Saijo Eri, Groveman Bradley R, Kraus Allison, Metrick Michael, Orrù Christina D, Hughson Andrew G, Caughey Byron
Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA.
Methods Mol Biol. 2019;1873:19-37. doi: 10.1007/978-1-4939-8820-4_2.
The abnormal assembly of tau, α-synuclein (αSyn), or prion protein into oligomers and multimers underpins the molecular pathogenesis of multiple neurodegenerative diseases. Such pathological aggregates can often grow by seeded polymerization mechanisms. We and others have taken advantage of these mechanisms to amplify seeding activities in vitro and devise ultrasensitive, specific and quantitative assays for these etiological biomarkers. Real-time quaking-induced conversion (RT-QuIC) assays are performed in multiwell plates with fluorescent readouts, facilitating efficient throughput. Prion RT-QuIC assays on cerebrospinal fluid (CSF) samples are being widely used for antemortem diagnosis of human prion diseases. Recently, we have also described a tau RT-QuIC prototype that has been optimized for Pick disease (with predominant 3R tau pathology) that detects 3R tau seeds in postmortem CSF, and brain tissue dilutions as extreme as a billion-fold. αSyn RT-QuIC prototypes have also been developed, providing ~92% diagnostic sensitivity and 100% specificity for Parkinson's disease and dementia with Lewy bodies using antemortem CSF. Here we provide detailed protocols for our 3R tau and αSyn RT-QuIC assays and refer the reader to published up-to-date protocols for prion RT-QuIC assays (Orru et al. Methods Mol Biol 1658:185-203, 2017; Schmitz et al. Nat Protoc 11:2233-2242, 2016).
tau蛋白、α-突触核蛋白（αSyn）或朊病毒蛋白异常组装成寡聚体和多聚体是多种神经退行性疾病分子发病机制的基础。这种病理性聚集体通常可通过种子聚合机制生长。我们和其他研究人员利用这些机制在体外扩增种子活性，并设计了针对这些病因生物标志物的超灵敏、特异且定量的检测方法。实时震颤诱导转化（RT-QuIC）检测在多孔板中进行，采用荧光读数，便于高效高通量检测。针对脑脊液（CSF）样本的朊病毒RT-QuIC检测已广泛用于人类朊病毒疾病的生前诊断。最近，我们还描述了一种针对匹克病（以3R tau病理为主）优化的tau RT-QuIC原型，该原型可检测死后脑脊液中的3R tau种子，以及稀释至十亿分之一的脑组织样本。αSyn RT-QuIC原型也已开发出来，使用生前脑脊液对帕金森病和路易体痴呆的诊断敏感性约为92%，特异性为100%。在此，我们提供3R tau和αSyn RT-QuIC检测的详细方案，并请读者参考已发表的朊病毒RT-QuIC检测的最新方案（Orru等人，《分子生物学方法》1658:185 - 203，2017；Schmitz等人，《自然方法》11:2233 - 2242，2016）。
