Chronic blockade of extrasynaptic NMDA receptors ameliorates synaptic dysfunction and pro-death signaling in Huntington disease transgenic mice

Neurobiology of Disease 62 (2014) 533–542

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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

Chronic blockade of extrasynaptic NMDA receptors ameliorates synaptic dysfunction and pro-death signaling in Huntington disease transgenic mice Alejandro Dau, Clare M. Gladding, Marja D. Sepers, Lynn A. Raymond ⁎ Department of Psychiatry, Division of Neuroscience, Brain Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

a r t i c l e

i n f o

Article history: Received 5 August 2013 Revised 17 October 2013 Accepted 12 November 2013 Available online 19 November 2013 Keywords: Huntington disease NMDA receptor GluN2B Extrasynaptic p38 pCREB Calpain Calcium signaling Striatum Memantine

a b s t r a c t In the YAC128 mouse model of Huntington disease (HD), elevated extrasynaptic NMDA receptor (Ex-NMDAR) expression contributes to the onset of striatal dysfunction and atrophy. A shift in the balance of synaptic– extrasynaptic NMDAR signaling and localization is paralleled by early stage dysregulation of intracellular calcium signaling pathways, including calpain and p38 MAPK activation, that couple to pro-death cascades. However, whether aberrant calcium signaling is a consequence of elevated Ex-NMDAR expression in HD is unknown. Here, we aimed to identify calcium-dependent pathways downstream of Ex-NMDARs in HD. Chronic (2month) treatment of YAC128 and WT mice with memantine (1 and 10 mg/kg/day), which at a low dose selectively blocks Ex-NMDARs, reduced striatal Ex-NMDAR expression and current in 4-month old YAC128 mice without altering synaptic NMDAR levels. In contrast, calpain activity was not affected by memantine treatment, and was elevated in untreated YAC128 mice at 1.5 months but not 4 months of age. In YAC128 mice, memantine at 1 mg/kg/day rescued CREB shut-off, while both doses suppressed p38 MAPK activation to WT levels. Taken together, our results indicate that Ex-NMDAR activity perpetuates increased extrasynaptic NMDAR expression and drives dysregulated p38 MAPK and CREB signaling in YAC128 mice. Elucidation of the pathways downstream of Ex-NMDARs in HD could help provide novel therapeutic targets for this disease. © 2013 Elsevier Inc. All rights reserved.

Introduction In Huntington disease (HD), progressive neurodegeneration is attributed to a polyglutamine (polyQ) expansion near the N-terminus of the protein huntingtin (mutant huntingtin, mtHtt) (Huntington's Disease Collaborative Research Group, 1993). Whereas wildtype huntingtin is vital for normal cellular function, mtHtt interferes with essential intracellular processes, including gene expression, Ca 2 + homeostasis and vesicular trafficking (Zuccato et al., 2010). The polyQ expansion primarily affects GABAergic medium-sized spiny projection neurons (SPNs) of the striatum, which exhibit up to 95% neuronal loss at late stages of the disease (Vonsattel et al., 1985). However, the mechanisms underlying selective vulnerability of the striatum to mtHtt-induced death remain unclear. Increased functional expression of N-methyl-D-aspartate glutamate receptors (NMDARs) in HD could contribute to selective striatal excitotoxicity (Cepeda et al., 2001; Chen et al., 1999; Fan et al., 2007; Li et al., 2003; Milnerwood et al., 2010; Starling et al., 2005;

⁎ Corresponding author at: Department of Psychiatry, University of British Columbia, 4N3-2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada. E-mail addresses: [email protected] (A. Dau), [email protected] (C.M. Gladding), [email protected] (M.D. Sepers), [email protected] (L.A. Raymond). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nbd.2013.11.013

Zeron et al., 2001, 2002; Zhang et al., 2008). Previously, we reported elevated expression of GluN2B-containing extrasynaptic NMDARs (Ex-NMDARs) in presymptomatic YAC128 mice (Milnerwood et al., 2010), in which the yeast artificial chromosome is used to express full-length human huntingtin with 128 CAG repeats (Slow et al., 2003). Synaptic NMDARs activate pro-survival pathways, while Ex-NMDARs trigger cell death (Hardingham and Bading, 2010; Hardingham et al., 2002). A shift in the balance of synaptic to Ex-NMDAR signaling contributes to HD pathology, as chronic Ex-NMDAR blockade attenuates mtHttinduced striatal atrophy and motor learning deficits in YAC128 mice (Milnerwood et al., 2010; Okamoto et al., 2009). Along with elevated Ex-NMDAR activity, intracellular Ca2+ signaling pathways that couple to survival or death are also dysregulated early in HD. Activity of the Ca2 +-dependent protease calpain is elevated in striatal tissue of post-mortem HD human brains and presymptomatic 1–2 month old YAC128 mice (Cowan et al., 2008; Gafni and Ellerby, 2002; Gladding et al., 2012). Calpain potentiates HD-associated striatal degeneration by cleaving mtHtt into toxic fragments and triggering pro-apoptotic cascades in parallel with caspases (Gafni et al., 2004; Kim et al., 2001). Calpain also contributes to Ex-NMDAR surface mislocalization in 1–2 month old YAC128 mice by cleaving the GluN2B C-terminus and thus altering NMDAR surface stability (Gladding et al., 2012). Activity of the p38 mitogen-activated protein kinase (MAPK), shown previously to be downstream of Ex-NMDARs (Xu

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et al., 2009), is also elevated in YAC128 mouse striatum at 1–2 months of age (Fan et al., 2012). The p38 MAPK mediates enhanced NMDAinduced toxicity in YAC128 cultured striatal neurons (Fan et al., 2012) and thus contributes to susceptibility of mtHtt-expressing striatal neurons to excitotoxic death. In addition, activity of the pro-survival transcription factor cAMP response element binding protein (CREB) is reduced in striatal tissue of 1 and 4 month-old YAC128 mice (Milnerwood et al., 2010). While synaptic NMDAR signaling promotes CREB activity, Ex-NMDARs trigger dephosphorylation and inactivation of CREB via dominant pathways (Hardingham et al., 2002). Additionally, CREB signaling is restored by chronic suppression of Ex-NMDAR activity in YAC128 mice (Milnerwood et al., 2010), suggesting a link between Ex-NMDARs and CREB shut-off. Together, elevated Ex-NMDAR activity and dysregulated intracellular signaling could contribute to mtHtt-induced striatal degeneration. However, whether aberrant Ca2 + signaling in HD is a direct result of enhanced Ex-NMDAR activity or a consequence of other effects of mtHtt remains unclear. Here, we examined the role of ExNMDARs in HD-associated aberrant Ca2 + signaling.

