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Coupling extrasynaptic NMDA receptors to aberrant intracellular signaling in Huntington disease Dau, Alejandro 2013

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COUPLING EXTRASYNAPTIC NMDA RECEPTORS TO ABERRANT INTRACELLULAR SIGNALING IN HUNTINGTON DISEASE  by ALEJANDRO DAU  B.Sc. (Hons), The University of Victoria, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2013  © Alejandro Dau, 2013  Abstract  N-methyl-D-aspartate glutamate receptors (NMDARs) play dichotomous roles on neuronal survival, depending on their surface localization: while synaptic NMDARs promote pro-survival pathways, those expressed at extrasynaptic sites (Ex-NMDARs) trigger pro-death cascades. In the YAC128 transgenic mouse model of Huntington disease (HD), elevated ExNMDAR expression contributes to the onset of cognitive dysfunction and striatal death. A shift in the balance of synaptic-extrasynaptic NMDAR signaling and localization is paralleled by dysregulation of intracellular Ca++ signaling pathways that couple to pro-death cascades. However, whether aberrant Ca++ signaling is a consequence of elevated Ex-NMDAR expression in HD is unknown. Here, we examined Ca++-dependent pathways downstream of Ex-NMDARs in HD. Chronic (2-month) treatment of YAC128 and WT mice with memantine (1 and 10mg/kg/d), which at a low dose selectively blocks Ex-NDMARs, reduced striatal Ex-NMDAR expression in YAC128 mice without altering synaptic NMDAR levels. In contrast, calpain activity was not affected by memantine treatment, and was elevated in untreated YAC128 mice at 1.5 months but not 4 months of age. In YAC128 mice, memantine at 1mg/kg/d rescued CREB shut-off, while both doses suppressed p38 MAPK activation to WT levels. In contrast, extrasynaptic PSD-95 expression was not affected by memantine in YAC128 mice but was increased by memantine at 10mg/kg/d in WT littermates. Hence, Ex-NMDAR activity drives increased extrasynaptic receptor expression as well as dysregulated p38 MAPK and CREB signaling in HD. Elucidation of the pathways centered around Ex-NMDARs in HD could help provide novel therapeutic targets for this disease.  ii  Preface  Experiments in this thesis were performed in accordance with animal care guidelines from the UBC Animal Care Committee and the Canadian Council on Animal Care (certificate number A11-0012). Lily Zhang helped remove brains during subcellular fractionations and performed SDSPAGE for n=2 samples in Figure 5. She also helped administer memantine treatments on some occasions, along with Liang Wang. Both Lily Zhang and Liang Wang are members of the Raymond lab.  iii  Table of Contents  Abstract ........................................................................................................................................... ii Preface…........................................................................................................................................ iii Table of Contents........................................................................................................................... iv List of Figures ............................................................................................................................... vii List of Abbreviations ................................................................................................................... viii Acknowledgements...................................................................................................................... xiii Chapter 1: Introduction .................................................................................................................. 1 1.1  NMDA Receptors ........................................................................................................... 1  1.1.1  Structure and subunit composition.......................................................................... 1  1.1.2  Diversity and regional distribution ......................................................................... 2  1.1.3  Forward trafficking of NMDARs ........................................................................... 4  1.1.4  Synaptic NMDAR stability..................................................................................... 5  1.1.5  Extrasynaptic NMDARs ......................................................................................... 6  1.1.6  NMDAR surface distribution.................................................................................. 7  1.2  Glutamate excitotoxicity................................................................................................. 8  1.2.1  Role of NMDARs in excitotoxicity ........................................................................ 8  1.2.2  Synaptic vs. extrasynaptic NMDAR signaling ....................................................... 9  1.3  Huntington disease........................................................................................................ 12  1.3.1  Clinical and pathological features......................................................................... 12  1.3.2  Wildtype vs. mutant huntingtin............................................................................. 13  1.3.3  Mouse models of HD ............................................................................................ 14 iv  1.3.4  MtHtt-induced calcium dyshomeostasis ............................................................... 15  1.3.5  Glutamate excitotoxicity in HD ............................................................................ 16  1.3.6  Synaptic dysfunction in HD: Role of Ex-NMDARs............................................. 17  1.3.7  Memantine: An uncompetitive Ex-NMDAR blocker........................................... 18  1.4  Coupling Ex-NMDARs to aberrant signaling in HD.................................................... 20  1.4.1  Calpain signaling .................................................................................................. 20  1.4.2  CREB-dependent gene expression........................................................................ 23  1.4.3  p38 MAPK signaling ............................................................................................ 25  1.5  Rationale and hypothesis .............................................................................................. 28  Chapter 2: Methods...................................................................................................................... 29 2.1  Memantine treatment .................................................................................................... 29  2.2  Subcellular fractionation............................................................................................... 30  2.3  Nuclear fractionation .................................................................................................... 31  2.4  Western blotting............................................................................................................ 32  2.5  Brain slice preparation .................................................................................................. 34  2.6  Slice electrophysiology................................................................................................. 34  2.7  Statistical analysis......................................................................................................... 36  Chapter 3: Results ........................................................................................................................ 37 3.1  Memantine decreases Ex-NMDAR expression in YAC128 mice................................ 37  3.2  Memantine reduces functional Ex-NMDARs in SPNs from YAC128 mice................ 41  3.3  Synaptic NMDAR levels are not different between genotypes or treatments .............. 44  3.4 Calpain activity is not different between 4 month-old YAC128 and WT mice, and is unaffected by memantine.................................................................................................. 46 v  3.5  Low-dose memantine rescues mtHtt-induced CREB shutoff....................................... 49  3.6  Memantine rescues elevated p38 MAPK activity in YAC128 mice ............................ 51  3.7  Memantine increases extrasynaptic PSD-95 levels in WT mice .................................. 53  Chapter 4: Discussion .................................................................................................................. 57 4.1  Assessment of NMDARs in HD ................................................................................... 58  4.2  Ex-NMDAR surface expression is Ex-NMDAR-activity dependent ........................... 59  4.3  Putative mechanisms of elevated Ex-NMDAR expression in HD ............................... 61  4.3.1  STEP signaling...................................................................................................... 62  4.3.2  CREB-PGC1α pathway ........................................................................................ 62  4.3.3  GluN2B-S1480 phosphorylation .......................................................................... 63  4.3.4  PKC signaling ....................................................................................................... 63  4.4  Ex-NMDARs couple to pro-death signaling in HD...................................................... 63  4.5  Memantine decreases body weight in female YAC128 mice....................................... 66  4.6  Experimental limitations............................................................................................... 67  4.7  Future directions and unanswered questions ................................................................ 69  References..................................................................................................................................... 71 Appendices.................................................................................................................................... 94 Appendix A: Effect of memantine on daily solution intake and body weights ........................ 94 Appendix B: Accuracy of subcellular fractionation ................................................................. 98  vi  List of Figures  Figure 1. Memantine decreases Ex-NMDAR expression in YAC128 mice................................ 39 Figure 2. Memantine reduces functional Ex-NMDARs in SPNs from YAC128 mice ............... 42 Figure 3. Synaptic NMDAR expression is not altered between genotypes or treatments ........... 45 Figure 4. Calpain activity and is unaffected by memantine treatment and is elevated in 1.5 month- but not 4 month-old YAC128 mice ........................................................... 48 Figure 5. Low-dose memantine rescues mtHtt-induced CREB shutoff....................................... 50 Figure 6. Memantine rescues elevated p38 MAPK activity in YAC128 mice ............................ 52 Figure 7. Memantine increases extrasynaptic PSD-95 levels in WT mice ................................. 55 Figure 8. Solution intake does not differ between memantine-treated and untreated mice......... 95 Figure 9. Memantine reduces body weight in female YAC128 mice.......................................... 96 Figure 10. Accuracy of subcellular fractionation ........................................................................ 98  vii  List of Abbreviations  aCSF  artificial cerebrospinal fluid  AMPA  α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid  AMPAR  AMPA receptor  AP  alkaline phosphatase  AP-2  adaptor protein 2  ATF-1  activating transcription factor 1  BAC  bacterial artificial chromosome  BAD  Bcl-2 associated death promoter  BCA  bicinchoninic acid  BSA  bovine serum albumin  BDNF  brain-derived neurotrophic factor  bZIP  basic leucine zipper  C-terminal  carboxy-terminal  Ca++  calcium ion  cAMP  cyclic adenosine monophosphate  CKII  casein-kinase II  CaMKII (or IV)  Calcium/calmodulin-dependent kinase II (or IV)  CNS  central nervous system  CREB  cAMP response element binding protein  CRE  cAMP response element  DIC-IR  differential interference contrast infrared viii  DL-TBOA  DL-threo--benzyloxyaspartic acid  DNA  deoxyribonucleic acid  DTT  dithiothreitol  ECL  enhanced chemiluminescence  EDTA  ethylenediaminetetraacetic acid  eEPSC  evoked excitatory postsynaptic current  EGTA  ethylene glycol tetraacetic acid  EPSC  excitatory postsynaptic current  ER  endoplasmic reticulum  ERK1/2  extracellular signal-regulated kinase 1/2  Ex-NMDAR  extrasynaptic N-methyl-D-aspartate receptor  FOXO  forkhead box protein O  GABA  γ-aminobutyric acid  GLT-1  glutamate transporter 1  Glu  glutamate  HAP 1  huntingtin associated protein 1  HAT  histone acetyltransferase  HD  huntington disease  HDAC  histone deacetylase  HEK293  human embryonic kidney 293 cells  HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  HRP  horseradish peroxidase  Htt  huntingtin ix  IC50  half-maximal inhibitory concentration  INMDA  NMDA-mediated current  KDa  kilo daltons  IP3  inositol triphosphate  IP3R  inositol triphosphate receptor  JNK  c-Jun N-terminal kinase  K+  potassium ion  KID  kinase inducible domain  LTD  long-term depression  LTN  lateral tuberal nucleus  LTP  long-term potentiation  Mg++  magnesium ions  MAPK  mitogen-activated protein kinase  MEF2C  myocyte specific enhancer factor 2C  Mem  memantine  mg/kg/d  milligrams per kilogram body weight per day  MPT  mitochondrial permeability transition  MK-801  5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine  MKK3/6  mitogen activated protein kinase kinase 3/6  MSK1/2  mitogen and stress activated protein kinase 1/2  mtHtt  mutant huntingtin  N-terminal  amino-terminal  Na+  sodium ions x  NF- κb  nuclear factor κ-light-chain-enhancer of activated B cells  NMDA  N-methyl-D-aspartate  NMDAR  N-methyl-D-aspartate receptor  nNOS  neuronal nitric oxide synthase  non-PSD  non-postsynaptic density  NBXQ  2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione  NOX  NADPH oxidase  OM  outer membrane  PI3K  phosphatidylinositol 3 kinase  PKA  protein kinase A  PKC  protein kinase C  PTX  picrotoxin  pCREBSer133  CREB phosphorylated at serine residue 133  polyQ  polyglutamine  PP1  protein phosphatase 1  PP2A  protein phosphatase 2A  PSB  protein sample buffer  PSD  postsynaptic density  PSD-95  postsynaptic density-95  PTX  picrotoxin  PVDF  polyvinylidene fluoride  QUIN  quinolinate  ROS  reactive oxygen species xi  RT  room temperature  RYR1  ryanodine receptor type 1  SAP-102  synapse-associated protein-102  SDS-PAGE  sodium dodecyl sulfate-polyacrylamide gel electrophoresis  S1480  serine residue 1480  Ser  serine residue  SPN  spiny projection neuron  STEP  striatal-enriched tyrosine phosphatase  SynGAP  synaptic Ras-GTPase activating protein  Thr  threonine residue  Tyr  tyrosine residue  TBS  tris-buffered saline  TBST  tris-buffered saline with 0.5% Tween  WT  wildtype  wtHtt  wildtype huntingtin  Y1472  tyrosine residue 1472  YAC  yeast artificial chromosome  YAC128  yeast artificial chromosome with 128 CAG  xii  Acknowledgements  My deepest thanks goes to my supervisor, Dr. Lynn Raymond. Her patience, caring attitude, and continuous support have made for an excellent learning environment and have been fundamental in my maturation as a graduate student. I have been inspired by her scientific expertise and professionalism and could not have asked for a better mentor. During my tenure in the lab, I was lucky to be surrounded by a group of talented postdoctoral fellows, who have been kind and supporting. In particular, I’d like to thank Clare Gladding for her mentorship during the start of my project, and for helping me attain the physical stamina required to carry out a subcellular fractionation. I am indebted also to Marja Sepers for taking time off from her experiments to teach me electrophysiology. Thanks also to Matt Parsons and Karolina Kolodziejczyk for their continuous support. I would also like to thank other members of the Raymond Lab, including Lily Zhang and Liang Wang for technical support, as well as Dr. Tim Murphy and his lab members for helping create a motivating research environment. Also, I’d like to thank my supervisory committee - Drs. Blair Leavitt, Brian MacVicar, and Yu Tian Wang, as well as my external examiner - Dr. Kurt Haas, for their time. A special thanks also to my parents Alvaro and Maria, my sister Catalina, and to my friends, especially Connor Keller for his support during the writing of this thesis.  xiii  Chapter 1: Introduction  Expression of N-methyl-D-aspartate receptors (NMDARs) at synaptic or extrasynaptic sites of neurons determines whether they activate pro-survival or pro-death pathways, respectively. In Huntington disease (HD), elevated extrasynaptic NMDA receptor (Ex-NMDAR) expression and activity occurs prior to phenotype onset and contributes to excitotoxic neuronal death. Intacellular Ca++-dependent pathways closely linked to cell death are also dysregulated early in HD, and help mediate cellular dysfunction and NMDAR mislocalization. However, whether aberrant intracellular signaling in HD is a direct consequence of Ca++ influx through ExNMDARs is unknown.  