CELLULAR SIGNALING AND ALZHEIMER-LIKE NEURODEGENERATION

细胞信号传导和阿尔茨海默样神经退行性变

• 批准号: 6098062
• 负责人: MARK P MATTSON
• 金额: $ 20.51万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6098062
• 关键词: Alzheimer's disease; amyloid proteins; antioxidants; antisense nucleic acid; biological signal transduction; calcium transporting ATPase; cell cycle proteins; cell death; embryo /fetus tissue /cell culture; enzyme activity; enzyme induction /repression; free radicals; glutamates; hippocampus; human tissue; laboratory rat; mitogen activated protein kinase; neural degeneration; neuroprotectants; neurotoxins; neurotrophic factors; oxidative stress; phosphorylation; sodium potassium exchanging ATPase; tau proteins; western blottings

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.

该提案测试了有关机制的几个相关假设 与阿尔茨海默病发病机制相关的神经元损伤和死亡 疾病（AD）。 在大鼠海马细胞培养物中进行的研究将 检验：（1）谷氨酸和Abeta损害大脑功能的假设 自由基介导的质膜 Na+/K+ ATP 酶和 Ca2+ ATP 酶 机制涉及肌醇磷脂水解。 这将是 使用灵敏的 ATP 酶活性测定和荧光来完成 成像技术来表征自由基的时间变化 [Na+]i 和 [Ca2+]i 水平。 (2)神经营养因子假说 通过增加保护神经元免受 Abeta 和谷氨酸毒性 抗氧化酶的表达，抑制自由基积累， 并防止 Na+/K+ 和 Ca2+ ATP 酶活性受损。 (3) 的 假设 MAP 激酶介导神经营养因子的影响 抗氧化酶水平、自由基积累、离子损失 体内平衡和细胞死亡。 这将通过操作来完成 使用酪氨酸激酶抑制剂和激活剂测定 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能导致 tau 的定位和磷酸化类似于 神经原纤维缠结 - 神经元将在以下条件下被诱导退化 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剂 AD 控制大脑。 离子动力 ATP 酶受损的假设 将对AD特别脆弱的大脑区域进行测试 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： 海马细胞 文化;突触膜制剂； ATP酶活性测定； 自由基水平和细胞内 Na+ 和 Ca2+ 水平的测量 使用荧光指示剂染料；反义技术来抑制 MAP 激酶的表达；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 有关自由基在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抵抗力的机制 兴奋性毒性和Abeta毒性。 此类机制的相关性 AD的发病机制将通过离子动力研究来确定 AD 死后突触膜中的 ATP 酶并控制人脑。 这项研究可能会确定细胞目标 AD 及相关疾病的预防和治疗干预措施的设计 神经退行性疾病。