Ultrasensitive RT-QuIC Seed Amplification Assays for Disease-Associated Tau, α-Synuclein, and Prion Aggregates
Ultrasensitive RT-QuIC Seed Amplification Assays for Disease-Associated Tau, α-Synuclein, and Prion Aggregates
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DOI：
10.1007/978-1-4939-8820-4_2
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Caughey, Byron
中科院分区：
文献类型：
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作者：
Saijo, Eri;Groveman, Bradley R.;Caughey, Byron
tau、α-突触核蛋白（α-Syn）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的分子发病机制的基础。这种病理性聚集体通常可以通过接种聚合机制生长。我们和其他人已经利用这些机制在体外扩增接种活性，并为这些病原学生物标志物设计超灵敏，特异性和定量测定。实时振荡诱导转化（RT-QuIC）测定在具有荧光读数的多孔板中进行，促进有效的通量。对脑脊液（CSF）样品的朊病毒RT-QuIC测定被广泛用于人类朊病毒疾病的死前诊断。最近，我们还描述了一种tau RT-QuIC原型，其已针对皮克病（具有主要的3R tau病理）进行了优化，其检测死后CSF中的3R tau种子，以及极端至十亿倍的脑组织稀释。还开发了alpha Syn RT-QuIC原型，使用死前CSF对帕金森病和路易体痴呆提供了类似于92%的诊断灵敏度和100%的特异性。在这里，我们提供了我们的3R tau和alpha Syn RT-QuIC测定的详细方案，并请读者参考已发表的朊病毒RT-QuIC测定的最新方案（Orru et al. Methods Mol Biol 1658：185-203，2017; Schmitz et al. Nat Protoc 11：2233-2242，2016）。
The abnormal assembly of tau, alpha-synuclein (alpha Syn), 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. alpha Syn RT-QuIC prototypes have also been developed, providing similar to 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 alpha Syn 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).