Distinct Effects of Familial Parkinson's Disease-Associated Mutations on alpha-Synuclein Phase Separation and Amyloid Aggregation

The Lewy bodies and Lewy neurites are key pathological hallmarks of Parkinson's disease (PD). Single-point mutations associated with familial PD cause alpha-synuclein (alpha-Syn) aggregation, leading to the formation of Lewy bodies and Lewy neurites. Recent studies suggest alpha-Syn nucleates through liquid-liquid phase separation (LLPS) to form amyloid aggregates in a condensate pathway. How PD-associated mutations affect alpha-Syn LLPS and its correlation with amyloid aggregation remains incompletely understood. Here, we examined the effects of five mutations identified in PD, A30P, E46K, H50Q, A53T, and A53E, on the phase separation of alpha-Syn. All other alpha-Syn mutants behave LLPS similarly to wild-type (WT) alpha-Syn, except that the E46K mutation substantially promotes the formation of alpha-Syn condensates. The mutant alpha-Syn droplets fuse to WT alpha-Syn droplets and recruit alpha-Syn monomers into their droplets. Our studies showed that alpha-Syn A30P, E46K, H50Q, and A53T mutations accelerated the formation of amyloid aggregates in the condensates. In contrast, the a alpha Syn A53E mutant retarded the aggregation during the liquid-to-solid phase transition. Finally, we observed that WT and mutant alpha-Syn formed condensates in the cells, whereas the E46K mutation apparently promoted the formation of condensates. These findings reveal that familial PD-associated mutations have divergent effects on alpha-Syn LLPS and amyloid aggregation in the phase-separated condensates, providing new insights into the pathogenesis of PD-associated alpha-Syn mutations.