\t \u00C2\u00A0adding\t \u00C2\u00A08-\u00C2\u00AD\u00E2\u0080\u009010mL\t \u00C2\u00A0of\t \u00C2\u00A0warm\t \u00C2\u00A0DMEM\t \u00C2\u00A0media\t \u00C2\u00A0(with\t \u00C2\u00A010%\t \u00C2\u00A0FBS)\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0mixture\t \u00C2\u00A0was\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0sterile\t \u00C2\u00A015mL\t \u00C2\u00A0Falcon\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0sit\t \u00C2\u00A0for\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00903min\t \u00C2\u00A0for\t \u00C2\u00A0dissociated\t \u00C2\u00A0cells\t \u00C2\u00A0to\t \u00C2\u00A0settle\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0bottom\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0tube.\t \u00C2\u00A0The\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0replaced\t \u00C2\u00A0with\t \u00C2\u00A010mL\t \u00C2\u00A0of\t \u00C2\u00A0fresh\t \u00C2\u00A0DMEM\t \u00C2\u00A0(with\t \u00C2\u00A010%\t \u00C2\u00A0FBS)\t \u00C2\u00A0twice.\t \u00C2\u00A0After\t \u00C2\u00A0the\t \u00C2\u00A0last\t \u00C2\u00A0wash,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0gently\t \u00C2\u00A0titrated\t \u00C2\u00A0and\t \u00C2\u00A0centrifuged\t \u00C2\u00A0for\t \u00C2\u00A045-\u00C2\u00AD\u00E2\u0080\u009050s\t \u00C2\u00A0at\t \u00C2\u00A02,500rpm.\t \u00C2\u00A0The\t \u00C2\u00A0wash\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0removed,\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090suspended\t \u00C2\u00A0in\t \u00C2\u00A0Neurobasal\t \u00C2\u00A0Plating\t \u00C2\u00A0media\t \u00C2\u00A0(NP).\t \u00C2\u00A0The\t \u00C2\u00A0recipe\t \u00C2\u00A0of\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0as\t \u00C2\u00A0follows:\t \u00C2\u00A0 63 Neurobasal\t \u00C2\u00A0Plating\t \u00C2\u00A0media\t \u00C2\u00A0(NP)\t \u00C2\u00A0487.75\t \u00C2\u00A0mL\t \u00C2\u00A0Neurobasal\t \u00C2\u00A0Media\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#21103)\t \u00C2\u00A00.5\t \u00C2\u00A0mM\t \u00C2\u00A0GlutaMAXTM-\u00C2\u00AD\u00E2\u0080\u0090I\t \u00C2\u00A0Supplement\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#\t \u00C2\u00A035050-\u00C2\u00AD\u00E2\u0080\u009006)\t \u00C2\u00A02%\t \u00C2\u00A0B27\t \u00C2\u00A0supplement\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#17504044)\t \u00C2\u00A025\u00CE\u00BCM\t \u00C2\u00A0glutamic\t \u00C2\u00A0acid\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#G8415)\t \u00C2\u00A0\t \u00C2\u00A0Cell\t \u00C2\u00A0number\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0determined\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0hemocytometer,\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0plated\t \u00C2\u00A0onto\t \u00C2\u00A0poly-\u00C2\u00AD\u00E2\u0080\u0090D-\u00C2\u00AD\u00E2\u0080\u0090lysine\t \u00C2\u00A0(PDL;\t \u00C2\u00A0Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#P7280)-\u00C2\u00AD\u00E2\u0080\u0090coated\t \u00C2\u00A0tissue\t \u00C2\u00A0culture\t \u00C2\u00A0plates\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0densities:\t \u00C2\u00A06.0\u00C3\u0097106\t \u00C2\u00A0for\t \u00C2\u00A010cm\t \u00C2\u00A0dishes,\t \u00C2\u00A09.0\u00C3\u0097105\t \u00C2\u00A0for\t \u00C2\u00A035mm\t \u00C2\u00A0dishes\t \u00C2\u00A01.5\u00C3\u0097105\t \u00C2\u00A0for\t \u00C2\u00A012-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plates,\t \u00C2\u00A0and\t \u00C2\u00A02.0\u00C3\u0097105\t \u00C2\u00A0for\t \u00C2\u00A024-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plates.\t \u00C2\u00A0After\t \u00C2\u00A02d\t \u00C2\u00A0in\t \u00C2\u00A0culture,\t \u00C2\u00A0two-\u00C2\u00AD\u00E2\u0080\u0090thirds\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0replaced\t \u00C2\u00A0with\t \u00C2\u00A0equal\t \u00C2\u00A0volume\t \u00C2\u00A0of\t \u00C2\u00A0Neurobasal\t \u00C2\u00A0Feeding\t \u00C2\u00A0media\t \u00C2\u00A0(NF:\t \u00C2\u00A0Neurobasal\t \u00C2\u00A0Media\t \u00C2\u00A0488.75\t \u00C2\u00A0mL,\t \u00C2\u00A0GlutaMAXTM-\u00C2\u00AD\u00E2\u0080\u0090I\t \u00C2\u00A00.5\t \u00C2\u00A0mM,\t \u00C2\u00A0B27\t \u00C2\u00A02%).\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0maintained\t \u00C2\u00A0by\t \u00C2\u00A0performing\t \u00C2\u00A0a\t \u00C2\u00A0media\t \u00C2\u00A0swap\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0above\t \u00C2\u00A0every\t \u00C2\u00A04\t \u00C2\u00A0days\t \u00C2\u00A0until\t \u00C2\u00A0use.\t \u00C2\u00A02.1.5 DRUG\t \u00C2\u00A0TREATMENT\t \u00C2\u00A0OF\t \u00C2\u00A0PRIMARY\t \u00C2\u00A0CULTURED\t \u00C2\u00A0NEURONS\t \u00C2\u00A0Primary\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0cultured\t \u00C2\u00A0until\t \u00C2\u00A014d\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0before\t \u00C2\u00A0treatment\t \u00C2\u00A0with\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090methyl\t \u00C2\u00A0D-\u00C2\u00AD\u00E2\u0080\u0090aspartate\t \u00C2\u00A0(NMDA;\t \u00C2\u00A0Tocris,\t \u00C2\u00A0cat.\t \u00C2\u00A0#Asc-\u00C2\u00AD\u00E2\u0080\u0090052),\t \u00C2\u00A0H2O2\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#H1009),\t \u00C2\u00A0D\u00E2\u0080\u0093(-\u00C2\u00AD\u00E2\u0080\u0090)-\u00C2\u00AD\u00E2\u0080\u00902-\u00C2\u00AD\u00E2\u0080\u0090Amino-\u00C2\u00AD\u00E2\u0080\u00905-\u00C2\u00AD\u00E2\u0080\u0090phosphonopentanoic\t \u00C2\u00A0acid\t \u00C2\u00A0(D-\u00C2\u00AD\u00E2\u0080\u0090AP5;\t \u00C2\u00A0Ascent\t \u00C2\u00A0Sci,\t \u00C2\u00A0cat.\t \u00C2\u00A0#Asc-\u00C2\u00AD\u00E2\u0080\u0090003),\t \u00C2\u00A0Catalase\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#C1345)\t \u00C2\u00A0or\t \u00C2\u00A0saline\t \u00C2\u00A0control.\t \u00C2\u00A0Before\t \u00C2\u00A0treatment,\t \u00C2\u00A0conditioned\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0saved\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A050mL\t \u00C2\u00A0Falcon\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0fresh\t \u00C2\u00A0Neurobasal\t \u00C2\u00A0Feeding\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0added.\t \u00C2\u00A0In\t \u00C2\u00A0experiments\t \u00C2\u00A0involving\t \u00C2\u00A0peptide\t \u00C2\u00A0treatment,\t \u00C2\u00A0the\t \u00C2\u00A0peptides\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0culture\t \u00C2\u00A0media\t \u00C2\u00A0for\t \u00C2\u00A01h\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0 64 penetration\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cells.\t \u00C2\u00A0D-\u00C2\u00AD\u00E2\u0080\u0090AP5\t \u00C2\u00A0and\t \u00C2\u00A0Catalase\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A030min\t \u00C2\u00A0prior\t \u00C2\u00A0H2O2\t \u00C2\u00A0treatment\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0penetration.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A0NMDA\t \u00C2\u00A0(50\u00CE\u00BCM)\t \u00C2\u00A0or\t \u00C2\u00A0H2O2\t \u00C2\u00A0(300mM)\t \u00C2\u00A0for\t \u00C2\u00A030min\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0incubator.\t \u00C2\u00A0To\t \u00C2\u00A0end\t \u00C2\u00A0the\t \u00C2\u00A0treatment,\t \u00C2\u00A0the\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0aspirated\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0once\t \u00C2\u00A0with\t \u00C2\u00A0warm\t \u00C2\u00A0PBS.\t \u00C2\u00A0Finally,\t \u00C2\u00A0conditioned\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0back\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0cultures\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0kept\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0incubator\t \u00C2\u00A0at\t \u00C2\u00A037\u00C2\u00B0C,\t \u00C2\u00A05%\t \u00C2\u00A0CO2\t \u00C2\u00A0until\t \u00C2\u00A0harvesting.\t \u00C2\u00A02.2 CELL\t \u00C2\u00A0AND\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A02.2.1 HEK\t \u00C2\u00A0293\t \u00C2\u00A0CELL\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0WHOLE\t \u00C2\u00A0CELL\t \u00C2\u00A0LYSATE\t \u00C2\u00A0To\t \u00C2\u00A0collect\t \u00C2\u00A0HEK\t \u00C2\u00A0cells\t \u00C2\u00A0for\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis,\t \u00C2\u00A0DMEM\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0with\t \u00C2\u00A01mL\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0phosphate-\u00C2\u00AD\u00E2\u0080\u0090buffered\t \u00C2\u00A0saline\t \u00C2\u00A0(PBS)\t \u00C2\u00A0for\t \u00C2\u00A0each\t \u00C2\u00A0well\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A06-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plate.\t \u00C2\u00A0PBS\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0replaced\t \u00C2\u00A0with\t \u00C2\u00A0500\u00CE\u00BCL\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0radio-\u00C2\u00AD\u00E2\u0080\u0090immuno-\u00C2\u00AD\u00E2\u0080\u0090precipitation\t \u00C2\u00A0assay\t \u00C2\u00A0(RIPA)\t \u00C2\u00A0buffer.\t \u00C2\u00A0Phosphate-\u00C2\u00AD\u00E2\u0080\u0090buffered\t \u00C2\u00A0saline\t \u00C2\u00A0(PBS)\t \u00C2\u00A0137mM\t \u00C2\u00A0NaCl\t \u00C2\u00A02.7mM\t \u00C2\u00A0KCl\t \u00C2\u00A010mM\t \u00C2\u00A0Na2HPO4\t \u00C2\u00A0\t \u00C2\u00A01.8mM\t \u00C2\u00A0KH2PO4\t \u00C2\u00A0Adjust\t \u00C2\u00A0to\t \u00C2\u00A0pH7.4,\t \u00C2\u00A0autoclave\t \u00C2\u00A0and\t \u00C2\u00A0cool\t \u00C2\u00A0prior\t \u00C2\u00A0use\t \u00C2\u00A0Radio-\u00C2\u00AD\u00E2\u0080\u0090immuno-\u00C2\u00AD\u00E2\u0080\u0090precipitation\t \u00C2\u00A0assay\t \u00C2\u00A0(RIPA)\t \u00C2\u00A0buffer\t \u00C2\u00A0150mM\t \u00C2\u00A0NaCl\t \u00C2\u00A00.3%\t \u00C2\u00A0deoxycholic\t \u00C2\u00A0acid\t \u00C2\u00A0sodium\t \u00C2\u00A050mM\t \u00C2\u00A0Tris\t \u00C2\u00A0(pH\t \u00C2\u00A08)\t \u00C2\u00A0 65 1mM\t \u00C2\u00A0EDTA\t \u00C2\u00A01.0%\t \u00C2\u00A0Triton\t \u00C2\u00A0X-\u00C2\u00AD\u00E2\u0080\u0090100\t \u00C2\u00A0Adjust\t \u00C2\u00A0to\t \u00C2\u00A0pH7.4\t \u00C2\u00A0\t \u00C2\u00A0Halt\t \u00C2\u00A0Protease\t \u00C2\u00A0Phosphatase\t \u00C2\u00A0Inhibitor\t \u00C2\u00A0(ThermoFisher,\t \u00C2\u00A0cat.\t \u00C2\u00A0#PI78442)\t \u00C2\u00A0\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0immediately\t \u00C2\u00A0scraped\t \u00C2\u00A0off\t \u00C2\u00A0the\t \u00C2\u00A0plate\t \u00C2\u00A0with\t \u00C2\u00A0specialized\t \u00C2\u00A0cell\t \u00C2\u00A0scrapers\t \u00C2\u00A0and\t \u00C2\u00A0collected\t \u00C2\u00A0into\t \u00C2\u00A01.5mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tubes.\t \u00C2\u00A0The\t \u00C2\u00A0tubes\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0kept\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0for\t \u00C2\u00A030min\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A014,000rpm\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0to\t \u00C2\u00A0precipitate\t \u00C2\u00A0cell\t \u00C2\u00A0debris.\t \u00C2\u00A0The\t \u00C2\u00A0supernatant\t \u00C2\u00A0was\t \u00C2\u00A0transferred\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A01.5mL\t \u00C2\u00A0Eppondorf\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0stored\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0further\t \u00C2\u00A0use.\t \u00C2\u00A02.2.2 PRIMARY\t \u00C2\u00A0NEURON\t \u00C2\u00A0CULTURE\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0WHOLE\t \u00C2\u00A0CELL\t \u00C2\u00A0LYSATE\t \u00C2\u00A0To\t \u00C2\u00A0collect\t \u00C2\u00A0primary\t \u00C2\u00A0neurons\t \u00C2\u00A0for\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis,\t \u00C2\u00A0Neurobasal\t \u00C2\u00A0Feeding\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0with\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0PBS,\t \u00C2\u00A0lysed\t \u00C2\u00A0in\t \u00C2\u00A0400\u00CE\u00BCL\t \u00C2\u00A0RIPA\t \u00C2\u00A0buffer\t \u00C2\u00A0supplemented\t \u00C2\u00A0with\t \u00C2\u00A00.1%SDS\t \u00C2\u00A0and\t \u00C2\u00A0centrifuged\t \u00C2\u00A0to\t \u00C2\u00A0remove\t \u00C2\u00A0cell\t \u00C2\u00A0debris\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A02.2.1.\t \u00C2\u00A0The\t \u00C2\u00A0supernatant\t \u00C2\u00A0was\t \u00C2\u00A0transferred\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A01.5mL\t \u00C2\u00A0Eppondorf\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0stored\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0further\t \u00C2\u00A0use.\t \u00C2\u00A02.2.3 PRIMARY\t \u00C2\u00A0NEURON\t \u00C2\u00A0CULTURE\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0NUCLEAR\t \u00C2\u00A0FRACTIONATION\t \u00C2\u00A0To\t \u00C2\u00A0collect\t \u00C2\u00A0neuronal\t \u00C2\u00A0tissue\t \u00C2\u00A0for\t \u00C2\u00A0nuclear\t \u00C2\u00A0fractionation\t \u00C2\u00A0and\t \u00C2\u00A0subsequent\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis,\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0cultured\t \u00C2\u00A0in\t \u00C2\u00A010cm\t \u00C2\u00A0dishes\t \u00C2\u00A0for\t \u00C2\u00A014d.\t \u00C2\u00A0Plates\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A02X\t \u00C2\u00A0with\t \u00C2\u00A07mL\t \u00C2\u00A0ice\t \u00C2\u00A0cold\t \u00C2\u00A0PB\t \u00C2\u00A0each\t \u00C2\u00A0time,\t \u00C2\u00A0and\t \u00C2\u00A0900\u00CE\u00BCL\t \u00C2\u00A0Buffer\t \u00C2\u00A0A\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0plate.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 66 Buffer\t \u00C2\u00A0A\t \u00C2\u00A010mM\t \u00C2\u00A0HEPES-\u00C2\u00AD\u00E2\u0080\u0090KOH\t \u00C2\u00A0\t \u00C2\u00A010mM\t \u00C2\u00A0KCl\t \u00C2\u00A010mM\t \u00C2\u00A0EDTA\t \u00C2\u00A01mM\t \u00C2\u00A0DTT\t \u00C2\u00A0\t \u00C2\u00A00.4%\t \u00C2\u00A0NP40\t \u00C2\u00A01.5mM\t \u00C2\u00A0MgCl2\t \u00C2\u00A0Halt\t \u00C2\u00A0Protease\t \u00C2\u00A0Phosphatase\t \u00C2\u00A0Inhibitor\t \u00C2\u00A0(ThermoFisher,\t \u00C2\u00A0cat.\t \u00C2\u00A0#PI78442)\t \u00C2\u00A0\t \u00C2\u00A0Adjust\t \u00C2\u00A0to\t \u00C2\u00A0pH7.9\t \u00C2\u00A0Plates\t \u00C2\u00A0were\t \u00C2\u00A0rocked\t \u00C2\u00A0at\t \u00C2\u00A0150rpm\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A0platform\t \u00C2\u00A0rotator\t \u00C2\u00A0for\t \u00C2\u00A020min\t \u00C2\u00A0and\t \u00C2\u00A0lysates\t \u00C2\u00A0were\t \u00C2\u00A0collected\t \u00C2\u00A0into\t \u00C2\u00A01.5mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tubes.\t \u00C2\u00A0For\t \u00C2\u00A0each\t \u00C2\u00A0sample,\t \u00C2\u00A0100\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0lysate\t \u00C2\u00A0was\t \u00C2\u00A0saved\t \u00C2\u00A0as\t \u00C2\u00A0whole-\u00C2\u00AD\u00E2\u0080\u0090cell\t \u00C2\u00A0lysate\t \u00C2\u00A0control.\t \u00C2\u00A0The\t \u00C2\u00A0remainder\t \u00C2\u00A0was\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A014,000rpm\t \u00C2\u00A0for\t \u00C2\u00A03min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0to\t \u00C2\u00A0precipitate\t \u00C2\u00A0the\t \u00C2\u00A0crude\t \u00C2\u00A0nuclear\t \u00C2\u00A0fraction\t \u00C2\u00A0into\t \u00C2\u00A0a\t 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\u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0further\t \u00C2\u00A0use.\t \u00C2\u00A0To\t \u00C2\u00A0purify\t \u00C2\u00A0the\t \u00C2\u00A0nuclear\t \u00C2\u00A0fraction,\t \u00C2\u00A0the\t \u00C2\u00A0original\t \u00C2\u00A0pellet\t \u00C2\u00A0was\t \u00C2\u00A0washed\t \u00C2\u00A0with\t \u00C2\u00A01mL\t \u00C2\u00A0Buffer\t \u00C2\u00A0A\t \u00C2\u00A0by\t \u00C2\u00A0gently\t \u00C2\u00A0pipetting\t \u00C2\u00A0up-\u00C2\u00AD\u00E2\u0080\u0090and-\u00C2\u00AD\u00E2\u0080\u0090down,\t \u00C2\u00A0and\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A014,000g\t \u00C2\u00A0for\t \u00C2\u00A03min\t \u00C2\u00A0at\t \u00C2\u00A04\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0to\t \u00C2\u00A0pellet\t \u00C2\u00A0the\t \u00C2\u00A0nuclei.\t \u00C2\u00A0The\t \u00C2\u00A0washing\t \u00C2\u00A0buffer\t \u00C2\u00A0was\t \u00C2\u00A0discarded\t \u00C2\u00A0and\t \u00C2\u00A0150\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0Buffer\t \u00C2\u00A0B\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0pellet\t \u00C2\u00A0and\t \u00C2\u00A0gently\t \u00C2\u00A0vortexed\t \u00C2\u00A0to\t \u00C2\u00A0detach\t \u00C2\u00A0and\t \u00C2\u00A0homogenize\t \u00C2\u00A0the\t \u00C2\u00A0pellet.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 67 Buffer\t \u00C2\u00A0B\t \u00C2\u00A020mM\t \u00C2\u00A0HEPES-\u00C2\u00AD\u00E2\u0080\u0090KOH\t \u00C2\u00A0400mM\t \u00C2\u00A0NaCl\t \u00C2\u00A01mM\t \u00C2\u00A0EDTA\t \u00C2\u00A010%\t \u00C2\u00A0glycerol\t \u00C2\u00A01mM\t \u00C2\u00A0DTT\t \u00C2\u00A0Halt\t \u00C2\u00A0Protease\t \u00C2\u00A0Phosphatase\t \u00C2\u00A0Inhibitor\t \u00C2\u00A0(ThermoFisher,\t \u00C2\u00A0cat.\t \u00C2\u00A0#PI78442)\t \u00C2\u00A0\t \u00C2\u00A0Adjust\t \u00C2\u00A0to\t \u00C2\u00A0pH7.9\t \u00C2\u00A0Each\t \u00C2\u00A0nuclei\t \u00C2\u00A0sample\t \u00C2\u00A0was\t \u00C2\u00A0further\t \u00C2\u00A0lysed\t \u00C2\u00A0by\t \u00C2\u00A0rocking\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0at\t \u00C2\u00A0150rpm\t \u00C2\u00A0for\t \u00C2\u00A02h\t \u00C2\u00A0in\t \u00C2\u00A0Buffer\t \u00C2\u00A0B,\t \u00C2\u00A0and\t \u00C2\u00A0vortexed\t \u00C2\u00A0gently\t \u00C2\u00A0for\t \u00C2\u00A010s\t \u00C2\u00A0every\t \u00C2\u00A030min.\t \u00C2\u00A0Finally,\t \u00C2\u00A0the\t \u00C2\u00A0soluble\t \u00C2\u00A0nuclear\t \u00C2\u00A0fraction\t \u00C2\u00A0was\t \u00C2\u00A0obtained\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0the\t \u00C2\u00A0sample\t \u00C2\u00A0at\t \u00C2\u00A014,000rpm\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0and\t \u00C2\u00A0stored\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0use.\t \u00C2\u00A02.2.4 PRIMARY\t \u00C2\u00A0NEURON\t \u00C2\u00A0CULTURE\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0MITOCHONDRIAL\t \u00C2\u00A0FRACTIONATION\t \u00C2\u00A0To\t \u00C2\u00A0collect\t \u00C2\u00A0neuronal\t \u00C2\u00A0tissue\t \u00C2\u00A0for\t \u00C2\u00A0mitochondrial\t \u00C2\u00A0fractionation\t \u00C2\u00A0and\t \u00C2\u00A0subsequent\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis,\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0cultured\t \u00C2\u00A0in\t \u00C2\u00A010cm\t \u00C2\u00A0dishes\t \u00C2\u00A0for\t \u00C2\u00A014d.\t \u00C2\u00A0Mitochondria\t \u00C2\u00A0isolation\t \u00C2\u00A0was\t \u00C2\u00A0performed\t \u00C2\u00A0using\t \u00C2\u00A0the\t \u00C2\u00A0ThermoScientific\t \u00C2\u00A0kit\t \u00C2\u00A0(cat.\t \u00C2\u00A0#89874)\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0instructions.\t \u00C2\u00A0Briefly,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A0trypsin\t \u00C2\u00A0and\t \u00C2\u00A0collected\t \u00C2\u00A0into\t \u00C2\u00A02mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tubes,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0pelleted\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0at\t \u00C2\u00A0850g\t \u00C2\u00A0for\t \u00C2\u00A02min\t \u00C2\u00A0at\t \u00C2\u00A04\t\u00CC\u008A \u00C2\u00A0C.\t \u00C2\u00A0To\t \u00C2\u00A0lyse\t \u00C2\u00A0the\t \u00C2\u00A0cells,\t \u00C2\u00A0800\u00CE\u00BCL\t \u00C2\u00A0Reagent\t \u00C2\u00A0A\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0sample\t \u00C2\u00A0and\t \u00C2\u00A0subsequently\t \u00C2\u00A0vortexed\t \u00C2\u00A0at\t \u00C2\u00A0medium\t \u00C2\u00A0speed\t \u00C2\u00A0for\t \u00C2\u00A05s\t \u00C2\u00A0before\t \u00C2\u00A0a\t \u00C2\u00A02min\t \u00C2\u00A0incubation\t \u00C2\u00A0period\t \u00C2\u00A0on\t \u00C2\u00A0ice.\t \u00C2\u00A0Cell\t \u00C2\u00A0lysis\t \u00C2\u00A0was\t \u00C2\u00A0terminated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0Reagent\t \u00C2\u00A0B\t \u00C2\u00A0and\t \u00C2\u00A0vortexing\t \u00C2\u00A0at\t \u00C2\u00A0maximum\t \u00C2\u00A0speed\t \u00C2\u00A0for\t \u00C2\u00A05s.\t \u00C2\u00A0Each\t \u00C2\u00A0sample\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0incubated\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0and\t \u00C2\u00A0vortexed\t \u00C2\u00A0at\t \u00C2\u00A0maximum\t \u00C2\u00A0speed\t \u00C2\u00A0every\t \u00C2\u00A0minute.\t \u00C2\u00A0To\t \u00C2\u00A0yield\t \u00C2\u00A0the\t \u00C2\u00A0crude\t \u00C2\u00A0cytosolic\t \u00C2\u00A0 68 fraction,\t \u00C2\u00A0800\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0Reagent\t \u00C2\u00A0C\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0sample,\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0each\t \u00C2\u00A0sample\t \u00C2\u00A0at\t \u00C2\u00A0700g\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0and\t \u00C2\u00A0transferring\t \u00C2\u00A0the\t \u00C2\u00A0supernatant\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A0tube.\t \u00C2\u00A0To\t \u00C2\u00A0pellet\t \u00C2\u00A0the\t \u00C2\u00A0mitochondria,\t \u00C2\u00A0the\t \u00C2\u00A0supernatant\t \u00C2\u00A0was\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A03,000g\t \u00C2\u00A0for\t \u00C2\u00A015min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0The\t \u00C2\u00A0pellet\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0additional\t \u00C2\u00A0500\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0Reagent\t \u00C2\u00A0C\t \u00C2\u00A0and\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A012,000g\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0The\t \u00C2\u00A0mitochondria\t \u00C2\u00A0pellet\t \u00C2\u00A0was\t \u00C2\u00A0immediately\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A04X\t \u00C2\u00A0sample\t \u00C2\u00A0buffer\t \u00C2\u00A0boiled\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A095\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0heating\t \u00C2\u00A0block\t \u00C2\u00A0for\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis.\t \u00C2\u00A04X\t \u00C2\u00A0Sample\t \u00C2\u00A0Buffer\t \u00C2\u00A050%\t \u00C2\u00A0glycerol\t \u00C2\u00A0125mM\t \u00C2\u00A0Tris-\u00C2\u00AD\u00E2\u0080\u0090HCl\t \u00C2\u00A0(pH\t \u00C2\u00A06.8)\t \u00C2\u00A04%\t \u00C2\u00A0SDS\t \u00C2\u00A00.08%\t \u00C2\u00A0Bromophenol\t \u00C2\u00A0Blue\t \u00C2\u00A0Add\t \u00C2\u00A05%\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090mercaptomethanol\t \u00C2\u00A0prior\t \u00C2\u00A0use\t \u00C2\u00A02.2.5 ANIMAL\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0LYSATE\t \u00C2\u00A0To\t \u00C2\u00A0obtain\t \u00C2\u00A0brain\t \u00C2\u00A0tissue\t \u00C2\u00A0for\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis,\t \u00C2\u00A0each\t \u00C2\u00A0rat\t \u00C2\u00A0was\t \u00C2\u00A0sacrificed\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0overdose\t \u00C2\u00A0of\t \u00C2\u00A0urethane\t \u00C2\u00A0(3g/kg,\t \u00C2\u00A0i.p.).\t \u00C2\u00A0To\t \u00C2\u00A0minimize\t \u00C2\u00A0blood\t \u00C2\u00A0flow\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0brain,\t \u00C2\u00A0the\t \u00C2\u00A0heart\t \u00C2\u00A0was\t \u00C2\u00A0stopped\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0blunt\t \u00C2\u00A0force\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0chest\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0head\t \u00C2\u00A0was\t \u00C2\u00A0quickly\t \u00C2\u00A0displaced\t \u00C2\u00A0with\t \u00C2\u00A0scissors.\t \u00C2\u00A0The\t \u00C2\u00A0skull\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0removed\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0bone\t \u00C2\u00A0cutter\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0gently\t \u00C2\u00A0harvested\t \u00C2\u00A0and\t \u00C2\u00A0place\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090chilled\t \u00C2\u00A0brain\t \u00C2\u00A0mold.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0brain\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0coronally\t \u00C2\u00A0sectioned\t \u00C2\u00A0into\t \u00C2\u00A02mm-\u00C2\u00AD\u00E2\u0080\u0090thick\t \u00C2\u00A0slices\t \u00C2\u00A0and\t \u00C2\u00A0stained\t \u00C2\u00A0with\t \u00C2\u00A02,3,5-\u00C2\u00AD\u00E2\u0080\u0090triphenyltetrazolium\t \u00C2\u00A0chloride\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A02.5.1\t \u00C2\u00A0to\t \u00C2\u00A0identify\t \u00C2\u00A0areas\t \u00C2\u00A0damaged\t \u00C2\u00A0by\t \u00C2\u00A0middle\t \u00C2\u00A0cerebral\t \u00C2\u00A0arterial\t \u00C2\u00A0occlusion.\t \u00C2\u00A0Infarct\t \u00C2\u00A0areas\t \u00C2\u00A0and\t \u00C2\u00A0contralateral\t \u00C2\u00A0control\t \u00C2\u00A0areas\t \u00C2\u00A0were\t \u00C2\u00A0 69 excised\t \u00C2\u00A0with\t \u00C2\u00A0surgical\t \u00C2\u00A0scissors\t \u00C2\u00A0and\t \u00C2\u00A0homogenized\t \u00C2\u00A0in\t \u00C2\u00A01mL\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0lysis\t \u00C2\u00A0buffer\t \u00C2\u00A0(RIPA\t \u00C2\u00A0buffer\t \u00C2\u00A0with\t \u00C2\u00A00.1%SDS\t \u00C2\u00A0and\t \u00C2\u00A0protease\t \u00C2\u00A0inhibitor\t \u00C2\u00A0cocktail)\t \u00C2\u00A0via\t \u00C2\u00A0roughly\t \u00C2\u00A030\t \u00C2\u00A0strokes\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0plastic\t \u00C2\u00A0homogenizer\t \u00C2\u00A0until\t \u00C2\u00A0the\t \u00C2\u00A0tissue\t \u00C2\u00A0completely\t \u00C2\u00A0dispersed.\t \u00C2\u00A0The\t \u00C2\u00A0samples\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0sequentially\t \u00C2\u00A0passed\t \u00C2\u00A0five\t \u00C2\u00A0times\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A016-\u00C2\u00AD\u00E2\u0080\u0090,\t \u00C2\u00A018-\u00C2\u00AD\u00E2\u0080\u0090,\t \u00C2\u00A021-\u00C2\u00AD\u00E2\u0080\u0090,\t \u00C2\u00A023-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A025-\u00C2\u00AD\u00E2\u0080\u0090gauge\t \u00C2\u00A0needle\t \u00C2\u00A0(with\t \u00C2\u00A0a\t \u00C2\u00A01mL\t \u00C2\u00A0syringe),\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0left\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0refrigerator\t \u00C2\u00A0overnight\t \u00C2\u00A0for\t \u00C2\u00A0further\t \u00C2\u00A0lysis.\t \u00C2\u00A0Cellular\t \u00C2\u00A0debris\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0at\t \u00C2\u00A014,000rpm\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0the\t \u00C2\u00A0next\t \u00C2\u00A0day,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0supernatant\t \u00C2\u00A0was\t \u00C2\u00A0collected\t \u00C2\u00A0and\t \u00C2\u00A0kept\t \u00C2\u00A0in\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009080\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0use.\t \u00C2\u00A02.2.6 ANIMAL\t \u00C2\u00A0TISSUE\t \u00C2\u00A0COLLECTION\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0PERFUSION\t \u00C2\u00A0AND\t \u00C2\u00A0FIXATION\t \u00C2\u00A0Each\t \u00C2\u00A0animal\t \u00C2\u00A0was\t \u00C2\u00A0anesthetized\t \u00C2\u00A0with\t \u00C2\u00A0urethane\t \u00C2\u00A0(1.5g/kg,\t \u00C2\u00A0i.p.)\t \u00C2\u00A0and\t \u00C2\u00A0brain\t \u00C2\u00A0tissue\t \u00C2\u00A0was\t \u00C2\u00A0fixed\t \u00C2\u00A0via\t \u00C2\u00A0an\t \u00C2\u00A0intra-\u00C2\u00AD\u00E2\u0080\u0090cardiac\t \u00C2\u00A0perfusion\t \u00C2\u00A0procedure.\t \u00C2\u00A0A\t \u00C2\u00A0V-\u00C2\u00AD\u00E2\u0080\u0090shaped\t \u00C2\u00A0incision\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0skin\t \u00C2\u00A0and\t \u00C2\u00A0underlying\t \u00C2\u00A0muscles\t \u00C2\u00A0was\t \u00C2\u00A0made\t \u00C2\u00A0starting\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0sternum\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0abdominal\t \u00C2\u00A0cavity\t \u00C2\u00A0and\t \u00C2\u00A0diaphragm,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0part\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0chest\t \u00C2\u00A0cavity.\t \u00C2\u00A0The\t \u00C2\u00A0diaphragm\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0cut\t \u00C2\u00A0away\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0beating\t \u00C2\u00A0heart.\t \u00C2\u00A0The\t \u00C2\u00A0chest\t \u00C2\u00A0wall\t \u00C2\u00A0was\t \u00C2\u00A0deflected\t \u00C2\u00A0upwards\t \u00C2\u00A0over\t \u00C2\u00A0the\t \u00C2\u00A0head\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0animal\t \u00C2\u00A0and\t \u00C2\u00A0held\t \u00C2\u00A0in\t \u00C2\u00A0place\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0large\t \u00C2\u00A0hemostat\t \u00C2\u00A0to\t \u00C2\u00A0fully\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0chest\t \u00C2\u00A0cavity.\t \u00C2\u00A0The\t \u00C2\u00A0heart\t \u00C2\u00A0was\t \u00C2\u00A0held\t \u00C2\u00A0in\t \u00C2\u00A0place\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0hemostat\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0cannula\t \u00C2\u00A0running\t \u00C2\u00A0saline\t \u00C2\u00A0was\t \u00C2\u00A0gently\t \u00C2\u00A0inserted\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0left\t \u00C2\u00A0ventricle\t \u00C2\u00A0and\t \u00C2\u00A0clamped\t \u00C2\u00A0in\t \u00C2\u00A0place.\t \u00C2\u00A0The\t \u00C2\u00A0right\t \u00C2\u00A0atrium\t \u00C2\u00A0was\t \u00C2\u00A0quickly\t \u00C2\u00A0cut\t \u00C2\u00A0open\t \u00C2\u00A0by\t \u00C2\u00A0small\t \u00C2\u00A0surgical\t \u00C2\u00A0scissors\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0outflow\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0circulating\t \u00C2\u00A0blood.\t \u00C2\u00A0A\t \u00C2\u00A0small\t \u00C2\u00A0incision\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0tip\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0tail\t \u00C2\u00A0was\t \u00C2\u00A0made\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0observation\t \u00C2\u00A0of\t \u00C2\u00A0blood\t \u00C2\u00A0outflow,\t \u00C2\u00A0which\t \u00C2\u00A0typically\t \u00C2\u00A0clears\t \u00C2\u00A0after\t \u00C2\u00A0perfusing\t \u00C2\u00A0with\t \u00C2\u00A0250-\u00C2\u00AD\u00E2\u0080\u0090300mL\t \u00C2\u00A0of\t \u00C2\u00A0saline.\t \u00C2\u00A0The\t \u00C2\u00A0saline\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0switched\t \u00C2\u00A0to\t \u00C2\u00A0paraformaldehyde\t \u00C2\u00A0(PFA)\t \u00C2\u00A0solution\t \u00C2\u00A0(4%\t \u00C2\u00A0PFA\t \u00C2\u00A0in\t \u00C2\u00A0PBS)\t \u00C2\u00A0and\t \u00C2\u00A0each\t \u00C2\u00A0rat\t \u00C2\u00A0is\t \u00C2\u00A0perfused\t \u00C2\u00A0with\t \u00C2\u00A0250mL\t \u00C2\u00A0PFA\t \u00C2\u00A0solution\t \u00C2\u00A0or\t \u00C2\u00A0until\t \u00C2\u00A0completely\t \u00C2\u00A0stiff.\t \u00C2\u00A0\t \u00C2\u00A0 70 The\t \u00C2\u00A0head\t \u00C2\u00A0was\t \u00C2\u00A0displaced\t \u00C2\u00A0with\t \u00C2\u00A0scissors.\t \u00C2\u00A0The\t \u00C2\u00A0skull\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0removed\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0bone\t \u00C2\u00A0cutter\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0gently\t \u00C2\u00A0harvested\t \u00C2\u00A0and\t \u00C2\u00A0place\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A050mL\t \u00C2\u00A0Falcon\t \u00C2\u00A0tube\t \u00C2\u00A0filled\t \u00C2\u00A0with\t \u00C2\u00A04%\t \u00C2\u00A0PFA\t \u00C2\u00A0solution\t \u00C2\u00A0and\t \u00C2\u00A0kept\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0refrigerator\t \u00C2\u00A0overnight.\t \u00C2\u00A0The\t \u00C2\u00A0brains\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A030%\t \u00C2\u00A0sucrose\t \u00C2\u00A0solution\t \u00C2\u00A0(in\t \u00C2\u00A04%\t \u00C2\u00A0PFA)\t \u00C2\u00A0in\t \u00C2\u00A050mL\t \u00C2\u00A0Falcon\t \u00C2\u00A0tubes\t \u00C2\u00A0until\t \u00C2\u00A0they\t \u00C2\u00A0sunk\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0bottom\t \u00C2\u00A0to\t \u00C2\u00A0achieve\t \u00C2\u00A0cryoprotection.\t \u00C2\u00A0The\t \u00C2\u00A0brains\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0carefully\t \u00C2\u00A0rinsed\t \u00C2\u00A0with\t \u00C2\u00A0PBS,\t \u00C2\u00A0wrapped\t \u00C2\u00A0in\t \u00C2\u00A0aluminum\t \u00C2\u00A0foil\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009080\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A01h\t \u00C2\u00A0prior\t \u00C2\u00A0slicing.\t \u00C2\u00A0\t \u00C2\u00A0Cryoprotected\t \u00C2\u00A0brains\t \u00C2\u00A0were\t \u00C2\u00A0covered\t \u00C2\u00A0in\t \u00C2\u00A0Optimal\t \u00C2\u00A0Cutting\t \u00C2\u00A0Temperature\t \u00C2\u00A0(OCT)\t \u00C2\u00A0compound\t \u00C2\u00A0(Tissue-\u00C2\u00AD\u00E2\u0080\u0090Tek\t \u00C2\u00A0CRYO-\u00C2\u00AD\u00E2\u0080\u0090OCT,\t \u00C2\u00A0cat.\t \u00C2\u00A0#4583)\t \u00C2\u00A0and\t \u00C2\u00A0sliced\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0cryostat\t \u00C2\u00A0into\t \u00C2\u00A030\u00CE\u00BCm\t \u00C2\u00A0thick\t \u00C2\u00A0coronal\t \u00C2\u00A0sections\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0chamber\t \u00C2\u00A0temperature.\t \u00C2\u00A0The\t \u00C2\u00A0sections\t \u00C2\u00A0were\t \u00C2\u00A0kept\t \u00C2\u00A0in\t \u00C2\u00A00.1M\t \u00C2\u00A0PB\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0they\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0immunohistochemistry\t \u00C2\u00A0staining\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Chapter\t \u00C2\u00A02.5.5)\t \u00C2\u00A0or\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090Jade\t \u00C2\u00A0B\t \u00C2\u00A0staining\t \u00C2\u00A0for\t \u00C2\u00A0degenerating\t \u00C2\u00A0neurons\t \u00C2\u00A0(Chapter\t \u00C2\u00A02.5.2).\t \u00C2\u00A02.3 PROTEIN\t \u00C2\u00A0AND\t \u00C2\u00A0PEPTIDE\t \u00C2\u00A0ANALYSIS\t \u00C2\u00A02.3.1 PROTEIN\t \u00C2\u00A0CONCENTRATION\t \u00C2\u00A0Protein\t \u00C2\u00A0concentration\t \u00C2\u00A0was\t \u00C2\u00A0determined\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0Lowry\t \u00C2\u00A0method,\t \u00C2\u00A0using\t \u00C2\u00A0Bio-\u00C2\u00AD\u00E2\u0080\u0090Rad\t \u00C2\u00A0DC\t \u00C2\u00A0Protein\t \u00C2\u00A0Assay\t \u00C2\u00A0Kit\t \u00C2\u00A0(Bio-\u00C2\u00AD\u00E2\u0080\u0090Rad,\t \u00C2\u00A0cat.\t \u00C2\u00A0#500-\u00C2\u00AD\u00E2\u0080\u00900112)\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0instructions.\t \u00C2\u00A0Briefly,\t \u00C2\u00A0protein\t \u00C2\u00A0standards\t \u00C2\u00A0were\t \u00C2\u00A0made\t \u00C2\u00A0from\t \u00C2\u00A0powered\t \u00C2\u00A0BSA\t \u00C2\u00A0solubilized\t \u00C2\u00A0in\t \u00C2\u00A0RIPA\t \u00C2\u00A0buffer\t \u00C2\u00A0through\t \u00C2\u00A0serial\t \u00C2\u00A0dilutions,\t \u00C2\u00A0yielding\t \u00C2\u00A0BSA\t \u00C2\u00A0solutions\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0concentrations:\t \u00C2\u00A010mg/mL,\t \u00C2\u00A05mg/mL,\t \u00C2\u00A02.5mg/mL,\t \u00C2\u00A01.25mg/mL,\t \u00C2\u00A00.625mg/mL\t \u00C2\u00A0and\t \u00C2\u00A00.31mg/mL.\t \u00C2\u00A0To\t \u00C2\u00A0calculate\t \u00C2\u00A0a\t \u00C2\u00A0standard\t \u00C2\u00A0curve,\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0protein\t \u00C2\u00A0standard\t \u00C2\u00A0was\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0Reagent\t \u00C2\u00A0S,\t \u00C2\u00A0100\u00CE\u00BCL\t \u00C2\u00A0Reagent\t \u00C2\u00A0A\t \u00C2\u00A0and\t \u00C2\u00A0890\u00CE\u00BCL\t \u00C2\u00A0Reagent\t \u00C2\u00A0B\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0plastic\t \u00C2\u00A0cuvette.\t \u00C2\u00A0Similarly,\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0Reagent\t \u00C2\u00A0S,\t \u00C2\u00A0100\u00CE\u00BCL\t \u00C2\u00A0Reagent\t \u00C2\u00A0A\t \u00C2\u00A0and\t \u00C2\u00A0800\u00CE\u00BCL\t \u00C2\u00A0 71 Reagent\t \u00C2\u00A0B\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0protein\t \u00C2\u00A0sample.\t \u00C2\u00A0The\t \u00C2\u00A0preparations\t \u00C2\u00A0were\t \u00C2\u00A0rested\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0for\t \u00C2\u00A020min\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0color\t \u00C2\u00A0to\t \u00C2\u00A0develop,\t \u00C2\u00A0and\t \u00C2\u00A0subsequently\t \u00C2\u00A0measured\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0Biochrom\t \u00C2\u00A0Ultrospec\t \u00C2\u00A060\t \u00C2\u00A0at\t \u00C2\u00A0750nm\t \u00C2\u00A0to\t \u00C2\u00A0obtain\t \u00C2\u00A0the\t \u00C2\u00A0standard\t \u00C2\u00A0curve\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0concentration\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0protein\t \u00C2\u00A0sample.\t \u00C2\u00A02.3.2 ELECTROPHORESIS\t \u00C2\u00A0AND\t \u00C2\u00A0WESTERN\t \u00C2\u00A0BLOT\t \u00C2\u00A0ANALYSIS\t \u00C2\u00A0Proteins\t \u00C2\u00A0were\t \u00C2\u00A0separated\t \u00C2\u00A0by\t \u00C2\u00A0sodium\t \u00C2\u00A0dodecyl\t \u00C2\u00A0sulfate\t \u00C2\u00A0polyacrylamide\t \u00C2\u00A0gel\t \u00C2\u00A0electrophoresis\t \u00C2\u00A0(SDS-\u00C2\u00AD\u00E2\u0080\u0090PAGE)\t \u00C2\u00A0and\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0polyvinyl\t \u00C2\u00A0difluoride\t \u00C2\u00A0(PVDF)\t \u00C2\u00A0membranes.\t \u00C2\u00A0The\t \u00C2\u00A0membranes\t \u00C2\u00A0were\t \u00C2\u00A0blocked\t \u00C2\u00A0with\t \u00C2\u00A05%\t \u00C2\u00A0skim\t \u00C2\u00A0milk,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0sequentially\t \u00C2\u00A0incubated\t \u00C2\u00A0with\t \u00C2\u00A0primary\t \u00C2\u00A0and\t \u00C2\u00A0secondary\t \u00C2\u00A0antibodies\t \u00C2\u00A0prior\t \u00C2\u00A0detection\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0bands\t \u00C2\u00A0by\t \u00C2\u00A0chemiluminescent\t \u00C2\u00A0imaging.\t \u00C2\u00A0SDS-\u00C2\u00AD\u00E2\u0080\u0090PAGE\t \u00C2\u00A0gels\t \u00C2\u00A0(1.5mm-\u00C2\u00AD\u00E2\u0080\u0090thick,\t \u00C2\u00A010-\u00C2\u00AD\u00E2\u0080\u0090wells)\t \u00C2\u00A0were\t \u00C2\u00A0made\t \u00C2\u00A0using\t \u00C2\u00A0standard\t \u00C2\u00A0cast\t \u00C2\u00A0racks\t \u00C2\u00A0(BIO-\u00C2\u00AD\u00E2\u0080\u0090RAD,\t \u00C2\u00A0cat.\t \u00C2\u00A0#165-\u00C2\u00AD\u00E2\u0080\u00908000).\t \u00C2\u00A0Each\t \u00C2\u00A0gel\t \u00C2\u00A0consisted\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0bottom\t \u00C2\u00A010%\t \u00C2\u00A0acrylamide\t \u00C2\u00A0separating\t \u00C2\u00A0gel\t \u00C2\u00A0(4.0mL\t \u00C2\u00A0of\t \u00C2\u00A0distilled\t \u00C2\u00A0water,\t \u00C2\u00A03.3mL\t \u00C2\u00A0of\t \u00C2\u00A030%\t \u00C2\u00A0acrylamide\t \u00C2\u00A0solution,\t \u00C2\u00A02.5mL\t \u00C2\u00A0of\t \u00C2\u00A01.5M\t \u00C2\u00A0Tris\t \u00C2\u00A0pH8.8,\t \u00C2\u00A0100\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A010%\t \u00C2\u00A0SDS,\t \u00C2\u00A050\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A010%\t \u00C2\u00A0APS,\t \u00C2\u00A0and\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0TEMED)\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0top\t \u00C2\u00A05%\t \u00C2\u00A0stacking\t \u00C2\u00A0gel\t \u00C2\u00A0(2.82mL\t \u00C2\u00A0of\t \u00C2\u00A0distilled\t \u00C2\u00A0water,\t \u00C2\u00A00.83mL\t \u00C2\u00A0of\t \u00C2\u00A030%\t \u00C2\u00A0acrylamide\t \u00C2\u00A0solution,\t \u00C2\u00A01.25mL\t \u00C2\u00A0of\t \u00C2\u00A00.5M\t \u00C2\u00A0Tris\t \u00C2\u00A0pH\t \u00C2\u00A06.8,\t \u00C2\u00A050\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A010%\t \u00C2\u00A0SDS,\t \u00C2\u00A050\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A010%\t \u00C2\u00A0APS,\t \u00C2\u00A0and\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0TEMED).\t \u00C2\u00A0A\t \u00C2\u00A010-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0comb\t \u00C2\u00A0was\t \u00C2\u00A0inserted\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0stacking\t \u00C2\u00A0gel\t \u00C2\u00A0prior\t \u00C2\u00A0solidifying\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0loading\t \u00C2\u00A0wells.\t \u00C2\u00A0Gels\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A0with\t \u00C2\u00A0distilled\t \u00C2\u00A0water,\t \u00C2\u00A0wrapped\t \u00C2\u00A0in\t \u00C2\u00A0wet\t \u00C2\u00A0tissue\t \u00C2\u00A0and\t \u00C2\u00A0stored\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0use.\t \u00C2\u00A0\t \u00C2\u00A0Protein\t \u00C2\u00A0samples\t \u00C2\u00A0for\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A0were\t \u00C2\u00A0prepared\t \u00C2\u00A0by\t \u00C2\u00A0mixing\t \u00C2\u00A030\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0sample\t \u00C2\u00A0with\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A04X\t \u00C2\u00A0sample\t \u00C2\u00A0buffer\t \u00C2\u00A0and\t \u00C2\u00A0boiled\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A095\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0heat\t \u00C2\u00A0block\t \u00C2\u00A0for\t \u00C2\u00A05min.\t \u00C2\u00A0Samples\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A014,000rpm,\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01min\t \u00C2\u00A0and\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0cool\t \u00C2\u00A0prior\t \u00C2\u00A0loading\t \u00C2\u00A0onto\t \u00C2\u00A0SDS-\u00C2\u00AD\u00E2\u0080\u0090 72 PAGE\t \u00C2\u00A0gels.\t \u00C2\u00A0To\t \u00C2\u00A0facilitate\t \u00C2\u00A0protein\t \u00C2\u00A0identification,\t \u00C2\u00A08\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0PageRuler\t \u00C2\u00A0Prestained\t \u00C2\u00A0Protein\t \u00C2\u00A0Ladder\t \u00C2\u00A0(ThermoScientific,\t \u00C2\u00A0cat.\t \u00C2\u00A0#SM0671)\t \u00C2\u00A0was\t \u00C2\u00A0also\t \u00C2\u00A0loaded\t \u00C2\u00A0onto\t \u00C2\u00A0the\t \u00C2\u00A0gel.\t \u00C2\u00A0Gels\t \u00C2\u00A0were\t \u00C2\u00A0subsequently\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0electrophoresis\t \u00C2\u00A0in\t \u00C2\u00A0running\t \u00C2\u00A0buffer\t \u00C2\u00A0(25mM\t \u00C2\u00A0Tris\t \u00C2\u00A0base,\t \u00C2\u00A0190mM\t \u00C2\u00A0glycine,\t \u00C2\u00A0and\t \u00C2\u00A00.1%\t \u00C2\u00A0SDS\t \u00C2\u00A0at\t \u00C2\u00A0pH\t \u00C2\u00A08.3)\t \u00C2\u00A0at\t \u00C2\u00A0100V\t \u00C2\u00A0for\t \u00C2\u00A0stacking\t \u00C2\u00A0gel\t \u00C2\u00A0and\t \u00C2\u00A0140V\t \u00C2\u00A0for\t \u00C2\u00A0separating\t \u00C2\u00A0gel.\t \u00C2\u00A0\t \u00C2\u00A0Gels\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0rinsed\t \u00C2\u00A0with\t \u00C2\u00A0distilled\t \u00C2\u00A0water\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0into\t \u00C2\u00A0transfer\t \u00C2\u00A0buffer\t \u00C2\u00A0(25mM\t \u00C2\u00A0Tris\t \u00C2\u00A0base,\t \u00C2\u00A0190mM\t \u00C2\u00A0glycine,\t \u00C2\u00A0and\t \u00C2\u00A020%\t \u00C2\u00A0methanol\t \u00C2\u00A0at\t \u00C2\u00A0pH\t \u00C2\u00A08.3).\t \u00C2\u00A0PVDF\t \u00C2\u00A0membranes\t \u00C2\u00A0(Immobilon-\u00C2\u00AD\u00E2\u0080\u0090P\t \u00C2\u00A0Transfer\t \u00C2\u00A0Membrane,\t \u00C2\u00A0Millipore,\t \u00C2\u00A0#IPVH00010)\t \u00C2\u00A0were\t \u00C2\u00A0activated\t \u00C2\u00A0by\t \u00C2\u00A0incubating\t \u00C2\u00A0in\t \u00C2\u00A0methanol\t \u00C2\u00A0for\t \u00C2\u00A02min,\t \u00C2\u00A0after\t \u00C2\u00A0which\t \u00C2\u00A0they\t \u00C2\u00A0were\t \u00C2\u00A0immersed\t \u00C2\u00A0in\t \u00C2\u00A0transfer\t \u00C2\u00A0buffer\t \u00C2\u00A0until\t \u00C2\u00A0use.\t \u00C2\u00A0Each\t \u00C2\u00A0gel\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0secured\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0transfer\t \u00C2\u00A0cassette\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0transfer\t \u00C2\u00A0chamber\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0ice\t \u00C2\u00A0pack.\t \u00C2\u00A0The\t \u00C2\u00A0transfer\t \u00C2\u00A0apparatus\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0moved\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0cold\t \u00C2\u00A0room\t \u00C2\u00A0and\t \u00C2\u00A0proteins\t \u00C2\u00A0were\t \u00C2\u00A0transferred\t \u00C2\u00A0at\t \u00C2\u00A0110V\t \u00C2\u00A0for\t \u00C2\u00A060-\u00C2\u00AD\u00E2\u0080\u009090min.\t \u00C2\u00A0\t \u00C2\u00A0After\t \u00C2\u00A0completing\t \u00C2\u00A0the\t \u00C2\u00A0transfer,\t \u00C2\u00A0PVDF\t \u00C2\u00A0membranes\t \u00C2\u00A0were\t \u00C2\u00A0rinsed\t \u00C2\u00A0in\t \u00C2\u00A0Tris-\u00C2\u00AD\u00E2\u0080\u0090buffered\t \u00C2\u00A0saline\t \u00C2\u00A0with\t \u00C2\u00A0Triton\t \u00C2\u00A0X-\u00C2\u00AD\u00E2\u0080\u0090100\t \u00C2\u00A0(TBST)\t \u00C2\u00A0and\t \u00C2\u00A0blocked\t \u00C2\u00A0with\t \u00C2\u00A05%\t \u00C2\u00A0skim\t \u00C2\u00A0milk\t \u00C2\u00A0in\t \u00C2\u00A0TBST\t \u00C2\u00A0for\t \u00C2\u00A01h.\t \u00C2\u00A0The\t \u00C2\u00A0membranes\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0rinsed\t \u00C2\u00A0with\t \u00C2\u00A0TBST\t \u00C2\u00A0and\t \u00C2\u00A0incubated\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0primary\t \u00C2\u00A0antibody\t \u00C2\u00A0(diluted\t \u00C2\u00A0in\t \u00C2\u00A03%\t \u00C2\u00A0BSA\t \u00C2\u00A0in\t \u00C2\u00A0PBS\t \u00C2\u00A0and\t \u00C2\u00A00.01%\t \u00C2\u00A0NaN3)\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0cold\t \u00C2\u00A0room\t \u00C2\u00A0overnight\t \u00C2\u00A0with\t \u00C2\u00A0constant\t \u00C2\u00A0agitation.\t \u00C2\u00A0\t \u00C2\u00A0TBST\t \u00C2\u00A050mM\t \u00C2\u00A0Tris-\u00C2\u00AD\u00E2\u0080\u0090HCl\t \u00C2\u00A0150mM\t \u00C2\u00A0NaCl\t \u00C2\u00A00.01%\t \u00C2\u00A0TritonX-\u00C2\u00AD\u00E2\u0080\u0090100\t \u00C2\u00A0Adjust\t \u00C2\u00A0to\t \u00C2\u00A0pH7.4\t \u00C2\u00A0Following\t \u00C2\u00A0primary\t \u00C2\u00A0antibody\t \u00C2\u00A0incubation,\t \u00C2\u00A0each\t \u00C2\u00A0membrane\t \u00C2\u00A0was\t \u00C2\u00A0washed\t \u00C2\u00A03X\t \u00C2\u00A05min\t \u00C2\u00A0with\t \u00C2\u00A0TBST\t \u00C2\u00A0 73 and\t \u00C2\u00A0incubated\t \u00C2\u00A0with\t \u00C2\u00A0HRP-\u00C2\u00AD\u00E2\u0080\u0090conjugated\t \u00C2\u00A0secondary\t \u00C2\u00A0antibodies\t \u00C2\u00A0for\t \u00C2\u00A01h\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0with\t \u00C2\u00A0agitation\t \u00C2\u00A0(mouse\t \u00C2\u00A0IgG-\u00C2\u00AD\u00E2\u0080\u0090HRP:\t \u00C2\u00A0Perkin-\u00C2\u00AD\u00E2\u0080\u0090Elmer,\t \u00C2\u00A0cat.\t \u00C2\u00A0#NEF8822001EA;\t \u00C2\u00A0rabbit\t \u00C2\u00A0IgG-\u00C2\u00AD\u00E2\u0080\u0090HRP:\t \u00C2\u00A0Perkin-\u00C2\u00AD\u00E2\u0080\u0090Elmer,\t \u00C2\u00A0cat.\t \u00C2\u00A0#NEF812001EA;\t \u00C2\u00A0rat\t \u00C2\u00A0IgG-\u00C2\u00AD\u00E2\u0080\u0090HRP:\t \u00C2\u00A0Amersham,\t \u00C2\u00A0cat.\t \u00C2\u00A0#NA935;\t \u00C2\u00A0diluted\t \u00C2\u00A01:5000\t \u00C2\u00A0in\t \u00C2\u00A03%\t \u00C2\u00A0skim\t \u00C2\u00A0milk\t \u00C2\u00A0in\t \u00C2\u00A0TBST).\t \u00C2\u00A0After\t \u00C2\u00A0another\t \u00C2\u00A03X5min\t \u00C2\u00A0washes\t \u00C2\u00A0with\t \u00C2\u00A0TBST,\t \u00C2\u00A0each\t \u00C2\u00A0membrane\t \u00C2\u00A0was\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A01mL\t \u00C2\u00A0Luminata\t \u00C2\u00A0Crescendo\t \u00C2\u00A0Western\t \u00C2\u00A0HRP\t \u00C2\u00A0substrate\t \u00C2\u00A0(Fischer;\t \u00C2\u00A0cat.\t \u00C2\u00A0#WBLUR0500)\t \u00C2\u00A0to\t \u00C2\u00A0detect\t \u00C2\u00A0protein\t \u00C2\u00A0bands\t \u00C2\u00A0via\t \u00C2\u00A0chemiluminescent\t \u00C2\u00A0imaging.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0primary\t \u00C2\u00A0antibodies\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0work\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0are\t \u00C2\u00A0listed\t \u00C2\u00A0in\t \u00C2\u00A0Table\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00901.\t \u00C2\u00A0\t \u00C2\u00A02.3.3 CO-\u00C2\u00AD\u00E2\u0080\u0090IMMUNOPRECIPITATION\t \u00C2\u00A0(CO-\u00C2\u00AD\u00E2\u0080\u0090IP)\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0cultured\t \u00C2\u00A0on\t \u00C2\u00A010cm\t \u00C2\u00A0dishes\t \u00C2\u00A0in\t \u00C2\u00A0preparation\t \u00C2\u00A0for\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090IP.\t \u00C2\u00A0Each\t \u00C2\u00A0dish\t \u00C2\u00A0was\t \u00C2\u00A0washed\t \u00C2\u00A02X\t \u00C2\u00A0with\t \u00C2\u00A05mL\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0PBS,\t \u00C2\u00A0and\t \u00C2\u00A01mL\t \u00C2\u00A0RIPA\t \u00C2\u00A0buffer\t \u00C2\u00A0(with\t \u00C2\u00A0protease\t \u00C2\u00A0inhibitor\t \u00C2\u00A0cocktail)\t \u00C2\u00A0was\t \u00C2\u00A0subsequently\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0plate.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0scraped\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0dishes\t \u00C2\u00A0with\t \u00C2\u00A0specialized\t \u00C2\u00A0cell\t \u00C2\u00A0scrapers,\t \u00C2\u00A0collected\t \u00C2\u00A0into\t \u00C2\u00A01.5mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tubes\t \u00C2\u00A0and\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0rest\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0for\t \u00C2\u00A030min.\t \u00C2\u00A0The\t \u00C2\u00A0lysates\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0passed\t \u00C2\u00A03X\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A027-\u00C2\u00AD\u00E2\u0080\u0090gauge\t \u00C2\u00A0needle\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A014,000rpm\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0at\t \u00C2\u00A04\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0to\t \u00C2\u00A0precipitate\t \u00C2\u00A0cell\t \u00C2\u00A0debris.\t \u00C2\u00A0Protein\t \u00C2\u00A0concentration\t \u00C2\u00A0was\t \u00C2\u00A0measured\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A02.3.1.\t \u00C2\u00A0Protein\t \u00C2\u00A0G\t \u00C2\u00A0Sepharose\t \u00C2\u00A0beads\t \u00C2\u00A0(GE\t \u00C2\u00A0Healthcare,\t \u00C2\u00A0cat.\t \u00C2\u00A0#17-\u00C2\u00AD\u00E2\u0080\u00900618-\u00C2\u00AD\u00E2\u0080\u009001)\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0monoclonal\t \u00C2\u00A0and/or\t \u00C2\u00A0mouse\t \u00C2\u00A0primary\t \u00C2\u00A0antibodies,\t \u00C2\u00A0while\t \u00C2\u00A0Protein\t \u00C2\u00A0A\t \u00C2\u00A0Sepharose\t \u00C2\u00A0CL-\u00C2\u00AD\u00E2\u0080\u00904B\t \u00C2\u00A0(GE\t \u00C2\u00A0Healthcare,\t \u00C2\u00A0cat.\t \u00C2\u00A0#17-\u00C2\u00AD\u00E2\u0080\u00900780-\u00C2\u00AD\u00E2\u0080\u009001)\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0polyclonal\t \u00C2\u00A0and/or\t \u00C2\u00A0rabbit\t \u00C2\u00A0antibodies.\t \u00C2\u00A0To\t \u00C2\u00A0prepare\t \u00C2\u00A0Protein\t \u00C2\u00A0A\t \u00C2\u00A0beads,\t \u00C2\u00A0100mg\t \u00C2\u00A0of\t \u00C2\u00A0powder\t \u00C2\u00A0was\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A01mL\t \u00C2\u00A0PBS\t \u00C2\u00A0and\t \u00C2\u00A0rotated\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A04h.\t \u00C2\u00A0To\t \u00C2\u00A0precipitate\t \u00C2\u00A0the\t \u00C2\u00A0beads,\t \u00C2\u00A0each\t \u00C2\u00A0tube\t \u00C2\u00A0was\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A02,000rpm\t \u00C2\u00A0for\t \u00C2\u00A01min.\t \u00C2\u00A0The\t \u00C2\u00A0beads\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0three\t \u00C2\u00A0 74 times\t \u00C2\u00A0by\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090suspending\t \u00C2\u00A0in\t \u00C2\u00A01mL\t \u00C2\u00A0of\t \u00C2\u00A0PBS,\t \u00C2\u00A0incubating\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0centrifuging\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0above\t \u00C2\u00A0each\t \u00C2\u00A0time.\t \u00C2\u00A0Protein\t \u00C2\u00A0G\t \u00C2\u00A0beads\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0way\t \u00C2\u00A0prior\t \u00C2\u00A0use.\t \u00C2\u00A0To\t \u00C2\u00A0eliminate\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0directly\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0beads,\t \u00C2\u00A0samples\t \u00C2\u00A0were\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090cleared\t \u00C2\u00A0by\t \u00C2\u00A0adding\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0appropriate\t \u00C2\u00A0beads\t \u00C2\u00A0to\t \u00C2\u00A01\u00CE\u00BCg\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0sample\t \u00C2\u00A0and\t \u00C2\u00A0rotated\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01h.\t \u00C2\u00A0Samples\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A02,000rpm\t \u00C2\u00A0for\t \u00C2\u00A01min\t \u00C2\u00A0and\t \u00C2\u00A0transferred\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A0tube.\t \u00C2\u00A04\u00CE\u00BCg\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0appropriate\t \u00C2\u00A0normal\t \u00C2\u00A0IgG\t \u00C2\u00A0(rabbit:\t \u00C2\u00A0Santa\t \u00C2\u00A0cruz,\t \u00C2\u00A0cat.\t \u00C2\u00A0#sc-\u00C2\u00AD\u00E2\u0080\u00902027;\t \u00C2\u00A0mouse:\t \u00C2\u00A0Santa\t \u00C2\u00A0cruz,\t \u00C2\u00A0cat.\t \u00C2\u00A0#sc-\u00C2\u00AD\u00E2\u0080\u00902025)\t \u00C2\u00A0or\t \u00C2\u00A04\u00CE\u00BCg\t \u00C2\u00A0of\t \u00C2\u00A0primary\t \u00C2\u00A0antibody\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0sample\t \u00C2\u00A0and\t \u00C2\u00A0rotated\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0overnight.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0following\t \u00C2\u00A0day\t \u00C2\u00A060\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0Protein\t \u00C2\u00A0A\t \u00C2\u00A0or\t \u00C2\u00A0G\t \u00C2\u00A0beads\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0samples\t \u00C2\u00A0were\t \u00C2\u00A0rotated\t \u00C2\u00A0for\t \u00C2\u00A04h\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0Beads\t \u00C2\u00A0were\t \u00C2\u00A0precipitated\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0each\t \u00C2\u00A0tube\t \u00C2\u00A0at\t \u00C2\u00A02,000rpm\t \u00C2\u00A0for\t \u00C2\u00A01min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0and\t \u00C2\u00A0discarding\t \u00C2\u00A0the\t \u00C2\u00A0supernatant.\t \u00C2\u00A0The\t \u00C2\u00A0beads\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A0three\t \u00C2\u00A0times\t \u00C2\u00A0as\t \u00C2\u00A0follows:\t \u00C2\u00A0500\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0RIPA\t \u00C2\u00A0buffer\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0tube\t \u00C2\u00A0to\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090suspend\t \u00C2\u00A0the\t \u00C2\u00A0beads,\t \u00C2\u00A0which\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0incubated\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0prior\t \u00C2\u00A0centrifuging\t \u00C2\u00A0at\t \u00C2\u00A02,000rpm\t \u00C2\u00A0for\t \u00C2\u00A01min\t \u00C2\u00A0to\t \u00C2\u00A0collect\t \u00C2\u00A0the\t \u00C2\u00A0beads.\t \u00C2\u00A0A\t \u00C2\u00A030-\u00C2\u00AD\u00E2\u0080\u0090gauge\t \u00C2\u00A0needle\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0completely\t \u00C2\u00A0remove\t \u00C2\u00A0wash\t \u00C2\u00A0buffer\t \u00C2\u00A0each\t \u00C2\u00A0time.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0elute\t \u00C2\u00A0the\t \u00C2\u00A0proteins\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0beads,\t \u00C2\u00A026\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0PBS\t \u00C2\u00A0and\t \u00C2\u00A012.5\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A04X\t \u00C2\u00A0sample\t \u00C2\u00A0buffer\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0heated\t \u00C2\u00A0at\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A0heating\t \u00C2\u00A0block.\t \u00C2\u00A0Beads\t \u00C2\u00A0were\t \u00C2\u00A0deposited\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0at\t \u00C2\u00A02,000rpm\t \u00C2\u00A0for\t \u00C2\u00A01min\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0supernatant\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis.\t \u00C2\u00A02.3.4 HIS-\u00C2\u00AD\u00E2\u0080\u0090TAGGED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0PURIFICATION\t \u00C2\u00A0AND\t \u00C2\u00A0COOMASSIE\t \u00C2\u00A0BLUE\t \u00C2\u00A0STAINING\t \u00C2\u00A0The\t \u00C2\u00A06X\t \u00C2\u00A0his-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0peptides\t \u00C2\u00A0Tat-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0and\t \u00C2\u00A0Tat-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0were\t \u00C2\u00A0purified\t \u00C2\u00A0with\t \u00C2\u00A0HisPur\t \u00C2\u00A0Ni-\u00C2\u00AD\u00E2\u0080\u0090NTA\t \u00C2\u00A0Resin\t \u00C2\u00A0(Thermo\t \u00C2\u00A0Scientific,\t \u00C2\u00A0cat.\t \u00C2\u00A0#88221)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0gravity-\u00C2\u00AD\u00E2\u0080\u0090flow\t \u00C2\u00A0column\t \u00C2\u00A0according\t \u00C2\u00A0 75 to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0protocol.\t \u00C2\u00A0Briefly,\t \u00C2\u00A0BL21\t \u00C2\u00A0bacteria\t \u00C2\u00A0were\t \u00C2\u00A0transformed\t \u00C2\u00A0with\t \u00C2\u00A0plasmid\t \u00C2\u00A0DNA\t \u00C2\u00A0and\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0shaker\t \u00C2\u00A0spinning\t \u00C2\u00A0at\t \u00C2\u00A0250rpm\t \u00C2\u00A0until\t \u00C2\u00A0the\t \u00C2\u00A0absorbance\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0solution\t \u00C2\u00A0reached\t \u00C2\u00A00.6,\t \u00C2\u00A0measured\t \u00C2\u00A0at\t \u00C2\u00A0600nm.\t \u00C2\u00A0Isopropyl\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090D-\u00C2\u00AD\u00E2\u0080\u00901-\u00C2\u00AD\u00E2\u0080\u0090thiogalactopyranoside\t \u00C2\u00A0(IPTG;\t \u00C2\u00A0BioShop,\t \u00C2\u00A0cat.\t \u00C2\u00A0#IPT001)\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0final\t \u00C2\u00A0concentration\t \u00C2\u00A0of\t \u00C2\u00A01mM\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0flask\t \u00C2\u00A0was\t \u00C2\u00A0returned\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0shaker\t \u00C2\u00A0for\t \u00C2\u00A05h.\t \u00C2\u00A0The\t \u00C2\u00A0bacteria\t \u00C2\u00A0pellet\t \u00C2\u00A0was\t \u00C2\u00A0obtained\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0at\t \u00C2\u00A04,000rpm\t \u00C2\u00A0for\t \u00C2\u00A020min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0and\t \u00C2\u00A0discarding\t \u00C2\u00A0the\t \u00C2\u00A0supernatant.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0purify\t \u00C2\u00A06X\t \u00C2\u00A0his-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0peptides,\t \u00C2\u00A0bacteria\t \u00C2\u00A0pellets\t \u00C2\u00A0were\t \u00C2\u00A0lysed\t \u00C2\u00A0via\t \u00C2\u00A0sonication\t \u00C2\u00A0in\t \u00C2\u00A010mL\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0PBS\t \u00C2\u00A0containing\t \u00C2\u00A01%\t \u00C2\u00A0protease-\u00C2\u00AD\u00E2\u0080\u0090phosphatase\t \u00C2\u00A0inhibitor\t \u00C2\u00A0cocktail\t \u00C2\u00A0(Halt\t \u00C2\u00A0Protease\t \u00C2\u00A0Phosphatase\t \u00C2\u00A0Inhibitor,\t \u00C2\u00A0ThermoFisher,\t \u00C2\u00A0cat.\t \u00C2\u00A0#PI78442).\t \u00C2\u00A0Protein\t \u00C2\u00A0extract\t \u00C2\u00A0was\t \u00C2\u00A0purified\t \u00C2\u00A0by\t \u00C2\u00A0centrifuging\t \u00C2\u00A0at\t \u00C2\u00A013,000rpm\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0to\t \u00C2\u00A0remove\t \u00C2\u00A0debris,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A0Equilibration\t \u00C2\u00A0Buffer\t \u00C2\u00A0(20mM\t \u00C2\u00A0sodium\t \u00C2\u00A0phosphate,\t \u00C2\u00A0300mM\t \u00C2\u00A0sodium\t \u00C2\u00A0chloride,\t \u00C2\u00A010mM\t \u00C2\u00A0imidazole\t \u00C2\u00A0at\t \u00C2\u00A0pH7.4)\t \u00C2\u00A0so\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0final\t \u00C2\u00A0volume\t \u00C2\u00A0reached\t \u00C2\u00A010mL.\t \u00C2\u00A0At\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0time,\t \u00C2\u00A0resin\t \u00C2\u00A0columns\t \u00C2\u00A0were\t \u00C2\u00A0equilibrated\t \u00C2\u00A0with\t \u00C2\u00A03mL\t \u00C2\u00A0of\t \u00C2\u00A0Equilibration\t \u00C2\u00A0Buffer.\t \u00C2\u00A0The\t \u00C2\u00A0protein\t \u00C2\u00A0extract\t \u00C2\u00A0mixture\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0column\t \u00C2\u00A0and\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0drain\t \u00C2\u00A0by\t \u00C2\u00A0gravity\t \u00C2\u00A0flow.\t \u00C2\u00A0The\t \u00C2\u00A0flow-\u00C2\u00AD\u00E2\u0080\u0090through\t \u00C2\u00A0was\t \u00C2\u00A0collected\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0tube.\t \u00C2\u00A0The\t \u00C2\u00A0resin\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A02X\t \u00C2\u00A0with\t \u00C2\u00A05mL\t \u00C2\u00A0Wash\t \u00C2\u00A0Buffer\t \u00C2\u00A0(25mM\t \u00C2\u00A0imidazole\t \u00C2\u00A0in\t \u00C2\u00A0PBS,\t \u00C2\u00A0pH7.4)\t \u00C2\u00A0each\t \u00C2\u00A0time\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0flow-\u00C2\u00AD\u00E2\u0080\u0090through\t \u00C2\u00A0was\t \u00C2\u00A0collected\t \u00C2\u00A0in\t \u00C2\u00A0new\t \u00C2\u00A0collection\t \u00C2\u00A0tubes.\t \u00C2\u00A0His-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0proteins\t \u00C2\u00A0were\t \u00C2\u00A0eluted\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0resin\t \u00C2\u00A0three\t \u00C2\u00A0times,\t \u00C2\u00A0each\t \u00C2\u00A0time\t \u00C2\u00A0using\t \u00C2\u00A03mL\t \u00C2\u00A0of\t \u00C2\u00A0Elution\t \u00C2\u00A0Buffer\t \u00C2\u00A0(250mM\t \u00C2\u00A0imidazole\t \u00C2\u00A0in\t \u00C2\u00A0PBS,\t \u00C2\u00A0pH7.4).\t \u00C2\u00A0Each\t \u00C2\u00A0fraction\t \u00C2\u00A0was\t \u00C2\u00A0collected\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0separate\t \u00C2\u00A0tube.\t \u00C2\u00A0 76 To\t \u00C2\u00A0test\t \u00C2\u00A0for\t \u00C2\u00A0peptide\t \u00C2\u00A0purity,\t \u00C2\u00A0the\t \u00C2\u00A0eluted\t \u00C2\u00A0samples\t \u00C2\u00A0were\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A04X\t \u00C2\u00A0sample\t \u00C2\u00A0buffer\t \u00C2\u00A0and\t \u00C2\u00A0separated\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A0SDS-\u00C2\u00AD\u00E2\u0080\u0090PAGE\t \u00C2\u00A0gel\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A02.3.2.\t \u00C2\u00A0The\t \u00C2\u00A0gel\t \u00C2\u00A0was\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0100mL\t \u00C2\u00A0of\t \u00C2\u00A0Coomassie\t \u00C2\u00A0Blue\t \u00C2\u00A0solution\t \u00C2\u00A0\t \u00C2\u00A0(0.1%\t \u00C2\u00A0Coomassie\t \u00C2\u00A0R-\u00C2\u00AD\u00E2\u0080\u0090250\t \u00C2\u00A0in\t \u00C2\u00A040%\t \u00C2\u00A0ethanol\t \u00C2\u00A0and\t \u00C2\u00A010%\t \u00C2\u00A0acetic\t \u00C2\u00A0acid)\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0for\t \u00C2\u00A02h\t \u00C2\u00A0with\t \u00C2\u00A0constant\t \u00C2\u00A0agitation,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0rinsed\t \u00C2\u00A0with\t \u00C2\u00A0distilled\t \u00C2\u00A0water\t \u00C2\u00A0and\t \u00C2\u00A0subsequently\t \u00C2\u00A0destained\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0overnight\t \u00C2\u00A0in\t \u00C2\u00A0100mL\t \u00C2\u00A0destaining\t \u00C2\u00A0solution\t \u00C2\u00A0(10%\t \u00C2\u00A0ethanol\t \u00C2\u00A0and\t \u00C2\u00A07.5%\t \u00C2\u00A0acetic\t \u00C2\u00A0acid).\t \u00C2\u00A0The\t \u00C2\u00A0gel\t \u00C2\u00A0was\t \u00C2\u00A0photographed\t \u00C2\u00A0once\t \u00C2\u00A0the\t \u00C2\u00A0desired\t \u00C2\u00A0background\t \u00C2\u00A0was\t \u00C2\u00A0achieved.\t \u00C2\u00A02.3.5 SEQUENCES\t \u00C2\u00A0OF\t \u00C2\u00A0SYNTHETIC\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0Synthetic\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0were\t \u00C2\u00A0synthesized\t \u00C2\u00A0in\t \u00C2\u00A0house\t \u00C2\u00A0using\t \u00C2\u00A0the\t \u00C2\u00A0Prelude\t \u00C2\u00A0peptide\t \u00C2\u00A0synthesizer\t \u00C2\u00A0or\t \u00C2\u00A0obtained\t \u00C2\u00A0from\t \u00C2\u00A0GL\t \u00C2\u00A0Biochem\t \u00C2\u00A0(Shanghai)\t \u00C2\u00A0in\t \u00C2\u00A0powered\t \u00C2\u00A0form.\t \u00C2\u00A0Each\t \u00C2\u00A0peptide\t \u00C2\u00A0stock\t \u00C2\u00A0was\t \u00C2\u00A0stored\t \u00C2\u00A0in\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009080\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0prior\t \u00C2\u00A0use.\t \u00C2\u00A0Stock\t \u00C2\u00A0peptide\t \u00C2\u00A0solutions\t \u00C2\u00A0were\t \u00C2\u00A0obtained\t \u00C2\u00A0by\t \u00C2\u00A0dissolving\t \u00C2\u00A0a\t \u00C2\u00A0certain\t \u00C2\u00A0amount\t \u00C2\u00A0of\t \u00C2\u00A0peptide\t \u00C2\u00A0in\t \u00C2\u00A0sterile\t \u00C2\u00A0ddH2O,\t \u00C2\u00A0and\t \u00C2\u00A0each\t \u00C2\u00A0stock\t \u00C2\u00A0solution\t \u00C2\u00A0stored\t \u00C2\u00A0in\t \u00C2\u00A0aliquots\t \u00C2\u00A0to\t \u00C2\u00A0avoid\t \u00C2\u00A0freeze-\u00C2\u00AD\u00E2\u0080\u0090thaw\t \u00C2\u00A0cycles.\t \u00C2\u00A0A\t \u00C2\u00A0list\t \u00C2\u00A0of\t \u00C2\u00A0peptides\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0work\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0found\t \u00C2\u00A0in\t \u00C2\u00A0Table\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00902.\t \u00C2\u00A02.4 CELL\t \u00C2\u00A0DEATH\t \u00C2\u00A0ANALYSIS\t \u00C2\u00A0To\t \u00C2\u00A0control\t \u00C2\u00A0for\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0toxicity,\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0was\t \u00C2\u00A0measured\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0Sigma\t \u00C2\u00A0In\t \u00C2\u00A0Vitro\t \u00C2\u00A0Toxicology\t \u00C2\u00A0Assay\t \u00C2\u00A0Kit\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0lactate\t \u00C2\u00A0dehydrogenase\t \u00C2\u00A0(LDH)\t \u00C2\u00A0release\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#TOX7)\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0protocol.\t \u00C2\u00A0Briefly,\t \u00C2\u00A0the\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0mixture\t \u00C2\u00A0was\t \u00C2\u00A0made\t \u00C2\u00A0by\t \u00C2\u00A0mixing\t \u00C2\u00A0equal\t \u00C2\u00A0volumes\t \u00C2\u00A0of\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0substrate\t \u00C2\u00A0solution,\t \u00C2\u00A0LHD\t \u00C2\u00A0assay\t \u00C2\u00A0dye\t \u00C2\u00A0solution\t \u00C2\u00A0and\t \u00C2\u00A01X\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0cofactor\t \u00C2\u00A0solution\t \u00C2\u00A0(prepared\t \u00C2\u00A0with\t \u00C2\u00A025mL\t \u00C2\u00A0ddH2O/bottle\t \u00C2\u00A0and\t \u00C2\u00A0stored\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009080\t\u00CC\u008A \u00C2\u00A0C).\t \u00C2\u00A0The\t \u00C2\u00A0 77 mixture\t \u00C2\u00A0was\t \u00C2\u00A0kept\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0before\t \u00C2\u00A0use.\t \u00C2\u00A0To\t \u00C2\u00A0initiate\t \u00C2\u00A0the\t \u00C2\u00A0reaction,\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0Costar\t \u00C2\u00A096-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0EIA/RIA\t \u00C2\u00A0plate\t \u00C2\u00A0(Fisher,\t \u00C2\u00A0cat.#0720035),\t \u00C2\u00A0100\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0solution\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A050\u00CE\u00BCL\t \u00C2\u00A0cell\t \u00C2\u00A0culture\t \u00C2\u00A0media.\t \u00C2\u00A0Each\t \u00C2\u00A0sample\t \u00C2\u00A0was\t \u00C2\u00A0tested\t \u00C2\u00A0in\t \u00C2\u00A0duplicates.\t \u00C2\u00A050\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0ddH2O\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0negative\t \u00C2\u00A0control.\t \u00C2\u00A0For\t \u00C2\u00A0positive\t \u00C2\u00A0control,\t \u00C2\u00A0100\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0lysis\t \u00C2\u00A0solution\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0cells\t \u00C2\u00A0in\t \u00C2\u00A01mL\t \u00C2\u00A0cell\t \u00C2\u00A0culture\t \u00C2\u00A0media\t \u00C2\u00A0and\t \u00C2\u00A0returned\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0incubator\t \u00C2\u00A0for\t \u00C2\u00A045min.\t \u00C2\u00A0Media\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0collected\t \u00C2\u00A0and\t \u00C2\u00A0briefly\t \u00C2\u00A0spun\t \u00C2\u00A0down\t \u00C2\u00A0to\t \u00C2\u00A0pellet\t \u00C2\u00A0the\t \u00C2\u00A0debris,\t \u00C2\u00A0and\t \u00C2\u00A050\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0supernatant\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0positive\t \u00C2\u00A0control.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0plate\t \u00C2\u00A0was\t \u00C2\u00A0covered\t \u00C2\u00A0with\t \u00C2\u00A0aluminum\t \u00C2\u00A0foil\t \u00C2\u00A0and\t \u00C2\u00A0placed\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A0heated\t \u00C2\u00A0shaker\t \u00C2\u00A0(300rpm,\t \u00C2\u00A025\t\u00CC\u008A \u00C2\u00A0C)\t \u00C2\u00A0for\t \u00C2\u00A030-\u00C2\u00AD\u00E2\u0080\u009060min.\t \u00C2\u00A0Absorbance\t \u00C2\u00A0was\t \u00C2\u00A0measure\t \u00C2\u00A0spectrophotometrically\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0microplate\t \u00C2\u00A0reader\t \u00C2\u00A0(mQuant,\t \u00C2\u00A0Bio-\u00C2\u00AD\u00E2\u0080\u0090TEK\t \u00C2\u00A0instruments)\t \u00C2\u00A0at\t \u00C2\u00A0a\t \u00C2\u00A0wavelength\t \u00C2\u00A0of\t \u00C2\u00A0490nm,\t \u00C2\u00A0and\t \u00C2\u00A0background\t \u00C2\u00A0absorbance\t \u00C2\u00A0was\t \u00C2\u00A0measured\t \u00C2\u00A0at\t \u00C2\u00A0690nm.\t \u00C2\u00A02.5 HISTOLOGY,\t \u00C2\u00A0IMMUNOCYTOCHEMISTRY,\t \u00C2\u00A0AND\t \u00C2\u00A0IMMUNOHISTOCHEMISTRY\t \u00C2\u00A02.5.1 2,3,5-\u00C2\u00AD\u00E2\u0080\u0090TRIPHENYLTETRAZOLIUM\t \u00C2\u00A0CHLORIDE\t \u00C2\u00A0(TTC)\t \u00C2\u00A0STAINING\t \u00C2\u00A0To\t \u00C2\u00A0aid\t \u00C2\u00A0the\t \u00C2\u00A0visualization\t \u00C2\u00A0of\t \u00C2\u00A0ischemic\t \u00C2\u00A0damage,\t \u00C2\u00A0coronal\t \u00C2\u00A0brain\t \u00C2\u00A0slices\t \u00C2\u00A0(2mm\t \u00C2\u00A0thick)\t \u00C2\u00A0were\t \u00C2\u00A0placed\t \u00C2\u00A0sequentially\t \u00C2\u00A0(rostral\t \u00C2\u00A0to\t \u00C2\u00A0caudal)\t \u00C2\u00A0into\t \u00C2\u00A0each\t \u00C2\u00A0well\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A06-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plate\t \u00C2\u00A0containing\t \u00C2\u00A0freshly\t \u00C2\u00A0made\t \u00C2\u00A01%\t \u00C2\u00A0TTC\t \u00C2\u00A0solution\t \u00C2\u00A0in\t \u00C2\u00A0ddH2O.\t \u00C2\u00A0The\t \u00C2\u00A0tissue\t \u00C2\u00A0plate\t \u00C2\u00A0was\t \u00C2\u00A0wrapped\t \u00C2\u00A0in\t \u00C2\u00A0aluminum\t \u00C2\u00A0foil\t \u00C2\u00A0and\t \u00C2\u00A0brain\t \u00C2\u00A0slices\t \u00C2\u00A0were\t \u00C2\u00A0stained\t \u00C2\u00A0for\t \u00C2\u00A015min\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0without\t \u00C2\u00A0agitation,\t \u00C2\u00A0after\t \u00C2\u00A0which\t \u00C2\u00A0they\t \u00C2\u00A0were\t \u00C2\u00A0removed\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0solution\t \u00C2\u00A0and\t \u00C2\u00A0photographed.\t \u00C2\u00A0\t \u00C2\u00A0 78 2.5.2 FLUORO-\u00C2\u00AD\u00E2\u0080\u0090JADE\t \u00C2\u00A0B\t \u00C2\u00A0STAINING\t \u00C2\u00A0PFA-\u00C2\u00AD\u00E2\u0080\u0090fixed\t \u00C2\u00A0coronal\t \u00C2\u00A0brain\t \u00C2\u00A0slices\t \u00C2\u00A0(30\u00CE\u00BCm\t \u00C2\u00A0thick)\t \u00C2\u00A0were\t \u00C2\u00A0float-\u00C2\u00AD\u00E2\u0080\u0090stained\t \u00C2\u00A0in\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090jade\t \u00C2\u00A0B\t \u00C2\u00A0solution\t \u00C2\u00A0(Millipore,\t \u00C2\u00A0cat.\t \u00C2\u00A0#AG310)\t \u00C2\u00A0to\t \u00C2\u00A0label\t \u00C2\u00A0degenerating\t \u00C2\u00A0neurons.\t \u00C2\u00A0\t \u00C2\u00A0Slices\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A03X\t \u00C2\u00A010min\t \u00C2\u00A0with\t \u00C2\u00A0PBS\t \u00C2\u00A0and\t \u00C2\u00A0equilibrated\t \u00C2\u00A0in\t \u00C2\u00A0100%\t \u00C2\u00A0ethanol\t \u00C2\u00A0for\t \u00C2\u00A03min,\t \u00C2\u00A0and\t \u00C2\u00A0subsequently\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090hydrated\t \u00C2\u00A0with\t \u00C2\u00A070%\t \u00C2\u00A0ethanol\t \u00C2\u00A0for\t \u00C2\u00A01min\t \u00C2\u00A0and\t \u00C2\u00A0ddH2O\t \u00C2\u00A0for\t \u00C2\u00A01\t \u00C2\u00A0min.\t \u00C2\u00A0To\t \u00C2\u00A0decrease\t \u00C2\u00A0background\t \u00C2\u00A0staining,\t \u00C2\u00A0slices\t \u00C2\u00A0were\t \u00C2\u00A0floated\t \u00C2\u00A0in\t \u00C2\u00A00.06%\t \u00C2\u00A0KMnO4\t \u00C2\u00A0solution\t \u00C2\u00A0(prepared\t \u00C2\u00A0in\t \u00C2\u00A0ddH2O)\t \u00C2\u00A0for\t \u00C2\u00A015min\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0with\t \u00C2\u00A0gentle\t \u00C2\u00A0shaking\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0rinsed\t \u00C2\u00A0with\t \u00C2\u00A0ddH2O\t \u00C2\u00A0for\t \u00C2\u00A01min.\t \u00C2\u00A0Slices\t \u00C2\u00A0were\t \u00C2\u00A0subsequently\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A00.001%\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090Jade\t \u00C2\u00A0B\t \u00C2\u00A0solution\t \u00C2\u00A0(diluted\t \u00C2\u00A0from\t \u00C2\u00A0a\t \u00C2\u00A00.01%\t \u00C2\u00A0stock\t \u00C2\u00A0solution\t \u00C2\u00A0in\t \u00C2\u00A0180\u00CE\u00BCL\t \u00C2\u00A00.1%\t \u00C2\u00A0acetic\t \u00C2\u00A0acid)\t \u00C2\u00A0for\t \u00C2\u00A040min\t \u00C2\u00A0with\t \u00C2\u00A0gentle\t \u00C2\u00A0agitation,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A03X\t \u00C2\u00A01min\t \u00C2\u00A0with\t \u00C2\u00A0ddH2O\t \u00C2\u00A0prior\t \u00C2\u00A0mounting\t \u00C2\u00A0onto\t \u00C2\u00A0glass\t \u00C2\u00A0slides\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0dark\t \u00C2\u00A0room.\t \u00C2\u00A0The\t \u00C2\u00A0slides\t \u00C2\u00A0were\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0dry\t \u00C2\u00A0overnight\t \u00C2\u00A0away\t \u00C2\u00A0from\t \u00C2\u00A0light,\t \u00C2\u00A0and\t \u00C2\u00A0were\t \u00C2\u00A0cleared\t \u00C2\u00A0in\t \u00C2\u00A03X\t \u00C2\u00A02min\t \u00C2\u00A0xylene\t \u00C2\u00A0and\t \u00C2\u00A0mounted\t \u00C2\u00A0with\t \u00C2\u00A0Permount\t \u00C2\u00A0media\t \u00C2\u00A0(Fischer\t \u00C2\u00A0Scientific,\t \u00C2\u00A0cat.\t \u00C2\u00A0#SP15-\u00C2\u00AD\u00E2\u0080\u0090500)\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0day.\t \u00C2\u00A0Images\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0hippocampus\t \u00C2\u00A0were\t \u00C2\u00A0taken\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0fluorescent\t \u00C2\u00A0microscope,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0Fluoro\t \u00C2\u00A0Jade-\u00C2\u00AD\u00E2\u0080\u0090positive\t \u00C2\u00A0neurons\t \u00C2\u00A0was\t \u00C2\u00A0counted\t \u00C2\u00A0at\t \u00C2\u00A010X\t \u00C2\u00A0magnification\t \u00C2\u00A0using\t \u00C2\u00A0the\t \u00C2\u00A0Image\t \u00C2\u00A0J\t \u00C2\u00A0particle\t \u00C2\u00A0analyzer.\t \u00C2\u00A02.5.3 HEMATOXYLIN\t \u00C2\u00A0(MAYER\u00E2\u0080\u0099S)\t \u00C2\u00A0AND\t \u00C2\u00A0EOSIN\t \u00C2\u00A0Y\t \u00C2\u00A0STAINING\t \u00C2\u00A0PFA-\u00C2\u00AD\u00E2\u0080\u0090fixed\t \u00C2\u00A0coronal\t \u00C2\u00A0brain\t \u00C2\u00A0slices\t \u00C2\u00A0(30\u00CE\u00BCm\t \u00C2\u00A0thick)\t \u00C2\u00A0were\t \u00C2\u00A0mounted\t \u00C2\u00A0and\t \u00C2\u00A0dried\t \u00C2\u00A0on\t \u00C2\u00A0glass\t \u00C2\u00A0slides\t \u00C2\u00A0prior\t \u00C2\u00A0staining.\t \u00C2\u00A0Slides\t \u00C2\u00A0were\t \u00C2\u00A0stained\t \u00C2\u00A0with\t \u00C2\u00A00.1%\t \u00C2\u00A0Mayer\u00E2\u0080\u0099s\t \u00C2\u00A0Hematoxylin\t \u00C2\u00A0solution\t \u00C2\u00A0(Sigma,\t \u00C2\u00A0cat.\t \u00C2\u00A0#MHS1)\t \u00C2\u00A0for\t \u00C2\u00A015\t \u00C2\u00A0min\t \u00C2\u00A0away\t \u00C2\u00A0from\t \u00C2\u00A0light\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A050mL\t \u00C2\u00A0Falcon\t \u00C2\u00A0tube.\t \u00C2\u00A0Slices\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0rinsed\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0under\t \u00C2\u00A0tap\t \u00C2\u00A0water\t \u00C2\u00A0before\t \u00C2\u00A0they\t \u00C2\u00A0were\t \u00C2\u00A0briefly\t \u00C2\u00A0tapped\t \u00C2\u00A0dry\t \u00C2\u00A0and\t \u00C2\u00A0dipped\t \u00C2\u00A012\t \u00C2\u00A0times\t \u00C2\u00A0into\t \u00C2\u00A00.5%\t \u00C2\u00A0Eosin\t \u00C2\u00A0Y\t \u00C2\u00A0 79 (Sigma,\t \u00C2\u00A0E4009-\u00C2\u00AD\u00E2\u0080\u00905G)\t \u00C2\u00A0solution\t \u00C2\u00A0in\t \u00C2\u00A095%\t \u00C2\u00A0ethanol.\t \u00C2\u00A0Slides\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A0by\t \u00C2\u00A0dipping\t \u00C2\u00A0into\t \u00C2\u00A0ddH2O\t \u00C2\u00A0until\t \u00C2\u00A0the\t \u00C2\u00A0eosin\t \u00C2\u00A0stopped\t \u00C2\u00A0streaking,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0sequentially\t \u00C2\u00A0dehydrated\t \u00C2\u00A0with\t \u00C2\u00A050%\t \u00C2\u00A0and\t \u00C2\u00A070%\t \u00C2\u00A0ethanol\t \u00C2\u00A0at\t \u00C2\u00A010\t \u00C2\u00A0dips\t \u00C2\u00A0per\t \u00C2\u00A0concentration.\t \u00C2\u00A0Slides\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0equilibrated\t \u00C2\u00A0in\t \u00C2\u00A095%\t \u00C2\u00A0ethanol\t \u00C2\u00A0for\t \u00C2\u00A030s\t \u00C2\u00A0and\t \u00C2\u00A0100%\t \u00C2\u00A0ethanol\t \u00C2\u00A0for\t \u00C2\u00A01min,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0cleared\t \u00C2\u00A02X\t \u00C2\u00A01min\t \u00C2\u00A0with\t \u00C2\u00A0xylene\t \u00C2\u00A0before\t \u00C2\u00A0mounting\t \u00C2\u00A0with\t \u00C2\u00A0Permount\t \u00C2\u00A0(Fischer\t \u00C2\u00A0Scientific,\t \u00C2\u00A0cat.\t \u00C2\u00A0#SP15-\u00C2\u00AD\u00E2\u0080\u0090500)\t \u00C2\u00A0media.\t \u00C2\u00A02.5.4 IMMUNOCYTOCHEMISTRY\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0COS7\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0cultured\t \u00C2\u00A0on\t \u00C2\u00A0PDL-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0coverslips\t \u00C2\u00A0in\t \u00C2\u00A024-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0tissue\t \u00C2\u00A0plates\t \u00C2\u00A0and\t \u00C2\u00A0transfected\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0or\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A02.1.2.\t \u00C2\u00A0One\t \u00C2\u00A0day\t \u00C2\u00A0after\t \u00C2\u00A0transfection\t \u00C2\u00A0the\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0twice\t \u00C2\u00A0with\t \u00C2\u00A0ice-\u00C2\u00AD\u00E2\u0080\u0090cold\t \u00C2\u00A0PBS\t \u00C2\u00A0and\t \u00C2\u00A0fixed\t \u00C2\u00A0with\t \u00C2\u00A04%\t \u00C2\u00A0PFA\t \u00C2\u00A0solution\t \u00C2\u00A0for\t \u00C2\u00A01h\t \u00C2\u00A0at\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0They\t \u00C2\u00A0were\t \u00C2\u00A0subsequently\t \u00C2\u00A0washed\t \u00C2\u00A0three\t \u00C2\u00A0times\t \u00C2\u00A0with\t \u00C2\u00A0PBS\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0each\t \u00C2\u00A0time.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0permeablized\t \u00C2\u00A0with\t \u00C2\u00A00.25%\t \u00C2\u00A0TritonX-\u00C2\u00AD\u00E2\u0080\u0090100\t \u00C2\u00A0in\t \u00C2\u00A0PBS\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0without\t \u00C2\u00A0agitation\t \u00C2\u00A0and\t \u00C2\u00A0rinsed\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0with\t \u00C2\u00A0PBS.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0coverslips\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0transferred\t \u00C2\u00A0onto\t \u00C2\u00A0a\t \u00C2\u00A0container\t \u00C2\u00A0lined\t \u00C2\u00A0with\t \u00C2\u00A0parafilm.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0blocked\t \u00C2\u00A0with\t \u00C2\u00A010%\t \u00C2\u00A0BSA\t \u00C2\u00A0in\t \u00C2\u00A0PBS\t \u00C2\u00A0for\t \u00C2\u00A030min\t \u00C2\u00A0at\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0without\t \u00C2\u00A0agitation,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0incubated\t \u00C2\u00A0with\t \u00C2\u00A0primary\t \u00C2\u00A0antibody\t \u00C2\u00A0(anti\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00901,\t \u00C2\u00A0Abcam,\t \u00C2\u00A0cat.\t \u00C2\u00A0#ab13523)\t \u00C2\u00A0diluted\t \u00C2\u00A01:100\t \u00C2\u00A0in\t \u00C2\u00A03%\t \u00C2\u00A0BSA\t \u00C2\u00A0in\t \u00C2\u00A0PBS\t \u00C2\u00A0overnight\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0with\t \u00C2\u00A0agitation.\t \u00C2\u00A0The\t \u00C2\u00A0next\t \u00C2\u00A0day,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0six\t \u00C2\u00A0times\t \u00C2\u00A0with\t \u00C2\u00A0PBS\t \u00C2\u00A0for\t \u00C2\u00A02min\t \u00C2\u00A0each\t \u00C2\u00A0time\t \u00C2\u00A0with\t \u00C2\u00A0gentle\t \u00C2\u00A0agitation.\t \u00C2\u00A0They\t \u00C2\u00A0were\t \u00C2\u00A0next\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0Molecular\t \u00C2\u00A0Probes\t \u00C2\u00A0Alexa\t \u00C2\u00A0Fluor\t \u00C2\u00A0555\t \u00C2\u00A0secondary\t \u00C2\u00A0antibody\t \u00C2\u00A0(1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0in\t \u00C2\u00A03%\t \u00C2\u00A0BSA,\t \u00C2\u00A0Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#21428)\t \u00C2\u00A0at\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A045min,\t \u00C2\u00A0and\t \u00C2\u00A0washed\t \u00C2\u00A0again\t \u00C2\u00A06X\t \u00C2\u00A02min\t \u00C2\u00A0with\t \u00C2\u00A0PBS.