Mitigation of augmented extrasynaptic NMDAR signaling and apoptosis in cortico-striatal co-cultures from Huntington's disease mice

Neurobiology of Disease 48 (2012) 40–51

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Mitigation of augmented extrasynaptic NMDAR signaling and apoptosis in cortico-striatal co-cultures from Huntington's disease mice Austen J. Milnerwood a, b, 1, 2, Alexandra M. Kaufman a, c, 1, Marja D. Sepers a, b, Clare M. Gladding a, b, Lily Zhang a, Liang Wang a, Jing Fan a, b, Ainsley Coquinco b, c, Joy Yi Qiao a, Hwan Lee a, Yu Tian Wang b, d, Max Cynader a, e, Lynn A. Raymond a, b, d,⁎ a

Department of Psychiatry, University of British Columbia, Vancouver, B.C., Canada Brain Research Centre, University of British Columbia, Vancouver, B.C., Canada c Graduate Program in Neuroscience, University of British Columbia, Vancouver, B.C., Canada d Department of Medicine, University of British Columbia, Vancouver, B.C., Canada e Department of Opthalmology, University of British Columbia, Vancouver, B.C., Canada b

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Article history: Received 22 November 2011 Revised 6 May 2012 Accepted 24 May 2012 Available online 2 June 2012 Keywords: NMDA receptor GluN2A GluN2B Huntington's disease Neurodegeneration Striatal medium spiny neuron Excitotoxicity Glutamate Extrasynaptic Corticostriatal Coculture pCREB Microfluidic isolation chamber Electrophysiology YAC128 Striatum

a b s t r a c t We recently reported evidence for disturbed synaptic versus extrasynaptic NMDAR transmission in the early pathogenesis of Huntington's disease (HD), a late-onset neurodegenerative disorder caused by CAG repeat expansion in the gene encoding huntingtin. Studies in glutamatergic cells indicate that synaptic NMDAR transmission increases phosphorylated cyclic-AMP response element binding protein (pCREB) levels and drives neuroprotective gene transcription, whereas extrasynaptic NMDAR activation reduces pCREB and promotes cell death. By generating striatal and cortical neuronal co-cultures to investigate the glutamatergic innervation of striatal neurons, we demonstrate that dichotomous synaptic and extrasynaptic NMDAR signaling also occurs in GABAergic striatal medium-sized spiny neurons (MSNs), which are acutely vulnerable in HD. Further, we show that wild-type (WT) and HD transgenic YAC128 MSNs co-cultured with cortical cells have similar levels of glutamatergic synapses, synaptic NMDAR currents and synaptic GluN2B and GluN2A subunit-containing NMDARs. However, NMDAR whole-cell, and especially extrasynaptic, current is elevated in YAC128 MSNs. Moreover, GluN2B subunit-containing NMDAR surface expression is markedly increased, irrespective of whether or not the co-cultured cortical cells express mutant huntingtin. The data suggest that MSN cell-autonomous increases in extrasynaptic NMDARs are driven by the HD mutation. Consistent with these results, we find that extrasynaptic NMDAR-induced pCREB reductions and apoptosis are also augmented in YAC128 MSNs. Moreover, both NMDAR-mediated apoptosis and CREB-off signaling are blocked by co-application of either memantine or the GluN2B subunit-selective antagonist ifenprodil in YAC128 MSNs. GluN2A-subunit-selective concentrations of the antagonist NVP-AAM077 did not reduce cell death in either genotype. Cortico-striatal co-cultures provide an in vitro model system in which to better investigate striatal neuronal dysfunction in disease than mono-cultured striatal cells. Results from the use of this system, which partially recapitulates the cortico-striatal circuit and is amenable to acute genetic and pharmacological manipulations, suggest that pathophysiological NMDAR signaling is an intrinsic frailty in HD MSNs that can be successfully targeted by pharmacological interventions. © 2012 Elsevier Inc. All rights reserved.

Introduction Huntington's disease (HD) is a terminal condition for which there is currently no cure. The disorder is characterized by late-onset motor dysfunction, dementia and the degeneration of striatal GABAergic ⁎ Corresponding author at: Department of Psychiatry, University of British Columbia, Vancouver, B.C., Canada. E-mail address: [email protected] (L.A. Raymond). 1 Equal contribution. 2 Present address: Department of Medical Genetics, University of British Columbia, Canada. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2012.05.013

medium-sized spiny neurons (MSNs) and, to a lesser extent, cortical neurons (Cowan and Raymond, 2006). HD is caused by a CAG repeat expansion in the gene encoding the protein huntingtin (htt); 36 CAG repeats or more lead to HD, and longer repeats are associated with earlier disease onset. Although HD is a severe neurodegenerative disease, many studies suggest that early cognitive deficits occur in patients years prior to cell death or overt neurological symptoms, probably due to synaptic and cellular dysfunction (Gladding and Raymond, 2011; Milnerwood and Raymond, 2010; Orth et al., 2010; Paulsen et al., 2008; Raymond et al., 2011; Schippling et al., 2009). Striatal injections of glutamatergic excitants (Beal et al., 1986; Coyle and Schwarcz, 1976; McGeer and McGeer, 1976) and global

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metabolic compromise (Beal et al., 1993) both result in degeneration of MSNs; the agents that produce the most specific MSN loss (Beal et al., 1990) are agonists of NMDA-type glutamate receptors (NMDARs). The NMDAR plays a critical role in neuronal transmission, signaling for synaptic plasticity, gene transcription, and survival; however, excessive (or inappropriate) calcium entry through NMDARs is excitotoxic (Greer and Greenberg, 2008; Hardingham and Bading, 2010). There are several explanations for the dichotomy of NMDAR signaling: low versus high levels of receptor activation, specific protein–protein interactions, NMDAR modulatory subunit (GluN2A–D) composition, and the subcellular location of the receptor (Greer and Greenberg, 2008; Hardingham and Bading, 2010; Kohr, 2006). Studies investigating the subcellular location of NMDAR activation have produced a compelling and well-characterized model in which synaptically-localized NMDARs signal cell survival, while those located extrasynaptically dominantly oppose synaptic activity and promote cell death (Hardingham and Bading, 2010). We recently found evidence for increased GluN2B-subunit containing extrasynaptic NMDARs in brain slices from YAC72 and YAC128 transgenic HD mice (Milnerwood et al., 2010). Furthermore, we and others also reported that early treatment with low-dose memantine, which is believed to preferentially reduce extrasynaptic NMDAR signaling in vivo (Chen and Lipton, 2006; Hardingham and Bading, 2010), prevents late-stage neurodegeneration (Okamoto et al., 2009) and alleviates early cognitive and synaptic signaling deficits (Milnerwood et al., 2010) in YAC128 HD mice. Primary striatal monocultures (predominantly GABAergic cells lacking glutamatergic input) prepared from YAC72 and YAC128 mice exhibit polyglutamine length-dependent increases in NMDAinduced toxicity (Shehadeh et al., 2006; Zeron et al., 2002, 2004). In YAC72 MSNs, increased toxicity was associated with increased whole-cell NMDAR current and surface GluN2B expression (Fan et al., 2007); however, YAC128 MSNs in monoculture exhibited augmented toxicity with no increase in NMDAR whole-cell current or surface GluN2B expression (Fernandes et al., 2007). The data suggest that a qualitative difference in NMDAR signaling must underlie increased toxicity (Fernandes et al., 2007). Primary neuronal culture models of disease are beneficial, as they are more amenable to rapid and flexible investigations of receptor signaling and trafficking than whole animals. In order to investigate synaptic and extrasynaptic NMDAR localization and signaling in GABAergic MSNs, and to further determine the effects of mutant huntingtin (mhtt) upon NMDAR regulation, we developed cocultures that partially recapitulate the cortico-striatal pathway in vitro, enabling the study of excitatory transmission onto GABAergic MSNs. Methods Transgenic mice and culture preparation Wild-type (WT) FVB/N mice and transgenic YAC128 (line 55) mice (Slow et al., 2003), expressing full-length human htt containing 128 CAG repeats on an FVB/N background, were maintained at the UBC Medicine Animal Resource Unit according to Canadian Council on Animal Care regulations. Cultures were prepared as previously described (Fan et al., 2009); briefly, striatal and cortical neurons were isolated from mouse pups at postnatal day 0, or else the fetuses of timedpregnant rat or mouse dames at E18 and E16.5, respectively. Brains were removed and placed on ice in Hank's Balanced Salt Solution (HBSS, GIBCO). For production of cortico-striatal co-cultures, isolated striatal cells, 80–90% of which exhibit morphological and immunocytochemical characteristics of MSNs (Shehadeh et al., 2006) were nucleofected, as in Kaufman et al. (2012), with enhanced yellow fluorescent protein (YFP): 3–5 million cells were suspended in 100 μl of electroporation buffer (Mirus Bio) with 1–5 μg of endonuclease-free DNA, placed

