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Abstract：
Transcribed nucleotide repeat expansions form detectable RNA foci in patient cells that contribute to disease pathogenesis. The most widely used method for detecting RNA foci, fluorescence in situ hybridization (FISH), is powerful but can suffer from issues related to signal above background. Here we developed a repeat-specific form of hybridization chain reaction (R-HCR) as an alternative method for detection of repeat RNA foci in two neurodegenerative disorders: C9orf72 associated ALS and frontotemporal dementia (C9 ALS/FTD) and Fragile X-associated tremor/ataxia syndrome. R-HCR to both G4C2 and CGG repeats exhibited comparable specificity but > 40 × sensitivity compared to FISH, with better detection of both nuclear and cytoplasmic foci in human C9 ALS/FTD fibroblasts, patient iPSC derived neurons, and patient brain samples. Using R-HCR, we observed that integrated stress response (ISR) activation significantly increased the number of endogenous G4C2 repeat RNA foci and triggered their selective nuclear accumulation without evidence of stress granule co-localization in patient fibroblasts and patient derived neurons. These data suggest that R-HCR can be a useful tool for tracking the behavior of repeat expansion mRNA in C9 ALS/FTD and other repeat expansion disorders.
摘要：
转录的核苷酸重复扩增在患者细胞中形成可检测的RNA病灶，这有助于疾病的发病机理。最广泛使用的检测RNA病灶的方法，荧光原位杂交（FISH），功能强大，但可能会遇到与背景信号相关的问题。在这里，我们开发了一种重复特异性形式的杂交链反应（R-HCR），作为检测两种神经退行性疾病中重复RNA病灶的替代方法：C9orf72相关的ALS和额颞叶痴呆（C9ALS/FTD）和脆性X相关的震颤/共济失调综合征。与FISH相比，对G4C2和CGG重复的R-HCR表现出相当的特异性，但>40倍的灵敏度，在人C9ALS/FTD成纤维细胞中更好地检测细胞核和细胞质灶，患者iPSC衍生的神经元，和病人的大脑样本.使用R-HCR,我们观察到整合应激反应(ISR)激活显著增加了内源性G4C2重复RNA病灶的数量,并触发了它们的选择性核积累,而没有证据表明应激颗粒在患者成纤维细胞和患者来源的神经元中共定位.这些数据表明R-HCR可以是追踪C9ALS/FTD和其他重复扩增障碍中重复扩增mRNA行为的有用工具。
