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Palmitoylation on NMDA receptor trafficking and function in corticostriatal co-culture from HD mouse… Wang, Liang 2015

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	  PALMITOYLATION ON NMDA RECEPTOR TRAFFICKING AND FUNCTION IN CORTICOSTRIATAL CO-CULTURE FROM HD MOUSE MODEL    by LIANG WANG   B.Sc., The University of British Columbia, 2009    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF     MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Neuroscience)        THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)   November 2015  © Liang Wang, 2015  	   ii	  Abstract    Huntington’s Disease (HD) is a neurodegenerative disorder in which the medium-sized spiny neurons (MSNs) of the striatum are earliest and most severely affected. Selective striatal degeneration in HD has been caused, in part, by altered N-methyl-D-aspartate receptor (NMDAR) activity. Our lab has found in the YAC128 transgenic mouse model of HD that GluN2B-containing NMDARs (2B-NMDARs) at extrasynaptic (Ex) sites are increased in striatum at an early stage; however, the mechanism underlying altered NMDAR trafficking in HD remains unknown. Palmitoylation at the C-terminus of 2B-NMDAR regulates its surface expression and synaptic targeting. Notably, a palmitoyl transferase (PAT) enzyme – ZDHHC17 (HIP14) – interacts with huntingtin, the protein mutated in HD. Pilot data in the lab suggests reduced 2B-NMDAR palmitoylation may contribute to increased Ex-NMDAR in striatal MSN from YAC128 HD mice. However, a potential role for HIP14 in regulating 2B-NMDAR trafficking has not been explored. On the other hand, suppression of acyl-protein thioesterases 1/2 (APT1/2), the depalmitoylation enzymes, by a small molecule inhibitor PalmB leads to increased protein palmitoylation level, which may affect NMDAR distribution. Here, we examined the effect of changing palmitoylation on NMDAR trafficking and function in YAC128 HD mice. Knockdown of endogenous HIP14 did not change 2B-NMDAR surface expression and total NMDAR current. The treatment of DMSO (vehicle) and PalmB reduced synaptic NMDAR current and increased Ex-NMDAR current, and the main difference was found in FVB/N (control) mouse. In contrast, after 4 hours of PalmB treatment, miniature excitatory postsynaptic current (mEPSC) frequency significantly increased at YAC128 but not FVB/N corticostriatal synapse. Investigation of palmitoylation on NMDAR activity is useful for clinical application.	   iii	  Preface    Experiments in this thesis were performed in accordance with animal care guidelines from the University of British Columbia Animal Care Committee and the Canadian Council on Animal Care (certificate number A15-0069). In Figure. 3.1 and 3.2, the analysis of surface vs. internal expression ratio were performed by Rujun Kang, a member of the Raymond Lab. In Figure. 3.7, the analysis of western blots was performed by Rujun Kang.                           	   iv	  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, composition, and basic functions ........................................................... 1 1.1.2 NMDAR assembly and trafficking ......................................................................... 4 1.2 Palmitoylaiton ................................................................................................................. 5 1.2.1 General overview and basic functions .................................................................... 5 1.2.2 Enzymes and Substrates: PATs and APTs .............................................................. 6 1.2.3 NMDAR palmitoylation ......................................................................................... 9 1.2.4 Diseases affecting the nervous system .................................................................. 11 1.3 Huntington’s disease ..................................................................................................... 12 1.3.1 Overview ............................................................................................................... 12 1.3.2 Mouse models of HD ............................................................................................ 13 1.3.3 Corticostriatal synaptic dysfunction in HD   .......................................................... 15 1.4 NMDAR in HD ............................................................................................................. 17 1.4.1 NMDAR dysfunction in early HD ......................................................................... 17 1.4.2 GluN2B-type NMDARs in HD ............................................................................. 18 	   v	  1.4.3 Extrasynaptic NMDARs in HD ............................................................................. 19 1.4.4 Palmitoylation on altered 2B-NMDAR subcellular distribution ........................... 20 1.5 HIP14 in HD .................................................................................................................. 24 1.5.1 General overview: discovery, structure, and expression in the CNS .................... 24 1.5.2 Substrates of HIP14 and HIP14L .......................................................................... 25 1.5.3 HIP14, HIP14L deficient mouse models ............................................................... 26 1.6 Palmostatin B ................................................................................................................. 27 1.6.1 Synthesis and application ...................................................................................... 27 1.6.2 Effect of PalmB on depalmitoylation of synaptic proteins .................................... 28 1.7 Rationale and hypothesis    ............................................................................................ 28 1.7.1 Hypothesis: HIP14 regulates NMDAR trafficking and affects NMDAR function     ............................................................................................................................... 28 1.7.2 Hypothesis: PalmB affects NMDAR trafficking and function .............................. 29 Chapter 2: Methods ....................................................................................................................... 30 2.1 Primary neuronal culture and transfection ..................................................................... 30 2.2 HIP14 ASO and PalmB treatments ............................................................................... 31 2.3 Immunocytochemistry ................................................................................................... 33 2.4 Microscopy and image analysis ..................................................................................... 33 2.5 Acyl-biotin exchange (ABE) assay ............................................................................... 34 2.6 Western blot analysis ..................................................................................................... 35 2.7 Electrophysiology .......................................................................................................... 36 2.8 Data analysis and statistics ............................................................................................ 38 Chapter 3: Results .......................................................................................................................... 39 3.1 Role of HIP14 in 2B-NMDAR trafficking and NMDAR current in HD mouse model    . 39 	   vi	  3.1.1 Knock-down of endogenous HIP14 with antisense oligonucleotide (ASO) does not alter 2B-NMDAR surface expression .................................................................... 39 3.1.2 HIP14 ASO does not affect the level of functional surface NMDARs in MSNs  . 45 3.2 Effect of general increase in protein palmitoylation on NMDAR current in HD striatal neurons .......................................................................................................................... 47 3.2.1 Increased Ex-NMDAR current in DIV18 MSNs of YAC128 corticostriatal co-cultures  ................................................................................................................. 47 3.2.2 DMSO increases functional Ex-NMDARs in FVB/N MSNs  ............................. 48 3.2.3 DMSO and PalmB reduce synaptic NMDAR but not synaptic AMPAR current in FVB/N and YAC128 MSNs ................................................................................. 53 3.2.4 PalmB increases mEPSC frequency in YAC128 MSNs  ..................................... 57 3.2.5 PalmB increases GluN2B and PSD-95 palmitoylation in corticostriatal co-cultures  .............................................................................................................................. 60 Chapter 4: Discussion  .................................................................................................................... 63 4.1 HIP14 and regulation of 2B-NMDAR trafficking  ....................................................... 64 4.2 HIP14 and HIP14L ....................................................................................................... 67 4.3 PTMs beyond palmitoylation ........................................................................................ 68 4.4 PalmB effect on NMDAR current and palmitoylation  ................................................ 70 4.5 DMSO effect on NMDAR distribution  ........................................................................ 73 4.6 Genotype-specific PalmB effect on mEPSC frequency ................................................ 76 4.7 Limitations  ................................................................................................................... 77 4.8 Future directions   ......................................................................................................... 80 References  ..................................................................................................................................... 83	  	   vii	  List of Figures    Figure 1.1: Palmitoylation sites at C-terminal of GluN2A and GluN2B subunits ........................ 10  Figure 1.2: Reduced palmitoylation in 1- to 3-month-old YAC128 HD mice striatum ................. 21  Figure 1.3: Rapid turnover rate of GluN2B palmitoylation ........................................................... 22  Figure 1.4: Enhanced GluN2B 5CS surface expression in FVB/N striatal neurons ...................... 23  Figure 2.1: HIP14 ASO dose response time course curve in FVB/N cortical neurons .................. 32  Figure 3.1: No effect of HIP14 knockdown on 2B-NMDAR surface expression in FVB/N striatal neurons ............................................................................................................................................ 42  Figure 3.2: No effect of HIP14 knockdown on 2B-NMDAR surface expression in YAC128 striatal neurons ............................................................................................................................................ 44  Figure 3.3: No effect of HIP14 knockdown on whole-cell NMDA-evoked current ...................... 46  Figure 3.4: DMSO and PalmB on NMDAR distribution in FVB/N and YAC128 MSNs ............. 52  Figure 3.5: Effects of DMSO and PalmB on synaptic NMDAR and AMPAR in FVB/N and YAC128 MSNs ............................................................................................................................... 56  Figure 3.6: Increased mEPSC frequency in PalmB-treated YAC128 MSNs ................................ 60  Figure 3.7: PalmB increases GluN2B and PSD95 palmitoylation in coticostriatal co-cultures ..... 62      	   viii	  List of Abbreviations    2A-NMDAR GluN2A type NMDAR 2B 5CS Cys Cluster II palmitoylation-resistant mutant GluN2B  2B-NMDAR GluN2B type NMDAR 2BP 2-bromopalmitate 4-AP 4-aminopyridine ABE acyl-biotin exchange aCSF artificial cerebrospinal fluid AD Alzheimer’s disease AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid  AMPAR AMPA receptor ANOVA analysis of variance APT acyl protein thioesterase APT1 acyl-protein thioesterase 1 (LYPLA1) APT2 lysophospholipase II (LYPLA2) APTL1 APT like 1 ASO antisense oligonucleotides BAC bacterial artificial chromosome BSA bovine serum albumin BDNF brain-derived neurotrophic factor C-terminal carboxy-terminal Ca2+ calcium ion 	   ix	  CM chloroform–methanol CNS central nervous system COS CV-1 (simian) in Origin carrying the SV40 genetic material   CREB cAMP response element binding protein CSP Cysteine string protein D1 dopamine receptor 1 D2 dopamine receptor 2 DA dopamine DHHC Asp-His-His-Cys DIV day in vitro DMEM D minimum essential medium dMSN MSNs of the direct pathway DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DTT dithiothreitol EGTA ethylene glycol tetraacetic acid EPSC excitatory postsynaptic current ER endoplasmic reticulum Ex-NMDAR extrasynaptic N-methyl-D-aspartate receptor  GABA γ-aminobutyric acid GAD65 glutamic acid decarboxylase 65 GAP43 growth associated protein 43 GFP green fluorescent protein 	   x	  HAM hydroxylamine HBSS Hank’s balanced salt solution HD Huntington’s disease HEK-293 human embryonic kidney 293 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIP14 huntingtin interacting protein 14 (ZDHHC17) HIP14L huntingtin interacting protein 14-like (ZDHHC13)   HPDP hexadecyl fluorophosphonate Htt huntingtin iMSN MSNs of the indirect pathway INMDA NMDA-mediated current K+ potassium ion MAGUK membrane associated guanylate kinase Mg2+ magnesium ions MK-801 5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine mHtt mutant huntingtin MSN medium-sized spiny neuron Na+ sodium ion NEM N-ethylmaleimide NMDA N-methyl-D-aspartate NMDAR N-methyl-D-aspartate receptor nNOS neuronal nitric oxide synthase NO nitric oxide 	   xi	  N-Htt N-terminal domain of Htt N-terminal amino-terminal PalmB Palmostatin B PAT palmitoyl acyl-transferases PBS phosphate buffered saline PBST phosphate buffered saline plus triton X-100 pCREB phosphorylated CREB PD Parkinson’s disease PDL poly-d-lysine PFA paraformaldehyde PM plasma membranes PPT protein palmitoyl thioesterase PPT1 protein palmitoyl thioesterase 1 PSD postsynaptic density PSD-95 postsynaptic density-95 PTM post-translational modification PTX picrotoxin ROI region of interest RT room temperature SAP97 synapse associated protein 97 SAP102 synapse associated protein 102 SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SNAP23 Synaptosomal-associated protein 23 	   xii	  SNAP25 Synaptosomal-associated protein 25 SNARE Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor STEP striatal-enriched tyrosine phosphatase Stx1b syntaxin 1b Stx7 syntaxin 7 TBS tris-buffered saline TBST tris-buffered saline with 0.5% Tween TMD transmembrane domains TrkB Tropomyosin receptor kinase B TTX tetrodotoxin WT wild-type YAC yeast artificial chromosome YAC128 yeast artificial chromosome with 128 CAG YFP yellow fluorescent protein ZDHHC zinc finger, Asp-His-His-Cys Zn+ zinc ions     	   xiii	  Acknowledgements    I owe my thanks to so many people, without whom I would’ve not been even close to finish the thesis. First, I would like to give my deepest gratitude to my devoted supervisor, Dr. Lynn A. Raymond, whose kindness and patience, support and encouragement has been guiding me for more than seven years. You are my role model with a profound inspiration for me not only in academic research and professionalism, but more importantly, in personality and life. I would like to acknowledge my supervisory committee members Dr. Ann Marie Craig, Dr. Shernaz Bamji, and Dr. Yutian Wang for their guidance. I would like to thank Dr. Elizabeth Conibear for taking time out to serve as the external examiner for my oral defense. I also wish to express my sincere thanks to Dr. Tim Murphy for his encouragement. I am obligated to give my great appreciation to all my lovely colleagues in the lab before and now: Ashley, Jing, Ali, Austen, Clare, Lily, Rujun, Marja, Matt, Karolina, Alex, Caodu, Amy, and Cam. I came to the lab as an undergrad clueless about pretty much everything. I have been so lucky for having your support, witness, and accompanying in all these wonderful years at the fourth floor of Detwiller. I would also like to thank Kim, Cindy, Yichen, Shaun, David, and Mandi for your support in both academic and personal perspectives. Last but not least, I would like to thank my family. You are the best could happen in my life and I couldn’t asked for more. Thank you mom and dad for your unconditioned caring and listening. Thank you Xiaoxiao for your tireless love and always believing in me. Thank you Kevin for still accept me as your friend even I was not able to make it when you took your first step. And finally, the family, the entire world, and myself just cannot wait to meet you, Alethea, in less than six weeks.  	   1	  Chapter 1: Introduction    Evidence indicates that the trafficking and distribution of the N-methyl-D-aspartate receptor (NMDAR) plays a critical role in a wide range of neurological diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and ischemic stroke (Parsons and Raymond, 2014; Fan and Raymond 2007). In early HD, increased expression and signaling of NMDARs at extrasynaptic sites contributes to phenotype onset and excitotoxic neuronal death (Milnerwood et al., 2010). On the other hand, palmitoylation is one of the key lipid post-translational modifications and regulates cellular processes like protein trafficking, stability, interactions, and membrane associations (Young et al., 2012). Although altered palmitoylation of key synaptic proteins has been implicated in HD (Singaraja et al., 2011), to date, changes of NMDAR palmitoylation in HD remain largely unknown and a deeper understanding may provide insights on both scientific and clinical applications.  