nuclear matrix fraction (not shown). Fractions were stored at −80 °C until use. Western blotting

WT and YAC128 (line 55) mice (Slow et al., 2003) bred on the FVB/N background were maintained at the University of British Columbia (UBC) Faculty of Medicine Animal Resource Unit, according to guidelines of the Canadian Council on Animal Care. Animals were housed in identical conditions (2–4 mice/cage) in a 12-h light/dark cycle, with full access to food and water. WT and YAC128 mice were treated with memantine as described previously (Milnerwood et al., 2010; Okamoto et al., 2009). Briefly, memantine at 1 or 10 mg/kg/day was provided to mice ad libitum in their drinking bottles, starting at 2 months of age (+ 10 days) for 2 months. Control mice received water (vehicle). Memantine solution concentrations were adjusted on a semi-monthly basis according to mouse body weight and daily solution intake to ensure consistent dosing throughout the treatment period. Cohorts alternated between male and female mice (a total of 4 female cohorts and 6 male cohorts). No differences in daily solution intake were detected between treatment groups compared to control groups for either genotype or sex (data not shown); thus, the mice had no apparent aversion to the taste of memantine.

Protein concentration was assessed by a BCA protein assay (Pierce). Freshly-thawed samples were prepared for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) by heating (3–5 min, 80–85 °C), in 3X protein sample buffer (PSB) (6% SDS, 0.4 mM Tris (pH 6.8), 30% glycerol, pyronin Y, 70 mg/mL DTT). Equal amounts of protein (5–15 μg for non-PSD, PSD, nuclear matrix fractions, or 20–40 μg for cytosolic fractions) were separated in 10% (w/v) SDSpolyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes by semi-dry electrophoresis (BioRad). Membranes were blocked in TBS with 0.5% Tween-20 (TBST) and 3% BSA (or 5% milk for spectrin blots) (1 h, RT), incubated in primary antibodies (overnight, 4 °C), then washed and incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (2 h, RT). Blots were then washed and visualized using an enhanced chemiluminescence substrate (ECL, Amersham) and developed by exposure to film (Amersham), except for pCREBSer13 and CREB blots which were developed using an automated ChemiDoc XRS Molecular Imager (BioRad). Blots for which total p38 was probed were subsequently reprobed to quantify phosphorylated p38 (P-p38), using alkaline phosphatase (AP)-conjugated secondary antibodies and a Lumi-phosWB Chemiluminescent Substrate detection system (Pierce). The following primary antibodies were used: rabbit N-terminal anti-GluN2B (AGC-003; Alomone, 1:500), rabbit anti-spectrin (cleaved) (AB38, gift from Dr. David Lynch, University of Pennsylvania, Philadelphia, PA, 1:2000), rabbit anti- pCREBSer133 (06-519, Millipore, 1:500), rabbit anti-CREB (9197, Cell Signaling, 1:500), rabbit anti-P-p38 MAPK (4511S, Cell Signaling, 1:200), mouse anti-p38 MAPK (sc-7972, Santa Cruz, 1:200), mouse anti-PSD-95 (MA1-045, Pierce, 1:500), goat anti-β-actin (sc-1616, Santa Cruz, 1:1500), goat anti-α-tubulin (sc9935, Santa Cruz, 1:1500), goat anti-HDAC (sc-6268, Santa Cruz, 1:500), and mouse anti-synaptophysin (S5768, Sigma, 1:1000). All primary antibodies were diluted in TBST with 3% BSA, except for anti-spectrin, which was diluted in TBST with 5% milk. The following secondary antibodies were used: anti-mouse HRP-conjugated (NA931V, Amersham, 1:5000), anti-rabbit HRP-conjugated (NA934V, Amersham; 1:5000), anti-rabbit AP-conjugated (S372, Promega, 1:5000), and anti-goat HRP-conjugated (sc-2020; Santa Cruz, 1:5000). All secondary antibodies were diluted in TBST with 1% BSA.

Striatal dissection, subcellular and nuclear fractionations

Image analysis

Striatal tissue from memantine-treated (1 and 10 mg/kg/day) and untreated WT and YAC128 mice was collected after the treatment period and paired on the day of dissection. Mice were decapitated following halothane vapour anesthesia. Brains were rapidly removed and striatal sections were dissected and homogenized in 200 μL ice-cold sucrose buffer (0.32 M sucrose, 10 mM HEPES, pH 7.4). Striatal cytosolic, synaptosomal, and nuclear fractions were obtained by subcellular and nuclear fractionation as described previously (Milnerwood et al., 2010). Synaptic (postsynaptic density, PSD) and extrasynaptic-enriched (non-PSD) synaptosomal fractions were isolated based on the principle that the non-PSD is Triton-X-soluble, whereas the PSD is Triton-X-insoluble. All buffers contained ‘complete’ protease and phosphatase inhibitor cocktails (Roche), as well as 15 μM calpeptin (Calbiochem), 1 mM EDTA, 1 mM EGTA, 40 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 30 mM sodium fluoride. The purity of the subcellular fractionation was confirmed by enrichment of the PSD marker PSD-95 in the PSD fraction, and the presynaptic marker synaptophysin in the non-PSD (not shown). The purity of the nuclear fractionation was confirmed by enrichment of the nuclear marker histone deacetylase (HDAC) in the

For blots developed using film, the optical density of bands was quantified using Image J software (NIH) after background subtraction. Band intensities of GluN2B in synaptosomal fractions were normalized to β-actin (loading control), whereas bands for cytosolic spectrin were normalized to α-tubulin, which gave a clearer signal in the cytosolic fraction. P-p38 bands were normalized to p38 bands probed on the same membrane. For quantification of pCREBSer133/ CREB ratios, CREB and pCREBSer133 levels were probed on separate gels, and each was normalized to HDAC (loading control), as probing for CREB and pCREBSer133 on the same membrane did not provide clear results. pCREBSer133 and CREB blots, which were developed using the ChemiDoc XRS Molecular Imager, were quantified using Image-Lab Analysis Software (4.1, BioRad).