1.1  1.1.1  NMDA Receptors  Structure and subunit composition The N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor (NMDAR) is a  cationic channel that mediates excitatory synaptic transmission throughout the CNS. It is both ligand- and voltage-gated. In resting conditions, extracellular Mg++ blocks the channel pore. Channel activation requires binding of its endogenous agonist glutamate and the coagonist glycine or D-serine, simultaneously with membrane depolarization to relieve the voltagedependent Mg++ block (Sanz-Clemente et al., 2013). Hence, the channel acts as a unique “coincidence detector”: it is activated only during periods of simultaneous presynaptic and postsynaptic stimulation, an important requirement for the induction of Hebbian synaptic plasticity. The NMDAR is permeable to Na+, K+, and Ca++. Na+/K+ flux through the NMDAR 1  mediates fast synaptic transmission by regulating postsynaptic voltage changes. In contrast, Ca++ influx through the channel can activate a variety of Ca++-dependent signaling cascades involved in physiological and pathophysiological cellular processes (Mayer and Armstrong, 2004). Seven NMDAR subunits exist, namely GluN1, GluN2 (A-D), and GluN3 (A-B). Fully assembled receptors generally consist of a tetrameric complex composed of two GluN1 subunits and two GluN2 subunits, with binding sites for glycine and glutamate, respectively (Wenthold et al., 2003). In some cases, triheteromeric GluN1/GluN2/GluN3 receptors also form, although GluN3 subunits are generally regulatory (Das et al., 1998; Pérez-Otaño et al., 2006). Each subunit consists of an N-terminal extracellular domain, 3 transmembrane domains (M1, M3, and M4), a long extracellular loop (M3-M4), and a C-terminal intracellular domain. The ionpermeable channel pore is formed by a re-entrant M2-transmembrane loop, with a highly conserved asparagine residue involved in Mg++ blocking and Ca++ permeability (Mayer and Armstrong, 2004). While the extracellular domains mediate ligand binding, the C-terminal intracellular tails regulate receptor biophysical properties, as well as surface trafficking, localization, and stability. This is, at least in part, accomplished by direct binding to various intracellular signaling proteins, including calmodulin, CamKII, yotiao, actinin, tubulin, and PSD-95, among others (Gladding and Raymond, 2011).  1.1.2  Diversity and regional distribution Individual NMDAR subunits exhibit distinct biophysical properties, subcellular  localizations, and regional distribution patterns in the CNS. GluN1 subunits are obligatory, as conditional GluN1 knock-out mice do not form functional NMDARs (Fukaya et al., 2003). These subunits are alternatively spliced into 8 variants, each with distinct kinetic profiles and 2  trafficking properties (Horak and Wenthold, 2009). GluN2A and GluN2B subunits also exhibit distinct properties: First, GluN2A-containing NMDARs have a higher open probability (Chen et al., 1999; Erreger et al., 2005), faster deactivation kinetics (Vicini et al., 1998), and greater Ca++dependent inactivation (Krupp et al., 1996) than GluN2B-containing NMDARs. Second, experiments applying GluN2B-selective inhibitors suggest that GluN2B and GluN2A subunits predominate at extrasynaptic and synaptic sites, respectively (Kew et al., 1998; Tovar and Westbrook, 1999; Barria and Malinow, 2002), although this has been disputed (Thomas et al., 2006). Third, GluN2B-expressing NMDARs are enriched in the striatum (Landwehrmeyer et al., 1995; Watanabe et al., 1993) and predominate early in development, while GluN2A expression is highest in the hippocampus and cortex (Wang et al., 1995; Jin et al., 1997) and predominates over GluN2B as the brain matures (Hestrin, 1992; Dumas, 2005; Bellone and Nicoll, 2007). This developmental shift may be important in fine-tuning the differential Ca++ requirements of developing and mature neurons (Dumas, 2005). Particular properties also distinguish GluN2C and D subunits from the others: GluN2C subunits are mainly expressed in the cerebellum, and have low channel conductance, open probability, and sensitivity to magnesium, relative to others (Farrant et al., 1994). In contrast, GluN2D subunits exhibit particularly slow decay kinetics and are mainly expressed in the midbrain (Monyer et al., 1994). The functional diversity between NMDAR subtypes has profound physiological implications. For instance, while GluN2B-containing NMDARs are implicated in cell-death signaling, GluN2A-containing receptors promote pro-survival signaling (Liu et al., 2007; Tu et al., 2010; Lujan et al., 2012; Martel et al., 2012). Similarly, while GluN2B-containing NMDARs have been proposed to promote long-term depression (LTD), GluN2A-containing receptors  3  mediate long-term potentiation (LTP) (Liu et al., 2004; Brigman et al., 2010; Dalton et al., 2012; Yang et al., 2012).  1.1.3  Forward trafficking of NMDARs NMDARs are assembled in the ER (McIlhinney et al., 1998, 2003), advance along  secretory pathways, and are primarily targeted to the postsynaptic membrane (Wenthold et al., 2003). Receptor assembly is an important prerequisite for their ER release and surface expression: when expressed alone, GluN2 or GluN1 subunits do not form functional receptors (McIlhinney et al., 1998). An ER retention signal (RRR) in the GluN1 C-terminus (Scott et al., 2001) acts as a quality control mechanism to prevent forward trafficking of unassembled subunits. Binding of GluN2 subunits to GluN1 masks this retention signal, promoting ER release of fully assembled receptors (Horak et al., 2008). Receptor release from the ER is also regulated by interactions with PDZ-containing proteins (Standley et al., 2000), and is induced in an activity-dependent manner by PKC-mediated phosphorylation of the GluN1 RRR (Scott et al., 2001). Assembled NDMARs are trafficked to the synapse in vesicular packets along microtubules. This process is mediated by binding of the receptor to the kinesin motor KIF17 via the LIN10 adaptor (Setou et al., 2000). At the ER and golgi, NMDARs also bind scaffolding proteins such as SAP-102 and PSD-95, which additionally participate in forward receptor trafficking (Elias and Nicoll, 2007). For instance, SAP-102 binds SEC8, a component of the exocyst complex, which targets the NMDAR to synapses and mediates receptor exocytosis (Sans et al., 2003).  4  1.1.4  Synaptic NMDAR stability Postsynaptic clustering of glutamate receptors is critical for efficient excitatory synaptic  transmission. On spine heads, NMDARs and other glutamate receptors form a highly stable macromolecular complex (postsynaptic density, or PSD) directly opposed to presynaptic glutamate release sites. NMDARs are anchored at the PSD by cytoskeletal proteins such as spectrin and -actinin (Wechsler and Teichberg, 1998; Dunah et al., 2000). Additionally, scaffolding proteins including PSD-95 and SAP102 physically tether the NMDAR to other ion channels, cytoskeletal elements, and cell-adhesion molecules, thus linking PSD components together (Müller et al., 1996; Kim and Sheng, 2004). Synaptic receptor retention is mainly controlled at the NMDAR C-terminus, which contains key domains that regulate its surface stability (Gladding and Raymond, 2011). Truncated NMDAR subunits lacking the C-terminus are excluded from the synapse, while mutations in C-terminal NMDAR internalization motifs increase synaptic receptor stability (Prybylowski et al., 2005). Kinases and phosphatases can also control NMDAR synaptic expression by altering the phosphorylation state of C-terminal Tyrosine residues. For instance, GluN2B phosphorylation at Y1472 by the kinase Fyn occludes the NMDAR YEKL internalization motif, and thus blocks receptor endocytosis (Prybylowski et al., 2005). By binding the NMDAR C-terminus, scaffolding proteins occlude internalization motifs and also promote NMDAR synaptic stability by physically coupling the NMDAR to kinases such as Fyn (Tezuka et al., 1999).  5  1.1.5  Extrasynaptic NMDARs NMDARs are also stably expressed at extrasynaptic sites (Ex-NMDARs). Immunogold  EM imaging studies have detected receptor pools along dendritic shafts and spine-necks, which are concentrated at these locations via associations with scaffolding proteins and adhesion factors (Petralia et al., 2010, 2012). Ex-NMDARs can be activated by glutamate spillover following synchronous afferent stimulation (Rusakov and Kullmann, 1998), or by neurotransmitter release from glia (Fellin et al., 2004), particularly in neuropathological states. Although the exact role of Ex-NMDARs in neuronal signaling remains elusive, several physiological functions have been proposed. Some reports suggest that Ex-NMDAR signaling is most critical during early neuronal development. Approximately 75% of surface NMDARs are located extrasynaptically at 1 week in vitro (Tovar and Westbrook, 1999, 2002), and this percentage rapidly declines with synaptic maturation (Harris and Pettit, 2007). Ex-NMDARs concentrate at points of contact with adjacent axon terminals and shafts, and may thus help form novel excitatory synapses (Petralia et al., 2010). Other groups suggest that synaptic and extrasynaptic NMDARs have opposing roles in synaptic plasticity: while synaptic NMDAR activation induces LTP, Ex-NMDARs mediate LTD (Massey et al., 2004; Papouin et al., 2012). Alternatively, Ex-NMDARs may provide a readily-available receptor pool that can be quickly mobilized to the synapse upon demand during metaplasticity and synaptic remodelling (Carroll and Zukin, 2002). Finally, since endocytic zones are primaily located extrasynaptically, ExNMDARs could consist of receptors in transit to or from synaptic sites, at an intermediate stage between endocytosis/exocytosis and synaptic targeting (Blanpied et al., 2002; Lau and Zukin, 2007).  6  1.1.6  NMDAR surface distribution The balance between synaptic and extrasynaptic NMDAR activity can have profound  effects on neuronal function, and NMDAR surface localization is thus tightly regulated in an activity-dependent manner (Groc and Choquet, 2006; Hardingham and Bading, 2010). Surface NMDAR distribution is regulated by lateral diffusion of the receptor along the plasma membrane, as well as by endocytic/exocytic mechanisms (Tovar and Westbrook, 2002; Groc et al., 2004, 2006). Both these processes are modulated by post-translational modifications on the NMDAR C-terminus (Gladding and Raymond, 2011). For instance, GluN2B C-terminal phosphorylation at S1480 by casein-kinase II disrupts NMDAR-PSD-95 interactions, thus decreasing NMDAR synaptic anchoring and promoting its internalization or lateral diffusion (Chung et al., 2004; Sanz-Clemente et al., 2010). Activation of synaptic NMDARs elevates GluNB-S1480 phosphorylation and suppresses NMDAR surface expression, whereas selective Ex-NMDAR stimulation reduces S1480 phosphorylation, suggesting that NMDARs differentially modulate GluN2B surface stability via activity-dependent feedback mechanisms. The Ca++-dependent STriatal-Enriched tyrosine Phosphatase (STEP) also promotes clathrinmediated NMDAR endocytosis by dephosphorylating the C-terminal GluN2B-Y1472 residue, which exposes the YEKL internalization motif for AP-2 binding (Braithwaite et al., 2006). Alternatively, C-terminal NMDAR cleavage by calpain removes domains associated with synaptic receptor retention and exposes membrane-proximal internalization signals (Scott et al., 2004), thus promoting NMDAR endocytosis. Once internalized, receptors are either degraded or recycled to other sites on the plasma membrane, which alters their surface localization. Activation of both STEP and calpain are Ca++-dependent (Braithwaite et al., 2006; Doshi and Lynch, 2009) further demonstrating that surface receptor expression can be modulated by 7  NMDAR activity. The surface mobility of GluN2A and GluN2B subunits is differentially regulated (Sanz-Clemente et al., 2013). GluN2B-type NMDARs have higher diffusion rates than GluN2A subunits (Groc et al., 2006) and are subject to clathrin-mediated endocytosis to a greater extent than GluN2A-containing NMDARs (Lavezzari et al., 2004). These differences are attributed to sequence diversity in the C-terminal domains of each subunit (Cousins et al., 2009), and may help explain the relative extrasynaptic enrichment of GluN2B-containing NMDARs.  1.2  1.2.1  Glutamate excitotoxicity  Role of NMDARs in excitotoxicity Cytosolic Ca++ is an important second messenger. During synaptic transmission, transient  rises in intracellular Ca++ stimulate pathways involved in synaptic plasticity, development, and cell survival or death (Bading, 2004). Due to its incomplete desensitization and high Ca++ permeability, the NMDAR contributes substantially to activity-dependent rises in cytosolic Ca++ (Hardingham, 2009). However, the degree of Ca++ entry through the NMDAR dictates downstream cellular outcomes: while NMDAR blockade induces cell death (Gould et al., 1994), high or sustained receptor stimulation is also neurotoxic, by a phenomenon termed ‘glutamate excitotoxicity’ (Olney, 1969; Choi, 1987). Hence, cellular responses to NMDAR stimulation follow a bell-shape curve depending on the degree of Ca++ entry, such that only moderate levels of NMDAR activity are neuroprotective (Hardingham and Bading, 2003, 2010). During periods of prolonged NMDAR stimulation, excess mitochondrial Ca++ activates the mitochondrial permeability transition (MPT). This induces mitochondrial depolarization and bioenergetic failure, release of pro-apoptotic factors, and synthesis of reactive oxygen species (ROS) (White 8  and Reynolds, 1996; Dong et al., 2009; Seo et al., 2009). Ca++ can also generate ROS directly, by activating enzymes such as NOX (NADPH oxidase) and nNOS (neuronal nitric oxide synthase) (Brennan et al., 2009; Forder and Tymianski, 2009). ROS in turn induces DNA damage, ER and mitochondrial stress, and pro-apoptotic signaling (Forder and Tymianski, 2009). Finally, damaging proteases, DNAases, and lipases are also activated by high cytosolic Ca++ levels, and these contribute to excitotoxicity by degrading cellular components (Forder and Tymianski, 2009). Recent work suggests that it is not only the degree of Ca++ influx, but also the site of its entry, that determines whether neuroprotective or neurotoxic pathways are activated. While Ca++ entry through synaptic NMDARs induces pro-survival signaling cascades, comparable levels of Ca++ influx through Ex-NMDARs triggers pro-death signaling (Hardingham et al., 2002; Léveillé et al., 2008).  