1.1 NMDA Receptors 	  1.1.1 Structure, composition, and basic functions The NMDAR is an ionotropic glutamate receptor and a cationic channel permeable to Na+, K+ and Ca2+. It is both ligand-gated and voltage-gated. In resting conditions, the channel pore is blocked by extracellular Mg2+. There is a simultaneous dual requirement for channel activation: 1) glutamate as the endogenous agonist and glycine as the co-agonist bind to the receptor; 2) voltage-dependent Mg2+ blockade needs to be removed by membrane depolarization (Dingledine et al., 1999; Sanz-Clemente et al., 2013). This unique feature makes the NMDAR a ‘coincidence 	   2	  detector’ and activation happens when presynaptic glutamate release and postsynaptic depolarization occur at the same time. Seven NMDAR subunits have been identified: one GluN1, four GluN2 (A-D), and two GluN3 (A-B). The majority of functional NMDARs are tetrameric complexes composed of two GluN1 and two GluN2 subunits (Chen and Lipton, 2006; Wenthold et al., 2003; Laube et al., 1998). The GluN1 subunit contains the glycine binding site and is obligatory for function. GluN2 subunits contain the glutamate binding site and define NMDAR subtypes and channel properties like regional expression profiles, electrophysiological and pharmacological properties, downstream signaling and trafficking mechanisms (Cull-Candy and Leszkiewicz, 2004; Sheng and Kim, 2002; Dingledine et al., 1999).  Another difference between GluN1 and GluN2 subunits is their expression level during development. GluN1 expression is high throughout development in most areas of the central nervous system (CNS). GluN2A levels tend to be low early and increase in adulthood, whereas GluN2B expression dominates early and declines with maturation (Cull-Candy et al., 2001; Sheng et al., 1994). GluN2A expression is relatively high in the CNS, GluN2B is highly expressed in forebrain and particularly enriched in the striatum, and GluN2C expression is profound in the cerebellum (Jin et al., 1997; Monyer et al., 1994). The difference of these GluN2 subunits in their subcellular localization and protein interactions and signaling may have an important impact on the balance between neuronal pro-survival and pro-death pathways (Cull-Candy et al., 2001; Dingledine et al., 1999; Hardingham and Bading, 2010; Kohr, 2006; Prybylowski and Wenthold, 2004). In minor cases, triheteromeric receptors (GluN1/GluN2/GluN3) are also formed and GluN3 subunits are usually regulatory (Das et al., 1998; Pérez-Otaño et al., 2006; Cavara and Hollmann, 2008). All NMDAR subunits contain an extracellular N-terminus mediating ligand binding, three transmembrane domains (M1, M3, and M4), an extracellular loop 	   3	  between M3 and M4, and intracellular C-terminus regulating receptor biophysical properties, surface trafficking, localization and stability. The C-terminal tail also facilitates cytoplasmic protein-protein interactions and possesses binding sites for different intracellular proteins key for signaling like calmodulin, and the scaffolding protein postsynaptic density protein 95 (PSD-95) that links Ca2+ influx to regulatory enzymes for various post-translational modifications (Paoletti and Neyton, 2007; Gladding and Raymond 2011). The channel pore is formed by a transmembrane P loop (M2) that dips into the membrane from the cytoplasm with a highly conserved asparagine residue vital for Mg2+ block and Ca2+ permeability (Mayer and Armstrong, 2004; Cull-Candy and Leszkiewicz, 2004). The NMDAR contributes to excitatory synaptic transmission throughout the CNS. After activation, Na+/K+ flux conducts fast synaptic transmission by mediating postsynaptic membrane potential changes. In contrast, Ca2+ influx through the channel can activate a variety of Ca2+-dependent signalling cascades involved in physiological and pathophysiological cellular processes (Mayer and Armstrong, 2004). This Ca2+ permeability feature of the NMDAR is critical for its role in synapse formation and plasticity (Bliss and Collingridge, 1993; Sattler and Tymianski, 2001; Waxman and Lynch, 2005). Besides endogenous agonist glutamate and co-agonist glycine, NMDA, after which the receptor is named, is also an agonist to the channel. MK-801 ((+)-5-methyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) is an open-channel blocker that is very slowly reversible over hours at resting membrane potential (Waxman and Lynch, 2005).    	   4	  1.1.2 NMDAR assembly and trafficking The expression level and subunit composition of NMDARs depends on a variety of factors: cell type, specific subcellular site, developmental stage, activity-dependent regulation, and protein interactions (Collingridge, 2003; Lau and Zukin, 2007; Pérez-Otaño and Ehlers, 2004). If expressed alone, GluN1 or GlunN2 subunits remain in the endoplasmic reticulum (ER) of non-neuronal cells and cannot form functional receptors (McIlhinney et al., 1998, 2003). Usually there is a large pool of GluN1 subunits in the ER available, and the number of GluN2 subunits appears to be the rate-limiting step of NMDAR assembly (Prybylowski et al., 2002). Binding of GluN2 subunits to GluN1 masks an ER retention signal in the GluN1 C-terminus and promotes ER release of fully assembled receptors primarily to the postsynaptic membrane (Scott et al., 2001; Horak et al., 2008). GluN2 subunits are also involved with the accurate delivery of the receptor to synaptic sites, as evidence has shown that truncated GluN2 subunits without the C-terminal tail impair synaptic localization of NMDARs (Mohrmann et al., 2002; Mori et al., 1998). One widely accepted mechanism for this forward trafficking is via association of NMDAR to the family of membrane-associated guanylate kinases (MAGUKs) like PSD-95 and synapse associated protein 102 (SAP102), during the receptor assembly in the ER (Hung and Sheng, 2002; McGee and Bredt, 2003; Elias and Nicoll, 2007). Upon binding to the kinesin KIF17-LIN10 motor complex, newly assembled receptors are transported to the cell membrane along microtubules (Guillaud et al., 2003; Setou et al., 2000). The fact that each subtype of GluN2 has a different C-terminal sequence may impact on the receptor delivery to specific synapses.    	   5	  1.2 Palmitoylation  1.2.1 General overview and basic functions Among post-translational modifications (PTMs), attachment of lipid to a protein increases its hydrophobicity, thus promoting its insertion into intracellular or plasma membranes (PMs). There are three common lipid modifications: myristoylation, prenylation, and palmitoylation (Conibear and Davis, 2010; Young et al., 2012; Fukata and Fukata, 2010), among which palmitoylation is the most common form in the brain. Palmitoylation usually refers to the addition of saturated 16-carbon palmitic acid to specific cysteine residues via a thioester linkage (S-palmitoylation). The most distinguishing feature of S-palmitoylation compared to myristoylation and prenylation is its reversibility. Less common is N-palmitoylation, the irreversible and stable addition of palmitic acid to an N-terminal cysteine via an amide rather than a thioester bond (Nadolski and Linder 2007). For convenience, the term palmitoylation in this thesis refers to S-palmitoylation. Some features of palmitoylation make it a key mechanism underlying a wide range of neuronal processes like tethering and associating signaling proteins to membranes, protein trafficking and stability, protein-protein interactions, and segregation of substrates to particular protein subdomains (Fukata and Fukata, 2010; Linder and Deschenes, 2007; Salaun et al., 2010). First, palmitoylation increases the hydrophobicity of a protein therefore serves to tether and stabilize cytoplasmic proteins to membranes (El-Husseini et al., 2000a; Fukata and Fukata, 2010; Shahinian and Silvius, 1995). Compared to the best-studied PTM, phosphorylation, which is a charge-based regulatory mechanism, palmitoylation is well suited in regulating the countless budding and fusion events between the trafficking vesicles and the PMs in a highly hydrophobic 	   6	  lipid-rich environment. In other cases, palmitoylation promotes protein shuttling and directs their localization between intracellular compartments and specific membrane microdomains, where these proteins are in proximity with others, thus interactions and signaling events can occur (Brown, 2006; Levental et al., 2010). Palmitoylation may only change the hydrophobicity of a particular domain in a complex protein like integral membrane proteins. It then leads not to a major shift on localization, but a conformational change of the substrate protein followed by downstream signaling events (Charollais and Van Der Goot, 2009). Second, unlike the other two major lipid modifications, palmitoylation is reversible due to the labile nature of the thioester bond (Conibear and Davis, 2010). The on/off feature makes it possible to act as a switch in regulating different states of the same substrate. This characteristic provides potential mechanisms for dynamic cellular events like neuronal development and synaptic signaling (Fukata and Fukata, 2010). Third, the addition and removal of palmitic acid can happen rapidly. The de-/palmitoylation of some synaptic proteins like PSD-95 and Ras has a short turnover cycle. This makes palmitoylation an interesting candidate in certain scenarios involved with fast events, for example synaptic formation and functions, and responding to transient and specific signals.  1.2.2 Enzymes and Substrates: PATs and APTs Palmitoyl Acyl-Transferases Palmitoylation was first described more than 35 years ago (Schmidt et al, 1979). However, the enzymes that regulate palmitoylation were only identified after two decades of numerous trials on isolating and characterizing these enzymes (Lobo et al., 2002; Roth et al., 2002; Dietrich and Ungermann, 2004). One reason underlying this difficult in identifying the enzymes may be that 	   7	  some, yet not all, proteins could undergo spontaneous autocatalytic palmitoylation without the need for an enzyme. Finally, two yeast proteins Akr1 and Erf2/4 (Roth et al., 2002), and huntingtin interacting protein 14 (HIP14), the mammalian ortholog of Akr1 (Singaraja et al., 2002; Huang et al., 2004) were proven to have enzymatic activity for palmitoylation. HIP14, the first mammalian palmitoylation enzyme turns out to be number 17 (ZDHHC17) in a family of proteins that share a highly conserved core DHHC (Asp-His-His-Cys) motif and are now collectively named palmitoyl acyl-transferases (PATs) (Fukata et al., 2004; Huang et al., 2004; Roth et al., 2006). To date, 23 DHHC PAT genes have been discovered in humans (zdhhc1-9 and 11-24) and 24 in mice (zdhhc1-9 and 11-25) (Fukata et al., 2004; Huang et al., 2009). PATs are multipass transmembrane proteins with 4 to 6 predicted transmembrane domains (TMDs). All 23 PATs share a common sequence of a 50-residue, zinc-finger-like cysteine rich domain that contains the DHHC core essential for catalytic function (Conibear and Davis, 2010). Most of the PATs are located in endomembrane compartments like the ER, Golgi, or endosomes with the exception of a few at the PM (Ohno et al., 2006). All DHHC PATs demonstrate distinct yet overlapping substrate specificities (Fukata et al., 2004; Huang et al., 2009) and autopalmitoylation, which allows the PAT to transiently enter an intermediate state and then transfer palmitate to the substrate (Lobo et al., 2002; Mitchell et al., 2010; Roth et al., 2002). Similarly, some substrates show remarkable dependency on a particular PAT (Roth et al., 2006), while others are non-selective and interact with multiple PATs (Fukata et al., 2004; Huang et al., 2009). Deletion of a particular PAT may drive the palmitoylation by another PAT that would not occur under physiological circumstances (Salaun et al., 2010).   	   8	  Acyl-Protein Thioesterases  Unlike the large array of PATs, only two acyl-protein thioesterases (APTs), enzymes that can catalyze depalmitoylation processes, have been discovered so far: APT1 and PPT1 (protein palmitoyl thioesterase 1) (Tomatis et al., 2010; Zeidamn et al., 2009). The involvement of APT1 in synaptic function was proven by an experiment in which downregulation of APT1 expression resulted in suppression of hippocampal dendritic spine enlargement (Siegel et al., 2009). APT1 is a cytoplasmic thioesterase (Hirano et al., 2009) and does not contain TMDs (Zeidman et al., 2009). It is suggested that APT1 first gets palmitoylated in order to interact with membrane-associated substrates (Yang et al., 2010). It is the only thioesterase that has been proven to regulate protein depalmitoylation in vivo (Conibear and Davis, 2010). In addition to APT1, mammals express two other APT1 homologs: APT2 (64% amino acid sequence identity to APT1) (Toyoda et al., 1999) and APTL1 (31% identity) (Zeidman et al, 2009). The second thioesterase, PPT1, resides in the lysosome and cleaves fatty acids, including palmitic acid, without discriminating and serves as part of protein degradation pathways (Verkruyse and Hofman, 1996; Zeidman et al., 2009). Aside from its lysosomal localization, there are reports of its existence in synaptosomes and synaptic vesicles, supporting its potential role in neurotransmission (Heinonen et al., 2000; Lehtovirta et al., 2001; Cho et al., 2000).  Synaptic PAT substrates Over the past 15 years, the specificity, reversibility, and rapidity of palmitoylation has drawn a great deal of interest as to its possible involvement in synaptic protein trafficking in and out of synapses. Indeed, the list of synaptic proteins as palmitoylation substrates keeps growing. The assembly and compartmentalization of many neuronal proteins are regulated by palmitoylation 	   9	  at both presynaptic terminal and postsynaptic site (El-Husseini and Bredt, 2002; Fukata and Fukata, 2010; Huang and El-Husseini, 2005; Prescott et al., 2009; Kang et al., 2008). To name some examples, at presynaptic sites, proteins regulating vesicle fusion and neurotransmitter release, like the SNAREs and synaptotagmins (Kang et al., 2008), are found palmitoylated. At postsynaptic sites, the list of plamitoylated proteins includes neurotransmitter receptors (NMDAR, AMPAR, and GABAR), scaffolding proteins involved in excitatory synapse development and plasticity (PSD-95 and SAP97) (Topinka and Bredt, 1998; El-Husseini et al., 2000a, 2002; Kang et al., 2008), and downstream signaling proteins (H/N-Ras) (Hancock et al., 1989). Synaptic adhesion molecules also require palmitoylation for their normal function, which makes palmitoylation critical for neuronal developmental processes and synaptic signal transduction (Brigidi et al., 2014, 2015).  1.2.3 NMDAR palmitoylation Palmitoylation of the NMDAR was first described among more than 200 proteins identified in a global characterization of the rat neural palmitoyl-proteome in 2008 (Kang et al., 2008). Both GluN2A and GluN2B subunits were found palmitoylated in their long intracellular tails. In 2009, another study in cultured cortical neurons showed that GluN2A and GluN2B are palmitoylated at two separate C-terminal cysteine clusters (Hayashi et al., 2009; Fig. 1.1). Palmitoylation at each cluster has remarkably different consequences. In both GluN2A and GluN2B subunits, Cluster I is located on a membrane-proximal region and its palmitoylation enhances Src-family tyrosine phosphorylation thereby increasing the stability of synaptic NMDAR expression (Prybylowski et al., 2005). Mutation of cluster I cysteines leads to increased rates of NMDAR internalization. In contrast, Cluster II is located in the middle of the C-terminus and plays an opposite role to Cluster 	   10	  I. A Golgi apparatus-specific protein with a DHHC zinc finger domain (GODZ/ZDHHC3) has been found to have PAT activity toward Cluster II. Mutation of cysteines in GluN2A and GluN2B Cluster II accelerates release of NMDARs from the Golgi apparatus to the PM, which reveals that Cluster II palmitoylation may play a part in retaining the receptor in the Golgi and potentially serve as a quality-control step in receptor maturation (Hayashi et al., 2009; Thomas and Huganir, 2013). A similar phenomenon was then observed by another lab using an electrophysiological approach in 2012 (Mattison et al., 2012).   Figure 1.1. Schematic illustration of palmitoylation sites at C-terminal of GluN2A and GluN2B subunits. Consensus cysteine clusters are localized at C-terminal of GluN2A and GluN2B subunits. Boxes indicate conserved cysteine residues (Cys Cluster I and II). Reprint from (Hayashi et al. 2009) with permission.  Besides the direct effect of palmitoylation on the NMDAR, palmitoylation might also affect the receptor indirectly by changing the state of other proteins that closely interact with the receptor and are required for the normal distribution of the receptor. For example, palmitoylation is found necessary for PSD-95 oligomerization, synaptic targeting, and its role of clustering neurotransmitter receptors (Christopherson et al., 2003; Craven et al., 1999; El-Husseini et al., 	   11	  2000a,b). Palmitoylation also regulates the Golgi-PM trafficking cycle of PSD95 to compensate changes in strength of synaptic stimulation. Strong excitatory stimulation tends to increase PSD-95 depalmitoylation, thus lessen the number of PSD95 at the PM of the postsynaptic site (El-Husseini et al., 2002), whereas reducing synaptic activity leads to an increase in PSD-95 palmitoylation and localization to the postsynaptic PM (Noritake et al., 2009; Conibear and Davis, 2010). Recently, the first antibody specific for the palmitoylated conformation of a protein was developed (Fukata et al., 2013). It is a single-chain variable fragment PSD-95 antibody and was able to demonstrate that palmitoylated PSD-95 is localized almost exclusively at the PSD of excitatory synapses (Fukata et al, 2013). Notably, previous work in our lab showed that PSD-95 palmitoylation has an effect on NMDAR desensitization (Li et al., 2003). Thus, palmitoylation is a powerful PTM in regulating the effect of NMDAR and PSD-95 localization and function on excitatory synaptic signaling.  