College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Theranostics. 2019 Aug 1;9(19):5672-5680. doi: 10.7150/thno.33867. eCollection 2019.
Autophagy is a dynamic and complicated catabolic process. Imaging autophagic flux can clearly advance knowledge of its pathophysiology significance. While the most common way autophagy is imaged relies on fluorescent protein-based probes, this method requires substantial genetic manipulation that severely restricts the application. Small fluorescent probes capable of tracking autophagic flux with good spatiotemporal resolution are highly demanable. In this study, we developed a small-molecule fluorogenic probe () that facilitates real-time imaging of autophagic flux in both intact cells and live mice. is inspired by the cascading nitrosative and acidic microenvironments evolving during autophagy. It operates over two sequential steps. In the first step, responds to the up-regulated peroxynitrite at the initiation of autophagy by its diphenylamino group being oxidatively dearylated to yield a daughter probe. In the second step, the daughter probe responds to the acidic autolysosomes at the late stage of autophagy by being protonated. This pathway-dependent mechanism has been confirmed first by sequentially sensing ONOO and acid in aqueous solution, and then by imaging autophagic flux in live cells. Furthermore, has been successfully applied to visualize autophagic flux in real-time in live mice following brain ischemic injury, justifying its robustness. Due to the specificity, easy operation, and the dynamic information yielded, should serve as a potential tool to explore the roles of autophagy under various pathological settings.
自噬是一个动态而复杂的分解代谢过程。对自噬流的成像可以清楚地提高对其病理生理学意义的认识。虽然最常见的自噬成像方法依赖于基于荧光蛋白的探针，但这种方法需要大量的遗传操作，严重限制了其应用。能够以良好的时空分辨率跟踪自噬流的小分子荧光探针是非常需要的。在本研究中，我们开发了一种小分子荧光探针（），可以在完整细胞和活小鼠中实时成像自噬流。受自噬过程中不断演变的级联硝化和酸性微环境的启发，它通过两个连续的步骤进行工作。在第一步中，探针的二苯氨基基团在自噬起始时被过氧亚硝酸盐氧化脱芳基化，生成一个子探针，从而响应上调的过氧亚硝酸盐。在第二步中，子探针在自噬的晚期通过质子化响应酸性自溶酶体。这种依赖于途径的机制首先通过在水溶液中依次感应 ONOO 和酸得到证实，然后通过在活细胞中成像自噬流得到证实。此外，已经成功地应用于实时可视化脑缺血损伤后活小鼠中的自噬流，证明了其稳健性。由于其特异性、易于操作和提供的动态信息，应该可以作为一种潜在的工具，用于在各种病理环境下探索自噬的作用。