\t \u00C2\u00A0\t \u00C2\u00A0 80 To\t \u00C2\u00A0help\t \u00C2\u00A0visualize\t \u00C2\u00A0the\t \u00C2\u00A0nuclei,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0stained\t \u00C2\u00A0with\t \u00C2\u00A0DAPI\t \u00C2\u00A0(4',6-\u00C2\u00AD\u00E2\u0080\u0090diamidino-\u00C2\u00AD\u00E2\u0080\u00902-\u00C2\u00AD\u00E2\u0080\u0090phenylindole;\t \u00C2\u00A01:500\t \u00C2\u00A0in\t \u00C2\u00A0PBS)\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0without\t \u00C2\u00A0agitation,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A06X\t \u00C2\u00A02min\t \u00C2\u00A0before\t \u00C2\u00A0mounting\t \u00C2\u00A0on\t \u00C2\u00A0glass\t \u00C2\u00A0slides\t \u00C2\u00A0with\t \u00C2\u00A0ProLong\t \u00C2\u00A0Gold\t \u00C2\u00A0Antifade\t \u00C2\u00A0Reagent\t \u00C2\u00A0(Molecular\t \u00C2\u00A0Probes,\t \u00C2\u00A0cat.\t \u00C2\u00A0#P36930).\t \u00C2\u00A0The\t \u00C2\u00A0slides\t \u00C2\u00A0were\t \u00C2\u00A0dried\t \u00C2\u00A0overnight\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0cabinet\t \u00C2\u00A0away\t \u00C2\u00A0from\t \u00C2\u00A0light\t \u00C2\u00A0before\t \u00C2\u00A0imaging\t \u00C2\u00A0with\t \u00C2\u00A0Leica\t \u00C2\u00A0DMIRE2.\t \u00C2\u00A02.5.5 IMMUNOHISTOCHEMISTRY\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0DAPK1\t \u00C2\u00A0PFA-\u00C2\u00AD\u00E2\u0080\u0090fixed\t \u00C2\u00A0coronal\t \u00C2\u00A0brain\t \u00C2\u00A0slices\t \u00C2\u00A0(30\u00CE\u00BCm\t \u00C2\u00A0thick)\t \u00C2\u00A0were\t \u00C2\u00A0float-\u00C2\u00AD\u00E2\u0080\u0090stained\t \u00C2\u00A0for\t \u00C2\u00A0immunohistochemical\t \u00C2\u00A0labeling.\t \u00C2\u00A0Slices\t \u00C2\u00A0were\t \u00C2\u00A0washed\t \u00C2\u00A0with\t \u00C2\u00A00.1M\t \u00C2\u00A0PBS\t \u00C2\u00A0for\t \u00C2\u00A03X\t \u00C2\u00A010min\t \u00C2\u00A0with\t \u00C2\u00A0gentle\t \u00C2\u00A0agitation,\t \u00C2\u00A0before\t \u00C2\u00A0being\t \u00C2\u00A0simultaneously\t \u00C2\u00A0blocked\t \u00C2\u00A0and\t \u00C2\u00A0permeablized\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0for\t \u00C2\u00A030min\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0solution\t \u00C2\u00A0containing\t \u00C2\u00A00.1M\t \u00C2\u00A0PBS,\t \u00C2\u00A01%\t \u00C2\u00A0BSA\t \u00C2\u00A0and\t \u00C2\u00A00.2%\t \u00C2\u00A0Triton\t \u00C2\u00A0X-\u00C2\u00AD\u00E2\u0080\u0090100.\t \u00C2\u00A0They\t \u00C2\u00A0were\t \u00C2\u00A0subsequently\t \u00C2\u00A0rinsed\t \u00C2\u00A0with\t \u00C2\u00A00.1M\t \u00C2\u00A0PBS/0.5%\t \u00C2\u00A0BSA\t \u00C2\u00A0for\t \u00C2\u00A03X\t \u00C2\u00A010min\t \u00C2\u00A0with\t \u00C2\u00A0agitation,\t \u00C2\u00A0and\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0antibody\t \u00C2\u00A0(diluted\t \u00C2\u00A01:100\t \u00C2\u00A0in\t \u00C2\u00A00.1M\t \u00C2\u00A0PBS\t \u00C2\u00A0and\t \u00C2\u00A00.5%BSA,\t \u00C2\u00A0Sigma,\t \u00C2\u00A0cat.#\t \u00C2\u00A0D1319x)\t \u00C2\u00A0for\t \u00C2\u00A03d\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A04\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0cold\t \u00C2\u00A0room\t \u00C2\u00A0with\t \u00C2\u00A0constant\t \u00C2\u00A0gentle\t \u00C2\u00A0agitation.\t \u00C2\u00A0The\t \u00C2\u00A0slices\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A03X\t \u00C2\u00A010min\t \u00C2\u00A0in\t \u00C2\u00A00.1M\t \u00C2\u00A0PBS/0.5%BSA\t \u00C2\u00A0and\t \u00C2\u00A0subsequently\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0Molecular\t \u00C2\u00A0Probes\t \u00C2\u00A0Alexa\t \u00C2\u00A0Fluor\t \u00C2\u00A0488\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#11034)\t \u00C2\u00A0secondary\t \u00C2\u00A0antibody,\t \u00C2\u00A0diluted\t \u00C2\u00A01:1000\t \u00C2\u00A0in\t \u00C2\u00A00.1M\t \u00C2\u00A0PBS/0.5%\t \u00C2\u00A0BSA\t \u00C2\u00A0overnight\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0cold\t \u00C2\u00A0room\t \u00C2\u00A0with\t \u00C2\u00A0gentle\t \u00C2\u00A0agitation.\t \u00C2\u00A0Slices\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0washed\t \u00C2\u00A03X\t \u00C2\u00A010min\t \u00C2\u00A0in\t \u00C2\u00A00.1M\t \u00C2\u00A0PBS/0.5%\t \u00C2\u00A0BSA\t \u00C2\u00A0with\t \u00C2\u00A0agitation,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0mounted\t \u00C2\u00A0onto\t \u00C2\u00A0glass\t \u00C2\u00A0slides\t \u00C2\u00A0and\t \u00C2\u00A0air-\u00C2\u00AD\u00E2\u0080\u0090dried\t \u00C2\u00A0overnight\t \u00C2\u00A0away\t \u00C2\u00A0from\t \u00C2\u00A0light.\t \u00C2\u00A0Finally,\t \u00C2\u00A0slides\t \u00C2\u00A0were\t \u00C2\u00A0mounted\t \u00C2\u00A0with\t \u00C2\u00A0coverslips\t \u00C2\u00A0with\t \u00C2\u00A0Fluoromount-\u00C2\u00AD\u00E2\u0080\u0090G\t \u00C2\u00A0(SoutherBiotech,\t \u00C2\u00A0cat.\t \u00C2\u00A0#0100-\u00C2\u00AD\u00E2\u0080\u009001).\t \u00C2\u00A0Whole-\u00C2\u00AD\u00E2\u0080\u0090brain\t \u00C2\u00A0images\t \u00C2\u00A0were\t \u00C2\u00A0obtained\t \u00C2\u00A0under\t \u00C2\u00A0a\t \u00C2\u00A0fluorescent\t \u00C2\u00A0microscope.\t \u00C2\u00A0 81 2.6 SIRNA\t \u00C2\u00A0KNOCKOUT,\t \u00C2\u00A0RNA\t \u00C2\u00A0ISOLATION\t \u00C2\u00A0AND\t \u00C2\u00A0RT-\u00C2\u00AD\u00E2\u0080\u0090PCR\t \u00C2\u00A02.6.1 SIRNA\t \u00C2\u00A0KNOCKOUT\t \u00C2\u00A0AND\t \u00C2\u00A0POLY-\u00C2\u00AD\u00E2\u0080\u0090CHAIN\t \u00C2\u00A0REACTION\t \u00C2\u00A0(PCR)\t \u00C2\u00A0To\t \u00C2\u00A0silence\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0expression,\t \u00C2\u00A0siRNA\t \u00C2\u00A0targeting\t \u00C2\u00A0the\t \u00C2\u00A0cytosolic\t \u00C2\u00A0tail\t \u00C2\u00A0of\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0were\t \u00C2\u00A0synthesized\t \u00C2\u00A0through\t \u00C2\u00A0Invitrogen\t \u00C2\u00A0Custom\t \u00C2\u00A0Primers\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0sequence:\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0siRNA:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AUA\t \u00C2\u00A0UCC\t \u00C2\u00A0AGU\t \u00C2\u00A0AUG\t \u00C2\u00A0AUG\t \u00C2\u00A0GCG\t \u00C2\u00A0CUU\t \u00C2\u00A0[dT][dT]\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Duplex\t \u00C2\u00A0siRNA\t \u00C2\u00A0powder\t \u00C2\u00A0was\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090suspended\t \u00C2\u00A0in\t \u00C2\u00A0RNase-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0water\t \u00C2\u00A0to\t \u00C2\u00A0make\t \u00C2\u00A0a\t \u00C2\u00A020\u00CE\u00BCM\t \u00C2\u00A0solution\t \u00C2\u00A0and\t \u00C2\u00A0stored\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0until\t \u00C2\u00A0further\t \u00C2\u00A0use.\t \u00C2\u00A0Primary\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0cultured\t \u00C2\u00A0in\t \u00C2\u00A024-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plates\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A02.1.4\t \u00C2\u00A0until\t \u00C2\u00A06\t \u00C2\u00A0div.\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0siRNA\t \u00C2\u00A0(60pmol)\t \u00C2\u00A0and\t \u00C2\u00A0Silencer\t \u00C2\u00A0negative\t \u00C2\u00A0control\t \u00C2\u00A0siRNA\t \u00C2\u00A0(60pmol,\t \u00C2\u00A0Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#AM4611)\t \u00C2\u00A0were\t \u00C2\u00A0transfected\t \u00C2\u00A0into\t \u00C2\u00A0neurons\t \u00C2\u00A0using\t \u00C2\u00A0Genlantis\t \u00C2\u00A0GeneSilencer\t \u00C2\u00A0siRNA\t \u00C2\u00A0transfection\t \u00C2\u00A0reagent\t \u00C2\u00A0(cat.\t \u00C2\u00A0#T500750)\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0protocol\t \u00C2\u00A0as\t \u00C2\u00A0follows.\t \u00C2\u00A0In\t \u00C2\u00A0one\t \u00C2\u00A01.5mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tube,\t \u00C2\u00A01.8\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0reagent\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A018.2\u00CE\u00BCL\t \u00C2\u00A0Opti-\u00C2\u00AD\u00E2\u0080\u0090MEM;\t \u00C2\u00A0in\t \u00C2\u00A0another\t \u00C2\u00A0tube,\t \u00C2\u00A060pmol\t \u00C2\u00A0siRNA\t \u00C2\u00A0was\t \u00C2\u00A0diluted\t \u00C2\u00A0with\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0diluent\t \u00C2\u00A0and\t \u00C2\u00A0Opti-\u00C2\u00AD\u00E2\u0080\u0090MEM\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0total\t \u00C2\u00A0volume\t \u00C2\u00A0of\t \u00C2\u00A020\u00CE\u00BCL.\t \u00C2\u00A0The\t \u00C2\u00A0two\t \u00C2\u00A0solutions\t \u00C2\u00A0were\t \u00C2\u00A0mixed\t \u00C2\u00A0together\t \u00C2\u00A0and\t \u00C2\u00A0equilibrated\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0for\t \u00C2\u00A010min,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0overlayed\t \u00C2\u00A0onto\t \u00C2\u00A0neurons\t \u00C2\u00A0in\t \u00C2\u00A01mL\t \u00C2\u00A0NF.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0returned\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0incubator\t \u00C2\u00A0for\t \u00C2\u00A06h,\t \u00C2\u00A0after\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0transfection\t \u00C2\u00A0reagents\t \u00C2\u00A0were\t \u00C2\u00A0replaced\t \u00C2\u00A0with\t \u00C2\u00A0complete\t \u00C2\u00A0growth\t \u00C2\u00A0media.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0harvested\t \u00C2\u00A0for\t \u00C2\u00A0immunoblotting\t \u00C2\u00A0and\t \u00C2\u00A0PCR\t \u00C2\u00A0analysis\t \u00C2\u00A072h\t \u00C2\u00A0later.\t \u00C2\u00A0\t \u00C2\u00A0 82 The\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0transcripts\t \u00C2\u00A0were\t \u00C2\u00A0tested\t \u00C2\u00A0with\t \u00C2\u00A0PCR\t \u00C2\u00A0to\t \u00C2\u00A0confirm\t \u00C2\u00A0successful\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0primers\t \u00C2\u00A0(10\u00CE\u00BCM)\t \u00C2\u00A0sequences\t \u00C2\u00A0were\t \u00C2\u00A0as\t \u00C2\u00A0follows:\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\u00E2\u0080\u0093\t \u00C2\u00A0GGT\t \u00C2\u00A0CTC\t \u00C2\u00A0AAG\t \u00C2\u00A0CGC\t \u00C2\u00A0CAT\t \u00C2\u00A0CAT\t \u00C2\u00A0AC\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0antisense:\t \u00C2\u00A05\u00E2\u0080\u0099\u00E2\u0080\u0093\t \u00C2\u00A0GAT\t \u00C2\u00A0GCC\t \u00C2\u00A0CCT\t \u00C2\u00A0CTG\t \u00C2\u00A0GGA\t \u00C2\u00A0AGT\t \u00C2\u00A0TC\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0PCR\t \u00C2\u00A0reaction\t \u00C2\u00A0solution\t \u00C2\u00A0contained\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0components:\t \u00C2\u00A010X\t \u00C2\u00A0Taq\t \u00C2\u00A0Buffer\t \u00C2\u00A0(1\u00CE\u00BCL),\t \u00C2\u00A0cDNA\t \u00C2\u00A0(1\u00CE\u00BCL),\t \u00C2\u00A0Actb\t \u00C2\u00A0primers\t \u00C2\u00A0(0.2\u00CE\u00BCL\t \u00C2\u00A0each),\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0primers\t \u00C2\u00A0(0.2\u00CE\u00BCL\t \u00C2\u00A0each),\t \u00C2\u00A025mM\t \u00C2\u00A0MgCl2\t \u00C2\u00A0solution\t \u00C2\u00A0(0.6\u00CE\u00BCL),\t \u00C2\u00A010mM\t \u00C2\u00A0dNTP\t \u00C2\u00A0solution\t \u00C2\u00A0(0.2\u00CE\u00BCL),\t \u00C2\u00A05000U/mL\t \u00C2\u00A0Taq\t \u00C2\u00A0polymerase\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#10342-\u00C2\u00AD\u00E2\u0080\u0090053)\t \u00C2\u00A0(0.1\u00CE\u00BCL)\t \u00C2\u00A0and\t \u00C2\u00A0ddH2O\t \u00C2\u00A0up\t \u00C2\u00A0to\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0final\t \u00C2\u00A0volume.\t \u00C2\u00A0The\t \u00C2\u00A0PCR\t \u00C2\u00A0program\t \u00C2\u00A0was\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A0follows:\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A03min;\t \u00C2\u00A020\t \u00C2\u00A0cycles\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A030s,\t \u00C2\u00A050\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A030s,\t \u00C2\u00A072\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01min;\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A072\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A010min.\t \u00C2\u00A0PCR\t \u00C2\u00A0products\t \u00C2\u00A0were\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0electrophoresis\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A02%\t \u00C2\u00A0agarose\t \u00C2\u00A0DNA\t \u00C2\u00A0gel\t \u00C2\u00A0in\t \u00C2\u00A0TBE\t \u00C2\u00A0buffer\t \u00C2\u00A0(0.045M\t \u00C2\u00A0Tris-\u00C2\u00AD\u00E2\u0080\u0090borate,\t \u00C2\u00A00.001M\t \u00C2\u00A0EDTA)\t \u00C2\u00A0at\t \u00C2\u00A0100V\t \u00C2\u00A0for\t \u00C2\u00A010min.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0DNA\t \u00C2\u00A0gel\t \u00C2\u00A0was\t \u00C2\u00A0made\t \u00C2\u00A0by\t \u00C2\u00A0dissolving\t \u00C2\u00A0agarose\t \u00C2\u00A0powder\t \u00C2\u00A0in\t \u00C2\u00A0TBE\t \u00C2\u00A0buffer\t \u00C2\u00A0by\t \u00C2\u00A0microwaving\t \u00C2\u00A0the\t \u00C2\u00A0mixture\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0Erlenmeyer\t \u00C2\u00A0flask\t \u00C2\u00A0for\t \u00C2\u00A01.5min;\t \u00C2\u00A0once\t \u00C2\u00A0slightly\t \u00C2\u00A0cooled,\t \u00C2\u00A0SYBR\t \u00C2\u00A0Safe\t \u00C2\u00A0DNA\t \u00C2\u00A0Gel\t \u00C2\u00A0Stain\t \u00C2\u00A0(Life\t \u00C2\u00A0technologies,\t \u00C2\u00A0cat.\t \u00C2\u00A0#S33102)\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0(1:10,000\t \u00C2\u00A0dilution)\t \u00C2\u00A0and\t \u00C2\u00A0mixed\t \u00C2\u00A0by\t \u00C2\u00A0swirling\t \u00C2\u00A0the\t \u00C2\u00A0solution.\t \u00C2\u00A0The\t \u00C2\u00A0mixture\t \u00C2\u00A0was\t \u00C2\u00A0poured\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0standard\t \u00C2\u00A0gel\t \u00C2\u00A0box\t \u00C2\u00A0and\t \u00C2\u00A0covered\t \u00C2\u00A0with\t \u00C2\u00A0aluminum\t \u00C2\u00A0foil\t \u00C2\u00A0while\t \u00C2\u00A0the\t \u00C2\u00A0gels\t \u00C2\u00A0set.\t \u00C2\u00A02.6.2 TOTAL\t \u00C2\u00A0RNA\t \u00C2\u00A0ISOLATION\t \u00C2\u00A0WITH\t \u00C2\u00A0TRIZOL\t \u00C2\u00A0Prior\t \u00C2\u00A0to\t \u00C2\u00A0RNA\t \u00C2\u00A0extraction,\t \u00C2\u00A0all\t \u00C2\u00A0solutions\t \u00C2\u00A0and\t \u00C2\u00A0pipette\t \u00C2\u00A0tips\t \u00C2\u00A0were\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A00.1%\t \u00C2\u00A0diethylpyrocarbonate\t \u00C2\u00A0(DEPC)\t \u00C2\u00A0and\t \u00C2\u00A0autoclaved\t \u00C2\u00A0to\t \u00C2\u00A0remove\t \u00C2\u00A0RNases.\t \u00C2\u00A0Primary\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0cultured\t \u00C2\u00A0in\t \u00C2\u00A06-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0plates\t \u00C2\u00A0until\t \u00C2\u00A014\t \u00C2\u00A0div.\t \u00C2\u00A0Complete\t \u00C2\u00A0growth\t \u00C2\u00A0media\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0 83 were\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A01mL\t \u00C2\u00A0TRIzol\t \u00C2\u00A0reagent\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#15596)\t \u00C2\u00A0per\t \u00C2\u00A0well\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0with\t \u00C2\u00A0agitation\t \u00C2\u00A0to\t \u00C2\u00A0homogenize\t \u00C2\u00A0the\t \u00C2\u00A0tissue.\t \u00C2\u00A0Samples\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0collected\t \u00C2\u00A0into\t \u00C2\u00A01.5mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tubes\t \u00C2\u00A0and\t \u00C2\u00A00.2mL\t \u00C2\u00A0of\t \u00C2\u00A0chloroform\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0tube,\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A015s\t \u00C2\u00A0of\t \u00C2\u00A0shaking\t \u00C2\u00A0to\t \u00C2\u00A0mix\t \u00C2\u00A0and\t \u00C2\u00A02min\t \u00C2\u00A0of\t \u00C2\u00A0incubation\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature.\t \u00C2\u00A0Samples\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A012,000g\t \u00C2\u00A0for\t \u00C2\u00A015min\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0phase\t \u00C2\u00A0separation.\t \u00C2\u00A0The\t \u00C2\u00A0upper\t \u00C2\u00A0aqueous\t \u00C2\u00A0phase\t \u00C2\u00A0(containing\t \u00C2\u00A0total\t \u00C2\u00A0RNA)\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0transferred\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0fresh\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A00.5mL\t \u00C2\u00A0of\t \u00C2\u00A0isopropyl\t \u00C2\u00A0alcohol\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0sample.\t \u00C2\u00A0After\t \u00C2\u00A010min\t \u00C2\u00A0of\t \u00C2\u00A0incubation\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature,\t \u00C2\u00A0the\t \u00C2\u00A0samples\t \u00C2\u00A0were\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A012,000g\t \u00C2\u00A0for\t \u00C2\u00A020min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0to\t \u00C2\u00A0pellet\t \u00C2\u00A0total\t \u00C2\u00A0RNA.\t \u00C2\u00A0After\t \u00C2\u00A0removing\t \u00C2\u00A0the\t \u00C2\u00A0supernatant,\t \u00C2\u00A0the\t \u00C2\u00A0pellet\t \u00C2\u00A0was\t \u00C2\u00A0washed\t \u00C2\u00A0with\t \u00C2\u00A01mL\t \u00C2\u00A075%\t \u00C2\u00A0ethanol\t \u00C2\u00A0by\t \u00C2\u00A0vortexing,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0wash\t \u00C2\u00A0solution\t \u00C2\u00A0was\t \u00C2\u00A0discarded\t \u00C2\u00A0after\t \u00C2\u00A0centrifuging\t \u00C2\u00A0at\t \u00C2\u00A07,500g\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0total\t \u00C2\u00A0RNA\t \u00C2\u00A0pellet\t \u00C2\u00A0was\t \u00C2\u00A0air-\u00C2\u00AD\u00E2\u0080\u0090dried\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0and\t \u00C2\u00A0dissolved\t \u00C2\u00A0in\t \u00C2\u00A040mL\t \u00C2\u00A0RNase-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0water\t \u00C2\u00A0by\t \u00C2\u00A0passing\t \u00C2\u00A0the\t \u00C2\u00A0solution\t \u00C2\u00A0a\t \u00C2\u00A0few\t \u00C2\u00A0times\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0pipette\t \u00C2\u00A0tip\t \u00C2\u00A0and\t \u00C2\u00A0incubating\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A060\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0water\t \u00C2\u00A0bath.\t \u00C2\u00A02.6.3 DNA\t \u00C2\u00A0ELECTROPHORESIS\t \u00C2\u00A0AND\t \u00C2\u00A0REAL-\u00C2\u00AD\u00E2\u0080\u0090TIME\t \u00C2\u00A0PCR\t \u00C2\u00A0(RT-\u00C2\u00AD\u00E2\u0080\u0090PCR)\t \u00C2\u00A0DNA\t \u00C2\u00A0digestion,\t \u00C2\u00A0reverse\t \u00C2\u00A0transcription\t \u00C2\u00A0and\t \u00C2\u00A0real-\u00C2\u00AD\u00E2\u0080\u0090time\t \u00C2\u00A0PCR\t \u00C2\u00A0were\t \u00C2\u00A0completed\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0SuperScript\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090Step\t \u00C2\u00A0RT-\u00C2\u00AD\u00E2\u0080\u0090PCR\t \u00C2\u00A0System\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#10928-\u00C2\u00AD\u00E2\u0080\u0090042)\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0instructions.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0digest\t \u00C2\u00A0DNA,\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0RNase-\u00C2\u00AD\u00E2\u0080\u0090free,\t \u00C2\u00A00.5mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tube:\t \u00C2\u00A0RNA\t \u00C2\u00A0sample\t \u00C2\u00A0(1mg),\t \u00C2\u00A010X\t \u00C2\u00A0DNase\t \u00C2\u00A0I\t \u00C2\u00A0Reaction\t \u00C2\u00A0Buffer\t \u00C2\u00A0(1\u00CE\u00BCL),\t \u00C2\u00A0DNase\t \u00C2\u00A0I\t \u00C2\u00A0Amp\t \u00C2\u00A0Grade,\t \u00C2\u00A01U/mL\t \u00C2\u00A0(1\u00CE\u00BCL)\t \u00C2\u00A0and\t \u00C2\u00A0 84 DEPC-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0water\t \u00C2\u00A0to\t \u00C2\u00A010\u00CE\u00BCL.\t \u00C2\u00A0Samples\t \u00C2\u00A0were\t \u00C2\u00A0incubated\t \u00C2\u00A0for\t \u00C2\u00A015min\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature.\t \u00C2\u00A0To\t \u00C2\u00A0inactivate\t \u00C2\u00A0DNase\t \u00C2\u00A0I,\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A025mM\t \u00C2\u00A0ETDA\t \u00C2\u00A0solution\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0reaction\t \u00C2\u00A0mixture\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0heating\t \u00C2\u00A0for\t \u00C2\u00A010min\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A065\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0water\t \u00C2\u00A0bath.\t \u00C2\u00A0For\t \u00C2\u00A0reverse\t \u00C2\u00A0transcription,\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0reagents\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0nuclease-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A00.5mL\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0tube:\t \u00C2\u00A0500\u00CE\u00BCg/mL\t \u00C2\u00A0OligodT12-\u00C2\u00AD\u00E2\u0080\u009018\t \u00C2\u00A0(1\u00CE\u00BCL),\t \u00C2\u00A0total\t \u00C2\u00A0RNA\t \u00C2\u00A0(1\u00CE\u00BCg),\t \u00C2\u00A010mM\t \u00C2\u00A0dNTP\t \u00C2\u00A0mix\t \u00C2\u00A0(1\u00CE\u00BCL)\t \u00C2\u00A0and\t \u00C2\u00A0nuclease-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0water\t \u00C2\u00A0up\t \u00C2\u00A0to\t \u00C2\u00A012\u00CE\u00BCL.\t \u00C2\u00A0The\t \u00C2\u00A0mixture\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0heated\t \u00C2\u00A0to\t \u00C2\u00A065\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0water\t \u00C2\u00A0bath\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0and\t \u00C2\u00A0quickly\t \u00C2\u00A0chilled\t \u00C2\u00A0on\t \u00C2\u00A0ice.\t \u00C2\u00A0Following\t \u00C2\u00A0brief\t \u00C2\u00A0centrifuging,\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0mixture:\t \u00C2\u00A05X\t \u00C2\u00A0First-\u00C2\u00AD\u00E2\u0080\u0090Strand\t \u00C2\u00A0Buffer\t \u00C2\u00A0(4\u00CE\u00BCL),\t \u00C2\u00A00.1M\t \u00C2\u00A0DTT\t \u00C2\u00A0(2\u00CE\u00BCL),\t \u00C2\u00A040U/\u00CE\u00BCL\t \u00C2\u00A0RNaseOUT\t \u00C2\u00A0(1\u00CE\u00BCL).\t \u00C2\u00A0The\t \u00C2\u00A0tubes\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A042\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0water\t \u00C2\u00A0bath\t \u00C2\u00A0for\t \u00C2\u00A02min,\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0SuperScript\t \u00C2\u00A0II\t \u00C2\u00A0RT\t \u00C2\u00A0(200U)\t \u00C2\u00A0and\t \u00C2\u00A0gentle\t \u00C2\u00A0mixing.\t \u00C2\u00A0The\t \u00C2\u00A0samples\t \u00C2\u00A0were\t \u00C2\u00A0once\t \u00C2\u00A0again\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A042\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0water\t \u00C2\u00A0bath\t \u00C2\u00A0for\t \u00C2\u00A050min,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0reaction\t \u00C2\u00A0was\t \u00C2\u00A0halted\t \u00C2\u00A0by\t \u00C2\u00A0heating\t \u00C2\u00A0samples\t \u00C2\u00A0to\t \u00C2\u00A070\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A015min\t \u00C2\u00A0to\t \u00C2\u00A0obtain\t \u00C2\u00A0the\t \u00C2\u00A0cDNA.\t \u00C2\u00A0Prior\t \u00C2\u00A0to\t \u00C2\u00A0RT-\u00C2\u00AD\u00E2\u0080\u0090PCR,\t \u00C2\u00A0the\t \u00C2\u00A0cDNA\t \u00C2\u00A0quality\t \u00C2\u00A0was\t \u00C2\u00A0first\t \u00C2\u00A0confirmed\t \u00C2\u00A0by\t \u00C2\u00A0conventional\t \u00C2\u00A0PCR\t \u00C2\u00A0by\t \u00C2\u00A0testing\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0transcripts.\t \u00C2\u00A0Actb\t \u00C2\u00A0primers\t \u00C2\u00A0(10\u00CE\u00BCM)\t \u00C2\u00A0each\t \u00C2\u00A0were\t \u00C2\u00A0generous\t \u00C2\u00A0gifts\t \u00C2\u00A0from\t \u00C2\u00A0Dr.\t \u00C2\u00A0Max\t \u00C2\u00A0Cynader.\t \u00C2\u00A0The\t \u00C2\u00A0following\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0Eppendorf\t \u00C2\u00A0PCR\t \u00C2\u00A0tube:\t \u00C2\u00A010X\t \u00C2\u00A0Taq\t \u00C2\u00A0Buffer\t \u00C2\u00A0(1\u00CE\u00BCL),\t \u00C2\u00A0cDNA\t \u00C2\u00A0(1\u00CE\u00BCL),\t \u00C2\u00A0Actb\t \u00C2\u00A0primers\t \u00C2\u00A0(0.4\u00CE\u00BCL\t \u00C2\u00A0each),\t \u00C2\u00A025mM\t \u00C2\u00A0MgCl2\t \u00C2\u00A0solution\t \u00C2\u00A0(0.6\u00CE\u00BCL),\t \u00C2\u00A010mM\t \u00C2\u00A0dNTP\t \u00C2\u00A0solution\t \u00C2\u00A0(0.2\u00CE\u00BCL),\t \u00C2\u00A05000U/mL\t \u00C2\u00A0Taq\t \u00C2\u00A0polymerase\t \u00C2\u00A0(0.1\u00CE\u00BCL)\t \u00C2\u00A0and\t \u00C2\u00A0ddH2O\t \u00C2\u00A0to\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0final\t \u00C2\u00A0volume.\t \u00C2\u00A0The\t \u00C2\u00A0PCR\t \u00C2\u00A0program\t \u00C2\u00A0was\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A0follows:\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A03min;\t \u00C2\u00A035\t \u00C2\u00A0cycles\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A030s,\t \u00C2\u00A050\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A030s,\t \u00C2\u00A072\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01min;\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A072\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A010min.\t \u00C2\u00A0PCR\t \u00C2\u00A0products\t \u00C2\u00A0(7\u00CE\u00BCL/well)\t \u00C2\u00A0were\t \u00C2\u00A0ran\t \u00C2\u00A0along\t \u00C2\u00A0with\t \u00C2\u00A03\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0100bp\t \u00C2\u00A0DNA\t \u00C2\u00A0ladder\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#15628-\u00C2\u00AD\u00E2\u0080\u0090019)\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A01%\t \u00C2\u00A0agarose\t \u00C2\u00A0DNA\t \u00C2\u00A0gel\t \u00C2\u00A0in\t \u00C2\u00A0TBE\t \u00C2\u00A0buffer\t \u00C2\u00A0(0.045M\t \u00C2\u00A0Tris-\u00C2\u00AD\u00E2\u0080\u0090borate,\t \u00C2\u00A00.001M\t \u00C2\u00A0EDTA)\t \u00C2\u00A0at\t \u00C2\u00A0100V\t \u00C2\u00A0for\t \u00C2\u00A010min.\t \u00C2\u00A0\t \u00C2\u00A0 85 For\t \u00C2\u00A0real-\u00C2\u00AD\u00E2\u0080\u0090time\t \u00C2\u00A0PCR,\t \u00C2\u00A0primers\t \u00C2\u00A0for\t \u00C2\u00A0Dapk1\t \u00C2\u00A0and\t \u00C2\u00A0Actb\t \u00C2\u00A0were\t \u00C2\u00A0first\t \u00C2\u00A0prepared\t \u00C2\u00A0as\t \u00C2\u00A010\u00CE\u00BCM\t \u00C2\u00A0solutions.\t \u00C2\u00A0Sequences\t \u00C2\u00A0for\t \u00C2\u00A0Dapk1\t \u00C2\u00A0primers\t \u00C2\u00A0were:\t \u00C2\u00A0Dapk1\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CTC\t \u00C2\u00A0AGT\t \u00C2\u00A0GGT\t \u00C2\u00A0GTC\t \u00C2\u00A0CCG\t \u00C2\u00A0GTG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Dapk1\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GGA\t \u00C2\u00A0AGG\t \u00C2\u00A0ACT\t \u00C2\u00A0GGT\t \u00C2\u00A0GCC\t \u00C2\u00A0TTC\t \u00C2\u00A0TG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0For\t \u00C2\u00A0each\t \u00C2\u00A0sample,\t \u00C2\u00A02\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0cDNA\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0mixture:\t \u00C2\u00A0SYBR\t \u00C2\u00A0Green\t \u00C2\u00A0(Applied\t \u00C2\u00A0Biosystems,\t \u00C2\u00A0cat.\t \u00C2\u00A0#4509155,\t \u00C2\u00A012.5\u00CE\u00BCL),\t \u00C2\u00A0primers\t \u00C2\u00A00.75\u00CE\u00BCL\t \u00C2\u00A0each\t \u00C2\u00A0and\t \u00C2\u00A0ddH2O\t \u00C2\u00A09\u00CE\u00BCL.\t \u00C2\u00A0The\t \u00C2\u00A0mixture\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0MicroAmp\t \u00C2\u00A0Optical\t \u00C2\u00A096-\u00C2\u00AD\u00E2\u0080\u0090well\t \u00C2\u00A0Reaction\t \u00C2\u00A0Plate\t \u00C2\u00A0(Applied\t \u00C2\u00A0Biosystems,\t \u00C2\u00A0cat.\t \u00C2\u00A0#N801-\u00C2\u00AD\u00E2\u0080\u00900560)\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0and\t \u00C2\u00A0tightly\t \u00C2\u00A0sealed\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0plastic\t \u00C2\u00A0cover.\t \u00C2\u00A0Real-\u00C2\u00AD\u00E2\u0080\u0090time\t \u00C2\u00A0PCR\t \u00C2\u00A0was\t \u00C2\u00A0performed\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0ABI\t \u00C2\u00A07300\t \u00C2\u00A0Real\t \u00C2\u00A0Time\t \u00C2\u00A0PCR\t \u00C2\u00A0system\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0parameters:\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0at\t \u00C2\u00A050\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A02min;\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A010min;\t \u00C2\u00A040\t \u00C2\u00A0cycles\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A015s;\t \u00C2\u00A040\t \u00C2\u00A0cycles\t \u00C2\u00A0of\t \u00C2\u00A060\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01min;\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A015s,\t \u00C2\u00A060\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01min,\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A015s\t \u00C2\u00A0and\t \u00C2\u00A060\t\u00CC\u008A \u00C2\u00A0C\t \u00C2\u00A0for\t \u00C2\u00A015s.\t \u00C2\u00A02.7 GENETIC\t \u00C2\u00A0ENGINEERING\t \u00C2\u00A02.7.1 PLASMID\t \u00C2\u00A0AND\t \u00C2\u00A0MINIGENE\t \u00C2\u00A0SOURCE\t \u00C2\u00A0pEGFP-\u00C2\u00AD\u00E2\u0080\u0090N2\t \u00C2\u00A0vector\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0from\t \u00C2\u00A0Clontech\t \u00C2\u00A0(cat.\t \u00C2\u00A0#6081-\u00C2\u00AD\u00E2\u0080\u00901).\t \u00C2\u00A0The\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0pEGFP-\u00C2\u00AD\u00E2\u0080\u0090N2\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0contructed\t \u00C2\u00A0by\t \u00C2\u00A0Drs.\t \u00C2\u00A0J.\t \u00C2\u00A0Wang\t \u00C2\u00A0and\t \u00C2\u00A0J.\t \u00C2\u00A0Lu\t \u00C2\u00A0from\t \u00C2\u00A0UBC.\t \u00C2\u00A0The\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0vector\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0from\t \u00C2\u00A0Invitrogen\t \u00C2\u00A0(now\t \u00C2\u00A0discontinued).\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0T7\t \u00C2\u00A0pTAT-\u00C2\u00AD\u00E2\u0080\u0090HA\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0a\t \u00C2\u00A0kind\t \u00C2\u00A0gift\t \u00C2\u00A0from\t \u00C2\u00A0Dr.\t \u00C2\u00A0S.F.\t \u00C2\u00A0Dowdy\t \u00C2\u00A0of\t \u00C2\u00A0Washington\t \u00C2\u00A0University.\t \u00C2\u00A0The\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0(Origin:\t \u00C2\u00A0Rattus\t \u00C2\u00A0norvegicus)\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0a\t \u00C2\u00A0kind\t \u00C2\u00A0gift\t \u00C2\u00A0from\t \u00C2\u00A0Dr.\t \u00C2\u00A0Y.M.\t \u00C2\u00A0Lu\t \u00C2\u00A0of\t \u00C2\u00A0Louisiana\t \u00C2\u00A0State\t \u00C2\u00A0University\t \u00C2\u00A0Health\t \u00C2\u00A0Sciences\t \u00C2\u00A0Center\t \u00C2\u00A0School\t \u00C2\u00A0of\t \u00C2\u00A0Medicine.\t \u00C2\u00A0The\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0plasmid\t \u00C2\u00A0(Origin:\t \u00C2\u00A0Homo\t \u00C2\u00A0Sapiens)\t \u00C2\u00A0was\t \u00C2\u00A0 86 a\t \u00C2\u00A0kind\t \u00C2\u00A0gift\t \u00C2\u00A0from\t \u00C2\u00A0Dr.\t \u00C2\u00A0Qin\t \u00C2\u00A0Hong.\t \u00C2\u00A0\t \u00C2\u00A0All\t \u00C2\u00A0minigenes\t \u00C2\u00A0were\t \u00C2\u00A0custom\t \u00C2\u00A0synthesized\t \u00C2\u00A0and\t \u00C2\u00A0purified\t \u00C2\u00A0by\t \u00C2\u00A0Integrated\t \u00C2\u00A0DNA\t \u00C2\u00A0Technologies.\t \u00C2\u00A02.7.2 PLASMID\t \u00C2\u00A0AMPLIFICATION,\t \u00C2\u00A0PURIFICATION\t \u00C2\u00A0AND\t \u00C2\u00A0STORAGE\t \u00C2\u00A0Upon\t \u00C2\u00A0receipt\t \u00C2\u00A0or\t \u00C2\u00A0modification,\t \u00C2\u00A0each\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0transformed,\t \u00C2\u00A0amplified\t \u00C2\u00A0and\t \u00C2\u00A0purified\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0MiniPrep\t \u00C2\u00A0kit\t \u00C2\u00A0from\t \u00C2\u00A0QIAGEN\t \u00C2\u00A0or\t \u00C2\u00A0the\t \u00C2\u00A0PureYield\t \u00C2\u00A0MaxiPrep\t \u00C2\u00A0system\t \u00C2\u00A0from\t \u00C2\u00A0Promega\t \u00C2\u00A0(see\t \u00C2\u00A0List\t \u00C2\u00A0of\t \u00C2\u00A0Plasmids\t \u00C2\u00A0on\t \u00C2\u00A0page\t \u00C2\u00A0xxii).\t \u00C2\u00A0Transformation\t \u00C2\u00A0was\t \u00C2\u00A0achieved\t \u00C2\u00A0by\t \u00C2\u00A0incubating\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0plasmid\t \u00C2\u00A0in\t \u00C2\u00A050-\u00C2\u00AD\u00E2\u0080\u009075\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0E.\t \u00C2\u00A0Coli\t \u00C2\u00A0DH5\u00CE\u00B1\t \u00C2\u00A0competent\t \u00C2\u00A0cells\t \u00C2\u00A0for\t \u00C2\u00A040min\t \u00C2\u00A0on\t \u00C2\u00A0ice,\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A060-\u00C2\u00AD\u00E2\u0080\u009090s\t \u00C2\u00A0heat\t \u00C2\u00A0shock\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0water\t \u00C2\u00A0bath.\t \u00C2\u00A0The\t \u00C2\u00A0transformed\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0immediately\t \u00C2\u00A0placed\t \u00C2\u00A0back\t \u00C2\u00A0on\t \u00C2\u00A0ice\t \u00C2\u00A0for\t \u00C2\u00A02min,\t \u00C2\u00A0after\t \u00C2\u00A0which\t \u00C2\u00A0250\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0LB\t \u00C2\u00A0base\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0tube\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0bacteria\t \u00C2\u00A0were\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0grow\t \u00C2\u00A0for\t \u00C2\u00A01h\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0shaker\t \u00C2\u00A0set\t \u00C2\u00A0at\t \u00C2\u00A037\t \u00C2\u00A0C\u00CC\u008A\t \u00C2\u00A0and\t \u00C2\u00A0250rpm.\t \u00C2\u00A0Thereafter,\t \u00C2\u00A020\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0transformed\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0plated\t \u00C2\u00A0evenly\t \u00C2\u00A0onto\t \u00C2\u00A0a\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090dried\t \u00C2\u00A0(20min\t \u00C2\u00A0inside\t \u00C2\u00A0a\t \u00C2\u00A0laminar\t \u00C2\u00A0flow\t \u00C2\u00A0cabinet)\t \u00C2\u00A0agar\t \u00C2\u00A0plate\t \u00C2\u00A0containing\t \u00C2\u00A0the\t \u00C2\u00A0appropriate\t \u00C2\u00A0antibiotic\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0U-\u00C2\u00AD\u00E2\u0080\u0090shaped\t \u00C2\u00A0sterile\t \u00C2\u00A0glass\t \u00C2\u00A0rod\t \u00C2\u00A0in\t \u00C2\u00A0close\t \u00C2\u00A0vicinity\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0Bunsen\t \u00C2\u00A0burner\t \u00C2\u00A0to\t \u00C2\u00A0ensure\t \u00C2\u00A0sterility.\t \u00C2\u00A0The\t \u00C2\u00A0plate\t \u00C2\u00A0was\t \u00C2\u00A0placed\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0chamber\t \u00C2\u00A0upside-\u00C2\u00AD\u00E2\u0080\u0090down\t \u00C2\u00A0and\t \u00C2\u00A0incubated\t \u00C2\u00A0overnight.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0next\t \u00C2\u00A0morning,\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0colony\t \u00C2\u00A0was\t \u00C2\u00A0picked\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0agar\t \u00C2\u00A0plate\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0sterile\t \u00C2\u00A0pipette\t \u00C2\u00A0tip\t \u00C2\u00A0and\t \u00C2\u00A0dipped\t \u00C2\u00A0into\t \u00C2\u00A05mL\t \u00C2\u00A0of\t \u00C2\u00A0LB\t \u00C2\u00A0base\t \u00C2\u00A0(with\t \u00C2\u00A0the\t \u00C2\u00A0appropriate\t \u00C2\u00A0antibiotic,\t \u00C2\u00A0either\t \u00C2\u00A0ampicillin\t \u00C2\u00A0or\t \u00C2\u00A0kanamycin)\t \u00C2\u00A0and\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0amplify\t \u00C2\u00A0overnight\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0loose-\u00C2\u00AD\u00E2\u0080\u0090capped\t \u00C2\u00A0tube\t \u00C2\u00A0at\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0with\t \u00C2\u00A0agitation\t \u00C2\u00A0at\t \u00C2\u00A0250rpm.\t \u00C2\u00A0This\t \u00C2\u00A0culture\t \u00C2\u00A0was\t \u00C2\u00A0either\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0MiniPrep\t \u00C2\u00A0(QIAGEN,\t \u00C2\u00A0QIAprep\t \u00C2\u00A0Spin\t \u00C2\u00A0Miniprep\t \u00C2\u00A0Kit;\t \u00C2\u00A0cat.\t \u00C2\u00A0#27106)\t \u00C2\u00A0the\t \u00C2\u00A0next\t \u00C2\u00A0day\t \u00C2\u00A0or\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0grow\t \u00C2\u00A0further\t \u00C2\u00A0in\t \u00C2\u00A0250mL\t \u00C2\u00A0of\t \u00C2\u00A0LB\t \u00C2\u00A0base\t \u00C2\u00A0(with\t \u00C2\u00A0the\t \u00C2\u00A0appropriate\t \u00C2\u00A0antibiotic)\t \u00C2\u00A0for\t \u00C2\u00A012-\u00C2\u00AD\u00E2\u0080\u009016h\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0manner.\t \u00C2\u00A0This\t \u00C2\u00A0larger\t \u00C2\u00A0amplified\t \u00C2\u00A0 87 bacterial\t \u00C2\u00A0culture\t \u00C2\u00A0was\t \u00C2\u00A0purified\t \u00C2\u00A0the\t \u00C2\u00A0next\t \u00C2\u00A0day\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0MaxiPrep\t \u00C2\u00A0kit\t \u00C2\u00A0(PureYield\u00E2\u0084\u00A2\t \u00C2\u00A0Plasmid\t \u00C2\u00A0Maxiprep\t \u00C2\u00A0System,\t \u00C2\u00A0cat.\t \u00C2\u00A0#A2393).\t \u00C2\u00A0Prior\t \u00C2\u00A0purification,\t \u00C2\u00A00.8mL\t \u00C2\u00A0of\t \u00C2\u00A0bacterial\t \u00C2\u00A0culture\t \u00C2\u00A0was\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A00.8mL\t \u00C2\u00A060%\t \u00C2\u00A0glycerol,\t \u00C2\u00A0wrapped\t \u00C2\u00A0in\t \u00C2\u00A0Tena\t \u00C2\u00A0pads\t \u00C2\u00A0and\t \u00C2\u00A0gradually\t \u00C2\u00A0cooled\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009080\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0freezer\t \u00C2\u00A0for\t \u00C2\u00A0long-\u00C2\u00AD\u00E2\u0080\u0090term\t \u00C2\u00A0storage.\t \u00C2\u00A0After\t \u00C2\u00A0purification,\t \u00C2\u00A0the\t \u00C2\u00A0concentration\t \u00C2\u00A0and\t \u00C2\u00A0quality\t \u00C2\u00A0(A260nm/280nm)\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0measured\t \u00C2\u00A0by\t \u00C2\u00A0spectrometry.\t \u00C2\u00A0The\t \u00C2\u00A0plasmids\t \u00C2\u00A0were\t \u00C2\u00A0then\t \u00C2\u00A0sequence\t \u00C2\u00A0by\t \u00C2\u00A0NAPS\t \u00C2\u00A0at\t \u00C2\u00A0UBC,\t \u00C2\u00A0and\t \u00C2\u00A0upon\t \u00C2\u00A0confirmation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0correct\t \u00C2\u00A0sequence\t \u00C2\u00A0each\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0documented\t \u00C2\u00A0and\t \u00C2\u00A0stored\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009020\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A02.7.3 MINIGENE\t \u00C2\u00A0ANNEALING\t \u00C2\u00A0Each\t \u00C2\u00A0pair\t \u00C2\u00A0of\t \u00C2\u00A0sense\t \u00C2\u00A0and\t \u00C2\u00A0antisense\t \u00C2\u00A0minigene\t \u00C2\u00A0was\t \u00C2\u00A0reconstituted\t \u00C2\u00A0with\t \u00C2\u00A0nuclease-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0ddH2O\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0final\t \u00C2\u00A0concentration\t \u00C2\u00A0of\t \u00C2\u00A01\u00CE\u00BCg/\u00CE\u00BCL.\t \u00C2\u00A0In\t \u00C2\u00A0one\t \u00C2\u00A0tube,\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0minigene\t \u00C2\u00A0solution\t \u00C2\u00A0was\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0RNase-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0ddH2O.\t \u00C2\u00A0The\t \u00C2\u00A0mixture\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0heated\t \u00C2\u00A0to\t \u00C2\u00A065\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A05min\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0water\t \u00C2\u00A0bath,\t \u00C2\u00A0and\t \u00C2\u00A0gradually\t \u00C2\u00A0cooled\t \u00C2\u00A0to\t \u00C2\u00A0below\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0Once\t \u00C2\u00A0cooled,\t \u00C2\u00A030\u00CE\u00BCL\t \u00C2\u00A0distilled\t \u00C2\u00A0water,\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A03M\t \u00C2\u00A0sodium\t \u00C2\u00A0acetate\t \u00C2\u00A0and\t \u00C2\u00A0150\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0100%\t \u00C2\u00A0ethanol\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0each\t \u00C2\u00A0sample.\t \u00C2\u00A0The\t \u00C2\u00A0tubes\t \u00C2\u00A0were\t \u00C2\u00A0placed\t \u00C2\u00A0at\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u009080\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01h\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0centrifuged\t \u00C2\u00A0at\t \u00C2\u00A014,000rpm\t \u00C2\u00A0for\t \u00C2\u00A020min\t \u00C2\u00A0at\t \u00C2\u00A04\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0The\t \u00C2\u00A0supernatant\t \u00C2\u00A0was\t \u00C2\u00A0completely\t \u00C2\u00A0removed\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0pellet\t \u00C2\u00A0(containing\t \u00C2\u00A0the\t \u00C2\u00A0DNA)\t \u00C2\u00A0was\t \u00C2\u00A0air-\u00C2\u00AD\u00E2\u0080\u0090dried\t \u00C2\u00A0for\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u009010min\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090dissolved\t \u00C2\u00A0in\t \u00C2\u00A060\u00CE\u00BCL\t \u00C2\u00A0nuclease-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0water.\t \u00C2\u00A0To\t \u00C2\u00A0verify\t \u00C2\u00A0annealing,\t \u00C2\u00A02\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0minigene\t \u00C2\u00A0was\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0electrophoresis\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A01%\t \u00C2\u00A0agarose\t \u00C2\u00A0DNA\t \u00C2\u00A0gel\t \u00C2\u00A0at\t \u00C2\u00A0100V\t \u00C2\u00A0for\t \u00C2\u00A05min.\t \u00C2\u00A02.7.4 SITE-\u00C2\u00AD\u00E2\u0080\u0090DIRECTED\t \u00C2\u00A0MUTAGENESIS\t \u00C2\u00A0Site-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0was\t \u00C2\u00A0achieved\t \u00C2\u00A0by\t \u00C2\u00A0performing\t \u00C2\u00A0PCR\t \u00C2\u00A0with\t \u00C2\u00A0primers\t \u00C2\u00A0that\t \u00C2\u00A0contained\t \u00C2\u00A0the\t \u00C2\u00A0appropriate\t \u00C2\u00A0nucleotide\t \u00C2\u00A0substitutions.\t \u00C2\u00A0Each\t \u00C2\u00A0PCR\t \u00C2\u00A0reaction\t \u00C2\u00A0contained\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0Pfu\t \u00C2\u00A0Turbo\t \u00C2\u00A0 88 (2.5U/\u00CE\u00BCL,\t \u00C2\u00A0Invitrogen,\t \u00C2\u00A0cat.#600250),\t \u00C2\u00A0100ng\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cDNA\t \u00C2\u00A0template,\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0each\t \u00C2\u00A0primer\t \u00C2\u00A0(100ng/mL),\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A010mM\t \u00C2\u00A0dNTP\t \u00C2\u00A0solution\t \u00C2\u00A0(Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#\t \u00C2\u00A018427-\u00C2\u00AD\u00E2\u0080\u0090013),\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A010x\t \u00C2\u00A0Cloned\t \u00C2\u00A0Pfu\t \u00C2\u00A0DNA\t \u00C2\u00A0polymerase\t \u00C2\u00A0reaction\t \u00C2\u00A0buffer,\t \u00C2\u00A0and\t \u00C2\u00A0ddH2O\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0total\t \u00C2\u00A0volume\t \u00C2\u00A0of\t \u00C2\u00A040\u00CE\u00BCL.\t \u00C2\u00A0The\t \u00C2\u00A0PCR\t \u00C2\u00A0reaction\t \u00C2\u00A0was\t \u00C2\u00A0performed\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0automated\t \u00C2\u00A0thermo-\u00C2\u00AD\u00E2\u0080\u0090cycler\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0parameters:\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A02min;\t \u00C2\u00A030\t \u00C2\u00A0cycles\t \u00C2\u00A0of\t \u00C2\u00A095\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A030s,\t \u00C2\u00A055\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A030s,\t \u00C2\u00A072\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A01min/kb;\t \u00C2\u00A01\t \u00C2\u00A0cycle\t \u00C2\u00A0of\t \u00C2\u00A072\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0for\t \u00C2\u00A010min.\t \u00C2\u00A0To\t \u00C2\u00A0digest\t \u00C2\u00A0the\t \u00C2\u00A0parent-\u00C2\u00AD\u00E2\u0080\u0090DNA,\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0DpnI\t \u00C2\u00A0restriction\t \u00C2\u00A0enzyme\t \u00C2\u00A0(Thermo\t \u00C2\u00A0Scientific,\t \u00C2\u00A0cat.\t \u00C2\u00A0#FD1704)\t \u00C2\u00A0was\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A050\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0PCR\t \u00C2\u00A0sample\t \u00C2\u00A0and\t \u00C2\u00A0incubated\t \u00C2\u00A0for\t \u00C2\u00A01h\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC\t \u00C2\u00A0water\t \u00C2\u00A0bath.\t \u00C2\u00A0The\t \u00C2\u00A0reaction\t \u00C2\u00A0was\t \u00C2\u00A0stopped\t \u00C2\u00A0by\t \u00C2\u00A0placing\t \u00C2\u00A0the\t \u00C2\u00A0PCR\t \u00C2\u00A0tube\t \u00C2\u00A0on\t \u00C2\u00A0ice,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0contents\t \u00C2\u00A0were\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0electrophoresis\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A01%\t \u00C2\u00A0agarose\t \u00C2\u00A0gel.\t \u00C2\u00A0Plasmids\t \u00C2\u00A0were\t \u00C2\u00A0purified\t \u00C2\u00A0using\t \u00C2\u00A0QIAprep\t \u00C2\u00A0Spin\t \u00C2\u00A0Miniprep\t \u00C2\u00A0Kit\t \u00C2\u00A0(QIAGEN,\t \u00C2\u00A0cat.\t \u00C2\u00A0#27106)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0microcentrifudge\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0instructions.\t \u00C2\u00A0Plasmids\t \u00C2\u00A0were\t \u00C2\u00A0sent\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0NAPS\t \u00C2\u00A0Unit\t \u00C2\u00A0at\t \u00C2\u00A0UBC\t \u00C2\u00A0for\t \u00C2\u00A0sequencing\t \u00C2\u00A0to\t \u00C2\u00A0confirm\t \u00C2\u00A0the\t \u00C2\u00A0mutation.\t \u00C2\u00A02.7.5 RESTRICTION,\t \u00C2\u00A0PURIFICATION\t \u00C2\u00A0AND\t \u00C2\u00A0LIGATION\t \u00C2\u00A0Restriction\t \u00C2\u00A0digestion,\t \u00C2\u00A0plasmid\t \u00C2\u00A0purification\t \u00C2\u00A0and\t \u00C2\u00A0DNA\t \u00C2\u00A0ligation\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0insert\t \u00C2\u00A0additional\t \u00C2\u00A0DNA\t \u00C2\u00A0sequences\t \u00C2\u00A0(i.e.\t \u00C2\u00A0PCR\t \u00C2\u00A0products\t \u00C2\u00A0and/or\t \u00C2\u00A0minigenes)\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0plasmid\t \u00C2\u00A0vector\t \u00C2\u00A0for\t \u00C2\u00A0expression.\t \u00C2\u00A0For\t \u00C2\u00A0PCR\t \u00C2\u00A0products,\t \u00C2\u00A0digestion\t \u00C2\u00A0was\t \u00C2\u00A0performed\t \u00C2\u00A0with\t \u00C2\u00A0restriction\t \u00C2\u00A0enzymes\t \u00C2\u00A0purchased\t \u00C2\u00A0from\t \u00C2\u00A0New\t \u00C2\u00A0England\t \u00C2\u00A0BioLabs\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0restriction\t \u00C2\u00A0sites:\t \u00C2\u00A0EcoRI\t \u00C2\u00A0(cat.\t \u00C2\u00A0#R3101S),\t \u00C2\u00A0the\t \u00C2\u00A0BglII\t \u00C2\u00A0(cat.\t \u00C2\u00A0#R0144S)\t \u00C2\u00A0or\t \u00C2\u00A0NcoI\t \u00C2\u00A0(cat.\t \u00C2\u00A0#R0193S).\t \u00C2\u00A0Each\t \u00C2\u00A0reaction\t \u00C2\u00A0contained\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0components:\t \u00C2\u00A048\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0PCR\t \u00C2\u00A0product,\t \u00C2\u00A02.5\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0EcoRI\t \u00C2\u00A0(20,000U/mL),\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0BglII\t \u00C2\u00A0or\t \u00C2\u00A0NcoI\t \u00C2\u00A0 89 (10,000U/mL),\t \u00C2\u00A010\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0NE#3\t \u00C2\u00A0reaction\t \u00C2\u00A0buffer\t \u00C2\u00A0and\t \u00C2\u00A034.5\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0ddH2O.\t \u00C2\u00A0PCR\t \u00C2\u00A0products\t \u00C2\u00A0were\t \u00C2\u00A0digested\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A037\u00C2\u00B0C\t \u00C2\u00A0water\t \u00C2\u00A0bath\t \u00C2\u00A0for\t \u00C2\u00A020h.\t \u00C2\u00A0\t \u00C2\u00A0For\t \u00C2\u00A0vectors,\t \u00C2\u00A0each\t \u00C2\u00A0reaction\t \u00C2\u00A0solution\t \u00C2\u00A0contained\t \u00C2\u00A05\u00CE\u00BCg\t 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\u00C2\u00A0restriction\t \u00C2\u00A0sites\t \u00C2\u00A0were\t \u00C2\u00A0digested\t \u00C2\u00A0for\t \u00C2\u00A02.5h.\t \u00C2\u00A0Following\t \u00C2\u00A0digestion,\t \u00C2\u00A0PCR\t \u00C2\u00A0products\t \u00C2\u00A0and\t \u00C2\u00A0plasmids\t \u00C2\u00A0were\t \u00C2\u00A0purified\t \u00C2\u00A0with\t \u00C2\u00A0QIAprep\t \u00C2\u00A0Spin\t \u00C2\u00A0Miniprep\t \u00C2\u00A0Kit\t \u00C2\u00A0(QIAGEN,\t \u00C2\u00A0cat.\t \u00C2\u00A0#27106)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0microcentrifudge\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0manufacturer\u00E2\u0080\u0099s\t \u00C2\u00A0instructions.\t \u00C2\u00A0Finally,\t \u00C2\u00A0the\t \u00C2\u00A0purified\t \u00C2\u00A0PCR\t \u00C2\u00A0products\t \u00C2\u00A0and\t \u00C2\u00A0vectors\t \u00C2\u00A0were\t \u00C2\u00A0eluded\t \u00C2\u00A0in\t \u00C2\u00A030\u00CE\u00BCL\t \u00C2\u00A0and\t \u00C2\u00A045\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0DNase-\u00C2\u00AD\u00E2\u0080\u0090free\t \u00C2\u00A0water,\t \u00C2\u00A0respectively.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0prepare\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0vectors\t \u00C2\u00A0for\t \u00C2\u00A0minigene\t \u00C2\u00A0insertion,\t \u00C2\u00A0the\t \u00C2\u00A0vector\t \u00C2\u00A0was\t \u00C2\u00A0digested\t \u00C2\u00A0with\t \u00C2\u00A0FastDigest\t \u00C2\u00A0NotI\t \u00C2\u00A0(cat.\t \u00C2\u00A0#FD0594)\t \u00C2\u00A0and\t \u00C2\u00A0HindIII\t \u00C2\u00A0(cat.\t \u00C2\u00A0#FD0504),\t \u00C2\u00A0purchased\t \u00C2\u00A0from\t \u00C2\u00A0Invitrogen.\t \u00C2\u00A0The\t \u00C2\u00A0reaction\t \u00C2\u00A0mixture\t \u00C2\u00A0contained\t \u00C2\u00A01\u00CE\u00BCg\t \u00C2\u00A0of\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0vector,\t \u00C2\u00A010X\t \u00C2\u00A0FastDigest\t \u00C2\u00A0buffer\t \u00C2\u00A02\u00CE\u00BCL,\t \u00C2\u00A0NotI\t \u00C2\u00A01\u00CE\u00BCL,\t \u00C2\u00A0HindIII\t \u00C2\u00A01\u00CE\u00BCL\t \u00C2\u00A0and\t \u00C2\u00A0ddH2O\t \u00C2\u00A015\u00CE\u00BCL.\t \u00C2\u00A0The\t \u00C2\u00A0vector\t \u00C2\u00A0was\t \u00C2\u00A0digested\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A037\u00C2\u00B0C\t \u00C2\u00A0water\t \u00C2\u00A0bath\t \u00C2\u00A0for\t \u00C2\u00A01h\t \u00C2\u00A0and\t \u00C2\u00A0purified\t \u00C2\u00A0prior\t \u00C2\u00A0to\t \u00C2\u00A0ligation.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0induce\t \u00C2\u00A0ligation,\t \u00C2\u00A013\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0purified\t \u00C2\u00A0PCR\t \u00C2\u00A0solution\t \u00C2\u00A0was\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A00.5\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0T4\t \u00C2\u00A0Ligase\t \u00C2\u00A0(1U/\u00CE\u00BCL,\t \u00C2\u00A0Invitrogen,\t \u00C2\u00A0cat.\t \u00C2\u00A0#15224-\u00C2\u00AD\u00E2\u0080\u0090107),\t \u00C2\u00A04\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A05X\t \u00C2\u00A0ligation\t \u00C2\u00A0buffer\t \u00C2\u00A0and\t \u00C2\u00A02.5\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0purified\t \u00C2\u00A0plasmid\t \u00C2\u00A0vector.\t \u00C2\u00A0The\t \u00C2\u00A0ligation\t \u00C2\u00A0reaction\t \u00C2\u00A0occurred\t \u00C2\u00A0at\t \u00C2\u00A0room\t \u00C2\u00A0temperature\t \u00C2\u00A0for\t \u00C2\u00A01h.\t \u00C2\u00A0Finally,\t \u00C2\u00A05\u00CE\u00BCL\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0ligated\t \u00C2\u00A0DNA\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0transformation\t \u00C2\u00A0and\t \u00C2\u00A0amplification\t \u00C2\u00A0(See\t \u00C2\u00A0Chapter\t \u00C2\u00A02.7.2).\t \u00C2\u00A0 90 2.7.6 PLASMID\t \u00C2\u00A0PRODUCTION\t \u00C2\u00A02.7.6.1 PLASMIDS\t \u00C2\u00A0FOR\t \u00C2\u00A0GFP\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0To\t \u00C2\u00A0inactivate\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0sequence\t \u00C2\u00A0(KFERQ1KILDQ2RFFE)\t \u00C2\u00A0in\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0plasmid,\t \u00C2\u00A0the\t \u00C2\u00A0two\t \u00C2\u00A0glutamine\t \u00C2\u00A0residues\t \u00C2\u00A0(Q1\t \u00C2\u00A0and\t \u00C2\u00A0Q2)\t \u00C2\u00A0were\t \u00C2\u00A0individually\t \u00C2\u00A0replaced\t \u00C2\u00A0by\t \u00C2\u00A0alanine\t \u00C2\u00A0residues\t \u00C2\u00A0(A)\t \u00C2\u00A0through\t \u00C2\u00A0site-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090A1Q2-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0and\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090Q1A2-\u00C2\u00AD\u00E2\u0080\u0090GFP.\t \u00C2\u00A0The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0are\t \u00C2\u00A0as\t \u00C2\u00A0follows:\t \u00C2\u00A0A1\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GAA\t \u00C2\u00A0GTT\t \u00C2\u00A0TGA\t \u00C2\u00A0GCG\t \u00C2\u00A0GGC\t \u00C2\u00A0GAA\t \u00C2\u00A0GAT\t \u00C2\u00A0ACT\t \u00C2\u00A0C\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0A1\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GGT\t \u00C2\u00A0CGA\t \u00C2\u00A0GTA\t \u00C2\u00A0TCT\t \u00C2\u00A0TCG\t \u00C2\u00A0CCC\t \u00C2\u00A0GCT\t \u00C2\u00A0CAA\t \u00C2\u00A0AC\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0A2\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCT\t \u00C2\u00A0CGA\t \u00C2\u00A0AAA\t \u00C2\u00A0ATC\t \u00C2\u00A0GCG\t \u00C2\u00A0CGT\t \u00C2\u00A0CGA\t \u00C2\u00A0GTA\t \u00C2\u00A0TC\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0A2\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GAA\t \u00C2\u00A0GAT\t \u00C2\u00A0ACT\t \u00C2\u00A0CGA\t \u00C2\u00A0CGC\t \u00C2\u00A0GCG\t \u00C2\u00A0ATT\t \u00C2\u00A0TTT\t \u00C2\u00A0CG\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0newly\t \u00C2\u00A0generated\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090A1Q2-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0template\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090A1A2-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0both\t \u00C2\u00A0Q\t \u00C2\u00A0residues\t \u00C2\u00A0are\t \u00C2\u00A0mutated\t \u00C2\u00A0to\t \u00C2\u00A0A\t \u00C2\u00A0residues\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0A2\t \u00C2\u00A0primers\t \u00C2\u00A0described\t \u00C2\u00A0above.\t \u00C2\u00A02.7.6.2 PLASMIDS\t \u00C2\u00A0FOR\t \u00C2\u00A0\u00CE\u0091-\u00C2\u00AD\u00E2\u0080\u0090SYNUCLEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0strongly\t \u00C2\u00A0interacts\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0short\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0stretch\t \u00C2\u00A0(between\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A036-\u00C2\u00AD\u00E2\u0080\u009045)\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u00B2syn36)\t \u00C2\u00A0(Shaltiel-\u00C2\u00AD\u00E2\u0080\u0090Karyo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0To\t \u00C2\u00A0generate\t \u00C2\u00A0the\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM,\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0primers\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0PCR,\t \u00C2\u00A0with\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090r\u00CE\u00B2syn-\u00C2\u00AD\u00E2\u0080\u0090degron\t \u00C2\u00A0(YGRKKRRQRRRRTKSGVYLVGRRRG,\t \u00C2\u00A0a\t \u00C2\u00A0kind\t \u00C2\u00A0gift\t \u00C2\u00A0from\t \u00C2\u00A0 91 my\t \u00C2\u00A0colleague\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin)\t \u00C2\u00A0as\t \u00C2\u00A0template.\t \u00C2\u00A0\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM(HindIII)\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCC\t \u00C2\u00A0AAG\t \u00C2\u00A0CTT\t \u00C2\u00A0ATG\t \u00C2\u00A0GAC\t \u00C2\u00A0TAC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM(EcoRI)\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AAG\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0AGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CGC\t \u00C2\u00A0TTA\t \u00C2\u00A0CTC\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0TCG\t \u00C2\u00A0CTG\t \u00C2\u00A0GTC\t \u00C2\u00A0GAG\t \u00C2\u00A0TAT\t \u00C2\u00A0CTT\t \u00C2\u00A0CTG\t \u00C2\u00A0CCG\t \u00C2\u00A0CTC\t \u00C2\u00A0AAA\t \u00C2\u00A0CTT\t \u00C2\u00A0ACC\t \u00C2\u00A0AAC\t \u00C2\u00A0CAA\t \u00C2\u00A0ATA\t \u00C2\u00A0AAC\t \u00C2\u00A0ACC\t \u00C2\u00A0AG\t \u00C2\u00A0\u00E2\u0080\u00933\u00E2\u0080\u0099\t \u00C2\u00A0To\t \u00C2\u00A0generate\t \u00C2\u00A0a\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0containing\t \u00C2\u00A0the\t \u00C2\u00A0Flag\t \u00C2\u00A0tag\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B2syn36\t \u00C2\u00A0without\t \u00C2\u00A0CTM\t \u00C2\u00A0(Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36),\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0primers\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0site-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0with\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0degron\t \u00C2\u00A0as\t \u00C2\u00A0template\t \u00C2\u00A0to\t \u00C2\u00A0convert\t \u00C2\u00A0the\t \u00C2\u00A0first\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0of\t \u00C2\u00A0degron\t \u00C2\u00A0(RRRG)\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0stop\t \u00C2\u00A0codon.\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0TAT\t \u00C2\u00A0TTG\t \u00C2\u00A0GTT\t \u00C2\u00A0GGT\t \u00C2\u00A0TGA\t \u00C2\u00A0CGA\t \u00C2\u00A0CGA\t \u00C2\u00A0GGC\t \u00C2\u00A0T\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AGC\t \u00C2\u00A0CTC\t \u00C2\u00A0GTC\t \u00C2\u00A0GTC\t \u00C2\u00A0AAC\t \u00C2\u00A0CAA\t \u00C2\u00A0CCA\t \u00C2\u00A0AAT\t \u00C2\u00A0A\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0To\t \u00C2\u00A0see\t \u00C2\u00A0whether\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0repeat\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0knockdown,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0constructed\t \u00C2\u00A0a\t \u00C2\u00A0series\t \u00C2\u00A0of\t \u00C2\u00A0plasmids\t \u00C2\u00A0expressing\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0one\t \u00C2\u00A0or\t \u00C2\u00A0two\t \u00C2\u00A0CTM\t \u00C2\u00A0repeats.\t \u00C2\u00A0PCR\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0extract\t \u00C2\u00A0the\t \u00C2\u00A0appropriate\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0(1\t \u00C2\u00A0or\t \u00C2\u00A02\t \u00C2\u00A0repeats)\t \u00C2\u00A0sequence\t \u00C2\u00A0from\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM.\t \u00C2\u00A0The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0construct\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synKFERQ\t \u00C2\u00A0were:\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM\t \u00C2\u00A0F_HindIII:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCC\t \u00C2\u00A0AAG\t \u00C2\u00A0CTT\t \u00C2\u00A0ATG\t \u00C2\u00A0GAC\t \u00C2\u00A0TAC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u00933\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM1\t \u00C2\u00A0R_NotI:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AAG\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0AGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CGC\t \u00C2\u00A0TTA\t \u00C2\u00A0CTG\t \u00C2\u00A0CCG\t \u00C2\u00A0CTC\t \u00C2\u00A0AAA\t \u00C2\u00A0CTT\t \u00C2\u00A0ACC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0 92 The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0construct\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synQKILD\t \u00C2\u00A0were:\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM\t \u00C2\u00A0F_HindIII:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCC\t \u00C2\u00A0AAG\t \u00C2\u00A0CTT\t \u00C2\u00A0ATG\t \u00C2\u00A0GAC\t \u00C2\u00A0TAC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM2\t \u00C2\u00A0R_NotI:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AAG\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0AGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CGC\t \u00C2\u00A0TTA\t \u00C2\u00A0GTC\t \u00C2\u00A0GAG\t \u00C2\u00A0TAT\t \u00C2\u00A0CTT\t \u00C2\u00A0CTG\t \u00C2\u00A0ACC\t \u00C2\u00A0AAC\t \u00C2\u00A0CAA\t \u00C2\u00A0ATA\t \u00C2\u00A0AAC\t \u00C2\u00A0ACC\t \u00C2\u00A0AG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0construct\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synQRFFE\t \u00C2\u00A0were:\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM\t \u00C2\u00A0F_HindIII:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCC\t \u00C2\u00A0AAG\t \u00C2\u00A0CTT\t \u00C2\u00A0ATG\t \u00C2\u00A0GAC\t \u00C2\u00A0TAC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM3\t \u00C2\u00A0R_NotI:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AAG\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0AGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CGC\t \u00C2\u00A0TTA\t \u00C2\u00A0CTC\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0TCG\t \u00C2\u00A0CTG\t \u00C2\u00A0ACC\t \u00C2\u00A0AAC\t \u00C2\u00A0CAA\t \u00C2\u00A0ATA\t \u00C2\u00A0AAC\t \u00C2\u00A0ACC\t \u00C2\u00A0AG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0construct\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synKFERQKILD\t \u00C2\u00A0were:\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM\t \u00C2\u00A0F_HindIII:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCC\t \u00C2\u00A0AAG\t \u00C2\u00A0CTT\t \u00C2\u00A0ATG\t \u00C2\u00A0GAC\t \u00C2\u00A0TAC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM12\t \u00C2\u00A0R_NotI:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AAG\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0AGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CGC\t \u00C2\u00A0TTA\t \u00C2\u00A0GTC\t \u00C2\u00A0GAG\t \u00C2\u00A0TAT\t \u00C2\u00A0CTT\t \u00C2\u00A0CTG\t \u00C2\u00A0CCG\t \u00C2\u00A0CTC\t \u00C2\u00A0AAA\t \u00C2\u00A0CTT\t \u00C2\u00A0ACC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0construct\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synQKILDQRFFE\t \u00C2\u00A0were:\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM\t \u00C2\u00A0F_HindIII:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCC\t \u00C2\u00A0AAG\t \u00C2\u00A0CTT\t \u00C2\u00A0ATG\t \u00C2\u00A0GAC\t \u00C2\u00A0TAC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM23\t \u00C2\u00A0R_NotI:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AAG\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0AGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CGC\t \u00C2\u00A0TTA\t \u00C2\u00A0CTC\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0TCG\t \u00C2\u00A0CTG\t \u00C2\u00A0GTC\t \u00C2\u00A0GAG\t \u00C2\u00A0TAT\t \u00C2\u00A0CTT\t \u00C2\u00A0CTG\t \u00C2\u00A0ACC\t \u00C2\u00A0AAC\t \u00C2\u00A0CAA\t \u00C2\u00A0ATA\t \u00C2\u00A0AAC\t \u00C2\u00A0ACC\t \u00C2\u00A0AG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0construct\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synKFERQQRFFE\t \u00C2\u00A0were:\t \u00C2\u00A0 93 Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM\t \u00C2\u00A0F_HindIII:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCC\t \u00C2\u00A0AAG\t \u00C2\u00A0CTT\t \u00C2\u00A0ATG\t \u00C2\u00A0GAC\t \u00C2\u00A0TAC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2CTM13\t \u00C2\u00A0R_NotI:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0AAG\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0AGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CGC\t \u00C2\u00A0TTA\t \u00C2\u00A0CTC\t \u00C2\u00A0GAA\t \u00C2\u00A0AAA\t \u00C2\u00A0TCG\t \u00C2\u00A0CTG\t \u00C2\u00A0CTG\t \u00C2\u00A0CCG\t \u00C2\u00A0CTC\t \u00C2\u00A0AAA\t \u00C2\u00A0CTT\t \u00C2\u00A0ACC\t \u00C2\u00A0AA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0PCR\t \u00C2\u00A0products\t \u00C2\u00A0were\t \u00C2\u00A0double\t \u00C2\u00A0digested\t \u00C2\u00A0with\t \u00C2\u00A0HindIII\t \u00C2\u00A0and\t \u00C2\u00A0NotI\t \u00C2\u00A0and\t \u00C2\u00A0inserted\t \u00C2\u00A0into\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0vector.\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(A53T)\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0pathogenic\t \u00C2\u00A0variant\t \u00C2\u00A0of\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0implicated\t \u00C2\u00A0in\t \u00C2\u00A0familial\t \u00C2\u00A0PD.\t \u00C2\u00A0To\t \u00C2\u00A0see\t \u00C2\u00A0if\t \u00C2\u00A0the\t \u00C2\u00A0A53T\t \u00C2\u00A0variant\t \u00C2\u00A0can\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0targeted\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM,\t \u00C2\u00A0wild\t \u00C2\u00A0type\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0was\t \u00C2\u00A0mutated\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(A53T)\t \u00C2\u00A0via\t \u00C2\u00A0site-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0primers:\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1syn(A53T)\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GTG\t \u00C2\u00A0CAT\t \u00C2\u00A0GGT\t \u00C2\u00A0GTG\t \u00C2\u00A0ACA\t \u00C2\u00A0ACA\t \u00C2\u00A0GTG\t \u00C2\u00A0GCT\t \u00C2\u00A0G\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1syn(A53T)\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CAG\t \u00C2\u00A0CCA\t \u00C2\u00A0CTG\t \u00C2\u00A0TTG\t \u00C2\u00A0TCA\t \u00C2\u00A0CAC\t \u00C2\u00A0CAT\t \u00C2\u00A0GCA\t \u00C2\u00A0C\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0contains\t \u00C2\u00A0a\t \u00C2\u00A0native\t \u00C2\u00A0CTM\t \u00C2\u00A0(95VKKDQ99)\t \u00C2\u00A0and\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0machinery\t \u00C2\u00A0under\t \u00C2\u00A0normal\t \u00C2\u00A0conditions\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004;\t \u00C2\u00A0Vogiatzi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008;\t \u00C2\u00A0Mak\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0To\t \u00C2\u00A0see\t \u00C2\u00A0whether\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein,\t \u00C2\u00A0the\t \u00C2\u00A0native\t \u00C2\u00A0CTM\t \u00C2\u00A0(95VKKDQ99)\t \u00C2\u00A0mutated\t \u00C2\u00A0to\t \u00C2\u00A095VKKAA99\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0using\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0primers:\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1syn(\u00CE\u0094DQ)\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GCC\t \u00C2\u00A0ACT\t \u00C2\u00A0GGC\t \u00C2\u00A0TTT\t \u00C2\u00A0GTC\t \u00C2\u00A0AAA\t \u00C2\u00A0AAG\t \u00C2\u00A0GCC\t \u00C2\u00A0GCG\t \u00C2\u00A0TTG\t \u00C2\u00A0GGC\t \u00C2\u00A0AAG\t \u00C2\u00A0AAT\t \u00C2\u00A0GAA\t \u00C2\u00A0GAA\t \u00C2\u00A0GGA\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0 94 HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1syn(\u00CE\u0094DQ)\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0TCC\t \u00C2\u00A0TTC\t \u00C2\u00A0TTC\t \u00C2\u00A0ATT\t \u00C2\u00A0CTT\t \u00C2\u00A0GCC\t \u00C2\u00A0CAA\t \u00C2\u00A0CGC\t \u00C2\u00A0GGC\t \u00C2\u00A0CTT\t \u00C2\u00A0TTT\t \u00C2\u00A0GAC\t \u00C2\u00A0AAA\t \u00C2\u00A0GCC\t \u00C2\u00A0AGT\t \u00C2\u00A0GGC\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A02.7.6.3 PLASMIDS\t \u00C2\u00A0FOR\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0calcium-\u00C2\u00AD\u00E2\u0080\u0090calmodulin\t \u00C2\u00A0regulated\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0that\t \u00C2\u00A0is\t \u00C2\u00A0normally\t \u00C2\u00A0inactive\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0(Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0Under\t \u00C2\u00A0certain\t \u00C2\u00A0pathological\t \u00C2\u00A0conditions\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0damage\t \u00C2\u00A0or\t \u00C2\u00A0cerebral\t \u00C2\u00A0ischemia,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0activated\t \u00C2\u00A0and\t \u00C2\u00A0recruited\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR\t \u00C2\u00A0complex\t \u00C2\u00A0by\t \u00C2\u00A0interacting\t \u00C2\u00A0with\t \u00C2\u00A0residues\t \u00C2\u00A01292-\u00C2\u00AD\u00E2\u0080\u00901304\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0subunit\t \u00C2\u00A0carboxyl\t \u00C2\u00A0tail\t \u00C2\u00A0(GluN2Bct1292-\u00C2\u00AD\u00E2\u0080\u00901304)\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0A\t \u00C2\u00A0gene\t \u00C2\u00A0sequence\t \u00C2\u00A0encoding\t \u00C2\u00A0the\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A01242-\u00C2\u00AD\u00E2\u0080\u00901342\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0carboxyl\t \u00C2\u00A0tail\t \u00C2\u00A0was\t \u00C2\u00A0obtained\t \u00C2\u00A0via\t \u00C2\u00A0PCR,\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0cDNA\t \u00C2\u00A0library\t \u00C2\u00A0(Origin:\t \u00C2\u00A0Rattus\t \u00C2\u00A0Norvegicus)\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0template\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0pair\t \u00C2\u00A0of\t \u00C2\u00A0primers:\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0(1242)BglII:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GGA\t \u00C2\u00A0AGA\t \u00C2\u00A0TCT\t \u00C2\u00A0ATG\t \u00C2\u00A0TGT\t \u00C2\u00A0GAG\t \u00C2\u00A0GCC\t \u00C2\u00A0TGT\t \u00C2\u00A0AAG\t \u00C2\u00A0AAG\t \u00C2\u00A0GCT\t \u00C2\u00A0CG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0(1342)EcoRI:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCG\t \u00C2\u00A0GAA\t \u00C2\u00A0TTC\t \u00C2\u00A0TGG\t \u00C2\u00A0CAT\t \u00C2\u00A0CTC\t \u00C2\u00A0AAA\t \u00C2\u00A0CAT\t \u00C2\u00A0ATG\t \u00C2\u00A0GGC\t 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\u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0plasmid\t \u00C2\u00A06X\t \u00C2\u00A0His-\u00C2\u00AD\u00E2\u0080\u0090TAT-\u00C2\u00AD\u00E2\u0080\u0090HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0via\t \u00C2\u00A0PCR,\t \u00C2\u00A0which\t \u00C2\u00A0contains\t \u00C2\u00A0the\t \u00C2\u00A0smaller\t \u00C2\u00A0human\t \u00C2\u00A0influenza\t \u00C2\u00A0hemagglutinin\t \u00C2\u00A0(HA)\t \u00C2\u00A0tag\t \u00C2\u00A0in\t \u00C2\u00A0place\t \u00C2\u00A0of\t \u00C2\u00A0GFP\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0carboxyl-\u00C2\u00AD\u00E2\u0080\u0090terminal.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0inactivate\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0sequence\t \u00C2\u00A0(KFERQ1KILDQ2RFFE)\t \u00C2\u00A0in\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0plasmid,\t \u00C2\u00A0the\t \u00C2\u00A0two\t \u00C2\u00A0Q\t \u00C2\u00A0residues\t \u00C2\u00A0(Q1\t \u00C2\u00A0and\t \u00C2\u00A0Q2)\t \u00C2\u00A0were\t \u00C2\u00A0individually\t \u00C2\u00A0replaced\t \u00C2\u00A0by\t \u00C2\u00A0A\t \u00C2\u00A0residues\t \u00C2\u00A0through\t \u00C2\u00A0site\t \u00C2\u00A0directed\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm.\t \u00C2\u00A0The\t \u00C2\u00A0primers\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0mutate\t \u00C2\u00A0Q1\t \u00C2\u00A0were:\t \u00C2\u00A0A12\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GAA\t \u00C2\u00A0GTT\t \u00C2\u00A0TGA\t \u00C2\u00A0GCG\t \u00C2\u00A0GGC\t \u00C2\u00A0GAA\t \u00C2\u00A0GAT\t \u00C2\u00A0ACT\t \u00C2\u00A0C\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0A12\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GGT\t \u00C2\u00A0CGA\t \u00C2\u00A0GTA\t \u00C2\u00A0TCT\t \u00C2\u00A0TCG\t \u00C2\u00A0CCC\t \u00C2\u00A0GCT\t \u00C2\u00A0CAA\t \u00C2\u00A0AC\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0The\t \u00C2\u00A0newly\t \u00C2\u00A0generated\t \u00C2\u00A0plasmid\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0template\t \u00C2\u00A0to\t \u00C2\u00A0generate\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0both\t \u00C2\u00A0Q\t \u00C2\u00A0residues\t \u00C2\u00A0are\t \u00C2\u00A0mutated\t \u00C2\u00A0to\t \u00C2\u00A0A\t \u00C2\u00A0residues\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0primers:\t \u00C2\u00A0\t \u00C2\u00A0A3\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0GAA\t \u00C2\u00A0GAT\t \u00C2\u00A0ACT\t \u00C2\u00A0CGA\t \u00C2\u00A0CGC\t \u00C2\u00A0GCG\t \u00C2\u00A0ATT\t \u00C2\u00A0TTT\t \u00C2\u00A0CG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0A3\t \u00C2\u00A0antisense:\t \u00C2\u00A0\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CCT\t \u00C2\u00A0CGA\t \u00C2\u00A0AAA\t \u00C2\u00A0ATC\t \u00C2\u00A0GCG\t \u00C2\u00A0CGT\t \u00C2\u00A0CGA\t \u00C2\u00A0GTA\t \u00C2\u00A0TC\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0Under\t \u00C2\u00A0basal\t \u00C2\u00A0conditions\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0of\t \u00C2\u00A0GluN2Bct\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0interact\t \u00C2\u00A0with\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0inactive\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0To\t \u00C2\u00A0generate\t \u00C2\u00A0a\t \u00C2\u00A0plasmid\t \u00C2\u00A0that\t \u00C2\u00A0expresses\t \u00C2\u00A0constitutively\t \u00C2\u00A0active\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(cDAPK1)\t \u00C2\u00A0that\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0GluN2Bct,\t \u00C2\u00A0the\t \u00C2\u00A0CaM\t \u00C2\u00A0regulatory\t \u00C2\u00A0domain\t \u00C2\u00A0of\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0was\t \u00C2\u00A0removed\t \u00C2\u00A0by\t \u00C2\u00A0PCR\t \u00C2\u00A0using\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0primers:\t \u00C2\u00A0 97 (\u00CE\u0094CaM)\t \u00C2\u00A0sense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CAG\t \u00C2\u00A0CAT\t \u00C2\u00A0CCC\t \u00C2\u00A0TGG\t \u00C2\u00A0ATC\t \u00C2\u00A0AAG\t \u00C2\u00A0TCC\t \u00C2\u00A0AGA\t \u00C2\u00A0AGT\t \u00C2\u00A0AAC\t \u00C2\u00A0ATG\t \u00C2\u00A0AGT\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0(\u00CE\u0094CaM)\t \u00C2\u00A0antisense:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0ACT\t \u00C2\u00A0CAT\t \u00C2\u00A0GTT\t \u00C2\u00A0ACT\t \u00C2\u00A0TCT\t \u00C2\u00A0GGA\t \u00C2\u00A0CTT\t \u00C2\u00A0GAT\t \u00C2\u00A0CCA\t \u00C2\u00A0GGG\t \u00C2\u00A0ATG\t \u00C2\u00A0CTG\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A0To\t \u00C2\u00A0confirm\t \u00C2\u00A0the\t \u00C2\u00A0sequence\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0resulting\t \u00C2\u00A0plasmid,\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090DAPK1(\u00CE\u0094CaM),\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0primer\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0sequencing:\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090seq:\t \u00C2\u00A05\u00E2\u0080\u0099\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0CAG\t \u00C2\u00A0AGT\t \u00C2\u00A0TTG\t \u00C2\u00A0TCG\t \u00C2\u00A0CTC\t \u00C2\u00A0CTG\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A03\u00E2\u0080\u0099\t \u00C2\u00A02.8 ANIMAL\t \u00C2\u00A0MODELS\t \u00C2\u00A02.8.1 EXPERIMENTAL\t \u00C2\u00A0ANIMALS\t \u00C2\u00A0Male\t \u00C2\u00A0Sprague-\u00C2\u00AD\u00E2\u0080\u0090Dawley\t \u00C2\u00A0rats\t \u00C2\u00A0(Charles\t \u00C2\u00A0River,\t \u00C2\u00A0200-\u00C2\u00AD\u00E2\u0080\u0090250g\t \u00C2\u00A0for\t \u00C2\u00A0stereotaxic\t \u00C2\u00A0surgery,\t \u00C2\u00A0300-\u00C2\u00AD\u00E2\u0080\u0090350g\t \u00C2\u00A0for\t \u00C2\u00A0focal\t \u00C2\u00A0ischemia)\t \u00C2\u00A0were\t \u00C2\u00A0housed\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0Animal\t \u00C2\u00A0Research\t \u00C2\u00A0Unit\t \u00C2\u00A0(ARU)\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0UBC\t \u00C2\u00A0hospital\t \u00C2\u00A0in\t \u00C2\u00A0groups\t \u00C2\u00A0and\t \u00C2\u00A0housed\t \u00C2\u00A0separately\t \u00C2\u00A0following\t \u00C2\u00A0surgery.\t \u00C2\u00A0All\t \u00C2\u00A0rats\t \u00C2\u00A0had\t \u00C2\u00A0free\t \u00C2\u00A0access\t \u00C2\u00A0to\t \u00C2\u00A0standard\t \u00C2\u00A0rat\t \u00C2\u00A0pellet\t \u00C2\u00A0chow\t \u00C2\u00A0and\t \u00C2\u00A0water.\t \u00C2\u00A0All\t \u00C2\u00A0experiments\t \u00C2\u00A0conducted\t \u00C2\u00A0were\t \u00C2\u00A0approved\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0Animal\t \u00C2\u00A0Care\t \u00C2\u00A0Committee\t \u00C2\u00A0(ACC)\t \u00C2\u00A0of\t \u00C2\u00A0UBC\t \u00C2\u00A0and\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0protocol\t \u00C2\u00A0A12-\u00C2\u00AD\u00E2\u0080\u00900317\t \u00C2\u00A0for\t \u00C2\u00A0focal\t \u00C2\u00A0ischemia.\t \u00C2\u00A02.8.2 MIDDLE\t \u00C2\u00A0CEREBRAL\t \u00C2\u00A0ARTERIAL\t \u00C2\u00A0OCCLUSION\t \u00C2\u00A0(MCAO)\t \u00C2\u00A0All\t \u00C2\u00A0experiments\t \u00C2\u00A0were\t \u00C2\u00A0performed\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0ARU\t \u00C2\u00A0surgery\t \u00C2\u00A0room,\t \u00C2\u00A0in\t \u00C2\u00A0collaboration\t \u00C2\u00A0with\t \u00C2\u00A0my\t \u00C2\u00A0colleague\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin.\t \u00C2\u00A0Prior\t \u00C2\u00A0to\t \u00C2\u00A0surgery,\t \u00C2\u00A0rats\t \u00C2\u00A0were\t \u00C2\u00A0weighed\t \u00C2\u00A0and\t \u00C2\u00A0anesthetized\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0chamber\t \u00C2\u00A0with\t \u00C2\u00A04%\t \u00C2\u00A0isoflurane\t \u00C2\u00A0(PPC,\t \u00C2\u00A0cat.\t \u00C2\u00A0#M60303)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0nitrous\t \u00C2\u00A0oxide\t \u00C2\u00A0and\t \u00C2\u00A0oxygen\t \u00C2\u00A0(7:3)\t \u00C2\u00A0mixture,\t \u00C2\u00A0and\t \u00C2\u00A0thereafter\t \u00C2\u00A0isoflurane\t \u00C2\u00A0levels\t \u00C2\u00A0were\t \u00C2\u00A0decreased\t \u00C2\u00A0to\t \u00C2\u00A02%\t \u00C2\u00A0to\t \u00C2\u00A0maintain\t \u00C2\u00A0the\t \u00C2\u00A0anesthesia.\t \u00C2\u00A0Body\t \u00C2\u00A0temperature\t \u00C2\u00A0was\t \u00C2\u00A0monitored\t \u00C2\u00A0throughout\t \u00C2\u00A0the\t \u00C2\u00A0surgery\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0rectal\t \u00C2\u00A0probe,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0connected\t \u00C2\u00A0to\t \u00C2\u00A0an\t \u00C2\u00A0automated\t \u00C2\u00A0heating\t \u00C2\u00A0pad\t \u00C2\u00A0to\t \u00C2\u00A0maintain\t \u00C2\u00A0the\t \u00C2\u00A0animals\u00E2\u0080\u0099\t \u00C2\u00A0body\t \u00C2\u00A0temperature\t \u00C2\u00A0 98 around\t \u00C2\u00A037\t \u00C2\u00A0\u00CC\u008AC.\t \u00C2\u00A0Animals\t \u00C2\u00A0were\t \u00C2\u00A0closely\t \u00C2\u00A0monitored\t \u00C2\u00A0throughout\t \u00C2\u00A0the\t \u00C2\u00A0surgery\t \u00C2\u00A0to\t \u00C2\u00A0check\t \u00C2\u00A0for\t \u00C2\u00A0laboured\t \u00C2\u00A0breathing.\t \u00C2\u00A0\t \u00C2\u00A0Each\t \u00C2\u00A0rat\t \u00C2\u00A0was\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0reversible\t \u00C2\u00A0MCAo\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0suture-\u00C2\u00AD\u00E2\u0080\u0090insertion\t \u00C2\u00A0method.\t \u00C2\u00A0Fur\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0ventral\t \u00C2\u00A0neck\t \u00C2\u00A0region\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0shaved\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0skin.\t \u00C2\u00A0The\t \u00C2\u00A0surgical\t \u00C2\u00A0site\t \u00C2\u00A0was\t \u00C2\u00A0disinfected\t \u00C2\u00A0with\t \u00C2\u00A0three\t \u00C2\u00A0applications\t \u00C2\u00A0of\t \u00C2\u00A070%\t \u00C2\u00A0ethanol.\t \u00C2\u00A0Using\t \u00C2\u00A0sterile\t \u00C2\u00A0technique,\t \u00C2\u00A0a\t \u00C2\u00A0midline\t \u00C2\u00A0incision\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0skin\t \u00C2\u00A0and\t \u00C2\u00A0underlying\t \u00C2\u00A0tissue\t \u00C2\u00A0was\t \u00C2\u00A0made\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0digastric,\t \u00C2\u00A0the\t \u00C2\u00A0omohyoid,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0sternomastoid\t \u00C2\u00A0muscles,\t \u00C2\u00A0which\t \u00C2\u00A0are\t \u00C2\u00A0then\t \u00C2\u00A0carefully\t \u00C2\u00A0isolated\t \u00C2\u00A0and\t \u00C2\u00A0retracted\t \u00C2\u00A0using\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00900\t \u00C2\u00A0silk\t \u00C2\u00A0sutures\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0right\t \u00C2\u00A0common\t \u00C2\u00A0carotid\t \u00C2\u00A0artery\t \u00C2\u00A0(CCA),\t \u00C2\u00A0the\t \u00C2\u00A0external\t \u00C2\u00A0carotid\t \u00C2\u00A0artery\t \u00C2\u00A0(ECA),\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0internal\t \u00C2\u00A0carotid\t \u00C2\u00A0artery\t \u00C2\u00A0(ICA).\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0CCA\t \u00C2\u00A0was\t \u00C2\u00A0isolated\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0adjacent\t \u00C2\u00A0vagal\t \u00C2\u00A0nerve\t \u00C2\u00A0and\t \u00C2\u00A0pulled\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0proximal\t \u00C2\u00A0direction\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0loose\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00900\t \u00C2\u00A0silk\t \u00C2\u00A0suture\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0ECA\t \u00C2\u00A0and\t \u00C2\u00A0ICA.