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

长期摄入乙醇可上调Ca^2+通道活性，进而增加细胞内Ca^2+浓度，导致神经元细胞损伤。在阿尔茨海默病（AD）等痴呆症中也发现了通过激活Ca^<2+>通道而引起的细胞内Ca^<2+>升高。在本研究中，发现乙醇暴露于培养的神经元细胞增加了膜联蛋白A4的表达，并伴随着NF-κB的激活。膜联蛋白A4直接与NF-κB结合，膜联蛋白A4上的Ca^2+结合位点参与了这一过程。我们进一步用神经元培养细胞研究了Ca^<2+>-应激对分泌蛋白的定量改变。在细胞活力与未处理对照相当的条件下，研究了暴露于Ca^2+-离子载体（A23187）后分泌蛋白的量。A23187可增加几种蛋白质。双向凝胶电泳后，通过LC-MS光谱鉴定蛋白质。数据库检索表明，这三个点是annexin A2，胎球蛋白，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，并使用夹心ELISA分析痴呆患者血浆中的膜联蛋白A5。结果显示，痴呆患者（AD、血管性痴呆和酒精性痴呆）的血浆膜联蛋白A5水平显著高于健康对照。然而，很难通过这种方法检测早期AD。这些蛋白质在AD病因学中的生理特征将在下一步中被揭示。

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.