Methods Memantine treatment

Brain slice preparation Ex-NMDAR currents were recorded from coronal brain slices made from memantine-treated (1 mg/kg/day) and untreated WT and YAC128 mice. Mice were halothane-anesthetized and decapitated. Brains were immersed in ice-cold oxygenated (95% O2 , 5% CO 2 )

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low-calcium artificial cerebrospinal fluid (aCSF), containing (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 MgCl2, 0.5 CaCl2, 25 glucose, pH 7.3–7.4, 300–310 mosmol L−1. 300 μm thick coronal slices were cut on a vibratome (Leica VT1000), placed in a holding chamber with continuously oxygenated standard aCSF (as above, but with 1 mM MgCl2 and 2 mM CaCl2) at 37 °C for 45mins-1 h, then at RT until time of recording. Slice electrophysiology Slice electrophysiological recordings were conducted as described previously (Milnerwood and Raymond, 2007; Milnerwood et al., 2010). Following incubation in a holding chamber, slices were perfused continuously (1.0–1.5 ml/min, RT) with oxygenated standard aCSF containing 10 μM glycine, 2 μM strychnine, and 100 μM picrotoxin (Tocris), and allowed to equilibrate for 15–20 min prior to recording. EPSCs were evoked (eEPSCs) in the center of the striatum (150 μs, 25–150 μA, every 20 s) using a glass micropipette (2–5MΩ) filled with aCSF. NMDAR currents were recorded at a holding potential of +40mV from a randomly-selected SPN 150–250μm ventral to the site of stimulation, with a micropipette filled with (in mM): 130 cesium methanesulfonate, 5 CsCl, 4 NaCl, 1 MgCl 2 , 5 EGTA, 10 HEPES, 5 QX-314, 0.5 GTP, 10 Na 2 -phosphocreatine and 5 Mg ATP, pH 7.3, 280–290 mosmol/L. All recordings were made in 2,3-dihydroxy-6-nitro7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, 10 μM, Tocris) to block AMPA receptors. After a stable baseline was established (N 5 min), DL-threo-β-benzyloxyaspartic acid (DL-TBOA, 30 μM, Tocris) was bath-applied to block glutamate transporters and to induce extrasynaptic glutamate spillover. NMDAR-mediated eEPSCs were monitored in the presence of TBOA for 5 min, then TBOA was washed off for 25 min. Signals were filtered at 1 kHz, digitized at 10 kHz and analyzed in Clampfit 10 (Axon Instruments). The area under the curve for current in the first 3 s of each NMDAR-eEPSC was normalized to peak current amplitude for that trace. Pipette resistance was 3–7 MΩ. Series resistance (Rs) was b25 MΩ and uncompensated, and monitored throughout the experiment; tolerance for ΔRs was b50% provided Rs b 30MΩ. Statistical analysis Statistical analyses were conducted using Prism 6 software (GraphPad). Data are presented as mean + SEM. All analyses were performed using a two-way ANOVA. For each treatment condition, significant differences between genotypes or age groups were tested by Bonferroni's multiple comparisons post-hoc test. For each genotype, significant differences between treatment dose (1 or 10 mg/kg/day) and the control (H2O) were examined by Dunnett's multiple comparisons post-hoc test, unless indicated otherwise. Dunnett's post-hoc test was selected for the latter analyses as it primarily compares differences between a set of test groups and a control. Overall significant effects of interaction, treatment, genotype or age are indicated in the text. Results Memantine decreases Ex-NMDAR expression in YAC128 mice Elevated expression of GluN2B-containing Ex-NMDARs contributes to enhanced susceptibility of striatal neurons to excitotoxic death in YAC128 mice (Milnerwood et al., 2010, 2012). However, the mechanisms underlying NMDAR mislocalization in HD remain unclear. Previous studies indicate that Ex-NMDAR currents activate signaling pathways, including calpains and protein phosphatases/kinases, that can in turn modulate synaptic vs. extrasynaptic targeting of GluN2Bcontaining receptors (Gladding and Raymond, 2011; Gladding et al., 2012; Xu et al., 2009). To determine whether Ex-NMDAR activity regulates GluN2B-NMDAR localization, 2 month-old WT and YAC128 mice

