Excitotoxic Superoxide Production and Neuronal Death Require Both Ionotropic and Non-ionotropic NMDA Receptor Signaling

NMDA-type glutamate receptors (NMDAR) trigger superoxide production by neuronal NADPH oxidase-2 (NOX2), which if sustained leads to cell death. This process involves Ca influx through NMDAR channels. By contrast, comparable Ca influx by other routes does not induce NOX2 activation or cell death. This contrast has been attributed to site-specific effects of Ca flux through NMDAR. Here we show instead that it stems from non-ionotropic signaling by NMDAR GluN2B subunits. To evaluate non-ionotropic effects, mouse cortical neurons were treated with NMDA together with 7-chlorokynurenate, L-689,560, or MK-801, which block Ca influx through NMDAR channels but not NMDA binding. NMDA-induced superoxide formation was prevented by the channel blockers, restored by concurrent Ca influx through ionomycin or voltage-gated calcium channels, and not induced by the Ca influx in the absence of NMDAR ligand binding. Neurons expressing either GluN2B subunits or chimeric GluN2A/GluN2B C-terminus subunits exhibited NMDA-induced superoxide production, whereas neurons expressing chimeric GluN2B/GluN2A C-terminus subunits did not. Neuronal NOX2 activation requires phosphoinositide 3-kinase (PI3K), and NMDA binding to NMDAR increased PI3K association with NMDA GluN2B subunits independent of Ca influx. These findings identify a non-ionotropic signaling pathway that links NMDAR to NOX2 activation through the C-terminus domain of GluN2B.