CELLULAR SIGNALING AND ALZHEIMER-LIKE NEURODEGENERATION

该提案测试了关于 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 测试：（1）假设谷氨酸和Abeta损害功能， 质膜Na+/K+ ATP酶和Ca ~（2+）ATP酶的活性 涉及肌醇磷脂水解的机制。 这将是 使用敏感的ATP酶活性测定和荧光 表征自由基时间变化的成像技术 水平[Na+]i和[Ca 2 +]i。(2)假设神经营养因子 保护神经元免受Abeta和谷氨酸毒性，通过增加 抗氧化酶的表达，抑制自由基积累， 防止Na+/K+和Ca 2 + ATP酶活性受损。(3)的 假设MAP激酶介导神经营养因子对 抗氧化酶水平，自由基积累，离子损失 稳态和细胞死亡。 这将通过操纵 使用酪氨酸激酶抑制剂和激活剂的MAP激酶活性， 和反义寡脱氧核苷酸以消耗MAP激酶水平。(4)的 假设神经退行性和 神经保护信号通路可导致 tau蛋白的定位和磷酸化类似于 神经元缠结-神经元将被诱导退化， MAP激酶被激活的条件。 最终目标将采用 来自脆弱和非脆弱区域的突触膜制备物 AD控制组的大脑 离子运动型ATP酶受损的假说 在大脑中特别容易受到AD影响的区域将被测试， 将确定Abeta对ATP酶活性的影响。 这 研究将采用一系列技术，包括： 培养;突触膜制备; ATP酶活性测定; 自由基水平和细胞内Na+和Ca 2+水平的测量 使用荧光指示剂染料;反义技术抑制 MAP激酶表达;蛋白质印迹分析;色谱分析 肌醇磷脂代谢物。 生成的数据将提供 关于自由基作用的基本信息 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 兴奋性毒性和Abeta毒性。 这些机制与下列方面的相关性： AD的发病机制将在离子动力学的研究中建立， 死后AD和对照人脑突触膜中的ATP酶。 这项研究可能会确定细胞靶点，以瞄准在 设计AD及相关疾病的预防和治疗干预措施 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.