Nox4-dependent H2O2 production contributes to chronic glutamate toxicity in primary cortical neurons

Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H(2)O(2)) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca(2+)-free medium or treated with BAPTA showed reduced glutamate-induced H(2)O(2) generation, indicating that H(2)O(2) generation is Ca(2+)-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H(2)O(2) generation and that activation of NMDA and AMPA receptors is involved in this H(2)O(2) generation. The Nox4 siRNA reduced NMDA-induced H(2)O(2) production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H(2)O(2) production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases.