Generic placeholder image

Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Letter Article

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*

Volume 28 , Issue 1 , 2021

Published on: 20 April, 2020

Page: [115 - 120] Pages: 6

DOI: 10.2174/0929866527666200420105352

Price: $65


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.

Keywords: α-Synuclein, α-synucleinopathy, RT-QuIC, assay protein, aggregation diagnosis, cerebrospinal fluid.

Graphical Abstract
Spillantini, M.G.; Crowther, R.A.; Jakes, R.; Hasegawa, M.; Goedert, M. α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with lewy bodies. Proc. Natl. Acad. Sci. USA, 1998, 95(11), 6469-6473.
[] [PMID: 9600990]
Martí, M.J.; Tolosa, E.; Campdelacreu, J. Clinical overview of the synucleinopathies. Mov. Disord., 2003, 18(Suppl. 6), S21-S27.
[] [PMID: 14502652]
Mollenhauer, B.; Cullen, V.; Kahn, I.; Krastins, B.; Outeiro, T.F.; Pepivani, I.; Ng, J.; Schulz-Schaeffer, W.; Kretzschmar, H.A.; McLean, P.J.; Trenkwalder, C.; Sarracino, D.A.; Vonsattel, J.P.; Locascio, J.J.; El-Agnaf, O.M.; Schlossmacher, M.G. Direct quantification of CSF α-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration. Exp. Neurol., 2008, 213(2), 315-325.
[] [PMID: 18625222]
Williams, S.M.; Schulz, P.; Sierks, M.R. Oligomeric α-synuclein and β-amyloid variants as potential biomarkers for Parkinson’s and Alzheimer’s diseases. Eur. J. Neurosci., 2016, 43(1), 3-16.
[] [PMID: 26332448]
Ohrfelt, A.; Grognet, P.; Andreasen, N.; Wallin, A.; Vanmechelen, E.; Blennow, K.; Zetterberg, H. Cerebrospinal fluid alpha-synuclein in neurodegenerative disorders-a marker of synapse loss? Neurosci. Lett., 2009, 450(3), 332-335.
[] [PMID: 19022350]
Tokuda, T.; Qureshi, M.M.; Ardah, M.T.; Varghese, S.; Shehab, S.A.; Kasai, T.; Ishigami, N.; Tamaoka, A.; Nakagawa, M.; El-Agnaf, O.M. Detection of elevated levels of α-synuclein oligomers in CSF from patients with Parkinson disease. Neurology, 2010, 75(20), 1766-1772.
[] [PMID: 20962290]
Park, J.H.; Choi, Y.G.; Lee, Y.J.; Park, S.J.; Choi, H.S.; Choi, K.C.; Choi, E.K.; Kim, Y.S. Real-Time quaking-induced conversion analysis for the diagnosis of sporadic creutzfeldt-jakob disease in korea. J. Clin. Neurol., 2016, 12(1), 101-106.
[] [PMID: 26541494]
Atarashi, R.; Satoh, K.; Sano, K.; Fuse, T.; Yamaguchi, N.; Ishibashi, D.; Matsubara, T.; Nakagaki, T.; Yamanaka, H.; Shirabe, S.; Yamada, M.; Mizusawa, H.; Kitamoto, T.; Klug, G.; McGlade, A.; Collins, S.J.; Nishida, N. Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat. Med., 2011, 17(2), 175-178.
[] [PMID: 21278748]
Fairfoul, G.; McGuire, L.I.; Pal, S.; Ironside, J.W.; Neumann, J.; Christie, S.; Joachim, C.; Esiri, M.; Evetts, S.G.; Rolinski, M.; Baig, F.; Ruffmann, C.; Wade-Martins, R.; Hu, M.T.; Parkkinen, L.; Green, A.J. Alpha-synuclein RT-QuIC in the CSF of patients with alpha-synucleinopathies. Ann. Clin. Transl. Neurol., 2016, 3(10), 812-818.
[] [PMID: 27752516]
Sano, K.; Atarashi, R.; Satoh, K.; Ishibashi, D.; Nakagaki, T.; Iwasaki, Y.; Yoshida, M.; Murayama, S.; Mishima, K.; Nishida, N. Prion-like seeding of misfolded α-synuclein in the brains of dementia with lewy body patients in RT-QUIC. Mol. Neurobiol., 2018, 55(5), 3916-3930.
[PMID: 28550528]
Groveman, B.R.; Orrù, C.D.; Hughson, A.G.; Raymond, L.D.; Zanusso, G.; Ghetti, B.; Campbell, K.J.; Safar, J.; Galasko, D.; Caughey, B. Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC. Acta Neuropathol. Commun., 2018, 6(1), 7.
[] [PMID: 29422107]
Manne, S.; Kondru, N.; Hepker, M.; Jin, H.; Anantharam, V.; Lewis, M.; Huang, X.; Kanthasamy, A.; Kanthasamy, A.G. Ultrasensitive detection of aggregated α-synuclein in glial cells, human cerebrospinal fluid, and brain tissue using the RT-QuIC assay: New high-throughput neuroimmune biomarker assay for Parkinsonian disorders. J. Neuroimmune Pharmacol., 2019, 14(3), 423-435.
[] [PMID: 30706414]
van Rumund, A.; Green, A.J.E.; Fairfoul, G.; Esselink, R.A.J.; Bloem, B.R.; Verbeek, M.M. α-Synuclein real-time quaking-induced conversion in the cerebrospinal fluid of uncertain cases of parkinsonism. Ann. Neurol., 2019, 85(5), 777-781.
[] [PMID: 30801759]
Coelho-Cerqueira, E.; Carmo-Gonçalves, P.; Pinheiro, A.S.; Cortines, J.; Follmer, C. α-Synuclein as an intrinsically disordered monomer--fact or artefact? FEBS J., 2013, 280(19), 4915-4927.
[] [PMID: 23927048]
Huang, C.; Ren, G.; Zhou, H.; Wang, C.C. A new method for purification of recombinant human alpha-synuclein in Escherichia coli. Protein Expr. Purif., 2005, 42(1), 173-177.
[] [PMID: 15939304]

Rights & Permissions Print Export Cite as
© 2022 Bentham Science Publishers | Privacy Policy