Pseudophosphorylation of tau modulates its function and induces AD-like changes

Abstract

The microtubule associate protein tau, in a hyperphosphorylated form, loses its normal function and aggregates into insoluble paired-helical filaments (PHFs) in Alzheimer's disease (AD) and other tauopathies. The stoichiometry of phosphorylation is increased from 2-3 mol of phosphate per mole of tau in normal brain to 5-9 mol of phosphate per mole of tau in AD. In AD, the deregulation of kinases, such as glycogen synthase kinase-3beta (GSK-3beta), is believed to be associated with the generation of hyperphosphorylated tau. However, the functional contribution of hyperphosphorylation on AD is not well understood. Therefore, pseudohyperphosphorylation mutants at six or sever GSK-3beta phosphorylation sites were generated by amino acid substitution. In addition, several single, double and triple pseudophosphorylation mutants at these sites were also generated and used as controls. I studied the changes on mobility on SDS-PAGE, microtubule (MT) binding and arachidonic acid (ARA) induced polymerization. Four pseudophosphorylation mutants induced an SDS-resistant mobility change. All but three mutants had a reduced MT binding affinity and pseudohyperphosphorylation mutants did not have a greater effect compared with pseudophosphorylation mutants at single or double sites. Three pseudohyperphosphorylation mutants with retarded SDS mobility had a greater effect on ARA-induced polymerization, with reduced nucleation and elongation rate. Some pseudophosphorylation mutants had a significantly increased polymerization at high ARA concentrations compared with wild type tau. The tangle like aggregates similar to those isolated from AD brain were formed in the mixture of six pseudophosphorylation mutants. These observations suggest the increased toxicity of hyperphosphorylated tau may be induced by decreased MT binding affinity and reduced nucleation and polymerization rate. These observations also explain the potentially beneficial role of tau polymerization and NFT formation. I also studied the mechanism of ARA-induced tau polymerization. The results suggest that ARA can induce tau polymerization both as large micelles and monomers (or small micelles) and molecular nature of tau can affect the morphology of tau filament. The amount and morphology of tau filament can also be affected by surface area : inducer ratio.