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Overactivation of NMDA receptors (NMDARs) is believed to play a role in degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). This hereditary disorder is caused by an expansion >35 in the polyglutamine (polyQ) region of the protein huntingtin (htt). Previous work has shown that NMDAR current, calcium signaling, and/or toxicity are enhanced in striatal MSNs in a variety of transgenic mice and cellular models of HD, but whether the enhancement is specific for MSNs or correlated with mutant htt (mhtt) polyQ length is not known. Furthermore, the mechanism underlying the increase in NMDAR activity has not been elucidated. Here we report polyQ length-dependent enhancement of peak NMDAR current density by mhtt in cultured MSNs, but not cortical neurons, from the yeast artificial chromosome (YAC) transgenic HD mouse model. We also observed a shift of NMDAR subunits NR1 and NR2B from internal pools to the plasma membrane and a significantly faster rate of NMDAR insertion to the surface in YAC72 MSNs. In comparing YAC72 with wild-type striatal tissue, subcellular fractionation revealed a relative enrichment of NR1 C2'-containing NMDARs in the vesicle/microsome-enriched fraction, and coimmunoprecipitation experiments demonstrated an increased proportion of NR1 C2' isoforms associated with NR2 subunits, which may contribute to faster forward trafficking of these receptors. Our results suggest that altered NMDAR trafficking may underlie potentiation of NMDAR-mediated current and toxicity in the YAC72 HD mouse model. This polyQ length-dependent, neuronal-specific change in NMDAR activity induced by mhtt may contribute to selective neuronal degeneration in HD.

Huntington disease (HD), caused by expansion >35 of a polyglutamine tract in huntingtin, results in degeneration of striatal medium spiny neurons (MSNs). Previous studies demonstrated mitochondrial dysfunction, altered intracellular calcium release, and enhanced NMDAR-mediated current and apoptosis in cellular and mouse models of HD. Here, we exposed cultured MSNs from YAC transgenic mice, expressing full-length human huntingtin with 18, 72, or 128 repeats, to a variety of apoptosis-inducing compounds that inhibit mitochondrial function or increase intracellular calcium, and assessed apoptosis 24 h later. All compounds produced a polyglutamine length-dependent increase in apoptosis, but NMDA produced the largest potentiation in apoptosis of YAC72 and YAC128 versus YAC18 MSNs. Moreover, reduction of NMDAR-mediated current and calcium influx in YAC72 MSNs to levels seen in wild-type reduced NMDAR-mediated apoptosis proportionately to wild-type levels. Our results suggest that increased NMDAR signaling plays a major role in enhanced excitotoxic MSN death in this HD mouse model.

Evidence suggests N-methyl-D-aspartate receptor (NMDAR) activation is involved in the degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). We tested the hypothesis that enhanced NMDAR-mediated excitotoxicity is mediated by the mitochondrial-associated apoptotic pathway in cultured MSNs from YAC transgenic mice expressing full-length huntingtin (htt) with a polyglutamine (polyQ) expansion of 46 or 72 (YAC46 or YAC72). NMDAR-mediated Ca(2+) transients and mitochondrial membrane depolarization were significantly increased in YAC compared to wild-type mice MSNs. Inhibitors of the mitochondrial permeability transition (mPT), cyclosporin A and bongkrekic acid, and coenzyme Q10 (an anti-oxidant involved in bioenergetic metabolism) dramatically diminished NMDA-induced cell death and eliminated genotypic differences. In YAC46 MSNs, NMDA stimulated significantly higher activation of caspase-3 and caspase-9 but not caspase-8, and NMDA-induced caspase-3 and -9 activation was markedly attenuated by cyclosporin A. Agents that improve mitochondrial function or inhibit the permeability transition may eliminate increased caspase activation and cell death associated with enhanced NMDAR activity in HD.

NMDA-type glutamate receptors play a critical role in neuronal synaptogenesis, plasticity, and excitotoxic death. Recent studies indicate that functional NMDA receptors are also expressed in certain glial populations in the normal brain. Using immunohistochemical methods, we detected the presence of the NMDA receptor 2B (NR2B) subunit of the NMDA receptor in neurons but not astrocytes in the CA1 and subicular regions of the rat hippocampus. However, after ischemia-induced neuronal death in these regions, double immunohistochemical labeling revealed that NR2B subunits colocalized with the astrocyte marker glial fibrillary acid protein and with NR1 subunits that are required for functional NMDA receptors. NR2B expression was first observed 3 d after ischemia and reached a peak at 28 d. At 56 d, only a few NR2B-expressing astrocytes were still present. In vitro, when postnatal hippocampal cultures were subjected to 5 min of anoxia, it resulted in NR2B expression on astrocytes in the glial feed layer. Imaging of intracellular calcium with postanoxic cultures and astrocytes isolated acutely from the ischemic hippocampus revealed a rise in intracellular [Ca2+] after stimulation with the specific agonist NMDA. The response could be blocked reversibly with the competitive antagonist 2-amino-5-phosphonovalerate and attenuated by the NR2B-selective antagonist ifenprodil. Control astrocytes were not responsive to NMDA but responded to glutamate. An understanding of the role of astrocytes that express functional NMDA receptors in response to ischemia may guide development of novel stroke therapies.