Suppressing aberrant GluN3A expression rescues synaptic and behavioral impairments in Huntington's disease models

Huntington's disease is a neurodegenerative disorder associated with glutamate receptor dysfunction. Now Isabel Pérez-Otaño and colleagues report that the HTT protein that aggregates in the brains of individuals with the disease disrupts the ability of the adaptor protein PACSIN1 to keep the glutamate receptor subunit GluN3A away from the surface of neurons. Huntington's disease is caused by an expanded polyglutamine repeat in the huntingtin protein (HTT), but the pathophysiological sequence of events that trigger synaptic failure and neuronal loss are not fully understood. Alterations in N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) have been implicated. Yet, it remains unclear how the HTT mutation affects NMDAR function, and direct evidence for a causative role is missing. Here we show that mutant HTT redirects an intracellular store of juvenile NMDARs containing GluN3A subunits to the surface of striatal neurons by sequestering and disrupting the subcellular localization of the endocytic adaptor PACSIN1, which is specific for GluN3A. Overexpressing GluN3A in wild-type mouse striatum mimicked the synapse loss observed in Huntington's disease mouse models, whereas genetic deletion of GluN3A prevented synapse degeneration, ameliorated motor and cognitive decline and reduced striatal atrophy and neuronal loss in the YAC128 Huntington's disease mouse model. Furthermore, GluN3A deletion corrected the abnormally enhanced NMDAR currents, which have been linked to cell death in Huntington's disease and other neurodegenerative conditions. Our findings reveal an early pathogenic role of GluN3A dysregulation in Huntington's disease and suggest that therapies targeting GluN3A or pathogenic HTT-PACSIN1 interactions might prevent or delay disease progression.

the polyglutamine expansion 17 , fulfilling a key criterion for pathogenic interactions 18 , and PACSIN1 overexpression suppresses mHTT toxicity in Drosophila screens 4 . Thus we hypothesized that mHTT might interfere with the endocytic removal of GluN3A-containing NMDARs by PACSIN1, leading to age-inappropriate synapse destabilization during Huntington's disease pathogenesis.
We confirm that mHTT binds and sequesters PACSIN1 away from its normal cellular locations, causing the aberrant accumulation of juvenile GluN3A-containing NMDARs at the surface of striatal neurons. We also show that GluN3A expression is abnormally elevated across mouse models of Huntington's disease and in human Huntington's disease striatum, and that GluN3A overexpression in mice drives the degeneration of afferent synapses onto MSNs. Importantly, suppressing GluN3A reactivation corrected the early enhancement of NMDAR currents in MSNs from YAC128 mice, prevented both early stage and progressive dendritic spine pathology and ameliorated later motor and cognitive decline. Our results reveal a new mechanism that mediates NMDAR dysfunction and synapse loss in Huntington's disease-dysregulation of the expression of NMDARs that contain GluN3A subunits by altered endocytic trafficking-and identify a potential safe target for pharmacological therapy.

PACSIN1 binds to mHTT and is sequestered into aggregates
We began by verifying the interaction between HTT and PACSIN1 (refs. 4,17) with coimmunoprecipitation assays on striatal lysates from wild-type and YAC128 mice, which express full-length human HTT with 128 CAG repeats 19 . Although PACSIN1 interacted with both HTT variants, the interaction was stronger in the YAC128 striatum (2.23 ± 0.3-fold (mean ± s.e.m.) increase in PACSIN1 bound to HTT relative to wild type, P = 0.005; Fig. 1a). Coimmunoprecipitation assays from HEK293 cells cotransfected with PACSIN1 and GFPtagged HTT exon 1 fragments spanning the proline-rich domain that binds PACSIN1 (ref. 17) and a normal or expanded polyglutamine tract (HTT ex1 17Q-GFP and HTT ex1 46Q-GFP, respectively) confirmed the polyglutamine dependence of the interaction and additionally showed that exon 1 is sufficient for PACSIN1 binding (Fig. 1b,c). We demonstrated specificity for HTT rather than the polyglutamine tract through experiments in which PACSIN1 did not interact with either normal or expanded ataxin 1, a polyglutamine repeat protein that is involved in spinocerebellar ataxia (Fig. 1c).
