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

 描述(申请人提供)：C9orf72非编码区的六核苷酸重复扩增最近被确定为肌萎缩侧索硬化症(ALS)和额叶颞叶痴呆(FTD)的最常见遗传原因。该病机制的主要假说是从扩展的重复序列获得毒性，有两种非互斥机制：1)六核苷酸重复序列形成的RNA焦点，可以隔离RNA结合蛋白并破坏RNA加工；2)重复序列相关的非ATG翻译(RAN翻译)产生的二肽重复蛋白(Dprs)积累。此外，重复序列在正义和反义两个方向上都被转录。这些不同的产品究竟如何或在多大程度上导致了疾病，目前还没有确定。在这个项目中，建议结合新的基因组、生物化学和细胞和小鼠建模工具来确定疾病发病的分子机制，并确定治疗开发的候选靶点。我将在来自大量患者成纤维细胞的人类神经元中定义C9orf72重复扩展依赖的RNA签名，包括使用全基因组测序方法在RNA表达和选择性剪接方面的变化。我将确定通过反义寡核苷酸(ASO)治疗减少正义和/或包含反义重复序列的转录本是否逆转C9orf72患者神经元中的RNA签名。我还将通过基因组工程控制具有个别潜在有毒产物的细胞，破译包含重复序列的RNA和RAN翻译编码的多肽的功能贡献。我将使用体内核糖核酸标记系统在完整的细胞中识别与正义或反义六核苷酸重复相关的特定核糖核酸结合蛋白(S)，并确定其中任何一个的“功能丧失”是否导致C9orf72神经元的核糖核酸签名。然后，我将通过操纵候选基因变化、受干扰的途径和RNA结合蛋白来破译神经元中的损伤途径。最后，我将通过结合小鼠模型来确定C9orf72重复扩张在体内引起的细胞类型特异性、年龄和重复长度依赖的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同培养，来确定胶质细胞中的重复扩张是否通过非细胞自主机制对神经元产生毒性影响。总体而言，我相信我的建议有可能测试几个关于C9orf72介导的ALS/FTD发病机制的关键假说，并确定一个疾病依赖的分子特征，使治疗策略的发展成为可能。

 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.