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were treated with memantine (1 and 10 mg/kg/day) for two months, as described previously (Milnerwood et al., 2010; Okamoto et al., 2009). The 1mg/kg/day dose is estimated to result in an effective concentration at the NMDAR channel mouth of ~5–10 μM, which selectively blocks Ex-NMDARs, relatively sparing synaptic receptors in cultured neurons, and closely mimics the therapeutic dose of 10–20 mg/day in humans (Okamoto et al., 2009; Raina et al., 2008; Xia et al., 2010). We additionally selected the higher 10 mg/kg/day dose as it has been shown to be neuroprotective in several neuroinflammatory disorders (Rammes et al., 2008; Rosi et al., 2006). After maintaining mice with either memantine in the drinking water or water alone (untreated control), we isolated striatal tissue and examined extrasynaptic GluN2B subunit levels by subcellular fractionation and western blotting. The subcellular fractionation separates synaptic (postsynaptic density, PSD) from extrasynaptic-enriched (non-PSD) membranes, and thus allowed us to directly examine protein expression in each synaptosomal compartment (Gladding et al., 2012; GoebelGoody et al., 2009; Pacchioni et al., 2009). We used an N-terminal GluN2B antibody that detects both calpain-cleaved (115kDa) and full-length (180kDa) subunits. There was an enrichment of calpaincleaved relative to full-length GluN2B in the non-PSD fraction in all groups examined, as reported previously (Gladding et al., 2012) (Fig. 1A). Total (full-length plus cleaved) GluN2B levels were significantly increased in the non-PSD fraction of untreated YAC128 compared to WT mice (p b 0.01) (GluN2B/β-actin ratios for untreated mice: WT, 1.51 + 0.12; YAC128, 2.04 + .11) (Fig. 1B). Interestingly, YAC128 extrasynaptic GluN2B expression was significantly reduced to WT levels by memantine at both doses (p b 0.01) (GluN2B/β-actin ratios for treated YAC128 mice: 1 mg/kg/day, 1.61 + 0.13; 10 mg/kg/day, 1.56 + 0.l7). This trend was also observed when full-length and calpain-cleaved GluN2B bands were quantified separately (Fig. 1C,D). However, significant differences were only detected in full-length GluN2B levels between memantine-treated (1 mg/kg/day) and untreated YAC128 mice (p b 0.05), as separate quantification of full-length and calpain-cleaved GluN2B bands yielded higher variability. Moreover, when normalized to total receptor levels, calpain-cleaved GluNB was not different between genotypes or treatments, suggesting that the mtHtt- and memantine-induced changes in Ex-NMDAR localization were not associated with changes in the extent of C-terminal GluN2B cleavage (calpain-cleaved/total GluN2B levels: WTH2O, 0.55 + .04; YAC128H2O, 0.53 + .05; WT1 mg/kg/day, 0.53 + .05; YAC1281 mg/kg/day, 0.56 + .04; WT10 mg/kg/day, 0.56 + .05; YAC12810 mg/kg/day, 0.60 + 0.04). To more definitively determine the effect of the 2-month memantine treatment on functional Ex-NMDAR expression, we examined striatal spiny projection neurons (SPNs) from untreated and memantine-treated (1 mg/kg/day) WT and YAC128 mice by wholecell electrophysiology in acute cortico-striatal slices. As brain slices were incubated in recording solution lacking memantine for at least an hour prior to recording, any residual memantine should have been removed from the tissue. We evoked NMDAR-mediated EPSCs before and during application of the glutamate transporter inhibitor DL-TBOA (30 μM), to induce glutamate spillover and activate extrasynaptic receptors (Tzingounis and Wadiche, 2007) as described previously (Milnerwood et al., 2010). The effect of TBOA on NMDAR chargetransfer reflects activation of Ex-NMDARs (Milnerwood et al., 2010). In TBOA, NMDAR peak-normalized charge increased (Fig. 1E), as previously observed (Milnerwood et al., 2010), and this effect was more pronounced for untreated YAC128 mice compared to other groups. In fact, maximal TBOA effects were significantly greater in untreated YAC128 mice compared to WT (p b 0.05) (Fig. 1F), consistent with increased Ex-NMDAR levels (maximal TBOA increase over baseline for untreated mice: WT, 23.8 + 6.0%; YAC128, 46.1 + 5.7%). Moreover, YAC128 maximal TBOA effects were significantly reduced by 1mg/kg/day memantine treatment (p b 0.05), to levels similar to WT, in agreement with our biochemical analysis of non-PSD GluN2B expression (maximal TBOA increase for memantine-treated mice: WT, 29.29 + 9.2%;

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Fig. 1. Memantine decreases functional Ex-NMDAR expression in YAC128 mice. WT and YAC128 mice were treated with water (H2O) only or memantine (1 and 10 mg/kg/day) in the drinking water for 2 months, beginning at 2 months of age. A) Representative blots of non-PSD fractions probed for full-length (180kDa) and calpain-cleaved (115 kDa) GluN2B and βactin (loading control), in memantine-treated (1 and 10 mg/kg/day) and untreated WT and YAC128 mice. Calpain-cleaved GluN2B bands were highly enriched relative to full-length GluN2B bands in the non-PSD; thus higher (top panel) and lower (bottom panel) exposures were used to quantify full-length and cleaved GluN2B levels, respectively. B–D) Quantification of total (cleaved plus full-length) (B), full-length (C), and calpain-cleaved (D) GluN2B subunit levels normalized to β-actin. Analyzed by a two-way ANOVA (**p b 0.01, Bonferroni's post-hoc test; #p b 0.05, ## p b 0.01, Dunnett's post-hoc test). The number of independent experiments is indicated inside each bar. B) The interaction between groups was significant (F(2,55) = 5.462, p b 0.01). E) Representative peak-normalized NMDAR-mediated EPSCs in SPNs of acute coronal brain slices before (gray) and during (black) bath application of DL-TBOA (30 μM), for untreated WT (i) and YAC128 (ii) and 1 mg/kg/day memantine-treated WT (iii) and YAC128 (iv) mice. F) Quantification of maximal TBOA effects on peak-normalized NMDAR charge as a percentage of baseline (*p b 0.05; two-way ANOVA, Bonferroni's post-hoc test). WTH20, n = 14; YAC128H2O, n = 17; WT1 mg/kg/day, n = 9; YAC1281 mg/kg/day, n = 12; 5–7 mice per group. The interaction between groups was significant (F(1,48) = 4.984, p b 0.05).

YAC128, 22.9 + 4.6%). Together, these results suggest that chronic blockade of Ex-NMDARs with memantine corrects the elevated functional expression of Ex-NMDARs in striatal neurons from YAC128 mice to WT levels. Synaptic NMDAR levels are unaffected by mtHtt or memantine The mtHtt- and memantine-induced changes in Ex-NMDAR expression could be mediated by shifts in synaptic-extrasynaptic localization

of surface receptors. To observe whether synaptic NMDAR levels are inversely correlated with Ex-NMDAR expression, we examined synaptic GluN2B expression by western blotting of the striatal PSD fraction from memantine-treated (1 and 10 mg/kg/day) and untreated WT and YAC128 mice. In contrast to the non-PSD, fulllength GluN2B levels were enriched compared to calpain-cleaved subunits in the PSD of all groups tested, as reported previously (Gladding et al., 2012) (Fig. 2A). However, no average changes were detected in total (full-length plus cleaved) synaptic GluN2B

