Optical pulse labeling studies reveal exogenous seeding slows α-synuclein clearance

The accumulation of alpha-synuclein (alpha-syn) in intracellular formations known as Lewy bodies (LBs) is associated with several neurodegenerative diseases including Parkinson's disease and Lewy Body Dementia. There is still limited understanding of how alpha-syn and LB formation is associated with cellular dysfunction and degeneration in these diseases. To examine the clearance and production dynamics of alpha-syn we transduced organotypic murine brain slice cultures (BSCs) with recombinant adeno-associated viruses (rAAVs) to express Dendra2-tagged human wild-type (WT) and mutant A53T alpha-syn, with and without the addition of exogenous alpha-syn fibrillar seeds and tracked them over several weeks in culture using optical pulse labeling. We found that neurons expressing WT or mutant A53T human alpha-syn show similar rates of alpha-syn turnover even when insoluble, phosphorylated Ser129 alpha-syn has accumulated. Taken together, this data reveals alpha-syn aggregation and overexpression, pSer129 alpha-syn, nor the A53T mutation affect alpha-syn dynamics in this system. Prion-type seeding with exogenous alpha-syn fibrils significantly slows alpha-syn turnover, in the absence of toxicity but is associated with the accumulation of anti-p62 immunoreactivity and Thiazin Red positivity. Prion-type induction of alpha-syn aggregation points towards a potential protein clearance deficit in the presence of fibrillar seeds and the ease of this system to explore precise mechanisms underlying these processes. This system facilitates the exploration of alpha-syn protein dynamics over long-term culture periods. This platform can further be exploited to provide mechanistic insight on what drives this slowing of alpha-syn turnover and how therapeutics, other genes or different alpha-syn mutations may affect alpha-syn protein dynamics.

Automated summary

The study investigates the dynamics of alpha-synuclein accumulation and turnover in neurodegenerative diseases, finding that aggregation, overexpression, phosphorylation, and mutation do not affect alpha-syn dynamics, while exogenous alpha-syn fibril seeds significantly slow turnover.