Laser Analytics Group, Department of Chemical Engineering and Biotechnology , University of Cambridge , Cambridge CB3 0AS , United Kingdom.
Centre for Misfolding Diseases, Department of Chemistry , University of Cambridge , Cambridge CB2 1EW , United Kingdom.
ACS Chem Biol. 2019 Jul 19;14(7):1628-1636. doi: 10.1021/acschembio.9b00354. Epub 2019 Jun 27.
The nematode worm has emerged as an important model organism in the study of the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation, and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterization of different types of aggregates. Specifically, in longitudinal studies of models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetized worms moving in specially designed agarose microchambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living that can be used to study mechanisms of protein aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.
线虫已成为研究与淀粉样蛋白形成相关的蛋白质错误折叠疾病的分子机制的重要模式生物，因为它体积小、遗传操作容易且光学透明。然而，在该系统中获得蛋白质聚集的可靠和定量读数仍然是一个挑战。为了解决这个问题，我们在这里提出了一种快速时间门控荧光寿命成像（TG-FLIM）方法，并通过快速表征不同类型的聚集体，证明它为研究活体动物中蛋白质聚集的过程提供了功能见解。具体来说，在帕金森病和亨廷顿病模型的纵向研究中，我们观察到α-突触核蛋白和聚谷氨酰胺形成的蛋白包涵体的聚集动力学和性质存在明显差异。特别是，我们发现α-突触核蛋白包涵体直到蠕虫生命的后期才显示出类淀粉样特征，而聚谷氨酰胺在成年早期迅速形成淀粉样特征。此外，我们还表明，TG-FLIM 方法能够对在专门设计的琼脂糖微室中移动的活体和非麻醉的蠕虫进行成像。总之，我们的研究结果表明，TG-FLIM 方法能够对活体进行高通量功能成像，可用于研究蛋白质聚集的机制，并且有可能有助于寻找蛋白质聚集和毒性的治疗修饰剂。