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ratios: WTH2O, 0.82 + 0.06; YAC128H2O, 0.88 + 0.08; WT1 mg/kg/day, 0.99 + 0.07; YAC1281 mg/kg/day, 0.89 + 0.09; WT10 mg/kg/day, 0.74 + 0.07; YAC12810 mg/kg/day, 0.83 + 0.09; p N 0.05) (Fig. 3A,B). We previously observed elevated calpain activity in untreated 1–2 month-old YAC128 mice (Cowan et al., 2008; Gladding et al., 2012); this apparent discrepancy with the lack of difference between 4 month-old YAC128 and WT mice could be due to differences in the ages of mice examined in each study. To confirm that calpain activity is elevated in 1–2 month-old but not 4 month-old YAC128 mice, we compared spectrin cleavage in untreated animals at both ages. Calpain-cleaved spectrin levels were significantly elevated in YAC128 compared to WT mice at 1.5 months (p b 0.05) but not 4 months of age (cleaved spectrin/α-tubulin ratios at 1.5 months: WT, 0.98 + 0.05; YAC128, 1.31 + 0.10; at 4 months: WT, 0.87 + 0.13; YAC128, 0.82 + 0.11) (Fig. 3C,D). In fact, YAC128 calpain-cleaved spectrin levels were significantly decreased at 4 months compared to 1.5 months (p b 0.01). Low-dose memantine rescues mtHtt-induced CREB shut-off

Fig. 2. Synaptic NMDAR expression is not altered between genotypes or treatments. PSD fractions of memantine-treated (1 and 10 mg/kg/day) and untreated WT and YAC128 mice were isolated from striatal tissue and probed for synaptic GluN2B subunit levels. A) Representative blots of PSD fractions probed for full-length and calpain-cleaved GluN2B and β-actin (loading control). An enrichment of full-length GluN2B was detected in the PSD subcellular fraction. B) Quantification of total (cleaved plus full-length) GluN2B/β-actin ratios. Analyzed by a two-way ANOVA. The number of independent experiments is indicated inside each bar.

expression between YAC128 and WT mice with or without memantine treatment (Fig. 2B) (total GluN2B/β-actin ratios: WTH2O, 1.64 + .11; YAC128H2O, 1.82 + .09; WT1 mg/kg/day, 1.69 + 0.10; YAC1281 mg/kg/day, 1.61 + 0.21; WT10 mg/kg/day, 1.84 + 0.21; YAC12810 mg/kg/day, 1.79 + 0.15). Moreover, when quantified separately, full-length and cleaved GluN2B bands were not different between genotypes or treatments (not shown). Striatal calpain activity is not different between 4 month-old YAC128 and WT mice, and is unaffected by memantine The modulation of YAC128 Ex-NMDAR expression by memantine suggests that receptor mislocalization in HD is Ex-NMDAR-activity dependent. The protease calpain is activated by Ca2 + influx through Ex-NMDARs (Xu et al., 2009), and elevated calpain activity contributes to Ex-NMDAR mislocalization in presymptomatic (1–2 month-old) YAC128 mice (Gladding et al., 2012). Hence, calpain signaling is a putative Ex-NMDAR activity-dependent pathway driving HD-associated receptor mislocalization. To determine whether calpain contributes to elevated Ex-NMDAR expression in 4 month-old YAC128 mice, we examined calpain activity in the cytosolic fraction of striatal tissue from memantine-treated and untreated WT and YAC128 mice. We quantified cleavage levels of the calpain substrate spectrin with a neo-epitope antibody specific for its calpain cleavage product (150 kDa) (a gift from Dr. David Lynch, University of Pennsylvania, Philadelphia, PA), as used previously (Cowan et al., 2008). Interestingly, the level of calpain-cleaved spectrin was not different between 4 month-old untreated WT and YAC128 mice, and was not significantly altered by memantine in either genotype (cleaved spectrin/α-tubulin

While synaptic NMDARs promote CREB activation by stimulating pathways that result in phosphorylation of the active site Ser133 residue (pCREBSer133), Ex-NMDARs trigger dominant CREB shut-off pathways (Hardingham and Bading, 2010; Hardingham et al., 2002; Papadia and Hardingham, 2007). In a previous study, suppression of Ex-NMDAR activity with low-dose memantine (1 mg/kg/day, 2 months) fully rescued mtHtt-associated CREB shut-off in 4 month-old YAC128 mice to WT levels (Milnerwood et al., 2010). However, at higher doses memantine also blocks synaptic receptors (Okamoto et al., 2009) and could thereby suppress synaptic NMDAR-mediated CREB phosphorylation. We thus aimed to confirm that low-dose (1 mg/kg/day) memantine fully restores striatal CREB activity as previously reported, and to investigate whether the higher dose (10 mg/kg/day) rescues or further suppresses CREBSer133 phosphorylation. We compared nuclear pCREB Ser133 relative to total CREB levels between untreated and memantine-treated WT and YAC128 mice. In agreement with previous data (Milnerwood et al., 2010), untreated YAC128 pCREBSer133/CREB ratios were significantly decreased compared to untreated WT mice at 4 months of age (p b 0.05) and were rescued to WT levels by memantine treatment at 1 mg/kg/day (p b 0.05) (Fig. 4A,B). At the 10 mg/kg/day dose, however, YAC128 pCREBSer133/CREB levels remained reduced. Additionally, there was a trend towards a decrease in WT pCREB Ser133 /CREB levels in a dose-dependent manner with memantine treatment, although this did not reach significance (pCREB Ser133 /CREB ratios: WT H2O , 1.00 + 0.05; YAC128H2O , 0.70 + 0.05; WT 1mg/kg/day , 0.85 + 0.09; YAC128 1 mg/kg/day , 0.99 + 0.12; WT 10 mg/kg/day , 0.69 + 0.15; YAC1281 mg/kg/day, 0.69 +0.08). Expression levels of HDAC also appeared to vary between conditions in the blot shown in Fig. 4A; however, this variability was not consistent between experiments. Moreover, average HDAC levels were not significantly different between genotypes or treatments (average HDAC band intensity (×106): WTH2O, 1.83 + 0.21; YAC128H2O, 1.70 + 0.28; WT1mg/kg/day, 2.06 + 0.34; 1.71 + 0.15; WT10 mg/kg/day, 2.15 + 0.34; YAC1281 mg/kg/day, YAC1281 mg/kg/day, 1.92 +0.25). Memantine reduces elevated p38 MAPK activity in YAC128 mice Ex-NMDARs activate p38 MAPK (Xu et al., 2009), which is associated with neuronal death (Soriano et al., 2008). Additionally, p38 MAPK activity is elevated in 1–2 month-old YAC128 mice, and contributes to NMDA-induced toxicity in mtHtt-expressing SPNs (Fan et al., 2012). We thus examined whether YAC128 p38 MAPK activity is also elevated at 4 months of age, and whether Ex-NMDAR activity drives mtHtt-induced p38 MAPK activation in vivo. As a measure of p38 MAPK activity, phosphorylation levels of p38 MAPK at Thr180 and Tyr182 (herein P-p38) were quantified in cytosolic fractions

