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Role of SAP102 in mutant Huntingtin-mediated regulation of NMDA receptor function in the YAC transgenic… Fan, Jing 2007

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ROLE OF SAP102 IN MUTANT HUNTINGTIN-MEDIATED REGULATION OF NMDA RECEPTOR FUNCTION IN THE YAC TRANSGENIC MOUSE MODEL OF HUNTINGTON DISEASE by Jing Fan B . S c , Peking University, Beijing, China, 2003 A THESIS S U B M I T T E D I N P A R T I A L F U L L F I L L M E N T OF T H E R E Q U I R E M E N T F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Neuroscience) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A June 2007 ©Jing Fan, 2007 ABSTRACT Huntington disease (HD) is a dominantly inherited neurodegenerative disease, which is caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Increasing evidence suggests that the N-methyl-D-aspartate (NMDA)-type glutamate receptor plays a role in mediating death of striatal medium-sized spiny neurons (MSNs) observed in HD. For example, previous results from our laboratory demonstrate that NMDA receptor (NMDAR)-mediated current and toxicity is increased in neurons from the Yeast Artificial Chromosome (YAC) transgenic mouse model expressing huntingtin with 72 polyQ (YAC72), and suggest that an increase in surface NMDAR number is the trigger for increased cytosolic calcium, ultimately resulting in activation of caspases and leading to neuronal death. Here, I hypothesize that potentiation of NMDAR surface expression and excitotoxicity by mutant htt (mhtt) may be mediated in part by interaction with membrane-associated guanylate kinases (MAGUKs) such as PSD-95 or SAP102. I report the novel finding that htt is co-immunoprecipitated with SAP102 in HEK293T cells and in striatal tissue from wild-type and YAC transgenic mice, but that the association of SAP102 with the NMDAR NR2B subunit or htt is not altered by htt polyQ length: However, by using a Tat-NR2B9c peptide to block binding between NR2B and either SAP 102 or PSD-95 in cultured striatal neurons, I show that this interaction plays a key role in mediating the increase in NMDAR surface expression and NMDA-induced toxicity observed with mhtt expression. Moreover, the Tat-NR2B9c peptide has no effect on glutamate receptor 1 (GluRl) surface expression or staurosporine-induced toxicity. The ii mechanism by which SAP 102 and/or PSD-95 mediates the altered NMDAR trafficking and function observed with mhtt expression remains to be explored. My research may help to elucidate further the mechanisms underlying enhanced excitotoxicity in HD. in TABLE OF CONTENTS Abstract i i Table of Contents iv List of Figures '. v i i i List of Abbreviations -x Acknowledgements xiv Chapter 1 Introduction 1 1.1 Huntington's disease 1 1.1.1 Huntington's disease and huntingtin 1 1.1.2 Function of wild-type huntingtin 3 1.1.3 Selective Neurodegeneration in Huntington's disease 3 1.1.4 Proteins interact with Huntingtin 4 1.1.5 Mouse models of Huntington's disease 5 1.1.5.1 YAC transgenic mouse model 6 1.1.5.2 R6/2 and R6/1 mouse model 7 1.1.5.3 Knock-in mouse models 7 1.2 Excitotoxicity hypothesis of Huntington's disease 8 1.2.1 Glutamate and Glutamate receptors 8 1.2.2 Glutamate and Huntington's disease 9 1.2.3 NMDA Receptors and Huntington's disease 10 1.2.3.1 NR2B-type NMDA Receptors 11 1.2.4 AMPA and Kainate Receptors and Huntington's disease 13 1.2.5 Metatropic Glutamate Receptors and Huntington's disease.... 13 1.2.6 Calcium Homeostasis and Huntington's Disease 14 1.2.7 Apoptosis and Huntington's Disease 16 1.3 M A G U K proteins and Huntington's Disease 18 1.3.1 PSD-95... 21 iv 1.3.2 SAP102 22 1.3.3 SAP97 and PSD-93/Chapsyn-110 24 1.3.4 PSD-95 and Huntington's Disease 24 1.3.5 Tat-NR2B9c peptide 25 1.4 Hypothesis 26 Chapter 2 Materials and Methods 27 2.1 Material 27 2.1.1 Plasmids 27 2.1.2 Antibodies 27 2.1.3 Peptides 28 2.2 HEK293T cell line and transfection 28 2.3 Transgenic mice 30 2.4 Primary striatal neuronal culture 30 2.5 Brain lysates and solubilization 31 2.6 Immunoprecipitation 32 2.7 Western blotting 33 2.8 Immunocytochemistry and surface NR1 and GluRl staining 33 2.9 NMDA and staurosporine induced cytotoxicity 34 2.10 TUNEL Assay and assessment of apoptosis 35 2.11 Data analysis 36 Chapter 3 Results 37 3.1 The expression level of SAP 102 in 4 week-old YAC 18, YAC72, and YAC 128 mice striatal tissues 37 3.2 Association of SAP 102 and huntingtin 40 3.2.1 Association of SAP102 and huntingtin in httl38 and httl5 transfected HEK293T cells 40 3.2.2 Association of SAP 102 and huntingtin in WT and YAC mice striatal tissues 43 3.3 Interaction of SAP 102 and NR2B 46 3.3.1 Interaction of SAP102 and NR2B in htt 138 and htt 15 transfected HEK293T cells 46 3.3.2 Interaction of SAP102 and NR2B in YAC mice striatal tissues 46 3.4 Tat-NR2B9c interferes with the interaction of SAP102 and NR2B 50 3.4.1 l uM Tat-NR2B9c interferes with the interaction of SAP 102 and NR2B in transfected HEK cells 50 3.4.2 l u M Tat-NR2B9c interferes with the interaction of SAP102 and NR2B in cultured YAC 128 MSNs 51 3.5 Tat-NR2B9c reduces the surface NMDAR expression in cultured MSNs 58 3.5.1 l u M Tat-NR2B9c reduces the surface NR1 expression in cultured MSNs 58 3.5.2 l u M Tat-NR2B9c does not change the surface GluRl expression in cultured MSNs 59 3.5.3 200nM Tat-NR2B9c reduces the surface NR1 expression in cultured MSNs 60 3.6 200nM Tat-NR2B9c reduces NMDA-induced toxicity in YAC72, YAC 128, but not WT MSNs 65 3.6.1 200nM Tat-NR2B9c reduces the NMDA-induced toxicity in cultured YAC mice MSNs 65 3.6.2 200nM Tat-NR2B9c has no significant effect on the staurosporine-induced toxicity in cultured YAC72 MSNs 67 Chapter 4 Discussion 73 4.1 Tat-NR2B9c peptide's specificity 75 4.2 Controls 76 4.3 Possible mechanisms of htt regulation of NMDAR surface expression 77 VI 4.4 NR2B versus NR2A; 78 4.5 Concentration of Tat-NR2B9c peptide used in different studies 82 4.6 Possible mechanisms of PSD-95 and/or SAP102 mediated mutant htt-regulation of N M D A R function 85 4.7 Future directions.. 86 References 89 vii List of Figures Figure 1. Interaction and organization of PDZ domain-containing proteins at a mammalian excitatory synapse 20 Figure 2. Representative western blot showing expression level of SAP 102 in 4 week-old YAC mice striatal tissues... 38 Figure 3. Expression level of SAP102 in 4 week-old YAC mice striatal tissues....39 'Figure 4. SAP102 associates with huntingtin in transfected 293T cells 42 Figure 5. SAP102 associates with huntingtin in 4 week-old YAC mice striatal tissues 45 Figure 6. The associations of SAP102 and NR2B are similar in httl5 and htt 138 transfected 293T cells 48 Figure 7. The associations of SAP102 and NR2B are similar in 4 week-old YAC18, YAC72 and YAC128 mice striatal tissues 49 Figure 8. Tat-NR2B9c (luM) perturbs co-IP of NR2B with SAP102 in HEK293T cells.: 53 Figure 9. Quantitatively measured SAP102/NR2B co-IP ratio in HEK293T cells.54 Figure 10. Tat-NR2B9c (luM) perturbs co-IP of NR2B with SAP102 in YAC128 cultured MSNs ..56 Figure 11. Quantitatively measured SAP102/NR2B co-IP ratio in YAC128 cultured MSNs 57 Figure 12. Representative photomicrographs of surface and internal NR1 staining of 1 uM Tat-NR2B peptides pretreated YAC72 MSNs 61 Figure 13. Effect of luM Tat-NR2B9c peptide pretreatment on surface/internal NR1 ratio of WT and YAC72 MSNs 62 Figure 14. Effect of luM Tat-NR2B9c peptide pretreatment on surface/internal GluRl ratio of WT and YAC72 MSNs 63 Figure 15. Effect of 200nM and luM Tat-NR2B9c peptide pretreatment on surface/internal NR1 ratio of WT and YAC72 MSNs. 64 Figure 16. Representative photomicrographs showing TUNEL and PI stained YAC128 MSNs pretreated with 200nM Tat-NR2B9c for lhr (which has not been exposed to NMDA)... .......68 Figure 17. Effect of 200nM Tat-NR2B peptides pretreatment on NMDA-induced toxicity in WT, YAC72, and YAC128 cultured MSNs 70 Figure 18. Effect of 200nM Tat-NR2B peptides pretreatment on basal cell death of Wt, Y72Q and Y128Q MSNs 71 Figure 19. 200nM Tat-NR2B peptides lhr pretreatment has no effect on lOuM staurosporine induced cell death of cultured Y72Q MSNs 72 Figure 20. Proposed relationships between di-subunit-containing and tri-subunit-containing NMDA receptor tetramers, scaffolding complexes and trafficking proteins 81 Figure 21. The hypothesis that NMDAR-PSD-95 complex maybe dissociated using Tat peptides fused to the COOH-terminus of NR2B (Tat-NR2B9c), thus reducing the efficiency of excitotoxic signaling through nNOS 84 LIST OF ABBREVIATION Aa amino acid A M P A a -amino-3-hydroxy-5-methyl-4-isoxazolepropionate A M P A R A M P A receptor B A C Bacterial artificial chromosomes BDNF Brain-derived neurotrophic factor BES N, N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid BSA Bovine serum albumin BSS Balanced salt solution °C Degrees centigrade CaMKII Ca2+/calmodulin-dependent protein kinase II C a 2 + Calcium CaCb Calcium chloride CBP C R E B binding protein cDNA Complementary deoxyribonucleic acid Chapsyn 110 Channel-associated proteins of the synapses-110 C M V Cytomegalovirus C 0 2 Carbon dioxide CNS Central nervous system C R E B c A M P response element binding protein Ctrl Control DARPP-32 Dopamine- and cyclic AMP-regulated phosphoprotein-DIV Days in vitro Dig Discs large D M S O Dimethyl sulfoxide D T T Dithiothreitol E A A Excitatory amino acid E C L Enhanced chemiluminescence system E D T A Ethylenediaminetetraacetic acid EGTA Ethyleneglycol-bis[ 0 -aminoethyl ether]-N, N , N ' N ' -tetraacetic acid ER Endoplasmic reticulum ERK Ras-extracellular signal-regulated kinase EtOH Ethanol FBS Fetal bovine serum GABA y -aminobutyric acid GAD-65 Glutamic acid decarboxylase-65 GFP Green fluorescent protein GKAP Guanylate kinase-associated protein GluR Glutamate receptor GLT1 Glutamate transporter-1 GST Glutathione-S-transferase h (hr) hour HAP Huntingtin-associated protein HBSS Hank's balanced salt solution HD Huntington's disease HEK293T cells Human embryonic kidney 293T cells . HIP-1 Huntingtin-interacting protein-1 HIPPI HIP 1-protein interactor 3-HK 3-hydroxykynurenine HRP Horse radish peroxidase Htt Huntingtin iGluRs ionotropic glutamate receptors . IP Immunoprecipitation IP3 Inositol (1,4,5) - trisphosphate IP31 Type 1 inositol (1,4,5) - trisphosphate receptor KARs Kainate receptors KC1 Potassium chloride LTD Long-term depression xi LTP Long-term potentiation Lys Lysate M Molar MAGUK Membrane-associated guanylate kinases M g 2 + Magnesium M E M Minimum essential medium mGluRs metabotropic glutamate receptors mL Milliliter uL Microliter mM Millimolar uM Micromolar mPTP Mitochondria permeability transition pore mRNA Messenger ribonucleic acid ms Milliseconds MSNs GABAergic medium-sized spiny striatal neurons mV Millivolts NaCl Sodium chloride Na 2 HP0 4 Sodium phosphate, dibasic NaH 2 P0 4 Sodium phosphate, monobasic NGS Normal goat serum NMDA N-methyl-D-aspartate NMDAR NMDA receptor nNOS Neuronal nitric oxide synthase NO Nitric oxide NOS Nitric oxide synthase NR1 NMDA receptor subunit-1 NR2 NMDA receptor subunit-2 NRSE Neuron restrictive silencer element P Postnatal day PFA Paraformaldehyde PI Propidium Iodide PKA Protein Kinase A PKC Protein Kinase C PBS Phosphate-buffered saline PDZ domain PSD/Discs-large/ZO-1 domain PMSF Phenylmethylsulfonyl fluoride PolyQ Polyglutamine expansion PSD-93 Postsynaptic density-93 PSD-95 Postsynaptic density-95 PVDF Polyvinylidene difluoride Q Glutamine (PolyQ repeats) REST Repressor element-1 transcription factor ROIs Regions of interest SAP97 Synapse-associated protein-97 SAP102 . Synapse-associated protein-102 SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis sec Seconds SEM Standard error of the mean SH3 Src homology 3 Shank SH3 and ankyrin repeat-containing protein Spl Specificity protein-1 SPN Supernatant Stauro Staurosporine SynGAP Synaptic RAS GTPase-activating protein TBS Tris-buffered saline TUNEL Terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling WT Wild-type (FVB/N mice) YAC Yeast artificial chromosome xm ACKNOWLEDGEMENTS My deepest gratitude goes first and foremost to Dr. Lynn Raymond, my supervisor, for her constant encouragement and guidance on my research. She has walked me through all the stages of the work and writing for this thesis. Without her knowledge and supportiveness, I would never have such a good chance to explore and confirm my interest in this area, and improve my research abilities for future career. I would also like to thank all the past and present members of our laboratory, all of whom have provided selfless assistance, warm encouragements and precious insights. Special thanks to Mannie Fan, who has helped me through many experimental and literature details with her kindness and patience. Thanks to Lily Zhang and Esther Yu not only for neuron cultures or help on experiments, but also for the care and support. Great thanks to Tao Luo, Dr. Catherine Cowan, Herman Fernandes, Dr. Austen Milnerwood, Lavan Sornarajah, Christine Sutton, Tulin Okbinoglu, Cristina Vasuta, Jacqueline Shehadeh, who have made our laboratory truly a warm and comfortable work place. I would also like to thank Dr. Tim Murphy, Kun Huang, Rujun Kang, Zhi Liu, and all the other members of Kinsmen Tower for their friendship and kind helps in the past 3 years. Numerous thanks to the members of my supervisory committee, Dr. Alaa El-Husseini, Dr. Brian Mac Vicar, and Dr. Yutian Wang, who have given me lots of helpful guidance and suggestions on this project. Thanks our director Dr. Steve Vincent, and dear secretary Liz Wong and Veronica. Dr. Michael R. Hayden, Simon Warby, Rona Graham, Martina Metzler, Dr. Robert Wenthold, and Dr. Michael Tymianski have been great collaborators. Without their assistant, this work cannot be possibly done. Special thanks to Dr. Andy Shi, who has been a wonderful and inspirational friend, and encouraged me to pursue a scientific career. I would also like to thank Shanshan Zhu, Ning Zhou, Chao Tai, Dongchuan Wu, Yi Yan, Shu Zhang, Guang Yang, Xiaojian Sun, and many other friends, who have been very important and caring to me. My husband Xiaojun Xie and my parents are always understanding and, supportive to me through all these years. Their love and care give me the strength to continue this journey no matter how tough and lonely it could be. xiv CHAPTER 1 Introduction 1.1 Huntington's disease 1.1.1 Huntington's disease and huntingtin HD is an autosomal dominant hereditary neurodegenerative condition affecting approximately 5 to 10 in 100,000 people (Vonsattel and DiFiglia 1998). It is characterized by involuntary movements, cognitive decline, and mood disturbance (Harper 1991). HD is caused by an expansion (>36) of a CAG repeat near the 5' end in the HD gene, leading to enlargement of a glutamine repeat (polyQ) in the N-terminus of a 350 kDa protein - huntingtin (htt) (Huntington's Disease Collaborative Research Group, 1993). This mutation results in selective degeneration of striatal medium-sized spiny GABAergic projection neurons (MSNs), with lesser losses of pyramidal neurons in certain layers of the cortex and CA1 of the hippocampus, and some neuronal populations in the hypothalamus (Vonsattel and DiFiglia 1998). The longer the length of CAG repeat is, the earlier the age of disease onset will be (Andrew, Goldberg et al. 1993; Brinkman, Mezei et al. 1997; Sieradzan, Mann et al. 1997). HD patients with CAG repeats < 60 usually show adult onset, normally at