Peroxynitrite-Mediated tau Modifications Stabilize Preformed Filaments and Destabilize Microtubules through Distinct Mechanisms

摘要

Alzheimer's disease (AD) is a progressive amnestic dementia typified by abnormal modifications of the microtubule (MT)-associated r 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 T 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 r pathobiology:(1) the stability of preformed tau filaments and (2) the ability of monomeric tau to promote tubulin assembly.Here,we report that treatment of synthetic tau 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 tau,filamentous tau 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,Tyrl97,and Tyr394,events known to stabilize the pathological Alz-50 conformation [Reynolds et al.(2005) Biochemistry 44,13997-14009],inhibits the ability of monomeric tau to promote tubulin assembly.This effect is specific for the 3-NT modification,as mutant tau proteins pseudophosphorylated at each Tyr residue are fully competent to stabilize MTs.Collectively,our results suggest that ONOO~--mediated modifications stabilize tau filaments via 3,3'-DT bonding and destabilize MTs by site-selective nitration of tau monomers.Moreover,assumption of the Alz-50 conformation may be the mechanism through which tau nitration modulates MT stability.