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in a cuvette and electroporated (AMAXA nucleofector I: program 05). Solution was removed from the cuvette and striatal cells were re-suspended in D minimum essential (DMEM, GIBCO) plus 10% fetal bovine serum (DMEM+) and plated at medium density (herein standard cultures) with non-transfected cortical cells at a ratio of 1:1. After 2–4 h, DMEM+ was replaced with 500 μl plating medium (PM, 2% B27, Invitrogen; penicillin/streptomycin; 2 mM α-glutamine; neurobasal medium, GIBCO). At div 4, 500 μl was added, then 50% of media exchanged every 3–5 days. DNA constructs for YFP on a β-actin promoter were a gift from A.M. Craig, University of British Columbia (originally from S. Kaech and G. Banker, Oregon Health Sciences University, Portland on a CAG promoter from J. Miyazaki (Niwa et al., 1991; Kaech and Banker, 2006)). The construct for YFP fused to the N-terminus of GluN2B (YFP-GluN2B) contained YFP followed by the linker LVPRGSRSR, inserted by sitedirected mutagenesis and PCR into rat GluN2B; this construct is expressed in a modified pLentiLox 3.7 vector on a synapsin promoter and was a gift from K. She and A.M. Craig, originally from M. Sheng, Genentech, San Francisco (Kim et al., 2005). YFP-GluN2A was developed by K. She and A.M. Craig from rat GluN2A (also a gift of M. Sheng, Genentech) by inserting the enhanced YFP from YFP-GluN2B, including the signal sequence and linker, in frame upstream of GluN2A alanine 24 and subcloned into pVIVO2 expression plasmid (InvivoGen). For some experiments cells were plated in microfluidic isolation chambers (Kaufman et al., 2012; Park et al., 2006) to physically separate MSNs from cortical cells while retaining their synaptic connectivity. Briefly, chambers were prepared as in Park et al. (2006) and cells were plated at a density of 4–5 million cells/ml; 10 μl was added to the top, and 5 μl to the bottom wells causing flux through the chamber. After 45 min at 37 °C to allow cells to adhere, 150 μl of PM was added to the top and bottom wells. Cells were maintained as above. Biotinylation of surface GluN2 receptors At div 14, cortico-striatal co-cultures from WT and YAC128 mice were subject to surface biotinylation prior to harvesting cells for western blot to determine the relative surface expression of GluN2A and GluN2B subunits, as in Fan et al. (2007). Briefly, co-cultures were precooled to 10 °C to halt biological processes, washed twice with 10 °C PBS and incubated with NHS-SS-Biotin (1.5 mg/ml in PBS, supplemented with 0.1 mM Ca2+, 1 mM Mg2+, Fishersci, PI-21331) to label surface proteins. Excess biotin was removed by two washes of 0.1% BSA in PBS then harvested. Cells were solubilized with 1% Triton X-100 (total soluble lysate), from which 20% was set aside as “lysate” sample and 80% was incubated with pre-equilibrated NeutrAvidin biotin-binding resin (Fishersci, PI-53150) for 2.5 h at 4 °C. The resin was then washed with solubilization buffer, and bound biotin-tagged proteins were eluted from resin by incubation with 3× protein sample buffer (30 min at 4 °C). Samples were then resolved by SDS-PAGE and subjected to immunoblotting. The biotinylation eluate fraction represents the portion of proteins present on the cell surface during the biotin-labeling step. Primary antibodies were: mouse anti-GluN2B (Fishersci, MA1-2014, 1:1000), rabbit anti-GluN2A (Upstate, Cat: 07‐ 632, 1:1000), and goat anti-Tubulin (Santa Cruz Biotechnology, sc9935, 1:1500). Electrophysiology Whole-cell patch-clamp recordings were performed on WT or YAC128 MSNs identified by YFP fluorescence in co-culture with cortical cells at div 13–14 as in Kaufman et al. (2012). Voltage clamp recordings were obtained at Vh −70 mV and uncompensated, and the membrane function was used to determine intrinsic membrane properties ~1 min after obtaining a whole-cell configuration, as described previously (Fernandes et al., 2007; Kaufman et al., 2012). Briefly, neurons were perfused at room temperature with (in mM unless stated):

