Phosphoinositide 3-kinase couples NMDA receptors to superoxide release in excitotoxic neuronal death

A M Brennan-Minnella,R A Swanson,Y Shen,J El-Benna

doi:10.1038/cddis.2013.111

Abstract

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.

Highlights

NOX2 is the isoform of NADPH oxidase most abundantly expressed by brain neurons.[19]
Given that Phosphoinositide 3-kinase (PI3K) activation can be triggered by calcium entry through N-methly D-aspartate (NMDA) receptors,[22,39] we evaluated the role of PI3K in NMDAinduced NOX2 activation
Superoxide production is a requisite event in the excitotoxic cell death process triggered by NMDA receptor activation,[6,7,12] and superoxide is a major source of oxidative stress in brain ischemia and other neurological disorders.[16,17,18]

Summary

Introduction

NOX2 is the isoform of NADPH oxidase most abundantly expressed by brain neurons.[19]. Phosphoinositide 3-kinase (PI3K) is induced by NMDA receptor activation.[21,22,23] PI3K phosphorylates the three position of hydroxyl groups on the inositol ring of specific phosphatidylinositol membrane phospholipids.[24] PI3K activation engages prosurvival signaling pathways in many cell types, and in the central nervous system PI3K is involved in neuronal plasticity.[21,25] PI3K activation has been associated with neuronal death in some settings,[26,27] by mechanisms yet to be established. Inhibition of the PI3K signaling pathway blocks NOX2 activation, superoxide formation, and cell death, without blocking NMDA-induced calcium influx, cell swelling, or mitochondrial depolarization. These findings demonstrate a novel role for PI3K in regulating neuronal superoxide production, and suggest a shared signaling pathway underlying plasticity and excitotoxic death in the nervous system

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