1.2.4 Diseases affecting the nervous system Given how important palmitoylation is to maintain the normal trafficking and distribution of a long list of synaptic proteins, it is not surprising that aberrant palmitoylation has also been found to underlie the pathophysiology of multiple diseases of the nervous system, like AD (ZDHHC12) (Mizumaru et al., 2009), HD (HIP14 and HIP14L, ZDHHC17 and ZDHHC13, respectively) (Singaraja et al., 2002; Huang et al., 2004; Saleem et al., 2010; Sanders et al., 2015b), schizophrenia (ZDHHC8) (Mukai et al., 2004), and mental retardation (ZDHHC9 and ZDHHC15) (Raymond et al., 2007; Mansouri et al., 2005). Palmitoylation is critical not only for the neuronal proteins that control firing at the synapse, but also for proteins important in neurodevelopmental processes and neuronal survival. Thus, aberrant palmitoylation may result in misregulation of neuronal proteins and neuropsychiatric disease. 	   12	  1.3 Huntington’s Disease 	  1.3.1 Overview First described thoroughly by George Huntington in 1872 (Huntington, 1872), Huntington’s disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder for which there is currently few symptomatic treatments and no cure (Cowan and Raymond, 2006). This inherited disease is caused by a dominant mutation in the HD gene, located in the 4p16.3 region of the short arm of chromosome 4, which leads to the expansion of CAG repeats producing an elongated polyglutamine tract in the N-terminal region of the huntingtin (Htt) protein (The Huntington’s Disease Collaborative Research Group, 1993). Non-affected individuals have less than 36 CAG repeats, and longer repeats are associated with earlier disease onset. With a 50% risk of inheritance, HD equally affects both men and women and has a prevalence of ∼14 per 100,000 people (Fisher and Hayden, 2014). HD is a typical middle-age onset disease (35-50 years old) with the exception of individuals carrying CAG repeats exceeding 60, which leads to juvenile onset. Emotional and cognitive changes usually happen earlier than major motor abnormalities. The former include mood disorder, particularly depression, and personality changes, and the latter include involuntary dance-like movements (chorea), postural imbalance, speech and swallowing difficulties, and akinesia and rigidity at later stage (Raymond et al., 2011). Neuropathologically, although a wide range of different brain regions and neuronal types are affected by the disease, for instance globus pallidus, thalamus, hypothalamus, subthalamic nucleus, substantia nigra, and cerebellum, HD most severely affects the striatum of the basal ganglia and certain layers of cortex (Raymond et al., 2011). It was found that HD shows a pattern of selective neurodegeneration, which affects the GABAergic medium-sized spiny projection 	   13	  neurons (MSNs) of the striatum earliest and most severely. MSNs comprise more than 95% of the striatal neuronal population and project mainly to substantia nigra and globus pallidus (Raymond et al., 2011). The mechanisms of selective vulnerability of MSNs in HD are not fully understood. Wild-type (WT) Htt is a large protein (∼350 kDa) and ubiquitously expressed throughout the body (Strong et al., 1993) with highest levels in the CNS (Trottier et al., 1995). However, the highest levels of Htt in the CNS are found in the cerebellum, whereas there is no particular enrichment of Htt in striatum (Milnerwood and Raymond, 2010). It shows a spectrum of different functions of WT Htt that has not been fully explored. Previous research has linked WT Htt with diverse activities (Cattaneo et al., 2005): neuronal protection (Leavitt et al., 2006; Buren et al., 2014) and anti-apoptosis, vesicle transport (Caviston and Holzbaur, 2009), transcriptional regulation, synaptic transmission and post-synaptic density (PSD) organization (Sun et al., 2001). Three different lines of Htt-knockout mice die in utero before the formation of the nervous system, which also proves Htt is essential for embryonic development (Nasir et al., 1995). It is established that mutant Htt (mHtt) leads to HD through a toxic gain-of-function, which particularly targets MSNs (Milnerwood and Raymond, 2010). In addition, accumulating studies show that loss of the normal cellular function of WT Htt also contributes to HD pathogenesis (Cattaneo et al., 2005; Zuccato et al., 2010).   1.3.2 Mouse models of HD Animal models of HD have been assisting researchers with insight on understanding HD for over 30 years. Especially after the identification of the HD gene in 1993, a variety of genetic mouse and rat models have been developed (Fan and Raymond, 2007). These mouse models can be generally classified into three groups (Raymond et al., 2011): 1) Htt-fragment transgenic mice, 	   14	  expressing truncated mHtt under the control of a CMV promoter (R6/1 and R6/2) (Mangiarini et al., 1996; Davies et al., 1997); 2) transgenic mice expressing full-length mutant human Htt with its endogenous 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); and 3) knock-in mice with expanded CAG repeats into the endogenous mouse HD gene (HdhQ111 and HdhQ150) (Wheeler et al, 2000; Lin et al., 2001). Each of the models has strengths and weaknesses. The mutant Htt-fragment transgenic mice (R6/1 and R6/2) show rapid onset of motor deficits and progression of the disease, as R6/1 mice die within 8-10 months and R6/2 in 4 months. The accelerated HD-like disease makes these mice good models to study new therapies. The major limitation of these mice is the lack of full-length Htt and no evidence of striatal neuronal loss, the distinctive feature of human HD. The problem with knock-in mice is different. They have the most accurate replication of the mHtt gene yet only mild motor deficits and neuropathological changes as late as 2 years of age. Among these animal models, the YAC mouse is the most widely studied full-length transgenic model (Hodgson et al., 1999; Slow et al., 2003). The YAC lines were developed on the FVB/N mouse strain background, expressing full-length human Htt, including the upstream and downstream endogenous regulatory sequences. The YAC128 line used in my study contains a pathological 128 CAG repeats and shows age-dependent striatal and subsequent cortical degeneration consistent with clinical manifestations in HD patients; these changes occur at an earlier age compared to other YAC HD strains like YAC46 and YAC72 (Slow et al., 2003). All of the HD mouse models exhibit changes in striatal electrophysiological (currents, capacitance, and/or resistance) and neuropathological (striatal and cortical atrophy at 9 and 12 months respectively, cell loss, and/or nuclear inclusions) properties similar to those found in 	   15	  human HD. The YAC128 HD mice also show progressive cognitive and motor deficits in behavioral tests (open field activity and rotarod performance): hyperkinesia as early as 3 months of age and hypokinesia at 12 months of age (Slow et al., 2003; Van Raamsdonk et al., 2005a, b; Levine et al., 2004). One inconsistency with human HD patients is that YAC128 mice tend to gain weight (Van Raamsdonk et al., 2006) instead of losing it, which may be caused by over expression of human Htt in the YAC128 mice (Van Raamsdonk et al., 2006).  1.3.3 Corticostriatal synaptic dysfunction in HD Striatal cells, especially MSNs, are the cells affected by the disease both the earliest and most severely. However, Htt is not only ubiquitously expressed throughout the body but also shows no regional correlation with the neuronal vulnerability (Fusco et al., 1999). The mechanisms underlying this striatal selectivity have not been fully elucidated. However, after decades of research on genetic mouse HD models, along with advanced clinical studies, accumulating evidence indicates that corticostriatal network dysfunction occurs prior to the onset of overt motor and neuropathological symptoms, and contributes to the progressive degeneration of the striatum in HD (Sepers and Raymond, 2014; Milnerwood and Raymond 2010; Plotkin and Surmeier, 2015). Glutamate excitotoxicity, disrupted brain-derived neurotrophic factor (BDNF) expression and signaling, as well as altered dopamine and endocannabinoid signaling may all underlie dysfunction of the complex corticostriatal network. Excitotoxicity is defined as the overactivation of receptors by excitatory amino acid neurotransmitters, resulting inneuronal death (Olney, 1969; Choi, 1987). In the mammalian CNS, glutamate is the main excitatory neurotransmitter (Hebb, 1970). The hypothesis of glutamate excitotoxicity in HD includes the possible contributions from excessive release of glutamate from 	   16	  cortical afferents, impaired glutamate uptake, and/or hypersensitivity of glutamate receptors, together leading to excessive influx of ions (especially Ca2+ through NMDARs). The dysfunction then results in death of MSNs in the striatum (DiFiglia 1990). Ca2+ must be under delicate control and excessive Ca2+ influx is neurotoxic, activating multiple pro-death cascades such as activation of degradative enzymes (caspases and calpains), generation of reactive oxygen species, and mitochondrial energy failure (Albin and Greenamyre, 1992). Intrastriatal injections of glutamate receptor agonists, especially those selective for NMDAR, lead to striatal lesions and motor dysfunction in both rodents and non-human primates similar to that of HD patients (McGeer and McGeer, 1976; Hantraye et al., 1990; Ferrante et al., 1993). Elevated glutamate release is found in early stages of HD (Raymond et al., 2011). Decreased glutamate uptake from the extracellular space at corticostriatal synapses by glutamate transporters in astrocytes has also been reported in symptomatic rodent HD models (Miller et al., 2012). BDNF is a neuron-released growth factor critical for neurite growth and spine formation. Striatal neurons rely on BDNF produced by other brain regions. In mouse, loss of BDNF from the cortex and substantia nigra causes severe motor deficits. Furthermore, loss of TrkB, the tyrosine kinase receptor of BDNF, leads to reduced striatal volume (Li et al., 2012). WT Htt is required for the expression and vesicular transport of BDNF (Gauthier et al., 2004), which is disrupted by mHtt, weakening the trophic support to the striatum in both animal models and HD patients (Zuccato et al., 2010). mHtt also reduces the transport of TrkB receptor to the striatal dendrites (Liot et al., 2013). On the other hand, overexpression of BDNF in the cortex of YAC128 mice increased BDNF and TrkB levels in the striatum and improved motor deficits (Xie et al., 2010). Dopamine (DA) signaling dysfunction is found in many movement and psychiatric disorders. In striatum, complex outputs are conventionally classified into two complementary 	   17	  pathways according to their different functional outcomes. The direct pathway projects to the internal segment of the globus pallidus and substantia nigra pars reticulata, and facilitates movement by promoting cortical action selection. The indirect pathway projects to the external segment of the globus pallidus and inhibits movement by suppressing unwanted action selection. MSNs of the direct pathway (dMSNs) express dopamine receptor 1 (D1) and MSNs of the indirect pathway (iMSNs) express dopamine receptor 2 (D2) (Gittis and Kreitzer, 2012). DA has a higher affinity at D2 receptors, and in HD, an altered tonic DA level may affect the iMSNs earlier than it does to the dMSNs. Since the iMSNs of the striatum are responsible for suppressing unwanted movement, increased DA acts at the iMSNs and may result in chorea by reducing ability to inhibit movement in early HD (Eidelberg and Surmeier, 2011). Later in HD, dMSNs are affected and patients lose the ability to promote purposeful movements thereby showing hypokinesia. Endogenous cannabinoids are lipophilic neuromodulators. mHtt in animal models reduces G protein-coupled cannabinoid receptors and may result in hyperkinesia (Sepers and Raymond, 2014).  1.4 NMDAR in HD 	  1.4.1 NMDAR dysfunction in early HD More than three decades ago quinolinic acid (a selective NMDAR agonist) was used to produce the behavioral and neuropathological manifestations of HD in murine and non-human primate models (Coyle, 1979; Ferrante et al., 1993). Previous studies in our lab showed that elevated NMDAR currents were recorded in HEK-293 cells transfected with the mHtt gene, in cultured and acutely dissociated mHtt-expressing MSNs, and in brain tissue from HD mice (Chen 	   18	  et al., 1999; Zeron et al., 2001, 2002; Li et al., 2002; Li et al., 2003; Fan et al., 2007). Later on, larger evoked NMDAR excitatory postsynaptic current (EPSC) amplitudes were recorded in striatal neurons from acute corticostriatal brain slices of 1-/2-month-old YAC128 mice (Milnerwood and Raymond, 2007, 2010; Graham et al., 2009). These studies along with enhanced mitochondrial depolarization, activation of pro-death pathways, and increased NMDA-induced toxicity (Zeron et al., 2001, 2002, 2004; Fan et al., 2009, 2010, 2012) are consistent with the increased striatal neuronal vulnerability to NMDA toxicity (Shehadeh et al., 2006), suggesting NMDAR dysfunction is one early mechanism behind the selective vulnerability of the striatum in HD. Actually an increased NMDAR current in MSNs was found in most of the HD mouse models (Raymond et al., 2011).  1.4.2 GluN2B-type NMDARs in HD In most brain regions GluN2B-type NMDARs (2B-NMDARs) predominate early in development, before they are decreased and GluN2A (2A-NMDARs) expression is increased as the brain matures (Dumas, 2005; Bellone and Nicoll 2007). However, in the adult brain it is interesting to note that there are relatively higher expression levels of 2B-NMDARs in striatal MSNs than other NMDAR subtypes and brain regions (Landwehrmever et al., 1995; Watanabe et al., 1993). Over recent years, independent groups found that 2B-NMDARs are preferentially involved with pro-death signaling pathways, whereas 2A-NMDARs have been more closely associated with pro-survival pathways (Kim et al., 2005; Liu et al., 2007; Tu et al., 2010). Previous findings from our lab also demonstrated that 2B- and not 2A-NMDARs are linked to early dysfunction of NMDARs in HD. The enhanced NMDAR current and NMDA-induced toxicity found in mHtt-expressing MSN could be rescued by treating cultured neurons and acute striatal brain slice with the selective 	   19	  2B-NMDAR antagonist ifenprodil (Chen et al., 1999; Zeron et al., 2002; Fan et al., 2007; Milnerwood et al., 2010). Furthermore, another group showed that 2B-NMDAR contribute proportionally more to total NMDA-evoked current in D2 iMSNs than in D1 dMSNs, consistent with the enhanced vulnerability of iMSNs in early HD (Jocoy et al., 2011).  1.4.3 Extrasynaptic NMDARs in HD Besides subunit composition, studies in last decade showed that a disrupted balance between synaptic and extrasynaptic (Ex-) NMDARs activation contributes to the dichotomy that a single receptor has dual roles in triggering pro-survival and pro-death pathways. To elucidate, synaptic NMDAR activation leads to survival and plasticity pathways, via increasing the expression and activity of a master transcriptional regulator, cyclic-AMP responsive element binding protein (CREB) (Hardingham et al., 2001; Papadia et al., 2005; Zhang et al., 2009). When phosphorylated, CREB is activated and promotes expression of a variety of pro-survival proteins, including BDNF (Zuccato et al., 2010; Papadia et al., 2008). In contrast, Ex-NMDAR activity triggers CREB shut-off by dephosphorylation, pro-death gene expression, Ca2+ overload in mitochondria, and activation of apoptotic cascades (Xu et al., 2009; Hardingham and Bading, 2010).  It still needs to be explored whether the pattern that 2A-NMDAR is targeted to synapses and 2B-NMDAR to extrasynaptic sites holds true, or which hypothesis — subunit composition, subcellular localization, or even a ‘unified hypothesis’ that combines the two — account more for the neuropathogenesis of HD. A recent study used a knock-in mouse model expressing a chimeric form of NMDARs, in which the C-terminal tails of 2A- and 2B-NMDARs are switched. NMDARs with the GluN2B C-terminus preferentially contribute to NMDA-induced toxicity (Martel et al., 	   20	  2012). Adding to the complexity, other recent studies demonstrated that 2A-NMDAR and synaptic NMDAR could both induce excitotoxicity under certain experimental circumstances (Papouin et al., 2012; Zhou et al., 2013a, 2013b). The linkage of Ex-NMDARs to mHtt-associated striatal neuronal toxicity in early HD was demonstrated in a 2010 study from our lab. My colleagues demonstrated that the abundance of Ex-NMDARs rises in MSNs of young YAC128 mice (Milnerwood et al., 2010), and this pathological effect is mediated predominantly by 2B-NMDARs. A 2-month treatment of YAC128 mice with low doses of memantine to preferentially block Ex-NMDARs restored CREB activity and behavioural abnormalities to levels found in FVB/N mice (Dau et al., 2014; Milnerwood et al., 2010; Okamoto et al., 2009). Subsequently, our lab showed that both calpain and striatal enriched protein tyrosine phosphatase (STEP) contribute to up-regulation of Ex-NMDARs at least in pre-manifest YAC128 mice; those results were obtained by subcellular fractionation on striatal tissue and isolation of extrasynaptic and synaptic synaptosomal components (Milnerwood et al., 2010; Gladding et al., 2012, 2014). In primary neuronal culture, the proportion of NMDAR current remained after blockade of synaptic NMDAR was larger in YAC128 MSNs compared to FVB/N MSNs (Milnerwood et al., 2012).  