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167 NaCl, 2.4 KCl, 10 μM MgCl2, 10 glucose, 10 HEPES, 2 CaCl2, pH 7.3, 300 mOsm. Tetrodotoxin (TTX, 0.3 μM), glycine (10 μM), and picrotoxin (PTX, 100 μM) were added before use. Intracellular recording solution contained (in mM): 130 Caesium methanesulphonate, 5 CsCl, 4 NaCl, 1 MgCl, 10 HEPES, 5 EGTA, 5 lidocaine, 0.5 GTP, 10 Naphosphocreatine, 5 MgATP, pH 7.2, 290 mOsm. Data were acquired using the Axopatch 200B amplifier running pClamp 10.2 software (Molecular Devices, Palo Alto, CA). Rapid application of 1 mM NMDA was achieved by pClamp (Axon Instruments) triggering of a pressurized perfusion system (Harvard Apparatus, Saint-Laurent, Quebec). NMDA was applied for 3 s and pulsed ≥five times with a 60 s inter-pulse interval. Whole-cell current density was calculated as peak NMDA current (pA)/cell capacitance (pF). Steady-state current density was measured as mean current amplitude over 200 ms at the end of NMDA application, divided by capacitance. Charge transfer density was calculated as the area under the curve (nA ms) normalized to capacitance. For augmentation of spontaneous synaptic bursting and blockade of synaptic NMDARs, cells were held at −80 mV in the presence of 4-aminopyridine (4-AP, 10 μM, Tocris) for 5 min of TTX wash-out and emergent burst generation, prior to addition of MK-801 (10 μM, Sigma) for a further 5 min; subsequently, extrasynaptic NMDAR currents were then assayed after wash-out of unbound MK-801, reapplication of TTX and whole-cell NMDA application as in Bengtson et al. (2008) and Kaufman et al. (2012). Data are presented as mean ± S.E.M. where n is cells from a minimum of 3 separate cultures. Drug treatments To stimulate synaptic NMDARs for nuclear signaling assays, neurons were treated with a synaptic stimulation cocktail containing: bicuculline (50 μM, Tocris), 4AP (2.5 mM, Tocris), nifedipine (5 μM, Tocris), glycine (10 μM, Sigma) and strychnine (2 μM, Tocris) mixed in conditioned media as in Hardingham et al. (2002) for 15 min. Control medium contained nifedipine, glycine and strychnine at the same concentrations. Following synaptic activation, the cocktail was replaced with conditioned media or conditioned media containing 15 μM NMDA +/− memantine (1 μM, Tocris) or ifenprodil (3 μM, Tocris), for a further 15 min. In some experiments the level of cAMP response element binding protein (CREB) activity, quantified by nuclear CREB phosphorylation (pCREB), was analyzed directly after this period, to determine the immediate effects of synaptic stimulation. For NMDA and rescue experiments a further 30-min (postNMDA treatment) incubation was conducted to determine the downstream effects of NMDAR activation. For toxicity assays, neurons were pre-treated for 1 h with conditioned media +/− memantine (3 or 30 μM), ifenprodil (3 μM) or NVP-AAM077 (0.1 or 0.4 μM) then with varying concentrations of NMDA (15 μM, 30 μM, 60 μM) +/−antagonists for 10 min. Drugs were removed and the cells were incubated for 6 h in conditioned media before fixation (in 4% PFA with 4% sucrose). Immunocytochemistry At div 13–16 cells were fixed in 4% paraformaldehyde (PFA) + 4% sucrose for 10 min before 3× rinse with phosphate buffered saline (PBS) then washed (5 min, PBS plus 0.3% triton X-100; Sigma, PBST) and blocked (30 min, 10% normal goat serum, NGS, in PBS). Primary antibodies were incubated overnight with agitation at 4 °C in PBST plus 2% NGS, incubated at RT for 1 h, then washed 3× with PBST before 1.5 h at RT with secondary antibodies (Alexa-488 and Alexa 568 conjugated α-mouse and α-rabbit, Molecular probes; antiGuinea pig AMCA, Jackson Laboratories). For live staining of surface YFP-GluN2B/GluN2A, cells were incubated for 10 min at 37 °C in conditioned media with an α-GFP primary (which recognizes eYFP) before being rinsed 2× with media, then

fixed in PFA-sucrose for 10 min, rinsed 3 times with PBS, and incubated with secondary antibody for 1.5 h. Live-cell incubation with antiGFP antibodies may cause artificial aggregation of diffuse YFPGluN2B (Mammen et al., 1997), but it allows amplification of the extrasynaptic receptor signal that may otherwise be too low to assess. Surface staining was confirmed by a clear outer membrane halo-like staining on the cell body, at all z-planes above the basal (glass attached) membrane up to the outer membrane at the apex of the soma; no clusters were observed inside of the somatic outer membrane. For labeling of internal YFP-GluN2B/GluN2A, YFP, PSD-95 and VGLUT1, cells were permeabilized with methanol for 5 min at −20 °C, rinsed 3 times with PBS, washed for 5 min with PBST, then treated with primary antibodies overnight, washed 3 times with PBST, and incubated with secondary antibodies as above. Controls for surface (green anti-GFP) and internal (red anti-GFP) GluN2B staining in non-transfected neurons showed no apparent fluorescent signal. Furthermore, following live-staining with anti-GFP and green secondary antibodies, when the second round of anti-GFP and red secondary antibodies were added with the permeabilization step omitted, only minimal red fluorescence was observed and there was no appreciable red punctate colocalization with surface GluN2B clusters. For nuclear staining, cells were treated with 5 μM Hoechst 33342 (Invitrogen) for 10 min and washed 3 times with PBST. Coverslips were slide mounted with fluoromount (SouthernBiotech). Primary antibodies included: α-phosphorylated cyclic-AMP response element binding protein, Serine-133 (pCREB Ser133, mouse, Millipore 05‐667, 1:750), chicken α-green fluorescent protein (GFP, chicken, AbCam ab13970, 1:1000), rabbit α-green fluorescent protein (GFP, rabbit, Synaptic Systems 132‐002, 1:500), α-post synaptic density protein 95 (PSD-95, mouse, Thermo Scientific, MA 1‐045, 1:1000) and α-vesicular glutamate transporter 1 (VGLUT1, guinea pig, Chemicon, AB 5905, 1:4000). Microscopy and image analysis Images were acquired using a Zeiss Axiovert 200 M fluorescence microscope. Nuclear pCREB Ser133 staining was imaged by acquisition of 0.4 μm z-stacks (3 centered on nuclei) at 63× magnification then flattened using the extended focus projection function (Axiovision 4.6). Exposure times were constrained and analyses were conducted on unprocessed (raw) images. Nuclear and cytoplasmic pCREB Ser133 fluorescence was quantified with ImageJ (NIH) as nuclear mean intensity and a mean of three regions of interest within the perinuclear cytoplasm. A nuclear/cytoplasmic ratio was calculated for each cell to control for background fluorescence and data expressed as mean ± S.E.M., where n is cells from a minimum of 4 separate cultures, with 10–15 cells per condition. For analysis of toxicity, the proportion of apoptotic eYFP-transfected neurons in each condition (200 cells assessed per condition) was scored based on dendritic and nuclear morphology and data are expressed as mean ± S.E.M. where n is culture (Fan et al., 2010). For co-localization and intensity analysis, images (63×, 8–15 z-stacks of 0.4 μm) were acquired and extended focus projections created from 4 to 5 slices containing all visible dendritic surface staining and the glassattached somatic membrane. For cluster analyses images were manually thresholded with experimenter blind to condition, and quantification was conducted in dendritic masks covering secondary dendrites. Colocalization was calculated using an ImageJ colocalization plugin (http://rsb.info.nih.gov/ij/plugins/colocalization.html) as in Kaufman et al. (2012) and Tapia et al. (2011); data are expressed as mean ±S.E.M., where n is cells with 5–10 cells from each of 3 separate cultures. For surface and internal GluN2B and GluN2A intensity measurements, raw images (fixed exposures and no post-processing) were used to measure mean gray intensity level in region of interest masks drawn around 3 (primary and secondary) dendrites, and 3 adjacent areas for background subtraction, to create a cell average dendritic intensity.

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Statistical analyses Statistical analyses were performed using Prism software (Graphpad, Inc.). Direct comparisons were made by Student's t-test (2tailed, herein t-test) and multiple comparisons by appropriate analyses of variance (ANOVA) and post-tests, as detailed in the text.

Results MSNs in co-culture with glutamatergic input Striatal MSNs were YFP-nucleofected immediately following isolation, prior to plating with non-transfected cortical cells at a ratio of 1:1. The long-lasting neuronal expression of the YFP construct enabled identification of striatal cells in medium density co-cultures at >2 weeks in vitro (Figs. 4A and 5A). In striatal monoculture, in which striatal cells are devoid of glutamatergic cells and resultant excitatory input (Shehadeh et al., 2006), cultured neurones show a

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decline in viability at ~div 11. Here, we found that growing striatal cells in the presence of glutamatergic input increases MSN viability, such that experiments can be routinely conducted at div 14–21.

