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

项目摘要 衰老是神经退行性疾病中最大的风险因素，应在成人发病中重现。 疾病研究模型然而，目前的模型利用从诱导分化的人类神经元， 多能干细胞，消除衰老的细胞特征。Yoo实验室开创了一个系统， 利用microRNA-9/9 * 和-124（miR-9/9 *-124）的异位表达， 将成纤维细胞（HAF）转化为microRNA诱导的神经元（MiN）。miN维持分子年龄相关的 这些特征包括表观遗传时钟、端粒长度和氧化应激特征。额外 转录因子可以与miR-9/9 *-124协同作用，产生神经元细胞亚型，包括microRNA-1。 诱导中等多刺神经元（MSN），即在亨廷顿病（HD）中丢失的原代细胞。这些亚型 可以模拟成人发病的神经退行性疾病中的细胞病理，例如内源性聚集， DNA损伤、线粒体功能障碍和细胞死亡。多种疾病中的退化神经元，包括 HD也显示了增加的长基因表达（LGE），染色质失调，和高兴奋性。 与健康的神经元相比，这些HD相关的特征尚未成功靶向于 治疗这个建议建立在我发表的第一作者研究的基础上，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。这些神经元位点和LGE的相当大一部分对应于 与对照MSN相比，HD-MSN中失调的基因。在这里，我建议使用我以前的 研究HD-MSNs中7SK表达的缺失如何可能改善HD神经退行性变 表型在目标1中，我计划测试是否击倒7SK可以使HD-MSN中的LGE达到控制水平， 可以调节HD相关的染色质特征。在目标2之后，我建议评估是否抑制 7SK可以改善HD相关的神经退行性表型，包括过度兴奋、代谢紊乱、 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 病理学分析，我将评估是否减少7SK可以恢复这些HD相关的分子特征， 细胞表型与健康对照MSN水平。这些目标的实现将揭示 神经元染色质和转录调节，以减轻HD相关的表型。这些数据将 为成人发病神经退行性疾病建模的进步提供基础知识， 探索用于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.