Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation.
Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.
Sugawara
Taku
T
Department of Neurosurgery, Stanford University School of Medicine, Stanford, California 94305-5487, USA.
Noshita
Nobuo
N
Lewén
Anders
A
Gasche
Yvan
Y
Ferrand-Drake
Michel
M
Fujimura
Miki
M
Morita-Fujimura
Yuiko
Y
Chan
Pak H
PH
D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D002545:Brain Ischemia / Q000201:enzymology* / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D019610:Cytoprotection / Q000502:physiology*; D004249:DNA Damage / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006624:Hippocampus / Q000098:blood supply / Q000201:enzymology / Q000473:pathology; D007150:Immunohistochemistry; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000378:metabolism; D009474:Neurons / Q000201:enzymology* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics; D000072105:Superoxide Dismutase-1; D013481:Superoxides / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; C489013:DIABLO protein, rat; D004791:Enzyme Inhibitors; D024101:Mitochondrial Proteins; D013481:Superoxides; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases