Abnormal Hyperphosphorylation of Tau

这项建议的长期目标是了解神经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。我们的工作假说是：(1)AD脑中蛋白质的磷酸化/去磷酸化失衡，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，导致微管破裂，继而轴浆流受损和逆行神经元变性；以及(3)抑制神经纤维变性将阻止AD的进展。针对这一假说，我们建议：(1)从人脑中研究PP-2A的两种生理抑制物I1PP2A和I2PP2A的结构和活性，方法包括：(1)从人脑中产生针对这些蛋白独特区域的合成肽的兔亲和纯化抗体，通过柱层析和制备SDS-PAGE从脑中分离这些蛋白，对与肾脏有很大不同的I2PP2A进行氨基酸序列测定，从人脑cDNA文库中克隆这些磷酸酶抑制物，产生和纯化重组脑抑制物，并通过这些抑制物抑制磷酸酶活性；(2)通过~(125)I-免疫斑点印迹法检测AD和年龄匹配对照脑各核区和胞浆中I1PP2A、I2PP2A和DARPP-32的含量和活性，并通过免疫细胞化学方法和细胞定位，研究PP-2A和PP-1在AD和年龄匹配对照脑组织中的活性调节；以及(3)通过生成稳定表达磷酸酶抑制剂的海马区神经前体细胞，研究PP-2A和PP-1活性下调在tau的磷酸化和细胞变性中的作用；这些研究将包括：每种磷酸酶抑制剂的过表达对细胞存活率(四甲基偶氮唑盐比色试验)和形态(相位对比和免疫荧光)的影响，与位点特异的磷酸化依赖抗体对特定位置tau的磷酸化(125i Western Blots)，通过光散射实验得到的磷酸化的tau促进/抑制微管组装的能力，以及结合和沉淀实验对正常MAP的分离。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.