Danish Research Institute of Translational Neuroscience-DANDRITE, Aarhus University, Aarhus, Denmark.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
PLoS One. 2019 Oct 11;14(10):e0216764. doi: 10.1371/journal.pone.0216764. eCollection 2019.
The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type α-synuclein (α-syn) aggregates within degenerating brain cells. α-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating α-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized α-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific α-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked α-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked α-syn oligomers could serve as important calibrators to facilitate comparative and standardized α-syn biomarker studies going forward.
神经退行性疾病包括帕金森病（PD）、路易体痴呆（DLB）和多系统萎缩（MSA），这些疾病的退化脑细胞中都含有包含淀粉样α-突触核蛋白（α-syn）聚集物的包涵体。α-syn 还存在可溶性寡聚体形式，这些寡聚体被认为是其天然状态和聚集状态之间的中间体。这些寡聚体存在于患有突触核蛋白病的患者的脑提取物中，具有作为生物标志物的巨大潜力。尽管在体外很容易制备，但寡聚体是亚稳态的，会随时间解离，从而使 α-syn 寡聚体研究变得复杂。我们使用小分子胺反应交联剂甲醛（FA）成功稳定了 α-syn 寡聚体，而不影响其大小、整体结构或对聚集构象特异性 α-syn 抗体 FILA 和 MJFR-14-6-4-2 的抗原性。此外，交联的 α-syn 寡聚体对变性剂如尿素和 SDS 的处理具有抗性，并且在与尿素孵育后仍保持作为聚合特异性酶联免疫吸附测定（ELISA）内标准的完全功能。我们提出，FA 交联的 α-syn 寡聚体可以作为重要的校准物，以促进未来的比较和标准化的 α-syn 生物标志物研究。
