Role of Glia in Neuronal Degeneration (Supplemental Application Binder Proj 2)

描述（由申请人提供）：iNOS的上调及其产物一氧化氮对蛋白酪氨酸的氧化硝化作用是大多数神经退行性疾病的关键特征，尽管这些改变在tau发病机制中的作用尚不清楚。自提交原始提案以来，我们已经大大提高了我们对氧化应激对tau的影响及其在神经退行性过程中的潜在作用的理解，使其成为本提案的主要焦点。在过去的两年中，我们已经确定tau蛋白的五种酪氨酸存在分层硝化作用，并且我们已经开始研究这些位点的硝化作用对tau蛋白聚集成细丝的能力的影响。我们现在建议继续这些正在进行的研究，以进一步了解位点特异性tau硝化在其自我结合到病理内含物及其与自然结合靶标微管的关联中的作用。我们建议研究在阿尔茨海默病进行性大脑的神经元和胶质细胞中硝化tau蛋白的出现

DESCRIPTION (provided by applicant): Upregulation of iNOS and the consequent oxidative nitration of protein tyrosines by its product, nitric oxide, is a key feature of most neurodegenerative diseases, although the role of these alterations in tau pathogenesis is not understood. Since submission of the original proposal, we have significantly advanced our understanding of the effect of oxidative stress on tau and its potential role in the neurodegenerative process, making this a prime focus of the present proposal. Over the past two years, we have determined that there is a hierarchical nitration of the five tyrosines of tau and we have begun to study the effects of nitration at these sites on tau's ability to aggregate into filaments. We now propose to continue these ongoing studies with the goals of further understanding the role of site-specific tau nitration on its self-association into pathological inclusions and on its association with its natural binding target, the microtubule. We propose to study the appearance of nitrated tau in both neurons and glia in the brains of Alzheimer's disease, Progressive Supranuclear Palsy, Corticobasal Degeneration and Pick Disease patients immunohistochemically by producing novel antibodies that recognize only site-specific nitration of tau. Based on results gathered over the past two years, we hypothesize that certain tau nitration events prevent aggregation and cause tau to bind more tightly to microtubules while nitration at other sites are somewhat neutral in their effects. We will test these hypotheses by accomplishing the following specific aims: 1. We will continue to determine the effects of site-specific nitration on the aggregation of tau into filaments using individual tau isoforms nitrated at each of the relevant tyrosine residues. 2. We will determine the effect of nitration at each relevant tyrosine residue on the binding of the six tau isoforms to pre-formed, taxol-stabilized microtubules. This will determine which nitrotyrosine residues are involved in mediating tau's affinity for microtubules. 3. We will produce novel monoclonal antibodies specific for nitrated peptides of tau beginning with those tyrosine residues most readily nitrated. 4. We will stain human brain sections with our novel site-specific nitrotyrosine antibodies to determine when during the course of NFT evolution in AD neuronal tau is nitrated at specific tyrosines. Although it is not possible to do progression studies in PSP, CBD, and PiD, we will also check the glial and neuronal inclusions in these diseases to determine whether similar site-specific alterations occur.