Abnormal Hyperphosphorylation of Tau

本研究的长期目标是了解神经原纤维变性的分子机制，然后在此基础上开发阿尔茨海默病（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和其他一些神经元蛋白异常过度磷酸化；(2)这种缺陷部分是由于磷酸蛋白磷酸酶(PP) -2A和-1的缺陷导致微管的破坏，从而导致轴浆血流受损和逆行性神经元变性；(3)抑制神经原纤维变性可以阻止AD的进展。针对这一假设，我们提出：(1)通过生成兔亲和纯化抗体合成与PP-2A蛋白独特区域对应的肽段，对人脑中两种PP-2A生理抑制剂I1PP2A和I2PP2A的结构和活性进行研究，并通过柱层析和制备SDS-PAGE从人脑中分离出这两种蛋白，对与肾脏中分子质量差异很大的I2PP2A进行氨基酸测序。从人脑cDNA文库中克隆这些磷酸酶抑制剂，生成和纯化重组脑抑制剂，以及这些抑制剂对磷酸酶活性的抑制作用；(2)通过测定AD和年龄匹配对照脑核区和胞质区I1PP2A、I2PP2A和DARPP-32的水平（采用125i -免疫斑点法）和活性（采用放射酶测定法），以及抑制剂在AD各组织病理病变区和未病变区及对照脑相应区域的免疫细胞化学分布和细胞定位，研究AD和年龄匹配对照脑中PP-2A和PP-1活性的调控；(3)通过产生稳定转染的海马分离和繁殖的神经祖细胞，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；这些研究将包括每个磷酸酶抑制剂的过表达对细胞活力（MTT测定）和细胞形态（相对比和免疫荧光）的影响，使用特异性磷酸化依赖抗体在特定位点磷酸化tau蛋白（125I Western blots），通过光散射测定产生的磷酸化tau蛋白促进/抑制微管组装的能力，以及通过结合和沉淀测定对正常MAPS的隔离。这些研究将有助于阐明PP-2A-和PP-1-抑制剂在AD中发生的tau异常过度磷酸化和神经退行性变中的作用。

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.