1.2.2  Synaptic vs. extrasynaptic NMDAR signaling Synaptic NMDAR activation induces antioxidant defenses, suppression of cell death  pathways, and the maintenance of mitochondrial health (Hardingham and Bading, 2003). This is accomplished either by post-translational protein modifications on signaling proteins or changes in neuronal gene expression. For instance, Ca++ influx through synaptic NMDARs activates the cAMP Response Element Binding Protein (CREB) via CaMKIV- or Ras-ERK1/2-mediated phosphorylation at its activator site (Ser133) (Matthews et al., 1994; Wu et al., 2001). CREB is an activity-dependent transcription factor; it induces pro-survival gene expression, and thus mediates long-lasting neuroprotection even after NMDAR activity is ceased (Lonze and Ginty, 2002; Papadia et al., 2005). In culture, selective activation of synaptic NMDARs leads to sustained phosphorylation of the ERK1/2 MAP kinase, which promotes CREB activation and 9  inhibits the pro-death protein BAD (Chandler et al., 2001; Adams and Sweatt, 2002; Ivanov et al., 2006; Xu et al., 2009). Synaptic NMDARs also promote neuroprotective pathways by inducing anti-oxidant gene expression (Papadia et al., 2008) and suppressing intrinsic apoptotic signaling (Léveillé et al., 2010). Prolonged activation of the PI3K-Akt pathway by synaptic NMDAR stimulation induces nuclear export and inactivation of the forkhead box protein O (FOXO) family of transcription factors, which induce the expression of pro-apoptotic genes such as FasL and Bim (Brunet et al., 1999; Gilley et al., 2003). Akt also directly phosphorylates and inhibits apoptotic pathway components, such as BAD and Caspase 9 (Brunet et al., 1999). In contrast, Ex-NMDARs couple to pro-death signaling pathways, induce ROS synthesis, and promote mitochondrial depolarization, resulting in Ca++-dyshomeostasis, energy depletion, and subsequent cell death (Hardingham, 2009). This is in part mediated by direct suppression of synaptic NMDAR mediated pro-survival signaling. For instance, extrasynaptic Ca++ influx shuts off CREB via Ser133 dephosphorylation (Hardingham et al., 2002). In culture, selective ExNMDAR activation shuts off the pro-survival Ras-ERK1/2 MAP kinase and the PI3K-Akt pathways, thus inhibiting their neuroprotective functions (Ivanov et al., 2006; Papadia et al., 2008). On the other hand, Ex-NMDARs can activate a distinct set of pathways not associated with synaptic receptors. Stimulation of extrasynaptic but not synaptic NMDARs induces sustained activation of p38 MAPK pro-death signaling, via calpain activation, and selective inhibition of this pathway is neuroprotective in cortical neuronal cultures (Xu et al., 2009). ExNMDARs also induce a selective program of pro-death gene expression, leading to long-lasting activation of neuronal death mechanisms (Zhang et al., 2007). Ex-NMDARs promote nuclear translocation of FoxO3a, a member of the FOXO pro-death transcription factors, via p38, JNK, and PP1 activation (Dick and Bading, 2010). During hypoxic or ischemic conditions, Ex10  NMDAR activation triggers expression of Clca 1, which encodes the neurotoxic calciumactivated chloride channel (Wahl et al., 2009). Notably, simultaneous activation of synaptic and Ex-NMDARs by bath application of glutamate induces pro-death signaling, suggesting that ExNMDAR signaling dominates over synaptic pathways (Hardingham et al., 2002). The molecular basis for the differences in synaptic/extrasynaptic NMDAR signaling may be attributed to subcellular compartmentalization of signaling components. For instance, p38 MAPK, but not ERK1/2, is extrasynaptically enriched (Xu et al., 2009). Signaling proteins associated with synaptic NMDARs are tethered within the PSD meshwork, whereas extrasynaptic components may not have access to the synapse due to structural shielding by the PSD (Papadia and Hardingham, 2007). Second, differences in subunit composition could help explain the synaptic/extrasynaptic NMDAR dichotomy. GluN2A-containing receptors are synaptically enriched and are proposed to couple to pro-survival signaling, whereas GluN2Bcontaining receptors are primarily extrasynaptic and preferentially induce pro-death pathways (Tovar and Westbrook, 1999; Liu et al., 2007) . Finally, the dichotomy of synaptic/extrasynaptic NMDARs signaling could be attributed to differences in the way each receptor pool is activated. While synaptic NMDARs are transiently activated by trans-synaptic glutamate release, ExNMDARs are tonically activated by elevated levels of ambient glutamate. Differences in receptor stimulation patterns could induce distinct intracellular calcium transients, which could lead to the activation of distinct downstream pathways (Hardingham and Bading, 2010).  11  1.3  1.3.1  Huntington disease  Clinical and pathological features Huntington disease (HD) is a late onset neurodegenerative condition for which there is no  effective cure or treatment. Prevalence of the disease is relatively rare, ranging from 1/10,000 to 1/20,000 in the Caucasian population (Roos, 2010). The average age of onset is 30-50 years, although in rare cases (10%), symptoms may present as early as the first or second decade (Roos, 2010). The disease is clinically characterized by a triad of motor, cognitive, and psychiatric symptoms. Motor disturbances are manifested as uncontrolled involuntary movements (termed “chorea”), coupled with rigidity, postural instability, impaired motor coordination, difficulty initiating movements, and difficulty talking or swallowing (Margolis and Ross, 2003). Cognitive and psychiatric disturbances include memory deficits, confusion, as well as irritability, apathy, anxiety, and mood swings (van Duijn et al., 2007). The disease is fatal, although the primary cause of death results from secondary complications arising from these symptoms, including choking, falls, pneumonia, or suicide (Sørensen and Fenger, 1992; Di Maio et al., 1993). Neuronal atrophy in HD predominates in the striatum (Graveland et al., 1985; Vonsattel et al., 1985), a region responsible for integrating and modulating motor inputs from cortical and thalamic afferents to control and fine-tune motor behaviour (Albin et al., 1989). GABAergic medium Spiny Projection Neurons (SPNs), which encompass >90% of the neurons in the striatum, are primarily vulnerable to death, exhibiting up to ~95% of neuronal loss in advanced stages of HD (Vonsattel et al., 1985). The remaining 5-10% of neurons, primarily consisting of interneurons, are relatively spared (Ferrante et al., 1985, 1987). Cortical and hippocampal regions also degenerate although less severely (Vonsattel et al., 1985; Reddy et al., 1999). 12  1.3.2  Wildtype vs. mutant huntingtin HD is caused by a polyglutamine (polyQ) expansion near the N-terminus of the protein  huntingtin, encoded by expanded (> 35) CAG triplet repeats in exon 1 of the Htt gene (Huntington's Disease Collaborative Research Group, 1993). As the mutation is inherited in an autosomal dominant manner, a person with HD has a 50% likelihood of transmitting the mutant gene to his/her offspring. Repeat length is inversely correlated with age of onset (Rosenblatt et al., 2006). The wildtype form of huntingtin (wtHtt, <35 polyQ) interacts intimately with intracellular processes and thus plays critical roles in normal cellular function (Zuccato et al., 2010). This is reflected in the fact that homozygous deletion of the wtHtt gene results in early embryonic lethality (Nasir et al., 1995). WtHtt promotes neuronal development (Reiner et al., 2003), blocks pro-death pathways (Rigamonti et al., 2000), facilitates transcription of prosurvival factors such as BDNF (Zuccato et al., 2001), regulates vesicular and axonal transport (Caviston and Holzbaur, 2009), and modulates synaptic activity (Smith et al., 2005). MtHtt contains a polyQ expansion of >35, which results in protein misfolding, aggregation, and malfunction (Zuccato et al., 2010). Proteolysis of the mutant protein into Nterminal fragments by calpains and caspases is an important prerequisite for mtHtt-induced toxicity, as mutation of the calpain-cleavage sites of Htt reduces cell death in vitro (Wellington et al., 2002; Gafni et al., 2004). mtHtt interferes with vital cellular processes, resulting in cell death and tissue degeneration (Zuccato et al., 2010; Eidelberg and Surmeier, 2011). Particularly, mtHtt impairs normal gene transcription (Kim et al., 2008), Ca++ homeostasis and mitochondrial function (Panov et al., 2003), protein trafficking (Trushina et al., 2004), endocytotic/exocytotic mechanisms (Trushina et al., 2006), and the ubiquitin-proteasomal system (Bence et al., 2001), by directly interfering with these processes. 13  1.3.3  Mouse models of HD Genetic mouse models of HD have been useful in elucidating the natural history and  mechanisms of the disease, as well as in testing novel therapies. Three main types of HD mouse models have been developed (Levine et al., 2004; Zuccato et al., 2010). In HD ‘knock-in’ models (ex. HdhQ111 and HdhQ150), expanded CAG repeats are introduced into the mouse Htt gene (Wheeler et al., 2000; Lin et al., 2001), most accurately replicating the genetic mutation in humans. However, knock-in models do not express robust motor or neuropathological deficits until ~2 years of age, and are thus mainly helpful for studying early disease mechanisms (Heng et al., 2007). In contrast, transgenic mice expressing exon 1 of the human Htt gene with 115 or 150 CAG (R6/1 and R6/2, respectively), exhibit rapid onset of motor deficits, and die at ~8-10 months and 4 months respectively (Mangiarini et al., 1996; Davies et al., 1997). However, R6/1 and R6/2 mice lack full-length Htt and do not exhibit the striatal neuronal loss that is a hallmark of the human disease; hence, these models are mainly useful for therapeutic screening (Gil and Rego, 2009). A third category of HD mouse models express full-length human huntingtin with its promoter, intronic, and upstream/downstream regulatory elements in the Bacterial or Yeast Artificial Chromosome (BAC or YAC) (Hodgson et al., 1999; Gray et al., 2008). YAC mice have been developed that express either 18 (normal) or 46, 72, and 128 (pathological) CAG repeats, in an identical tissue- and developmental-specific manner as the murine Htt gene (Hodgson et al., 1999). The YAC model accurately mirrors the natural history of the human disease, with synaptic and cognitive deficits that precede neurodegeneration (Hodgson et al., 1999; Van Raamsdonk et al., 2005, 2007; Milnerwood and Raymond, 2007). Among these, the YAC128 line on the FVB/N strain is of particular interest, as it exhibits the lowest inter-animal behavioural variability, and cognitive and behavioural deficits occur at an earlier age than other 14  YAC strains (Slow et al., 2003) These mice display selective striatal neuronal loss that correlates with motor dysfunction, a biphasic activity profile with hyperkinesia at 3 months followed by hypokinesia at 12 months, and striatal and cortical atrophy at 9 and 12 months, respectively (Slow et al., 2003). However, in contrast to the loss in body weight observed in human HD patients (Aziz et al., 2008), YAC128 mice exhibit enhanced weight gain (Van Raamsdonk et al., 2006), which has been attributed to a higher expression level of humant Htt in the YAC128 model (Van Raamsdonk et al., 2006; Pouladi et al., 2010).  1.3.4  MtHtt-induced calcium dyshomeostasis Ca++ dyshomeostasis is a central component of HD-associated striatal pathology. It is in  part attributed to mtHtt-mediated disruption of intracellular Ca++ handling processes (Zuccato et al., 2010). In the cytosol, regulated mitochondrial and ER-mediated Ca++ uptake mechanisms maintain intracellular Ca++ levels within a physiological range. mtHtt directly interferes with these processes, resulting in Ca++ dyshomeostasis and cellular toxicity. First, mtHtt associates aberrantly with the mitochondrial outer membrane (OM) and forms cation-selective pores, or interacts directly with mitochondrial OM proteins (Panov et al., 2003; Wang et al., 2009; Oliveira, 2010). This induces mitochondrial dysfunction, membrane depolarization, and release of Ca++ and pro-apoptotic factors. Second, mtHtt binds to huntingtin-associated protein 1 (HAP1) and thereby associates with inositol triphosphate receptors (IP3Rs), which regulate Ca++ mobilization from ER stores. This aberrant interaction sensitizes the receptors to activation by IP3, thus enhancing their open-probability and promoting Ca++ mobilization from the ER to the cytosol (Tang et al., 2003). Finally, oligonucleotide DNA arrays demonstrate that mtHtt suppresses the transcription of proteins involved in Ca++ homeostasis, including the calcium 15  ATPase and the type 1 ryanodine receptor (RYR1) (Luthi-Carter et al., 2000), thus suppressing physiological Ca++ handling processes.  1.3.5  Glutamate excitotoxicity in HD Htt is ubiquitously expressed, and regional mtHtt expression patterns do not correlate  with neuronal vulnerability, even within the striatum (Fusco et al., 1999). This confounding observation suggests that non cell-autonomous factors are additionally involved in the selective vulnerability of mtHtt-expressing SPNs to neuronal death (Palop et al., 2006; Eidelberg and Surmeier, 2011). Selective striatal degeneration in HD has been recently attributed to corticostriatal network dysfunction, and in particular glutamate excitotoxicity (Eidelberg and Surmeier, 2011). Early experiments demonstrated that intrastriatal injections of glutamate receptor agonists in rodents and primates induced striatal lesions and motor dysfunction similar to that of HD patients (McGeer and McGeer, 1976; Hantraye et al., 1990; Ferrante et al., 1993). Additionally, levels of the NMDAR agonist quinolinate (QUIN) are elevated in human HD striatum (Guidetti et al., 2004, 2006). Together, these early studies suggested a role of aberrant glutamatergic signaling in HD-assocaited striatal death. Importantly, corticostriatal dysfunction occurs prior to onset of overt motor or neuropathological symptoms, and may thus be an important trigger leading to disease progression (Milnerwood et al., 2006, 2010; Milnerwood and Raymond, 2007).  16  1.3.6  Synaptic dysfunction in HD: Role of Ex-NMDARs Recent work has focused on delineating the mechanisms associated with corticostriatal  network dysfunction in HD (Eidelberg and Surmeier, 2011). Selective striatal excitotoxicity can be attributed to presynaptic, postsynaptic, or astrocytic processes, or a combination of the three. First, presynaptic glutamate release is elevated at early stages of HD (Joshi et al., 2009), although mtHtt has also been shown to dirsupt axonal vesicular transport and exocytosis (Li et al., 2001, 2003; Gunawardena et al., 2003; Szebenyi et al., 2003). Second, mtHtt decreases functional expression of the glial glutamate transporter GLT-1, either by suppressing its transcription or mediating post-translational modifications on the transporter (Behrens et al., 2002; Shin et al., 2005; Huang et al., 2010). This is proposed to reduce the efficacy of glial glutamate uptake and elevate neurotransmitter spillover to extrasynaptic sites, resulting in aberrant Ex-NMDAR activation. Third, postsynaptic processes, particularly those associated with NMDAR overactivation, could be involved in the selective death of SPNs. Early studies from our lab reported elevated NMDAR currents in mtHtt-transfected HEK293 cells, acutely dissociated and cultured mtHtt-expressing SPNs, and HD mouse brain tissue (Chen et al., 1999; Zeron et al., 2001, 2002; Li et al., 2003). These changes are specific to striatal GluN2B- but not GluN2A-containing NMDARs, are present from birth (Fan et al., 2007), and are associated with enhanced mitochondrial depolarization, activation of pro-apoptotic pathways, and enhanced susceptibility to NMDA-induced toxicity ( Zeron et al., 2001, 2002, 2004; Fan et al., 2009, 2012). Notably, mtHtt-induced NMDAR potentiation does not correlate with changes in protein expression (Chen et al., 1999; Li et al., 2003; Fan et al., 2007), but rather, with enhanced NMDAR surface expression as a result of accelerated forward trafficking (Fan et al., 2007).  17  Most recently, a shifted balance of synaptic to extrasynaptic NMDAR surface localization has been shown to contribute to increased susceptibility of striatal SPN to NMDA-induced toxicity in HD (Milnerwood et al., 2010). Elevated GluN2B-type Ex-NMDAR expression and currents are observed in presymptomatic (1 month-old) YAC128 mice, and these changes were confirmed by subcellular fractionation on striatal tissue to isolate extrasynaptic and synaptic synaptosomal components (Milnerwood et al., 2010; Gladding et al., 2012). Elevated ExNMDAR signaling could contribute to the selective loss of SPNs in HD. In fact, Ex-NMDAR blockade attenuates striatal mtHtt toxicity, pathology, and behavioural abnormalities in the YAC128 mice ( Okamoto et al., 2009; Milnerwood et al., 2010). The observation that elevated Ex-NMDAR expression is GluN2B subunit-specific is consistent with reports that these subunits exhibit high surface mobility rates with short dwell times in the synapse (Tovar and Westbrook, 2002; Groc et al., 2006), and are the most abundant NMDAR subtype in the striatum (Watanabe et al., 1993; Landwehrmeyer et al., 1995). Additionally, the role of GluN2B-type Ex-NMDARs in HD-associated excitotoxicity is consistent with the notion that GluN2B subunits couple preferentially to pro-death pathways (Lujan et al., 2012; Martel et al., 2012).  1.3.7  Memantine: An uncompetitive Ex-NMDAR blocker The use of NMDAR blockers to modulate glutamate excitotoxicity has yielded little  therapeutic promise, due to the receptor’s critical role in excitatory synaptic transmission. In light of the causal link between Ex-NMDARs and excitotoxicity, pharmacological advances have aimed to directly block neurotoxic Ex-NMDAR signaling, while preserving neuroprotective synaptic receptor activity (Papadia and Hardingham, 2007). A potential pharmaceutical candidate is memantine: at low concentrations (1-10μM) this drug mildly attenuates NMDAR 18  signaling by blocking pathological (extrasynaptic) NMDAR signaling, while relatively sparing physiological (synaptic) receptor activity (Chen and Lipton, 2006; Lipton, 2007). The selectivity of memantine for Ex-NMDARs is primarily attributed to the drug’s particular pharmacological properties: memantine is an open-channel blocker with a fast off-rate, voltage-dependent blocking/unblocking kinetics, and a relatively low affinity (IC50 of ~1μM) in comparison to other conventional NMDAR blockers such as MK-801 (Lipton, 2004, 2007; Lipton and Chen, 2005). Due to these attributes, memantine does not accumulate in the channel pore, and is primarily effective at blocking NMDARs that are tonically open by ambiently elevated glutamate, as occurs extrasynptically. In contrast, it has relatively less access to synaptic NMDARs, which are activated very transiently by presynaptic glutamate release during transsynaptic signaling (Lipton, 2004, 2007; Hardingham and Bading, 2010). However, the selectivity of memantine for Ex-NMDARs is highly dependent on its effective concentration: at higher concentrations (30μM), memantine blocks both synaptic and Ex-NMDARs, and induces neuronal death (Okamoto et al., 2009). The selectivity of memantine for Ex-NMDARs has been elegantly demonstrated in culture (Okamoto et al., 2009; Xia et al., 2010) but was not confirmed in vivo. However, when administered to mice at a low dose, memantine was shown to effectively uncouple the NMDAR from pro-death, but not pro-survival, signaling pathways, suggestive of selective Ex-NMDAR blockade as observed in vitro. In fact, treatment of 2 month-old YAC128 mice with low-dose (1mg/kg/d) memantine for 2 months fully rescues CREB signaling and deficits in motor learning (Milnerwood et al., 2010). In a similar study, low-dose (1mg/kg/d) memantine treatment for 10 months reversed late-stage striatal pathology, and improved rotarod performance in 12 monthold YAC128 mice, whereas high-dose (30mg/kg/d) treatment worsened the HD phenotype. 