ALZHEIMER'S NEUROFIBRILLARY TANGLES--BIOCHEMICAL STUDIES

这个项目的长期目标是学习病因和 阿尔茨海默病/老年性痴呆的发病机制 类型(AD)构成了我们的主要公共卫生问题之一 自那以来，有200多万人受到影响。越来越多的证据 提示微管相关蛋白tau异常 AD脑中的磷酸化，是阿尔茨海默病的主要成分 成对螺旋丝(PHF)。我们的工作假设是(1) 阿尔茨海默病患者脑内蛋白质磷酸化-去磷酸化系统缺陷 导致异常磷酸化的tau，以及(2)这种异常 磷酸化至少部分是由于磷蛋白的缺失。 磷酸酶系统。对于这一假设，我们建议：(1)确定 阿尔茨海默病患者和对照组脑组织中磷蛋白的活性水平 使用体外磷酸化磷酸化酶激酶和LIGH的磷酸酶 肌球蛋白链作为外源底物，并在体外磷酸化 正常tau和AD脑内异常磷酸化tau 底物；(2)分离AD和对照的磷酸化蛋白磷酸酶 并测定它们对标准外源的酶动力学 肌球蛋白的底物、磷酸化酶和轻链及朝向 PHF，未聚合，异常磷酸化的tau，正常tau和体外 磷酸化tau蛋白；(3)产生兔抗体以分离 磷酸酶，并确定免疫细胞化学分布，以及 免疫测定法检测AD患者不同脑区各磷酸酶水平 对照大脑；(4)微管蛋白刺激微管组装的研究 在PHF-tau、未聚合的异常tau和正常tau的前后 用来自特定目标2的不同的磷酸酶去磷酸化。 特定目标#1的酶活性将用放射计量学方法进行测定 朝向体外磷酸化的[32P]底物。磷酸酶 从特定目标#1指示的将通过组织分离来分离 其次是盐渍或乙醇沉淀，液体和亲和力 色谱图。磷酸酶的免疫细胞化学分布(目标3) 将通过光学显微镜进行研究。特定靶点的微管组装 #4将由浊度测量和负值两种方法确定 染色电子显微镜。本申请中建议的研究是关于 导致异常的蛋白磷酸酶/S的鉴定 阿尔茨海默病大脑中哪些信息是关键信息 设计一种合理的方法来纠正这一缺陷。

The long term objective of this project is to learn the etiology and the pathogenesis of Alzheimer's disease/senile dementia of the Alzheimer's type (AD) which constitutes one of the major public health problems in our country since over two million people are affected. Increasing evidence suggests that microtubule-associated protein tau is abnormally phosphorylated in AD brain and is a major component of the Alzheimer paired helical filaments (PHF). Our working hypothesis is (1) that the protein phosphorylation-dephosphorylation system is defective in AD brain leading to abnormally phosphorylated tau, and (2) that this abnormal phosphorylation is at least partly due to a deficit in the phosphoprotein phosphatase system. Towards this hypothesis we propose to: (1) determine in AD and control brains the levels of activities of phosphoprotein phosphatases using in vitro phosphorylated phosphorylase kinase and light chain of myosin as exogenous substrates, and in vitro phosphorylated normal tau and AD brain abnormally phosphorylated tau as endogenous substrates; (2) isolate phosphoprotein phosphatases from AD and control brains and determine their enzyme kinetics towards standard exogenous substrates, phosphorylase kinase and light chain of myosin and towards PHF, unpolymerized ,abnormally phosphorylated tau, normal tau and in vitro phosphorylated tau; (3) generate rabbit antibodies to isolated phosphatases, and determine immunocytochemical distribution, and immunoassay the levels of each phosphatase in various areas of AD and control brains; (4) study stimulation of microtubule assembly from tubulin with PHF-tau, unpolymerized abnormal tau, and normal tau, before and after dephosphorylation with different phosphatases from Specific Aim #2. The enzyme activities in Specific Aim #1 will be assayed radiometrically towards in vitro phosphorylated [32P] substrates. The phosphatases indicated from Specific Aim #1 will be isolated by tissue fractionation followed by salting or ethanol precipitation, liquid and affinity chromatographies. Immunocytochemical distribution of phosphatases (Aim #3) will be studied by light microscopy. Microtubule assembly in Specific Aim #4 will be determined both by turbidimetric measurements and by negative stain electron microscopy. Studies proposed in this application are on the identification of the protein phosphatase/s responsible for the abnormal phosphorylation in Alzheimer disease brain which information is critical to devise a rational approach in correcting this defect.