Disease Related Point Mutations and Solution Conditions Determine Fibrillization Behavior of α-Synuclein

Abstract

In vitro fibrillization of proteins into amyloid fibrils provides critical insights into the factors influencing protein aggregation and has a key role in understanding the molecular basis of several neurodegenerative diseases caused by amyloids. α-Synuclein (αSyn), an intrinsically disordered protein implicated in Parkinson's disease, aggregates readily in vitro into fibrils that exhibit appreciable structural polymorphism. This inherent polymorphism is a major obstacle in elucidating structural features and understanding the fibrillization process. By selecting specific solution conditions we were able to produce morphologically homogeneous fibrils of wt and disease mutant αSyn at the plateau phase of Thioflavin-T (ThT) assays, as evident from atomic force microscopy (AFM) imaging and analyses. Our results indicate that the in vitro aggregation conditions as well as the disease related point mutations of the protein determine the dominant morphology and the maturation behavior of the fibrils produced. Specifically, the morphology of wt αSyn fibrils appears to be dictated by two distinct mechanisms that is competitive growth of different polymorphs during the fibrillization phase followed by structural rearrangements during the process of aging. In contrast, the disease mutant αSyn variants aggregate with faster kinetics and result in fibrils with well defined and stable morphology over time. Additional cross seeding experiments of wt αSyn with disease mutant proteins have shown faithful transmission of the mutant fibril morphologies across two generations. The aggregation into homogeneous fibril populations with mutant-specific morphology is characterized by distinct fibrillization kinetics in ThT assays. Moreover, our experiments indicate differential interaction of ThT with morphologically different αSyn amyloid fibrils.