Delayed mitochondrial dysfunction in excitotoxic neuron death: Cytochrome c release and a secondary increase in superoxide production

An increased production of superoxide has been shown to mediate glutamate-i nduced neuron death. We monitored intracellular superoxide production of hi ppocampal neurons during and after exposure to the glutamate receptor agoni st NMDA (300 mu M). 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 rema ined elevated for 10 min, but then rapidly returned to baseline. Mitochondr ial membrane potential also recovered after wash-out of NMDA. However, reco very of mitochondria was transient and followed by delayed mitochondrial de polarization, loss of cytochrome c, and a secondary rise in superoxide prod uction 4-8 hr after NMDA exposure. Treatment with a superoxide dismutase mi metic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibite d by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relat ionship between cytochrome c release and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (rho(-) cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochrome c, caspase activ ation, and cell death in control and rho(-) cells. However, a delayed incre ase in superoxide production was only observed in control cells. Our data s uggest that the delayed superoxide production in excitotoxicity and apoptos is occurs secondary to a defect in mitochondrial electron transport and tha t mitochondrial cytochrome c release occurs upstream of this defect.