the age of ~ 39 (Harper 1991), while persons with CAG repeats > 60 have juvenile onset and accelerated clinical progression (Brandt, Bylsma et al. 1996). Humans homozygous or heterozygous for the HD mutation show no significant differences in disease onset or progression (Wexler, Young et al. 1987; Myers, Leavitt et al. 1989; MacDonald and Gusella 1996). Results of many studies suggest that mutant huntingtin is responsible for mitochondrial dysfunction, impaired energy metabolism, oxidative stress, impaired ubiquitination and proteasomal function, dysregulation of the transcriptional machinery, as well as compromised endocytosis and axonal transport (Tobin and Signer 2000; Davies and Ramsden 2001; Menalled, Sison et al. 2002; Ross 2002; Rubinsztein 2002; Harjes and Wanker 2003; Sugars and Rubinsztein 2003; L i and L i 2004). A l l these changes together or sequentially lead to neuronal dysfunction and eventually neurodegeneration. Additionally, there is a hypothesis that neuronal dysfunction results, at least in part, from altered astrocyte or glia glutamate uptake function caused by mutant huntingtin. Microdialysis studies have suggested a downregulated glial glutamate transporter-1 (GLT1) function in R6/2 mice, and a deficient clearance of glutamate by the GLT1 in the striatum of R6 transgenic mice (Lievens, Woodman et al. 2001; Behrens, Franz et al. 2002; Shin, Fang et al. 2005). Another possibility is that soluble mutant htt might alter neuronal gene expression through interaction with several transcription factors, such as c A M P response element binding protein (CREB)-binding protein (CBP) and specificity protein-1 (Spl) (Nucifora, Sasaki et al. 2001; Steffan, Bodai et al. 2001; Dunah, Jeong et al. 2002; L i , Cheng et al. 2002; Luthi-Carter, Hanson et al. 2002). In one study, wild-type htt was found to interact with the repressor element-1 transcription factor (REST) in the cytoplasm, which associates with the neuron restrictive silencer element (NRSE) to affect transcription of neuronal genes, including brain-derived neurotrophic factor (BDNF). However, mutant htt interacts weakly with R E S T - N R S E , leading to increased accumulation of R E S T - N R S E in the nucleus, which subsequently inhibits the expression of B D N F and other genes and thus contributes to neuronal dysfunction(Zuccato, Tartari et al. 2003). 2 1.1.2. Funct ion o f w i ld - type hunt ing t in Huntingtin is widely expressed in the central nervous system (CNS) (Vonsattel and DiFiglia 1998; Sieradzan and Mann 2001), and has been shown to be critical for development. The huntingtin knock-out mice are embryonic-lethal (Duyao, Auerbach et al. 1995; Nasir, Floresco et al. 1995; Zeitlin, Liu et al. 1995; Hodgson, Smith et al. 1996), and lethality can be rescued by introducing the human HD gene in the YAC transgenic mice (Hodgson, Smith et al. 1996). A study shows that htt controls neuronal gene (including BDNF) transcription through its association with the repressor element-1 transcription factor REST-NRSE (neuron restrictive silencer element) complex (Zuccato, Tartari et al. 2003). Moreover, both in vivo and in vitro studies show that htt plays an anti-apoptotic role in response to a variety of insults (Rigamonti, Bauer et al. 2000; Cattaneo, Rigamonti et al. 2001; Leavitt, Guttman et al. 2001; Rigamonti, Sipione et al. 2001; Zhang, Ona et al. 2003). Some evidence suggests a role in vesicle transport, protein trafficking and endocytosis, as htt associates with vesicular and trans-Golgi-network membranes and microtubules (DiFiglia, Sapp et al. 1995; Sharp, Loev et al. 1995; Wood, MacMillan et al. 1996; Velier, Kim et al. 1998). 1.1.3. Selective neurodegenerat ion in Hunt ing ton 's disease Early findings in autopsy brain tissue from humans with HD showed that the most severe degeneration (up to -95% loss of neurons) occurs in the neostriatum, particularly the caudate and putamen (Vonsattel, Myers et al. 1985). In these regions, the most affected neuronal population is MSNs, which comprise -90% of neurons in the striatum (Surmeier, Bargas et al. 1988), while the interneurons are spared (Sieradzan and Mann 2001). In severe cases and late stages, -30% neuron loss was found in neocortical regions, and -35% loss for hippocampal CA1 pyramidal neurons. 3 Thus, striatum and MSNs will be our focus of H D study. 1.1.4 Proteins interact with huntingtin The expression levels of htt in the central nervous system (CNS) and in different types of striatal neurons are similar, without any enrichment in the striatum or MSNs as might be expected given the selective degeneration of this neuronal population (Li, Schilling et al. 1993; DiFiglia, Sapp et al. 1995; Sharp, Loev et al. 1995; Ferrante, Gutekunst et al. 1997; Kumar, Asotra et al. 1997). Thus, this evidence suggests that the level of htt expression may not be the reason for selective degeneration, but that htt interactions with proteins which are selectively expressed in brain regions or neurons and whose interactions are changed by the htt polyQ expansion may lead to the selective neuronal death. Discovery of several proteins that interact with huntingtin in a polyQ-dependent manner has provided clues to the role of wild-type htt in normal neuronal function, as well as to the possible mechanisms by which polyQ-expanded htt ("mutant htt") results in neurodegeneration (Li and L i 2004). Huntingtin-associated proteins (HAP-1,2) and huntingtin-interacting proteins (HIP-1,2,3) are two groups of proteins reported to interact with the N-terminus of htt in a polyQ length-dependent manner, by yeast two-hybrid screens. H A P and HIP families are ubiquitously expressed cytoskeletal proteins that interact with vesicle or membrane associated proteins. As interaction of H A P 1 with htt is enhanced by polyQ expansion, and HAP1 was recently found to interact with the type 1 inositol (1,4,5) - trisphosphate receptor (IP3I), it is suggested that mutant htt enhances the sensitivity of IP3I to inositol (1,4,5) -triphosphate through the IP31 - HAP1A - Htt ternary complex (Tang, Tu et al. 2004). 4 In addition, HAP1 mediates the interaction of htt with a transcription factor NeuroD, which is important for neuronal survival and development (Marcora, Gowan et al. 2003). On the other hand, it was reported that decreased binding of mutant htt to HIP1 might increase association of HIP1 and HIPPI (HIPl-protein interactor), which induces caspase-8 cleavage and apoptosis (Gervais, Singaraja et al. 2002). Another interesting htt-interacting protein is postsynaptic density protein-95 (PSD-95) (Sun, Savanenin et al. 2001), which has been shown to cluster and stabilize ion channels and receptors at the synapse (Kim and Sheng 2004). PSD-95 directly binds to the proline-rich region adjacent to the polyQ domain in the N-terminus of wild-type htt, through its type-II Src homology 3 (SH3) domain (Sun, Savanenin et al. 2001). As polyQ length increases, PSD-95 fails to bind mutant htt, and the authors speculate that this could result in more ion channels staying at synapses and thus make neurons more vulnerable to excitation insults (Sun, Savanenin et al. 2001). 1.1.5 Mouse models of Huntington's disease Recently, many genetic mouse models of HD have been generated and used to reveal the mechanisms and suggest possible therapies of HD. A l l mouse models of H D can be classified into three major groups, one of which expresses full-length mutant human huntingtin and is generated by using yeast or bacterial artificial chromosomes (YAC or B A C ) ; the other two are the knock-in mouse models with expanded C A G repeats introduced into the endogenous mouse H D gene, and transgenic mouse models expressing truncated mutant huntingtin under the control of promoters (Levine, Cepeda et al. 2004). A l l these mouse models exhibit changes in electrophysiological (e.g. currents, capacitance, and resistance), neuropathological (e.g. atrophy, cell loss, 5 neuropil aggregates, and nuclear inclusions) properties of striatal neurons and/or impairments in behavioral tests (e.g. open field activity and rotarod performance) that are consistent with clinical manifestations in HD patients (Levine, Cepeda et al. 2004). The advantages and disadvantages of each type of HD mouse models will be briefly discussed below. 1.1.5.1 Y A C transgenic mouse model Yeast artificial chromosome (YAC) transgenic mice are established in the FVB/N mouse strain background, expressing full-length human htt with the endogenous human HD promoter, containing either 18 CAG repeats (termed "YAC 18") or a pathological number of repeats (YAC46, YAC72 and YAC 128), in a developmental and tissue-specific manner (Hodgson, Smith et al. 1996; Hodgson, Agopyan et al. 1999; Slow, van Raamsdonk et al. 2003). In the YAC72 line 2511 mice, expressing mutant human htt at low levels, electrophysiological abnormalities appear in hippocampal slices at 6 months followed by behavioral impairments, while selective degeneration of MSNs in the striatum begins at about 12 months and no intraneuronal htt aggregation is observed (Hodgson, Agopyan et al. 1999). YAC128 mice also display selective striatal degeneration, but at earlier ages than for YAC72 and correlating with well-defined motor deficits (Slow, van Raamsdonk et al. 2003; Van Raamsdonk, Pearson et al. 2005). Huntingtin neuronal intranuclear inclusions are also detected in YAC 128 mice after the onset of behavioral and neuropathological changes (Slow, van Raamsdonk et al. 2003). Accelerated death is only observed in a subgroup of the most extreme YAC128 model, depending on the level of expression of human mutant htt (Van Raamsdonk, Murphy et al. 2005). 6 Many features make Y A C transgenic mice a favorable model for studying mechanisms of pathogenesis of HD. Some of these features include: expression of human huntingtin in a manner similar to endogenous htt in H D patients, due to inclusion of the 5' and 3' regulatory regions of the H D gene; behavioral and neuropathological changes that are similar to those found in human H D ; and a more severe phenotype that can be more easily measured than the knock-in mouse models (Hodgson, Agopyan et al. 1999; Hickey and Chesselet 2003; Slow, van Raamsdonk et al. 2003). 1.1.5.2 R6/2 and R6/1 mouse model The R6/1 and R6/2 lines of transgenic mice are the first mouse HD models, which express exon 1 of human H D gene with -115 or -155 C A G repeats controlled by 1 kB of the human H D promoter region. These mice show certain motor defects typical of H D earlier than found in the Y A C models and death at the age of 4-5 months, but no specific neuronal loss (Davies, Turmaine et al. 1997). The accelerated phenotype is more useful for therapeutic trials in the symptomatic stages of disease. 1.1.5.3 Knock-in mouse models The H D knock-in mouse models include the CAG94 and C A G 140 mouse, as well as the HdhQ92 and Q l 11, in which the human exon 1 with C A G repeats are inserted into the mouse H D gene. These mice exhibit some of the typical early neuropathological changes found in human H D brains, such as neuritic aggregates in striatal MSNs. But the behavioural phenotype of these mice is very mild, and these mice generally lack neuronal degeneration (Albin, Young et al. 1990; Albin and Greenamyre 1992; L i , L i et al. 2001; Menalled, Sison et al. 2002; Menalled, Sison et al. 2003 2006). 7 1.2 Excitotoxic hypothesis of Huntington's disease Based on experiments from the 1950s, excitotoxicity is defined as overactivation of receptors by excitatory amino acid (EAA) neurotransmitters, which induces neuronal death (Olney, Ho et al. 1971; Choi 1992; Doble 1999). Glutamate is the major excitatory neurotransmitter in the mammalian CNS (Hebb 1970; Engelsen 1986), and disrupted glutamate homeostasis has been seen in many neurological disorders, thereby making the glutamatergic system of great interest for neuronal excitotoxicity. The "excitotoxic hypothesis" of HD usually refers to the dysfunction and death of MSNs in the striatum caused by excessive glutamate from cortical afferents, impaired uptake of glutamate, and/or hyper-sensitivity of glutamate receptors, resulting in excessive influx of ions (especially calcium) into the cells (Schwarcz, Bennett et al. 1977; DiFiglia 1990). The excessive calcium entry is particularly neurotoxic, leading to activation of degradative enzymes like caspases and calpains, and production of reactive oxygen species (Albin and Greenamyre 1992; Coyle and Puttfarcken 1993; Berliocchi, Bano et al. 2005). 1.2.1 Glutamate and Glutamate receptors Glutamate is widely expressed in the adult CNS as it mediates most fast excitatory neurotransmission in the brain (Bergles, Diamond et al. 1999). During stress or disease, glutamate can reach very high concentration (100-300 uM) in the synaptic cleft due to excessive glutamate release or impaired glutamate uptake ability of astrocytes (Schubert and Piasecki 2001) There are two categories of glutamate receptors: ionotropic glutamate receptors 8 (iGluRs), which allow cationic ions to enter the cell upon ligand binding (Dingledine, Borges et al. 1999), and metabotropic glutamate receptors (mGluRs), which are linked to many downstream effectors through G-proteins (Conn and Pin 1997). Activation of iGluRs leads to membrane depolarization and increases the probability of action potential firing; both types of GluRs are involved in synaptic plasticity in the adult brain, e.g. long-term potentiation (LTP) and long-term depression (LTD) (Asztely and Gustafsson 1996; Bortolotto, Fitzjohn et al. 1999; Gubellini, Pisani et al. 2004). 