A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha-synuclein to lysosomes

A key question in PD research relates to the mechanism by which alpha-synuclein (α-syn) fibrils enter cells. It is generally thought that α-syn preformed fibrils (PFF), a toxic form of α-syn, are trafficked to lysosomes through the endosomal system over the course of hours, although many conflicting results exist in the literature. We believe that some of the conflicting evidence arises from examining α-syn internalization at longer time courses and not immediately after its addition to cells. We also believe that dynamin inhibition, employed in many studies to inhibit PFF internalization, should not be treated as synonymous with inhibition of clathrin-mediated endocytosis (CME), as the role of dynamin spans beyond CME and is involved in multiple endocytic pathways and actin polymerization. We thus sought to examine PFF endocytosis immediately after its addition to cells. Remarkably, PFF are internalized rapidly and appear in lysosomes within 2 min, completely bypassing conventional endosomal trafficking pathways. We confirmed this result in multiple cell lines, primary human cells, and neurons derived from human induced pluripotent stem cells (hiPSC). The internalization is not dependent on clathrin but instead utilizes macropinocytosis.All the raw data collected in our manuscript, has been organized and included in the .xlsx file attached. Additionally, the statistical calculations computed, with the use of Prism (graphpad.com) have also been included.