We next assessed whether binding to mHTT interferes with PACSIN1 localization or function in a cellular model of Huntington's disease 20 . We transfected cultured rat striatal neurons with HTT ex1 fragments and evaluated PACSIN1 localization by immunofluorescence. HTT ex1 46Q-GFP formed detectable intracellular aggregates in ~25% of neurons within 24-48 h, whereas HTT ex1 17Q-GFP showed diffuse expression (Supplementary Fig. 1). PACSIN1 immunostaining was punctate and distributed evenly in somatodendritic compartments of untransfected and HTT ex1 17Q-GFP-expressing neurons (Fig. 1d), but was enriched in somatodendritic HTT aggregates in neurons expressing HTT ex1 46Q-GFP (Fig. 1d,e and Supplementary  Fig. 1). A quantitative colocalization analysis 21 yielded highly significant intensity correlation coefficient (ICQ) values for HTT ex1 46Q-GFP and PACSIN1 (0.26 ± 0.02 (mean ± s.e.m.), P sign test < 0.005). We found lower ICQ values for HTT ex1 17Q-GFP (0.065 ± 0.01), which is consistent with weaker binding in biochemical assays. Other endocytic proteins were not enriched in aggregates (Fig. 1f), ruling out nonspecific sequestration.  I  I  I  I  I  IP  IP  IP  IP Figure 1 PACSIN1 binds to and colocalizes with mHTT. (a) Coimmunoprecipitation of HTT and PACSIN1 from striatal lysates of 3-month-old wild-type (WT) and YAC128 mice. HTT was immunoprecipitated with an antibody that recognizes both wild-type and mutant HTT. Immunoprecipitates (IP) were immunoblotted with the indicated antibodies. Additionally, 10% of the lysate (input) used for immunoprecipitation was loaded. (b) Scheme of the PACSIN1 structure indicating the F-BAR membrane deformation domain, the NPF (AsnProPhe) motifs responsible for GluN3A binding and the C-terminal SH3 domain. The SH3 domain mediates association with the proline-rich domain (PRD) of HTT but also links PACSIN1 to proteins of the endocytic machinery such as dynamin 1 and N-WASP. The location of GFP within the constructs used in this study is indicated. aa, amino acids. (c) Immunoblot using the indicated antibodies of lysates of HEK293 cells transfected with the indicated constructs and immunoprecipitated with a GFP-specific antibody. I, input lysates. (d) Representative images of striatal neurons transfected with HTT ex1 17Q-GFP or HTT ex1 46Q-GFP and stained for endogenous PACSIN1 48 h later. Arrows point to the localization of mHTT ex1 and PACSIN1 in cytoplasmic aggregates. (e) Colocalization of mHTT ex1 and PACSIN1 in dendritic aggregates as shown by line scan analysis of PACSIN1 and HTT ex1 46Q-GFP fluorescence (aggregate flanked by arrowheads in the images and shown by the gray bar in the graph). AFU, arbitrary fluorescence units. (f) Representative images of striatal neurons transfected with HTT ex1 46Q-GFP and immunostained for endogenous α-adaptin (AP-2), the early endosome marker EEA1 or transferrin receptor (TfR), which labels recycling endosomes. All scale bars, 5 µm. Redistribution into mHTT ex1 aggregates was associated with reductions of endogenous PACSIN1 amounts within their normal postsynaptic location, as quantified by decreases in dendriteto-soma fluorescence intensity ratios and consistent with dendritic depletion of PACSIN1 in humans with Huntington's disease 17 (Supplementary Fig. 2a,b). The total amounts of PACSIN1 were unchanged (Supplementary Fig. 2c), indicating that reduced dendritic availability reflects altered subcellular distribution.
