A robust assay to monitor ataxin-3 amyloid fibril assembly

Abstract

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation—a starting point for further studies with cell and animal models of SCA3.