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: 6457020
• 负责人: MARK P MATTSON
• 金额: $ 25.46万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6457020
• 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.

该提案检验了几种有关机制的相关假设 与阿尔茨海默氏病的发病机理有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 测试：（1）谷氨酸和Abeta损害功能的假设 质膜Na+/K+ ATPase和Ca2+ ATPase通过自由基介导的 涉及肌醇磷脂水解的机制。 这将是 使用敏感的ATPase活性测定和荧光完成 成像技术表征自由基的时间变化 级别[Na+] i和[Ca2+] i。 （2）神经营养因素的假设 通过增加神经元免受ABETA和谷氨酸毒性的侵害 抗氧化剂的表达，抑制自由基积累， 并防止Na+/K+和Ca2+ ATPase活动的损害。 （3） MAP激酶介导神经营养因素对的假设 抗氧化剂酶水平，自由基积累，离子损失 稳态和细胞死亡。 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激酶活性的 和反义寡脱氧核苷酸以耗尽地图激酶水平。 （4） 假设神经退行性的激活和 神经保护信号通路可能会导致改变 Tau的定位和磷酸化类似于 神经原纤维缠结 - 神经元将被诱导在下面 激活MAP激酶的条件。 最终目标将采用 来自脆弱和不可吸引区域的突触膜制剂 广告控制大脑。 关于离子 - 摩托ATPases受损的假设 在广告中特别容易受到攻击的大脑区域中，将进行测试，并且 将确定Abeta对ATPase活动的影响。 这 研究将采用一系列技术，包括：海马细胞 文化;突触膜制剂； ATPase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；抑制反义技术 地图激酶的表达； Western印迹分析；色谱分析 肌醇磷脂代谢产物。 生成的数据将提供 关于自由基在 谷氨酸和ABETA对离子稳态机制的损害，以及 神经营养因素增加神经元的抗性的机制 兴奋性和ABETA毒性。 这种机制与 AD的发病机理将在离子 - 动力的研究中建立 来自验尸广告和控制人大脑的突触膜中的ATPases。 这项研究可能会确定要针对的细胞靶标 AD和相关的预防性和治疗干预措施的设计 神经退行性疾病。