1.2.2 Glutamate and Huntington's disease MSNs are central to the function of the striatum because they provide the major output from the caudate nucleus and putamen, and receive a diverse input from the cerebral cortex, thalamus, and dopaminergic neurons of the substantia nigra (Braak and Braak 1982). The cortex and thalamus send glutamatergic outputs to the striatum (DiFiglia 1990; Beal 1992). Results of radiolabeled ligand binding assays in human HD postmortem brain tissue indicated a profound 50-60% loss of glutamate receptors specifically in striatum whereas no loss was observed in cortex (Young, Greenamyre et al. 1988; Albin, Young et al. 1990). Recent studies suggest that quinolinate, kynurenic acid and 3-hydroxykynurenine (3-HK) show increased levels in low-grade human HD and three mouse models of HD (R6/2, YAC128, Hdh-Qll l and Hdh-Q92) brains (Guidetti, Luthi-Carter et al. 2004; Guidetti, Bates et al. 2006). However, the elevated neuroactive kynurenine pathway metabolites cannot fully explain the mutant htt-mediated cellular changes, and may itself be a byproduct of the early pathological changes in HD. 9 1.2.3 NMDA Receptors and Huntington's Disease N-methyl-D-aspartate receptors (NMDAR) are glutamate receptors localized mainly to postsynaptic membranes, and are key mediators of excitatory neurotransmission in the brain (Dingledine, Borges et al. 1999). They are tetrameric transmembrane protein complexes mainly containing two NR1 subunits (with eight splice variants: NR1A-H) and two NR2 subunits (four subtypes: NR2A-D) (Kutsuwada, Kashiwabuchi et al. 1992; Monyer, Sprengel et al. 1992; Sugihara, Moriyoshi et al. 1992; Hollmann, Boulter et al. 1993; Dingledine, Borges et al. 1999). Different NR2 subunits confer different pharmacological and physiological properties and functions on NMDARs (Chazot, Cik et al. 1992; Buller, Larson et al. 1994; Wyllie, Behe et al. 1998; Chen, Luo et al. 1999; Chen, Luo et al. 1999; Dingledine, Borges et al. 1999). The NMDARs have received much attention because of their high permeability to calcium ions, voltage-dependent block by extracellular magnesium and slow activation and deactivation kinetics (Mayer, Westbrook et al. 1984; Nowak, Bregestovski et al. 1984; Cull-Candy, Brickley et al. 2001). Normal calcium entry through NMDARs has important physiologic functions, as it is required for synapse formation during development and also for synaptic plasticity, which is required for learning and memory (Maren and Baudry 1995; Asztely and Gustafsson 1996). However, overactivation of NMDARs has been proposed to be involved in neuronal dysfunction and excitotoxic neuronal death in HD. Firstly, quinolinic acid injection into the striatum of the rat or non-human primate replicates some of the features of HD (Beat, Kowall et al. 1986; Sanberg, Calderon et al. 1989). Secondly, postmortem analysis of brain tissue from patients with HD suggests that neurons with high expression of NMDARs are particularly vulnerable to degeneration (Young, Greenamyre et al. 1988; Albin, Young et al. 1990). As well, NMDARs are more effective and selective than 10 other glutamate receptor subclasses in mediating excitotoxic damage of striatal medium-sized spiny neurons (MSNs) (DiFiglia 1990). Furthermore, data from our laboratory and another group suggested that NMDAR activation may be involved in degeneration of MSNs in HD (Chen, Luo et al. 1999; Levine, Klapstein et ai. 1999; Zeron, Chen et al. 2001; Zeron, Hansson et al. 2002). Moreover, the report of increased NMDAR currents, caspase-3 activity and excitotoxicity in striatal neurons of YAC72 mice compared to WT controls provides additional evidence to show that mutant huntingtin can affect the function and signaling of NMDARs (Zeron, Hansson et al. 2002). More interestingly, we found a -50% increase of NR1 and a -30% increase of NR2B surface expression in cultured striatal MSNs from YAC72 mice compared to WT (Fan, Fernandes et al. 2007), while overall NR1/NR2 expression is similar in striatal tissues from both genotypes (Li, Fan et al. 2003). While these findings might explain the increased NMDAR-mediated current in MSNs from YAC mice expressing expanded polyQs (Zeron, Hansson et al. 2002), how mutant htt causes more NMDARs to be trafficked to or remain at the cell surface is unknown. Increased surface expression of NMDARs could theoretically result from a number of possible changes including: differential splicing of NR1; altered phosphorylation of NMDARs, including increased tyrosine 1472 (Y1472) phosphorylation of NR2B, decreased serine 1480 (SI480) phosphorylation of NR2B, and/or increased serine phosphorylation of NR1; and increased interaction with anchoring proteins like the PSD-95 family. 1.2.3.1 NR2B-type NMDA Receptors NR2 subunits are differentially distributed in the brain, and NR2B is more enriched in mature striatal MSNs compared with other NR2 subunits and mature neurons in other brain regions (Landwehrmeyer, Standaert et al. 1995; Christie, Jane et al. 2000; Li , Fan et al. 2003). In addition, NR2B-containing NMDARs predominate at extra-synaptic sites on the plasma membrane, whereas NR2A-containing NMDARs are enriched at synapses (Li, Wang et al. 1998; Stocca and Vicini 1998; Tovar and Westbrook 1999; Barria and Malinow 2002). A recent study in whole genome expression revealed that the location of the activated NMDA receptor (calcium signal initiation sites) decides the neuronal fate at transcription levels: synaptic NMDA receptors induce up-regulation of pro-survival genes and down-regulation of pro-death genes, while extrasynaptic NMDA receptors work in the opposite way (Zhang, Steijaert et al. 2007). ' Studies in HEK293 cells from our laboratory have shown that when mutant huntingtin and NMDARs are expressed, NMDAR-mediated current and apoptosis is increased, and the extent of cell death is larger for cells expressing NR1/NR2B compared to cells expressing the NR1/NR2A subtype (Chen, Luo et al. 1999; Zeron, Chen et al. 2001). These results suggest a selective interaction of huntingtin with the NR1/NR2B NMDAR subtype. In MSNs from YAC72 mice, excitotoxic death was increased after intrastriatal injection of quinolinate in vivo, and after NMDA but not AMPA exposure in cultured MSNs, and the NMDA-induced cell death was abolished by an NR2B subtype-selective antagonist (Zeron, Hansson et al. 2002). In contrast, NMDAR-mediated death of cerebellar granule neurons was not enhanced by mutant htt expression, which is consistent with cell-type and NMDAR subtype specificity (cerebellar granule neurons do not express NR2B). 12 1.2.4 AMPA and Kainate Receptors and Huntington's Disease The a -amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) and kainate receptors (KARs) are two subtypes of ionotropic GluRs that mediate fast excitatory neurotransmission at synapses. The majority of these receptors are selectively permeable to monovalent cations, except for a minor subset which show moderate calcium permeability as well. The glutamate-evoked currents of these receptors deactivate and desensitize rapidly (Dingledine, Borges et al. 1999). The GluRl subtype is predominantly expressed in the striosome and in aspiny interneurons (Vonsattel and DiFiglia 1998; Nansen, Jokel et al. 2000), while GluR2/GluR3 subunits, which form non-calcium permeable channels, are expressed at high levels in MSNs but are absent in interneurons (Martin, Blackstone et al. 1993; Tallaksen-Greene and Albin 1996; Bernard, Somogyi et al. 1997; Kim, Chung et al. 2001). Administration of AMPAR and KAR agonists directly into the striatum results in excitotoxic lesions and a phenotype resembling HD, but not as closely as phenotypes induced by NMDAR agonists (Coyie and Schwarcz 1976; McGeer and McGeer 1976; Coyle, Ferkany et al. 1983). 1.2.5 Metatropic Glutamate Receptors and Huntington's Disease MSNs mainly express mGIuR5 and mGluR3 receptors (Vonsattel and DiFiglia 1998), and there is evidence showing that NMDARs and mGluR5 (or mGluRl) are linked via PKC activation only in MSNs but not in striatal interneurons (Calabresi, Centonze et al. 1999). The role of mGluRs in HD pathology will not be discussed in this study and remains to be explored. 1.2.6 Calcium Homeostasis and Huntington's Disease Calcium is perhaps the most studied physiological ion because of its vital intracellular j messenger role, regulating cellular functions like exocytosis including transmitter release, membrane excitability and synaptic plasticity, and even cell growth and differentiation (Clapham 1995; Berridge 1998). Efficient calcium signaling relies on cells maintaining basal intracellular calcium concentration about 20,000 times lower than that of the extracellular fluid (Carafoli 1987; Clapham 1995). Calcium homeostasis is normally adjusted by calcium influx processes (through voltage-gated calcium channels, NMDARs or AMPARs), calcium efflux processes (through the calcium/ATPase pump or sodium/calcium antiporter), calcium storage organelles (mitochondria, endoplasmic reticulum and nucleus), and calcium binding proteins (like calbindin and calmodulin) (Sattler and Tymianski 2000). Increased intracellular calcium has been found to play a role in both necrosis and apoptosis (Yu, Canzoniero et al. 2001). Calcium influx through NMDARs after intensive glutamate stimulation is thought to be uniquely toxic and cause neuronal degeneration (Choi, Peters et al. 1987), due to the receptor's subcellular localization (Tymianski, Charlton et al. 1993; Hardingham, Fukunaga et al. 2002), especially the extrasynaptic NR2B-containing receptors, or the receptor's high permeability to calcium (Hartley, Kurth et al. 1993). It has been shown that increased intracellular calcium is an important trigger for caspase activation and mitochondrial dysfunction in striatal cultures (Petersen, Castilho et al. 2000). Calcium-induced excitotoxicity may be mediated by many calcium-dependent enzymes, such as calpains and nitric oxide synthase (NOS) (Siman, Noszek et al. 1989; Dawson, Dawson et al. 1991), or other enzymes involved in cell death, such as 14 proteases and endonucleases (Orrenius, Ankarcrona et al. 1996; Leist and Nicotera 1998). Another excitotoxic-inducing pathway is triggered by calcium overload in mitochondria, resulting in energetic failure and production of reactive oxygen species, as well as mitochondrial membrane depolarization with more calcium released through the mitochondria permeability transition pore (mPTP) (Nicholls and Budd 2000). Calcium homeostasis has been found to be compromised in many HD models. Immortalized striatal cells from STHdhQl l l /Ql l l knock-in mice (Seong, Ivanova et al. 2005), striatal neurons from R6/2 mice (Hansson, Guatteo et al. 2001), and hippocampal neurons from symptomatic YAC72 mice (Hodgson, Agopyan et al. 1999), all showed elevated basal calcium concentration. Altered NMDAR-mediated calcium signaling was observed in striatal cells cultured from YAC transgenic mouse expressing expanded htt at birth (Zeron, Fernandes et al. 2004; Oliveira, Chen et al. 2006). Striatal slices from pre-symptomatic and symptomatic R6/2 mice (Cepeda, Ariano et al. 2001), as well as cultured striatal MSNs from YAC46 and YAC72 mice (Zeron, Fernandes et al. 2004) all displayed larger increases in intracellular calcium levels in response to NMDA. Similarly, cytosolic calcium levels in cultured MSNs from YAC 128 mice were enhanced after repeated glutamate application (Tang, Slow et al. 2005). Moreover, IP3 receptor (IP3R)-mediated calcium release from ER stores was potentiated by polyQ-expanded htt in cultured MSNs from both rat and mouse (Tang, Tu et al. 2003; Tang, Tu et al. 2004). Taken together, all these studies suggest that dysregulation of calcium homeostasis may be caused by mhtt, and it plays a key role in the dysfunction and neuronal loss of striatal neurons in HD. 1.2.7 Apoptosis and Huntington's Disease Apoptosis, also called "programmed cell death" or "cell suicide", is different from necrosis in many ways, including the absence of toxic factor release into the extracellular space during cell death, and morphological changes such as cell shrinkage, condensed nuclear chromatin, and break down of nucleus and cytoplasm into "apoptotic bodies" (Kanduc, Mittelman et al. 2002; Burlacu 2003; Derradji and Baatout 2003). In addition, apoptosis takes place in individual cells and leaves surrounding healthy cells unaffected, whereas necrosis induced by injury and trauma involves neighboring cells by releasing inflammatory factors (Sastry and Rao 2000; Kanduc, Mittelman et al. 2002). Excitotoxic stimuli, environmental toxins, and metabolic or oxidative stresses, could initiate apoptosis by activating calpains, initiator caspases (e.g. caspases-8 or -9) or the effector caspases (e.g. caspases-3) (Thornberry 1997; Chan and Mattson 1999; Sastry and Rao 2000). Increased apoptosis has been reported in several HD models expressing expanded htt. We have shown before that NMDA-induced caspases-3 and -9 activities are elevated in striatal MSNs from YAC46 and YAC72 mice compared to WT mice (Zeron, Hansson et al. 2002; Zeron, Fernandes et al. 2004). There is also evidence showing increased calpain (Gafni and Ellerby 2002), caspase-1 (Ona, L i et al. 1999) and caspase-8 (Sanchez, Xu et al. 1999) activity in the striatum of HD patients. Furthermore, the increased apoptosis in HD has been linked to NMDARs. In one study, the glutamate-induced apoptosis seen in the hippocampal HN33 cell line expressing htt-48Q, but not htt-16Q, could be prevented by AP-5 treatment, which is an NMDAR antagonist (Sun, Savanenin et al. 2001). The enhancement of excitotoxicity by polyQ expansion in mhtt seems to be NMDAR-specific, since 16 AMPA-induced apoptosis in MSNs is unaffected by genotype (Zeron, Hansson et al. 2002). Data from our lab has also shown that NMDA-induced apoptosis was increased only in cells that co-expressed htt-138Q with NR1/NR2B but not NR1/NR2A (Zeron, Chen et al. 2001). Further experiments indicated that enhancement of NMDA-induced apoptosis in cultured MSNs showed YAC72 > YAC46 > WT > YAC 18, i.e., the NMDA-induced apoptosis is proportional to the polyQ length of mutant htt (Zeron, Hansson et al. 2002; Shehadeh, Fernandes et al. 2006). Treatment with the NMDAR antagonist AP-5 at concentrations shown to decrease NMDA-induced calcium influx in YAC72 MSNs to WT levels resulted in a proportional reduction of NMDA-induced apoptosis to WT levels (Shehadeh, Fernandes et al. 2006). The toxic effect of increased calcium from both extracellular space (application of KCI and an L-type voltage-gated calcium channel agonist) and intracellular stores (caffeine and DHPG treatments) were also explored. The potentiation of apoptosis induced by these non-NMDAR dependent stimuli in YAC72 and YAC128 MSNs relative to YAC 18 MSNs was much smaller than that observed with NMDA (Shehadeh, Fernandes et al. 2006). Additionally, high concentrations of staurosporine induced similar apoptosis in cultured MSNs from YAC72 and WT mice, and there was only a small potentiation in YAC128 MSNs compared to WT (Shehadeh, Fernandes et al. 2006). Staurosporine induces apoptosis by inhibiting protein kinase C and/or activating the mPT via suppression of Bcl-2 expression, resulting in caspase-3 activation (Nicotera, Leist et al. 1999; Roucou, Antonsson et al. 2001; Swannie and Kaye 2002). These results suggest that cell death, and especially NMDAR-dependent apoptosis, is augmented in mhtt-expressing neurons. As well, mitochondria are more vulnerable to calcium influx through NMDARs than through other ways of increasing cytosolic calcium, which may be due to microstructure or different pathways (Peng and Greenamyre 1998; 17 Sattler and Tymianski 2000). 