Differential Effects on In Vitro Tau Aggregation Due to 7, 11, and 14 Pseudophosphorylated Sites

Abstract

Neurofibrillary tangles are intracellular aggregates composed of the microtubule-associated protein tau. These insoluble aggregates are found in the brain of those affected by Alzheimer’s disease and other related tauopathies. Hyperphosphorylation of tau in disease has been hypothesized to cause tau to dissociate from microtubules and form amyloid-like oligomers and fibrils. Under normal conditions, there is 2–3 mol of phosphate per mole of tau; however, studies have found 2–3 times more phosphate per mole of tau in diseased conditions. The in vitro arachidonic acid induction of aggregation of different combinations of pseudophosphorylated sites up to 7 sites has previously been shown to result in differences in aggregation properties, characterized by increasing lengths of filaments with increasing numbers of pseudophosphorylation sites. Because several other sites of tau are also phosphorylated in disease, tau aggregation of protein variants with 11 and 14 sites mimicking hyperphosphorylation was compared to the 7 pseudophosphorylation sites previously studied using arachidonic acid and polyphosphate as fibrillization inducers. An increase in filament length, along with a decrease in the number of shorter filaments, was observed with increasing numbers of pseudophosphorylation sites regardless of the inducer employed. Variants displayed differential aggregation kinetics depending on the number of pseudophosphorylation sites and the inducer used. Although the rate of tubulin polymerization decreased as the number of pseudophosphorylation sites increased, microtubule stability was maintained across all pseudophosphorylated variants compared with unmodified wild-type tau. These results demonstrate that increasing levels of hyperphosphorylation can continue to have increased effects on tau aggregation and microtubule stabilization.