CELLULAR SIGNALING AND ALZHEIMER-LIKE NEURODEGENERATION

这一提议检验了几个与此相关的假说。 阿尔茨海默病发病机制中的神经元损伤和死亡 疾病(AD)。在大鼠海马细胞培养中进行的研究将 检验：(1)假设谷氨酸和β-天冬氨酸会损害脑细胞的功能。 自由基介导的质膜Na~+/K~+-ATPase和Ca~(2+)-ATPase 涉及肌醇磷脂水解的机制。这将是 使用灵敏的ATPase活性分析和荧光 表征自由基时间变化的成像技术 [Na+]i和[Ca2+]i。(2)神经营养因子的假说 通过增加Abeta和谷氨酸的毒性保护神经元 抗氧化酶的表达，抑制自由基的积累， 防止Na+/K+和Ca~(2+)-ATPase活性受损。(3) 关于MAP激酶介导神经营养因子影响的假说 抗氧化酶水平、自由基积聚、离子损失 动态平衡和细胞死亡。这将使用操作来完成 使用酪氨酸激酶抑制剂和激活剂的MAP激酶活性， 和反义寡核苷酸以耗尽MAP激酶水平。(4) 一种假说是伴随着神经退行性变和 神经保护性信号通路可导致脑内 Tau的定位和磷酸化类似于在 神经原纤维缠结-神经元将被诱导退化 激活贴图激活酶的条件。最终目标将使用 易损区和非易损区的突触膜制备 控制阿尔茨海默病的大脑。离子动力ATPase受损的假说 在阿尔茨海默病特别脆弱的大脑区域将进行测试和 Abeta对ATPase活性的影响将被确定。这 这项研究将采用一系列技术，包括：海马细胞 培养；突触膜制剂；ATPase活性测定； 自由基水平和细胞内Na+、Ca~(2+)水平的测定 使用荧光指示剂染料；反义技术抑制 MAP激酶的表达；蛋白质印迹分析；层析分析 肌醇磷脂代谢物。生成的数据将提供 关于自由基在人体内的作用的基本信息 谷氨酸和Abeta对离子稳态机制的损害，以及 神经营养因子增加神经元对药物敏感性的机制 兴奋性毒性和Aβ毒性。这些机制与以下方面的相关性 阿尔茨海默病的发病机制将在离子动力研究中确定 死后AD和对照人脑突触膜中的ATPase。 这项研究很可能确定细胞目标，以在 阿尔茨海默病及相关疾病的预防和治疗干预设计 神经退行性疾病。

This proposal tests several related hypotheses concerning mechanisms of neuronal injury and death relevant to the pathogenesis of Alzheimer's disease (AD). Studies performed in rat hippocampal cell cultures will test: (1) The hypothesis that glutamate and Abeta impair function of the plasma membrane Na+/K+ ATPase and Ca2+ ATPase by a free radical-mediated mechanism involving inositol phospholipid hydrolysis. This will be accomplished using sensitive ATPase activity assays, and fluorescence imaging technologies to characterize temporal changes in free radical levels [Na+]i, and [Ca2+]i. (2) The hypothesis that neurotrophic factors protect neurons against Abeta and glutamate toxicities by increasing expression of antioxidant enzymes, suppressing free radical accumulation, and preventing impairment of Na+/K+ and Ca2+ ATPase activities. (3) The hypothesis that MAP kinases mediate the effects of neurotrophic factors on antioxidant enzyme levels, free radical accumulation, loss of ion homeostasis and cell death. This will be accomplished using manipulations of MAP kinase activity using tyrosine kinase inhibitors and activators, and antisense oligodeoxynucleotides to deplete MAP kinase levels. (4) The hypothesis that concomitant activation of neurodegenerative and neuroprotective signaling pathways can result in alterations in localization and phosphorylation of tau similar to those seen in neurofibrillary tangles - neurons will be induced to degenerate under conditions where MAP kinases are activated. The final aim will employ synaptic membrane preparations from vulnerable and non-vulnerable regions of AD control brains. The hypothesis that ion-motive ATPases are impaired in brain regions that are particularly vulnerable in AD will be tested and the effects of Abeta on ATPase activities will be determined. This research will employ a battery of technologies including: hippocampal cell culture; synaptic membrane preparations; ATPase activity assays; measurements of free radical levels and intracellular Na+ and Ca2+ levels using fluorescent indicator dyes; antisense techniques to suppress expression of MAP kinases; Western blot analysis; chromatographic analyses of inositol phospholipid metabolites. The data generated will provide fundamental information concerning the roles of free radicals in the impairment of ion homeostatic mechanisms by glutamate and Abeta, and the mechanism whereby neurotrophic factors increase resistance of neurons to excitotoxicity and Abeta toxicity. The relevance of such mechanisms to the pathogenesis of AD will be established in studies of ion-motive ATPases in synaptic membranes from postmortem AD and control human brains. This research is likely to identify cellular targets to aim at in the design of prophylactic and therapeutic interventions in AD and related neurodegenerative disorders.