19  Memantine is clinically well-tolerated, and small clinical studies for its treatment of human HD patients have been promising (Beister et al., 2004; Ondo et al., 2007).  1.4  Coupling Ex-NMDARs to aberrant signaling in HD The signaling pathways downstream of Ex-NMDARs in HD remain widely unexplored.  Our lab and others have identified potential candidate pathways associated with Ex-NMDARinduced toxicity in HD, including activation of the protease calpain, p38 MAPK signaling, and reduction of nuclear CREB activity. However, whether these pathways are downstream of mtHttmediated Ca++ dysregulation or aberrant Ex-NMDAR activity remains unclear.  1.4.1  Calpain signaling Calpain is a cytosolic cysteine protease activated by Ca++. Two calpain isozymes exist,  namely μ-calpain (or calpain I), and m-calpain (or calpain II), with identical substrate specificity but differential sensitivity to Ca++: μ-calpain is activated by μM concentrations of cytosolic Ca++, and is therefore sensitive to supra-physiological (1-5 μM) rises in Ca++, as occurs during excitotoxicity (Goll et al., 2003; Vosler et al., 2008). In contrast, m-calpain is activated by mM Ca++ concentrations and is thus likely activated via more complex pathways, such as ERK1/2MAPK and BDNF signaling (Glading et al., 2000; Vosler et al., 2008; Briz et al., 2013). Calpain activity is modulated endogenously by calpastatin (Todd et al., 2003). Calpains participate in cytoskeletal remodelling, cell division and migration (Huttenlocher et al., 1997), neuronal development (Kaczmarek et al., 2012), and synaptic plasticity (Amini et al., 2013; Briz et al., 2013). In particular, the role of calpains in pro-death signaling is a hallmark of several neuropathological states (Vosler et al., 2008). A high degree of 20  crosstalk exists between caspases and calpains, and the parallel action of both proteins accelerates apoptotic cell death (Nakagawa and Yuan, 2000). Like caspases, calpain hydrolyses (and inactivates) the pro-survival factor Bcl-2, and cleaves (and activates) the pro-apoptotic factors Bax (Choi et al., 2001) and Bid (Mandic et al., 2002), as well as pro-caspases (Nakagawa and Yuan, 2000). The protease also induces cell death by modulating downstream Ca++dependent pathways. During excitotoxic stress, calpain indirectly activates calcineurin, a phosphatase involved in Ca++-induced cell death, by cleaving and inactivating its endogenous inhibitor cain/cabinI (Kim et al., 2002). Similarly, calpain activity shuts off pro-survival CREB signaling by cleaving and inactivating CaMKIV (McGinnis et al., 1998). Finally, calpains cleave and inactivate the striatal-enriched tyrosine phosphatase (STEP), facilitating p38 MAPK prodeath signaling (Xu et al., 2009). Interestingly, calpain can potentiate its own activity in a feedforward loop: the protease cleaves Ryanodine and IP3 receptors (IP3Rs) (Kopil et al., 2012; Pedrozo et al., 2010), mobilizing Ca++ into the cytosol and further inducing calpain activation. NMDARs couple to calpain in both physiological and pathological states. Selective activation of NMDARs but not voltage-gated Ca++ channels is sufficient to activate calpain in cultured hippocampal neurons (Adamec et al., 1998). Important developmental signaling processes depend on NMDAR-mediated calpain activity (Abe and Takeichi, 2007). Additionally, calpain activation during excitotoxicity is downstream of NMDARs (Brustovetsky et al., 2010), and its selective inhibition prevents NMDAR-induced excitotoxicity in vivo (Nimmrich et al., 2008). Notably, selective activation of extrasynaptic but not synaptic NMDARs activates calpain in cultured neurons (Xu et al., 2009). Calpains can couple Ca++ influx through the receptor by activity-dependent surface translocation (Saido et al., 1992), or by forming direct associations with the NMDAR complex via Cdk5 ( Husi et al., 2000; Hawasli et al., 2007). 21  Accumulating evidence has implicated aberrant calpain activity in HD pathogenesis. Increased activation of μ-calpain and m-calpain has been detected in post-mortem human HD caudate and putamen (Saito et al., 1993; Gafni and Ellerby, 2002). A similar trend is observed in HD animal models: striatal calpain activity is elevated in a rat model of HD treated with 3nitropropionic acid (3-NP), and its inhibition reduced striatal neuropathology in this model (Bizat et al., 2003). Elevated expression and activity of calpain has also been reported in HD knock-in models (Gafni et al., 2004). Finally, our group detected elevated calpain activity in the striatum of 1-2month-old YAC128 mice (Cowan et al., 2008; Gladding et al., 2012). Calpains participate in mtHtt-induced toxicity by cleaving the mutant protein in a polyQ length-dependent manner, either in parallel or sequentially with caspases (Kim et al., 2001; Gafni and Ellerby, 2002; Sun et al., 2002; Bizat et al., 2003). Calpains also contribute to HD-associated synaptic dysfunction, by altering NMDAR functional expression. Calpain-mediated C-terminal cleavage of GluN2(A-C) subunits removes regions involved in synaptic NMDAR retention (Guttmann et al., 2002; Simpkins et al., 2003). This alters receptor surface stability and localization, although calpain-cleaved receptors remain functional when expressed at the surface (Guttmann et al., 2002; Simpkins et al., 2003; Doshi and Lynch, 2009; Gladding et al., 2012). Removal of the GluN2B C-terminus by calpain exposes membrane-proximal binding sites for the endocytic AP-2 complex, promoting clathrin-dependent receptor endocytosis ( Roche et al., 2001; Cowan et al., 2008; Scott et al., 2004). On the other hand, calpain hydrolyses the AP-2 subunits α and β2, which broadly disrupts receptor endocytosis and thus potentiates NMDAR activity (Rudinskiy et al., 2009; Gladding and Raymond, 2011). Our group has reported elevated calpain cleavage of GluN2B subunits in presymptomatic YAC128 mice (Cowan et al., 2008, Gladding et al., 2012). In a recent study, selective calpain inhibition suppressed mtHtt-induced 22  Ex-NMDAR expression, directly implicating the enzyme in HD-associated Ex-NMDAR mislocalization (Gladding et al., 2012). However, whether calpain is a direct cause or consequence of aberrant Ex-NMDAR signaling in vivo remains unclear.  1.4.2  CREB-dependent gene expression The cAMP-response element (CRE) binding protein (CREB) is a stimulus-inducible bZIP  transcription factor which is critical to normal neuronal function. It mediates expression of a broad range of genes involved in synaptic plasticity, neuronal development, circadian rhythms, and cell survival (Lonze and Ginty, 2002). CREB activity is induced by phosphorylation at Ser133 within its kinase-inducible domain (KID), which facilitates interactions with the transcriptional coactivator CREB-binding protein (CBP) (Chrivia et al., 1993). Once recruited to the CRE, CBP directly interacts with basal transcriptional machinery, promotes formation of the pre-initiation complex, and increases DNA accessibility via its endogenous histone acetyltransferase (HAT) activity ( Chrivia et al., 1993; Kwok et al., 1994; Bannister and Kouzarides, 1996; Martinez-Balbas et al., 1998). CREB-mediated gene expression is induced by extracellular stimuli such as growth factors and neuropeptides, as well as synaptic stimulation (Lonze and Ginty, 2002). Activation of adenylyl cyclase or receptor tyrosine kinases stimulates PKA and Ras-Erk/ PI3K-Akt pathways, respectively, both of which converge upon CREBSer133 phosphorylation (Gonzalez and Montminy, 1989; Patapoutian and Reichardt, 2001; Johannessen et al., 2004). Alternatively, glutamatergic synaptic transmission activates CREB-mediated gene expression. Stimulation of NMDARs or L-type Ca++ channels activates Ca++-mediated RasERK1/2 MAPK or CaMKIV, which can both phosphorylate CREB and CBP at Ser 133 and 301, respectively ( Dolmetsch et al., 2001; Impey et al., 2002; Bengtson and Bading, 2012). Although 23  these cascades converge upon a common substrate, their differential activation kinetics modulate the temporal profile of CREB-mediated transcription. While CaMKIV-induced CREB phosphorylation is highly transient and occurs seconds after synaptic stimulation, activation of CREB via Ras-ERK pathways is more delayed and mediates prolongued CREB-mediated gene expression (Wu et al., 2001). Interestingly, NMDARs have opposing roles on CREB activity, depending on the site of Ca++ entry: while synaptic NMDARs trigger CREB phosphorylation, Ex-NMDARs activate dominant CREB shut-off pathways (Hardingham et al., 2002; Léveillé et al., 2008; Kaufman et al., 2012), likely via PP2A activation (Wadzinski et al., 1993). CREB regulates critical neuroprotective processes, and its dysfunction is implicated in several neuropathologies (Saura and Valero, 2011). CREB-induced transcription promotes normal mitochondrial biogenesis and function (Lee et al., 2005), triggers the expression of prosurvival genes and neurotrophins such as BDNF (Wilson et al., 1996; Obrietan et al., 2002), and modulates neuroprotective synaptic transmission (Puddifoot et al., 2012). In HD, dysregulation of CREB-mediated gene expression could contribute to disease pathology. Genetic suppression of CREB signaling induces HD-like striatal atrophy, and exacerbates behavioural deficits in YAC128 mice (Mantamadiotis et al., 2002; Choi et al., 2009), whereas its activation ameliorates the HD phenotype (DeMarch et al., 2008; Choi et al., 2009). Consistent with these observations, striatal CREB activity is decreased in HD prior to neuronal death (Gines et al., 2003; Sugars et al., 2004; Cui et al., 2006; Milnerwood et al., 2010), and could thus contribute to disease pathology (Choi et al., 2009; Milnerwood et al., 2010). In particular, our lab has detected decreased nuclear CREB phosphorylation levels in the striatum of 1 and 4 month-old YAC128 mice, an age that precedes striatal atrophy (Milnerwood et al., 2010).  24  MtHtt may dysregulate CREB signaling by physically interfering with CREB-associated transcriptional mechanisms. By directly binding CBP and other coactivators, mtHtt can disrupt formation of the transcriptional activator complex ( Steffan et al., 2000; Cui et al., 2006). mtHtt can also sterically hinder gene expression by associating with CRE DNA promoter elements (Cui et al., 2006). Alternatively, decreased CREB signaling could be attributed to mtHtt-induced dysregulation of upstream pathways, such as suppressed PKA-cAMP signaling (Gines et al., 2003), or elevated Ex-NMDAR activity (Okamoto et al. 2009, Milnerwood et al. 2010). Our lab among others has proposed a causal link between Ex-NMDAR signaling and HD-associated suppression of CREB activity. Selective Ex-NMDAR blockade with low concentration (5μM) memantine fully rescues suppressed CREB signaling induced by blockade of physiological synaptic transmission (Okamoto et al. 2009). Additionally, low-dose (1mg/kg/d) memantine treatment for 2 months fully rescues nuclear CREB phosphorylation deficits in 4 month-old YAC128 mice (Milnerwood et al., 2010). Together, these studies suggest that aberrant ExNMDAR signaling drives HD-associated deficits in CREB activity.  1.4.3  p38 MAPK signaling The p38 mitogen-activated protein kinase (MAPK) belongs to a superfamily of  serine/threonine protein kinases, which also includes the extracellular-signal regulated kinases (ERK1/2, and 5), and c-Jun N-terminal kinases (JNK). MAPK pathways transduce extracellular signals to the nucleus by the sequential phosphorylation of three kinases, which together regulate the timing and amplitude of the signal (Kyriakis and Avruch, 2001). MAPKs are responsive to hormones, growth factors, vasoactive peptides, and pro-inflammatory factors. In particular, p38 and JNK MAPKs are termed stress-activated protein kinases (SAPKs), as they mediate responses 25  to cellular stress, including DNA damage, heat shock, oxidative stress, and inflammation (Wada and Penninger, 2004; Haddad, 2005). Four isoforms of p38 exist (α, β, γ, and δ) and are activated by dual phosphorylation at Thr 180 and Tyr 182 by the kinases MKK3 and MKK6 (Raingeaud et al., 1995; Wada and Penninger, 2004). In turn, p38 activates downstream kinases involved in regulating stress-response pathways or gene expression. For instance, p38 activates the mitogen and stress-activated kinase 1 and 2 (MSK1/2), which can then activate pro-inflammatory transcription factors, such as NF-Kβ (Deak et al., 1998; Reber et al., 2009). p38 also regulates gene expression directly, by activating transcription factors involved in stress-response mechanisms, such as activating transcription factor –1 (ATF-1) and myocyte enhancer factor 2C (MEF2C) (Wada and Penninger, 2004). p38 MAPKs also mediate pro-apoptotic signaling and contribute to glutamate excitotoxicity (Kawasaki et al., 1997; Takeda and Ichijo, 2002; Porras et al., 2004; Soriano et al., 2008). During periods of oxidative stress, p38 potentiates pro-apoptotic signaling by stimulating caspase activity, Bid truncation (Choi et al., 2004), as well as mitochondrial translocation of Bax and cytochrome-c release (Park et al., 2003). Several groups have demonstrated a direct causal link between NMDAR-mediated Ca++-influx and p38 MAPK activity ( Kawasaki et al., 1997; Vincent et al., 1998). NMDARs induce activation of the RasGTPase activating protein (SynGAP) (Rumbaugh et al., 2006), or nNOS-mediated ROS synthesis (Cao et al., 2005; Soriano et al., 2008), both of which contribute to p38 activation. Notably, both nNOS and SynGAP are coupled to the NMDAR via PSD-95 (Cao et al., 2005; Rumbaugh et al., 2006), and dirsupting GluN2B-PSD-95 interactions with a synthetic peptide suppresses p38 MAPK activity and excitotoxic death (Aarts et al., 2002; Soriano et al., 2008; Fan et al., 2012). Interestingly, p38 is enriched at extrasynaptic sites, and is selectively activated by extrasynaptic but not synaptic NMDARs in culture (Xu et al., 2009), consistent with a role of 26  Ex-NMDARs in excitotoxicity. However, nuclear p38 expression has also been detected (Raingeaud et al., 1995). Our lab has proposed a causal link between p38 MAPKs and mtHtt-induced enhanced susceptibility of striatal SPNs to NMDA excitotoxicity. Fan and colleagues (Fan et al., 2012) reported elevated p38 MAPK activity in striatal tissue from 1-2 month-old YAC128 mice. Furthermore, p38 inhibition attenuated NMDA-induced toxicity in cultured YAC128 SPNs, suggesting an involvement of aberrant p38 MAPK activity in mtHtt-enhanced excitotoxic cell death. Notably, PSD-95 expression and GluN2B-PSD-95 interactions were elevated at extrasynaptic sites in YAC128 mice, and disruption of these interactions decreased mtHttassociated p38 activation and cell death, consistent with a proposed role of GluN2B-PSD-95 interactions in downstream p38 signaling and mtHtt-induced enhanced excitotoxicity (Fan et al., 2009, 2010). However, whether Ex-NMDARs drive HD-associated p38 MAPK activation in vivo remains to be determined.  27  1.5  Rationale and hypothesis MtHtt profoundly alters intracellular signaling pathways that are essential for cell  survival (Zuccato et al., 2010). In YAC128 mouse striatum, dysregulated activity of calpain, p38 MAPK, and CREB-mediated signaling pathways occurs prior the onset of HD symptoms and could contribute to the induction of pro-apoptotic pathways and subsequent neurodegeneration (Milnerwood et al., 2010; Fan et al., 2012; Gladding et al., 2012). Although these pathways share the common feature of Ca++-dependence, the exact upstream mechanisms that mediate aberrant Ca++ signaling in HD remain unclear. First, aberrant Ca++-dependent signaling could be directly attributed to mtHtt-induced disruption of intracellular Ca++-homeostasis, as discussed above (see section 1.3.4). On the other hand, Ex-NMDAR activity, which is elevated early in HD, also contributes to intracellular Ca++ dyshomeostasis, the preferential induction of pro-apoptotic pathways, and HD-associated striatal atrophy and motor deficits ( Okamoto et al., 2009; Milnerwood et al., 2010). This study aims to examine whether aberrant Ca++ signaling in mtHtt-expressing striatal neurons is a direct consequence of enhanced Ex-NMDAR activity or other effects of mtHtt. Due to the proposed role of Ex-NMDARs in HD-associated intracellular Ca++ dyshomeostasis, prodeath signaling, and striatal excitotoxicity, we hypothesize that the dysregulation of intracellular signaling in YAC128 HD striatum at early stages is primarily driven by Ca++ influx through ExNMDARs rather than mtHtt-induced Ca++ dyshomeostasis.  