1.3 M A G U K proteins The membrane-associated guanylate kinases (PSD-93/Chapsyn-110, PSD-95/synapse-associated protein (SAP)-90, SAP-97 and SAP102) share sequence homology in their three PSD/Discs-large/ZO-1 (PDZ), one Src homology 3 (SH3) and one guanylate kinase-like domains. They are believed to cluster and anchor glutamate receptors and K+ channels at the synapse, as well as facilitate efficient signaling by keeping key enzymes in close proximity, which is why they are referred to as scaffolding proteins (Fujita and Kurachi 2000). PSD-95, SAP102, and PSD-93/Chapsynll0 are abundant in the CNS and mainly distributed to the postsynaptic density (PSD), while SAP102 and SAP-97 could localize in the presynaptic termini of excitatory synapses or along unmyelinated axons, and SAP-97 is also expressed in epithelial cells (Muller, Kistner et al. 1995; Fujita and Kurachi 2000). A study of MAGUK expression in rat hippocampus showed that SAP102 was highly expressed at P2 and dominated at the synapse in the early postnatal period when PSD-93 and PSD-95 were expressed at low levels, and later PSD-93 and PSD-95 expression increased around P10 and largely replaced SAP 102 at synapses (Sans, Petralia et al. 2000). Evidence suggests that the N-terminal domain of PSD-95 regulates postsynaptic protein clustering (Chetkovich, Bunn et al. 2002), and that different splice variants within the N-terminal domains of PSD-95 and SAP-97 govern their roles in activity-dependent regulation of synaptic AMPA receptor function (Schluter, Xu et al. 2006). The palmitoylation of two N-terminal cysteines (Cys3 and Cys5) is required for 18 the synaptic accumulation of PSD-95 (Craven, EI-Husseini et al. 1999). PSD-93 is also palmitoylated, while SAP102 and SAP-97 are not (El-Husseini, Topinka et al. 2000), but the latter two are still found at synapses. Another study found that the postsynaptic targeting of PSD-95 is maintained independently of palmitoylation when the N-terminus of PSD-95 is replaced by the N-terminus of PSD-93/Chapsyn-110 or SAP102 (Firestein, Craven et al. 2000) which suggests that MAGUKs contain diverse signals within their N-termini for postsynaptic targeting. SAP-97 binds directly with the GluRl subunit of AMPAR, while all the MAGUK proteins interact with the NR2 subunitsof NMDARs (Kornau, Schenker et al. 1995; Kim, Cho et al. 1996; Niethammer, Kim et al. 1996; Leonard, Davare et al. 1998; Kim and Sheng 2004). It is suggested that PSD-95 and PSD-93 are more involved in synaptic functions, whereas SAP-97 and SAP102 are more important in trafficking (Kim and Sheng 2004). It has been previously reported that chapsyn-110 and PSD-95 can heteromultimerize with each other and are recruited into the same NMDA receptor and K+ channel clusters (Kim, Cho et al. 1996). Receptor clustering depends on N-terminal domain-mediated multimerization and disulfide linkage of PSD-95 monomers, and PSD-95 has been found to exist with chapsyn-110 as disulfide-linked complexes in rat brain (Hsueh, Kim et al. 1997). In addition, a recent study demonstrated a direct interaction between SAP-97 and PSD-95 and suggested that SAP-97 and PSD-95 heteromeric complexes may play a functional role in the trafficking and clustering of AMPA receptors (Cai, L i et al. 2006). No evidence of heteromultimerized SAP102 has been reported yet 19 Figure 1. Interaction and organization of PDZ domain-containing proteins at a mammalian excitatory synapse. Nature Reviews | N e u r o s c i e n c © Nature Reviews - Neurosciences, 2004, by permission (Kim and Sheng, 2004, review) 20 1.3.1. PSD-95 PSD-95 has been reported to interact with a wide variety of membrane protein and cytoplasmic signalling molecules (Fig. 1). The PDZ1-2 domain in PSD-95 has been shown to bind to the tSXV motif in the COOH-terminal domain of NR2 subunits of NMDAR (Kornau, Schenker et al. 1995; Niethammer, Kim et al. 1996). Furthermore, c PSD-95 has been shown to play an important role in stabilizing NMDARs at the cell surface (Roche, Standley et al. 2001). PSD-95 can couple NMDARs to Src-family kinases and thereby promote phosphorylation of NR2B at the tyrosine-1472 site near the C-terminus, which increases localization of NR2B to the synaptic membrane and inhibits its endocytosis (Prybylowski, Chang et al. 2005). Co-expression of mhtt, PSD-95, NR1 and NR2B in 293T cells has been shown to increase Src-family kinase-dependent tyrosine phosphorylation of NR2B, and NMDAR-mediated toxicity (Song, Zhang et al. 2003). Increased NR2B binding to PSD-95 can reduce the levels of SI 480 phosphorylation of NR2B, which also promotes stabilization of NMDAR at the surface (Chung, Huang et al. 2004). The neuronal nitric oxide synthase (nNOS), which is a Ca2+/calmodulin-activated enzyme that produces nitric oxide (NO), binds directly to the PDZ-2 domain of PSD-95 (Brenman, Chao et al. 1996). The ternary NMDAR-PSD-95-nNOS complex could functionally couple NMDAR-dependent calcium entry to nNOS activation and production of NO, which mediates NMDAR-dependent excitotoxicity (Dawson, Dawson et al. 1991). Aarts et al., postulated that this NMDAR-PSD-95-nNOS complex is critical for mediating excitotoxicity, since disruption of the N M D A R -PSD-95 interaction with synthetic peptides resulted in reduced NMDAR-mediated excitotoxicity without affecting NMDAR function (Aarts, Liu et al. 2002). 21 It is known that PSD-95 interacts with the membrane protein stargazin, which binds directly to AMPAR subunits and is essential for the surface expression and synaptic accumulation of AMPARs (Chen, Chetkovich et al. 2000). There is also evidence that AMPAR activity depends on an interaction of the C-terminus of stargazin with the PDZ domains of PSD-95 (Schnell, Sizemore et al. 2002). An abundant PSD protein that binds to PSD-95 is synaptic RAS GTPase-activating protein (SynGAP) (Chen, Rojas-Soto et al. 1998; Kim, Liao et al. 1998), which is activated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Oh, Manzerra et al. 2004) and suppresses the Ras-extracellular signal-regulated kinase (ERK) pathway, thereby regulating synaptic plasticity (Zhu, Qin et al. 2002). PSD-95 also interacts with guanylate kinase-associated protein (GKAP), SH3 and ankyrin repeat-containing protein (Shank/ProSAP), as well as many other cytoskeletal-associated proteins, and is thus linked to other scaffolds in the PSD (Naisbitt, Valtschanoff et al. 2000). 1.3.2 SAP102 SAP102 is enriched in the postsynaptic density and has also been reported to interact with the carboxy-terminus of the NR2B subunit of NMDA receptors in vivo (Muller, Kistner et al. 1996). Studies suggest that during development, the expression of NR2B coincides with the level of SAP102 at hippocampal synapses, and there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP102 (Sans, Petralia et al. 2000). It is suggested that NR2B-SAP102 complexes may inhibit the ERK/MAPK pathway, through synaptic Ras-GTPase-activating protein, to facilitate loss of AMPARs from the postsynaptic membrane, whereas NR2A-PSD-95 complexes work in the opposite way (Kim, Dunah et al. 2005). 22 Recently, it was found that SAP 102 is upregulated in adult PSD-95 and PSD-93 double knockout mice, and compensates for the loss of these proteins in maintenance of synaptic AMPA receptors (Elias, Funke et al. 2006). SAP102 dominates in synaptic AMPAR clustering at immature synapses, while PSD-95 and PSD-93 play only minor roles at this stage (Elias, Funke et al. 2006). Another recent study showed that SAP 102 interacts with Sec8 subunit of the exocyst complex, which targets secretory vesicles to the cell surface, and that dominant-negative Sec8 inhibits NMDAR currents in neurons (Sans, Prybylowski et al. 2003). This result indicates that the synaptic trafficking of NMDARs is regulated by interaction of SAP102 with Sec8. Additionally, the interaction of SAP102 with the protein mPins (which mediates G-protein signaling) was shown to influence the trafficking of NMDA receptors between the ER and the trans-Golgi network (Sans, Wang etal. 2005). It has been reported that humans with loss-of-function mutations in the SAP 102 gene (dlg3/dlgh3/NE-dlg) show non-syndromic (i.e., cognitive impairment is the sole definable clinical feature) X-linked mental retardation (Tarpey, Parnau et al. 2004). A more recent study has shown that SAP 102 knock-out mice exhibit specific impairments in synaptic plasticity induced by selective frequencies of stimulation and accompanied by impairment in spatial learning (Cuthbert, Stanford et al. 2007). Futhermore, this study also reported a robust elevation of SAP 102 in PSD-95 mutant mice, and that more PSD-95 is associated with NR1 in SAP102 mutant mice. The fact that mice carrying a double mutation of PSD-95. and SAP102 die perinatally indicates partial overlapping functions of these two MAGUKs and that the presence of at least 23 one is required for postnatal development. They concluded that specific MAGUK proteins may couple the NMDA receptor to distinct downstream signaling pathways (Cuthbert, Stanford et al. 2007). 1.3.3 SAP-97 and PSD-93/Chapsyn-110 SAP-97 and PSD-93/Chapsynll0 have been reported to interact with the C-terminus of NMDAR NR2 subunits and play a role in NMDAR clustering (Kim, Cho et al. 1996; Niethammer, Kim et al. 1996). Furthermore, CaMKII-dependent phosphorylation of the SAP-97 N-terminus at different developmental stages and locations controls the trafficking and postsynaptic membrane insertion of the NR2A subunit of NMDA receptors (Mauceri, Gardoni et al. 2007). SAP-97 is also associated with the C-terminus of the GluRl subunit of AMPA receptors (Leonard, Davare et al. 1998). A recent paper suggests that SAP-97 may play a central role in the growth of synapses during development and plasticity by recruiting postsynaptic proteins that enhance presynaptic terminal growth and function via multiple trans-synaptic molecular interactions (Regalado, Terry-Lorenzo et al. 2006). 1.3.4 PSD-95 and Huntington's Disease Evidence indicates that huntingtin is associated with PSD-95 in transfected 293T cells and human cortical tissue by co-IP experiments. Furthermore, Glutathione-S-transferase (GST)-based pull-down experiments show that the SH3 domain of PSD-95 mediates its binding to htt (Sun, Savanenin et al. 2001). These authors also reported decreased binding of PSD-95 to a GST-fusion protein expressing N-terminal htt with 56Q (GST-Nhtt-56Q) relative to GST-Nhtt-16Q (Sun, Savanenin et al. 2001). Furthermore, NMDAR-dependent apoptosis induced by over-expression 24 of mhtt with 48Q in HN33 hippocampal cell line could be reduced by over-expressing htt-16Q, and this rescue was abolished by co-expression with PSD-95 (Sun, Savanenin et al. 2001). Sun and colleagues thus concluded that htt and NMDA receptors are associated via PSD-95, and polyglutamine expansion interferes with the ability of htt to interact with PSD-95 (Sun, Savanenin et al. 2001). Moreover, they hypothesized that diminished binding of polyQ-expanded htt to PSD-95 might increase the free PSD-95 available for binding to NMDARs and thereby augment NMDAR-dependent toxicity (Sun, Savanenin et al. 2001), since previous studies have suggested that coupling of NMDARs with PSD-95 leads to, activation of neuronal nitric oxide synthase (nNOS) and is required for NMDA-induced toxicity (Sattler, Xiong etal. 1999). 1.3.5 Tat-NR2B9c peptide The Tat-NR2B9c is a 20 amino acid fused protein constructed with the nine C-terminal residues of NR2B (where the PSD-95 PDZ1&2 domains bind) and the cell-membrane transduction domain of the HIV-1 Tat protein. This peptide has been shown to perturb the interaction of NR2B and PSD-95, thus attenuating NMDA toxicity and even reducing stroke damage in a rat model, while the control peptide Tat-NR2BAA (two serine residues in Tat-NR2B9c peptide have been substituted with alanines to eliminate the possibility of PDZ domain binding) does not (Aarts, Liu et al. 2002). Given the similarity of PDZ1 and PDZ2 domains of SAP102 and PSD-95, and that Tat-NR2B9c peptide disturbs the NR2B interaction with SAP102 and PSD-95 more than with the other MAGUK members (Cui et al. Society for Neuroscience abstract 679.9, from 36th Annual Meeting), Tat-NR2B9c peptide will help us to explore the roles of PSD-95 and SAP102 in NMDAR functional changes in the YAC 25 transgenic mouse model. 1.4 Hypothesis As much evidence suggests that NMDAR (especially NR2B-type)-induced excitotoxicity plays a role in the pathogenesis of HD, the emerging question is what molecules link NMDARs and huntingtin together in a htt polyQ length-dependent manner to alter NMDAR function and eventually lead to enhanced neuronal death? Given that the M A G U K family proteins play crucial roles in NMDAR trafficking and anchoring, the report of association of PSD-95 and htt, as well as recent studies on SAP102 specific function, we hypothesize that SAP102 together with PSD-95 play important role in mutant htt-mediated regulation of NMDAR function in HD pathogenesis. 26 CHAPTER 2 Materials and Methods 2.1 Material 2.1.1. Plasmids As gifts from S. Nakanishi, Kyoto University, Kyoto, Japan, N R l - l a (NR1A) and NR2B cDNAs were from rat brain and previously subcloned into the mammalian expression vector pRK5 with the cytomegalovirus promotor (Raymond, Moshaver et al. 1996; Chen, Moshaver et al. 1997). The cDNAs of full-length htt with 15 or 138 glutamine repeats (htt-15Q or htt-138Q) were subcloned into a vector with the cytomegalovirus promoter, as described previously (Goldberg, Nicholson et al. 1996). The myc-tagged SAP 102 in pCMVneo construct was a gift from Dr. R. Huganir, Johns Hopkins University, Baltimore, U.S.A. (Muller, Kistner et al. 1996). 2.1.2 Antibodies The immunocytochemistry was done with a mouse monoclonal antibody against NR1A (2ug/ml, Chemicon, #MAB363), rabbit polyclonal anti-GluRl antibody (lug/ml, Upstate, #06-306), anti-mouse Alexa Fluor 488 (Molecular Probes, 1:2000, A-11029), anti-rabbit Alexa Fluor 488 (Molecular Probes, 1:2000, A - l 1008), anti-rabbit Alexa Fluor 594 (Molecular Probes, 1:2000, A - l 1012), anti-mouse Cy3 (Jackson Immuno Research, 1:200, 715-165-150). The western blot was done with rabbit polyclonal anti-NR2B (lug/ml, Upstate, #06-600), mouse monoclonal anti-NR2B (1:250, Transduction Lab, #610417), mouse monoclonal anti-NR2B (lug/ml, Affinity BioReagents, #MA1-2014), anti-huntingtin mouse monoclonal antibody (lug/ml, Chemicon, #MAB2166), anti-huntingtin human-specific mouse monoclonal antibody (HD650, used at 1:500) from Dr. Michael Hayden's lab (CMMT, Vancouver, Canada), anti-SAP102 mouse monoclonal antibody (1 ug/ml, Stressgene, 27 #VAM PS006), anti-SAP102 rabbit polyclonal antibody (1:400, Chemicon, #AB5170), anti-SAP102 mouse monoclonal antibody (1 ug/ml, NeuroMAB, #75-058), anti-SAP 102 rabbit polyclonal antibody (used at 8 ug/ml) from Dr. Robert Wenthold's lab (NIH, MD, U.S.A.), or anti-myc MAB-4A6 (1 ug/ml, Upstate#05-724). Horseradish Peroxidase-linked secondary antibodies for western blotting are anti-mouse IgG antibody (NA931V) and anti-rabbit IgG antibody (NA934V), both from Amersham, and used at 1:5000. 2.1.3 Peptides The Tat-NR2B9c (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser -Ile-Glu-Ser-Asp-Val) and Tat-NR2BAA (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ala-Asp-Ala) peptides are gifts from Dr. Michael Tymianski's lab (Toronto Western Hospital Research Institute, Toronto, Canada). The peptides were dissolved in sterile Milli-Q water to make ImM stock solutions and stored at -20°C. All chemicals, unless specified, were purchased from Sigma-Aldrich Company (St. Louis, MO, U.S.A.). 2.2 HEK293T cell line and transfection HEK293T cells were obtained from Dr. Weihong Song's lab (University of British Columbia, Vancouver, Canada). Cells were cultured in minimum essential medium (MEM) (Gibco # 11700) containing 10% fetal bovine serum (FBS, qualified) from Gibco (#12483-020), lOOU/ml penicillin/streptomycin, 2 mM a -glutamine and 1 mM sodium pyruvate (MEM+), and maintained in 37°C, 5% CO2 incubator with 28 proper humidity. When cell density reached ~ 90%, and 2-3 days after last splitting, cells were propagated by washing with warm phosphate-buffered saline (PBS) and then digesting with 1ml 1 x 0.25% trypsin-EDTA (Gibco#25200) for approximately lmin. Cells then were suspended in 9 ml of fresh media and re-plated at a dilution of 1:5 or 1:10 in fresh medium. To prepare cells for transfection, the new plates were pre-coated with Poly-D-Lysine (Mol.Wt.30,000-70,000, Sigma#P-7280) for lhr and washed with sterile Milli-Q water. Cells were grown for 12-16hrs, and reached 50%-70% density before transfection. Cells were transfected using the calcium-phosphate precipitation method (Chen and Okayama 1987). Plasmid cDNAs encoding NR1A, NR2B, SAP102, htt-15Q, htt-138Q, and/or green fluorescent protein (GFP) were mixed at a ratio of NR1A: NR2B: htt-15Q/htt-138Q: SAP102: GFP = 1:1:2:2:1, and total cDNA was 10ug-'l2pg /10cm dish. The cDNA mixture was mixed with 1:10 v/v 3M sodium acetate followed by addition of 3 times the volume of 100% ethanol and then precipitated by brief centrifugation. The rest of the tube was then filled with 100% ethanol. After centrifuging at 14000 rpm for 8min, the ethanol was decanted and the DNA pellet was air-dried and then dissolved in 500uL of 250mM CaCb; 500uL of 2 x BES solution (50 mM BES, 280 mM NaCl, 1.5 mM Na 2HP0 4 , pH 6.98) was then added and the mixture allowed to sit for 20 min while cells for transfection were pre-incubated at 37 °C, 3% CO2. The DNA mixture was then added into the medium of the cell culture and mixed by mild swirling. Cells were transfected for 6-8 hrs in 37°C, 3% CO2 incubator and then washed once with warm PBS and incubated in fresh MEM+ medium with lOOuM memantine for 29-30 hrs in a 37°C, 5% CO2 incubator. Then cells were taken for assessment of transfection efficiency (GFP expression) by microscopy. If the GFP 29 positive cells were >60% of the whole population, then cells were used for further experiments. 