Investigating non-coding RNA 7SK in Huntington's Disease neurodegeneration using direct neuronal conversion

项目总结 衰老是神经退行性疾病中最大的风险因素，应该在成人发病时总结一下 疾病研究模型。然而，目前的模型使用的是从诱导分化而来的人神经元 多能干细胞，可以消除衰老的细胞信号。Yoo实验室率先开发了一种系统 利用microRNAs-9/9*和-124(miR-9/9*-124)的异位表达直接将人类成年 成纤维细胞(HAFs)转化为microRNA诱导的神经元(MIN)。MIN保持分子年龄相关 特性，包括表观遗传时钟、端粒长度和氧化应激信号。其他内容 转录因子可以与miR-9/9*-124协同作用，产生包括microRNA在内的神经细胞亚型。 诱导的中等刺神经元(MSN)是亨廷顿病(HD)的原代细胞。这些子类型 可以模拟成人起病的神经退行性疾病的细胞病理，如内源性聚集， DNA损伤、线粒体功能障碍和细胞死亡。多种疾病中的变性神经元，包括 HD，还表现出长基因表达(LGE)增加，染色质失调，以及超兴奋性 与健康的神经元相比，这些与HD相关的特征尚未成功地定位于 治疗。这一提议建立在我发表的第一作者研究成果的基础上，该研究表明，小核RNA RN7SK (7SK)是神经元染色质可及性和基因组转录激活所必需的 MIR-9/9*-124介导的重编程。这些神经元基因的很大一部分和LGE对应于 与对照MSN相比，HD-MSN中调控失调的基因。在这里，我建议用我以前的 研究HD-MSN中7SK表达缺失如何可能改善HD神经退变的研究结果 表型。在目标1中，我计划测试敲击7SK是否可以使HD-MSN中的LGE达到控制水平，以及是否 可以调制HD相关的染色质信号。在目标2之后，我建议评估是否压制 7SK可改善HD相关神经退行性变表型，包括过度兴奋、代谢 功能障碍和自发性细胞死亡。结合分子、基因组、电生理和细胞 病理分析，我将评估7SK的减少是否可以恢复这些HD相关的分子特征和 细胞表型对健康对照MSN水平的影响。完成这些目标将揭示 减轻HD相关表型的神经元染色质和转录调节。这些数据将 为成人起病神经退行性疾病模型的发展提供基础知识 探索HD治疗开发的假定表观基因组靶点。

PROJECT SUMMARY Aging is the greatest risk factor across neurodegenerative diseases and should be recapitulated in adult-onset disease research models. However, current models utilize human neurons differentiated from induced pluripotent stem cells, which erase cellular signatures of aging. The Yoo Laboratory has pioneered a system that uses ectopic expression of microRNAs-9/9* and -124 (miR-9/9*-124) to directly convert human adult fibroblasts (HAFs) into microRNA-induced neurons (miNs). The miNs maintain molecular age-associated properties, including the epigenetic clock, telomere lengths and oxidative stress signatures. Additional transcription factors can synergize with miR-9/9*-124 to generate neuronal cell subtypes including microRNA- induced medium spiny neurons (MSNs), the primary cells lost in Huntington’s Disease (HD). These subtypes can model cellular pathologies in adult-onset neurodegenerative disease, such as endogenous aggregation, DNA damage, mitochondrial dysfunction, and cell death. Degenerating neurons in multiple disorders, including HD, also demonstrate increased long gene expression (LGE), chromatin dysregulation, and hyperexcitability in comparison to healthy neurons, yet these HD-associated features have not been successfully targeted for treatment. This proposal builds on my published first-author study showing that the small nuclear RNA RN7SK (7SK) is required for neuronal chromatin accessibility and transcription activation across the genome during miR-9/9*-124 mediated reprogramming. A substantial portion of these neuronal loci and LGE correspond to genes that are dysregulated in HD-MSNs compared to control MSNs. Here, I propose to use my previous findings to study how depletion of 7SK expression in HD-MSNs can possibly improve HD neurodegenerative phenotypes. In Aim 1, I plan to test if knocking down 7SK can bring LGE in HD-MSNs to control levels and if it can modulate HD-associated chromatin signatures. Following in Aim 2, I propose to assess if repression of 7SK can ameliorate HD-associated neurodegenerative phenotypes, including hyperexcitability, metabolic dysfunction, and spontaneous cell death. Combining molecular, genomic, electrophysiological, and cell pathology assays, I will evaluate if reduction of 7SK can restore these HD-associated molecular features and cellular phenotypes to healthy control MSN levels. Completion of these aims will reveal implications of neuronal chromatin and transcriptional regulation for alleviating HD-associated phenotypes. These data will provide foundational knowledge for the advancement of adult-onset neurodegenerative disease modeling and explore putative epigenomic targets for HD therapeutic development.