Increased surface GluN3A expression in neurons expressing mHTT Because PACSIN1 controls the endocytic removal of GluN3Acontaining NMDARs 8 , stronger binding, subcellular sequestration or both by mHTT might be expected to interfere with ongoing endocytosis and affect plasma membrane expression. To test this idea, we cotransfected striatal neurons with hemagglutinin (HA)-tagged GluN3A and HTT ex1 variants and measured surface HA-GluN3A expression 48 h later using fluorescence-based antibody uptake assays. HTT ex1 46Q-GFP expression resulted in an accumulation of GluN3A at the neuronal surface (Fig. 2a,b) and concomitant increases in surface expression of the obligatory NMDAR subunit GluN1 (Fig. 2c), probably reflecting GluN1 assembly with GluN3A subunits which is required for their plasma-membrane transport and localization 22 . Notably, we observed increases in surface GluN3A expression in both neurons with diffuse HTT ex1 46Q-GFP expression and those with aggregated expression (Fig. 2b), suggesting that aggregation is not required and that binding to soluble mHTT species may be sufficient to alter PACSIN1 function. Supporting specificity for mHTT, polyglutamine expansions in ataxin 1 did not alter the surface expression of GluN3A (Fig. 2d). Because alterations in bulk endocytic recycling have been reported after mHTT expression in PC12 cells 23 , we analyzed transferrin uptake but found no alterations in neurons transfected with HTT ex1 46Q-GFP by 48 h, a time when surface GluN3A levels were already elevated (Supplementary Fig. 2d,e). HTT ex1 46Q-GFP also did not affect the surface expression of the AMPA receptor subunit GluA1 (Fig. 2e), which is highly reliant on endocytic recycling 24 . These results indicate that GluN3A accumulation is not due to a general impairment in neuronal endocytosis.
Knockdown of PACSIN1 using a PACSIN1-specific shRNA (shPAC-SIN1; Supplementary Fig. 3) also increased surface GluN3A expression ( Fig. 2f,g), demonstrating that PACSIN1 depletion can account for the mHTT-induced increase in surface GluN3A. We then asked whether an exogenous supply of PACSIN1 could counteract the effect of mHTT. Indeed, cotransfection of PACSIN1 rescued the surface accumulation of GluN3A subunits, which returned to control levels in HTT ex1 46Q-GFP-expressing neurons (Fig. 2a,h). These results show that loss of PACSIN1 function, probably due to mHTT binding, increases the surface expression of GluN3A-containing NMDARs in cultured striatal neurons.
Increased GluN3A in human Huntington's disease and mouse models GluN3A protein is highly expressed in the brain during early postnatal and juvenile stages (postnatal day (P) 8-15 in mice and the first years of life in humans), but expression declines thereafter 25,26 . Quantitative immunoblot analysis showed that GluN3A undergoes a sharper downregulation in mouse striatum relative to other brain regions and is nearly absent by adulthood (Supplementary Fig. 4). In contrast, we found no developmental changes in GluN2B, which is the predominant NMDAR subunit type in striatum (Supplementary Fig. 4). These results support a physiological role for GluN3A downregulation in striatal NMDAR function and predict substantial effects of the disruption  (a) Representative images of striatal neurons cotransfected with GFP-HTT ex1 and HA-GluN3A with or without Myc-PACSIN1. Surface (red) and internal receptors (white) were labeled with HA-specific antibody 48 h after transfection. Scale bar, 20 µm. (b-e) Quantification of the ratios of surface to internal GluN3A, GluN1 and GluA1 in neurons transfected with GFP-HTT ex1 or GFP-ataxin 1 (n = 20-54 neurons per condition from 2-3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 compared to HTT ex1 17Q-GFP, analysis of variance (ANOVA) followed by Bonferroni multiple comparison test). Diff, diffuse expression; aggr, aggregated expression. (f) Striatal neurons cotransfected with HA-GluN3A and PACSIN1-specific shRNA (shPACSIN1) or scrambled control shRNA (shSCR). Scale bar, 20 µm. (g) Quantification of the surface to internal ratios of HA-GluN3A in neurons transfected with shPACSIN1 (n = 17-52 neurons per condition from 2 independent experiments; ***P < 0.001, ANOVA followed by Bonferroni test). (h) Quantification of HA-GluN3A surface to internal ratios in HTT ex1 -GFP-transfected neurons after cotransfection with Myc-PACSIN1 (n = 51-69 neurons from 4 independent experiments; **P < 0.01, ***P < 0.001, ANOVA followed by Bonferroni test). All data are shown as the mean ± s.e.m. npg of this downregulation. To evaluate whether GluN3A dysregulation occurs in humans, we analyzed postmortem brain tissue from controls and individuals with Huntington's disease. GluN3A protein expression in control human putamen, a subregion of the striatum, was low, but we detected a significant increase in GluN3A expression in affected individuals ( Fig. 3a and Supplementary Table 1).