2.3 Transgenic mice Offspring of crosses between two homozygous YAC transgenic mice on a pure FVB/NJ strain background (Hodgson, Agopyan et al. 1999) were used for the experiments. YAC 18 (line 212; similar to line 29) (Leavitt, Guttman et al. 2001), YAC72 (line 2511) (Hodgson, Agopyan et al. 1999) and YAC128 (line55) (Graham, Slow et al. 2006) mice were used as models expressing full-length human htt containing 18, 72, or 128 polyglutamine repeats (18Q, 72Q, 128Q) and compared to FVB/N WT mice. Cultured neurons from YAC 18, YAC72 and YAC 128 mice are called "18Q", "72Q" and "128Q" MSNs, respectively. Al l mice were housed, tested and tissue harvested according to Canadian Council of Animal Care guidelines and animal protocols approved by the University of British Columbia. 2.4 Primary striatal neuronal culture Nitric-acid treated 12mm glass coverslips were placed in 24-well plates, coated with poly-D-Lysine (250 ug/ml) for at least one hour, rinsed once with sterile Milli-Q water and allowed to dry. Striatal tissues were dissected from postnatal day 0-1 (P0-P1) mice in Hank's Balanced Salt Solution (HBSS, Gibco) on ice, chopped, spun down, and then digested in warm papain (lOuL IM CaCl 2, lOuL 500uM EDTA, 0.475mL Papain, 0.002g L-Cystine in lOmL HBSS, adjust pH to 7.35) for ten minutes. Cells were further dissociated in warm trypsin inhibitor solution (0.025g BSA, 0.025g Trypsin inhibitor, lOOuL DNAse in lOmL Neuro Basal Media) by passage through Pasteur pipettes with successively reduced bore size. MSNs were then spun down and 30 transferred to warm serum-free plating medium (1 X B27, 50U/ml penicillin/streptomycin, 2 mM a-glutamine in Neurobasal medium), and plated at a density of approximately 2.0 X 105 cells / well in 0.5mL plating medium, as described (Zeron, Hansson et al. 2002). MSN cultures were then placed in a 37°C, 5% CO2 incubator and used at 9-10 days in vitro (DIV). Approximately 80-90% of the cells cultured in serum-free medium have the characteristics of MSNs (Kovacs, Cebers et al. 2001; Zeron, Fernandes et al. 2004), as evidenced by morphology (e.g. ovoid soma), dopamine- and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) staining (the marker for MSNs), and/or staining for the GABAergic neuronal marker glutamic acid decarboxylase-65 (GAD-65) (Petersen and Brundin 1999; Shehadeh, Fernandes et al. 2006). In experiments, measurements were taken from cells with morphological features consistent with medium-sized spiny neurons, including ovoid soma with diameter of about 10 urn and 2-4 closely projecting processes (Shi and Rayport 1994). 2.5 Brain lysates and solubilization 4 week-old (postnatal day 24-32) YAC or WT mice were anesthetized and sacrificed following the Canadian Council of Animal Care standards with halothane, (UBC animal protocol A03-0089 and A06-0261) and the whole brains were dissociated and placed in ice-cold PBS. Striatal tissues were dissected in ice-cold PBS, and transferred into homogenizing tubes with 700uL - lmL ice-cold Harvest Buffer (HB; ImM EDTA, ImM EGTA, ImM PMSF, 2ug/mL aprotinin, 2ug/mL leupeptin, 4ug/mL pepstatin A, 30mM NaF, 40mM 3 -glycerophosphate, 20mM sodium pyrophosphate, ImM sodium orthovanadate in PBS). Samples were homogenized with up to 8 X up and down strokes at 2000rpm and centrifuged at 7000rpm for lmin at 4°C. Supernatants 31 were discarded and each pair of striatal tissues were resuspended in 350uL ice-cold Lysis Buffer (LB; 50mM Tris-pH8.0, 150mMNaCl, ImM EDTA, ImM EGTA, ImM PMSF, 2pg/mL aprotinin, 2pg/mL leupeptin, 4pg/mL pepstatin A, 30mM NaF, 40mM f3-glycerophosphate, 20mM sodium pyrophosphate, ImM sodium orthovanadate, lOuM ZVAD, 1% NP40, in Milli-Q water). DNAse was added into LB to'O.lmg/mL final concentration and samples were sonicated using a Ultrasonics-model W-10 sonicator at setting 3 for 10 seconds or until no solid tissues could be seen. 2.6 Immunoprecipitation Then 20uL of an equilibrated 50% Protein A/G beads (totally 40uL) slurry was added into the homogenate (lysate) and rotated for lhr at 4°C to pre-clear lysates. Lysates were then centrifuged at 5000rpm for 5min at 4°C, and 40uL of each supernatant was saved as "lysate" (Lys); the remainder was transferred to fresh tubes containing 20pL of an equilibrated 50% Protein A/G beads (totally 40uL) slurry and lOpg anti-huntingtin or anti-NR2B antibodies, and then rotated slowly overnight (-16 hrs) at 4°C. Samples were then centrifuged at 5000rpm for lmin at 4"C, and 40pL of each supernatant was saved as "supernatant" (SPN). Beads of each sample were then washed 4 times with 500uL 1 XTris Wash Buffer (50mM Tris-pH 7.4, 150mM NaCl, ImM EDTA, ImM EGTA, 1% Triton in Milli-Q water), then centrifuged at 5000rpm for lmin at 4°C. Supernatants were discarded and 45uL 3X Protein Sample Buffer (PSB; 0.125 M Tris-pH6.8, 2% SDS, 10% Glycerol, 72 mg/mL DTT, with Pyronin Y in Milli-Q water) was added to the "IP" samples while 20uL PSB was added to the Lys and SPN samples. All the samples were then heated for 5min at 95-99°C then rapidly cooled and stored at -20°C until they were forwarded to SDS-PAGE. 32 2.7 Western blotting Paired samples were run on 8% SDS-PAGE (sodium dodecyl-sulphate polyacrylamide gel electrophoresis) at 80-100mV for 2-3hrs. Proteins were then transferred from gels to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) in transfer buffer (12.5mM TRIS, lOOmM Glycine, 20% methanol in Milli-Q water) for lhr at 80mV and then 30mV overnight at 4°C. Blots were incubated with 3% bovine serum albumin (BSA) in TBS-T (Tris buffered saline with Tween-20; 200mM NaCl, 50mM TRIS, 0.5% Tween-20 in Milli-Q water, adjust pH to 7.4) for lhr at room temperature (RT). The blots were cut as needed and forwarded to different primary antibody probing, usually for 1.5 hr at RT and then overnight at 4°C. Blots were then rinsed 3 times for 7min each with TBS-T and then incubated with the secondary antibody in TBS-T with 1% BSA for 45min to lhr at RT. Blots were rinsed 5 times for 7min each with TBS-T and then incubated with ECL (enhanced chemiluminescence; Amersham) solution (1:1 ratio of reagents 1 and 2 mixture) for 1 min. Blots were then placed in a cassette and covered by film (Kodak) in the dark room for timed exposure. Films were developed for 1 min and fixed for 1 min and then rinsed in water. 2.8 Immunocytochemistry and surface NR1 and GluRl staining Cultured MSNs were treated with 200nM or l^iM Tat-NR2B peptides for 1 hr on DIV 9, washed with PBS once and fixed with 4 % paraformaldehyde (PFA) for 12min. Then coverslips were washed with PBS for 3 times lmin and stored at 4°C before staining. Fixed MSNs were numbered according to different conditions and randomized by lab-mates before staining. To determine the proportion of NMDARs or AMPARs on the cell surface MSNs were 33 blocked with 10% normal goat serum (NGS) in PBS for 30min and incubated with anti-NRl or anti-GluRl primary antibody for 1.5 hr at RT and then overnight at 4°C. MSNs were then washed with PBS for 3 times lmin and incubated with green secondary antibody (anti-mouse or anti-rabbit) for 45 min to 1 hr at RT. MSNs were again washed with PBS for 3 times lmin and permeabilized with 0.5% TritonX-100 in PBS for 5 min. Then MSNs were blocked again with 10% NGS in PBS for 30min and incubated with anti-NRl or anti-GluRl primary antibody for 1.5 hr at RT. MSNs were then washed with PBS for 3 times lmin and incubated with red secondary antibody (anti-mouse or anti-rabbit) for 45 min to 1 hr at RT. MSNs were again washed with PBS for 3 times lmin, mounted on slides with Fluoromount-G (Southern Biotech Associates, Inc.) and dried overnight. Ten to twelve pictures of stained single MSNs per each coverslip were taken using a 63 X lens in the red and green fluorescent channels by Northern Eclipse software, with each condition done in duplicate. Pictures were analyzed by ImageJ by measuring the mean fluorescence intensity of processes and soma of each MSN after background subtraction. The surface/internal ratio of NR1 or GluRl was calculated as the green/red fluorescent channel intensity for each MSN. 2.9 NMDA- and staurosporine-induced cytotoxicity Cultured MSNs were pretreated with 200nM Tat-NR2B peptide added directly to the medium for 1 hr on DIV 9, then the medium was removed, cells were incubated in 500uL of warm balanced salt solution (BSS; 139mM NaCl, 3.5mM KCI, 2mM NaHC03, lOmM HEPES, 3mM Na 2HP0 4 in Milli-Q water, adjust pH to 7.4) with or without 500uM NMDA for 10 min. MSNs were washed twice with 500uL warm plating medium (PM) and then incubated in the conditioned PM (saved from other 34 untreated wells) in a 37°C, 5% CO2 incubator for 24 hrs. For staurosporine-induced cytotoxicity, lOuM staurosporine was added into the medium after peptide pretreatments and incubated for 24 hrs. Then MSNs were washed with PBS once and fixed with 4 % PFA for 15 min at RT and 45 min at 4°C. MSNs were then washed 3 times lmin with PBS and stored at 4°C before staining. 2.10 TUNEL assay and assessment of apoptosis Fixed MSNs were numbered according to different conditions and randomized by lab-mates before staining. MSNs were permeabilized with 500pL ice-cold 0.25% TritonX-100 in PBS with 0.5% sodium citrate on ice for 2 min. MSNs were then washed once with PBS and incubated in 500pL PBS with 16pg/mL RNAse at RT for 30 min. MSNs were washed 3 times lmin with PBS and incubated in 17.5pL TUNEL (Roche) reagent (at a ratio of labebenzyme = 9:1) per well in the dark. Then the whole plate was sealed with parafilm and incubated at 37°C for 1 hr in the dark. MSNs were washed once with PBS and then stained with 500uL of 8uM propidium iodide (PI). MSNs were then washed with PBS for 3 times lmin, mounted on slides with Fluoromount-G and dried overnight. The second day after staining, the percentage of apoptotic cell death was assessed by counting the numbers of TUNEL positive cells (which should also show condensed and blebbed nuclear morphology) in the green fluorescent channel, then dividing by the total number of Pl-positive cell nuclei in the red fluorescent channel and multiplying by 100. Only cells with nuclear morphological features of MSNs were counted. A total of 1000 neurons were counted per condition, usually from two different coverslips. The percentage of apoptotic cell death of MSNs exposed to BSS alone was subtracted as a baseline from each of the other conditions. 3 5 2.11 Data analysis Figures, tables, and statistical analyses were generated using Microsoft Excel, Northern Eclipse, ImagJ, Origin 6.0, Prism 3.0, or Adobe Photoshop 7.0 software. Data or bars are presented as the mean ± SEM. Significant differences were determined using the unpaired or paired, two-tail student's t-test, one-way ANOVA or two-way ANOVA, as appropriate. 36 CHAPTER 3 Results 3.1 The expression level of SAP102 is similar in YAC 18, YAC72, and YAC128 mice striatal tissues First of all, we explored the expression level of SAP 102 in 4 week-old YAC mice striatal tissue as part of our co-immunoprecipitation study. For each experiment, one-tenth volume of lysate collected from the striatal tissue of two mice was separated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with anti-SAP 102 antibody, then stripped and probed with anti-actin antibody as a loading control. As shown in Fig. 2 and Fig. 3, the SAP 102 expression levels were similar in striatal tissue of 4 week-old YAC 18, YAC72 and YAC 128 mice (n=5 for YAC 18 and YAC 128, n=4 for YAC72; SAP102/actin ratio is not significantly different across all 3 genotypes, by one-way ANOVA, P>0.05). The data suggest that mutant huntingtin does not affect SAP 102 expression at the transcriptional or translational levels. 37 Figure 2. Representative western blot showing expression level of SAP 102 in 4 week-old YAC mice striatal tissues. Blots were probed with anti-SAP102 and anti-actin antibodies separately. The blot shown here is representative of a total of n=5 independent experiments (except n=4 for YAC72). YAC 18 YAC 72 YAC 128 ant i-SAP102 « 102 kDa anti-actin ^ 42 kDa 38 Figure 3. Expression level of SAP102 in 4 week-old YAC mice striatal tissues. SAP102/actin band density ratio was calculated after subtracting the background. SAP102/actin band density ratio of each genotype was normalized to SAP102/actin band density ratio of YAC 18. Expression level of SAP102 is not significantly different across all 3 genotypes, n=5 for YAC 18 and YAC 128, n=4 for YAC72, by one-way ANOVA, p>0.05. .2 1.4 -»—< CD " 1.2 c CD Q_ < CO 1.0 0.8 o 0.6 CD § 0.4 0.2 0.0 39 3.2 Association of SAP102 and huntingtin 3.2.1 Association of SAP102 and huntingtin in httl38- and httl5-transfected HEK293T cells The non-neuronal HEK293T cell line is widely used to study interactions between transfected neuronal proteins. By using co-immunoprecipitation, we studied the association of SAP102 with full-length huntingtin in HEK293T cells transiently expressing NRl/NR2B/SAP102/httl5 or NRl/NR2B/SAP102/httl38. 