Abnormal Hyperphosphorylation of Tau

该提案的长期目标是了解神经原纤维变性的分子机制，然后根据这些知识开发阿尔茨海默病（AD）和相关疾病的治疗方法。 我们的工作假设是（1）AD大脑中蛋白质磷酸化/去磷酸化不平衡，导致tau蛋白和其他一些神经元蛋白质异常过度磷酸化； (2) 这种缺陷部分是由于磷蛋白磷酸酶 (PP) -2A 和 -1 的缺陷导致微管破裂，从而导致轴浆流受损和逆行神经元变性； (3)抑制神经原纤维变性将阻止AD的进展。 针对这一假设，我们建议：（1）研究人脑中两种 PP-2A 生理抑制剂（称为 I1PP2A 和 I2PP2A）的结构和活性，方法是生成针对与这些蛋白质的独特区域相对应的合成肽的兔亲和纯化抗体，通过柱色谱法和制备型 SDS-PAGE 从脑中分离这些蛋白质，对 I2PP2A 进行氨基酸测序（其分子量与肾脏中的分子量差异很大），克隆这些蛋白质来自人脑cDNA文库的磷酸酶抑制剂，重组脑抑制剂的产生和纯化以及这些抑制剂对磷酸酶活性的抑制； (2) 通过测定这些大脑不同区域的核和细胞质区室中 I1PP2A、I2PP2A 和 DARPP-32 的水平（通过 125I 免疫点印迹）和活性（通过放射性酶测定），以及通过抑制剂在不同部位的免疫细胞化学分布和细胞定位，研究 AD 和年龄匹配对照大脑中 PP-2A 和 PP-1 活性的调节。 AD 中受组织病理学影响和未受影响的区域以及对照大脑的相应区域； (3) 通过生成从海马分离和增殖的稳定转染的神经祖细胞，以可调节的方式过度表达磷酸酶抑制剂，研究 PP-2A 和 PP-1 活性下调对 tau 磷酸化和细胞变性的影响；这些研究将包括每种磷酸酶抑制剂的过表达对细胞活力（MTT 测定）和形态（相差和免疫荧光）的影响，使用位点特异性磷酸化依赖性抗体在特定位点磷酸化 tau（125I 蛋白质印迹），通过光散射测定产生的磷酸化 tau 促进/抑制微管组装的能力，以及通过结合和沉降测定隔离正常 MAPS。 这些研究将有助于阐明 PP-2A 和 PP-1 抑制剂在 tau 异常过度磷酸化和 AD 中发生的神经变性中的作用。

The long term objective of this proposal is to understand the molecular mechanism of neurofibrillary degeneration and then based on this knowledge develop therapeutic treatment for Alzheimer disease (AD) and related disorders. Our working hypothesis is (1) that the protein phosphorylation/dephosphorylation is imbalanced in AD brains, leading to abnormal hyperphosphorylation of tau and some other neuronal proteins; (2) that this defect is in part due to a deficit in the phosphoprotein phosphatase (PP) -2A and -1 which leads to the breakdown of microtubules and consequent impairment of axoplasmic flow and retrograde neuronal degeneration; and (3) that inhibition of neurofibrillary degeneration will arrest the progression of AD. Towards this hypothesis we propose to: (1) study the structures and the activities of the two physiological inhibitors of PP-2A, called I1PP2A and I2PP2A from human brain by generation of rabbit affinity purified antibodies to synthetic peptides corresponding to the unique regions of these proteins, isolation of these proteins from brain by column chromatographies and preparative SDS-PAGE, amino acid sequencing of I2PP2A which very much differs in its molecular mass from that in kidney, cloning of these phosphatase inhibitors from a human brain cDNA library, generation and purification of the recombinant brain inhibitors and inhibition of the phosphatase activities by these inhibitors; (2) study the regulation of the activities of PP-2A and PP-1 in AD and age-matched control brains by assaying the levels (by 125I-immuno-dot-blots) and the activities (by radiometric enzyme assays) of I1PP2A, I2PP2A and DARPP-32 in nuclear and cytoplasmic compartments of various areas of these brains, and by immunocytochemical distribution and cellular localization of the inhibitors in various histopathologically affected and unaffected areas of AD and corresponding areas of control brains; and (3) study the effect of the downregulation of PP-2A and PP-1 activities in the phosphorylation of tau and cellular degeneration by generating stably transfected neural progenitor cells isolated and propagated from hippocampus that overexpress the phosphatase inhibitors in a regulatable manner; these studies will include the effect of the overexpression of each phosphatase inhibitor on viability (MTT assay) and morphology of the cells (phase contrast and immunofluorescence), phosphorylation of tau at specific sites with site specific phosphorylation dependent antibodies (125I Western blots), ability of the resulting phosphorylated tau to promote/inhibit microtubule assembly by light scattering assay and sequestration of normal MAPS by binding and sedimentation assays. These studies will help elucidate the role of PP-2A- and PP-1- inhibitors in the abnormal hyperphosphorylation of tau and the neurodegeneration that occurs in AD.