\t \u00C2\u00A0The\t \u00C2\u00A0ECA\t \u00C2\u00A0was\t \u00C2\u00A0further\t \u00C2\u00A0dissected\t \u00C2\u00A0distally\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0its\t \u00C2\u00A0two\t \u00C2\u00A0branches\t \u00C2\u00A0(superior\t \u00C2\u00A0thyroid\t \u00C2\u00A0artery\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0occipital\t \u00C2\u00A0artery),\t \u00C2\u00A0and\t \u00C2\u00A0both\t \u00C2\u00A0were\t \u00C2\u00A0severed\t \u00C2\u00A0and\t \u00C2\u00A0coagulated\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0Bovie\t \u00C2\u00A0Low\t \u00C2\u00A0Temperature\t \u00C2\u00A0Cautery\t \u00C2\u00A0(microfine\t \u00C2\u00A0tip,\t \u00C2\u00A0cat.\t \u00C2\u00A0#AA90).\t \u00C2\u00A0Thereafter,\t \u00C2\u00A0the\t \u00C2\u00A0ECA\t \u00C2\u00A0was\t \u00C2\u00A0tied\t \u00C2\u00A0off\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00900\t \u00C2\u00A0silk\t \u00C2\u00A0suture\t \u00C2\u00A0at\t \u00C2\u00A0its\t \u00C2\u00A0distal\t \u00C2\u00A0end.\t \u00C2\u00A0Further\t \u00C2\u00A0distal\t \u00C2\u00A0dissection\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0ICA\t \u00C2\u00A0exposed\t \u00C2\u00A0its\t \u00C2\u00A0pterygopalatine\t \u00C2\u00A0artery\t \u00C2\u00A0branch,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0also\t \u00C2\u00A0tied\t \u00C2\u00A0off\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00900\t \u00C2\u00A0silk\t \u00C2\u00A0suture.\t \u00C2\u00A0To\t \u00C2\u00A0prevent\t \u00C2\u00A0blood\t \u00C2\u00A0flow,\t \u00C2\u00A0the\t \u00C2\u00A0CCA\t \u00C2\u00A0was\t \u00C2\u00A0pulled\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0loose\t \u00C2\u00A0suture\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0ICA\t \u00C2\u00A0was\t \u00C2\u00A0clipped\t \u00C2\u00A0with\t \u00C2\u00A0an\t \u00C2\u00A0aneurysm\t \u00C2\u00A0clip.\t \u00C2\u00A0A\t \u00C2\u00A0small\t \u00C2\u00A0incision\t \u00C2\u00A0was\t \u00C2\u00A0made\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0ECA\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0tied\t \u00C2\u00A0suture\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0carotid\t \u00C2\u00A0bifurcation.\t \u00C2\u00A0A\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00900\t \u00C2\u00A0nylon\t \u00C2\u00A0suture\t \u00C2\u00A0(the\t \u00C2\u00A0occluder)\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0rounded\t \u00C2\u00A0tip\t \u00C2\u00A0was\t \u00C2\u00A0carefully\t \u00C2\u00A0inserted\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0incision.\t \u00C2\u00A0The\t \u00C2\u00A0ECA\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0tied\t \u00C2\u00A0shut\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00900\t \u00C2\u00A0silk\t \u00C2\u00A0suture\t \u00C2\u00A0to\t \u00C2\u00A0 99 prevent\t \u00C2\u00A0bleeding,\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0CCA\t \u00C2\u00A0and\t \u00C2\u00A0ICA\t \u00C2\u00A0were\t \u00C2\u00A0released.\t \u00C2\u00A0Next,\t \u00C2\u00A0the\t \u00C2\u00A0ECA\t \u00C2\u00A0was\t \u00C2\u00A0cut\t \u00C2\u00A0and\t \u00C2\u00A0reversed\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0the\t \u00C2\u00A0entry\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0occluder\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0ICA\t \u00C2\u00A0until\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0resistance\t \u00C2\u00A0was\t \u00C2\u00A0felt,\t \u00C2\u00A0which\t \u00C2\u00A0indicates\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0occluder\t \u00C2\u00A0is\t \u00C2\u00A0in\t \u00C2\u00A0place\t \u00C2\u00A0to\t \u00C2\u00A0block\t \u00C2\u00A0the\t \u00C2\u00A0origin\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0right\t \u00C2\u00A0MCA.\t \u00C2\u00A0The\t \u00C2\u00A0surgical\t \u00C2\u00A0incisions\t \u00C2\u00A0were\t \u00C2\u00A0sutured\t \u00C2\u00A0closed\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0rats\t \u00C2\u00A0were\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0recover\t \u00C2\u00A0during\t \u00C2\u00A0the\t \u00C2\u00A060min\t \u00C2\u00A0occlusion\t \u00C2\u00A0period.\t \u00C2\u00A0To\t \u00C2\u00A0remove\t \u00C2\u00A0the\t \u00C2\u00A0suture\t \u00C2\u00A0and\t \u00C2\u00A0initiate\t \u00C2\u00A0reperfusion,\t \u00C2\u00A0rats\t \u00C2\u00A0were\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090anesthetized\t \u00C2\u00A0and\t \u00C2\u00A0monitored\t \u00C2\u00A0for\t \u00C2\u00A0body\t \u00C2\u00A0temperature\t \u00C2\u00A0and\t \u00C2\u00A0breathing\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0above.\t \u00C2\u00A0The\t \u00C2\u00A0sutures\t \u00C2\u00A0were\t \u00C2\u00A0removed\t \u00C2\u00A0to\t \u00C2\u00A0expose\t \u00C2\u00A0the\t \u00C2\u00A0occluder,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0gently\t \u00C2\u00A0pulled\t \u00C2\u00A0back\t \u00C2\u00A0and\t \u00C2\u00A0removed.\t \u00C2\u00A0The\t \u00C2\u00A0ECA\t \u00C2\u00A0was\t \u00C2\u00A0tied\t \u00C2\u00A0tightly\t \u00C2\u00A0to\t \u00C2\u00A0prevent\t \u00C2\u00A0bleeding\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0wound\t \u00C2\u00A0was\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090closed\t \u00C2\u00A0with\t \u00C2\u00A0sutures.\t \u00C2\u00A0The\t \u00C2\u00A0rats\t \u00C2\u00A0were\t \u00C2\u00A0allowed\t \u00C2\u00A0to\t \u00C2\u00A0recover\t \u00C2\u00A0in\t \u00C2\u00A0individual\t \u00C2\u00A0cages.\t \u00C2\u00A0One\t \u00C2\u00A0hour\t \u00C2\u00A0after\t \u00C2\u00A0reperfusion,\t \u00C2\u00A0rats\t \u00C2\u00A0were\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A0saline,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0(10mg/kg)\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(10mg/kg)\t \u00C2\u00A0peptide\t \u00C2\u00A0via\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0i.v.\t \u00C2\u00A0injection.\t \u00C2\u00A0The\t \u00C2\u00A0brains\t \u00C2\u00A0were\t \u00C2\u00A0harvested\t \u00C2\u00A06h\t \u00C2\u00A0after\t \u00C2\u00A0peptide\t \u00C2\u00A0treatment\t \u00C2\u00A0for\t \u00C2\u00A0further\t \u00C2\u00A0processing.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 100 Table\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00901.\t \u00C2\u00A0Primary\t \u00C2\u00A0antibodies\t \u00C2\u00A0Primary\t \u00C2\u00A0antibody\t \u00C2\u00A0 Company\t \u00C2\u00A0 Catalogue\t \u00C2\u00A0#\t \u00C2\u00A0 Dilution\t \u00C2\u00A0\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0 Clontech\t \u00C2\u00A0 cat.\t \u00C2\u00A0#632381\t \u00C2\u00A0 1:5000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090GAPDH\t \u00C2\u00A0 Abcam\t \u00C2\u00A0 cat.\t \u00C2\u00A0#ab9485\t \u00C2\u00A0 1:2000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0 Abcam\t \u00C2\u00A0 cat.\t \u00C2\u00A0#ab8227\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti\u00E2\u0080\u0093\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0 BD\t \u00C2\u00A0Transduction\t \u00C2\u00A0Laboratories\t \u00C2\u00A0cat.\t \u00C2\u00A0#610786\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090HA\t \u00C2\u00A0 Roche\t \u00C2\u00A0Applied\t \u00C2\u00A0Science\t \u00C2\u00A0 cat.\t \u00C2\u00A0#11867431001\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0monoclonal\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090Flag\t \u00C2\u00A0M2\t \u00C2\u00A0Sigma-\u00C2\u00AD\u00E2\u0080\u0090Aldrich\t \u00C2\u00A0 cat.\t \u00C2\u00A0#F1804\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0 Sigma\t \u00C2\u00A0 cat.\t \u00C2\u00A0#D1319x\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0monoclonal\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090phospho-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0(pSer308)\t \u00C2\u00A0Sigma\t \u00C2\u00A0 cat.\t \u00C2\u00A0#D4941\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0 Wang\t \u00C2\u00A0laboratory\t \u00C2\u00A0home\t \u00C2\u00A0made\t \u00C2\u00A0N/A\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti\u00E2\u0080\u0093LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0 Abcam\t \u00C2\u00A0 cat.\t \u00C2\u00A0#ab18528\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0 101 Primary\t \u00C2\u00A0antibody\t \u00C2\u00A0 Company\t \u00C2\u00A0 Catalogue\t \u00C2\u00A0#\t \u00C2\u00A0 Dilution\t \u00C2\u00A0\t \u00C2\u00A0anti\u00E2\u0080\u0093lamin\t \u00C2\u00A0B1\t \u00C2\u00A0 Abcam\t \u00C2\u00A0 cat.\t \u00C2\u00A0#ab16048\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090HSP90\t \u00C2\u00A0 BD\t \u00C2\u00A0Transduction\t \u00C2\u00A0Laboratories\t \u00C2\u00A0cat.\t \u00C2\u00A0#610418\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090VDAC1\t \u00C2\u00A0(porin)\t \u00C2\u00A0 MitoSciences\t \u00C2\u00A0 cat.\t \u00C2\u00A0#MSA03\t \u00C2\u00A0 1:1000\t \u00C2\u00A0dilution\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 102 Table\t \u00C2\u00A02-\u00C2\u00AD\u00E2\u0080\u00902.\t \u00C2\u00A0Synthetic\t \u00C2\u00A0peptide\t \u00C2\u00A0sequences\t \u00C2\u00A0Synthetic\t \u00C2\u00A0peptide\t \u00C2\u00A0 Target\t \u00C2\u00A0protein\t \u00C2\u00A0 Sequence\t \u00C2\u00A0GluN2B-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0 DAPK1\t \u00C2\u00A0 KKNRNKLRRQHSY-\u00C2\u00AD\u00E2\u0080\u0090KFERQKILDQRFFE\t \u00C2\u00A0Pep-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0 N/A\t \u00C2\u00A0(CPP)\t \u00C2\u00A0 KETWWETWWTEWSQPKKKRKV\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0 DAPK1\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0KKNRNKLRRQHSY-\u00C2\u00AD\u00E2\u0080\u0090KFERQKILDQRFFE\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0 N/A\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0KKNRNKLRRQHSY\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0 \u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0GVLYVGSKTR-\u00C2\u00AD\u00E2\u0080\u0090KFERQKILDQRFFE\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn\t \u00C2\u00A0 N/A\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090GVLYVGSKTR\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9cCTM\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090KLSSIESDV-\u00C2\u00AD\u00E2\u0080\u0090KFERQKILDQRFFE\t \u00C2\u00A0\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9c\t \u00C2\u00A0 N/A\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090KLSSIESDV\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090r\u00CE\u00B2synCTM1\t \u00C2\u00A0 \u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0RTKSGVYLVG\t \u00C2\u00A0\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090QKILD\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090r\u00CE\u00B2synCTM2\t \u00C2\u00A0 \u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0 YGRKKRRQRRR-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0RTKSGVYLVG\t \u00C2\u00A0\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090QKILDQRFFE\t \u00C2\u00A0\t \u00C2\u00A0 103 CHAPTER\t \u00C2\u00A03: SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0PROTEINS\t \u00C2\u00A03.1 INTRODUCTION\t \u00C2\u00A0The\t \u00C2\u00A0plethora\t \u00C2\u00A0of\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0allow\t \u00C2\u00A0the\t \u00C2\u00A0targeted\t \u00C2\u00A0manipulation\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0have\t \u00C2\u00A0greatly\t \u00C2\u00A0advanced\t \u00C2\u00A0our\t \u00C2\u00A0understanding\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0functions\t \u00C2\u00A0in\t \u00C2\u00A0health\t \u00C2\u00A0and\t \u00C2\u00A0disease.\t \u00C2\u00A0However,\t \u00C2\u00A0as\t \u00C2\u00A0discussed\t \u00C2\u00A0in\t \u00C2\u00A0detail\t \u00C2\u00A0in\t \u00C2\u00A0Chapters\t \u00C2\u00A01.2-\u00C2\u00AD\u00E2\u0080\u00901.5,\t \u00C2\u00A0existing\t \u00C2\u00A0strategies\t \u00C2\u00A0are\t \u00C2\u00A0often\t \u00C2\u00A0limited\t \u00C2\u00A0in\t \u00C2\u00A0speed,\t \u00C2\u00A0specificity,\t \u00C2\u00A0spatiotemporal\t \u00C2\u00A0control\t \u00C2\u00A0and\t \u00C2\u00A0reversibility.\t \u00C2\u00A0\t \u00C2\u00A0Degrons,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0degron\t \u00C2\u00A0and\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domains,\t \u00C2\u00A0offer\t \u00C2\u00A0a\t \u00C2\u00A0means\t \u00C2\u00A0to\t \u00C2\u00A0directly\t \u00C2\u00A0harness\t \u00C2\u00A0cellular\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0The\t \u00C2\u00A0introduction\t \u00C2\u00A0of\t \u00C2\u00A0degron-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0molecules\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0through\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0allows\t \u00C2\u00A0the\t \u00C2\u00A0direct\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0(e.g.\t \u00C2\u00A0PROTACs,\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.5.2).\t \u00C2\u00A0However,\t \u00C2\u00A0whether\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0is\t \u00C2\u00A0also\t \u00C2\u00A0feasible\t \u00C2\u00A0within\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0CMA\t \u00C2\u00A0system\t \u00C2\u00A0is\t \u00C2\u00A0unclear.\t \u00C2\u00A0\t \u00C2\u00A0Naturally\t \u00C2\u00A0occurring\t \u00C2\u00A0CTMs\t \u00C2\u00A0are\t \u00C2\u00A0pentapeptide\t \u00C2\u00A0motifs\t \u00C2\u00A0biochemically\t \u00C2\u00A0related\t \u00C2\u00A0to\t \u00C2\u00A0KFERQ.\t \u00C2\u00A0Although\t \u00C2\u00A0there\t \u00C2\u00A0is\t \u00C2\u00A0evidence\t \u00C2\u00A0that\t \u00C2\u00A0one\t \u00C2\u00A0motif\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0lysosomal\t \u00C2\u00A0targeting\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012),\t \u00C2\u00A0\t \u00C2\u00A0we\t \u00C2\u00A0engineered\t \u00C2\u00A0a\t \u00C2\u00A0multivalent\t \u00C2\u00A0CTM\t \u00C2\u00A0to\t \u00C2\u00A0potentially\t \u00C2\u00A0enhance\t \u00C2\u00A0its\t \u00C2\u00A0interaction\t \u00C2\u00A0with\t \u00C2\u00A0hsc70\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0enhance\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0efficiency.\t \u00C2\u00A0Our\t \u00C2\u00A0chimeric\t \u00C2\u00A0CTM\t \u00C2\u00A0contains\t \u00C2\u00A0three\t \u00C2\u00A0different\t \u00C2\u00A0CTMs\t \u00C2\u00A0derived\t \u00C2\u00A0from\t \u00C2\u00A0endogenous\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0RNase\t \u00C2\u00A0A\t \u00C2\u00A0(KFERQ)\t \u00C2\u00A0(Backer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01983),\t \u00C2\u00A0hsc70\t \u00C2\u00A0(QKILD)\t \u00C2\u00A0(Cuervo\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02000a)\t \u00C2\u00A0and\t \u00C2\u00A0hemoglobin\t \u00C2\u00A0(QRFFE)\t \u00C2\u00A0(Slot\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01986).\t \u00C2\u00A0 104 In\t \u00C2\u00A0this\t \u00C2\u00A0first\t \u00C2\u00A0set\t \u00C2\u00A0of\t \u00C2\u00A0experiments,\t \u00C2\u00A0we\t \u00C2\u00A0first\t \u00C2\u00A0demonstrate\t \u00C2\u00A0that\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0transferable.\t \u00C2\u00A0In\t \u00C2\u00A0other\t \u00C2\u00A0words,\t \u00C2\u00A0the\t \u00C2\u00A0direct\t \u00C2\u00A0attachment\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0proposed\t \u00C2\u00A0CTM\t \u00C2\u00A0suffices\t \u00C2\u00A0to\t \u00C2\u00A0make\t \u00C2\u00A0a\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0CMA\t \u00C2\u00A0degradation.\t \u00C2\u00A0More\t \u00C2\u00A0importantly,\t \u00C2\u00A0we\t \u00C2\u00A0present\t \u00C2\u00A0the\t \u00C2\u00A0first\t \u00C2\u00A0evidence\t \u00C2\u00A0that\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0can\t \u00C2\u00A0direct\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions.\t \u00C2\u00A03.2 RESULTS\t \u00C2\u00A03.2.1 KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090LINKED\t \u00C2\u00A0GREEN\t \u00C2\u00A0FLUORESCENT\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0(GFP)\t \u00C2\u00A0IN\t \u00C2\u00A0HUMAN\t \u00C2\u00A0CELLS\t \u00C2\u00A0GFP\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A027kDa\t \u00C2\u00A0protein\t \u00C2\u00A0known\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0resistant\t \u00C2\u00A0against\t \u00C2\u00A0degradation\t \u00C2\u00A0via\t \u00C2\u00A0CMA\t \u00C2\u00A0(Majeski\t \u00C2\u00A0and\t \u00C2\u00A0Dice,\t \u00C2\u00A02004).\t \u00C2\u00A0In\t \u00C2\u00A0an\t \u00C2\u00A0overexpression\t \u00C2\u00A0system,\t \u00C2\u00A0we\t \u00C2\u00A0first\t \u00C2\u00A0tested\t \u00C2\u00A0whether\t \u00C2\u00A0genetic\t \u00C2\u00A0fusion\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0CTM\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0of\t \u00C2\u00A0GFP\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0to\t \u00C2\u00A0direct\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0(CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901a).\t \u00C2\u00A0Consistent\t \u00C2\u00A0with\t \u00C2\u00A0GFP\t \u00C2\u00A0being\t \u00C2\u00A0a\t \u00C2\u00A0constitutively\t \u00C2\u00A0expressed\t \u00C2\u00A0cytosolic\t \u00C2\u00A0protein,\t \u00C2\u00A0immunofluorescence\t \u00C2\u00A0staining\t \u00C2\u00A0revealed\t \u00C2\u00A0diffuse\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0in\t \u00C2\u00A0most\t \u00C2\u00A0compartments\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cell,\t \u00C2\u00A0including\t \u00C2\u00A0the\t \u00C2\u00A0nucleus\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901b).\t \u00C2\u00A0In\t \u00C2\u00A0marked\t \u00C2\u00A0contrast,\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0appeared\t \u00C2\u00A0to\t \u00C2\u00A0cluster\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0perinuclear\t \u00C2\u00A0area\t \u00C2\u00A0and\t \u00C2\u00A0showed\t \u00C2\u00A0a\t \u00C2\u00A0high\t \u00C2\u00A0degree\t \u00C2\u00A0of\t \u00C2\u00A0colocalization\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0protein\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00901,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0is\t \u00C2\u00A0being\t \u00C2\u00A0directed\t \u00C2\u00A0towards\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0compartment.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0assess\t \u00C2\u00A0the\t \u00C2\u00A0metabolic\t \u00C2\u00A0stability\t \u00C2\u00A0of\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0and\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0we\t \u00C2\u00A0transiently\t \u00C2\u00A0expressed\t \u00C2\u00A0these\t \u00C2\u00A0constructs\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0for\t \u00C2\u00A024h\t \u00C2\u00A0and\t \u00C2\u00A0measured\t \u00C2\u00A0their\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0with\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0showed\t \u00C2\u00A0markedly\t \u00C2\u00A0 105 reduced\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00902),\t \u00C2\u00A0an\t \u00C2\u00A0effect\t \u00C2\u00A0that\t \u00C2\u00A0was\t \u00C2\u00A0fully\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0treatment\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0inhibitors\t \u00C2\u00A0ammonium\t \u00C2\u00A0choloride\t \u00C2\u00A0(inhibits\t \u00C2\u00A0lysosomal\t \u00C2\u00A0acidification)\t \u00C2\u00A0and\t \u00C2\u00A0pepstatin\t \u00C2\u00A0A\t \u00C2\u00A0(inhibits\t \u00C2\u00A0two\t \u00C2\u00A0primary\t \u00C2\u00A0lysosomal\t \u00C2\u00A0proteases,\t \u00C2\u00A0cathepsins\t \u00C2\u00A0D\t \u00C2\u00A0and\t \u00C2\u00A0E)\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0proteasomal\t \u00C2\u00A0inhibitor\t \u00C2\u00A0MG132\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0This\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0is\t \u00C2\u00A0being\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0system.\t \u00C2\u00A0As\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation\t \u00C2\u00A0can\t \u00C2\u00A0occur\t \u00C2\u00A0through\t \u00C2\u00A0macroautophagy,\t \u00C2\u00A0microautophagy\t \u00C2\u00A0or\t \u00C2\u00A0CMA,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0clarify\t \u00C2\u00A0which\t \u00C2\u00A0pathway\t \u00C2\u00A0mediates\t \u00C2\u00A0the\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0levels.\t \u00C2\u00A0Treatment\t \u00C2\u00A0with\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090methyladenine\t \u00C2\u00A0(3-\u00C2\u00AD\u00E2\u0080\u0090MA),\t \u00C2\u00A0an\t \u00C2\u00A0inhibitor\t \u00C2\u00A0for\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0CMA,\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0prevent\t \u00C2\u00A0the\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0serum\t \u00C2\u00A0withdrawal,\t \u00C2\u00A0which\t \u00C2\u00A0enhances\t \u00C2\u00A0CMA\t \u00C2\u00A0activity,\t \u00C2\u00A0significantly\t \u00C2\u00A0increased\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0degradation\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0These\t \u00C2\u00A0two\t \u00C2\u00A0lines\t \u00C2\u00A0of\t \u00C2\u00A0evidence\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0major\t \u00C2\u00A0pathway\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP.\t \u00C2\u00A0The\t \u00C2\u00A0Q\t \u00C2\u00A0residue\t \u00C2\u00A0in\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0crucial\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0normal\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0motif.\t \u00C2\u00A0To\t \u00C2\u00A0further\t \u00C2\u00A0provide\t \u00C2\u00A0evidence\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0main\t \u00C2\u00A0degradation\t \u00C2\u00A0route\t \u00C2\u00A0for\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0we\t \u00C2\u00A0mutated\t \u00C2\u00A0the\t \u00C2\u00A0two\t \u00C2\u00A0Q\t \u00C2\u00A0residues\t \u00C2\u00A0in\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0to\t \u00C2\u00A0A\t \u00C2\u00A0residues.\t \u00C2\u00A0As\t \u00C2\u00A0expected,\t \u00C2\u00A0the\t \u00C2\u00A0mutant\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0was\t \u00C2\u00A0resistant\t \u00C2\u00A0against\t \u00C2\u00A0degradation\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0Together,\t \u00C2\u00A0these\t \u00C2\u00A0data\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0experimental\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0to\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0proteins\t \u00C2\u00A0renders\t \u00C2\u00A0it\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0pathway.\t \u00C2\u00A0 106 3.2.2 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0\u00CE\u0091-\u00C2\u00AD\u00E2\u0080\u0090SYNUCLEIN\t \u00C2\u00A0\t \u00C2\u00A0We\t \u00C2\u00A0next\t \u00C2\u00A0examined\t \u00C2\u00A0whether\t \u00C2\u00A0a\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0protein\t \u00C2\u00A0could\t \u00C2\u00A0be\t \u00C2\u00A0indirectly\t \u00C2\u00A0targeted\t \u00C2\u00A0for\t \u00C2\u00A0CMA\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0through\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction.\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0implicated\t \u00C2\u00A0in\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0synucleinopathies\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0PD\t \u00C2\u00A0(Clayton\t \u00C2\u00A0and\t \u00C2\u00A0George,\t \u00C2\u00A01998).\t \u00C2\u00A0The\t \u00C2\u00A0protein\t \u00C2\u00A0was\t \u00C2\u00A0recently\t \u00C2\u00A0found\t \u00C2\u00A0to\t \u00C2\u00A0strongly\t \u00C2\u00A0interact\t \u00C2\u00A0with\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A036-\u00C2\u00AD\u00E2\u0080\u009045\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u00B2syn36)\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0(Shaltiel-\u00C2\u00AD\u00E2\u0080\u0090Karyo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010),\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0\u00CE\u00B2syn36\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0a\t \u00C2\u00A0suitable\t \u00C2\u00A0PBD\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein.\t \u00C2\u00A0We\t \u00C2\u00A0hypothesized\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0containing\t \u00C2\u00A0\u00CE\u00B2syn36\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0CTM,\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM,\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0sufficient\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0for\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation.\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0a\t \u00C2\u00A0peptide\t \u00C2\u00A0(FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36)\t \u00C2\u00A0containing\t \u00C2\u00A0\u00CE\u00B2syn36\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0the\t \u00C2\u00A0CTM,\t \u00C2\u00A0while\t \u00C2\u00A0having\t \u00C2\u00A0the\t \u00C2\u00A0ability\t \u00C2\u00A0to\t \u00C2\u00A0interact\t \u00C2\u00A0with\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein,\t \u00C2\u00A0would\t \u00C2\u00A0not\t \u00C2\u00A0be\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0knockdown.\t \u00C2\u00A0As\t \u00C2\u00A0expected,\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090expression\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0with\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein,\t \u00C2\u00A0significantly\t \u00C2\u00A0decreased\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0levels\t \u00C2\u00A048h\t \u00C2\u00A0following\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfection.\t \u00C2\u00A0Notably,\t \u00C2\u00A0by\t \u00C2\u00A0increasing\t \u00C2\u00A0the\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090dose\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0relative\t \u00C2\u00A0to\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein,\t \u00C2\u00A0we\t \u00C2\u00A0found\t \u00C2\u00A0that\t \u00C2\u00A0this\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0degradation\t \u00C2\u00A0was\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent,\t \u00C2\u00A0as\t \u00C2\u00A0opposed\t \u00C2\u00A0to\t \u00C2\u00A0all-\u00C2\u00AD\u00E2\u0080\u0090or-\u00C2\u00AD\u00E2\u0080\u0090none\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00903a).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0required\t \u00C2\u00A0normal\t \u00C2\u00A0lysosome\t \u00C2\u00A0function,\t \u00C2\u00A0as\t \u00C2\u00A0treatment\t \u00C2\u00A0with\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0completely\t \u00C2\u00A0abolished\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0knockdown\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00903a).\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36\t \u00C2\u00A0had\t \u00C2\u00A0no\t \u00C2\u00A0effect\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0even\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0highest\t \u00C2\u00A0transfected\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090dose\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00903b).\t \u00C2\u00A0 107 3.2.3 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0ACTIVATED\t \u00C2\u00A0DAPK1\t \u00C2\u00A0\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0interact\t \u00C2\u00A0constitutively\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0under\t \u00C2\u00A0basal\t \u00C2\u00A0conditions.\t \u00C2\u00A0However,\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0many\t \u00C2\u00A0cellular\t \u00C2\u00A0processes\t \u00C2\u00A0are\t \u00C2\u00A0regulated\t \u00C2\u00A0by\t \u00C2\u00A0transient\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0that\t \u00C2\u00A0binds\t \u00C2\u00A0conditionally\t \u00C2\u00A0to\t \u00C2\u00A0its\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0knockdown\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0condition-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner.\t \u00C2\u00A0As\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept,\t \u00C2\u00A0we\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0recombinant\t \u00C2\u00A0DAPK1\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death,\t \u00C2\u00A0calmodulin\u00E2\u0080\u0093regulated,\t \u00C2\u00A0serine-\u00C2\u00AD\u00E2\u0080\u0090threonine\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0that\t \u00C2\u00A0promotes\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A0following\t \u00C2\u00A0brain\t \u00C2\u00A0injury\t \u00C2\u00A0(Velentza\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005;\t \u00C2\u00A0Henshall\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004,\t \u00C2\u00A02005;\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0DAPK1\t \u00C2\u00A0was\t \u00C2\u00A0recently\t \u00C2\u00A0validated\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0CNS\t \u00C2\u00A0drug\t \u00C2\u00A0discovery\t \u00C2\u00A0target\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0observations.\t \u00C2\u00A0First,\t \u00C2\u00A0using\t \u00C2\u00A0an\t \u00C2\u00A0animal\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0ischemic\t \u00C2\u00A0stroke,\t \u00C2\u00A0the\t \u00C2\u00A0enzymatic\t \u00C2\u00A0activity\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0was\t \u00C2\u00A0found\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0after\t \u00C2\u00A0injury\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0therapeutically\t \u00C2\u00A0relevant\t \u00C2\u00A0time\t \u00C2\u00A0frame\t \u00C2\u00A0(Schumacher\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002),\t \u00C2\u00A0and\t \u00C2\u00A0inhibition\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activity\t \u00C2\u00A0attenuates\t \u00C2\u00A0brain\t \u00C2\u00A0injury\t \u00C2\u00A0(Velentza\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Second,\t \u00C2\u00A0increased\t \u00C2\u00A0DAPK1\t \u00C2\u00A0expression\t \u00C2\u00A0was\t \u00C2\u00A0also\t \u00C2\u00A0found\t \u00C2\u00A0to\t \u00C2\u00A0correlate\t \u00C2\u00A0with\t \u00C2\u00A0epileptic\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage\t \u00C2\u00A0in\t \u00C2\u00A0both\t \u00C2\u00A0animal\t \u00C2\u00A0models\t \u00C2\u00A0(Henshall\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003)\t \u00C2\u00A0and\t \u00C2\u00A0human\t \u00C2\u00A0patients\t \u00C2\u00A0(Henshall\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004).\t \u00C2\u00A0Finally,\t \u00C2\u00A0a\t \u00C2\u00A0recent\t \u00C2\u00A0genetic\t \u00C2\u00A0study\t \u00C2\u00A0in\t \u00C2\u00A0human\t \u00C2\u00A0patients\t \u00C2\u00A0uncovered\t \u00C2\u00A0a\t \u00C2\u00A0linkage\t \u00C2\u00A0between\t \u00C2\u00A0genetic\t \u00C2\u00A0variation\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1\t \u00C2\u00A0gene\t \u00C2\u00A0locus\t \u00C2\u00A0and\t \u00C2\u00A0susceptibility\t \u00C2\u00A0to\t \u00C2\u00A0late-\u00C2\u00AD\u00E2\u0080\u0090onset\t \u00C2\u00A0Alzheimer\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0(Li,\t \u00C2\u00A02006).\t \u00C2\u00A0Despite\t \u00C2\u00A0the\t \u00C2\u00A0therapeutic\t \u00C2\u00A0potential\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0inhibition,\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0currently\t \u00C2\u00A0no\t \u00C2\u00A0clinically\t \u00C2\u00A0promising\t \u00C2\u00A0small-\u00C2\u00AD\u00E2\u0080\u0090molecule\t \u00C2\u00A0inhibitors\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0kinase\t \u00C2\u00A0(Chico\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Huang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014),\t \u00C2\u00A0making\t \u00C2\u00A0it\t \u00C2\u00A0an\t \u00C2\u00A0attractive\t \u00C2\u00A0target\t \u00C2\u00A0for\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0knockdown.\t \u00C2\u00A0 108 Under\t \u00C2\u00A0basal\t \u00C2\u00A0conditions,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0phosphorylated\t \u00C2\u00A0and\t \u00C2\u00A0inactive\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0CNS\t \u00C2\u00A0(Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0Neurotoxic\t \u00C2\u00A0insults\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0ischemia\t \u00C2\u00A0activates\t \u00C2\u00A0DAPK1\t \u00C2\u00A0via\t \u00C2\u00A0dephosphorylation\t \u00C2\u00A0at\t \u00C2\u00A0serine\t \u00C2\u00A0308,\t \u00C2\u00A0whereby\t \u00C2\u00A0the\t \u00C2\u00A0kinase\t \u00C2\u00A0is\t \u00C2\u00A0recruited\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR\t \u00C2\u00A0complex\t \u00C2\u00A0via\t \u00C2\u00A0direct\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A01292\u00E2\u0080\u00931304\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0residues\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0subunit\t \u00C2\u00A0of\t \u00C2\u00A0NMDAR\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0GluN2B\t \u00C2\u00A0can\t \u00C2\u00A0bind\t \u00C2\u00A0only\t \u00C2\u00A0the\t \u00C2\u00A0active\t \u00C2\u00A0(dephosphorylated)\t \u00C2\u00A0and\t \u00C2\u00A0not\t \u00C2\u00A0the\t \u00C2\u00A0inactive\t \u00C2\u00A0(phosphorylated)\t \u00C2\u00A0form\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0we\t \u00C2\u00A0reasoned\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0peptide\t \u00C2\u00A0composed\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1\t \u00C2\u00A0binding\t \u00C2\u00A0domain\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0and\t \u00C2\u00A0CTM\t \u00C2\u00A0would\t \u00C2\u00A0specifically\t \u00C2\u00A0knockdown\t \u00C2\u00A0active\t \u00C2\u00A0DAPK1\t \u00C2\u00A0but\t \u00C2\u00A0leave\t \u00C2\u00A0the\t \u00C2\u00A0inactive\t \u00C2\u00A0DAPK1\t \u00C2\u00A0intact.\t \u00C2\u00A0\t \u00C2\u00A0We\t \u00C2\u00A0first\t \u00C2\u00A0inserted\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0GluN2Bct1242\u00E2\u0080\u00931342\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0of\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP)\t \u00C2\u00A0or\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP)\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904a).\t \u00C2\u00A0Following\t \u00C2\u00A0transfection\t \u00C2\u00A0and\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0either\t \u00C2\u00A0plasmid\t \u00C2\u00A0into\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells,\t \u00C2\u00A0we\t \u00C2\u00A0found\t \u00C2\u00A0that\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0was\t \u00C2\u00A0significantly\t \u00C2\u00A0degraded\t \u00C2\u00A0as\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0lysosomal\t \u00C2\u00A0inhibition\t \u00C2\u00A0(n=4;\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904b).\t \u00C2\u00A0To\t \u00C2\u00A0confirm\t \u00C2\u00A0binding\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0peptides\t \u00C2\u00A0and\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0we\t \u00C2\u00A0performed\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090IP,\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfecting\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0or\t \u00C2\u00A0its\t \u00C2\u00A0control\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0the\t \u00C2\u00A0constitutively\t \u00C2\u00A0active\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0or\t \u00C2\u00A0inactive\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1.\t \u00C2\u00A0Subsequent\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A0confirmed\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0immunoprecipitate\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904c).\t \u00C2\u00A0\t \u00C2\u00A0Notably,\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090dose\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0are\t \u00C2\u00A0noticeably\t \u00C2\u00A0lower\t \u00C2\u00A0than\t \u00C2\u00A0that\t \u00C2\u00A0of\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0itself\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0via\t \u00C2\u00A0CMA.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0are\t \u00C2\u00A0lower\t \u00C2\u00A0when\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfected\t \u00C2\u00A0with\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090 109 CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0as\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904c).\t \u00C2\u00A0This\t \u00C2\u00A0initial\t \u00C2\u00A0observation\t \u00C2\u00A0raises\t \u00C2\u00A0the\t \u00C2\u00A0possibility\t \u00C2\u00A0that\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0is\t \u00C2\u00A0facilitating\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1.\t \u00C2\u00A0To\t \u00C2\u00A0further\t \u00C2\u00A0investigate,\t \u00C2\u00A0we\t \u00C2\u00A0increased\t \u00C2\u00A0the\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090dose\t \u00C2\u00A0of\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0relative\t \u00C2\u00A0to\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0and\t \u00C2\u00A0found\t \u00C2\u00A0a\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0levels,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0significantly\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0concomitant\t \u00C2\u00A0treatment\t \u00C2\u00A0with\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904d).\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0the\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0had\t \u00C2\u00A0no\t \u00C2\u00A0effect\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904e).\t \u00C2\u00A0Taken\t \u00C2\u00A0together,\t \u00C2\u00A0these\t \u00C2\u00A0observations\t \u00C2\u00A0indicate\t \u00C2\u00A0that\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0its\t \u00C2\u00A0binding\t \u00C2\u00A0control\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0specifically\t \u00C2\u00A0directs\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0for\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation.\t \u00C2\u00A03.2.4 PLACING\t \u00C2\u00A0CTM\t \u00C2\u00A0AT\t \u00C2\u00A0THE\t \u00C2\u00A0TERMINUS\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER\t \u00C2\u00A0INCREASES\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0EFFICIENCY\t \u00C2\u00A0\t \u00C2\u00A0Given\t \u00C2\u00A0our\t \u00C2\u00A0discovery\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0facilitates\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0its\t \u00C2\u00A0interacting\t \u00C2\u00A0protein\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0pathway,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0optimize\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0to\t \u00C2\u00A0maximize\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0efficiency.\t \u00C2\u00A0The\t \u00C2\u00A0location\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0is\t \u00C2\u00A0generally\t \u00C2\u00A0thought\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0inconsequential,\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0only\t \u00C2\u00A0requirement\t \u00C2\u00A0being\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0exposed\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0cytosol\t \u00C2\u00A0so\t \u00C2\u00A0it\t \u00C2\u00A0can\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0hsc70\t \u00C2\u00A0and\t \u00C2\u00A0other\t \u00C2\u00A0chaperones\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012).\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0(14aa)\t \u00C2\u00A0is\t \u00C2\u00A0sandwiched\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0100aa\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A027kDa\t \u00C2\u00A0GFP\t \u00C2\u00A0tag.\t \u00C2\u00A0This\t \u00C2\u00A0increases\t \u00C2\u00A0the\t \u00C2\u00A0likelihood\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0obstructed\t \u00C2\u00A0by\t \u00C2\u00A0local\t \u00C2\u00A0bulky\t \u00C2\u00A0groups\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0become\t \u00C2\u00A0inaccessible\t \u00C2\u00A0to\t \u00C2\u00A0chaperone\t \u00C2\u00A0binding.\t \u00C2\u00A0We\t \u00C2\u00A0thus\t \u00C2\u00A0reasoned\t \u00C2\u00A0that\t \u00C2\u00A0 110 replacing\t \u00C2\u00A0GFP\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0smaller\t \u00C2\u00A0epitope\t \u00C2\u00A0tag\t \u00C2\u00A0HA\t \u00C2\u00A0(influenza\t \u00C2\u00A0hemagglutinin,\t \u00C2\u00A09aa)\t \u00C2\u00A0and\t \u00C2\u00A0transferring\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0terminal\t \u00C2\u00A0would\t \u00C2\u00A0increase\t \u00C2\u00A0the\t \u00C2\u00A0efficiency\t \u00C2\u00A0of\t \u00C2\u00A0lysosomal\t \u00C2\u00A0targeting\t \u00C2\u00A0and\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0We\t \u00C2\u00A0therefore\t \u00C2\u00A0engineered\t \u00C2\u00A0two\t \u00C2\u00A0new\t \u00C2\u00A0constructs\t \u00C2\u00A0that\t \u00C2\u00A0contained\t \u00C2\u00A0the\t \u00C2\u00A0HA\t \u00C2\u00A0epitope\t \u00C2\u00A0tag\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0100aa\t \u00C2\u00A0DAPK1\t \u00C2\u00A0binding\t \u00C2\u00A0domain\t \u00C2\u00A0(HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct)\t \u00C2\u00A0at\t \u00C2\u00A0their\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal,\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0either\t \u00C2\u00A0a\t \u00C2\u00A0functional\t \u00C2\u00A0CTM\t \u00C2\u00A0(HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM)\t \u00C2\u00A0or\t \u00C2\u00A0inactive\t \u00C2\u00A0CTM\t \u00C2\u00A0(HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm)\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905a).\t \u00C2\u00A0\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090expression\t \u00C2\u00A0and\t \u00C2\u00A0immunocytochemistry\t \u00C2\u00A0revealed\t \u00C2\u00A0significant\t \u00C2\u00A0overlap\t \u00C2\u00A0between\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(stained\t \u00C2\u00A0with\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090HA)\t \u00C2\u00A0and\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0(stained\t \u00C2\u00A0with\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090FLAG)\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905b).\t \u00C2\u00A0Consistent\t \u00C2\u00A0with\t \u00C2\u00A0this\t \u00C2\u00A0result,\t \u00C2\u00A0overexpression\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0degradation-\u00C2\u00AD\u00E2\u0080\u0090resistant\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0or\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0and\t \u00C2\u00A0subsequent\t \u00C2\u00A0reciprocal\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090IP\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905c).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0confirms\t \u00C2\u00A0our\t \u00C2\u00A0previous\t \u00C2\u00A0observation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090activity\t \u00C2\u00A0dependent\t \u00C2\u00A0interaction\t \u00C2\u00A0with\t \u00C2\u00A0GluN2Bct.\t \u00C2\u00A0\t \u00C2\u00A0Next,\t \u00C2\u00A0we\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090expressed\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0with\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0at\t \u00C2\u00A0increasing\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090dose\t \u00C2\u00A0ratios,\t \u00C2\u00A0and\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A0revealed\t \u00C2\u00A0a\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0that\t \u00C2\u00A0was\t \u00C2\u00A0reversed\t \u00C2\u00A0by\t \u00C2\u00A0lysosomal\t \u00C2\u00A0inhibition\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905d).\t \u00C2\u00A0Importantly,\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0which\t \u00C2\u00A0triggered\t \u00C2\u00A0~50%\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0at\t \u00C2\u00A0an\t \u00C2\u00A08:1\t \u00C2\u00A0transfection\t \u00C2\u00A0ratio\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904d),\t \u00C2\u00A0the\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090designed\t \u00C2\u00A0peptide\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0reduced\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0by\t \u00C2\u00A0~90%\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090dose\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905d).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0 111 clearly\t \u00C2\u00A0highlights\t \u00C2\u00A0the\t \u00C2\u00A0importance\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0freely\t \u00C2\u00A0available\t \u00C2\u00A0CTM\t \u00C2\u00A0for\t \u00C2\u00A0increasing\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0potency.\t \u00C2\u00A0This\t \u00C2\u00A0is\t \u00C2\u00A0consistent\t \u00C2\u00A0with\t \u00C2\u00A0previous\t \u00C2\u00A0studies\t \u00C2\u00A0that\t \u00C2\u00A0employ\t \u00C2\u00A0degron-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0chimeric\t \u00C2\u00A0molecules\t \u00C2\u00A0for\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0UPS,\t \u00C2\u00A0which\t \u00C2\u00A0show\t \u00C2\u00A0that\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0efficiency\t \u00C2\u00A0is\t \u00C2\u00A0increased\t \u00C2\u00A0when\t \u00C2\u00A0the\t \u00C2\u00A0degron\t \u00C2\u00A0is\t \u00C2\u00A0located\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0terminus\t \u00C2\u00A0and/or\t \u00C2\u00A0separated\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0rest\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0molecule\t \u00C2\u00A0by\t \u00C2\u00A0long\t \u00C2\u00A0glycine-\u00C2\u00AD\u00E2\u0080\u0090serine\t \u00C2\u00A0linkers\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0flexible\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0their\t \u00C2\u00A0target\t \u00C2\u00A0components\t \u00C2\u00A0(Su\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090expression\t \u00C2\u00A0of\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0with\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0significantly\t \u00C2\u00A0affect\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0transfected\t \u00C2\u00A0alone,\t \u00C2\u00A0once\t \u00C2\u00A0again\t \u00C2\u00A0illustrating\t \u00C2\u00A0the\t \u00C2\u00A0need\t \u00C2\u00A0of\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0in\t \u00C2\u00A0mediating\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0cDAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905e).\t \u00C2\u00A0Finally,\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090expression\t \u00C2\u00A0of\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0mutated,\t \u00C2\u00A0with\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0significantly\t \u00C2\u00A0alter\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0protein\t \u00C2\u00A0levels,\t \u00C2\u00A0demonstrating\t \u00C2\u00A0that\t \u00C2\u00A0an\t \u00C2\u00A0intact\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0necessary\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0(Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905f).\t \u00C2\u00A0Of\t \u00C2\u00A0note,\t \u00C2\u00A0because\t \u00C2\u00A0the\t \u00C2\u00A0only\t \u00C2\u00A0difference\t \u00C2\u00A0between\t \u00C2\u00A0wtDAPK1\t \u00C2\u00A0and\t \u00C2\u00A0cDAPK1\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0former\t \u00C2\u00A0protein\t \u00C2\u00A0contains\t \u00C2\u00A0a\t \u00C2\u00A047\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0CaM\t \u00C2\u00A0regulatory\t \u00C2\u00A0domain,\t \u00C2\u00A0the\t \u00C2\u00A0selective\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0cDAPK1\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0wtDAPK1\t \u00C2\u00A0demonstrates\t \u00C2\u00A0the\t \u00C2\u00A0superior\t \u00C2\u00A0selectivity\t \u00C2\u00A0of\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0as\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0conventional\t \u00C2\u00A0genetic\t \u00C2\u00A0techniques\t \u00C2\u00A0like\t \u00C2\u00A0gene\t \u00C2\u00A0knockout\t \u00C2\u00A0or\t \u00C2\u00A0RNAi\t \u00C2\u00A0(see\t \u00C2\u00A0Chapter\t \u00C2\u00A01.2\t \u00C2\u00A0and\t \u00C2\u00A01.3).\t \u00C2\u00A0\t \u00C2\u00A0 112 Taken\t \u00C2\u00A0together,\t \u00C2\u00A0these\t \u00C2\u00A0data\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0specific\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1,\t \u00C2\u00A0depends\t \u00C2\u00A0on\t \u00C2\u00A0three\t \u00C2\u00A0factors:\t \u00C2\u00A01)\t \u00C2\u00A0peptide\u00E2\u0080\u0093protein\t \u00C2\u00A0interaction;\t \u00C2\u00A02)\t \u00C2\u00A0a\t \u00C2\u00A0functional\t \u00C2\u00A0CTM\t \u00C2\u00A0that\t \u00C2\u00A0is\t \u00C2\u00A0exposed\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0cytosol\t \u00C2\u00A0and\t \u00C2\u00A03)\t \u00C2\u00A0the\t \u00C2\u00A0lysosome.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 113 Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0\t \u00C2\u00A0(a)\t \u00C2\u00A0Linear\t \u00C2\u00A0representation\t \u00C2\u00A0of\t \u00C2\u00A0constructs\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0and\t \u00C2\u00A0mCTM-\u00C2\u00AD\u00E2\u0080\u0090GFP.\t \u00C2\u00A0(b)\t \u00C2\u00A0Representative\t \u00C2\u00A0images\t \u00C2\u00A0of\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0of\t \u00C2\u00A0GFP\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0marker\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0in\t \u00C2\u00A0COS7\t \u00C2\u00A0cells\t \u00C2\u00A0transfected\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0wild\t \u00C2\u00A0type\t \u00C2\u00A0GFP\t \u00C2\u00A0(WT-\u00C2\u00AD\u00E2\u0080\u0090GFP;\t \u00C2\u00A0top;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A017)\t \u00C2\u00A0or\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0GFP\t \u00C2\u00A0(CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP;\t \u00C2\u00A0bottom;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A016).\t \u00C2\u00A0Quantification\t \u00C2\u00A0of\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0demonstrated\t \u00C2\u00A0a\t \u00C2\u00A0significant\t \u00C2\u00A0increase\t \u00C2\u00A0in\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0of\t \u00C2\u00A0GFP\t \u00C2\u00A0and\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0signals\t \u00C2\u00A0from\t \u00C2\u00A033.6\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A03.6%\t \u00C2\u00A0(WT-\u00C2\u00AD\u00E2\u0080\u0090GFP)\t \u00C2\u00A0to\t \u00C2\u00A079.1\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A01.4%\t \u00C2\u00A0(CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP)\t \u00C2\u00A0by\t \u00C2\u00A0inclusion\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CTM.\t \u00C2\u00A0Mann-\u00C2\u00AD\u00E2\u0080\u0090Whitney\t \u00C2\u00A0rank-\u00C2\u00AD\u00E2\u0080\u0090sum\t \u00C2\u00A0test,\t \u00C2\u00A0t\t \u00C2\u00A0=\t \u00C2\u00A0408,\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001.\t \u00C2\u00A0Scale\t \u00C2\u00A0bar,\t \u00C2\u00A020\u00CE\u00BCm.\t \u00C2\u00A0(c)\t \u00C2\u00A0Quantification\t \u00C2\u00A0of\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0and\t \u00C2\u00A0mCTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0relative\t \u00C2\u00A0to\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0cells\t \u00C2\u00A024\t \u00C2\u00A0h\t \u00C2\u00A0after\t \u00C2\u00A0individual\t \u00C2\u00A0transfections\t \u00C2\u00A0with\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0mCTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(n\t \u00C2\u00A0=\t \u00C2\u00A06)\t \u00C2\u00A0or\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP.\t \u00C2\u00A0Cells\t \u00C2\u00A0transfected\t \u00C2\u00A0with\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0or\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0were\t \u00C2\u00A0either\t \u00C2\u00A0untreated\t \u00C2\u00A0(no\t \u00C2\u00A0treatment;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A013)\t \u00C2\u00A0or\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A0serum\t \u00C2\u00A0deprivation\t \u00C2\u00A0(+\t \u00C2\u00A0SD;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A05),\t \u00C2\u00A0the\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0inhibitor\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090methyladenine\t \u00C2\u00A0(+\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA;\t \u00C2\u00A010mM;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A05),\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0inhibitor\t \u00C2\u00A0(+\t \u00C2\u00A0MG132;\t \u00C2\u00A05\u00CE\u00BCM;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A06),\t \u00C2\u00A0or\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0inhibitor\t \u00C2\u00A0ammonium\t \u00C2\u00A0chloride\t \u00C2\u00A0(+\t \u00C2\u00A0NH4Cl;\t \u00C2\u00A020mM;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A06)\t \u00C2\u00A0or\t \u00C2\u00A0pepstatin\t \u00C2\u00A0A\t \u00C2\u00A0(+\t \u00C2\u00A0PepA;\t \u00C2\u00A010\u00CE\u00BCM;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A05).\t \u00C2\u00A0Top\t \u00C2\u00A0panels\t \u00C2\u00A0are\t \u00C2\u00A0representative\t \u00C2\u00A0immunoblots\t \u00C2\u00A0of\t \u00C2\u00A0cell\t \u00C2\u00A0lysates\t \u00C2\u00A0for\t \u00C2\u00A0GFP.\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobed\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0Bars\t \u00C2\u00A0in\t \u00C2\u00A0bottom\t \u00C2\u00A0panel\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0protein\t \u00C2\u00A0levels,\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01;\t \u00C2\u00A0white\t \u00C2\u00A0bar).\t \u00C2\u00A0Sample\t \u00C2\u00A0size\t \u00C2\u00A0(n)\t \u00C2\u00A0represents\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0independent\t \u00C2\u00A0experiments\t \u00C2\u00A0from\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A04\t \u00C2\u00A0separate\t \u00C2\u00A0cultures.\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0with\t \u00C2\u00A0Fisher\t \u00C2\u00A0Least\t \u00C2\u00A0Significant\t \u00C2\u00A0Difference\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0comparison,\t \u00C2\u00A0F(12,73)\t \u00C2\u00A0=\t \u00C2\u00A020.939.\t \u00C2\u00A0***P\t \u00C2\u00A0<\t \u00C2\u00A00.001\t \u00C2\u00A0versus\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(white\t \u00C2\u00A0bar);\t \u00C2\u00A0\u00CE\u0094\u00CE\u0094\u00CE\u0094P\t \u00C2\u00A0<\t \u00C2\u00A00.001\t \u00C2\u00A0relative\t \u00C2\u00A0to\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0levels\t \u00C2\u00A0(gray\t \u00C2\u00A0bar).\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0mean\t \u00C2\u00A0values\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A0s.e.m.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0 114 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 115 Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0Time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0GFP.\t \u00C2\u00A0\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner,\t \u00C2\u00A0without\t \u00C2\u00A0affecting\t \u00C2\u00A0the\t \u00C2\u00A0stability\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0CMA-\u00C2\u00AD\u00E2\u0080\u0090substrate\t \u00C2\u00A0GAPDH\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0transiently\t \u00C2\u00A0transfected\t \u00C2\u00A0with\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP.\t \u00C2\u00A0N=3\t \u00C2\u00A0from\t \u00C2\u00A03\t \u00C2\u00A0separate\t \u00C2\u00A0cultures\t \u00C2\u00A0and\t \u00C2\u00A0transfections.\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0F(9,20)=12.418,\t \u00C2\u00A0p<0.001.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A012h\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0and\t \u00C2\u00A0compared\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A012h\t \u00C2\u00A0WT-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(white\t \u00C2\u00A0bar,\t \u00C2\u00A0*)\t \u00C2\u00A0or\t \u00C2\u00A012h\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(grey\t \u00C2\u00A0bar,\t \u00C2\u00A0\u00CE\u0094)\t \u00C2\u00A0*,\u00CE\u0094\t \u00C2\u00A0p<0.05,\t \u00C2\u00A0**,\u00CE\u0094\u00CE\u0094\t \u00C2\u00A0p<0.01\t \u00C2\u00A0and\t \u00C2\u00A0***,\t \u00C2\u00A0\u00CE\u0094\u00CE\u0094\u00CE\u0094p<0.001.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 116 Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein.\t \u00C2\u00A0\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0was\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090expressed\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0(a;\t \u00C2\u00A0n=8;\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0H=25.033,\t \u00C2\u00A0p<0.001)\t \u00C2\u00A0or\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36\t \u00C2\u00A0(b;\t \u00C2\u00A0n=4)\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0indicated\t \u00C2\u00A0gene-\u00C2\u00AD\u00E2\u0080\u0090dose\t \u00C2\u00A0ratios\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0and\t \u00C2\u00A0immunoblots\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0were\t \u00C2\u00A0performed\t \u00C2\u00A048h\t \u00C2\u00A0after\t \u00C2\u00A0transfection.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0transfected\t \u00C2\u00A0with\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0(ratio\t \u00C2\u00A00:1;\t \u00C2\u00A0white\t \u00C2\u00A0bar).\t \u00C2\u00A0**\t \u00C2\u00A0p<0.01\t \u00C2\u00A0and\t \u00C2\u00A0***\t \u00C2\u00A0p<0.001.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0My\t \u00C2\u00A0colleague\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin\t \u00C2\u00A0constructed\t \u00C2\u00A0the\t \u00C2\u00A0vectors\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0experiments\t \u00C2\u00A0shown\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0figure,\t \u00C2\u00A0and\t \u00C2\u00A0performed\t \u00C2\u00A0the\t \u00C2\u00A0experiments\t \u00C2\u00A0for\t \u00C2\u00A0Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00903b.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 117 Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00904\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1.\t \u00C2\u00A0\t \u00C2\u00A0(a)\t \u00C2\u00A0Linear\t \u00C2\u00A0representation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090functional\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP.\t \u00C2\u00A0(b)\t \u00C2\u00A0Similar\t \u00C2\u00A0to\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP,\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0following\t \u00C2\u00A0transfection\t \u00C2\u00A0into\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0(n=4;\t \u00C2\u00A0t=-\u00C2\u00AD\u00E2\u0080\u00903.340,\t \u00C2\u00A0p=0.012).\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0ratio\t \u00C2\u00A0of\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0to\t \u00C2\u00A0control\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0under\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0treatment\t \u00C2\u00A0condition.\t \u00C2\u00A0(c)\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090IP\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0immunoblotting\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0GluN2Bct\t \u00C2\u00A0specifically\t \u00C2\u00A0interacts\t \u00C2\u00A0with\t \u00C2\u00A0cDAPK1,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0wtDAPK1.\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0wild\t \u00C2\u00A0type\t \u00C2\u00A0(inactive)\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(wtDAPK1)\t \u00C2\u00A0or\t \u00C2\u00A0constitutively\t \u00C2\u00A0active\t \u00C2\u00A0mutant\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(cDAPK1)\t \u00C2\u00A0was\t \u00C2\u00A0expressed\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0or\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells,\t \u00C2\u00A0and\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090IP\t \u00C2\u00A0was\t \u00C2\u00A0performed\t \u00C2\u00A024h\t \u00C2\u00A0thereafter.\t \u00C2\u00A0(d)\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependently\t \u00C2\u00A0decreased\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0(n=4;\t \u00C2\u00A0F(5,21)=6.976,\t \u00C2\u00A0p=0.001).\t \u00C2\u00A0(e)\t \u00C2\u00A0GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0knockdown\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0(n=5;\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0Ranks;\t \u00C2\u00A0H=2.325;\t \u00C2\u00A0p=0.676).\t \u00C2\u00A0Levels\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfected\t \u00C2\u00A0with\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0vector\t \u00C2\u00A0(0:1,\t \u00C2\u00A0white\t \u00C2\u00A0bar)\t \u00C2\u00A0represent\t \u00C2\u00A0the\t \u00C2\u00A0control\t \u00C2\u00A0values\t \u00C2\u00A0arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01.\t \u00C2\u00A0*\t \u00C2\u00A0p<\t \u00C2\u00A00.05,\t \u00C2\u00A0**\t \u00C2\u00A0p<\t \u00C2\u00A00.01,\t \u00C2\u00A0***\t \u00C2\u00A0p<\t \u00C2\u00A00.001.\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobed\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0with\t \u00C2\u00A0Fisher\t \u00C2\u00A0Least\t \u00C2\u00A0Significant\t \u00C2\u00A0Difference.\t \u00C2\u00A0My\t \u00C2\u00A0colleague\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin\t \u00C2\u00A0constructed\t \u00C2\u00A0the\t \u00C2\u00A0vectors\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0experiments\t \u00C2\u00A0shown\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0figure.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 118 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 119 Figure\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u00905\t \u00C2\u00A0Enhancing\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0CTM\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide.\t \u00C2\u00A0(a)\t \u00C2\u00A0Linear\t \u00C2\u00A0representation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090functional\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm.\t \u00C2\u00A0(b)\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090immunocytochemistry\t \u00C2\u00A0revealed\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090localization\t \u00C2\u00A0(yellow\t \u00C2\u00A0arrows)\t \u00C2\u00A0between\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(green)\t \u00C2\u00A0with\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0(red),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0(red).\t \u00C2\u00A0Scale\t \u00C2\u00A0bar,\t \u00C2\u00A020\u00CE\u00BCm.\t \u00C2\u00A0(c)\t \u00C2\u00A0Reciprocal\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090IP\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0immunoblotting\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0GluN2Bct\t \u00C2\u00A0specifically\t \u00C2\u00A0interacted\t \u00C2\u00A0with\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1.\t \u00C2\u00A0(d)\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0specifically\t \u00C2\u00A0and\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependently\t \u00C2\u00A0decreased\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090cDAPK1\t \u00C2\u00A0(n\t \u00C2\u00A0=\t \u00C2\u00A04;\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001;\t \u00C2\u00A0F(5,18)\t \u00C2\u00A0=\t \u00C2\u00A018.27),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090wtDAPK1\t \u00C2\u00A0(e;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A03;\t \u00C2\u00A0P\t \u00C2\u00A0=\t \u00C2\u00A00.933;\t \u00C2\u00A0F(5,12)\t \u00C2\u00A0=\t \u00C2\u00A00.249).\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\u00E2\u0080\u0093mediated\t \u00C2\u00A0cDAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0was\t \u00C2\u00A0significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(d;\t \u00C2\u00A020\t \u00C2\u00A0mM;\t \u00C2\u00A0n\t \u00C2\u00A0=\t \u00C2\u00A04;\t \u00C2\u00A0\u00CE\u0094\u00CE\u0094\u00CE\u0094P\t \u00C2\u00A0<\t \u00C2\u00A00.001\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM:cDAPK1\t \u00C2\u00A08:1\t \u00C2\u00A0group)\t \u00C2\u00A0and\t \u00C2\u00A0by\t \u00C2\u00A0mutational\t \u00C2\u00A0inactivation\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0(f;\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTMm;\t \u00C2\u00A0P\t \u00C2\u00A0=\t \u00C2\u00A00.785;\t \u00C2\u00A0F(4,35)\t \u00C2\u00A0=\t \u00C2\u00A00.432,\t \u00C2\u00A0n=8).\t \u00C2\u00A0Levels\t \u00C2\u00A0of\t \u00C2\u00A0cDAPK1\t \u00C2\u00A0or\t \u00C2\u00A0wtDAPK1\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfected\t \u00C2\u00A0with\t \u00C2\u00A0pcDNA3.0\t \u00C2\u00A0vector\t \u00C2\u00A0(0:1,\t \u00C2\u00A0white\t \u00C2\u00A0bar)\t \u00C2\u00A0represent\t \u00C2\u00A0the\t \u00C2\u00A0control\t \u00C2\u00A0values\t \u00C2\u00A0arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01.\t \u00C2\u00A0*P\t \u00C2\u00A0<\t \u00C2\u00A00.05,\t \u00C2\u00A0**\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.01,\t \u00C2\u00A0***,\u00CE\u0094\u00CE\u0094\u00CE\u0094P\t \u00C2\u00A0<\t \u00C2\u00A00.001.\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobed\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0with\t \u00C2\u00A0Fisher\t \u00C2\u00A0Least\t \u00C2\u00A0Significant\t \u00C2\u00A0Difference.\t \u00C2\u00A0(Source\t \u00C2\u00A0for\t \u00C2\u00A0all\t \u00C2\u00A0except\t \u00C2\u00A0panel\t \u00C2\u00A0b:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 120 \t \u00C2\u00A0\t \u00C2\u00A0 121 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 122 CHAPTER\t \u00C2\u00A04: SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0ENDOGENOUS\t \u00C2\u00A0PROTEINS\t \u00C2\u00A04.1 INTRODUCTION\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0previous\t \u00C2\u00A0chapter\t \u00C2\u00A0we\t \u00C2\u00A0verified\t \u00C2\u00A0the\t \u00C2\u00A0effectiveness\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0core\t \u00C2\u00A0strategy\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0recombinant\t \u00C2\u00A0protein\t \u00C2\u00A0expression\t \u00C2\u00A0system.\t \u00C2\u00A0However,\t \u00C2\u00A0as\t \u00C2\u00A0our\t \u00C2\u00A0ultimate\t \u00C2\u00A0goal\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0translate\t \u00C2\u00A0this\t \u00C2\u00A0method\t \u00C2\u00A0for\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0animal\t \u00C2\u00A0research\t \u00C2\u00A0and\t \u00C2\u00A0potential\t \u00C2\u00A0clinical\t \u00C2\u00A0use\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0SNIPER\t \u00C2\u00A0can\t \u00C2\u00A0efficiently\t \u00C2\u00A0knockdown\t \u00C2\u00A0endogenous,\t \u00C2\u00A0unmodified\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0situ.\t \u00C2\u00A0There\t \u00C2\u00A0are\t \u00C2\u00A0two\t \u00C2\u00A0main\t \u00C2\u00A0strategies\t \u00C2\u00A0to\t \u00C2\u00A0possibly\t \u00C2\u00A0realize\t \u00C2\u00A0this\t \u00C2\u00A0goal.\t \u00C2\u00A0\t \u00C2\u00A0First,\t \u00C2\u00A0plasmids\t \u00C2\u00A0encoding\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0could\t \u00C2\u00A0be\t \u00C2\u00A0packaged\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0virus\t \u00C2\u00A0and\t \u00C2\u00A0delivered\t \u00C2\u00A0to\t \u00C2\u00A0tissues\t \u00C2\u00A0through\t \u00C2\u00A0viral\t \u00C2\u00A0infection,\t \u00C2\u00A0similar\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0current\t \u00C2\u00A0approach\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0deliver\t \u00C2\u00A0shRNAs.\t \u00C2\u00A0For\t \u00C2\u00A0research\t \u00C2\u00A0purposes,\t \u00C2\u00A0this\t \u00C2\u00A0strategy\t \u00C2\u00A0has\t \u00C2\u00A0considerable\t \u00C2\u00A0advantages:\t \u00C2\u00A01)\t \u00C2\u00A0a\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0or\t \u00C2\u00A0tissue-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0specific\t \u00C2\u00A0promoter\t \u00C2\u00A0could\t \u00C2\u00A0limit\t \u00C2\u00A0the\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0to\t \u00C2\u00A0certain\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090lineages;\t \u00C2\u00A02)\t \u00C2\u00A0a\t \u00C2\u00A0small-\u00C2\u00AD\u00E2\u0080\u0090chemical\t \u00C2\u00A0regulated\t \u00C2\u00A0promoter\t \u00C2\u00A0could\t \u00C2\u00A0allow\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0to\t \u00C2\u00A0occur\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0inducible\t \u00C2\u00A0manner,\t \u00C2\u00A0thus\t \u00C2\u00A0enabling\t \u00C2\u00A0the\t \u00C2\u00A0study\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0protein\t \u00C2\u00A0at\t \u00C2\u00A0different\t \u00C2\u00A0time\t \u00C2\u00A0points\t \u00C2\u00A0(i.e.\t \u00C2\u00A0throughout\t \u00C2\u00A0development);\t \u00C2\u00A03)\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0RNAi,\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0has\t \u00C2\u00A0the\t \u00C2\u00A0added\t \u00C2\u00A0benefit\t \u00C2\u00A0of\t \u00C2\u00A0directly\t \u00C2\u00A0targeting\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0knockdown\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0mRNA.\t \u00C2\u00A0That\t \u00C2\u00A0said,\t \u00C2\u00A0viral\t \u00C2\u00A0delivery\t \u00C2\u00A0is\t \u00C2\u00A0problematic\t \u00C2\u00A0in\t \u00C2\u00A0two\t \u00C2\u00A0main\t \u00C2\u00A0ways:\t \u00C2\u00A01)\t \u00C2\u00A0viruses\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0adenoviruses\t \u00C2\u00A0and\t \u00C2\u00A0adeno-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0viruses\t \u00C2\u00A0infect\t \u00C2\u00A0cells\t \u00C2\u00A0at\t \u00C2\u00A0multiple\t \u00C2\u00A0copies\t \u00C2\u00A0per\t \u00C2\u00A0cell,\t \u00C2\u00A0which\t \u00C2\u00A0makes\t \u00C2\u00A0dosing\t \u00C2\u00A0hard\t \u00C2\u00A0to\t \u00C2\u00A0control,\t \u00C2\u00A0and\t \u00C2\u00A02)\t \u00C2\u00A0the\t \u00C2\u00A0translation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0into\t \u00C2\u00A0clinical\t \u00C2\u00A0application\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0hindered.\t \u00C2\u00A0 123 An\t \u00C2\u00A0alternative\t \u00C2\u00A0strategy\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0develop\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0biomimetic\t \u00C2\u00A0drug.\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0previous\t \u00C2\u00A0experiments\t \u00C2\u00A0aimed\t \u00C2\u00A0at\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0recombinant\t \u00C2\u00A0proteins,\t \u00C2\u00A0in\t \u00C2\u00A0which\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0was\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090expressed\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0directly\t \u00C2\u00A0inside\t \u00C2\u00A0the\t \u00C2\u00A0cell,\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0requires\t \u00C2\u00A0an\t \u00C2\u00A0additional\t \u00C2\u00A0step:\t \u00C2\u00A0the\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cell.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0if\t \u00C2\u00A0targeting\t \u00C2\u00A0a\t \u00C2\u00A0CNS\t \u00C2\u00A0protein,\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0would\t \u00C2\u00A0also\t \u00C2\u00A0need\t \u00C2\u00A0to\t \u00C2\u00A0penetrate\t \u00C2\u00A0the\t \u00C2\u00A0BBB\t \u00C2\u00A0in\t \u00C2\u00A0sufficient\t \u00C2\u00A0amounts\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0effective.\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0detail\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.6.3,\t \u00C2\u00A0CPPs\t \u00C2\u00A0are\t \u00C2\u00A0highly\t \u00C2\u00A0effective\t \u00C2\u00A0at\t \u00C2\u00A0delivering\t \u00C2\u00A0peptide\t \u00C2\u00A0cargo\t \u00C2\u00A0across\t \u00C2\u00A0the\t \u00C2\u00A0BBB\t \u00C2\u00A0and\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane.\t \u00C2\u00A0Given\t \u00C2\u00A0their\t \u00C2\u00A0demonstrated\t \u00C2\u00A0safety\t \u00C2\u00A0in\t \u00C2\u00A0clinical\t \u00C2\u00A0trials\t \u00C2\u00A0(Hill\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012),\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0delivered\t \u00C2\u00A0via\t \u00C2\u00A0CPPs\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0much\t \u00C2\u00A0more\t \u00C2\u00A0easily\t \u00C2\u00A0adopted\t \u00C2\u00A0for\t \u00C2\u00A0clinical\t \u00C2\u00A0use\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0viral\t \u00C2\u00A0infection.\t \u00C2\u00A0There\t \u00C2\u00A0are\t \u00C2\u00A0currently\t \u00C2\u00A0two\t \u00C2\u00A0main\t \u00C2\u00A0ways\t \u00C2\u00A0to\t \u00C2\u00A0engineer\t \u00C2\u00A0peptides\t \u00C2\u00A0for\t \u00C2\u00A0experimental\t \u00C2\u00A0use.\t \u00C2\u00A0First,\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0bacterial\t \u00C2\u00A0protein\t \u00C2\u00A0expression\t \u00C2\u00A0system,\t \u00C2\u00A0recombinant\t \u00C2\u00A0CPP-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0peptides\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0purified\t \u00C2\u00A0in\t \u00C2\u00A0large\t \u00C2\u00A0amounts\t \u00C2\u00A0in\t \u00C2\u00A0house.\t \u00C2\u00A0Second,\t \u00C2\u00A0the\t \u00C2\u00A0synthesis\t \u00C2\u00A0and\t \u00C2\u00A0purification\t \u00C2\u00A0of\t \u00C2\u00A0peptides\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0outsourced\t \u00C2\u00A0to\t \u00C2\u00A0commercial\t \u00C2\u00A0facilities,\t \u00C2\u00A0which\t \u00C2\u00A0are\t \u00C2\u00A0often\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0synthesize\t \u00C2\u00A0peptides\t \u00C2\u00A0up\t \u00C2\u00A0to\t \u00C2\u00A0200\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0using\t \u00C2\u00A0solid-\u00C2\u00AD\u00E2\u0080\u0090phase\t \u00C2\u00A0synthesis\t \u00C2\u00A0(personal\t \u00C2\u00A0communication).\t \u00C2\u00A0This\t \u00C2\u00A0second\t \u00C2\u00A0approach\t \u00C2\u00A0offers\t \u00C2\u00A0a\t \u00C2\u00A0way\t \u00C2\u00A0for\t \u00C2\u00A0scientists\t \u00C2\u00A0not\t \u00C2\u00A0familiar\t \u00C2\u00A0with\t \u00C2\u00A0molecular\t \u00C2\u00A0biology\t \u00C2\u00A0to\t \u00C2\u00A0easily\t \u00C2\u00A0obtain\t \u00C2\u00A0synthetic\t \u00C2\u00A0peptides\t \u00C2\u00A0for\t \u00C2\u00A0their\t \u00C2\u00A0own\t \u00C2\u00A0research\t \u00C2\u00A0purposes.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0chapter,\t \u00C2\u00A0we\t \u00C2\u00A0present\t \u00C2\u00A0evidence\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0can\t \u00C2\u00A0directly\t \u00C2\u00A0knock\t \u00C2\u00A0down\t \u00C2\u00A0various\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0neuronal\t \u00C2\u00A0cells,\t \u00C2\u00A0using\t \u00C2\u00A0both\t \u00C2\u00A0recombinant\t \u00C2\u00A0and\t \u00C2\u00A0synthetic\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides.\t \u00C2\u00A0\t \u00C2\u00A0 124 4.2 KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0DAPK1\t \u00C2\u00A0WITH\t \u00C2\u00A0RECOMBINANT\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0IN\t \u00C2\u00A0CULTURED\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CELLS\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A03.2.3,\t \u00C2\u00A0following\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0stimuli\t \u00C2\u00A0or\t \u00C2\u00A0a\t \u00C2\u00A0stroke\t \u00C2\u00A0insult,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0becomes\t \u00C2\u00A0activated\t \u00C2\u00A0via\t \u00C2\u00A0dephosphorylation\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0couples\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0subunit\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0We\t \u00C2\u00A0first\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0verify\t \u00C2\u00A0this\t \u00C2\u00A0activity-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0association\t \u00C2\u00A0in\t \u00C2\u00A0neurons\t \u00C2\u00A0by\t \u00C2\u00A0using\t \u00C2\u00A0NMDA\t \u00C2\u00A0to\t \u00C2\u00A0treat\t \u00C2\u00A0primary\t \u00C2\u00A0cultured\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons.\t \u00C2\u00A0As\t \u00C2\u00A0expected,\t \u00C2\u00A0immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A0showed\t \u00C2\u00A0a\t \u00C2\u00A0notable\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0phosphorylated,\t \u00C2\u00A0inactive\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00901a),\t \u00C2\u00A0and\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090IP\t \u00C2\u00A0revealed\t \u00C2\u00A0considerable\t \u00C2\u00A0interaction\t \u00C2\u00A0between\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0GluN2B\t \u00C2\u00A0following\t \u00C2\u00A0NMDA\t \u00C2\u00A0stimuli,\t \u00C2\u00A0but\t \u00C2\u00A0no\t \u00C2\u00A0obvious\t \u00C2\u00A0association\t \u00C2\u00A0prior\t \u00C2\u00A0treatment\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00901b).\t \u00C2\u00A0As\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0depends\t \u00C2\u00A0on\t \u00C2\u00A0its\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide,\t \u00C2\u00A0these\t \u00C2\u00A0results\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0possible\t \u00C2\u00A0to\t \u00C2\u00A0selectively\t \u00C2\u00A0knockdown\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0while\t \u00C2\u00A0leaving\t \u00C2\u00A0the\t \u00C2\u00A0inactivate\t \u00C2\u00A0subpopulation\t \u00C2\u00A0intact.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0test\t \u00C2\u00A0this\t \u00C2\u00A0hypothesis,\t \u00C2\u00A0we\t \u00C2\u00A0first\t \u00C2\u00A0engineered\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting,\t \u00C2\u00A0membrane\t \u00C2\u00A0permeant\t \u00C2\u00A0recombinant\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0via\t \u00C2\u00A0bacterial\t \u00C2\u00A0expression\t \u00C2\u00A0and\t \u00C2\u00A0His-\u00C2\u00AD\u00E2\u0080\u0090tag\t \u00C2\u00A0purification.\t \u00C2\u00A0Coomassie\t \u00C2\u00A0blue\t \u00C2\u00A0staining\t \u00C2\u00A0validated\t \u00C2\u00A0the\t \u00C2\u00A0purity\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0preparations\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00901c,d).\t \u00C2\u00A0When\t \u00C2\u00A0bath\t \u00C2\u00A0applied\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0culture\t \u00C2\u00A0medium\t \u00C2\u00A0on\t \u00C2\u00A0its\t \u00C2\u00A0own,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0had\t \u00C2\u00A0no\t \u00C2\u00A0observable\t \u00C2\u00A0effect\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0basal\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902a).\t \u00C2\u00A0However,\t \u00C2\u00A0when\t \u00C2\u00A0NMDA\t \u00C2\u00A0was\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090applied\t \u00C2\u00A0to\t \u00C2\u00A0activate\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(but\t \u00C2\u00A0not\t \u00C2\u00A0the\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct)\t \u00C2\u00A0significantly\t \u00C2\u00A0decreased\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902a).\t \u00C2\u00A0Concurrent\t \u00C2\u00A0treatment\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0inhibitor\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0blocked\t \u00C2\u00A0this\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902a).\t \u00C2\u00A0These\t \u00C2\u00A0 125 results\t \u00C2\u00A0strongly\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0observed\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0result\t \u00C2\u00A0of\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation,\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0requires\t \u00C2\u00A0the\t \u00C2\u00A0specific\t \u00C2\u00A0interaction\t \u00C2\u00A0between\t \u00C2\u00A0of\t \u00C2\u00A0GluN2Bct\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide.