Abstract Body

A prevailing hypothesis in the field of neurodegenerative diseases, and Parkinson’s Disease (PD) in particular, is that pathogenic protein conformations may seed physiological endogenous proteins, leading to further protein aggregation and disease propagation. Consistent with this notion, the seeding ability of alpha-synuclein (AS) Pre-Formed Fibrils (PFFs) on endogenous AS has been well demonstrated in various cellular and animal systems, yet the mechanisms that lead to the generation and dissolution of such aggregates are unclear. Accordingly, we have applied human AS PPFs to a neuronally differentiated human neuroblastoma cell system with inducible tetracycline-dependent overexpression of human Wild Type (WT) AS. We observe time- and PFF dose-dependent seeding of endogenous AS in the Tet-off state, seeding that can be demonstrated with various AS antibodies. Antibodies directed against phosphorylated AS identified seeding only with much higher PFF concentrations. Experiments using pharmacological agents suggested that the lysosomal, and in particular the macroautophagic pathway, was responsible for the clearance of seeded WT AS, while the proteasomal system was involved specifically in the degradation of seeded phosphorylated AS. Clearance of seeded protein aggregates may also occur through extracellular release, although this could potentially lead to disease transmission. We examined accordingly if we could detect seeded AS load in released extracellular vesicles termed exosomes. Indeed, exosomes derived from PFF-treated Tet-off cells contained abundant seeded aggregated AS; however, levels of such species within exosomes did not change following lysosomal or proteasomal inhibition. We conclude that exosomal release represents another possible mechanism for the clearance of seeded aggregated AS.