Endogenous Levels of Alpha-Synuclein Modulate Seeding and Aggregation in Cultured Cells.

Parkinson's disease is a progressive neurodegenerative disorder characterized by the accumulation of misfolded alpha-synuclein in intraneuronal inclusions known as Lewy bodies and Lewy neurites. Multiple studies strongly implicate the levels of alpha-synuclein as a major risk factor for the onset and progression of Parkinson's disease. Alpha-synuclein pathology spreads progressively throughout interconnected brain regions but the precise molecular mechanisms underlying the seeding of alpha-synuclein aggregation are still unclear. Here, using stable cell lines expressing alpha-synuclein, we examined the correlation between endogenous alpha-synuclein levels and the seeding propensity by exogenous alpha-synuclein preformed fibrils. We applied biochemical approaches and imaging methods in stable cell lines expressing alpha-synuclein and in primary neurons to determine the impact of alpha-synuclein levels on seeding and aggregation. Our results indicate that the levels of alpha-synuclein define the pattern and severity of aggregation and the extent of p-alpha-synuclein deposition, likely explaining the selective vulnerability of different cell types in synucleinopathies. The elucidation of the cellular processes involved in the pathological aggregation of alpha-synuclein will enable the identification of novel targets and the development of therapeutic strategies for Parkinson's disease and other synucleinopathies.

Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells.

A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the 'α-Syn burden' underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. This study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6 days, and is still prominent after 12 days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6 days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13 days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7 days of incubation with a higher amount of PFFs. Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the proteasome system is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.