\t \u00C2\u00A0We\t \u00C2\u00A0next\t \u00C2\u00A0investigated\t \u00C2\u00A0the\t \u00C2\u00A0various\t \u00C2\u00A0characteristics\t \u00C2\u00A0of\t \u00C2\u00A0this\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0First,\t \u00C2\u00A0to\t \u00C2\u00A0test\t \u00C2\u00A0if\t \u00C2\u00A0the\t \u00C2\u00A0degree\t \u00C2\u00A0of\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0easily\t \u00C2\u00A0controllable,\t \u00C2\u00A0we\t \u00C2\u00A0treated\t \u00C2\u00A0primary\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons\t \u00C2\u00A0with\t \u00C2\u00A0increasing\t \u00C2\u00A0doses\t \u00C2\u00A0(25-\u00C2\u00AD\u00E2\u0080\u0090200\u00CE\u00BCM)\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0and\t \u00C2\u00A0found\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902b).\t \u00C2\u00A0Second,\t \u00C2\u00A0to\t \u00C2\u00A0determine\t \u00C2\u00A0the\t \u00C2\u00A0kinetics\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation,\t \u00C2\u00A0we\t \u00C2\u00A0followed\t \u00C2\u00A0the\t \u00C2\u00A0changes\t \u00C2\u00A0in\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0after\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0treatment\t \u00C2\u00A0and\t \u00C2\u00A0found\t \u00C2\u00A0significant\t \u00C2\u00A0knockdown\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902c).\t \u00C2\u00A0To\t \u00C2\u00A0the\t \u00C2\u00A0best\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0knowledge,\t \u00C2\u00A0such\t \u00C2\u00A0rapid\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0rarely\t \u00C2\u00A0achievable\t \u00C2\u00A0with\t \u00C2\u00A0DNA-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0or\t \u00C2\u00A0RNA-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods.\t \u00C2\u00A0Third,\t \u00C2\u00A0as\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0itself\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0via\t \u00C2\u00A0CMA,\t \u00C2\u00A0we\t \u00C2\u00A0reasoned\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0knockdown\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0reversible,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0indeed\t \u00C2\u00A0the\t \u00C2\u00A0case\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902c).\t \u00C2\u00A0Although\t \u00C2\u00A0reversibility\t \u00C2\u00A0is\t \u00C2\u00A0advantageous\t \u00C2\u00A0for\t \u00C2\u00A0studying\t \u00C2\u00A0protein\t \u00C2\u00A0function,\t \u00C2\u00A0in\t \u00C2\u00A0some\t \u00C2\u00A0situations,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0treatment\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0accumulation\t \u00C2\u00A0disorders,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0more\t \u00C2\u00A0desirable\t \u00C2\u00A0to\t \u00C2\u00A0achieve\t \u00C2\u00A0long-\u00C2\u00AD\u00E2\u0080\u0090term\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090causing\t \u00C2\u00A0protein.\t \u00C2\u00A0Thus,\t \u00C2\u00A0we\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0extend\t \u00C2\u00A0the\t \u00C2\u00A0duration\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0by\t \u00C2\u00A0administering\t \u00C2\u00A0a\t \u00C2\u00A0second\t \u00C2\u00A0dose\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A01.5h\t \u00C2\u00A0after\t \u00C2\u00A0the\t \u00C2\u00A0initial\t \u00C2\u00A0treatment,\t \u00C2\u00A0which\t \u00C2\u00A0indeed\t \u00C2\u00A0prolonged\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902d).\t \u00C2\u00A0\t \u00C2\u00A0Taken\t \u00C2\u00A0together,\t \u00C2\u00A0these\t \u00C2\u00A0experiments\t \u00C2\u00A0provide\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0degradation\t \u00C2\u00A0is\t \u00C2\u00A0suitable\t \u00C2\u00A0for\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0cells\t \u00C2\u00A0using\t \u00C2\u00A0recombinant\t \u00C2\u00A0peptides.\t \u00C2\u00A0Notably,\t \u00C2\u00A0our\t \u00C2\u00A0results\t \u00C2\u00A0indicate\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0degree\t \u00C2\u00A0and\t \u00C2\u00A0duration\t \u00C2\u00A0of\t \u00C2\u00A0POI\t \u00C2\u00A0 126 degradation\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0easily\t \u00C2\u00A0controlled\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0dose\t \u00C2\u00A0and/or\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0applications\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide.\t \u00C2\u00A0The\t \u00C2\u00A0specific\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0may\t \u00C2\u00A0enable\t \u00C2\u00A0scientists\t \u00C2\u00A0to\t \u00C2\u00A0selectively\t \u00C2\u00A0target\t \u00C2\u00A0a\t \u00C2\u00A0certain\t \u00C2\u00A0subpopulation\t \u00C2\u00A0of\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translationally\t \u00C2\u00A0modified\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0degradation,\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0enable\t \u00C2\u00A0the\t \u00C2\u00A0study\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0functions\t \u00C2\u00A0associated\t \u00C2\u00A0with\t \u00C2\u00A0specific\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modification\t \u00C2\u00A0events.\t \u00C2\u00A04.3 KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0DAPK1\t \u00C2\u00A0WITH\t \u00C2\u00A0SYNTHETIC\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0IN\t \u00C2\u00A0CULTURED\t \u00C2\u00A0NEURONAL\t \u00C2\u00A0CELLS\t \u00C2\u00A0A\t \u00C2\u00A0complimentary\t \u00C2\u00A0approach\t \u00C2\u00A0to\t \u00C2\u00A0engineering\t \u00C2\u00A0recombinant\t \u00C2\u00A0peptides\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0directly\t \u00C2\u00A0synthesize\t \u00C2\u00A0peptides\t \u00C2\u00A0using\t \u00C2\u00A0solid-\u00C2\u00AD\u00E2\u0080\u0090phase\t \u00C2\u00A0synthesis\t \u00C2\u00A0technology,\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0verification\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0sequence\t \u00C2\u00A0and\t \u00C2\u00A0purification\t \u00C2\u00A0with\t \u00C2\u00A0high\t \u00C2\u00A0performance\t \u00C2\u00A0liquid\t \u00C2\u00A0chromatography-\u00C2\u00AD\u00E2\u0080\u0090mass\t \u00C2\u00A0spectrometry\t \u00C2\u00A0(HPLC-\u00C2\u00AD\u00E2\u0080\u0090MS).\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0recombinant\t \u00C2\u00A0peptides,\t \u00C2\u00A0synthetic\t \u00C2\u00A0peptides\t \u00C2\u00A0are\t \u00C2\u00A0generally\t \u00C2\u00A0higher\t \u00C2\u00A0in\t \u00C2\u00A0purity\t \u00C2\u00A0and\t \u00C2\u00A0exhibit\t \u00C2\u00A0less\t \u00C2\u00A0batch-\u00C2\u00AD\u00E2\u0080\u0090to-\u00C2\u00AD\u00E2\u0080\u0090batch\t \u00C2\u00A0variation.\t \u00C2\u00A0\t \u00C2\u00A0However,\t \u00C2\u00A0the\t \u00C2\u00A0difficulty\t \u00C2\u00A0and\t \u00C2\u00A0cost\t \u00C2\u00A0of\t \u00C2\u00A0peptide\t \u00C2\u00A0synthesis\t \u00C2\u00A0substantially\t \u00C2\u00A0increases\t \u00C2\u00A0with\t \u00C2\u00A0its\t \u00C2\u00A0total\t \u00C2\u00A0length,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0mostly\t \u00C2\u00A0determined\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0length\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0PBD.\t \u00C2\u00A0The\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0peptide\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0experiments\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapters\t \u00C2\u00A04.2\t \u00C2\u00A0contains\t \u00C2\u00A0a\t \u00C2\u00A0100aa-\u00C2\u00AD\u00E2\u0080\u0090stretch\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0carboxyl\t \u00C2\u00A0tail\t \u00C2\u00A0as\t \u00C2\u00A0its\t \u00C2\u00A0PBD.\t \u00C2\u00A0Although\t \u00C2\u00A0this\t \u00C2\u00A0sequence\t \u00C2\u00A0contains\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0site,\t \u00C2\u00A0it\t \u00C2\u00A0also\t \u00C2\u00A0includes\t \u00C2\u00A0multiple\t \u00C2\u00A0proteolytic\t \u00C2\u00A0sites.\t \u00C2\u00A0Notably,\t \u00C2\u00A0the\t \u00C2\u00A0binding\t \u00C2\u00A0between\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0GluN2B\t \u00C2\u00A0is\t \u00C2\u00A0thought\t \u00C2\u00A0to\t \u00C2\u00A0only\t \u00C2\u00A0require\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A01292-\u00C2\u00AD\u00E2\u0080\u00901304\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0carboxyl\t \u00C2\u00A0tail\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0flanking\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0of\t \u00C2\u00A0GluN2B\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0(or\t \u00C2\u00A0modulate)\t \u00C2\u00A0the\t \u00C2\u00A0interaction\t \u00C2\u00A0between\t \u00C2\u00A0these\t \u00C2\u00A0two\t \u00C2\u00A0proteins,\t \u00C2\u00A0we\t \u00C2\u00A0 127 decided\t \u00C2\u00A0to\t \u00C2\u00A0initially\t \u00C2\u00A0incorporate\t \u00C2\u00A0a\t \u00C2\u00A0larger\t \u00C2\u00A0PBD\t \u00C2\u00A0in\t \u00C2\u00A0our\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0for\t \u00C2\u00A0our\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0experiments.\t \u00C2\u00A0To\t \u00C2\u00A0facilitate\t \u00C2\u00A0peptide\t \u00C2\u00A0synthesis,\t \u00C2\u00A0increase\t \u00C2\u00A0peptide\t \u00C2\u00A0stability\t \u00C2\u00A0and\t \u00C2\u00A0reduce\t \u00C2\u00A0potential\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0effects,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0opted\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0the\t \u00C2\u00A0minimal\t \u00C2\u00A0binding\t \u00C2\u00A0sequence\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0our\t \u00C2\u00A0synthetic\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides.\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.6.3,\t \u00C2\u00A0CPPs\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0either\t \u00C2\u00A0covalently\t \u00C2\u00A0or\t \u00C2\u00A0noncovalently\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0their\t \u00C2\u00A0peptide\t \u00C2\u00A0cargo\t \u00C2\u00A0for\t \u00C2\u00A0both\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0delivery.\t \u00C2\u00A0Having\t \u00C2\u00A0the\t \u00C2\u00A0option\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0either\t \u00C2\u00A0type\t \u00C2\u00A0of\t \u00C2\u00A0CPP\t \u00C2\u00A0would\t \u00C2\u00A0greatly\t \u00C2\u00A0increase\t \u00C2\u00A0the\t \u00C2\u00A0generalizability\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0method.\t \u00C2\u00A0Thus,\t \u00C2\u00A0we\t \u00C2\u00A0synthesized\t \u00C2\u00A0two\t \u00C2\u00A0new\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptides:\t \u00C2\u00A01)\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM,\t \u00C2\u00A0which\t \u00C2\u00A0uses\t \u00C2\u00A0TAT\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0delivery\t \u00C2\u00A0domain\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00903a),\t \u00C2\u00A0and\t \u00C2\u00A02)\t \u00C2\u00A0GluN2BCTM,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0used\t \u00C2\u00A0concurrently\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0amphipathic\t \u00C2\u00A0CPP\t \u00C2\u00A0Pep-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0(ChariotTM)\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00905a).\t \u00C2\u00A0We\t \u00C2\u00A0also\t \u00C2\u00A0synthesized\t \u00C2\u00A0their\t \u00C2\u00A0respective\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0control\t \u00C2\u00A0peptides\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0and\t \u00C2\u00A0GluN2B.\t \u00C2\u00A0Bath\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B,\t \u00C2\u00A0to\t \u00C2\u00A0cultured\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0resulted\t \u00C2\u00A0in\t \u00C2\u00A0significant\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0following\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatment,\t \u00C2\u00A0an\t \u00C2\u00A0effect\t \u00C2\u00A0that\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00903b).\t \u00C2\u00A0Importantly,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0alter\t \u00C2\u00A0Dapk1\t \u00C2\u00A0mRNA\t \u00C2\u00A0levels,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0observed\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0is\t \u00C2\u00A0in\t \u00C2\u00A0fact\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0direct\t \u00C2\u00A0proteolysis\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0via\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation,\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0inhibition\t \u00C2\u00A0of\t \u00C2\u00A0translation\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00904).\t \u00C2\u00A0Consistent\t \u00C2\u00A0with\t \u00C2\u00A0our\t \u00C2\u00A0results\t \u00C2\u00A0using\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM,\t \u00C2\u00A0treatment\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0GluN2BCTM/Pep-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0membrane-\u00C2\u00AD\u00E2\u0080\u0090permeant\t \u00C2\u00A0complex\t \u00C2\u00A0also\t \u00C2\u00A0efficiently\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00905b)\t \u00C2\u00A0and\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00905c)\t \u00C2\u00A0manner,\t \u00C2\u00A0 128 although\t \u00C2\u00A0neither\t \u00C2\u00A0peptide\t \u00C2\u00A0had\t \u00C2\u00A0any\t \u00C2\u00A0observable\t \u00C2\u00A0effects\t \u00C2\u00A0on\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0without\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatment.\t \u00C2\u00A0\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0present\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0nuclear,\t \u00C2\u00A0mitochondrial\t \u00C2\u00A0and\t \u00C2\u00A0cytosolic\t \u00C2\u00A0compartments\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cell.\t \u00C2\u00A0This\t \u00C2\u00A0raises\t \u00C2\u00A0the\t \u00C2\u00A0question\t \u00C2\u00A0of\t \u00C2\u00A0whether\t \u00C2\u00A0SNIPER\t \u00C2\u00A0can\t \u00C2\u00A0efficiently\t \u00C2\u00A0reduce\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0all\t \u00C2\u00A0subcellular\t \u00C2\u00A0compartments.\t \u00C2\u00A0Notably,\t \u00C2\u00A0previous\t \u00C2\u00A0experiments\t \u00C2\u00A0utilizing\t \u00C2\u00A0UPS-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0destabilizing\t \u00C2\u00A0domains\t \u00C2\u00A0(Chapter\t \u00C2\u00A01.5.1)\t \u00C2\u00A0showed\t \u00C2\u00A0that\t \u00C2\u00A0although\t \u00C2\u00A0cytoplasmic\t \u00C2\u00A0and\t \u00C2\u00A0nuclear\t \u00C2\u00A0fusion\t \u00C2\u00A0proteins\t \u00C2\u00A0were\t \u00C2\u00A0degraded,\t \u00C2\u00A0targeting\t \u00C2\u00A0the\t \u00C2\u00A0fusion\t \u00C2\u00A0POI\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0mitochondrial\t \u00C2\u00A0matrix\t \u00C2\u00A0or\t \u00C2\u00A0endoplasmic\t \u00C2\u00A0reticulum\t \u00C2\u00A0lumen\t \u00C2\u00A0resulted\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0accumulation\t \u00C2\u00A0(Sellmyer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0To\t \u00C2\u00A0see\t \u00C2\u00A0if\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0preferentially\t \u00C2\u00A0degrades\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0certain\t \u00C2\u00A0subcellular\t \u00C2\u00A0organelle,\t \u00C2\u00A0we\t \u00C2\u00A0treated\t \u00C2\u00A0primary\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A04.3,\t \u00C2\u00A0and\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0time\t \u00C2\u00A0of\t \u00C2\u00A0harvest\t \u00C2\u00A0(2h\t \u00C2\u00A0post\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090washout)\t \u00C2\u00A0fractionated\t \u00C2\u00A0the\t \u00C2\u00A0cellular\t \u00C2\u00A0lysate\t \u00C2\u00A0into\t \u00C2\u00A0nuclear,\t \u00C2\u00A0mitochondrial\t \u00C2\u00A0and\t \u00C2\u00A0cytosolic\t \u00C2\u00A0fractions.\t \u00C2\u00A0Interestingly,\t \u00C2\u00A0despite\t \u00C2\u00A0a\t \u00C2\u00A0significant\t \u00C2\u00A0increase\t \u00C2\u00A0in\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0mitochondria\t \u00C2\u00A0following\t \u00C2\u00A0NMDA\t \u00C2\u00A0stimulation\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00906),\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0was\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0reduce\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0all\t \u00C2\u00A0three\t \u00C2\u00A0fractions\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00906).\t \u00C2\u00A0\t \u00C2\u00A04.4 ELUCIDATING\t \u00C2\u00A0THE\t \u00C2\u00A0MECHANISM\t \u00C2\u00A0OF\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0\t \u00C2\u00A0Thus\t \u00C2\u00A0far,\t \u00C2\u00A0our\t \u00C2\u00A0experiments\t \u00C2\u00A0have\t \u00C2\u00A0used\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0lysosomal\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0effort\t \u00C2\u00A0to\t \u00C2\u00A0rescue\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Our\t \u00C2\u00A0reasoning\t \u00C2\u00A0for\t \u00C2\u00A0using\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0was\t \u00C2\u00A0three-\u00C2\u00AD\u00E2\u0080\u0090fold:\t \u00C2\u00A01)\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0widely\t \u00C2\u00A0accepted\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0literature\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0potent\t \u00C2\u00A0inhibitor\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0ALS;\t \u00C2\u00A02)\t \u00C2\u00A0it\t \u00C2\u00A0has\t \u00C2\u00A0relatively\t \u00C2\u00A0low\t \u00C2\u00A0toxicity\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0dose\t \u00C2\u00A0that\t \u00C2\u00A0we\t \u00C2\u00A0used\t \u00C2\u00A0(Bauer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010);\t \u00C2\u00A03)\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0easily\t \u00C2\u00A0water\t \u00C2\u00A0 129 soluble.\t \u00C2\u00A0Despite\t \u00C2\u00A0these\t \u00C2\u00A0advantages,\t \u00C2\u00A0the\t \u00C2\u00A0drug\t \u00C2\u00A0inhibits\t \u00C2\u00A0all\t \u00C2\u00A0endosome-\u00C2\u00AD\u00E2\u0080\u0090lysosome\t \u00C2\u00A0degradation\t \u00C2\u00A0pathways,\t \u00C2\u00A0including\t \u00C2\u00A0CMA,\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0and\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0(see\t \u00C2\u00A0Chapter\t \u00C2\u00A01.4.2\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0detailed\t \u00C2\u00A0description).\t \u00C2\u00A0Our\t \u00C2\u00A0previous\t \u00C2\u00A0experiments\t \u00C2\u00A0with\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0(Chapter\t \u00C2\u00A03.2.1)\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown,\t \u00C2\u00A0as\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0inhibit\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP.\t \u00C2\u00A0Nevertheless,\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0primary\t \u00C2\u00A0degradation\t \u00C2\u00A0route\t \u00C2\u00A0may\t \u00C2\u00A0change\t \u00C2\u00A0following\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0coupling,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0further\t \u00C2\u00A0validate\t \u00C2\u00A0CMA\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0main\t \u00C2\u00A0pathway\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0In\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0providing\t \u00C2\u00A0supplementary\t \u00C2\u00A0evidence\t \u00C2\u00A0to\t \u00C2\u00A0support\t \u00C2\u00A0our\t \u00C2\u00A0proposed\t \u00C2\u00A0mechanism\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER,\t \u00C2\u00A0i.e.\t \u00C2\u00A0CMA,\t \u00C2\u00A0elucidating\t \u00C2\u00A0the\t \u00C2\u00A0exact\t \u00C2\u00A0degradation\t \u00C2\u00A0route(s)\t \u00C2\u00A0is\t \u00C2\u00A0also\t \u00C2\u00A0of\t \u00C2\u00A0therapeutic\t \u00C2\u00A0importance\t \u00C2\u00A0as\t \u00C2\u00A0each\t \u00C2\u00A0pathway\t \u00C2\u00A0is\t \u00C2\u00A0differentially\t \u00C2\u00A0affected\t \u00C2\u00A0by\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090causing\t \u00C2\u00A0proteins\t \u00C2\u00A0(Chapter\t \u00C2\u00A01.4.2.4).\t \u00C2\u00A0We\t \u00C2\u00A0first\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0using\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA,\t \u00C2\u00A0which\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0rescue\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00907b).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0a\t \u00C2\u00A0major\t \u00C2\u00A0proteolysis\t \u00C2\u00A0route\t \u00C2\u00A0for\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation.\t \u00C2\u00A0We\t \u00C2\u00A0next\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0directly\t \u00C2\u00A0inhibit\t \u00C2\u00A0CMA\t \u00C2\u00A0function.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0no\t \u00C2\u00A0specific\t \u00C2\u00A0inhibitors\t \u00C2\u00A0for\t \u00C2\u00A0CMA,\t \u00C2\u00A0we\t \u00C2\u00A0used\t \u00C2\u00A0siRNA\t \u00C2\u00A0targeting\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0receptor\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0to\t \u00C2\u00A0down-\u00C2\u00AD\u00E2\u0080\u0090regulate\t \u00C2\u00A0this\t \u00C2\u00A0crucial\t \u00C2\u00A0component\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0system\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00908a).\t \u00C2\u00A0This\t \u00C2\u00A0approach\t \u00C2\u00A0is\t \u00C2\u00A0frequently\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0literature\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0CMA\t \u00C2\u00A0function\t \u00C2\u00A0(Vogiatzi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02008;\t \u00C2\u00A0Xilouri\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Alvarez-\u00C2\u00AD\u00E2\u0080\u0090Erviti\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Qi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012;\t \u00C2\u00A0Seki\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0knockdown\t \u00C2\u00A0significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00908b,c),\t \u00C2\u00A0further\t \u00C2\u00A0supporting\t \u00C2\u00A0CMA\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0main\t \u00C2\u00A0proteolytic\t \u00C2\u00A0route\t \u00C2\u00A0in\t \u00C2\u00A0our\t \u00C2\u00A0method.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 130 4.5 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IS\t \u00C2\u00A0GENERALIZABLE\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0be\t \u00C2\u00A0accepted\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0tool\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown,\t \u00C2\u00A0a\t \u00C2\u00A0method\t \u00C2\u00A0needs\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0easily\t \u00C2\u00A0generalizable\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0wide\t \u00C2\u00A0variety\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0of\t \u00C2\u00A0different\t \u00C2\u00A0sizes,\t \u00C2\u00A0functions,\t \u00C2\u00A0subcellular\t \u00C2\u00A0locations,\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0chemical\t \u00C2\u00A0and\t \u00C2\u00A0structural\t \u00C2\u00A0properties.\t \u00C2\u00A0To\t \u00C2\u00A0begin\t \u00C2\u00A0to\t \u00C2\u00A0address\t \u00C2\u00A0this\t \u00C2\u00A0key\t \u00C2\u00A0issue,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0tested\t \u00C2\u00A0SNIPER\t \u00C2\u00A0on\t \u00C2\u00A0two\t \u00C2\u00A0other\t \u00C2\u00A0endogenous\t \u00C2\u00A0neuronal\t \u00C2\u00A0proteins,\t \u00C2\u00A0postsynaptic\t \u00C2\u00A0density\t \u00C2\u00A0protein\t \u00C2\u00A095\t \u00C2\u00A0(PSD-\u00C2\u00AD\u00E2\u0080\u009095)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein.\t \u00C2\u00A04.5.1 PEPTIDE-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0NATIVE\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0membrane-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0guanylate\t \u00C2\u00A0kinase\t \u00C2\u00A0concentrated\t \u00C2\u00A0at\t \u00C2\u00A0glutamatergic\t \u00C2\u00A0synapses.\t \u00C2\u00A0As\t \u00C2\u00A0a\t \u00C2\u00A0key\t \u00C2\u00A0structural\t \u00C2\u00A0component\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0postsynaptic\t \u00C2\u00A0density\t \u00C2\u00A0(PSD),\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0helps\t \u00C2\u00A0to\t \u00C2\u00A0form,\t \u00C2\u00A0maintain\t \u00C2\u00A0and\t \u00C2\u00A0stabilize\t \u00C2\u00A0the\t \u00C2\u00A0synapse.\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0is\t \u00C2\u00A0relatively\t \u00C2\u00A0abundant,\t \u00C2\u00A0with\t \u00C2\u00A0roughly\t \u00C2\u00A0300\t \u00C2\u00A0copies\t \u00C2\u00A0present\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0PSD\t \u00C2\u00A0(Chen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005;\t \u00C2\u00A0Sugiyama\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005;\t \u00C2\u00A0Cheng\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006),\t \u00C2\u00A0and\t \u00C2\u00A0is\t \u00C2\u00A0highly\t \u00C2\u00A0stable\t \u00C2\u00A0(Sturgill\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009).\t \u00C2\u00A0In\t \u00C2\u00A0addition\t \u00C2\u00A0to\t \u00C2\u00A0organizing\t \u00C2\u00A0synaptic\t \u00C2\u00A0components,\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0also\t \u00C2\u00A0participates\t \u00C2\u00A0in\t \u00C2\u00A0synaptic\t \u00C2\u00A0plasticity\t \u00C2\u00A0and\t \u00C2\u00A0maturation\t \u00C2\u00A0(El-\u00C2\u00AD\u00E2\u0080\u0090Husseini\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02000),\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0(Christopherson,\t \u00C2\u00A01999;\t \u00C2\u00A0Sattler\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01999).\t \u00C2\u00A0Thus,\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0represents\t \u00C2\u00A0an\t \u00C2\u00A0interesting,\t \u00C2\u00A0if\t \u00C2\u00A0challenging,\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0nine\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0subunit\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0(GluN2B9c)\t \u00C2\u00A0(Aarts\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002).\t \u00C2\u00A0We\t \u00C2\u00A0therefore\t \u00C2\u00A0synthesized\t \u00C2\u00A0the\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9cCTM\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9c\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00909a).\t \u00C2\u00A0Four\t \u00C2\u00A0hours\t \u00C2\u00A0following\t \u00C2\u00A0bath\t \u00C2\u00A0application\t \u00C2\u00A0to\t \u00C2\u00A0cultured\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9cCTM,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9c,\t \u00C2\u00A0 131 significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00909a).\t \u00C2\u00A0In\t \u00C2\u00A0contrast\t \u00C2\u00A0to\t \u00C2\u00A0NH4Cl,\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0affect\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0the\t \u00C2\u00A0primary\t \u00C2\u00A0route\t \u00C2\u00A0of\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0knockdown\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00907c).\t \u00C2\u00A0To\t \u00C2\u00A0see\t \u00C2\u00A0whether\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9cCTM\t \u00C2\u00A0is\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0its\t \u00C2\u00A0target,\t \u00C2\u00A0we\t \u00C2\u00A0further\t \u00C2\u00A0immunoblotted\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090targeted\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B2\u00E2\u0080\u0093actin,\t \u00C2\u00A0and\t \u00C2\u00A0found\t \u00C2\u00A0that\t \u00C2\u00A0their\t \u00C2\u00A0levels\t \u00C2\u00A0were\t \u00C2\u00A0unchanged\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00909a).\t \u00C2\u00A0Thus,\t \u00C2\u00A0despite\t \u00C2\u00A0its\t \u00C2\u00A0stability\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0synapse,\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0is\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0rapid\t \u00C2\u00A0and\t \u00C2\u00A0specific\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0degradation.\t \u00C2\u00A04.5.2 PEPTIDE-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0OF\t \u00C2\u00A0\u00CE\u0091-\u00C2\u00AD\u00E2\u0080\u0090SYNUCLEIN\t \u00C2\u00A0AND\t \u00C2\u00A0ITS\t \u00C2\u00A0MUTANTS\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0(19kDa)\t \u00C2\u00A0unstructured\t \u00C2\u00A0cytosolic\t \u00C2\u00A0protein\t \u00C2\u00A0implicated\t \u00C2\u00A0in\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0synucleinopathies\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0PD\t \u00C2\u00A0(Irwin\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0Multiple\t \u00C2\u00A0lines\t \u00C2\u00A0of\t \u00C2\u00A0evidence\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0elimination\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0attenuates\t \u00C2\u00A0cellular\t \u00C2\u00A0pathology\t \u00C2\u00A0and\t \u00C2\u00A0slows\t \u00C2\u00A0disease\t \u00C2\u00A0progression\t \u00C2\u00A0in\t \u00C2\u00A0animal\t \u00C2\u00A0models\t \u00C2\u00A0of\t \u00C2\u00A0synucleinopathies\t \u00C2\u00A0(Dauer\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002;\t \u00C2\u00A0Hayashita-\u00C2\u00AD\u00E2\u0080\u0090Kinoh\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02006;\t \u00C2\u00A0Xilouri\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0Hence,\t \u00C2\u00A0a\t \u00C2\u00A0clinically\t \u00C2\u00A0applicable\t \u00C2\u00A0method\t \u00C2\u00A0that\t \u00C2\u00A0degrades\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0is\t \u00C2\u00A0highly\t \u00C2\u00A0desirable.\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0native\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0normal\t \u00C2\u00A0clearance\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0is\t \u00C2\u00A0tightly\t \u00C2\u00A0regulated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0pathway\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0(Mak\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Ebrahimi-\u00C2\u00AD\u00E2\u0080\u0090Fakhari\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0Under\t \u00C2\u00A0conditions\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0stress\t \u00C2\u00A0or\t \u00C2\u00A0increased\t \u00C2\u00A0protein\t \u00C2\u00A0burden,\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0clearance\t \u00C2\u00A0via\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0is\t \u00C2\u00A0increased\t \u00C2\u00A0in\t \u00C2\u00A0part\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0CMA\t \u00C2\u00A0induction\t \u00C2\u00A0(Mak\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Interestingly,\t \u00C2\u00A0the\t \u00C2\u00A0native\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A095VKKDQ99\t \u00C2\u00A0is\t \u00C2\u00A0located\t \u00C2\u00A0near\t \u00C2\u00A0the\t \u00C2\u00A0aggregation-\u00C2\u00AD\u00E2\u0080\u0090prone\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090 132 amyloid\t \u00C2\u00A0component\t \u00C2\u00A0(NAC)\t \u00C2\u00A0region\t \u00C2\u00A0(Surguchov,\t \u00C2\u00A02008).\t \u00C2\u00A0This\t \u00C2\u00A0raises\t \u00C2\u00A0the\t \u00C2\u00A0possibility\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0native\t \u00C2\u00A0CTM\t \u00C2\u00A0may\t \u00C2\u00A0become\t \u00C2\u00A0obstructed\t \u00C2\u00A0during\t \u00C2\u00A0aggregation,\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0adding\t \u00C2\u00A0more\t \u00C2\u00A0easily\t \u00C2\u00A0accessible\t \u00C2\u00A0CTMs\t \u00C2\u00A0to\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0may\t \u00C2\u00A0promote\t \u00C2\u00A0its\t \u00C2\u00A0clearance\t \u00C2\u00A0via\t \u00C2\u00A0CMA.\t \u00C2\u00A0Thus\t \u00C2\u00A0we\t \u00C2\u00A0explored\t \u00C2\u00A0whether\t \u00C2\u00A0SNIPER\t \u00C2\u00A0can\t \u00C2\u00A0further\t \u00C2\u00A0facilitate\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0knockdown\t \u00C2\u00A0by\t \u00C2\u00A0providing\t \u00C2\u00A0additional\t \u00C2\u00A0terminally\t \u00C2\u00A0located\t \u00C2\u00A0CTMs.\t \u00C2\u00A0\t \u00C2\u00A0We\t \u00C2\u00A0synthesized\t \u00C2\u00A0a\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0that\t \u00C2\u00A0contained\t \u00C2\u00A0\u00CE\u00B2syn36\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0(TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM),\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0its\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0our\t \u00C2\u00A0experiments\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A03.2.2.\t \u00C2\u00A0Bath\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn,\t \u00C2\u00A0to\t \u00C2\u00A0cultured\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0significantly\t \u00C2\u00A0decreased\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00909b).\t \u00C2\u00A0As\t \u00C2\u00A0with\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0and\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0the\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0inhibitor\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA\t \u00C2\u00A0failed\t \u00C2\u00A0to\t \u00C2\u00A0rescue\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0knockdown,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0process\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00907a).\t \u00C2\u00A0Membrane\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090targeted\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0their\t \u00C2\u00A0levels\t \u00C2\u00A0were\t \u00C2\u00A0not\t \u00C2\u00A0altered\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0is\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0its\t \u00C2\u00A0intended\t \u00C2\u00A0target\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00909b).\t \u00C2\u00A0Given\t \u00C2\u00A0our\t \u00C2\u00A0observation\t \u00C2\u00A0that\t \u00C2\u00A0additional\t \u00C2\u00A0CTMs\t \u00C2\u00A0provided\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0enhanced\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0knockdown,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0if\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0enhanced\t \u00C2\u00A0the\t \u00C2\u00A0cytosolic\t \u00C2\u00A0accessibility\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0native\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0additive\t \u00C2\u00A0manner,\t \u00C2\u00A0or\t \u00C2\u00A0if\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0itself\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0degradation.\t \u00C2\u00A0We\t \u00C2\u00A0inactivated\t \u00C2\u00A0the\t \u00C2\u00A0endogenous\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0CTM\t \u00C2\u00A0and\t \u00C2\u00A0expressed\t \u00C2\u00A0this\t \u00C2\u00A0mutant\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(\u00CE\u0094DQ)\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells.\t \u00C2\u00A0Treatment\t \u00C2\u00A0with\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(\u00CE\u0094DQ),\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(n=4)\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009010).\t \u00C2\u00A0Similarly,\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfection\t \u00C2\u00A0of\t \u00C2\u00A0plasmids\t \u00C2\u00A0expressing\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090 133 \u00CE\u00B2syn36CTM\t \u00C2\u00A0with\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(\u00CE\u0094DQ)\t \u00C2\u00A0also\t \u00C2\u00A0resulted\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(\u00CE\u0094DQ)\t \u00C2\u00A0levels\t \u00C2\u00A0(n=4)\t \u00C2\u00A0\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009011a).\t \u00C2\u00A0This\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0CTM\t \u00C2\u00A0motifs\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0are\t \u00C2\u00A0sufficient\t \u00C2\u00A0to\t \u00C2\u00A0direct\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0for\t \u00C2\u00A0knockdown.\t \u00C2\u00A0\t \u00C2\u00A0Various\t \u00C2\u00A0mutation\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0ASYN\t \u00C2\u00A0gene\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0familial\t \u00C2\u00A0forms\t \u00C2\u00A0of\t \u00C2\u00A0Parkinson\u00E2\u0080\u0099s\t \u00C2\u00A0disease,\t \u00C2\u00A0with\t \u00C2\u00A0A53T\t \u00C2\u00A0being\t \u00C2\u00A0a\t \u00C2\u00A0prominent\t \u00C2\u00A0and\t \u00C2\u00A0well-\u00C2\u00AD\u00E2\u0080\u0090characterized\t \u00C2\u00A0variant\t \u00C2\u00A0(Conway\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01998).\t \u00C2\u00A0Thus,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0the\t \u00C2\u00A0pathogenic\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0variant\t \u00C2\u00A0is\t \u00C2\u00A0also\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0degradation.\t \u00C2\u00A0\t \u00C2\u00A0Following\t \u00C2\u00A0overexpression\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0(n=4)\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009010).\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090transfection\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0with\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0similarly\t \u00C2\u00A0revealed\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0levels\t \u00C2\u00A0(n=4)\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009011b).\t \u00C2\u00A0That\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0reduced\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0levels\t \u00C2\u00A0is\t \u00C2\u00A0somewhat\t \u00C2\u00A0surprising,\t \u00C2\u00A0as\t \u00C2\u00A0there\t \u00C2\u00A0is\t \u00C2\u00A0evidence\t \u00C2\u00A0that\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0is\t \u00C2\u00A0poorly\t \u00C2\u00A0degraded\t \u00C2\u00A0via\t \u00C2\u00A0CMA\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0decreased\t \u00C2\u00A0internalization\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0lumen\t \u00C2\u00A0(Cuervo,\t \u00C2\u00A02004)\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0ex\t \u00C2\u00A0vivo\t \u00C2\u00A0isolated\t \u00C2\u00A0lysosome\t \u00C2\u00A0preparation.\t \u00C2\u00A0However,\t \u00C2\u00A0a\t \u00C2\u00A0recent\t \u00C2\u00A0study\t \u00C2\u00A0using\t \u00C2\u00A0human\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0transgenic\t \u00C2\u00A0mice\t \u00C2\u00A0found\t \u00C2\u00A0decreased\t \u00C2\u00A0association\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0mutant\t \u00C2\u00A0protein\t \u00C2\u00A0with\t \u00C2\u00A0lysosomes\t \u00C2\u00A0in\t \u00C2\u00A0brain\t \u00C2\u00A0areas\t \u00C2\u00A0vulnerable\t \u00C2\u00A0to\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0aggregation\t \u00C2\u00A0(Malkus\t \u00C2\u00A0and\t \u00C2\u00A0Ischiropoulos,\t \u00C2\u00A02012).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0rate\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0degradation\t \u00C2\u00A0was\t \u00C2\u00A0not\t \u00C2\u00A0impaired\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T),\t \u00C2\u00A0even\t \u00C2\u00A0following\t \u00C2\u00A0the\t \u00C2\u00A0aggregation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0(Malkus\t \u00C2\u00A0and\t \u00C2\u00A0Ischiropoulos,\t \u00C2\u00A02012).\t \u00C2\u00A0Thus,\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(A53T)\t \u00C2\u00A0via\t \u00C2\u00A0CMA\t \u00C2\u00A0appears\t \u00C2\u00A0to\t \u00C2\u00A0differ\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0than\t \u00C2\u00A0what\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0found\t \u00C2\u00A0in\t \u00C2\u00A0vitro,\t \u00C2\u00A0and\t \u00C2\u00A0 134 future\t \u00C2\u00A0experiments\t \u00C2\u00A0will\t \u00C2\u00A0have\t \u00C2\u00A0to\t \u00C2\u00A0confirm\t \u00C2\u00A0the\t \u00C2\u00A0validity\t \u00C2\u00A0of\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0mutants\t \u00C2\u00A0in\t \u00C2\u00A0vivo.\t \u00C2\u00A04.6 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IS\t \u00C2\u00A0SPECIFIC\t \u00C2\u00A0Specificity\t \u00C2\u00A0is\t \u00C2\u00A0of\t \u00C2\u00A0utmost\t \u00C2\u00A0importance\t \u00C2\u00A0for\t \u00C2\u00A0any\t \u00C2\u00A0method\t \u00C2\u00A0that\t \u00C2\u00A0seeks\t \u00C2\u00A0to\t \u00C2\u00A0perturb\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0protein.\t \u00C2\u00A0Our\t \u00C2\u00A0previous\t \u00C2\u00A0results\t \u00C2\u00A0showed\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0were\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0their\t \u00C2\u00A0intended\t \u00C2\u00A0target.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0was\t \u00C2\u00A0able\t \u00C2\u00A0to\t \u00C2\u00A0selectively\t \u00C2\u00A0degrade\t \u00C2\u00A0the\t \u00C2\u00A0active,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0inactive\t \u00C2\u00A0form\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0is\t \u00C2\u00A0highly\t \u00C2\u00A0specific.\t \u00C2\u00A0However,\t \u00C2\u00A0these\t \u00C2\u00A0results\t \u00C2\u00A0do\t \u00C2\u00A0not\t \u00C2\u00A0address\t \u00C2\u00A0the\t \u00C2\u00A0issue\t \u00C2\u00A0of\t \u00C2\u00A0whether\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0alter\t \u00C2\u00A0the\t \u00C2\u00A0turnover\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates,\t \u00C2\u00A0or\t \u00C2\u00A0whether\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0are\t \u00C2\u00A0also\t \u00C2\u00A0degraded.\t \u00C2\u00A0We\t \u00C2\u00A0therefore\t \u00C2\u00A0performed\t \u00C2\u00A0the\t \u00C2\u00A0following\t \u00C2\u00A0set\t \u00C2\u00A0of\t \u00C2\u00A0control\t \u00C2\u00A0experiments\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0first\t \u00C2\u00A0step\t \u00C2\u00A0to\t \u00C2\u00A0assess\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0method.\t \u00C2\u00A0GAPDH\t \u00C2\u00A0is\t \u00C2\u00A0perhaps\t \u00C2\u00A0the\t \u00C2\u00A0best-\u00C2\u00AD\u00E2\u0080\u0090characterized\t \u00C2\u00A0endogenous\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate,\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0degradation\t \u00C2\u00A0is\t \u00C2\u00A0widely\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0literature\t \u00C2\u00A0to\t \u00C2\u00A0monitor\t \u00C2\u00A0changes\t \u00C2\u00A0in\t \u00C2\u00A0CMA\t \u00C2\u00A0activity\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011b;\t \u00C2\u00A0Dohi\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012;\t \u00C2\u00A0Rodriguez-\u00C2\u00AD\u00E2\u0080\u0090Navarro\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012;\t \u00C2\u00A0Seki\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0To\t \u00C2\u00A0test\t \u00C2\u00A0if\t \u00C2\u00A0GAPDH\t \u00C2\u00A0levels\t \u00C2\u00A0are\t \u00C2\u00A0altered\t \u00C2\u00A0following\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM,\t \u00C2\u00A0we\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090probed\t \u00C2\u00A0immunoblot\t \u00C2\u00A0membranes\t \u00C2\u00A0from\t \u00C2\u00A0experiments\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A04.3\t \u00C2\u00A0and\t \u00C2\u00A0found\t \u00C2\u00A0no\t \u00C2\u00A0apparent\t \u00C2\u00A0changes\t \u00C2\u00A0in\t \u00C2\u00A0GAPDH\t \u00C2\u00A0levels\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009012),\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0disrupt\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates.\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0is\t \u00C2\u00A0 135 unsurprising\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0perturbed\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0exogenously\t \u00C2\u00A0introduced\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0proteins\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011b;\t \u00C2\u00A0Seki\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0To\t \u00C2\u00A0see\t \u00C2\u00A0if\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090interacting\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0also\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0SNIPER,\t \u00C2\u00A0we\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090probed\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0immunoblot\t \u00C2\u00A0membranes\t \u00C2\u00A0for\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0interact\t \u00C2\u00A0with\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0including\t \u00C2\u00A014-\u00C2\u00AD\u00E2\u0080\u00903-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0(Henshall\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003)\t \u00C2\u00A0and\t \u00C2\u00A0calcineurin\t \u00C2\u00A0(CaN,\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0The\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0proteins\t \u00C2\u00A0were\t \u00C2\u00A0not\t \u00C2\u00A0altered\t \u00C2\u00A0following\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0application\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009012).\t \u00C2\u00A0This\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0cellular\t \u00C2\u00A0turnover\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090interacting\t \u00C2\u00A0proteins\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0altered\t \u00C2\u00A0following\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0time\t \u00C2\u00A0point\t \u00C2\u00A0(2h\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout)\t \u00C2\u00A0when\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0first\t \u00C2\u00A0became\t \u00C2\u00A0significant.\t \u00C2\u00A0However,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0possible\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0particular\t \u00C2\u00A0protein\t \u00C2\u00A0may\t \u00C2\u00A0trigger\t \u00C2\u00A0changes\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0its\t \u00C2\u00A0interacting\t \u00C2\u00A0proteins\t \u00C2\u00A0through\t \u00C2\u00A0cellular\t \u00C2\u00A0homeostasis\t \u00C2\u00A0mechanisms\t \u00C2\u00A0at\t \u00C2\u00A0a\t \u00C2\u00A0later\t \u00C2\u00A0time\t \u00C2\u00A0point.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0on\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0is\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0carboxyl\t \u00C2\u00A0tail,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0couple\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0tail\t \u00C2\u00A0are\t \u00C2\u00A0perturbed.\t \u00C2\u00A0In\t \u00C2\u00A0its\t \u00C2\u00A0activated\t \u00C2\u00A0state,\t \u00C2\u00A0calcium/calmodulin-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0kinase\t \u00C2\u00A0II\t \u00C2\u00A0(CaMKII)\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0residues\t \u00C2\u00A01290-\u00C2\u00AD\u00E2\u0080\u00901309\t \u00C2\u00A0of\t \u00C2\u00A0GluN2B\t \u00C2\u00A0(Strack\t \u00C2\u00A0and\t \u00C2\u00A0Colbran,\t \u00C2\u00A01998),\t \u00C2\u00A0which\t \u00C2\u00A0covers\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0domain\t \u00C2\u00A0(1292-\u00C2\u00AD\u00E2\u0080\u00901304aa).\t \u00C2\u00A0However,\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090probing\t \u00C2\u00A0for\t \u00C2\u00A0CaMKII\t \u00C2\u00A0revealed\t \u00C2\u00A0no\t \u00C2\u00A0apparent\t \u00C2\u00A0change\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0protein,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0is\t \u00C2\u00A0selective\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009012).\t \u00C2\u00A0Although\t \u00C2\u00A0seemingly\t \u00C2\u00A0paradoxical,\t \u00C2\u00A0this\t \u00C2\u00A0finding\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0explained\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0observation\t \u00C2\u00A0that\t \u00C2\u00A0ischemic\t \u00C2\u00A0insults\t \u00C2\u00A0induce\t \u00C2\u00A0the\t \u00C2\u00A0aggregation\t \u00C2\u00A0and\t \u00C2\u00A0inactivation\t \u00C2\u00A0of\t \u00C2\u00A0CaMKII\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0limits\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0from\t \u00C2\u00A0coupling\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B\t \u00C2\u00A0carboxyl\t \u00C2\u00A0tail\t \u00C2\u00A0(Hanson\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01994;\t \u00C2\u00A0Waxham\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A01996;\t \u00C2\u00A0Petersen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Coultrap\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011).\t \u00C2\u00A0 136 This\t \u00C2\u00A0result\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0biological\t \u00C2\u00A0context\t \u00C2\u00A0can\t \u00C2\u00A0greatly\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0via\t \u00C2\u00A0condition-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions.\t \u00C2\u00A0\t \u00C2\u00A0Finally,\t \u00C2\u00A0we\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0the\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090targeted\t \u00C2\u00A0POIs\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0result\t \u00C2\u00A0of\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0cytotoxic\t \u00C2\u00A0responses\t \u00C2\u00A0stemming\t \u00C2\u00A0from\t \u00C2\u00A0impurities\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0solution\t \u00C2\u00A0(such\t \u00C2\u00A0as\t \u00C2\u00A0trifluoroacetic\t \u00C2\u00A0acid,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0added\t \u00C2\u00A0during\t \u00C2\u00A0peptide\t \u00C2\u00A0synthesis\t \u00C2\u00A0(Amblard\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005)).\t \u00C2\u00A0Treatment\t \u00C2\u00A0of\t \u00C2\u00A0cultured\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0with\t \u00C2\u00A0peptides\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0our\t \u00C2\u00A0previous\t \u00C2\u00A0experiments\t \u00C2\u00A0(including\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0and\t \u00C2\u00A0their\t \u00C2\u00A0controls)\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0result\t \u00C2\u00A0in\t \u00C2\u00A0overt\t \u00C2\u00A0cytotoxicity\t \u00C2\u00A0as\t \u00C2\u00A0measured\t \u00C2\u00A0by\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0for\t \u00C2\u00A024h\t \u00C2\u00A0after\t \u00C2\u00A0treatment\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009013).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0reversibility\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902c,\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00905c)\t \u00C2\u00A0also\t \u00C2\u00A0argues\t \u00C2\u00A0against\t \u00C2\u00A0an\t \u00C2\u00A0indiscriminative\t \u00C2\u00A0cytotoxic\t \u00C2\u00A0response\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0reason\t \u00C2\u00A0behind\t \u00C2\u00A0the\t \u00C2\u00A0observed\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation.\t \u00C2\u00A0Together,\t \u00C2\u00A0these\t \u00C2\u00A0results\t \u00C2\u00A0strongly\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0its\t \u00C2\u00A0intended\t \u00C2\u00A0target,\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0disrupt\t \u00C2\u00A0the\t \u00C2\u00A0normal\t \u00C2\u00A0turnover\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0and\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0degrade\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein.\t \u00C2\u00A0However,\t \u00C2\u00A0as\t \u00C2\u00A0specificity\t \u00C2\u00A0is\t \u00C2\u00A0paramount\t \u00C2\u00A0to\t \u00C2\u00A0any\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0approach,\t \u00C2\u00A0care\t \u00C2\u00A0must\t \u00C2\u00A0be\t \u00C2\u00A0taken\t \u00C2\u00A0to\t \u00C2\u00A0assess\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0each\t \u00C2\u00A0individual\t \u00C2\u00A0case.\t \u00C2\u00A0\t \u00C2\u00A0 137 4.7 A\t \u00C2\u00A0SINGLE\t \u00C2\u00A0CTM\t \u00C2\u00A0REPEAT\t \u00C2\u00A0IN\t \u00C2\u00A0SNIPER\t \u00C2\u00A0PEPTIDES\t \u00C2\u00A0IS\t \u00C2\u00A0SUFFICIENT\t \u00C2\u00A0FOR\t \u00C2\u00A0PROTEIN\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0\t \u00C2\u00A0As\t \u00C2\u00A0previously\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.6,\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0employed\t \u00C2\u00A0in\t \u00C2\u00A0our\t \u00C2\u00A0studies\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0hybrid\t \u00C2\u00A0of\t \u00C2\u00A0three\t \u00C2\u00A0natural\t \u00C2\u00A0CTM\t \u00C2\u00A0sequences\t \u00C2\u00A0(KFERQ,\t \u00C2\u00A0QKILD,\t \u00C2\u00A0QRFFE).\t \u00C2\u00A0We\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0or\t \u00C2\u00A0double\t \u00C2\u00A0CTM\t \u00C2\u00A0repeat\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0If\t \u00C2\u00A0so,\t \u00C2\u00A0the\t \u00C2\u00A0total\t \u00C2\u00A0length\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0greatly\t \u00C2\u00A0reduced\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0facilitate\t \u00C2\u00A0its\t \u00C2\u00A0synthesis.\t \u00C2\u00A0We\t \u00C2\u00A0therefore\t \u00C2\u00A0engineered\t \u00C2\u00A0plasmids\t \u00C2\u00A0expressing\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0one\t \u00C2\u00A0or\t \u00C2\u00A0two\t \u00C2\u00A0CTM\t \u00C2\u00A0repeats\t \u00C2\u00A0and\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfected\t \u00C2\u00A0each\t \u00C2\u00A0plasmid\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein-\u00C2\u00AD\u00E2\u0080\u0090expressing\t \u00C2\u00A0plasmid\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells.\t \u00C2\u00A0Immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A048h\t \u00C2\u00A0later\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0all\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides,\t \u00C2\u00A0save\t \u00C2\u00A0for\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synKFERQQRFFE,\t \u00C2\u00A0significant\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009014a).\t \u00C2\u00A0However,\t \u00C2\u00A0the\t \u00C2\u00A0knockdown\t \u00C2\u00A0efficiency\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0with\t \u00C2\u00A0one\t \u00C2\u00A0or\t \u00C2\u00A0two\t \u00C2\u00A0CTM\t \u00C2\u00A0repeats\t \u00C2\u00A0seemed\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0lower\t \u00C2\u00A0than\t \u00C2\u00A0that\t \u00C2\u00A0of\t \u00C2\u00A0Flag-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM,\t \u00C2\u00A0which\t \u00C2\u00A0contains\t \u00C2\u00A0three\t \u00C2\u00A0CTM\t \u00C2\u00A0repeats.\t \u00C2\u00A0We\t \u00C2\u00A0next\t \u00C2\u00A0synthesized\t \u00C2\u00A0two\t \u00C2\u00A0novel\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0containing\t \u00C2\u00A0either\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0CTM\t \u00C2\u00A0(QKILD)\t \u00C2\u00A0or\t \u00C2\u00A0double\t \u00C2\u00A0CTM\t \u00C2\u00A0repeats\t \u00C2\u00A0(QKILDQRFFE)\t \u00C2\u00A0and\t \u00C2\u00A0bath\t \u00C2\u00A0applied\t \u00C2\u00A0these\t \u00C2\u00A0peptides\t \u00C2\u00A0to\t \u00C2\u00A0primary\t \u00C2\u00A0neuronal\t \u00C2\u00A0culture.\t \u00C2\u00A0Immunoblotting\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0showed\t \u00C2\u00A0that\t \u00C2\u00A0both\t \u00C2\u00A0peptides\t \u00C2\u00A0efficiently\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0when\t \u00C2\u00A0assessed\t \u00C2\u00A024h\t \u00C2\u00A0later\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009014b,c).\t \u00C2\u00A0Similar\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0results\t \u00C2\u00A0observed\t \u00C2\u00A0with\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfection,\t \u00C2\u00A0the\t \u00C2\u00A0potency\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0knockdown\t \u00C2\u00A0seemed\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0repeats\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide,\t \u00C2\u00A0suggesting\t \u00C2\u00A0possible\t \u00C2\u00A0multivalent\t \u00C2\u00A0effects\t \u00C2\u00A0(Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009014).\t \u00C2\u00A0Together,\t \u00C2\u00A0these\t \u00C2\u00A0results\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0CTM\t \u00C2\u00A0repeat\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0 138 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0Activation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0GluN2B\t \u00C2\u00A0following\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatment.\t \u00C2\u00A0(a)\t \u00C2\u00A0NMDA\t \u00C2\u00A0(50\u00CE\u00BCM;\t \u00C2\u00A030\t \u00C2\u00A0min)\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0resulting\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0its\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0levels\t \u00C2\u00A0(pDAPK1,\t \u00C2\u00A0n=4).\t \u00C2\u00A0(b)\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090IP\t \u00C2\u00A0with\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0and\t \u00C2\u00A0sequential\t \u00C2\u00A0immunoblotting\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0GluN2B\t \u00C2\u00A0showed\t \u00C2\u00A0an\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0association\t \u00C2\u00A0between\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0GluN2B\t \u00C2\u00A0(n=3).\t \u00C2\u00A0(c)\t \u00C2\u00A0Design\t \u00C2\u00A0and\t \u00C2\u00A0production\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0and\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0peptides\t \u00C2\u00A0using\t \u00C2\u00A0E.\t \u00C2\u00A0coli\t \u00C2\u00A0expression\t \u00C2\u00A0system.\t \u00C2\u00A0Coomassie\t \u00C2\u00A0blue\t \u00C2\u00A0staining\t \u00C2\u00A0of\t \u00C2\u00A0SDS-\u00C2\u00AD\u00E2\u0080\u0090PAGE\t \u00C2\u00A0assessed\t \u00C2\u00A0their\t \u00C2\u00A0purity\t \u00C2\u00A0(d).\t \u00C2\u00A0Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00901d\t \u00C2\u00A0was\t \u00C2\u00A0a\t \u00C2\u00A0collaborative\t \u00C2\u00A0effort\t \u00C2\u00A0with\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 139 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0A\t \u00C2\u00A0recombinant\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0specifically\t \u00C2\u00A0degrades\t \u00C2\u00A0activated\t \u00C2\u00A0endogenous\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0culture.\t \u00C2\u00A0(a)\t \u00C2\u00A0Bath\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(200\u00CE\u00BCM;\t \u00C2\u00A0n=9),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0(200\u00CE\u00BCM;\t \u00C2\u00A0n=6)\t \u00C2\u00A01h\t \u00C2\u00A0before\t \u00C2\u00A0and\t \u00C2\u00A0during\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatment\t \u00C2\u00A0(50\u00CE\u00BCM,\t \u00C2\u00A030min),\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0prevented\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(20mM;\t \u00C2\u00A0n=\t \u00C2\u00A05;\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA;\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001,\t \u00C2\u00A0F(5,36)\t \u00C2\u00A0=\t \u00C2\u00A010.891),\t \u00C2\u00A0and\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0(b;\t \u00C2\u00A0n=4;\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001;\t \u00C2\u00A0F(6,21)\t \u00C2\u00A0=\t \u00C2\u00A018.14)\t \u00C2\u00A0and\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0(c;\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001;\t \u00C2\u00A0F(8,44)\t \u00C2\u00A0=\t \u00C2\u00A012.074).\t \u00C2\u00A0(d)\t \u00C2\u00A0A\t \u00C2\u00A0single\t \u00C2\u00A0pretreatment\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(sing;\t \u00C2\u00A0200\u00CE\u00BCM,\t \u00C2\u00A060min\t \u00C2\u00A0before\t \u00C2\u00A0and\t \u00C2\u00A0during\t \u00C2\u00A0the\t \u00C2\u00A030min\t \u00C2\u00A0NMDA\t \u00C2\u00A0stimulation)\t \u00C2\u00A0produced\t \u00C2\u00A0a\t \u00C2\u00A0transient\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0returning\t \u00C2\u00A0to\t \u00C2\u00A0baseline\t \u00C2\u00A0within\t \u00C2\u00A07h\t \u00C2\u00A0(n=4;\t \u00C2\u00A0P\t \u00C2\u00A0=\t \u00C2\u00A00.888)\t \u00C2\u00A0and\t \u00C2\u00A0an\t \u00C2\u00A0additional\t \u00C2\u00A0dose\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0(200\u00CE\u00BCM)\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout\t \u00C2\u00A0resulted\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0persistent\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0DAPK1\t \u00C2\u00A0up\t \u00C2\u00A0to\t \u00C2\u00A07h\t \u00C2\u00A0(mult;\t \u00C2\u00A0n=4;\t \u00C2\u00A0\u00CE\u0094\u00CE\u0094P\t \u00C2\u00A0=\t \u00C2\u00A00.002).\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA;\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001,\t \u00C2\u00A0F(4,15)\t \u00C2\u00A0=\t \u00C2\u00A010.389.\t \u00C2\u00A0Relative\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0were\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0those\t \u00C2\u00A0in\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0naive\t \u00C2\u00A0and\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0naive\t \u00C2\u00A0(white\t \u00C2\u00A0bar,\t \u00C2\u00A0*)\t \u00C2\u00A0or\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0group\t \u00C2\u00A0(gray\t \u00C2\u00A0bar,\t \u00C2\u00A0\u00CE\u0094).\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobed\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0loading\t \u00C2\u00A0control,\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A0s.e.m.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 140 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 141 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0Dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0synthetic\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM.\t \u00C2\u00A0(a)\t \u00C2\u00A0Schematic\t \u00C2\u00A0illustration\t \u00C2\u00A0of\t \u00C2\u00A0synthetic\t \u00C2\u00A0peptides\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0and\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM.\t \u00C2\u00A0(b)\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(25\u00CE\u00BCM;\t \u00C2\u00A0n=5;\t \u00C2\u00A0p=0.001),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0control\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0(25\u00CE\u00BCM;\t \u00C2\u00A0n=4;\t \u00C2\u00A0p=0.223)\t \u00C2\u00A0decreased\t \u00C2\u00A0native\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0prevented\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(20mM;\t \u00C2\u00A0n=5;\t \u00C2\u00A0p=0.302).\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(5,24)\t \u00C2\u00A0=\t \u00C2\u00A013.591.\t \u00C2\u00A0Relative\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0were\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0those\t \u00C2\u00A0in\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0naive\t \u00C2\u00A0and\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0naive\t \u00C2\u00A0(white\t \u00C2\u00A0bar,\t \u00C2\u00A0*)\t \u00C2\u00A0or\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0group\t \u00C2\u00A0(gray\t \u00C2\u00A0bar,\t \u00C2\u00A0\u00CE\u0094).\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobed\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0were\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0loading\t \u00C2\u00A0control,\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A0s.e.m.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 142 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00904\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0alter\t \u00C2\u00A0DAPK1\t \u00C2\u00A0mRNA\t \u00C2\u00A0levels.\t \u00C2\u00A0Cultured\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0with\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(25\u00CE\u00BCM,\t \u00C2\u00A060min\t \u00C2\u00A0prior\t \u00C2\u00A0to\t \u00C2\u00A0and\t \u00C2\u00A0during\t \u00C2\u00A0NMDA\t \u00C2\u00A0application)\t \u00C2\u00A0and\t \u00C2\u00A0NMDA\t \u00C2\u00A0(50\u00CE\u00BCM;\t \u00C2\u00A030min).\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0harvested\t \u00C2\u00A0at\t \u00C2\u00A02h\t \u00C2\u00A0(n=5),\t \u00C2\u00A04h\t \u00C2\u00A0(n=4)\t \u00C2\u00A0and\t \u00C2\u00A07h\t \u00C2\u00A0(n=4)\t \u00C2\u00A0following\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout,\t \u00C2\u00A0and\t \u00C2\u00A0total\t \u00C2\u00A0RNA\t \u00C2\u00A0was\t \u00C2\u00A0extracted\t \u00C2\u00A0and\t \u00C2\u00A0reverse-\u00C2\u00AD\u00E2\u0080\u0090transcribed\t \u00C2\u00A0into\t \u00C2\u00A0cDNA.\t \u00C2\u00A0dapk1\t \u00C2\u00A0mRNA\t \u00C2\u00A0levels\t \u00C2\u00A0were\t \u00C2\u00A0measured\t \u00C2\u00A0by\t \u00C2\u00A0q-\u00C2\u00AD\u00E2\u0080\u0090PCR\t \u00C2\u00A0with\t \u00C2\u00A0actb\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0an\t \u00C2\u00A0internal\t \u00C2\u00A0control,\t \u00C2\u00A0and\t \u00C2\u00A0subsequently\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0control\t \u00C2\u00A0levels.\t \u00C2\u00A0F(5,25)=0.134,\t \u00C2\u00A0p=0.983.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0mRNA\t \u00C2\u00A0was\t \u00C2\u00A0collected\t \u00C2\u00A0from\t \u00C2\u00A0primary\t \u00C2\u00A0neurons\t \u00C2\u00A0from\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A03\t \u00C2\u00A0separate\t \u00C2\u00A0cultures.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0\t \u00C2\u00A0Relative dapk1 fold change normalized to actb 0\t \u00C2\u00A00.2\t \u00C2\u00A00.4\t \u00C2\u00A00.6\t \u00C2\u00A00.8\t \u00C2\u00A01\t \u00C2\u00A01.2\t \u00C2\u00A01.4\t \u00C2\u00A01.6\t \u00C2\u00A01.8\t \u00C2\u00A02\t \u00C2\u00A02h 4h 7h 143 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00905\t \u00C2\u00A0Pep1-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0intracellular\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0synthetic\t \u00C2\u00A0GluN2BCTM\t \u00C2\u00A0peptide\t \u00C2\u00A0specifically\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons.\t \u00C2\u00A0\t \u00C2\u00A0(a)\t \u00C2\u00A0Schematic\t \u00C2\u00A0illustration\t \u00C2\u00A0of\t \u00C2\u00A0synthetic\t \u00C2\u00A0peptide\t \u00C2\u00A0GluN2BCTM.\t \u00C2\u00A0(b)\t \u00C2\u00A0GluN2BCTM,\t \u00C2\u00A0when\t \u00C2\u00A0delivered\t \u00C2\u00A0into\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons,\t \u00C2\u00A0specifically\t \u00C2\u00A0decreases\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0native\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090stimulation\t \u00C2\u00A0dependent\t \u00C2\u00A0manner.\t \u00C2\u00A0GluN2BCTM\t \u00C2\u00A0was\t \u00C2\u00A0first\t \u00C2\u00A0mixed\t \u00C2\u00A0with\t \u00C2\u00A0intracellular\t \u00C2\u00A0delivering\t \u00C2\u00A0carrier\t \u00C2\u00A0peptide\t \u00C2\u00A0Pep-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0at\t \u00C2\u00A0a\t \u00C2\u00A01:4\t \u00C2\u00A0ratio\t \u00C2\u00A0for\t \u00C2\u00A030min\t \u00C2\u00A0to\t \u00C2\u00A0form\t \u00C2\u00A0a\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane\t \u00C2\u00A0permeable\t \u00C2\u00A0peptide\t \u00C2\u00A0complex,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0then\t \u00C2\u00A0bath\t \u00C2\u00A0applied\t \u00C2\u00A0to\t \u00C2\u00A0neurons\t \u00C2\u00A060min\t \u00C2\u00A0prior\t \u00C2\u00A0to\t \u00C2\u00A0and\t \u00C2\u00A0during\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatments\t \u00C2\u00A0(50\u00CE\u00BCM;\t \u00C2\u00A030min).\t \u00C2\u00A0The\t \u00C2\u00A0combination\t \u00C2\u00A0of\t \u00C2\u00A0GluN2BCTM\t \u00C2\u00A0and\t \u00C2\u00A0Pep-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0(but\t \u00C2\u00A0not\t \u00C2\u00A0either\t \u00C2\u00A0peptide\t \u00C2\u00A0alone)\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependently\t \u00C2\u00A0(b,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(7,22)=6.993)\t \u00C2\u00A0and\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependently\t \u00C2\u00A0(c,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(7,28)=10.034)\t \u00C2\u00A0decreased\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0following\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatment.\t \u00C2\u00A0The\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0required\t \u00C2\u00A0NMDA\t \u00C2\u00A0stimulation\t \u00C2\u00A0(b)\t \u00C2\u00A0and\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0inhibiting\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0with\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(d;\t \u00C2\u00A0NH4Cl\t \u00C2\u00A020mM;\t \u00C2\u00A0n=5;\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(3,16)=15.129).\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0na\u00C3\u00AFve\t \u00C2\u00A0group\t \u00C2\u00A0(arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01),\t \u00C2\u00A0and\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0both\t \u00C2\u00A0saline-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0(white\t \u00C2\u00A0bar,\t \u00C2\u00A0*)\t \u00C2\u00A0or\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0group\t \u00C2\u00A0(grey\t \u00C2\u00A0bar,\t \u00C2\u00A0\u00CE\u0094).\t \u00C2\u00A0Membrane\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090probing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0*,\u00CE\u0094\t \u00C2\u00A0p<0.05,\t \u00C2\u00A0**,\u00CE\u0094\u00CE\u0094\t \u00C2\u00A0p<0.01\t \u00C2\u00A0and\t \u00C2\u00A0***,\t \u00C2\u00A0\u00CE\u0094\u00CE\u0094\u00CE\u0094p<0.001;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 144 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 145 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00906\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\u00E2\u0080\u0093induced\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0reduces\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0various\t \u00C2\u00A0subcellular\t \u00C2\u00A0compartments\t \u00C2\u00A0in\t \u00C2\u00A0neuron\t \u00C2\u00A0cultures.\t \u00C2\u00A0\t \u00C2\u00A0Bath\t \u00C2\u00A0applications\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(25\u00CE\u00BCM,\t \u00C2\u00A01hr\t \u00C2\u00A0prior\t \u00C2\u00A0to\t \u00C2\u00A0and\t \u00C2\u00A0during\t \u00C2\u00A030min\t \u00C2\u00A0NMDA\t \u00C2\u00A0treatment)\t \u00C2\u00A0with\t \u00C2\u00A0NMDA\t \u00C2\u00A0(50\u00CE\u00BCM)\t \u00C2\u00A0(labeled\t \u00C2\u00A0\u00E2\u0080\u009CTAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\u00E2\u0080\u009D)\t \u00C2\u00A0significantly\t \u00C2\u00A0decreased\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0nuclear\t \u00C2\u00A0(a,\t \u00C2\u00A0n=4,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(2,9)=19.139),\t \u00C2\u00A0cytosolic\t \u00C2\u00A0(b,\t \u00C2\u00A0n=4,\t \u00C2\u00A0p=0.006,\t \u00C2\u00A0F(2,8)=10.417)\t \u00C2\u00A0and\t \u00C2\u00A0mitochondrial\t \u00C2\u00A0(c,\t \u00C2\u00A0n=4,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(2,9)=18.597)\t \u00C2\u00A0subcellular\t \u00C2\u00A0fractions\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout,\t \u00C2\u00A0as\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0saline\t \u00C2\u00A0control\t \u00C2\u00A0(*)\t \u00C2\u00A0and\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0group\t \u00C2\u00A0(\u00CE\u0094,\t \u00C2\u00A0grey\t \u00C2\u00A0bar).\t \u00C2\u00A0Lamin\t \u00C2\u00A0B1\t \u00C2\u00A0(LB1)\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0marker\t \u00C2\u00A0for\t \u00C2\u00A0nuclear\t \u00C2\u00A0fractions,\t \u00C2\u00A0hsp90\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0marker\t \u00C2\u00A0for\t \u00C2\u00A0cytosolic\t \u00C2\u00A0fractions,\t \u00C2\u00A0and\t \u00C2\u00A0VDAC1\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0marker\t \u00C2\u00A0for\t \u00C2\u00A0mitochondrial\t \u00C2\u00A0fractions.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0collected\t \u00C2\u00A0from\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A03\t \u00C2\u00A0separate\t \u00C2\u00A0primary\t \u00C2\u00A0cultures.\t \u00C2\u00A0Lysates\t \u00C2\u00A0were\t \u00C2\u00A0collected\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0mixture\t \u00C2\u00A0of\t \u00C2\u00A0all\t \u00C2\u00A0treatment\t \u00C2\u00A0samples.\t \u00C2\u00A0\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0with\t \u00C2\u00A0Tukey\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090hoc\t \u00C2\u00A0was\t \u00C2\u00A0used.\t \u00C2\u00A0*\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0control,\t \u00C2\u00A0\u00CE\u0094\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0group\t \u00C2\u00A0(grey\t \u00C2\u00A0bar).\t \u00C2\u00A0*p<0.05,\t \u00C2\u00A0\u00CE\u0094\u00CE\u0094p<0.01;\t \u00C2\u00A0***p<0.001.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0na\u00C3\u00AFve\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0control\t \u00C2\u00A0(arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01).\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0 146 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 147 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00907\t \u00C2\u00A0Macroautophagy\t \u00C2\u00A0inhibition\t \u00C2\u00A0cannot\t \u00C2\u00A0rescue\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090peptide\t \u00C2\u00A0mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0(a)\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0(25\u00CE\u00BCM)\t \u00C2\u00A0significantly\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0native\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA\t \u00C2\u00A0(10mM).\t \u00C2\u00A0Basal\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0showed\t \u00C2\u00A0a\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090significant\t \u00C2\u00A0trend\t \u00C2\u00A0towards\t \u00C2\u00A0increase\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA.\t \u00C2\u00A0n=5,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(3,16)=47.013.\t \u00C2\u00A0(b)\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0alter\t \u00C2\u00A0either\t \u00C2\u00A0the\t \u00C2\u00A0basal\t \u00C2\u00A0levels\t \u00C2\u00A0or\t \u00C2\u00A0NMDA\t \u00C2\u00A0activation-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0by\t \u00C2\u00A0its\t \u00C2\u00A0targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(25\u00CE\u00BCM).\t \u00C2\u00A0n=4,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(3,12)=21.675.\t \u00C2\u00A0NMDA\t \u00C2\u00A0(50\u00CE\u00BCM,\t \u00C2\u00A030min)\t \u00C2\u00A0is\t \u00C2\u00A0added\t \u00C2\u00A0to\t \u00C2\u00A0all\t \u00C2\u00A0groups\t \u00C2\u00A0except\t \u00C2\u00A0control.\t \u00C2\u00A0(c)\t \u00C2\u00A0Similarly,\t \u00C2\u00A03-\u00C2\u00AD\u00E2\u0080\u0090MA\t \u00C2\u00A0failed\t \u00C2\u00A0to\t \u00C2\u00A0affect\t \u00C2\u00A0basal\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0and\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0rescue\t \u00C2\u00A0the\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9cCTM\t \u00C2\u00A0(25\u00CE\u00BCM)\t \u00C2\u00A0faciliated\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0degradation.\t \u00C2\u00A0n=4,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(3,12)=14.836.\t \u00C2\u00A0\t \u00C2\u00A0Membrane\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0with\t \u00C2\u00A0Tukey\u00E2\u0080\u0099s\t \u00C2\u00A0test.\t \u00C2\u00A0\t \u00C2\u00A0**p<0.01\t \u00C2\u00A0and\t \u00C2\u00A0***p<0.001;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0na\u00C3\u00AFve\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0control\t \u00C2\u00A0(arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01).\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0collected\t \u00C2\u00A0from\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A03\t \u00C2\u00A0separate\t \u00C2\u00A0primary\t \u00C2\u00A0cultures.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0 148 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 149 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00908\t \u00C2\u00A0siRNA-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0reduces\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0induced\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation.\t \u00C2\u00A0\t \u00C2\u00A0(a)\t \u00C2\u00A0DNA\t \u00C2\u00A0electrophoresis\t \u00C2\u00A0of\t \u00C2\u00A0Lamp2a\t \u00C2\u00A0showing\t \u00C2\u00A0siRNA\t \u00C2\u00A0(60pmol)-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A03d\t \u00C2\u00A0after\t \u00C2\u00A0siRNA\t \u00C2\u00A0treatment.\t \u00C2\u00A0Scrb-\u00C2\u00AD\u00E2\u0080\u0090siRNA:\t \u00C2\u00A0scrambled\t \u00C2\u00A0siRNA,\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A-\u00C2\u00AD\u00E2\u0080\u0090siRNA:\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0siRNA.\t \u00C2\u00A0n=3.\t \u00C2\u00A0(b-\u00C2\u00AD\u00E2\u0080\u0090c)\t \u00C2\u00A0Sequential\t \u00C2\u00A0immunoblotting\t \u00C2\u00A0showing\t \u00C2\u00A0the\t \u00C2\u00A0inhibition\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0induced\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0by\t \u00C2\u00A0specifically\t \u00C2\u00A0blocking\t \u00C2\u00A0CMA\t \u00C2\u00A0with\t \u00C2\u00A0siRNA-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0knockdown.\t \u00C2\u00A03d\t \u00C2\u00A0following\t \u00C2\u00A0siRNA\t \u00C2\u00A0treatment,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0incubated\t \u00C2\u00A0in\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(25\u00CE\u00BCM)\t \u00C2\u00A0for\t \u00C2\u00A01hr\t \u00C2\u00A0before\t \u00C2\u00A0and\t \u00C2\u00A0during\t \u00C2\u00A0NMDA\t \u00C2\u00A0(50\u00CE\u00BCM;\t \u00C2\u00A030min).\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0harvested\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout.\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0knockdown:\t \u00C2\u00A0n=7,\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(3,24)=13.455;\t \u00C2\u00A0DAPK1:\t \u00C2\u00A0n=7,\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0Ranks\t \u00C2\u00A0with\t \u00C2\u00A0Tukey\u00E2\u0080\u0099s\t \u00C2\u00A0test,\t \u00C2\u00A0p=0.002,\t \u00C2\u00A0H(3)=15.047;\t \u00C2\u00A0\t \u00C2\u00A0*\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0na\u00C3\u00AFve\t \u00C2\u00A0control,\t \u00C2\u00A0\u00CE\u0094\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0group\t \u00C2\u00A0(grey\t \u00C2\u00A0bar).\t \u00C2\u00A0*,\u00CE\u0094\t \u00C2\u00A0p<0.05,\t \u00C2\u00A0***p<0.001;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0na\u00C3\u00AFve\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0control\t \u00C2\u00A0(arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01).\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0collected\t \u00C2\u00A0from\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A03\t \u00C2\u00A0separate\t \u00C2\u00A0primary\t \u00C2\u00A0cultures.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 150 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 151 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u00909\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons.\t \u00C2\u00A0\t \u00C2\u00A0(a)\t \u00C2\u00A0Top:\t \u00C2\u00A0Schematics\t \u00C2\u00A0of\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9cCTM\t \u00C2\u00A0and\t \u00C2\u00A0control\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9c.\t \u00C2\u00A0Middle:\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9cCTM\t \u00C2\u00A0(25\u00CE\u00BCM;\t \u00C2\u00A0n=4),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9c\t \u00C2\u00A0(25\u00CE\u00BCM;\t \u00C2\u00A0n=4),\t \u00C2\u00A0effectively\t \u00C2\u00A0degraded\t \u00C2\u00A0endogenous\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0(One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0Tukey\u00E2\u0080\u0099s\t \u00C2\u00A0test,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(3,12)\t \u00C2\u00A0=\t \u00C2\u00A018.154)\t \u00C2\u00A0without\t \u00C2\u00A0perturbing\t \u00C2\u00A0untargeted\t \u00C2\u00A0protein\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(bottom).\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0rescued\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0degradation.