1.4.4 Palmitoylation on altered 2B-NMDAR subcellular distribution  Notably, mHtt seems not to affect the total expression level of NMDAR, or the stability of surface receptors (Chen et al., 1999; Li et al., 2003); rather, it enhances NMDAR surface expression by accelerating forward trafficking (Fan et al., 2007). The detailed molecular mechanisms have not been fully elucidated until recent studies from our lab revealed the potential connection between altered PTM, especially palmitoylation, and 2B-NMDAR dysfunction and 	   21	  abnormal surface distribution in early HD. Previous study in our lab showed that after cleavage of the GluN2B C-terminus by calpain, the synaptic/extrasynaptic balance of 2B-NMDARs is changed in MSNs from presymptomatic YAC128 mice (Gladding et al., 2012), which suggests the involvement of the GluN2B C-terminus in the trafficking of 2B-NMDARs. The two cysteine clusters located within the GluN2B C-terminal region represent palmitoylation sites by which 2B-NMDAR trafficking can be regulated; therefore, the specific effect of each cluster has been examined in our lab (Kang et al., manuscript in preparation). The unpublished data demonstrate that palmitoylation of 2B-NMDAR is significantly reduced in striatal tissues of 1- to 3-month-old YAC128 mice (Fig. 1.2).    Figure 1.2. Significant reduction of 2B-NMDAR palmitoylation level in striatum from 1- to 3-month-old YAC128 HD mice. (A) Striatum tissues from 1- to 3-month-old FVB/N and YAC128 mice. (B) Quantitative analysis of 2B-NMDAR palmitoylation level in FVB/N and YAC128 striatum. Figure contributed by R. Kang.   	   22	  Furthermore, GluN2B palmitoylation shows a relatively rapid turnover rate in mouse cortical cultures (Fig. 1.3), indicating it can be dynamically regulated. In addition, NMDARs containing the cluster II (but not cluster I) palmitoylation-resistant mutant GluN2B (2B 5CS) show significantly enhanced surface expression in striatal neurons in FVB/N corticostriatal co-cultures (Fig. 1.4), mimicking the increased striatal GluN2B surface expression observed in YAC128 co-cultures; in contrast, there is little effect of 2B 5CS expression in YAC128 MSNs, suggesting the effect of reduced cluster II palmitoylation to increase surface 2B-NMDAR is occluded in YAC128 mice. Moreover, the increased striatal surface 2B 5CS in FVB/N MSNs is restricted to extrasynaptic membranes, suggesting that reduced GluN2B Cluster II palmitoylation contributes to mHtt-induced early alterations in 2B-NMDAR trafficking in YAC128 mouse striatum (Kang et al., manuscript in preparation).   Figure 1.3. Rapid turnover rate of GluN2B palmitoylation in mouse cortical neurons. (A) Mouse cortical neurons were treated with 2-bromo-palmitate (2BP) for 4 hours to inhibit palmitoylation at day in vitro (DIV) 14. (B) Graph showing that 4-h treatment of 2BP significantly reduced GluN2B palmitoylation level. Figure contributed by R. Kang. 	   23	    Figure 1.4. Significant enhancement of mutant GluN2B Cys Cluster II (GluN2B 5CS) in surface expression in FVB/N striatal neurons. (A, B) Example surface/internal expression of 2B-NMDAR in FVB/N and YAC128 MSNs transfected with GFP-tagged GluN2B WT and Cys Cluster II mutant form (5CS). (C) Quantitative analysis for surface over internal 2B-NMDAR intensity ratio. In FVB/N but not YAC128 MSNs, surface GluN2B 5CS was significantly increased compared to neurons expressing GluN2B WT. Figure contributed by R. Kang. 	   24	   HIP14 has been identified as the major PAT for Htt. Furthermore, altered interaction between mHtt and HIP14 has been shown to affect palmitoylation and trafficking of key proteins at the synapse, like PSD-95 and synaptotagmin (Singaraja et al., 2002; Huang et al., 2004). In addition, other studies in our lab found that the interaction between 2B-NMDAR and PSD-95 is increased at extrasynaptic sites in YAC128 striatal tissue, resulting in enhanced Ex-2B-NMDAR stability (Fan et al., 2009, 2012; Kang, unpublished data). Together, these findings suggest that altered GluN2B palmitoylation, possibly involved with HIP14, contributes to mis-trafficking of 2B-NMDAR in YAC128 striatum.  1.5 HIP14 in HD  1.5.1 General overview: discovery, structure, and expression in the CNS  HIP14 (ZDHHC17) first drew attention during a yeast two-hybrid screen for Htt interactors. Its interaction with Htt inversely correlated with CAG length (Singaraja et al., 2002). It is also found enriched in the brain and expressed in MSNs. In the same study, a HIP14 homolog, HIP14L (ZDHHC13), was also identified in a database search (Singaraja et al., 2002). HIP14L is more similar to HIP14 than any of the other 21 PATs (Fukata and Fukata, 2010). These two share 48% amino acid identity and 57% similarity at the protein level in human, and 46% identity and 63% similarity in mice (Sutton et al., 2013). In 2004, the first study to explore the PAT function of HIP14 discovered that the palmitoylation-dependent trafficking of several neuronal proteins including Htt were enhanced by HIP14 (Huang et al., 2004). A series of studies following this discovery revealed the HIP14-related aberrant palmitoylation in HD (Slow et al., 2003; Yanai et 	   25	  al., 2006; Huang et al., 2011; Singaraja et al., 2011). In cultured cortical cells from YAC128 mice, mHtt inclusion formation is increased when HIP14 is downregulated by siRNA, while inclusions are reduced when HIP14 is overexpressed (Yanai et al., 2006). It is not the expression level of HIP14, but the palmitoylation of HIP14 that is reduced in YAC128 and R6/2 mice brain tissue. Since autopalmitoylation is a necessary functional step, it was concluded that HIP14 PAT activity was reduced in the presence of mHtt (Huang et al., 2011; Singaraja et al., 2011). In addition to HIP14, 19 proteins were found to have a greater than 10% reduction in palmitoylation in brain tissue from 1-year old YAC128 mice, ranging from glial-specific proteins to glutamate transporters (Wan et al., 2013; Huang et al., 2010). In 2014, the binding sites in Htt for HIP14 and HIP14L were reported (Sanders, et al., 2014).  1.5.2 Substrates of HIP14 and HIP14L The list of HIP14 substrates keeps growing: SNAP25, GAD65, Htt, GluA1/2 AMPAR, subunits, Synaptotagmin I, and others (Fukata et al, 2004; Huang et al., 2004, 2009; Butland et al., 2014 Singaraja et al., 2011), among which some proteins, like PSD-95, showed inconsistency across different studies. PSD-95 was found as a substrate of HIP14 in vitro (Huang et al., 2004) which was supported by a later study using shRNA-mediated knockdown of HIP14 in cultured neurons (Huang et al., 2009). However, the other study showed that palmitoylation of PSD-95 was not increased by HIP14 in HEK-293 and COS-7 cells transfected with PSD-95 and HIP14 DNAs (Fukata et al., 2004). These seemingly contentious findings should not be surprising given how complex and sensitive palmitoylation is in different physiological contexts, and the redundancy and substrate overlap of different PATs. Unlike HIP14, so far only six HIP14L substrates have been identified (Huang et al., 2009, 2011; Sutton et al., 2013). HIP14 and HIP14L are the only two 	   26	  PATs that catalyze Htt palmitoylation in COS cells. Interestingly, in addition to being a substrate of HIP14, WT Htt in turn facilitates the palmitoylation of HIP14 itself (Huang et al., 2011; Milnerwood et al., 2013). Palmitoylation of HIP14 was decreased after antisense oligonucleotides (ASO)-mediated knockdown of Htt. Furthermore, palmitoylation of HIP14 substrates was increased in the presence of Htt but not mHtt, and was decreased when Htt expression was reduced (Huang et al., 2011).  1.5.3 HIP14, HIP14L deficient mouse models HIP14 and HIPL mouse models have been developed on the background of FVB/N to better study their possible involvement in HD. HIP14 knock-out (Hip14-/-) mice show normal survival and appearance in general. Some deficits of Hip14-/- mice include: early loss of body weight at 3 months and brain weight at 1 month; profound reduction of striatal volume, striatal neuron counts, two MSN markers, and striatal excitatory synapses per neuron; early impairments of motor coordination and balance at 3 months; and altered corticostriatal circuits at 12 months: all of which are similar to HD patients and/or YAC128 mice (Singaraja et al., 2011; Estrada-Sanchez et al., 2013). Intriguingly, Htt palmitoylation showed no change in Hip14-/- mice, implying possible compensation by other PATs like HIP14L in such condition. Other substrates of HIP14 but not HIP14L, like SNAP25 and PSD-95, showed reduced plamitoylation in Hip14-/- mice (Singaraja et al., 2011). A following electrophysiological characterization of these mice provided further confirmation of the involvement of HIP14 in HD pathogenesis (Milnerwood et al., 2013). Neuronal surface membrane area, number of excitatory synapses, excitability and probability of release at corticostriatal synapses are all found reduced in the MSNs. As well, Hip14-/- mice show severe impairment of hippocampal long-term potentiation. 	   27	   HIP14L mutant (Hip14l-/-) mice have also been generated on the FVB/N background (Sutton et al., 2013). These mice show deficits such as mild abnormalities of skin and fur, as well as progressive reduction of brain and body weight, striatal volume, and striatal neuron counts at 6 months. Behavioral deficits are similar to those observed in the YAC128 (Sutton et al., 2013; Van Raamsdonk et al., 2005b). Similar to Hip14-/- mice, Htt palmitoylation is not altered in Hip14l-/- mice. Most notably, double knock-out of Hip14 and Hip14L leads to embryonic lethality between embryonic day 10-11, indicating the critical overlap in function of these two PATs (Sanders et al., 2015a). Hip14-/-;Hip14l-/- mouse embryonic fibroblasts show a 25% decrease in Htt palmitoylation and those embryos have morphological similarities to embryonic lethal Htt deficient embryos.  A more recent yeast two-hybrid screen for HIP14 interactors discovered that HIP14 and Htt share 36 interactors in common and 17 of those are found involved in HD (Butland et al., 2014). It is interesting to know the mechanism of how the altered activity of HIP14 may contribute to HD pathogenesis.  1.6 Palmostatin B 	  1.6.1 Synthesis and application Palmostatin B (PalmB) is a newly developed small molecule inhibitor of APT1 (Dekker et al., 2010). It is a competitive inhibitor that reversibly modifies a serine residue at the active site of APT1, causing interruption of palmitate dynamic cycling. It was first shown to have a profound inhibition on Ras depalmitoylation in vivo, thereby disrupting its localization (Dekker and Hedberg, 2011). Due to the high efficiency and accuracy of PalmB in targeting APT1 and APT2 (Dekker et al., 2010; Rusch et al., 2011), PalmB has been used widely (Lin and Conibear, 2015). 	   28	  1.6.2 Effect of PalmB on depalmitoylation of synaptic proteins A recent study revealed more details of PalmB beyond an APT1/2-selective inhibitor. Results showed that PSD-95 depalmitoylation, usually not regulated by APT1/2-selective inhibitors, is also affected by PalmB, suggesting the existence of additional thioesterases, besides APT1/2, that may be inhibited by PalmB (D. Lin, unpublished data; Conibear lab, UBC). In COS cells, application of PalmB alters the distribution of PSD-95-GFP and inhibits palmitate turnover but not protein turnover of PSD-95 (D. Lin, unpublished data). N-terminal domain of Htt (N-Htt) palmitoylation was also affected by PalmB treatment. The depalmitoylation of N-Htt was potently inhibited by PalmB treatment through inhibition of APT1 and APT2. It would be interesting to study the effect of PalmB on the function and/or distribution of additional HD-related synaptic proteins like the NMDAR, especially since PSD-95 binds the NMDAR and is a key scaffolding protein in both physiological and HD pathological conditions.  1.7 Rationale and hypothesis 	  1.7.1 Hypothesis: HIP14 regulates NMDAR trafficking and affects NMDAR function Since its discovery over a decade ago, HIP14 has proven to be the key PAT involved in HD pathogenesis (Sanders and Hayden, 2015; Huang et al., 2004). Although the necessity of HIP14 for normal palmitoylation of a list of HD-related proteins has been well demonstrated by multiple approaches, including neuropathological and electrophysiological assays in cells, and/or behavioural assays (Huang et al., 2004), primary neuronal cultures from HD mice models (Yanai et al, 2006), and transgenic knockout mice models (Singaraja et al., 2011; Milnerwood et al., 2013), the exact mechanism of HIP14 contribution to early HD pathogenesis remains largely unknown at 	   29	  the molecular level. Given that HIP14 is a PAT critical for specific PM domain targeting of its substrates, and that abnormal distribution of 2B-NMDARs at extrasynaptic sites is a major cause leading to MSNs loss in early HD, it will be useful to investigate the effect of HIP14 on 2B-NMDAR trafficking in HD. We hypothesize that HIP14 regulates 2B-NMDARs trafficking and function in MSNs of striatum.  1.7.2 Hypothesis: PalmB affects NMDAR trafficking and function In the second part of this study, I aim to examine the potential effect of PalmB on NMDAR trafficking and synaptic activity in general. As a small molecule inhibitor of APT1/2, PalmB is a powerful tool to suppress depalmitoylation in a large number of proteins, considering APT1/2 are the only major APTs to be discovered so far that catalytically remove palmitate from proteins (Dekker et al., 2010; Lin and Conibear, 2015). PalmB has already been shown to have a potent effect on a list of synaptic proteins like Ras, PSD-95, and N-Htt (Dekker and Hedberg, 2011; Lin, unpublished data). Due to the proposed role of PalmB, treatment with it would potentially prolong the palmitoylation state of GluN2B and may thereby impact 2B-NMDARs trafficking and function. We hypothesize that treatment with PalmB affects NMDAR trafficking and synaptic activity in general.   	   30	  Chapter 2: Methods    2.1 Primary neuronal culture and transfection Major glutamatergic inputs projecting into the striatum are from the cortex and thalamus, and dopaminergic modulation from the substantia nigra. Given that over 90% of striatal cells are GABAergic, striatal mono-culture is a suboptimal system for studying NMDAR trafficking due to lack of excitatory synapses. With significant improvement compared to striatal mono-culture (Kaufman et al., 2012), our lab and other groups have developed and refined the corticostriatal co-cultures (Milnerwood et al., 2012; Fan et al., 2012; Segel et al., 2003) that recapitulate the corticostriatal pathway in a simple in vitro system. MSNs benefit from the glutamatergic inputs from the cortical cells and develop spines (Segal et al., 2003; Kaufman et al. 2012). Procedures of the co-culture prepared in the Raymond Lab were described previously (Fan et al., 2012). Briefly, at DIV0, pregnant FVB/N and YAC128 mice were anesthetized with halothane followed by immediate decapitation. The brains from 17- to 18-day-old mice embryos were removed and placed in ice-cold Hank’s Balanced Salt Solution (HBSS, Invitrogen). The striatum and the cortex were dissected from the brains and placed in tubes separately, followed by trypsin digestion at 37°C for 13-15 minutes. Striatal and cortical neurons were then isolated by adding trypsin inhibitor and briefly centrifuging, and then placed in transfecting media DMEM+ (D minimum essential medium plus 10% fetal bovine serum – Invitrogen). To identify MSNs, 2 million striatal cells were immediately centrifuged and suspended in 100 µl electroporation buffer (Mirus Bio.) with 2-5 µg of plasmid DNA construct, placed in a cuvette (0.2 µm, Biorad) and electroporated within an AMAXA nucleofector I (Amaxa, Lonza Bio.), using program O-05. 	   31	  Transfected striatal cells were then mixed with untransfected cortical cells at a ratio of 1:1, re-suspended in DMEM+ and placed in a Costar 24-well plate (Corning) with poly-D-lysine (Sigma) pre-coated 12 mm glass coverslips (Marlenfeld, Germany). After 2-4 hours DMEM+ was replaced with 500 µl of serum-free plating media per well (2% B27, 200 unit/ml penicillin/streptomycin, and 2 mM α-glutamine in Neurobasal Medium – Invitrogen) and another 500 µl of plating medium was added at DIV 4. All cultures were maintained in a humidified 37 °C incubator with 5% CO2 and refreshed every 3-5 days by changing half of the plating medium. The constructs used in this study include high quality endotoxin-free yellow fluorescent protein (YFP) plasmid on a β-actin promoter (a gift from Dr. A.M. Craig, University of British Columbia), and a GFP-tagged GluN2B construct, which was kindly provided by Dr. Richard Huganir, Johns Hopkins University.  2.2 HIP14 ASO and PalmB treatments A phosphorothioate backbone ASO with five 2’O-methoxyethyl ribose sugars in the wings against the Hip14 3’UTR was designed by ISIS Pharmaceuticals (515084, 5’-TGCTTTATTTTCAGACCGTG-3’) and kindly provided by Dr. Shaun Sanders (Hayden lab, UBC). Hip14 ASO was resuspended in sterilized PBS to a concentration of 0.5 -1 µM. The potency of this Hip14 ASO was tested by bath application into the media of DIV3 FVB/N mouse primary cortical neurons, which freely take up ASOs from the media (Carroll et al., 2011). Based on the results of the Hip14 ASO dose response time course, the 250 nM final concentration for 10 days was determined to be optimal for HIP14 knockdown (Fig. 2.1). For HIP14 ASO treatment, corticostriatal co-cultures from FVB/N and YAC128 mice were maintained with 1 mL/well plating medium. Hip14 ASO stock was added to a final concentration 250 nM at DIV4. Neurons were fed with 50µL plating medium per well every 3-4 days, and then followed with immunocytochemistry 	   32	  or electrophysiology experiments on DIV14.   