We next used the R6/1 and YAC128 mouse models of Huntington's disease to evaluate the timing of changes in GluN3A expression and their association with pathogenic events. R6/1 mice, which express N-terminally truncated human HTT carrying 115 CAG repeats 27 , showed a progressive increase in striatal GluN3A expression relative to wild-type mice starting at around 12-16 weeks of age ( Supplementary  Fig. 5a). Because PACSIN1 is enriched at synapses where it mediates GluN3A removal 8,28 , we biochemically isolated synaptic plasma membranes (SPMs) to determine whether increased GluN3A expression reflects increased residency at synaptic compartments associated with changes in PACSIN1 localization. Indeed, we detected a large increase in GluN3A expression (~2.5 fold) in SPMs from 20-week-old R6/1 mice, which matched a reduction in the synaptic abundance of PACSIN1 (Fig. 3b,c).
We obtained similar results in 3-month-old YAC128 mice (Fig. 3d,e and Supplementary Fig. 5b). Increased GluN3A expression in striatal SPMs was paralleled by increases in GluN1 and GluN2B, suggesting that GluN3A forms heteromultimeric NMDAR complexes that include these subunits (Fig. 3d), and was associated with reduced synaptic PACSIN1 expression (Fig. 3e). We did not detect changes in GluN2A, GluA2 or GluA3, in presynaptic proteins such as synaptophysin or in the trafficking regulators Rab4 or Rab11 (Fig. 3d,e). Further fractionation of striatal synaptic membranes showed increased GluN3A expression in both detergent-soluble (non-postsynaptic density (PSD)) and insoluble (PSD) fractions, which is consistent with the subsynaptic localization pattern that is characteristic of GluN3A 8 (Supplementary Fig. 6). Overall PACSIN1 protein expression was unchanged (93% ± 3% (mean ± s.e.m.) of wild type, P = 0.19), corroborating our culture data and further indicating that lower synaptic expression of PACSIN1 reflects subcellular redistribution. At this early disease stage, GluN3A expression was not altered in brain regions that are affected later in the progression of Huntington's disease, such as the cortex and hippocampus (Supplementary Fig. 5b). Immunolabeling further showed that increases in GluN3A expression occurred mainly in DARPP-32-labeled MSNs (Supplementary Fig. 5c,d).
To test whether elevated GluN3A expression reflects a defect in endocytosis, as would be predicted if subcellular sequestration of PACSIN1 by mHTT is the underlying mechanism, we incubated striatal slices from control and YAC128 mice with the membraneimpermeable cross-linker bissulfosuccinimidyl suberate (BS3). This reagent cross-links surface receptors to form high-molecularweight complexes that can be separated from unlinked intracellular receptors by electrophoresis, allowing a quantification of the internal receptor pool (Fig. 3f). These experiments revealed a significantly reduced fraction of intracellular GluN3A in YAC128 striatum but no changes in GluA2 or GluA3 (Fig. 3f,g). We additionally looked for contributions of altered transcription but found no significant changes in GluN3A mRNA levels in either R6/1 or YAC128 striatum at times when the synaptic and surface expression of GluN3A protein was increased by approximately two-fold (Supplementary Fig. 5e). These results confirm that GluN3A is redistributed to the plasma membrane and synaptic compartments in mouse models of Huntington's disease. They further show that striatal GluN3A dysregulation begins before or around the onset of motor or cognitive symptoms [29][30][31][32] , long preceding neuronal loss, and may thus be an early disease mechanism in Huntington's disease pathophysiology. We also observed increases in GluN3A  npg expression preceding symptom onset in a third Huntington's disease model, Hdh Q111 (where Hdh is the murine gene homolog of HTT) knock-in mice, in which the phenotype is much delayed 33 (Supplementary Fig. 5f).