28  Chapter 2: Methods  2.1  Memantine treatment WT and YAC128 (line HD55) mice (Slow et al. 2003) bred on the FVB/N background  were housed and handled at the University of British Columbia (UBC) Faculty of Medicine Animal Resource Unit, according to guidelines of the UBC Animal Care Committee and the Canadian Council on Animal Care (protocol A11-0012). Animals were housed in identical conditions (2-4mice/cage) in a 12-hr light/dark cycle, with full access to food and water. Memantine treatments on WT and YAC128 mice were performed as described previously (Okamoto et al., 2009; Milnerwood et al., 2010). Memantine solutions (Tocris) were prepared in water, and solution concentrations adjusted according to daily water intake and mouse weights in each cage, to yield 1 and 10mg per kg body weight per day (mg/kg/d) doses. Memantine solutions were provided ad libitum to WT and YAC128 mice in their drinking blottles at 1 or 10mg/kg/d, starting at 2months (+ 10 days) of age, for 2 months. Control mice received water (vehicle). The 1mg/kg/d dose is estimated to result in an effective concentration at the NMDAR channel mouth of ~5-10 μM, which selectively blocks Ex-NMDARs in culture, and mimics the therapeutic dose of 10mg twice per day in humans (Okamoto et al., 2009). The 10mg/kg/d dose has not been confirmed to selectively block Ex-NMDARs, but has been shown to be neuroprotective (Hare et al. 2001, Rosi et al. 2006, Rammes et al. 2008). Drinking solutions were replaced with fresh stock twice per week. Daily solution intake and mouse weights were monitored semiweekly and semimonthly, respectively. Fresh memantine solutions were prepared on a semimonthly basis and adjusted accordingly to ensure dosing consistency throughout the treatment period. Cohorts alternated between male and female 29  mice (n=4 female cohorts, n=6 male cohorts). No differences in daily solution intake were observed between genotypes or treatment groups for either sex (Fig 8, Appendix). However, memantine treatment at both doses significantly reduced body weight in female but not male YAC128 mice (Fig 9, Appendix).  2.2  Subcellular fractionation Tissue from memantine-treated and untreated WT and YAC128 mice was collected after  memantine treatment, at which time the animals were 4 months (+ 10 days) of age. Dissections and subcellular fractionations were paired on the same day for all six treatment conditions (1 animal per condition). Fractionations were performed according to a modified protocol adapted from Pacchioni et al. (2009), courtesy of Dr. P Kalivas (Medical University of South Carolina, Charleston, SC USA), as done previously (Goebel-Goody et al., 2009; Milnerwood et al., 2010; Fan et al., 2012; Gladding et al., 2012). All procedures were performed at 4°C or on ice to minimize enzymatic activity. After drug treatment, mice were killed by decapitation following deep halothane vapour anesthesia to prevent suffering, in accordance with the UBC Animal Care Committee and the Canadian Council on Animal Care. Brains were rapidly removed and corpus striatum was dissected and homogenized in cold 0.32M sucrose buffer (0.32M sucrose, 10mM HEPES, pH 7.4). Samples were centrifuged (1,000g, 10min), then resuspended in sucrose buffer, and centrifuged again (1,000g, 10min). The resulting nuclear-enriched pellet fraction (P1) was stored at -80°C for nuclear fractionation on a later day. Supernatants (S1) from both spins were pooled and centrifuged (1,000g, 6min), and the resulting pellet (nuclear debris) was discarded. The supernatant (S1) was then centrifuged (12,000g, 20min), to separate the synaptosomal membrane 30  fraction (P2) from the microsomal and cytosolic fraction (S2). The S2 (supernatant) was stored at -80°C until later use, and the P2 (pellet) was washed twice by resuspension in 150uL HEPES/ EDTA buffer (4mM HEPES, 1mM EDTA, pH7.4) and centrifugation (12,000g, 20min). After washing, the pellet was resuspended in G-actin buffer (20mM HEPES, 100mM NaCl, 0.5% Triton X-100, pH=7.2), rotated slowly for 15-18min to solubilize the non-PSD synaptosomal fraction, and then centrifuged (12,000g, 20min). The supernatant (non-PSD) was stored at -80°C until use, and the pellet (PSD, synaptic membrane fraction) was resuspended in F-actin buffer (20mM HEPES, 0.15mM NaCl, 1% Triton X-100, 1% SDS, 1% deoxycholic acid, 1mM dithiothreitol (DTT)), solubilized by slow rotating for 45-50min, and stored at -80°C until use. All buffers contained ‘complete’ protease and phosphatase inhibitor cocktails (Roche), 15 M calpeptin (Calbiochem), 1mM EDTA, 1mM EGTA, 40mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1mM sodium orthovanadate, and 30mM sodium fluoride. The purity of the fractionation was confirmed by enrichment of the PSD marker PSD-95 in the PSD fraction, and the presynaptic marker synaptophysin in the non-PSD fraction, as assessed by western blotting (Fig 10A, Appendix).  2.3  Nuclear fractionation Nuclear fractionations were performed on striatal tissue to assess nuclear CREB activity,  as described in Milnerwood et al. (2010). As per subcellular fractionations, all procedures were performed at 4°C or on ice. Nuclear-enriched pellets (P1) obtained from subcellular fractionations were solubilized in Nuclear Buffer A (10mM HEPES-KOH, 10mM KCl, 10mM EDTA, 1.5mM MgCl2, 0.2% BSA, 1mM DTT, 0.4% NP40, pH 7.9) with gentle rotating (2022min). Samples were then centrifuged (15,000g, 10min) to isolate the crude nuclear fraction. 31  Supernatants were centrifuged (15,000g, 3min), the pellet (nuclear debris) discarded, and the resulting supernatant was stored at -80°C as the “non-nuclear supernatant” (NS) fraction. Pellets were washed in Nuclear Buffer A (without disturbing the pellet), centrifuged (15,000g, 3min), resuspended in nuclear buffer B (20mM HEPES-KOH, 400mM NaCl, 1mM EDTA, 10% Glycerol, 1mM DTT), and rotated gently (2hr) with gentle vortexing (3sec) every 30min. Samples were then centrifuged (15,000g, 15min) to isolate the nuclear matrix/extract (NE, supernatant), from the nuclear envelope (pellet). Both fractions were stored at -80°C until western blotting. As in subcellular fractionations, buffers contained protease and phosphatase inhibitor cocktails, 15M calpeptin, 1mM EDTA, 1mM EGTA, 40mM β-glycerophosphate, 20mM sodium pyrophosphate, 1mM sodium orthovanadate, and 30mM sodium fluoride. The purity of the nuclear fractionation was confirmed by enrichment of the nuclear marker histone deacetylase (HDAC) in the NE but not the NS fraction (Fig 10B, Appendix). pCREB/CREB levels were examined in the NE fraction only.  2.4  Western blotting Protein concentration was assessed by a BCA protein assay (Pierce). Freshly-thawed  samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) by heating (3-5 min, 80-85°C), in 3X protein sample buffer (PSB) (6% SDS, 0.4 mM Tris (pH 6.8), 30% glycerol, pyronin Y, 70 mg/mL DTT). Equal amounts of protein (5-15μg for non-PSD, PSD, NE, and NS, or 20-40μg for S2) were separated in 10% (w/v) SDSpolyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes by semi-dry electrophoresis (BioRad). Membranes were blocked for 1-2hrs at RT in TBS with 0.5% Tween – 20 (TBST) and 3% BSA or 5% milk, incubated in primary antibodies overnight at 4°C, then for 32  1-2hrs at RT in HRP-conjugated secondary antibodies. Blots were washed in TBST (1hour, 4x 15min washes) between incubations and prior to exposure. Blots were then visualized using an enhanced chemiluminescence substrate (ECL, Amersham) and developed by exposure to Kodak film (BioLab), except for CREB/pCREB blots, which were developed using an automated ChemiDoc XRSTM Molecular Imager (BioRad). Blots for which total p38 was probed were reprobed to quantify phosphorylated p38 bands, using alkaline-phosphatase (AP)-conjugated secondary antibodies and a Lumi-phosWB Chemiluminescent Substrate detection system (Pierce). This was done to avoid signal contamination from the first ECL probe. The following primary antibodies were used: rabbit N-terminal anti-GluN2B (AGC-003; Alomone, 1:500), rabbit anti-spectrin (cleaved) (AB38, gift from Dr. David Lynch, University of Pennsylvania, Philadelphia, PA, 1:2000), rabbit anti-pCREB (06-519, Millipore, 1:500), rabbit anti-CREB (9197, Cell Signaling, 1:500), rabbit anti-P-p38 MAPK (4511S, Cell Signaling, 1:200), mouse anti-p38 MAPK (sc-7972, 1:200 Santa Cruz, 1:200), mouse anti-PSD-95 (MA1045 Pierce, 1:500), goat anti--actin (sc-1616, Santa Cruz, 1:1500), goat anti-α-tubulin (sc-9935, Santa Cruz, 1:1500), goat anti-HDAC (sc-6268, Santa Cruz, 1:500), and mouse antisynaptophysin (S5768; Sigma, 1:1000). All primary antibodies were diluted in TBST with 3% BSA, except for anti-spectrin, which was diluted in TBST with 5% milk. The following secondary antibodies were used: anti-mouse HRP-conjugated (NA931V, Amersham, 1:5000), anti-rabbit HRP-conjugated (NA934V, Amersham; 1:5000), anti-rabbit AP-conjugated (S372, Promega, 1:5000), and anti-goat HRP-conjugated (sc-2020; Santa Cruz, 1:5000). All secondary antibodies were diluted in TBST with 1% BSA. For blots developed using Kodak film, the optical density of bands was quantified using Image J software (NIH) after background subtraction. pCREB/CREB bands were quantified with 33  Image-Lab Analysis Software (4.1, BioRad), as these were developed using the ChemiDoc XRS Molecular Imager (BioRad). -actin was used to normalize band intensities for GluN2B and PSD-95 in the synaptosomal fractions, and α-tubulin was used to normalize bands for cytosolic spectrin, as α-tubulin bands yielded a clearer signal in the cytosol. P-p38 bands were normalized to p38 bands probed on the same membrane. In the nuclear fraction, CREB and pCREB bands (probed on different membranes) were each normalized to HDAC.  2.5  Brain slice preparation Mice were anesthesized and decapitated as described previously. Brains were immersed  in ice-cold oxygenated (95% O2, 5% CO2) artificial low-calcium (cutting) cerebrospinal fluid (cutting aCSF), containing (mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 MgCl2, 0.5 CaCl2, 25 glucose, (pH 7.3–7.4, 300–310 mosmol L−1). 300 μm thick coronal slices were cut on a vibratome (Leica VT1000) and placed in a holding chamber with continuously oxygenated standard aCSF (cutting aCSF, but with 1 mM MgCl2 and 2 mM CaCl2 instead) at 37°C for 45mins-1hr.  2.6  Slice electrophysiology Electrophysiological assesment of Ex-NMDAR currents was performed on WT and  YAC128 untreated (H2O) and treated (1mg/kg/d memantine) mice, immediately after the memantine treatment period. All mice were aged 4 months + 10 days at the time of recording. Electrophysiological procedures were conducted as previously described (Milnerwood and Raymond 2007, Milnerwood et al. 2010), on 300 μm thick coronal slices. After incubation in a holding chamber, slices were transferred to a recording chamber on a DIC-IR microscope 34  (Axioskop, Zeiss) at RT, and held in place with a harp. Slices were perfused continuously (1.01.5ml/min) with oxygenated standard aCSF containing 10µM glycine, 2µM strychnine, and 100µM picrotoxin (PTX, Tocris), to potentiate NMDARs, block glycine receptors, and block GABAA receptors, respectively. Slices were allowed to equilibrate ~15-20min prior to recording. Recordings were made in standard aCSF. EPSCs were evoked (eEPSCs) by intrastriatal electrical stimulation (150µs, 25-150µA, every 20seconds) using a glass micropipette (2-5M), placed in the center of the striatum. Whole-cell voltage-clamp NMDA current (INMDA) recordings were made on a randomly-selected SPN 150-250µm ventral to the site of stimulation, with a micropipette filled with (in mM): 130 caesium methanesulphonate, 5 CsCl, 4 NaCl, 1 MgCl2, 5 EGTA, 10 Hepes, 5 QX-314, 0.5 GTP, 10 Na2-phosphocreatine, and 5 MgATP, pH 7.3, 280– 290 mosmol/L. Pipette resistance (Rp) was 3-7M. Series resistance was <25M and uncompensated, and was monitored throughout the experiment; tolerance for Rs was <50% provided Rs<30M. All recordings were made in 10µM 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) (Tocris) to block AMPA receptors (AMPARs). Once effective AMPAR blockade was confirmed, cells were depolarized to +40mV to remove voltagedependent Mg2+ block of NMDARs. A stable baseline was established (>5min), then DL-threo-benzyloxyaspartic acid (TBOA, 30µM, Tocris) was bath-applied to block GLT-1 glial uptake of glutamate and induce neurotransmitter spillover to extrasynaptic sites. NMDAR-mediated eEPSCs were tracked in the presence of TBOA for 5mins, then TBOA was washed off for 25min. Signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed in Clampfit10 (Axon Instruments). The first 3 seconds of each eEPSC (NMDA current) was normalized to peak amplitude. 35  2.7  Statistical analysis Statistical analyses were conducted using Prism 6 software (GraphPad). Data are  presented as mean + SEM. All analyses were performed using Two-Way ANOVA. For each treatment condition, significant differences between genotypes or age-groups were tested by Bonferroni’s multiple comparisons post hoc test. For each genotype, significant differences were examined between treatment dose (1 or 10mg/kg/d) and the control (H2O) by Dunnett’s multiple comparisons post hoc test, unless indicated otherwise. Overall significant effects of interaction, treatment, genotype, or age are indicated in the text.  36  Chapter 3: Results  3.1  Memantine decreases Ex-NMDAR expression in YAC128 mice A shift in the balance of synaptic-extrasynaptic GluN2B-type NMDAR expression  contributes to the selective vulnerability of YAC128 striatum to excitotoxicity (Milnerwood et al., 2010). However, the mechanisms underlying elevated Ex-NMDAR expression in HD remain elusive. To determine whether Ex-NDMAR currents themselves play a role in GluN2B-NMDAR mislocalization, we treated 2-month-old WT and YAC128 mice with memantine (1 and 10mg/kg/day) for two months to chronically block Ex-NMDAR activity, as in (Okamoto et al. 2009; Milnerwood et al., 2010). We then examined extrasynaptic GluN2B subunit levels by subcellular fractionation and western blotting on striatal tissue from memantine-treated and untreated mice. The subcellular fractionation isolates synaptic (postsynaptic density, PSD) from extrasynaptic-enriched (non-PSD) subcellular regions, and thus allowed us to directly examine protein expression in each individual surface compartment (Goebel-Goody et al., 2009). We used an N-terminal GluN2B antibody that detects both calpain-cleaved (115KDa) and full-length (180KDa) subunits, which enabled us to directly compare expression levels between cleaved and uncleaved GluN2B in each fraction. There was an enrichment of calpain-cleaved relative to fulllength GluN2B subunits in the non-PSD fraction (Fig 1A). Total (full-length plus cleaved) GluN2B levels were significantly increased in the non-PSD fraction of untreated YAC128 mice compared to WT (p<0.01) (GluN2B/β-actin ratios for untreated mice: WT, 1.51 + 0.12; YAC128, 2.04 + .11) (Fig 1B). Interestingly, this increase was significantly reduced to WT levels by memantine at both doses (p<0.01) (GluN2B/β-actin ratios for treated YAC128 mice: 1mg/kg/d, 1.61 + 0.13; 10mg/kg/d, 1.56 + 0.l7). In contrast, non-PSD GluN2B expression was 37  not significantly altered in WT littermates with or without treatment. A similar trend was observed when full-length bands were quantified alone: full-length GluN2B levels were elevated in untreated YAC128 mice compared to WT, and reduced to WT levels by memantine (Fig 1C). Calpain-mediated GluN2B cleavage contributes to NMDAR mislocalization in 1-2 month-old YAC128 mice, by decreasing synaptic NMDAR stability and promoting its lateral mobility to extrasynaptic sites (Guttmann et al., 2001, 2002; Simpkins et al., 2003; Gladding et al. 2012). We thus hypothesized that the mtHtt- and memantine-induced changes in Ex-NMDAR expression may be a result, in part, of changes in calpain-mediated GluN2B cleavage. We detected a trend towards increased cleaved GluN2B in untreated YAC128 mice, which was reduced to WT levels by both doses of memantine (Fig 1D), as was observed for full-length and total GluN2B levels. However, when normalized to total GluN2B substrate expression, calpaincleaved GluN2B levels did not differ between genotypes or treatments (Fig 1E). Additionally, calpain cleavage of GluN2B was not inversely associated with full-length receptor levels. Thus, in 4 month-old YAC128 mice calpain-mediated NMDAR cleavage is not selectively altered by mtHtt, nor affected by memantine.  38  39  Figure 1. Memantine decreases Ex-NMDAR expression in YAC128 mice. WT and YAC128 mice were treated with H2O or memantine (1 and 10mg/kg/day) for 2 months, beginning at 2 months of age. Following treatment, striatal tissues were subjected to subcellular fractionation to isolate the non-PSD synaptosomal fraction, and non-PSD GluN2B levels were quantified by western analysis. A) Representative blots of non-PSD fractions probed for full-length (180KDa) and calpain-cleaved (115KDa) GluN2B and β-actin (loading control), in memantine-treated (1 and 10mg/kg/d) and untreated WT and YAC128 mice. An N-terminal GluN2B antibody was used, which detects both cleaved and full-length subunits. Calpain-cleaved GluN2B bands were highly enriched relative to full-length bands in the non-PSD; thus, higher (top panel) and lower (bottom panel) exposures were used to quantify full-length and cleaved GluN2B levels, respectively. B) Quantification of total (cleaved + full-length) GluN2B subunit levels normalized to β-actin. YAC128 total GluN2B levels are significantly elevated in the untreated condition (**p<0.01, 2-way ANOVA, Bonferroni’s post-test), and are restored to WT levels by both doses of memantine (## p<0.01. 2-way ANOVA, Dunnet’s post-test). The interaction between groups was significant (F(2,55)= 5.462, p<0.01). C, D) Individual quantification of full-length (C) and calpain-cleaved (D) GluN2B levels. A trend towards increased cleaved and full-length GluN2B subunits was detected in untreated YAC128 mice, and restored to WT levels by memantine. The effect was significant for full-length GluN2B bewteen YAC128 H2O and 1mg/kg/d (*p<0.05, 2way ANOVA, Dunnet’s post-test). The interaction between full-length GluN2B groups was significant (F(2,56)=3.2121, p<0.05). E) Normalization of cleaved/total GluN2B subunit ratios. No changes in the ratio of calpain-cleaved/total GluN2B subunits were detected between genotypes or treatments.  40  3.2  Memantine reduces functional Ex-NMDARs in SPNs from YAC128 mice As it is isolated on the basis of Triton-X solubility, the non-PSD fraction not only  contains receptors associated with extrasynaptic membranes at the postsynapse, but also presynaptic and endosomal structures not tightly bound to the PSD (Goebel-Goody et al. 2009, Pachioni et al. 2009). Thus, to more exclusively determine the effect of memantine on extrasynaptic surface receptors, we examined Ex-NMDAR functional expression in untreated and memantine-treated (1mg/kg/d) WT and YAC128 mice by whole-cell slice electrophysiology. We recorded NMDAR-eEPSCs in striatal SPNs before and during application of the glutamate transporter GLT-1 inhibitor DL-TBOA (30μM), to induce glutamate spillover and activate extrasynaptic receptors (Tzingounis and Wadiche, 2007) (Fig 2A). The effect of TBOA on NMDAR charge-transfer and current kinetics reflects Ex-NMDAR levels (Milnerwood et al. 2010). NMDAR-eEPSC kinetics slowed in the presence of TBOA (Fig 2B), as shown previously (Milnerwood et al., 2010). Slowing of INMDA was specifically pronounced in untreated YAC128 mice in comparison to other groups. In TBOA, peak-normalized charge was more enhanced in untreated YAC128 mice compared to WT (Fig 2C) suggestive of elevated Ex-NMDAR levels (peak TBOA increase over baseline for untreated mice: WT, 26.8 + 7.4%; YAC128, 43.7 + 5.8%). However, this trend was not significant (p= 0.136). YAC128 peak TBOA effects were significantly reduced to WT levels by 1mg/kg/d memantine treatment, in agreement with our biochemical data (peak TBOA increase for memantine-treated mice: WT, 29.29 + 9.2%; YAC128, 22.9 + 4.6%). No differences were detected in TBOA effects between memantinetreated and untreated WT mice, confirming that the effect of memantine on Ex-NMDAR expression is specific to mtHtt-expressing neurons. Hence, Ex-NMDAR functional expression is significantly suppressed by memantine treatment in YAC128 but not WT mice. 41  42  Figure 2. Memantine reduces functional Ex-NMDARs in SPNs from YAC128 mice. NMDAR-eEPSCs were recorded in SPNs of acute coronal brain slices following intrastriatal stimulation of glutamatergic afferents. Recordings were made at +40mV in aCSF with NBQX (10μM), PTX (100μM), glycine (10μM), and strychnine (2μM) to isolate and potentiate NMDAR responses. NMDAR-eEPSCs were compared before and during bath application of DL-TBOA (30μM), to quantify functional Ex-NMDAR levels. A) Cartoon depicting TBOAinduced glutamate spillover. In the baseline condition (i), glial glutamate transporters prevent neurotransmitter spillover beyond the synaptic cleft. In TBOA (ii), GLT-1 blockade induces neurotransmitter diffusion beyond the synapse and concomitant Ex-NMDAR activation. B) Representative NMDAR-eEPSCs before (gray) and during (black) TBOA application, for untreated WT (i) and YAC128 (ii) and 1mg/kg/d memantine-treated WT (iii) and YAC128 (iv) mice. Charge was normalized to peak current amplitude, as there was a progressive decline in peak INMDA throughout the experiment. TBOA slowed INMDA kinetics in all conditions, but slowing of INMDA was most pronounced for untreated YAC128 mice (ii). C) Quantification of maximal TBOA effects on peak-normalized NMDA charge as a percentage of baseline. Responses were analyzed to 3 seconds. TBOA effects were significantly different between memantine-treated and untreated YAC128 mice (*p<0.05; Two-Way ANOVA, Bonferroni’s post-test). WTH20, n=11; YAC128H2O, n=15; WT1mg/kg/d, n=9; YAC1281mg/kg/d, n=12; 4 -7 animals per group.  43  3.3  Synaptic NMDAR levels are not different between genotypes or treatments NMDARs dynamically traffic between synaptic and extrasynaptic sites of neurons (Tovar  and Westbrook, 2002; Groc et al. 2004, 2006). Thus, we hypothesized that the mtHtt- and memantine-induced changes in Ex-NMDAR expression could be associated with shifts in synaptic-extrasynaptic receptor localization. To observe whether synaptic NMDAR levels are inversely associated with Ex-NMDAR expression, we examined synaptic GluN2B expression by western blotting on the postsynaptic density (PSD) subcellular fraction of memantine-treated (1 and 10mg/kg/d) and untreated WT and YAC128 mice. In contrast to the enrichment in calpaincleaved GluN2B in the non-PSD, full-length GluN2B levels were enriched compared to calpaincleaved subunits in the PSD of all groups tested (Fig 3A). However, no significant changes were detected in total, full-length, or calpain-cleaved synaptic GluN2B expression of YAC128 compared to WT untreated mice (Fig 3B-D) (total GluN2B/β-actin ratios for untreated mice: WT, 1.64 + .11; YAC128, 1.82 + .09). Additionally, chronic memantine had no effect on synaptic GluN2B levels of either WT or YAC128 mice at either dose (total GluN2B/β-actin ratios for treated mice: WT1mg/kg/d, 1.69+ 0.10; YAC1281mg/kg/d, 1.61+ 0.21; WT10mg/kg/d, 1.84+ 0.21; YAC12810mg/kg/d, 1.79+0.15). Therefore, the mtHtt- and memantine-induced changes in NMDAR surface expression are specific to extrasynaptic sites of neurons, and are not associated with alterations in synaptic NMDAR localization.  44  Figure 3. Synaptic NMDAR expression is not altered between genotypes or treatments. PSD fractions were isolated from striatal tissue and probed for synaptic GluN2B subunit levels. A) Representative blots of PSD fractions probed for full-length (180KDa) and calpain-cleaved (115KDa) GluN2B and β-actin (loading control) in memantine-treated (1 and 10mg/kg/d) and untreated WT and YAC128 mice. As in non-PSD fractions, an N-terminal antibody was used to probe for GluN2B subunits. An enrichment of full-length GluN2B was detected in the PSD subcellular fraction. (B,C,D) Quantification of total (cleaved + full) (B), full-length (C), and calpain-cleaved (D) GluN2B/β-actin ratios. No differences in total, full-length, or calpaincleaved GluN2B expression were detected between YAC128 and WT mice + memantine. 45  3.4  Calpain activity is not different between 4 month-old YAC128 and WT mice, and is  unaffected by memantine The modulation of YAC128 Ex-NMDAR expression by memantine suggests that receptor mislocalization in HD is Ex-NMDAR-activity dependent. We thus aimed to delineate signaling pathways downstream of Ex-NMDARs that may contribute to receptor mislocalization. We focused on calpain signaling as a primary candidate: this protease is activated by Ca++ influx through Ex-NMDARs (Xu et al., 2009), and elevated calpain activity contributes to Ex-NMDAR mislocalization in presymptomatic (1-2 month-old) YAC128 mice (Gladding et al., 2012). To examine whether calpain drives a potential feed-forward loop of Ex-NMDAR expression in HD, we probed for striatal calpain activity in the cytosolic fraction of memantine-treated and untreated WT and YAC128 mice. We quantified cleavage levels of the calpain substrate spectrin, with a neo-epitope antibody specific for its calpain cleavage product (150KDa) (a gift from Dr. David Lynch, University of Pennsylvania, Philadelphia, PA), as used previously (Cowan et al., 2008). Interestingly, calpain-cleaved spectrin levels were not different between 4 month-old untreated WT and YAC128 mice, and were not significantly altered by memantine treatment in either genotype (cleaved-spectrin/ α-tubulin ratios: WTH2O, 0.82 + 0.06; YAC128H2O, 0.88 + 0.08; WT1mg/kg/d, 0.99 + 0.07; YAC1281mg/kg/d, 0.89 + 0.09; WT10mg/kg/d, 0.74 + 0.07; YAC12810mg/kg/d, 0.83 + 0.09; p>0.05) (Fig 4 A,B). In WT mice, we detected a small increase in spectrin cleavage at the 1mg/kg/d dose, and a decrease at the 10mg/kg/d dose; however, these changes were not significant. Hence, calpain activity is not affected by mtHtt or memantine treatment in 4 month-old YAC128 mice. This observation contradicts previous reports of elevated calpain activity in untreated 1-2 month-old YAC128 mice (Cowan et al. 2008, Gladding et al. 2012), which could be due to differences in the ages of mice examined in each study. To 46  confirm whether calpain activity is elevated in 1-2 month- but not 4 month-old YAC128 mice, we compared spectrin cleavage in untreated animals at both ages. Calpain-cleaved spectrin levels were significantly elevated in YAC128 mice compared to WT at 1.5 months (p<0.05) but not 4 months of age (cleaved-spectrin/α-tubulin ratios at 1.5 months: WT, 0.98 + 0.05; YAC128, 1.31 + 0.10; at 4 months: WT, 0.87 + 0.13; YAC128, 0.82 + 0.11) (Fig 4 C,D). In fact, YAC128 calpain-cleaved spectrin levels were significantly decreased in 4 month- compared to 1.5 monthold animals (p<0.01). No changes were detected between 1.5 month- and 4 month-old WT mice. Hence, YAC128 calpain activity is elevated at presymptomatic stages of HD (1-2 months), but is attenuated to WT levels as the disease progresses (4 months).  47  Figure 4. Calpain activity is is unaffected by memantine treatment, and is elevated in 1.5 month- but not 4 month-old YAC128 mice. Striatal cytosolic fractions were probed for calpain-cleaved spectrin (150KDa). A) Representative blots of calpain-cleaved spectrin and αtubulin (loading control) in memantine-treated (1 and 10mg/kg/d) and untreated WT and YAC128 mice. B) Quantification of calpain-cleaved spectrin/α-tubulin ratios. No significant changes in calpain-cleaved spectrin levels were detected between genotypes or treatments at 4 months. C) Representative blots for calpain-cleaved spectrin and α-tubulin in untreated 1.5 and 4month-old mice. D) Quantification of calpain-cleaved spectrin/α-tubulin ratios between 1.5 and 4 months. Calpain activity is elevated in YAC128 compared to WT mice at 1.5 months but is decreased to WT levels at 4 months (*p<0.05, **p<0.01, Two-Way ANOVA, Bonferroni’s posttest). The main effect of age was significant (F(1,21)=8.664, p<0.01). 48  3.5  Low-dose memantine rescues mtHtt-induced CREB shutoff We next aimed to identify signaling pathways downstream of Ex-NMDARs in HD.  While synaptic NMDARs promote CREB activation by phosphorylating the active site Ser133 residue (pCREBSer133), Ex-NMDARs trigger dominant CREB shut-off pathways (Hardingham et al., 2002; Milnerwood et al., 2010, 2012; Kaufman et al., 2012). Suppression of Ex-NMDAR activity with low-dose memantine (1mg/kg/d, 2 months) fully rescues mtHtt-associated CREB shutoff in 4 month-old YAC128 mice to WT levels (Milnerwood et al., 2010). However, at higher doses memantine also blocks synaptic receptors (Okamoto et al., 2009) and could thus prevent synaptic NMDAR-mediated CREB phosphorylation. We thus examined whether lowdose memantine fully restores striatal CREB activation as previously reported, and whether the higher 10mg/kg/d dose rescues or further suppresses CREBSer133 phosphorylation. We compared nuclear pCREBSer133 relative to total CREB levels between untreated and memantine-treated WT and YAC128 mice, by western blotting on striatal nuclear fractions. In agreement with previous data (Milnerwood et al., 2010), YAC128 pCREBSer133/CREB ratios were significantly decreased compared to WT at 4 months of age (p<0.05) and were fully rescued to WT levels by memantine treatment at 1mg/kg/d (p<0.05) (Fig 5B). At the 10mg/kg/d dose, however, YAC128 pCREBSer133/CREB levels remained reduced. Additionally, WT pCREBSer133/CREB levels trended toward a decrease in a dose-dependent manner with memantine treatment, although this did not reach significance. (pCREB/CREB ratios: WTH2O, 1.00+ 0.05; YAC128H2O, 0.70+ 0.05; WT1mg/kg/d, 0.85+ 0.09; YAC1281mg/kg/d, 0.99+ 0.12; WT10mg/kg/d, 0.69+ 0.15; YAC1281mg/kg/d, 0.69+ 0.08). Hence, low-dose (1mg/kg/d) but not high-dose (10mg/kg/d) memantine rescues mtHtt-induced CREB shut-off, and shows a trend toward decreasing WT CREB activity in a dose-dependent manner. 49  Figure 5. Low-dose memantine rescues mtHtt-induced CREB shutoff. Nuclear fractions were isolated from striatal tissue and probed for pCREBSer133 and total CREB protein levels by western blotting. A) Representative blots for pCREB Ser133 (i) and total CREB (ii) in memantinetreated (1 and 10mg/kg/d) and untreated WT and YAC128 mice. pCREBSer133 and total CREB protein were probed in separate gels and normalized to HDAC (loading control). Two bands were detected by both the pCREBSer133 and CREB antibodies which likely corresponded to phosphorylated (top band) and unphosphorylated (bottom band) CREB. While only the top band was analyzed to quantify pCREB Ser133 levels, both bands were analyzed for total CREB quantification. B) Quantification of pCREBSer133/CREB levels. The pCREB Ser133/CREB ratio was significantly decreased in untreated YAC128 mice compared to WT (*p<0.05, Two-Way ANOVA, Bonferroni’s post-test), and restored to WT levels by memantine at 1mg/kg/d (#p<0.05, Two-Way ANOVA, Dunnet’s post-test), but not at 10mg/kg/d.  50  3.6  Memantine rescues elevated p38 MAPK activity in YAC128 mice Ex-NMDARs selectively activate p38 MAPK (Xu et al., 2009), which is associated with  neuronal death (Soriano et al., 2008). Additionally, p38 MAPK activity is elevated in 1-2 monthold YAC128 mice, and contributes to NMDA-induced toxicity in mtHtt-expressing SPNs (Fan et al., 2012). We thus examined whether YAC128 p38 MAPK activity is also elevated by mtHtt at 4 months of age, and whether Ca++ influx through Ex-NMDARs drives mtHtt-induced p38 MAPK activation in vivo. As a measure of p38 MAPK activity, we quantified relative phosphorylation levels of p38 MAPK at Thr180 and Tyr182 (herein P-p38) in cytosolic subcellular fractions from memantine-treated and untreated WT and YAC128 mice. As previously reported in 1-2 month-old mice (Fan et al. 2012), the ratio of P-p38/p38 was significantly elevated in untreated 4 month-old YAC128 compared to WT mice (p<0.05) (Fig 6A,B). Notably, YAC128 p38 MAPK activity was significantly attenuated to WT levels by memantine treatment at both doses (p<0.01), while no changes were detected between memantine-treated and untreated WT mice (P-p38/p38 ratios: WTH2O, 1.03 + 0.04; YAC128H2O, 1.39 + 0.12; WT1mg/kg/d, 0.94 + 0.10; YAC1281mg/kg/d, 0.88 + 0.10; WT10mg/kg/d, 1.04 + 0.15; YAC12810mg/kg/d, 0.92 + 0.10). Therefore, p38 MAPK activity is elevated in 4 month-old YAC128 mice, and this effect is restored to WT levels by chronic memantine treatment at both low (1mg/kg/d) and high (10mg/kg/d) doses.  51  Figure 6. Memantine rescues elevated p38 MAPK activity in YAC128 mice. Cytosolic fractions isolated from striatal tissue were probed for P-p38 and total p38 levels, as a measure of relative p38 MAPK activity. A) Representative blots for P-p38 and p38 levels in memantinetreated (1 and 10mg/kg/d) and untreated WT and YAC128 mice. After probing for total p38 protein, blots were reprobed for P-p38 using an antibody directed against p38 phosphorylated at Thr180 and Tyr182. P-p38 levels were normalized to total p38 expression in each lane. B) Quantification of P-p38/p38 ratios. A significant increase in the P-p38/p38 ratio was detected in untreated YAC128 compared to WT mice (*p<0.05, Two-Way ANOVA, Bonferroni’s post-test), which was restored to WT levels by memantine at both doses (**p<0.01, Two-Way ANOVA, Dunnet’s post-test). The main effect of treatment was significant (F(2,46)=4.085, p<0.05).  