Delayed Mitochondrial Dysfunction in Excitotoxic Neuron Death: CytochromecRelease and a Secondary Increase in Superoxide Production

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Abstract
An increased production of superoxide has been shown to mediate glutamate-induced neuron death. We monitored intracellular superoxide production of hippocampal neurons during and after exposure to the glutamate receptor agonist NMDA (300 μm). During a 30 min NMDA exposure, intracellular superoxide production increased significantly and remained elevated for several hours after wash-out of NMDA. After a 5 min exposure, superoxide production remained elevated for 10 min, but then rapidly returned to baseline. Mitochondrial membrane potential also recovered after wash-out of NMDA. However, recovery of mitochondria was transient and followed by delayed mitochondrial depolarization, loss of cytochromec, and a secondary rise in superoxide production 4–8 hr after NMDA exposure. Treatment with a superoxide dismutase mimetic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibited by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relationship between cytochromecrelease and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (ρ−cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochromec, caspase activation, and cell death in control and ρ−cells. However, a delayed increase in superoxide production was only observed in control cells. Our data suggest that the delayed superoxide production in excitotoxicity and apoptosis occurs secondary to a defect in mitochondrial electron transport and that mitochondrial cytochromecrelease occurs upstream of this defect.