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Fig. 3. Calpain activity is unaffected by memantine treatment, and is elevated in 1.5 month- but not 4 month-old YAC128 mice. Striatal cytosolic fractions were probed for calpain-cleaved spectrin (150kDa). A) Representative blots of calpain-cleaved spectrin and α-tubulin (loading control) in memantine-treated (1 and 10 mg/kg/day) and untreated 4 month-old WT and YAC128 mice. B) Quantification of calpain-cleaved spectrin/α-tubulin ratios. C) Representative blots for calpain-cleaved spectrin and α-tubulin in untreated 1.5 and 4 month-old mice. D) Quantification of calpain-cleaved spectrin/α-tubulin ratios between 1.5 and 4 months. Analyzed by a two-way ANOVA (*p b 0.05, **p b 0.01, Bonferroni's post-hoc test). The main effect of age was significant (F(1,21) = 8.664, p b 0.01). B, D) The number of independent experiments is indicated inside each bar.

from memantine-treated and untreated WT and YAC128 mice. The ratio of P-p38/p38 was significantly elevated in untreated 4 month-old YAC128 compared to WT mice (p b 0.05) (Fig. 5A,B), as previously reported in 1–2 month-old mice (Fan et al., 2012). Notably, YAC128 p38 MAPK activity was significantly attenuated to WT levels by memantine treatment at both doses (p b 0.01). (P-p38/p38 ratios: WT H2O , 1.03 + 0.04; YAC128H2O, 1.39 + 0.12; WT1 mg/kg/day, 0.94 + 0.10; YAC128 1 mg/kg/day , 0.88 + 0.10; WT10 mg/kg/day, 1.04 + 0.15; YAC12810 mg/kg/day, 0.92 + 0.10).

Discussion Identification of the pathways downstream of Ex-NMDARs may help to develop new targets for therapy in HD. Elevated ExNMDAR activity occurs early in the YAC128 mouse model of HD and contributes to mtHtt-induced striatal pathology (Milnerwood et al., 2010; Okamoto et al., 2009). Ca2 + dependent pathways closely linked to cell death are also dysregulated prior to phenotype onset and contribute to neuronal toxicity in HD (Cowan et al., 2008; Fan et al., 2012; Gafni and Ellerby, 2002; Gladding et al., 2012; Milnerwood et al., 2010; Wu et al., 2011; Zhang et al., 2008). Here, we aimed to demonstrate a causal link between Ex-NMDAR activity and aberrant intracellular Ca2 + signaling in HD. Somewhat surprisingly, we found that chronic suppression of Ex-NMDAR activity with memantine reduced striatal extrasynaptic GluN2B subunit expression as well as Ex-NMDAR current in YAC128 mice to WT levels without altering synaptic GluN2B-NMDAR expression. These changes were not associated with calpain signaling, which was unaffected by memantine treatment, and was only elevated in YAC128 mice at 1.5 months but not 4 months of age. Finally, memantine treatment rescued YAC128 CREB shut-off at the lower but not higher dose, while both doses decreased mtHtt-induced p38 MAPK activation to WT levels.

Considerations for in vivo memantine treatment Ex-NMDAR blockade with low-dose memantine is a valuable tool to discriminate pathways mediated by Ex-NMDAR activity from those which are a direct result of mtHtt-induced Ca2 + dyshomeostasis. Prevous dose-finding pilot studies have determined that memantine at 1 mg/kg/day in mice results in an approximate concentration of 5–10 μM at the NMDAR channel mouth (Okamoto et al., 2009; Xia et al., 2010). To confirm the selectivity of this concentration for ExNMDAR blockade, Okamoto et al. (2009) compared the effect of lowdose memantine on extrasynpatic vs. synaptic NMDAR-mediated EPSCs in cultured cortical neurons. They isolated Ex-NMDAR currents using MK-801 to block synaptic NMDAR activity, followed by bath application of NMDA to activate extrasynaptic receptors, as done previously (Hardingham et al., 2002). Bath application of memantine at 1– 10 μM blocked N85% of isolated Ex-NMDAR-mediated evoked EPSCs. Moreover, memantine at the same concentration had no effect on NMDAR-mediated sEPSCs in a separate experiment. Together, these experiments suggest that memantine preferentially blocks extrasynaptic over synaptic NMDARs. Using a similar experimental approach, Xia et al. (2010) compared the effect of memantine on isolated extrasynaptic and synaptic NMDAR-mediated EPSCs. At physiological Mg2+ concentrations, bath application of low-dose memantine (10μM) blocked significantly more extrasynaptic than synaptic NMDAR-mediated currents in the same neuron (1.5-fold higher blockade of extrasynaptic over synaptic NMDARs). Notably, the selectivity of memantine for Ex-NMDAR blockade is highly dose-dependent: at higher concentrations (30 μM) memantine blocks similar proportions of synaptic and extrasynpatic NMDAR currents in culture (Okamoto et al., 2009). Moreover, treatment of YAC128 mice with high-dose memantine (30 mg/kg/day) for 10 months exacerbates mtHtt-associated neuropathology and behavioral deficits at 12 months of age (Okamoto et al., 2009). Although less is known about the 10 mg/kg/day dose used in our study, our

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Fig. 4. Low-dose memantine rescues mtHtt-induced CREB shut-off. Nuclear fractions from striatal tissue were probed for pCREBSer133 and total CREB protein levels. A) Representative blots for pCREB Ser133 and total CREB in memantine-treated (1 and 10 mg/kg/d) and untreated WT and YAC128 mice. pCREBSer133 and total CREB protein were probed in separate gels and normalized to HDAC (loading control). The top band was analyzed to quantify pCREB Ser133 levels. B) Quantification of pCREBSer133/CREB levels. Analyzed by a two-way ANOVA (*p b 0.05, Bonferroni's post-hoc test; #p b 0.05, Dunnett's post-hoc test). The number of independent experiments is indicated inside each bar.

