March 1, 2021 Journal article Open Access

Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in Neuronal Cells

Pantazopoulou, Marina; Brembati, Vivianna; Kanellidi, Angeliki; Bousset, Luc; Melki, Ronald; Stefanis, Leonidas

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 Ubiquitin Proteasome System (UPS) and the Autophagy-Lysosome Pathway (ALP), contributes to the intracellular degradation of α‐Syn. The current 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 (tet-off system). Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding of endogenous α-Syn 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 PFF-induced SDS-soluble α-Syn 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. Fibrillar phosphorylated α-Syn at S129 was only apparent at 7 days of incubation with a higher amount of PFFs. Pharmacological inhibition of the proteasome resulted in further accumulation of PFF-induced SDS-soluble phosphorylated α-Syn at S129, without affecting total α-Syn. 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 PFF-induced SDS-soluble phosphorylated α-Syn at S129.