Domain-specific modulatory effects of phosphomimetic substitutions on liquid-liquid phase separation of tau protein.

Aggregation of tau is one of the major pathogenic events in Alzheimer's disease and several other neurodegenerative disorders. Recent reports demonstrated that tau can condense into liquid droplets that undergo time-dependent transition to a solid-like state, suggesting that liquid condensates may be on the pathway to pathological aggregation of tau. While hyperphosphorylation is a key feature of tau isolated from brains of patients with Alzheimer's disease and other tauopathies, the mechanistic role of phosphorylation in tau liquid-liquid phase separation (LLPS) remains largely unexplored. In an attempt to bridge this gap, here we performed systematic studies by introducing phosphomimetic substitutions of Ser/Thr residues with negatively charged Asp/Glu residues in different regions of the protein. Our data indicate that the phosphorylation patterns that increase the polarization of charge distribution in full-length tau (tau441) promote protein LLPS, whereas those that decrease charge polarization have an opposite effect. Overall, this study further supports the notion that tau LLPS is driven by attractive intermolecular electrostatic interactions between the oppositely charged domains. We also show that the phosphomimetic tau variants with low intrinsic propensity for LLPS can be efficiently recruited to droplets formed by the variants with high LLPS propensity. Furthermore, the present data demonstrate that phosphomimetic substitutions have a major effect on time-dependent material properties of tau droplets, generally slowing down their aging. The latter effect is most dramatic for the tau variant with substitutions within the repeat domain, which correlates with the decreased fibrillation rate of this variant.