observation that memantine at this higher dose did not rescue YAC128 CREB activity could indicate that this dose also blocks a substantial proportion of synaptic NMDARs. Future studies will be required to determine the selectivity of memantine at both doses for Ex-NMDARs in vivo, and to examine the efficacy of both doses in ameliorating striatal excitotoxicity and cognitive dysfunction in HD. Certain considerations should be taken into account when using memantine treatment in vivo. First, several studies examining the affinity of memantine for the NMDAR have been largely performed in vitro, in the absence of Mg 2 + (Dravid et al., 2007; Johnson and Kotermanski, 2006). However, in vivo at physiological Mg2 + concentrations (~ 1 mM), memantine likely exhibits a higher IC50 than that reported in vitro (Kotermanski and Johnson, 2009; Otton et al., 2011), as memantine and Mg2 + compete for NMDAR channel binding (Sobolevsky et al., 1998). As indicated above, more recent studies have confirmed that low-dose memantine (10 μM) effectively blocks the majority of Ex-NDMAR currents in culture and remains selective for these receptors at physiological Mg 2 + concentrations (1mM) (Xia et al., 2010). Second, memantine blocks 5-HT3 and acetylcholine receptors (Aracava et al., 2005; Rammes et al., 2001) and activates dopamine D2 receptors (D2Rs) by binding them with similar affinity as Ex-NMDARs (Seeman et al., 2008). Agonism of modulatory dopaminergic signaling in D2R-expressing indirect pathway SPNs could

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Fig. 5. Memantine reduces elevated p38 MAPK activity in YAC128 mice. Cytosolic fractions isolated from striatal tissue were probed for P-p38 and total p38 levels, as a measure of relative p38 MAPK activity. A) Representative blots for P-p38 and p38 levels in memantinetreated (1 and 10 mg/kg/day) and untreated WT and YAC128 mice. After probing for total p38 protein, blots were reprobed for P-p38 using an antibody directed against p38 phosphorylated at Thr180 and Tyr182. P-p38 levels were normalized to total p38 expression in each lane. B) Quantification of P-p38/p38 ratios. Analyzed by a two-way ANOVA (*p b 0.05, Bonferroni's post-hoc test; ##p b 0.01, Dunnett's post-hoc test). The main effect of treatment was significant (F(2,46) = 4.085, p b 0.05). The number of independent experiments is indicated inside each bar.

suppress excitatory corticostriatal neurotransmission in this pathway (Gerfen and Surmeier, 2011). However, whether this would result in a net neuroprotective or neurotoxic effect in HD remains to be determined. Moreover, the potential off-target effects of memantine are likely indirect and may not affect Ex-NMDAR-mediated Ca2+ signaling. Ex-NMDAR surface expression is Ex-NMDAR activity-dependent Previously, we reported elevated striatal expression of GluN2Bcontaining Ex-NMDARs in YAC128 mice both at early presymptomatic (1–2 months of age) and late stages of HD (1 year of age) (Gladding et al., 2012; Milnerwood et al., 2010). Supporting these findings, we observed a significant increase in total (calpain-cleaved and full-length) GluN2B expression in the non-PSD of 4 month-old untreated YAC128 compared to WT mice. Hence, increased Ex-NMDAR expression in HD is present at early, mid, and late stages of the disease, and could contribute to striatal excitotoxicity at both early and more advanced periods of disease progression. Although the non-PSD fraction contains presynaptic, endosomal, and extrasynaptic membranes, our electrophysiological data showing elevated Ex-NMDAR currents in untreated YAC128 mice suggest that the increase in non-PSD GluN2B subunits likely involves functional receptors. Interestingly, memantine (1 and 10 mg/kg/day) reduced functional Ex-NMDAR expression in YAC128 mice to WT levels. This suggests

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that the mtHtt-induced increase in extrasynaptic receptor expression is Ex-NMDAR activity-dependent. Hence, Ex-NMDAR mislocalization could be driven by a feed-forward loop, whereby Ex-NMDAR activity triggers pathways that in turn potentiate expression of extrasynaptic receptors. The protease calpain, which is activated early in HD, contributes to Ex-NMDAR surface mislocalization in 1–2 month old YAC128 mice (Gladding et al., 2012). Notably, calpain activity is downstream of ExNMDARs (Xu et al., 2009), and could thus drive Ex-NMDAR-mediated receptor mislocalization. Although we detected an increase in calpaincleaved spectrin levels in 1.5 month-old YAC128 mice compared to WT littermates, calpain cleavage of spectrin was similar between 4 month old YAC128 and WT mice, and was unaffected by memantine treatment. Hence, in contrast to presymptomatic stages of HD, calpain activity is attenuated with HD progression and may not be associated with receptor mislocalization or striatal excitotoxicity at later stages of the disease. The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a transcriptional coactivator that regulates mitochondrial function (Finck and Kelly, 2006), has recently been reported to modulate Ex-NMDAR surface expression: reduced levels of PGC-1α are associated with a shift in NMDAR distribution to extrasynaptic sites, whereas increased levels reduce Ex-NMDARs (Puddifoot et al., 2012). CREB-mediated PGC-1α expression is reduced in HD, which may in part result from Ex-NMDAR-mediated CREB shut-off (Cui et al., 2006; Okamoto et al., 2009; Puddifoot et al., 2012). Thus, suppressed PGC1α signaling could be a putative mechanism linking Ex-NMDAR activity to receptor mislocalization. Synaptic (PSD) GluN2B levels were unchanged between untreated YAC128 and WT mice and were unaffected by memantine treatment. Hence, the effect of mtHtt and memantine on NMDAR distribution is specific to an increase at extrasynaptic sites of neurons and may not involve a shift in synaptic–extrasynaptic NMDAR localization. This is consistent with reports that synaptic NMDARs are less mobile than Ex-NMDARs due to tight anchoring in the PSD (Gladding and Raymond, 2011). Moreover, in agreement with these data, we have previously detected similar levels of synaptic NMDAR activity and GluN2B expression between WT and YAC128 SPNs (Milnerwood et al., 2010, 2012). Elevated Ex-NMDAR expression in HD could be attributed to an accelerated rate of NMDAR forward trafficking and translocation of synaptic receptors to extrasynaptic sites, or increased surface retention of Ex-NMDARs (Fan et al., 2007; Gladding and Raymond, 2011; Gladding et al., 2012; Tovar and Westbrook, 2002). As we did not detect changes in synaptic NMDAR levels between YAC128 mice compared to WT, a shift in NMDAR localization from synaptic to extrasynaptic sites would have to occur simultaneously with increased surface delivery at the PSD, such that net synaptic NMDAR levels remain unchanged. Further studies will be required to elucidate the pathways driving activity-dependent Ex-NMDAR mislocalization in HD. Calcium signaling is age-dependent in HD The observation that elevated calpain activity in YAC128 mice is attenuated with age suggests that Ca2 +-dependent signaling mechanisms in HD may evolve with disease progression. Previous work has also detected age-dependent alterations in Ca2 + signaling pathways in HD. For instance, activity and expression of the Ca 2 +dependent STriatal-Enriched tyrosine Phosphatase (STEP) is decreased with age in a late-stage HD mouse model (Saavedra et al., 2011). Additionally, striatal neurons in an excitotoxin-resistant mouse model develop enhanced cytosolic Ca2 + buffering with age, which could account for their resistant phenotype (Hansson et al., 2001). Notably, these age-dependent alterations in Ca2 + signaling appear to be specific to particular pathways, as Ex-NMDAR expression, as well as p38 MAPK and CREB activity, remain dysregulated at both early (1–2 months) and later (4 months) stages of HD, as

