Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system atrophy

Abstract

Multiple system atrophy (MSA) and Parkinsons disease (PD) are caused by misfolded -synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in -synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in -synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo, providing key evidence to support the hypothesis that -synuclein adopts unique strains in patients. Here, to further interrogate -synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate -synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of -synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support -synuclein misfolding. Overall, our studies use a structure-based approach to investigate -synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.