\t \u00C2\u00A0(b)\t \u00C2\u00A0Top:\t \u00C2\u00A0Schematics\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0synthetic\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090penetrating\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0targeting\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0control\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn.\t \u00C2\u00A0Middle:\t \u00C2\u00A0Immunoblots\t \u00C2\u00A0demonstrate\t \u00C2\u00A0that\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0(25\u00CE\u00BCM;\t \u00C2\u00A0n=5),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0the\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090lacking\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn\t \u00C2\u00A0(25\u00CE\u00BCM;\t \u00C2\u00A0n=5),\t \u00C2\u00A0specifically\t \u00C2\u00A0decreased\t \u00C2\u00A0the\t \u00C2\u00A0targeted\t \u00C2\u00A0endogenous\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0Tukey\u00E2\u0080\u0099s\t \u00C2\u00A0test,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0F(3,16)\t \u00C2\u00A0=\t \u00C2\u00A012.435),\t \u00C2\u00A0without\t \u00C2\u00A0affecting\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0unrelated\t \u00C2\u00A0control\t \u00C2\u00A0proteins\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0at\t \u00C2\u00A04h\t \u00C2\u00A0(bottom),\t \u00C2\u00A0and\t \u00C2\u00A0this\t \u00C2\u00A0reduction\t \u00C2\u00A0was\t \u00C2\u00A0prevented\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0lysosomal\t \u00C2\u00A0inhibitor\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(20mM;\t \u00C2\u00A0n=5).\t \u00C2\u00A0Membrane\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0additional\t \u00C2\u00A0specificity\t \u00C2\u00A0and\t \u00C2\u00A0loading\t \u00C2\u00A0controls.\t \u00C2\u00A0\u00CE\u0094p<\t \u00C2\u00A00.05,\t \u00C2\u00A0**,\u00CE\u0094\u00CE\u0094p<0.01\t \u00C2\u00A0and\t \u00C2\u00A0***p<0.001;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A0s.e.m.\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0naive\t \u00C2\u00A0untreated\t \u00C2\u00A0control\t \u00C2\u00A0(arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01).\t \u00C2\u00A0(Adapted\t \u00C2\u00A0from:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0 152 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 153 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009010\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0are\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0endogenous\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0CTM\t \u00C2\u00A0was\t \u00C2\u00A0eliminated\t \u00C2\u00A0by\t \u00C2\u00A0mutating\t \u00C2\u00A095VKKDQ99\t \u00C2\u00A0into\t \u00C2\u00A095VKKAA99.\t \u00C2\u00A0Plasmids\t \u00C2\u00A0expressing\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein,\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0were\t \u00C2\u00A0transfected\t \u00C2\u00A0into\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0and\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn\t \u00C2\u00A0(50\u00CE\u00BCM)\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0(50\u00CE\u00BCM)\t \u00C2\u00A0starting\t \u00C2\u00A0at\t \u00C2\u00A024h\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090transfection.\t \u00C2\u00A0Two\t \u00C2\u00A0additional\t \u00C2\u00A0doses\t \u00C2\u00A0of\t \u00C2\u00A0peptide\t \u00C2\u00A0were\t \u00C2\u00A0added\t \u00C2\u00A04h\t \u00C2\u00A0and\t \u00C2\u00A08h\t \u00C2\u00A0later,\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0harvested\t \u00C2\u00A0at\t \u00C2\u00A048h\t \u00C2\u00A0after\t \u00C2\u00A0transfection.\t \u00C2\u00A0Wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0was\t \u00C2\u00A0significantly\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0inhibiting\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0with\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(n=4,\t \u00C2\u00A0H(2)=8.290,\t \u00C2\u00A0p=0.005,\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0Ranks).\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(\u00CE\u0094DQ)\t \u00C2\u00A0was\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0(n=4,\t \u00C2\u00A0F(2,11)=9.482,\t \u00C2\u00A0p=0.006,\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA),\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(n=4,\t \u00C2\u00A0p=0.001).\t \u00C2\u00A0Similarly,\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0was\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM\t \u00C2\u00A0(n=4,\t \u00C2\u00A0F(2,11)=6.340,\t \u00C2\u00A0p=0.019,\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA),\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(n=4,\t \u00C2\u00A0p=0.019).\t \u00C2\u00A0Student-\u00C2\u00AD\u00E2\u0080\u0090Newman-\u00C2\u00AD\u00E2\u0080\u0090Keuls\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090hoc\t \u00C2\u00A0analysis.\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0*\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn,\t \u00C2\u00A0\u00CE\u0094\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synCTM.\t \u00C2\u00A0*,\u00CE\u0094\t \u00C2\u00A0p<0.05,\t \u00C2\u00A0**p<0.01;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0collected\t \u00C2\u00A0from\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A04\t \u00C2\u00A0separate\t \u00C2\u00A0cultures\t \u00C2\u00A0and\t \u00C2\u00A0transfections.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 154 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 155 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009011\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0are\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfected\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0in\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells.\t \u00C2\u00A0\t \u00C2\u00A0Plasmids\t \u00C2\u00A0expressing\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0(\u00CE\u0094DQ)\t \u00C2\u00A0and\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0were\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090transfected\t \u00C2\u00A0into\t \u00C2\u00A0HEK\t \u00C2\u00A0293\t \u00C2\u00A0cells\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36\t \u00C2\u00A0(1:8)\t \u00C2\u00A0or\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0(1:8),\t \u00C2\u00A0and\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0harvested\t \u00C2\u00A0at\t \u00C2\u00A048h\t \u00C2\u00A0after\t \u00C2\u00A0transfection.\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein(\u00CE\u0094DQ)\t \u00C2\u00A0was\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0(n=4,\t \u00C2\u00A0H(3)=11.216,\t \u00C2\u00A0p=0.011,\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0Ranks),\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(n=4,\t \u00C2\u00A0p<0.05).\t \u00C2\u00A0Similarly,\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0A53T\t \u00C2\u00A0was\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2syn36CTM\t \u00C2\u00A0(n=4,\t \u00C2\u00A0H(3)=10.836,\t \u00C2\u00A0p=0.013,\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0Ranks),\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0also\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(n=4,\t \u00C2\u00A0p<0.05).\t \u00C2\u00A0Student-\u00C2\u00AD\u00E2\u0080\u0090Newman-\u00C2\u00AD\u00E2\u0080\u0090Keuls\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090hoc\t \u00C2\u00A0analysis.\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0*,\u00CE\u0094\t \u00C2\u00A0p<0.05;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0The\t \u00C2\u00A0plasmids\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0this\t \u00C2\u00A0experiment\t \u00C2\u00A0were\t \u00C2\u00A0generated\t \u00C2\u00A0by\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 156 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 157 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009012\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0is\t \u00C2\u00A0specific\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1.\t \u00C2\u00A0Primary\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A025\u00CE\u00BCM\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0for\t \u00C2\u00A01h,\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A0NMDA\t \u00C2\u00A0(50\u00CE\u00BCM;\t \u00C2\u00A030min).\t \u00C2\u00A0Cells\t \u00C2\u00A0were\t \u00C2\u00A0harvested\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0lysates\t \u00C2\u00A0were\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0sequential\t \u00C2\u00A0immunoblot\t \u00C2\u00A0to\t \u00C2\u00A0detect\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0(n=3).\t \u00C2\u00A0\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 158 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009013\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0were\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090toxic\t \u00C2\u00A024h\t \u00C2\u00A0after\t \u00C2\u00A0treatment.\t \u00C2\u00A0\t \u00C2\u00A0Cortical\t \u00C2\u00A0neurons\t \u00C2\u00A0were\t \u00C2\u00A0treated\t \u00C2\u00A0with\t \u00C2\u00A025\u00CE\u00BCM\t \u00C2\u00A0of\t \u00C2\u00A0recombinant\t \u00C2\u00A0(left,\t \u00C2\u00A0n=4,\t \u00C2\u00A0p=0.005\t \u00C2\u00A0H(3)=12.706)\t \u00C2\u00A0or\t \u00C2\u00A0synthetic\t \u00C2\u00A0(right,\t \u00C2\u00A0n=4,\t \u00C2\u00A0p<0.001,\t \u00C2\u00A0H(7)=28.074)\t \u00C2\u00A0peptides,\t \u00C2\u00A0and\t \u00C2\u00A024h\t \u00C2\u00A0later,\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0was\t \u00C2\u00A0assessed\t \u00C2\u00A0LDH\t \u00C2\u00A0assay.\t \u00C2\u00A0For\t \u00C2\u00A0positive\t \u00C2\u00A0control,\t \u00C2\u00A0cells\t \u00C2\u00A0were\t \u00C2\u00A0lysed\t \u00C2\u00A0with\t \u00C2\u00A0Triton-\u00C2\u00AD\u00E2\u0080\u0090X\t \u00C2\u00A0100\t \u00C2\u00A0prior\t \u00C2\u00A0assay\t \u00C2\u00A0per\t \u00C2\u00A0the\t \u00C2\u00A0manufacture\u00E2\u0080\u0099s\t \u00C2\u00A0instruction.\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090Way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0Ranks\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0analysis.\t \u00C2\u00A0*p<0.05.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0normalized\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0na\u00C3\u00AFve\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0control\t \u00C2\u00A0(arbitrarily\t \u00C2\u00A0set\t \u00C2\u00A0as\t \u00C2\u00A01).\t \u00C2\u00A0n=4\t \u00C2\u00A0individual\t \u00C2\u00A0experiments\t \u00C2\u00A0from\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A02\t \u00C2\u00A0separate\t \u00C2\u00A0primary\t \u00C2\u00A0cultures.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 159 Figure\t \u00C2\u00A04-\u00C2\u00AD\u00E2\u0080\u009014\t \u00C2\u00A0A\t \u00C2\u00A0single\t \u00C2\u00A0CTM\t \u00C2\u00A0repeat\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0(a)\t \u00C2\u00A0Co-\u00C2\u00AD\u00E2\u0080\u0090transfection\t \u00C2\u00A0of\t \u00C2\u00A0plasmids\t \u00C2\u00A0expressing\t \u00C2\u00A0various\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0with\t \u00C2\u00A0HA-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0shows\t \u00C2\u00A0that\t \u00C2\u00A0all\t \u00C2\u00A0peptides,\t \u00C2\u00A0except\t \u00C2\u00A0for\t \u00C2\u00A0FLAG-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2synKFERQQRFFE,\t \u00C2\u00A0efficiently\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0(n=4).\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0F(7,31)=2.956,\t \u00C2\u00A0p=0.022.\t \u00C2\u00A0Similarly,\t \u00C2\u00A0the\t \u00C2\u00A0synthetic\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0containing\t \u00C2\u00A0either\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0(b;\t \u00C2\u00A0F(7,31)=7.251,\t \u00C2\u00A0p<0.001)\t \u00C2\u00A0or\t \u00C2\u00A0double\t \u00C2\u00A0(c;\t \u00C2\u00A0F(7,31)=22.181,\t \u00C2\u00A0p<0.001)\t \u00C2\u00A0CTM\t \u00C2\u00A0repeat(s)\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0endogenous\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0in\t \u00C2\u00A0primary\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0(n=4).\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0with\t \u00C2\u00A0Fisher\t \u00C2\u00A0LSD\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090hoc.\t \u00C2\u00A0*p<0.05.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0mean\t \u00C2\u00A0values\u00C2\u00B1s.e.m.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 160 \t \u00C2\u00A0\t \u00C2\u00A0 161 CHAPTER\t \u00C2\u00A05: NEUROPROTECTION\t \u00C2\u00A0BY\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IN\t \u00C2\u00A0VITRO\t \u00C2\u00A0AND\t \u00C2\u00A0IN\t \u00C2\u00A0VIVO\t \u00C2\u00A05.1 INTRODUCTION\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0previous\t \u00C2\u00A0chapters\t \u00C2\u00A0we\t \u00C2\u00A0used\t \u00C2\u00A0multiple\t \u00C2\u00A0recombinant\t \u00C2\u00A0and\t \u00C2\u00A0endogenous\t \u00C2\u00A0protein\t \u00C2\u00A0targets\t \u00C2\u00A0to\t \u00C2\u00A0demonstrate\t \u00C2\u00A0the\t \u00C2\u00A0efficacy\t \u00C2\u00A0and\t \u00C2\u00A0robustness\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0chapter,\t \u00C2\u00A0we\t \u00C2\u00A0examine\t \u00C2\u00A0the\t \u00C2\u00A0downstream\t \u00C2\u00A0functional\t \u00C2\u00A0consequences\t \u00C2\u00A0of\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0vivo.\t \u00C2\u00A0Specifically,\t \u00C2\u00A0we\t \u00C2\u00A0ask\t \u00C2\u00A0whether\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0observable\t \u00C2\u00A0phenotypic\t \u00C2\u00A0outcomes.\t \u00C2\u00A0Moreover,\t \u00C2\u00A0we\t \u00C2\u00A0explore\t \u00C2\u00A0the\t \u00C2\u00A0potential\t \u00C2\u00A0of\t \u00C2\u00A0utilizing\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090promoting\t \u00C2\u00A0proteins\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A0avenue\t \u00C2\u00A0to\t \u00C2\u00A0develop\t \u00C2\u00A0therapeutics\t \u00C2\u00A0for\t \u00C2\u00A0ischemic\t \u00C2\u00A0stroke.\t \u00C2\u00A0\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.6.4,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0crucial\t \u00C2\u00A0mediator\t \u00C2\u00A0of\t \u00C2\u00A0stroke-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neuronal\t \u00C2\u00A0injury\t \u00C2\u00A0(Fujita\t \u00C2\u00A0and\t \u00C2\u00A0Yamashita,\t \u00C2\u00A02014).\t \u00C2\u00A0As\t \u00C2\u00A0a\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090cell\t \u00C2\u00A0death\t \u00C2\u00A0kinase,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0convergence\t \u00C2\u00A0point\t \u00C2\u00A0for\t \u00C2\u00A0multiple\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0pathways\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0stroke\t \u00C2\u00A0(Pei\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014),\t \u00C2\u00A0including\t \u00C2\u00A0excitotoxicity\t \u00C2\u00A0(Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005;\t \u00C2\u00A0Hainsworth\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010),\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0(Eisenberg-\u00C2\u00AD\u00E2\u0080\u0090Lerner\t \u00C2\u00A0and\t \u00C2\u00A0Kimchi,\t \u00C2\u00A02007)\t \u00C2\u00A0and\t \u00C2\u00A0inflammation\t \u00C2\u00A0(Chuang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Turner-\u00C2\u00AD\u00E2\u0080\u0090Brannen\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Lai\t \u00C2\u00A0and\t \u00C2\u00A0Chen,\t \u00C2\u00A02014).\t \u00C2\u00A0The\t \u00C2\u00A0inhibition\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0an\t \u00C2\u00A0attractive\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0strategy\t \u00C2\u00A0against\t \u00C2\u00A0stroke\t \u00C2\u00A0damage,\t \u00C2\u00A0as\t \u00C2\u00A0shRNA-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0significantly\t \u00C2\u00A0protects\t \u00C2\u00A0cells\t \u00C2\u00A0against\t \u00C2\u00A0hydrogen\t \u00C2\u00A0peroxide\t \u00C2\u00A0(H2O2)-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0(Eisenberg-\u00C2\u00AD\u00E2\u0080\u0090Lerner\t \u00C2\u00A0and\t \u00C2\u00A0Kimchi,\t \u00C2\u00A02007),\t \u00C2\u00A0and\t \u00C2\u00A0administration\t \u00C2\u00A0of\t \u00C2\u00A0an\t \u00C2\u00A0aminopyridazine-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0small\t \u00C2\u00A0chemical\t \u00C2\u00A0inhibitor\t \u00C2\u00A0reduces\t \u00C2\u00A0infarct\t \u00C2\u00A0volume\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0mouse\t \u00C2\u00A0 162 model\t \u00C2\u00A0of\t \u00C2\u00A0ischemia\t \u00C2\u00A0(Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005;\t \u00C2\u00A0Valentza\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0dissociation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR\t \u00C2\u00A0by\t \u00C2\u00A0administration\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0not\t \u00C2\u00A0only\t \u00C2\u00A0prevents\t \u00C2\u00A0NMDA-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0damage\t \u00C2\u00A0but\t \u00C2\u00A0also\t \u00C2\u00A0partially\t \u00C2\u00A0reduces\t \u00C2\u00A0ischemic\t \u00C2\u00A0brain\t \u00C2\u00A0injuries\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0MCAo\t \u00C2\u00A0stroke\t \u00C2\u00A0model\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010),\t \u00C2\u00A0and\t \u00C2\u00A0inhibiting\t \u00C2\u00A0DAPK1\t \u00C2\u00A0dephosphorylation\t \u00C2\u00A0via\t \u00C2\u00A0G-\u00C2\u00AD\u00E2\u0080\u0090protein-\u00C2\u00AD\u00E2\u0080\u0090coupled\t \u00C2\u00A0receptor\t \u00C2\u00A030\t \u00C2\u00A0stimulation\t \u00C2\u00A0also\t \u00C2\u00A0conferred\t \u00C2\u00A0rapid\t \u00C2\u00A0neuroprotection\t \u00C2\u00A0(Liu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activates\t \u00C2\u00A0downstream\t \u00C2\u00A0of\t \u00C2\u00A0NMDAR,\t \u00C2\u00A0it\t \u00C2\u00A0stands\t \u00C2\u00A0to\t \u00C2\u00A0reason\t \u00C2\u00A0that\t \u00C2\u00A0targeting\t \u00C2\u00A0the\t \u00C2\u00A0kinase\t \u00C2\u00A0may\t \u00C2\u00A0offer\t \u00C2\u00A0a\t \u00C2\u00A0longer\t \u00C2\u00A0therapeutic\t \u00C2\u00A0window\t \u00C2\u00A0than\t \u00C2\u00A0directly\t \u00C2\u00A0inhibiting\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR.\t \u00C2\u00A0Indeed,\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0administration\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0small\t \u00C2\u00A0chemical\t \u00C2\u00A0DAPK1\t \u00C2\u00A0inhibitor\t \u00C2\u00A06h\t \u00C2\u00A0after\t \u00C2\u00A0hypoxia-\u00C2\u00AD\u00E2\u0080\u0090ischemia\t \u00C2\u00A0insult\t \u00C2\u00A0attenuated\t \u00C2\u00A0neuronal\t \u00C2\u00A0loss\t \u00C2\u00A0assessed\t \u00C2\u00A0one\t \u00C2\u00A0week\t \u00C2\u00A0later\t \u00C2\u00A0in\t \u00C2\u00A0mice\t \u00C2\u00A0(Velentza\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Unfortunately,\t \u00C2\u00A0the\t \u00C2\u00A0undesirable\t \u00C2\u00A0molecular\t \u00C2\u00A0properties\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0drug\t \u00C2\u00A0preclude\t \u00C2\u00A0its\t \u00C2\u00A0further\t \u00C2\u00A0development\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0neuroprotectant\t \u00C2\u00A0for\t \u00C2\u00A0stroke\t \u00C2\u00A0(Velentza\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Based\t \u00C2\u00A0on\t \u00C2\u00A0analysis\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1\t \u00C2\u00A0catalytic\t \u00C2\u00A0domain,\t \u00C2\u00A0thus\t \u00C2\u00A0far\t \u00C2\u00A08\t \u00C2\u00A0DAPK1\t \u00C2\u00A0chemical\t \u00C2\u00A0inhibitors\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0identified,\t \u00C2\u00A0but\t \u00C2\u00A0few\t \u00C2\u00A0are\t \u00C2\u00A0validated\t \u00C2\u00A0in\t \u00C2\u00A0cells\t \u00C2\u00A0(Huang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0Given\t \u00C2\u00A0the\t \u00C2\u00A0lack\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0viable\t \u00C2\u00A0small\t \u00C2\u00A0chemical\t \u00C2\u00A0DAPK1\t \u00C2\u00A0inhibitor,\t \u00C2\u00A0we\t \u00C2\u00A0reasoned\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0animal\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0stroke\t \u00C2\u00A0would\t \u00C2\u00A0not\t \u00C2\u00A0only\t \u00C2\u00A0illustrate\t \u00C2\u00A0the\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0applicability\t \u00C2\u00A0and\t \u00C2\u00A0functional\t \u00C2\u00A0relevance\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0method,\t \u00C2\u00A0but\t \u00C2\u00A0also\t \u00C2\u00A0present\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0therapeutic\t \u00C2\u00A0strategy\t \u00C2\u00A0for\t \u00C2\u00A0neuroprotection\t \u00C2\u00A0in\t \u00C2\u00A0stroke.\t \u00C2\u00A0 163 5.2 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090FACILITATED\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0PROTECTS\t \u00C2\u00A0AGAINST\t \u00C2\u00A0OXIDATIVE\t \u00C2\u00A0STRESS\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0We\t \u00C2\u00A0first\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0investigate\t \u00C2\u00A0whether\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0protective\t \u00C2\u00A0against\t \u00C2\u00A0toxicity\t \u00C2\u00A0from\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress.\t \u00C2\u00A0To\t \u00C2\u00A0investigate\t \u00C2\u00A0whether\t \u00C2\u00A0NMDAR-\u00C2\u00AD\u00E2\u0080\u0090independent\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0can\t \u00C2\u00A0activate\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0we\t \u00C2\u00A0exposed\t \u00C2\u00A0cultured\t \u00C2\u00A0primary\t \u00C2\u00A0neurons\t \u00C2\u00A0to\t \u00C2\u00A0H2O2,\t \u00C2\u00A0a\t \u00C2\u00A0potent\t \u00C2\u00A0generator\t \u00C2\u00A0of\t \u00C2\u00A0reactive\t \u00C2\u00A0oxygen\t \u00C2\u00A0species\t \u00C2\u00A0(ROS).\t \u00C2\u00A0Immunoblot\t \u00C2\u00A0analysis\t \u00C2\u00A0showed\t \u00C2\u00A0a\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0decrease\t \u00C2\u00A0in\t \u00C2\u00A0phosphorylated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0H2O2\t \u00C2\u00A0can\t \u00C2\u00A0indeed\t \u00C2\u00A0induce\t \u00C2\u00A0substantial\t \u00C2\u00A0activation\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00901a).\t \u00C2\u00A0\t \u00C2\u00A0Next,\t \u00C2\u00A0to\t \u00C2\u00A0investigate\t \u00C2\u00A0whether\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation,\t \u00C2\u00A0we\t \u00C2\u00A0pretreated\t \u00C2\u00A0the\t \u00C2\u00A0neurons\t \u00C2\u00A0with\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0for\t \u00C2\u00A0an\t \u00C2\u00A0hour\t \u00C2\u00A0prior\t \u00C2\u00A0to\t \u00C2\u00A0H2O2\t \u00C2\u00A0insult\t \u00C2\u00A0and\t \u00C2\u00A0assessed\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A02h\t \u00C2\u00A0later.\t \u00C2\u00A0As\t \u00C2\u00A0expected,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct,\t \u00C2\u00A0significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0lysosome-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00901b).\t \u00C2\u00A0To\t \u00C2\u00A0determine\t \u00C2\u00A0if\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0induced\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0neuroprotective,\t \u00C2\u00A0we\t \u00C2\u00A0used\t \u00C2\u00A0LDH\t \u00C2\u00A0release\t \u00C2\u00A0as\t \u00C2\u00A0our\t \u00C2\u00A0endpoint\t \u00C2\u00A0to\t \u00C2\u00A0assess\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage.\t \u00C2\u00A0Exposure\t \u00C2\u00A0to\t \u00C2\u00A0H2O2\t \u00C2\u00A0(300\u00CE\u00BCM,\t \u00C2\u00A030min)\t \u00C2\u00A0significantly\t \u00C2\u00A0increased\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A0as\t \u00C2\u00A0evidenced\t \u00C2\u00A0by\t \u00C2\u00A0increased\t \u00C2\u00A0LDH\t \u00C2\u00A0release,\t \u00C2\u00A0and\t \u00C2\u00A0pretreatment\t \u00C2\u00A0with\t \u00C2\u00A0catalase,\t \u00C2\u00A0an\t \u00C2\u00A0enzyme\t \u00C2\u00A0that\t \u00C2\u00A0breaks\t \u00C2\u00A0down\t \u00C2\u00A0H2O2,\t \u00C2\u00A0completely\t \u00C2\u00A0rescued\t \u00C2\u00A0this\t \u00C2\u00A0neurotoxicity\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0the\t \u00C2\u00A0selective\t \u00C2\u00A0NMDAR\t \u00C2\u00A0competitive\t \u00C2\u00A0antagonist\t \u00C2\u00A0(2R)-\u00C2\u00AD\u00E2\u0080\u0090amino-\u00C2\u00AD\u00E2\u0080\u00905-\u00C2\u00AD\u00E2\u0080\u0090phosphonopentanoate\t \u00C2\u00A0(APV,\t \u00C2\u00A01\t \u00C2\u00A0mM)\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0protect\t \u00C2\u00A0against\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neurotoxicity,\t \u00C2\u00A0suggesting\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0was\t \u00C2\u00A0not\t \u00C2\u00A0mediated\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR.\t \u00C2\u00A0Consistent\t \u00C2\u00A0with\t \u00C2\u00A0this\t \u00C2\u00A0 164 idea,\t \u00C2\u00A0the\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0(50\u00CE\u00BCM)\t \u00C2\u00A0had\t \u00C2\u00A0no\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0effects\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0is\t \u00C2\u00A0expected\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0only\t \u00C2\u00A0protects\t \u00C2\u00A0against\t \u00C2\u00A0NMDAR-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0excitotoxicity\t \u00C2\u00A0by\t \u00C2\u00A0disrupting\t \u00C2\u00A0the\t \u00C2\u00A0binding\t \u00C2\u00A0between\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0GluN2B.\t \u00C2\u00A0In\t \u00C2\u00A0stark\t \u00C2\u00A0contrast,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(50\u00CE\u00BCM)\t \u00C2\u00A0significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neurotoxicity,\t \u00C2\u00A0and\t \u00C2\u00A0concurrent\t \u00C2\u00A0lysosome\t \u00C2\u00A0inhibition\t \u00C2\u00A0with\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(20mM)\t \u00C2\u00A0abolished\t \u00C2\u00A0this\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0effect\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00901c).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0strongly\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0observed\t \u00C2\u00A0neuroprotection\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0result\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation.\t \u00C2\u00A0\t \u00C2\u00A0Taken\t \u00C2\u00A0together,\t \u00C2\u00A0these\t \u00C2\u00A0results\t \u00C2\u00A0demonstrate\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0crucial\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0signaling\t \u00C2\u00A0molecule\t \u00C2\u00A0that\t \u00C2\u00A0leads\t \u00C2\u00A0to\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0after\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress\t \u00C2\u00A0in\t \u00C2\u00A0vitro.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0also\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0NMDAR-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0excitotoxicity,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0possible\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0may\t \u00C2\u00A0simultaneously\t \u00C2\u00A0inhibit\t \u00C2\u00A0multiple\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0signaling\t \u00C2\u00A0pathways.\t \u00C2\u00A0\t \u00C2\u00A05.3 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090PEPTIDE\t \u00C2\u00A0KNOCKS\t \u00C2\u00A0DOWN\t \u00C2\u00A0DAPK1\t \u00C2\u00A0IN\t \u00C2\u00A0THE\t \u00C2\u00A0CORTEX\t \u00C2\u00A0FOLLOWING\t \u00C2\u00A0EXCITOTOXIC\t \u00C2\u00A0STIMULI\t \u00C2\u00A0\t \u00C2\u00A0Having\t \u00C2\u00A0confirmed\t \u00C2\u00A0the\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0effects\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0from\t \u00C2\u00A0ROS,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0the\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0could\t \u00C2\u00A0efficiently\t \u00C2\u00A0knock\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0following\t \u00C2\u00A0excitotoxicity.\t \u00C2\u00A0Kainic\t \u00C2\u00A0acid\t \u00C2\u00A0(KA)\t \u00C2\u00A0is\t \u00C2\u00A0often\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0animal\t \u00C2\u00A0models\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0damage\t \u00C2\u00A0and\t \u00C2\u00A0inflammation,\t \u00C2\u00A0and\t \u00C2\u00A0was\t \u00C2\u00A0previously\t \u00C2\u00A0shown\t \u00C2\u00A0to\t \u00C2\u00A0induce\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activation\t \u00C2\u00A0in\t \u00C2\u00A0rats\t \u00C2\u00A0(Henshall\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Thus,\t \u00C2\u00A0we\t \u00C2\u00A0pre-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0rats\t \u00C2\u00A0with\t \u00C2\u00A010mg/kg\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(n=7)\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0(n=4)\t \u00C2\u00A0(i.v.)\t \u00C2\u00A0to\t \u00C2\u00A0allow\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0to\t \u00C2\u00A0penetrate\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0brain,\t \u00C2\u00A0and\t \u00C2\u00A0then\t \u00C2\u00A0injected\t \u00C2\u00A010mg/kg\t \u00C2\u00A0KA\t \u00C2\u00A0(i.p.)\t \u00C2\u00A030min\t \u00C2\u00A0later.\t \u00C2\u00A0Four\t \u00C2\u00A0hours\t \u00C2\u00A0 165 after\t \u00C2\u00A0KA\t \u00C2\u00A0injection,\t \u00C2\u00A0we\t \u00C2\u00A0harvested\t \u00C2\u00A0the\t \u00C2\u00A0brains\t \u00C2\u00A0and\t \u00C2\u00A0detected\t \u00C2\u00A0DAPK1\t \u00C2\u00A0with\t \u00C2\u00A0immunoblot.\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0animals\t \u00C2\u00A0injected\t \u00C2\u00A0with\t \u00C2\u00A0KA\t \u00C2\u00A0alone\t \u00C2\u00A0or\t \u00C2\u00A0the\t \u00C2\u00A0control\t \u00C2\u00A0peptide\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0significantly\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cortex\t \u00C2\u00A0of\t \u00C2\u00A0KA-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0rats\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00902).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0is\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0penetrating\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0following\t \u00C2\u00A0systemic\t \u00C2\u00A0delivery\t \u00C2\u00A0and\t \u00C2\u00A0knock\t \u00C2\u00A0down\t \u00C2\u00A0its\t \u00C2\u00A0intended\t \u00C2\u00A0target\t \u00C2\u00A0protein.\t \u00C2\u00A05.4 SNIPER-\u00C2\u00AD\u00E2\u0080\u0090MEDIATED\t \u00C2\u00A0DAPK1\t \u00C2\u00A0KNOCKDOWN\t \u00C2\u00A0IS\t \u00C2\u00A0A\t \u00C2\u00A0NEUROPROTECTIVE\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0ischemic\t \u00C2\u00A0stroke\t \u00C2\u00A0induces\t \u00C2\u00A0damage\t \u00C2\u00A0through\t \u00C2\u00A0multiple\t \u00C2\u00A0cytotoxic\t \u00C2\u00A0signaling\t \u00C2\u00A0pathways,\t \u00C2\u00A0we\t \u00C2\u00A0reasoned\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0a\t \u00C2\u00A0better\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0strategy\t \u00C2\u00A0than\t \u00C2\u00A0simply\t \u00C2\u00A0blocking\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0interaction,\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0latter\t \u00C2\u00A0mainly\t \u00C2\u00A0inhibits\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0damage.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0as\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activation\t \u00C2\u00A0occurs\t \u00C2\u00A0downstream\t \u00C2\u00A0of\t \u00C2\u00A0NMDAR\t \u00C2\u00A0overactivation,\t \u00C2\u00A0it\t \u00C2\u00A0stands\t \u00C2\u00A0to\t \u00C2\u00A0reason\t \u00C2\u00A0that\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0is\t \u00C2\u00A0potentially\t \u00C2\u00A0effective\t \u00C2\u00A0at\t \u00C2\u00A0reducing\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage\t \u00C2\u00A0when\t \u00C2\u00A0administered\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090stroke\t \u00C2\u00A0insult.\t \u00C2\u00A0Such\t \u00C2\u00A0a\t \u00C2\u00A0characteristic\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0highly\t \u00C2\u00A0advantageous\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0clinical\t \u00C2\u00A0setting,\t \u00C2\u00A0as\t \u00C2\u00A0prophylactic\t \u00C2\u00A0treatment\t \u00C2\u00A0is\t \u00C2\u00A0often\t \u00C2\u00A0not\t \u00C2\u00A0possible.\t \u00C2\u00A0\t \u00C2\u00A0First,\t \u00C2\u00A0we\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0investigate\t \u00C2\u00A0whether\t \u00C2\u00A0systemic\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0could\t \u00C2\u00A0efficiently\t \u00C2\u00A0knockdown\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0MCAo\t \u00C2\u00A0rat\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0focal\t \u00C2\u00A0ischemia,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0previously\t \u00C2\u00A0shown\t \u00C2\u00A0to\t \u00C2\u00A0reliably\t \u00C2\u00A0activate\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0(Shamloo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A0Rats\t \u00C2\u00A0were\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0relative\t \u00C2\u00A0mild\t \u00C2\u00A0insult\t \u00C2\u00A0of\t \u00C2\u00A01h\t \u00C2\u00A0of\t \u00C2\u00A0unilateral\t \u00C2\u00A0occlusion\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0MCA.\t \u00C2\u00A0One\t \u00C2\u00A0hour\t \u00C2\u00A0after\t \u00C2\u00A0reperfusion,\t \u00C2\u00A0rats\t \u00C2\u00A0were\t \u00C2\u00A0administered\t \u00C2\u00A0saline,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(10mg/kg,\t \u00C2\u00A0i.v.)\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090 166 GluN2B\t \u00C2\u00A0(10mg/kg,\t \u00C2\u00A0i.v.),\t \u00C2\u00A0and\t \u00C2\u00A0sacrificed\t \u00C2\u00A06h\t \u00C2\u00A0later\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0histological\t \u00C2\u00A0examination\t \u00C2\u00A0of\t \u00C2\u00A0infarct\t \u00C2\u00A0areas\t \u00C2\u00A0and\t \u00C2\u00A0bioassays\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0(schematic\t \u00C2\u00A0in\t \u00C2\u00A0Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903a).\t \u00C2\u00A02,3,5-\u00C2\u00AD\u00E2\u0080\u0090Triphenyltetrazolium\t \u00C2\u00A0chloride\t \u00C2\u00A0(TTC)\t \u00C2\u00A0staining\t \u00C2\u00A0of\t \u00C2\u00A0transverse\t \u00C2\u00A0brain\t \u00C2\u00A0sections\t \u00C2\u00A0from\t \u00C2\u00A0saline-\u00C2\u00AD\u00E2\u0080\u0090injected\t \u00C2\u00A0animals\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0to\t \u00C2\u00A0confirm\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0stroke\t \u00C2\u00A0damage.\t \u00C2\u00A0The\t \u00C2\u00A0surgery\t \u00C2\u00A0induced\t \u00C2\u00A0reliable\t \u00C2\u00A0unilateral\t \u00C2\u00A0ischemic\t \u00C2\u00A0damage\t \u00C2\u00A0primarily\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0ipsilateral\t \u00C2\u00A0striatum\t \u00C2\u00A0with\t \u00C2\u00A0limited\t \u00C2\u00A0cortical\t \u00C2\u00A0damage\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903b).\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0MCAo\t \u00C2\u00A0activates\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0activity-\u00C2\u00AD\u00E2\u0080\u0090dependent,\t \u00C2\u00A0we\t \u00C2\u00A0reasoned\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0maximally\t \u00C2\u00A0activated\t \u00C2\u00A0and\t \u00C2\u00A0degraded\t \u00C2\u00A0in\t \u00C2\u00A0regions\t \u00C2\u00A0most\t \u00C2\u00A0affected\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0ischemic\t \u00C2\u00A0insult.\t \u00C2\u00A0Using\t \u00C2\u00A0TTC-\u00C2\u00AD\u00E2\u0080\u0090stained\t \u00C2\u00A0sections\t \u00C2\u00A0to\t \u00C2\u00A0help\t \u00C2\u00A0identify\t \u00C2\u00A0stroke-\u00C2\u00AD\u00E2\u0080\u0090affected\t \u00C2\u00A0areas,\t \u00C2\u00A0we\t \u00C2\u00A0excised\t \u00C2\u00A0brain\t \u00C2\u00A0tissue\t \u00C2\u00A0from\t \u00C2\u00A0both\t \u00C2\u00A0ipsilateral\t \u00C2\u00A0(MCAo-\u00C2\u00AD\u00E2\u0080\u0090damaged)\t \u00C2\u00A0and\t \u00C2\u00A0contralateral\t \u00C2\u00A0sides\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0striatum\t \u00C2\u00A0and\t \u00C2\u00A0nearby\t \u00C2\u00A0cortex\t \u00C2\u00A0for\t \u00C2\u00A0immunoblotting\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903c).\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0contralateral\t \u00C2\u00A0side,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0had\t \u00C2\u00A0no\t \u00C2\u00A0observable\t \u00C2\u00A0effect\t \u00C2\u00A0on\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0MCAo-\u00C2\u00AD\u00E2\u0080\u0090damaged\t \u00C2\u00A0side\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903d).\t \u00C2\u00A0In\t \u00C2\u00A0stark\t \u00C2\u00A0contrast,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0significantly\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0ipsilateral\t \u00C2\u00A0side,\t \u00C2\u00A0with\t \u00C2\u00A0DAPK1\t \u00C2\u00A0being\t \u00C2\u00A0reduced\t \u00C2\u00A0to\t \u00C2\u00A0~40%\t \u00C2\u00A0of\t \u00C2\u00A0that\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0contralateral\t \u00C2\u00A0side\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903d).\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0some\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0collected\t \u00C2\u00A0tissue\t \u00C2\u00A0also\t \u00C2\u00A0contained\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090infarct\t \u00C2\u00A0areas,\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0possible\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0actual\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0even\t \u00C2\u00A0higher\t \u00C2\u00A0than\t \u00C2\u00A0that\t \u00C2\u00A0measured\t \u00C2\u00A0here.\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0also\t \u00C2\u00A0strongly\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0is\t \u00C2\u00A0specifically\t \u00C2\u00A0degraded\t \u00C2\u00A0in\t \u00C2\u00A0areas\t \u00C2\u00A0damaged\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0ischemic\t \u00C2\u00A0insult.\t \u00C2\u00A0 167 We\t \u00C2\u00A0next\t \u00C2\u00A0sought\t \u00C2\u00A0to\t \u00C2\u00A0verify\t \u00C2\u00A0the\t \u00C2\u00A0observed\t \u00C2\u00A0region-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0using\t \u00C2\u00A0immunohistochemistry.\t \u00C2\u00A0A\t \u00C2\u00A0new\t \u00C2\u00A0group\t \u00C2\u00A0of\t \u00C2\u00A0rats\t \u00C2\u00A0was\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0MCAo\t \u00C2\u00A0protocol\t \u00C2\u00A0and\t \u00C2\u00A0injected\t \u00C2\u00A0with\t \u00C2\u00A0either\t \u00C2\u00A0saline,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0using\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0dose\t \u00C2\u00A0and\t \u00C2\u00A0regimen\t \u00C2\u00A0as\t \u00C2\u00A0described\t \u00C2\u00A0above.\t \u00C2\u00A0Six\t \u00C2\u00A0hours\t \u00C2\u00A0following\t \u00C2\u00A0peptide\t \u00C2\u00A0injection,\t \u00C2\u00A0the\t \u00C2\u00A0animals\t \u00C2\u00A0were\t \u00C2\u00A0sacrificed\t \u00C2\u00A0and\t \u00C2\u00A0perfused\t \u00C2\u00A0with\t \u00C2\u00A04%\t \u00C2\u00A0PFA.\t \u00C2\u00A0In\t \u00C2\u00A0transverse\t \u00C2\u00A0brain\t \u00C2\u00A0slices,\t \u00C2\u00A0immunohistochemical\t \u00C2\u00A0staining\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A0and\t \u00C2\u00A0H&E\t \u00C2\u00A0staining\t \u00C2\u00A0in\t \u00C2\u00A0adjacent\t \u00C2\u00A0slices\t \u00C2\u00A0clearly\t \u00C2\u00A0showed\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0was\t \u00C2\u00A0specific\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0striatum\t \u00C2\u00A0and\t \u00C2\u00A0surrounding\t \u00C2\u00A0cortex,\t \u00C2\u00A0i.e.\t \u00C2\u00A0regions\t \u00C2\u00A0that\t \u00C2\u00A0would\t \u00C2\u00A0have\t \u00C2\u00A0otherwise\t \u00C2\u00A0been\t \u00C2\u00A0damaged\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0stroke\t \u00C2\u00A0insult\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903e).\t \u00C2\u00A0To\t \u00C2\u00A0the\t \u00C2\u00A0best\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0knowledge,\t \u00C2\u00A0this\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0first\t \u00C2\u00A0demonstration\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0systemic\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0agent\t \u00C2\u00A0can\t \u00C2\u00A0trigger\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090related\t \u00C2\u00A0subpopulation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090affected\t \u00C2\u00A0areas\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0brain.\t \u00C2\u00A0\t \u00C2\u00A0Having\t \u00C2\u00A0verified\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0vivo,\t \u00C2\u00A0we\t \u00C2\u00A0next\t \u00C2\u00A0asked\t \u00C2\u00A0whether\t \u00C2\u00A0such\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0against\t \u00C2\u00A0stroke\t \u00C2\u00A0damage.\t \u00C2\u00A0Previous\t \u00C2\u00A0evidence\t \u00C2\u00A0clearly\t \u00C2\u00A0demonstrate\t \u00C2\u00A0that\t \u00C2\u00A0stroke\t \u00C2\u00A0can\t \u00C2\u00A0induce\t \u00C2\u00A0both\t \u00C2\u00A0acute\t \u00C2\u00A0and\t \u00C2\u00A0delayed\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage,\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0latter\t \u00C2\u00A0occurring\t \u00C2\u00A0a\t \u00C2\u00A0week\t \u00C2\u00A0(or\t \u00C2\u00A0longer)\t \u00C2\u00A0after\t \u00C2\u00A0the\t \u00C2\u00A0initial\t \u00C2\u00A0insult\t \u00C2\u00A0(Lai\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0our\t \u00C2\u00A0goal\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0investigate\t \u00C2\u00A0the\t \u00C2\u00A0link\t \u00C2\u00A0between\t \u00C2\u00A0region-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0and\t \u00C2\u00A0region-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0neuroprotection,\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0is\t \u00C2\u00A0reversible\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0temporary,\t \u00C2\u00A0we\t \u00C2\u00A0opted\t \u00C2\u00A0to\t \u00C2\u00A0assess\t \u00C2\u00A0acute\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A0rather\t \u00C2\u00A0than\t \u00C2\u00A0delayed\t \u00C2\u00A0damage\t \u00C2\u00A0to\t \u00C2\u00A0enable\t \u00C2\u00A0visualization\t \u00C2\u00A0of\t \u00C2\u00A0brain\t \u00C2\u00A0areas\t \u00C2\u00A0with\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Transverse\t \u00C2\u00A0brain\t \u00C2\u00A0slices\t \u00C2\u00A0stained\t \u00C2\u00A0with\t \u00C2\u00A0H&E\t \u00C2\u00A0showed\t \u00C2\u00A0that\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0treatment\t \u00C2\u00A0reduced\t \u00C2\u00A0the\t \u00C2\u00A0infarct\t \u00C2\u00A0area,\t \u00C2\u00A0most\t \u00C2\u00A0likely\t \u00C2\u00A0by\t \u00C2\u00A0inhibiting\t \u00C2\u00A0NMDAR-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0coupling\t \u00C2\u00A0(Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0 168 However,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0treatment\t \u00C2\u00A0led\t \u00C2\u00A0to\t \u00C2\u00A0a\t \u00C2\u00A0much\t \u00C2\u00A0more\t \u00C2\u00A0significant\t \u00C2\u00A0reduction\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0area\t \u00C2\u00A0of\t \u00C2\u00A0infart\t \u00C2\u00A0as\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B.\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0is\t \u00C2\u00A0in\t \u00C2\u00A0line\t \u00C2\u00A0with\t \u00C2\u00A0our\t \u00C2\u00A0idea\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0blocks\t \u00C2\u00A0multiple\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0signaling\t \u00C2\u00A0pathways.\t \u00C2\u00A0\t \u00C2\u00A0To\t \u00C2\u00A0further\t \u00C2\u00A0confirm\t \u00C2\u00A0that\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0treatment\t \u00C2\u00A0is\t \u00C2\u00A0more\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0than\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B,\t \u00C2\u00A0we\t \u00C2\u00A0quantified\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0degenerating\t \u00C2\u00A0neurons\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0striatum\t \u00C2\u00A0and\t \u00C2\u00A0cortex\t \u00C2\u00A0using\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090Jade\t \u00C2\u00A0B\t \u00C2\u00A0staining.\t \u00C2\u00A0Consistent\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0results\t \u00C2\u00A0from\t \u00C2\u00A0H&E\t \u00C2\u00A0staining,\t \u00C2\u00A0although\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0also\t \u00C2\u00A0reduced\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0degenerating\t \u00C2\u00A0neurons\t \u00C2\u00A0in\t \u00C2\u00A0both\t \u00C2\u00A0the\t \u00C2\u00A0striatum\t \u00C2\u00A0and\t \u00C2\u00A0cortex\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0saline,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0was\t \u00C2\u00A0far\t \u00C2\u00A0more\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0in\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0almost\t \u00C2\u00A0completely\t \u00C2\u00A0eliminated\t \u00C2\u00A0signs\t \u00C2\u00A0of\t \u00C2\u00A0acutely\t \u00C2\u00A0degenerating\t \u00C2\u00A0neurons\t \u00C2\u00A0(Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903f).\t \u00C2\u00A0\t \u00C2\u00A0Altogether,\t \u00C2\u00A0in\t \u00C2\u00A0terms\t \u00C2\u00A0of\t \u00C2\u00A0developing\t \u00C2\u00A0methodology,\t \u00C2\u00A0these\t \u00C2\u00A0results\t \u00C2\u00A0provide\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0can\t \u00C2\u00A0efficiently\t \u00C2\u00A0knockdown\t \u00C2\u00A0CNS\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0freely\t \u00C2\u00A0moving\t \u00C2\u00A0animals.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0depending\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0nature\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction,\t \u00C2\u00A0it\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0possible\t \u00C2\u00A0to\t \u00C2\u00A0achieve\t \u00C2\u00A0region-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0undesirable\t \u00C2\u00A0subpopulation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0target\t \u00C2\u00A0protein.\t \u00C2\u00A0In\t \u00C2\u00A0terms\t \u00C2\u00A0of\t \u00C2\u00A0advancing\t \u00C2\u00A0neuroprotectants\t \u00C2\u00A0for\t \u00C2\u00A0stroke,\t \u00C2\u00A0these\t \u00C2\u00A0results\t \u00C2\u00A0suggest\t \u00C2\u00A0that\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0comparatively\t \u00C2\u00A0more\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0than\t \u00C2\u00A0simply\t \u00C2\u00A0uncoupling\t \u00C2\u00A0the\t \u00C2\u00A0GluN2B-\u00C2\u00AD\u00E2\u0080\u0090DAPK1\t \u00C2\u00A0association,\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0proteins\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0an\t \u00C2\u00A0attractive\t \u00C2\u00A0strategy\t \u00C2\u00A0to\t \u00C2\u00A0reduce\t \u00C2\u00A0damage\t \u00C2\u00A0after\t \u00C2\u00A0insults\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0brain.\t \u00C2\u00A0\t \u00C2\u00A0 169 Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090activated\t \u00C2\u00A0DAPK1,\t \u00C2\u00A0protecting\t \u00C2\u00A0neurons\t \u00C2\u00A0against\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neurotoxicity\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0cultures.\t \u00C2\u00A0\t \u00C2\u00A0(a)\t \u00C2\u00A0Immunoblotting\t \u00C2\u00A0for\t \u00C2\u00A0phosphorylated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(pDAPK1)\t \u00C2\u00A0revealed\t \u00C2\u00A0a\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0activation\t \u00C2\u00A0(dephosphorylation)\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0by\t \u00C2\u00A0H2O2\t \u00C2\u00A0treatment\t \u00C2\u00A0(300\u00CE\u00BCM;\t \u00C2\u00A030min;\t \u00C2\u00A0n=4).\t \u00C2\u00A0(b)\t \u00C2\u00A0Bath\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0100\u00CE\u00BCM\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(n=8;\t \u00C2\u00A0p=0.001),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0(n=7;\t \u00C2\u00A0p=0.311),\t \u00C2\u00A01h\t \u00C2\u00A0before\t \u00C2\u00A0and\t \u00C2\u00A0during\t \u00C2\u00A0H2O2\t \u00C2\u00A0treatment\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0at\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0washout,\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0(20mM;\t \u00C2\u00A0n=8).\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0F(4,34)\t \u00C2\u00A0=\t \u00C2\u00A011.628,\t \u00C2\u00A0P<0.001.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0relative\t \u00C2\u00A0to\t \u00C2\u00A0untreated\t \u00C2\u00A0group.\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0(c)\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0revealed\t \u00C2\u00A0that\t \u00C2\u00A0H2O2\t \u00C2\u00A0treatment\t \u00C2\u00A0(300\u00CE\u00BCM;\t \u00C2\u00A030min)\t \u00C2\u00A0resulted\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0significant\t \u00C2\u00A0increase\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A012h\t \u00C2\u00A0after\t \u00C2\u00A0treatment\t \u00C2\u00A0(n=8),\t \u00C2\u00A0which\t \u00C2\u00A0was\t \u00C2\u00A0rescued\t \u00C2\u00A0by\t \u00C2\u00A0breaking\t \u00C2\u00A0down\t \u00C2\u00A0H2O2\t \u00C2\u00A0with\t \u00C2\u00A0catalase\t \u00C2\u00A0(100U;\t \u00C2\u00A0n=4).\t \u00C2\u00A0H2O2-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neurotoxicity\t \u00C2\u00A0was\t \u00C2\u00A0significantly\t \u00C2\u00A0reduced\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(50\u00CE\u00BCM;\t \u00C2\u00A0n\t \u00C2\u00A0=9),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct\t \u00C2\u00A0(50\u00CE\u00BCM;\t \u00C2\u00A0n=9)\t \u00C2\u00A0or\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR\t \u00C2\u00A0antagonist\t \u00C2\u00A0APV\t \u00C2\u00A0(1mM;\t \u00C2\u00A0n=4).\t \u00C2\u00A0NH4Cl\t \u00C2\u00A0abolished\t \u00C2\u00A0the\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0effect\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0(n=4).\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA,\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001,\t \u00C2\u00A0F(6,41)\t \u00C2\u00A0=\t \u00C2\u00A026.842.\t \u00C2\u00A0**P\t \u00C2\u00A0<\t \u00C2\u00A00.01\t \u00C2\u00A0and\t \u00C2\u00A0***,\u00CE\u0094\u00CE\u0094\u00CE\u0094P\t \u00C2\u00A0<\t \u00C2\u00A00.001;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A0s.e.m.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nature\t \u00C2\u00A0Neuroscience\t \u00C2\u00A02014)\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 170 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 171 Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00902\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cortex\t \u00C2\u00A0of\t \u00C2\u00A0animals\t \u00C2\u00A0following\t \u00C2\u00A0KA\t \u00C2\u00A0treatment.\t \u00C2\u00A0Rats\t \u00C2\u00A0were\t \u00C2\u00A0injected\t \u00C2\u00A0with\t \u00C2\u00A010mg/kg\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0(i.v.),\t \u00C2\u00A0followed\t \u00C2\u00A0by\t \u00C2\u00A010mg/kg\t \u00C2\u00A0KA\t \u00C2\u00A0(i.p.)\t \u00C2\u00A030min\t \u00C2\u00A0later.\t \u00C2\u00A0Immunoblotting\t \u00C2\u00A0for\t \u00C2\u00A0DAPK1\t \u00C2\u00A04h\t \u00C2\u00A0after\t \u00C2\u00A0KA\t \u00C2\u00A0injection\t \u00C2\u00A0showed\t \u00C2\u00A0that\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(n=7),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0(n=4)\t \u00C2\u00A0or\t \u00C2\u00A0KA\t \u00C2\u00A0treatment\t \u00C2\u00A0alone\t \u00C2\u00A0(n=4),\t \u00C2\u00A0efficiently\t \u00C2\u00A0knocked\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cortex\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0animals.\t \u00C2\u00A0Bars\t \u00C2\u00A0represent\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0relative\t \u00C2\u00A0to\t \u00C2\u00A0untreated\t \u00C2\u00A0group.\t \u00C2\u00A0Membranes\t \u00C2\u00A0reprobing\t \u00C2\u00A0for\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0loading\t \u00C2\u00A0control.\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0one-\u00C2\u00AD\u00E2\u0080\u0090way\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0Ranks,\t \u00C2\u00A0H=17.284,\t \u00C2\u00A0p=<0.001.\t \u00C2\u00A0*p<\t \u00C2\u00A00.05;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A0s.e.m.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 172 Figure\t \u00C2\u00A05-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0specifically\t \u00C2\u00A0knocks\t \u00C2\u00A0down\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0ischemic\t \u00C2\u00A0brain\t \u00C2\u00A0areas\t \u00C2\u00A0and\t \u00C2\u00A0reduces\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0MCAo\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0focal\t \u00C2\u00A0ischemia\t \u00C2\u00A0in\t \u00C2\u00A0rats.\t \u00C2\u00A0\t \u00C2\u00A0(a)\t \u00C2\u00A0Timeline\t \u00C2\u00A0of\t \u00C2\u00A0tissue\t \u00C2\u00A0collection\t \u00C2\u00A0for\t \u00C2\u00A0analysis\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0rats.\t \u00C2\u00A0(b)\t \u00C2\u00A0TTC\t \u00C2\u00A0staining\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0series\t \u00C2\u00A0of\t \u00C2\u00A0transverse\t \u00C2\u00A0brain\t \u00C2\u00A0sections\t \u00C2\u00A0showed\t \u00C2\u00A0reliable\t \u00C2\u00A0damage\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0ipsilateral\t \u00C2\u00A0side\t \u00C2\u00A0following\t \u00C2\u00A0unilateral\t \u00C2\u00A0MCAo.\t \u00C2\u00A0(c)\t \u00C2\u00A0Black-\u00C2\u00AD\u00E2\u0080\u0090dashed\t \u00C2\u00A0lines\t \u00C2\u00A0represent\t \u00C2\u00A0brain\t \u00C2\u00A0areas\t \u00C2\u00A0removed\t \u00C2\u00A0for\t \u00C2\u00A0immunoblotting.\t \u00C2\u00A0(d)\t \u00C2\u00A0Immunoblots\t \u00C2\u00A0demonstrate\t \u00C2\u00A0specific\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0ipsilateral,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0contralateral\t \u00C2\u00A0side\t \u00C2\u00A0following\t \u00C2\u00A0application\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(10mg/kg,\t \u00C2\u00A0i.v.;\t \u00C2\u00A0n=3;\t \u00C2\u00A0t(4)\t \u00C2\u00A0=\t \u00C2\u00A014.459,\t \u00C2\u00A0p<0.001),\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0(10mg/kg,\t \u00C2\u00A0i.v.;\t \u00C2\u00A0n=3;\t \u00C2\u00A0t(4)\t \u00C2\u00A0=\t \u00C2\u00A00.739,\t \u00C2\u00A0p=\t \u00C2\u00A00.501).\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090actin\t \u00C2\u00A0was\t \u00C2\u00A0used\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0loading\t \u00C2\u00A0control,\t \u00C2\u00A0two-\u00C2\u00AD\u00E2\u0080\u0090tailed\t \u00C2\u00A0student\u00E2\u0080\u0099s\t \u00C2\u00A0t-\u00C2\u00AD\u00E2\u0080\u0090test\t \u00C2\u00A0***p<0.001\t \u00C2\u00A0(e)\t \u00C2\u00A0H&E\t \u00C2\u00A0(left)\t \u00C2\u00A0and\t \u00C2\u00A0immunohistochemical\t \u00C2\u00A0DAPK1\t \u00C2\u00A0staining\t \u00C2\u00A0(right)\t \u00C2\u00A0of\t \u00C2\u00A0adjacent\t \u00C2\u00A0brain\t \u00C2\u00A0sections.\t \u00C2\u00A0Compared\t \u00C2\u00A0with\t \u00C2\u00A0saline\t \u00C2\u00A0(top)\t \u00C2\u00A0and\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0treated\t \u00C2\u00A0(middle)\t \u00C2\u00A0controls,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0treatment\t \u00C2\u00A0(bottom)\t \u00C2\u00A0selectively\t \u00C2\u00A0reduced\t \u00C2\u00A0infarct\t \u00C2\u00A0area\t \u00C2\u00A0(left)\t \u00C2\u00A0and\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0(right)\t \u00C2\u00A0ipsilaterally.\t \u00C2\u00A0(f)\t \u00C2\u00A0Left:\t \u00C2\u00A0brain\t \u00C2\u00A0sections\t \u00C2\u00A0stained\t \u00C2\u00A0with\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090jade\t \u00C2\u00A0B\t \u00C2\u00A0in\t \u00C2\u00A0rats\t \u00C2\u00A0injected\t \u00C2\u00A0with\t \u00C2\u00A0saline\t \u00C2\u00A0(n=6),\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B\t \u00C2\u00A0(n=5)\t \u00C2\u00A0or\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0(n=5)\t \u00C2\u00A0after\t \u00C2\u00A0treatment\t \u00C2\u00A0as\t \u00C2\u00A0shown\t \u00C2\u00A0in\t \u00C2\u00A0a;\t \u00C2\u00A0scale\t \u00C2\u00A0bar,\t \u00C2\u00A020\u00CE\u00BCm.\t \u00C2\u00A0Right:\t \u00C2\u00A0quantification\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0damage\t \u00C2\u00A0by\t \u00C2\u00A0counting\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090jade\t \u00C2\u00A0B-\u00C2\u00AD\u00E2\u0080\u0090positive\t \u00C2\u00A0cells\t \u00C2\u00A0in\t \u00C2\u00A0each\t \u00C2\u00A0image\t \u00C2\u00A0at\t \u00C2\u00A010X\t \u00C2\u00A0magnification.\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0displayed\t \u00C2\u00A0more\t \u00C2\u00A0prominent\t \u00C2\u00A0neuroprotection\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cortex\t \u00C2\u00A0(p<\t \u00C2\u00A00.001)\t \u00C2\u00A0and\t \u00C2\u00A0striatum\t \u00C2\u00A0(p<\t \u00C2\u00A00.001)\t \u00C2\u00A0as\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B.\t \u00C2\u00A0Cortex:\t \u00C2\u00A0H(2)\t \u00C2\u00A0=\t \u00C2\u00A041.235;\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001;\t \u00C2\u00A0striatum:\t \u00C2\u00A0H(2)\t \u00C2\u00A0=\t \u00C2\u00A038.808;\t \u00C2\u00A0P\t \u00C2\u00A0<\t \u00C2\u00A00.001.\t \u00C2\u00A0Kruskal-\u00C2\u00AD\u00E2\u0080\u0090Wallis\t \u00C2\u00A0ANOVA\t \u00C2\u00A0on\t \u00C2\u00A0ranks\t \u00C2\u00A0with\t \u00C2\u00A0Dunn\u00E2\u0080\u0099s\t \u00C2\u00A0post\t \u00C2\u00A0hoc;\t \u00C2\u00A0bars\t \u00C2\u00A0represent\t \u00C2\u00A0relative\t \u00C2\u00A0mean\t \u00C2\u00A0values\t \u00C2\u00A0\u00C2\u00B1\t \u00C2\u00A0s.e.m.\t \u00C2\u00A0***,\u00CE\u0094\u00CE\u0094\u00CE\u0094P\t \u00C2\u00A0<\t \u00C2\u00A00.001.\t \u00C2\u00A0MCAo\t \u00C2\u00A0surgery\t \u00C2\u00A0and\t \u00C2\u00A0TTC\t \u00C2\u00A0staining\t \u00C2\u00A0was\t \u00C2\u00A0a\t \u00C2\u00A0combined\t \u00C2\u00A0effort\t \u00C2\u00A0with\t \u00C2\u00A0my\t \u00C2\u00A0colleague\t \u00C2\u00A0Wu\t \u00C2\u00A0Yang\t \u00C2\u00A0Jin.\t \u00C2\u00A0(Source:\t \u00C2\u00A0Fan\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A0Nat\t \u00C2\u00A0Neurosci\t \u00C2\u00A02014)\t \u00C2\u00A0 173 174 \t \u00C2\u00A0 175 CHAPTER\t \u00C2\u00A06: DISCUSSION\t \u00C2\u00A0AND\t \u00C2\u00A0CONCLUSIONS\t \u00C2\u00A06.1 OVERALL\t \u00C2\u00A0SIGNIFICANCE\t \u00C2\u00A0Manipulating\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0POI\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0widely\t \u00C2\u00A0used\t \u00C2\u00A0strategy\t \u00C2\u00A0in\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0to\t \u00C2\u00A0interrogate\t \u00C2\u00A0its\t \u00C2\u00A0cellular\t \u00C2\u00A0function.\t \u00C2\u00A0As\t \u00C2\u00A0such,\t \u00C2\u00A0efforts\t \u00C2\u00A0over\t \u00C2\u00A0the\t \u00C2\u00A0past\t \u00C2\u00A030\t \u00C2\u00A0years\t \u00C2\u00A0have\t \u00C2\u00A0targeted\t \u00C2\u00A0every\t \u00C2\u00A0step\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0central\t \u00C2\u00A0dogma\t \u00C2\u00A0\u00E2\u0080\u009Cgene\t \u00C2\u00A0to\t \u00C2\u00A0RNA\t \u00C2\u00A0to\t \u00C2\u00A0protein\u00E2\u0080\u009D\t \u00C2\u00A0to\t \u00C2\u00A0develop\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0allow\t \u00C2\u00A0scientists\t \u00C2\u00A0to\t \u00C2\u00A0inactivate\t \u00C2\u00A0the\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0genes\t \u00C2\u00A0and\t \u00C2\u00A0their\t \u00C2\u00A0products.\t \u00C2\u00A0Without\t \u00C2\u00A0a\t \u00C2\u00A0doubt,\t \u00C2\u00A0nuclei\t \u00C2\u00A0acid-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0approaches,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0gene\t \u00C2\u00A0knockout\t \u00C2\u00A0and\t \u00C2\u00A0RNAi,\t \u00C2\u00A0have\t \u00C2\u00A0revolutionized\t \u00C2\u00A0basic\t \u00C2\u00A0and\t \u00C2\u00A0applied\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0and\t \u00C2\u00A0propelled\t \u00C2\u00A0our\t \u00C2\u00A0understanding\t \u00C2\u00A0of\t \u00C2\u00A0health\t \u00C2\u00A0and\t \u00C2\u00A0disease\t \u00C2\u00A0(Castanotto\t \u00C2\u00A0and\t \u00C2\u00A0Rossi,\t \u00C2\u00A02009;\t \u00C2\u00A0Kole\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0The\t \u00C2\u00A0recent\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0techniques\t \u00C2\u00A0that\t \u00C2\u00A0allow\t \u00C2\u00A0precise\t \u00C2\u00A0and\t \u00C2\u00A0efficient\t \u00C2\u00A0genome\t \u00C2\u00A0editing,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0TALEN\t \u00C2\u00A0and\t \u00C2\u00A0CRISPR-\u00C2\u00AD\u00E2\u0080\u0090Cas,\t \u00C2\u00A0will\t \u00C2\u00A0no\t \u00C2\u00A0doubt\t \u00C2\u00A0further\t \u00C2\u00A0facilitate\t \u00C2\u00A0the\t \u00C2\u00A0functional\t \u00C2\u00A0elucidation\t \u00C2\u00A0of\t \u00C2\u00A0genetic\t \u00C2\u00A0variants\t \u00C2\u00A0and\t \u00C2\u00A0modalities\t \u00C2\u00A0(Kim\t \u00C2\u00A0and\t \u00C2\u00A0Kim,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0Although\t \u00C2\u00A0invaluable,\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0DNA\t \u00C2\u00A0or\t \u00C2\u00A0RNA\t \u00C2\u00A0to\t \u00C2\u00A0manipulate\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0are\t \u00C2\u00A0inherently\t \u00C2\u00A0limited\t \u00C2\u00A0in\t \u00C2\u00A0speed\t \u00C2\u00A0and\t \u00C2\u00A0spatiotemporal\t \u00C2\u00A0control\t \u00C2\u00A0as\t \u00C2\u00A0they\t \u00C2\u00A0do\t \u00C2\u00A0not\t \u00C2\u00A0target\t \u00C2\u00A0the\t \u00C2\u00A0genetic\t \u00C2\u00A0end\t \u00C2\u00A0product\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0the\t \u00C2\u00A0protein.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0these\t \u00C2\u00A0methods\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0is\t \u00C2\u00A0still\t \u00C2\u00A0a\t \u00C2\u00A0relatively\t \u00C2\u00A0slow\t \u00C2\u00A0and\t \u00C2\u00A0cumbersome\t \u00C2\u00A0process.\t \u00C2\u00A0To\t \u00C2\u00A0overcome\t \u00C2\u00A0these\t \u00C2\u00A0problems,\t \u00C2\u00A0a\t \u00C2\u00A0complementary\t \u00C2\u00A0approach\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0directly\t \u00C2\u00A0manipulate\t \u00C2\u00A0protein\t \u00C2\u00A0levels\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level\t \u00C2\u00A0by\t \u00C2\u00A0guiding\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0intracellular\t \u00C2\u00A0proteolytic\t \u00C2\u00A0systems\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0Although\t \u00C2\u00A0a\t \u00C2\u00A0plethora\t \u00C2\u00A0of\t \u00C2\u00A0such\t \u00C2\u00A0methods\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0successfully\t \u00C2\u00A0used\t \u00C2\u00A0for\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0(detailed\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.5),\t \u00C2\u00A0many\t \u00C2\u00A0require\t \u00C2\u00A0genetic\t \u00C2\u00A0engineering\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0 176 and/or\t \u00C2\u00A0viral\t \u00C2\u00A0infection\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0knockdown\t \u00C2\u00A0agents,\t \u00C2\u00A0thus\t \u00C2\u00A0severely\t \u00C2\u00A0limiting\t \u00C2\u00A0their\t \u00C2\u00A0potential\t \u00C2\u00A0for\t \u00C2\u00A0research\t \u00C2\u00A0and\t \u00C2\u00A0clinical\t \u00C2\u00A0use.\t \u00C2\u00A0\t \u00C2\u00A0By\t \u00C2\u00A0building\t \u00C2\u00A0upon\t \u00C2\u00A0the\t \u00C2\u00A0successes\t \u00C2\u00A0and\t \u00C2\u00A0circumventing\t \u00C2\u00A0the\t \u00C2\u00A0limitations\t \u00C2\u00A0of\t \u00C2\u00A0existing\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0technologies,\t \u00C2\u00A0the\t \u00C2\u00A0central\t \u00C2\u00A0aim\t \u00C2\u00A0of\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0develop\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0method\t \u00C2\u00A0that\t \u00C2\u00A0specifically\t \u00C2\u00A0targets\t \u00C2\u00A0unmodified,\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0cells\t \u00C2\u00A0and\t \u00C2\u00A0animals.\t \u00C2\u00A0We\t \u00C2\u00A0envision\t \u00C2\u00A0such\t \u00C2\u00A0a\t \u00C2\u00A0technology\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0useful\t \u00C2\u00A0for\t \u00C2\u00A0both\t \u00C2\u00A0basic\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0and\t \u00C2\u00A0therapeutic\t \u00C2\u00A0development.\t \u00C2\u00A06.2 METHODOLOGY\t \u00C2\u00A06.2.1 CTM\t \u00C2\u00A0IS\t \u00C2\u00A0A\t \u00C2\u00A0DEGRON\t \u00C2\u00A0FOR\t \u00C2\u00A0CMA\t \u00C2\u00A0To\t \u00C2\u00A0complement\t \u00C2\u00A0existing\t \u00C2\u00A0direct\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0rely\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0UPS,\t \u00C2\u00A0we\t \u00C2\u00A0opted\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation\t \u00C2\u00A0pathway\t \u00C2\u00A0for\t \u00C2\u00A0SNIPER.\t \u00C2\u00A0Facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0achieved\t \u00C2\u00A0by\t \u00C2\u00A0applying\t \u00C2\u00A0a\t \u00C2\u00A0targeting\t \u00C2\u00A0agent\t \u00C2\u00A0that\t \u00C2\u00A0recognizes\t \u00C2\u00A0both\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0cellular\t \u00C2\u00A0degradation\t \u00C2\u00A0machinery\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0degron\t \u00C2\u00A0motif\t \u00C2\u00A0(Chapter\t \u00C2\u00A01.5.2).\t \u00C2\u00A0Many\t \u00C2\u00A0different\t \u00C2\u00A0types\t \u00C2\u00A0of\t \u00C2\u00A0degrons\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0identified\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0UPS\t \u00C2\u00A0pathway.\t \u00C2\u00A0In\t \u00C2\u00A0contrast,\t \u00C2\u00A0although\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0necessary\t \u00C2\u00A0and\t \u00C2\u00A0sufficient\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012),\t \u00C2\u00A0it\t \u00C2\u00A0was\t \u00C2\u00A0not\t \u00C2\u00A0known\t \u00C2\u00A0whether\t \u00C2\u00A0CTM\t \u00C2\u00A0could\t \u00C2\u00A0confer\t \u00C2\u00A0susceptibility\t \u00C2\u00A0to\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrates,\t \u00C2\u00A0i.e.\t \u00C2\u00A0whether\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0degron.\t \u00C2\u00A0In\t \u00C2\u00A0Chapter\t \u00C2\u00A03.2.1,\t \u00C2\u00A0we\t \u00C2\u00A0showed\t \u00C2\u00A0that\t \u00C2\u00A0genetic\t \u00C2\u00A0tagging\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0to\t \u00C2\u00A0GFP,\t \u00C2\u00A0a\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0protein,\t \u00C2\u00A0rendered\t \u00C2\u00A0it\t \u00C2\u00A0susceptible\t \u00C2\u00A0to\t \u00C2\u00A0degradation\t \u00C2\u00A0through\t \u00C2\u00A0CMA.\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0demonstrates\t \u00C2\u00A0the\t \u00C2\u00A0transferability\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0 177 conferring\t \u00C2\u00A0metabolic\t \u00C2\u00A0instability,\t \u00C2\u00A0and\t \u00C2\u00A0together\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0findings\t \u00C2\u00A0from\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011b),\t \u00C2\u00A0confirms\t \u00C2\u00A0CTM\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0degron\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0system.\t \u00C2\u00A0\t \u00C2\u00A0More\t \u00C2\u00A0importantly,\t \u00C2\u00A0we\t \u00C2\u00A0demonstrated\t \u00C2\u00A0that\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0can\t \u00C2\u00A0degrade\t \u00C2\u00A0a\t \u00C2\u00A0cytosolic\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090CMA\t \u00C2\u00A0substrate\t \u00C2\u00A0protein\t \u00C2\u00A0by\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0and\t \u00C2\u00A0recruiting\t \u00C2\u00A0the\t \u00C2\u00A0necessary\t \u00C2\u00A0chaperones\t \u00C2\u00A0to\t \u00C2\u00A0initiate\t \u00C2\u00A0degradation.\t \u00C2\u00A0To\t \u00C2\u00A0the\t \u00C2\u00A0best\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0knowledge,\t \u00C2\u00A0this\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0first\t \u00C2\u00A0evidence\t \u00C2\u00A0that\t \u00C2\u00A0CTMs\t \u00C2\u00A0are\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting,\t \u00C2\u00A0and\t \u00C2\u00A0raises\t \u00C2\u00A0interesting\t \u00C2\u00A0questions\t \u00C2\u00A0regarding\t \u00C2\u00A0the\t \u00C2\u00A0turnover\t \u00C2\u00A0of\t \u00C2\u00A0natural\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0and\t \u00C2\u00A0their\t \u00C2\u00A0interacting\t \u00C2\u00A0proteins.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0are\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0degraded\t \u00C2\u00A0simultaneously\t \u00C2\u00A0via\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting?\t \u00C2\u00A0\t \u00C2\u00A0Using\t \u00C2\u00A0pharmacological\t \u00C2\u00A0inhibitors\t \u00C2\u00A0and\t \u00C2\u00A0RNAi\t \u00C2\u00A0to\t \u00C2\u00A0inhibit\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0and\t \u00C2\u00A0CMA,\t \u00C2\u00A0respectively,\t \u00C2\u00A0we\t \u00C2\u00A0demonstrated\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0occurs\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0pathway\t \u00C2\u00A0(Chapter\t \u00C2\u00A04.4).\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0detail\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A01.4.2.2,\t \u00C2\u00A0substrate\t \u00C2\u00A0delivery\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0requires\t \u00C2\u00A0four\t \u00C2\u00A0main\t \u00C2\u00A0steps:\t \u00C2\u00A01)\t \u00C2\u00A0binding\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0cytosolic\t \u00C2\u00A0tail\t \u00C2\u00A0via\t \u00C2\u00A0electrostatic\t \u00C2\u00A0interactions;\t \u00C2\u00A02)\t \u00C2\u00A0transient\t \u00C2\u00A0oligomerization\t \u00C2\u00A0of\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A;\t \u00C2\u00A03)\t \u00C2\u00A0unfolding\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0substrate;\t \u00C2\u00A0and\t \u00C2\u00A04)\t \u00C2\u00A0internalization\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0substrate\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0multicomplex\t \u00C2\u00A0pore.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0our\t \u00C2\u00A0method\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction,\t \u00C2\u00A0it\t \u00C2\u00A0remains\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0seen\t \u00C2\u00A0how\t \u00C2\u00A0the\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090substrate\t \u00C2\u00A0protein\t \u00C2\u00A0is\t \u00C2\u00A0internalized\t \u00C2\u00A0once\t \u00C2\u00A0directed\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0receptors.\t \u00C2\u00A0One\t \u00C2\u00A0possibility\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0survives\t \u00C2\u00A0protein\t \u00C2\u00A0unfolding.\t \u00C2\u00A0Unlike\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions\t \u00C2\u00A0that\t \u00C2\u00A0often\t \u00C2\u00A0include\t \u00C2\u00A0multiple\t \u00C2\u00A0binding\t \u00C2\u00A0sites\t \u00C2\u00A0in\t \u00C2\u00A0globular\t \u00C2\u00A0domains,\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions\t \u00C2\u00A0rely\t \u00C2\u00A0on\t \u00C2\u00A0short\t \u00C2\u00A0linear\t \u00C2\u00A0motifs\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0local\t \u00C2\u00A0properties\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0protein,\t \u00C2\u00A0and\t \u00C2\u00A0contain\t \u00C2\u00A0significantly\t \u00C2\u00A0more\t \u00C2\u00A0 178 hydrogen\t \u00C2\u00A0bonds\t \u00C2\u00A0involving\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0backbone\t \u00C2\u00A0(Stein\t \u00C2\u00A0and\t \u00C2\u00A0Aloy,\t \u00C2\u00A02008).\t \u00C2\u00A0Another\t \u00C2\u00A0possibility,\t \u00C2\u00A0perhaps\t \u00C2\u00A0more\t \u00C2\u00A0likely,\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090substrate\t \u00C2\u00A0directly\t \u00C2\u00A0binds\t \u00C2\u00A0to\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0once\t \u00C2\u00A0shuttled\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0by\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides.\t \u00C2\u00A0Once\t \u00C2\u00A0bound,\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0is\t \u00C2\u00A0processed\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0similar\t \u00C2\u00A0way\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0native\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate.\t \u00C2\u00A0It\t \u00C2\u00A0should\t \u00C2\u00A0be\t \u00C2\u00A0noted\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0exact\t \u00C2\u00A0mechanisms\t \u00C2\u00A0of\t \u00C2\u00A0substrate\t \u00C2\u00A0internalization\t \u00C2\u00A0is\t \u00C2\u00A0still\t \u00C2\u00A0not\t \u00C2\u00A0well\t \u00C2\u00A0understood,\t \u00C2\u00A0and\t \u00C2\u00A0proteins\t \u00C2\u00A0(e.g.\t \u00C2\u00A0chaperones)\t \u00C2\u00A0other\t \u00C2\u00A0than\t \u00C2\u00A0LAMP-\u00C2\u00AD\u00E2\u0080\u00902A\t \u00C2\u00A0are\t \u00C2\u00A0likely\t \u00C2\u00A0involved.\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0generally\t \u00C2\u00A0believed\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0CTMs\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0correlate\t \u00C2\u00A0with\t \u00C2\u00A0its\t \u00C2\u00A0degradation\t \u00C2\u00A0efficacy,\t \u00C2\u00A0i.e.\t \u00C2\u00A0there\t \u00C2\u00A0are\t \u00C2\u00A0no\t \u00C2\u00A0multivalent\t \u00C2\u00A0effects\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990).\t \u00C2\u00A0Our\t \u00C2\u00A0results\t \u00C2\u00A0contradict\t \u00C2\u00A0this\t \u00C2\u00A0finding\t \u00C2\u00A0by\t \u00C2\u00A0showing\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0terminal\t \u00C2\u00A0CTMs\t \u00C2\u00A0to\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein,\t \u00C2\u00A0a\t \u00C2\u00A0CMA\t \u00C2\u00A0substrate,\t \u00C2\u00A0enhances\t \u00C2\u00A0its\t \u00C2\u00A0degradation\t \u00C2\u00A0(Chapter\t \u00C2\u00A04.5.2).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0the\t \u00C2\u00A0potency\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0seems\t \u00C2\u00A0to\t \u00C2\u00A0correlate\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0number\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0repeats\t \u00C2\u00A0incorporated\t \u00C2\u00A0(Chapter\t \u00C2\u00A04.7).\t \u00C2\u00A0A\t \u00C2\u00A0potential\t \u00C2\u00A0explanation\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0provide\t \u00C2\u00A0unobstructed\t \u00C2\u00A0CTMs\t \u00C2\u00A0that\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0more\t \u00C2\u00A0effective\t \u00C2\u00A0at\t \u00C2\u00A0recruiting\t \u00C2\u00A0cytosolic\t \u00C2\u00A0chaperones\t \u00C2\u00A0to\t \u00C2\u00A0initiate\t \u00C2\u00A0degradation.\t \u00C2\u00A0Indeed,\t \u00C2\u00A0native\t \u00C2\u00A0CTMs\t \u00C2\u00A0are\t \u00C2\u00A0often\t \u00C2\u00A0buried\t \u00C2\u00A0within\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0and\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0subjected\t \u00C2\u00A0to\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modifications\t \u00C2\u00A0that\t \u00C2\u00A0inhibit\t \u00C2\u00A0their\t \u00C2\u00A0function.\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0positioning\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0native\t \u00C2\u00A0substrates\t \u00C2\u00A0is\t \u00C2\u00A0generally\t \u00C2\u00A0thought\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0inconsequential\t \u00C2\u00A0to\t \u00C2\u00A0CMA\t \u00C2\u00A0degradation.\t \u00C2\u00A0However,\t \u00C2\u00A0we\t \u00C2\u00A0clearly\t \u00C2\u00A0demonstrated\t \u00C2\u00A0that\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0as\t \u00C2\u00A0effective\t \u00C2\u00A0when\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0sandwiched\t \u00C2\u00A0between\t \u00C2\u00A0two\t \u00C2\u00A0large\t \u00C2\u00A0protein\t \u00C2\u00A0domains\t \u00C2\u00A0(GluN2B-\u00C2\u00AD\u00E2\u0080\u0090CTM-\u00C2\u00AD\u00E2\u0080\u0090GFP)\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0when\t \u00C2\u00A0placed\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0carboxyl\t \u00C2\u00A0terminal\t \u00C2\u00A0(HA-\u00C2\u00AD\u00E2\u0080\u0090GluN2B-\u00C2\u00AD\u00E2\u0080\u0090CTM)\t \u00C2\u00A0(Chapters\t \u00C2\u00A03.2.3\t \u00C2\u00A0and\t \u00C2\u00A03.2.4).\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0again\t \u00C2\u00A0points\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0critical\t \u00C2\u00A0role\t \u00C2\u00A0of\t \u00C2\u00A0an\t \u00C2\u00A0exposed\t \u00C2\u00A0CTM\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0for\t \u00C2\u00A0initiating\t \u00C2\u00A0and/or\t \u00C2\u00A0enhancing\t \u00C2\u00A0the\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0its\t \u00C2\u00A0binding\t \u00C2\u00A0partner.\t \u00C2\u00A0The\t \u00C2\u00A0 179 relative\t \u00C2\u00A0effectiveness\t \u00C2\u00A0of\t \u00C2\u00A0CTM\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0or\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0in\t \u00C2\u00A0directing\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0remains\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0seen.\t \u00C2\u00A0Sequence\t \u00C2\u00A0analysis\t \u00C2\u00A0reveals\t \u00C2\u00A0that\t \u00C2\u00A0~30%\t \u00C2\u00A0of\t \u00C2\u00A0soluble\t \u00C2\u00A0cytosolic\t \u00C2\u00A0proteins\t \u00C2\u00A0contain\t \u00C2\u00A0one\t \u00C2\u00A0or\t \u00C2\u00A0more\t \u00C2\u00A0CTMs,\t \u00C2\u00A0whereas\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins\t \u00C2\u00A0generally\t \u00C2\u00A0do\t \u00C2\u00A0not\t \u00C2\u00A0(Kaushik\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo,\t \u00C2\u00A02012).\t \u00C2\u00A0The\t \u00C2\u00A0only\t \u00C2\u00A0known\t \u00C2\u00A0exceptions\t \u00C2\u00A0are\t \u00C2\u00A0certain\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins\t \u00C2\u00A0that\t \u00C2\u00A0contain\t \u00C2\u00A0CTMs\t \u00C2\u00A0that\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0released\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cytosol\t \u00C2\u00A0upon\t \u00C2\u00A0cleavage.\t \u00C2\u00A0However,\t \u00C2\u00A0our\t \u00C2\u00A0analysis\t \u00C2\u00A0revealed\t \u00C2\u00A0the\t \u00C2\u00A0presence\t \u00C2\u00A0of\t \u00C2\u00A0putative\t \u00C2\u00A0CTMs\t \u00C2\u00A0in\t \u00C2\u00A0multiple\t \u00C2\u00A0glutamatergic\t \u00C2\u00A0receptors,\t \u00C2\u00A0whereas\t \u00C2\u00A0GABAergic\t \u00C2\u00A0and\t \u00C2\u00A0glycine\t \u00C2\u00A0receptors\t \u00C2\u00A0generally\t \u00C2\u00A0lack\t \u00C2\u00A0the\t \u00C2\u00A0motif\t \u00C2\u00A0(Appendix\t \u00C2\u00A0B;\t \u00C2\u00A0Python\t \u00C2\u00A0code\t \u00C2\u00A0in\t \u00C2\u00A0Appendix\t \u00C2\u00A0A).\t \u00C2\u00A0Whether\t \u00C2\u00A0CTMs\t \u00C2\u00A0in\t \u00C2\u00A0transmembrane\t \u00C2\u00A0proteins\t \u00C2\u00A0have\t \u00C2\u00A0functional\t \u00C2\u00A0consequences\t \u00C2\u00A0remains\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0seen.\t \u00C2\u00A0\t \u00C2\u00A0Interestingly,\t \u00C2\u00A0the\t \u00C2\u00A0\u00E2\u0080\u009CKFERQ\u00E2\u0080\u009D-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0pentapeptide\t \u00C2\u00A0motif\t \u00C2\u00A0can\t \u00C2\u00A0also\t \u00C2\u00A0direct\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0substrate\t \u00C2\u00A0to\t \u00C2\u00A0late\t \u00C2\u00A0endosomes\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0hsc70-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0manner\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0process\t \u00C2\u00A0called\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0(Sahu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A0Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011b).