30 hrs after transfection of plasmids, 293T cell lysates were prepared and co-immunoprecipitation was carried out using the anti-huntingtin antibody, MAB2166, or beads only as a negative control. The western blots were cut along 160 kDa and then probed with anti-huntingtin antibody, two different anti-SAP102 antibodies or an anti-myc antibody (the transfected SAP102 construct has a myc tag). As shown in Fig. 4, SAP102 was detected in htt immunoprecipitates (IPs) of both httl5- and httl38-transfected 293T cell lysates. The two different anti-SAP102 antibodies and the anti-myc antibody all recognized a band around MW 102 kDa in IP lanes as well as in lysates and supernatants (not shown). There are two bands around 102 kDa in panel iii of Fig.4: one is higher than the 105 kDa ladder band and could represent a modified form of SAP102 or else another non-specific protein recognized by the antibody, and the other band below 105 kDa is considered to be SAP102 and was used in quantitative analysis; two bands were occasionally seen with other anti-SAP 102 antibodies as well as in experiments in which anti-NR2B antibodies were used to co-immunoprecipitate SAP102. No SAP 102 or huntingtin bands were detected in the beads-only immunoprecipitates lane (labelled as "noAb" in the figure and showing one example of anti-myc probing). This is the first evidence showing htt and SAP102 are associated in a heterologous system, which suggests this interaction might be 40 direct. Even though the blots in Fig. 4 showed a difference in amounts of SAP 102 co-immunoprecipitated with huntingtin in httl38- and htt 15-transfected cells, the average SAP102/huntingtin band density ratio was not significantly different in paired comparisons across n = 9 different experiments (the SAP102/htt band density ratio is 0.50 + 0.06 for httl38 expressing cells and 0.72 + 0.14 for httl5 expressing cells; P>0.05 by paired t-test). The blots in Fig. 4 panel i and ii, which are representative of most of our blots, show an expected, small molecular weight difference between httl38 and httl5. Although the blot shown in panel iii exhibits no apparent molecular weight difference between htt 138 and htt 15 bands, this may be a result of a shorter running time of the SDS-PAGE gel. Additionally, we used an 8% SDS-PAGE gel to separate proteins, which makes the molecular weight difference of httl38 and httl5 difficult to detect. ' 41 Figure 4. SAP102 associates with huntingtin in transfected 293T cells. Lysates from 239T cells transfected with full-length htt 15 or httl38 and full-length SAP102 were precipitated with anti-huntingtin antibody (MAB2166). The blot was cut into two parts along the 160 kDa marker. The upper part was probed with anti-htt (MAB2166) and the other part was probed with anti-SAP 102 antibodies (i. Stressgene MAB #VAM PS006, n=14; i i . Rabbit polyclonal anti-SAP102 Ab from Dr. Robert Wenthold's lab, n=2; iii . anti-myc MAB-4A6 from Upstate, n=3). htt15 htt138 htt15 htt138 noAb htt15 htt138 i ii iii 42 3.2.2 Association of SAP102 and huntingtin in WT and YAC mice striatal tissues Based on the result in HEK cells, we then investigated whether huntingtin and SAP102 co-associate in 4 week-old WT or YAC mice striatal tissues, an age at which SAP 102 is expressed at high levels. Brain lysates were immunoprecipitated with MAB2166, or anti-human huntingtin antibody HD650. As a negative control, protein A or G Sepharose beads alone were also incubated with tissue lysates, and no SAP 102 or htt bands were detected (not shown). The blots were cut along 160 kDa and probed with anti-htt antibodies and anti-SAP 102 antibodies separately. As shown in Fig. 5, htt and SAP102 were detected in htt immunoprecipitates in all 3 genotypes (lysates and supernatant not shown). The data suggest for the first time that huntingtin is associated with SAP102 in vivo in mouse striatum. Notably, the representative blot in Fig. 5 showed no molecular weight difference of htt bands in immunoprecipitates between genotypes, using the anti-human htt (HD650) antibody for immunoprecipitation and probing. The reason is unknown, but we do see a molecular weight difference of htt bands between genotypes when using the anti- htt (MAB2166, Chemicon) antibody for immunoprecipitation and probing. However, a study using this HD650 antibody for immunoprecipitation and MAB2166 for probing has demonstrated the appropriate molecular weight difference for htt detected in brain tissues from YAC 128 and YAC18 (Warby, Chan et al. 2005). By quantitative analysis, we found no significant difference (by one-way ANOVA, n=6 for YAC 18 and YAC72, n=4 for YAC128) of the SAP102/htt ratio across all 3 genotypes of YAC mice striatal tissues. The SAP102/htt ratio data for YAC 18, YAC72 43 and YAC128 is 2.77+1.38, 3.14+1.83, 6.12 + 5.02 (mean + SEM), respectively. The large standard error of the mean is likely a result of variability between individual mice within each genotype and the errors inherent in immunoprecipitation manipulation and quantitative western blotting, which might obscure a potentially significant difference of SAP102-htt association between genotypes. 44 Figure 5. SAP102 associates with huntingtin in 4 week-old YAC mice striatal tissues. Lysates from 4 week-old YAC transgenic mice striatal tissues were incubated with HD650, an anti-human htt specific antibody. The representative blot was probed with anti-human htt and anti-SAP 102 (NeuroMAB) antibodies. Representative blot showing one of six independent experiments. YAC18 YAC72 YAC128 htt *~~350 kDa S A P 102 m± ^ . A « _ 102 kDa 45 3.3 Association of SAP102 and NR2B is similar in YAC18, YAC72, and YAC128 striatal tissues, or httl38- and httl5-transfected 293T cells 3.3.1 Interaction of SAP102 and NR2B in httl38 and httl5 transfected HEK293T cells SAP102 is known to bind to the C-terminus of the NMDA receptor NR2B subunit (Muller, Kistner et al. 1996). Since the three proteins might be associated together as a group, I set out to determine whether the SAP102-NR2B interaction is altered by the expanded polyQ of htt. Lysates of HEK293T cells expressing NRl/NR2B/SAP102/httl5 or NRl/NR2B/SAP102/httl38 were prepared, and co-immunoprecipitation was conducted using anti-NR2B antibodies or beads only as a control. The blots were cut along 160 kDa and probed with anti-NR2B antibodies and anti-SAP 102 antibodies separately. As shown in Fig. 6, SAP 102 was detected in NR2B immunoprecipitates from both httl 5- and httl38-transfected 293T cells, but not in the beads-only immunoprecipitates (labeled as "noAb"). We found no significant difference (by paired, 2-tailed student's t-test, n=6) of the ratio of SAP102/NR2B between httl 5- or httl38-expressing 293T cells (the SAP102/NR2B band density ratio is 0.53 + 0.20 for httl38 transfected cells and 0.89 + 0.28 for httl5 transfected cells). 3.3.2 Interaction of SAP102 and NR2B in YAC mice striatal tissues We also explored the association of NR2B with SAP102 in YAC mice striatal tissues. Striatal lysates from 4 week-old YAC mice were immunoprecipitated with anti-NR2B antibodies (Upstate or Bioreagents). Protein A or G Sepharose beads alone were also incubated with tissue lysates as a negative control (not shown). As shown in Fig. 7, NR2B and SAP 102 were detected in immunoprecipitates of all three genotypes. Again, we found no significant difference (by one-way ANOVA, n=6 for YAC 18 and 46 YAC 128, n=5 for YAC72) of the ratio of SAP102/NR2B across YAC 18, YAC72, and YAC 128. The data suggest that polyglutamine-expanded htt does not alter the interaction of SAP 102 with NR2B to affect NMDAR functions. However, the SAP102/NR2B ratio data for YAC 18, YAC72 and YAC 128 is 1.23 + 0.64, 0.75 + 0.21, 1.11+0.47 (mean + SEM) respectively. As mentioned in section 2 with the htt immunoprecipitation, the large standard error of mean may obscure small differences of SAP102-NR2B association between genotypes. 47 Figure 6. The association of SAP102 and NR2B are similar in httl5- and httl38-transfected 293T cells. Lysates from 239T cells transfected with NR1A, NR2B, full-length httl5 or httl38 and full-length SAP102 were immunoprecipitated with anti-NR2B antibody (Upstate or Bioreagents). The blot was cut into two parts along the 160 kDa marker. The upper part was probed with anti-NR2B (Upstate) and the other part was probed with anti-SAP102 antibodies (Chemicon) or anti-myc antibody. The blot shown here is representative of 6 independent experiments. noAb htt 15 httl 38 ant i -NR2B anti-SAP102 102 kDa Figure 7. The association of SAP 102 and NR2B is similar in 4 week-old YAC 18, YAC72 and YAC 128 mice striatal tissues. Lysates of 4 week-old YAC mice striatal tissues were immunoprecipitated with anti-NR2B antibody (Upstate or Bioreagents). The blot was cut into two parts along the 160 kDa marker. The upper part was probed with anti-NR2B (Upstate or Transduction lab) and the other part was probed with anti-SAP 102 antibodies (Stressgene, Chemicon, NeuroMAB). The blot shown here is representative of 6 independent experiments. YAC 18 YAC72 YAC 128 NR2B S A P 102 -180 KDa -102 KDa 49 3.4 Tat-NR2B9c reduces co-immunoprecipitation of NR2B with SAP102 in HEK293T and YAC128 cultured MSNs The Tat-NR2B9c peptide has been shown to interfere with the PSD-95 and NR2B interaction in rat hippocampal tissues (Aarts, Liu et al. 2002). Given the similarity of MAGUK proteins, and a recent report (Cui et al. Society for Neuroscience abstract 679.9, from 36th Annual Meeting) that the Tat-NR2B9c peptide binds PSD-95 and SAP 102 equally, and both more strongly than other MAGUKs in vitro, we hypothesized that the Tat-NR2B9c peptide could also interfere with the binding of SAP102 andNR2B. 3.4.1 luM Tat-NR2B9c interferes with the interaction of SAP102 and NR2B in transfected HEK cells We used l u M Tat-NR2B9c or l u M control Tat-NR2BAA peptides to pre-treat HEK293T cells expressing NR1/NR2B/SAP102 for lhr. The cell lysates were then immunoprecipitated with anti-NR2B antibodies as described in section 3.2. Protein A or G Sepharose beads alone were also incubated with lysates as a negative control. As shown in Fig. 8 and Fig. 9, the ratio of SAP 102 immunoprecipitated with NR2B in l u M Tat-NR2B9c-treated cells was significantly reduced by 49+11% (PO.05 by paired, 2-tailed student's t-test, n=5), comparing to l u M Tat-NR2BAA-treated cells. No SAP 102 or NR2B bands were detected in beads-only controls. In addition to the previously reported interference of Tat-NR2B9c with PSD-95-NR2B interaction in rat hippocampal neurons, our data indicate that the SAP102-NR2B interaction is also disrupted by Tat-NR2B9c in transfected 293T cells. 50 3.4.2 l u M Tat-NR2B9c interferes with the interaction of SAP102 and NR2B in cultured YAC128 MSNs We then confirmed that lhr pre-treatment of l u M Tat-NR2B9c also interferes with the SAP102-NR2B interaction in YAC128 cultured MSNs, using the same method. As shown in Fig. 10 and Fig. 11, the ratio of SAP102 immunoprecipitated with NR2B in l u M Tat-NR2B9c-treated YAC 128 MSNs was significantly reduced by 58+11% (PO.05 by paired, 1-tailed student t-test, n=8) comparing to l u M Tat-NR2BAA-treated cells. No SAP102 or NR2B bands were detected in beads-only controls. The data suggest that the Tat-NR2B9c peptide also interferes with the SAP102-NR2B interaction in cultured MSNs. We have also tested lhr pre-treatment of Tat-NR2B9c peptides at the lower concentration of 200nM, but the amount of SAP102 immunoprecipitated with NR2B was not significantly decreased after 3 trials (data not shown). However, the l p M concentration of Tat-NR2B9c we used to demonstrate significant uncoupling of NR2B and SAP 102 in cultured 293T cells and murine MSNs is much lower than the reported 1 OOuM concentration required to perturb the NR2B-PSD-95 co-immunoprecipitation from rat forebrain lysates (Aarts, Liu et al. 2002), which will be discussed later in chapter 4. 51 Figure 8. Tat-NR2B9c (luM) perturbs co-IP of NR2B with SAP102 in HEK293T cells. 293T cells expressing NR2B and SAP102 were pretreated with l p M Tat-NR2B9c or l u M Tat-NR2BAA peptides for lhr. Then cell lysates were harvested and incubated with anti-NR2B antibody (Upstate or Bioreagents). The blot was cut into two parts along the 160 kDa marker. The upper part was probed with anti-NR2B (Upstate or Transduction lab) and the other part was probed with anti-SAP102 antibodies (Stressgene, Chemicon, or NeuroMAB). The blot shown here is representative of 5 independent experiments. 52 SPN Lys IP r >> f -\ f s noAb Tat-2BAA Tat-2B9c noAb Tat-2BAA Tat-2B9c noAb Tat-2BAA Tat-2B9c anti-NR2B anti-SAP102 180 kDa 102 kDa Figure 9. Quantitatively measured SAP102/NR2B co-IP ratio was decreased 49 ± 11% by l u M Tat-NR2B9c comparing to l u M Tat-NR2BAA lhr pretreatment in transfected HEK cells (n=5; * P< 0.05 by paired 2-tailed student's t-test). 54 Figure 1 0 . Tat-NR2B9c (luM) perturbs co-IP of NR2B with SAP102 in YAC128 cultured MSNs. Cultured YAC 128 MSNs were pre-treated with l u M Tat-NR2B9c or l p M Tat-NR2BAA peptides for lhr at DIV 9. Then cell lysates were harvested and incubated with anti-NR2B antibody (Upstate or Bioreagents) or beads only. The blot was cut into two parts along the 160 kDa marker. The upper part was probed with anti-NR2B (Upstate or Transduction lab) and the other part was probed with anti-SAP102 antibodies (Chemicon or NeuroMAB). The blot shown here is representative of 8 independent experiments. 55 SPN Lys IP f N. ( \ f \ noAbTat-2B9c Tat-2BAA noAb Tat-2B9c Tat-2BAA noAb Tat-2B9c Tat-2BAA anti-NR2B L 180 kDa anti-SAP102 - 102 kDa Figure 11. Quantitatively measured SAP102/NR2B co-IP ratio decreased 58 11 % by 1 hr pre-treatment with lpM Tat-NR2B9c compared to l u M Tat-NR2BAAin cultured YAC128 MSNs (n=8; * P< 0.05 by paired 1-tailed student's t-test). (0 (0 c o T3 DQ CM DC z o y— CL < 1 [iM Tat-NR2BAA 1 |j M Tat-NR2B9c 57 3.5 Tat-NR2B9c peptide reduces surface NMDAR levels in cultured MSN neurons Given that the Tat-NR2B9c peptide could interfere with the interaction of SAP102 with NR2B (my data), as well as with the PSD-95 interaction with NR2B previously reported (Aarts, Liu et al. 2002), and that these interactions are crucial for trafficking and/or anchoring of NMDA receptors at the synapse, we examined surface expression of NMDARs following treatment with the Tat-NR2B9c or Tat-NR2BAA peptides. 3.5.1 luM Tat-NR2B9c reduces the surface NR1 expression in cultured MSNs In pilot experiments we found no effect of 50nM Tat-NR2B peptides on surface NR1 expression in cultured MSNs (data not shown), even though this peptide concentration was previously shown to reduce apoptosis in cultured hippocampal neurons (Aarts, Liu et al. 2002). Next, cultured WT or YAC72 MSNs were pretreated with luM Tat-NR2B peptides for 1 hr on DIV 9, then fixed and immunostained for surface and internal NR1 using anti-NRl antibody, as described in the Methods; immunostaining and fluorescence imaging were performed in parallel on pairs of WT and YAC72 cultured MSNs. The surface/internal ratio of NR1 was calculated as the green/red fluorescence channel intensity of each MSN (representative photomicrographs shown in Fig. 12). As seen in the photomicrographs, the green fluorescence intensity (surface NR1 staining) is substantially dimmer for l uM Tat-NR2B9c-treated MSNs than for the 1 uM Tat-NR2BAA-treated MSNs, whereas the red fluorescence intensity (internal NR1 staining) is almost the same for both treatments. This difference in fluorescence intensity usually was visible to the operator before carrying out any analysis, even though the operator was blinded to the conditions and genotypes. The difference was more obvious in the neuronal processes than the soma, which is consistent with 58 preferential dendritic localization of PSD-95 and SAP102. After analyzing, we found that the luM Tat-NR2B9c peptide could reduce the level of surface NMDAR by 15.3 + 4.6% in YAC72 and 8.9 + 3.4% in WT MSNs relative to luM Tat-NR2BAA peptide-treated MSNs, (shown in Fig. 13, n=4 for both genotypes; Fi;,8=10.35 for genotype, P<0.005; F2,18=1.80, P=0A9 for treatment; F2,i8=0.71 for interaction, P=.0.50; after normalizing each peptide-treated group to the untreated group, FU 2=0.50 for genotype, P=0.49; FU2=32.80, PO.0001 for treatment; F112—8.37 for interaction, P=.0.0135, by two-way ANOVA). It is worth mentioning that, in Fig. 13, we also showed a significant, approximately 30% increase of surface/internal NR1 ratio in YAC72 compared with WT MSNs in the untreated group (the ratio was 0.81+0.07 versus 0.62 + 0.03, respectively), which is consistent with our previous data (Fan et al. 2007). Additionally, we found no significant difference in NR1 surface to internal ratio between groups treated with the control peptide Tat-NR2BAA and the untreated groups. 