Figure 2.1. HIP14 ASO dose response time course curve in FVB/N mouse cortical neurons. Neurons were treated on DIV3 with various doses of a Hip14 ASO (in nM): 0, 31.3, 62.5, 125, 250, or 500 and were harvested at different time points afterwards: 2, 4, 7, or 11 days post-treatment. HIP14 expression levels were assessed by western blots. Representative blot shows knock-down of endogenous HIP14 protein expression in a dose- and time-dependent manner. Figure contributed by R. Kang.  PalmB, a β-lactone-core containing APT1/2 inhibitor (Dekker et al., 2010), was purchased from EMD Millipore. PalmB was solubilized in DMSO upon arrival to make a 15mM stock solutions and was kept at -20°C until use. At the treatment day, corticostriatal co-cultures from FVB/N and YAC128 mice were treated with PalmB in a final concentration of 15µM, and the treatment was then topped up every hour over a 4-hour period  (total 4 treatments). DMSO-only treatment was used as a vehicle control (final concentration in the medium was 0.4%). Freeze and thaw cycles were minimized.  	   33	  2.3 Immunocytochemistry Procedures of for live staining of cultured neurons were described previously (Milnerwood et al., 2012; Parsons et al., 2014). Briefly, GFP-tagged GluN2B transfected cells at DIV 13-15 were incubated for 10 min at 37°C with chicken anti-GFP polyclonal antibody (AbCam ab13970; 1:1000) in conditioned plating media, fixed in 4% PFA (Sigma) with 4% sucrose (Sigma) for 10 min and then rinsed 3 times with phosphate-buffered saline (PBS; Sigma) solution. Cells were then incubated with goat anti-chicken secondary antibody conjugated to Alexa 488 fluorophore (Invitrogen A11039; 1:2000 in PBS with 2% Normal Goat Serum; Vector Lab) for 1 hour at room temperature (RT). For internal GluN2B labeling, cells were permeabilized by adding 0.3% Triton X-1000 (Sigma) in PBS (PBST), then incubated with chicken anti-GFP antibody followed by incubation with secondary antibody conjugated to the Alexa 568 fluorophore (1:2000 in PBST with 2% NGS) for 1 hour at RT. Coverslips were then washed a final three times with PBST prior to being mounted on slides (Frost Plus; Fisher) with Fluoromount-G (Southern Biotechnology).  2.4 Microscopy and image analysis Images were acquired using a Zeiss Axiovert 200 M fluorescence microscope. For GluN2B surface/internal expression (green/red fluorescence ratio) analysis, cells transfected with GFP-tagged GluN2B WT were immunostained as above. 12 images (63X, 0.4µm z-stacks) of one transfected cell were acquired; all exposure times were constrained at the same level within experiments and analysis was conducted on unprocessed (raw) TIFF images. Z-stack images were flattened using the extended focus projection function of Axiovision 4.6 (objective was 1.4 NA; Zeiss, ZEN system), from 4 to 5 focal planes containing all of the visible dendritic surface staining. GluN2B surface and internal expression fluorescence was quantified with ImageJ (National 	   34	  Institutes of Health) as mean grey intensity level within three regions of interest (ROI), which included one primary dendrite and two secondary dendrites. The mean intensity was measured, subtracting background adjacent to the individual ROI, to create a cell average dendritic intensity, and the ratio of surface fluorescence to internal signal was calculated for each cell. Data are expressed as mean ± S.E.M., where n indicates the number of cells analyzed from 2-6 separate cultures.  2.5 Acyl-biotin exchange (ABE) assay Corticostriatal co-cultures from FVB/N and YAC128 mice were treated with 15µM PalmB or vehicle for 4 hours at DIV14; neurons were harvested and saved at -800C. Protein palmitoylation of GluN2B and PSD-95 was assessed using an Acyl-Biotin-Exchange (ABE) method, an in vitro chemistry that exchanges biotin moieties onto sites of protein palmitoylation (Drisdel and Green, 2004; Brigidi and Bamji, 2013). The harvested cells were suspended in lysis buffer containing: 150mM NaCl, 50mM Tris (pH7.4), 5mM EGTA, 1M N-ethylmaleimide (NEM, to block free cysteine residues; Sigma), 0.5mM PMSF and 1 tablet (Roche) protease inhibitor cocktail (leupeptin, chymostatin, pepstatin, aprotinin)/10ml buffer; Triton X-100 was subsequently added to 1.7% followed by incubation with end-over-end rotation at 40C for 1 hour. Particulates and unbroken cells were removed by centrifugation (10,000g, 40C, 10 min). The protein extracts were subjected to the three-step Chloroform–methanol (CM) precipitation as described before (Wan et al., 2007). Briefly, with one volume of sample, 4 volumes of methanol were added and then vortexed to mix; 1.5 volumes of chloroform then 3 volumes of water were added and then vortexed to mix. After centrifugation (6,000g, RT, 15 min), two separated phases were clearly visible with protein precipitated at the interphase. The top, aqueous (water and methanol) phase was removed 	   35	  with care to avoid removal of the interphase protein; then 3 volumes of methanol were added and mixed gently. After another centrifugation (6,000g, RT, 15 min), protein was precipitated to the tube bottom as a loose pellet; the supernatant was removed and protein pellet was air-dried. After completely drying the pellet in air, pellets were dissolved in lysis buffer containing 0.2% SDS, 0.2% triton X-100 and 1M NEM and subjected to end-over-end rotation at 40C overnight. The next day, after 2 times CM precipitation, one half of the sample was treated with 1M hydroxylamine (HAM, to cleave the thioester bond between cysteine and palmitate) (Sigma) for 1 hour to remove palmitate from Cys residues (+HAM) and the other half was the control (-HAM) for non-specific biotynlation. Simultaneously, samples were treated with the biotinylated Cys-crosslinking reagent 1mM HPDP-biotin (Thermo Scientific) to label the newly free cysteines. After two-times CM precipitation to completely remove HAM and HPDP-biotin, biotinylated proteins were then isolated by pull-down using streptavidin sepharose (GE) beads and forwarded to western blot analysis.  2.6 Western blot analysis The protein samples were resuspended in Laemmli buffer (4% SDS, 20% glycerol, 0.004% bromphenol blue, 0.125 M Tris HCl, pH 6.8) with 10mM dithiothreitol (DTT; Sigma), and samples were analysed by SDS-PAGE (NuPAGE 4-12% Bis-Tris Gel, Invitrogen). SDS-PAGE gels were transferred onto nitrocellulose membranes (Amersham, 0.5A for 90 min at 4°C), blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) in TBST (0.5% Tween 20 in Tris-Buffered Saline) for 1hr at RT, and probed with specific primary antibodies: anti-GluN2B (Millipore, 1:1000), anti-PSD-95 (Thermo Scientific, 1:1000), and anti-actin (Sigma, 1:500). Primary antibodies were applied to the immunoblots at 4°C overnight in Odyssey blocking buffer (Li-COR Bioscience). 	   36	  The secondary antibodies (anti-rabbit IRD800, Anti-Mouse Alexa Fluor 680; Rockland), used at a dilution of 1:10,000, were applied in 3% BSA TBST for an hour at RT. The immunoblots were visualized using the Odyssey Infrared Imaging System (Li-COR Bioscience), and the optical density of bands was quantified by ImageJ software (NIH). Palmitoylation levels, measured from the purified palmitoyl-protein samples (palmitoylation), were normalized to protein levels measured from the corresponding unpurified extracts (total). Results were from six independent experiments. Data were analyzed using the Student’s t-test. Error bars show S.E.M.  2.7 Electrophysiology For assessing NMDA current (INMDA), coverslips with DIV14 or DIV18 corticostriatal co-cultures were transferred to a chamber for whole-cell patch clamp recording from FVB/N or YAC128 YFP-positive striatal neurons, as described previously (Milnerwood et al., 2012). Neurons were superperfused at room temperature with solution containing (in mM unless stated): 167 NaCl, 2.4 KCl, 0.01 MgCl2, 10 glucose, 10 HEPES, 2 CaCl2, pH 7.3, 300 mOsm, tetrodotoxin (TTX, 0.3 µM) to block voltage-gated sodium channels, glycine (10 µM) to potentiate NMDARs, strychnine (2µM) to block glycine receptors and picrotoxin (PTX, 100 µM) to block GABAA receptors. Patch electrodes (3-4MΩ) were made from borosilicate glass (Warner Instruments) and filled with intracellular recording solution contained the following (in mM): 130 CsMe, 5 CsCl, 4 NaCl, 1 MgCl2, 10 HEPES, 5 EGTA, 5 lidocaine, 0.5 GTP, 10 Na-phosphocreatine, 5 MgATP, titrated to pH 7.2, 290 mOsm. Briefly, voltage clamp recordings were conducted at Vh -70 mV, and intrinsic membrane properties were determined by the membrane function ~30s after obtaining the whole-cell configuration. Series resistance was typically 10–20MΩ; cells were discarded if resistance exceeded 20MΩ. Cells in which the series resistance changed by >25% during recording 	   37	  were also discarded. Data were obtained using the Axopatch 200B amplifier running pClamp 10.2 software (Molecular Devices). Rapid application of 1 mM NMDA (Sigma) was achieved by pClamp (Axon Instruments) triggering of a pressurized perfusion system (Harvard Apparatus) for 3s and repeated at least five times at intervals of 30s. NMDAR-mediated whole-cell peak current density was calculated as peak current (pA) normalized to cell capacitance (pF). Total charge transfer density was calculated as the area under the curve (nA × ms)/cell capacitance (pF). For augmentation of spontaneous synaptic bursting, cells were held at -80mV with 4-aminopyridine (4-AP, 10 µM, Tocris) added in the external solution to widen action potentials, together with PTX to induce synaptic bursting by network disinhibition. At least 3 min of recordings were made under these conditions, to ensure complete wash-out of TTX as indicated by emergence of bursts of excitatory synaptic activity; then MK-801 (10 µM, Sigma), an irreversible, stable, open-channel NMDAR inhibitor , was added to the external solution for another 3 min to block synaptic NMDARs. After wash-out of unbound MK-801 for 2 min with addition of TTX, a second round of NMDA-evoked current mainly mediated by Ex-NMDARs was assayed (Kaufman et al., 2012). For measurement of miniature excitatory postsynaptic currents, cells were held at -80mV in the presence of TTX with the same internal and external solution used in the experiments of whole-cell NMDA recording except that a normal (1 mM) concentration of MgCl2, and no glycine or strychinine, was added to exteral solution. Signals were filtered at 1 kHz and digitized at 10 kHz. Traces were analysed in Clampfit10 (Axon Instruments). Data are presented as mean ± S.E.M. where n is cell number from a minimum of 5 different cultures. 	   38	  2.8 Data analysis and statistics Figures and statistical analyses were generated using Microsoft Excel, ImageJ, Prism, Adobe Photoshop or Adobe Illustrator software. Data or bar graphs are presented as the mean ± S.E.M. Significant differences were determined using Prism software (Graphpad, Inc.); direct comparisons were made by the unpaired or paired, two-tail Student’s t-test (2-tailed, Mann-Whitney U) or Two-Way ANOVA with significant difference between genotypes or treatments tested by Bonferroni’s multiple comparisons post hoc test, as appropriate.   	   39	  Chapter 3: Results    3.1 Role of HIP14 in 2B-NMDAR trafficking and NMDAR current in HD mouse model  Previous studies in our lab showed that NMDAR activity is altered in striatal neurons of YAC128 HD mice by one month of age, and that elevated NMDAR current and GluN2B surface expression are localized to extrasynaptic sites (Milnerwood and Raymond, 2007; Milnerwood et al., 2010). Palmitoylation of GluN2B on two C-terminal cysteine clusters regulates the stability of surface expression and rate of forward trafficking of GluN2B-containing NMDARs (Hayashi et al., 2009). Pilot study in our lab found a reduction of GluN2B palmitoylation in striatum of YAC128 mice as early as one month of age (Kang et al., manuscript in preparation). HIP14 (ZDHHC17) is the major palmitoyl acyl transferase (PAT) that interacts with the Htt protein (Huang et al., 2011), and its activity is significantly reduced in brains of YAC128 mice (Huang et al., 2011; Singaraja et al., 2011). Therefore, I hypothesized that a reduction in HIP14 activity could play a role in altering 2B-NMDAR trafficking in YAC128 MSNs.  3.1.1 Knock-down of endogenous HIP14 with antisense oligonucleotide (ASO) does not alter 2B-NMDAR surface expression To determine whether decreased HIP14 activity contributes to the altered 2B-NMDAR surface expression and function in striatal neurons of YAC128 mice, we treated FVB/N and YAC128 DIV4 corticostriatal co-cultures with HIP14-specific ASO (250 nM) or vehicle (medium change) for ten days and then examined the surface/internal ratio of GFP-tagged 2B-NMDARs. 	   40	  The surface/internal ratios could only be compared within, but not between, genotypes because the genotypes were not paired in these two sets of experiments; therefore, data from the two genotypes are presented on separate graphs (Fig. 3.1, 3.2). I found that the surface to internal intensity ratio of GluN2B was not affected by treatment with HIP14 ASO in striatal neurons from either FVB/N (Fig. 3.1B) or YAC128 (Fig. 3.2B) mice.                   	   41	  (A)  	   42	    Figure 3.1. Knock-down of endogenous HIP14 with antisense oligonucleotide (ASO) does not affect 2B-NMDAR surface distribution in FVB/N striatal neurons. (A) Representative examples of surface/internal expression of 2B-NMDARs in FVB/N MSNs transfected with GFP-tagged GluN2B. Transfected MSNs grown in corticostriatal co-culture were treated with 250 nM HIP14 ASO for 10 days. At DIV14, co-cultures were live-stained for surface GluN2B (green) with GFP antibody, then fixed, permeabilized and stained for internal GluN2B (red). Merged images show the total GluN2B expression. (Scale bar, 20 µm for whole cell; 10 µm for detail). (B) Quantitative analysis for the ratio of surface to internal GluN2B intensity after 10 days of HIP14 ASO treatment. The surface expression of GluN2B in MSNs showed similar levels with or without HIP14 ASO treatment in FVB/N corticostriatal co-cultures (N = 38(6); number of cells (number of culture batches)). Bars represent means ± S.E.M. (n.s. p =	  0.0676,	  Nonparametric	  test,	  Mann-­‐Whitney	  U).  	   43	  (A)  	   44	     Figure 3.2. Knock-down of endogenous HIP14 with ASO does not affect 2B-NMDAR surface distribution in YAC128 striatal neurons. (A) Representative examples of surface/internal expression of 2B-NMDARs in YAC128 MSNs transfected with GPF-tagged GluN2B. Methods as in Fig. 3.1. (Scale bar, 20 µm for whole cell; 10 µm for detail). (B) Quantitative analysis for the ratio of surface to internal GluN2B intensity after 10 days of HIP14 ASO treatment. The surface expression of GluN2B in MSNs showed similar levels with or without HIP14 ASO treatment in YAC128 corticostriatal co-cultures (N = 42(5)). Bars represent means ± S.E.M. (n.s. p = 0.0770, Nonparametric test, Mann-Whitney U). 	   45	  3.1.2 HIP14 ASO does not affect the level of functional surface NMDARs in MSNs  We then further examined whether HIP14 is necessary for surface expression of functional NMDAR using a simple and quantitative electrophysiological assay. We performed whole-cell patch clamp recordings of YFP transfected MSNs in corticostriatal co-cultures with/without knockdown of endogenous HIP14 by ASO. Whole-cell NMDAR current was measured in response to fast application of NMDA at DIV13-14 (Fig. 3.3A). Consistent with previous results (Milnerwood, et al., 2012), whole-cell NMDA-induced peak current density was significantly increased in vehicle-treated (control) YAC128 vs. FVB/N MSNs (59.8 ± 4.7 and 77.8 ± 6.4 pA/pF, n=22 and 22 different neurons, WT and YAC128 respectively, p=0.029 by t-test, Fig. 3.3B); however, consistent with the immunocytochemical results, HIP14 ASO treatment did not affect NMDAR-induced peak current density either in FVB/N MSNs (59.8 ± 4.7 and 58.3 ± 5.1 pA/pF, n=22 and 20, CTL and HIP14 ASO respectively, Fig. 3.3B) or in YAC128 MSNs (77.8 ± 6.4 and 87.0 ± 7.4 pA/pF, n=22 and 22, CTL and HIP14 ASO respectively, Fig. 3.3B).  Similarly, total charge transfer was significantly increased in untreated YAC128 vs. FVB/N MSNs (109.3 ± 9.0 and 142.6 ± 10.0 nA*ms/pF, FVB/N and YAC128 respectively, p=0.0003, Fig. 3.3C); however, total charge transfer was not affected by HIP14 ASO in FVB/N MSNs (109.3 ± 9.0. and 119.3 ± 10.6 nA*ms/pF, n=22 and 20, CTL and HIP14 respectively) or in YAC128 MSNs (142.6 ± 10.0 and 160.8 ± 13.7 nA*ms/pF, CTL and HIP14 ASO respectively, Fig. 3.3C). There were no significant differences in the steady-state to peak current (Iss/Ipeak) ratio with HIP14 ASO treatment between WT and YAC128 MSNs (data not shown). 	   46	    Figure 3.3. Knock-down of endogenous HIP14 with ASO does not affect whole-cell NMDA-evoked current. (A) Representative traces of whole-cell currents induced by fast application of 1 mM NMDA onto MSNs in FVB/N and YAC128 corticostriatal DIV14 co-cultures treated without (CTL) or with HIP14 ASO (ASO) for previous 10 days. (B, C) Quantification of peak current density (B) and total charge transfer (C) reveals that NMDAR currents are similar with or without HIP14 ASO treatment in MSNs in co-cultures from both genotypes, although control treatment reveals a significant increase in NMDAR current measured from YAC128 vs. FVB/N MSNs. 	   47	  Significant by two-way ANOVA, p=0.0002 for Ipeak, p=0.0003 for total charge, p=0.5348 for HIP14 ASO, p=0.3785 for interaction; ** p< 0.01 by Bonferroni post hoc test.  3.2 Effect of general increase in protein palmitoylation on NMDAR current in HD striatal neurons  After suppressing the level of HIP14, the major PAT in HD, we then were interested to examine the effect on NMDAR current at corticostriatal synapses from the opposite perspective: stimulating a general increase of protein palmitoylation in the co-culture. To achieve that, I chose PalmB, a recently synthesized small molecule inhibitor (Dekker et al., 2010), to target APT1, the only enzyme confirmed to regulate depalmitoylation in vivo (Conibear and Daivs, 2010; Lin and Conibear, 2015) and its homolog, APT2. APT1 has been shown to be involved in synaptic function, and a number of APT1 substrates have been identified in vivo or in vitro, like eNOS (Yeh et al., 1999), SNAP23 (Veit and Schmidt, 2001; Flaumenhaft et al., 2007), Ras (Dekker et al., 2010), and Gα13 (Siegel et al., 2009). Based on previous findings of PalmB-induced suppression on APT1/2 function, I hypothesized that the distribution and the current of NMDARs in striatal neurons could be affected after PalmB treatment. The results of PalmB treatment study could potentially provide a primary understanding on the effect of changing palmitoylation level on NMDAR activity.  