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α-Synuclein (α-syn) phosphorylation at serine 129 (pS129-α-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-α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-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-α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129-α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129-α-syn (WT-α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129-α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129-α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129-α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129-α-syn as a measure of efficacy in clinical trials. [ABSTRACT FROM AUTHOR]

Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson's disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology. [ABSTRACT FROM AUTHOR]

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading. [ABSTRACT FROM AUTHOR]