Suppressing GluN3A corrects NMDAR dysfunction in YAC128 mice Alterations in NMDAR function have been reported in MSNs of YAC128 mice by 1 month of age. These defects involve long-lasting responses to glutamate release and have been attributed to increased activation of extrasynaptic NMDARs 13 . Because a distinguishing feature of GluN3A-containing receptors is their perisynaptic and extrasynaptic localization 8,34 , and GluN3A expression was increased early in synaptosomal subfractions from YAC128 striatum that are enriched in extrasynaptic plasma membrane receptors (non-PSD), we asked whether the electrophysiological defect could be explained by elevated GluN3A expression.
We crossed YAC128 mice with Grin3a −/− (Grin3a is the gene encoding GluN3A) mice and compared NMDAR responses after intense paired-pulse afferent stimulation in wild-type, YAC128 and YAC128 mice lacking GluN3A 13 . We obtained isolated NMDAR responses by calculating the difference between the responses recorded in the absence and presence of the NMDAR blocker (2R)-amino-5-phosphonovaleric acid (AP5). We detected no changes in the AMPAR-mediated component obtained in AP5 but found large differences in the decay kinetics of the isolated NMDAR component (P < 0.005 for YAC128 compared to wild type; Fig. 3h,i), confirming previous observations 13 . These differences were largely abolished in YAC128 mice lacking GluN3A (Fig. 3h,i), indicating that GluN3A is required for the mHTT-induced enhancement in NMDAR currents and consistent with the possibility that aberrant GluN3A expression underlies the earliest events of Huntington's disease pathophysiology.
Suppressing GluN3A rescues synapse pathology in YAC128 mice Dendritic dystrophy and loss of glutamatergic spiny synapses are early degenerative changes in Huntington's disease 7,35,36 that might underlie symptom onset. Because enhancement of GluN3A function in the hippocampus drives synapse elimination 16 , we asked whether aberrant GluN3A expression could contribute to the synaptic pathology. We first used Golgi impregnation techniques to measure dendritic spines, which are sites where excitatory synapses form, in YAC128 mice at times when GluN3A expression was elevated (Fig. 4a). Spine numbers were decreased in dendrites of YAC128 MSNs by 3 months of age (12-15% decrease compared to wild type), and this decay worsened with age (Supplementary Fig. 7).
To test whether abnormal accumulation of GluN3A in adult MSNs could explain the progressive spine loss induced by mHTT, we measured spine densities in transgenic mice that overexpress GluN3A into adulthood (double-transgenic GFP-GluN3A mice). These mice were generated by crossing mice expressing GFP-tagged GluN3A under the control of the tetO promoter with mice expressing the tetracyclinecontrolled transactivator (tTA) under the Camk2a promoter 16 ; the Camk2a promoter drives transgene expression into MSNs 34 . MSNs from double-transgenic GFP-GluN3A mice showed a pattern of spine loss that was remarkably similar to that observed in YAC128 mice (~11% decrease in double-transgenic mice relative to control littermates by 3 months of age, with larger spine losses at later stages; Fig. 4b,c, compare with Supplementary Fig. 7).
