Department of Neurology, Graduate School of Medicine, Osaka University, Yamadaoka 2-2, Suita, Osaka, 565-0871, Japan.
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan.
Sci Rep. 2019 Apr 12;9(1):6001. doi: 10.1038/s41598-019-42399-0.
α-Synuclein aggregates, a key hallmark of the pathogenesis of Parkinson's disease, can be amplified by using their seeding activity, and the evaluation of the seeding activity of cerebrospinal fluid (CSF) is reportedly useful for diagnosis. However, conventional shaking-based assays are time-consuming procedures, and the clinical significance of the diversity of seeding activity among patients remains to be clarified. Previously, we reported a high-throughput ultrasonication-induced amyloid fibrillation assay. Here, we adapted this assay to amplify and detect α-synuclein aggregates from CSF, and investigated the correlation between seeding activity and clinical indicators. We confirmed that this assay could detect α-synuclein aggregates prepared in vitro and also aggregates released from cultured cells. The seeding activity of CSF correlated with the levels of α-synuclein oligomers measured by an enzyme-linked immunosorbent assay. Moreover, the seeding activity of CSF from patients with Parkinson's disease was higher than that of control patients. Notably, the lag time of patients with Parkinson's disease was significantly correlated with the MIBG heart-to-mediastinum ratio. These findings showed that our ultrasonication-based assay can rapidly amplify misfolded α-synuclein and can evaluate the seeding activity of CSF.
α-突触核蛋白聚集物是帕金森病发病机制的一个关键标志，可以通过利用其种子活性进行放大，据报道脑脊液（CSF）的种子活性评估对诊断有用。然而，传统的基于摇动的测定方法是耗时的过程，并且患者之间种子活性的多样性的临床意义仍有待阐明。先前，我们报道了一种高通量超声诱导的淀粉样纤维形成测定法。在这里，我们将该测定法进行了改编，以放大和检测 CSF 中的α-突触核蛋白聚集物，并研究了种子活性与临床指标之间的相关性。我们证实该测定法可以检测体外制备的α-突触核蛋白聚集物以及从培养细胞中释放的聚集物。CSF 的种子活性与通过酶联免疫吸附测定法测量的α-突触核蛋白寡聚物的水平相关。此外，帕金森病患者的 CSF 种子活性高于对照组患者。值得注意的是，帕金森病患者的滞后时间与 MIBG 心脏与纵隔比值显著相关。这些发现表明，我们的基于超声的测定法可以快速放大错误折叠的α-突触核蛋白，并可以评估 CSF 的种子活性。
