Cellular and molecular mechanisms of N-methyl-D-aspartate receptor mediated calcium-dependent neurotoxicity in cortical cell cultures

grantor: University of TorontoThe N-methyl-D-aspartate receptors (NMDARs) have been ascribed a key role in excitotoxicity owing to their ubiquitous distribution in central neurons, and to their high permeability to Calcium (Ca2+) ions. NMDAR overactivation was thought to trigger excitotoxicity by excessive intracellular Ca2+ accumulation that exceeds the cell's regulatory capacity. Here, we established that the chief determinant of Ca2+ neurotoxicity is the route of Ca2+ entry, and not the total Ca2+ load. Whereas neurons were rapidly damaged when loaded with Ca2+ ions through NMDARs, a similar Ca2+ load incurred through alternative pathways, such as voltage sensitive calcium channels (VSCCs), was innocuous. First, we investigated whether it is the synaptic localization of NMDARs that is responsible for the toxic potential of NMDAR-mediated Ca2+ loading. Perturbing F-actin, a cytoskeletal protein that participates in targeting synaptic NMDARs in dendritic spines, reduced the number of dendritic NMDAR clusters and the NMDAR-mediated component of spontaneous miniature EPSCs, while the AMPA receptor-mediated component was unchanged. This selective perturbation of synaptic NMDARs had no effect on neuronal death or the accumulation of 45Ca2+ evoked by applying exogenous NMDA, which reaches both synaptic and extrasynaptic receptors. However, it increased survival and decreased 45Ca2+ accumulation in neurons exposed to oxygen-glucose deprivation, which causes excitotoxicity by synaptic glutamate release, and causes toxicity mainly by activation of synaptic receptors. Thus, it is not the synaptic localization of the NMDARs that determines their toxic potential. We next examined whether the association of NMDARs with distinct membrane-associated macromolecular complexes initiate and/or propagate neurotoxic Ca2+ signaling. We studied excitotoxic NMDAR signaling by suppressing the expression of the NMDAR scaffolding protein PSD-95, which selectively attenuated excitotoxicity triggered via NMDARs, but not by other glutamate or Ca2+ channels. NMDAR function was unaffected, as receptor expression, NMDA-currents and 45Ca2+ loading via NMDARs were unchanged. Suppressing PSD-95 selectively blocked Ca 2+-activated nitric oxide production by NMDARs, but not by other pathways, without affecting neuronal nitric oxide synthase (nNOS) expression or function. Thus, PSD-95 is required for the efficient coupling of NMDAR activity to nitric oxide toxicity, and imparts specificity to NMDAR-mediated excitotoxic Ca 2+ signaling.Ph.D