Multiple neurodegenerative diseases are characterized by aggregation of tau molecules. Adult humans express six isoforms of tau that contain either 3 or 4 microtubule binding repeats (3R or 4R tau). Different diseases involve preferential aggregation of 3R (e.g Pick disease), 4R (e.g. progressive supranuclear palsy), or both 3R and 4R tau molecules [e.g. Alzheimer disease and chronic traumatic encephalopathy]. Three ultrasensitive cell-free seed amplification assays [called tau real-time quaking induced conversion (tau RT-QuIC) assays] have been developed that preferentially detect 3R, 4R, or 3R/4R tau aggregates in biospecimens. In these reactions, low-fg amounts of a given self-propagating protein aggregate (the seed) are incubated with a vast excess of recombinant tau monomers (the substrate) in multi-well plates. Over time, the seeds incorporate the substrate to grow into amyloids that can then be detected using thioflavin T fluorescence. Here we describe a tau RT-QuIC assay (K12 RT-QuIC) that, using a C-terminally extended recombinant 3R tau substrate (K12CFh), enables sensitive detection of Pick disease, Alzheimer disease, and chronic traumatic encephalopathy seeds in brain homogenates. The discrimination of Pick disease from Alzheimer disease and chronic traumatic encephalopathy cases is then achieved through the quantitative differences in K12 RT-QuIC assay thioflavin T responses, which correlate with structural properties of the reaction products. In particular, Fourier transform infrared spectroscopy analysis of the respective K12CFh amyloids showed distinct β-sheet conformations, suggesting at least partial propagation of the original seed conformations in vitro. Thus, K12 RT-QuIC provides a single assay for ultrasensitive detection and discrimination of tau aggregates comprised mainly of 3R, or both 3R and 4R, tau isoforms.

中文翻译：

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用于检测和区分阿尔茨海默病和匹克病的 tau 蛋白聚集体的单一超灵敏测定。

多种神经退行性疾病的特征是 tau 分子聚集。成年人表达六种 tau 亚型，其中包含 3 或 4 个微管结合重复序列（3R 或 4R tau）。不同的疾病涉及3R（例如皮克病）、4R（例如进行性核上性麻痹）或3R和4R tau分子的优先聚集[例如阿尔茨海默病和慢性创伤性脑病]。现已开发出三种超灵敏无细胞种子扩增测定法（称为 tau 实时振动诱导转化 (tau RT-QuIC) 测定法），可优先检测生物样本中的 3R、4R 或 3R/4R tau 聚集体。在这些反应中，低 fg 量的给定自繁殖蛋白聚集体（种子）与大量过量的重组 tau 单体（底物）在多孔板中孵育。随着时间的推移，种子结合底物生长成淀粉样蛋白，然后可以使用硫代黄素 T 荧光进行检测。在这里，我们描述了一种 tau RT-QuIC 测定 (K12 RT-QuIC)，该测定使用 C 端延伸的重组 3R tau 底物 (K12CFh)，能够灵敏地检测脑匀浆中的匹克病、阿尔茨海默病和慢性创伤性脑病种子。然后通过 K12 RT-QuIC 测定硫黄素 T 反应的定量差异来区分匹克病与阿尔茨海默病和慢性创伤性脑病病例，这与反应产物的结构特性相关。特别是，对各个 K12CFh 淀粉样蛋白的傅里叶变换红外光谱分析显示出不同的 β-折叠构象，表明原始种子构象在体外至少部分传播。因此，K12 RT-QuIC 提供了一种用于超灵敏检测和区分主要由 3R 或 3R 和 4R tau 同工型组成的 tau 聚集体的单一测定方法。