Pigazzini Maria Lucia, Gallrein Christian, Iburg Manuel, Kaminski Schierle Gabriele, Kirstein Janine
Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund Berlin; NeuroCure Cluster of Excellence, Charité - Universitätsmedizin Berlin.
Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund Berlin.
J Vis Exp. 2020 Mar 27(157). doi: 10.3791/61004.
Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington's, Parkinson's, or Alzheimer's disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.
淀粉样纤维与多种神经退行性疾病相关，如亨廷顿病、帕金森病或阿尔茨海默病。这些淀粉样纤维可隔离内源性亚稳态蛋白以及蛋白质稳态网络（PN）的组分，从而加剧细胞内的蛋白质错误折叠。在动物体内，用于评估淀粉样蛋白聚集过程的工具数量有限。我们提出了一种荧光寿命显微镜（FLIM）方案，该方案能够以非侵入性方式，随着衰老进程以及在PN受到扰动时，监测并量化特定细胞（如神经元）中的淀粉样纤维化。FLIM与荧光团的表达水平无关，无需进一步染色或漂白就能对聚集过程进行分析。当荧光团靠近淀粉样结构时会发生淬灭，这导致荧光寿命缩短。淬灭与淀粉样蛋白的聚集直接相关。FLIM是一种通用技术，可用于以非侵入性方式在体内比较不同淀粉样蛋白的纤维化过程、环境刺激或遗传背景。