\t \u00C2\u00A0Unlike\t \u00C2\u00A0CMA,\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0require\t \u00C2\u00A0protein\t \u00C2\u00A0unfolding\t \u00C2\u00A0prior\t \u00C2\u00A0degradation,\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0the\t \u00C2\u00A0process\t \u00C2\u00A0may\t \u00C2\u00A0potentially\t \u00C2\u00A0be\t \u00C2\u00A0harnessed\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0aggregated\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0manner\t \u00C2\u00A0similar\t \u00C2\u00A0to\t \u00C2\u00A0macroautophagy.\t \u00C2\u00A0The\t \u00C2\u00A0question\t \u00C2\u00A0remains\t \u00C2\u00A0how\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0degron\t \u00C2\u00A0(CTM)\t \u00C2\u00A0can\t \u00C2\u00A0shuttle\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0to\t \u00C2\u00A0either\t \u00C2\u00A0CMA\t \u00C2\u00A0or\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI\t \u00C2\u00A0proteolytic\t \u00C2\u00A0pathway,\t \u00C2\u00A0but\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0likely\t \u00C2\u00A0that\t \u00C2\u00A0different\t \u00C2\u00A0co-\u00C2\u00AD\u00E2\u0080\u0090chaperones\t \u00C2\u00A0are\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0process.\t \u00C2\u00A0Identifying\t \u00C2\u00A0these\t \u00C2\u00A0molecules\t \u00C2\u00A0may\t \u00C2\u00A0facilitate\t \u00C2\u00A0the\t \u00C2\u00A0design\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0specific\t \u00C2\u00A0protein\t \u00C2\u00A0aggregates\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0via\t \u00C2\u00A0e-\u00C2\u00AD\u00E2\u0080\u0090MI.\t \u00C2\u00A0 180 6.2.2 GENERALIZABILITY\t \u00C2\u00A0AND\t \u00C2\u00A0PREDICTABILITY\t \u00C2\u00A0\t \u00C2\u00A0Our\t \u00C2\u00A0central\t \u00C2\u00A0hypothesis\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0containing\t \u00C2\u00A0CTM\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0PBD\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0universal\t \u00C2\u00A0approach\t \u00C2\u00A0that\t \u00C2\u00A0directly\t \u00C2\u00A0depletes\t \u00C2\u00A0an\t \u00C2\u00A0endogenous\t \u00C2\u00A0protein\t \u00C2\u00A0via\t \u00C2\u00A0CMA.\t \u00C2\u00A0In\t \u00C2\u00A0Chapters\t \u00C2\u00A03\t \u00C2\u00A0and\t \u00C2\u00A04,\t \u00C2\u00A0we\t \u00C2\u00A0demonstrated\t \u00C2\u00A0the\t \u00C2\u00A0reliability\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0with\t \u00C2\u00A0three\t \u00C2\u00A0target\t \u00C2\u00A0proteins\t \u00C2\u00A0(DAPK1,\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0and\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0recombinant\t \u00C2\u00A0protein\t \u00C2\u00A0expression\t \u00C2\u00A0system\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0neuronal\t \u00C2\u00A0cells.\t \u00C2\u00A0Theoretically,\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0should\t \u00C2\u00A0perform\t \u00C2\u00A0for\t \u00C2\u00A0any\t \u00C2\u00A0cytosolic\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0which\t \u00C2\u00A0a\t \u00C2\u00A0binding\t \u00C2\u00A0domain\t \u00C2\u00A0is\t \u00C2\u00A0available\t \u00C2\u00A0or\t \u00C2\u00A0identifiable.\t \u00C2\u00A0\t \u00C2\u00A0Whether\t \u00C2\u00A0SNIPER\t \u00C2\u00A0is\t \u00C2\u00A0effective\t \u00C2\u00A0at\t \u00C2\u00A0degrading\t \u00C2\u00A0transmembrane\t \u00C2\u00A0proteins\t \u00C2\u00A0remains\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0seen.\t \u00C2\u00A0Unfolded\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0known\t \u00C2\u00A0to\t \u00C2\u00A0recruit\t \u00C2\u00A0the\t \u00C2\u00A0peripheral\t \u00C2\u00A0protein\t \u00C2\u00A0quality\t \u00C2\u00A0control\t \u00C2\u00A0system,\t \u00C2\u00A0including\t \u00C2\u00A0chaperones\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0hsc70,\t \u00C2\u00A0CHIP,\t \u00C2\u00A0hop\t \u00C2\u00A0and\t \u00C2\u00A0hsp90,\t \u00C2\u00A0which\t \u00C2\u00A0results\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0receptor\t \u00C2\u00A0cytosolic\t \u00C2\u00A0tail,\t \u00C2\u00A0endocytosis\t \u00C2\u00A0and\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0(Okiyoneda\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0interesting\t \u00C2\u00A0to\t \u00C2\u00A0note\t \u00C2\u00A0that\t \u00C2\u00A0these\t \u00C2\u00A0multi-\u00C2\u00AD\u00E2\u0080\u0090functional\t \u00C2\u00A0chaperones\t \u00C2\u00A0also\t \u00C2\u00A0participate\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0shuttling\t \u00C2\u00A0of\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0(Chapter\t \u00C2\u00A01.4.2.2).\t \u00C2\u00A0It\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0interesting\t \u00C2\u00A0to\t \u00C2\u00A0see\t \u00C2\u00A0whether\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0that\t \u00C2\u00A0bind\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0cytosolic\t \u00C2\u00A0tails\t \u00C2\u00A0of\t \u00C2\u00A0transmembrane\t \u00C2\u00A0proteins\t \u00C2\u00A0can\t \u00C2\u00A0induce\t \u00C2\u00A0their\t \u00C2\u00A0internalization\t \u00C2\u00A0and\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation.\t \u00C2\u00A0A\t \u00C2\u00A0major\t \u00C2\u00A0drawback\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0targeting\t \u00C2\u00A0and\t \u00C2\u00A0RNAi\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0difficult\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0if\t \u00C2\u00A0not\t \u00C2\u00A0impossible\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0to\t \u00C2\u00A0control\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0In\t \u00C2\u00A0Chapter\t \u00C2\u00A04.2,\t \u00C2\u00A0we\t \u00C2\u00A0demonstrate\t \u00C2\u00A0a\t \u00C2\u00A0clear\t \u00C2\u00A0dose-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0time-\u00C2\u00AD\u00E2\u0080\u0090response\t \u00C2\u00A0relationship\t \u00C2\u00A0between\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2Bct-\u00C2\u00AD\u00E2\u0080\u0090CTM\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Importantly,\t \u00C2\u00A0DAPK1\t \u00C2\u00A0knockdown\t \u00C2\u00A0became\t \u00C2\u00A0significant\t \u00C2\u00A0within\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0NMDA\t \u00C2\u00A0washout.\t \u00C2\u00A0Such\t \u00C2\u00A0rapid\t \u00C2\u00A0kinetics\t \u00C2\u00A0is\t \u00C2\u00A0unattainable\t \u00C2\u00A0with\t \u00C2\u00A0nucleotide-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0 181 methods\t \u00C2\u00A0and\t \u00C2\u00A0reduces\t \u00C2\u00A0the\t \u00C2\u00A0chance\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0compensation.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0we\t \u00C2\u00A0observed\t \u00C2\u00A0that\t \u00C2\u00A0washout\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0allows\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0translation\t \u00C2\u00A0system\t \u00C2\u00A0to\t \u00C2\u00A0restore\t \u00C2\u00A0DAPK1\t \u00C2\u00A0levels\t \u00C2\u00A0to\t \u00C2\u00A0baseline\t \u00C2\u00A0after\t \u00C2\u00A07h,\t \u00C2\u00A0and\t \u00C2\u00A0that\t \u00C2\u00A0re-\u00C2\u00AD\u00E2\u0080\u0090applying\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0prolongs\t \u00C2\u00A0the\t \u00C2\u00A0knockdown\t \u00C2\u00A0period.\t \u00C2\u00A0These\t \u00C2\u00A0properties\t \u00C2\u00A0allow\t \u00C2\u00A0predictable\t \u00C2\u00A0control\t \u00C2\u00A0over\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0and\t \u00C2\u00A0duration\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cell,\t \u00C2\u00A0and\t \u00C2\u00A0enable\t \u00C2\u00A0the\t \u00C2\u00A0observation\t \u00C2\u00A0of\t \u00C2\u00A0phenotypes\t \u00C2\u00A0associated\t \u00C2\u00A0with\t \u00C2\u00A0both\t \u00C2\u00A0protein\t \u00C2\u00A0depletion\t \u00C2\u00A0and\t \u00C2\u00A0recovery.\t \u00C2\u00A0\t \u00C2\u00A06.2.3 SPECIFICITY\t \u00C2\u00A0\t \u00C2\u00A0Specificity\t \u00C2\u00A0is\t \u00C2\u00A0of\t \u00C2\u00A0upmost\t \u00C2\u00A0importance\t \u00C2\u00A0in\t \u00C2\u00A0any\t \u00C2\u00A0method\t \u00C2\u00A0designed\t \u00C2\u00A0to\t \u00C2\u00A0manipulate\t \u00C2\u00A0the\t \u00C2\u00A0levels\t \u00C2\u00A0of\t \u00C2\u00A0specific\t \u00C2\u00A0proteins.\t \u00C2\u00A0In\t \u00C2\u00A0this\t \u00C2\u00A0study\t \u00C2\u00A0we\t \u00C2\u00A0assessed\t \u00C2\u00A0several\t \u00C2\u00A0potential\t \u00C2\u00A0sources\t \u00C2\u00A0of\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0effects,\t \u00C2\u00A0including:\t \u00C2\u00A01)\t \u00C2\u00A0saturation\t \u00C2\u00A0or\t \u00C2\u00A0alternations\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0pathway;\t \u00C2\u00A02)\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090targeted\t \u00C2\u00A0proteins;\t \u00C2\u00A0and\t \u00C2\u00A03)\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0POI-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0proteins.\t \u00C2\u00A0\t \u00C2\u00A0Our\t \u00C2\u00A0results\t \u00C2\u00A0indicate\t \u00C2\u00A0that\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0peptides\t \u00C2\u00A0do\t \u00C2\u00A0not\t \u00C2\u00A0indiscriminately\t \u00C2\u00A0upregulate\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0system.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0given\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0is\t \u00C2\u00A0highly\t \u00C2\u00A0adaptive\t \u00C2\u00A0to\t \u00C2\u00A0increased\t \u00C2\u00A0substrate\t \u00C2\u00A0load\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011a),\t \u00C2\u00A0thus\t \u00C2\u00A0far\t \u00C2\u00A0saturation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0system\t \u00C2\u00A0has\t \u00C2\u00A0not\t \u00C2\u00A0yet\t \u00C2\u00A0been\t \u00C2\u00A0demonstrated.\t \u00C2\u00A0Our\t \u00C2\u00A0efforts\t \u00C2\u00A0to\t \u00C2\u00A0degrade\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0and\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0clearly\t \u00C2\u00A0show\t \u00C2\u00A0that\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090targeted\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0particular\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0(Chapter\t \u00C2\u00A04.5),\t \u00C2\u00A0and\t \u00C2\u00A0our\t \u00C2\u00A0experiments\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0also\t \u00C2\u00A0show\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0proteins\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0amenable\t \u00C2\u00A0to\t \u00C2\u00A0degradation\t \u00C2\u00A0(Chapter\t \u00C2\u00A04.6).\t \u00C2\u00A0Thus,\t \u00C2\u00A0similar\t \u00C2\u00A0to\t \u00C2\u00A0trans-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0UPS,\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0are\t \u00C2\u00A0capable\t \u00C2\u00A0of\t \u00C2\u00A0specifically\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0complex\t \u00C2\u00A0while\t \u00C2\u00A0leaving\t \u00C2\u00A0the\t \u00C2\u00A0other\t \u00C2\u00A0components\t \u00C2\u00A0intact.\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0currently\t \u00C2\u00A0unclear\t \u00C2\u00A0to\t \u00C2\u00A0what\t \u00C2\u00A0extent\t \u00C2\u00A0 182 SNIPER\t \u00C2\u00A0alters\t \u00C2\u00A0the\t \u00C2\u00A0entire\t \u00C2\u00A0proteome;\t \u00C2\u00A0although\t \u00C2\u00A0a\t \u00C2\u00A0proteomics\t \u00C2\u00A0approach\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0used,\t \u00C2\u00A0such\t \u00C2\u00A0an\t \u00C2\u00A0experiment\t \u00C2\u00A0may\t \u00C2\u00A0not\t \u00C2\u00A0be\t \u00C2\u00A0ideal\t \u00C2\u00A0as\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0impossible\t \u00C2\u00A0to\t \u00C2\u00A0assess\t \u00C2\u00A0the\t \u00C2\u00A0level\t \u00C2\u00A0of\t \u00C2\u00A0every\t \u00C2\u00A0single\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0proteome\t \u00C2\u00A0after\t \u00C2\u00A0SNIPER\t \u00C2\u00A0treatment.\t \u00C2\u00A0A\t \u00C2\u00A0more\t \u00C2\u00A0accessible\t \u00C2\u00A0approach\t \u00C2\u00A0to\t \u00C2\u00A0assess\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0knockdown\t \u00C2\u00A0would\t \u00C2\u00A0be\t \u00C2\u00A0to\t \u00C2\u00A0synthesize\t \u00C2\u00A0multiple\t \u00C2\u00A0SNIPERs\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0different\t \u00C2\u00A0domains\t \u00C2\u00A0on\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0and\t \u00C2\u00A0compare\t \u00C2\u00A0the\t \u00C2\u00A0phenotypic\t \u00C2\u00A0outcome.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0our\t \u00C2\u00A0method\t \u00C2\u00A0is\t \u00C2\u00A0perhaps\t \u00C2\u00A0best\t \u00C2\u00A0illustrated\t \u00C2\u00A0by\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0dephosphorylated\t \u00C2\u00A0(active)\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0phosphorylated\t \u00C2\u00A0(inactive)\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(Chapter\t \u00C2\u00A04.2\t \u00C2\u00A0and\t \u00C2\u00A04.3).\t \u00C2\u00A0The\t \u00C2\u00A0selective\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translationally\t \u00C2\u00A0modified\t \u00C2\u00A0protein\t \u00C2\u00A0variant\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0achievable\t \u00C2\u00A0with\t \u00C2\u00A0gene\t \u00C2\u00A0knockout\t \u00C2\u00A0or\t \u00C2\u00A0RNAi,\t \u00C2\u00A0both\t \u00C2\u00A0of\t \u00C2\u00A0which\t \u00C2\u00A0eliminates\t \u00C2\u00A0all\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translationally\t \u00C2\u00A0modified\t \u00C2\u00A0forms\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0target\t \u00C2\u00A0protein.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0different\t \u00C2\u00A0variants\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0same\t \u00C2\u00A0protein\t \u00C2\u00A0often\t \u00C2\u00A0participate\t \u00C2\u00A0in\t \u00C2\u00A0diverse\t \u00C2\u00A0biological\t \u00C2\u00A0processes,\t \u00C2\u00A0SNIPER\t \u00C2\u00A0may\t \u00C2\u00A0enable\t \u00C2\u00A0the\t \u00C2\u00A0examination\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0protein\u00E2\u0080\u0099s\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0particular\t \u00C2\u00A0cellular\t \u00C2\u00A0pathway\t \u00C2\u00A0by\t \u00C2\u00A0eradicating\t \u00C2\u00A0the\t \u00C2\u00A0responsible\t \u00C2\u00A0variant.\t \u00C2\u00A0\t \u00C2\u00A0Of\t \u00C2\u00A0note,\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0and\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0method\t \u00C2\u00A0relies\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0specific\t \u00C2\u00A0binding\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein,\t \u00C2\u00A0hence\t \u00C2\u00A0identifying\t \u00C2\u00A0a\t \u00C2\u00A0suitable\t \u00C2\u00A0PBD\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0essential\t \u00C2\u00A0step\t \u00C2\u00A0in\t \u00C2\u00A0designing\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide.\t \u00C2\u00A0Peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0obtained\t \u00C2\u00A0through\t \u00C2\u00A0multiple\t \u00C2\u00A0avenues.\t \u00C2\u00A0First,\t \u00C2\u00A0candidate\t \u00C2\u00A0PBDs\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0directly\t \u00C2\u00A0obtained\t \u00C2\u00A0through\t \u00C2\u00A0a\t \u00C2\u00A0simple\t \u00C2\u00A0literature\t \u00C2\u00A0search,\t \u00C2\u00A0as\t \u00C2\u00A0demonstrated\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis.\t \u00C2\u00A0The\t \u00C2\u00A0increasing\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0interference\t \u00C2\u00A0peptides\t \u00C2\u00A0in\t \u00C2\u00A0studies\t \u00C2\u00A0provides\t \u00C2\u00A0a\t \u00C2\u00A0rich\t \u00C2\u00A0resource\t \u00C2\u00A0from\t \u00C2\u00A0which\t \u00C2\u00A0a\t \u00C2\u00A0PBD\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0designed.\t \u00C2\u00A0Second,\t \u00C2\u00A0the\t \u00C2\u00A0PBD\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0identified\t \u00C2\u00A0from\t \u00C2\u00A0known\t \u00C2\u00A0binding\t \u00C2\u00A0partners\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein.\t \u00C2\u00A0Such\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0obtained\t \u00C2\u00A0by\t \u00C2\u00A0searching\t \u00C2\u00A0literature\t \u00C2\u00A0 183 or\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction\t \u00C2\u00A0databases\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0BioGRID.\t \u00C2\u00A0It\t \u00C2\u00A0is\t \u00C2\u00A0then\t \u00C2\u00A0possible\t \u00C2\u00A0to\t \u00C2\u00A0design\t \u00C2\u00A0a\t \u00C2\u00A0custom\t \u00C2\u00A0peptide\t \u00C2\u00A0microarray\t \u00C2\u00A0based\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0binding\t \u00C2\u00A0partner\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0POI\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0identify\t \u00C2\u00A0key\t \u00C2\u00A0stretches\t \u00C2\u00A0of\t \u00C2\u00A0residues\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0interaction.\t \u00C2\u00A0The\t \u00C2\u00A0interaction\t \u00C2\u00A0affinity\t \u00C2\u00A0and\t \u00C2\u00A0dissociation\t \u00C2\u00A0constant\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0measured\t \u00C2\u00A0with\t \u00C2\u00A0Biacore\t \u00C2\u00A0and\t \u00C2\u00A0fluorescence\t \u00C2\u00A0resonance\t \u00C2\u00A0energy\t \u00C2\u00A0transfer,\t \u00C2\u00A0and\t \u00C2\u00A0candidate\t \u00C2\u00A0peptides\t \u00C2\u00A0may\t \u00C2\u00A0then\t \u00C2\u00A0be\t \u00C2\u00A0screened\t \u00C2\u00A0as\t \u00C2\u00A0potential\t \u00C2\u00A0binding\t \u00C2\u00A0partners\t \u00C2\u00A0in\t \u00C2\u00A0vitro.\t \u00C2\u00A0\t \u00C2\u00A0Once\t \u00C2\u00A0a\t \u00C2\u00A0PBD\t \u00C2\u00A0has\t \u00C2\u00A0been\t \u00C2\u00A0identified,\t \u00C2\u00A0additional\t \u00C2\u00A0measures\t \u00C2\u00A0may\t \u00C2\u00A0help\t \u00C2\u00A0enhance\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0and\t \u00C2\u00A0affinity\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interaction.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0incorporating\t \u00C2\u00A0multiple\t \u00C2\u00A0PBD\t \u00C2\u00A0repeats\t \u00C2\u00A0often\t \u00C2\u00A0increases\t \u00C2\u00A0interaction\t \u00C2\u00A0affinity,\t \u00C2\u00A0while\t \u00C2\u00A0separating\t \u00C2\u00A0the\t \u00C2\u00A0PBD\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0CPP\t \u00C2\u00A0and/or\t \u00C2\u00A0CTM\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0glycine-\u00C2\u00AD\u00E2\u0080\u0090serine\t \u00C2\u00A0linker\t \u00C2\u00A0allows\t \u00C2\u00A0for\t \u00C2\u00A0more\t \u00C2\u00A0flexible\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0target\t \u00C2\u00A0protein\t \u00C2\u00A0and\t \u00C2\u00A0chaperones\t \u00C2\u00A0(Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Matsuzawa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02005).\t \u00C2\u00A06.2.4 TOXICITY\t \u00C2\u00A0\t \u00C2\u00A0In\t \u00C2\u00A0contrast\t \u00C2\u00A0to\t \u00C2\u00A0small\t \u00C2\u00A0chemical\t \u00C2\u00A0inhibitors\t \u00C2\u00A0that\t \u00C2\u00A0are\t \u00C2\u00A0often\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0nanomolar\t \u00C2\u00A0range,\t \u00C2\u00A0our\t \u00C2\u00A0synthetic\t \u00C2\u00A0knockdown\t \u00C2\u00A0peptides\t \u00C2\u00A0are\t \u00C2\u00A0dosed\t \u00C2\u00A0between\t \u00C2\u00A05\u00CE\u00BCM\t \u00C2\u00A0and\t \u00C2\u00A050\u00CE\u00BCM\t \u00C2\u00A0for\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0studies\t \u00C2\u00A0and\t \u00C2\u00A010mg/kg\t \u00C2\u00A0for\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0use.\t \u00C2\u00A0Such\t \u00C2\u00A0dosages\t \u00C2\u00A0are\t \u00C2\u00A0in\t \u00C2\u00A0line\t \u00C2\u00A0with\t \u00C2\u00A0those\t \u00C2\u00A0described\t \u00C2\u00A0for\t \u00C2\u00A0interference\t \u00C2\u00A0peptides\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0literature\t \u00C2\u00A0(Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013;\t \u00C2\u00A0Brown\t \u00C2\u00A0and\t \u00C2\u00A0Liu,\t \u00C2\u00A02014).\t \u00C2\u00A0Of\t \u00C2\u00A0note,\t \u00C2\u00A0LDH\t \u00C2\u00A0assay\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0reveal\t \u00C2\u00A0overt\t \u00C2\u00A0toxicity\t \u00C2\u00A024h\t \u00C2\u00A0after\t \u00C2\u00A0treatment\t \u00C2\u00A0in\t \u00C2\u00A0primary\t \u00C2\u00A0cultured\t \u00C2\u00A0neurons\t \u00C2\u00A0(Chapter\t \u00C2\u00A04.6),\t \u00C2\u00A0and\t \u00C2\u00A0H&E\t \u00C2\u00A0staining\t \u00C2\u00A0and\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090Jade\t \u00C2\u00A0B\t \u00C2\u00A0staining\t \u00C2\u00A0in\t \u00C2\u00A0brain\t \u00C2\u00A0slices\t \u00C2\u00A0from\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM-\u00C2\u00AD\u00E2\u0080\u0090treated\t \u00C2\u00A0animals\t \u00C2\u00A0did\t \u00C2\u00A0not\t \u00C2\u00A0display\t \u00C2\u00A0signs\t \u00C2\u00A0of\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0toxicity,\t \u00C2\u00A0i.e.\t \u00C2\u00A0lesions\t \u00C2\u00A0or\t \u00C2\u00A0Fluoro-\u00C2\u00AD\u00E2\u0080\u0090Jade\t \u00C2\u00A0B\t \u00C2\u00A0positive\t \u00C2\u00A0cells,\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090infarcted\t \u00C2\u00A0side\t \u00C2\u00A0(Chapter\t \u00C2\u00A05.4).\t \u00C2\u00A0\t \u00C2\u00A0 184 Although\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0increases\t \u00C2\u00A0CMA\t \u00C2\u00A0load,\t \u00C2\u00A0the\t \u00C2\u00A0treatment\t \u00C2\u00A0does\t \u00C2\u00A0not\t \u00C2\u00A0seem\t \u00C2\u00A0to\t \u00C2\u00A0disrupt\t \u00C2\u00A0the\t \u00C2\u00A0normal\t \u00C2\u00A0turnover\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0CMA\t \u00C2\u00A0substrates,\t \u00C2\u00A0as\t \u00C2\u00A0illustrated\t \u00C2\u00A0by\t \u00C2\u00A0GAPDH\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A04.6.\t \u00C2\u00A0This\t \u00C2\u00A0result\t \u00C2\u00A0is\t \u00C2\u00A0consistent\t \u00C2\u00A0with\t \u00C2\u00A0previous\t \u00C2\u00A0findings\t \u00C2\u00A0that\t \u00C2\u00A0show\t \u00C2\u00A0CTM-\u00C2\u00AD\u00E2\u0080\u0090tagged\t \u00C2\u00A0fluorescent\t \u00C2\u00A0proteins\t \u00C2\u00A0do\t \u00C2\u00A0not\t \u00C2\u00A0disrupt\t \u00C2\u00A0CMA\t \u00C2\u00A0function\t \u00C2\u00A0when\t \u00C2\u00A0introduced\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0(Koga\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011b).\t \u00C2\u00A0It\t \u00C2\u00A0should\t \u00C2\u00A0be\t \u00C2\u00A0noted\t \u00C2\u00A0that\t \u00C2\u00A0CMA\t \u00C2\u00A0activity\t \u00C2\u00A0is\t \u00C2\u00A0upregulated\t \u00C2\u00A0in\t \u00C2\u00A0times\t \u00C2\u00A0of\t \u00C2\u00A0increased\t \u00C2\u00A0substrate\t \u00C2\u00A0load\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0normal\t \u00C2\u00A0physiological\t \u00C2\u00A0response\t \u00C2\u00A0and\t \u00C2\u00A0serves\t \u00C2\u00A0to\t \u00C2\u00A0maintain\t \u00C2\u00A0homeostasis.\t \u00C2\u00A0\t \u00C2\u00A06.3 CLINICAL\t \u00C2\u00A0APPLICABILITY\t \u00C2\u00A0The\t \u00C2\u00A0pathophysiology\t \u00C2\u00A0of\t \u00C2\u00A0many\t \u00C2\u00A0human\t \u00C2\u00A0diseases\t \u00C2\u00A0involves\t \u00C2\u00A0the\t \u00C2\u00A0aberrant\t \u00C2\u00A0accumulation,\t \u00C2\u00A0aggregation\t \u00C2\u00A0and/or\t \u00C2\u00A0activation\t \u00C2\u00A0of\t \u00C2\u00A0normal\t \u00C2\u00A0proteins,\t \u00C2\u00A0or\t \u00C2\u00A0the\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0mutant\t \u00C2\u00A0protein\t \u00C2\u00A0variants.\t \u00C2\u00A0As\t \u00C2\u00A0such,\t \u00C2\u00A0eliminating\t \u00C2\u00A0these\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090related\t \u00C2\u00A0proteins\t \u00C2\u00A0to\t \u00C2\u00A0slow\t \u00C2\u00A0down\t \u00C2\u00A0or\t \u00C2\u00A0reverse\t \u00C2\u00A0disease\t \u00C2\u00A0progression\t \u00C2\u00A0is\t \u00C2\u00A0an\t \u00C2\u00A0attractive\t \u00C2\u00A0strategy\t \u00C2\u00A0for\t \u00C2\u00A0therapy.\t \u00C2\u00A0The\t \u00C2\u00A0experiments\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A05\t \u00C2\u00A0provide\t \u00C2\u00A0proof-\u00C2\u00AD\u00E2\u0080\u0090of-\u00C2\u00AD\u00E2\u0080\u0090concept\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0potential\t \u00C2\u00A0utility\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic\t \u00C2\u00A0approach.\t \u00C2\u00A06.3.1 PEPTIDE\t \u00C2\u00A0DELIVERY\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0agents\t \u00C2\u00A0in\t \u00C2\u00A0vivo\t \u00C2\u00A0has\t \u00C2\u00A0traditionally\t \u00C2\u00A0relied\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0viral\t \u00C2\u00A0vectors.\t \u00C2\u00A0Although\t \u00C2\u00A0impressive\t \u00C2\u00A0advances\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0made\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0field\t \u00C2\u00A0of\t \u00C2\u00A0viral\t \u00C2\u00A0delivery,\t \u00C2\u00A0such\t \u00C2\u00A0strategies\t \u00C2\u00A0are\t \u00C2\u00A0not\t \u00C2\u00A0easily\t \u00C2\u00A0feasible\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0clinical\t \u00C2\u00A0setting.\t \u00C2\u00A0Our\t \u00C2\u00A0approach\t \u00C2\u00A0dramatically\t \u00C2\u00A0differs\t \u00C2\u00A0from\t \u00C2\u00A0these\t \u00C2\u00A0previous\t \u00C2\u00A0approaches\t \u00C2\u00A0in\t \u00C2\u00A0that\t \u00C2\u00A0we\t \u00C2\u00A0incorporated\t \u00C2\u00A0a\t \u00C2\u00A0peptidergic\t \u00C2\u00A0delivery\t \u00C2\u00A0system\t \u00C2\u00A0into\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0to\t \u00C2\u00A0facilitate\t \u00C2\u00A0their\t \u00C2\u00A0entry\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0and\t \u00C2\u00A0cell\t \u00C2\u00A0interior.\t \u00C2\u00A0 185 Specifically,\t \u00C2\u00A0we\t \u00C2\u00A0opted\t \u00C2\u00A0to\t \u00C2\u00A0use\t \u00C2\u00A0several\t \u00C2\u00A0CPPs\t \u00C2\u00A0that\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0repeatedly\t \u00C2\u00A0validated\t \u00C2\u00A0for\t \u00C2\u00A0efficient\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0peptide\t \u00C2\u00A0cargos\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0and\t \u00C2\u00A0in\t \u00C2\u00A0vivo.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0the\t \u00C2\u00A0TAT\t \u00C2\u00A0domain\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0synthetic\t \u00C2\u00A0peptides\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A04\t \u00C2\u00A0was\t \u00C2\u00A0found\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0safe\t \u00C2\u00A0and\t \u00C2\u00A0effective\t \u00C2\u00A0at\t \u00C2\u00A0delivering\t \u00C2\u00A0a\t \u00C2\u00A0conjugate\t \u00C2\u00A0peptide\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0human\t \u00C2\u00A0brain\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0recently\t \u00C2\u00A0completed\t \u00C2\u00A0Phase\t \u00C2\u00A0II\t \u00C2\u00A0clinical\t \u00C2\u00A0trial\t \u00C2\u00A0(Hill\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012).\t \u00C2\u00A0This\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0peptide\t \u00C2\u00A0(TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2B9c\t \u00C2\u00A0or\t \u00C2\u00A0NA-\u00C2\u00AD\u00E2\u0080\u00901)(Aarts\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02002;\t \u00C2\u00A0Cook\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012;\t \u00C2\u00A0Hill\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012),\t \u00C2\u00A0similar\t \u00C2\u00A0in\t \u00C2\u00A0size\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0employed\t \u00C2\u00A0in\t \u00C2\u00A0our\t \u00C2\u00A0studies,\t \u00C2\u00A0is\t \u00C2\u00A0now\t \u00C2\u00A0undergoing\t \u00C2\u00A0a\t \u00C2\u00A0Phase\t \u00C2\u00A0III\t \u00C2\u00A0clinical\t \u00C2\u00A0trial.\t \u00C2\u00A0The\t \u00C2\u00A0success\t \u00C2\u00A0of\t \u00C2\u00A0NA-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0illustrates\t \u00C2\u00A0the\t \u00C2\u00A0feasibility\t \u00C2\u00A0of\t \u00C2\u00A0using\t \u00C2\u00A0TAT\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0peptidergic\t \u00C2\u00A0therapeutics\t \u00C2\u00A0in\t \u00C2\u00A0humans.\t \u00C2\u00A06.3.2 STROKE\t \u00C2\u00A0\t \u00C2\u00A0Stroke\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0third\t \u00C2\u00A0leading\t \u00C2\u00A0cause\t \u00C2\u00A0of\t \u00C2\u00A0death\t \u00C2\u00A0in\t \u00C2\u00A0Canada\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0significant\t \u00C2\u00A0burden\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0Canadian\t \u00C2\u00A0economy.\t \u00C2\u00A0Despite\t \u00C2\u00A0its\t \u00C2\u00A0heavy\t \u00C2\u00A0toll,\t \u00C2\u00A0tissue\t \u00C2\u00A0plasminogen\t \u00C2\u00A0activator\t \u00C2\u00A0(tPA)\t \u00C2\u00A0is\t \u00C2\u00A0the\t \u00C2\u00A0only\t \u00C2\u00A0pharmaceutical\t \u00C2\u00A0that\t \u00C2\u00A0is\t \u00C2\u00A0currently\t \u00C2\u00A0approved\t \u00C2\u00A0for\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0stroke.\t \u00C2\u00A0Unfortunately,\t \u00C2\u00A0the\t \u00C2\u00A0effectiveness\t \u00C2\u00A0of\t \u00C2\u00A0tPA\t \u00C2\u00A0is\t \u00C2\u00A0limited\t \u00C2\u00A0to\t \u00C2\u00A0cases\t \u00C2\u00A0of\t \u00C2\u00A0ischemic\t \u00C2\u00A0stroke\t \u00C2\u00A0(as\t \u00C2\u00A0opposed\t \u00C2\u00A0to\t \u00C2\u00A0hemorrhagic\t \u00C2\u00A0stroke,\t \u00C2\u00A0which\t \u00C2\u00A0contribute\t \u00C2\u00A0to\t \u00C2\u00A0roughly\t \u00C2\u00A020%\t \u00C2\u00A0of\t \u00C2\u00A0all\t \u00C2\u00A0stroke\t \u00C2\u00A0cases)\t \u00C2\u00A0and\t \u00C2\u00A0its\t \u00C2\u00A0therapeutic\t \u00C2\u00A0window\t \u00C2\u00A0is\t \u00C2\u00A0limited\t \u00C2\u00A0to\t \u00C2\u00A04.5\t \u00C2\u00A0hours\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090stroke\t \u00C2\u00A0onset.\t \u00C2\u00A0Due\t \u00C2\u00A0to\t \u00C2\u00A0delays\t \u00C2\u00A0in\t \u00C2\u00A0seeking\t \u00C2\u00A0medical\t \u00C2\u00A0treatment,\t \u00C2\u00A0many\t \u00C2\u00A0patients\t \u00C2\u00A0do\t \u00C2\u00A0not\t \u00C2\u00A0qualify\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0treatment.\t \u00C2\u00A0Given\t \u00C2\u00A0these\t \u00C2\u00A0limitations\t \u00C2\u00A0of\t \u00C2\u00A0tPA,\t \u00C2\u00A0a\t \u00C2\u00A0major\t \u00C2\u00A0goal\t \u00C2\u00A0in\t \u00C2\u00A0designing\t \u00C2\u00A0novel\t \u00C2\u00A0therapeutics\t \u00C2\u00A0for\t \u00C2\u00A0stroke\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0identify\t \u00C2\u00A0effective\t \u00C2\u00A0neuroprotectants\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0wider\t \u00C2\u00A0therapeutic\t \u00C2\u00A0window\t \u00C2\u00A0(Lai\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0Overactivation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0NMDAR\t \u00C2\u00A0triggers\t \u00C2\u00A0rapid\t \u00C2\u00A0Ca2+\t \u00C2\u00A0influx\t \u00C2\u00A0that\t \u00C2\u00A0leads\t \u00C2\u00A0to\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A0following\t \u00C2\u00A0a\t \u00C2\u00A0stroke\t \u00C2\u00A0insult.\t \u00C2\u00A0Yet\t \u00C2\u00A0direct\t \u00C2\u00A0blockade\t \u00C2\u00A0of\t \u00C2\u00A0NMDARs\t \u00C2\u00A0has\t \u00C2\u00A0failed\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0 186 neuroprotective\t \u00C2\u00A0strategy\t \u00C2\u00A0in\t \u00C2\u00A0stroke,\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A0in\t \u00C2\u00A0part\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0intolerable\t \u00C2\u00A0psychosomatic\t \u00C2\u00A0side\t \u00C2\u00A0effects\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0result\t \u00C2\u00A0of\t \u00C2\u00A0blocking\t \u00C2\u00A0normal\t \u00C2\u00A0NMDAR\t \u00C2\u00A0function\t \u00C2\u00A0and/or\t \u00C2\u00A0relatively\t \u00C2\u00A0narrow\t \u00C2\u00A0windows\t \u00C2\u00A0for\t \u00C2\u00A0effective\t \u00C2\u00A0intervention\t \u00C2\u00A0(Ikonomidou\t \u00C2\u00A0and\t \u00C2\u00A0Turski,\t \u00C2\u00A02002;\t \u00C2\u00A0Lai\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011;\t \u00C2\u00A02014).\t \u00C2\u00A0An\t \u00C2\u00A0emerging\t \u00C2\u00A0approach\t \u00C2\u00A0in\t \u00C2\u00A0designing\t \u00C2\u00A0neuroprotectants\t \u00C2\u00A0for\t \u00C2\u00A0stroke\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0focus\t \u00C2\u00A0on\t \u00C2\u00A0key\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090signaling\t \u00C2\u00A0proteins\t \u00C2\u00A0and\t \u00C2\u00A0druggable\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0interactions\t \u00C2\u00A0downstream\t \u00C2\u00A0of\t \u00C2\u00A0NMDAR\t \u00C2\u00A0activation\t \u00C2\u00A0that\t \u00C2\u00A0specifically\t \u00C2\u00A0lead\t \u00C2\u00A0to\t \u00C2\u00A0neuronal\t \u00C2\u00A0death\t \u00C2\u00A0(Bonny\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001;\t \u00C2\u00A0Borsello\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003;\t \u00C2\u00A0Zhang\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013;\t \u00C2\u00A0Tu\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Taghibiglou\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A0Tymianski,\t \u00C2\u00A02013;\t \u00C2\u00A0Lai\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0\t \u00C2\u00A0As\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0detail\t \u00C2\u00A0in\t \u00C2\u00A0Chapter\t \u00C2\u00A05.4,\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090GluN2BCTM\t \u00C2\u00A0reduced\t \u00C2\u00A0stroke\t \u00C2\u00A0damage\t \u00C2\u00A0when\t \u00C2\u00A0given\t \u00C2\u00A02h\t \u00C2\u00A0after\t \u00C2\u00A0MCAo\t \u00C2\u00A0onset\t \u00C2\u00A0by\t \u00C2\u00A0specifically\t \u00C2\u00A0knocking\t \u00C2\u00A0down\t \u00C2\u00A0activated\t \u00C2\u00A0DAPK1\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0infarct\t \u00C2\u00A0side\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0rat\t \u00C2\u00A0brain.\t \u00C2\u00A0This\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0prophylactic\t \u00C2\u00A0treatment\t \u00C2\u00A0is\t \u00C2\u00A0not\t \u00C2\u00A0required\t \u00C2\u00A0to\t \u00C2\u00A0realize\t \u00C2\u00A0the\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0effects\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activation\t \u00C2\u00A0occurs\t \u00C2\u00A0downstream\t \u00C2\u00A0of\t \u00C2\u00A0NMDAR\t \u00C2\u00A0activation,\t \u00C2\u00A0strategies\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0DAPK1\t \u00C2\u00A0(as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0other\t \u00C2\u00A0key\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090signaling\t \u00C2\u00A0proteins)\t \u00C2\u00A0have\t \u00C2\u00A0a\t \u00C2\u00A0wider\t \u00C2\u00A0therapeutic\t \u00C2\u00A0window\t \u00C2\u00A0than\t \u00C2\u00A0the\t \u00C2\u00A0classical\t \u00C2\u00A0approach\t \u00C2\u00A0of\t \u00C2\u00A0inhibiting\t \u00C2\u00A0NMDARs.\t \u00C2\u00A0Indeed,\t \u00C2\u00A0inhibiting\t \u00C2\u00A0DAPK1\t \u00C2\u00A0activity\t \u00C2\u00A06h\t \u00C2\u00A0after\t \u00C2\u00A0brain\t \u00C2\u00A0injury\t \u00C2\u00A0was\t \u00C2\u00A0found\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0in\t \u00C2\u00A0rats\t \u00C2\u00A0one\t \u00C2\u00A0week\t \u00C2\u00A0later\t \u00C2\u00A0(Velentza\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0Together,\t \u00C2\u00A0these\t \u00C2\u00A0data\t \u00C2\u00A0point\t \u00C2\u00A0to\t \u00C2\u00A0DAPK1\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0key\t \u00C2\u00A0target\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0intervention\t \u00C2\u00A0of\t \u00C2\u00A0neurotoxicity\t \u00C2\u00A0and\t \u00C2\u00A0illustrate\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090promoting\t \u00C2\u00A0proteins\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A0avenue\t \u00C2\u00A0to\t \u00C2\u00A0develop\t \u00C2\u00A0novel\t \u00C2\u00A0therapeutics\t \u00C2\u00A0for\t \u00C2\u00A0stroke.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 187 6.3.3 APPLICATION\t \u00C2\u00A0AND\t \u00C2\u00A0SAFETY\t \u00C2\u00A0\t \u00C2\u00A0Compared\t \u00C2\u00A0to\t \u00C2\u00A0small\t \u00C2\u00A0chemical\t \u00C2\u00A0drugs,\t \u00C2\u00A0peptidergic\t \u00C2\u00A0therapeutics\t \u00C2\u00A0are\t \u00C2\u00A0limited\t \u00C2\u00A0in\t \u00C2\u00A0their\t \u00C2\u00A0routes\t \u00C2\u00A0of\t \u00C2\u00A0administration\t \u00C2\u00A0and\t \u00C2\u00A0have\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0immunorejection.\t \u00C2\u00A0Nevertheless,\t \u00C2\u00A0these\t \u00C2\u00A0limitations\t \u00C2\u00A0should\t \u00C2\u00A0not\t \u00C2\u00A0necessarily\t \u00C2\u00A0deter\t \u00C2\u00A0their\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0clinical\t \u00C2\u00A0setting.\t \u00C2\u00A0Although\t \u00C2\u00A0peptidergic\t \u00C2\u00A0drugs\t \u00C2\u00A0are\t \u00C2\u00A0most\t \u00C2\u00A0commonly\t \u00C2\u00A0administered\t \u00C2\u00A0intravenously\t \u00C2\u00A0to\t \u00C2\u00A0minimize\t \u00C2\u00A0proteolytic\t \u00C2\u00A0degradation,\t \u00C2\u00A0they\t \u00C2\u00A0can\t \u00C2\u00A0also\t \u00C2\u00A0be\t \u00C2\u00A0delivered\t \u00C2\u00A0via\t \u00C2\u00A0an\t \u00C2\u00A0intranasal\t \u00C2\u00A0(i.n.)\t \u00C2\u00A0route.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0an\t \u00C2\u00A0interference\t \u00C2\u00A0peptide\t \u00C2\u00A0that\t \u00C2\u00A0disrupts\t \u00C2\u00A0D1\t \u00C2\u00A0and\t \u00C2\u00A0D2\t \u00C2\u00A0dopamine\t \u00C2\u00A0receptor\t \u00C2\u00A0interaction\t \u00C2\u00A0was\t \u00C2\u00A0found\t \u00C2\u00A0to\t \u00C2\u00A0alleviate\t \u00C2\u00A0depression-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0symptoms\t \u00C2\u00A0when\t \u00C2\u00A0administered\t \u00C2\u00A0i.n.\t \u00C2\u00A0in\t \u00C2\u00A0rats\t \u00C2\u00A0(Brown\t \u00C2\u00A0and\t \u00C2\u00A0Liu,\t \u00C2\u00A02014).\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0there\t \u00C2\u00A0is\t \u00C2\u00A0considerable\t \u00C2\u00A0interest\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0peptide\t \u00C2\u00A0formulations\t \u00C2\u00A0that\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0administered\t \u00C2\u00A0through\t \u00C2\u00A0oral\t \u00C2\u00A0routes,\t \u00C2\u00A0and\t \u00C2\u00A0advancements\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0made\t \u00C2\u00A0(Gupta\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0For\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0acute\t \u00C2\u00A0diseases\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0stroke,\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0intravenous\t \u00C2\u00A0dose\t \u00C2\u00A0is\t \u00C2\u00A0sufficient\t \u00C2\u00A0to\t \u00C2\u00A0achieve\t \u00C2\u00A0substantial\t \u00C2\u00A0neuroprotection,\t \u00C2\u00A0thus\t \u00C2\u00A0minimizing\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0activating\t \u00C2\u00A0the\t \u00C2\u00A0host\t \u00C2\u00A0immune\t \u00C2\u00A0response.\t \u00C2\u00A0The\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0immunorejection\t \u00C2\u00A0is\t \u00C2\u00A0higher\t \u00C2\u00A0with\t \u00C2\u00A0chronic\t \u00C2\u00A0treatment,\t \u00C2\u00A0for\t \u00C2\u00A0example\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0case\t \u00C2\u00A0of\t \u00C2\u00A0treating\t \u00C2\u00A0protein\t \u00C2\u00A0accumulation\t \u00C2\u00A0disorders\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0HD\t \u00C2\u00A0or\t \u00C2\u00A0PD.\t \u00C2\u00A0However,\t \u00C2\u00A0peptides\t \u00C2\u00A0are\t \u00C2\u00A0only\t \u00C2\u00A0weakly\t \u00C2\u00A0immunogenic\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0protein\t \u00C2\u00A0drugs,\t \u00C2\u00A0and\t \u00C2\u00A0modifications\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0using\t \u00C2\u00A0D-\u00C2\u00AD\u00E2\u0080\u0090amino\t \u00C2\u00A0acids\t \u00C2\u00A0or\t \u00C2\u00A0adding\t \u00C2\u00A0polyethylene\t \u00C2\u00A0glycol\t \u00C2\u00A0(PEG)\t \u00C2\u00A0chains\t \u00C2\u00A0further\t \u00C2\u00A0reduce\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0triggering\t \u00C2\u00A0allergic\t \u00C2\u00A0reactions\t \u00C2\u00A0(Bellmann-\u00C2\u00AD\u00E2\u0080\u0090Sickert\t \u00C2\u00A0and\t \u00C2\u00A0Beck-\u00C2\u00AD\u00E2\u0080\u0090Sickinger,\t \u00C2\u00A02010).\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0degradation\t \u00C2\u00A0products\t \u00C2\u00A0of\t \u00C2\u00A0peptides\t \u00C2\u00A0are\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090toxic\t \u00C2\u00A0amino\t \u00C2\u00A0acids,\t \u00C2\u00A0which\t \u00C2\u00A0minimizes\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0drug-\u00C2\u00AD\u00E2\u0080\u0090drug\t \u00C2\u00A0interactions\t \u00C2\u00A0as\t \u00C2\u00A0well\t \u00C2\u00A0as\t \u00C2\u00A0systemic\t \u00C2\u00A0toxicity.\t \u00C2\u00A0Furthermore,\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0their\t \u00C2\u00A0 188 relatively\t \u00C2\u00A0short\t \u00C2\u00A0half-\u00C2\u00AD\u00E2\u0080\u0090life,\t \u00C2\u00A0peptides\t \u00C2\u00A0rarely\t \u00C2\u00A0accumulate\t \u00C2\u00A0in\t \u00C2\u00A0tissues\t \u00C2\u00A0and\t \u00C2\u00A0thereby\t \u00C2\u00A0reduce\t \u00C2\u00A0the\t \u00C2\u00A0risk\t \u00C2\u00A0of\t \u00C2\u00A0complications\t \u00C2\u00A0caused\t \u00C2\u00A0by\t \u00C2\u00A0their\t \u00C2\u00A0metabolites\t \u00C2\u00A0(Vlieghe\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A06.4 POTENTIAL\t \u00C2\u00A0LIMITATIONS\t \u00C2\u00A0AND\t \u00C2\u00A0FUTURE\t \u00C2\u00A0DIRECTIONS\t \u00C2\u00A06.4.1 PEPTIDE\t \u00C2\u00A0STABILITY\t \u00C2\u00A0\t \u00C2\u00A0Perhaps\t \u00C2\u00A0the\t \u00C2\u00A0largest\t \u00C2\u00A0deterrent\t \u00C2\u00A0in\t \u00C2\u00A0using\t \u00C2\u00A0peptides\t \u00C2\u00A0as\t \u00C2\u00A0drug\t \u00C2\u00A0candidates\t \u00C2\u00A0is\t \u00C2\u00A0their\t \u00C2\u00A0metabolic\t \u00C2\u00A0instability,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0due\t \u00C2\u00A0in\t \u00C2\u00A0part\t \u00C2\u00A0to\t \u00C2\u00A0high\t \u00C2\u00A0biodegradability\t \u00C2\u00A0by\t \u00C2\u00A0gastrointestinal,\t \u00C2\u00A0plasma\t \u00C2\u00A0and\t \u00C2\u00A0tissue\t \u00C2\u00A0peptidases\t \u00C2\u00A0(Mason,\t \u00C2\u00A02010;\t \u00C2\u00A0Vlieghe\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010).\t \u00C2\u00A0Most\t \u00C2\u00A0peptides\t \u00C2\u00A0have\t \u00C2\u00A0a\t \u00C2\u00A0half-\u00C2\u00AD\u00E2\u0080\u0090life\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0few\t \u00C2\u00A0hours\t \u00C2\u00A0at\t \u00C2\u00A0best.\t \u00C2\u00A0Nevertheless,\t \u00C2\u00A0advancements\t \u00C2\u00A0in\t \u00C2\u00A0peptide\t \u00C2\u00A0technologies\t \u00C2\u00A0have\t \u00C2\u00A0identified\t \u00C2\u00A0many\t \u00C2\u00A0chemical\t \u00C2\u00A0optimizations\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0resistance\t \u00C2\u00A0against\t \u00C2\u00A0proteolysis,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0the\t \u00C2\u00A0use\t \u00C2\u00A0of\t \u00C2\u00A0cyclic\t \u00C2\u00A0peptides,\t \u00C2\u00A0pseudo\t \u00C2\u00A0peptides,\t \u00C2\u00A0peptides\t \u00C2\u00A0formed\t \u00C2\u00A0from\t \u00C2\u00A0unnatural\t \u00C2\u00A0amino\t \u00C2\u00A0acids,\t \u00C2\u00A0and\t \u00C2\u00A0retro-\u00C2\u00AD\u00E2\u0080\u0090inverso\t \u00C2\u00A0peptides\t \u00C2\u00A0(Gentilucci\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02010;\t \u00C2\u00A0Craik\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0One\t \u00C2\u00A0potential\t \u00C2\u00A0means\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0intracellular\t \u00C2\u00A0peptide\t \u00C2\u00A0levels\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0enhance\t \u00C2\u00A0its\t \u00C2\u00A0delivery\t \u00C2\u00A0into\t \u00C2\u00A0cells\t \u00C2\u00A0and\t \u00C2\u00A0increase\t \u00C2\u00A0retainability\t \u00C2\u00A0once\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0is\t \u00C2\u00A0inside\t \u00C2\u00A0the\t \u00C2\u00A0cell.\t \u00C2\u00A0The\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090domain\t \u00C2\u00A0allows\t \u00C2\u00A0its\t \u00C2\u00A0conjugated\t \u00C2\u00A0cargo\t \u00C2\u00A0to\t \u00C2\u00A0penetrate\t \u00C2\u00A0into\t \u00C2\u00A0and\t \u00C2\u00A0out\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0until\t \u00C2\u00A0a\t \u00C2\u00A0chemical\t \u00C2\u00A0equilibrium\t \u00C2\u00A0is\t \u00C2\u00A0established.\t \u00C2\u00A0However,\t \u00C2\u00A0the\t \u00C2\u00A0bidirectional\t \u00C2\u00A0movement\t \u00C2\u00A0of\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0peptides\t \u00C2\u00A0should\t \u00C2\u00A0not\t \u00C2\u00A0deter\t \u00C2\u00A0its\t \u00C2\u00A0accumulation\t \u00C2\u00A0inside\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0because\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0creates\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A0gradient\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0allows\t \u00C2\u00A0more\t \u00C2\u00A0to\t \u00C2\u00A0penetrate\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cell.\t \u00C2\u00A0Nevertheless,\t \u00C2\u00A0the\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090domain\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0rest\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide\t \u00C2\u00A0via\t \u00C2\u00A0a\t \u00C2\u00A0disulfide\t \u00C2\u00A0bound,\t \u00C2\u00A0which\t \u00C2\u00A0is\t \u00C2\u00A0generally\t \u00C2\u00A0cleaved\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0reducing\t \u00C2\u00A0environment\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0cytosol\t \u00C2\u00A0and\t \u00C2\u00A0thus\t \u00C2\u00A0traps\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide\t \u00C2\u00A0inside\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0(Saito\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02003).\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0peptides\t \u00C2\u00A0enter\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0through\t \u00C2\u00A0both\t \u00C2\u00A0direct\t \u00C2\u00A0 189 penetration\t \u00C2\u00A0and\t \u00C2\u00A0endocytosis\t \u00C2\u00A0(Patel\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02007).\t \u00C2\u00A0In\t \u00C2\u00A0the\t \u00C2\u00A0latter\t \u00C2\u00A0case,\t \u00C2\u00A0escape\t \u00C2\u00A0from\t \u00C2\u00A0endosomes\t \u00C2\u00A0is\t \u00C2\u00A0necessary\t \u00C2\u00A0to\t \u00C2\u00A0avoid\t \u00C2\u00A0direct\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0lysosome.\t \u00C2\u00A0Although\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0peptides\t \u00C2\u00A0are\t \u00C2\u00A0normally\t \u00C2\u00A0released\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0cytosol\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0lowering\t \u00C2\u00A0pH\t \u00C2\u00A0of\t \u00C2\u00A0endosomes\t \u00C2\u00A0as\t \u00C2\u00A0they\t \u00C2\u00A0mature,\t \u00C2\u00A0the\t \u00C2\u00A0addition\t \u00C2\u00A0of\t \u00C2\u00A0endosome\t \u00C2\u00A0disrupters,\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0HA2,\t \u00C2\u00A0may\t \u00C2\u00A0further\t \u00C2\u00A0enhance\t \u00C2\u00A0endosomal\t \u00C2\u00A0escape\t \u00C2\u00A0(Patel\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02007).\t \u00C2\u00A0Another\t \u00C2\u00A0potential\t \u00C2\u00A0means\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0the\t \u00C2\u00A0stability\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0mask\t \u00C2\u00A0its\t \u00C2\u00A0CTM\t \u00C2\u00A0domain,\t \u00C2\u00A0so\t \u00C2\u00A0that\t \u00C2\u00A0they\t \u00C2\u00A0are\t \u00C2\u00A0no\t \u00C2\u00A0longer\t \u00C2\u00A0recognized\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0CMA\t \u00C2\u00A0system\t \u00C2\u00A0as\t \u00C2\u00A0substrates.\t \u00C2\u00A0One\t \u00C2\u00A0potential\t \u00C2\u00A0strategy\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0make\t \u00C2\u00A0a\t \u00C2\u00A0cryptic\t \u00C2\u00A0CTM,\t \u00C2\u00A0which\t \u00C2\u00A0lacks\t \u00C2\u00A0a\t \u00C2\u00A0necessary\t \u00C2\u00A0residue\t \u00C2\u00A0that\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0provided\t \u00C2\u00A0through\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modification.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0can\t \u00C2\u00A0provide\t \u00C2\u00A0a\t \u00C2\u00A0missing\t \u00C2\u00A0negative\t \u00C2\u00A0charge,\t \u00C2\u00A0while\t \u00C2\u00A0acetylation\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0lysine\t \u00C2\u00A0residue\t \u00C2\u00A0can\t \u00C2\u00A0provide\t \u00C2\u00A0a\t \u00C2\u00A0missing\t \u00C2\u00A0glutamine.\t \u00C2\u00A0However,\t \u00C2\u00A0this\t \u00C2\u00A0approach\t \u00C2\u00A0requires\t \u00C2\u00A0the\t \u00C2\u00A0modified\t \u00C2\u00A0peptide\t \u00C2\u00A0to\t \u00C2\u00A0contain\t \u00C2\u00A0specific\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0modification\t \u00C2\u00A0sites\t \u00C2\u00A0for\t \u00C2\u00A0enzymes\t \u00C2\u00A0to\t \u00C2\u00A0target,\t \u00C2\u00A0which\t \u00C2\u00A0may\t \u00C2\u00A0increase\t \u00C2\u00A0the\t \u00C2\u00A0size\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0peptide.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0Another\t \u00C2\u00A0strategy\t \u00C2\u00A0to\t \u00C2\u00A0increase\t \u00C2\u00A0stability\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0add\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0cap\t \u00C2\u00A0that\t \u00C2\u00A0temporarily\t \u00C2\u00A0masks\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0until\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0released.\t \u00C2\u00A0Such\t \u00C2\u00A0an\t \u00C2\u00A0approach\t \u00C2\u00A0has\t \u00C2\u00A0previously\t \u00C2\u00A0been\t \u00C2\u00A0demonstrated\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0crytic\t \u00C2\u00A0proteasome-\u00C2\u00AD\u00E2\u0080\u0090targeting\t \u00C2\u00A0degron\t \u00C2\u00A0(Bonger\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02011,\t \u00C2\u00A02014).\t \u00C2\u00A0One\t \u00C2\u00A0advantage\t \u00C2\u00A0with\t \u00C2\u00A0this\t \u00C2\u00A0approach\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0release\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0controlled,\t \u00C2\u00A0so\t \u00C2\u00A0that\t \u00C2\u00A0it\t \u00C2\u00A0is\t \u00C2\u00A0limited\t \u00C2\u00A0to\t \u00C2\u00A0certain\t \u00C2\u00A0disease\t \u00C2\u00A0states.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0if\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0cap\t \u00C2\u00A0is\t \u00C2\u00A0removed\t \u00C2\u00A0by\t \u00C2\u00A0calpain\t \u00C2\u00A0cleavage,\t \u00C2\u00A0the\t \u00C2\u00A0CTM\t \u00C2\u00A0is\t \u00C2\u00A0only\t \u00C2\u00A0released\t \u00C2\u00A0when\t \u00C2\u00A0calpain\t \u00C2\u00A0is\t \u00C2\u00A0activated\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0for\t \u00C2\u00A0example,\t \u00C2\u00A0under\t \u00C2\u00A0conditions\t \u00C2\u00A0such\t \u00C2\u00A0as\t \u00C2\u00A0stroke\t \u00C2\u00A0(Neumar\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02001).\t \u00C2\u00A0\t \u00C2\u00A0 190 6.4.2 TARGETED\t \u00C2\u00A0DELIVERY\t \u00C2\u00A0\t \u00C2\u00A0A\t \u00C2\u00A0major\t \u00C2\u00A0drawback\t \u00C2\u00A0of\t \u00C2\u00A0using\t \u00C2\u00A0TAT\t \u00C2\u00A0is\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0CPP\t \u00C2\u00A0cannot\t \u00C2\u00A0specifically\t \u00C2\u00A0target\t \u00C2\u00A0certain\t \u00C2\u00A0tissues\t \u00C2\u00A0and/or\t \u00C2\u00A0cell\t \u00C2\u00A0types\t \u00C2\u00A0for\t \u00C2\u00A0delivery.\t \u00C2\u00A0For\t \u00C2\u00A0use\t \u00C2\u00A0in\t \u00C2\u00A0basic\t \u00C2\u00A0research\t \u00C2\u00A0in\t \u00C2\u00A0vivo,\t \u00C2\u00A0this\t \u00C2\u00A0limitation\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0circumvented\t \u00C2\u00A0by\t \u00C2\u00A0packaging\t \u00C2\u00A0a\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090expressing\t \u00C2\u00A0plasmid\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0viral\t \u00C2\u00A0vector,\t \u00C2\u00A0and\t \u00C2\u00A0controlling\t \u00C2\u00A0the\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0plasmid\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0specific\t \u00C2\u00A0and/or\t \u00C2\u00A0inducible\t \u00C2\u00A0promoter.\t \u00C2\u00A0As\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0occurs\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0post-\u00C2\u00AD\u00E2\u0080\u0090translational\t \u00C2\u00A0level,\t \u00C2\u00A0SNIPER\t \u00C2\u00A0would\t \u00C2\u00A0retain\t \u00C2\u00A0its\t \u00C2\u00A0highly\t \u00C2\u00A0specific\t \u00C2\u00A0and\t \u00C2\u00A0rapid\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0target\t \u00C2\u00A0proteins.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0Tissue-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0or\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0specific\t \u00C2\u00A0targeting\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0clinical\t \u00C2\u00A0setting\t \u00C2\u00A0is\t \u00C2\u00A0more\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0challenge\t \u00C2\u00A0(Lalatsa\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02014).\t \u00C2\u00A0Previous\t \u00C2\u00A0efforts\t \u00C2\u00A0have\t \u00C2\u00A0mostly\t \u00C2\u00A0focused\t \u00C2\u00A0on\t \u00C2\u00A0tumour-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0delivery,\t \u00C2\u00A0by\t \u00C2\u00A0utilizing\t \u00C2\u00A0the\t \u00C2\u00A0pathological\t \u00C2\u00A0local\t \u00C2\u00A0milieu.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0one\t \u00C2\u00A0group\t \u00C2\u00A0utilized\t \u00C2\u00A0the\t \u00C2\u00A0hypoxic\t \u00C2\u00A0environment\t \u00C2\u00A0of\t \u00C2\u00A0tumours\t \u00C2\u00A0by\t \u00C2\u00A0incorporating\t \u00C2\u00A0an\t \u00C2\u00A0oxygen\t \u00C2\u00A0dependent\t \u00C2\u00A0domain\t \u00C2\u00A0(ODD)\t \u00C2\u00A0of\t \u00C2\u00A0hypoxia\t \u00C2\u00A0inducible\t \u00C2\u00A0factor\t \u00C2\u00A0(HIF)\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0TAT-\u00C2\u00AD\u00E2\u0080\u0090linked\t \u00C2\u00A0caspase\t \u00C2\u00A0protein.\t \u00C2\u00A0ODD\t \u00C2\u00A0stabilizes\t \u00C2\u00A0the\t \u00C2\u00A0fusion\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0tumour\t \u00C2\u00A0environment\t \u00C2\u00A0allowing\t \u00C2\u00A0its\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090death\t \u00C2\u00A0function,\t \u00C2\u00A0but\t \u00C2\u00A0degrades\t \u00C2\u00A0it\t \u00C2\u00A0in\t \u00C2\u00A0normal\t \u00C2\u00A0tissues\t \u00C2\u00A0(Inoue\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02004).\t \u00C2\u00A0The\t \u00C2\u00A0recent\t \u00C2\u00A0identification\t \u00C2\u00A0of\t \u00C2\u00A0tumour-\u00C2\u00AD\u00E2\u0080\u0090selective\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090penetrating\t \u00C2\u00A0peptides\t \u00C2\u00A0suggests\t \u00C2\u00A0that\t \u00C2\u00A0targeted\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090knockdown\t \u00C2\u00A0peptides\t \u00C2\u00A0may\t \u00C2\u00A0be\t \u00C2\u00A0feasible,\t \u00C2\u00A0at\t \u00C2\u00A0least\t \u00C2\u00A0for\t \u00C2\u00A0some\t \u00C2\u00A0cell\t \u00C2\u00A0lineages.\t \u00C2\u00A0For\t \u00C2\u00A0example,\t \u00C2\u00A0the\t \u00C2\u00A0cyclic\t \u00C2\u00A0peptide\t \u00C2\u00A0iRGD\t \u00C2\u00A0only\t \u00C2\u00A0penetrates\t \u00C2\u00A0cells\t \u00C2\u00A0with\t \u00C2\u00A0\u00CE\u00B1v\t \u00C2\u00A0intergrins\t \u00C2\u00A0and\t \u00C2\u00A0neurophilin-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0receptors\t \u00C2\u00A0(such\t \u00C2\u00A0as\t \u00C2\u00A0certain\t \u00C2\u00A0prostate\t \u00C2\u00A0cancer\t \u00C2\u00A0cells)\t \u00C2\u00A0(Sugahara\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02009;\t \u00C2\u00A02010),\t \u00C2\u00A0while\t \u00C2\u00A0Xentry\t \u00C2\u00A0permeates\t \u00C2\u00A0only\t \u00C2\u00A0syndecan-\u00C2\u00AD\u00E2\u0080\u0090expressing\t \u00C2\u00A0adherent\t \u00C2\u00A0cells\t \u00C2\u00A0(Montrose\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02013).\t \u00C2\u00A0Many\t \u00C2\u00A0other\t \u00C2\u00A0tumour-\u00C2\u00AD\u00E2\u0080\u0090lineage\t \u00C2\u00A0specific\t \u00C2\u00A0CPPs\t \u00C2\u00A0have\t \u00C2\u00A0been\t \u00C2\u00A0identified\t \u00C2\u00A0(Kondo\t \u00C2\u00A0et\t \u00C2\u00A0al.,\t \u00C2\u00A02012),\t \u00C2\u00A0 191 and\t \u00C2\u00A0high-\u00C2\u00AD\u00E2\u0080\u0090throughput\t \u00C2\u00A0phage\t \u00C2\u00A0display\t \u00C2\u00A0technology\t \u00C2\u00A0may\t \u00C2\u00A0aid\t \u00C2\u00A0the\t \u00C2\u00A0discovery\t \u00C2\u00A0of\t \u00C2\u00A0other\t \u00C2\u00A0novel\t \u00C2\u00A0tissue-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0and\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090selective\t \u00C2\u00A0CPPs\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0delivery.\t \u00C2\u00A0\t \u00C2\u00A06.5 CONCLUDING\t \u00C2\u00A0REMARKS\t \u00C2\u00A0A\t \u00C2\u00A0major\t \u00C2\u00A0goal\t \u00C2\u00A0in\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0is\t \u00C2\u00A0to\t \u00C2\u00A0develop\t \u00C2\u00A0methods\t \u00C2\u00A0that\t \u00C2\u00A0rapidly,\t \u00C2\u00A0reversibly\t \u00C2\u00A0and\t \u00C2\u00A0specifically\t \u00C2\u00A0eliminate\t \u00C2\u00A0a\t \u00C2\u00A0given\t \u00C2\u00A0POI.\t \u00C2\u00A0The\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090facilitated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0method\t \u00C2\u00A0(SNIPER)\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0dissertation\t \u00C2\u00A0provides\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0tool\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0research\t \u00C2\u00A0community\t \u00C2\u00A0to\t \u00C2\u00A0examine\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0functions\t \u00C2\u00A0of\t \u00C2\u00A0native\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0situ\t \u00C2\u00A0and\t \u00C2\u00A0to\t \u00C2\u00A0potentially\t \u00C2\u00A0develop\t \u00C2\u00A0novel\t \u00C2\u00A0therapeutics\t \u00C2\u00A0that\t \u00C2\u00A0aim\t \u00C2\u00A0to\t \u00C2\u00A0eliminate\t \u00C2\u00A0disease-\u00C2\u00AD\u00E2\u0080\u0090promoting\t \u00C2\u00A0proteins.\t \u00C2\u00A0The\t \u00C2\u00A0protocols\t \u00C2\u00A0developed\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0lay\t \u00C2\u00A0out\t \u00C2\u00A0a\t \u00C2\u00A0roadmap\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0rapid\t \u00C2\u00A0screening\t \u00C2\u00A0of\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptides\t \u00C2\u00A0in\t \u00C2\u00A0cells\t \u00C2\u00A0lines\t \u00C2\u00A0(Chapter\t \u00C2\u00A03),\t \u00C2\u00A0the\t \u00C2\u00A0assessment\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0candidate\t \u00C2\u00A0peptides\t \u00C2\u00A0in\t \u00C2\u00A0primary\t \u00C2\u00A0cultured\t \u00C2\u00A0cells\t \u00C2\u00A0(Chapter\t \u00C2\u00A04),\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0verification\t \u00C2\u00A0of\t \u00C2\u00A0their\t \u00C2\u00A0use\t \u00C2\u00A0as\t \u00C2\u00A0therapeutics\t \u00C2\u00A0in\t \u00C2\u00A0animal\t \u00C2\u00A0models\t \u00C2\u00A0of\t \u00C2\u00A0diseases\t \u00C2\u00A0(Chapter\t \u00C2\u00A05).\t \u00C2\u00A0\t \u00C2\u00A0SNIPER\t \u00C2\u00A0not\t \u00C2\u00A0only\t \u00C2\u00A0has\t \u00C2\u00A0potential\t \u00C2\u00A0for\t \u00C2\u00A0propelling\t \u00C2\u00A0research\t \u00C2\u00A0in\t \u00C2\u00A0neuroscience,\t \u00C2\u00A0but\t \u00C2\u00A0may\t \u00C2\u00A0also\t \u00C2\u00A0impact\t \u00C2\u00A0other\t \u00C2\u00A0biomedical\t \u00C2\u00A0research\t \u00C2\u00A0fields.\t \u00C2\u00A0Given\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0system\t \u00C2\u00A0is\t \u00C2\u00A0highly\t \u00C2\u00A0conserved\t \u00C2\u00A0in\t \u00C2\u00A0mammals,\t \u00C2\u00A0the\t \u00C2\u00A0SNIPER\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0method\t \u00C2\u00A0described\t \u00C2\u00A0in\t \u00C2\u00A0this\t \u00C2\u00A0thesis\t \u00C2\u00A0may\t \u00C2\u00A0potentially\t \u00C2\u00A0be\t \u00C2\u00A0used\t \u00C2\u00A0in\t \u00C2\u00A0experimental\t \u00C2\u00A0animals\t \u00C2\u00A0for\t \u00C2\u00A0which\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0is\t \u00C2\u00A0difficult\t \u00C2\u00A0to\t \u00C2\u00A0achieve\t \u00C2\u00A0with\t \u00C2\u00A0conventional\t \u00C2\u00A0genetic\t \u00C2\u00A0knockout.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 192 REFERENCES\t \u00C2\u00A0 Aarts\t \u00C2\u00A0M,\t \u00C2\u00A0Liu\t \u00C2\u00A0Y,\t \u00C2\u00A0Liu\t \u00C2\u00A0L,\t \u00C2\u00A0Besshoh\t \u00C2\u00A0S,\t \u00C2\u00A0Arundine\t \u00C2\u00A0M,\t \u00C2\u00A0Gurd\t \u00C2\u00A0JW,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT,\t \u00C2\u00A0Salter\t \u00C2\u00A0MW,\t \u00C2\u00A0Tymianski\t \u00C2\u00A0M\t \u00C2\u00A0(2002)\t \u00C2\u00A0Treatment\t \u00C2\u00A0of\t \u00C2\u00A0ischemic\t \u00C2\u00A0brain\t \u00C2\u00A0damage\t \u00C2\u00A0by\t \u00C2\u00A0perturbing\t \u00C2\u00A0NMDA\t \u00C2\u00A0receptor-\u00C2\u00AD\u00E2\u0080\u0090PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0protein\t \u00C2\u00A0interactions.\t \u00C2\u00A0Science\t \u00C2\u00A0298:846-\u00C2\u00AD\u00E2\u0080\u0090850.\t \u00C2\u00A0Agarraberes\t \u00C2\u00A0FA,\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(2001)\t \u00C2\u00A0A\t \u00C2\u00A0molecular\t \u00C2\u00A0chaperone\t \u00C2\u00A0complex\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0is\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0translocation.\t \u00C2\u00A0J\t \u00C2\u00A0Cell\t \u00C2\u00A0Sci\t \u00C2\u00A0114:2491\u00E2\u0080\u00932499.\t \u00C2\u00A0Agarraberes\t \u00C2\u00A0FA,\t \u00C2\u00A0Terlecky\t \u00C2\u00A0SR,\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(1997)\t \u00C2\u00A0An\t \u00C2\u00A0intralysosomal\t \u00C2\u00A0hsp70\t \u00C2\u00A0is\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A0selective\t \u00C2\u00A0pathway\t \u00C2\u00A0of\t \u00C2\u00A0lysosomal\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0The\t \u00C2\u00A0Journal\t \u00C2\u00A0of\t \u00C2\u00A0Cell\t \u00C2\u00A0Biology\t \u00C2\u00A0137:825\u00E2\u0080\u0093834.\t \u00C2\u00A0Ahmadian\t \u00C2\u00A0G,\t \u00C2\u00A0Ju\t \u00C2\u00A0W,\t \u00C2\u00A0Liu\t \u00C2\u00A0L,\t \u00C2\u00A0Wyszynski\t \u00C2\u00A0M,\t \u00C2\u00A0Lee\t \u00C2\u00A0SH,\t \u00C2\u00A0Dunah\t \u00C2\u00A0AW,\t \u00C2\u00A0Taghibiglou\t \u00C2\u00A0C,\t \u00C2\u00A0Wang\t \u00C2\u00A0Y,\t \u00C2\u00A0Lu\t \u00C2\u00A0J,\t \u00C2\u00A0Wong\t \u00C2\u00A0TP,\t \u00C2\u00A0Sheng\t \u00C2\u00A0M,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2004)\t \u00C2\u00A0Tyrosine\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0of\t \u00C2\u00A0GluR2\t \u00C2\u00A0is\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0insulin-\u00C2\u00AD\u00E2\u0080\u0090stimulated\t \u00C2\u00A0AMPA\t \u00C2\u00A0receptor\t \u00C2\u00A0endocytosis\t \u00C2\u00A0and\t \u00C2\u00A0LTD.\t \u00C2\u00A0EMBO\t \u00C2\u00A0J\t \u00C2\u00A023:1040\u00E2\u0080\u00931050.\t \u00C2\u00A0Alvarez-\u00C2\u00AD\u00E2\u0080\u0090Erviti\t \u00C2\u00A0L,\t \u00C2\u00A0Rodriguez-\u00C2\u00AD\u00E2\u0080\u0090Oroz\t \u00C2\u00A0MC,\t \u00C2\u00A0Cooper\t \u00C2\u00A0JM,\t \u00C2\u00A0Caballero\t \u00C2\u00A0C,\t \u00C2\u00A0Ferrer\t \u00C2\u00A0I,\t \u00C2\u00A0Obeso\t \u00C2\u00A0JA,\t \u00C2\u00A0Schapira\t \u00C2\u00A0AHV\t \u00C2\u00A0(2010)\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0markers\t \u00C2\u00A0in\t \u00C2\u00A0Parkinson\t \u00C2\u00A0disease\t \u00C2\u00A0brains.\t \u00C2\u00A0Arch\t \u00C2\u00A0Neurol\t \u00C2\u00A067:1464\u00E2\u0080\u00931472.\t \u00C2\u00A0Amblard\t \u00C2\u00A0M,\t \u00C2\u00A0Fehrentz\t \u00C2\u00A0J-\u00C2\u00AD\u00E2\u0080\u0090A,\t \u00C2\u00A0Martinez\t \u00C2\u00A0J,\t \u00C2\u00A0Subra\t \u00C2\u00A0G\t \u00C2\u00A0(2005)\t \u00C2\u00A0Fundamentals\t \u00C2\u00A0of\t \u00C2\u00A0modern\t \u00C2\u00A0peptide\t \u00C2\u00A0synthesis.\t \u00C2\u00A0Methods\t \u00C2\u00A0Mol\t \u00C2\u00A0Biol\t \u00C2\u00A0298:3\u00E2\u0080\u009324.\t \u00C2\u00A0\t \u00C2\u00A0 193 Araki\t \u00C2\u00A0T,\t \u00C2\u00A0Shinoda\t \u00C2\u00A0S,\t \u00C2\u00A0Schindler\t \u00C2\u00A0CK,\t \u00C2\u00A0Quan-\u00C2\u00AD\u00E2\u0080\u0090Lan\t \u00C2\u00A0J,\t \u00C2\u00A0Meller\t \u00C2\u00A0R,\t \u00C2\u00A0Taki\t \u00C2\u00A0W,\t \u00C2\u00A0Simon\t \u00C2\u00A0RP,\t \u00C2\u00A0Henshall\t \u00C2\u00A0DC\t \u00C2\u00A0(2004)\t \u00C2\u00A0Expression,\t \u00C2\u00A0interaction,\t \u00C2\u00A0and\t \u00C2\u00A0proteolysis\t \u00C2\u00A0of\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0and\t \u00C2\u00A0p53\t \u00C2\u00A0within\t \u00C2\u00A0vulnerable\t \u00C2\u00A0and\t \u00C2\u00A0resistant\t \u00C2\u00A0hippocampal\t \u00C2\u00A0subfields\t \u00C2\u00A0following\t \u00C2\u00A0seizures.\t \u00C2\u00A0Hippocampus\t \u00C2\u00A014:326\u00E2\u0080\u0093336.\t \u00C2\u00A0Arndt\t \u00C2\u00A0V,\t \u00C2\u00A0Dick\t \u00C2\u00A0N,\t \u00C2\u00A0Tawo\t \u00C2\u00A0R,\t \u00C2\u00A0Dreiseidler\t \u00C2\u00A0M,\t \u00C2\u00A0Wenzel\t \u00C2\u00A0D,\t \u00C2\u00A0Hesse\t \u00C2\u00A0M,\t \u00C2\u00A0F\u00C3\u00BCrst\t \u00C2\u00A0DO,\t \u00C2\u00A0Saftig\t \u00C2\u00A0P,\t \u00C2\u00A0Saint\t \u00C2\u00A0R,\t \u00C2\u00A0Fleischmann\t \u00C2\u00A0BK,\t \u00C2\u00A0Hoch\t \u00C2\u00A0M,\t \u00C2\u00A0H\u00C3\u00B6hfeld\t \u00C2\u00A0J\t \u00C2\u00A0(2010).\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090assisted\t \u00C2\u00A0selective\t \u00C2\u00A0autophagy\t \u00C2\u00A0is\t \u00C2\u00A0essential\t \u00C2\u00A0for\t \u00C2\u00A0muscle\t \u00C2\u00A0maintenance.\t \u00C2\u00A0Current\t \u00C2\u00A0Biology\t \u00C2\u00A020(2):143-\u00C2\u00AD\u00E2\u0080\u00908.\t \u00C2\u00A0Austin\t \u00C2\u00A0CP\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2004)\t \u00C2\u00A0The\t \u00C2\u00A0knockout\t \u00C2\u00A0mouse\t \u00C2\u00A0project.\t \u00C2\u00A0Nat\t \u00C2\u00A0Genet\t \u00C2\u00A036:921\u00E2\u0080\u0093924.\t \u00C2\u00A0Backer\t \u00C2\u00A0J,\t \u00C2\u00A0Bourret\t \u00C2\u00A0L,\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(1983)\t \u00C2\u00A0Regulation\t \u00C2\u00A0of\t \u00C2\u00A0catabolism\t \u00C2\u00A0of\t \u00C2\u00A0microinjected\t \u00C2\u00A0ribonuclease\t \u00C2\u00A0A\t \u00C2\u00A0requires\t \u00C2\u00A0the\t \u00C2\u00A0amino-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A020\t \u00C2\u00A0amino\t \u00C2\u00A0acids.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A080:2166\u00E2\u0080\u00932170.\t \u00C2\u00A0Banaszynski\t \u00C2\u00A0LA,\t \u00C2\u00A0Liu\t \u00C2\u00A0CW,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2005)\t \u00C2\u00A0Characterization\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0FKBP\u00C2\u009F\u00EF\u0082\u009FRapamycin\u00C2\u009F\u00EF\u0082\u009FFRB\t \u00C2\u00A0ternary\t \u00C2\u00A0complex.\t \u00C2\u00A0J.\t \u00C2\u00A0Am.\t \u00C2\u00A0Chem.\t \u00C2\u00A0Soc.\t \u00C2\u00A0127:4715-\u00C2\u00AD\u00E2\u0080\u00904721.\t \u00C2\u00A0Banaszynski\t \u00C2\u00A0LA,\t \u00C2\u00A0Chen\t \u00C2\u00A0L-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Maynard-\u00C2\u00AD\u00E2\u0080\u0090Smith\t \u00C2\u00A0LA,\t \u00C2\u00A0Ooi\t \u00C2\u00A0AGL,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2006)\t \u00C2\u00A0A\t \u00C2\u00A0Rapid,\t \u00C2\u00A0Reversible,\t \u00C2\u00A0and\t \u00C2\u00A0Tunable\t \u00C2\u00A0Method\t \u00C2\u00A0to\t \u00C2\u00A0Regulate\t \u00C2\u00A0Protein\t \u00C2\u00A0Function\t \u00C2\u00A0in\t \u00C2\u00A0Living\t \u00C2\u00A0Cells\t \u00C2\u00A0Using\t \u00C2\u00A0Synthetic\t \u00C2\u00A0Small\t \u00C2\u00A0Molecules.\t \u00C2\u00A0Cell\t \u00C2\u00A0126:995\u00E2\u0080\u00931004.\t \u00C2\u00A0Banaszynski\t \u00C2\u00A0LA,\t \u00C2\u00A0Sellmyer\t \u00C2\u00A0MA,\t \u00C2\u00A0Contag\t \u00C2\u00A0CH,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ,\t \u00C2\u00A0Thorne\t \u00C2\u00A0SH\t \u00C2\u00A0(2008)\t \u00C2\u00A0Chemical\t \u00C2\u00A0control\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0stability\t \u00C2\u00A0and\t \u00C2\u00A0function\t \u00C2\u00A0in\t \u00C2\u00A0living\t \u00C2\u00A0mice.\t \u00C2\u00A0Nat\t \u00C2\u00A0Med\t \u00C2\u00A014:1123\u00E2\u0080\u00931127.\t \u00C2\u00A0Banaszynski\t \u00C2\u00A0LA,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2006)\t \u00C2\u00A0Conditional\t \u00C2\u00A0Control\t \u00C2\u00A0of\t \u00C2\u00A0Protein\t \u00C2\u00A0Function.\t \u00C2\u00A0Chemistry\t \u00C2\u00A0&\t \u00C2\u00A0Biology\t \u00C2\u00A013:11\u00E2\u0080\u009321.\t \u00C2\u00A0 194 Bandyopadhyay\t \u00C2\u00A0U,\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Varticovski\t \u00C2\u00A0L,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2008)\t \u00C2\u00A0The\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy\t \u00C2\u00A0Receptor\t \u00C2\u00A0Organizes\t \u00C2\u00A0in\t \u00C2\u00A0Dynamic\t \u00C2\u00A0Protein\t \u00C2\u00A0Complexes\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0Lysosomal\t \u00C2\u00A0Membrane.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A028:5747\u00E2\u0080\u00935763.\t \u00C2\u00A0Bandyopadhyay\t \u00C2\u00A0U,\t \u00C2\u00A0Sridhar\t \u00C2\u00A0S,\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Kiffin\t \u00C2\u00A0R,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2010)\t \u00C2\u00A0Identification\t \u00C2\u00A0of\t \u00C2\u00A0Regulators\t \u00C2\u00A0of\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy.\t \u00C2\u00A0Molecular\t \u00C2\u00A0Cell\t \u00C2\u00A039:535\u00E2\u0080\u0093547.\t \u00C2\u00A0Barbaric\t \u00C2\u00A0I,\t \u00C2\u00A0Miller\t \u00C2\u00A0G,\t \u00C2\u00A0Dear\t \u00C2\u00A0TN\t \u00C2\u00A0(2007)\t \u00C2\u00A0Appearances\t \u00C2\u00A0can\t \u00C2\u00A0be\t \u00C2\u00A0deceiving:\t \u00C2\u00A0phenotypes\t \u00C2\u00A0of\t \u00C2\u00A0knockout\t \u00C2\u00A0mice.\t \u00C2\u00A0Briefings\t \u00C2\u00A0in\t \u00C2\u00A0Functional\t \u00C2\u00A0Genomics\t \u00C2\u00A0and\t \u00C2\u00A0Proteomics\t \u00C2\u00A06:91\u00E2\u0080\u0093103.\t \u00C2\u00A0Bauer\t \u00C2\u00A0PO,\t \u00C2\u00A0Goswami\t \u00C2\u00A0A,\t \u00C2\u00A0Wong\t \u00C2\u00A0HK,\t \u00C2\u00A0Okuno\t \u00C2\u00A0M,\t \u00C2\u00A0Kurosawa\t \u00C2\u00A0M,\t \u00C2\u00A0Yamada\t \u00C2\u00A0M,\t \u00C2\u00A0Miyazaki\t \u00C2\u00A0H,\t \u00C2\u00A0Matsumoto\t \u00C2\u00A0G,\t \u00C2\u00A0Kino\t \u00C2\u00A0Y,\t \u00C2\u00A0Nagai\t \u00C2\u00A0Y,\t \u00C2\u00A0Nukina\t \u00C2\u00A0N\t \u00C2\u00A0(2010)\t \u00C2\u00A0Harnessing\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0selective\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0mutant\t \u00C2\u00A0huntingtin\t \u00C2\u00A0protein.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A028:256\u00E2\u0080\u0093263.\t \u00C2\u00A0Baumeister\t \u00C2\u00A0W,\t \u00C2\u00A0Walz\t \u00C2\u00A0J,\t \u00C2\u00A0Z\u00C3\u00BChl\t \u00C2\u00A0F,\t \u00C2\u00A0Seem\u00C3\u00BCller\t \u00C2\u00A0E\t \u00C2\u00A0(1998)\t \u00C2\u00A0The\t \u00C2\u00A0proteasome:\t \u00C2\u00A0paradigm\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0self-\u00C2\u00AD\u00E2\u0080\u0090compartmentalizing\t \u00C2\u00A0protease.\t \u00C2\u00A0Cell\t \u00C2\u00A092:367\u00E2\u0080\u0093380.\t \u00C2\u00A0Bedell\t \u00C2\u00A0VM,\t \u00C2\u00A0Wang\t \u00C2\u00A0Y,\t \u00C2\u00A0Campbell\t \u00C2\u00A0JM,\t \u00C2\u00A0Poshusta\t \u00C2\u00A0TL,\t \u00C2\u00A0Starker\t \u00C2\u00A0CG,\t \u00C2\u00A0Krug\t \u00C2\u00A0RG\t \u00C2\u00A0II,\t \u00C2\u00A0Tan\t \u00C2\u00A0W,\t \u00C2\u00A0Penheiter\t \u00C2\u00A0SG,\t \u00C2\u00A0Ma\t \u00C2\u00A0AC,\t \u00C2\u00A0Leung\t \u00C2\u00A0AYH,\t \u00C2\u00A0Fahrenkrug\t \u00C2\u00A0SC,\t \u00C2\u00A0Carlson\t \u00C2\u00A0DF,\t \u00C2\u00A0Voytas\t \u00C2\u00A0DF,\t \u00C2\u00A0Clark\t \u00C2\u00A0KJ,\t \u00C2\u00A0Essner\t \u00C2\u00A0JJ,\t \u00C2\u00A0Ekker\t \u00C2\u00A0SC\t \u00C2\u00A0(2013)\t \u00C2\u00A0In\t \u00C2\u00A0vivo\t \u00C2\u00A0genome\t \u00C2\u00A0editing\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0high-\u00C2\u00AD\u00E2\u0080\u0090efficiency\t \u00C2\u00A0TALEN\t \u00C2\u00A0system.\t \u00C2\u00A0Nature\t \u00C2\u00A0490:114\u00E2\u0080\u0093118.\t \u00C2\u00A0Bellmann-\u00C2\u00AD\u00E2\u0080\u0090Sickert\t \u00C2\u00A0K,\t \u00C2\u00A0Beck-\u00C2\u00AD\u00E2\u0080\u0090Sickinger\t \u00C2\u00A0AG\t \u00C2\u00A0(2010)\t \u00C2\u00A0Peptide\t \u00C2\u00A0drugs\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0G\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090coupled\t \u00C2\u00A0receptors.\t \u00C2\u00A0Trends\t \u00C2\u00A0in\t \u00C2\u00A0Pharmacological\t \u00C2\u00A0Sciences\t \u00C2\u00A031:434\u00E2\u0080\u0093441.\t \u00C2\u00A0 195 Bennett\t \u00C2\u00A0CF,\t \u00C2\u00A0Swayze\t \u00C2\u00A0EE\t \u00C2\u00A0(2010)\t \u00C2\u00A0RNA\t \u00C2\u00A0Targeting\t \u00C2\u00A0Therapeutics:\t \u00C2\u00A0Molecular\t \u00C2\u00A0Mechanisms\t \u00C2\u00A0of\t \u00C2\u00A0Antisense\t \u00C2\u00A0Oligonucleotides\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0Therapeutic\t \u00C2\u00A0Platform.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Pharmacol\t \u00C2\u00A0Toxicol\t \u00C2\u00A050:259\u00E2\u0080\u0093293.\t \u00C2\u00A0Bhattacharyya\t \u00C2\u00A0S,\t \u00C2\u00A0Yu\t \u00C2\u00A0H,\t \u00C2\u00A0Mim\t \u00C2\u00A0C,\t \u00C2\u00A0Matouschek\t \u00C2\u00A0A\t \u00C2\u00A0(2014)\t \u00C2\u00A0Regulated\t \u00C2\u00A0protein\t \u00C2\u00A0turnover:\t \u00C2\u00A0snapshots\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0in\t \u00C2\u00A0action.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A015:122\u00E2\u0080\u0093133.\t \u00C2\u00A0Bibikova\t \u00C2\u00A0M,\t \u00C2\u00A0Golic\t \u00C2\u00A0M,\t \u00C2\u00A0Golic\t \u00C2\u00A0KG,\t \u00C2\u00A0Carroll\t \u00C2\u00A0D\t \u00C2\u00A0(2002)\t \u00C2\u00A0Targeted\t \u00C2\u00A0chromosomal\t \u00C2\u00A0cleavage\t \u00C2\u00A0and\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0in\t \u00C2\u00A0Drosophila\t \u00C2\u00A0using\t \u00C2\u00A0zinc-\u00C2\u00AD\u00E2\u0080\u0090finger\t \u00C2\u00A0nucleases.\t \u00C2\u00A0Genetics\t \u00C2\u00A0161:1169\u00E2\u0080\u00931175.\t \u00C2\u00A0Bonger\t \u00C2\u00A0KM,\t \u00C2\u00A0Chen\t \u00C2\u00A0L-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Liu\t \u00C2\u00A0CW,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2011)\t \u00C2\u00A0Small-\u00C2\u00AD\u00E2\u0080\u0090molecule\t \u00C2\u00A0displacement\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0cryptic\t \u00C2\u00A0degron\t \u00C2\u00A0causes\t \u00C2\u00A0conditional\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0Nat\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A07:531\u00E2\u0080\u0093537.\t \u00C2\u00A0Bonger\t \u00C2\u00A0KM,\t \u00C2\u00A0Rakhit\t \u00C2\u00A0R,\t \u00C2\u00A0Payumo\t \u00C2\u00A0AY,\t \u00C2\u00A0Chen\t \u00C2\u00A0JK,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2014)\t \u00C2\u00A0General\t \u00C2\u00A0Method\t \u00C2\u00A0for\t \u00C2\u00A0Regulating\t \u00C2\u00A0Protein\t \u00C2\u00A0Stability\t \u00C2\u00A0with\t \u00C2\u00A0Light.\t \u00C2\u00A0ACS\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A09:111\u00E2\u0080\u0093115.\t \u00C2\u00A0Bonny\t \u00C2\u00A0C,\t \u00C2\u00A0Oberson\t \u00C2\u00A0A,\t \u00C2\u00A0Negri\t \u00C2\u00A0S,\t \u00C2\u00A0Sauser\t \u00C2\u00A0C,\t \u00C2\u00A0Schorderet\t \u00C2\u00A0DF\t \u00C2\u00A0(2001)\t \u00C2\u00A0Cell-\u00C2\u00AD\u00E2\u0080\u0090permeable\t \u00C2\u00A0peptide\t \u00C2\u00A0inhibitors\t \u00C2\u00A0of\t \u00C2\u00A0JNK:\t \u00C2\u00A0novel\t \u00C2\u00A0blockers\t \u00C2\u00A0of\t \u00C2\u00A0beta-\u00C2\u00AD\u00E2\u0080\u0090cell\t \u00C2\u00A0death.\t \u00C2\u00A0Diabetes\t \u00C2\u00A050,\t \u00C2\u00A077\u00E2\u0080\u009382.\t \u00C2\u00A0\t \u00C2\u00A0Borsello\t \u00C2\u00A0T,\t \u00C2\u00A0Clarke\t \u00C2\u00A0PG,\t \u00C2\u00A0Hirt\t \u00C2\u00A0L,\t \u00C2\u00A0Vercelli\t \u00C2\u00A0A,\t \u00C2\u00A0Repici\t \u00C2\u00A0M,\t \u00C2\u00A0Schorderet\t \u00C2\u00A0DF,\t \u00C2\u00A0Bogous-\u00C2\u00AD\u00E2\u0080\u0090slavsky\t \u00C2\u00A0J,\t \u00C2\u00A0Bonny\t \u00C2\u00A0C\t \u00C2\u00A0(2003)\t \u00C2\u00A0A\t \u00C2\u00A0peptide\t \u00C2\u00A0inhibitor\t \u00C2\u00A0of\t \u00C2\u00A0c-\u00C2\u00AD\u00E2\u0080\u0090Jun\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090terminal\t \u00C2\u00A0kinase\t \u00C2\u00A0protects\t \u00C2\u00A0against\t \u00C2\u00A0excitotoxicity\t \u00C2\u00A0and\t \u00C2\u00A0cerebral\t \u00C2\u00A0ischemia.\t \u00C2\u00A0Nat\t \u00C2\u00A0Med\t \u00C2\u00A09,\t \u00C2\u00A01180\u00E2\u0080\u00931186.\t \u00C2\u00A0\t \u00C2\u00A0Bradley\t \u00C2\u00A0A\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2012)\t \u00C2\u00A0The\t \u00C2\u00A0mammalian\t \u00C2\u00A0gene\t \u00C2\u00A0function\t \u00C2\u00A0resource:\t \u00C2\u00A0the\t \u00C2\u00A0international\t \u00C2\u00A0knockout\t \u00C2\u00A0mouse\t \u00C2\u00A0consortium.\t \u00C2\u00A0Mamm\t \u00C2\u00A0Genome\t \u00C2\u00A023:580\u00E2\u0080\u0093586.\t \u00C2\u00A0\t \u00C2\u00A0 196 Brebner\t \u00C2\u00A0K\t \u00C2\u00A0(2005)\t \u00C2\u00A0Nucleus\t \u00C2\u00A0Accumbens\t \u00C2\u00A0Long-\u00C2\u00AD\u00E2\u0080\u0090Term\t \u00C2\u00A0Depression\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0Expression\t \u00C2\u00A0of\t \u00C2\u00A0Behavioral\t \u00C2\u00A0Sensitization.\t \u00C2\u00A0Science\t \u00C2\u00A0310:1340\u00E2\u0080\u00931343.\t \u00C2\u00A0Bright\t \u00C2\u00A0R,\t \u00C2\u00A0Mochly-\u00C2\u00AD\u00E2\u0080\u0090Rosen\t \u00C2\u00A0D\t \u00C2\u00A0(2005)\t \u00C2\u00A0The\t \u00C2\u00A0Role\t \u00C2\u00A0of\t \u00C2\u00A0Protein\t \u00C2\u00A0Kinase\t \u00C2\u00A0C\t \u00C2\u00A0in\t \u00C2\u00A0Cerebral\t \u00C2\u00A0Ischemic\t \u00C2\u00A0and\t \u00C2\u00A0Reperfusion\t \u00C2\u00A0Injury.\t \u00C2\u00A0Stroke\t \u00C2\u00A036:2781\u00E2\u0080\u00932790.\t \u00C2\u00A0Brown\t \u00C2\u00A0V,\t \u00C2\u00A0Liu\t \u00C2\u00A0F\t \u00C2\u00A0(2014)\t \u00C2\u00A0Intranasal\t \u00C2\u00A0Delivery\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0Peptide\t \u00C2\u00A0with\t \u00C2\u00A0Antidepressant-\u00C2\u00AD\u00E2\u0080\u0090Like\t \u00C2\u00A0Effect.\t \u00C2\u00A0Neuropharmacology\t \u00C2\u00A039:2131\u00E2\u0080\u00932141.\t \u00C2\u00A0Capecchi\t \u00C2\u00A0MR\t \u00C2\u00A0(2005)\t \u00C2\u00A0Gene\t \u00C2\u00A0targeting\t \u00C2\u00A0in\t \u00C2\u00A0mice:\t \u00C2\u00A0functional\t \u00C2\u00A0analysis\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0mammalian\t \u00C2\u00A0genome\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0twenty-\u00C2\u00AD\u00E2\u0080\u0090first\t \u00C2\u00A0century.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Genet\t \u00C2\u00A06:507\u00E2\u0080\u0093512.\t \u00C2\u00A0Carmony\t \u00C2\u00A0KC,\t \u00C2\u00A0Kim\t \u00C2\u00A0K-\u00C2\u00AD\u00E2\u0080\u0090B\t \u00C2\u00A0(2012)\t \u00C2\u00A0PROTAC-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0proteolytic\t \u00C2\u00A0targeting.\t \u00C2\u00A0Methods\t \u00C2\u00A0Mol\t \u00C2\u00A0Biol\t \u00C2\u00A0832:627\u00E2\u0080\u0093638.\t \u00C2\u00A0Castanotto\t \u00C2\u00A0D,\t \u00C2\u00A0Rossi\t \u00C2\u00A0JJ\t \u00C2\u00A0(2009)\t \u00C2\u00A0The\t \u00C2\u00A0promises\t \u00C2\u00A0and\t \u00C2\u00A0pitfalls\t \u00C2\u00A0of\t \u00C2\u00A0RNA-\u00C2\u00AD\u00E2\u0080\u0090interference-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0therapeutics.\t \u00C2\u00A0Nature\t \u00C2\u00A0457:426\u00E2\u0080\u0093433.\t \u00C2\u00A0Cataldo\t \u00C2\u00A0AM,\t \u00C2\u00A0Nixon\t \u00C2\u00A0RA\t \u00C2\u00A0(1990)\t \u00C2\u00A0Enzymatically\t \u00C2\u00A0active\t \u00C2\u00A0lysosomal\t \u00C2\u00A0proteases\t \u00C2\u00A0are\t \u00C2\u00A0associated\t \u00C2\u00A0with\t \u00C2\u00A0amyloid\t \u00C2\u00A0deposits\t \u00C2\u00A0in\t \u00C2\u00A0Alzheimer\t \u00C2\u00A0brain.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A087:3861\u00E2\u0080\u00933865.\t \u00C2\u00A0Caussinus\t \u00C2\u00A0E,\t \u00C2\u00A0Kanca\t \u00C2\u00A0O,\t \u00C2\u00A0Affolter\t \u00C2\u00A0M\t \u00C2\u00A0(2011)\t \u00C2\u00A0Fluorescent\t \u00C2\u00A0fusion\t \u00C2\u00A0protein\t \u00C2\u00A0knockout\t \u00C2\u00A0mediated\t \u00C2\u00A0by\t \u00C2\u00A0anti-\u00C2\u00AD\u00E2\u0080\u0090GFP\t \u00C2\u00A0nanobody.\t \u00C2\u00A0Nat\t \u00C2\u00A0Struct\t \u00C2\u00A0Mol\t \u00C2\u00A0Biol\t \u00C2\u00A019:117\u00E2\u0080\u0093121.