The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrial cytochrome c in ischemic brain after transient focal cerebral ischemia in mice.
Release of mitochondrial cytochrome c into the cytosol is a critical step in apoptosis. We have reported that early release of cytochrome c in vivo occurs after permanent focal cerebral ischemia (FCI) and is mediated by the mitochondrial antioxidant manganese superoxide dismutase (SOD). However, the role of reactive oxygen species produced after ischemia-reperfusion in the mitochondrial apoptosis process is still unknown, although overexpression of copper/zinc-SOD (SOD1), a cytosolic isoenzyme, protects against ischemia-reperfusion. We now hypothesize that the overexpression of SOD1 also prevents apoptosis after FCI. To address this issue, we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and in SOD1 transgenic (Tg) mice after transient FCI. Cytosolic cytochrome c was detected as early as 2 hr after reperfusion, and correspondingly, mitochondrial cytochrome c was significantly reduced after FCI. Cytosolic cytochrome c was significantly lower in the SOD1 Tg mice compared with wild types 2 (p < 0.0001) and 4 (p < 0.05) hr after FCI. Apaf-1, which interacts with cytochrome c and activates caspases, was constitutively expressed in both groups of animals, with no alteration after FCI. Double staining with cytochrome c immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between cytosolic cytochrome c expression and DNA fragmentation. A significant amount of DNA laddering was detected 24 hr after ischemia and was reduced in SOD1 Tg mice. These data suggest that SOD1 blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after transient FCI.
Fujimura
M
M
Department of Neurosurgery, Program in Neurosciences, Stanford University School of Medicine, Palo Alto, California 94304, USA.
Morita-Fujimura
Y
Y
Noshita
N
N
Sugawara
T
T
Kawase
M
M
Chan
P H
PH
D000818:Animals; D053477:Apoptotic Protease-Activating Factor 1; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D053938:DNA Fragmentation / Q000502:physiology; D006801:Humans; D002546:Ischemic Attack, Transient / Q000201:enzymology*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000201:enzymology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000276:immunology / Q000378:metabolism*; D000072105:Superoxide Dismutase-1
C506592:APAF1 protein, human; C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D011506:Proteins; C000606290:SOD1 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1