Sugawara Taku, Noshita Nobuo, Lewén Anders, Gasche Yvan, Ferrand-Drake Michel, Fujimura Miki, Morita-Fujimura Yuiko, Chan Pak H
Department of Neurosurgery, Stanford University School of Medicine, Stanford, California 94305-5487, USA.
J Neurosci. 2002 Jan 1;22(1):209-17. doi: 10.1523/JNEUROSCI.22-01-00209.2002.
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.
已知线粒体通过释放细胞色素c、半胱天冬酶-9和第二种线粒体衍生的半胱天冬酶激活剂（Smac）参与细胞凋亡的早期阶段。我们曾报道，铜/锌超氧化物歧化酶（SOD1）的过表达可减少超氧化物的产生，并改善全脑缺血后海马CA1亚区的神经元损伤。然而，缺血/再灌注后产生的氧自由基在线粒体信号通路中的作用尚未阐明。对雄性SOD1转基因（Tg）和野生型（Wt）同窝大鼠进行5分钟的全脑缺血。通过免疫组织化学、蛋白质印迹和半胱天冬酶活性测定来评估细胞色素c和Smac的胞质表达以及半胱天冬酶的激活情况。通过DNA缺口末端和单链DNA标记来表征凋亡细胞死亡。在Wt动物中，缺血后观察到早期超氧化物产生、细胞色素c、Smac的线粒体释放以及半胱天冬酶-9的裂解。随后活性半胱天冬酶-3增加，并且85%的海马CA1神经元在缺血3天后显示出凋亡性DNA损伤。Tg动物显示出较少的超氧化物产生以及细胞色素c和Smac释放。随后活性半胱天冬酶-3的表达不明显，并且只有45%的神经元显示出凋亡性DNA损伤。一种半胱天冬酶-3抑制剂（N-苄氧羰基-缬氨酰-丙氨酰-天冬氨酸-氟甲基酮）仅在Wt动物中减少细胞死亡。这些结果表明，SOD1的过表达降低了氧化应激，从而减弱了细胞色素c和Smac的线粒体释放，导致较少的半胱天冬酶激活和凋亡细胞死亡。氧自由基可能在全脑缺血后海马CA1神经元凋亡细胞死亡的线粒体信号通路中起关键作用。
