Mechanisms of C9orf72 hexanucleotide repeat expansion caused neurodegeneration in ALS and FTD

 描述（由申请人提供）：最近确定C9 orf 72非编码区中的六核苷酸重复扩增是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的最常见遗传原因。疾病机制的主要假设是来自扩增的重复序列的毒性获得，具有两种非相互排斥的机制：1）由六核苷酸重复序列形成的RNA焦点，其可以螯合RNA结合蛋白并破坏RNA加工;和2）由重复序列相关的非ATG翻译（RAN翻译）产生的二肽重复序列蛋白（DPR）的积累。此外，重复序列在有义和反义方向上转录。这些不同的产品究竟如何或在多大程度上导致疾病尚未确定。在本项目中，建议结合联合收割机新的基因组、生物化学和细胞及小鼠建模工具，以确定疾病发病机制的分子机制，并确定治疗开发的候选靶点。我将使用全基因组测序方法，在来自大量患者成纤维细胞的人类神经元中定义C9 orf 72重复扩增依赖性RNA签名，包括RNA表达和选择性剪接的改变。本发明人将确定通过反义寡核苷酸（阿索）处理减少含有正义和/或反义重复的转录物是否逆转C9 orf 72患者神经元中的RNA特征。我还将通过基因组工程控制细胞与个别潜在的有毒产品破译功能的贡献，从重复含有RNA和RAN防御编码的聚二肽。我将使用体内RNA标记系统在完整细胞中鉴定与有义或反义六核苷酸重复相关的特异性RNA结合蛋白，并确定这些蛋白中任何一种的“功能丧失”是否有助于C9 orf 72神经元中的RNA特征。然后，我将通过操纵候选基因的变化、干扰的通路和RNA结合蛋白来破译神经元中的破坏性通路。最后，我将确定是否在神经胶质细胞中的重复扩增通过非细胞自主机制对神经元产生毒性作用，通过结合小鼠建模来识别由C9 orf 72重复扩增在体内引起的细胞类型特异性、年龄和重复长度依赖性RNA变化，以及将iPSC分化的神经元与星形胶质细胞或少突胶质细胞共培养。总体而言，我认为我的提案有可能检验关于C9 orf 72介导的ALS/FTD发病机制的几个关键假设，并确定一种疾病依赖性分子特征，从而能够开发治疗策略。

 DESCRIPTION (provided by applicant): Hexanucleotide repeat expansion in a non-coding region of C9orf72 was recently identified to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The leading hypothesis for the disease mechanism is gain of toxicity from the expanded repeats, with two non- mutually exclusive mechanisms: 1) RNA foci formed by hexanucleotide repeats that could sequester RNA binding proteins and disrupt RNA processing; and 2) accumulation of dipeptide repeat proteins (DPRs) produced by repeat-associated non-ATG translation (RAN translation). Furthermore, the repeats are transcribed in both sense and antisense directions. How exactly or to what extent these different products contribute to disease is not established. In this project, propose to combine new genomic, biochemistry and cellular and mouse modeling tools to determine the molecular mechanism of disease pathogenesis and identify candidate targets for therapeutic development. I will define a C9orf72 repeat expansion-dependent RNA signature in human neurons derived from a large number of patient fibroblasts, including alterations in both RNA expression and alternative splicing using genome-wide sequencing approaches. I will determine whether decreasing sense and/or antisense repeat- containing transcripts by antisense oligonucleotide (ASO) treatment reverses the RNA signature in C9orf72 patient neurons. I will also decipher the functional contribution from repeat-containing RNAs and RAN translation-encoded poly-dipeptides by genome engineering control cells with individual potentially toxic product. I will identify specific RNA-binding protein(s) associated with either sense or antisense hexanucleotide repeats in intact cells using an in vivo RNA tagging system, and determine whether "loss of function" of any of these contributes to the RNA signature in C9orf72 neurons. I will then decipher the damaging pathways in neurons by manipulating the candidate gene changes, perturbed pathways and RNA-binding proteins. Finally, I will determine whether the repeat expansion in glial cells have toxic effects on neurons through a non-cell autonomous mechanism by combining mouse modeling for identification of cell type-specific, age- and repeat length-dependent RNA changes caused by C9orf72 repeat expansion in vivo, and co-culturing of iPSC- differentiated neurons with astrocytes or oligodendrocytes. Overall, I believe that my proposal has the potential to test several key hypotheses regarding C9orf72-mediated pathogenesis of ALS/FTD and identify a disease- dependent molecular signature that enables the development of therapeutic strategies.