3.2.1 Increased Ex-NMDAR current in DIV18 MSNs of YAC128 corticostriatal co-cultures  Multiple lines of experimental evidence indicate that Ex-NMDAR activity is involved in HD pathogenesis (Hardingham and Bading, 2010). Previous study in our lab revealed elevated Ex-NMDAR activity in striatal neurons in acute brain slices and DIV14 corticostriatal co-cultures 	   48	  from YAC128 mice (Milnerwood et al., 2010, 2012). Before further testing the effect of treatments, I first investigated the Ex-NMDAR activity in untreated MSNs from more mature, DIV18 corticostriatal co-cultures, using the same electrophysiological assay as in previous studies from our lab. After recording the NMDA-evoked whole cell current that is mediated by both synaptic and extrasynaptic NMDARs, I applied a two-step stimulation-blockade pharmacological treatment: 1) to augment the network activity, thereby maximizing synaptic NMDAR activation, by blocking A-type K+ channels with 4-AP and removing inhibition with PTX (GABAergic) and strychnine (Glycinergic); and then 2) to block the activated synaptic NMDARs with the open-channel blocker MK-801. I then applied a second round of NMDA to elicit whole-cell current, and at this step the NMDAR current is selectively mediated by Ex-NMDARs since the synaptic receptors remain blocked by MK-801 (Fig. 3.4A). The ratio of the 2nd NMDAR current (after MK801) over the 1st NMDAR current (before MK801) indicates the functional Ex-NMDAR proportion in MSNs. Consistent with previous findings, Ex-NMDAR current in DIV18 MSNs of YAC128 co-cultures was significantly increased in both peak current density (FVB/N, 37.32 ± 3.47%; YAC128, 55.61 ± 3.58%) and total charge transfer (FVB/N, 35.81 ± 3.42%; YAC128, 54.13 ± 3.07%; Fig. 3.4D). There was also a significant increase in the steady state current density (data not shown).  3.2.2 DMSO increases functional Ex-NMDARs in FVB/N MSNs  I then investigated the potential impact on distribution and function of NMDARs of a treatment with a drug expected to elevate the palmitoylation level of a wide range of pre-/post-synaptic proteins. I used PalmB to suppress the enzymatic activity of the depalmitoylating enzyme APT1, thereby increasing the palmitoylation level of APT1 substrates (Dekker et al., 2010). At 	   49	  DIV18, I treated the FVB/N and YAC128 corticostriatal co-cultures with PalmB in DMSO or DMSO alone (vehicle) or medium change only (untreated) for 4 hours. At the beginning of every hour, 15 µM PalmB in 1:1000 DMSO or 1:1000 DMSO alone was added to the co-cultures, for a total of 4 applications (so that total DMSO concentration in the medium at the end of the treatment protocol was 0.4%). The 4-hour long treatment was followed immediately by electrophysiological assays to examine a variety of synaptic and extrasynaptic current components. I found that there was no significant difference in whole-cell NMDA current mediated by both synaptic and extrasynaptic NMDARs (1st NMDAR current, before MK-801 blockade) either by treatment (untreated, DMSO, and PalmB) or by genotypes (FVB/N and YAC128) (peak current density in pA/pF: FVB/N, 98.38 ± 7.30 for untreated, 100.46 ± 12.48 for DMSO, 85.59 ± 7.09 for PalmB; YAC128, 88.70 ± 7.48 for untreated, 100.01 ± 12.01 for DMSO, 113.55 ± 8.85 for PalmB; Fig. 3.4B.i; and total charge transfer in nA/pF: FVB/N, 185.63 ± 11.74 for untreated, 213.91 ± 27.21 for DMSO, 189.14 ± 15.31 for PalmB; YAC128, 188.75 ± 11.82 for untreated, 218.33 ± 22.26 for DMSO; 251.85 ± 21.17 for PalmB; Fig. 3.4C.i). The proportion of Ex-NMDAR current (2nd NMDAR current, after MK-801 blockade) to the initial whole-cell (total) NMDAR current was significantly increased by treatment in both genotypes (peak current density: FVB/N, 37.32 ± 3.48% for untreated, 54.61 ± 3.30% for DMSO, 59.61 ± 2.22% for PalmB; YAC128, 55.61 ± 3.58% for untreated, 62.05 ± 2.17% for DMSO, 68.46 ± 3.85% for PalmB; Fig. 3.4B.iii; and total charge transfer: FVB/N, 35.81 ± 3.4% for untreated, 57.16 ± 3.43% for DMSO, 60.13 ± 2.88% for PalmB; YAC128, 54.13 ± 3.07% for untreated, 64.82 ± 2.48% for DMSO, 69.04 ± 4.08% for PalmB; Fig. 3.4C.iii). However, the biggest difference was observed between DMSO-treated and untreated MSNs of FVB/N co-cultures (Fig. 3.4B.iii, 3.4C.iii) and there was no significant difference between DMSO-treated and PalmB-treated groups in either genotype. The statistical 	   50	  analysis of the steady state current density was consistent with those of peak current and total charge transfer (data not shown).     	   51	    	   52	     Figure 3.4. Effect of decreased APT1-mediated depalmitoylation by PalmB treatment on functional NMDARs in MSNs in corticostriatal co-cultures from FVB/N and YAC128 mice. (A) Representative traces of normalized NMDA-induced whole cell currents before and after the irreversible blockade of synaptic NMDARs by 10 µM MK801, recorded from YFP-transfected FVB/N or YAC128 MSNs in DIV18 corticostriatal co-cultures. (B, C) Quantification of peak current density (B) and total charge transfer (C) both reveal the genotype difference between FVB/N and YAC128 Ex-NMDAR current in untreated MSNs of DIV18 corticostriatal co-cultures (better demonstrated in D). Two-way ANOVA test demonstrates that treatment is highly significant compared to untreated condition in both genotypes (***p<0.0001 for peak current density Biii; ***p<0.0001 for total charge transfer Ciii). However, the biggest difference for Ex-NMDAR current is between untreated and DMSO- treated (both DMSO-only and PalmB in DMSO) conditions in FVB/N (***p<0.001 for peak current density Biii;  ***p<0.001 for total charge transfer Ciii; by Bonferroni post-test), with no significant difference between DMSO-only 	   53	  and PalmB treated conditions. In (B, C), i) bars represent the actual values of NMDA current in peak density and total charge transfer, respectively, before MK801 treatment, reflecting total NMDAR current; ii) bars represent the actual values of NMDAR current in peak density and total charge transfer, respectively, after MK801 treatment, dominated by Ex-NMDARs; iii) bars represent the proportion of Ex-NMDAR current in ii) normalized to total NMDAR current in i) for peak density and total charge transfer, respectively. (D) Data from (Biii) and (Ciii) focuses solely on Ex-NMDAR proportion in untreated MSNs from both genotypes at DIV18 (**p=0.0025 for peak current density and **p=0.0013 for total charge transfer, unpaired t test). FVB/N untreated, N = 8(4); FVB/N DMSO treated, N = 10(5); FVB/N PalmB treated, N = 11(5); YAC128 untreated, N = 8(5); YAC128 DMSO treated, N = 10(3); YAC128 PalmB treated, N = 10(5).  3.2.3 DMSO and PalmB reduce synaptic NMDAR but not synaptic AMPAR current in FVB/N and YAC128 MSNs With the finding that DMSO and PalmB treatments increase the proportion of Ex-NMDAR current, I was then interested in the effect of these treatments on excitatory synaptic transmission. The post-synaptic part of excitatory synaptic transmission contains two major components: a relatively faster AMPAR activation corresponding to the peak part of the burst, and a relatively slow NMDAR activation corresponding to the tail part of the current. Before the blockade byMK-801, the augmented spontaneous bursting recorded during the stimulation of network activity (by adding 4-AP and strychnine, and removing TTX, in the external solution) is a mixture of both NMDAR and AMPAR components (Fig. 3.5A). With the NMDAR blockade by MK-801, the burst is mainly dominated by AMPAR. In order to individually examine each component, I analyzed NMDAR and AMPAR total charge transfer separately. To specifically measure synaptic NMDAR 	   54	  activity, I first obtained the time duration of the AMPAR-dominated burst at the MK-801 step; then, at the 4-AP step before MK-801 blockade, I only measured the ‘tail’ charge after that time duration to avoid the AMPAR ‘contamination’ and to ‘dissect’ out the synaptic NMDAR component in the compound burst. The AMPAR component was relatively easier to measure, as MK-801 blocked synaptic NMDARs that were activated at the 4-AP step, and so the burst recorded at the MK-801 step was mainly mediated by AMPARs. For the synaptic NMDARs, firstly there was no genotype difference in the untreated conditions (Fig. 3.5B) and this finding is consistent with previous studies on DIV14 co-cultures in our lab (Milnerwood et al., 2012). Secondly, there was a significant overall effect of treatment (p=0.0276*, two-way ANOVA) but not of genotype or interaction. Thirdly, the biggest difference lay between untreated and treated (DMSO and PalmB) FVB/N MSNs, consistent with findings in 3.2.2 (NMDAR-dominated tail charge in nA*s/pF: FVB/N, 0.532 ± 0.118 for untreated, 0.283 ± 0.041 for DMSO, 0.337 ± 0.067 for PalmB; YAC128, 0.546 ± 0.110 for untreated, 0.284 ± 0.064 for DMSO, 0.478 ± 0.111 for PalmB; Fig. 3.5B). Together, these findings of a significantly increased Ex-NMDAR and a trend toward reduction of synaptic NMDAR current suggested a loss of synaptic NMDAR upon treatment with DMSO, mainly in FVB/N MSNs. For the synaptic AMPARs, no significant difference on total charge transfer of the AMPA-dominated burst at the MK-801 step was detected across all 6 conditions (Fig. 3.5C). It revealed that neither genotype nor treatment has an effect on synaptic AMPAR activity (AMPAR-dominated total charge at MK-801 step in nA*s/pF: FVB/N, 1.179 ± 0.204 for untreated, 1.090 ± 0.078 for DMSO, 0.997 ± 0.158 for PalmB; YAC128, 1.218 ± 0.221 for untreated, 1.096 ± 0.135 for DMSO, 1.182 ± 0.177 for PalmB).  	   55	   (A)                   	   56	  (B)  (C)         Figure 3.5. Treatment of DMSO and PalmB tends to reduce synaptic NMDAR component yet not to affect synaptic AMPAR component in FVB/N and YAC128 MSNs. (A) Example traces of spontaneous glutamatergic synaptic bursts before and after application of the irreversible NMDAR antagonist MK801 for untreated and 0.4 % DMSO (vehicle) treated FVB/N MSNs in DIV18 corticostriatal co-cultures. (B) Quantification of the normalized tail charge transfer 	   57	  (NMDAR component of synaptic transmission) as average synaptic burst charge transfer before MK801-mediated synaptic NMDAR block, calculated by eliminating its AMPAR-dominated part from the synaptic burst charge transfer. Synaptic NMDAR component of MSNs shows no difference between two genotypes in untreated DIV18 corticostriatal co-cultures. There is an overall significant difference detected by two-way ANOVA in treatment; however, only a trend of reduction was found when comparing DMSO or PalmB group to the untreated group in both genotypes. This trend is relatively more obvious in DMSO treatment than in PalmB treatment and also more in FVB/N than in YAC128 MSNs. (C) Quantification of the spontaneous synaptic burst charge transfer after MK-801 blockade. The burst now is dominated by AMPAR after the removal of NMDAR component by MK-801. There is no difference across all 6 conditions. It reveals that neither genotype nor treatment has an effect on synaptic functional AMPAR. FVB/N untreated, N = 13(5); FVB/N DMSO treated, N = 15(8); FVB/N PalmB treated, N = 19(8); YAC128 untreated, N = 12(5); YAC128 DMSO treated, N = 9(5); YAC128 PalmB treated, N = 12(5).  3.2.4 PalmB increases mEPSC frequency in YAC128 MSNs As the only thioesterase that is experimentally proven in vivo (Conibear and Davis, 2010), APT1 regulates the depalmitoylation of over one hundred proteins in synaptosomes (Lin and Conibear, 2015; Siegel et al., 2009). Therefore, suppressing the enzymatic activity of APT1 by PalmB treatment might well affect synaptic transmission from both pre- and post-synaptic sites. Work from our lab has shown reduction in frequency of miniature excitatory postsynaptic currents (mEPSCs) in striatal MSNs in YAC128 corticostriatal co-cultures (Buren, Parsons et al., manuscript in revision), so it is also of interest to examine whether mEPSCs in YAC128 MSNs are differentially impacted by PalmB treatment compared to FVB/N MSNs. 	   58	  I recorded mEPSCs from YFP-transfected MSNs in corticostriatal co-cultures under conditions that selected for AMPAR-only synaptic activation, by increasing the external Mg2+ concentration to 1mM while recording at a holding potential of -80mV. Recordings were obtained at DIV18 from FVB/N and YAC128 untreated, DMSO- and PalmB-treated MSNs (Fig. 3.6A). The most interesting finding is that mEPSC frequency was significantly increased only in YAC128 PalmB-treated MSNs (**p<0.01 compared to YAC128 untreated MSNs; **p<0.01 compared to YAC128 DMSO-only treated MSNs). This phenomenon was only found in YAC128 MSNs. In FVB/N MSNs, all three conditions showed no difference in mEPSC frequency (mEPSC frequency in Hz: FVB/N, 10.92 ± 1.15 for untreated, 9.17 ± 2.23 for DMSO, 11.53 ± 2.28 for PalmB; YAC128, 10.03 ± 1.32 for untreated, 8.83 ± 1.40 for DMSO, 18.01 ± 2.13 for PalmB; Fig. 3.6B). Additionally, there was no difference in mEPSC amplitude in all 6 conditions (mEPSC amplitude in pA: FVB/N, 29.31 ± 2.00 for untreated, 30.33 ± 3.99 for DMSO, 30.58 ± 2.56 for PalmB; YAC128, 32.59 ± 2.22 for untreated, 30.58 ± 2.36 for DMSO, 38.36 ± 4.48 for PalmB; Fig. 3.6C).                      	   59	  (A)    (B)       	   60	  (C)   Figure 3.6. mEPSC frequency is increased only in PalmB-treated YAC128 MSNs. (A) Representative traces showing mEPSC events recorded from MSNs in DIV18 corticostriatal co-culture. (B) Quantification of mEPSC frequency in all six conditions: untreated, DMSO, and PalmB treatment in FVB/N and YAC128 DIV18 YFP-transfected MSNs. Two-way ANOVA reveals a significant treatment effect (**p=0.0058) but no interaction (p=0.0624) or genotype (p=0.2421) effect. In FVB/N MSNs, there is no difference across all 3 conditions. However, in YAC128 MSNs, mEPSC frequency of PalmB-treated MSNs is significantly larger than untreated (**p<0.01) and DMSO-treated conditions (**p<0.01). (C) Quantification of mEPSC amplitude in all six conditions: untreated, DMSO, and PalmB treatment in FVB/N and YAC128 DIV18 YFP-transfected MSNs. Two-way ANOVA reveals no difference in interaction (p=0.05017), treatment (p=0.3582), or genotype (p=0.1424) effects. FVB/N untreated, N = 26(6); FVB/N DMSO treated, N = 12(4); FVB/N PalmB treated, N = 19(5); YAC128 untreated, N = 21(4); YAC128 DMSO treated, N = 14(3); YAC128 PalmB treated, N = 21(4).  	   61	  3.2.5 PalmB increases GluN2B and PSD-95 palmitoylation in corticostriatal co-culture  Besides investigating the change in multiple synaptic components from a functional perspective, I also confirmed the effect of PalmB on the palmitoylation level of GluN2B (Fig. 3.7A.i) and PSD-95 (Fig. 3.7B.ii) in DIV14 corticostriatal co-cultures. After the treatment of DMSO and PalmB, the co-cultures were harvested. Then the palmitoylation levels of GluN2B and PSD-95 were examined by ABE assay and visualized by western blotting. I found that the palmitoylation levels of both GluN2B and PSD95 were significantly increased after PalmB treatment (GluN2B palmitoylation / total in ratio: 0.611 ± 0.109 for DMSO and 0.938 ± 0.160 for PalmB; PSD-95 palmitoylation / total in ratio: 1.087 ± 0.271 for DMSO and 1.990 ± 0.594 for PalmB; Fig. 3.7A.ii, B.ii). The protein expression levels of GluN2B and PSD-95 were also examined and there was no difference between DMSO and PalmB groups (data not shown). 	   62	    Figure 3.7. Palmitoylation level of GluN2B and PSD95 is significantly increased after APT1-mediated depalmitoylation is repressed by PalmB treatment. Neurons in corticostriatal co-cultures at DIV14 were treated with PalmB or DMSO for 4 hours. At the beginning of each hour 15 µM PalmB or 1:1000 DMSO was added. Then neurons were harvested and saved at -800C for ABE/western analysis (A, B). Representative western blots show palmitoylation levels of GluN2B (A.i) and PSD-95 (B.i) in conditions of DMSO (vehicle) and PalmB treatment. The palmitoylation levels were detected in the purified proteins (palmitoylation), and the total protein was measured from the corresponding unpurified extracts (total).  (A.ii and B.ii) Quantification of palmitate levels 	   63	  on GluN2B (A.ii) and PSD-95 (B.ii).  Bar indicates the corresponding ratio of each antibody’s band intensity in hydroxylamine plus (+HAM) for purified proteins (palmitoylation), normalized to the total, unpurified protein extract (total). The palmitoylation of GluN2B (A.ii) and PSD95 (B.ii) is significantly increased in the condition of PalmB treatment. Data is presented from 6 independent co-cultures for GluN2B and PSD-95 (3 from FVB/N and 3 from YAC128). Bars represent mean ± S.E.M. *p < 0.05 (paired t-test).   	   