Elevated surface GluN2B subunits in YAC128 cortico-striatal co-cultures In order to determine whether the presence of mhtt induces an alteration in NMDA receptor localization in vitro, similar to that observed in vivo (Milnerwood et al., 2010), we conducted surface biotinylation experiments in cortico-striatal co-cultures prepared from paired WT and YAC128 timed pregnancy mice (Fig. 1A). Despite a strong trend, increases in surface GluN2A localization were not significant (paired t-test; p = 0.22, n = 4 cultures). Contrastingly, the surface expression of GluN2B NMDAR subunits was significantly (paired t-test; p = 0.014) elevated in co-cultures from YAC128 mice, suggesting a robust increase in surface membrane GluN2B subunits in the presence of mhtt. The data are consistent with our previous observation of subtle elevations in GluN2A, concomitant with robust

Fig. 1. Increased surface NMDARs in YAC128 co-cultures and specifically augmented extrasynaptic current in YAC128 MSNs. A) Biochemical immunodetection of surface NMDAR subunits. A.i) Representative western blots showing increased GluN2A and GluN2B band density in biotinylated fractions of YAC128 vs WT cortico-striatal co-cultures (Lysate = total cell lysate, Biot. = biotinylated surface fraction, SN = supernatant). A.ii) Quantification of surface (biotinylated) GluN2 subunits relative to total cell lysates demonstrates that GluN2B surface levels are ~30% greater in YAC128 than WT cells. B Top) Representative traces of whole cell currents induced by fast application of 1 mM NMDA onto WT and YAC128 MSNs in co-culture with CTX cells at div 14. Bottom) Quantification of peak and steady state current density (left) and total charge transfer (right) reveals that NMDAR currents are greater in YAC128 than WT MSNs. C Top) Example traces of spontaneous glutamatergic synaptic bursts before and after application of the use-dependent, irreversible NMDAR antagonist MK-801. C Bottom) Quantification of NMDAR-sensitive component of synaptic transmission as average synaptic burst peak (left) and charge transfer (right) percent remaining after MK801-mediated synaptic NMDAR block. NMDAR component of the burst charge transfer is similar in WT and YAC128 MSNs. D Top) Example traces of normalized whole-cell NMDAR currents before (WC) and after (Exsyn. = extrasynaptic) the irreversible blockade of synaptic NMDARs by MK-801. D Bottom) Quantification demonstrates significantly elevated extrasynaptic NMDAR current peak and charge transfer in co-cultured YAC128 MSNs. *p b 0.05, **p b 0.01.

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increases in GluN2B, NMDAR subunits in extrasynaptic fractions of striatal and cortical tissue from YAC128 mice (Milnerwood et al., 2010). Increased whole-cell, but not synaptic, NMDA receptor currents In order to determine whether increased NMDAR surface expression was present in MSNs in these co-cultures, we conducted whole-cell patch clamp recordings of co-cultured MSNs identified by YFP expression. There were no significant differences in access resistance (Ra, 10.6±0.6 and 9.9±1.0 MΩ, n=19 and 18, WT and YAC128 respectively, p=0.6 by t-test), membrane resistance (Rm, 356.5±30.4 and 279.9±28.5 MΩ, WT and YAC128 respectively, p=0.08 by t-test) or whole cell capacitance (Cm, 59.1±4.9 and 64.2±5.9 pF, WT and YAC128 respectively, p=0.5 by t-test) values between WT and YAC128 MSNs. Whole-cell (WC) NMDAR current, carried by both synaptic and extrasynaptic receptors, was measured in response to fast application of NMDA (Fig. 1B). There was a 25% increase in NMDA-induced peak current density in YAC128 MSNs that did not reach significance, likely due to one outlier (67.7 ± 5.0 and 84.5 ± 7.7 pA/pF, n = 19 and 18, WT and YAC128 respectively, p = 0.07 by t-test, Fig. 1B); however, NMDAR-mediated steady state current density was significantly increased by 47% (ss, 29.1 ± 2.5 and 42.8 ± 3.8 pA/pF, n = 19 and 18, WT and YAC128 respectively, p = 0.004 by t-test, Fig. 1B) and total charge transfer by 37% (C, 99.8 ± 8.0 and 136.4 ± 10.9 nA*ms/pF, n = 19 and 16, WT and YAC128 respectively, p = 0.01 by t-test, Fig. 1B) in YAC128 MSNs. There were no significant differences in the steady-state to peak current (Iss/Ipeak) ratio between WT and YAC128 MSNs (not shown). In hippocampal and cortical primary neuronal cultures, cells form excitatory synaptic networks, in which the addition of GABAA receptor antagonists produces disinhibition and glutamatergic synaptic burst firing, by removing the inhibitory activity of GABAergic cells in the culture (Hardingham et al., 2002). By including cortical cells in the co-culture system, we were able to use network disinhibition to study glutamatergic synaptic activity onto MSNs; in addition, we included 4-AP (a K + channel blocker) to increase presynaptic transmitter release (Hardingham et al., 2002; Milnerwood et al., 2010). In low Mg 2+ ACSF, synaptic bursts (silenced by TTX or AMPA-type glutamate antagonists; data not shown) were readily observed in MSNs. The slow components of synaptic bursts were rapidly eliminated by the addition of the NMDAR antagonist MK-801 (10 μM), whereas peak burst amplitudes were largely unaffected (Fig. 1C). Quantification of NMDAR-mediated synaptic charge transfer (% initial charge blocked by MK-801) in burst events revealed no difference between WT and YAC128 MSNs (69.17 ± 3.9 and 72.1 ± 4.9%, n = 6 and 6, WT and YAC128 respectively, p = 0.7 by t-test, Fig. 1C). Increased NMDAR current suggests that the number of surface NMDARs are increased in co-cultured YAC128 MSNs, similar to observations in YAC128 and YAC72 mouse striatum (Milnerwood et al., 2010) and YAC72 striatal monocultures (Fan et al., 2007). Moreover, also in agreement with observations in slices from WT and YAC18/ 72/128 mice (Milnerwood et al., 2010), similar NMDAR components of synaptic burst charge transfer in WT and YAC128 MSNs in vitro suggests that synaptic NMDAR activity is similar, regardless of mhtt expression. Following the irreversible blockade of synaptic NMDARs, we then re-assayed the remaining WC current, carried by extrasynaptic NMDARs. Both extrasynaptic peak and charge were significantly (t-test; p = 0.04 and 0.02, n = 7 and 8 for WT and YAC128, respectively) greater in co-cultured MSNs from YAC128 mice (Fig. 1D). Synaptogenesis and NMDA receptor localization To further investigate excitatory synapse formation and NMDAR surface expression levels, we conducted immunofluorescence imaging of striatal cells nucleofected with constructs to drive the expression of

