Alpha-synuclein (α-Syn), an intrinsically disordered protein (IDP), is associated with neurodegenerative disorders, including Parkinson's disease (PD or other α-synucleinopathies. Recent investigations propose the transmission of α-Syn protein fibrils, in a prion-like manner, by entering proximal cells to seed further fibrillization in PD. Despite the recent advances, the mechanisms by which extracellular protein aggregates internalize into the cells remain poorly understood. Using a simple cell-based model of human neuroblastoma-derived differentiated neurons, we present the cellular internalization of α-Syn PFF to check cellular uptake and recycling kinetics along with the standard endocytic markers Transferrin (Tf) marking clathrin-mediated endocytosis (CME) and Galectin3 (Gal3) marking clathrin-independent endocytosis (CIE). Specific inhibition of endocytic pathways using chemical inhibitors reveals no significant involvement of CME, CIE and caveolae-mediated endocytosis (CvME). A substantial reduction in cellular uptake was observed after perturbation of actin polymerization and treatment with macropinosomes inhibitor. Our results show that α-Syn PFF mainly internalizes into the SH-SY5Y cells and differentiated neurons via the macropinocytosis pathway. The elucidation of the molecular and cellular mechanism involved in the α-Syn PFF internalization will help improve the understanding of α-synucleinopathies including PD, and further design specific inhibitors for the same.

α‐Synuclein fibrils explore actin‐mediated macropinocytosis for cellular entry into model neuroblastoma neurons