3.5.2 l u M Tat-NR2B9c does not change the surface GluRl expression in cultured MSNs As a control, the surface/internal GluRl ratio was also analyzed in both WT and YAC72 MSNs, and we found no significant difference between groups treated with luM Tat-NR2B9c versus l u M Tat-NR2BAA (shown in Fig. 14; not significant for genotype and treatment, two-way ANOVA, n=4 for YAC72, n=3 for WT; Fi_i5=1.39 for genotype, P>0.05; F2j5=0.03, P>0.05 for treatment; F2ji5=0.02 for interaction, P>0.05, by two-way ANOVA). These data indicate that the Tat-NR2B9c peptide specifically affects the surface expression of NMDA receptors, but not AMPA 59 receptors. 3.5.3 200nM Tat-NR2B9c reduces the surface NR1 expression in cultured MSNs We also tested the effect of the Tat-NR2B9c peptide at a concentration of 200nM on the surface expression of NMDARs, and found a 17.6 ± 2.6% reduction of surface/internal NR1 ratio in YAC72 MSNs, similar to the reduction found for treatment with the l u M peptide concentration. Surprisingly however, the surface/internal NR1 ratio was reduced by 26.9 + 4.2% WT MSNs, which was a significantly larger reduction than found for the higher peptide concentration (Fig. 15). The data are presented together with the luM Tat-NR2B treatment data, after each Tat-NR2B9c data is normalized to the relative Tat-NR2BAA data (9c/AA ratio) to be comparable (shown in Fig. 15, significant for 200nM and l u M Tat-NR2B treatment on WT MSNs, tested by 2-way ANOVA, n=4 for all conditions; F u 2=0.14 for genotype, P=0.059; Fi,i2=7.14, P=0.02 for concentration; FU 2=04.34 for interaction, P>0.05). It is unexpected as the higher concentration of this Tat-NR2B9c peptide should reduce surface NMDARs more, or have similar effect as 200nM at-NR2B9c peptide if 200nM is already saturated for interfering at synapses (further discussion deferred to chapter 4). Nevertheless, both concentrations (200nM and 1 uM) Tat-NR2B9c reduced surface NMDARs in both WT and YAC72 MSNs by -10-30%, which would be expected to alter NMDAR current and downstream signaling. 60 Figure 12. Representative photomicrographs of surface and internal NR1 staining of luM Tat-NR2B peptides pretreated YAC72 MSNs. Representative photomicrographs showing luM Tat-NR2B9c peptide reduces surface NR1 expression in YAC72 MSNs, comparing to 1 uM Tat-NR2BAA peptide. fi> 7J ro 00 CD o fi) 73 ro 00 > > Surface NR1 (Green) Internal NR1 (Red) 61 Figure 13. Effect of l u M Tat-NR2B9c peptide pretreatment on surface/internal NR1 ratio of WT and YAC72 MSNs. Average surface/internal NR1 ratio data of WT and YAC72 MSNs representing 4 independent experiments with or without l uM Tat-NR2B peptides pretreatment. *P<0.05 by Bonferroni posttests. I I W t l~ I Y 7 2 Q .2 1 0 1 re ^ 0.8 ] DC 1 0.6 re 0.2 t ^ 0.0 Untreated AA 9c AA 9c 62 Figure 14. Effect of l u M Tat-NR2B9c peptide pretreatment on surface/internal GluRl ratio of WT and YAC72 MSNs. Average surface/internal GluRl ratio data representing 3 batches of WT and 4 batches of YAC72 MSNs independent experiments with or without 1 uM Tat-NR2B peptides pretreatment. ESI11Y72Q O ro o "ro c i _ <D •*-> C "rB o 3 CO CD +-> PQ <N i o ON OQ CN OQ CN i -4-> f3 O ON OQ CN 63 Figure 15. Effect of 200nM and l u M Tat-NR2B9c peptide pretreatment on surface/internal NR1 ratio of WT and YAC72 MSNs. Average Tat-NR2B9c-treated surface/internal NR1 ratio data normalized to Tat-NR2BAA treated groups, representing 4 independent experiments with or without 200nM or luM Tat-NR2B peptides on WT and YAC72 MSNs. *P<0.05 by Bonferroni posttests. O VP (0 5 ro c 0) c u I 3 CO < o 200nM Tat-NR2B 1 Tat-NR2B 6 4 3.6 200nM Tat-NR2B9c reduces NMDA-induced toxicity in YAC72, YAC128, but not WT MSNs 3.6.1 200nM Tat-NR2B9c reduces the NMDA-induced toxicity in cultured YAC mice MSN Since a reduced number of surface NMDARs (especially the extrasynaptic, NR2B-containing ones) is expected to lead to reduced calcium influx in response to excitotoxic insults and therefore diminished apoptosis, we tested the Tat-NR2B9c peptide on cultured MSNs to see whether it could protect MSNs from NMDA-induced toxicity. We chose to use a concentration of 200 nM rather than 1 uM, since we found 200nM to be the lowest effective concentration for reducing surface NMDAR expression (see Fig. 15; in pilot experiments, 50nM Tat-NR2B9c.showed no effect on NMDA-induced toxicity, consistent with the lack of effect on NMDAR surface expression - data not shown). Cultured WT, YAC72, and YAC 128 MSNs were pretreated with 200nM Tat-NR2B peptides for 1 hr on DIV 9, then exposed to 500uM NMDA for lOmin and fixed 24 hrs later. Plates of WT, YAC72 and YAC128 MSNs were then processed in parallel, using the TUNEL stain to identify apoptotic neurons and PI staining to identify all nuclei. The proportion of apoptotic nuclei was then assessed by operators blinded to conditions and genotypes (representative photomicrographs shown in Fig. 16). As illustrated in the photomicrographs, only TUNEL-positive and blebbed nuclei were counted as apoptotic neurons, while all nuclei in the PI (red channel) field were counted as total number of neurons (except for unusually large nuclei, which likely represented astrocytes that make up -9% of the cells in our cultures). The proportion of apoptotic MSNs exposed to BSS alone was subtracted as a baseline from the other conditions for statistical analysis only; the apoptotic death rate shown in Fig. 17 is uncorrected for background. 65 As shown in Fig. 17, we found that 200nM Tat-NR2B9c reduced NMDA-induced cell death in YAC72 and YAC 128 MSNs down to the same level observed with WT MSNs (a 37.4 + 2.7% reduction for YAC72, and 33.6 + 6.6% reduction for YAC128, tested by two-way ANOVA, significant for both genotypes and treatments, n=4; F2,5o=6.34 for genotype, PO.005; Fs . s r r^^ , PO.0001 for treatment; F10,5o=l-42 for interaction, P>0.05). On the other hand, the 200nM Tat-NR2B9c treatment had no significant protective effect on WT MSNs (which will be discussed in chapter 4). Similar to previous findings from our lab (Shehadeh, Fernandes et al. 2006), we also found an NMDA-induced apoptosis rate in the groups treated with -NMDA alone (without peptides) of 18.7+1.1% for WT MSNs, 31.2 + 2.7% for YAC72 MSNs, and 30.8 + 0.6% for YAC 128 MSNs. As a control, Tat-NR2BAA had no significant additive toxic effect on NMDA-treated MSNs. Furthermore, 200nM Tat-NR2B9c or Tat-NR2BAA alone did not cause significant apoptosis (as shown in Fig. 18; n=4, by two-way ANOVA, F2,23=0.61 for genotype, 7>0.05; F2>23=1.26, P>0.05 for treatment; F4,23=0.21 for interaction, i>>0.05). This result indicated that Tat-NR2B9c peptide could fully rescue the increased NMDA-induced toxicity observed in MSNs expressing mhtt with an expanded polyQ tract, and supports the idea that the interaction of SAP102/PSD-95 with NR2B might play an important role in the enhanced sensitivity to excitotoxicity observed in the YAC transgenic HD mouse models. 66 3.6.2 200nM Tat-NR2B9c has no significant effect on the staurosporine-induced toxicity in cultured YAC72 MSN In order to prove that 200nM Tat-NR2B9c selectively reduces NMDAR-dependent apoptosis, I choose to explore the effect of 200nM Tat-NR2B9c on staurosporine-induced apoptosis. Staurosporine is known to induce apoptosis through mitochondrial pathway via suppression of Bcl-2 expression, resulting in caspase-3 activation (Nicbtera, Leist et al. 1999; Roucou, Antonsson et al. 2001; Swannie and Kaye 2002). Thus, Tat-NR2B9c, which disrupts the NMDAR and PDZ domain protein binding, should not be able to rescue the staurosporine-induced apoptosis. I find that 200nM Tat-NR2B peptides lhr pretreatment has no effect on lOuM staurosporine induced cell death of cultured Y72Q MSNs as shown in Fig. 19 (n=3, no significant difference for all staurosporine treated groups by one-way ANOVA; significant for staurosporine treatment effect, *P<0.05). 67 Figure 16. Representative photomicrographs showing TUNEL and PI stained YAC72 MSNs, which have been pretreated with 200nM Tat-NR2BAA or 200nM Tat-NR2B9c for lhr and then exposed to 500uM NMDA for 10 min. Figure 17. Effect of 200nM Tat-NR2B peptides pretreatment on NMDA-induced toxicity in WT, YAC72, and YAC 128 cultured MSNs. Pre-treatment with 200nM Tat-NR2B9c peptides reduces NMDA-induced toxicity in YAC72, YAC 128, but not WT cultured MSNs. Average proportion of apoptotic cells following pre-treatment with Tat-NR2B peptides then exposure to 500uM NMDA (BSS control baseline subtracted of each genotype), representing 4 independent experiments for WT, YAC72 and YAC128 MSNs. (.*P<0.05, ** P<0.01, by Bonferroni posttests.) 69 r i — 1 — i — r n — 1 — i — - < -o CM CD co co CM o o o O o o + I + + + - + ° < 5 o Q rn o ^ <L 0 5 O Q § OQ (pejoejjqns euneseq) SNSIAI ojiojdode jo uoijjodojd § o (—' CM ™ Figure 18. Effect of 200nM Tat-NR2B peptides pretreatment on basal cell death of Wt, Y72Q and Y128Q MSNs. Average proportion of apoptotic cells data representing 4 independent experiments (except n=2 for WT Tat-NR2B peptides treatment) of WT, YAC72 and YAC128 MSNs with or without pre-treatment with 200nM Tat-NR2B peptides. No significant difference between genotypes or treatments by 2-way ANOVA. ] w t Figure 19. 200nM Tat-NR2B peptides lhr pretreatment has no effect on lOuM staurosporine induced cell death of cultured Y72Q MSNs. N=3. Significant for effect of staurosporine treatment by one-way ANOVA, T<0.05. No significant difference across staurosporine treated 3 groups by posttests. co CO o '•4—" o -»—' CL o C L ro H— O c o •c o Q . O 10 y M Staurosporine 200nM Tat-NR2BAA 200nM Tat-NR2B9c + + + 72 CHAPTER 4 Discussion A variety of evidence suggests that striatal neurons expressing mhtt have higher levels of NMDAR activity (especially that mediated by the NRl/NR2B-subtype), and thus are more vulnerable to excitotoxicity. Great attention has been paid to how this NMDAR hyperactivity results in increased intracellular calcium entry and catabolic enzyme activity that may trigger a cascade of events leading to neuronal dysfunction and death. However, how mhtt causes the elevated NMDA receptor activity and through what molecule(s) are still mysteries. As reviewed in the introduction, the PSD-95 family of MAGUK proteins can regulate NMDAR surface expression and signaling to cell death pathways; therefore, the evidence showing polyglutamine expansion perturbs the interaction between htt and PSD-95 (Sun, Savanenin et al. 2001) provided a candidate for the linking protein. However, this study did not prove that the altered interaction between polyQ-expanded htt and PSD-95 is related to enhanced NMDAR function. In this thesis, I chose to investigate SAP102 as a possible candidate because of its similarity to PSD-95 in function and interaction with NR2 subunits, and also because of its role in NMDAR trafficking and early neuronal development. My immunoprecipitation results in cultured neurons and transfected HEK cells have shown that SAP102 also interacts with htt. Whether this interaction is direct needs to be confirmed by GST-pull down experiments. In my studies, I used the Tat-NR2B9c peptide to disrupt NR2B binding to MAGUKs. This peptide has been shown to reduce NMDAR-mediated excitotoxicity in cultured 73 rat hippocampal neurons as well as in a rat stroke model, although the peptide concentration required to disrupt NR2B/PSD-95 binding was 1000 times higher than used to rescue cell death (Aarts, Liu et al. 2002). Here, I have shown that at peptide concentrations that substantially disrupt NR2B binding with SAP 102, NMDAR surface expression is significantly reduced in both WT and YAC72 MSNs, while cell death is decreased in YAC72 MSNs only. These data suggest that the interaction of SAP 102 with htt and NR2B contributes to the elevated NMDAR surface expression and cell excitotoxicity caused by mhtt. It is interesting that the Tat-NR2B9c peptide resulted in only an -15% decrease of surface NMDAR level whereas the reduction in NMDA-induced cell death was -35% in YAC72 MSNs. This discrepancy suggests that Tat-NR2B9c interferes with cell death signaling downstream of NMDAR activation in addition to its effect on NMDAR surface expression, as has been suggested previously (Aarts, Liu et al. 2002). The fact that in WT MSNs, the Tat-NR2B9c peptide reduced surface NMDAR expression yet had no effect on the NMDA-induced cell death suggests that it is the signaling downstream of NMDAR activation mediated by the NR2B/MAGUK interaction, rather than the M A G UK-regulated level of NMDAR surface expression, that is differentially affected by mhtt expression. In the surface NR1 expression study, I did not include YAC 128 MSNs, as we have data in the lab showing that WT and YAC 128 MSNs exhibit the same NMDAR current density and surface NR1 expression level (Fernandes et al., submitted; Zhang L and Raymond LA, unpublished data). Additionally, others in the lab have demonstrated that the increased NMDA-induced toxicity seen in YAC128 MSNs is 74 largely caused by impaired calcium handling by mitochondria in these neurons, and that this alteration in mitochondrial function is selective for NMDAR-induced toxicity (Fernandes et al., submitted). As a result, I only tested the effect of Tat-NR2B peptides on NMDAR surface expression in WT and YAC72 MSNs, because these two genotypes show a polyQ-dependent difference in NR1 surface expression. To confirm that the perturbed interaction of SAP102 and NR2B by l u M Tat-NR2B9c peptide in HEK cells is also true in MSNs, I repeated this peptide treatment experiment in YAC 128 MSNs, and observed a similar -50% reduction of SAP 102 immunoprecipitated with NR2B. On the other hand, I found that Tat-NR2B9c peptide reduces surface NR1 expression and especially NMDA-induced toxicity to different degrees in MSNs from different genotypes. 4.1 Tat-NR2B9c peptide's specificity It should be noted that although the Tat-NR2B9c peptide binds SAP102 and PSD-95 with highest affinity, it also binds with three other members of the M A G U K family of proteins (PSD-93, SAP97, Tipl), as reported by Cui et al., Society for Neuroscience abstract 679.9, from 36th Annual Meeting. It is even possible that Tat-NR2B9c peptide could disrupt the binding of MAGUK proteins to other PDZ domain-interacting proteins, such as Shank and nNOS, and thus interfere with down-stream signaling pathways other than NMDAR-dependent pathway, and contribute to the peptide protective effect on cell death. Thus, the effect of Tat-NR2B9c peptide in this research is complicated and cannot be solely attributed to the disruption of interaction between PSD-95/SAP102 and NMDARs. However, with relatively low concentration, Tat-NR2B9c did not show any effect in our staurosporine 75 experiment. More studies in different apoptotic pathways will be helpful to determine whether the protective effect of 200nM Tat-NR2B9c on cell death is mainly NMDAR-dependent. Besides, it is possible that low peptide concentrations may differentially disrupt NR2B binding with SAP102 and PSD-95. I have shown that luJVI Tat-NR2B9c is effective in uncoupling NR2B and SAP102, in the same range of concentrations required to alter receptor surface expression and toxicity. Although previous work suggested that much higher peptide concentrations (50-100 uM) are required to disrupt NR2B/PSD-95 binding (Aarts, Liu et al. 2002), further work is required to determine the effective peptide concentration for NR2B/PSD-95 uncoupling in our model system of cultured striatal MSNs. However, it is interesting that recent co-immunoprecipitation experiments from our lab have shown that interaction between PSD-95 and NR2B is enhanced in striatal tissue from YAC transgenic compared with WT mice (Zhang, Cowan and Raymond, unpublished data), whereas my experiments have not revealed an altered interaction between NR2B and SAP102 with mhtt expression. 