We then reasoned that if the spine loss in MSNs was caused by enhanced GluN3A function, it should be prevented by lowering GluN3A expression. To test this idea, we re-examined spine densities in wild-type and YAC128 mice in the presence and absence of GluN3A. Lack of GluN3A prevented both the early reductions in spine numbers in YAC128 mice and the progressive synaptic disconnection detected as a larger loss of MSN spines npg at later stages of disease (Fig. 4d,e). In contrast, spine densities were not altered in MSNs from adult Grin3a −/− mice (Fig. 4d,e), indicating that blocking GluN3A expression targets the synaptic pathology in Huntington's disease without affecting basal spine numbers in controls. This result differs from the increased spine density reported in Grin3a −/− cortical neurons 15 and probably reflects the lower GluN3A expression retained in adult striatum (Supplementary Fig. 4). At high resolution, electron microscopy revealed a marked loss of asymmetric glutamatergic synapses onto MSNs of YAC128 mice, which was rescued by GluN3A deletion (Fig. 4f,g). In addition, postsynaptic densities were smaller in YAC128 mice (Fig. 4g), which is consistent with earlier studies in which GluN3A overexpression decreased synapse size 16 , and this deficit was also rescued by suppressing GluN3A (Fig. 4g). These results show that diminished synaptic connectivity in MSNs from YAC128 mice begins at early stages, matching the timing of GluN3A dysregulation, and can be rescued by deleting GluN3A or replicated by transgenically overexpressing GluN3A.
Suppressing GluN3A ameliorates motor and cognitive dysfunction We next assessed the impact of GluN3A on Huntington's diseaselike symptoms using striatum-dependent motor and cognitive tasks. YAC128 mice showed impaired motor learning by 10 months of age, as indicated by increased failures when learning a fixed-speed rotarod task, and impaired coordination, as indicated by a shorter latency to fall from an accelerating rotarod. Both deficits were improved in YAC128 mice lacking GluN3A (Fig. 5a,b). A vertical pole test, another measure of motor coordination, showed that YAC128 mice spent less time on the pole than wild-type mice, an effect that was again rescued by GluN3A deletion (Fig. 5c). Nevertheless, the hypokinetic phenotype shown by YAC128 mice in an open field was still apparent in YAC128 mice lacking GluN3A (Fig. 5d). Neither weight gain nor loss of muscle tone was rescued by GluN3A deletion (Supplementary  Fig. 8), ruling out the possibility that improvements in coordination resulted from reversal of these parameters.
We evaluated cognitive function using a swimming T-maze task 37 (Fig. 5e-g). YAC128 mice showed a pronounced impairment during the learning phase, whereas YAC128 mice lacking GluN3A performed as well as wild-type mice (Fig. 5e,g). On day 4, we switched the platform to the opposite arm of the T maze to probe reversal (strategyshifting) learning. YAC128 mice required longer than wild-type mice to reach the new platform location in all trials, and this defect was rescued by GluN3A deletion (Fig. 5f,g).

Suppressing GluN3A rescues mHTT toxicity
To investigate whether abnormally increased GluN3A expression contributes to later striatal atrophy and MSN degeneration, we performed stereological analyses in 16-month-old mice, an age at which neuronal death can be reproducibly detected in the YAC128 model 19 . Suppressing GluN3A expression in YAC128 mice rescued striatal atrophy and reductions in NeuN-positive and DARPP-32-positive neurons and normalized DARPP-32 expression in surviving MSNs (Fig. 6a-d); DARPP-32 is a key component of MSN dopamine signaling cascades that is downregulated in Huntington's disease mouse models from early stages 38 .
Analogous analyses in control and double-transgenic GFP-GluN3A mice showed that GluN3A overexpression does not cause striatal atrophy or neuronal loss per se by 16 months of age (Fig. 6e,f) but mimics other neuropathological features of Huntington's disease, including reductions in the size of MSNs 39 (Fig. 6g) and striatal DARPP-32 expression 40 (100 ± 16 (mean ± s.e.m., integrated density values) in control mice compared to 61 ± 5 in double-transgenic GFP-GluN3A mice; n = 6; P < 0.05; Fig. 6h). We next conducted experiments in a rat corticostriatal slice model 41 to test whether increased GluN3A expression influences the susceptibility of MSNs to mHTT toxicity. We cotransfected slice explants with expanded HTT ex1 (HTT ex1 73Q-GFP) along with GluN3A and assessed MSN degeneration by coexpression of yellow fluorescent protein (YFP).
The number of surviving MSNs was decreased in brain slices cotransfected with mHTT and GluN3A relative to slices transfected with mHTT alone (Fig. 6i). Thus, increased GluN3A expression recapitulates features of Huntington's disease neuronal dysfunction, such as MSN shrinkage and disturbed signaling, and renders MSNs more vulnerable to mHTT toxicity.

