Objective: To apply an ultrasonication-based misfolded  protein amplification to the evaluation of α-synuclein aggregates in CSF, and to investigate their clinical significances in Parkinson’s disease

Background: α-synuclein aggregate is a key hallmark in the pathogenesis of Parkinson’s disease(PD) and other synucleinopathy. Some groups have reported the cyclic amplification of α-synuclein aggregates in CSF with shaking-based assays, and its usefulness for diagnosis of PD (Ref.1-3). However, conventional shaking-based assays needs several days, and it remains to be elucidated the clinical significance of the diversity of seeding activity among patients with PD.

Method: Recently, we developed an ultrasonication-based amyloid fibrillation assay (HANdai Amyloid Burst Inducer; HANABI) (Ref. 4,5). This assay was adapted to the amplification and detection of α-synuclein aggregates. First, the assay was implemented by pre-formed fibril in vitro. Second, the medium of cells induced aggregation was analyzed, mimicking CSF from patients. Finally, we evaluated the seeding activity of CSF from the cohort of 44 PD patients and 17 control patients. We investigated the correlation of seeding activity with oligomers in CSF measured by enzyme-linked immunosorbent assay (ELISA) and oligomerization activity of CSF. We analyzed the difference between patients with PD and controls, and compared the results of HANABI assay with clinical scores and imaging data.

Results: The assay could detect α-synuclein aggregates prepared in vitro and also increasing aggregates released from cultured cells to their medium (figure.1). The seeding activity of CSF reflected the amounts of α-synuclein oligomers measured by ELISA and oligomerization activity of CSF (figure.2). Although there are overlaps, CSF from patients with PD had higher seeding activity than that of controls (figure. 3). Notably, the lag time of patients with Parkinson’s disease was significantly correlated with the MIBG heart-to-mediastinum ratio (figure, 4).

Conclusion: The ultrasonication-based assay rapidly detected α-synuclein aggregates. We showed the correlation between seeding activity and reduced MIBG uptake. Further improvement in the control system of ultrasonication are needed for clinical use.

References: Ref. 1: Shahnawaz, M. et al. Development of a Biochemical Diagnosis of Parkinson Disease by Detection of alpha-Synuclein Misfolded Aggregates in Cerebrospinal Fluid. JAMA Neurol, doi:10.1001/jamaneurol.2016.4547 (2016). Ref. 2: Fairfoul, G. et al. Alpha-synuclein RT-QuIC in the CSF of patients with alpha-synucleinopathies. Annals of clinical and translational neurology 3, 812-818, doi:10.1002/acn3.338 (2016). Ref. 3: Groveman, B. R. et al. Rapid and ultra-sensitive quantitation of disease-associated alpha-synuclein seeds in brain and cerebrospinal fluid by alphaSyn RT-QuIC. Acta Neuropathol Commun 6, 7, doi:10.1186/s40478-018-0508-2 (2018). Ref. 4: So, M. et al. Ultrasonication-dependent acceleration of amyloid fibril formation. J Mol Biol 412, 568-577, doi:10.1016/j.jmb.2011.07.069 (2011). 27 Ref. 5: Umemoto, A., Yagi, H., So, M. & Goto, Y. High-throughput analysis of ultrasonication-forced amyloid fibrillation reveals the mechanism underlying the large fluctuation in the lag time. J Biol Chem 289, 27290-27299, doi:10.1074/jbc.M114.569814 (2014).

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MDS Abstracts - https://www.mdsabstracts.org/abstract/amplification-of-%ce%b1-synuclein-aggregates-in-cerebrospinal-fluid-by-rapid-ultrasonication-based-assay/