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Delayed mitochondrial dysfunction in excitotoxic neuron death: cytochrome c release and a secondary increase in
C M Luetjens1, N T Bui, B Sengpiel
1Interdisciplinary Center for Clinical Research, Research Group "Apoptosis and Cell Death", Westphalian Wilhelms-University, Münster, Germany.
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 microm). 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 cytochrome c, 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 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 activation, and cell death in control and rho(-) 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 cytochrome c release occurs upstream of this defect.
Insights
Glutamate exposure increases superoxide production, leading to neuron death. A delayed rise in superoxide, linked to mitochondrial dysfunction, also contributes to cell death in excitotoxicity and apoptosis.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Glutamate receptor overactivation can cause neuron death.
- Superoxide production is implicated in excitotoxicity.
Purpose of the Study:
- To investigate the role of superoxide production in NMDA-induced excitotoxicity.
- To explore the mechanisms underlying delayed superoxide production and its link to mitochondrial function.
Main Methods:
- Monitoring intracellular superoxide production in hippocampal neurons exposed to NMDA.
- Assessing mitochondrial membrane potential, cytochrome c release, and caspase activation.
- Utilizing rotenone and antimycin A to probe mitochondrial electron transport chain involvement.
- Comparing superoxide production in control and respiration-deficient cells during apoptosis induction.
Main Results:
- NMDA exposure caused a sustained increase in superoxide production.
- Delayed mitochondrial depolarization and cytochrome c release preceded a secondary rise in superoxide.
- Inhibition of mitochondrial complex I or III affected superoxide production.
- Delayed superoxide increase was observed in control cells but not in respiration-deficient cells during staurosporine treatment.
Conclusions:
- Delayed superoxide production in excitotoxicity and apoptosis is secondary to mitochondrial electron transport defects.
- Mitochondrial cytochrome c release occurs upstream of this defect.
- Targeting superoxide production can protect neurons from excitotoxicity.