shown here and elsewhere (Fan et al., 2012; Milnerwood et al., 2010). Overall, more work is required to further examine the timedependent alterations in aberrant Ca2 + signaling in HD.

Ex-NMDARs couple to pro-death signaling in HD In culture, activation of Ex-NMDARs induces CREB shut-off (Hardingham et al., 2002; Kaufman et al., 2012; Papadia and Hardingham, 2007). Here, we observed a significant decrease in nuclear CREB activity in 4 month-old YAC128 mice compared to WT littermates, which was rescued by memantine treatment at the lower dose (1 mg/kg/day), as reported previously (Milnerwood et al., 2010). Thus, our data confirms that Ex-NMDAR activity drives suppression of neuroprotective CREB activity, which could contribute to striatal neurodegeneration in HD. Strikingly, memantine at the higher dose did not rescue YAC128 CREB activity and trended to decrease pCREBSer133/ CREB levels in WT mice. Synaptic NMDARs, which activate CREB via Ras-ERK or CaMKIV pathways (Impey et al., 2002; Wu et al., 2001), are blocked by memantine at higher doses (Okamoto et al., 2009). Thus, partial inhibition of synaptic NMDARs by memantine at the 10 mg/kg/day dose could explain its inability to rescue CREB signaling in YAC128 mice. P-p38/p38 levels were significantly increased in untreated 4 month-old YAC128 mice compared to WT. This is consistent with reports of elevated p38 MAPK activity in 1–2 month old YAC128 mice (Fan et al., 2012), and suggests that unlike calpain, aberrant p38 signaling remains elevated as HD progresses. Moreover, p38 phosphorylation in YAC128 mice was suppressed to WT levels by memantine, indicating that p38 signaling is downstream of ExNMDAR activity in HD. In fact, stimulation of Ex-NMDARs in culture induces a rapid and prolonged increase in p38 activity (Xu et al., 2009). During periods of oxidative stress, p38 activation stimulates mitochondrial translocation of Bax, cytochrome-c release and caspase activation thus potentiating pro-apoptotic signaling (Ghatan et al., 2000; Gomez-Lazaro et al., 2007). Hence, p38 MAPK signaling could be a pathway by which Ex-NMDARs facilitate later striatal neurodegeneration. In neurons, Ex-NMDARs could couple to p38 MAPK activity via distinct pathways. In cultured cortical neurons, Ex-NMDARs potentiate p38 phosphorylation by inducing calpain-mediated cleavage and inactivation of the striatal enriched tyrosine phosphatase (STEP), which dephosphorylates p38 (Xu et al., 2009). We did not detect changes in calpain activity in 4 month-old YAC128 mice; thus, other mechanisms must mediate Ex-NMDAR-dependent enhanced p38 activation at this age. Alternatively, GluN2B-PSD-95 interactions couple Ca2+ influx through the receptor to SynGAP and nNOS, both of which trigger p38 signaling (Aarts et al., 2002; Cao et al., 2005; Rumbaugh et al., 2006; Soriano et al., 2008). Indeed, associations between Ex-NMDARs and PSD-95 are increased in YAC128 mice, and these interactions mediate p38 activity and acute excitotoxic death in mtHtt-expressing SPNs (Fan et al., 2009, 2012). Hence, Ex-NMDAR-induced p38 MAPK activation in HD could be associated with aberrant GluN2B-PSD-95mediated signaling.

Conclusions By chronically blocking Ex-NMDARs in vivo, we show that ExNMDAR activity contributes to receptor mislocalization as well as aberrant p38 and CREB signaling pathways in the YAC128 mouse model of HD. As these mechanisms promote mtHtt-induced neuronal dysfunction and death (Fan et al., 2012; Milnerwood et al., 2010; Okamoto et al., 2009; Zeron et al., 2004), our data shed light on the signaling pathways linking Ex-NMDARs to cognitive decline and striatal atrophy in HD, and help clarify why low-dose memantine treatment is neuroprotective in YAC128 HD pathology.

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Acknowledgments We would like to thank D. Lynch for the anti-spectrin AB28 antibody. We would also like to thank L. Wang and L. Zhang for technical support, as well as M. Parsons and K. Kolodziejczyk for experimental advice and input. Funding for LAR was provided by the Cure Huntington's Disease Initiative (CHDI) and the Canadian Institutes of Health Research (CIHR) (MOP-12699). Funding for CMG was provided by a CIHR–Huntington Society of Canada Fellowship, and CMG also held a Hereditary Disease Foundation Fellowship.

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