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. ? ? ?
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