Kawase M, Fujimura M, Morita-Fujimura Y, Chan P H
Department of Neurosurgery, Neurology, Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Palo Alto, CA, USA.
Stroke. 1999 Feb;30(2):441-8; discussion 449. doi: 10.1161/01.str.30.2.441.
To clarify the relationship between apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, and delayed neuronal cell death associated with apoptosis, we examined the expression of APE/Ref-1 before and after transient global ischemia in rats.
Global ischemia was induced by bilateral common carotid artery occlusion and hypotension. Expression of the APE/Ref-1 protein was evaluated by Western blot and immunohistochemical analyses. Apoptosis after global ischemia was observed by DNA electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining.
Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the control brains. Nuclear immunoreactivity of APE/Ref-1 was significantly decreased 2 days after 10 minutes of ischemia in the hippocampal CA1 subregion. Western blot analysis of a sample from the normal brains showed a characteristic 37-kDa band, which was reduced in the hippocampal CA1 subregion after ischemia. A significant amount of DNA fragmentation was observed at 3 days but not at 1 day after ischemia. Double staining with APE/Ref-1 and TUNEL clearly showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive.
Our data provide evidence that APE/Ref-1 decreased in hippocampal CA1 neurons after transient global ischemia and that this reduction precedes DNA fragmentation, which is destined to cause apoptosis. Our results suggest the possibility that a decrease of APE/Ref-1 activity and the failure of DNA repair may underlie the mechanism of apoptosis after transient focal ischemia.
为阐明DNA碱基切除修复途径中的多功能蛋白脱嘌呤/脱嘧啶内切酶（APE/Ref-1）与凋亡相关的迟发性神经元细胞死亡之间的关系，我们检测了大鼠短暂性全脑缺血前后APE/Ref-1的表达。
通过双侧颈总动脉闭塞和低血压诱导全脑缺血。采用蛋白质免疫印迹法和免疫组织化学分析法评估APE/Ref-1蛋白的表达。通过DNA电泳和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）染色观察全脑缺血后的凋亡情况。
免疫组织化学显示，对照脑内APE/Ref-1呈核表达。缺血10分钟后2天，海马CA1亚区APE/Ref-1的核免疫反应性显著降低。正常脑样本的蛋白质免疫印迹分析显示有一条特征性的37 kDa条带，缺血后海马CA1亚区该条带减少。缺血后3天观察到大量DNA片段化，但1天时未观察到。APE/Ref-1与TUNEL双重染色清楚显示，失去APE/Ref-1免疫反应性的神经元变为TUNEL阳性。
我们的数据表明，短暂性全脑缺血后海马CA1神经元中的APE/Ref-1减少，且这种减少先于注定导致凋亡的DNA片段化。我们的结果提示，APE/Ref-1活性降低和DNA修复失败可能是短暂性局灶性缺血后凋亡机制的基础。
