Abstract T P216: Regulation of Neuronal Survival by NMDA Receptor Subunit GluN2C Following Ischemia

Abstract

BACKGROUND: Glutamate-mediated excitotoxicity is a key mechanism underlying neuronal death following ischemia. Postischemic activation of NMDA receptors has been linked to subunit-specific effects that can be attributed to factors such as unique channel kinetics and protein-protein interactions. GluN2C-containing NMDARs have lower calcium permeability and open probability compared to GluN2A/2B and behave as a functional NMDAR under physiological conditions. Hypothesis: We hypothesize that GluN2C plays a neuroprotective role following ischemia in the hippocampus. METHODS: To determine the level of endogenous GluN2C mRNA following oxygen-glucose deprivation (OGD), qPCR was performed on acute hippocampal slices dissected from P30-45 Sprague Dawley rats. Slices were treated with 4-30 min of OGD, followed by 90 min of recovery. To test the survival outcome of GluN2C expression, hippocampal neurons were transfected with SEP-GluN2C and 30 min time-lapse imaging was performed following 200uM NMDA application. Neuronal injury was assessed by determination of cellular morphology (cell body swelling and dendritic varicosities) and DAPI staining. Yeast two-hybrid assay was used to determine 14-3-3 isoform interactions with GluN2C. RESULTS: We found an initial 20% increase in GluN2C mRNA following 4 min exposure to OGD (normalized expression=1.193**; n=4). At 10, 15 and 30 min OGD, GluN2C progressively decreased (0.709, 0.739*, 0.301** respectively; n=9). GluN2A expression remained stable and GluN2B significantly increased at 10 min* and 30 min**. GluN2C expressed neurons showed marked resistance to NMDA toxicity (n=6). To characterize pro-survival binding partners of GluN2C, yeast two-hybrid assay revealed distinct 14-3-3 isoforms bind specifically to GluN2C. (**p<0.001, *p< 0.05) CONCLUSION: These results support our hypothesis that GluN2C plays a neuroprotective role in ischemia. Following ischemia, a reduction in GluN2C leads to increased calcium permeability through GluN2A/2B and greater cell death. Its association with 14-3-3 provides additional evidence of its neuroprotective role. This study can lead to the development of subunit-specific therapeutic strategies to minimize ischemic cell death.