Crespo Rosa, Koudstaal Wouter, Apetri Adrian
Janssen Prevention Center, Janssen Pharmaceutical Companies of Johnson and Johnson.
Janssen Prevention Center, Janssen Pharmaceutical Companies of Johnson and Johnson;
J Vis Exp. 2018 Nov 20(141). doi: 10.3791/58570.
Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer's disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.
tau蛋白的聚集和双螺旋丝的形成是阿尔茨海默病和其他tau蛋白病的标志。与其他与神经退行性疾病相关的蛋白质相比，报道的tau蛋白体外聚集动力学不太一致，呈现出相对较高的变异性。在这里，我们描述了一种体外聚集测定方法的开发，该方法模拟了体内与tau蛋白错误折叠和聚集相关的预期步骤。该测定使用最长的tau异构体（huTau441），其既包含N端酸性插入片段，又包含四个微管结合结构域（MBD）。体外聚集通过添加肝素触发，并在96孔微孔板中通过硫黄素T荧光连续监测。tau聚集测定在不同孔、实验运行和蛋白质批次之间具有高度可重复性。聚集导致tau PHF样形态，这在播种从头形成的纤维状结构方面非常有效。除了应用于研究tau蛋白错误折叠和聚集的机制外，当前的测定方法还是筛选可能干扰tau蛋白发病机制的药物的强大工具。