DISCUSSION
We identify a new early stage disease mechanism that underlies NMDAR dysfunction and synapse loss in Huntington's disease: subcellular sequestration of the endocytic adaptor PACSIN1 by mHTT leaves an overabundance of NMDARs containing GluN3A subunits in the plasma membrane and at postsynaptic sites of striatal neurons.
The new mechanism provides a missing link between two major phenomena previously known to be impaired by HTT mutations, defects in protein trafficking and impaired NMDAR transmission. GluN3A dysregulation targeted MSNs, the vulnerable population in Huntington's disease striatum, and was an early feature across Huntington's disease mouse models. Knocking out GluN3A in Huntington's disease mice corrected a full sequence of early-to-late pathophysiological events, demonstrating a crucial role for GluN3A in disease pathogenesis in vivo: it normalized NMDAR currents; fully prevented synapse degeneration; rescued motor and cognitive decline; and reduced striatal cell death.
Our data extend previous findings pointing toward GluN3A subunits as a synapse destabilizing or pruning factor 16 and show the pathogenic effects of aberrant reactivation in the adult brain of a pruning mechanism that is normally restricted to developmental stages. In the normal brain, PACSIN1-mediated retrieval of GluN3A from synapses contributes to receptor downregulation and is thought to provide a crucial signal for synaptic plasticity and growth and for robust information storage by allowing the replacement of immature with mature NMDAR subtypes 8,16 . Factors that inhibit PACSIN1 function, such as dominant-negative variants of PACSIN1 (ref. 8) or mHTT, increase surface and synaptic GluN3A expression, yielding a higher proportion of small, immature synapses. Although the precise mechanisms by which mHTT alters PACSIN1 distribution remain unclear, our observations of reduced synaptic PACSIN1 expression in young YAC128 mice, months before aggregates are present 42 , and surface accumulation of GluN3A in striatal neurons with no detectable HTT aggregates argue against a requirement for aggregation. This fits with current concepts linking soluble mHTT species, rather than aggregates, to cytotoxicity 20,43,44 . But PACSIN1 redistribution into microaggregates not visible by light microscopy cannot be ruled out, and the question of whether PACSIN1 binds to particular conformations of mHTT remains open.
Regarding the stoichiometry of GluN3A-containing NMDARs in Huntington's disease striatum, our biochemical fractionation results indicate the presence of GluN1 and GluN2B subunits. This is consistent with biochemical evidence for assembly of GluN3A and GluN2B subunits in striatal tissue 34 and reports attributing the enhanced NMDAR responses in YAC128 mice to GluN2B-containing NMDARs 13,45 , as well as our finding that suppressing GluN3A corrects the alteration. GluN3A-containing subtypes are less anchored to PSDs than conventional NMDARs (GluN1-GluN2 heteromers), and GluN3A overexpression has been shown to impair the postsynaptic stabilization of GluN2B-containing NMDARs 34 , which might facilitate their diffusion toward extrasynaptic sites, providing a testable cell-biological mechanism for the increased extrasynaptic GluN2Bmediated currents in YAC models 13 . Along with synapse loss and npg enhanced extrasynaptic NMDARs, elevated GluN3A expression could explain features of altered glutamatergic transmission that have been observed in presymptomatic Huntington's disease mice and cannot be accounted for by the increased GluN2B-mediated currents without considering the presence of additional subunits in the receptor complex. These include anomalously reduced long-term potentiation [46][47][48] and decreased magnesium sensitivity of NMDAR currents 11,49 , both of which have been reported in GluN3A-overexpressing mice 16 .
