α-Synuclein Seeding Assay Using RT-QuIC

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein (αSyn). They include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In each disease, it has been proposed that aggregates of αSyn represent different conformational strains of αSyn, leading to self-propagation and spreading from cell to cell. It has been considered that αSyn aggregates grow by seeded polymerization mechanisms. Previously, the mechanism of seed conversion in prion protein aggregation has been exploited by real-time quaking-induced conversion (RT-QuIC) assay. It was further refined by incorporating the fluorescent dye thioflavin-T, which enabled the real-time monitoring of kinetic changes with a highly sensitive detection of seed aggregates present at an extremely low level. In an application for diagnostics, it has been reported that αSyn RT-QuIC exhibits specificity between 82% and 100%, while its sensitivity varies between 70% and 100%, on the basis of a study in which this assay was performed at multiple different laboratories. Furthermore, it has been suggested that the αSyn RT-QuIC method can be applied to study the biochemical characteristics of different αSyn strains among synucleinopathies. In this article, we describe the detailed protocols for αSyn RT-QuIC assays.