\t \u00C2\u00A0Chen\t \u00C2\u00A0CH,\t \u00C2\u00A0Wang\t \u00C2\u00A0WJ,\t \u00C2\u00A0Kuo\t \u00C2\u00A0JC,\t \u00C2\u00A0Tsai\t \u00C2\u00A0HC,\t \u00C2\u00A0Lin\t \u00C2\u00A0JR,\t \u00C2\u00A0Chang\t \u00C2\u00A0ZF,\t \u00C2\u00A0Chen\t \u00C2\u00A0RH\t \u00C2\u00A0(2005)\t \u00C2\u00A0Bidirectional\t \u00C2\u00A0signals\t \u00C2\u00A0transduced\t \u00C2\u00A0by\t \u00C2\u00A0DAPK\u00E2\u0080\u0093ERK\t \u00C2\u00A0interaction\t \u00C2\u00A0promote\t \u00C2\u00A0the\t \u00C2\u00A0apoptotic\t \u00C2\u00A0effect\t \u00C2\u00A0of\t \u00C2\u00A0DAPK.\t \u00C2\u00A0EMBO\t \u00C2\u00A0J.\t \u00C2\u00A024,\t \u00C2\u00A0294\u00E2\u0080\u0093304.\t \u00C2\u00A0\t \u00C2\u00A0 197 Chen\t \u00C2\u00A0X,\t \u00C2\u00A0Vinade\t \u00C2\u00A0L,\t \u00C2\u00A0Leapman\t \u00C2\u00A0RD,\t \u00C2\u00A0Petersen\t \u00C2\u00A0JD,\t \u00C2\u00A0Nakagawa\t \u00C2\u00A0T,\t \u00C2\u00A0Phillips\t \u00C2\u00A0TM,\t \u00C2\u00A0Sheng\t \u00C2\u00A0M,\t \u00C2\u00A0Reese\t \u00C2\u00A0TS\t \u00C2\u00A0(2005)\t \u00C2\u00A0Mass\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0postsynaptic\t \u00C2\u00A0density\t \u00C2\u00A0and\t \u00C2\u00A0enumeration\t \u00C2\u00A0of\t \u00C2\u00A0three\t \u00C2\u00A0key\t \u00C2\u00A0molecules.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0102:11551\u00E2\u0080\u009311556.\t \u00C2\u00A0Cheng\t \u00C2\u00A0D,\t \u00C2\u00A0Hoogenraad\t \u00C2\u00A0CC,\t \u00C2\u00A0Rush\t \u00C2\u00A0J,\t \u00C2\u00A0Ramm\t \u00C2\u00A0E,\t \u00C2\u00A0Schlager\t \u00C2\u00A0MA,\t \u00C2\u00A0Duong\t \u00C2\u00A0DM,\t \u00C2\u00A0Xu\t \u00C2\u00A0P,\t \u00C2\u00A0Wijayawardana\t \u00C2\u00A0SR,\t \u00C2\u00A0Hanfelt\t \u00C2\u00A0J,\t \u00C2\u00A0Nakagawa\t \u00C2\u00A0T,\t \u00C2\u00A0Sheng\t \u00C2\u00A0M,\t \u00C2\u00A0Peng\t \u00C2\u00A0J\t \u00C2\u00A0(2006)\t \u00C2\u00A0Relative\t \u00C2\u00A0and\t \u00C2\u00A0absolute\t \u00C2\u00A0quantification\t \u00C2\u00A0of\t \u00C2\u00A0postsynaptic\t \u00C2\u00A0density\t \u00C2\u00A0proteome\t \u00C2\u00A0isolated\t \u00C2\u00A0from\t \u00C2\u00A0rat\t \u00C2\u00A0forebrain\t \u00C2\u00A0and\t \u00C2\u00A0cerebellum.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Proteomics\t \u00C2\u00A05:1158\u00E2\u0080\u00931170.\t \u00C2\u00A0Chiang\t \u00C2\u00A0HL,\t \u00C2\u00A0Terlecky\t \u00C2\u00A0SR,\t \u00C2\u00A0Plant\t \u00C2\u00A0CP,\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(1989)\t \u00C2\u00A0A\t \u00C2\u00A0role\t \u00C2\u00A0for\t \u00C2\u00A0a\t \u00C2\u00A070-\u00C2\u00AD\u00E2\u0080\u0090kilodalton\t \u00C2\u00A0heat\t \u00C2\u00A0shock\t \u00C2\u00A0protein\t \u00C2\u00A0in\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0intracellular\t \u00C2\u00A0proteins.\t \u00C2\u00A0Science\t \u00C2\u00A0246:382\u00E2\u0080\u0093385.\t \u00C2\u00A0Chico\t \u00C2\u00A0LK,\t \u00C2\u00A0Van\t \u00C2\u00A0Eldik\t \u00C2\u00A0LJ,\t \u00C2\u00A0Watterson\t \u00C2\u00A0DM\t \u00C2\u00A0(2009)\t \u00C2\u00A0Targeting\t \u00C2\u00A0protein\t \u00C2\u00A0kinases\t \u00C2\u00A0in\t \u00C2\u00A0central\t \u00C2\u00A0nervous\t \u00C2\u00A0system\t \u00C2\u00A0disorders.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A08:892\u00E2\u0080\u0093909.\t \u00C2\u00A0Christopherson\t \u00C2\u00A0KS\t \u00C2\u00A0(1999)\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0Assembles\t \u00C2\u00A0a\t \u00C2\u00A0Ternary\t \u00C2\u00A0Complex\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090Methyl-\u00C2\u00AD\u00E2\u0080\u0090D-\u00C2\u00AD\u00E2\u0080\u0090aspartic\t \u00C2\u00A0Acid\t \u00C2\u00A0Receptor\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0Bivalent\t \u00C2\u00A0Neuronal\t \u00C2\u00A0NO\t \u00C2\u00A0Synthase\t \u00C2\u00A0PDZ\t \u00C2\u00A0Domain.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0274:27467\u00E2\u0080\u009327473.\t \u00C2\u00A0Chuang\t \u00C2\u00A0Y-\u00C2\u00AD\u00E2\u0080\u0090T,\t \u00C2\u00A0Lin\t \u00C2\u00A0Y-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Lin\t \u00C2\u00A0K-\u00C2\u00AD\u00E2\u0080\u0090H,\t \u00C2\u00A0Chou\t \u00C2\u00A0T-\u00C2\u00AD\u00E2\u0080\u0090F,\t \u00C2\u00A0Kuo\t \u00C2\u00A0W-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Yang\t \u00C2\u00A0K-\u00C2\u00AD\u00E2\u0080\u0090T,\t \u00C2\u00A0Wu\t \u00C2\u00A0P-\u00C2\u00AD\u00E2\u0080\u0090R,\t \u00C2\u00A0Chen\t \u00C2\u00A0R-\u00C2\u00AD\u00E2\u0080\u0090H,\t \u00C2\u00A0Kimchi\t \u00C2\u00A0A,\t \u00C2\u00A0Lai\t \u00C2\u00A0M-\u00C2\u00AD\u00E2\u0080\u0090Z\t \u00C2\u00A0(2011)\t \u00C2\u00A0Tumor\t \u00C2\u00A0suppressor\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0is\t \u00C2\u00A0required\t \u00C2\u00A0for\t \u00C2\u00A0full\t \u00C2\u00A0IL-\u00C2\u00AD\u00E2\u0080\u00901\u00CE\u00B2\t \u00C2\u00A0production.\t \u00C2\u00A0Blood\t \u00C2\u00A0117:960\u00E2\u0080\u0093970.\t \u00C2\u00A0Clayton\t \u00C2\u00A0DF,\t \u00C2\u00A0George\t \u00C2\u00A0JM\t \u00C2\u00A0(1998)\t \u00C2\u00A0The\t \u00C2\u00A0synucleins:\t \u00C2\u00A0a\t \u00C2\u00A0family\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0involved\t \u00C2\u00A0in\t \u00C2\u00A0synaptic\t \u00C2\u00A0function,\t \u00C2\u00A0plasticity,\t \u00C2\u00A0neurodegeneration\t \u00C2\u00A0and\t \u00C2\u00A0disease.\t \u00C2\u00A0Trends\t \u00C2\u00A0Neurosci\t \u00C2\u00A021:249\u00E2\u0080\u0093254.\t \u00C2\u00A0 198 Coffey\t \u00C2\u00A0ET\t \u00C2\u00A0(2014)\t \u00C2\u00A0Nuclear\t \u00C2\u00A0and\t \u00C2\u00A0cytosolic\t \u00C2\u00A0JNK\t \u00C2\u00A0signalling\t \u00C2\u00A0in\t \u00C2\u00A0neurons.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Neuro\t \u00C2\u00A015:285\u00E2\u0080\u0093299.\t \u00C2\u00A0Cong\t \u00C2\u00A0F,\t \u00C2\u00A0Zhang\t \u00C2\u00A0J,\t \u00C2\u00A0Pao\t \u00C2\u00A0W,\t \u00C2\u00A0Zhou\t \u00C2\u00A0P,\t \u00C2\u00A0Varmus\t \u00C2\u00A0H\t \u00C2\u00A0(2003)\t \u00C2\u00A0A\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0strategy\t \u00C2\u00A0to\t \u00C2\u00A0study\t \u00C2\u00A0the\t \u00C2\u00A0function\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin\t \u00C2\u00A0in\t \u00C2\u00A0tumorigenesis.\t \u00C2\u00A0BMC\t \u00C2\u00A0Mol\t \u00C2\u00A0Biol\t \u00C2\u00A04:10.\t \u00C2\u00A0Cong\t \u00C2\u00A0L,\t \u00C2\u00A0Ran\t \u00C2\u00A0FA,\t \u00C2\u00A0Cox\t \u00C2\u00A0D,\t \u00C2\u00A0Lin\t \u00C2\u00A0S,\t \u00C2\u00A0Barretto\t \u00C2\u00A0R,\t \u00C2\u00A0Habib\t \u00C2\u00A0N,\t \u00C2\u00A0Hsu\t \u00C2\u00A0PD,\t \u00C2\u00A0Wu\t \u00C2\u00A0X,\t \u00C2\u00A0Jiang\t \u00C2\u00A0W,\t \u00C2\u00A0Marraffini\t \u00C2\u00A0LA,\t \u00C2\u00A0Zhang\t \u00C2\u00A0F\t \u00C2\u00A0(2013)\t \u00C2\u00A0Multiplex\t \u00C2\u00A0Genome\t \u00C2\u00A0Engineering\t \u00C2\u00A0Using\t \u00C2\u00A0CRISPR/Cas\t \u00C2\u00A0Systems.\t \u00C2\u00A0Science\t \u00C2\u00A0339:819\u00E2\u0080\u0093823.\t \u00C2\u00A0Conway\t \u00C2\u00A0KA,\t \u00C2\u00A0Harper\t \u00C2\u00A0JD,\t \u00C2\u00A0Lansbury\t \u00C2\u00A0PT\t \u00C2\u00A0(1998)\t \u00C2\u00A0Accelerated\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0fibril\t \u00C2\u00A0formation\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0mutant\t \u00C2\u00A0alpha-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0early-\u00C2\u00AD\u00E2\u0080\u0090onset\t \u00C2\u00A0Parkinson\t \u00C2\u00A0disease.\t \u00C2\u00A0Nat\t \u00C2\u00A0Med\t \u00C2\u00A04:1318\u00E2\u0080\u00931320.\t \u00C2\u00A0Cook\t \u00C2\u00A0DJ,\t \u00C2\u00A0Teves\t \u00C2\u00A0L,\t \u00C2\u00A0Tymianski\t \u00C2\u00A0M\t \u00C2\u00A0(2012)\t \u00C2\u00A0Treatment\t \u00C2\u00A0of\t \u00C2\u00A0stroke\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0inhibitor\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0gyrencephalic\t \u00C2\u00A0primate\t \u00C2\u00A0brain.\t \u00C2\u00A0Nature\t \u00C2\u00A0483:213\u00E2\u0080\u0093217.\t \u00C2\u00A0Coultrap\t \u00C2\u00A0SJ,\t \u00C2\u00A0Freund\t \u00C2\u00A0RK,\t \u00C2\u00A0O'Leary\t \u00C2\u00A0H,\t \u00C2\u00A0Sanderson\t \u00C2\u00A0JL,\t \u00C2\u00A0Roche\t \u00C2\u00A0KW,\t \u00C2\u00A0Dell'Acqua\t \u00C2\u00A0ML,\t \u00C2\u00A0Bayer\t \u00C2\u00A0KU\t \u00C2\u00A0(2014)\t \u00C2\u00A0Autonomous\t \u00C2\u00A0CaMKII\t \u00C2\u00A0mediates\t \u00C2\u00A0both\t \u00C2\u00A0LTP\t \u00C2\u00A0and\t \u00C2\u00A0LTD\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0mechanism\t \u00C2\u00A0for\t \u00C2\u00A0differential\t \u00C2\u00A0substrate\t \u00C2\u00A0site\t \u00C2\u00A0selection.\t \u00C2\u00A0Cell\t \u00C2\u00A0Reports\t \u00C2\u00A06:431\u00E2\u0080\u0093437.\t \u00C2\u00A0Coultrap\t \u00C2\u00A0SJ,\t \u00C2\u00A0Vest\t \u00C2\u00A0RS,\t \u00C2\u00A0Ashpole\t \u00C2\u00A0NM,\t \u00C2\u00A0Hudmon\t \u00C2\u00A0A,\t \u00C2\u00A0Bayer\t \u00C2\u00A0KU\t \u00C2\u00A0(2011)\t \u00C2\u00A0CaMKII\t \u00C2\u00A0in\t \u00C2\u00A0cerebral\t \u00C2\u00A0ischemia.\t \u00C2\u00A0Acta\t \u00C2\u00A0Pharm\t \u00C2\u00A0Sinic\t \u00C2\u00A032:861\u00E2\u0080\u0093872.\t \u00C2\u00A0Crabbe\t \u00C2\u00A0JC,\t \u00C2\u00A0Wahlsten\t \u00C2\u00A0D,\t \u00C2\u00A0Dudek\t \u00C2\u00A0BC\t \u00C2\u00A0(1999)\t \u00C2\u00A0Genetics\t \u00C2\u00A0of\t \u00C2\u00A0mouse\t \u00C2\u00A0behavior:\t \u00C2\u00A0interactions\t \u00C2\u00A0with\t \u00C2\u00A0laboratory\t \u00C2\u00A0environment.\t \u00C2\u00A0Science\t \u00C2\u00A0284:1670\u00E2\u0080\u00931672.\t \u00C2\u00A0Craik\t \u00C2\u00A0DJ,\t \u00C2\u00A0Fairlie\t \u00C2\u00A0DP,\t \u00C2\u00A0Liras\t \u00C2\u00A0S,\t \u00C2\u00A0Price\t \u00C2\u00A0D\t \u00C2\u00A0(2013)\t \u00C2\u00A0The\t \u00C2\u00A0future\t \u00C2\u00A0of\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0drugs.\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A0 199 Drug\t \u00C2\u00A0Des\t \u00C2\u00A081:136\u00E2\u0080\u0093147.\t \u00C2\u00A0Crews\t \u00C2\u00A0CM\t \u00C2\u00A0(2010)\t \u00C2\u00A0Targeting\t \u00C2\u00A0the\t \u00C2\u00A0Undruggable\t \u00C2\u00A0Proteome:\t \u00C2\u00A0The\t \u00C2\u00A0Small\t \u00C2\u00A0Molecules\t \u00C2\u00A0of\t \u00C2\u00A0My\t \u00C2\u00A0Dreams.\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A017:551\u00E2\u0080\u0093555.\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(2000a)\t \u00C2\u00A0Unique\t \u00C2\u00A0properties\t \u00C2\u00A0of\t \u00C2\u00A0lamp2a\t \u00C2\u00A0compared\t \u00C2\u00A0to\t \u00C2\u00A0other\t \u00C2\u00A0lamp2\t \u00C2\u00A0isoforms.\t \u00C2\u00A0J\t \u00C2\u00A0Cell\t \u00C2\u00A0Sci\t \u00C2\u00A0113:4441\u00E2\u0080\u00934450.\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(2000b)\t \u00C2\u00A0Regulation\t \u00C2\u00A0of\t \u00C2\u00A0lamp2a\t \u00C2\u00A0levels\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane.\t \u00C2\u00A0Traffic\t \u00C2\u00A01:570\u00E2\u0080\u0093583.\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(2000c)\t \u00C2\u00A0Age-\u00C2\u00AD\u00E2\u0080\u0090related\t \u00C2\u00A0decline\t \u00C2\u00A0in\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0JBC\t \u00C2\u00A0275:31505\u00E2\u0080\u009331513.\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Stefanis\t \u00C2\u00A0L,\t \u00C2\u00A0Fredenburg\t \u00C2\u00A0R,\t \u00C2\u00A0Lansbury\t \u00C2\u00A0PT,\t \u00C2\u00A0Sulzer\t \u00C2\u00A0D\t \u00C2\u00A0(2004)\t \u00C2\u00A0Impaired\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0Mutant\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090Synuclein\t \u00C2\u00A0by\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy.\t \u00C2\u00A0Science\t \u00C2\u00A0305:1292\u00E2\u0080\u00931295.\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Palmer\t \u00C2\u00A0A,\t \u00C2\u00A0Rivett\t \u00C2\u00A0AJ,\t \u00C2\u00A0Knecht\t \u00C2\u00A0E\t \u00C2\u00A0(1995)\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0proteasomes\t \u00C2\u00A0by\t \u00C2\u00A0lysosomes\t \u00C2\u00A0in\t \u00C2\u00A0rat\t \u00C2\u00A0liver.\t \u00C2\u00A0Eur\t \u00C2\u00A0J\t \u00C2\u00A0Biochem\t \u00C2\u00A0227:792\u00E2\u0080\u0093800.\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Wong\t \u00C2\u00A0E\t \u00C2\u00A0(2013)\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy:\t \u00C2\u00A0roles\t \u00C2\u00A0in\t \u00C2\u00A0disease\t \u00C2\u00A0and\t \u00C2\u00A0aging.\t \u00C2\u00A0Cell\t \u00C2\u00A0Research\t \u00C2\u00A024:92\u00E2\u0080\u0093104.\t \u00C2\u00A0Cyrus\t \u00C2\u00A0K,\t \u00C2\u00A0Wehenkel\t \u00C2\u00A0M,\t \u00C2\u00A0Choi\t \u00C2\u00A0E-\u00C2\u00AD\u00E2\u0080\u0090Y,\t \u00C2\u00A0Swanson\t \u00C2\u00A0H,\t \u00C2\u00A0Kim\t \u00C2\u00A0K-\u00C2\u00AD\u00E2\u0080\u0090B\t \u00C2\u00A0(2010)\t \u00C2\u00A0Two-\u00C2\u00AD\u00E2\u0080\u0090headed\t \u00C2\u00A0PROTAC:\t \u00C2\u00A0an\t \u00C2\u00A0effective\t \u00C2\u00A0new\t \u00C2\u00A0tool\t \u00C2\u00A0for\t \u00C2\u00A0targeted\t \u00C2\u00A0protein\t \u00C2\u00A0degradation.\t \u00C2\u00A0Chem\t \u00C2\u00A0Bio\t \u00C2\u00A0Chem\t \u00C2\u00A011:1531\u00E2\u0080\u00931534.\t \u00C2\u00A0 200 Dantuma\t \u00C2\u00A0NP,\t \u00C2\u00A0Bott\t \u00C2\u00A0LC\t \u00C2\u00A0(2014)\t \u00C2\u00A0The\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090proteasome\t \u00C2\u00A0system\t \u00C2\u00A0in\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0diseases:\t \u00C2\u00A0precipitating\t \u00C2\u00A0factor,\t \u00C2\u00A0yet\t \u00C2\u00A0part\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0solution.\t \u00C2\u00A0Front\t \u00C2\u00A0Mol\t \u00C2\u00A0Neurosci\t \u00C2\u00A07:70.\t \u00C2\u00A0Das\t \u00C2\u00A0AA,\t \u00C2\u00A0Sharma\t \u00C2\u00A0OP,\t \u00C2\u00A0Kumar\t \u00C2\u00A0MS,\t \u00C2\u00A0Krishna\t \u00C2\u00A0R,\t \u00C2\u00A0Mathur\t \u00C2\u00A0PP\t \u00C2\u00A0(2013)\t \u00C2\u00A0PepBind:\t \u00C2\u00A0A\t \u00C2\u00A0Comprehensive\t \u00C2\u00A0Database\t \u00C2\u00A0and\t \u00C2\u00A0Computational\t \u00C2\u00A0Tool\t \u00C2\u00A0for\t \u00C2\u00A0Analysis\t \u00C2\u00A0of\t \u00C2\u00A0Protein\u00E2\u0080\u0093peptide\t \u00C2\u00A0Interactions.\t \u00C2\u00A0Genomics,\t \u00C2\u00A0Proteomics\t \u00C2\u00A0&\t \u00C2\u00A0Bioinformatics\t \u00C2\u00A011:241\u00E2\u0080\u0093246.\t \u00C2\u00A0Dauer\t \u00C2\u00A0W,\t \u00C2\u00A0Kholodilov\t \u00C2\u00A0N,\t \u00C2\u00A0Vila\t \u00C2\u00A0M,\t \u00C2\u00A0Trillat\t \u00C2\u00A0A-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Goodchild\t \u00C2\u00A0R,\t \u00C2\u00A0Larsen\t \u00C2\u00A0KE,\t \u00C2\u00A0Staal\t \u00C2\u00A0R,\t \u00C2\u00A0Tieu\t \u00C2\u00A0K,\t \u00C2\u00A0Schmitz\t \u00C2\u00A0Y,\t \u00C2\u00A0Yuan\t \u00C2\u00A0CA,\t \u00C2\u00A0Rocha\t \u00C2\u00A0M,\t \u00C2\u00A0Jackson-\u00C2\u00AD\u00E2\u0080\u0090Lewis\t \u00C2\u00A0V,\t \u00C2\u00A0Hersch\t \u00C2\u00A0S,\t \u00C2\u00A0Sulzer\t \u00C2\u00A0D,\t \u00C2\u00A0Przedborski\t \u00C2\u00A0S,\t \u00C2\u00A0Burke\t \u00C2\u00A0R,\t \u00C2\u00A0Hen\t \u00C2\u00A0R\t \u00C2\u00A0(2002)\t \u00C2\u00A0Resistance\t \u00C2\u00A0of\t \u00C2\u00A0alpha-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0null\t \u00C2\u00A0mice\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0parkinsonian\t \u00C2\u00A0neurotoxin\t \u00C2\u00A0MPTP.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A099:14524\u00E2\u0080\u009314529.\t \u00C2\u00A0Davidson\t \u00C2\u00A0BL,\t \u00C2\u00A0McCray\t \u00C2\u00A0PB\t \u00C2\u00A0(2011)\t \u00C2\u00A0Current\t \u00C2\u00A0prospects\t \u00C2\u00A0for\t \u00C2\u00A0RNA\t \u00C2\u00A0interference-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0therapies.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Genet\t \u00C2\u00A012:329\u00E2\u0080\u0093340.\t \u00C2\u00A0Deshayes\t \u00C2\u00A0S,\t \u00C2\u00A0Heitz\t \u00C2\u00A0A,\t \u00C2\u00A0Morris\t \u00C2\u00A0MC,\t \u00C2\u00A0Charnet\t \u00C2\u00A0P,\t \u00C2\u00A0Divita\t \u00C2\u00A0G,\t \u00C2\u00A0Heitz\t \u00C2\u00A0F\t \u00C2\u00A0(2004)\t \u00C2\u00A0Insight\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0Mechanism\t \u00C2\u00A0of\t \u00C2\u00A0Internalization\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0Cell-\u00C2\u00AD\u00E2\u0080\u0090Penetrating\t \u00C2\u00A0Carrier\t \u00C2\u00A0Peptide\t \u00C2\u00A0Pep-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0through\t \u00C2\u00A0Conformational\t \u00C2\u00A0Analysis.\t \u00C2\u00A0Biochem\t \u00C2\u00A043:1449\u00E2\u0080\u00931457.\t \u00C2\u00A0Dev\t \u00C2\u00A0KK\t \u00C2\u00A0(2004)\t \u00C2\u00A0Making\t \u00C2\u00A0protein\t \u00C2\u00A0interactions\t \u00C2\u00A0druggable:\t \u00C2\u00A0targeting\t \u00C2\u00A0PDZ\t \u00C2\u00A0domains.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A03:1047\u00E2\u0080\u00931056.\t \u00C2\u00A0Dias\t \u00C2\u00A0N,\t \u00C2\u00A0Stein\t \u00C2\u00A0CA\t \u00C2\u00A0(2002)\t \u00C2\u00A0Antisense\t \u00C2\u00A0oligonucleotides:\t \u00C2\u00A0basic\t \u00C2\u00A0concepts\t \u00C2\u00A0and\t \u00C2\u00A0mechanisms.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cancer\t \u00C2\u00A0Ther\t \u00C2\u00A01:347\u00E2\u0080\u0093355.\t \u00C2\u00A0\t \u00C2\u00A0 201 Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(1987)\t \u00C2\u00A0Molecular\t \u00C2\u00A0determinants\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0half-\u00C2\u00AD\u00E2\u0080\u0090lives\t \u00C2\u00A0in\t \u00C2\u00A0eukaryotic\t \u00C2\u00A0cells.\t \u00C2\u00A0FASEB\t \u00C2\u00A0J\t \u00C2\u00A01:349\u00E2\u0080\u0093357.\t \u00C2\u00A0Dice\t \u00C2\u00A0JF\t \u00C2\u00A0(1990)\t \u00C2\u00A0Peptide\t \u00C2\u00A0sequences\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0cytosolic\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0lysosomal\t \u00C2\u00A0proteolysis.\t \u00C2\u00A0Trends\t \u00C2\u00A0Biochem\t \u00C2\u00A0Sci\t \u00C2\u00A015:305\u00E2\u0080\u0093309.\t \u00C2\u00A0Disterer\t \u00C2\u00A0P,\t \u00C2\u00A0Kryczka\t \u00C2\u00A0A,\t \u00C2\u00A0Liu\t \u00C2\u00A0Y,\t \u00C2\u00A0Badi\t \u00C2\u00A0YE,\t \u00C2\u00A0Wong\t \u00C2\u00A0JJ,\t \u00C2\u00A0Owen\t \u00C2\u00A0JS,\t \u00C2\u00A0Khoo\t \u00C2\u00A0B\t \u00C2\u00A0(2014)\t \u00C2\u00A0Development\t \u00C2\u00A0of\t \u00C2\u00A0therapeutic\t \u00C2\u00A0splice-\u00C2\u00AD\u00E2\u0080\u0090switching\t \u00C2\u00A0oligonucleotides.\t \u00C2\u00A0Hum\t \u00C2\u00A0Gene\t \u00C2\u00A0Ther\t \u00C2\u00A025:587\u00E2\u0080\u0093598.\t \u00C2\u00A0Doetschman\t \u00C2\u00A0T\t \u00C2\u00A0(2009)\t \u00C2\u00A0Influence\t \u00C2\u00A0of\t \u00C2\u00A0Genetic\t \u00C2\u00A0Background\t \u00C2\u00A0on\t \u00C2\u00A0Genetically\t \u00C2\u00A0Engineered\t \u00C2\u00A0Mouse\t \u00C2\u00A0Phenotypes.\t \u00C2\u00A0In:\t \u00C2\u00A0Gene\t \u00C2\u00A0knockout\t \u00C2\u00A0protocols,\t \u00C2\u00A02nd\t \u00C2\u00A0ed.,\t \u00C2\u00A0pp\t \u00C2\u00A0423\u00E2\u0080\u0093433\t \u00C2\u00A0Methods\t \u00C2\u00A0in\t \u00C2\u00A0Molecular\t \u00C2\u00A0Biology.\t \u00C2\u00A0New\t \u00C2\u00A0York,\t \u00C2\u00A0NY:\t \u00C2\u00A0Humana\t \u00C2\u00A0Press.\t \u00C2\u00A0Dohi\t \u00C2\u00A0E,\t \u00C2\u00A0Tanaka\t \u00C2\u00A0S,\t \u00C2\u00A0Seki\t \u00C2\u00A0T,\t \u00C2\u00A0Miyagi\t \u00C2\u00A0T,\t \u00C2\u00A0Hide\t \u00C2\u00A0I,\t \u00C2\u00A0Takahashi\t \u00C2\u00A0T,\t \u00C2\u00A0Matsumoto\t \u00C2\u00A0M,\t \u00C2\u00A0Sakai\t \u00C2\u00A0N\t \u00C2\u00A0(2012)\t \u00C2\u00A0Hypoxic\t \u00C2\u00A0stress\t \u00C2\u00A0activates\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0and\t \u00C2\u00A0modulates\t \u00C2\u00A0neuronal\t \u00C2\u00A0cell\t \u00C2\u00A0survival.\t \u00C2\u00A0Neurochem\t \u00C2\u00A0Int\t \u00C2\u00A060:431\u00E2\u0080\u0093442.\t \u00C2\u00A0Dohmen\t \u00C2\u00A0RJ\t \u00C2\u00A0(2000)\t \u00C2\u00A0Primary\t \u00C2\u00A0destruction\t \u00C2\u00A0signals.\t \u00C2\u00A0IN:\t \u00C2\u00A0Proteasomes:\t \u00C2\u00A0the\t \u00C2\u00A0world\t \u00C2\u00A0of\t \u00C2\u00A0regulatory\t \u00C2\u00A0proteolysis,\t \u00C2\u00A01st\t \u00C2\u00A0ed.,\t \u00C2\u00A0pp\t \u00C2\u00A0188\u00E2\u0080\u0093205\t \u00C2\u00A0Molecular\t \u00C2\u00A0Biology\t \u00C2\u00A0Intelligence\t \u00C2\u00A0Unit.\t \u00C2\u00A0Georgetown,\t \u00C2\u00A0TX:\t \u00C2\u00A0RG\t \u00C2\u00A0Landes\t \u00C2\u00A0Biosciences.\t \u00C2\u00A0Dohmen\t \u00C2\u00A0RJ,\t \u00C2\u00A0Wu\t \u00C2\u00A0P,\t \u00C2\u00A0Varshavsky\t \u00C2\u00A0A\t \u00C2\u00A0(1994)\t \u00C2\u00A0Heat-\u00C2\u00AD\u00E2\u0080\u0090inducible\t \u00C2\u00A0degron:\t \u00C2\u00A0a\t \u00C2\u00A0method\t \u00C2\u00A0for\t \u00C2\u00A0constructing\t \u00C2\u00A0temperature-\u00C2\u00AD\u00E2\u0080\u0090sensitive\t \u00C2\u00A0mutants.\t \u00C2\u00A0Science\t \u00C2\u00A0263:1273\u00E2\u0080\u00931276.\t \u00C2\u00A0Drews\t \u00C2\u00A0J\t \u00C2\u00A0(1998)\t \u00C2\u00A0Biotechnology's\t \u00C2\u00A0metamorphosis\t \u00C2\u00A0into\t \u00C2\u00A0a\t \u00C2\u00A0drug\t \u00C2\u00A0discovery\t \u00C2\u00A0industry.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A016:22\u00E2\u0080\u009324.\t \u00C2\u00A0 202 Dykxhoorn\t \u00C2\u00A0DM,\t \u00C2\u00A0Lieberman\t \u00C2\u00A0J\t \u00C2\u00A0(2005)\t \u00C2\u00A0The\t \u00C2\u00A0Silent\t \u00C2\u00A0Revolution:\t \u00C2\u00A0RNA\t \u00C2\u00A0Interference\t \u00C2\u00A0as\t \u00C2\u00A0Basic\t \u00C2\u00A0Biology,\t \u00C2\u00A0Research\t \u00C2\u00A0Tool,\t \u00C2\u00A0and\t \u00C2\u00A0Therapeutic.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Med\t \u00C2\u00A056:401\u00E2\u0080\u0093423.\t \u00C2\u00A0Ebrahimi-\u00C2\u00AD\u00E2\u0080\u0090Fakhari\t \u00C2\u00A0D,\t \u00C2\u00A0Cantuti-\u00C2\u00AD\u00E2\u0080\u0090Castelvetri\t \u00C2\u00A0I,\t \u00C2\u00A0Fan\t \u00C2\u00A0Z,\t \u00C2\u00A0Rockenstein\t \u00C2\u00A0E,\t \u00C2\u00A0Masliah\t \u00C2\u00A0E,\t \u00C2\u00A0Hyman\t \u00C2\u00A0BT,\t \u00C2\u00A0McLean\t \u00C2\u00A0PJ,\t \u00C2\u00A0Unni\t \u00C2\u00A0VK\t \u00C2\u00A0(2011)\t \u00C2\u00A0Distinct\t \u00C2\u00A0Roles\t \u00C2\u00A0In\t \u00C2\u00A0Vivo\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0Ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090Proteasome\t \u00C2\u00A0System\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0Autophagy-\u00C2\u00AD\u00E2\u0080\u0090Lysosomal\t \u00C2\u00A0Pathway\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090Synuclein.\t \u00C2\u00A0J\t \u00C2\u00A0Neurosci\t \u00C2\u00A031:14508\u00E2\u0080\u009314520.\t \u00C2\u00A0Eisenberg-\u00C2\u00AD\u00E2\u0080\u0090Lerner\t \u00C2\u00A0A,\t \u00C2\u00A0Kimchi\t \u00C2\u00A0A\t \u00C2\u00A0(2007)\t \u00C2\u00A0DAP\t \u00C2\u00A0kinase\t \u00C2\u00A0regulates\t \u00C2\u00A0JNK\t \u00C2\u00A0signaling\t \u00C2\u00A0by\t \u00C2\u00A0binding\t \u00C2\u00A0and\t \u00C2\u00A0activating\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0D\t \u00C2\u00A0under\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress.\t \u00C2\u00A0Cell\t \u00C2\u00A0Death\t \u00C2\u00A0Differ\t \u00C2\u00A014:1908\u00E2\u0080\u00931915.\t \u00C2\u00A0El-\u00C2\u00AD\u00E2\u0080\u0090Husseini\t \u00C2\u00A0AE,\t \u00C2\u00A0Schnell\t \u00C2\u00A0E,\t \u00C2\u00A0Chetkovich\t \u00C2\u00A0DM,\t \u00C2\u00A0Nicoll\t \u00C2\u00A0RA,\t \u00C2\u00A0Bredt\t \u00C2\u00A0DS\t \u00C2\u00A0(2000)\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0involvement\t \u00C2\u00A0in\t \u00C2\u00A0maturation\t \u00C2\u00A0of\t \u00C2\u00A0excitatory\t \u00C2\u00A0synapses.\t \u00C2\u00A0Science\t \u00C2\u00A0290:1364\u00E2\u0080\u00931368.\t \u00C2\u00A0Erales\t \u00C2\u00A0J,\t \u00C2\u00A0Coffino\t \u00C2\u00A0P\t \u00C2\u00A0(2014)\t \u00C2\u00A0Ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090independent\t \u00C2\u00A0proteasomal\t \u00C2\u00A0degradation.\t \u00C2\u00A0Biochimica\t \u00C2\u00A0et\t \u00C2\u00A0Biophysica\t \u00C2\u00A0Acta\t \u00C2\u00A01843:\t \u00C2\u00A0216-\u00C2\u00AD\u00E2\u0080\u0090221.\t \u00C2\u00A0Eskelinen\t \u00C2\u00A0E-\u00C2\u00AD\u00E2\u0080\u0090L,\t \u00C2\u00A0Tanaka\t \u00C2\u00A0Y,\t \u00C2\u00A0Saftig\t \u00C2\u00A0P\t \u00C2\u00A0(2003)\t \u00C2\u00A0At\t \u00C2\u00A0the\t \u00C2\u00A0acidic\t \u00C2\u00A0edge:\t \u00C2\u00A0emerging\t \u00C2\u00A0functions\t \u00C2\u00A0for\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins.\t \u00C2\u00A0Trends\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A013:137\u00E2\u0080\u0093145.\t \u00C2\u00A0Fan\t \u00C2\u00A0X,\t \u00C2\u00A0Jin\t \u00C2\u00A0WY,\t \u00C2\u00A0Lu\t \u00C2\u00A0J,\t \u00C2\u00A0Wang\t \u00C2\u00A0J,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2014)\t \u00C2\u00A0Rapid\t \u00C2\u00A0and\t \u00C2\u00A0reversible\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0endogenous\t \u00C2\u00A0proteins\t \u00C2\u00A0by\t \u00C2\u00A0peptide-\u00C2\u00AD\u00E2\u0080\u0090directed\t \u00C2\u00A0lysosomal\t \u00C2\u00A0degradation.\t \u00C2\u00A0Nat\t \u00C2\u00A0Neurosci\t \u00C2\u00A017:471\u00E2\u0080\u0093480.\t \u00C2\u00A0Feng\t \u00C2\u00A0Y,\t \u00C2\u00A0He\t \u00C2\u00A0D,\t \u00C2\u00A0Yao\t \u00C2\u00A0Z,\t \u00C2\u00A0Klionsky\t \u00C2\u00A0DJ\t \u00C2\u00A0(2013)\t \u00C2\u00A0The\t \u00C2\u00A0machinery\t \u00C2\u00A0of\t \u00C2\u00A0macroautophagy.\t \u00C2\u00A0Cell\t \u00C2\u00A0Research\t \u00C2\u00A024:24\u00E2\u0080\u009341.\t \u00C2\u00A0 203 Fire\t \u00C2\u00A0A,\t \u00C2\u00A0Xu\t \u00C2\u00A0S,\t \u00C2\u00A0Montgomery\t \u00C2\u00A0MK,\t \u00C2\u00A0Kostas\t \u00C2\u00A0SA,\t \u00C2\u00A0Driver\t \u00C2\u00A0SE,\t \u00C2\u00A0Mello\t \u00C2\u00A0CC\t \u00C2\u00A0(1998)\t \u00C2\u00A0Potent\t \u00C2\u00A0and\t \u00C2\u00A0specific\t \u00C2\u00A0genetic\t \u00C2\u00A0interference\t \u00C2\u00A0by\t \u00C2\u00A0double-\u00C2\u00AD\u00E2\u0080\u0090stranded\t \u00C2\u00A0RNA\t \u00C2\u00A0in\t \u00C2\u00A0Caenorhabditis\t \u00C2\u00A0elegans.\t \u00C2\u00A0Nature\t \u00C2\u00A0391:806\u00E2\u0080\u0093811.\t \u00C2\u00A0Flight\t \u00C2\u00A0MH\t \u00C2\u00A0(2013)\t \u00C2\u00A0New\t \u00C2\u00A0kinase\t \u00C2\u00A0targets\t \u00C2\u00A0for\t \u00C2\u00A0Alzheimer\u00E2\u0080\u0099s\t \u00C2\u00A0disease.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A012:739\u00E2\u0080\u0093739.\t \u00C2\u00A0Fu\t \u00C2\u00A0Y,\t \u00C2\u00A0Foden\t \u00C2\u00A0JA,\t \u00C2\u00A0Khayter\t \u00C2\u00A0C,\t \u00C2\u00A0Maeder\t \u00C2\u00A0ML,\t \u00C2\u00A0Reyon\t \u00C2\u00A0D,\t \u00C2\u00A0Joung\t \u00C2\u00A0JK,\t \u00C2\u00A0Sander\t \u00C2\u00A0JD\t \u00C2\u00A0(2013)\t \u00C2\u00A0High-\u00C2\u00AD\u00E2\u0080\u0090frequency\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0induced\t \u00C2\u00A0by\t \u00C2\u00A0CRISPR-\u00C2\u00AD\u00E2\u0080\u0090Cas\t \u00C2\u00A0nucleases\t \u00C2\u00A0in\t \u00C2\u00A0human\t \u00C2\u00A0cells.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A031:822\u00E2\u0080\u0093826.\t \u00C2\u00A0Fujita\t \u00C2\u00A0Y,\t \u00C2\u00A0Yamashita\t \u00C2\u00A0T\t \u00C2\u00A0(2014)\t \u00C2\u00A0Role\t \u00C2\u00A0of\t \u00C2\u00A0DAPK\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0cell\t \u00C2\u00A0death.\t \u00C2\u00A0Apoptosis\t \u00C2\u00A019:339-\u00C2\u00AD\u00E2\u0080\u0090345.\t \u00C2\u00A0Gaj\t \u00C2\u00A0T,\t \u00C2\u00A0Gersbach\t \u00C2\u00A0CA,\t \u00C2\u00A0Barbas\t \u00C2\u00A0CF\t \u00C2\u00A0III\t \u00C2\u00A0(2013)\t \u00C2\u00A0ZFN,\t \u00C2\u00A0TALEN,\t \u00C2\u00A0and\t \u00C2\u00A0CRISPR/Cas-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0methods\t \u00C2\u00A0for\t \u00C2\u00A0genome\t \u00C2\u00A0engineering.\t \u00C2\u00A0Trends\t \u00C2\u00A0Biotechnol\t \u00C2\u00A031:397\u00E2\u0080\u0093405.\t \u00C2\u00A0Garriga-\u00C2\u00AD\u00E2\u0080\u0090Canut\t \u00C2\u00A0M,\t \u00C2\u00A0Agust\u00C3\u00ADn-\u00C2\u00AD\u00E2\u0080\u0090Pav\u00C3\u00B3n\t \u00C2\u00A0C,\t \u00C2\u00A0Herrmann\t \u00C2\u00A0F,\t \u00C2\u00A0S\u00C3\u00A1nchez\t \u00C2\u00A0A,\t \u00C2\u00A0Dierssen\t \u00C2\u00A0M,\t \u00C2\u00A0Fillat\t \u00C2\u00A0C,\t \u00C2\u00A0Isalan\t \u00C2\u00A0M\t \u00C2\u00A0(2012)\t \u00C2\u00A0Synthetic\t \u00C2\u00A0zinc\t \u00C2\u00A0finger\t \u00C2\u00A0repressors\t \u00C2\u00A0reduce\t \u00C2\u00A0mutant\t \u00C2\u00A0huntingtin\t \u00C2\u00A0expression\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0brain\t \u00C2\u00A0of\t \u00C2\u00A0R6/2\t \u00C2\u00A0mice.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0109:3136\u00E2\u0080\u00933145.\t \u00C2\u00A0Gentilucci\t \u00C2\u00A0L,\t \u00C2\u00A0De\t \u00C2\u00A0Marco\t \u00C2\u00A0R,\t \u00C2\u00A0Cerisoli\t \u00C2\u00A0L\t \u00C2\u00A0(2010)\t \u00C2\u00A0Chemical\t \u00C2\u00A0modifications\t \u00C2\u00A0designed\t \u00C2\u00A0to\t \u00C2\u00A0improve\t \u00C2\u00A0peptide\t \u00C2\u00A0stability:\t \u00C2\u00A0incorporation\t \u00C2\u00A0of\t \u00C2\u00A0non-\u00C2\u00AD\u00E2\u0080\u0090natural\t \u00C2\u00A0amino\t \u00C2\u00A0acids,\t \u00C2\u00A0pseudo-\u00C2\u00AD\u00E2\u0080\u0090peptide\t \u00C2\u00A0bonds,\t \u00C2\u00A0and\t \u00C2\u00A0cyclization.\t \u00C2\u00A0Curr\t \u00C2\u00A0Pharm\t \u00C2\u00A0Des\t \u00C2\u00A016:3185\u00E2\u0080\u00933203.\t \u00C2\u00A0Giepmans\t \u00C2\u00A0BNG\t \u00C2\u00A0(2006)\t \u00C2\u00A0The\t \u00C2\u00A0Fluorescent\t \u00C2\u00A0Toolbox\t \u00C2\u00A0for\t \u00C2\u00A0Assessing\t \u00C2\u00A0Protein\t \u00C2\u00A0Location\t \u00C2\u00A0and\t \u00C2\u00A0Function.\t \u00C2\u00A0Science\t \u00C2\u00A0312:217\u00E2\u0080\u0093224.\t \u00C2\u00A0 204 Goldberg\t \u00C2\u00A0A,\t \u00C2\u00A0Dice\t \u00C2\u00A0J\t \u00C2\u00A0(1974)\t \u00C2\u00A0Intracellular\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0and\t \u00C2\u00A0bacterial\t \u00C2\u00A0cells.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Biochem\t \u00C2\u00A043:835\u00E2\u0080\u0093869.\t \u00C2\u00A0Goldberg\t \u00C2\u00A0AL\t \u00C2\u00A0(2003)\t \u00C2\u00A0Protein\t \u00C2\u00A0degradation\t \u00C2\u00A0and\t \u00C2\u00A0protection\t \u00C2\u00A0against\t \u00C2\u00A0misfolded\t \u00C2\u00A0or\t \u00C2\u00A0damaged\t \u00C2\u00A0proteins.\t \u00C2\u00A0Nature\t \u00C2\u00A0426:895\u00E2\u0080\u0093899.\t \u00C2\u00A0Gosink\t \u00C2\u00A0MM,\t \u00C2\u00A0Vierstra\t \u00C2\u00A0RD\t \u00C2\u00A0(1995)\t \u00C2\u00A0Redirecting\t \u00C2\u00A0the\t \u00C2\u00A0specificity\t \u00C2\u00A0of\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0by\t \u00C2\u00A0modifying\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090conjugating\t \u00C2\u00A0enzymes.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A092:9117\u00E2\u0080\u00939121.\t \u00C2\u00A0Gottardi\t \u00C2\u00A0CJ\t \u00C2\u00A0(2004)\t \u00C2\u00A0Distinct\t \u00C2\u00A0molecular\t \u00C2\u00A0forms\t \u00C2\u00A0of\t \u00C2\u00A0beta-\u00C2\u00AD\u00E2\u0080\u0090catenin\t \u00C2\u00A0are\t \u00C2\u00A0targeted\t \u00C2\u00A0to\t \u00C2\u00A0adhesive\t \u00C2\u00A0or\t \u00C2\u00A0transcriptional\t \u00C2\u00A0complexes.\t \u00C2\u00A0J\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A0167:339\u00E2\u0080\u0093349.\t \u00C2\u00A0Gozuacik\t \u00C2\u00A0D,\t \u00C2\u00A0Bialik\t \u00C2\u00A0S,\t \u00C2\u00A0Raveh\t \u00C2\u00A0T,\t \u00C2\u00A0Mitou\t \u00C2\u00A0G,\t \u00C2\u00A0Shohat\t \u00C2\u00A0G,\t \u00C2\u00A0Sabanay\t \u00C2\u00A0H,\t \u00C2\u00A0Mizushima\t \u00C2\u00A0N,\t \u00C2\u00A0Yoshimori\t \u00C2\u00A0T,\t \u00C2\u00A0Kimchi\t \u00C2\u00A0A\t \u00C2\u00A0(2008)\t \u00C2\u00A0DAP-\u00C2\u00AD\u00E2\u0080\u0090kinase\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0mediator\t \u00C2\u00A0of\t \u00C2\u00A0endoplasmic\t \u00C2\u00A0reticulum\t \u00C2\u00A0stress-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0caspase\t \u00C2\u00A0activation\t \u00C2\u00A0and\t \u00C2\u00A0autophagic\t \u00C2\u00A0cell\t \u00C2\u00A0death.\t \u00C2\u00A0Cell\t \u00C2\u00A0Death\t \u00C2\u00A0Differ\t \u00C2\u00A015(12):1875\u00E2\u0080\u00931886.\t \u00C2\u00A0Grigoryan\t \u00C2\u00A0G,\t \u00C2\u00A0Reinke\t \u00C2\u00A0AW,\t \u00C2\u00A0Keating\t \u00C2\u00A0AE\t \u00C2\u00A0(2009)\t \u00C2\u00A0Design\t \u00C2\u00A0of\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090interaction\t \u00C2\u00A0specificity\t \u00C2\u00A0gives\t \u00C2\u00A0selective\t \u00C2\u00A0bZIP-\u00C2\u00AD\u00E2\u0080\u0090binding\t \u00C2\u00A0peptides.\t \u00C2\u00A0Nature\t \u00C2\u00A0458:859\u00E2\u0080\u0093864.\t \u00C2\u00A0Grimm\t \u00C2\u00A0D,\t \u00C2\u00A0Streetz\t \u00C2\u00A0KL,\t \u00C2\u00A0Jopling\t \u00C2\u00A0CL,\t \u00C2\u00A0Storm\t \u00C2\u00A0TA,\t \u00C2\u00A0Pandey\t \u00C2\u00A0K,\t \u00C2\u00A0Davis\t \u00C2\u00A0CR,\t \u00C2\u00A0Marion\t \u00C2\u00A0P,\t \u00C2\u00A0Salazar\t \u00C2\u00A0F,\t \u00C2\u00A0Kay\t \u00C2\u00A0MA\t \u00C2\u00A0(2006)\t \u00C2\u00A0Fatality\t \u00C2\u00A0in\t \u00C2\u00A0mice\t \u00C2\u00A0due\t \u00C2\u00A0to\t \u00C2\u00A0oversaturation\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0microRNA/short\t \u00C2\u00A0hairpin\t \u00C2\u00A0RNA\t \u00C2\u00A0pathways.\t \u00C2\u00A0Nature\t \u00C2\u00A0441:537\u00E2\u0080\u0093541.\t \u00C2\u00A0Gu\t \u00C2\u00A0H,\t \u00C2\u00A0Marth\t \u00C2\u00A0JD,\t \u00C2\u00A0Orban\t \u00C2\u00A0PC,\t \u00C2\u00A0Mossmann\t \u00C2\u00A0H,\t \u00C2\u00A0Rajewsky\t \u00C2\u00A0K\t \u00C2\u00A0(1994)\t \u00C2\u00A0Deletion\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0DNA\t \u00C2\u00A0polymerase\t \u00C2\u00A0beta\t \u00C2\u00A0gene\t \u00C2\u00A0segment\t \u00C2\u00A0in\t \u00C2\u00A0T\t \u00C2\u00A0cells\t \u00C2\u00A0using\t \u00C2\u00A0cell\t \u00C2\u00A0type-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0gene\t \u00C2\u00A0targeting.\t \u00C2\u00A0Science\t \u00C2\u00A0265:103\u00E2\u0080\u0093106.\t \u00C2\u00A0 205 Gump\t \u00C2\u00A0JM,\t \u00C2\u00A0Dowdy\t \u00C2\u00A0SF\t \u00C2\u00A0(2007)\t \u00C2\u00A0TAT\t \u00C2\u00A0transduction:\t \u00C2\u00A0the\t \u00C2\u00A0molecular\t \u00C2\u00A0mechanism\t \u00C2\u00A0and\t \u00C2\u00A0therapeutic\t \u00C2\u00A0prospects.\t \u00C2\u00A0Trends\t \u00C2\u00A0Mol\t \u00C2\u00A0Med\t \u00C2\u00A013:443\u00E2\u0080\u0093448.\t \u00C2\u00A0Gupta\t \u00C2\u00A0S,\t \u00C2\u00A0Jain\t \u00C2\u00A0A,\t \u00C2\u00A0Chakraborty\t \u00C2\u00A0M,\t \u00C2\u00A0Sahni\t \u00C2\u00A0JK,\t \u00C2\u00A0Ali\t \u00C2\u00A0J,\t \u00C2\u00A0Dang\t \u00C2\u00A0S\t \u00C2\u00A0(2013)\t \u00C2\u00A0Oral\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0therapeutic\t \u00C2\u00A0proteins\t \u00C2\u00A0and\t \u00C2\u00A0peptides:\t \u00C2\u00A0a\t \u00C2\u00A0review\t \u00C2\u00A0on\t \u00C2\u00A0recent\t \u00C2\u00A0developments.\t \u00C2\u00A0Drug\t \u00C2\u00A0Deliv\t \u00C2\u00A020:237\u00E2\u0080\u0093246.\t \u00C2\u00A0Hainsworth\t \u00C2\u00A0AH,\t \u00C2\u00A0Allsopp\t \u00C2\u00A0RC,\t \u00C2\u00A0Jim\t \u00C2\u00A0A,\t \u00C2\u00A0Potter\t \u00C2\u00A0JF,\t \u00C2\u00A0Lowe\t \u00C2\u00A0J,\t \u00C2\u00A0Talbot\t \u00C2\u00A0CJ,\t \u00C2\u00A0Prettyman\t \u00C2\u00A0RJ\t \u00C2\u00A0(2010)\t \u00C2\u00A0Death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0(DAPK1)\t \u00C2\u00A0in\t \u00C2\u00A0cerebral\t \u00C2\u00A0cortex\t \u00C2\u00A0of\t \u00C2\u00A0late-\u00C2\u00AD\u00E2\u0080\u0090onset\t \u00C2\u00A0Alzheimer's\t \u00C2\u00A0disease\t \u00C2\u00A0patients\t \u00C2\u00A0and\t \u00C2\u00A0aged\t \u00C2\u00A0controls.\t \u00C2\u00A0Neuropath\t \u00C2\u00A0Appl\t \u00C2\u00A0Neuro\t \u00C2\u00A036:17\u00E2\u0080\u009324.\t \u00C2\u00A0Hanson\t \u00C2\u00A0SK,\t \u00C2\u00A0Grotta\t \u00C2\u00A0JC,\t \u00C2\u00A0Waxham\t \u00C2\u00A0MN,\t \u00C2\u00A0Aronowski\t \u00C2\u00A0J,\t \u00C2\u00A0Ostrow\t \u00C2\u00A0P\t \u00C2\u00A0(1994)\t \u00C2\u00A0Calcium/calmodulin-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0II\t \u00C2\u00A0activity\t \u00C2\u00A0in\t \u00C2\u00A0focal\t \u00C2\u00A0ischemia\t \u00C2\u00A0with\t \u00C2\u00A0reperfusion\t \u00C2\u00A0in\t \u00C2\u00A0rats.\t \u00C2\u00A0Stroke\t \u00C2\u00A025:466\u00E2\u0080\u0093473.\t \u00C2\u00A0Hara\t \u00C2\u00A0MR,\t \u00C2\u00A0Snyder\t \u00C2\u00A0SH\t \u00C2\u00A0(2007)\t \u00C2\u00A0Cell\t \u00C2\u00A0Signaling\t \u00C2\u00A0and\t \u00C2\u00A0Neuronal\t \u00C2\u00A0Death.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Pharmacol\t \u00C2\u00A0Toxicol\t \u00C2\u00A047:117\u00E2\u0080\u0093141.\t \u00C2\u00A0Harris\t \u00C2\u00A0H,\t \u00C2\u00A0Rubinsztein\t \u00C2\u00A0DC\t \u00C2\u00A0(2011)\t \u00C2\u00A0Control\t \u00C2\u00A0of\t \u00C2\u00A0autophagy\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapy\t \u00C2\u00A0for\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0disease.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Neurol\t \u00C2\u00A08:108\u00E2\u0080\u0093117.\t \u00C2\u00A0Hayashita-\u00C2\u00AD\u00E2\u0080\u0090Kinoh\t \u00C2\u00A0H,\t \u00C2\u00A0Yamada\t \u00C2\u00A0M,\t \u00C2\u00A0Yokota\t \u00C2\u00A0T,\t \u00C2\u00A0Mizuno\t \u00C2\u00A0Y,\t \u00C2\u00A0Mochizuki\t \u00C2\u00A0H\t \u00C2\u00A0(2006)\t \u00C2\u00A0Down-\u00C2\u00AD\u00E2\u0080\u0090regulation\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0expression\t \u00C2\u00A0can\t \u00C2\u00A0rescue\t \u00C2\u00A0dopaminergic\t \u00C2\u00A0cells\t \u00C2\u00A0from\t \u00C2\u00A0cell\t \u00C2\u00A0death\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0substantia\t \u00C2\u00A0nigra\t \u00C2\u00A0of\t \u00C2\u00A0Parkinson\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0rat\t \u00C2\u00A0model.\t \u00C2\u00A0Biochem\t \u00C2\u00A0Biophys\t \u00C2\u00A0Res\t \u00C2\u00A0Comms\t \u00C2\u00A0341:1088\u00E2\u0080\u00931095.\t \u00C2\u00A0\t \u00C2\u00A0 206 Heffner\t \u00C2\u00A0CS,\t \u00C2\u00A0Pratt\t \u00C2\u00A0CH,\t \u00C2\u00A0Babiuk\t \u00C2\u00A0RP,\t \u00C2\u00A0Sharma\t \u00C2\u00A0Y,\t \u00C2\u00A0Rockwood\t \u00C2\u00A0SF,\t \u00C2\u00A0Donahue\t \u00C2\u00A0LR,\t \u00C2\u00A0Eppig\t \u00C2\u00A0JT,\t \u00C2\u00A0Murray\t \u00C2\u00A0SA\t \u00C2\u00A0(2012)\t \u00C2\u00A0Supporting\t \u00C2\u00A0conditional\t \u00C2\u00A0mouse\t \u00C2\u00A0mutagenesis\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0comprehensive\t \u00C2\u00A0cre\t \u00C2\u00A0characterization\t \u00C2\u00A0resource.\t \u00C2\u00A0Nat\t \u00C2\u00A0Comms\t \u00C2\u00A03:1218\u00E2\u0080\u00931219.\t \u00C2\u00A0Henshall\t \u00C2\u00A0DC,\t \u00C2\u00A0Araki\t \u00C2\u00A0T,\t \u00C2\u00A0Schindler\t \u00C2\u00A0CK,\t \u00C2\u00A0Shinoda\t \u00C2\u00A0S,\t \u00C2\u00A0Lan\t \u00C2\u00A0J-\u00C2\u00AD\u00E2\u0080\u0090Q,\t \u00C2\u00A0Simon\t \u00C2\u00A0RP\t \u00C2\u00A0(2003)\t \u00C2\u00A0Expression\t \u00C2\u00A0of\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0and\t \u00C2\u00A0recruitment\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0tumor\t \u00C2\u00A0necrosis\t \u00C2\u00A0factor\t \u00C2\u00A0signaling\t \u00C2\u00A0pathway\t \u00C2\u00A0following\t \u00C2\u00A0brief\t \u00C2\u00A0seizures.\t \u00C2\u00A0J\t \u00C2\u00A0Neurochem\t \u00C2\u00A086:1260\u00E2\u0080\u00931270.\t \u00C2\u00A0Henshall\t \u00C2\u00A0DC,\t \u00C2\u00A0Schindler\t \u00C2\u00A0CK,\t \u00C2\u00A0So\t \u00C2\u00A0NK,\t \u00C2\u00A0Lan\t \u00C2\u00A0J-\u00C2\u00AD\u00E2\u0080\u0090Q,\t \u00C2\u00A0Meller\t \u00C2\u00A0R,\t \u00C2\u00A0Simon\t \u00C2\u00A0RP\t \u00C2\u00A0(2004)\t \u00C2\u00A0Death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0expression\t \u00C2\u00A0in\t \u00C2\u00A0human\t \u00C2\u00A0temporal\t \u00C2\u00A0lobe\t \u00C2\u00A0epilepsy.\t \u00C2\u00A0Ann\t \u00C2\u00A0Neurol\t \u00C2\u00A055:485\u00E2\u0080\u0093494.\t \u00C2\u00A0Hershko\t \u00C2\u00A0A,\t \u00C2\u00A0Ciechanover\t \u00C2\u00A0A\t \u00C2\u00A0(1998)\t \u00C2\u00A0The\t \u00C2\u00A0ubiquitin\t \u00C2\u00A0system.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Biochem\t \u00C2\u00A067:425\u00E2\u0080\u0093479.\t \u00C2\u00A0Hilgeroth\t \u00C2\u00A0A\t \u00C2\u00A0(2013)\t \u00C2\u00A0Recent\t \u00C2\u00A0developments\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0inhibitors\t \u00C2\u00A0as\t \u00C2\u00A0potential\t \u00C2\u00A0AD\t \u00C2\u00A0therapeutics.\t \u00C2\u00A0Front\t \u00C2\u00A0Cell\t \u00C2\u00A0Neurosci\t \u00C2\u00A07:189.\t \u00C2\u00A0Hill\t \u00C2\u00A0MD\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2012)\t \u00C2\u00A0Safety\t \u00C2\u00A0and\t \u00C2\u00A0efficacy\t \u00C2\u00A0of\t \u00C2\u00A0NA-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0in\t \u00C2\u00A0patients\t \u00C2\u00A0with\t \u00C2\u00A0iatrogenic\t \u00C2\u00A0stroke\t \u00C2\u00A0after\t \u00C2\u00A0endovascular\t \u00C2\u00A0aneurysm\t \u00C2\u00A0repair\t \u00C2\u00A0(ENACT):\t \u00C2\u00A0a\t \u00C2\u00A0phase\t \u00C2\u00A02,\t \u00C2\u00A0randomised,\t \u00C2\u00A0double-\u00C2\u00AD\u00E2\u0080\u0090blind,\t \u00C2\u00A0placebo-\u00C2\u00AD\u00E2\u0080\u0090controlled\t \u00C2\u00A0trial.\t \u00C2\u00A0Lancet\t \u00C2\u00A0Neurol\t \u00C2\u00A011:942\u00E2\u0080\u0093950.\t \u00C2\u00A0Hines\t \u00C2\u00A0J,\t \u00C2\u00A0Gough\t \u00C2\u00A0JD,\t \u00C2\u00A0Corson\t \u00C2\u00A0TW,\t \u00C2\u00A0Crews\t \u00C2\u00A0CM\t \u00C2\u00A0(2013).\t \u00C2\u00A0Posttranslational\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0coupled\t \u00C2\u00A0to\t \u00C2\u00A0receptor\t \u00C2\u00A0tyrosine\t \u00C2\u00A0kinase\t \u00C2\u00A0activation\t \u00C2\u00A0with\t \u00C2\u00A0phosphoPROTACs.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0110:8942-\u00C2\u00AD\u00E2\u0080\u00908947.\t \u00C2\u00A0Holland\t \u00C2\u00A0AJ,\t \u00C2\u00A0Fachinetti\t \u00C2\u00A0D,\t \u00C2\u00A0Han\t \u00C2\u00A0JS,\t \u00C2\u00A0Cleveland\t \u00C2\u00A0DW\t \u00C2\u00A0(2012)\t \u00C2\u00A0Inducible,\t \u00C2\u00A0reversible\t \u00C2\u00A0system\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0rapid\t \u00C2\u00A0and\t \u00C2\u00A0complete\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0 207 Sci\t \u00C2\u00A0USA\t \u00C2\u00A0109:3350\u00E2\u0080\u00933357.\t \u00C2\u00A0Hu\t \u00C2\u00A0J,\t \u00C2\u00A0Matsui\t \u00C2\u00A0M,\t \u00C2\u00A0Gagnon\t \u00C2\u00A0KT,\t \u00C2\u00A0Schwartz\t \u00C2\u00A0JC,\t \u00C2\u00A0Gabillet\t \u00C2\u00A0S,\t \u00C2\u00A0Arar\t \u00C2\u00A0K,\t \u00C2\u00A0Wu\t \u00C2\u00A0J,\t \u00C2\u00A0Bezprozvanny\t \u00C2\u00A0I,\t \u00C2\u00A0Corey\t \u00C2\u00A0DR\t \u00C2\u00A0(2009)\t \u00C2\u00A0Allele-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0silencing\t \u00C2\u00A0of\t \u00C2\u00A0mutant\t \u00C2\u00A0huntingtin\t \u00C2\u00A0and\t \u00C2\u00A0ataxin-\u00C2\u00AD\u00E2\u0080\u00903\t \u00C2\u00A0genes\t \u00C2\u00A0by\t \u00C2\u00A0targeting\t \u00C2\u00A0expanded\t \u00C2\u00A0CAG\t \u00C2\u00A0repeats\t \u00C2\u00A0in\t \u00C2\u00A0mRNAs.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A027:478\u00E2\u0080\u0093484.\t \u00C2\u00A0Huang\t \u00C2\u00A0Y,\t \u00C2\u00A0Chen\t \u00C2\u00A0L,\t \u00C2\u00A0Guo\t \u00C2\u00A0L,\t \u00C2\u00A0Hupp\t \u00C2\u00A0TR,\t \u00C2\u00A0Lin\t \u00C2\u00A0Y\t \u00C2\u00A0(2014)\t \u00C2\u00A0Evaluating\t \u00C2\u00A0DAPK\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic\t \u00C2\u00A0target.\t \u00C2\u00A0Apoptosis\t \u00C2\u00A019:371-\u00C2\u00AD\u00E2\u0080\u0090386.\t \u00C2\u00A0Hwang\t \u00C2\u00A0CS,\t \u00C2\u00A0Shemorry\t \u00C2\u00A0A,\t \u00C2\u00A0Varshavsky\t \u00C2\u00A0A\t \u00C2\u00A0(2010)\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090Terminal\t \u00C2\u00A0Acetylation\t \u00C2\u00A0of\t \u00C2\u00A0Cellular\t \u00C2\u00A0Proteins\t \u00C2\u00A0Creates\t \u00C2\u00A0Specific\t \u00C2\u00A0Degradation\t \u00C2\u00A0Signals.\t \u00C2\u00A0Science\t \u00C2\u00A0327:973\u00E2\u0080\u0093977.\t \u00C2\u00A0Ikonomidou\t \u00C2\u00A0C,\t \u00C2\u00A0Turski\t \u00C2\u00A0L\t \u00C2\u00A0(2002)\t \u00C2\u00A0Why\t \u00C2\u00A0did\t \u00C2\u00A0NMDA\t \u00C2\u00A0receptor\t \u00C2\u00A0antagonists\t \u00C2\u00A0fail\t \u00C2\u00A0clinical\t \u00C2\u00A0trials\t \u00C2\u00A0for\t \u00C2\u00A0stroke\t \u00C2\u00A0and\t \u00C2\u00A0traumatic\t \u00C2\u00A0brain\t \u00C2\u00A0injury?\t \u00C2\u00A0Lancet\t \u00C2\u00A0Neurol\t \u00C2\u00A01:383\u00E2\u0080\u0093386.\t \u00C2\u00A0Inoue\t \u00C2\u00A0M,\t \u00C2\u00A0Mukai\t \u00C2\u00A0M,\t \u00C2\u00A0Hamanaka\t \u00C2\u00A0Y,\t \u00C2\u00A0Tatsuta\t \u00C2\u00A0M,\t \u00C2\u00A0Hiraoka\t \u00C2\u00A0M,\t \u00C2\u00A0Kizaka-\u00C2\u00AD\u00E2\u0080\u0090Kondo\t \u00C2\u00A0S\t \u00C2\u00A0(2004)\t \u00C2\u00A0Targeting\t \u00C2\u00A0hypoxic\t \u00C2\u00A0cancer\t \u00C2\u00A0cells\t \u00C2\u00A0with\t \u00C2\u00A0a\t \u00C2\u00A0protein\t \u00C2\u00A0prodrug\t \u00C2\u00A0is\t \u00C2\u00A0effective\t \u00C2\u00A0in\t \u00C2\u00A0experimental\t \u00C2\u00A0malignant\t \u00C2\u00A0ascites.\t \u00C2\u00A0Int\t \u00C2\u00A0J\t \u00C2\u00A0Oncology\t \u00C2\u00A025:713-\u00C2\u00AD\u00E2\u0080\u0090720.\t \u00C2\u00A0Irwin\t \u00C2\u00A0DJ,\t \u00C2\u00A0Lee\t \u00C2\u00A0VMY,\t \u00C2\u00A0Trojanowski\t \u00C2\u00A0JQ\t \u00C2\u00A0(2013)\t \u00C2\u00A0Parkinson\u00E2\u0080\u0099s\t \u00C2\u00A0disease\t \u00C2\u00A0dementia:\t \u00C2\u00A0convergence\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein,\t \u00C2\u00A0tau\t \u00C2\u00A0and\t \u00C2\u00A0amyloid-\u00C2\u00AD\u00E2\u0080\u0090\u00CE\u00B2\t \u00C2\u00A0pathologies.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Neurosci\t \u00C2\u00A014:626\u00E2\u0080\u0093636.\t \u00C2\u00A0Itoh\t \u00C2\u00A0Y,\t \u00C2\u00A0Ishikawa\t \u00C2\u00A0M,\t \u00C2\u00A0Kitaguchi\t \u00C2\u00A0R,\t \u00C2\u00A0Okuhira\t \u00C2\u00A0K,\t \u00C2\u00A0Naito\t \u00C2\u00A0M,\t \u00C2\u00A0Hashimoto\t \u00C2\u00A0Y\t \u00C2\u00A0(2012)\t \u00C2\u00A0Double\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown\t \u00C2\u00A0of\t \u00C2\u00A0cIAP1\t \u00C2\u00A0and\t \u00C2\u00A0CRABP-\u00C2\u00AD\u00E2\u0080\u0090II\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0hybrid\t \u00C2\u00A0molecule\t \u00C2\u00A0consisting\t \u00C2\u00A0of\t \u00C2\u00A0ATRA\t \u00C2\u00A0and\t \u00C2\u00A0IAPs\t \u00C2\u00A0antagonist.\t \u00C2\u00A0Bioorg\t \u00C2\u00A0Med\t \u00C2\u00A0Chem\t \u00C2\u00A0Lett\t \u00C2\u00A022:4453\u00E2\u0080\u00934457.\t \u00C2\u00A0\t \u00C2\u00A0 208 Itoh\t \u00C2\u00A0Y,\t \u00C2\u00A0Ishikawa\t \u00C2\u00A0M,\t \u00C2\u00A0Kitaguchi\t \u00C2\u00A0R,\t \u00C2\u00A0Sato\t \u00C2\u00A0S,\t \u00C2\u00A0Naito\t \u00C2\u00A0M,\t \u00C2\u00A0Hashimoto\t \u00C2\u00A0Y\t \u00C2\u00A0(2011)\t \u00C2\u00A0Development\t \u00C2\u00A0of\t \u00C2\u00A0target\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090selective\t \u00C2\u00A0degradation\t \u00C2\u00A0inducer\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0knockdown.\t \u00C2\u00A0Bioorg\t \u00C2\u00A0Med\t \u00C2\u00A0Chem\t \u00C2\u00A019:3229\u00E2\u0080\u00933241.\t \u00C2\u00A0Itoh\t \u00C2\u00A0Y,\t \u00C2\u00A0Ishikawa\t \u00C2\u00A0M,\t \u00C2\u00A0Naito\t \u00C2\u00A0M,\t \u00C2\u00A0Hashimoto\t \u00C2\u00A0Y\t \u00C2\u00A0(2010)\t \u00C2\u00A0Protein\t \u00C2\u00A0Knockdown\t \u00C2\u00A0Using\t \u00C2\u00A0Methyl\t \u00C2\u00A0Bestatin\u00E2\u0088\u0092Ligand\t \u00C2\u00A0Hybrid\t \u00C2\u00A0Molecules:\t \u00C2\u00A0Design\t \u00C2\u00A0and\t \u00C2\u00A0Synthesis\t \u00C2\u00A0of\t \u00C2\u00A0Inducers\t \u00C2\u00A0of\t \u00C2\u00A0Ubiquitination-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0Cellular\t \u00C2\u00A0Retinoic\t \u00C2\u00A0Acid-\u00C2\u00AD\u00E2\u0080\u0090Binding\t \u00C2\u00A0Proteins.\t \u00C2\u00A0J\t \u00C2\u00A0Am\t \u00C2\u00A0Chem\t \u00C2\u00A0Soc\t \u00C2\u00A0132:5820\u00E2\u0080\u00935826.\t \u00C2\u00A0Iwamoto\t \u00C2\u00A0M,\t \u00C2\u00A0Bj\u00C3\u00B6rklund\t \u00C2\u00A0T,\t \u00C2\u00A0Lundberg\t \u00C2\u00A0C,\t \u00C2\u00A0Kirik\t \u00C2\u00A0D,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2010)\t \u00C2\u00A0A\t \u00C2\u00A0General\t \u00C2\u00A0Chemical\t \u00C2\u00A0Method\t \u00C2\u00A0to\t \u00C2\u00A0Regulate\t \u00C2\u00A0Protein\t \u00C2\u00A0Stability\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0Mammalian\t \u00C2\u00A0Central\t \u00C2\u00A0Nervous\t \u00C2\u00A0System.\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A017:981\u00E2\u0080\u0093988.\t \u00C2\u00A0Jackson\t \u00C2\u00A0AL,\t \u00C2\u00A0Bartz\t \u00C2\u00A0SR,\t \u00C2\u00A0Schelter\t \u00C2\u00A0J,\t \u00C2\u00A0Kobayashi\t \u00C2\u00A0SV,\t \u00C2\u00A0Burchard\t \u00C2\u00A0J,\t \u00C2\u00A0Mao\t \u00C2\u00A0M,\t \u00C2\u00A0Li\t \u00C2\u00A0B,\t \u00C2\u00A0Cavet\t \u00C2\u00A0G,\t \u00C2\u00A0Linsley\t \u00C2\u00A0PS\t \u00C2\u00A0(2003)\t \u00C2\u00A0Expression\t \u00C2\u00A0profiling\t \u00C2\u00A0reveals\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0gene\t \u00C2\u00A0regulation\t \u00C2\u00A0by\t \u00C2\u00A0RNAi.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A021:635\u00E2\u0080\u0093637.\t \u00C2\u00A0Jang\t \u00C2\u00A0ER,\t \u00C2\u00A0Lee\t \u00C2\u00A0W,\t \u00C2\u00A0Kim\t \u00C2\u00A0K-\u00C2\u00AD\u00E2\u0080\u0090B\t \u00C2\u00A0(2010)\t \u00C2\u00A0Targeted\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0Proteins\t \u00C2\u00A0by\t \u00C2\u00A0PROTACs.\t \u00C2\u00A0Curr\t \u00C2\u00A0Protoc\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A02:71\u00E2\u0080\u009387.\t \u00C2\u00A0Janse\t \u00C2\u00A0DM\t \u00C2\u00A0(2004a)\t \u00C2\u00A0Localization\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0Proteasome\t \u00C2\u00A0Is\t \u00C2\u00A0Sufficient\t \u00C2\u00A0for\t \u00C2\u00A0Degradation.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0279:21415\u00E2\u0080\u009321420.\t \u00C2\u00A0Jentsch\t \u00C2\u00A0S,\t \u00C2\u00A0Schlenker\t \u00C2\u00A0S\t \u00C2\u00A0(1995)\t \u00C2\u00A0Selective\t \u00C2\u00A0protein\t \u00C2\u00A0degradation:\t \u00C2\u00A0a\t \u00C2\u00A0journey's\t \u00C2\u00A0end\t \u00C2\u00A0within\t \u00C2\u00A0the\t \u00C2\u00A0proteasome.\t \u00C2\u00A0Cell\t \u00C2\u00A082:881\u00E2\u0080\u0093884.\t \u00C2\u00A0 209 Johansen\t \u00C2\u00A0T,\t \u00C2\u00A0Lamark\t \u00C2\u00A0T\t \u00C2\u00A0(2014)\t \u00C2\u00A0Selective\t \u00C2\u00A0autophagy\t \u00C2\u00A0goes\t \u00C2\u00A0exclusive.\t \u00C2\u00A0Nat\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A016:395\u00E2\u0080\u0093397.\t \u00C2\u00A0Johnson\t \u00C2\u00A0ES,\t \u00C2\u00A0Gonda\t \u00C2\u00A0DK,\t \u00C2\u00A0Varshavsky\t \u00C2\u00A0A\t \u00C2\u00A0(1990)\t \u00C2\u00A0cis-\u00C2\u00AD\u00E2\u0080\u0090trans\t \u00C2\u00A0recognition\t \u00C2\u00A0and\t \u00C2\u00A0subunit-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0short-\u00C2\u00AD\u00E2\u0080\u0090lived\t \u00C2\u00A0proteins.\t \u00C2\u00A0Nature\t \u00C2\u00A0346:287\u00E2\u0080\u0093291.\t \u00C2\u00A0Johnston\t \u00C2\u00A0JA,\t \u00C2\u00A0Johnson\t \u00C2\u00A0ES,\t \u00C2\u00A0Waller\t \u00C2\u00A0PR,\t \u00C2\u00A0Varshavsky\t \u00C2\u00A0A\t \u00C2\u00A0(1995)\t \u00C2\u00A0Methotrexate\t \u00C2\u00A0inhibits\t \u00C2\u00A0proteolysis\t \u00C2\u00A0of\t \u00C2\u00A0dihydrofolate\t \u00C2\u00A0reductase\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090end\t \u00C2\u00A0rule\t \u00C2\u00A0pathway.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0270:8172\u00E2\u0080\u00938178.\t \u00C2\u00A0Judge\t \u00C2\u00A0AD,\t \u00C2\u00A0Sood\t \u00C2\u00A0V,\t \u00C2\u00A0Shaw\t \u00C2\u00A0JR,\t \u00C2\u00A0Fang\t \u00C2\u00A0D,\t \u00C2\u00A0McClintock\t \u00C2\u00A0K,\t \u00C2\u00A0MacLachlan\t \u00C2\u00A0I\t \u00C2\u00A0(2005)\t \u00C2\u00A0Sequence-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0stimulation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0mammalian\t \u00C2\u00A0innate\t \u00C2\u00A0immune\t \u00C2\u00A0response\t \u00C2\u00A0by\t \u00C2\u00A0synthetic\t \u00C2\u00A0siRNA.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A023:457\u00E2\u0080\u0093462.\t \u00C2\u00A0Kanemaki\t \u00C2\u00A0M,\t \u00C2\u00A0Sanchez-\u00C2\u00AD\u00E2\u0080\u0090Diaz\t \u00C2\u00A0A,\t \u00C2\u00A0Gambus\t \u00C2\u00A0A,\t \u00C2\u00A0Labib\t \u00C2\u00A0K\t \u00C2\u00A0(2003)\t \u00C2\u00A0Functional\t \u00C2\u00A0proteomic\t \u00C2\u00A0identification\t \u00C2\u00A0of\t \u00C2\u00A0DNA\t \u00C2\u00A0replication\t \u00C2\u00A0proteins\t \u00C2\u00A0by\t \u00C2\u00A0induced\t \u00C2\u00A0proteolysis\t \u00C2\u00A0in\t \u00C2\u00A0vivo.\t \u00C2\u00A0Nature\t \u00C2\u00A0423:720\u00E2\u0080\u0093724.\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Bandyopadhyay\t \u00C2\u00A0U,\t \u00C2\u00A0Sridhar\t \u00C2\u00A0S,\t \u00C2\u00A0Kiffin\t \u00C2\u00A0R,\t \u00C2\u00A0Martinez-\u00C2\u00AD\u00E2\u0080\u0090Vicente\t \u00C2\u00A0M,\t \u00C2\u00A0Kon\t \u00C2\u00A0M,\t \u00C2\u00A0Orenstein\t \u00C2\u00A0SJ,\t \u00C2\u00A0Wong\t \u00C2\u00A0E,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2011)\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0at\t \u00C2\u00A0a\t \u00C2\u00A0glance.\t \u00C2\u00A0J\t \u00C2\u00A0Cell\t \u00C2\u00A0Sci\t \u00C2\u00A0124:495\u00E2\u0080\u0093499.\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2012)\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy:\t \u00C2\u00A0a\t \u00C2\u00A0unique\t \u00C2\u00A0way\t \u00C2\u00A0to\t \u00C2\u00A0enter\t \u00C2\u00A0the\t \u00C2\u00A0lysosome\t \u00C2\u00A0world.\t \u00C2\u00A0Trends\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A022:407\u00E2\u0080\u0093417.\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Massey\t \u00C2\u00A0AC,\t \u00C2\u00A0Mizushima\t \u00C2\u00A0N,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2008)\t \u00C2\u00A0Constitutive\t \u00C2\u00A0Activation\t \u00C2\u00A0of\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0Autophagy\t \u00C2\u00A0in\t \u00C2\u00A0Cells\t \u00C2\u00A0with\t \u00C2\u00A0Impaired\t \u00C2\u00A0Macroautophagy.\t \u00C2\u00A0Mol\t \u00C2\u00A0Biol\t \u00C2\u00A0 210 Cell\t \u00C2\u00A019:2179\u00E2\u0080\u00932192.\t \u00C2\u00A0Kernstock\t \u00C2\u00A0S,\t \u00C2\u00A0Davydova\t \u00C2\u00A0E,\t \u00C2\u00A0Jakobsson\t \u00C2\u00A0M,\t \u00C2\u00A0Moen\t \u00C2\u00A0A,\t \u00C2\u00A0Pettersen\t \u00C2\u00A0S,\t \u00C2\u00A0landsmo\t \u00C2\u00A0GMMA,\t \u00C2\u00A0Egge-\u00C2\u00AD\u00E2\u0080\u0090Jacobsen\t \u00C2\u00A0W,\t \u00C2\u00A0Falnes\t \u00C2\u00A0PALO\t \u00C2\u00A0(2012)\t \u00C2\u00A0Lysine\t \u00C2\u00A0methylation\t \u00C2\u00A0of\t \u00C2\u00A0VCP\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0member\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0novel\t \u00C2\u00A0human\t \u00C2\u00A0protein\t \u00C2\u00A0methyltransferase\t \u00C2\u00A0family.\t \u00C2\u00A0Nat\t \u00C2\u00A0Comms\t \u00C2\u00A03:1038\u00E2\u0080\u009311.\t \u00C2\u00A0Kiffin\t \u00C2\u00A0R,\t \u00C2\u00A0Christian\t \u00C2\u00A0C,\t \u00C2\u00A0Knecht\t \u00C2\u00A0E,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2004)\t \u00C2\u00A0Activation\t \u00C2\u00A0of\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0during\t \u00C2\u00A0oxidative\t \u00C2\u00A0stress.\t \u00C2\u00A0Mol\t \u00C2\u00A0Biol\t \u00C2\u00A0Cell\t \u00C2\u00A015:4829\u00E2\u0080\u00934840.\t \u00C2\u00A0Kim\t \u00C2\u00A0BM,\t \u00C2\u00A0You\t \u00C2\u00A0M-\u00C2\u00AD\u00E2\u0080\u0090H,\t \u00C2\u00A0Chen\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090H,\t \u00C2\u00A0Lee\t \u00C2\u00A0S,\t \u00C2\u00A0Hong\t \u00C2\u00A0Y,\t \u00C2\u00A0Hong\t \u00C2\u00A0Y,\t \u00C2\u00A0Kimchi\t \u00C2\u00A0A,\t \u00C2\u00A0Zhou\t \u00C2\u00A0XZ,\t \u00C2\u00A0Lee\t \u00C2\u00A0TH\t \u00C2\u00A0(2014)\t \u00C2\u00A0Death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A01\t \u00C2\u00A0has\t \u00C2\u00A0a\t \u00C2\u00A0critical\t \u00C2\u00A0role\t \u00C2\u00A0in\t \u00C2\u00A0aberrant\t \u00C2\u00A0tau\t \u00C2\u00A0protein\t \u00C2\u00A0regulation\t \u00C2\u00A0and\t \u00C2\u00A0function.\t \u00C2\u00A0Cell\t \u00C2\u00A0Death\t \u00C2\u00A0Dis\t \u00C2\u00A05:e1237\u00E2\u0080\u009312.\t \u00C2\u00A0Kim\t \u00C2\u00A0H,\t \u00C2\u00A0Kim\t \u00C2\u00A0J-\u00C2\u00AD\u00E2\u0080\u0090S\t \u00C2\u00A0(2014)\t \u00C2\u00A0A\t \u00C2\u00A0guide\t \u00C2\u00A0to\t \u00C2\u00A0genome\t \u00C2\u00A0engineering\t \u00C2\u00A0with\t \u00C2\u00A0programmable\t \u00C2\u00A0nucleases.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Genet\t \u00C2\u00A015:321\u00E2\u0080\u0093334.\t \u00C2\u00A0Klionsky\t \u00C2\u00A0DJ,\t \u00C2\u00A0Schulman\t \u00C2\u00A0BA\t \u00C2\u00A0(2014)\t \u00C2\u00A0Dynamic\t \u00C2\u00A0regulation\t \u00C2\u00A0of\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0by\t \u00C2\u00A0distinctive\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090like\t \u00C2\u00A0proteins.\t \u00C2\u00A0Nat\t \u00C2\u00A0Struct\t \u00C2\u00A0Molecul\t \u00C2\u00A0Bio\t \u00C2\u00A021:336\u00E2\u0080\u0093345.\t \u00C2\u00A0Koga\t \u00C2\u00A0H,\t \u00C2\u00A0Martinez-\u00C2\u00AD\u00E2\u0080\u0090Vicente\t \u00C2\u00A0M,\t \u00C2\u00A0Arias\t \u00C2\u00A0E,\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Sulzer\t \u00C2\u00A0D,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2011a)\t \u00C2\u00A0Constitutive\t \u00C2\u00A0Upregulation\t \u00C2\u00A0of\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy\t \u00C2\u00A0in\t \u00C2\u00A0Huntington's\t \u00C2\u00A0Disease.\t \u00C2\u00A0J\t \u00C2\u00A0Neuro\t \u00C2\u00A031:18492\u00E2\u0080\u009318505.\t \u00C2\u00A0Koga\t \u00C2\u00A0H,\t \u00C2\u00A0Martinez-\u00C2\u00AD\u00E2\u0080\u0090Vicente\t \u00C2\u00A0M,\t \u00C2\u00A0Macian\t \u00C2\u00A0F,\t \u00C2\u00A0Verkhusha\t \u00C2\u00A0VV,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2011b)\t \u00C2\u00A0A\t \u00C2\u00A0photoconvertible\t \u00C2\u00A0fluorescent\t \u00C2\u00A0reporter\t \u00C2\u00A0to\t \u00C2\u00A0track\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0Nat\t \u00C2\u00A0Comms\t \u00C2\u00A02:386\u00E2\u0080\u009310.\t \u00C2\u00A0 211 Kole\t \u00C2\u00A0R,\t \u00C2\u00A0Krainer\t \u00C2\u00A0AR,\t \u00C2\u00A0Altman\t \u00C2\u00A0S\t \u00C2\u00A0(2012)\t \u00C2\u00A0RNA\t \u00C2\u00A0therapeutics:\t \u00C2\u00A0beyond\t \u00C2\u00A0RNA\t \u00C2\u00A0interference\t \u00C2\u00A0and\t \u00C2\u00A0antisense\t \u00C2\u00A0oligonucleotides.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A011:125\u00E2\u0080\u0093140.\t \u00C2\u00A0Komander\t \u00C2\u00A0D,\t \u00C2\u00A0Rape\t \u00C2\u00A0M\t \u00C2\u00A0(2012)\t \u00C2\u00A0The\t \u00C2\u00A0Ubiquitin\t \u00C2\u00A0Code.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Biochem\t \u00C2\u00A081:203\u00E2\u0080\u0093229.\t \u00C2\u00A0Komatsu\t \u00C2\u00A0M,\t \u00C2\u00A0Ichimura\t \u00C2\u00A0Y\t \u00C2\u00A0(2010)\t \u00C2\u00A0Physiological\t \u00C2\u00A0significance\t \u00C2\u00A0of\t \u00C2\u00A0selective\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0p62\t \u00C2\u00A0by\t \u00C2\u00A0autophagy.\t \u00C2\u00A0FEBS\t \u00C2\u00A0Letters\t \u00C2\u00A0584:1374\u00E2\u0080\u00931378.\t \u00C2\u00A0Kondo\t \u00C2\u00A0E,\t \u00C2\u00A0Saito\t \u00C2\u00A0K,\t \u00C2\u00A0Tashiro\t \u00C2\u00A0Y,\t \u00C2\u00A0Kamide\t \u00C2\u00A0K,\t \u00C2\u00A0Uno\t \u00C2\u00A0S,\t \u00C2\u00A0Furuya\t \u00C2\u00A0T,\t \u00C2\u00A0Mashita\t \u00C2\u00A0M,\t \u00C2\u00A0Nakajima\t \u00C2\u00A0K,\t \u00C2\u00A0Tsumuraya\t \u00C2\u00A0T,\t \u00C2\u00A0Kobayashi\t \u00C2\u00A0N,\t \u00C2\u00A0Nishibori\t \u00C2\u00A0M,\t \u00C2\u00A0Tanimoto\t \u00C2\u00A0M,\t \u00C2\u00A0Matsushita\t \u00C2\u00A0M\t \u00C2\u00A0(2012)\t \u00C2\u00A0Tumour\t \u00C2\u00A0lineage-\u00C2\u00AD\u00E2\u0080\u0090homing\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090penetrating\t \u00C2\u00A0peptides\t \u00C2\u00A0as\t \u00C2\u00A0anticancer\t \u00C2\u00A0molecular\t \u00C2\u00A0delivery\t \u00C2\u00A0systems.\t \u00C2\u00A0Nat\t \u00C2\u00A0Comms\t \u00C2\u00A03:951\u00E2\u0080\u009313.\t \u00C2\u00A0Kordasiewicz\t \u00C2\u00A0HB,\t \u00C2\u00A0Stanek\t \u00C2\u00A0LM,\t \u00C2\u00A0Wancewicz\t \u00C2\u00A0EV,\t \u00C2\u00A0Mazur\t \u00C2\u00A0C,\t \u00C2\u00A0McAlonis\t \u00C2\u00A0MM,\t \u00C2\u00A0Pytel\t \u00C2\u00A0KA,\t \u00C2\u00A0Artates\t \u00C2\u00A0JW,\t \u00C2\u00A0Weiss\t \u00C2\u00A0A,\t \u00C2\u00A0Cheng\t \u00C2\u00A0SH,\t \u00C2\u00A0Shihabuddin\t \u00C2\u00A0LS,\t \u00C2\u00A0Hung\t \u00C2\u00A0G,\t \u00C2\u00A0Bennett\t \u00C2\u00A0CF,\t \u00C2\u00A0Cleveland\t \u00C2\u00A0DW\t \u00C2\u00A0(2012)\t \u00C2\u00A0Sustained\t \u00C2\u00A0Therapeutic\t \u00C2\u00A0Reversal\t \u00C2\u00A0of\t \u00C2\u00A0Huntington's\t \u00C2\u00A0Disease\t \u00C2\u00A0by\t \u00C2\u00A0Transient\t \u00C2\u00A0Repression\t \u00C2\u00A0of\t \u00C2\u00A0Huntingtin\t \u00C2\u00A0Synthesis.\t \u00C2\u00A0Neuron\t \u00C2\u00A074:1031\u00E2\u0080\u00931044.\t \u00C2\u00A0Koren\t \u00C2\u00A0E,\t \u00C2\u00A0Torchilin\t \u00C2\u00A0VP\t \u00C2\u00A0(2012)\t \u00C2\u00A0Cell-\u00C2\u00AD\u00E2\u0080\u0090penetrating\t \u00C2\u00A0peptides:\t \u00C2\u00A0breaking\t \u00C2\u00A0through\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0other\t \u00C2\u00A0side.\t \u00C2\u00A0Trends\t \u00C2\u00A0Mol\t \u00C2\u00A0Med\t \u00C2\u00A018:386\u00E2\u0080\u0093394.\t \u00C2\u00A0Korolchuk\t \u00C2\u00A0VI,\t \u00C2\u00A0Mansilla\t \u00C2\u00A0A,\t \u00C2\u00A0Menzies\t \u00C2\u00A0FM,\t \u00C2\u00A0Rubinsztein\t \u00C2\u00A0DC\t \u00C2\u00A0(2009)\t \u00C2\u00A0Autophagy\t \u00C2\u00A0Inhibition\t \u00C2\u00A0Compromises\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0Ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090Proteasome\t \u00C2\u00A0Pathway\t \u00C2\u00A0Substrates.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A033:517\u00E2\u0080\u0093527.\t \u00C2\u00A0Krieg\t \u00C2\u00A0AM,\t \u00C2\u00A0Yi\t \u00C2\u00A0AK,\t \u00C2\u00A0Matson\t \u00C2\u00A0S,\t \u00C2\u00A0Waldschmidt\t \u00C2\u00A0TJ,\t \u00C2\u00A0Bishop\t \u00C2\u00A0GA,\t \u00C2\u00A0Teasdale\t \u00C2\u00A0R,\t \u00C2\u00A0Koretzky\t \u00C2\u00A0GA,\t \u00C2\u00A0 212 Klinman\t \u00C2\u00A0DM\t \u00C2\u00A0(1995)\t \u00C2\u00A0CpG\t \u00C2\u00A0motifs\t \u00C2\u00A0in\t \u00C2\u00A0bacterial\t \u00C2\u00A0DNA\t \u00C2\u00A0trigger\t \u00C2\u00A0direct\t \u00C2\u00A0B-\u00C2\u00AD\u00E2\u0080\u0090cell\t \u00C2\u00A0activation.\t \u00C2\u00A0Nature\t \u00C2\u00A0374:546\u00E2\u0080\u0093549.\t \u00C2\u00A0Kuhn\t \u00C2\u00A0R,\t \u00C2\u00A0Schwenk\t \u00C2\u00A0F,\t \u00C2\u00A0Aguet\t \u00C2\u00A0M,\t \u00C2\u00A0Rajewsky\t \u00C2\u00A0K\t \u00C2\u00A0(1995)\t \u00C2\u00A0Inducible\t \u00C2\u00A0gene\t \u00C2\u00A0targeting\t \u00C2\u00A0in\t \u00C2\u00A0mice.\t \u00C2\u00A0Science\t \u00C2\u00A0269:1427-\u00C2\u00AD\u00E2\u0080\u00901429.\t \u00C2\u00A0Labella\t \u00C2\u00A0ML,\t \u00C2\u00A0Sigrist\t \u00C2\u00A0S,\t \u00C2\u00A0Jorgensen\t \u00C2\u00A0EM\t \u00C2\u00A0(2013)\t \u00C2\u00A0Putting\t \u00C2\u00A0genetics\t \u00C2\u00A0into\t \u00C2\u00A0optogenetics:\t \u00C2\u00A0knocking\t \u00C2\u00A0out\t \u00C2\u00A0proteins\t \u00C2\u00A0with\t \u00C2\u00A0light.\t \u00C2\u00A0In:\t \u00C2\u00A0Optogenetics,\t \u00C2\u00A0pp\t \u00C2\u00A079\u00E2\u0080\u009390.\t \u00C2\u00A0Dahlem\t \u00C2\u00A0Workshop\t \u00C2\u00A0Reports.\t \u00C2\u00A0Berlin:\t \u00C2\u00A0Walter\t \u00C2\u00A0de\t \u00C2\u00A0Gruyter.\t \u00C2\u00A0\t \u00C2\u00A0Lai\t \u00C2\u00A0M-\u00C2\u00AD\u00E2\u0080\u0090Z,\t \u00C2\u00A0Chen\t \u00C2\u00A0R-\u00C2\u00AD\u00E2\u0080\u0090H\t \u00C2\u00A0(2014)\t \u00C2\u00A0Regulation\t \u00C2\u00A0of\t \u00C2\u00A0inflammation\t \u00C2\u00A0by\t \u00C2\u00A0DAPK.\t \u00C2\u00A0Apoptosis\t \u00C2\u00A019:357-\u00C2\u00AD\u00E2\u0080\u0090363.\t \u00C2\u00A0Lai\t \u00C2\u00A0TW,\t \u00C2\u00A0Shyu\t \u00C2\u00A0W-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2011)\t \u00C2\u00A0Stroke\t \u00C2\u00A0intervention\t \u00C2\u00A0pathways:\t \u00C2\u00A0NMDA\t \u00C2\u00A0receptors\t \u00C2\u00A0and\t \u00C2\u00A0beyond.\t \u00C2\u00A0Trends\t \u00C2\u00A0Mol\t \u00C2\u00A0Med\t \u00C2\u00A017:266\u00E2\u0080\u0093275.\t \u00C2\u00A0Lai\t \u00C2\u00A0TW,\t \u00C2\u00A0Zhang\t \u00C2\u00A0S,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2014)\t \u00C2\u00A0Excitotoxicity\t \u00C2\u00A0and\t \u00C2\u00A0stroke:\t \u00C2\u00A0Identifying\t \u00C2\u00A0novel\t \u00C2\u00A0targets\t \u00C2\u00A0for\t \u00C2\u00A0neuroprotection.\t \u00C2\u00A0Prog\t \u00C2\u00A0Neurobiol\t \u00C2\u00A0115:157\u00E2\u0080\u0093188.\t \u00C2\u00A0Lalatsa\t \u00C2\u00A0A,\t \u00C2\u00A0Schatzlein\t \u00C2\u00A0AG,\t \u00C2\u00A0Uchegbu\t \u00C2\u00A0IF\t \u00C2\u00A0(2014)\t \u00C2\u00A0Strategies\t \u00C2\u00A0to\t \u00C2\u00A0deliver\t \u00C2\u00A0peptide\t \u00C2\u00A0drugs\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0brain.\t \u00C2\u00A0Mol\t \u00C2\u00A0Pharmaceutics\t \u00C2\u00A011:1081\u00E2\u0080\u00931093.\t \u00C2\u00A0Lecker\t \u00C2\u00A0SH\t \u00C2\u00A0(2006)\t \u00C2\u00A0Protein\t \u00C2\u00A0Degradation\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0Ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090Proteasome\t \u00C2\u00A0Pathway\t \u00C2\u00A0in\t \u00C2\u00A0Normal\t \u00C2\u00A0and\t \u00C2\u00A0Disease\t \u00C2\u00A0States.\t \u00C2\u00A0J\t \u00C2\u00A0Am\t \u00C2\u00A0Soc\t \u00C2\u00A0Nephrol\t \u00C2\u00A017:1807\u00E2\u0080\u00931819.\t \u00C2\u00A0Lewandoski\t \u00C2\u00A0M\t \u00C2\u00A0(2001)\t \u00C2\u00A0Conditional\t \u00C2\u00A0control\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0expression\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0mouse.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Genet\t \u00C2\u00A02:743\u00E2\u0080\u0093755.\t \u00C2\u00A0 213 L\u00C3\u00A9vy\t \u00C2\u00A0F,\t \u00C2\u00A0Johnston\t \u00C2\u00A0JA,\t \u00C2\u00A0Varshavsky\t \u00C2\u00A0A\t \u00C2\u00A0(1999)\t \u00C2\u00A0Analysis\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0conditional\t \u00C2\u00A0degradation\t \u00C2\u00A0signal\t \u00C2\u00A0in\t \u00C2\u00A0yeast\t \u00C2\u00A0and\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells.\t \u00C2\u00A0Eur\t \u00C2\u00A0J\t \u00C2\u00A0Biochem\t \u00C2\u00A0259:244\u00E2\u0080\u0093252.\t \u00C2\u00A0Li\t \u00C2\u00A0Y\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2006)\t \u00C2\u00A0DAPK1\t \u00C2\u00A0variants\t \u00C2\u00A0are\t \u00C2\u00A0associated\t \u00C2\u00A0with\t \u00C2\u00A0Alzheimer's\t \u00C2\u00A0disease\t \u00C2\u00A0and\t \u00C2\u00A0allele-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0expression.\t \u00C2\u00A0Hum\t \u00C2\u00A0Mol\t \u00C2\u00A0Gen\t \u00C2\u00A015:2560\u00E2\u0080\u00932568.\t \u00C2\u00A0Lim\t \u00C2\u00A0JP,\t \u00C2\u00A0Gleeson\t \u00C2\u00A0PA\t \u00C2\u00A0(2011)\t \u00C2\u00A0Macropinocytosis:\t \u00C2\u00A0an\t \u00C2\u00A0endocytic\t \u00C2\u00A0pathway\t \u00C2\u00A0for\t \u00C2\u00A0internalising\t \u00C2\u00A0large\t \u00C2\u00A0gulps.\t \u00C2\u00A0Immunol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A089:836\u00E2\u0080\u0093843.\t \u00C2\u00A0Lima\t \u00C2\u00A0WF,\t \u00C2\u00A0Prakash\t \u00C2\u00A0TP,\t \u00C2\u00A0Murray\t \u00C2\u00A0HM,\t \u00C2\u00A0Kinberger\t \u00C2\u00A0GA,\t \u00C2\u00A0Li\t \u00C2\u00A0W,\t \u00C2\u00A0Chappell\t \u00C2\u00A0AE,\t \u00C2\u00A0Li\t \u00C2\u00A0CS,\t \u00C2\u00A0Murray\t \u00C2\u00A0SF,\t \u00C2\u00A0Gaus\t \u00C2\u00A0H,\t \u00C2\u00A0Seth\t \u00C2\u00A0PP,\t \u00C2\u00A0Swayze\t \u00C2\u00A0EE,\t \u00C2\u00A0Crooke\t \u00C2\u00A0ST\t \u00C2\u00A0(2012)\t \u00C2\u00A0Single-\u00C2\u00AD\u00E2\u0080\u0090Stranded\t \u00C2\u00A0siRNAs\t \u00C2\u00A0Activate\t \u00C2\u00A0RNAi\t \u00C2\u00A0in\t \u00C2\u00A0Animals.\t \u00C2\u00A0Cell\t \u00C2\u00A0150:883\u00E2\u0080\u0093894.\t \u00C2\u00A0Liu\t \u00C2\u00A0J,\t \u00C2\u00A0Stevens\t \u00C2\u00A0J,\t \u00C2\u00A0Matsunami\t \u00C2\u00A0N,\t \u00C2\u00A0White\t \u00C2\u00A0RL\t \u00C2\u00A0(2004)\t \u00C2\u00A0Targeted\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin\t \u00C2\u00A0by\t \u00C2\u00A0chimeric\t \u00C2\u00A0F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0fusion\t \u00C2\u00A0proteins.\t \u00C2\u00A0Biochem\t \u00C2\u00A0Biophys\t \u00C2\u00A0Res\t \u00C2\u00A0Comms\t \u00C2\u00A0313:1023\u00E2\u0080\u00931029.\t \u00C2\u00A0Liu\t \u00C2\u00A0SB,\t \u00C2\u00A0Zhang\t \u00C2\u00A0N,\t \u00C2\u00A0Guo\t \u00C2\u00A0YY,\t \u00C2\u00A0Zhao\t \u00C2\u00A0R,\t \u00C2\u00A0Shi\t \u00C2\u00A0TY,\t \u00C2\u00A0Feng\t \u00C2\u00A0SF,\t \u00C2\u00A0Wang\t \u00C2\u00A0SQ,\t \u00C2\u00A0Yang\t \u00C2\u00A0Q,\t \u00C2\u00A0Li\t \u00C2\u00A0XQ,\t \u00C2\u00A0Wu\t \u00C2\u00A0YM,\t \u00C2\u00A0Ma\t \u00C2\u00A0L,\t \u00C2\u00A0Hou\t \u00C2\u00A0Y,\t \u00C2\u00A0Xiong\t \u00C2\u00A0LZ,\t \u00C2\u00A0Zhang\t \u00C2\u00A0W,\t \u00C2\u00A0Zhao\t \u00C2\u00A0MG\t \u00C2\u00A0(2012)\t \u00C2\u00A0G-\u00C2\u00AD\u00E2\u0080\u0090protein-\u00C2\u00AD\u00E2\u0080\u0090coupled\t \u00C2\u00A0receptor\t \u00C2\u00A030\t \u00C2\u00A0mediates\t \u00C2\u00A0rapid\t \u00C2\u00A0neuroprotective\t \u00C2\u00A0effects\t \u00C2\u00A0of\t \u00C2\u00A0estrogen\t \u00C2\u00A0via\t \u00C2\u00A0depression\t \u00C2\u00A0of\t \u00C2\u00A0NR2B-\u00C2\u00AD\u00E2\u0080\u0090containing\t \u00C2\u00A0NMDA\t \u00C2\u00A0receptors.\t \u00C2\u00A0J\t \u00C2\u00A0Neurosci\t \u00C2\u00A032(14):4887\u00E2\u0080\u00934900.\t \u00C2\u00A0Liu\t \u00C2\u00A0T,\t \u00C2\u00A0Daniels\t \u00C2\u00A0CK,\t \u00C2\u00A0Cao\t \u00C2\u00A0S\t \u00C2\u00A0(2012)\t \u00C2\u00A0Comprehensive\t \u00C2\u00A0review\t \u00C2\u00A0on\t \u00C2\u00A0the\t \u00C2\u00A0HSC70\t \u00C2\u00A0functions,\t \u00C2\u00A0interactions\t \u00C2\u00A0with\t \u00C2\u00A0related\t \u00C2\u00A0molecules\t \u00C2\u00A0and\t \u00C2\u00A0involvement\t \u00C2\u00A0in\t \u00C2\u00A0clinical\t \u00C2\u00A0diseases\t \u00C2\u00A0and\t \u00C2\u00A0therapeutic\t \u00C2\u00A0potential.\t \u00C2\u00A0Pharmacol\t \u00C2\u00A0Ther\t \u00C2\u00A0136:354\u00E2\u0080\u0093374.\t \u00C2\u00A0London\t \u00C2\u00A0N,\t \u00C2\u00A0Movshovitz-\u00C2\u00AD\u00E2\u0080\u0090Attias\t \u00C2\u00A0D,\t \u00C2\u00A0Schueler-\u00C2\u00AD\u00E2\u0080\u0090Furman\t \u00C2\u00A0O\t \u00C2\u00A0(2010)\t \u00C2\u00A0The\t \u00C2\u00A0Structural\t \u00C2\u00A0Basis\t \u00C2\u00A0of\t \u00C2\u00A0 214 Peptide-\u00C2\u00AD\u00E2\u0080\u0090Protein\t \u00C2\u00A0Binding\t \u00C2\u00A0Strategies.\t \u00C2\u00A0Fold\t \u00C2\u00A0Des\t \u00C2\u00A018:188\u00E2\u0080\u0093199.\t \u00C2\u00A0Lu\t \u00C2\u00A0K,\t \u00C2\u00A0Psakhye\t \u00C2\u00A0I,\t \u00C2\u00A0Jentsch\t \u00C2\u00A0S\t \u00C2\u00A0(2014)\t \u00C2\u00A0Autophagic\t \u00C2\u00A0Clearance\t \u00C2\u00A0of\t \u00C2\u00A0PolyQ\t \u00C2\u00A0Proteins\t \u00C2\u00A0Mediated\t \u00C2\u00A0by\t \u00C2\u00A0Ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090Atg8\t \u00C2\u00A0Adaptorsof\t \u00C2\u00A0the\t \u00C2\u00A0Conserved\t \u00C2\u00A0CUET\t \u00C2\u00A0Protein\t \u00C2\u00A0Family.\t \u00C2\u00A0Cell\t \u00C2\u00A0153:549-\u00C2\u00AD\u00E2\u0080\u0090563.\t \u00C2\u00A0Luzio\t \u00C2\u00A0JP,\t \u00C2\u00A0Pryor\t \u00C2\u00A0PR,\t \u00C2\u00A0Bright\t \u00C2\u00A0NA\t \u00C2\u00A0(2007)\t \u00C2\u00A0Lysosomes:\t \u00C2\u00A0fusion\t \u00C2\u00A0and\t \u00C2\u00A0function.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A08:622\u00E2\u0080\u0093632.\t \u00C2\u00A0Lv\t \u00C2\u00A0L,\t \u00C2\u00A0Li\t \u00C2\u00A0D,\t \u00C2\u00A0Di\t \u00C2\u00A0Zhao,\t \u00C2\u00A0Lin\t \u00C2\u00A0R,\t \u00C2\u00A0Chu\t \u00C2\u00A0Y,\t \u00C2\u00A0Zhang\t \u00C2\u00A0H,\t \u00C2\u00A0Zha\t \u00C2\u00A0Z,\t \u00C2\u00A0Liu\t \u00C2\u00A0Y,\t \u00C2\u00A0Li\t \u00C2\u00A0Z,\t \u00C2\u00A0Xu\t \u00C2\u00A0Y,\t \u00C2\u00A0Wang\t \u00C2\u00A0G,\t \u00C2\u00A0Huang\t \u00C2\u00A0Y,\t \u00C2\u00A0Xiong\t \u00C2\u00A0Y,\t \u00C2\u00A0Guan\t \u00C2\u00A0K-\u00C2\u00AD\u00E2\u0080\u0090L,\t \u00C2\u00A0Lei\t \u00C2\u00A0Q-\u00C2\u00AD\u00E2\u0080\u0090Y\t \u00C2\u00A0(2011)\t \u00C2\u00A0Acetylation\t \u00C2\u00A0Targets\t \u00C2\u00A0the\t \u00C2\u00A0M2\t \u00C2\u00A0Isoform\t \u00C2\u00A0of\t \u00C2\u00A0Pyruvate\t \u00C2\u00A0Kinase\t \u00C2\u00A0for\t \u00C2\u00A0Degradation\t \u00C2\u00A0through\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy\t \u00C2\u00A0and\t \u00C2\u00A0Promotes\t \u00C2\u00A0Tumor\t \u00C2\u00A0Growth.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A042:719\u00E2\u0080\u0093730.\t \u00C2\u00A0Majeski\t \u00C2\u00A0AE,\t \u00C2\u00A0Fred\t \u00C2\u00A0DJ\t \u00C2\u00A0(2004)\t \u00C2\u00A0Mechanisms\t \u00C2\u00A0of\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0Int\t \u00C2\u00A0J\t \u00C2\u00A0Biochem\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A036:2435\u00E2\u0080\u00932444.\t \u00C2\u00A0Mak\t \u00C2\u00A0SK,\t \u00C2\u00A0McCormack\t \u00C2\u00A0AL,\t \u00C2\u00A0Manning-\u00C2\u00AD\u00E2\u0080\u0090Bog\t \u00C2\u00A0AB,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Di\t \u00C2\u00A0Monte\t \u00C2\u00A0DA\t \u00C2\u00A0(2010)\t \u00C2\u00A0Lysosomal\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090Synuclein\t \u00C2\u00A0in\t \u00C2\u00A0Vivo.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0285:13621\u00E2\u0080\u009313629.\t \u00C2\u00A0Mali\t \u00C2\u00A0P,\t \u00C2\u00A0Yang\t \u00C2\u00A0L,\t \u00C2\u00A0Esvelt\t \u00C2\u00A0KM,\t \u00C2\u00A0Aach\t \u00C2\u00A0J,\t \u00C2\u00A0Guell\t \u00C2\u00A0M,\t \u00C2\u00A0DiCarlo\t \u00C2\u00A0JE,\t \u00C2\u00A0Norville\t \u00C2\u00A0JE,\t \u00C2\u00A0Church\t \u00C2\u00A0GM\t \u00C2\u00A0(2013)\t \u00C2\u00A0RNA-\u00C2\u00AD\u00E2\u0080\u0090Guided\t \u00C2\u00A0Human\t \u00C2\u00A0Genome\t \u00C2\u00A0Engineering\t \u00C2\u00A0via\t \u00C2\u00A0Cas9.\t \u00C2\u00A0Science\t \u00C2\u00A0339:823\u00E2\u0080\u0093826.\t \u00C2\u00A0Malkus\t \u00C2\u00A0KA,\t \u00C2\u00A0Ischiropoulos\t \u00C2\u00A0H\t \u00C2\u00A0(2012)\t \u00C2\u00A0Regional\t \u00C2\u00A0deficiencies\t \u00C2\u00A0in\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0underlie\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0aggregation\t \u00C2\u00A0and\t \u00C2\u00A0neurodegeneration.\t \u00C2\u00A0Neurobiol\t \u00C2\u00A0Dis\t \u00C2\u00A046:732\u00E2\u0080\u0093744.\t \u00C2\u00A0\t \u00C2\u00A0 215 Mason\t \u00C2\u00A0JM\t \u00C2\u00A0(2010)\t \u00C2\u00A0Design\t \u00C2\u00A0and\t \u00C2\u00A0development\t \u00C2\u00A0of\t \u00C2\u00A0peptides\t \u00C2\u00A0and\t \u00C2\u00A0peptide\t \u00C2\u00A0mimetics\t \u00C2\u00A0as\t \u00C2\u00A0antagonists\t \u00C2\u00A0for\t \u00C2\u00A0therapeutic\t \u00C2\u00A0intervention.\t \u00C2\u00A0Future\t \u00C2\u00A0Med\t \u00C2\u00A0Chem\t \u00C2\u00A02:1813\u00E2\u0080\u00931822.\t \u00C2\u00A0Massey\t \u00C2\u00A0A,\t \u00C2\u00A0Kiffin\t \u00C2\u00A0R,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2004)\t \u00C2\u00A0Pathophysiology\t \u00C2\u00A0of\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0Int\t \u00C2\u00A0J\t \u00C2\u00A0Biochem\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A036:2420\u00E2\u0080\u00932434.\t \u00C2\u00A0Massey\t \u00C2\u00A0AC,\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Sovak\t \u00C2\u00A0G,\t \u00C2\u00A0Kiffin\t \u00C2\u00A0R,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2006)\t \u00C2\u00A0Consequences\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0selective\t \u00C2\u00A0blockage\t \u00C2\u00A0of\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0103:5805\u00E2\u0080\u00935810.\t \u00C2\u00A0Matsuzawa\t \u00C2\u00A0S-\u00C2\u00AD\u00E2\u0080\u0090I,\t \u00C2\u00A0Cuddy\t \u00C2\u00A0M,\t \u00C2\u00A0Fukushima\t \u00C2\u00A0T,\t \u00C2\u00A0Reed\t \u00C2\u00A0JC\t \u00C2\u00A0(2005)\t \u00C2\u00A0Method\t \u00C2\u00A0for\t \u00C2\u00A0targeting\t \u00C2\u00A0protein\t \u00C2\u00A0destruction\t \u00C2\u00A0by\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090independent,\t \u00C2\u00A0proteasome-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0degradation\t \u00C2\u00A0pathway.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0102:14982\u00E2\u0080\u009314987.\t \u00C2\u00A0McCaffrey\t \u00C2\u00A0AP,\t \u00C2\u00A0Meuse\t \u00C2\u00A0L,\t \u00C2\u00A0Pham\t \u00C2\u00A0T-\u00C2\u00AD\u00E2\u0080\u0090TT,\t \u00C2\u00A0Conklin\t \u00C2\u00A0DS,\t \u00C2\u00A0Hannon\t \u00C2\u00A0GJ,\t \u00C2\u00A0Kay\t \u00C2\u00A0MA\t \u00C2\u00A0(2002)\t \u00C2\u00A0RNA\t \u00C2\u00A0interference\t \u00C2\u00A0in\t \u00C2\u00A0adult\t \u00C2\u00A0mice.\t \u00C2\u00A0Nature\t \u00C2\u00A0418:38\u00E2\u0080\u009339.\t \u00C2\u00A0Meister\t \u00C2\u00A0G,\t \u00C2\u00A0Tuschl\t \u00C2\u00A0T\t \u00C2\u00A0(2004)\t \u00C2\u00A0Mechanisms\t \u00C2\u00A0of\t \u00C2\u00A0gene\t \u00C2\u00A0silencing\t \u00C2\u00A0by\t \u00C2\u00A0double-\u00C2\u00AD\u00E2\u0080\u0090stranded\t \u00C2\u00A0RNA.\t \u00C2\u00A0Nature\t \u00C2\u00A0431:\t \u00C2\u00A0343-\u00C2\u00AD\u00E2\u0080\u0090349.\t \u00C2\u00A0Mijaljica\t \u00C2\u00A0D,\t \u00C2\u00A0Prescott\t \u00C2\u00A0M,\t \u00C2\u00A0Devenish\t \u00C2\u00A0RJ\t \u00C2\u00A0(2011)\t \u00C2\u00A0Microautophagy\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells:\t \u00C2\u00A0Revisiting\t \u00C2\u00A0a\t \u00C2\u00A040-\u00C2\u00AD\u00E2\u0080\u0090year-\u00C2\u00AD\u00E2\u0080\u0090old\t \u00C2\u00A0conundrum.\t \u00C2\u00A0Autophagy\t \u00C2\u00A07:673\u00E2\u0080\u0093682.\t \u00C2\u00A0Miyazaki\t \u00C2\u00A0Y,\t \u00C2\u00A0Imoto\t \u00C2\u00A0H,\t \u00C2\u00A0Chen\t \u00C2\u00A0L-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2012)\t \u00C2\u00A0Destabilizing\t \u00C2\u00A0Domains\t \u00C2\u00A0Derived\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0Human\t \u00C2\u00A0Estrogen\t \u00C2\u00A0Receptor.\t \u00C2\u00A0J\t \u00C2\u00A0Am\t \u00C2\u00A0Chem\t \u00C2\u00A0Soc\t \u00C2\u00A0134:3942\u00E2\u0080\u00933945.\t \u00C2\u00A0\t \u00C2\u00A0 216 Mizushima\t \u00C2\u00A0N,\t \u00C2\u00A0Komatsu\t \u00C2\u00A0M\t \u00C2\u00A0(2011)\t \u00C2\u00A0Autophagy:\t \u00C2\u00A0Renovation\t \u00C2\u00A0of\t \u00C2\u00A0Cells\t \u00C2\u00A0and\t \u00C2\u00A0Tissues.\t \u00C2\u00A0Cell\t \u00C2\u00A0147:728\u00E2\u0080\u0093741.\t \u00C2\u00A0Mizushima\t \u00C2\u00A0N,\t \u00C2\u00A0Levine\t \u00C2\u00A0B,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Klionsky\t \u00C2\u00A0DJ\t \u00C2\u00A0(2008)\t \u00C2\u00A0Autophagy\t \u00C2\u00A0fights\t \u00C2\u00A0disease\t \u00C2\u00A0through\t \u00C2\u00A0cellular\t \u00C2\u00A0self-\u00C2\u00AD\u00E2\u0080\u0090digestion.\t \u00C2\u00A0Nature\t \u00C2\u00A0451:1069\u00E2\u0080\u00931075.\t \u00C2\u00A0Montrose\t \u00C2\u00A0K,\t \u00C2\u00A0Yang\t \u00C2\u00A0Y,\t \u00C2\u00A0Sun\t \u00C2\u00A0X,\t \u00C2\u00A0Wiles\t \u00C2\u00A0S,\t \u00C2\u00A0Krissansen\t \u00C2\u00A0GW\t \u00C2\u00A0(2013)\t \u00C2\u00A0Xentry,\t \u00C2\u00A0a\t \u00C2\u00A0new\t \u00C2\u00A0class\t \u00C2\u00A0of\t \u00C2\u00A0cell-\u00C2\u00AD\u00E2\u0080\u0090penetrating\t \u00C2\u00A0peptide\t \u00C2\u00A0uniquely\t \u00C2\u00A0equipped\t \u00C2\u00A0for\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0drugs.\t \u00C2\u00A0Sci\t \u00C2\u00A0Rep\t \u00C2\u00A03:1661.\t \u00C2\u00A0Morini\t \u00C2\u00A0S,\t \u00C2\u00A0Carotti\t \u00C2\u00A0S,\t \u00C2\u00A0Carpino\t \u00C2\u00A0G,\t \u00C2\u00A0Franchitto\t \u00C2\u00A0A,\t \u00C2\u00A0Corradini\t \u00C2\u00A0SG,\t \u00C2\u00A0Merli\t \u00C2\u00A0M,\t \u00C2\u00A0Gaudio\t \u00C2\u00A0E\t \u00C2\u00A0(2005)\t \u00C2\u00A0GFAP\t \u00C2\u00A0expression\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0liver\t \u00C2\u00A0as\t \u00C2\u00A0an\t \u00C2\u00A0early\t \u00C2\u00A0marker\t \u00C2\u00A0of\t \u00C2\u00A0stellate\t \u00C2\u00A0cells\t \u00C2\u00A0activation.\t \u00C2\u00A0Ital\t \u00C2\u00A0J\t \u00C2\u00A0Anat\t \u00C2\u00A0Embryol.\t \u00C2\u00A0110:193\u00E2\u0080\u0093207.\t \u00C2\u00A0Morris\t \u00C2\u00A0MC,\t \u00C2\u00A0Chaloin\t \u00C2\u00A0L,\t \u00C2\u00A0Mery\t \u00C2\u00A0J,\t \u00C2\u00A0Heitz\t \u00C2\u00A0F,\t \u00C2\u00A0Divita\t \u00C2\u00A0G\t \u00C2\u00A0(1999)\t \u00C2\u00A0A\t \u00C2\u00A0novel\t \u00C2\u00A0potent\t \u00C2\u00A0strategy\t \u00C2\u00A0for\t \u00C2\u00A0gene\t \u00C2\u00A0delivery\t \u00C2\u00A0using\t \u00C2\u00A0a\t \u00C2\u00A0single\t \u00C2\u00A0peptide\t \u00C2\u00A0vector\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0carrier.\t \u00C2\u00A0Nucleic\t \u00C2\u00A0Acids\t \u00C2\u00A0Res\t \u00C2\u00A027:3510\u00E2\u0080\u00933517.\t \u00C2\u00A0Morris\t \u00C2\u00A0MC,\t \u00C2\u00A0Depollier\t \u00C2\u00A0J,\t \u00C2\u00A0Mery\t \u00C2\u00A0J,\t \u00C2\u00A0Heitz\t \u00C2\u00A0F,\t \u00C2\u00A0Divita\t \u00C2\u00A0G\t \u00C2\u00A0(2001)\t \u00C2\u00A0A\t \u00C2\u00A0peptide\t \u00C2\u00A0carrier\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0delivery\t \u00C2\u00A0of\t \u00C2\u00A0biologically\t \u00C2\u00A0active\t \u00C2\u00A0proteins\t \u00C2\u00A0into\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A019:1173\u00E2\u0080\u00931176.