An imbalance between synaptic and extrasynaptic NMDAR activity is thought to be crucial for neurodegeneration because the two receptor populations signal to cell-survival and death pathways, respectively 50 , and increased extrasynaptic activity has been linked to cell death in Huntington's disease 13,43 . Enhanced GluN3A expression could contribute to cell death by driving or aggravating this imbalance in two ways. First, it might trigger the activation of death pathways by increasing extrasynaptic NMDAR localization. Second, MSNs receive dense glutamatergic input from cortical axons, and the synaptic disconnection driven by GluN3A could inhibit prosurvival signaling pathways coupled to synaptic NMDAR activation 50,51 . But GluN3Acontaining subtypes flux less Ca 2+ than other NMDAR subtypes 22 , and data from our own laboratory support a neuroprotective action of GluN3A overexpression in acute excitotoxic cell-death assays 34,52 , which might explain the paradoxical resistance to acute excitotoxic insults observed in Huntington's disease models 53,54 . Nonetheless, aberrant GluN3A expression over the much longer time course relevant to Huntington's disease (that is, decades rather than minutes) could have deleterious effects due to inhibition of synaptotrophic NMDAR activity and/or chronically disturbed signaling by extrasynaptic NMDARs activated by ambient glutamate.
In summary, we uncover a role for GluN3A dysregulation in Huntington's disease and provide a rationale for the use of therapies targeting GluN3A or PACSIN1 early in the course of the disease to ameliorate cognitive or motor problems, halt disease progression or both. An advantage of targeting GluN3A (as compared to other NMDAR subunits) is that it is lacking in adult brains for the most part and would allow for the selective blocking of a pathological trait without hampering normal synaptic function.

METHODS
Methods and any associated references are available in the online version of the paper. with two trials per day spaced 1-2 h apart during 3 consecutive days. During this learning phase, mice falling from the rod were returned, and the number of falls was recorded until the addition of the latencies to fall reached a total time of 60 s per trial 13,29 . For the accelerating task, the rotarod was accelerated from 5 to 40 r.p.m. over 5 min. The latency to fall was recorded for each of two trials and averaged. For the vertical pole test, a metal pole (1.5 cm in diameter, 50 cm long) wrapped with cloth tape was used. The mouse was placed in the center of the pole, which was held in a horizontal position. The pole was then gradually lifted to a vertical position, and the latency to fall was measured. For the swimming T-maze test, a T maze (dimensions: arms, 38 × 14 cm; platform, 10 × 14 cm) was filled with water to a depth of 7 cm, and a submerged escape platform was located in the right arm of the maze. Mice were placed into the water at the base of the stem arm of the maze and learned the location of the escape platform. During the normal testing phase, mice received four trials per day spaced 45 min apart for 3 consecutive days. For reversal learning, the platform was switched to the left arm of the maze. Mice received four trials spaced 45 min apart. Times to reach the hidden platform were recorded.
HTT exon 1 brain-slice assay. Brain-slice assays for HTT ex1 Q73-induced neurodegeneration were as described 41 . Briefly, 250-µm thick corticostriatal brain slices from P10 Sprague-Dawley rat pups of both sexes were prepared on a vibratome and placed in an interface configuration over culture medium containing 15% heat-inactivated horse serum, 10 mM KCl, 10 mM HEPES, 100 U ml −1 penicillin and streptomycin, 1 mM sodium pyruvate and 1 mM l-glutamine in Neurobasal A (Invitrogen) under 5% CO 2 at 32 °C. Rat pups were euthanized in accordance with US National Institutes of Health guidelines and under Duke University Medical Center Institutional Animal Care and Use Committee approval. Slices were biolistically transfected (Helios gene gun, Bio-Rad) with DNA plasmids encoding YFP, HTT ex1 Q73, and/or GluN3A, all in gWiz (Genlantis). Four days after transfection, MSNs were identified by their location within the striatum and by their characteristic dendritic arborization and scored as healthy if they showed continuous YFP fluorescence throughout a cell body of normal diameter and at least two clear and unbroken primary dendrites that were at least two cell body-diameters long.
Statistics. Values are shown as the mean ± s.e.m. Student's t test was used for simple comparisons of one variable between two groups, and one-or two-way ANOVA followed by Bonferroni or Tukey post hoc tests were used to determine differences between more than two groups, unless otherwise indicated in the figure legends. The level of statistical significance was set at P < 0.05. No statistical method was used to predetermine sample size, and the animal experiments were not randomized.