alpha-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 (pS129-alpha-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129-alpha-syn remains controversial, so we sought to identify when pS129 modification occurs during alpha-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129-alpha-syn inhibits alpha-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129-alpha-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129-alpha-syn (WT-alpha-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of alpha-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with alpha-syn preformed fibrils, and we observed aggregation of nonphosphorylated a-syn followed by later pS129-alpha-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated alpha-syn and disease duration. These findings suggest that pS129-alpha-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129-alpha-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129-alpha-syn as a measure of efficacy in clinical trials.