NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation

Abstract

Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47 phox subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.Activation of the neuronal NMDAR initiates several downstream events, including cation influx, activation of nitric oxide synthase and formation of superoxide [1][2][3] . Superoxide functions as an inter-cellular messenger in long-term potentiation 4,5 and participates in redox inhibition of NMDAR channel function 6 ; however, superoxide can also promote neuronal death when NMDAR activation is sustained 1,7 . Notably, the primary source of superoxide induced by NMDAR activation remains unresolved.Initial studies suggested a mechanism in which Ca 2+ influx through NMDAR channels leads to mitochondrial depolarization 8,9 and subsequent mitochondrial production of superoxide 10,11 . However, a biochemical mechanism linking these events has not been identified and evidence supporting mitochondria as the primary source of neuronal superoxide production remains indirect 12,13 . Calcium was shown to induce superoxide production in isolated © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to R.A.S. (Raymond.swanson@ucsf.edu). AUTHOR CONTRIBUTIONS A.M.B. carried out the cell culture studies and data analysis and prepared the manuscript drafts. S.W.S. supervised the mouse surgical studies and analyzed these data. S.J.W. performed mouse surgery studies and mouse brain histology. P.N. maintained the Sod2 + mouse colony and prepared the Sod2 + cell cultures. T.M.K. and Y.E. assisted with the p47 phox translocation studies and data analysis. H.L. assisted in the analysis of the cell culture ethidium fluorescence results. P.H.C. assisted with the studies involving Sod2 + neurons. R.A.S. organized the studies and prepared the final manuscript.Note: Supplementary information is available on the Nature Neuroscience website. 14 , but more recent studies indicate that this effect is highly dependent on experimental conditions, especially the presence of bovine serum albumin in the medium and succinate as a metabolic substrate 13 . The important question is w...

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