Site-Specific Nitration and Oxidative Dityrosine Bridging of the τ Protein by Peroxynitrite: Implications for Alzheimer's Disease^†

Alzheimer's disease (AD) is a progressive amnestic disorder typified by the pathological misfolding and deposition of the microtubule-associated τ protein into neurofibrillary tangles (NFTs). While numerous post-translational modifications influence NFT formation, the molecular mechanisms responsible for τ aggregation remain enigmatic. Since nitrative and oxidative injury have previously been shown to play a mechanistic role in neurodegeneration, we examined whether these events influence τ aggregation. In this report, we characterize the effects of peroxynitrite (ONOO^-)-mediated nitration and oxidation on τ polymerization in vitro. Treatment of τ with ONOO^- results in 3-nitrotyrosine (3-NT) immunoreactivity and the formation of heat-stable, SDS-insoluble oligomers. Using ESI-MS and HPLC with fluorescent detection, we show that these higher-order aggregates contain 3,3‘-dityrosine (3,3‘-DT). Tyrosine (Tyr) residues are critical for ONOO^--mediated oligomerization, as τ proteins lacking all Tyr residues fail to generate oligomers upon ONOO^- treatment. Further, τ nitration targets residues Y18, Y29, and to a lesser degree Y197 and Y394, and nitration at these sites inhibits in vitro polymerization. The inhibitory effect of nitration on τ polymerization is specific for the 3-NT modification, as pseudophosphorylation at these same Tyr residues does not inhibit τ assembly. Our results suggest that the nitrative and oxidative roles of ONOO^- differentially affect τ polymerization and that ONOO^--mediated cross-linking could facilitate τ aggregation in AD.