Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry
Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry
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DOI：
10.3791/53205
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发表时间：
2015-12-01
影响因子：
1.2
通讯作者：
Diamond, Marc I.
中科院分区：
文献类型：
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作者：
Furman, Jennifer L.;Holmes, Brandon B.;Diamond, Marc I.
越来越多的证据支持毒性蛋白质聚集体或蛋白质病种子的跨细胞传播，作为几种神经退行性疾病（包括阿尔茨海默病和相关的tau蛋白病）中病理学的起始和进展的机制。tau种子在疾病进展中的潜在关键作用强烈支持了对容易检测生物样品中的种子活性的灵敏测定的需要。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，已经设计了一种超灵敏的接种测定法，其与来自重组或生物样品（包括人和小鼠脑匀浆）的种子检测相容。该试验采用单克隆HEK 293 T细胞，其稳定表达tau的易聚集重复结构域（RD），所述tau具有与CFP或YFP融合的疾病相关P301 S突变，其在蛋白质聚集时产生FRET信号。蛋白质病tau种子（而不是其他蛋白质）进入生物传感器细胞的摄取刺激RD-CFP和RD-YFP的聚集，流式细胞术灵敏地和定量地监测这种聚集诱导的FRET。该测定检测重组tau种子的飞摩尔浓度（单体当量），具有跨越三个数量级的动态范围，并且与来自tau蛋白病转基因小鼠和人tau蛋白病受试者的脑匀浆相容。通过轻微的修改，该测定还可以检测其他蛋白质病种子（例如α-突触核蛋白）的接种活性，并且还与原代神经元培养物相容。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广泛的蛋白质聚集疾病。
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 a-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.