YFP-fused GluN2B and GluN2A subunits. YFP-GluN2 subunits must assemble with endogenous GluN1/2 subunits to form functional receptor complexes and become trafficked to the surface membrane and reside at synaptic and extrasynaptic sites. At 14–16 div we performed a live stain to specifically label surface NMDAR receptors containing YFP-GluN2B or -GluN2A subunits, prior to permeabilization and immunostaining for the presynaptic vesicular glutamate transporter 1 (VGLUT1) and internal YFP-GluN2B or YFPGluN2A-containing NMDARs in MSNs grown in co-cultures (Fig. 2A). WT and YAC128 MSNs had similar numbers of both surface GluN2B and GluN2A clusters (Fig. 2B). Furthermore, excitatory synapses, identified by presynaptic VGLUT1 clusters on MSNs from both genotypes, were of similar densities in WT and YAC128 MSNs, regardless of whether MSNs were expressing YFP-GluN2B or -GluN2A (GluN2B WT n = 35 and YAC128 n = 49 cells from 6 independent cultures, GluN2A WT n = 13 and YAC128 n = 17 from 2 independent cultures, p > 0.05 Bonferroni post test Fig. 2B.i), suggesting that glutamatergic synapse formation and GluN2B- or GluN2A-containing surface NMDAR cluster numbers are equivalent between WT and YAC128 MSNs. The colocalization of VGLUT1 and GluN2B- or GluN2A-containing surface NMDARs was also similar between WT and YAC128 MSNs (Fig. 2B.ii), suggesting that synaptic incorporation of GluN2B- or GluN2Acontaining NMDARs is also not significantly altered by mhtt expression. Despite comparable synaptic clustering, investigation of GluN2B surface expression, by quantification of surface to internal YFP staining ratios, revealed robust and significantly increased surface expression of GluN2B in YAC128 MSNs (Fig. 2B.iii, WT 0.87 ± 0.05 and YAC128 1.74 ± 0.13, n = 46 and 39 from 6 cultures for WT and YAC128 respectively, p b 0.0001 by t-test). Contrastingly, despite a modest trend, there were no significant differences in GluN2A surface to internal ratios (Fig. 2B.iii, WT 0.95 ± 0.22 and YAC128 1.33 ± 0.23, n = 13 and 17 from 2 cultures, p = 0.25 by t-test). The data are supportive of the increased extrasynaptic NMDAR localization suggested by both the biotinylation and electrophysiological data (Fig. 1). Taken together, the data highlight a shift toward increased extrasynaptic GluN2Bcontaining NMDAR localization in the presence of mhtt, and suggest that less pronounced alterations in GluN2A subunits may also occur. To further investigate synapse density and the synaptic incorporation of GluN2B, and to determine whether alterations in surface expression are cell autonomous (i.e., driven by mhtt expression specifically within MSNs), we plated YFP-GluN2B expressing MSNs from paired WT and YAC128 timed pregnancy mice with cortical cells from the same animals, and separately produced chimeric cultures composed of YAC128 MSNs plated with cortical cells from the WT mice (Fig. 3A). Similar to the unpaired cultures (Fig. 2), there were no significant differences between WT and YAC128 MSNs in surface GluN2B cluster densities, VGLUT1 densities or colocalization of VGLUT1 with GluN2B; moreover there were also no significant differences in these measures for chimeric cultures of YAC128 MSNs and WT cortical cells (Fig. 3A.i and ii). As expected, surface to internal GluN2B ratios were markedly increased in MSNs in YAC128 co-cultures, and this increase was also observed in chimeric cultures (Fig. 3A.iii, WT 0.9± 0.1, YAC128 1.56 ± 0.2, Chimeric 1.36 ± 0.1, ANOVA p = 0.015 F2,70 = 4.5, WT v 128 and WT v Chimeric p b 0.05 by Newman–Keuls post test). The data demonstrate that increased extrasynaptic expression of GluN2B-containing NMDARs occurs in mhtt expressing MSNs regardless of whether or not network-associated cortical cells also express mhtt. As the data suggest that synapse densities are unaltered by mhtt expression in this system, and that MSN-specific alterations in NMDAR trafficking occur irrespective of the genotype of the cortical inputs, we plated GluN2B-expressing MSNs into microfluidic isolation platforms (Kaufman et al., 2012; Park et al., 2006) with glutamatergic input from non-transgenic rat cortical cells. In this way we were able to immunostain in relatively low culture densities desirable for triple colocalization analysis, and further test whether synaptic development and NMDAR localization are altered by mhtt expression specifically

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Fig. 2. Equivalent synapse number and synaptic NMDAR clusters, yet markedly increased surface GluN2B expression in co-cultured YAC128 MSNs. A) Representative examples of non-thresholded images of YFP-GluN2B transfected WT (top left) and YAC128 (lower left) MSNs co-cultured with cortical cells from the same animal. Cells were live-stained for GluN2B-containing surface NMDARs (green) prior to permeabilization and staining for VGLUT1 (blue) and internal GluN2B (red). A.ii) Higher magnification inserts (right) show surface and internal GluN2B staining from the areas indicated in the larger panels, and GluN2A staining from sister cultures. B) Quantification of thresholded (not shown, see Fig. 3 for comparison) dendritic cluster density (i) and colocalization (ii) demonstrates that VGLUT1 terminal number and colocalization with surface GluN2B (synapse formation and synaptic GluN2B incorporation) are similar in WT and YAC128 MSNs, similar to that seen in GluN2A expressing MSNs. B.iii). Significantly elevated surface to internal GluN2B raw signal intensity reveals increased surface localization of GluN2B — but not GluN2A-containing NMDARs in co-cultured YAC128 MSNs. ***p b 0.001.

within MSNs. At 14–16 div, live staining to label YFP-GluN2B subunitcontaining surface NMDA receptors, was conducted with staining for VGLUT1 and the postsynaptic scaffold protein, PSD-95 (Fig. 3B). Similar to MSNs co-cultured with cortical cells of the same genotype and chimeric cultures (Figs. 2 and 3A), there were no differences between WT and YAC128 MSNs in the mean density of surface GluN2Bcontaining NMDAR clusters, VGLUT1 clusters or PSD-95 clusters (Fig. 3B.ii. WT n = 23, YAC128 n = 32 ANOVA, p > 0.05 Bonferroni post test). Colocalization analysis similarly demonstrated no significant differences between WT and YAC128 MSNs in glutamatergic synapse formation (VGLUT1 + PSD-95), GluN2B-subunit colocalization with presynaptic terminals (VGLUT1 + GluN2B) or PSD-95 containing synapses (VGLUT1 + PSD-95 + GluN2B, Fig. 3B.iii ANOVA, p > 0.05 Bonferroni post test). VGLUT1 cluster density and GluN2B+ VGLUT1 colocalization were very similar, regardless of the culture conditions or genotype of excitatory cortical cells, further suggesting that GluN2B-containing clusters, synapse formation/maintenance and the incorporation of GluN2B into PSD-95 containing synapses are similar in WT and YAC128 MSNs, and are unaffected by the presence or not of mhtt in innervating cortical cells. Curiously, we also observed numerous somatic GluN2B clusters in MSNs and ~50% of these were associated with VGLUT1, suggesting somatic excitatory synapses, similar to our previous report in rat cultures (Kaufman et al., 2012). If these represent functional synapses, somatic GluN2B-mediated calcium signaling would occur near cellular

transcription machinery. However, this is likely a developmental/cell culture-specific phenomenon, as somatic excitatory synapses are not observed in adult striatum (Smith and Bolam, 1990). In agreement with the surface to internal alterations observed in standard cultures, raw image (prior to thresholding for cluster analysis) surface GluN2B intensity was increased in both dendrites and cell bodies of YAC128 MSNs (WT n = 21, YAC128 n = 31, p = 0.03 and WT n = 21, YAC128 n = 30, p = 0.04 by t-test for dendrites and soma respectively, Fig. 3B.iv), although significance was achieved only after exclusion of two WT and one YAC128 statistical outliers (>2 × s.d. from the mean). The data support the previous results and further suggest that relative surface levels of GluN2B-containing NMDARs are increased in YAC128 MSNs, even when co-cultured with non-transgenic rat cortical cells.

