Real-Time Quaking- Induced Conversion Assays for Prion Diseases, Synucleinopathies, and Tauopathies

Abstract

Prion diseases, synucleinopathies and tauopathies are neurodegenerative disorders characterized by deposition of abnormal protein aggregates in brain and other tissues. These aggregates consist of misfolded forms of prion, α-synuclein (αSyn), or tau proteins that cause neurodegeneration and represent hallmarks of these disorders. A main challenge in the management of these diseases is the accurate detection and differentiation of these abnormal proteins during the early stages of disease before the onset of severe clinical symptoms. Unfortunately, many clinical manifestations may occur only after neuronal damage is already advanced and definite diagnoses typically require post-mortem neuropathological analysis. Over the last decade, several methods have been developed to increase the sensitivity of prion detection with the aim of finding reliable assays for the accurate diagnosis of prion disorders. Among these, the real-time quaking-induced conversion (RT-QuIC) assay now provides a validated diagnostic tool for human patients, with positive results being accepted as an official criterion for a diagnosis of probable prion disease in multiple countries. In recent years, applications of this approach to the diagnosis of other prion-like disorders, such as synucleinopathies and tauopathies, have been developed. In this review, we summarize the current knowledge on the use of the RT-QuIC assays for human proteopathies.

Keywords: RT-QuIC assay; diagnosis; prion disorders; seeding activity; synucleinopathies; tauopathies.

FIGURE 1

Real-time quaking- induced conversion assay schematic. (A) Test specimens are added to multi-well plates along with solutions of substrate protein monomers and an amyloid sensitive dye such as thioflavin T. The plates are subjected to intermittent shaking and fluorescence readings in a temperature-controlled plate-reader. (B) Amplification cycle: Seeds in the specimen bind monomers and induce their conformational conversion as they are recruited into the growing fibril. Secondary nucleation may occur due to assembly and conversion of monomers on the sides of fibrils, contributing, along with fragmentation, to the generation of new seeding surfaces. New seeding surfaces are critical in obtaining exponential growth kinetics. (C) Fluorescence readout: Binding of the dye to amyloid fibrils enhances its fluorescence. In the absence of pre-existing seeds in the specimen, amyloid fibril formation requires spontaneous nucleation, which, under conditions optimized for these assays at least, is much slower than the growth and fragmentation of pre-existing seeds. In reactions provided with pre-existing seeds, the lag corresponds to the phase in which seeds are growing at sub-detectable levels. The fluorescence plateaus once the available monomer is consumed. Sometimes fluorescence can even decrease after peaking, an effect that is likely due to consolidation or redistribution of fibrils in the well in a way that affects the fluorescence measurement (not illustrated).