THE MOLECULAR MECHANISM FOR THE STRESS-INDUCED NEURONAL RESPONSE BY ALCOHOL AND DEPENDENT DRUG.

酒精和依赖性药物引起的应激神经元反应的分子机制。

基本信息

批准号：
15591238
负责人：
SOHMA Hitoshi
金额：
$ 1.92万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Chronic ingestion of ethanol upregulates the Ca^<2+> channel activity, which in turn increases the intracellular Ca^<2+>-concentration, resulting in neuronal cell damage. Intracellular elevation of Ca^<2+> via the activation of Ca^<2+> channel is also revealed in dementia such as Alzheimer's disease (AD), etc. In the present study, it was shown that ethanol exposure to cultured neuronal cells augments annexin A4 expression and accompanied by the activation of NF-κB. Annexin A4 directly bound to NF-κB and the Ca^<2+> binding sites on annexin A4 were involved in this event. We further investigated the quantitative alteration of the secreted proteins by Ca^<2+>-stress with neuronal culture cells. The amounts of secreted proteins were investigated after exposure to Ca^<2+>-ionophre (A23187) under the condition where cell viability was comparable with untreated control. Several proteins were shown to be increased by A23187. After two dimensional-gel electrophoresis, the proteins were identified by LC-MS spectrometry. The data base searching indicated that these three spots are annexin A2, fetuin, annexin A5 and SLUG. These proteins might be biological markers of dementia. We next focused on annexin A5 and analyzed it in blood plasma of dementia patients using sandwich ELISA. It was shown that plasma level of annexin A5 was significantly higher in dementia patients (AD, vascular and alcoholic dementia) than in healthy controls. However, it is hard to detect early stage of AD by this method. The physiological feature of these proteins in the etiology of AD is uncovered in the next step.

慢性摄入乙醇上调CA^<2+>通道活性，进而增加细胞内Ca^<2+> - 浓度，从而导致神经元细胞损伤。在痴呆症（例如阿尔茨海默氏病（AD）等）中，通过激活Ca^<2+>通道的细胞内升高也揭示了本研究中，这表明乙醇暴露于培养的神经元细胞上，增强了附属膜A4的表达并通过NF-κB的激活来实现。膜联蛋白A4直接与NF-κB结合，并参与了膜联蛋白A4上的Ca^<2+>结合位点。我们进一步研究了通过神经元培养细胞的Ca^<2+> - 胁迫对分泌蛋白的定量改变。暴露于Ca^<2+> - 离子磷中（A23187）后，研究了分泌蛋白的量，在细胞生存力与未处理的对照相当的情况下。显示了几种蛋白质增加了A23187。在二维凝胶电泳后，通过LC-MS光谱法鉴定蛋白质。搜索数据库表明这三个斑点是膜联蛋白A2，fetuin，Annexin A5和Slug。这些蛋白质可能是痴呆的生物标志物。接下来，我们专注于膜联蛋白A5，并使用三明治ELISA在痴呆症患者的血浆中进行了分析。结果表明，痴呆症患者（AD，血管和酒精性痴呆症）的血浆A5 A5水平明显高于健康对照。但是，很难通过这种方法检测AD的早期阶段。这些蛋白质在AD的病因中的物理特征在下一步中被发现。