64	  Chapter 4: Discussion    Medium-sized spiny projection neurons in the striatum are the population that suffer the earliest and most severe neurodegeneration in HD (Gladding and Raymond, 2011). After decades of research, we now understand that a series of dysfunction at the corticostriatal synapse occurs years before the diagnosis of HD, the overt symptoms and brain atrophy (Milnerwood and Raymond, 2011; Sepers and Raymond, 2014). Work from others and our lab demonstrated that altered distribution and elevated activity of 2B-NMDARs is one of the major causes leading to these dysfunctions (Raymond et al., 2011). Early increase in MSN Ex-NMDAR current and signaling, mainly GluN2B subtypes, is shown to be involved in multiple downstream cascades that disturb the overall balance of pro-survival and pro-death signaling (Milnerwood et al., 2010; Parsons and Raymond, 2014). Investigating mechanisms underlying this altered distribution of 2B-NMDARs would be critical in understanding early HD pathogenesis. Considerable progress in the last 15 years in understanding mechanisms of protein palmitoylation (Conibear and Davis, 2010; Young et al., 2012; Sanders and Hayden, 2015), including its unique features like reversibility, versatility, and hydrophobicity, makes this PTM a very promising and interesting perspective to explore the aberrant trafficking and surface distribution of NMDARs in HD. Here, we aimed to examine the effect of manipulating palmitoylation level on NMDAR surface expression in striatal MSN from an HD mouse background from two opposite angles: i) knocking down HIP14, the major PAT regulating the palmitoylation of some key, synaptic HD-related proteins, by ASO; and ii) increasing the palmitoylation of a wide range of pre- and post-synaptic proteins by using the small molecule inhibitor PalmB to suppress activity of APT1, the 	   65	  prime acyl protein thioesterase mediating protein depalmitoylation. Firstly, I showed that after 10-day treatment of HIP14 ASO, neither surface expression (Fig. 3.1, 3.2) nor functional whole-cell current (Fig. 3.3) of 2B-NMDAR had changed in MSNs in corticostriatal co-cultures from FVB/N or YAC128 mouse models. Secondly, after confirming the elevation of Ex-NMDAR current in untreated YAC128 vs. WT MSNs (Fig. 3.4D), I found that a 4-hour treatment of up to 0.4% DMSO alone or PalmB in DMSO significantly decreased synaptic NMDAR current in both genotypes. I found a general decrease on synaptic NMDAR current after treatment detected by two-way ANOVA and the main difference is between untreated group and treated groups in FVB/N MSNs (Fig. 3.5B). DMSO treatment also increased functional Ex-NMDARs in FVB/N MSNs (Fig. 3.4.B.iii, C.iii). On the other hand, the AMPAR current component at the post-synaptic site was found not affected either by genotype or treatments (Fig. 3.5C). Thirdly, a treatment- and genotype-specific increase of mEPSC frequency was revealed only in PalmB-treated YAC128 MSNs (Fig. 3.6B). A similar non-significant increase also showed up in mEPSC amplitude of PalmB-treated YAC128 MSNs (Fig. 3.6C). In contrast, neither frequency nor amplitude of mEPSC in FVB/N MSNs was affected by the treatments. Finally, 4-hour PalmB treatment increased the overall palmitoylation level of 2B-NMDARs and PSD-95 in corticostriatal co-cultures in both genotypes (Fig. 3.7).  4.1 HIP14 and regulation of 2B-NMDAR trafficking In 2009, the first dedicated study on NMDAR palmitoylation revealed the existence of two cysteines clusters located at the C-terminus of GluN2A and GluN2B subunits (Hayashi et al., 2009). Palmitoylation at cysteine Cluster I, close to the transmembrane domain, increases the stability of synaptic and surface expression of NMDAR; whereas palmitoylation at Cluster II, 	   66	  located in the middle of the intracellular C-terminal, is associated with the retention of NMDAR in the Golgi apparatus (Hayashi et al., 2009; Thomas and Huganir, 2013). A recent study in our lab (Kang et al., manuscript in preparation) found that GluN2B 5CS, a mutant form of GluN2B that eliminates palmitoylation on Cluster II, increased surface expression at extrasynaptic but not synaptic sites in FVB/N MSNs, which mimicked the effect of mHtt in YAC128 MSNs, and had no significant impact on 2B-NMDAR surface expression in YAC128. In contrast, Cluster I mutant form GluN2B 3CS did not affect GluN2B trafficking in MSNs from either FVB/N or YAC128 HD mice. After my experiments showing lack of effect with HIP14 ASO treatment on NMDAR current or 2B-NMDAR surface expression, subsequent experiments in the lab revealed that 2B-NMDAR, more specifically, Cys Cluster I but not Cluster II is a substrate of HIP14 (Kang et al., manuscript in preparation). Thus, it is not surprising that HIP14 knock-down did not impact 2B-NMDAR surface expression and function, since cluster II appears to be the major regulatory site for these processes in striatal neurons, and the cysteines in that cluster are not substrates of HIP14. On the other hand, our knowledge and technology on protein post-translational modification by palmitoylation is still at an early stage. For example, the very first antibody that specifically detects the palmitoylated form of a protein (PSD-95) was just developed in 2013 (Fukata et al., 2013). The lack of detailed understanding on the palmitoylation of NMDARs and its interactors at the synapse may further complicate our interpretation of the experimental results. First, Cluster I and II are not the only cysteine sites in the GluN2B subunit. There are 17 intracellular cysteine residues in CluN2B in total (Hayashi et al., 2009) and the interactions between these sites wait for future examination. Theoretically, cysteine sites in the transmembrane domains may also contribute to regulating NMDAR trafficking, given that palmitoylation is one of the major hydrophobicity-related PTMs, and experiments are required to test potential influence of 	   67	  those sites. Furthermore, the regulation of NMDAR trafficking by palmitoylation is more complex than only on the receptor itself. The precise delivery of NMDARs to the synapse requires the collaboration of a wide range of interactors that might also be regulated by HIP14. For example, PSD-95 was found to be palmitoylated and one of its PATs is HIP14 (El-Husseini et al., 2002; Huang et al., 2004). With the newly developed palmitoylated-state-specific antibody, it was then discovered that PSD-95 requires being palmitoylated in order to move to the postsynaptic density (Fukata et al., 2013). The potential effect of HIP14 on PSD95 and other synaptic proteins might multiply the complexity of the regulations of NMDAR trafficking by palmitoylation (Thomas and Huganir, 2013; Lussier et al., 2015).  4.2 HIP14 and HIP14L To date 24 DHHC PATs in mouse and 23 in human have been identified (Fukata et al., 2004; Huang et al., 2009). HIP14, also known as ZDHHC17, has a unique feature distinguishing it from most of the other PATs. It contains seven intracellular ankyrin repeat domains and six transmembrane domains. The only other PAT known to contain the ankyrin repeats is HIP14L or ZDHHC13. HIP14 and HIP14L are the only two PATs to interact with Htt, and this occurs through the ankyrin repeats (Sanders and Hayden, 2015). Based on previous findings, it is not uncommon for multiple PATs to demonstrate overlapping PAT-substrate specificity (Huang et al., 2009; Greaves et al., 2011). In this case, HIP14 and HIP14L both regulate the palmitoylation of Htt (Huang et al., 2004; Sutton et al., 2013). However, the details of the interaction between Htt and these two PATs still remain largely unknown. Transgenic knock-out mice of HIP14 or HIP14L both recapitulate features of HD to a certain degree (Singaraja et al., 2011; Milnerwood et al., 2013; Sutton et al., 2013). Nevertheless, the fact that both knock-out mouse models were able to 	   68	  survive to adulthood and Htt palmitoylation was not altered in either one suggests the possible compensation for loss of one by the other between these two PATs. Actually, double knock-out transgenic mice never survived beyond embryonic day 10-11 (Sanders et al., 2015). In our study, the corticostriatal co-culture was obtained from E17-18 mouse brain and the treatment with HIP14 ASO started at DIV4 and lasted for 10 days. It is reasonable to suspect that during the period of ASO treatment, HIP14L may take over enzymatic function of HIP14 and compensate the gradual loss of HIP14 as a PAT to a certain degree, which might lead to a false negative result in the experiment. Determining the profiles and details of how HIP14 and HIP14L overlap, and whether HIP14L has an enzymatic function on these two clusters, will provide valuable information. Lastly, at the end of 10-d 250 nM ASO treatment, about ~90% of HIP14 was knocked down (Fig. 2.1). However, it is possible that even 10% of remaining HIP14 is sufficient to maintain its enzymatic function. Therefore, it would also be important to assay the palmitoylation level of GluN2B to determine whether 90% knock-down of HIP14 significantly reduced 2B-NMDAR palmitoylation.  4.3 PTMs beyond palmitoylation After global knocking-down of HIP14, our understanding is still preliminary about the consequence not only of the palmitoylation of GluN2A or GluN2B subunits but also on other subunits or even other types of PTMs (Sanz-Clemente et al., 2013). For instance, a very recent study shows that in order to leave the ER, two asparagine residues in GluN1 subunits need to be N-glycosylated (Lichnerova et al., 2015). Cross-talk between palmitoylation on Cys Cluster II and phosphorylation on GluN2B C-terminal sites is also possible. In fact, this type of cross-talk has already been documented for AMPARs (Lin et al., 2009). Previous studies showed that the interaction between mHtt and GluN2B subunit altered the phosphorylation and trafficking of 	   69	  NMDAR (Fan et al., 2009; Dau et al., 2012; Metzler et al., 2010; Sanz-Clemente et al., 2010, 2012), therefore it would be interesting to determine whether a similar cross-talk exists for any of the GluN2B Cys Clusters between phosphorylation and palmitoylation. Another possible PTM that directly competes with palmitoylation for the cysteine residue and might replace palmitoylation under certain pathological circumstances is nitrosylation. Nitric oxide (NO) levels may be elevated in YAC128 MSNs (Nakamura et al., 2013) and our lab also found that nNOS inhibition rescues the enhanced vulnerability of YAC128 MSNs to NMDA-induced excitotoxicity (Fan et al., 2009). In 2011, the first detailed study about the dynamic interaction between nitrosylation and palmitoylation on one paradigm protein, PSD-95, showed that nitrosylation of PSD-95 not only reduced its palmitoylation level and synaptic expression but also decreased the expression of proteins associated with PSD95 like NMDARs and AMPARs (Ho et al., 2011). Later the same group showed that nitrosylation on the GluA1 subunit physiologically regulates AMPAR phosphorylation and endocytosis (Selvakumar et al., 2013). It will be intriguing to learn about the interaction of these PTMs on NMDAR trafficking and distribution, especially on GluN2B two cysteine clusters, in a HD animal model. A potential pro-death positive-feedback loop might be triggered by elevated Ex-GluN2B NMDAR activity leading to an increased production of nNOS and nitrosylation of certain cysteine sites, including Cluster II at GluN2B. It might reciprocally suppress the palmitoylation at Cluster II thus in turn increasing GluN2B surface expression in MSNs.  Together, these results suggest that under my experimental conditions, knocking-down endogenous HIP14 does not affect 2B-NMDAR surface expression or NMDAR whole-cell current. Further studies are required to provide more details before drawing any conclusion.  	   70	  4.4 PalmB effect on NMDAR current and palmitoylation Recent progress on developing small molecule inhibitors like Palmostatin B (Dekker et al., 2010), hexadecyl fluorophosphonate (HDFP, Martin et al., 2011), and piperazine amide inhibitor C83 (Adibekian et al., 2012), boosted the exploration of APT1 in protein depalmitoylation in vivo (Lin and Conibear, 2015). PalmB was identified from the screening of synthesized compounds for APT1 binding. It has been widely applied as a competitive, non-selective APT1 and APT2 blocker via reversible covalent modification of a serine residue at the APT1/2 active site (Dekker et al., 2010). However, evidence that PalmB exclusively targets APT1/2 and PPT1 is not definitive, and it is highly possible that other low abundance PalmB targets exist (Lin and Conibear, 2015; Rusch et al., 2011). For example, double knockdown of APT1 and APT2 by RNA interference showed no effect on PSD-95 depalmitoylation, whereas PalmB treatment robustly inhibited PSD-95 depalmitoylation (Lin, unpublished data). Meanwhile, there are very few in vivo studies of APT1 substrate specificity, especially focusing on either synaptic proteins or proteins associated with neurodegenerative diseases. To date, synaptosomal-associated protein 23 (SNAP23) is reported being depalmitoylated by APT1 in vitro (Flaumenhaft et al., 2007) and Gα13 in cultured rat hippocampal neurons (Siegel et al., 2009). Other substrates of potential interest include Ras (Dekker et al., 2010), glutamate decarboxylase 65 (GAD65), and Htt N-terminal (N-Htt) (D. Lin, unpublished data). A recent study curated 15 palmitoylation proteomics studies in rat, mouse, and human cells into one compendium and surprisingly revealed that 10% of the genome and 41% of synaptic genes encode proteins that are physiologically palmitoylated (Sanders et al., 2015). Along with cancers and other CNS disorders like schizophrenia, HD is among the top five diseases associated with aberrant palmitoylation. More specifically, the studies claimed that 273 palmitoylated proteins are 	   71	  related to neurodegenerative diseases and 419 synaptic proteins are palmitoylated based on the analysis of the compendium. Given the current gap between detailed knowledge of PalmB function or APT1 substrates, and the large number of synaptic proteins that are palmitoylated, our study on PalmB treatment should be interpreted with caution. On one hand, gross suppression of APT1 by PalmB treatment provides proof-of-concept information about its effect on NMDAR trafficking and function; while on the other hand, the lack of understanding of APT1 machinery and the diversity of PalmB targets also makes it difficult to interpret the experimental results. I found that the palmitoylation level of both GluN2B and PSD-95 was significantly increased in corticostriatal co-cultures from FVB/N and YAC128 mice. Without an advanced assay to further distinguish the potential difference of 2B-NMDAR palmitoylation between cortical neurons and striatal neurons in the co-cultures, it is not clear which type of neuron mainly drives this palmitoylation increase detected by ABE/western blotting assay. It is important to note that an increase in palmitoylation level of an APT1/2 substrate after a 4-h PalmB treatment depends upon a sufficiently rapid rate of palmitate turn-over on the specific protein; consistent with the time period of PalmB treatment used in my experiments, both PSD-95 (Kang et al., 2008) and GluN2B (Kang et al., manuscript in preparation) have been shown to undergo significant depalmitoylation after a 4-h or 5-h treatment with 2-bromopalmitate, which is a non-specific inhibitor of palmitoylation. After PalmB treatment, functional synaptic NMDARs showed a trend toward reduction while the proportion of Ex-NMDARs was increased in YFP-transfected MSNs from both genotypes; however, these trends were also found for DMSO-only (vehicle) treatment. Notably, the NMDA-evoked whole cell current before the application of the 4-AP/MK-801 drugs cocktail 	   72	  (reflecting total surface, functional NMDARs) showed no difference across treatments or genotypes. So although both genotypes appear to show a shift of functional NMDARs away from synapses and into extrasynaptic sites, DMSO may be contributing importantly to this effect, especially in FVB/N MSNs. On the other hand, the YAC128 MSNs show a strong trend to an increase (by ~70%) in synaptic NMDAR current when comparing PalmB treatment to DMSO alone. Thus, the dominant effect of DMSO in redistribution of surface NMDAR seen in FVB/N MSNs appears less robust in the YAC128 MSNs, and indeed there may be a specific increase in synaptic NMDARs induced by increased GluN2B palmitoylation in the PalmB-treated YAC128 MSNs. One caveat to interpreting the data from these PalmB experiments is the question of an effect of the vehicle DMSO itself. However, combining all three conditions together, PalmB still showed an increasing trend in Ex-NMDARs compared to the DMSO group; specifically, an incremental increase between the three conditions is clearly visible in Ex-NMDAR current peak, total charge (Fig. 3.4B.iii, C.iii), and steady state (data not shown). This suggests that even partially masked by DMSO, PalmB tends to increase Ex-NMDARs in YAC128 MSNs, although the effect is not significant between DMSO and PalmB. It will be interesting to confirm whether there is indeed an increase in synaptic or extrasynaptic 2B-containing NMDARs in PalmB vs. DMSO treatment, and if so, whether GluN2B Cys Cluster I or II is a substrate of APT1/2. Other proteins associated with NMDAR distribution such as PSD-95 could also be of interest. The incremental increase in Ex-NMDAR current between untreated, DMSO and PalmB treatments is not observed in FVB/N measurements. For the latter, the biggest difference lies between the DMSO and untreated conditions in both synaptic and Ex-NMDARs, and there is no visible difference between the two treatment groups (Fig. 3.4B.iii, C.iii for Ex-NMDARs; Fig. 	   73	  3.5B for synaptic NMDARs), suggesting PalmB itself may have little effect on top of the DMSO treatment in FVB/N MSNs. Another interesting comparison worthy of mention is that after treatment with DMSO or PalmB, all three measurements (peak, total, steady state) of Ex-NMDAR currents are now increased to a similar level as untreated YAC128 MSNs, yet still lower than PalmB-treated YAC128 MSNs. Due to the lack of relevant information on the detailed mechanism of APT1 or PalmB function, any further interpretation could be impractical. However, these preliminary results at least remind us that any attempt for a ‘universal’ solution on correcting altered NMDAR distribution in early HD should be cautiously examined, especially when dealing with a powerful inhibitor that possibly targets a wide range of synaptic proteins and with a vehicle that itself has effects on NMDAR surface distribution. Drugs with better ‘resolution’ than PalmB will be valuable to distinguish the specificity between different acyl protein thioesterases in future.  