Pseudo-hyperphosphorylation causing AD-like changes in tau has significant effects on its polymerization

Abstract

The microtubule-associated protein tau, in a hyperphosphorylated form, aggregates into insoluble paired-helical filaments (PHFs) in Alzheimer’s disease (AD) and other tauopathies. In AD, there are approximately 8 mol of phosphate per mol of tau distributed among approximately 30 PHF phosphorylation sites as compared to 2–3 phosphate per mol in normal brain. In AD, kinases such as glycogen synthase kinase-3β (GSK-3β) are believed to be involved in generation of hyperphosphorylated tau. However, the functional consequences of hyperphosphorylation on the microtubule binding and polymerization of tau are not well understood. To address this question, we have generated pseudo-hyperphosphorylation mutants consisting of six and seven sites in the proline rich region and carboxy-terminus of tau by amino acid substitution. In addition, several single, double and triple pseudo-phosphorylation mutants were also generated. Pseudo-phosphorylation of tau decreases its affinity for microtubules, and pseudo-hyperphosphorylated forms of tau do not have significantly decreased microtubule binding as compared to single and double-sites. Three pseudo-hyperphosphorylated forms of tau with altered SDS-PAGE migration have a greater effect on its inducer mediated polymerization, slowing the rate of nucleation and elongation. Based on the observations that pseudo-hyperphosphorylated tau has decreased affinity for microtubules and reduced inducer-initiated rates of nucleation and polymerization, we propose that this combination could be the cause for the increased cytotoxicity of hyperphosphorylated tau in Alzheimer’s disease and also explain the potentially beneficial role of tau polymerization and NFT formation.