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

Author(s):

Seok-Joo Park, Yun-Jung Lee, Jeong-Ho Park, Hyoung-Tae Jin, Myoung-Ju Choi, Cha-Gyun Jung, Hiroyasu Akatsu, Eun-Kyoung Choi and Yong-Sun Kim*   Pages 1 - 6 ( 6 )

Abstract:


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 RT-QuIC 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 substrate, reaction buffers, and fluorescence detectors. We observed that the addition of beads and the use of 6x His-tagged rαSyn as substrate help to obtain higher positive responses from αSyn RTQuIC 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 ~15–25 h, 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 disease in various biofluids such as CSF.

Keywords:

α-synuclein, α-synucleinopathy, RT-QuIC, assay protein, aggregation diagnosis, cerebrospinal fluid

Affiliation:

Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Department of Biochemistry, Nagoya City University Graduate School of Medical Sciences, Nagoya, Department of Community-based Medical Education, Nagoya City University Graduate School of Medical Sciences, Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea and Department of Biomedical Gerontology, Graduate School of Hallym University, Chuncheon, Gangwon-do, Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi-do, Korea and Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Gangwon-do



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