NMDA receptor signaling to pCREB and augmented extrasynaptic activity Signals mediated by synaptic and extrasynaptic NMDARs converge upon nuclear CREB activity by differential regulation of CREB phosphorylation at the serine 133 residue. Phosphorylated CREB (pCREB) levels are increased by synaptic NMDAR-mediated Ca2+ influx, and this process is dominantly opposed by extrasynaptic NMDAR activity. Within the nucleus pCREB drives plasticity, pro-survival and anti-apoptotic

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Fig. 3. Cell-autonomous increases in surface expression of GluN2B in YAC128 MSNs. A) Comparison of paired WT, YAC128 and chimeric cultures (YAC128 MSNs co-cultured with WT cortical cells) expressing YFP-GluN2B (stained as in Fig. 2) reveals equivalent surface GluN2B and VGLUT1 cluster density (i) and colocalization (ii) in all three co-culture combinations. A.iii) Surface to internal GluN2B ratios are significantly greater in both YAC128 and chimeric culture MSNs relative to WT. B) Representative immunofluorescence imaging of YFP-GluN2B expressing WT (top two panels) and YAC128 (lower two panels) MSNs grown in microfluidic isolation chambers with glutamatergic input from rat cortical cells (i). Higher magnification inserts show the result of thresholding for synaptic cluster analysis (zoom of areas indicated in larger panel). Excitatory presynaptic (VGLUT1, blue) and postsynaptic (PSD-95, red) markers reveal clear punctate immunofluorescence, distributed along dendrites and regularly co-localized (open arrow heads) to indicate the presence of closely associated pre- and postsynaptic structures (excitatory synapses). Surface GluN2B clusters (green) are visible on the outer somatic membrane and throughout dendrites. While many GluN2B clusters are not associated with VGLUT1 or PSD95 (filled stars), ~20% of VGLUT1clusters are colocalized with GluN2B (open stars), as are ~25% of synapses (as defined by both VGLUT1 and PSD-95 label, filled arrow heads). Quantification of dendritic cluster density (ii) and colocalization (iii) demonstrates that surface GluN2B, PSD-95 and VGLUT1 cluster densities and co-localization are equivalent in WT and YAC128 MSNs. The data suggest that synapse formation, maintenance and incorporation of GluN2B NMDAR subunits are unaltered by mhtt in MSNs. B.iv). Analysis of surface GluN2B intensity in unprocessed, non-thresholded, images reveals significantly increased signal on both the dendrites and soma of YAC128 MSNs, suggesting increased surface expression of GluN2B-containing NMDARs, despite non-transgenic glutamatergic input and similar cluster density. *p b 0.05.

gene transcription (Greer and Greenberg, 2008; Hardingham and Bading, 2010; Milnerwood and Raymond, 2010). We used a potent network disinhibition cocktail to increase glutamatergic synaptic transmission onto MSNs and promote nuclear pCREB (Fig. 4). We found that basal pCREB levels were quite variable from culture to culture, with a 1–2 fold nuclear to cytoplasmic signal; however, 15 min after a 15 minute treatment with the synaptic activation cocktail, nuclear pCREB levels were consistently high in both WT (2.1 ± 0.07 fold n = 54) and YAC128 MSNs (2.0 ± 0.1 fold n = 78) and this activation was significantly blocked by application of the NMDAR antagonist DAP5 (25 μM: WT 1.2 ± 0.07 fold n = 30, p b 0.0001; YAC128 1.3 ± 0.08

fold n = 52, p b 0.0001 by t-test). After synaptic stimulation of NMDARs, we tested the effects of extrasynaptic NMDAR activity upon pCREB by bath application of NMDA, as used elsewhere (Hardingham et al., 2002; Ivanov et al., 2006; Leveille et al., 2008, 2010; Papadia et al., 2008), with or without 1 μM memantine or 3 μM of the GluN2B subunit-selective antagonist ifenprodil. NMDA (15 μM) significantly reduced nuclear pCREB in WT MSNs (Fig. 4B.i) relative to both synaptic activation (Bicuculline then media, Kruskal–Wallis (KW)-ANOVA p b 0.0001, KW = 30.68, Bic– Media n = 128 vs Bic–NMDA n = 134 p b 0.001 by Bonferroni post test) and synaptic activation followed by memantine (Bicuculline–

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Fig. 4. Synaptic and extrasynaptic NMDAR signaling to nuclear pCREB. A) Representative examples of YFP-nucleofected co-cultured MSNs (green) from YAC128 mice at 14 div; without treatment (media, left), after synaptic activation cocktail (Bicuculline, middle), and with synaptic activation followed by bath NMDA to stimulate all NMDARs (Bicuculline then NMDA, right). Cells were stained for cell nuclei (Hoescht, blue) and pCREB (pCREBSer133, red). Inserts show nuclear (top, blue) and pCREB (middle, red) staining (bottom, merged image). Under basal conditions (left) pCREB staining is diffuse throughout the cytoplasm and slightly increased within the cell nucleus. Synaptic activation results in increased nuclear pCREB signal (middle) whereas subsequent NMDA treatment reduces nuclear pCREB (right). B) Quantification of nuclear/cytoplasmic pCREB intensity in WT (i), YAC128 (ii) and summary data for comparison (iii). Nuclear pCREB signal following synaptic activation (Bic–Media) is not significantly altered by successive memantine application (1 μM) in either WT or YAC128 MSNs. The subsequent activation of both synaptic and extrasynaptic NMDARs by NMDA bath application (Bic–NMDA) results in a reversal or shutoff of nuclear pCREB signaling in both WT and YAC128 MSNs. The presence of memantine significantly reduced NMDA-induced pCREB shut-off in YAC128, but not WT MSNs. Summarized pCREB intensity (iii, relative to synaptic activation followed by memantine) reveals that NMDA significantly reduces nuclear pCREB in both WT and YAC128 MSNs; an effect that is statistically much more pronounced in YAC128 MSNs. Memantine co-application with NMDA does not significantly reduce pCREB shut-off in WT MSNs, although in this condition pCREB levels are no longer significantly different from controls. C) Quantification of nuclear/cytoplasmic pCREB intensity (as in B) in experiments testing the GluN2B-subunit selective antagonist ifenprodil (3 μM). The presence of ifenprodil had no significant effects, and in this data subset extrasynaptic NMDAR activation did not significantly reduce pCREB (i) in WT MSNs. Ifenprodil also had no reductive effect upon synaptic pCREB activation in YAC128 MSNs (ii), whereas NMDA induced a robust pCREB shut-off, which was significantly greater than the same treatment of WT cells, and significantly reduced by ifenprodil co-application (ii and iii) to the extent that NMDA treatment + ifenprodil was not significantly different from controls (iii). *p b 0.05, **p b 0.01, ***p b 0.001 by ANOVA; #p b 0.05, ###p b 0.001 by 1 sample t-test.

memantine; Bic–Mem n=88 vs Bic–NMDA n=134 pb 0.001 by Bonferroni post test). The presence of memantine produced a small alleviation of pCREB shut-off that was not significantly different from NMDA treatment in WT MSNs; although the remaining pCREB was no longer significantly less than synaptic activation in the presence of memantine. NMDA treatment of YAC128 MSNs (Fig. 4B.ii) produced a highly significant reduction in nuclear pCREB relative to both synaptic activation (Bicuculline then media, KW-ANOVA pb 0.0001, KW=118.6, Bic–Media n=172 vs Bic–NMDA n=175 pb 0.001) and synaptic activation followed by memantine (Bicuculline–memantine; Bic–Mem n=100 vs Bic–NMDA n=175 pb 0.001 by Bonferroni post test). The presence of memantine produced a significant alleviation of NMDA-induced pCREB shut-off in YAC128 MSNs (Bic–NMDA+Mem n=113 pb 0.01 by Bonferroni post test). Normalized summary data (Fig. 4B.iii) demonstrate that NMDA treatment significantly reduced nuclear pCREB in both WT and YAC128 MSNs, and that the reduction was significantly greater in MSNs from YAC128 mice (KW-ANOVA p b 0.0001, KW = 29.15, p b 0.01 by Bonferroni post test). Memantine co-application prevented a significant reduction in WT nuclear pCREB by NMDA treatment, and significantly reduced pCREB shut-off in YAC128 MSNs.

