Furman Jennifer L, Holmes Brandon B, Diamond Marc I
Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center;
Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center.
J Vis Exp. 2015 Dec 8(106):e53205. doi: 10.3791/53205.
Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as α-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.
越来越多的证据支持毒性蛋白聚集体或蛋白病种子的跨细胞传播，这是包括阿尔茨海默病和相关tau蛋白病在内的几种神经退行性疾病发病和进展的一种机制。tau蛋白种子在疾病进展中可能起关键作用，这有力地支持了需要一种能够轻松检测生物样品中种子活性的灵敏检测方法。通过结合荧光共振能量转移（FRET）的特异性、流式细胞术的灵敏度和单克隆细胞系的稳定性，设计了一种超灵敏的种子检测方法，该方法与从重组或生物样品（包括人和小鼠脑匀浆）中检测种子兼容。该检测方法使用稳定表达携带疾病相关P301S突变的tau蛋白易聚集重复结构域（RD）的单克隆HEK 293T细胞，该结构域与CFP或YFP融合，在蛋白质聚集时产生FRET信号。蛋白病tau蛋白种子（而非其他蛋白质）被生物传感器细胞摄取会刺激RD-CFP和RD-YFP的聚集，流式细胞术可灵敏且定量地监测这种聚集诱导的FRET。该检测方法可检测飞摩尔浓度（单体当量）的重组tau蛋白种子，动态范围跨越三个数量级，并且与tau蛋白病转基因小鼠和人类tau蛋白病受试者的脑匀浆兼容。经过轻微修改，该检测方法还可以检测其他蛋白病种子（如α-突触核蛋白）的种子活性，并且也与原代神经元培养兼容。FRET流式细胞术的简便性、灵敏度和广泛适用性使其可用于研究多种蛋白质聚集性疾病。
