Monomeric α-Synuclein Real-Time Induced Conversion: A New Approach to the Diagnostics of Neurodegenerative Synucleinopathies with Weak RT-QuIC Responses.

Abstract

Neurodegenerative disorders classified as synucleinopathies (Parkinson's disease, dementia with Lewy bodies, and multiple-system atrophy) are characterized by the accumulation of aberrant α-synuclein aggregates in neurons and glial cells. These diseases manifest clinically several years after the initial formation of pathological protein aggregates in the brain, making early and accurate diagnosis challenging. In recent years, a new method, which is based on real-time quaking-induced conversion (RT-QuIC) of α-synuclein, has been developed and validated. This technology holds great promise as a powerful diagnostic tool for the early and precise identification of synucleinopathies, potentially opening new horizons in the study of neurodegenerative diseases. RT-QuIC detects misfolded α-synuclein aggregates in human physiological fluids by introducing an excess of recombinant α-synuclein, which undergoes conformational conversion in an exponential, prion-like manner. The production of high-quality recombinant α-synuclein is a critical step in the effective application of this method, as protein purity significantly affects the sensitivity and specificity of the assay - key factors in its diagnostic utility. Using a three-step chromatographic purification protocol, we produced recombinant monomeric α-synuclein with a purity exceeding 97% from the periplasmic fraction of bacterial cells. While higher purity increases the assay duration, it also reduces the background signal and permits extended incubation times, which are essential for reliably detecting synucleinopathies with weak RT-QuIC responses, such as the cerebellar subtype of multiple-system atrophy. The data presented support the conclusion that optimized components of the RT-QuIC system will enable an accurate diagnosis of neurodegenerative synucleinopathies.