We conducted separate experiments to investigate the potential of ifenprodil to ameliorate extrasynaptic NMDAR activity (Fig. 4C). Ifenprodil had no significant effect upon WT pCREB following synaptic activation (Fig. 4C.i, n=43 and 44 for Bic–Med and Bic–Ifen, respectively), and NMDA also failed to significantly reduce pCREB in this smaller data set (n=45). Co-application of ifenprodil with NMDA had no reductive or additive effect in WT MSNs (n=45). Ifenprodil also had no reductive effect upon synaptic pCREB signaling in YAC128 MSNs (Fig. 4C.ii, n=56 and 60 for Bic–Med and Bic–Ifen, respectively), but significantly ameliorated the clear reductive effects of extrasynaptic activity in YAC128 MSNs (KW-ANOVA pb 0.0001, KW=21.2, Bic–NMDA n=60 vs Bic– NMDA+Ifen, n=58 pb 0.05 by Bonferroni post test), to the extent that no significant reduction in nuclear pCREB was observed (Fig. 4C.iii). Together the data demonstrate that subtle reductions in WT co-cultured MSN pCREB levels, driven by extrasynaptic GluN2B-containing NMDAR activity, are pronounced in YAC128 MSNs. Elevated NMDA-induced toxicity Given the evidence for augmented extrasynaptic NMDAR current, localization and signaling in YAC128s, one would predict that sensitivity

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to NMDAR-mediated excitotoxicity would also be greater (Fig. 5). The concentration of NMDA used to induce pCREB shut-off (15 μM) did not induce significant apoptosis in either WT (0NMDA n = 22, 15NMDA n = 3) or YAC128 (0NMDA n = 23, 15NMDA n = 4) MSNs 6 h post-treatment (Fig. 5B). There was no significant difference in toxicity at 30 μM NMDA between genotypes (WT n = 23, YAC128 n = 21). However, robust apoptosis was observed in both WT and YAC128 MSNs at NMDA concentrations of 50 μM (WT n = 8, YAC128 n = 11) and 60 μM (WT n = 19, YAC128 n = 18), and YAC128 MSNs were significantly more sensitive to NMDA at these concentrations (25 and 20% respectively, ANOVA p b 0.0001, F9,142 = 130.4, 50 μM p b 0.05, 60 μM p b 0.01 by Bonferroni post test). We next tested the potential for pharmacological protection of cocultured MSNs from NMDA-mediated toxicity (Figs. 5C and D) by the uncompetitive NMDAR antagonist memantine (3 and 30 μM), the GluN2B-subunit preferring antagonist ifenprodil (3 μM), and selective and non-selective concentrations of the GluN2A-subunit preferring antagonist NVP-AAM077 (0.1 and 0.4 μM, respectively). Ifenprodil and both concentrations of memantine protected WT and YAC128 MSNs from cell death at both low and high NMDA concentrations, whereas NVP-AAM077 did not protect MSNs from toxicity at a concentration (0.1 μM) selective for GluN2A NMDARs (Figs. 5C and D. WT n = 20, 7, 5, 7, 5, 5, 16, 7, 5, 7, 5, 5 for N30, N30 + 3Mem, N30 +

30Mem, N30 + 3Ifen, N30 + 0.1NVP, N30 + 0.4NVP, N60, N60 + 3Mem, N60 + 30Mem, N60 + 3Ifen, N60 + 0.1NVP, N60 + 0.4NVP, respectively; ANOVA p b 0.0001, F11,82 = 39.65, *p b 0.05, **p b 0.01, ***p b 0.001 by Bonferroni post test. YAC128 n = 21, 6, 5, 6, 5, 5, 18, 6, 5, 6, 5, 5; ANOVA p b 0.0001, F11,92 = 120.9, *p b 0.05, ***p b 0.001 by Bonferroni post test). Discussion Here we demonstrate that co-culture of striatal MSNs and glutamatergic cortical cells provides a useful model for investigating the effects of mhtt upon synaptic and extrasynaptic NMDAR signaling. The partial recapitulation of the cortico-striatal pathway in vitro supports and extends recent results from acute brain slices and striatal monocultures (Fan et al., 2007; Fernandes et al., 2007; Graham et al., 2009; Milnerwood and Raymond, 2007; Milnerwood et al., 2006, 2010; Shehadeh et al., 2006; Zeron et al., 2002; Zeron et al., 2004; Zhang et al., 2008). Although synaptic NMDAR expression and signaling was equivalent between genotypes in this study, whole-cell and isolated extrasynaptic NMDAR currents were ~35% increased in YAC128 MSNs, as were GluN2B subunit-containing NMDAR surface levels. In general concordance with these results, we found that extrasynaptic NMDAR signaling was twice as effective (in terms of

Fig. 5. NMDA-induced toxicity is prevented by memantine and ifenprodil. A) Representative examples of WT and YAC128 YFP-nucleofected MSNs grown in co-culture with CTX cells from the same animal at div 14–15, stained to amplify transgene-expressed YFP (green) and cell nuclei (Hoescht, blue). Neurons were fixed 6 h after 10 min of control media change (no NMDA, N0) or a toxic dose of bath applied NMDA (60 μM NMDA, N60) with and without memantine (3 μM Memantine, N60 + Mem) or ifenprodil (3 μM ifenprodil, N60 + 3Ifen). Inserts show nuclei of MSNs, and nuclear morphology used for scoring of apoptosis; healthy nuclei are rounded and contain discrete areas of bright staining (open stars), whereas apoptotic nuclei are much smaller, brighter and condensed (closed stars). B) Quantification of NMDA-induced apoptosis by nuclear morphology. C and D) Quantification of NMDA-induced apoptosis and rescue by memantine (3 μM and 30 μM), ifenprodil (3 μM) and NVP-AAM077 (0.1 and 0.4 μM) in co-cultured MSNs in WT (C) and YAC128 (D) co-cultures.

