| Abstract |
Genetic, neuropathological and biochemical data all point to a major role of the presynaptic protein α-Syn in the pathogenesis of PD and related synucleinopathies. Moreover, PTMs, and in particular phosphorylation at serine 129, are thought to play a role in the formation of the aberrant conformations, i.e. oligomeric and fibrillar α-Syn, affecting neuronal homeostasis, although the valence of the effect of phosphorylation is still controversial. The mechanisms governing α-Syn degradation remain a subject of debate, but it is generally acknowledged that the degradation of α-Syn, and its multiple conformational states, depends on two major intracellular protein degradation pathways; the UPS, and the autophagy-lysosome pathway (macroautophagy, microautophagy and CMA). A major obstacle to elucidating the role of α-Syn pathology has been the lack of model systems to study the acute effects of α-Syn aggregation occurring in real-time. We describe here a novel cellular model based on the over‐expression of wild type α-Syn in which the formation of α-Syn inclusions was triggered by the addition of preformed-fibrils (PFFs). This cell model recapitulates the features of LB-like inclusions such as conversion of endogenous soluble α-Syn into insoluble, hyperphosphorylated pathological species. In our cell model, the seeding of endogenous α-Syn occurs within a time frame of 6 days, and was more prominent after 10 days of fibril-incubation. Moreover, our model gives us the ability to shut down the expression of the α-Syn by adding DOX and follow the clearance of α-Syn assemblies. More specifically, we showed that a switch-off of α-Syn expression on the 6th day of PFF-incubation, resulted in the clearance of α-Syn assemblies over a period of 6 days. Following this, we examined the clearance of both total and pS129 α-Syn. Our data showed that when the expression of α-Syn is shut down by DOX addition for 6 days, accumulation of PFF-induced α-Syn aggregates is detected upon bafilomycin treatment, suggesting that lysosomal processes are involved in the clearance of the seeded material. Additionally, we showed that proteasomal inhibition with epoxomicin did not affect the clearance of α-Syn. Concerning pS129, our findings showed that fibrillar pS129 α-Syn was only apparent relatively late, at 7 days of high-dose fibril-incubation and that phosphorylation occurred only in the endogenous α-
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Syn as depicted from control experiments using mutant S129A PFFs. Fibrillar phosphorylated α-Syn was degraded after two days of DOX incubation, while proteasomal inhibition reversed this turnover. Further, pS129 α-Syn was observed using fluorescence confocal microscopy, and was detected in aggregated puncta only upon 24-hour proteasomal inhibition with epoxomicin, suggesting that the proteasomal pathway is, at least in part, responsible for its clearance. To this end, our data strongly suggest a link between aggregation and the degradation machineries. This established model system could be used to decipher the role of the crosstalk between the different PTMs i.e. ubiquitylation, sumoylation and phosphorylation in α-Syn degradation and aggregation, and identify components that modulate this crosstalk and as a result the levels of α-Syn. Eventually, we hope to improve the understanding of the mechanisms ofα-Syn metabolismin the nervous system, intricately linked to PD development, accelerating druggability and therapeutic approaches.
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