Sustained activation of neuronal N-methly D-aspartate (NMDA)-type glutamate receptors leads to excitotoxic cell death in stroke, trauma, and neurodegenerative disorders. Excitotoxic neuronal death results in part from superoxide produced by neuronal NADPH oxidase (NOX2), but how NMDA receptors are coupled to neuronal NOX2 activation is not well understood. Here, we identify a signaling pathway coupling NMDA receptor activation to NOX2 activation in primary neuron cultures. Calcium influx through the NR2B subunit of NMDA receptors leads to the activation of phosphoinositide 3-kinase (PI3K). Formation of phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) by PI3K activates the atypical protein kinase C, PKC zeta (PKCζ), which in turn phosphorylates the p47phox organizing subunit of neuronal NOX2. Calcium influx through NR2B-containing NMDA receptors triggered mitochondrial depolarization, NOX2 activation, superoxide formation, and cell death. However, equivalent magnitude calcium elevations induced by ionomycin did not induce NOX2 activation or neuronal death, despite causing mitochondrial depolarization. The PI3K inhibitor wortmannin prevented NMDA-induced NOX2 activation and cell death, without preventing cell swelling, calcium elevation, or mitochondrial depolarization. The effects of wortmannin were circumvented by exogenous supply of the PI3K product, PI(3,4,5)P3, and by transfection with protein kinase M, a constitutively active form of PKCζ. These findings demonstrate that superoxide formation and excitotoxic neuronal death can be dissociated from mitochondrial depolarization, and identify a novel role for PI3K in this cell death pathway. Perturbations in this pathway may either increase or decrease superoxide production in response to NMDA receptor activation, and may thereby impact neurological disorders, in which excitotoxicity is a contributing factor.

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H + along with extracellular superoxide, and the intracellular H + must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDAinduced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na + /H + exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia-reperfusion was likewise prevented by inhibiting Na + /H + exchange and by reduced expression of the Na + /H + exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na + /H + exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.any metabolic processes generate hydrogen ions, and hydrogen ions in turn influence cell metabolism and survival (1). Cerebral ischemia in particular produces acidosis of variable degree, depending upon blood glucose levels, degree of blood flow reduction, and other factors. Severe acidosis, below pH 6.4, exacerbates ischemic injury (2) by mechanisms involving protein denaturation, acid-sensing calcium channels, and release of ferrous iron (3-5). Conversely, lesser degrees of acidosis, in the range of 7.0-6.5, reduce both ischemic injury (6) and glutamateinduced neuronal death (7). These neuroprotective effects have been attributed to an inhibitory effect of hydrogen ions on NMDA receptor activation (8-10), but a causal link has not been demonstrated.Excessive activation of N-methyl-D-aspartate (NMDA) type glutamate receptors leads to excitotoxic cell death in stroke and other neurological disorders (11,12). Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic cell death (13)(14)(15)(16)(17)(18)(19) Together, the pH sensitivity of NOX2 and the role of NOX2 in NMDA receptor-mediated cell death suggest the possibility that reduced intracellular pH might limit neurotoxicity by dissociating NMDA receptor activation from superoxide production. Findings presented here confirm that both the superoxide production and cell death resulting from neuronal NMDA receptor activation are highly pH sensitive. We show that neurons use Na + /H + exchange as a major route of proton efflux during NOX2 activation, and either genetic or pharmacologic inhibition of neuronal Na + /H + exchange prevent both excitotoxic superoxide production and cell death. ResultsMild Acidosis Blocks NMDA-Induced Superoxide Production and Cell Death. We first performed cell culture studies at a physiological medium pH ...

Significance Mitochondria are often considered a source of reactive oxygen species (ROS) that cause damage to cellular components including nuclear DNA. This endogenous damage is thought to underlie neurodegeneration, especially in diseases such as Cockayne syndrome (CS) that lack transcription-coupled DNA repair (TCR). We find no evidence, however, for any nuclear DNA damage from increased mitochondrial ROS. Our results indicate that the neurodegenerative symptoms in CS may be mitochondrial in origin, independent of nuclear DNA damage and repair.