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CREB shut-off) and NMDA-induced apoptosis was approximately 25% higher in co-cultured YAC128 relative to WT MSNs. This is in high concordance with our earlier results showing increased extrasynaptic NMDAR activity and GluN2B localization, and also reductions in pCREB, in striatal tissue from 1- to 2-month-old YAC128 mice (Milnerwood et al., 2010). Increased extrasynaptic NMDAR currents appear here to be predominantly carried by increases in GluN2B-containing NMDARs, as alterations to GluN2A were strongly trending, but not significant. It is likely that there are indeed alterations to GluN2A trafficking as a consequence of mhtt expression, but they were more variable and below detection here. In support of this we previously observed increases in GluN2A subunits in extrasynaptic fractions of striatal tissue from YAC128 mice, but they were less robust than those seen for GluN2B subunits (Milnerwood et al., 2010). Furthermore, in YAC128 striatal slices the GluN2B selective antagonist ifenprodil eliminated the differences in extrasynaptic currents (Milnerwood et al., 2010), while in the current study similar concentrations of ifenprodil reversed pCREB shut-off and reduced cell death by ~80%. Here, a concentration of the GluN2A-preferring antagonist NVP-AAM077 demonstrated to specifically block 90% of GluN2A current (0.1 μM) offered no protection from NMDAinduced apoptosis, whereas a higher concentration (0.4 μM), which also blocks ~70% of GluN2B (Berberich et al., 2005), offered moderate protection. Taken together the data suggest that although less pronounced alterations to GluN2A subunits likely occur, pharmacological targeting of GluN2B is sufficient to prevent toxic extrasynaptic signaling. It is possible that the GluN2 subunit type, rather than location, is the critical determinant for deleterious signaling i.e., that GluN2B subunits are pro-death and GluN2A are pro-survival, regardless of synaptic or extrasynaptic localisation. This is supported by the lack of significant reductions in pro-survival synaptic NMDAR pCREB activation in the presence of ifenprodil, and the lack of effect of GluN2A antagonism (despite some extrasynaptic localization) upon toxicity. Alternatively, observed synaptic GluN2B subunits may reside within GluN2A/GluN2B heterotetramers which are much less sensitive to ifenprodil (Hatton and Paoletti, 2005); in concordance with this suggestion, synaptic responses generated by stimulation in slices were only ~15% reduced by ifenprodil (Milnerwood et al., 2010). In acute slices from presymptomatic mice, synaptic NMDAR transmission was similar between WT, YAC18, YAC72 and YAC128 MSNs (Milnerwood et al., 2010); consistent with those findings, we have shown that the percent of excitatory synaptic current blocked by MK-801 (reflecting the proportion carried by synaptic NMDARs) is similar in WT and YAC128 co-cultured MSNs, as previously demonstrated in non-transfected vs. mhtt-transfected cortical cells in another recent report (Okamoto et al., 2009). In agreement with these findings, comparable densities of VGLUT1 presynaptic terminals and synapse-associated GluN2B subunits were observed in WT and YAC128 MSNs, regardless of the presence or not of mhtt in the associated glutamatergic cells. Together these data suggest that excitatory synapse formation, synaptic NMDAR transmission and synaptic GluN2B subunit localization are not primarily disturbed by the Huntington's disease mutation, although alterations in both GABA and AMPA receptor trafficking to/from synapses have been observed by others (Mandal et al., 2011; Twelvetrees et al., 2010). At 10 days in vitro, monocultured striatal MSNs (lacking glutamatergic synapses) from YAC72 and YAC128 MSNs exhibit 10 and 25% apoptosis 24 h after 100 μM NMDA, respectively (Shehadeh et al., 2006). At 500 μM NMDA, a maximal level of apoptosis is achieved in both genotypes of ~35%, compared with ~20% for WT MSNs (Shehadeh et al., 2006). Elevated toxicity in YAC72 vs.WT MSNs is associated with a ~30–40% increase in NMDAR surface levels and NMDA peak current (Fan et al., 2007); however, despite the fact that monocultured YAC128 MSNs are more sensitive than YAC72 MSNs to NMDA-induced apoptosis (Shehadeh et al., 2006), we have reported previously that they do not exhibit elevated NMDAR whole-cell

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currents (Fernandes et al., 2007), although another study has shown these currents were indeed increased in YAC128 mono-cultured MSNs (Zhang et al., 2008). In contrast to our earlier study in monocultured MSNs (Fernandes et al., 2007), we found increased NMDA-evoked whole-cell current and NMDAR surface levels in YAC128 MSNs when co-cultured with cortical cells for ≥2 weeks. Thus, growing MSNs with cortical input not only increases viability, likely due to the fact that excitatory synaptic signaling positively modulates neuronal survival (Hardingham et al., 2002; Ivanov et al., 2006; Leveille et al., 2008, 2010), but also reveals differences between WT and YAC128 MSNs that might have been missed in immature and less viable cultures at div 10. Although all NMDARs in div 10 monocultured striatal cells are in some way extrasynaptic, due to a lack of excitatory synaptic input, the predicted pattern of synaptic and extrasynaptic signaling may not be apparent; dominant extrasynaptic CREB-off signaling is not observed in young neurons (Hardingham and Bading, 2002). In support of this, the concentration of bath-applied NMDA required to induce MSN death in mature co-cultures is much lower than in div 10 monocultures, whereas the maximal apoptotic response is much greater. However, it is also likely that toxic effects of glutamate release from (and/or release of deleterious factors resulting from cell death of) cortical glutamatergic cells within the culture would contribute to elevated cell death in co-cultures. Increased NMDAR transmission has now been associated with the HD mutation in many reports (Cepeda et al., 2001; Chen et al., 1999; Fan et al., 2007, 2009; Fernandes et al., 2007; Heng et al., 2009; Levine et al., 1999; Li et al., 2004; Milnerwood and Raymond, 2007; Milnerwood et al., 2006, 2010; Okamoto et al., 2009; Starling et al., 2005), and there is evidence to suggest that the deleterious effects of disturbed NMDAR signaling may be an early event in the pathology of HD (Fan and Raymond, 2007; Gladding and Raymond, 2011; Milnerwood and Raymond, 2010; Raymond et al., 2011). Three major downstream effects of elevated extrasynaptic NMDAR transmission are reductions in BDNF, PGC1α, and pCREB (Greer and Greenberg, 2008; Hardingham and Bading, 2010; Milnerwood and Raymond, 2010; Okamoto et al., 2009), all key regulators of signal transduction leading to plasticity, pro-survival and anti-apoptotic gene transcription. Therefore, prolonged extrasynaptic NMDAR antagonism of such prosurvival signaling in HD might be predicted to have catastrophic longterm consequences, resulting in latent neurodegeneration. In agreement with literature in cortical and hippocampal neurons, we have shown that memantine (Gouix et al., 2009; Leveille et al., 2008; Papadia et al., 2008) and ifenprodil (Hardingham et al., 2002; Liu et al., 2007) reduce NMDA-induced cell death, and ameliorate extrasynaptic NMDAR-induced pCREB down-regulation in striatal MSNs from HD mice. Furthermore, this study highlights the importance of studying cultured GABAergic cells with appropriate sources of excitatory input. We are developing an improved understanding of deleterious NMDAR transmission in HD, and the data suggest that therapeutic strategies aimed at reducing the influence of extrasynaptic NMDAR transmission will prove beneficial for HD and other neurodegenerative disorders. Acknowledgments We would like to thank: Professor Ann Marie Craig, Mr. Kevin She for constructs and advice, Dr. Matt Parsons for contributions to transfected culture experiments, Dr. Jacqueline Shehadeh for contribution to analysis and Dr. Karolina Kolodziejczyk for comments on the manuscript. Funding for LAR was provided by the Cure Huntington's Disease Initiative and the CIHR: (MOP-12699) and emerging team grant (GPG-102165). AJM received fellowships from CIHR, Huntington's Society of Canada and the Huntington's Disease Society of America; CMG holds a CIHR-Huntington Society of Canada fellowship and a Hereditary Disease Foundation Scholarship.

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