Peroxynitrite-mediated τ modifications stabilize preformed filaments and destabilize microtubules through distinct mechanisms

Alzheimer's disease (AD) is a progressive amnestic dementia typified by abnormal modifications of the microtubule (MT)-associated τ protein that promote its pathological self-assembly and displacement from the MT lattice. Previously, we showed that peroxynitrite (ONOO^-) induces the oxidative 3,3′-dityrosine (3,3′-DT) cross-linking and site-selective nitration of τ monomers [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we examined the effects of ONOO ^--mediated modifications on two key elements of τ pathobiology: (1) the stability of preformed τ filaments and (2) the ability of monomeric τ to promote tubulin assembly. Here, we report that treatment of synthetic τ filaments with ONOO^- generates heat-stable, SDS-insoluble aggregates with a significantly reduced mobility by SDS-PAGE compared to that of nontreated filaments. Ultrastructurally, these aggregates appear to be cross-linked via interfilament bridges. Using LC-MS/MS and HPLC with fluorescent detection, we demonstrate that covalent 3,3′-DT linkages are present within these higher-order aggregates. Similar to monomeric τ, filamentous τ exhibits a hierarchical pattern of nitration following ONOO^- treatment with site selectivity toward the amino-terminal residues Tyr18 and Tyr29. Further, select nitration of residues Tyr18, Tyr29, Tyr197, and Tyr394, events known to stabilize the pathological Alz-50 conformation [Reynolds et al. (2005) Biochemistry 44, 13997-14009], inhibits the ability of monomeric τ to promote tubulin assembly. This effect is specific for the 3-NT modification, as mutant τ proteins pseudophosphorylated at each Tyr residue are fully competent to stabilize MTs. Collectively, our results suggest that ONOO ^--mediated modifications stabilize τ filaments via 3,3′-DT bonding and destabilize MTs by site-selective nitration of τ monomers. Moreover, assumption of the Alz-50 conformation may be the mechanism through which τ nitration modulates MT stability.