Department of Physiology, Biomedical Science Institute and Medical Research Center, School of Medicine, Kyung Hee University, Seoul, 130-701, South Korea.
Neurochem Res. 2013 Aug;38(8):1648-60. doi: 10.1007/s11064-013-1067-3. Epub 2013 May 14.
To elucidate the molecular events involved in early ischemic neuronal death, we performed two-dimensional proteome profiling of primary cultures of rat cortical neurons following chemical ischemia induced by the administration of sodium azide under glucose-free conditions. Using a lactic dehydrogenase assay and Western blot analysis of dephosporylation of the voltage-gated potassium channel Kv2.1, we determined duration of chemical ischemia of 2 h to be the relevant time-point for early ischemic neuronal death. Sixty-one proteins were differentially expressed, and 26 different proteins were identified by MALDI-TOF with Mascot database searching. The proteome data indicated that chemical ischemia altered the expression of 20 proteins that are involved in stress response/chaperone, brain development, cytoskeletal/structural proteins, metabolic enzymes, and calcium ion homeostasis. Western blotting and immunocytochemical studies of the 6-most functionally significant proteins showed that, in the ischemia-treated group, the expression of glucose-related protein 78, heat shock protein 90 alpha, and α-enolase was significantly increased, while the expression of inositol triphosphate receptor 1 and ATP synthase beta subunit was decreased. In addition, the expression of dihydropyrimidinase-like 3 showed a truncated pattern in the ischemia group. The changes in the expression of these proteins might be significant indicators of early ischemic neuronal death.
为了阐明早期缺血性神经元死亡所涉及的分子事件，我们在无糖条件下给予叠氮化钠诱导化学性缺血后，对原代培养的大鼠皮质神经元进行了二维蛋白质组谱分析。通过乳酸脱氢酶测定和电压门控钾通道 Kv2.1 的去磷酸化的 Western blot 分析，我们确定 2 小时的化学性缺血是早期缺血性神经元死亡的相关时间点。有 61 种蛋白质表达差异，通过 MALDI-TOF 并用 Mascot 数据库搜索鉴定了 26 种不同的蛋白质。蛋白质组数据表明，化学性缺血改变了与应激反应/伴侣蛋白、脑发育、细胞骨架/结构蛋白、代谢酶和钙离子稳态相关的 20 种蛋白质的表达。对 6 种最具功能意义的蛋白质的 Western blot 和免疫细胞化学研究表明，在缺血处理组中，葡萄糖相关蛋白 78、热休克蛋白 90α 和α-烯醇酶的表达显著增加，而三磷酸肌醇受体 1 和 ATP 合酶β亚基的表达减少。此外，在缺血组中，二氢嘧啶酶样 3 的表达呈截断模式。这些蛋白质表达的变化可能是早期缺血性神经元死亡的重要指标。