4.5 DMSO effect on NMDAR distribution The main changes, increased Ex-NMDARs and loss of synaptic NMDARs, appear to be a result of treatment with the vehicle, DMSO, in FVB/N MSNs. Unlike in YAC128 MSNs, the NMDA-evoked whole-cell current recorded before MK-801 from FVB/N MSNs did not change among treatments in FVB/N MSNs (Fig. 3.4B.i, C.i). Together, it suggests that DMSO seemingly triggers a redistribution of the receptors already existing on the surface rather than stimulating any internalization or expression of NMDARs in FVB/N MSNs. Synthesized in 1866 and first applied in medicine in 1963, DMSO is one of the most popular polar aprotic solvents used as a hydrophobic drug vehicle in the laboratory for in vivo and in vitro studies, and its possible interference with drug effects has been well documented (Kelava 	   74	  et al., 2011; Santos et al., 2003). In some cases, a low dose of DMSO showed neuroprotection in brain injuries (Bardutzky et al., 2005; Shimizu et al., 1997). Other studies showed a potentially harmful effect of DMSO with reduction of nerve conduction and myelin disruption (Cavaletti et al., 2000). Mechanisms of DMSO include blockade of Na+ and Ca2+ influx, anti-inflammatory and antioxidant effects, and suppression of excess glutamate release (Jacob and de la Torre, 2009). In 2001, a study directly showed that DMSO suppressed NMDAR and AMPAR activity and subsequent Ca2+ influx in primary rat hippocampal mono-culture (Lu and Mattson, 2001). 24-h exposure of 5% DMSO caused a 20% loss in neuron numbers and 2-h treatment with 1% DMSO resulted in a significant decrease in the peak of NMDA-induced whole-cell current (Lu and Mattson, 2001). Another study in 2009 showed that at DIV4, 6-d treatment of 0.5% DMSO produced 50% neuronal death in rat hippocampal mono-culture (Hanslick et al., 2009). In my study, the initial concentration of DMSO was 0.1% and with topping up every hour, the final concentration was 0.4% at the end of the 4-h treatment. DMSO is a hydrogen-bond disrupter and can penetrate the cell membrane easily (Santos et al., 2003). Therefore, it may be possible for DMSO, once beyond a certain concentration, to start interfering with the fluidity of the plasma member and disturbing the distribution of NMDARs. Previous work has shown that altered balance in synaptic/extrasynaptic NMDAR activation can shift survival/death signaling, with increased Ex-NMDAR activity or reduced synaptic NMDAR stimulation favoring cell death pathways (Hardingham and Bading, 2010). Thus, my result that the Ex-NMDAR proportion in the DMSO-treated FVB/N MSNs reached the level observed in untreated YAC128 MSNs (Fig. 3.4B.iii, C.iii), and that synaptic NMDARs showed a strong trend of reduction (Fig. 3.5B) by the end of 4-h hours, suggests a potential mechanism for inducing neuronal apoptosis over a longer period of time, as shown in previous studies (Lu and Mattson, 	   75	  2001; Hanslick et al., 2009). Notably, both synaptic AMPAR total charge transfer during MK-801 treatment (Fig. 3.5C) and mEPSC frequency and amplitude (Fig. 3.6) did not show any difference between DMSO and untreated groups in FVB/N (or YAC128) MSNs. This suggests that at least functional synaptic AMPARs did not change over the period of DMSO treatment. In addition, there must be an NMDAR-specific mechanism(s) involved in the DMSO treatment. Since the PalmB condition shows no difference from the DMSO condition in FVB/N MSNs, it is possible that other APTs besides APT1/2 or fast interaction beyond palmitoylation may be responsible. Phosphorylation at the C-terminal of GluN2 subunits also regulates receptor trafficking (Gladding and Raymond, 2011; Gladding et al., 2012; Dau et al., 2012), which could serve as another potential candidate of interest. On the other hand, it may be that NMDARs are more susceptible to DMSO-induced disruption of surface distribution compared to AMPARs, perhaps due to differential distribution in subcompartments of the plasma membrane, including lipid rafts (Allen et al, 2007). Two previous studies reported the involvement of lipid rafts for Ca2+ conductance by NMDARs in primary neuronal cultures. One study demonstrated the inhibition of NMDA-dependent Ca2+ influx by cholesterol perturbing agents in rat hippocampal neurons (Frank et al., 2004). The other study showed that after lowering cholesterol levels by an amphipathic compound, cyclodextrins, GluN2B subunits, PSD-95, and nNOS were all redistributed in cortical neurons (Abulrob et al., 2005). A more recent study found that cholesterol modulated NMDAR open probability and desensitization. Interestingly, neither AMPAR nor kainate receptors responded to cholesterol manipulation (Korinek et al., 2015).   	   76	  4.6 Genotype-specific PalmB effect on mEPSC frequency In contrast to FVB/N mEPSCs, there is a significant increase in mEPSC frequency in PalmB-treated MSNs compared to untreated or DMSO-treated ones from YAC128 co-culture (Fig. 3.6B). The increase of frequency is more likely to reflect a pre-synaptic change instead of a post-synaptic one, since no change was found in the post-synaptic AMPAR component of spontaneous bursts augmented by 4-AP treatment (Fig. 3.5C). A simple interpretation could be that by suppressing the activity of APT1/2, PalmB increased the palmitoylation level of certain pre-synaptic proteins associated with vesicle release. Due to our limited knowledge on the presynaptic substrates of APT1/2, there is little experimental evidence directly demonstrating the interaction between APT1/2 and pre-synaptic vesicle release. One possible candidate is Growth Associated Protein 43 (GAP43). In rat brain, GAP43 interacts with a series of vesicle exocytosis related proteins like syntaxin, SNAP25, and vesicle-associated membrane protein (VAMP) (Haruta et al., 1997). This interaction is coupled with the phosphorylation of GAP43 by protein kinase C (Haruta et al., 1997) and the association of GAP43 to the plasma membrane requires palmitoylation at its two cysteine residues (Arni et al., 1998; Denny, 2006; El-Husseini and Bredt, 2002). A more recent in vitro study showed that knockdown of APT2 but not APT1 altered GAP43 localization (Tomatis et al., 2010). Syntaxins, a family of proteins participating in exocytosis, are also candidates of interest. Unlike the relatively stable palmitoylation of SNAP25, syntaxin 1b (Stx1b) was found to have a rapid turnover in the 5-h 2-BP treatment in cortical neurons (Kang et al., 2008). Other syntaxins like syntaxin 7 (Stx7) and 8 were also found to be palmitoylated, and the palmitoylation of Stx7 is necessary for its association to the plasma membrane (He and Linder, 2009; Prescott et al., 2009). Another perspective to approach this result is to focus on the genotype difference since this 	   77	  increase only showed up in PalmB-treated YAC128 co-cultures yet not in FVB/N ones. A 2013 study profiled the palmitoylation change in YAC128 mouse and revealed that 19 proteins have been shown to have a greater than 10% change of palmitoylation in 12-month old YAC128 mouse (Wan et al., 2013). Although the majority of these 19 proteins are glial specific proteins, it will be insightful to have another look at proteins with a minor (<10%) change in palmitoylation level in the YAC128 mouse brain, and specifically in the striatum, since the Wan et al. study examined only whole-brain samples. There is a bigger pool of potential candidates from the 2015 compendium that provides 419 palmitoylated synaptic proteins (Sanders et al., 2015).  4.7 Limitations This study has several limitations. First, we do not know if the 2B-NMDAR palmitoylation level was actually reduced by HIP14 ASO. Biochemical assays such as the ABE/western blot approach would be required to obtain that information. Second, as mentioned above, I have noticed that even at the end of the ASO treatment, there is still about 10% of HIP14 protein left. Although a 90% reduction in HIP14 expression is significant, it is possible that the residual HIP14 is sufficient to maintain its enzymatic role as a PAT in regulating a fast reaction like palmitoylation. Third, in the immunocytochemical study, what I have actually stained was transfected YFP-tagged GluN2B subunits yet not the endogenous ones, due to the absence of any dependable external epitope GluN2B-specific antibodies. YFP-tagged 2B-NMDARs may not distribute in the same way as native ones, and over-expression of GluN2B may also have altered some physiological properties of MSNs. To minimize these possible exogenous effects, all conditions were examined on YFP-tagged 2B-NMDARs instead of native ones and at least obligatory GluN1 subunits are all endogenous, which served as a quality-checking step during receptor assembly. Fourth, another 	   78	  caveat is that in the HIP14 knock-down experiments, I did not separately examine synaptic vs. extrasynaptic NMDAR current, therefore I did not control for effects of HIP14 ASO on the NMDAR localization in the electrophysiological experiments. The surface distribution of NMDARs could be altered without a change in total whole-cell current. Fifth, the surface/internal assay was restricted to 2B-NMDARs; 2A-NMDARs, on the other hand, may be more sensitive to alterations in surface/internal distribution by HIP14. However, the lack of ASO effect on the total NMDAR current does not support this possibility. Finally, questions are always raised about the intrinsic limitations of using cell culture for a pathophysiological model. In my study, at least cortical and striatal neurons were co-cultured together to ensure the availability of an excitatory network of glutamatergic synapses. For the study of the effect of PalmB treatment on 2B-NMDAR palmitoylation, first I did not include a negative control. A good negative control should be some protein that is not a substrate of APT1/2. Cysteine string protein (CSP), a presynaptic protein bringing vesicles to the docking site, is one such protein. Furthermore, due to the limited amount of cells available for biochemical assays, I only had two conditions -- DMSO and PalmB treatments. However, based on the electrophysiological data showing an effect by DMSO, a third condition of untreated control is needed. Second, a major concern about the ABE assay is that it is vulnerable to false positives. An increased NO production in YAC128 striatum could lead to an increase of nitrosylation of GluN2B cysteines (Nakamura et al., 2013) and still be detected by ABE assay because it cannot distinguish between thioester bonds formed through palmitoylation and s-nitrosylation. Third, despite its wide application and strong effect, PalmB is a relatively non-specific inhibitor, in that it is non-selective between APT1 and APT2, which make the experimental outcomes more difficult to interpret. To address this issue, a more specific inhibitor should also be tested like C83 for APT1 and C115 for 	   79	  APT2 (Adibekian et al., 2012). Another problem with PalmB treatment is that different proteins may have varying palmitoylation turnover cycles, and not all can be detected by a single time window of 4 hours, as in my treatments. Time course tests are definitely needed for a list of interesting proteins. Moreover, my results show that DMSO itself has an effect compared to the untreated group, thus a better vehicle control is need. Some possible substitutes are propylene glycol, ethanol, or even a water dilution of DMSO (50:50). DMSO contains both highly polar domain and apolar methyl groups making it amphipathic and soluble in water (Santos et al., 2003). Finally, there are some caveats in the electrophysiological assay. MK-801 is only an open channel NMDAR blocker yet not specific to synaptic NMDARs. The assessment of Ex-NMDAR current by first pre-treating synaptically active neurons with MK-801 relies on the assumption that only synaptic NMDARs were activated during the 4-AP treatment. I am not certain that there is no spillover of glutamate at the 4-AP step, since there are no glial cells to prevent it, which could thereby activate (and subsequently block by MK-801) some of the Ex-NMDARs; on the other hand, the experiments are done in a relatively large volume of bathing solution, with continuous superfusion of aCSF, making glutamate spill-over and activation of Ex-NMDAR less likely. As well, I kept the experimental procedure standardized across genotypes and treatments. More importantly, even if there was glutamate spillover onto extrasynaptic sites leading to early activation of Ex-NMDARs (during the 4-AP step), it would result in an underestimation of the Ex-NMDAR proportion, which would reinforce our findings that the Ex-NMDAR proportion is increased in DMSO and PalmB treatments. As a final point, I would need to isolate GluN2B-containing NMDAR current from the total current (mediated by both 2A- and 2B-NMDAR) to make conclusions about the effect of PalmB on 2B-NMDAR functional surface distribution.  	   80	  4.8 Future direction This study raises important questions at several perspectives regarding palmitoylation of NMDAR and its effect on NMDAR distribution and function in early HD. First, more experiments are needed to elucidate the different mechanics of Cys Cluster I and II. The long-term goal could be a detailed map of PATs and APTs for each cluster. However for now, it would be interesting to start with feasible candidates. A study from our lab identified that Cys Cluster I but not Cluster II is a substrate of HIP14 in COS-7 cells. A similar test could be used to probe for the potential interaction between HIP14L and the two GluN2B cysteine clusters. Another interesting PAT is ZDHHC3 given that GluN2B and GluN2A were both found to be its substrates in cortical neurons (Hayashi et al., 2009). Also, the same electrophysiological assay in the PalmB part of this study could also be applied to directly examine the different consequences of possible NMDAR re-distribution with the two different cluster mutants (3CS and 5CS) in future. Currently such a study would have to be restricted to an immunocytochemical assay, since I could not separate the contribution of mutant 2B-NMDAR from endogenous 2B-NMDAR in whole-cell patch clamp recording. A recent study showed that the cluster II mutant form (GluN2B 5CS) did not increase synaptic 2B-NMDAR-mediated current (Mattison et al., 2012), which raises the question as to whether the Ex-NMDARs are affected or not. Since native 2B-NMDARs will interfere with any attempt to assess the current mediated by just the mutant forms, another approach such RNA or peptide interference targeting endogenous 2B-NMDARs, is needed prior to any functional studies. Another perspective to explore on more details about early HD pathogenesis in the striatum is to separately examine D1 and D2 neurons as increasing evidence indicates different neuropathological mechanisms in HD between these two types of striatal cells (Raymond et al., 2011; Sepers and Raymond, 2014). 	   81	  Second, our knowledge also needs to be expanded for HIP14 and HIP14L. Experimental results from genetics analysis, in vitro neuronal cultures, and transgenic knock-out mouse lines, all point to the possibility of a reciprocal compensation between these two PATs, at least to a certain degree (Sanders et al., 2014, 2015a, b; Sutton et al., 2012), and the necessity of a double loss-of-function study on HIP14 and HIP14L together. Although double knock-out of HIP14 and HIP14L causes embryonic lethality, there are ways to by-pass this obstacle. Given that both Hip14-/- and Hip14l-/- lines are available, in vitro study is possible after knocking down the existing PAT in cultures from mice lacking the other one. Another approach is to establish an inducible knock-out mouse line for in vivo studies. Third, study on PSD-95 demonstrated that the dynamic competition between s-nitrosylation and palmitoylation directly regulates the synaptic targeting of this protein (Ho et al., 2011). Both PTMs are also found to mediate AMPAR trafficking, distribution, and endocytosis (Hayashi et al., 2005; Lin et al., 2009; Selvakumar et al., 2013). Work from others and our labs have already shown that aberrant NO production is involved with HD pathogenesis (Nakamura et al., 2013; Fan et al., 2009). Future work is required to determine whether similar mechanisms are also applicable to NMDARs, especially on the altered distribution of NMDARs in HD. Finally, further work still needs to be done regarding APT1 and PalmB. A thorough examination of PalmB targets will be helpful for making a better interpretation of experimental outcomes. Meanwhile a follow-up study using a more specific inhibitor like C83 for APT1 and C115 for APT2 will be insightful (Adibekian et al., 2012). If knock-down of native 2B-NMDARs could be done by RNA interference, the effect of PalmB treatment on different cluster mutant forms should also help to answer some of the cluster-specific questions. On the other hand, studies on a PalmB-treated HD mouse model could also bring the experimental scale to systemic and 	   82	  behavioral levels. However, the side effects of DMSO or other vehicles need to be carefully examined based on the findings in this study. 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