52  3.7  Memantine increases extrasynaptic PSD-95 levels in WT mice NMDAR-PSD-95 interactions trigger p38 MAPK activation and subsequent neuronal  death (Soriano et al., 2008; Fan et al., 2012) by coupling Ca++ influx through the receptor to SynGAP activation (Kim et al., 1998, Rumbaugh et al., 2006), or nNOS-mediated ROS synthesis (Aarts et al., 2002). In YAC128 striatal tissue, extrasynaptic PSD-95 expression and GluN2BPSD-95 binding is elevated, and disrupting these interactions suppresses NMDA-induced p38 MAPK activation in YAC128 SPNs (Fan et al., 2012). We thus hypothesized that Ex-NMDAR mediated p38 MAPK activation could be associated with PSD-95 surface mislocalization. To determine whether memantine-induced suppression of p38 MAPK activity is associated with decreased extrasynaptic PSD-95 expression, we examined PSD-95 expression levels in the nonPSD fractions of memantine-treated and untreated WT and YAC128 mice. The antibody directed against PSD-95 detected two distinct bands at approximately 95KDa and 100KDa (Fig 7A), as has been observed previously (Fan et al., 2012). We first quantified the lower band, which more closely corresponded to the expected molecular weight of PSD-95 (95KDa). PSD-95 expression showed a trend toward an increase in the non-PSD of untreated YAC128 compared to WT mice, although this did not reach significance (Fig 7B). Interestingly, memantine treatment induced a dose-dependent increase in WT PSD-95 levels in the non-PSD fraction, which was significant at 10mg/kg/d (p<0.05). In contrast, no changes were detected between memantine-treated and untreated YAC128 mice (PSD-95/β-actin ratios: WTH2O, 0.76 + 0.06; YAC128H2O, 1.09 + 0.09; WT1mg/kg/d, 1.16 + 0.13; YAC1281mg/kg/d, 1.13 + 0.15; WT10mg/kg/d, 1.39 + 0.20; YAC12810mg/kg/d, 1.18 + 0.22). Although we could not confirm the identity of the upper band, a similar trend was observed when we quantified this band (Fig C). In fact, there were no differences when the upper band was normalized to the lower band (Fig 7D), confirming that both bands follow the same 53  expression pattern between genotypes and treatments. Furthermore, we did not detect any changes in PSD-95 expression in the PSD fraction (Fig 7 E,F), suggesting that the effect of memantine on extrasynaptic PSD-95 expression in WT mice is not associated with a shift of synaptic PSD-95 to extrasynaptic sites. An upper band was also detected by the PSD-95 antibody in the synaptic fraction, but was much weaker in intensity relative to the lower band, and was not quantifiable. Together, our data suggest that extrasynaptic PSD-95 levels are not affected by memantine treatment in YAC128 mice, whereas memantine elevated PSD-95 expression in the non-PSD in WT animals; additionally, this effect is not associated with any change in synaptic PSD-95.  54  55  Figure 7. Memantine increases extrasynaptic PSD-95 levels in WT mice. Non-PSD and PSD fractions isolated from striatal tissue were probed for PSD-95. A) Representative blots for PSD95 and β-actin (loading control) in the non-PSD fraction of memantine-treated and untreated WT and YAC128 mice. Two bands were detected (~95KDa and ~100KDa). (B,C) Quantification of extrasynaptic PSD-95/ β-actin ratios, performed individually for the lower (B) and upper (C) bands. PSD-95 expression trended toward an increase in untreated YAC compared to WT mice, and was significantly elevated by memantine at 10mg/kg/d in WT mice (*p<0.05, Two-Way ANOVA, Dunnet’s post-test). D) Ratio of upper PSD-95 band normalized to lower band. No changes in upper/lower band expression were detected between groups, confirming that both bands follow similar expression patterns. E) Representative blots of synaptic PSD-95 and βactin. As PSD-95 was highly enriched in the PSD relative to non-PSD fractions, a lower exposure was used than in (A). Upper (~100KDa) and lower (~95KDa) bands were also detected in the PSD; however, the lower band was highly enriched, and the upper band was not quantifiable. F) Quantification of PSD-95/β-actin ratios in the PSD fraction. No changes in synaptic PSD-95 expression were detected between genotypes or treatments.  56  Chapter 4: Discussion  Identification of the pathways downstream of Ex-NMDARs is a crutial step in understanding HD pathology. Elevated Ex-NMDAR activity occurs early in HD and contributes to mtHtt-induced cognitive dysfunction and striatal atrophy, likely by dysregulating intracellular Ca++ homeostasis (Papadia and Hardingham, 2007; Okamoto et al., 2009; Milnerwood et al., 2010). Ca++ dependent pathways closely linked to cell death are also dysregulated prior to phenotype onset, and include elevated calpain and p38 MAPK activity, as well as CREB shut-off (Gafni and Ellerby, 2002; Cowan et al., 2008; Milnerwood et al., 2010; Fan et al., 2012; Gladding et al., 2012). Ex-NMDAR blockade with low-dose memantine is a valuable tool to discriminate pathways mediated by Ex-NMDAR activity from those which are a direct result of mtHtt-induced Ca++ dyshomeostasis. Here, we aimed to demonstrate a causal link between ExNMDARs and aberrant intracellular Ca++ signaling in HD. Chronic in vivo treatment of YAC128 and WT mice with memantine at lower (1mg/kg/d) and higher (10mg/kg/d) doses reduced the increase in YAC128 striatal Ex-NMDAR expression without altering synaptic NMDAR levels. These changes were not associated with altered calpain activity, which was unaffected by memantine treatment, and only elevated in YAC128 mice at 1.5 months but not 4 months of age. Memantine treatment rescued YAC128 CREB shut-off at the lower but not higher dose, while both doses decreased mtHtt-induced p38 MAPK activation to WT levels. In contrast, extrasynaptic PSD-95 expression was not altered by memantine treatment in YAC128 mice but was increased by memantine in a dose-dependent manner in WT littermates.  57  4.1  Assessment of NMDARs in HD Previous studies reported elevated striatal GluN2B-containing Ex-NMDAR expression in  1-2 month-old YAC128 mice (Milnerwood et al., 2010; Gladding et al., 2012). Consistent with these studies, we observed a significant increase in total (calpain-cleaved and full-length) GluN2B expression in the non-PSD fraction of 4 month-old untreated YAC128 mice compared to WT littermates (Fig 1). This suggests that the increase in GluN2B-type Ex-NMDARs is not specific to early presymptomatic disease stages and may also mediate SPN excitotoxicity at later stages of HD. In fact, YAC128 Ex-NMDAR currents are elevated at 1 year of age, and chronic inhibition of Ex-NMDAR activity with low-dose memantine attenuates late-stage striatal atrophy at 12 months (Okamoto et al., 2009; Milnerwood et al., 2010). To confirm that the change in YAC128 GluN2B expression is representative of functional receptors, we assessed Ex-NMDAR activation following TBOA-induced glutamate spillover. A previous study reported significantly higher TBOA effects in 1 month-old YAC128 compared to WT mice (50% vs. 125% peak TBOA increase in NMDAR currents for WT and YAC128, respectively) (Milnerwood et al., 2010). Consistent with this study and our biochemical data, we detected a trend towards increased Ex-NMDAR currents in YAC128 mice compared to WT (Fig 2). However, this trend did not reach significance, as TBOA effects were highly variable between cells. Moreover, our TBOA effects for both genotypes (26% for WT and 44% for YAC128) were attenuated compared with those observed by Milnerwood et al. (2010). These differences could be attributed to changes in cortico-striatal synaptic function between 1 and 4 month-old mice. For instance, GLT-1 functional expression is reduced with age in both WT and YAC128 mice (Huang et al., 2010; Rujun Kang, unpublished work). This reduction 58  could attenuate glial glutamate uptake efficacy, facilitate basal glutamate spillover, and thus occlude TBOA effects in older mice. Synaptic (PSD) cleaved and full-length GluN2B levels were unchanged between untreated YAC128 and WT mice and were unaffected by memantine treatment (Fig 3), suggesting that the effect of mtHtt on NMDAR mislocalization is specific to extrasynaptic sites. This is not surprising, as synaptic NMDARs are tightly anchored within the PSD by interactions with scaffolding proteins, cell-adhesion factors, and cytoskeletal elements (Gladding and Raymond, 2011). However, our observation contradicts previous studies, which detected either a slight decrease (~10%) in full-length GluN2B (Milnerwood et al., 2010) or increased (~50%) calpain-cleaved GluN2B levels in the striatal PSD fraction of 1-2 month-old YAC128 mice (Gladding et al., 2012). The reason for this discrepancy is unclear, but it could be attributed to differences in the ages of the mice examined between studies. Other measurements could be used to confirm that the synaptic pool of NMDARs is unaltered in 4 month-old YAC128 mice with or without memantine, such as NMDA-mEPSCs or evoked NMDAR-EPSCs at low stimulation intensities (Prybylowski et al., 2005; Milnerwood et al., 2010).  4.2  Ex-NMDAR surface expression is Ex-NMDAR-activity dependent Memantine at both doses reduced functional YAC128 Ex-NMDAR expression to WT  levels (Fig 1-2), suggesting that the mtHtt-induced increase in extrasynaptic receptor expression is Ex-NMDAR activity-dependent. Thus, memantine may exert its neuroprotective effects not only by chronically blocking Ex-NMDAR activity, but also by reducing elevated Ex-NMDAR surface expression. In light of this observation, we hypothesize that Ex-NMDAR mislocalization in HD is driven by a feed-forward loop, in which Ex-NMDAR activity triggers pathways that in 59  turn potentiate extrasynaptic receptor expression. Such a feed-forward loop would be expected to gradually increase Ex-NMDAR accumulation, which could help drive progressive SPN cell death and striatal atrophy in HD. As synaptic NMDAR levels were unaltered by memantine treatment (Fig 3), the effect of memantine on Ex-NMDAR expression likely does not involve a shift in localization of extrasynaptic NMDARs to synaptic sites. Calpain signaling is elevated in 1-2 month-old YAC128 mice and contributes to ExNMDAR mislocalization (Cowan et al., 2008; Gladding et al., 2012). Consistent with these studies, we detected elevated calpain-cleaved spectrin levels in 1.5 month-old YAC128 mice compared to WT littermates (Fig 4). However, calpain-cleaved/total GluN2B and calpaincleaved spectrin levels were similar in 4 month-old YAC128 and WT mice, and were unaffected by memantine treatment (Fig 1E, 4). This suggests that although calpain activity is elevated in YAC128 mice at 1.5 months, at 4 months calpain signaling is not altered by mtHtt expression, is independent of Ex-NMDAR activity, and is not contributing to increased Ex-NMDAR surface expression. This observation is inconsistent with previous studies which detected elevated calpain signaling in human HD striatum and knock-in HD mouse models (Gafni and Ellerby, 2002; Gafni et al., 2004). Moreover, the fact that calpain activity was unaffected by memantine treatment is inconsistent with previous studies in cultured neurons demonstrating that calpain signaling is downstream of Ex-NMDARs (Xu et al., 2009). Our data suggest that Ca++-dependent signaling mechanisms in HD may evolve with disease progression. In turn, we propose that the signaling pathways that contribute to ExNMDAR mislocalization at early stages of HD may be distinct from those at later disease stages. With age, mtHtt-expressing SPNs develop resistance to excitotoxicity, which is in part attributed to enhanced cytosolic Ca++ buffering after NMDAR stimulation in R6/2 mice (Hansson et al., 60  2001), or decreased coticostriatal synaptic transmission in YAC128 mice (Graham et al., 2009; Joshi et al., 2009). Although full onset of the resistance phenotype occurs at 7-9 months in the YAC128 model (Graham et al., 2009), these changes are progressive, and it is possible that some resistance mechanisms may already be present at 4 months. If so, resitance to excitotoxicity could help explain an age-dependent decrease in Ca++-dependent calpain signaling. However, whether 4 month-old YAC128 mice indeed exhibit attenuated corticostriatal synaptic transmission as is observed at >7months (Graham et al., 2009; Joshi et al., 2009), and/or improved Ca++ buffering as is observed in the R6/2 model (Hansson et al., 2001), is speculative. Overall, further work is required to identify the pathways driving YAC128 Ex-NMDAR mislocalization at 4 months, and to elucidate the underlying cause of attenuated calpain signaling at this age.  4.3  Putative mechanisms of elevated Ex-NMDAR expression in HD Elevated Ex-NMDAR expression in HD could be attributed to an accelerated rate of  NMDAR forward trafficking, translocation of synaptic receptors to extrasynaptic sites, or increased surface retention of Ex-NMDARs (Tovar and Westbrook, 2002; Fan et al., 2007; Gladding and Raymond, 2011; Gladding et al., 2012). We did not detect changes in synaptic NMDAR levels between YAC128 mice compared to WT; therefore, a shift in NMDAR localization from synaptic to extrasynaptic sites would have to occur simultaneously with increased surface delivery at the PSD, such that net synaptic NMDAR levels remain unchanged. Putative Ca++-dependent pathways that could drive Ex-NMDAR expression are presented below.  61  4.3.1  STEP signaling The Striatal Enriched Tyrosine Phosphatase (STEP) dephosphorylates GluN2B at Y1472,  which promotes clathrin binding and receptor endocytosis, potentially leading to recycling of synaptic NMDARs at extrasynaptic sites (Braithwaite et al., 2006; Gladding and Raymond, 2011; Gladding et al., 2012). Whereas Ex-NMDARs trigger STEP cleavage and inactivation (Xu et al., 2009), mtHtt potentiates synaptic STEP activity in 1-2month-old YAC128 mice (Gladding et al., 2012). Therefore, synaptically increased and/or extrasynaptically decreased STEP activity in HD could elevate Ex-NMDAR expression via mtHtt- and Ex-NMDAR-mediated pathways.  4.3.2  CREB-PGC1α pathway The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a  neuroprotective transcriptional coactivator that regulates mitochondrial function (Fink and Kelly, 2006). It was recently proposed that PGC-1α modulates Ex-NMDAR surface expression (Puddifoot et al., 2012). Moreover, transcriptional expression of PGC-1α, which is mediated by CREB signaling, is suppressed in YAC128 mice, likely due to Ex-NMDAR-mediated CREB shut-off (Cui et al., 2006; Okamoto et al., 2009; Milnerwood et al., 2010). Together, these studies suggest that Ex-NMDAR activity could suppress CREB-induced PGC-1α expression, in turn enhancing extrasynaptic accumulation of the receptor. However, the mechanism by which PGC-1α modulates Ex-NMDAR expression is unclear.  62  4.3.3  GluN2B-S1480 phosphorylation GluN2B-S1480 phosphorylation by the Ca++-dependent casein-kinase II (CKII) disrupts  GluN2B-PSD-95 interactions, which facilitates NMDAR surface dispersal or internalization (Chung et al., 2004). Ex-NMDARs trigger GluN2B-S1480 dephosphorylation (Chung et al., 2004), which could contribute to increased extrasynaptic retention of NMDARs. CKII expression is increased in YAC128 mice (Fan et al., 2008). While increased CKII expression in HD could contribute to surface NMDAR dispersal or internalization, elevated Ex-NMDAR activity could locally decrease extrasynaptic GluN2B-S1480 phosphorylation, potentially leading to Ex-NMDAR surface accumulation.  4.3.4  PKC signaling Ca++-dependent PKC stimulation enhances NMDAR forward trafficking and  translocation from synaptic to extrasynaptic compartments (Lan et al., 2001; Fong et al., 2002; Yan et al., 2011). Notably, Ca++ influx through Ex-NMDARs activates PKC (Sun and June Liu, 2007). Therefore, in HD, elevated Ex-NMDAR activity could potentiate PKC, which would in turn promote NMDAR forward trafficking and extrasynaptic accumulation (Yan et al., 2011).  