Noshita N, Sugawara T, Fujimura M, Morita-Fujimura Y, Chan P H
Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, California, U.S.A.
J Cereb Blood Flow Metab. 2001 May;21(5):557-67. doi: 10.1097/00004647-200105000-00010.
Release of cytochrome c from mitochondria to cytosol is a critical step in the mitochondrial-dependent signaling pathways of apoptosis. The authors have reported that manganese superoxide dismutase (Mn-SOD) attenuated cytochrome c release and apoptotic cell death after focal cerebral ischemia (FCI). To investigate downstream to the cytochrome c-dependent pathway, the authors examined caspase-9 activation after transient FCI by immunohistochemistry and Western blotting in both wild-type and Sod2 -/+ mice. Mice were subjected to 60 minutes of middle cerebral artery occlusion followed by 1, 2, 4, or 24 hours of reperfusion. Two hours after reperfusion, cytochrome c and caspase-9 were observed in the cytosol and significantly increased in Sod2 -/+ mutants compared with wild-type mice as shown by Western blotting. Immunofluorescent double labeling for cytochrome c and caspase-9 showed cytosolic cytochrome c 1 hour after transient FCI. Cleaved caspase-9 first appeared in the cytosol at 2 hours and colocalized with cytochrome c. Terminal deoxynucleotidyl transferase-mediated uridine 5;-triphosphate-biotin nick and labeling (TUNEL) showed significant increase of positive cells in Sod2 -/+ mice compared with the wild-type in the cortex, but not in the caudate putamen. The current study revealed Mn-SOD might affect cytochrome c translocation and downstream caspase activation in the mitochondrial-dependent cell death pathway after transient FCI.
细胞色素c从线粒体释放到细胞质是线粒体依赖性凋亡信号通路中的关键步骤。作者报道，锰超氧化物歧化酶（Mn-SOD）可减轻局灶性脑缺血（FCI）后细胞色素c的释放和凋亡性细胞死亡。为了研究细胞色素c依赖性途径的下游情况，作者通过免疫组织化学和蛋白质印迹法，在野生型和Sod2 -/+小鼠中检测了短暂性FCI后caspase-9的激活情况。小鼠接受60分钟的大脑中动脉闭塞，随后再灌注1、2、4或24小时。再灌注2小时后，通过蛋白质印迹法显示，在细胞质中观察到细胞色素c和caspase-9，与野生型小鼠相比，Sod2 -/+突变体中的这些蛋白显著增加。细胞色素c和caspase-9的免疫荧光双重标记显示，短暂性FCI后1小时细胞质中存在细胞色素c。裂解的caspase-9在2小时时首次出现在细胞质中，并与细胞色素c共定位。末端脱氧核苷酸转移酶介导的尿苷5'-三磷酸生物素缺口末端标记（TUNEL）显示，与野生型相比，Sod2 -/+小鼠皮质中的阳性细胞显著增加，但尾状壳核中没有。当前研究表明，Mn-SOD可能会影响短暂性FCI后线粒体依赖性细胞死亡途径中细胞色素c的易位和下游caspase的激活。
