Real-Time Monitoring of Self-Aggregation of β-Amyloid by a Fluorescent Probe Based on Ruthenium Complex
Real-Time Monitoring of Self-Aggregation of β-Amyloid by a Fluorescent Probe Based on Ruthenium Complex
复制标题
基于钌配合物的荧光探针实时监测β-淀粉样蛋白的自聚集
DOI：
10.1021/acs.analchem.9b03566
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发表时间：
2020
影响因子：
7.4
通讯作者：
Yu Lin
中科院分区：
文献类型：
--
作者：
Yu Hui-juan;Zhao Wei;Xie Mengting;Li Xiaoqing;Sun Ming;He Jun;Wang Lu;Yu Lin
淀粉样β蛋白（Aβ）自身积累成不溶性纤维是阿尔茨海默病的主要标志。纤维生长的实时监测对于阐明聚集的机制和发现治疗靶点至关重要。已经探索了多种方法，包括NMR、电子显微镜（EM）、原子力显微镜（AFM）和全内反射荧光显微镜（TIRFM），以监测原纤维生长或揭示Aβ聚集体的形态。然而，没有一种方法允许在生理条件下实时观察，而没有任何扰动。在此，我们提出了一种荧光探针[Ru（phen）2（fipc）]2+（Ru-fipc）（phen = 1，10-菲咯啉，fipc = 5-氟-N-（1，10-菲咯啉-5-基）-1H-吲哚-2-甲酰胺），它可以结合所有的Aβ形式，即，单体、低聚物和原纤维，同时不干扰聚集。使用这种探针与激光共聚焦显微镜相结合，整个聚集过程可以清晰，准确地成像在单个原纤维水平。Ru-fipc与硫磺素T（ThT）共定位的结果证实了Ru-fipc的可靠性。重要的是，Ru-fipc可用于监测非常早期的成核和寡聚化过程，这被认为是神经毒性发展中的关键步骤，而ThT无法观察到。据我们所知，这是第一个开发用于实时监测Aβ聚集的荧光探针，特别是对于非常早期的组装阶段，在溶液中具有最小的扰动。该方法适用于体外和体内研究。我们相信这将为神经退行性疾病发病机制的研究和治疗靶点的发现提供有价值的补充工具。
Self-accumulation of amyloid-β protein (Aβ) into insoluble fibrils is the major hallmark of Alzheimer’s disease. Real-time monitoring of fibril growth is essential for clarifying the mechanism underlying aggregation and discovering therapeutic targets. A variety of approaches including NMR, electron microscopy (EM), atomic force microscopy (AFM), and total internal reflection fluorescence microscopy (TIRFM) have been explored to monitor the fibril growth or reveal morphology of Aβ aggregates. However, none of the methods allow real-time observation under physiological conditions while without any perturbations. Here, we present a fluorescent probe [Ru(phen)2(fipc)]2+(Ru-fipc) (phen = 1,10-phenanthroline, fipc = 5-fluoro-N-(1,10-phenanthrolin-5-yl)-1H-indole-2-carboxamide) that can bind to all the Aβ forms, i.e., monomers, oligomers, and fibrils, while not perturbing aggregation. Using this probe in combination with laser confocal microscopy, the entire aggregation process could be clearly and exactly imaged at the single fibril level. The reliability ofRu-fipcwas confirmed based on colocalization with thioflavin T (ThT). Importantly,Ru-fipccan be used to monitor the very early nucleation and oligomerization process, which is thought to be a critical step in the development of neurotoxicity while it cannot be visualized with ThT. To our knowledge, this is the first fluorescent probe developed for real-time monitoring of Aβ aggregation, especially for the very early assembly stage, in solution with minimal perturbation. This method is suitable forin vitroandin vivostudies. We believe this would provide a valuable complementary tool for the study of pathogenesis and discovery of therapeutic targets of neurodegenerative diseases.