4.4  Ex-NMDARs couple to pro-death signaling in HD Nuclear CREB activity, as reflected in the pCREB/CREB ratio, was significantly  decreased in 4 month-old YAC128 mice compared to WT and was fully rescued to WT levels by memantine at the lower dose (Fig 5). Thus, Ex-NMDAR activity drives mtHtt-associated CREB shut-off, further implicating these receptors in HD pathology. Similar results have been published (Milnerwood et al., 2010), and are consistent with reports that Ex-NMDARs mediate 63  CREB shut-off in cultured neurons (Harindgham et al., 2002). Additionally, the ability of lowdose memantine to rescue neuroprotective CREB signaling is concomitant with observations that the same dose ameliorates behavioural deficits at 4 months and striatal atrophy at 12 months (Okamoto et al., 2009; Milnerwood et al., 2010). Strikingly, memantine at the higher dose did not rescue YAC128 CREB activity and trended to decrease pCREB/CREB levels in WT mice. Synaptic NMDARs, which activate CREB via Ras-ERK or CaMK pathways (Hardingham and Bading, 2010), are blocked by higher doses of memantine (Okamoto et al., 2009). Thus, synaptic NMDARs may be blocked by memantine at the higher dose (10mg/kg/d), which could explain why this dose did not recue CREB shut-off in YAC128 mice and trended to decrease CREB signaling in WT littermates. However, the receptor subpopulation inhibited by memantine at 10mg/kg/d in vivo has not been identified. P-p38/p38 levels were significantly increased in untreated 4 month-old YAC128 mice compared to WT (Fig 6). This is consistent with previous reports of elevated YAC128 p38 MAPK activity at 1-2 months of age (Fan et al., 2012), and suggests that unlike calpain, aberrant p38 signaling remains elevated as HD progresses. p38 phosphorylation in YAC128 mice was suppressed to WT levels by memantine at both doses, indicating that mtHtt-induced p38 signaling is downstream of Ex-NMDAR activity. In agreement with this, stimulation of ExNMDARs in culture induces a rapid and prolonged increase in p38 activity (Xu et al., 2009). As p38 signaling mediates cell death (Soriano et al., 2008; Xu et al., 2009; Fan et al., 2012), ExNDMAR-mediated p38 activation could be another putative mechanism by which these receptors contribute to HD pathology. Interestingly, while memantine at the higher dose did not rescue mtHtt-associated CREB shut-off in YAC128 mice, both doses were equally effective at suppressing elevated p38 signaling. If synaptic NMDARs are indeed blocked by memantine at 64  10mg/kg/d as proposed, our data suggests that p38 activity is independent of Ca++ influx through these receptors. This would be consistent with reports that p38 is enriched at extrasynaptic sites and its activity is relatively unaltered by synaptic NMDAR stimulation (Xu et al., 2009). The pathways that link Ex-NMDAR activity to p38 MAPK phosphorylation remain elusive. In cultured cortical neurons, Ex-NMDARs potentiate p38 phosphorylation by inducing calpain-mediated cleavage and inactivation of the phosphatase STEP, which dephosphorylates p38 (Xu et al., 2009). However, as we did not detect changes in calpain activity in 4 month-old YAC128 mice, other pathways are likely involved that couple Ex-NMDARs to p38 activation at this age. NMDAR-PSD-95 interactions couple Ca++ influx through the receptor to SynGAP and nNOS, both which trigger p38 signaling (Aarts et al., 2002; Cao et al., 2005; Rumbaugh et al., 2006; Soriano et al., 2008). In YAC128 mice, increased associations between Ex-NMDARs and PSD-95 (Fan et al., 2012) could potentiate SynGAP and/or nNOS signaling, contributing to the increase in p38 activation. Moreover, elevated p38 activity in YAC128 mice is associated with enhanced extrasynaptic expression of PSD-95 (Fan et al., 2012). Hence, we examined whether the effect of memantine on p38 activity is associated with changes in the surface localization of PSD-95. Although extrasynaptic PSD-95 levels in untreated YAC128 mice trended to be increased compared to WT, they were unchanged by memantine treatment (Fig 7). Instead, memantine increased PSD-95 levels in the non-PSD fraction of WT mice, suggesting that ExNMDAR activity modulates extrasynaptic expression of PSD-95 in the absence of mtHtt. Further study is required to clarify the link between Ex-NDMAR activity and extrasynaptic PSD-95 localization in WT mice. We speculated that the age-dependent decrease in calpain activity in YAC128 mice could be associated with increased resistance to excitotoxicity, which could attenuate Ca++-dependent 65  aberrant signaling in HD (Hansson et al., 2001; Graham et al., 2009; Joshi et al., 2009). However, p38 and CREB signaling pathways, which are also Ca++-dependent, were dysregulated at 4 months in YAC128 mice. This discrepancy could be due to the differential sensitivity of each pathway for Ca++- dependent activation: while calpain requires supra-physiological Ca++ levels to be activated (Vosler et al., 2008), lower Ca++ levels may be sufficient to regulate p38 and CREB signaling. In fact, NMDAR-mediated p38 MAPK pathways are coupled to Ca++ influx through the NMDAR by interactions with PSD-95, and thus depend on submembranous rather than global rises in cytosolic Ca++ (Aarts et al., 2002; Soriano et al., 2008). Alternatively, Ex-NMDAR-induced CREB shut-off likely occurs via PP1, which has a higher affinity for Ca++ compared to calpain ( Rusnak and Mertz, 2000; Goll et al., 2003), and may thus be activated by lower levels of cytosolic Ca++. Hence, p38 and CREB signaling may still be dysregulated in 4 month-old YAC128 mice, even if these mice indeed exhibit resistance to excitotoxicity.  4.5  Memantine decreases body weight in female YAC128 mice From 2 to 4 months of age, body weight was progressively greater in untreated YAC128  compared to WT mice of both sexes (Fig 9, Appendix). Increased weight gain in >2 month-old YAC128 mice has been previously reported, and has been attributed to increased expression levels of full-length human htt rather than to expanded polyQ length (Van Raamsdonk et al., 2006; Pouladi et al., 2010). Interestingly, memantine treatment attenuated weight gain in female but not male YAC128 mice, suggesting that the Htt-induced body weight gain in females is ExNMDAR activity-dependent. A mechanistic link for the effect of memantine on body weights is unclear. Dysregulated body weight in HD could be associated with altered feeding behaviours or metabolism. The hypothalamic lateral tuberal nucleus (LTN), which regulates hunger, is 66  selectively degraded in HD ( Kremer et al., 1991; Kremer, 1992). This could be attributed to its particularly high NMDAR content (Kremer et al., 1993), which may enhance its vulnerability to excitotoxicity. Hence, chronic blockade of Ex-NMDARs with memantine could protect the LTN from excitotoxicity and ameliorate dysregulated feeding behaviours in HD. Another potential theory could be that memantine enhances energy expenditure in female YAC128 mice. However, neither food intake or energy expenditure were measured in the present study. An interesting observation was that the effect of memantine on body weights was specific to female mice. Both sexes were treated with the same memantine doses and self-administered memantine at similar rates (Fig 8, Appendix), so sex differences in dosing efficacy cannot account for the present observations. However, the basal difference in body weight between untreated YAC128 and WT mice was much more pronounced for females than for males; hence, an effect of memantine on male YAC128 body weight may be more difficult to detect than in females. Futher study is required to examine whether hormonal or behavioural differences between sexes in YAC128 mice can differentially affect the efficacy or downstream effects of memantine-induced ExNMDAR blockade. Nonetheless, the selective body weight gain in YAC128 mice is discordant with weight loss that is observed in HD patients (Aziz et al., 2008), so the effect of memantine on modulating body weight in this model may be clinically irrelevant.  4.6  Experimental limitations This study has several limitations. First, the efficacy of our memantine treatments rely on  the drug’s selectivity for Ex-NMDAR blockade in vivo, which we did not confirm. Previous reports have suggested that memantine at 1mg/kg/d is selective for Ex-NMDARs when administered to YAC128 mice ad libitum: at 1mg/kg/d, memantine blocked pro-death signaling 67  known to be downstream of Ex-NMDARs and was neuroprotective in YAC128 mice (Okamoto et al., 2009; Milnerwood et al., 2010). This dose is also estimated to result in a concentration of 5-10μM at the NMDAR channel mouth, which effectively blocks Ex-NMDARs in culture and mimics the dose used to treat Alzheimer’s disease in humans (10mg twice daily) (Okamoto et al., 2009). We selected the higher dose (10mg/kg/d) because it is also neuroprotective in various pathologies (Hare et al. 2001, Rosi et al. 2006, Rammes et al. 2008). However, it is unclear whether this dose is also selective for Ex-NMDARs. The observation that memantine at 10mg/kg/d did not rescue YAC128 CREB activity could suggest that this dose also blocks synaptic NMDARs, but further study is required to confirm this. Also, memantine treatment when administered ad libitum could be highly discontinous, and the drug has a short half-life in rodent brains (<4 hours) (Beconi et al., 2011). Together, these issues could cause abrupt changes in the effective brain concentration of memantine throughout treatment, which could affect its efficacy or selectivity for Ex-NMDAR blockade. This issue may be resolved in part by the drug’s lipophilic nature: as it preferentially accumulates within the brain’s lipid parenchyma, memantine is steadily released into the interstitial fluid, providing a continuous supply to neurons (Subczynski et al., 1998; Okamoto et al., 2009). Also, we selected a 2-month ad libitum treatment period (from 2 to 4 months of age) as this protocol was previously shown to rescue neuroprotective pCREB signaling (Milnerwood et al., 2010). However, this limited us to examining Ca++ signaling at 4 months of age. Since Ca++-dependent signaling pathways such as calpain activity could change with disease progression, we could not directly compare our results to those of previous studies in the lab, which mainly examined signaling at 1-2 months of age (Milnerwood et al., 2010; Fan et al. 2012; Gladding et al., 2012).  68  Our electrophysiological methodology also has some limitations. The assessment of ExNMDAR currents by TBOA relies on the assumption that only synaptic NMDARs are activated prior to TBOA application. However, we cannot determine whether afferent stimulation evokes basal glutamate spillover before TBOA application, which may occlude TBOA effects. Our biochemical methods also have potential pitfalls. First, the quantification of calpain activity by spectrin cleavage is indirect, and could be affected by proteases that also cleave spectrin, including caspases (Williams et al., 2003). Hence, it will be important to validate our assessment of calpain activity by more direct measurements, such as the quantification of proteolytically processed (active) vs. unprocessed (inactive) calpain subunits (Gafni and Ellerby, 2002). Moreover, the PSD-95 antibody detected two distinct bands, which may represent different posttranslational modifications of PSD-95. We did not detect differences between the expression levels of the lower and upper band between genotypes or treatments. Nonetheless, further biochemical studies such as siRNA silencing of PSD-95 and phosphatase/palmitoylation assays are required to confirm the identity of these bands, and determine whether the upper band represents PSD-95 with post-translational modifications.  4.7  Future directions and unanswered questions The observation that Ex-NMDARs drive their own surface mislocalization raises several  interesting questions. First, if elevated expression of extrasynaptic receptors is Ex-NMDARactivity dependent, what triggered their mislocalization in the first place? mtHtt could potentiate forward NMDAR trafficking and extrasynaptic translocation from an early age via Ex-NMDARindependent pathways, for example by aberrantly interacting with trafficking machinery (Trushina et al., 2004). Once Ex-NMDAR levels reach a certain threshold, they may in turn 69  trigger dominant feed-forward pathways that mediate Ex-NMDAR surface acumulation. Second, if memantine inhibits the feed-forward loop that drives Ex-NMDAR expression, could it potentially normalize Ex-NMDAR levels in the long-term, even after treatment is discontinued? If so, memantine could exert neuroprotective effects even after treatment is discontinued, which may be clinically relevant. In agreement with reports that memantine is only neuroprotective at low doses (Okamoto et al., 2009), we have demonstrated that memantine at 1mg/kg/d rescues dysregulated CREB and p38 MAPK signaling in HD, whereas the higher dose does not rescue CREB activity. To extend our study, future work should compare the effects of both doses of memantine on YAC128 motor and cognitive dysfunction as well as striatal atrophy and vulnerability to excitotoxic stress. In particular, identifying whether the higher dose is neuroprotective or deleterious could help clarify upper dosing limits for clinical trials of memantine treatment. Finally, we have proposed that YAC128 calpain activity is reduced in an age-dependent manner, potentially as a consequence of the development of excitotoxic resistance (Hansson et al., 2001; Graham et al., 2009; Joshi et al., 2009). However, this is speculative and more experiments are required to test this hypothesis. 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The Huntington’s Disease Collaborative Research Group. Cell 72, 971–983.  93  Appendices  Appendix A: Effect of memantine on daily solution intake and body weights Daily memantine intake was not statistically different from water (vehicle) intake in male and female YAC128 and WT mice (Fig 8), confirming that the mice drink memantine solutions at the same rate as they drink water. Both female and male untreated YAC128 mice exhibited significantly greater body weight than WT littermates, although this increase was clearer for females (Fig 9). The relative increase in YAC128 body weight was progressively greater with age, and was most significant 8 weeks after start of treatment, at which time the mice were 4 months old (p<0.001 for females; p<0.01 for males). In female YAC128 mice, the relative increase in body weight was significantly reduced to WT levels by memantine treatment at both doses (p<0.001), although this decrease was not dose-dependent. In contrast, body weight of male YAC128 mice was not affected by memantine treatment (Fig 9Bii, iii). Body weights of male and female WT mice were not altered by memantine, suggesting that the effect of memantine on body weight is specific to YAC128 mice.  94  Figure 8. Solution intake does not differ between memantine-treated and untreated mice. Female (A) and male (B) WT and YAC128 mice were treated with memantine (1 and 10mg/kg/d) or H2O (vehicle) starting at 2 months (+ 10 days) of age for 2 months. Solutions were administerd in the drinking bottles of each cage ad libitum. Drinking volumes per cage were measured twice per week throughout the dosing period and calculated to obtain average daily solution intake per mouse, representative of the entire dosing period. No differences in daily memantine or H2O intake were detected between female and male WT or YAC 128 mice. n=4 female cohorts, n=6 male cohorts.  95  Figure 9. Memantine reduces body weight in female YAC128 mice. Female (A) and male (B) WT and YAC128 mice were treated with memantine (1 and 10mg/kg/d) or H2O starting at 2 months (+ 10 days) of age for 2 months. Body weights of memantine-treated and untreated mice were calculated 0 weeks (i), 4 weeks (ii), or 8 weeks (iii) from the start of treatment. Statistical differences at each time point were examined by Two-Way ANOVA. A) In females, body weight of untreated YAC128 mice was significantly greater than WT littermates (**p<0.01, ***p<0.001, Bonferroni’s post-test), and was reduced to WT levels by memantine at 4-week and 8-week time-points (###p<0.001, Dunnet’s post-test). Ai) At 0 weeks, there was a significant effect of genotype (F(1,79)=4.897, p<0.05). Aii) At 4 weeks, there were significant effects of 96  genoytpe (F(1,79)=13.81, p<0.001), and interaction (F(2,79)= 10.30, p<0.001). Aiii) At 8 weeks, there were significant effects of genotype (F(1,53)=24.20, p<0.001), treatment (F(2,53)=4.076, p<0.05), and interaction (F(2,53)=12.92, p<0.001). B) In males, body weight of untreated YAC128 mice was also significantly greater than WT (*p<0.05, **p<0.01, ***p<0.001, Bonferroni’s post-test; ##p<0.01 Dunnet’s post-test), but was not restored to WT levels by memantine at either dose. Bi) At 0 weeks, there were significant effects of genotype (F(1,118)=40.56, p<0.001), treatment (F(2,118)=3.202, p<0.05), and interaction (F(2,118)=4.609, p<0.05). (Bii, Biii) At 4 and 8 weeks, there was a significant effect of genotype (4 weeks: F(1,96)=20.00, p<0.001; 8 weeks: F(1,68)=16.02, p<0.001).  97  Appendix B: Accuracy of subcellular fractionation  Figure 10. Accuracy of subcellular fractionation. Striatal tissue from memantine-treated and untreated WT and YAC128 mice was subjected to subcellular fractionation to isolate non-PSD and PSD synaptosomal membranes (A), or nuclear matrix (B) fractions. Equal protein amounts from all 6 treatment conditions were probed for subcellular markers in each fraction, to confirm the accuracy of the fractionations. A) Synaptophysin (presynaptic marker) and PSD-95 (postsynaptic marker) are enriched in non-PSD and PSD synaptosomal fractions, respectively. B) Histone deacetylase (HDAC, nuclear matrix marker) is enriched in the nuclear extract (matrix) fraction. Hence, synaptosomal and nuclear fractionations are accurate. 98  


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