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Mutant huntingtin enhanced sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity Zeron, Melinda M.


Previous work suggests N-methyl-D-aspartate receptor (NMDAR) activation may be involved in degeneration of medium spiny striatal neurons in Huntington's disease (HD). Here, we determined whether expression of huntingtin (htt) containing the polyglutamine expansion augments NMDAR-mediated excitotoxicity. First, human embryonic kidney (HEK) 293 cells co-expressing mutant huntingtin (htt-138Q) and either NR1A/NR2A- or NR1 A/NR2B-type NMDARs exposed to 1 mM NMDA showed a significant increase in excitotoxic cell death compared to controls [cells co-expressing htt-15Q or green fluorescent protein (GFP)], but the difference was larger for NR1A/NR2B. Moreover, agonist-dependent cell death showed apoptotic features for cells co-expressing htt-138Q and NR1A/NR2B, but not for cells expressing htt-138Q and NR1A/NR2A. Furthermore, NRlA/NR2B-mediated apoptosis was not seen with co-expression of an N-terminal fragment of mutant htt. Since NR1A/NR2B is the predominant NMDAR subtype in neostriatal medium-sized spiny neurons (MSNs), we hypothesized that enhancement of NMDA-induced apoptotic death in NRlA/NR2B-expressing cells by full-length mutant htt may contribute to selective neurodegeneration in HD. To test this hypothesis we compared NMDAR-induced cell death in striatal neurons from a yeast artificial chromosome (YAC) transgenic mouse model of HD expressing full-length mutant huntingtin with striatal neurons from the same strain of wild-type mice. Excitotoxic death of MSNs from the transgenic mice was increased after NMDA but not AMPA. NMDAR-mediated cell death was completely blocked by the NR2B subtype-selective antagonist ifenprodil, and was also associated with increased caspase-3 activity relative to wildtype (WT) MSNs. Importantly, there was no enhancement of NMDAR-mediated cell death in cerebellar granule neurons expressing mutant huntingtin, demonstrating cell type specificity and also consistent with NMDAR subtype specificity. Although caspase-3 can cleave huntingtin to form smaller fragments we did not observe an increase in the proteolysis of huntingtin after exposure to NMDA in WT or mutant htt expressing striatal cultures. Enhanced mitochondrial membrane depolarization and intracellular calcium levels were observed upon NMDAR application in MSNs expressing mutant htt. NMDAR excitotoxicity was partially inhibited by application of cyclosporin A in MSNs, and inhibition was greater in MSNs from transgenic mice than WT mice. Together, our data support a role for NR2B-subtype NMDAR activation as an upstream trigger for mitochondrial dysfunction and caspase-3 activation. These results may help explain selective neuronal degeneration in HD.

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