Mitochondrial porin required for ischemia-induced mitochondrial dysfunction and neuronal damage.
The precise molecular events of mitochondrial dysfunction, one of the last steps that irreversibly determines cellular degeneration and death, remain unknown. We introduce a novel strategy to isolate and assess the molecular mechanisms underlying mitochondrial dysfunction. Using an in vitro ischemia model, we obtained evidence for prolonged mitochondrial depolarization in rat organotypic hippocampal brain slices during reperfusion. Then, mitochondria were isolated from brain slices and mitochondrial proteins were purified on a cyclosporin-A affinity column. Cyclosporin-A is the most potent inhibitor of mitochondrial dysfunction, in particular the mitochondrial permeability transition, and therefore we hypothesized that it may interact with proteins involved in the permeability transition after mitochondria were subjected to manipulations that promote this event. Mitochondrial porin was reproducibly eluted from the affinity column using proteins from ischemic brain mitochondria, or from mitochondria exposed to oxidative stress that were used as a positive control. Anti-porin antibodies prevented mitochondrial depolarization and electrophysiological deterioration of hippocampal neurons during hypoxia-reperfusion, as measured by simultaneous fluorescence imaging and whole-cell recordings. These observations provide biochemical and functional evidence that porin is directly involved in mitochondrial dysfunction and neuronal impairment during ischemia-reperfusion, and indicate that porin could be a novel therapeutic target to prevent cellular degeneration.
Perez Velazquez
J L
JL
Playfair Neuroscience Unit, Department of Medicine (Neurology), Toronto Western Hospital, University of Toronto, 399 Bathurst Street, Toronto, Canada. jlpv@playfair.utoronto.ca
Frantseva
M V
MV
Huzar
D V
DV
Carlen
P L
PL
D000818:Animals; D002545:Brain Ischemia / Q000503:physiopathology; D002458:Cell Fractionation; D004622:Embryo, Mammalian; D006624:Hippocampus / Q000502:physiology* / Q000503:physiopathology; D008297:Male; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000473:pathology / Q000502:physiology*; D009924:Organ Culture Techniques; D010539:Permeability; D018272:Porins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000503:physiopathology*; D050994:Voltage-Dependent Anion Channels
D009419:Nerve Tissue Proteins; D018272:Porins; D050994:Voltage-Dependent Anion Channels