Abnormal Hyperphosphorylation of Tau

Tau 蛋白异常过度磷酸化

• 批准号: 7025063
• 负责人: KHALID IQBAL
• 金额: $ 36.71万
• 依托单位: INSTITUTE FOR BASIC RES IN DEV DISABIL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025063
• 关键词: Alzheimer' s disease; DARPP; SDS polyacrylamide gel electrophoresis; affinity chromatography; cytotoxicity; enzyme inhibitors; human tissue; immunocytochemistry; laboratory rabbit; laboratory rat; microtubule associated protein; molecular cloning; monoclonal antibody; nerve stem cell; neural degeneration; phosphoprotein phosphatase; phosphorylation; postmortem; protein kinase; protein sequence; protein structure function; tau proteins; western blottings

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.

本提案的长期目标是了解神经退行性变的分子机制，然后在此基础上开发阿尔茨海默病（AD）及相关疾病的治疗方法。 我们的工作假设是：（1）AD脑内蛋白磷酸化/去磷酸化失衡，导致tau蛋白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蛋白磷酸酶（PP）-2A和-1的缺陷，这导致微管的破坏，从而损害轴浆流和逆行神经元变性;（3）抑制神经元退行性变可阻止AD的进展。 针对这一假设，我们建议：（1）通过制备兔亲和纯化的抗人脑中PP-2A特异性区域合成肽的抗体，通过柱层析和制备性SDS-PAGE从脑中分离这两种蛋白质，研究脑中PP-2A的两种生理抑制剂I1 PP 2A和I2 PP 2A的结构和活性，I2 PP 2A的氨基酸序列测定，其分子量与肾脏中的I2 PP 2A的分子量有很大差异，从人脑cDNA文库中克隆这些磷酸酶抑制剂，产生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;（2）通过测定AD患者及正常对照组脑组织中PP-2A、PP-1活性的变化，探讨其在AD发病过程中的调节作用。（125 I-免疫斑点法）和活性（通过放射性酶测定）在这些脑的不同区域的细胞核和细胞质区室中的I1 PP 2A、I2 PP 2A和DARPP-32，以及通过抑制剂在AD的各种组织病理学影响和未影响的区域以及对照脑的相应区域中的免疫细胞化学分布和细胞定位;（3）研究PP-2A和PP-2A表达下调对心肌细胞凋亡的影响。1在tau的磷酸化和细胞变性中的活性，通过产生从海马分离和繁殖的稳定转染的神经祖细胞，所述神经祖细胞以可调节的方式过表达磷酸酶抑制剂;这些研究将包括每种磷酸酶抑制剂的过表达对生存力的影响（MTT测定）和细胞形态（相差和免疫荧光），用位点特异性磷酸化依赖性抗体在特定位点磷酸化tau（125 I蛋白质印迹）、通过光散射测定得到的磷酸化tau促进/抑制微管组装的能力以及通过结合和沉降测定螯合正常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。