Establishment of Method for the Determination of Aggregated α-Synuclein in DLB Patient Using RT-QuIC Assay

Background: The accumulation of aggregated α-synuclein (αSyn) is known as one of the critical reasons to exhibit their variable molecular pathologies and phenotypes in synucleinopathies. Recent studies suggested that the real-time quaking-induced conversion (RT-QuIC) assay is one of the potential methods to detect these αSyn aggregates and could detect the aggregated αSyn in the brain tissue and cerebrospinal fluid (CSF) using the propensity of the prion-like oligomerization. Objective: We tried to optimize the αSyn RT-QuIC assay based on the aggregation of αSyn in brain samples of synucleinopathies by comparing the conditions of the recently reported αSyn RTQuIC assays. Methods: This study applied a highly sensitive RT-QuIC assay using recombinant αSyn (rαSyn) to detect aggregated αSyn in the brain tissue from dementia with Lewy bodies (DLB). Results: This study compared αSyn RT-QuIC assays under conditions such as beads, rαSyn as a substrate, reaction buffers, and fluorescence detectors. We observed that the addition of beads and the use of 6x His-tagged rαSyn as a substrate help to obtain higher positive responses from αSyn RT-QuIC assay seeding with brain homogenate (BH) of DLB and phosphate buffer-based reaction showed higher positive responses than HEPES buffer-based reaction on both fluorescent microplate readers. We also observed that the DLB BHs gave positive responses within 15-25h, which is faster high positive responses than recently reported assays. Conclusion: This established αSyn RT-QuIC assay will be able to apply to the early clinical diagnosis of αSyn aggregates-related diseases in various biofluids such as CSF.