\t \u00C2\u00A0Morris\t \u00C2\u00A0MC,\t \u00C2\u00A0Deshayes\t \u00C2\u00A0S,\t \u00C2\u00A0Heitz\t \u00C2\u00A0F,\t \u00C2\u00A0Divita\t \u00C2\u00A0G\t \u00C2\u00A0(2012)\t \u00C2\u00A0Cell-\u00C2\u00AD\u00E2\u0080\u0090penetrating\t \u00C2\u00A0peptides:\t \u00C2\u00A0from\t \u00C2\u00A0molecular\t \u00C2\u00A0mechanisms\t \u00C2\u00A0to\t \u00C2\u00A0therapeutics.\t \u00C2\u00A0Biol\t \u00C2\u00A0Cell\t \u00C2\u00A0100:201\u00E2\u0080\u0093217.\t \u00C2\u00A0M\u00C3\u00BCller\t \u00C2\u00A0M\t \u00C2\u00A0(2011)\t \u00C2\u00A0Current\t \u00C2\u00A0issues\t \u00C2\u00A0in\t \u00C2\u00A0drug\t \u00C2\u00A0development.\t \u00C2\u00A0In:\t \u00C2\u00A0Clinical\t \u00C2\u00A0Pharmacology:\t \u00C2\u00A0Current\t \u00C2\u00A0Topics\t \u00C2\u00A0and\t \u00C2\u00A0Case\t \u00C2\u00A0Studies,\t \u00C2\u00A0pp\t \u00C2\u00A07\u00E2\u0080\u009318.\t \u00C2\u00A0Springer.\t \u00C2\u00A0Nagy\t \u00C2\u00A0A\t \u00C2\u00A0(2000)\t \u00C2\u00A0Cre\t \u00C2\u00A0recombinase:\t \u00C2\u00A0the\t \u00C2\u00A0universal\t \u00C2\u00A0reagent\t \u00C2\u00A0for\t \u00C2\u00A0genome\t \u00C2\u00A0tailoring.\t \u00C2\u00A0Genesis\t \u00C2\u00A026:99\u00E2\u0080\u0093109.\t \u00C2\u00A0 217 Nawy\t \u00C2\u00A0T\t \u00C2\u00A0(2012)\t \u00C2\u00A0Small\t \u00C2\u00A0RNAs:\t \u00C2\u00A0Leaner,\t \u00C2\u00A0meaner\t \u00C2\u00A0siRNAs.\t \u00C2\u00A0Nat\t \u00C2\u00A0Methods\t \u00C2\u00A09:1050\u00E2\u0080\u00931050.\t \u00C2\u00A0Neher\t \u00C2\u00A0SB,\t \u00C2\u00A0Sauer\t \u00C2\u00A0RT,\t \u00C2\u00A0Baker\t \u00C2\u00A0TA\t \u00C2\u00A0(2003)\t \u00C2\u00A0Distinct\t \u00C2\u00A0peptide\t \u00C2\u00A0signals\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0UmuD\t \u00C2\u00A0and\t \u00C2\u00A0UmuD\u0000\t \u00C2\u00A0subunits\t \u00C2\u00A0of\t \u00C2\u00A0UmuD/D\u0000\t \u00C2\u00A0mediate\t \u00C2\u00A0tethering\t \u00C2\u00A0and\t \u00C2\u00A0substrate\t \u00C2\u00A0processing\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0ClpXP\t \u00C2\u00A0protease.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0100:13219\u00E2\u0080\u009313224.\t \u00C2\u00A0Neklesa\t \u00C2\u00A0TK,\t \u00C2\u00A0Crews\t \u00C2\u00A0CM\t \u00C2\u00A0(2012)\t \u00C2\u00A0Greasy\t \u00C2\u00A0tags\t \u00C2\u00A0for\t \u00C2\u00A0protein\t \u00C2\u00A0removal.\t \u00C2\u00A0Nature\t \u00C2\u00A0487:308\u00E2\u0080\u0093309.\t \u00C2\u00A0Neklesa\t \u00C2\u00A0TK,\t \u00C2\u00A0Noblin\t \u00C2\u00A0DJ,\t \u00C2\u00A0Kuzin\t \u00C2\u00A0A,\t \u00C2\u00A0Lew\t \u00C2\u00A0S,\t \u00C2\u00A0Seetharaman\t \u00C2\u00A0J,\t \u00C2\u00A0Acton\t \u00C2\u00A0TB,\t \u00C2\u00A0Kornhaber\t \u00C2\u00A0G,\t \u00C2\u00A0Xiao\t \u00C2\u00A0R,\t \u00C2\u00A0Montelione\t \u00C2\u00A0GT,\t \u00C2\u00A0Tong\t \u00C2\u00A0L,\t \u00C2\u00A0Crews\t \u00C2\u00A0CM\t \u00C2\u00A0(2013)\t \u00C2\u00A0A\t \u00C2\u00A0Bidirectional\t \u00C2\u00A0System\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0Dynamic\t \u00C2\u00A0Small\t \u00C2\u00A0Molecule\t \u00C2\u00A0Control\t \u00C2\u00A0of\t \u00C2\u00A0Intracellular\t \u00C2\u00A0Fusion\t \u00C2\u00A0Proteins.\t \u00C2\u00A0ACS\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A08:2293\u00E2\u0080\u00932300.\t \u00C2\u00A0Neklesa\t \u00C2\u00A0TK,\t \u00C2\u00A0Tae\t \u00C2\u00A0HS,\t \u00C2\u00A0Schneekloth\t \u00C2\u00A0AR,\t \u00C2\u00A0Stulberg\t \u00C2\u00A0MJ,\t \u00C2\u00A0Corson\t \u00C2\u00A0TW,\t \u00C2\u00A0Sundberg\t \u00C2\u00A0TB,\t \u00C2\u00A0Raina\t \u00C2\u00A0K,\t \u00C2\u00A0Holley\t \u00C2\u00A0SA,\t \u00C2\u00A0Crews\t \u00C2\u00A0CM\t \u00C2\u00A0(2011)\t \u00C2\u00A0Small-\u00C2\u00AD\u00E2\u0080\u0090molecule\t \u00C2\u00A0hydrophobic\t \u00C2\u00A0tagging\u00E2\u0080\u0093induced\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0HaloTag\t \u00C2\u00A0fusion\t \u00C2\u00A0proteins.\t \u00C2\u00A0Nat\t \u00C2\u00A0Chem\t \u00C2\u00A0Biol\t \u00C2\u00A07:538\u00E2\u0080\u0093543.\t \u00C2\u00A0Neumar\t \u00C2\u00A0RW,\t \u00C2\u00A0Meng\t \u00C2\u00A0FH,\t \u00C2\u00A0Mills\t \u00C2\u00A0AM,\t \u00C2\u00A0Xu\t \u00C2\u00A0YA,\t \u00C2\u00A0Zhang\t \u00C2\u00A0C,\t \u00C2\u00A0Welsh\t \u00C2\u00A0FA,\t \u00C2\u00A0Siman\t \u00C2\u00A0R\t \u00C2\u00A0(2001)\t \u00C2\u00A0Calpain\t \u00C2\u00A0Activity\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0Rat\t \u00C2\u00A0Brain\t \u00C2\u00A0after\t \u00C2\u00A0Transient\t \u00C2\u00A0Forebrain\t \u00C2\u00A0Ischemia.\t \u00C2\u00A0Exp\t \u00C2\u00A0Neurol170:27\u00E2\u0080\u009335.\t \u00C2\u00A0Nero\t \u00C2\u00A0TL,\t \u00C2\u00A0Morton\t \u00C2\u00A0CJ,\t \u00C2\u00A0Holien\t \u00C2\u00A0JK,\t \u00C2\u00A0Wielens\t \u00C2\u00A0J,\t \u00C2\u00A0Parker\t \u00C2\u00A0MW\t \u00C2\u00A0(2014).\t \u00C2\u00A0Oncogenic\t \u00C2\u00A0protein\t \u00C2\u00A0interfaces:\t \u00C2\u00A0small\t \u00C2\u00A0molecules,\t \u00C2\u00A0big\t \u00C2\u00A0challenges.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Cancer.\t \u00C2\u00A014:248-\u00C2\u00AD\u00E2\u0080\u009062.\t \u00C2\u00A0\t \u00C2\u00A0Nishida\t \u00C2\u00A0Y,\t \u00C2\u00A0Arakawa\t \u00C2\u00A0S,\t \u00C2\u00A0Fujitani\t \u00C2\u00A0K,\t \u00C2\u00A0Yamaguchi\t \u00C2\u00A0H\t \u00C2\u00A0(2009)\t \u00C2\u00A0Discovery\t \u00C2\u00A0of\t \u00C2\u00A0Atg5/Atg7-\u00C2\u00AD\u00E2\u0080\u0090independent\t \u00C2\u00A0alternative\t \u00C2\u00A0macroautophagy.\t \u00C2\u00A0Nature\t \u00C2\u00A0461:\t \u00C2\u00A0654-\u00C2\u00AD\u00E2\u0080\u0090658.\t \u00C2\u00A0\t \u00C2\u00A0 218 Nishimura\t \u00C2\u00A0K,\t \u00C2\u00A0Fukagawa\t \u00C2\u00A0T,\t \u00C2\u00A0Takisawa\t \u00C2\u00A0H,\t \u00C2\u00A0Kakimoto\t \u00C2\u00A0T,\t \u00C2\u00A0Kanemaki\t \u00C2\u00A0M\t \u00C2\u00A0(2009)\t \u00C2\u00A0An\t \u00C2\u00A0auxin-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0degron\t \u00C2\u00A0system\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0rapid\t \u00C2\u00A0depletion\t \u00C2\u00A0of\t \u00C2\u00A0proteins\t \u00C2\u00A0in\t \u00C2\u00A0nonplant\t \u00C2\u00A0cells.\t \u00C2\u00A0Nat\t \u00C2\u00A0Methods\t \u00C2\u00A06:917\u00E2\u0080\u0093922.\t \u00C2\u00A0Niu\t \u00C2\u00A0Y\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2014)\t \u00C2\u00A0Generation\t \u00C2\u00A0of\t \u00C2\u00A0Gene-\u00C2\u00AD\u00E2\u0080\u0090Modified\t \u00C2\u00A0Cynomolgus\t \u00C2\u00A0Monkey\t \u00C2\u00A0via\t \u00C2\u00A0Cas9/RNA-\u00C2\u00AD\u00E2\u0080\u0090MediatedGene\t \u00C2\u00A0Targeting\t \u00C2\u00A0in\t \u00C2\u00A0One-\u00C2\u00AD\u00E2\u0080\u0090Cell\t \u00C2\u00A0Embryos.\t \u00C2\u00A0Cell\t \u00C2\u00A0156:836\u00E2\u0080\u0093843.\t \u00C2\u00A0Okazaki\t \u00C2\u00A0K,\t \u00C2\u00A0Sagata\t \u00C2\u00A0N\t \u00C2\u00A0(1995)\t \u00C2\u00A0The\t \u00C2\u00A0Mos/MAP\t \u00C2\u00A0kinase\t \u00C2\u00A0pathway\t \u00C2\u00A0stabilizes\t \u00C2\u00A0c-\u00C2\u00AD\u00E2\u0080\u0090Fos\t \u00C2\u00A0by\t \u00C2\u00A0phosphorylation\t \u00C2\u00A0and\t \u00C2\u00A0augments\t \u00C2\u00A0its\t \u00C2\u00A0transforming\t \u00C2\u00A0activity\t \u00C2\u00A0in\t \u00C2\u00A0NIH\t \u00C2\u00A03T3\t \u00C2\u00A0cells.\t \u00C2\u00A0EMBO\t \u00C2\u00A0J\t \u00C2\u00A014:5048-\u00C2\u00AD\u00E2\u0080\u00905059.\t \u00C2\u00A0Okiyoneda\t \u00C2\u00A0T,\t \u00C2\u00A0Barri\u00C3\u00A8re\t \u00C2\u00A0H,\t \u00C2\u00A0Bagd\u00C3\u00A1ny\t \u00C2\u00A0M,\t \u00C2\u00A0Rabeh\t \u00C2\u00A0WM,\t \u00C2\u00A0Du\t \u00C2\u00A0K,\t \u00C2\u00A0H\u00C3\u00B6hfeld\t \u00C2\u00A0J,\t \u00C2\u00A0Young\t \u00C2\u00A0JC,\t \u00C2\u00A0Lukacs\t \u00C2\u00A0GL\t \u00C2\u00A0(2010)\t \u00C2\u00A0Peripheral\t \u00C2\u00A0protein\t \u00C2\u00A0quality\t \u00C2\u00A0control\t \u00C2\u00A0removes\t \u00C2\u00A0unfolded\t \u00C2\u00A0CFTR\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane.\t \u00C2\u00A0Science\t \u00C2\u00A0329:805-\u00C2\u00AD\u00E2\u0080\u0090810.\t \u00C2\u00A0Osuga\t \u00C2\u00A0H,\t \u00C2\u00A0Osuga\t \u00C2\u00A0S,\t \u00C2\u00A0Wang\t \u00C2\u00A0F,\t \u00C2\u00A0Fetni\t \u00C2\u00A0R,\t \u00C2\u00A0Hogan\t \u00C2\u00A0MJ,\t \u00C2\u00A0Slack\t \u00C2\u00A0RS,\t \u00C2\u00A0Hakim\t \u00C2\u00A0AM,\t \u00C2\u00A0Ikeda\t \u00C2\u00A0JE,\t \u00C2\u00A0Park\t \u00C2\u00A0DS\t \u00C2\u00A0(2000)\t \u00C2\u00A0Cyclin-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0kinases\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic\t \u00C2\u00A0target\t \u00C2\u00A0for\t \u00C2\u00A0stroke.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A097:10254\u00E2\u0080\u009310259.\t \u00C2\u00A0Paddison\t \u00C2\u00A0PJ,\t \u00C2\u00A0Caudy\t \u00C2\u00A0AA,\t \u00C2\u00A0Bernstein\t \u00C2\u00A0E,\t \u00C2\u00A0Hannon\t \u00C2\u00A0GJ,\t \u00C2\u00A0Conklin\t \u00C2\u00A0DS\t \u00C2\u00A0(2002)\t \u00C2\u00A0Short\t \u00C2\u00A0hairpin\t \u00C2\u00A0RNAs\t \u00C2\u00A0\t \u00C2\u00A0induce\t \u00C2\u00A0sequence-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0silencing\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells.\t \u00C2\u00A0Genes\t \u00C2\u00A0Dev\t \u00C2\u00A016:948\u00E2\u0080\u0093958.\t \u00C2\u00A0Pan\t \u00C2\u00A0T,\t \u00C2\u00A0Kondo\t \u00C2\u00A0S,\t \u00C2\u00A0Zhu\t \u00C2\u00A0W,\t \u00C2\u00A0Xie\t \u00C2\u00A0W,\t \u00C2\u00A0Jankovic\t \u00C2\u00A0J,\t \u00C2\u00A0Le\t \u00C2\u00A0W\t \u00C2\u00A0(2008)\t \u00C2\u00A0Neuroprotection\t \u00C2\u00A0of\t \u00C2\u00A0rapamycin\t \u00C2\u00A0in\t \u00C2\u00A0lactacystin-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neurodegeneration\t \u00C2\u00A0via\t \u00C2\u00A0autophagy\t \u00C2\u00A0enhancement.\t \u00C2\u00A0Neurobio\t \u00C2\u00A0Dis\t \u00C2\u00A032:16\u00E2\u0080\u009325.\t \u00C2\u00A0 219 Park\t \u00C2\u00A0C,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2013)\t \u00C2\u00A0Selective\t \u00C2\u00A0Autophagy:\t \u00C2\u00A0Talking\t \u00C2\u00A0with\t \u00C2\u00A0the\t \u00C2\u00A0UPS.\t \u00C2\u00A0Cell\t \u00C2\u00A0Biochem\t \u00C2\u00A0Biophys\t \u00C2\u00A067:3\u00E2\u0080\u009313.\t \u00C2\u00A0Patel\t \u00C2\u00A0LN,\t \u00C2\u00A0Zaro\t \u00C2\u00A0JL,\t \u00C2\u00A0Shen\t \u00C2\u00A0WC\t \u00C2\u00A0(2007)\t \u00C2\u00A0Cell\t \u00C2\u00A0penetrating\t \u00C2\u00A0peptides:\t \u00C2\u00A0intracellular\t \u00C2\u00A0pathways\t \u00C2\u00A0and\t \u00C2\u00A0pharmaceutical\t \u00C2\u00A0perspectives.\t \u00C2\u00A0Pharm\t \u00C2\u00A0Res\t \u00C2\u00A024:\t \u00C2\u00A01977-\u00C2\u00AD\u00E2\u0080\u009092.\t \u00C2\u00A0Pattanayak\t \u00C2\u00A0V,\t \u00C2\u00A0Ramirez\t \u00C2\u00A0CL,\t \u00C2\u00A0Joung\t \u00C2\u00A0JK,\t \u00C2\u00A0Liu\t \u00C2\u00A0DR\t \u00C2\u00A0(2011)\t \u00C2\u00A0Revealing\t \u00C2\u00A0off-\u00C2\u00AD\u00E2\u0080\u0090target\t \u00C2\u00A0cleavage\t \u00C2\u00A0specificities\t \u00C2\u00A0of\t \u00C2\u00A0zinc-\u00C2\u00AD\u00E2\u0080\u0090finger\t \u00C2\u00A0nucleases\t \u00C2\u00A0by\t \u00C2\u00A0in\t \u00C2\u00A0vitro\t \u00C2\u00A0selection.\t \u00C2\u00A0Nat\t \u00C2\u00A0Methods\t \u00C2\u00A08:765\u00E2\u0080\u0093770.\t \u00C2\u00A0Patton\t \u00C2\u00A0EE,\t \u00C2\u00A0Willems\t \u00C2\u00A0AR,\t \u00C2\u00A0Sa\t \u00C2\u00A0D,\t \u00C2\u00A0Kuras\t \u00C2\u00A0L,\t \u00C2\u00A0Thomas\t \u00C2\u00A0D,\t \u00C2\u00A0Craig\t \u00C2\u00A0KL,\t \u00C2\u00A0Tyers\t \u00C2\u00A0M\t \u00C2\u00A0(1998)\t \u00C2\u00A0Cdc53\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0scaffold\t \u00C2\u00A0protein\t \u00C2\u00A0for\t \u00C2\u00A0multiple\t \u00C2\u00A0Cdc34/Skp1/F-\u00C2\u00AD\u00E2\u0080\u0090box\t \u00C2\u00A0protein\t \u00C2\u00A0complexes\t \u00C2\u00A0that\t \u00C2\u00A0regulate\t \u00C2\u00A0cell\t \u00C2\u00A0division\t \u00C2\u00A0and\t \u00C2\u00A0methionine\t \u00C2\u00A0biosynthesis\t \u00C2\u00A0in\t \u00C2\u00A0yeast.\t \u00C2\u00A0Genes\t \u00C2\u00A0Dev\t \u00C2\u00A012:692\u00E2\u0080\u0093705.\t \u00C2\u00A0Pei\t \u00C2\u00A0L,\t \u00C2\u00A0Shang\t \u00C2\u00A0Y,\t \u00C2\u00A0Jin\t \u00C2\u00A0H,\t \u00C2\u00A0Wang\t \u00C2\u00A0S,\t \u00C2\u00A0Wei\t \u00C2\u00A0N,\t \u00C2\u00A0Yan\t \u00C2\u00A0H,\t \u00C2\u00A0Wu\t \u00C2\u00A0Y,\t \u00C2\u00A0Yao\t \u00C2\u00A0C,\t \u00C2\u00A0Wang\t \u00C2\u00A0X,\t \u00C2\u00A0Zhu\t \u00C2\u00A0LQ,\t \u00C2\u00A0Lu\t \u00C2\u00A0Y\t \u00C2\u00A0(2014)\t \u00C2\u00A0DAPK1-\u00C2\u00AD\u00E2\u0080\u0090p53\t \u00C2\u00A0Interaction\t \u00C2\u00A0Converges\t \u00C2\u00A0Necrotic\t \u00C2\u00A0and\t \u00C2\u00A0Apoptotic\t \u00C2\u00A0Pathways\t \u00C2\u00A0of\t \u00C2\u00A0Ischemic\t \u00C2\u00A0Neuronal\t \u00C2\u00A0Death.\t \u00C2\u00A0J\t \u00C2\u00A0Neurosci\t \u00C2\u00A034:6546\u00E2\u0080\u00936556.\t \u00C2\u00A0Pelled\t \u00C2\u00A0D,\t \u00C2\u00A0Raveh\t \u00C2\u00A0T,\t \u00C2\u00A0Riebeling\t \u00C2\u00A0C,\t \u00C2\u00A0Fridkin\t \u00C2\u00A0M,\t \u00C2\u00A0Berissi\t \u00C2\u00A0H,\t \u00C2\u00A0Futerman\t \u00C2\u00A0AH,\t \u00C2\u00A0Kimchi\t \u00C2\u00A0A\t \u00C2\u00A0(2002)\t \u00C2\u00A0Death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0(DAP)\t \u00C2\u00A0kinase\t \u00C2\u00A0plays\t \u00C2\u00A0a\t \u00C2\u00A0central\t \u00C2\u00A0role\t \u00C2\u00A0in\t \u00C2\u00A0ceramide-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0apoptosis\t \u00C2\u00A0in\t \u00C2\u00A0cultured\t \u00C2\u00A0hippocampal\t \u00C2\u00A0neurons.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0277(3):1957\u00E2\u0080\u00931961.\t \u00C2\u00A0Petersen\t \u00C2\u00A0JD,\t \u00C2\u00A0Chen\t \u00C2\u00A0X,\t \u00C2\u00A0Vinade\t \u00C2\u00A0L,\t \u00C2\u00A0Dosemeci\t \u00C2\u00A0A,\t \u00C2\u00A0Lisman\t \u00C2\u00A0JE,\t \u00C2\u00A0Reese\t \u00C2\u00A0TS\t \u00C2\u00A0(2003)\t \u00C2\u00A0Distribution\t \u00C2\u00A0of\t \u00C2\u00A0postsynaptic\t \u00C2\u00A0density\t \u00C2\u00A0(PSD)-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0and\t \u00C2\u00A0Ca2+/calmodulin-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0II\t \u00C2\u00A0at\t \u00C2\u00A0the\t \u00C2\u00A0PSD.\t \u00C2\u00A0J\t \u00C2\u00A0Neurosci\t \u00C2\u00A023:11270\u00E2\u0080\u009311278.\t \u00C2\u00A0\t \u00C2\u00A0 220 Pfleger\t \u00C2\u00A0CM,\t \u00C2\u00A0Kirschner\t \u00C2\u00A0MW\t \u00C2\u00A0(2000)\t \u00C2\u00A0The\t \u00C2\u00A0KEN\t \u00C2\u00A0box:\t \u00C2\u00A0an\t \u00C2\u00A0APC\t \u00C2\u00A0recognition\t \u00C2\u00A0signal\t \u00C2\u00A0distinct\t \u00C2\u00A0from\t \u00C2\u00A0the\t \u00C2\u00A0D\t \u00C2\u00A0box\t \u00C2\u00A0targeted\t \u00C2\u00A0by\t \u00C2\u00A0Cdh1.\t \u00C2\u00A0Genes\t \u00C2\u00A0Dev\t \u00C2\u00A014:655\u00E2\u0080\u0093665.\t \u00C2\u00A0Pratt\t \u00C2\u00A0MR,\t \u00C2\u00A0Schwartz\t \u00C2\u00A0EC,\t \u00C2\u00A0Muir\t \u00C2\u00A0TW\t \u00C2\u00A0(2007)\t \u00C2\u00A0Small-\u00C2\u00AD\u00E2\u0080\u0090molecule-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0rescue\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0function\t \u00C2\u00A0by\t \u00C2\u00A0an\t \u00C2\u00A0inducible\t \u00C2\u00A0proteolytic\t \u00C2\u00A0shunt.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0104:11209\u00E2\u0080\u009311214.\t \u00C2\u00A0Qi\t \u00C2\u00A0L,\t \u00C2\u00A0Zhang\t \u00C2\u00A0X-\u00C2\u00AD\u00E2\u0080\u0090D,\t \u00C2\u00A0Wu\t \u00C2\u00A0J-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Lin\t \u00C2\u00A0F,\t \u00C2\u00A0Wang\t \u00C2\u00A0J,\t \u00C2\u00A0Difiglia\t \u00C2\u00A0M,\t \u00C2\u00A0Qin\t \u00C2\u00A0Z-\u00C2\u00AD\u00E2\u0080\u0090H\t \u00C2\u00A0(2012)\t \u00C2\u00A0The\t \u00C2\u00A0role\t \u00C2\u00A0of\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0in\t \u00C2\u00A0huntingtin\t \u00C2\u00A0degradation.\t \u00C2\u00A0PLoS\t \u00C2\u00A0ONE\t \u00C2\u00A07:e46834.\t \u00C2\u00A0Rechsteiner\t \u00C2\u00A0M,\t \u00C2\u00A0Rogers\t \u00C2\u00A0SW\t \u00C2\u00A0(1996)\t \u00C2\u00A0PEST\t \u00C2\u00A0sequences\t \u00C2\u00A0and\t \u00C2\u00A0regulation\t \u00C2\u00A0by\t \u00C2\u00A0proteolysis.\t \u00C2\u00A0Trends\t \u00C2\u00A0Biochem\t \u00C2\u00A0Sci\t \u00C2\u00A021:267\u00E2\u0080\u0093271.\t \u00C2\u00A0Roberts\t \u00C2\u00A0J,\t \u00C2\u00A0Palma\t \u00C2\u00A0E,\t \u00C2\u00A0Sazani\t \u00C2\u00A0P,\t \u00C2\u00A0\u00C3\u0098rum\t \u00C2\u00A0H,\t \u00C2\u00A0Cho\t \u00C2\u00A0M,\t \u00C2\u00A0Kole\t \u00C2\u00A0R\t \u00C2\u00A0(2006)\t \u00C2\u00A0Efficient\t \u00C2\u00A0and\t \u00C2\u00A0persistent\t \u00C2\u00A0splice\t \u00C2\u00A0switching\t \u00C2\u00A0by\t \u00C2\u00A0systemically\t \u00C2\u00A0delivered\t \u00C2\u00A0LNA\t \u00C2\u00A0oligonucleotides\t \u00C2\u00A0in\t \u00C2\u00A0mice.\t \u00C2\u00A0Mol\t \u00C2\u00A0Ther\t \u00C2\u00A014:471\u00E2\u0080\u0093475.\t \u00C2\u00A0Rodriguez-\u00C2\u00AD\u00E2\u0080\u0090Navarro\t \u00C2\u00A0JA,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2012)\t \u00C2\u00A0Dietary\t \u00C2\u00A0lipids\t \u00C2\u00A0and\t \u00C2\u00A0aging\t \u00C2\u00A0compromise\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0by\t \u00C2\u00A0similar\t \u00C2\u00A0mechanisms.\t \u00C2\u00A0Autophagy\t \u00C2\u00A08:1152-\u00C2\u00AD\u00E2\u0080\u00901154.\t \u00C2\u00A0Rodriguez-\u00C2\u00AD\u00E2\u0080\u0090Navarro\t \u00C2\u00A0JA,\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Koga\t \u00C2\u00A0H,\t \u00C2\u00A0Dall'Armi\t \u00C2\u00A0C,\t \u00C2\u00A0Shui\t \u00C2\u00A0G,\t \u00C2\u00A0Wenk\t \u00C2\u00A0MR,\t \u00C2\u00A0Di\t \u00C2\u00A0Paolo\t \u00C2\u00A0G,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2012)\t \u00C2\u00A0Inhibitory\t \u00C2\u00A0effect\t \u00C2\u00A0of\t \u00C2\u00A0dietary\t \u00C2\u00A0lipids\t \u00C2\u00A0on\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0109:705\u00E2\u0080\u0093714.\t \u00C2\u00A0Rosser\t \u00C2\u00A0MFN,\t \u00C2\u00A0Washburn\t \u00C2\u00A0E,\t \u00C2\u00A0Muchowski\t \u00C2\u00A0PJ,\t \u00C2\u00A0Patterson\t \u00C2\u00A0C,\t \u00C2\u00A0Cyr\t \u00C2\u00A0DM\t \u00C2\u00A0(2007)\t \u00C2\u00A0Chaperone\t \u00C2\u00A0Functions\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0E3\t \u00C2\u00A0Ubiquitin\t \u00C2\u00A0Ligase\t \u00C2\u00A0CHIP.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0282:22267\u00E2\u0080\u009322277.\t \u00C2\u00A0\t \u00C2\u00A0 221 Rothenberg\t \u00C2\u00A0C,\t \u00C2\u00A0Srinivasan\t \u00C2\u00A0D,\t \u00C2\u00A0Mah\t \u00C2\u00A0L,\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Peterhoff\t \u00C2\u00A0CM,\t \u00C2\u00A0Ugolino\t \u00C2\u00A0J,\t \u00C2\u00A0Fang\t \u00C2\u00A0S,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Nixon\t \u00C2\u00A0RA,\t \u00C2\u00A0Monteiro\t \u00C2\u00A0MJ\t \u00C2\u00A0(2010)\t \u00C2\u00A0Ubiquilin\t \u00C2\u00A0functions\t \u00C2\u00A0in\t \u00C2\u00A0autophagy\t \u00C2\u00A0and\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0Hum\t \u00C2\u00A0Mol\t \u00C2\u00A0Gen\t \u00C2\u00A019:3219\u00E2\u0080\u00933232.\t \u00C2\u00A0Rouet\t \u00C2\u00A0P,\t \u00C2\u00A0Smih\t \u00C2\u00A0F,\t \u00C2\u00A0Jasin\t \u00C2\u00A0M\t \u00C2\u00A0(1994)\t \u00C2\u00A0Introduction\t \u00C2\u00A0of\t \u00C2\u00A0double-\u00C2\u00AD\u00E2\u0080\u0090strand\t \u00C2\u00A0breaks\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0genome\t \u00C2\u00A0of\t \u00C2\u00A0mouse\t \u00C2\u00A0cells\t \u00C2\u00A0by\t \u00C2\u00A0expression\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0rare-\u00C2\u00AD\u00E2\u0080\u0090cutting\t \u00C2\u00A0endonuclease.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A014:8096\u00E2\u0080\u00938106.\t \u00C2\u00A0Rubinson\t \u00C2\u00A0DA,\t \u00C2\u00A0Dillon\t \u00C2\u00A0CP,\t \u00C2\u00A0Kwiatkowski\t \u00C2\u00A0AV,\t \u00C2\u00A0Sievers\t \u00C2\u00A0C\t \u00C2\u00A0(2003)\t \u00C2\u00A0A\t \u00C2\u00A0lentivirus-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0system\t \u00C2\u00A0to\t \u00C2\u00A0functionally\t \u00C2\u00A0silence\t \u00C2\u00A0genes\t \u00C2\u00A0in\t \u00C2\u00A0primary\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells,\t \u00C2\u00A0stem\t \u00C2\u00A0cells\t \u00C2\u00A0and\t \u00C2\u00A0transgenic\t \u00C2\u00A0mice\t \u00C2\u00A0by\t \u00C2\u00A0RNA\t \u00C2\u00A0interference.\t \u00C2\u00A0Nature\t \u00C2\u00A033:401-\u00C2\u00AD\u00E2\u0080\u0090406.\t \u00C2\u00A0Rubinsztein\t \u00C2\u00A0DC,\t \u00C2\u00A0Codogno\t \u00C2\u00A0P,\t \u00C2\u00A0Levine\t \u00C2\u00A0B\t \u00C2\u00A0(2012)\t \u00C2\u00A0Autophagy\t \u00C2\u00A0modulation\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0potential\t \u00C2\u00A0therapeutic\t \u00C2\u00A0target\t \u00C2\u00A0for\t \u00C2\u00A0diverse\t \u00C2\u00A0diseases.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A011:709\u00E2\u0080\u0093730.\t \u00C2\u00A0Russ\t \u00C2\u00A0AP,\t \u00C2\u00A0Lampel\t \u00C2\u00A0S\t \u00C2\u00A0(2005)\t \u00C2\u00A0The\t \u00C2\u00A0druggable\t \u00C2\u00A0genome:\t \u00C2\u00A0an\t \u00C2\u00A0update.\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A0Today\t \u00C2\u00A010:1607\u00E2\u0080\u00931610.\t \u00C2\u00A0Saftig\t \u00C2\u00A0P,\t \u00C2\u00A0Klumperman\t \u00C2\u00A0J\t \u00C2\u00A0(2009)\t \u00C2\u00A0Lysosome\t \u00C2\u00A0biogenesis\t \u00C2\u00A0and\t \u00C2\u00A0lysosomal\t \u00C2\u00A0membrane\t \u00C2\u00A0proteins:\t \u00C2\u00A0trafficking\t \u00C2\u00A0meets\t \u00C2\u00A0function.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A010:623-\u00C2\u00AD\u00E2\u0080\u0090635.\t \u00C2\u00A0Sahu\t \u00C2\u00A0R,\t \u00C2\u00A0Kaushik\t \u00C2\u00A0S,\t \u00C2\u00A0Clement\t \u00C2\u00A0CC,\t \u00C2\u00A0Cannizzo\t \u00C2\u00A0ES,\t \u00C2\u00A0Scharf\t \u00C2\u00A0B,\t \u00C2\u00A0Follenzi\t \u00C2\u00A0A,\t \u00C2\u00A0Potolicchio\t \u00C2\u00A0I,\t \u00C2\u00A0Nieves\t \u00C2\u00A0E,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM,\t \u00C2\u00A0Santambrogio\t \u00C2\u00A0L\t \u00C2\u00A0(2011)\t \u00C2\u00A0Microautophagy\t \u00C2\u00A0of\t \u00C2\u00A0Cytosolic\t \u00C2\u00A0Proteins\t \u00C2\u00A0by\t \u00C2\u00A0Late\t \u00C2\u00A0Endosomes.\t \u00C2\u00A0Dev\t \u00C2\u00A0Cell\t \u00C2\u00A020:131\u00E2\u0080\u0093139.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 222 Saito\t \u00C2\u00A0G,\t \u00C2\u00A0Swanson\t \u00C2\u00A0JA,\t \u00C2\u00A0Lee\t \u00C2\u00A0KD\t \u00C2\u00A0(2003)\t \u00C2\u00A0Drug\t \u00C2\u00A0delivery\t \u00C2\u00A0strategy\t \u00C2\u00A0utilizing\t \u00C2\u00A0conjugation\t \u00C2\u00A0via\t \u00C2\u00A0reversible\t \u00C2\u00A0disulfide\t \u00C2\u00A0linkages:\t \u00C2\u00A0role\t \u00C2\u00A0and\t \u00C2\u00A0site\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0reducing\t \u00C2\u00A0activities.\t \u00C2\u00A0Adv\t \u00C2\u00A0Drug\t \u00C2\u00A0Deliv\t \u00C2\u00A0Rev\t \u00C2\u00A055:199-\u00C2\u00AD\u00E2\u0080\u0090215.\t \u00C2\u00A0Sakamoto\t \u00C2\u00A0KM\t \u00C2\u00A0(2003)\t \u00C2\u00A0Development\t \u00C2\u00A0of\t \u00C2\u00A0Protacs\t \u00C2\u00A0to\t \u00C2\u00A0Target\t \u00C2\u00A0Cancer-\u00C2\u00AD\u00E2\u0080\u0090promoting\t \u00C2\u00A0Proteins\t \u00C2\u00A0for\t \u00C2\u00A0Ubiquitination\t \u00C2\u00A0and\t \u00C2\u00A0Degradation.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Proteomics\t \u00C2\u00A02:1350\u00E2\u0080\u00931358.\t \u00C2\u00A0Sakamoto\t \u00C2\u00A0KM,\t \u00C2\u00A0Kim\t \u00C2\u00A0KB,\t \u00C2\u00A0Kumagai\t \u00C2\u00A0A,\t \u00C2\u00A0Mercurio\t \u00C2\u00A0F,\t \u00C2\u00A0Crews\t \u00C2\u00A0CM,\t \u00C2\u00A0Deshaies\t \u00C2\u00A0RJ\t \u00C2\u00A0(2001)\t \u00C2\u00A0Protacs:\t \u00C2\u00A0chimeric\t \u00C2\u00A0molecules\t \u00C2\u00A0that\t \u00C2\u00A0target\t \u00C2\u00A0proteins\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0Skp1-\u00C2\u00AD\u00E2\u0080\u0090Cullin-\u00C2\u00AD\u00E2\u0080\u0090F\t \u00C2\u00A0box\t \u00C2\u00A0complex\t \u00C2\u00A0for\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0and\t \u00C2\u00A0degradation.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A098:8554\u00E2\u0080\u00938559.\t \u00C2\u00A0Salvador\t \u00C2\u00A0N,\t \u00C2\u00A0Aguado\t \u00C2\u00A0C,\t \u00C2\u00A0Horst\t \u00C2\u00A0M,\t \u00C2\u00A0and\t \u00C2\u00A0Knecht\t \u00C2\u00A0E\t \u00C2\u00A0(2000)\t \u00C2\u00A0Import\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0Cytosolic\t \u00C2\u00A0Protein\t \u00C2\u00A0into\t \u00C2\u00A0Lysosomes\t \u00C2\u00A0by\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy\t \u00C2\u00A0depends\t \u00C2\u00A0on\t \u00C2\u00A0its\t \u00C2\u00A0Folding\t \u00C2\u00A0State.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0\t \u00C2\u00A0Chem\t \u00C2\u00A0275:27447-\u00C2\u00AD\u00E2\u0080\u009027456.\t \u00C2\u00A0Sattler\t \u00C2\u00A0R,\t \u00C2\u00A0Xiong\t \u00C2\u00A0Z-\u00C2\u00AD\u00E2\u0080\u0090G,\t \u00C2\u00A0Lu\t \u00C2\u00A0W-\u00C2\u00AD\u00E2\u0080\u0090Y,\t \u00C2\u00A0Hafner\t \u00C2\u00A0M,\t \u00C2\u00A0MacDonald\t \u00C2\u00A0JF,\t \u00C2\u00A0Tymianski\t \u00C2\u00A0M\t \u00C2\u00A0(1999)\t \u00C2\u00A0Specific\t \u00C2\u00A0Coupling\t \u00C2\u00A0of\t \u00C2\u00A0NMDA\t \u00C2\u00A0Receptor\t \u00C2\u00A0Activation\t \u00C2\u00A0to\t \u00C2\u00A0Nitric\t \u00C2\u00A0Oxide\t \u00C2\u00A0Neurotoxicity\t \u00C2\u00A0by\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0Protein.\t \u00C2\u00A0Science\t \u00C2\u00A0284:1845\u00E2\u0080\u00931848.\t \u00C2\u00A0\t \u00C2\u00A0Sauer\t \u00C2\u00A0B,\t \u00C2\u00A0Henderson\t \u00C2\u00A0N\t \u00C2\u00A0(1988)\t \u00C2\u00A0Site-\u00C2\u00AD\u00E2\u0080\u0090specific\t \u00C2\u00A0DNA\t \u00C2\u00A0recombination\t \u00C2\u00A0in\t \u00C2\u00A0mammalian\t \u00C2\u00A0cells\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0Cre\t \u00C2\u00A0recombinase\t \u00C2\u00A0of\t \u00C2\u00A0bacteriophage\t \u00C2\u00A0P1.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A085:5166\u00E2\u0080\u00935170.\t \u00C2\u00A0Scaggiante\t \u00C2\u00A0B,\t \u00C2\u00A0Dapas\t \u00C2\u00A0B,\t \u00C2\u00A0Farra\t \u00C2\u00A0R,\t \u00C2\u00A0Grassi\t \u00C2\u00A0M,\t \u00C2\u00A0Pozzato\t \u00C2\u00A0G,\t \u00C2\u00A0Giansante\t \u00C2\u00A0C,\t \u00C2\u00A0Fiotti\t \u00C2\u00A0N,\t \u00C2\u00A0Grassi\t \u00C2\u00A0G\t \u00C2\u00A0(2011)\t \u00C2\u00A0Improving\t \u00C2\u00A0siRNA\t \u00C2\u00A0bio-\u00C2\u00AD\u00E2\u0080\u0090distribution\t \u00C2\u00A0and\t \u00C2\u00A0minimizing\t \u00C2\u00A0side\t \u00C2\u00A0effects.\t \u00C2\u00A0Curr\t \u00C2\u00A0Drug\t \u00C2\u00A0Metab\t \u00C2\u00A012:11\u00E2\u0080\u009323.\t \u00C2\u00A0 223 Scheffner\t \u00C2\u00A0M,\t \u00C2\u00A0Huibregtse\t \u00C2\u00A0JM,\t \u00C2\u00A0Vierstra\t \u00C2\u00A0RD,\t \u00C2\u00A0Howley\t \u00C2\u00A0PM\t \u00C2\u00A0(1993)\t \u00C2\u00A0The\t \u00C2\u00A0HPV-\u00C2\u00AD\u00E2\u0080\u009016\t \u00C2\u00A0E6\t \u00C2\u00A0and\t \u00C2\u00A0E6-\u00C2\u00AD\u00E2\u0080\u0090AP\t \u00C2\u00A0complex\t \u00C2\u00A0functions\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090protein\t \u00C2\u00A0ligase\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0of\t \u00C2\u00A0p53.\t \u00C2\u00A0Cell\t \u00C2\u00A075:495\u00E2\u0080\u0093505.\t \u00C2\u00A0Schmid\t \u00C2\u00A0B,\t \u00C2\u00A0Haass\t \u00C2\u00A0C\t \u00C2\u00A0(2013)\t \u00C2\u00A0Genomic\t \u00C2\u00A0editing\t \u00C2\u00A0opens\t \u00C2\u00A0new\t \u00C2\u00A0avenues\t \u00C2\u00A0for\t \u00C2\u00A0zebrafish\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0model\t \u00C2\u00A0for\t \u00C2\u00A0neurodegeneration.\t \u00C2\u00A0J\t \u00C2\u00A0Neurochem\t \u00C2\u00A0127:461\u00E2\u0080\u0093470.\t \u00C2\u00A0Schrader\t \u00C2\u00A0EK,\t \u00C2\u00A0Harstad\t \u00C2\u00A0KG,\t \u00C2\u00A0Matouschek\t \u00C2\u00A0A\t \u00C2\u00A0(2009)\t \u00C2\u00A0Targeting\t \u00C2\u00A0proteins\t \u00C2\u00A0for\t \u00C2\u00A0degradation.\t \u00C2\u00A0Nat\t \u00C2\u00A0Methods\t \u00C2\u00A05:815\u00E2\u0080\u0093822.\t \u00C2\u00A0Schumacher\t \u00C2\u00A0AM,\t \u00C2\u00A0Velentza\t \u00C2\u00A0AV,\t \u00C2\u00A0Watterson\t \u00C2\u00A0DM,\t \u00C2\u00A0Wainwright\t \u00C2\u00A0MS\t \u00C2\u00A0(2002)\t \u00C2\u00A0DAPK\t \u00C2\u00A0catalytic\t \u00C2\u00A0activity\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0hippocampus\t \u00C2\u00A0increases\t \u00C2\u00A0during\t \u00C2\u00A0the\t \u00C2\u00A0recovery\t \u00C2\u00A0phase\t \u00C2\u00A0in\t \u00C2\u00A0an\t \u00C2\u00A0animal\t \u00C2\u00A0model\t \u00C2\u00A0of\t \u00C2\u00A0brain\t \u00C2\u00A0hypoxic-\u00C2\u00AD\u00E2\u0080\u0090ischemic\t \u00C2\u00A0injury.\t \u00C2\u00A0Biochim\t \u00C2\u00A0Biophys\t \u00C2\u00A0Acta\t \u00C2\u00A01600:128\u00E2\u0080\u0093137.\t \u00C2\u00A0Schumacher\t \u00C2\u00A0AM,\t \u00C2\u00A0Schavocky\t \u00C2\u00A0JP,\t \u00C2\u00A0Velentza\t \u00C2\u00A0AV,\t \u00C2\u00A0Mirzoeva\t \u00C2\u00A0S,\t \u00C2\u00A0Watterson\t \u00C2\u00A0DM\t \u00C2\u00A0(2004)\t \u00C2\u00A0A\t \u00C2\u00A0calmodulin-\u00C2\u00AD\u00E2\u0080\u0090regulated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0linked\t \u00C2\u00A0to\t \u00C2\u00A0neuron\t \u00C2\u00A0survival\t \u00C2\u00A0is\t \u00C2\u00A0a\t \u00C2\u00A0substrate\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0calmodulin-\u00C2\u00AD\u00E2\u0080\u0090regulated\t \u00C2\u00A0death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase.\t \u00C2\u00A0Biochem\t \u00C2\u00A043:8116\u00E2\u0080\u00938124.\t \u00C2\u00A0Schwarze\t \u00C2\u00A0SR\t \u00C2\u00A0(1999)\t \u00C2\u00A0In\t \u00C2\u00A0Vivo\t \u00C2\u00A0Protein\t \u00C2\u00A0Transduction:\t \u00C2\u00A0Delivery\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0Biologically\t \u00C2\u00A0Active\t \u00C2\u00A0Protein\t \u00C2\u00A0into\t \u00C2\u00A0the\t \u00C2\u00A0Mouse.\t \u00C2\u00A0Science\t \u00C2\u00A0285:1569\u00E2\u0080\u00931572.\t \u00C2\u00A0Seki\t \u00C2\u00A0T,\t \u00C2\u00A0Yoshino\t \u00C2\u00A0K-\u00C2\u00AD\u00E2\u0080\u0090I,\t \u00C2\u00A0Tanaka\t \u00C2\u00A0S,\t \u00C2\u00A0Dohi\t \u00C2\u00A0E,\t \u00C2\u00A0Onji\t \u00C2\u00A0T,\t \u00C2\u00A0Yamamoto\t \u00C2\u00A0K,\t \u00C2\u00A0Hide\t \u00C2\u00A0I,\t \u00C2\u00A0Paulson\t \u00C2\u00A0HL,\t \u00C2\u00A0Saito\t \u00C2\u00A0N,\t \u00C2\u00A0Sakai\t \u00C2\u00A0N\t \u00C2\u00A0(2012)\t \u00C2\u00A0Establishment\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0Novel\t \u00C2\u00A0Fluorescence-\u00C2\u00AD\u00E2\u0080\u0090Based\t \u00C2\u00A0Method\t \u00C2\u00A0to\t \u00C2\u00A0Evaluate\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy\t \u00C2\u00A0in\t \u00C2\u00A0a\t \u00C2\u00A0Single\t \u00C2\u00A0Neuron.\t \u00C2\u00A0PLoS\t \u00C2\u00A0ONE\t \u00C2\u00A07:e31232.\t \u00C2\u00A0\t \u00C2\u00A0 224 Sellmyer\t \u00C2\u00A0MA,\t \u00C2\u00A0Chen\t \u00C2\u00A0L-\u00C2\u00AD\u00E2\u0080\u0090C,\t \u00C2\u00A0Egeler\t \u00C2\u00A0EL,\t \u00C2\u00A0Rakhit\t \u00C2\u00A0R,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ\t \u00C2\u00A0(2012)\t \u00C2\u00A0Intracellular\t \u00C2\u00A0Context\t \u00C2\u00A0Affects\t \u00C2\u00A0Levels\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0Chemically\t \u00C2\u00A0Dependent\t \u00C2\u00A0Destabilizing\t \u00C2\u00A0Domain.\t \u00C2\u00A0PLoS\t \u00C2\u00A0ONE\t \u00C2\u00A07:e43297.\t \u00C2\u00A0Shaltiel-\u00C2\u00AD\u00E2\u0080\u0090Karyo\t \u00C2\u00A0R,\t \u00C2\u00A0Frenkel-\u00C2\u00AD\u00E2\u0080\u0090Pinter\t \u00C2\u00A0M,\t \u00C2\u00A0Egoz-\u00C2\u00AD\u00E2\u0080\u0090Matia\t \u00C2\u00A0N,\t \u00C2\u00A0Frydman-\u00C2\u00AD\u00E2\u0080\u0090Marom\t \u00C2\u00A0A,\t \u00C2\u00A0Shalev\t \u00C2\u00A0DE,\t \u00C2\u00A0Segal\t \u00C2\u00A0D,\t \u00C2\u00A0Gazit\t \u00C2\u00A0E\t \u00C2\u00A0(2010)\t \u00C2\u00A0Inhibiting\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090Synuclein\t \u00C2\u00A0Oligomerization\t \u00C2\u00A0by\t \u00C2\u00A0Stable\t \u00C2\u00A0Cell-\u00C2\u00AD\u00E2\u0080\u0090Penetrating\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090Synuclein\t \u00C2\u00A0Fragments\t \u00C2\u00A0Recovers\t \u00C2\u00A0Phenotype\t \u00C2\u00A0of\t \u00C2\u00A0Parkinson's\t \u00C2\u00A0Disease\t \u00C2\u00A0Model\t \u00C2\u00A0Flies.\t \u00C2\u00A0PLoS\t \u00C2\u00A0ONE\t \u00C2\u00A05:e13863.\t \u00C2\u00A0Shamloo\t \u00C2\u00A0M,\t \u00C2\u00A0Soriano\t \u00C2\u00A0L,\t \u00C2\u00A0Wieloch\t \u00C2\u00A0T,\t \u00C2\u00A0Nikolich\t \u00C2\u00A0K,\t \u00C2\u00A0Urfer\t \u00C2\u00A0R,\t \u00C2\u00A0Oksenberg\t \u00C2\u00A0D\t \u00C2\u00A0(2005)\t \u00C2\u00A0Death-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0is\t \u00C2\u00A0activated\t \u00C2\u00A0by\t \u00C2\u00A0dephosphorylation\t \u00C2\u00A0in\t \u00C2\u00A0response\t \u00C2\u00A0to\t \u00C2\u00A0cerebral\t \u00C2\u00A0ischemia.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0280:42290\u00E2\u0080\u009342299.\t \u00C2\u00A0Shoji-\u00C2\u00AD\u00E2\u0080\u0090Kawata\t \u00C2\u00A0S\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2013)\t \u00C2\u00A0Identification\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0candidate\t \u00C2\u00A0therapeutic\t \u00C2\u00A0autophagy-\u00C2\u00AD\u00E2\u0080\u0090inducing\t \u00C2\u00A0peptide.\t \u00C2\u00A0Nature\t \u00C2\u00A0494:201\u00E2\u0080\u0093206.\t \u00C2\u00A0Skarnes\t \u00C2\u00A0WC,\t \u00C2\u00A0Rosen\t \u00C2\u00A0B,\t \u00C2\u00A0West\t \u00C2\u00A0AP,\t \u00C2\u00A0Koutsourakis\t \u00C2\u00A0M,\t \u00C2\u00A0Bushell\t \u00C2\u00A0W,\t \u00C2\u00A0Iyer\t \u00C2\u00A0V,\t \u00C2\u00A0Mujica\t \u00C2\u00A0AO,\t \u00C2\u00A0Thomas\t \u00C2\u00A0M,\t \u00C2\u00A0Harrow\t \u00C2\u00A0J,\t \u00C2\u00A0Cox\t \u00C2\u00A0T,\t \u00C2\u00A0Jackson\t \u00C2\u00A0D,\t \u00C2\u00A0Severin\t \u00C2\u00A0J,\t \u00C2\u00A0Biggs\t \u00C2\u00A0P,\t \u00C2\u00A0Fu\t \u00C2\u00A0J,\t \u00C2\u00A0Nefedov\t \u00C2\u00A0M,\t \u00C2\u00A0de\t \u00C2\u00A0Jong\t \u00C2\u00A0PJ,\t \u00C2\u00A0Stewart\t \u00C2\u00A0AF,\t \u00C2\u00A0Bradley\t \u00C2\u00A0A\t \u00C2\u00A0(2011)\t \u00C2\u00A0A\t \u00C2\u00A0conditional\t \u00C2\u00A0knockout\t \u00C2\u00A0resource\t \u00C2\u00A0for\t \u00C2\u00A0the\t \u00C2\u00A0genome-\u00C2\u00AD\u00E2\u0080\u0090wide\t \u00C2\u00A0study\t \u00C2\u00A0of\t \u00C2\u00A0mouse\t \u00C2\u00A0gene\t \u00C2\u00A0function.\t \u00C2\u00A0Nature\t \u00C2\u00A0474:337\u00E2\u0080\u0093342.\t \u00C2\u00A0Slot\t \u00C2\u00A0LA,\t \u00C2\u00A0Lauridsen\t \u00C2\u00A0A-\u00C2\u00AD\u00E2\u0080\u0090M,\t \u00C2\u00A0Hendil\t \u00C2\u00A0K\t \u00C2\u00A0(1986)\t \u00C2\u00A0Intracellular\t \u00C2\u00A0protein\t \u00C2\u00A0degradation\t \u00C2\u00A0in\t \u00C2\u00A0serum-\u00C2\u00AD\u00E2\u0080\u0090deprived\t \u00C2\u00A0human\t \u00C2\u00A0fibroblasts.\t \u00C2\u00A0J\t \u00C2\u00A0Biochem\t \u00C2\u00A0237:491\u00E2\u0080\u0093498.\t \u00C2\u00A0Soldner\t \u00C2\u00A0F\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2011)\t \u00C2\u00A0Generation\t \u00C2\u00A0of\t \u00C2\u00A0Isogenic\t \u00C2\u00A0Pluripotent\t \u00C2\u00A0Stem\t \u00C2\u00A0Cells\t \u00C2\u00A0Differing\t \u00C2\u00A0Exclusively\t \u00C2\u00A0at\t \u00C2\u00A0Two\t \u00C2\u00A0Early\t \u00C2\u00A0Onset\t \u00C2\u00A0Parkinson\t \u00C2\u00A0Point\t \u00C2\u00A0Mutations.\t \u00C2\u00A0Cell\t \u00C2\u00A0146:318\u00E2\u0080\u0093331.\t \u00C2\u00A0 225 Song\t \u00C2\u00A0E,\t \u00C2\u00A0Lee\t \u00C2\u00A0S-\u00C2\u00AD\u00E2\u0080\u0090K,\t \u00C2\u00A0Wang\t \u00C2\u00A0J,\t \u00C2\u00A0Ince\t \u00C2\u00A0N,\t \u00C2\u00A0Ouyang\t \u00C2\u00A0N,\t \u00C2\u00A0Min\t \u00C2\u00A0J,\t \u00C2\u00A0Chen\t \u00C2\u00A0J,\t \u00C2\u00A0Shankar\t \u00C2\u00A0P,\t \u00C2\u00A0Lieberman\t \u00C2\u00A0J\t \u00C2\u00A0(2003)\t \u00C2\u00A0RNA\t \u00C2\u00A0interference\t \u00C2\u00A0targeting\t \u00C2\u00A0Fas\t \u00C2\u00A0protects\t \u00C2\u00A0mice\t \u00C2\u00A0from\t \u00C2\u00A0fulminant\t \u00C2\u00A0hepatitis.\t \u00C2\u00A0Nat\t \u00C2\u00A0Med\t \u00C2\u00A09:347\u00E2\u0080\u0093351.\t \u00C2\u00A0Stankunas\t \u00C2\u00A0K,\t \u00C2\u00A0Bayle\t \u00C2\u00A0JH,\t \u00C2\u00A0Gestwicki\t \u00C2\u00A0JE,\t \u00C2\u00A0Lin\t \u00C2\u00A0YM,\t \u00C2\u00A0Wandless\t \u00C2\u00A0TJ,\t \u00C2\u00A0Crabtree\t \u00C2\u00A0GR\t \u00C2\u00A0(2003)\t \u00C2\u00A0Conditional\t \u00C2\u00A0protein\t \u00C2\u00A0alleles\t \u00C2\u00A0using\t \u00C2\u00A0knockin\t \u00C2\u00A0mice\t \u00C2\u00A0and\t \u00C2\u00A0a\t \u00C2\u00A0chemical\t \u00C2\u00A0inducer\t \u00C2\u00A0of\t \u00C2\u00A0dimerization.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A012:1615\u00E2\u0080\u00931624.\t \u00C2\u00A0Staub\t \u00C2\u00A0O\t \u00C2\u00A0(2006)\t \u00C2\u00A0Role\t \u00C2\u00A0of\t \u00C2\u00A0Ubiquitylation\t \u00C2\u00A0in\t \u00C2\u00A0Cellular\t \u00C2\u00A0Membrane\t \u00C2\u00A0Transport.\t \u00C2\u00A0Physiol\t \u00C2\u00A0Rev\t \u00C2\u00A086:669\u00E2\u0080\u0093707.\t \u00C2\u00A0Steentoft\t \u00C2\u00A0C,\t \u00C2\u00A0Vakhrushev\t \u00C2\u00A0SY,\t \u00C2\u00A0Vester-\u00C2\u00AD\u00E2\u0080\u0090Christensen\t \u00C2\u00A0MB,\t \u00C2\u00A0Schjoldager\t \u00C2\u00A0KT-\u00C2\u00AD\u00E2\u0080\u0090BG,\t \u00C2\u00A0Kong\t \u00C2\u00A0Y,\t \u00C2\u00A0Bennett\t \u00C2\u00A0EP,\t \u00C2\u00A0Mandel\t \u00C2\u00A0U,\t \u00C2\u00A0Wandall\t \u00C2\u00A0H,\t \u00C2\u00A0Levery\t \u00C2\u00A0SB,\t \u00C2\u00A0Clausen\t \u00C2\u00A0H\t \u00C2\u00A0(2011)\t \u00C2\u00A0Mining\t \u00C2\u00A0the\t \u00C2\u00A0O-\u00C2\u00AD\u00E2\u0080\u0090glycoproteome\t \u00C2\u00A0using\t \u00C2\u00A0zinc-\u00C2\u00AD\u00E2\u0080\u0090finger\t \u00C2\u00A0nuclease\u00E2\u0080\u0093glycoengineered\t \u00C2\u00A0SimpleCell\t \u00C2\u00A0lines.\t \u00C2\u00A0Nat\t \u00C2\u00A0Methods\t \u00C2\u00A08:977\u00E2\u0080\u0093982.\t \u00C2\u00A0Stefanis\t \u00C2\u00A0L,\t \u00C2\u00A0Larsen\t \u00C2\u00A0KE,\t \u00C2\u00A0Rideout\t \u00C2\u00A0HJ,\t \u00C2\u00A0Sulzer\t \u00C2\u00A0D,\t \u00C2\u00A0Greene\t \u00C2\u00A0LA\t \u00C2\u00A0(2001)\t \u00C2\u00A0Expression\t \u00C2\u00A0of\t \u00C2\u00A0A53T\t \u00C2\u00A0mutant\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0wild-\u00C2\u00AD\u00E2\u0080\u0090type\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0in\t \u00C2\u00A0PC12\t \u00C2\u00A0cells\t \u00C2\u00A0induces\t \u00C2\u00A0alterations\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0ubiquitin-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0degradation\t \u00C2\u00A0system,\t \u00C2\u00A0loss\t \u00C2\u00A0of\t \u00C2\u00A0dopamine\t \u00C2\u00A0release,\t \u00C2\u00A0and\t \u00C2\u00A0autophagic\t \u00C2\u00A0cell\t \u00C2\u00A0death.\t \u00C2\u00A0J\t \u00C2\u00A0Neurosci\t \u00C2\u00A021:9549\u00E2\u0080\u00939560.\t \u00C2\u00A0Stein\t \u00C2\u00A0A,\t \u00C2\u00A0Aloy\t \u00C2\u00A0P\t \u00C2\u00A0(2008)\t \u00C2\u00A0Contextual\t \u00C2\u00A0Specificity\t \u00C2\u00A0in\t \u00C2\u00A0Peptide-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Protein\t \u00C2\u00A0Interactions.\t \u00C2\u00A0PLoS\t \u00C2\u00A0ONE\t \u00C2\u00A03:e2524.\t \u00C2\u00A0Stewart\t \u00C2\u00A0E,\t \u00C2\u00A0Kobayashi\t \u00C2\u00A0H,\t \u00C2\u00A0Harrison\t \u00C2\u00A0D,\t \u00C2\u00A0Hunt\t \u00C2\u00A0T\t \u00C2\u00A0(1994)\t \u00C2\u00A0Destruction\t \u00C2\u00A0of\t \u00C2\u00A0Xenopus\t \u00C2\u00A0cyclins\t \u00C2\u00A0A\t \u00C2\u00A0and\t \u00C2\u00A0B2,\t \u00C2\u00A0but\t \u00C2\u00A0not\t \u00C2\u00A0B1,\t \u00C2\u00A0requires\t \u00C2\u00A0binding\t \u00C2\u00A0to\t \u00C2\u00A0p34cdc2.\t \u00C2\u00A0EMBO\t \u00C2\u00A0J\t \u00C2\u00A013:584\u00E2\u0080\u0093594.\t \u00C2\u00A0 226 Stirchak\t \u00C2\u00A0EP,\t \u00C2\u00A0Summerton\t \u00C2\u00A0JE,\t \u00C2\u00A0Weller\t \u00C2\u00A0DD\t \u00C2\u00A0(1989)\t \u00C2\u00A0Uncharged\t \u00C2\u00A0stereoregular\t \u00C2\u00A0nucleic\t \u00C2\u00A0acid\t \u00C2\u00A0analogs:\t \u00C2\u00A02.\t \u00C2\u00A0Morpholino\t \u00C2\u00A0nucleoside\t \u00C2\u00A0oligomers\t \u00C2\u00A0with\t \u00C2\u00A0carbamate\t \u00C2\u00A0internucleoside\t \u00C2\u00A0linkages.\t \u00C2\u00A0Nucleic\t \u00C2\u00A0Acids\t \u00C2\u00A0Res\t \u00C2\u00A017:6129\u00E2\u0080\u00936141.\t \u00C2\u00A0Strack\t \u00C2\u00A0S,\t \u00C2\u00A0Colbran\t \u00C2\u00A0RJ\t \u00C2\u00A0(1998)\t \u00C2\u00A0Autophosphorylation-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0targeting\t \u00C2\u00A0of\t \u00C2\u00A0calcium/calmodulin-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0II\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0NR2B\t \u00C2\u00A0subunit\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090methyl-\u00C2\u00AD\u00E2\u0080\u0090D-\u00C2\u00AD\u00E2\u0080\u0090aspartate\t \u00C2\u00A0receptor.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0273:20689\u00E2\u0080\u009320692.\t \u00C2\u00A0Sturgill\t \u00C2\u00A0JF,\t \u00C2\u00A0Steiner\t \u00C2\u00A0P,\t \u00C2\u00A0Czervionke\t \u00C2\u00A0BL,\t \u00C2\u00A0Sabatini\t \u00C2\u00A0BL\t \u00C2\u00A0(2009)\t \u00C2\u00A0Distinct\t \u00C2\u00A0Domains\t \u00C2\u00A0within\t \u00C2\u00A0PSD-\u00C2\u00AD\u00E2\u0080\u009095\t \u00C2\u00A0Mediate\t \u00C2\u00A0Synaptic\t \u00C2\u00A0Incorporation,\t \u00C2\u00A0Stabilization,\t \u00C2\u00A0and\t \u00C2\u00A0Activity-\u00C2\u00AD\u00E2\u0080\u0090Dependent\t \u00C2\u00A0Trafficking.\t \u00C2\u00A0J\t \u00C2\u00A0\t \u00C2\u00A0Neurosci\t \u00C2\u00A029:12845\u00E2\u0080\u009312854.\t \u00C2\u00A0Su\t \u00C2\u00A0Y,\t \u00C2\u00A0Ishikawa\t \u00C2\u00A0S,\t \u00C2\u00A0Kojima\t \u00C2\u00A0M,\t \u00C2\u00A0Liu\t \u00C2\u00A0B\t \u00C2\u00A0(2003)\t \u00C2\u00A0Eradication\t \u00C2\u00A0of\t \u00C2\u00A0pathogenic\t \u00C2\u00A0\u00CE\u00B2-\u00C2\u00AD\u00E2\u0080\u0090catenin\t \u00C2\u00A0by\t \u00C2\u00A0Skp1/Cullin/F\t \u00C2\u00A0box\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0machinery.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0100:12729\u00E2\u0080\u009312734.\t \u00C2\u00A0Sugahara\t \u00C2\u00A0KN,\t \u00C2\u00A0Teesalu\t \u00C2\u00A0T,\t \u00C2\u00A0Karmali\t \u00C2\u00A0PP,\t \u00C2\u00A0Kotamraju\t \u00C2\u00A0VR,\t \u00C2\u00A0Agemy\t \u00C2\u00A0L,\t \u00C2\u00A0Girard\t \u00C2\u00A0OM,\t \u00C2\u00A0Hanahan\t \u00C2\u00A0D,\t \u00C2\u00A0Mattrey\t \u00C2\u00A0RF,\t \u00C2\u00A0Ruoslahti\t \u00C2\u00A0E\t \u00C2\u00A0(2009)\t \u00C2\u00A0Tissue-\u00C2\u00AD\u00E2\u0080\u0090Penetrating\t \u00C2\u00A0Delivery\t \u00C2\u00A0of\t \u00C2\u00A0Compounds\t \u00C2\u00A0and\t \u00C2\u00A0Nanoparticles\t \u00C2\u00A0into\t \u00C2\u00A0Tumors.\t \u00C2\u00A0Cancer\t \u00C2\u00A0Cell\t \u00C2\u00A016:510\u00E2\u0080\u0093520.\t \u00C2\u00A0Sugahara\t \u00C2\u00A0KN,\t \u00C2\u00A0Teesalu\t \u00C2\u00A0T,\t \u00C2\u00A0Karmali\t \u00C2\u00A0PP,\t \u00C2\u00A0Kotamraju\t \u00C2\u00A0VR,\t \u00C2\u00A0Agemy\t \u00C2\u00A0L,\t \u00C2\u00A0Greenwald\t \u00C2\u00A0DR,\t \u00C2\u00A0Ruoslahti\t \u00C2\u00A0E\t \u00C2\u00A0(2010)\t \u00C2\u00A0Coadministration\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0Tumor-\u00C2\u00AD\u00E2\u0080\u0090Penetrating\t \u00C2\u00A0Peptide\t \u00C2\u00A0Enhances\t \u00C2\u00A0the\t \u00C2\u00A0Efficacy\t \u00C2\u00A0of\t \u00C2\u00A0Cancer\t \u00C2\u00A0Drugs.\t \u00C2\u00A0Science\t \u00C2\u00A0328:1031\u00E2\u0080\u00931035.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 227 Sugiyama\t \u00C2\u00A0Y,\t \u00C2\u00A0Kawabata\t \u00C2\u00A0I,\t \u00C2\u00A0Sobue\t \u00C2\u00A0K,\t \u00C2\u00A0Okabe\t \u00C2\u00A0S\t \u00C2\u00A0(2005)\t \u00C2\u00A0Determination\t \u00C2\u00A0of\t \u00C2\u00A0absolute\t \u00C2\u00A0protein\t \u00C2\u00A0numbers\t \u00C2\u00A0in\t \u00C2\u00A0single\t \u00C2\u00A0synapses\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0GFP-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0calibration\t \u00C2\u00A0technique.\t \u00C2\u00A0Nat\t \u00C2\u00A0Methods\t \u00C2\u00A02:677\u00E2\u0080\u0093684.\t \u00C2\u00A0Surguchov\t \u00C2\u00A0A\t \u00C2\u00A0(2008)\t \u00C2\u00A0Molecular\t \u00C2\u00A0and\t \u00C2\u00A0Cellular\t \u00C2\u00A0Biology\t \u00C2\u00A0of\t \u00C2\u00A0Synucleins.\t \u00C2\u00A0Int\t \u00C2\u00A0Rev\t \u00C2\u00A0Cell\t \u00C2\u00A0Mol\t \u00C2\u00A0Biol\t \u00C2\u00A0270:225-\u00C2\u00AD\u00E2\u0080\u0090318.\t \u00C2\u00A0\t \u00C2\u00A0Taghibiglou\t \u00C2\u00A0C,\t \u00C2\u00A0Martin\t \u00C2\u00A0HGS,\t \u00C2\u00A0Lai\t \u00C2\u00A0TW,\t \u00C2\u00A0Cho\t \u00C2\u00A0T,\t \u00C2\u00A0Prasad\t \u00C2\u00A0S,\t \u00C2\u00A0Kojic\t \u00C2\u00A0L,\t \u00C2\u00A0Lu\t \u00C2\u00A0J,\t \u00C2\u00A0Liu\t \u00C2\u00A0Y,\t \u00C2\u00A0Lo\t \u00C2\u00A0E,\t \u00C2\u00A0Zhang\t \u00C2\u00A0S,\t \u00C2\u00A0Wu\t \u00C2\u00A0JZZ,\t \u00C2\u00A0Li\t \u00C2\u00A0YP,\t \u00C2\u00A0Wen\t \u00C2\u00A0YH,\t \u00C2\u00A0Imm\t \u00C2\u00A0J-\u00C2\u00AD\u00E2\u0080\u0090H,\t \u00C2\u00A0Cynader\t \u00C2\u00A0MS,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2009)\t \u00C2\u00A0Role\t \u00C2\u00A0of\t \u00C2\u00A0NMDA\t \u00C2\u00A0receptor\u00E2\u0080\u0093dependent\t \u00C2\u00A0activation\t \u00C2\u00A0of\t \u00C2\u00A0SREBP1\t \u00C2\u00A0in\t \u00C2\u00A0excitotoxic\t \u00C2\u00A0and\t \u00C2\u00A0ischemic\t \u00C2\u00A0neuronal\t \u00C2\u00A0injuries.\t \u00C2\u00A0Nat\t \u00C2\u00A0Med\t \u00C2\u00A015:1399\u00E2\u0080\u00931407.\t \u00C2\u00A0Thompson\t \u00C2\u00A0LM\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2009)\t \u00C2\u00A0IKK\t \u00C2\u00A0phosphorylates\t \u00C2\u00A0Huntingtin\t \u00C2\u00A0and\t \u00C2\u00A0targets\t \u00C2\u00A0it\t \u00C2\u00A0for\t \u00C2\u00A0degradation\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0and\t \u00C2\u00A0lysosome.\t \u00C2\u00A0J\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A0187:1083\u00E2\u0080\u00931099.\t \u00C2\u00A0Tsurumi\t \u00C2\u00A0C,\t \u00C2\u00A0Ishida\t \u00C2\u00A0N,\t \u00C2\u00A0Tamura\t \u00C2\u00A0T,\t \u00C2\u00A0Kakizuka\t \u00C2\u00A0A,\t \u00C2\u00A0Nishida\t \u00C2\u00A0E,\t \u00C2\u00A0Okumura\t \u00C2\u00A0E,\t \u00C2\u00A0Kishimoto\t \u00C2\u00A0T,\t \u00C2\u00A0Inagaki\t \u00C2\u00A0M,\t \u00C2\u00A0Okazaki\t \u00C2\u00A0K,\t \u00C2\u00A0Sagata\t \u00C2\u00A0N\t \u00C2\u00A0(1995)\t \u00C2\u00A0Degradation\t \u00C2\u00A0of\t \u00C2\u00A0c-\u00C2\u00AD\u00E2\u0080\u0090Fos\t \u00C2\u00A0by\t \u00C2\u00A0the\t \u00C2\u00A026S\t \u00C2\u00A0proteasome\t \u00C2\u00A0is\t \u00C2\u00A0accelerated\t \u00C2\u00A0by\t \u00C2\u00A0c-\u00C2\u00AD\u00E2\u0080\u0090Jun\t \u00C2\u00A0and\t \u00C2\u00A0multiple\t \u00C2\u00A0protein\t \u00C2\u00A0kinases.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A015:5682\u00E2\u0080\u00935687.\t \u00C2\u00A0Tu\t \u00C2\u00A0W,\t \u00C2\u00A0Xu\t \u00C2\u00A0X,\t \u00C2\u00A0Peng\t \u00C2\u00A0L,\t \u00C2\u00A0Zhong\t \u00C2\u00A0X,\t \u00C2\u00A0Zhang\t \u00C2\u00A0W,\t \u00C2\u00A0Soundarapandian\t \u00C2\u00A0MM,\t \u00C2\u00A0Belal\t \u00C2\u00A0C,\t \u00C2\u00A0Wang\t \u00C2\u00A0M,\t \u00C2\u00A0Jia\t \u00C2\u00A0N,\t \u00C2\u00A0Zhang\t \u00C2\u00A0W,\t \u00C2\u00A0Lew\t \u00C2\u00A0F,\t \u00C2\u00A0Chan\t \u00C2\u00A0SL,\t \u00C2\u00A0Chen\t \u00C2\u00A0Y,\t \u00C2\u00A0Lu\t \u00C2\u00A0Y\t \u00C2\u00A0(2010)\t \u00C2\u00A0DAPK1\t \u00C2\u00A0Interaction\t \u00C2\u00A0with\t \u00C2\u00A0NMDA\t \u00C2\u00A0Receptor\t \u00C2\u00A0NR2B\t \u00C2\u00A0Subunits\t \u00C2\u00A0Mediates\t \u00C2\u00A0Brain\t \u00C2\u00A0Damage\t \u00C2\u00A0in\t \u00C2\u00A0Stroke.\t \u00C2\u00A0Cell\t \u00C2\u00A0140:222\u00E2\u0080\u0093234.\t \u00C2\u00A0Turlo\t \u00C2\u00A0KA,\t \u00C2\u00A0Gallaher\t \u00C2\u00A0SD,\t \u00C2\u00A0Vora\t \u00C2\u00A0R,\t \u00C2\u00A0Laski\t \u00C2\u00A0FA,\t \u00C2\u00A0Iruela-\u00C2\u00AD\u00E2\u0080\u0090Arispe\t \u00C2\u00A0ML\t \u00C2\u00A0(2010)\t \u00C2\u00A0When\t \u00C2\u00A0Cre-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Recombination\t \u00C2\u00A0in\t \u00C2\u00A0Mice\t \u00C2\u00A0Does\t \u00C2\u00A0Not\t \u00C2\u00A0Result\t \u00C2\u00A0in\t \u00C2\u00A0Protein\t \u00C2\u00A0Loss.\t \u00C2\u00A0Genetics\t \u00C2\u00A0186:959\u00E2\u0080\u0093967.\t \u00C2\u00A0 228 Turner-\u00C2\u00AD\u00E2\u0080\u0090Brannen\t \u00C2\u00A0E,\t \u00C2\u00A0Choi\t \u00C2\u00A0KYG,\t \u00C2\u00A0Arsenault\t \u00C2\u00A0R,\t \u00C2\u00A0El-\u00C2\u00AD\u00E2\u0080\u0090Gabalawy\t \u00C2\u00A0H,\t \u00C2\u00A0Napper\t \u00C2\u00A0S,\t \u00C2\u00A0Mookherjee\t \u00C2\u00A0N\t \u00C2\u00A0(2011)\t \u00C2\u00A0Inflammatory\t \u00C2\u00A0Cytokines\t \u00C2\u00A0IL-\u00C2\u00AD\u00E2\u0080\u009032\t \u00C2\u00A0and\t \u00C2\u00A0IL-\u00C2\u00AD\u00E2\u0080\u009017\t \u00C2\u00A0Have\t \u00C2\u00A0Common\t \u00C2\u00A0Signaling\t \u00C2\u00A0Intermediates\t \u00C2\u00A0despite\t \u00C2\u00A0Differential\t \u00C2\u00A0Dependence\t \u00C2\u00A0on\t \u00C2\u00A0TNF-\u00C2\u00AD\u00E2\u0080\u0090Receptor\t \u00C2\u00A01.\t \u00C2\u00A0J\t \u00C2\u00A0Immunol\t \u00C2\u00A0186:7127\u00E2\u0080\u00937135.\t \u00C2\u00A0Tymianski\t \u00C2\u00A0M\t \u00C2\u00A0(2013)\t \u00C2\u00A0Novel\t \u00C2\u00A0Approaches\t \u00C2\u00A0to\t \u00C2\u00A0Neuroprotection\t \u00C2\u00A0Trials\t \u00C2\u00A0in\t \u00C2\u00A0Acute\t \u00C2\u00A0Ischemic\t \u00C2\u00A0Stroke.\t \u00C2\u00A0Stroke\t \u00C2\u00A044:2942\u00E2\u0080\u00932950.\t \u00C2\u00A0Urnov\t \u00C2\u00A0FD,\t \u00C2\u00A0Miller\t \u00C2\u00A0JC,\t \u00C2\u00A0Lee\t \u00C2\u00A0Y-\u00C2\u00AD\u00E2\u0080\u0090L,\t \u00C2\u00A0Beausejour\t \u00C2\u00A0CM,\t \u00C2\u00A0Rock\t \u00C2\u00A0JM,\t \u00C2\u00A0Augustus\t \u00C2\u00A0S,\t \u00C2\u00A0Jamieson\t \u00C2\u00A0AC,\t \u00C2\u00A0Porteus\t \u00C2\u00A0MH,\t \u00C2\u00A0Gregory\t \u00C2\u00A0PD,\t \u00C2\u00A0Holmes\t \u00C2\u00A0MC\t \u00C2\u00A0(2005)\t \u00C2\u00A0Highly\t \u00C2\u00A0efficient\t \u00C2\u00A0endogenous\t \u00C2\u00A0human\t \u00C2\u00A0gene\t \u00C2\u00A0correction\t \u00C2\u00A0using\t \u00C2\u00A0designed\t \u00C2\u00A0zinc-\u00C2\u00AD\u00E2\u0080\u0090finger\t \u00C2\u00A0nucleases.\t \u00C2\u00A0Nature\t \u00C2\u00A0435:646\u00E2\u0080\u0093651.\t \u00C2\u00A0Urnov\t \u00C2\u00A0FD,\t \u00C2\u00A0Rebar\t \u00C2\u00A0EJ,\t \u00C2\u00A0Holmes\t \u00C2\u00A0MC,\t \u00C2\u00A0Zhang\t \u00C2\u00A0HS,\t \u00C2\u00A0Gregory\t \u00C2\u00A0PD\t \u00C2\u00A0(2010)\t \u00C2\u00A0Genome\t \u00C2\u00A0editing\t \u00C2\u00A0with\t \u00C2\u00A0engineeredzinc\t \u00C2\u00A0finger\t \u00C2\u00A0nucleases.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Genet\t \u00C2\u00A011:636\u00E2\u0080\u0093646.\t \u00C2\u00A0Utomo\t \u00C2\u00A0AR,\t \u00C2\u00A0Nikitin\t \u00C2\u00A0AY,\t \u00C2\u00A0Lee\t \u00C2\u00A0W-\u00C2\u00AD\u00E2\u0080\u0090H\t \u00C2\u00A0(1999)\t \u00C2\u00A0Temporal,\t \u00C2\u00A0spatial,\t \u00C2\u00A0and\t \u00C2\u00A0cell\t \u00C2\u00A0type\u00E2\u0080\u0093specific\t \u00C2\u00A0control\t \u00C2\u00A0of\t \u00C2\u00A0Cre-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0DNA\t \u00C2\u00A0recombination\t \u00C2\u00A0in\t \u00C2\u00A0transgenic\t \u00C2\u00A0mice.\t \u00C2\u00A0Nat\t \u00C2\u00A0Biotechnol\t \u00C2\u00A017:1091\u00E2\u0080\u00931096.\t \u00C2\u00A0Varshavsky\t \u00C2\u00A0A\t \u00C2\u00A0(1992)\t \u00C2\u00A0The\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090End\t \u00C2\u00A0Rule.\t \u00C2\u00A0Cell\t \u00C2\u00A069:725\u00E2\u0080\u0093735.\t \u00C2\u00A0Velentza\t \u00C2\u00A0AV,\t \u00C2\u00A0Wainwright\t \u00C2\u00A0MS,\t \u00C2\u00A0Zasadzki\t \u00C2\u00A0M,\t \u00C2\u00A0Mirzoeva\t \u00C2\u00A0S,\t \u00C2\u00A0Schumacher\t \u00C2\u00A0AM,\t \u00C2\u00A0Haiech\t \u00C2\u00A0J,\t \u00C2\u00A0Focia\t \u00C2\u00A0PJ,\t \u00C2\u00A0Egli\t \u00C2\u00A0M,\t \u00C2\u00A0Watterson\t \u00C2\u00A0DM\t \u00C2\u00A0(2003)\t \u00C2\u00A0An\t \u00C2\u00A0aminopyridazine-\u00C2\u00AD\u00E2\u0080\u0090based\t \u00C2\u00A0inhibitor\t \u00C2\u00A0of\t \u00C2\u00A0a\t \u00C2\u00A0pro-\u00C2\u00AD\u00E2\u0080\u0090apoptotic\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0attenuates\t \u00C2\u00A0hypoxia-\u00C2\u00AD\u00E2\u0080\u0090ischemia\t \u00C2\u00A0induced\t \u00C2\u00A0acute\t \u00C2\u00A0brain\t \u00C2\u00A0injury.\t \u00C2\u00A0Bioorg\t \u00C2\u00A0Med\t \u00C2\u00A0Chem\t \u00C2\u00A0Lett\t \u00C2\u00A013:3465\u00E2\u0080\u00933470.\t \u00C2\u00A0Vembar\t \u00C2\u00A0SS,\t \u00C2\u00A0Brodsky\t \u00C2\u00A0JL\t \u00C2\u00A0(2008)\t \u00C2\u00A0One\t \u00C2\u00A0step\t \u00C2\u00A0at\t \u00C2\u00A0a\t \u00C2\u00A0time:\t \u00C2\u00A0endoplasmic\t \u00C2\u00A0reticulum-\u00C2\u00AD\u00E2\u0080\u0090associated\t \u00C2\u00A0degradation.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A09:944-\u00C2\u00AD\u00E2\u0080\u0090957.\t \u00C2\u00A0 229 Vieth\t \u00C2\u00A0M,\t \u00C2\u00A0Sutherland\t \u00C2\u00A0JJ,\t \u00C2\u00A0Robertson\t \u00C2\u00A0DH,\t \u00C2\u00A0Campbell\t \u00C2\u00A0RM\t \u00C2\u00A0(2005)\t \u00C2\u00A0Kinomics:\t \u00C2\u00A0characterizing\t \u00C2\u00A0the\t \u00C2\u00A0therapeutically\t \u00C2\u00A0validated\t \u00C2\u00A0kinase\t \u00C2\u00A0space.\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A0Today\t \u00C2\u00A010:839\u00E2\u0080\u0093846.\t \u00C2\u00A0Viv\u00C3\u00A8s\t \u00C2\u00A0E,\t \u00C2\u00A0Brodin\t \u00C2\u00A0P,\t \u00C2\u00A0Lebleu\t \u00C2\u00A0B\t \u00C2\u00A0(1997)\t \u00C2\u00A0A\t \u00C2\u00A0truncated\t \u00C2\u00A0HIV-\u00C2\u00AD\u00E2\u0080\u00901\t \u00C2\u00A0Tat\t \u00C2\u00A0protein\t \u00C2\u00A0basic\t \u00C2\u00A0domain\t \u00C2\u00A0rapidly\t \u00C2\u00A0translocates\t \u00C2\u00A0through\t \u00C2\u00A0the\t \u00C2\u00A0plasma\t \u00C2\u00A0membrane\t \u00C2\u00A0and\t \u00C2\u00A0accumulates\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0cell\t \u00C2\u00A0nucleus.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0272:16010\u00E2\u0080\u009316017.\t \u00C2\u00A0Vlieghe\t \u00C2\u00A0P,\t \u00C2\u00A0Lisowski\t \u00C2\u00A0V,\t \u00C2\u00A0Martinez\t \u00C2\u00A0J,\t \u00C2\u00A0Khrestchatisky\t \u00C2\u00A0M\t \u00C2\u00A0(2010)\t \u00C2\u00A0Synthetic\t \u00C2\u00A0therapeutic\t \u00C2\u00A0peptides:\t \u00C2\u00A0science\t \u00C2\u00A0and\t \u00C2\u00A0market.\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A0Today\t \u00C2\u00A015:40\u00E2\u0080\u009356.\t \u00C2\u00A0Vogiatzi\t \u00C2\u00A0T,\t \u00C2\u00A0Xilouri\t \u00C2\u00A0M,\t \u00C2\u00A0Vekrellis\t \u00C2\u00A0K,\t \u00C2\u00A0Stefanis\t \u00C2\u00A0L\t \u00C2\u00A0(2008)\t \u00C2\u00A0Wild\t \u00C2\u00A0type\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein\t \u00C2\u00A0is\t \u00C2\u00A0degraded\t \u00C2\u00A0by\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy\t \u00C2\u00A0and\t \u00C2\u00A0macroautophagy\t \u00C2\u00A0in\t \u00C2\u00A0neuronal\t \u00C2\u00A0cells.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0283:23542\u00E2\u0080\u009323556.\t \u00C2\u00A0Wagner\t \u00C2\u00A0GP,\t \u00C2\u00A0Zhang\t \u00C2\u00A0J\t \u00C2\u00A0(2011)\t \u00C2\u00A0The\t \u00C2\u00A0pleiotropic\t \u00C2\u00A0structure\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0genotype\u00E2\u0080\u0093phenotype\t \u00C2\u00A0map:\t \u00C2\u00A0the\t \u00C2\u00A0evolvability\t \u00C2\u00A0of\t \u00C2\u00A0complex\t \u00C2\u00A0organisms.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Genet\t \u00C2\u00A012:204\u00E2\u0080\u0093213.\t \u00C2\u00A0Waxham\t \u00C2\u00A0MN,\t \u00C2\u00A0Grotta\t \u00C2\u00A0JC,\t \u00C2\u00A0Silva\t \u00C2\u00A0AJ,\t \u00C2\u00A0Strong\t \u00C2\u00A0R,\t \u00C2\u00A0Aronowski\t \u00C2\u00A0J\t \u00C2\u00A0(1996)\t \u00C2\u00A0Ischemia-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0neuronal\t \u00C2\u00A0damage:\t \u00C2\u00A0a\t \u00C2\u00A0role\t \u00C2\u00A0for\t \u00C2\u00A0calcium/calmodulin-\u00C2\u00AD\u00E2\u0080\u0090dependent\t \u00C2\u00A0protein\t \u00C2\u00A0kinase\t \u00C2\u00A0II.\t \u00C2\u00A0J\t \u00C2\u00A0Cereb\t \u00C2\u00A0Blood\t \u00C2\u00A0Flow\t \u00C2\u00A0Metab\t \u00C2\u00A016:1\u00E2\u0080\u00936.\t \u00C2\u00A0Webb\t \u00C2\u00A0JL,\t \u00C2\u00A0Ravikumar\t \u00C2\u00A0B,\t \u00C2\u00A0Atkins\t \u00C2\u00A0J,\t \u00C2\u00A0Skepper\t \u00C2\u00A0JN,\t \u00C2\u00A0Rubinsztein\t \u00C2\u00A0DC\t \u00C2\u00A0(2003)\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090Synuclein\t \u00C2\u00A0Is\t \u00C2\u00A0Degraded\t \u00C2\u00A0by\t \u00C2\u00A0Both\t \u00C2\u00A0Autophagy\t \u00C2\u00A0and\t \u00C2\u00A0the\t \u00C2\u00A0Proteasome.\t \u00C2\u00A0J\t \u00C2\u00A0Biol\t \u00C2\u00A0Chem\t \u00C2\u00A0278:25009\u00E2\u0080\u009325013.\t \u00C2\u00A0White\t \u00C2\u00A0JK\t \u00C2\u00A0et\t \u00C2\u00A0al.\t \u00C2\u00A0(2013)\t \u00C2\u00A0Genome-\u00C2\u00AD\u00E2\u0080\u0090wide\t \u00C2\u00A0Generation\t \u00C2\u00A0and\t \u00C2\u00A0Systematic\t \u00C2\u00A0Phenotyping\t \u00C2\u00A0of\t \u00C2\u00A0Knockout\t \u00C2\u00A0Mice\t \u00C2\u00A0Reveals\t \u00C2\u00A0New\t \u00C2\u00A0Roles\t \u00C2\u00A0for\t \u00C2\u00A0Many\t \u00C2\u00A0Genes.\t \u00C2\u00A0Cell\t \u00C2\u00A0154:452\u00E2\u0080\u0093464.\t \u00C2\u00A0 230 Whitehead\t \u00C2\u00A0KA,\t \u00C2\u00A0Langer\t \u00C2\u00A0R,\t \u00C2\u00A0Anderson\t \u00C2\u00A0DG\t \u00C2\u00A0(2009)\t \u00C2\u00A0Knocking\t \u00C2\u00A0down\t \u00C2\u00A0barriers:\t \u00C2\u00A0advances\t \u00C2\u00A0in\t \u00C2\u00A0siRNA\t \u00C2\u00A0delivery.\t \u00C2\u00A0Nat\t \u00C2\u00A0Rev\t \u00C2\u00A0Drug\t \u00C2\u00A0Discov\t \u00C2\u00A08:129\u00E2\u0080\u0093138.\t \u00C2\u00A0Wilson\t \u00C2\u00A0RC,\t \u00C2\u00A0Doudna\t \u00C2\u00A0JA\t \u00C2\u00A0(2013)\t \u00C2\u00A0Molecular\t \u00C2\u00A0Mechanisms\t \u00C2\u00A0of\t \u00C2\u00A0RNA\t \u00C2\u00A0Interference.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Biophys\t \u00C2\u00A042:217\u00E2\u0080\u0093239.\t \u00C2\u00A0Wolfer\t \u00C2\u00A0DP,\t \u00C2\u00A0Crusio\t \u00C2\u00A0WE,\t \u00C2\u00A0Lipp\t \u00C2\u00A0HP\t \u00C2\u00A0(2002)\t \u00C2\u00A0Knockout\t \u00C2\u00A0mice:\t \u00C2\u00A0simple\t \u00C2\u00A0solutions\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0problems\t \u00C2\u00A0of\t \u00C2\u00A0genetic\t \u00C2\u00A0background\t \u00C2\u00A0and\t \u00C2\u00A0flanking\t \u00C2\u00A0genes.\t \u00C2\u00A0Trends\t \u00C2\u00A0Neurosci\t \u00C2\u00A025:336\u00E2\u0080\u0093340.\t \u00C2\u00A0Wong\t \u00C2\u00A0E,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2010a)\t \u00C2\u00A0Autophagy\t \u00C2\u00A0gone\t \u00C2\u00A0awry\t \u00C2\u00A0in\t \u00C2\u00A0neurodegenerative\t \u00C2\u00A0diseases.\t \u00C2\u00A0Nat\t \u00C2\u00A0Neurosci\t \u00C2\u00A013:805-\u00C2\u00AD\u00E2\u0080\u0090811.\t \u00C2\u00A0Wong\t \u00C2\u00A0E,\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2010b)\t \u00C2\u00A0Integration\t \u00C2\u00A0of\t \u00C2\u00A0Clearance\t \u00C2\u00A0Mechanisms:\t \u00C2\u00A0The\t \u00C2\u00A0Proteasome\t \u00C2\u00A0and\t \u00C2\u00A0Autophagy.\t \u00C2\u00A0Cold\t \u00C2\u00A0Spring\t \u00C2\u00A0Harb\t \u00C2\u00A0Perspect\t \u00C2\u00A0Biol\t \u00C2\u00A02:a006734\u00E2\u0080\u0093a006734.\t \u00C2\u00A0Wong\t \u00C2\u00A0TP,\t \u00C2\u00A0Howland\t \u00C2\u00A0JG,\t \u00C2\u00A0Robillard\t \u00C2\u00A0JM,\t \u00C2\u00A0Ge\t \u00C2\u00A0Y,\t \u00C2\u00A0Yu\t \u00C2\u00A0W,\t \u00C2\u00A0Titterness\t \u00C2\u00A0AK,\t \u00C2\u00A0Brebner\t \u00C2\u00A0K,\t \u00C2\u00A0Liu\t \u00C2\u00A0L,\t \u00C2\u00A0Weinberg\t \u00C2\u00A0J,\t \u00C2\u00A0Christie\t \u00C2\u00A0BR,\t \u00C2\u00A0Phillips\t \u00C2\u00A0AG,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2007)\t \u00C2\u00A0Hippocampal\t \u00C2\u00A0long-\u00C2\u00AD\u00E2\u0080\u0090term\t \u00C2\u00A0depression\t \u00C2\u00A0mediates\t \u00C2\u00A0acute\t \u00C2\u00A0stress-\u00C2\u00AD\u00E2\u0080\u0090induced\t \u00C2\u00A0spatial\t \u00C2\u00A0memory\t \u00C2\u00A0retrieval\t \u00C2\u00A0impairment.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0104:11471\u00E2\u0080\u009311476.\t \u00C2\u00A0Xilouri\t \u00C2\u00A0M,\t \u00C2\u00A0Brekk\t \u00C2\u00A0OR,\t \u00C2\u00A0Kirik\t \u00C2\u00A0D,\t \u00C2\u00A0Stefanis\t \u00C2\u00A0L\t \u00C2\u00A0(2013)\t \u00C2\u00A0LAMP2A\t \u00C2\u00A0as\t \u00C2\u00A0a\t \u00C2\u00A0therapeutic\t \u00C2\u00A0target\t \u00C2\u00A0in\t \u00C2\u00A0Parkinson\t \u00C2\u00A0disease.\t \u00C2\u00A0Autophagy\t \u00C2\u00A09:2166\u00E2\u0080\u00932168.\t \u00C2\u00A0Xilouri\t \u00C2\u00A0M,\t \u00C2\u00A0Vogiatzi\t \u00C2\u00A0T,\t \u00C2\u00A0Vekrellis\t \u00C2\u00A0K,\t \u00C2\u00A0Park\t \u00C2\u00A0D,\t \u00C2\u00A0Stefanis\t \u00C2\u00A0L\t \u00C2\u00A0(2009)\t \u00C2\u00A0Abberant\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090Synuclein\t \u00C2\u00A0Confers\t \u00C2\u00A0Toxicity\t \u00C2\u00A0to\t \u00C2\u00A0Neurons\t \u00C2\u00A0in\t \u00C2\u00A0Part\t \u00C2\u00A0through\t \u00C2\u00A0Inhibition\t \u00C2\u00A0of\t \u00C2\u00A0Chaperone-\u00C2\u00AD\u00E2\u0080\u0090Mediated\t \u00C2\u00A0Autophagy.\t \u00C2\u00A0PLoS\t \u00C2\u00A0ONE\t \u00C2\u00A04:e5515.\t \u00C2\u00A0 231 Xu\t \u00C2\u00A0C,\t \u00C2\u00A0Kim\t \u00C2\u00A0N-\u00C2\u00AD\u00E2\u0080\u0090G,\t \u00C2\u00A0Gumbiner\t \u00C2\u00A0BM\t \u00C2\u00A0(2009)\t \u00C2\u00A0Regulation\t \u00C2\u00A0of\t \u00C2\u00A0protein\t \u00C2\u00A0stability\t \u00C2\u00A0by\t \u00C2\u00A0GSK3\t \u00C2\u00A0mediated\t \u00C2\u00A0phosphorylation.\t \u00C2\u00A0Cell\t \u00C2\u00A0Cycle\t \u00C2\u00A08:4032\u00E2\u0080\u00934039.\t \u00C2\u00A0Yaglom\t \u00C2\u00A0J,\t \u00C2\u00A0Linskens\t \u00C2\u00A0MH,\t \u00C2\u00A0Sadis\t \u00C2\u00A0S,\t \u00C2\u00A0Rubin\t \u00C2\u00A0DM,\t \u00C2\u00A0Futcher\t \u00C2\u00A0B,\t \u00C2\u00A0Finley\t \u00C2\u00A0D\t \u00C2\u00A0(1995)\t \u00C2\u00A0p34Cdc28-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0control\t \u00C2\u00A0of\t \u00C2\u00A0Cln3\t \u00C2\u00A0cyclin\t \u00C2\u00A0degradation.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A015:731\u00E2\u0080\u0093741.\t \u00C2\u00A0Yamamoto\t \u00C2\u00A0A,\t \u00C2\u00A0Yue\t \u00C2\u00A0Z\t \u00C2\u00A0(2014)\t \u00C2\u00A0Autophagy\t \u00C2\u00A0and\t \u00C2\u00A0Its\t \u00C2\u00A0Normal\t \u00C2\u00A0and\t \u00C2\u00A0Pathogenic\t \u00C2\u00A0States\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0Brain.\t \u00C2\u00A0Annu\t \u00C2\u00A0Rev\t \u00C2\u00A0Neurosci\t \u00C2\u00A037:55\u00E2\u0080\u009378.\t \u00C2\u00A0Yang\t \u00C2\u00A0F,\t \u00C2\u00A0Yang\t \u00C2\u00A0Y-\u00C2\u00AD\u00E2\u0080\u0090P,\t \u00C2\u00A0Mao\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090J,\t \u00C2\u00A0Liu\t \u00C2\u00A0L,\t \u00C2\u00A0Zheng\t \u00C2\u00A0H-\u00C2\u00AD\u00E2\u0080\u0090F,\t \u00C2\u00A0Hu\t \u00C2\u00A0L-\u00C2\u00AD\u00E2\u0080\u0090F,\t \u00C2\u00A0Liu\t \u00C2\u00A0C-\u00C2\u00AD\u00E2\u0080\u0090F\t \u00C2\u00A0(2013)\t \u00C2\u00A0Crosstalk\t \u00C2\u00A0between\t \u00C2\u00A0the\t \u00C2\u00A0proteasome\t \u00C2\u00A0system\t \u00C2\u00A0and\t \u00C2\u00A0autophagy\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0clearance\t \u00C2\u00A0of\t \u00C2\u00A0\u00CE\u00B1-\u00C2\u00AD\u00E2\u0080\u0090synuclein.\t \u00C2\u00A0Acta\t \u00C2\u00A0Pharm\t \u00C2\u00A0Sinica\t \u00C2\u00A034:674\u00E2\u0080\u0093680.\t \u00C2\u00A0Yang\t \u00C2\u00A0F,\t \u00C2\u00A0Wang\t \u00C2\u00A0Z,\t \u00C2\u00A0Wei\t \u00C2\u00A0X,\t \u00C2\u00A0Han\t \u00C2\u00A0H,\t \u00C2\u00A0Meng\t \u00C2\u00A0X,\t \u00C2\u00A0Zhang\t \u00C2\u00A0Y,\t \u00C2\u00A0Shi\t \u00C2\u00A0W,\t \u00C2\u00A0Li\t \u00C2\u00A0F,\t \u00C2\u00A0Xin\t \u00C2\u00A0T,\t \u00C2\u00A0Pang\t \u00C2\u00A0Q,\t \u00C2\u00A0Yi\t \u00C2\u00A0F\t \u00C2\u00A0(2014)\t \u00C2\u00A0NLRP3\t \u00C2\u00A0deficiency\t \u00C2\u00A0ameliorates\t \u00C2\u00A0neurovascular\t \u00C2\u00A0damage\t \u00C2\u00A0in\t \u00C2\u00A0experimental\t \u00C2\u00A0ischemic\t \u00C2\u00A0stroke.\t \u00C2\u00A0J\t \u00C2\u00A0Cereb\t \u00C2\u00A0Blood\t \u00C2\u00A0Flow\t \u00C2\u00A0Metab\t \u00C2\u00A034:660-\u00C2\u00AD\u00E2\u0080\u0090667.\t \u00C2\u00A0Yang\t \u00C2\u00A0Q,\t \u00C2\u00A0She\t \u00C2\u00A0H,\t \u00C2\u00A0Gearing\t \u00C2\u00A0M,\t \u00C2\u00A0Colla\t \u00C2\u00A0E,\t \u00C2\u00A0Lee\t \u00C2\u00A0M,\t \u00C2\u00A0Shacka\t \u00C2\u00A0JJ,\t \u00C2\u00A0Mao\t \u00C2\u00A0Z\t \u00C2\u00A0(2009)\t \u00C2\u00A0Regulation\t \u00C2\u00A0of\t \u00C2\u00A0neuronal\t \u00C2\u00A0survival\t \u00C2\u00A0factor\t \u00C2\u00A0MEF2D\t \u00C2\u00A0by\t \u00C2\u00A0chaperone-\u00C2\u00AD\u00E2\u0080\u0090mediated\t \u00C2\u00A0autophagy.\t \u00C2\u00A0Science\t \u00C2\u00A0323:124\u00E2\u0080\u0093127.\t \u00C2\u00A0Yang\t \u00C2\u00A0Z,\t \u00C2\u00A0Klionsky\t \u00C2\u00A0DJ\t \u00C2\u00A0(2010)\t \u00C2\u00A0Eaten\t \u00C2\u00A0alive:\t \u00C2\u00A0a\t \u00C2\u00A0history\t \u00C2\u00A0of\t \u00C2\u00A0macroautophagy.\t \u00C2\u00A0Nat\t \u00C2\u00A0Cell\t \u00C2\u00A0Biol\t \u00C2\u00A012:814-\u00C2\u00AD\u00E2\u0080\u0090822.\t \u00C2\u00A0Yano\t \u00C2\u00A0S,\t \u00C2\u00A0Tokumitsu\t \u00C2\u00A0H,\t \u00C2\u00A0Soderling\t \u00C2\u00A0TR\t \u00C2\u00A0(1998)\t \u00C2\u00A0Calcium\t \u00C2\u00A0promotes\t \u00C2\u00A0cell\t \u00C2\u00A0survival\t \u00C2\u00A0through\t \u00C2\u00A0CaM-\u00C2\u00AD\u00E2\u0080\u0090K\t \u00C2\u00A0kinase\t \u00C2\u00A0activation\t \u00C2\u00A0of\t \u00C2\u00A0the\t \u00C2\u00A0protein-\u00C2\u00AD\u00E2\u0080\u0090kinase-\u00C2\u00AD\u00E2\u0080\u0090B\t \u00C2\u00A0pathway.\t \u00C2\u00A0Nature\t \u00C2\u00A0396,\t \u00C2\u00A0584\u00E2\u0080\u0093587.\t \u00C2\u00A0\t \u00C2\u00A0 232 Yu\t \u00C2\u00A0D,\t \u00C2\u00A0Pendergraff\t \u00C2\u00A0H,\t \u00C2\u00A0Liu\t \u00C2\u00A0J,\t \u00C2\u00A0Kordasiewicz\t \u00C2\u00A0HB,\t \u00C2\u00A0Cleveland\t \u00C2\u00A0DW,\t \u00C2\u00A0Swayze\t \u00C2\u00A0EE,\t \u00C2\u00A0Lima\t \u00C2\u00A0WF,\t \u00C2\u00A0Crooke\t \u00C2\u00A0ST,\t \u00C2\u00A0Prakash\t \u00C2\u00A0TP,\t \u00C2\u00A0Corey\t \u00C2\u00A0DR\t \u00C2\u00A0(2012)\t \u00C2\u00A0Single-\u00C2\u00AD\u00E2\u0080\u0090Stranded\t \u00C2\u00A0RNAs\t \u00C2\u00A0Use\t \u00C2\u00A0RNAi\t \u00C2\u00A0to\t \u00C2\u00A0Potently\t \u00C2\u00A0and\t \u00C2\u00A0Allele-\u00C2\u00AD\u00E2\u0080\u0090Selectively\t \u00C2\u00A0Inhibit\t \u00C2\u00A0Mutant\t \u00C2\u00A0Huntingtin\t \u00C2\u00A0Expression.\t \u00C2\u00A0Cell\t \u00C2\u00A0150:895\u00E2\u0080\u0093908.\t \u00C2\u00A0Zamecnik\t \u00C2\u00A0PC,\t \u00C2\u00A0Stephenson\t \u00C2\u00A0ML\t \u00C2\u00A0(1978)\t \u00C2\u00A0Inhibition\t \u00C2\u00A0of\t \u00C2\u00A0Rous\t \u00C2\u00A0sarcoma\t \u00C2\u00A0virus\t \u00C2\u00A0replication\t \u00C2\u00A0and\t \u00C2\u00A0cell\t \u00C2\u00A0transformation\t \u00C2\u00A0by\t \u00C2\u00A0a\t \u00C2\u00A0specific\t \u00C2\u00A0oligodeoxynucleotide.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A075:280\u00E2\u0080\u0093284.\t \u00C2\u00A0Zhang\t \u00C2\u00A0C\t \u00C2\u00A0and\t \u00C2\u00A0Cuervo\t \u00C2\u00A0AM\t \u00C2\u00A0(2008)\t \u00C2\u00A0Nat\t \u00C2\u00A0Med\t \u00C2\u00A014,\t \u00C2\u00A0959\t \u00C2\u00A0-\u00C2\u00AD\u00E2\u0080\u0090\t \u00C2\u00A0965.\t \u00C2\u00A0\t \u00C2\u00A0Zhang\t \u00C2\u00A0J,\t \u00C2\u00A0Zheng\t \u00C2\u00A0N,\t \u00C2\u00A0Zhou\t \u00C2\u00A0P\t \u00C2\u00A0(2003)\t \u00C2\u00A0Exploring\t \u00C2\u00A0the\t \u00C2\u00A0functional\t \u00C2\u00A0complexity\t \u00C2\u00A0of\t \u00C2\u00A0cellular\t \u00C2\u00A0proteins\t \u00C2\u00A0by\t \u00C2\u00A0protein\t \u00C2\u00A0knockout.\t \u00C2\u00A0Proc\t \u00C2\u00A0Natl\t \u00C2\u00A0Acad\t \u00C2\u00A0Sci\t \u00C2\u00A0USA\t \u00C2\u00A0100:14127\u00E2\u0080\u009314132.\t \u00C2\u00A0Zhang\t \u00C2\u00A0S,\t \u00C2\u00A0Taghibiglou\t \u00C2\u00A0C,\t \u00C2\u00A0Girling\t \u00C2\u00A0K,\t \u00C2\u00A0Dong\t \u00C2\u00A0Z,\t \u00C2\u00A0Lin\t \u00C2\u00A0SZ,\t \u00C2\u00A0Lee\t \u00C2\u00A0W,\t \u00C2\u00A0Shyu\t \u00C2\u00A0WC,\t \u00C2\u00A0Wang\t \u00C2\u00A0YT\t \u00C2\u00A0(2013)\t \u00C2\u00A0Critical\t \u00C2\u00A0Role\t \u00C2\u00A0of\t \u00C2\u00A0Increased\t \u00C2\u00A0PTEN\t \u00C2\u00A0Nuclear\t \u00C2\u00A0Translocation\t \u00C2\u00A0in\t \u00C2\u00A0Excitotoxic\t \u00C2\u00A0and\t \u00C2\u00A0Ischemic\t \u00C2\u00A0Neuronal\t \u00C2\u00A0Injuries.\t \u00C2\u00A0J\t \u00C2\u00A0Neurosci\t \u00C2\u00A033:7997\u00E2\u0080\u00938008.\t \u00C2\u00A0Zhou\t \u00C2\u00A0P,\t \u00C2\u00A0Bogacki\t \u00C2\u00A0R,\t \u00C2\u00A0McReynolds\t \u00C2\u00A0L,\t \u00C2\u00A0Howley\t \u00C2\u00A0PM\t \u00C2\u00A0(2000)\t \u00C2\u00A0Harnessing\t \u00C2\u00A0the\t \u00C2\u00A0ubiquitination\t \u00C2\u00A0machinery\t \u00C2\u00A0to\t \u00C2\u00A0target\t \u00C2\u00A0the\t \u00C2\u00A0degradation\t \u00C2\u00A0of\t \u00C2\u00A0specific\t \u00C2\u00A0cellular\t \u00C2\u00A0proteins.\t \u00C2\u00A0Mol\t \u00C2\u00A0Cell\t \u00C2\u00A06:751\u00E2\u0080\u0093756.\t \u00C2\u00A0Zhuang\t \u00C2\u00A0S,\t \u00C2\u00A0Schnellmann\t \u00C2\u00A0RG\t \u00C2\u00A0(2006)\t \u00C2\u00A0A\t \u00C2\u00A0Death-\u00C2\u00AD\u00E2\u0080\u0090Promoting\t \u00C2\u00A0Role\t \u00C2\u00A0for\t \u00C2\u00A0Extracellular\t \u00C2\u00A0Signal-\u00C2\u00AD\u00E2\u0080\u0090Regulated\t \u00C2\u00A0Kinase.\t \u00C2\u00A0J\t \u00C2\u00A0Pharmacol\t \u00C2\u00A0Exp\t \u00C2\u00A0Ther\t \u00C2\u00A0319:991\u00E2\u0080\u0093997.\t \u00C2\u00A0\t \u00C2\u00A0 233 APPENDICES\t \u00C2\u00A0Appendix\t \u00C2\u00A0A\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0following\t \u00C2\u00A0program\t \u00C2\u00A0looks\t \u00C2\u00A0for\t \u00C2\u00A0all\t \u00C2\u00A0potential\t \u00C2\u00A0CTMs\t \u00C2\u00A0according\t \u00C2\u00A0to\t \u00C2\u00A0the\t \u00C2\u00A0biochemical\t \u00C2\u00A0makeup\t \u00C2\u00A0presented\t \u00C2\u00A0in\t \u00C2\u00A0(Dice,\t \u00C2\u00A01990).\t \u00C2\u00A0An\t \u00C2\u00A0exhaustive\t \u00C2\u00A0list\t \u00C2\u00A0is\t \u00C2\u00A0subsequently\t \u00C2\u00A0printed.\t \u00C2\u00A0Note\t \u00C2\u00A0that\t \u00C2\u00A0the\t \u00C2\u00A0program\t \u00C2\u00A0prints\t \u00C2\u00A0putative\t \u00C2\u00A0CTMs\t \u00C2\u00A0\u00E2\u0080\u0093\t \u00C2\u00A0whether\t \u00C2\u00A0these\t \u00C2\u00A0motifs\t \u00C2\u00A0are\t \u00C2\u00A0functional\t \u00C2\u00A0in\t \u00C2\u00A0the\t \u00C2\u00A0protein\t \u00C2\u00A0will\t \u00C2\u00A0have\t \u00C2\u00A0to\t \u00C2\u00A0be\t \u00C2\u00A0determined\t \u00C2\u00A0experimentally.\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0The\t \u00C2\u00A0program\t \u00C2\u00A0is\t \u00C2\u00A0written\t \u00C2\u00A0in\t \u00C2\u00A0Python\t \u00C2\u00A0by\t \u00C2\u00A0Andrej\t \u00C2\u00A0Karpathy\t \u00C2\u00A0and\t \u00C2\u00A0I.\t \u00C2\u00A0\t \u00C2\u00A0#\t \u00C2\u00A0All\t \u00C2\u00A0KFERQ-\u00C2\u00AD\u00E2\u0080\u0090non\t \u00C2\u00A0relating\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0are\t \u00C2\u00A0designated\t \u00C2\u00A0\"x\"\t \u00C2\u00A0#\t \u00C2\u00A0Basic\t \u00C2\u00A0\"+\",\t \u00C2\u00A0acidic\t \u00C2\u00A0\"-\u00C2\u00AD\u00E2\u0080\u0090\",\t \u00C2\u00A0bulky-\u00C2\u00AD\u00E2\u0080\u0090hydrophobic\t \u00C2\u00A0\"z\"\t \u00C2\u00A0a=\"\"\"\t \u00C2\u00A0\"\"\"\t \u00C2\u00A0#\t \u00C2\u00A0copy-\u00C2\u00AD\u00E2\u0080\u0090paste\t \u00C2\u00A0amino\t \u00C2\u00A0acid\t \u00C2\u00A0sequence\t \u00C2\u00A0AS\t \u00C2\u00A0IS\t \u00C2\u00A0from\t \u00C2\u00A0NCBI\t \u00C2\u00A0Protein\t \u00C2\u00A0database,\t \u00C2\u00A0include\t \u00C2\u00A0line\t \u00C2\u00A0breaks\t \u00C2\u00A0and\t \u00C2\u00A0numbering\t 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\u00C2\u00A0c='\t \u00C2\u00A0'\t \u00C2\u00A0for\t \u00C2\u00A0k\t \u00C2\u00A0in\t \u00C2\u00A0a:\t \u00C2\u00A0\t \u00C2\u00A0 if\t \u00C2\u00A0k\t \u00C2\u00A0in\t \u00C2\u00A0'krdefilvhagmpcstwyqn':\t \u00C2\u00A0#single\t \u00C2\u00A0letter\t \u00C2\u00A0code\t \u00C2\u00A0of\t \u00C2\u00A0amino\t \u00C2\u00A0acids\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 c+=k\t \u00C2\u00A0d=c\t \u00C2\u00A0 234 c=c.replace\t \u00C2\u00A0('k','+')\t \u00C2\u00A0c=c.replace('r','+')\t \u00C2\u00A0c=c.replace('d','-\u00C2\u00AD\u00E2\u0080\u0090')\t \u00C2\u00A0c=c.replace('e','-\u00C2\u00AD\u00E2\u0080\u0090')\t \u00C2\u00A0c=c.replace('f','z')\t \u00C2\u00A0c=c.replace('i','z')\t \u00C2\u00A0c=c.replace('l','z')\t \u00C2\u00A0c=c.replace('v','z')\t \u00C2\u00A0c=c.replace('h','x')\t \u00C2\u00A0c=c.replace('a','x')\t \u00C2\u00A0c=c.replace('g','x')\t \u00C2\u00A0c=c.replace('m','x')\t \u00C2\u00A0c=c.replace('p','x')\t \u00C2\u00A0c=c.replace('c','x')\t \u00C2\u00A0c=c.replace('s','x')\t \u00C2\u00A0c=c.replace('t','x')\t \u00C2\u00A0c=c.replace('w','x')\t \u00C2\u00A0c=c.replace('y','x')\t \u00C2\u00A0n=0\t \u00C2\u00A0 \t \u00C2\u00A0for\t \u00C2\u00A0b\t \u00C2\u00A0in\t \u00C2\u00A0lst:\t \u00C2\u00A0\t \u00C2\u00A0 s=0\t \u00C2\u00A0\t \u00C2\u00A0 e=d\t \u00C2\u00A0\t \u00C2\u00A0 found=False\t \u00C2\u00A0\t \u00C2\u00A0 while\t \u00C2\u00A0True:\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 f=c.find(b,s)\t \u00C2\u00A0 235 \t \u00C2\u00A0 \t \u00C2\u00A0 if\t \u00C2\u00A0f!=-\u00C2\u00AD\u00E2\u0080\u00901:\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 p1=\t \u00C2\u00A0e[:f]\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 p2=\t \u00C2\u00A0e[f+len(b):]\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 e=p1+e[f:f+len(b)].upper()+p2\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 print\t \u00C2\u00A0'match',f,e[f:f+len(b)].upper()\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 s=f+1\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 found=True\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 else:\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 break\t \u00C2\u00A0\t \u00C2\u00A0 if\t \u00C2\u00A0found:\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 n+=1\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 print\t \u00C2\u00A0'search\t \u00C2\u00A0for:\t \u00C2\u00A0'\t \u00C2\u00A0+\t \u00C2\u00A0b\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 t='\t \u00C2\u00A0'\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 i=0\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 for\t \u00C2\u00A0k\t \u00C2\u00A0in\t \u00C2\u00A0e:\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 if\t \u00C2\u00A0i%10==0:t+='\t \u00C2\u00A0'\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 if\t \u00C2\u00A0i%60==0:t+='\n'\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 t+=k\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 \t \u00C2\u00A0 i+=1\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 print\t \u00C2\u00A0t\t \u00C2\u00A0\t \u00C2\u00A0 \t \u00C2\u00A0 print\t \u00C2\u00A0'-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090-\u00C2\u00AD\u00E2\u0080\u0090'\t \u00C2\u00A0if\t \u00C2\u00A0n==0:\t \u00C2\u00A0\t \u00C2\u00A0 print\t \u00C2\u00A0'not\t \u00C2\u00A0found'\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0 236 Appendix\t \u00C2\u00A0B\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0Potential\t \u00C2\u00A0CTMs\t \u00C2\u00A0in\t \u00C2\u00A0excitatory\t \u00C2\u00A0and\t \u00C2\u00A0inhibitory\t \u00C2\u00A0receptors\t \u00C2\u00A0Membrane\t \u00C2\u00A0Receptor\t \u00C2\u00A0Species\t \u00C2\u00A0 Genebank\t \u00C2\u00A0\t \u00C2\u00A0 Potential\t \u00C2\u00A0CTM\t \u00C2\u00A0(no.\t \u00C2\u00A0of\t \u00C2\u00A0AAs\t \u00C2\u00A0from\t \u00C2\u00A0origin)\t \u00C2\u00A0NMDAR2A\t \u00C2\u00A0\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 EAW85187.1\t \u00C2\u00A0 666\t \u00C2\u00A0DKKFQ,\t \u00C2\u00A01293\t \u00C2\u00A0NIVDK\t \u00C2\u00A0NMDAR2B\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAI13621.1\t \u00C2\u00A0 90\t \u00C2\u00A0DRKIQ,\t \u00C2\u00A0667\t \u00C2\u00A0DKKFQ\t \u00C2\u00A0GABAAR\u00CE\u00B11\t \u00C2\u00A0\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA32874.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABAAR\u00CE\u00B12\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAB27278.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABAAR\u00CE\u00B13\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAB27279.1\t \u00C2\u00A0 38\t \u00C2\u00A0DFVKQ,\t \u00C2\u00A0171\t \u00C2\u00A0RLVDN\t \u00C2\u00A0GABAAR\u00CE\u00B14\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAB52519.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABAAR\u00CE\u00B15\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA01920.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABAAR\u00CE\u00B16\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA01921.1\t \u00C2\u00A0 33\t \u00C2\u00A0RILDN\t \u00C2\u00A0GABAAR\u00CE\u00B21\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA32875.1\t \u00C2\u00A0 371\t \u00C2\u00A0LEIRN,\t \u00C2\u00A0203\t \u00C2\u00A0NKIEL\t \u00C2\u00A0GABAAR\u00CE\u00B22\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAB33982.1\t \u00C2\u00A0 362\t \u00C2\u00A0KDIKQ,\t \u00C2\u00A0353\t \u00C2\u00A0RLDVN,\t \u00C2\u00A0408\t \u00C2\u00A0LEIKN\t \u00C2\u00A0GABAAR\u00CE\u00B23\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAA52511.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABAAR\u00CE\u00B32\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA33437.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0 237 Membrane\t \u00C2\u00A0Receptor\t \u00C2\u00A0Species\t \u00C2\u00A0 Genebank\t \u00C2\u00A0\t \u00C2\u00A0 Potential\t \u00C2\u00A0CTM\t \u00C2\u00A0(no.\t \u00C2\u00A0of\t \u00C2\u00A0AAs\t \u00C2\u00A0from\t \u00C2\u00A0origin)\t \u00C2\u00A0GABAAR\u00CE\u00B33\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAB39369.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABABR1a\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA09939.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABABR1b\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA09940.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABABR1c\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA09941.1\t \u00C2\u00A0 No\t \u00C2\u00A0match\t \u00C2\u00A0GABABR2\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAA09942.1\t \u00C2\u00A0 590\t \u00C2\u00A0IIKDQ,\t \u00C2\u00A0245\t \u00C2\u00A0NDVRI\t \u00C2\u00A0GluA1\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 AAI11735.1\t \u00C2\u00A0 217\t \u00C2\u00A0KLEKN,\t \u00C2\u00A0773\t \u00C2\u00A0DKLKN,\t \u00C2\u00A0309\t \u00C2\u00A0QRIDI,\t \u00C2\u00A0158\t \u00C2\u00A0QKVLD,\t \u00C2\u00A0342\t \u00C2\u00A0QVRFE,\t \u00C2\u00A0240\t \u00C2\u00A0NKFKE\t \u00C2\u00A0GluA2\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 EAX04865.1\t \u00C2\u00A0 359\t \u00C2\u00A0IKFDQ,\t \u00C2\u00A0212\t \u00C2\u00A0RDKVN,\t \u00C2\u00A0780\t \u00C2\u00A0DKLKN,\t \u00C2\u00A0316\t \u00C2\u00A0QRIEI,\t \u00C2\u00A0358\t \u00C2\u00A0NIKFD\t \u00C2\u00A0GluA3\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 P42263.2\t \u00C2\u00A0 195\t \u00C2\u00A0IKDVQ,\t \u00C2\u00A0791\t \u00C2\u00A0DKLKN,\t \u00C2\u00A0222\t \u00C2\u00A0VERIN,\t \u00C2\u00A0199\t \u00C2\u00A0QEFRR,\t \u00C2\u00A0326\t \u00C2\u00A0QRVDV,\t \u00C2\u00A0194\t \u00C2\u00A0NIKDV\t \u00C2\u00A0GluA4\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 P48058.2\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0279\t \u00C2\u00A0KKLDQ,\t \u00C2\u00A0200\t \u00C2\u00A0LDRRQ,\t \u00C2\u00A0214\t \u00C2\u00A0IERLQ,\t \u00C2\u00A0781\t \u00C2\u00A0DKLKN,\t \u00C2\u00A0204\t \u00C2\u00A0QEKKF,\t \u00C2\u00A0317\t \u00C2\u00A0QKIDI\t \u00C2\u00A0GluA5\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAC80546.1\t \u00C2\u00A0 237\t \u00C2\u00A0EILKQ,\t \u00C2\u00A0186\t \u00C2\u00A0QELIK,\t \u00C2\u00A0141\t \u00C2\u00A0NKDLF,\t \u00C2\u00A0 238 Membrane\t \u00C2\u00A0Receptor\t \u00C2\u00A0Species\t \u00C2\u00A0 Genebank\t \u00C2\u00A0\t \u00C2\u00A0 Potential\t \u00C2\u00A0CTM\t \u00C2\u00A0(no.\t \u00C2\u00A0of\t \u00C2\u00A0AAs\t \u00C2\u00A0from\t \u00C2\u00A0origin)\t \u00C2\u00A0361\t \u00C2\u00A0NLIKE\t \u00C2\u00A0Kainate\t \u00C2\u00A01\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 EAX09906.1\t \u00C2\u00A0 237\t \u00C2\u00A0EILKQ,\t \u00C2\u00A0186\t \u00C2\u00A0QELIK,\t \u00C2\u00A0141\t \u00C2\u00A0NKDLF,\t \u00C2\u00A0361\t \u00C2\u00A0NLIKE\t \u00C2\u00A0Kainate\t \u00C2\u00A02\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 EAW48446.1\t \u00C2\u00A0 185\t \u00C2\u00A0QELIK\t \u00C2\u00A0Kainate\t \u00C2\u00A03\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 CAI19119.1\t \u00C2\u00A0 185\t \u00C2\u00A0QELIK,\t \u00C2\u00A0897\t \u00C2\u00A0NDRRL,\t \u00C2\u00A0363\t \u00C2\u00A0NFIKE\t \u00C2\u00A0Kainate\t \u00C2\u00A04\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 EAW67511.1\t \u00C2\u00A0 750\t \u00C2\u00A0NRLEI,\t \u00C2\u00A0150\t \u00C2\u00A0NLEKL\t \u00C2\u00A0Kainate\t \u00C2\u00A05\t \u00C2\u00A0 Homo\t \u00C2\u00A0sapiens\t \u00C2\u00A0 EAW57090.1\t \u00C2\u00A0 360\t \u00C2\u00A0RVEFN,\t \u00C2\u00A0317\t \u00C2\u00A0VRELN,\t \u00C2\u00A0772\t \u00C2\u00A0NRLEI\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0\t \u00C2\u00A0"@en . "Thesis/Dissertation"@en . "2015-02"@en . "10.14288/1.0167106"@en . "eng"@en . "Neuroscience"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "Attribution-NonCommercial-NoDerivs 2.5 Canada"@en . "http://creativecommons.org/licenses/by-nc-nd/2.5/ca/"@en . "Graduate"@en . "Harnessing chaperone-mediated autophagy for the degradation of endogenous proteins"@en . "Text"@en . "http://hdl.handle.net/2429/51927"@en .