4.2 Controls In our studies, WT (FVB/N) mouse tissue and WT cultured MSNs are used as controls for the mhtt transgenic mice (in which the yeast artificial chromosome with human htt is expressed in FVB/N mice). However, the YAC 18 mouse tissue and YAC 18 cultured MSNs are better controls for any effect of YAC transgene expression, as well as the overexpression of human huntingtin on the base of mouse huntingtin. In my co-immunoprecipitation experiments, I paired striatal tissues of YAC 18 mice with those of YAC72 and YAC 128 mice to better determine any correlation of the polyQ length and the association of target proteins. Anti-htt and anti-NR2B 76 immunoprecipitation were also conducted in WT mouse striatal tissues for limited times, in order to confirm the association of SAP102 with these two proteins. However, in NR1 surface expression and NMDA-induced toxicity experiments, we only used WT MSNs as a control. This is based on previous data in our laboratory which has shown that WT and YAC 18 MSNs have similar surface NR1 expression level (Fan, Fernandes et al. 2007), whereas NMDA-induced apoptosis in YAC 18 MSNs is substantially less than in WT MSNs, as the human htt with non-toxic polyQ length expressed in YAC18 mice has been shown to be protective (Leavitt, van Raamsdonk et al. 2006). Thus, WT MSNs are a better control for toxicity studies as the YAC 18 MSNs are resistant to NMDA-induced cell death. As mentioned in the introduction, I chose the striatal tissue and cultured MSNs to study because of the selective loss of MSNs in striatum observed in HD. Besides, previous work in our lab has shown no difference of SAP 102 expression levels in striatal, hippocampal or cortex tissues (see Carolyn D. Icton 2001 MSc. thesis). It will be interesting to explore whether there is any tissue specific effect in my co-immunoprecipitation or Tat-NR2B9c peptide treatment studies. 4.3 Possible mechanisms of htt regulation of NMDAR surface expression Increased surface expression of NMDARs in YAC72 MSNs could result from a number of possible molecular changes including: increased interaction with PSD-95/SAP102; differential splicing of NR1; and/or altered phosphorylation of NMDARs. In this thesis, I have presented evidence to support a role for PSD-95 and/or SAP102 in the effect of mhtt on NMDAR surface expression and toxicity. 77 Evidence for other mechanisms is also reported. For example, differential splicing of NR1, favoring the C2'-containing isoforms rather than the C2-containing isoforms, promotes forward trafficking to the surface (Standley, Roche et al. 2000). Others in the lab have shown that the proportion of NR1 that contains the C2' cassette is indeed increased in YAC72 MSNs versus WT MSNs (Fan, Fernandes et al. 2007). On the other hand, co-ordinated phosphorylation of NR1 at serines 896 and 897, by PKC and PKA respectively, also has been shown to promote forward trafficking of NMDA receptors from the endoplasmic reticulum and hence increase levels of receptor surface expression (Scott, Blanpied et al. 2003). In our lab, we have found that the proportion of NR1 phosphorylated at NR1 S896 and S897 is significantly increased in YAC72 and YAC 128 cultured MSNs and striatal tissue compared to WT and YAC 18 (C. Cowan and L.A. Raymond, unpublished). Furthermore, treatment with a PKC inhibitor partially rescues the mhtt-containing MSNs from NMDA-induced excitotoxicity in vitro. However, the mechanisms underlying alterations in splicing and phosphorylation of NR1 by mhtt remain unknown. One important limitation of my surface NR1 study is that it is based on cultured MSNs. With no input from cortical neurons, these neurons lack distal dendrite and spine structures and may not have proper clustering of PSD proteins as in vivo, thus may not be representive of trafficking or surface receptor regulation in vivo. 4.4 NR2B versus NR2A Although SAP102 and PSD-95 interact with both NR2B and NR2A subunits, there is a preference for NR2B/SAP102 and NR2A/PSD-95, and SAP102 expression dominates in immature neurons with NR2B-type NMDARs (Sans, Petralia et al. 2000; 78 van Zundert, Yoshii et al. 2004; Elias, Funke et al. 2006). In my co-immunoprecipitation experiments, I studied the SAP102 interaction with the NR2B subunit, but not with NR2A because we have previously shown that the increased NMDA-induced apoptosis found with mhtt expression in transfected HEK293 cells is selective for cells expressing NR1/NR2B but not NR1/NR2A (Zeron, Chen et al. 2001). Additional evidence supports a greater role of the NR2B-type NMDAR in the excitotoxicity hypothesis of HD (please see Chapter 1, section 1.2.3.1). Nevertheless, I later conducted the surface expression studies using an NR1-specific antibody instead of assessing NR2B staining, due to the lack of availability of N-terminal NR2B antibodies. An in vitro study (Cui et al. Society for Neuroscience abstract 679.9, from 36th Annual Meeting) indicated that Tat-NR2B9c peptide perturbs the SAP102 interaction with NR2B and NR2A equally, whereas it disrupts the PSD-95 interaction with NR2A more strongly than with NR2B. Thus, based on my data, both NR2A- and NR2B-containing NMDARs may contribute to the decreased NR1 surface/internal ratio found after Tat-NR2B9c treatment. Specific anti-NR2A N-terminus or anti-NR2B N-terminus antibodies, if available, would help to determine the proportion of surface NR2B affected by Tat-NR2B9c. 79 Figure 20. Proposed relationships between di-subunit-containing and tri-subunit-containing NMDA receptor tetramers, scaffolding complexes and trafficking proteins. Downward arrows depict the sequence of appearance of the complexes at the postsynaptic density. The developmental switch from NR2B-rich to NR2A-rich NMDA receptors would also involve triheteromers that could be anchored by either SAP102 or PSD-95. Abbreviations: GK, guanylate kinase domain; SH3, src homology 3 domain; GKAP, guanylate kinase-associated protein; Dig, discs large. 80 © TRENDS in Neurosciences, 2004, by permission (van Zundert, Yoshii et al. 2004) 81 4.5 Concentration of Tat-NR2B9c peptide used in different studies It is interesting that co-immunoprecipitation of NR2B and SAP102 was apparently unaffected by 1-hr treatment with 200 nM Tat-NR2B9c in live, transfected HEK cells and cultured YAC 128 MSNs, while I found a robust effect of this peptide concentration on NMDAR surface expression and toxicity in cultured YAC72 and/or YAC128 MSNs. On the other hand, the luM concentration of Tat-NR2B9c that significantly uncoupled NR2B/SAP102 binding was much lower than the lOOuM concentration reported to perturb the NR2B-PSD-95 co-immunoprecipitation in rat forebrain lysates (Aarts, Liu et al. '2002). Furthermore, in my experiments the Tat-NR2B peptides were added into the medium of live cells, giving a more accurate correlation between data from co-IP experiments and functional effects on NMDAR surface expression and toxicity. Also in contrast to results reported in cultured rat cortical neurons (Aarts, Liu et al. 2002), I did not observe an effect of 50 nM Tat-NR2B9c on NMDA-induced excitotoxicity in striatal MSNs. Factors that may have contributed to these different results include use of different NMDA treatment protocols (Aarts, Liu et al. 2002. used 0-100uM NMDA, while we used 500uM NMDA and waited for 24 hrs) and different cell types. Indeed, MSNs do not express nNOS (Kubota, Mikawa et al. 1993; Kharazia, Schmidt et al. 1994), which has been reported to link NMDAR/PSD-95 with downstream apoptotic pathways (Aarts, Liu et al. 2002; also see Fig. 21). On the other hand, there's a study indicating that NO released from the nNOS-interneurons could promote release of calcium from mitochondria into the cytosol of MSNs (Horn, Wolf et al. 2002), and thus contribute to NMDA-induced cell death in MSNs. However, there are only -3% interneurons in our MSN culture system(Shehadeh, Fernandes et al. 2006), which would have limited effect on the rest MSNs, because any NO released would be massively diluted in the 82 culture medium. NOS inhibitor (N omega-nitro-l-arginine) could be used to rule out the effect of interneuron-released NO on apotosis of MSNs in our system. There may also exist NMDAR-dependent apoptotic pathways other than that mediated by nNOS in MSNs, and those pathways may require higher Tat-NR2B9c peptide concentrations for inhibition. The saturating NMDA concentration and longer waiting time for assessment of apoptosis used in my experiments will also challenge the effect of 50nM Tat-NR2B9c, while 200nM Tat-NR2B9c might have greater effect on reducing the surface NMDAR and protect MSNs from the more severe NMDA treatment. The result that a higher concentration of Tat-NR2B9c peptide (luM) causes a smaller decrease of surface NMDARs than 200nM Tat-NR2B9c in WT MSNs, is unexpected and hard to explain. It is possible that l uM Tat-NR2B9c strongly reduces surface NMDAR expression in both WT and YAC72 MSNs, but as a result, it activates compensatory mechanisms to speed up forward trafficking and insertion of new surface receptors in WT MSNs and not in YAC72 MSNs because of higher basal level of surface NMDARs in the latter. It is notable that l u M Tat-NR2B9c reduces co-immunoprecipitation of NR2B and SAP102 by 50% in MSNs, but only reduces surface NMDAR expression by 10-30%. This might be explained by disruption of NR2B/SAP102 binding in the ER/Golgi network (Sans, Prybylowski et al. 2003; Sans, Wang et al. 2005), which will be detected in co-immunoprecipitation studies, but may not be apparent in the surface'NRl expression studies after just one hour of exposure to Tat-NR2B9c. 83 Figure 21. The hypothesis that NMDAR-PSD-95 complex may be dissociated using Tat peptides fused to the COOH-terminus of NR2B (Tat-NR2B9c), thus reducing the efficiency of excitotoxic signaling through nNOS. j © Science, 2002, by permission (Aarts, Liu et al. 2002) 84 4.6 Possible mechanisms of PSD-95 and/or SAP102 mediated mutant htt-regulation of NMDAR function In this thesis, I have done a variety of experiments to address the hypothesis that SAP102 expression or the interaction between htt and SAP102, and/or between SAP102 and NR2B, is altered by expression of polyQ-expanded htt. The data indicate that SAP 102 expression levels and extent of interaction with htt and NR2B are unchanged by polyQ expansion in htt. However, my subsequent experiments using Tat-NR2B9c peptide indicate that disruption of the interaction between SAP102/PSD-95 and NMDARs is sufficient to reduce receptor surface expression and NMDA-induced cell death down to the levels seen in the presence of WT htt. Although there is evidence that interaction of NR2B with SAP102 and PSD-95 are more affected by the Tat-NR2B9c peptide than other MAGUKs, including PSD-93 and SAP-97 (Cui et al. Society for Neuroscience abstract 679.9, from 36th Annual Meeting), it is still possible that the other MAGUK family members also contribute to the altered NMDAR function in HD. Another possibility is that the aberrant NMDAR-MAGUK signaling is not due to an altered stoichiometry in the formation of the complex per se, but to the recruitment of some other protein to the NMDAR/MAGUK complex, whose regulation is perturbed by the polyQ expansion in htt. Further, it could be that a protein modification of SAP102 or PSD-95, such as phosphorylation or palmitoylation, is altered by the effect of mhtt, which could in turn change the targeting/subcellular localization of the NMDAR/MAGUK complex. In summary, I conclude that htt, NR2 subunits of NMDAR, and PSD-95/SAP102 are associated in a functional complex and that interaction of these proteins is critical in understanding how mhtt causes striatal MSN dysfunction in Huntington's disease. 85 Results of these studies also suggest that the Tat-NR2B9c peptide or another agent with similar function could be used as early treatment for Huntington's disease, which might prevent the early changes mediated by mhtt in NMDAR trafficking to the surface and cell death/survival signaling. 4.7 Future directions To answer whether SAP 102 or PSD-95 plays the dominant role in regulating surface NMDAR expression and NMDA-induced death, lenti-virus based shRNAi could be used to selectively knock down SAP 102 or PSD-95 expression in MSNs to see whether the same effect of Tat-NR2B9c on surface NMDAR expression and NMDA-induced death will be achieved. It will also be interesting to explore whether increased expression of SAP102 by lenti-virus infection will result in enhanced NMDAR surface expression. If so, will it cause more NMDA-induced apoptosis? To support our hypothesis that the reduced surface NMDAR expression by Tat-NR2B9c peptide will truly affect the NMDAR-dependent calcium influx, experiments to assess NMDAR-mediated currents and/or intracellular calcium responses, using whole-cell patch clamp recording or ratiometric dyes to image changes in intracellular calcium, will need to be done. GST pull-down experiments will be helpful to test whether the interaction of SAP102 and htt is direct, and whether it occurs through the SH3 domain. We will also confirm whether this interaction is affected by polyQ length. Once the interaction domains of SAP102 and htt have been elucidated,-we could design a Tat-fused peptide to interfere with this interaction and see whether the functional effects are the same as seen with 86 Tat-NR2B9c peptides. This will be a critical experiment to answer whether mhtt does alter NMDAR function through its interaction with SAP 102 and/or PSD-95. Another aspect we could look at is whether the co-localization of htt/SAP102, and/or NR2B/SAP102 has been changed on a subcellular level in MSNs with expression of mhtt. This will be more precise to uncover the changes of functional associations of these proteins than co-immunoprecipitation, although it requires higher resolution and also reliable antibodies. Even if the interaction of SAP 102 with NR2B and htt is unaltered with mhtt expression, we could still search for molecular modifications of SAP102 or PSD-95 which have been altered by mhtt, such as phosphorylation or palmitoylation. Those modifications may not alter the interaction ability of SAP102/PSD-95 with NR2B and htt, but could alter the anchoring or trafficking function of SAP102/PSD-95 or its stability in the PSD. There are recent studies to suggest that SAP-97 and PSD-93 might also play important roles in regulating glutamate receptor trafficking and surface expression (see chapter 1, section 1.3.3). It is possible that MAGUK proteins share overlapping functions but normally regulate different subsets, of glutamate receptors, or at different developmental stages, or respond to different upstream regulating signals (also see Fig. 20). Thus, whether SAP-97 or PSD-93/Chapsyn-110 also associate with htt in WT or YAC mice tissues, and whether their interaction with NR2B is altered by expanded polyQ could also be explored in the future. 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