ALZHEIMER'S NEUROFIBRILLARY TANGLES--BIOCHEMICAL STUDIES

这个项目的长期目标是了解病因和 Alzheimer病/Alzheimer's老年性痴呆的发病机制 这是本港其中一个主要的公共卫生问题。 全国有200多万人受到影响。越来越多的证据 表明微管相关蛋白tau异常 在AD脑中磷酸化，并且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）， AD脑内蛋白磷酸化-去磷酸化系统存在缺陷 导致异常磷酸化的tau，和（2）这种异常的 磷酸化至少部分是由于磷蛋白的缺陷 磷酸酶系统针对这一假设，我们建议：（1）确定 在AD和对照脑中， 使用体外磷酸化磷酸化酶激酶和光的磷酸酶 肌球蛋白链作为外源性底物，并在体外磷酸化 正常tau和AD脑异常磷酸化tau作为内源性 底物;（2）从AD和对照中分离磷蛋白磷酸酶 大脑，并确定它们对标准外源性 底物、磷酸化酶激酶和肌球蛋白轻链， PHF，未聚合的异常磷酸化tau，正常tau和体外 （3）产生兔抗体以分离的tau蛋白; 磷酸酶，并确定免疫细胞化学分布， 免疫测定AD不同区域中每种磷酸酶的水平， 对照脑;（4）研究微管蛋白对微管组装的刺激 PHF-tau，未聚合的异常tau和正常tau，前后 用来自特异性目标#2的不同磷酸酶去磷酸化。的 将通过放射性测定法测定特定目标#1中的酶活性 对体外磷酸化[32 P]底物的反应。磷酸酶 将通过组织分级分离从特定目标#1中分离 然后用盐析或乙醇沉淀，液相亲和 色谱法。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。微管的特异性组装 #4将通过浊度测量和负浊度测量来确定 染色电镜。本申请中提出的研究是关于 鉴定导致异常的蛋白磷酸酶 阿尔茨海默病大脑中的磷酸化信息是至关重要的 设计一个合理的方法来纠正这个缺陷。

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.