C9orf72 hexanucleotide repeat expansion caused neurodegeneration in ALS and FTD

C9orf72 六核苷酸重复扩增导致 ALS 和 FTD 中的神经变性

• 批准号: 9324797
• 负责人: Shuying Sun
• 金额: $ 24.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324797
• 关键词: Age; Aging; Alternative Splicing; Amyotrophic Lateral Sclerosis; Antisense Oligonucleotides; Antithymoglobulin; Astrocytes; Biochemistry; C9ORF72; Candidate Disease Gene; Cell model; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Coupled; Dipeptides; Disease; Fibroblasts; Frontotemporal Dementia; Genes; Genetic; Genetic Transcription; Genome engineering; Genomics; Goals; Health; Housekeeping Gene; Human; Individual; Introns; Length; Life; Ligation; Measures; Mediating; Messenger RNA; Methodology; Methods; Molecular; Molecular Profiling; Motor Neurons; Mus; Muscle Weakness; Mutation; Nerve Degeneration; Neuroglia; Neurons; Oligodendroglia; Oligonucleotides; Paralysed; Pathogenesis; Pathway interactions; Patients; Phase; Proteins; RNA; RNA Processing; RNA Sequences; RNA Splicing; RNA-Binding Proteins; System; Testing; Therapeutic Intervention; Toxic effect; Transcript; Transgenes; Translating; Translations; Untranslated RNA; Work; cell type; genome-wide; in vivo; induced pluripotent stem cell; loss of function; motor neuron degeneration; mouse model; targeted treatment; therapeutic development; therapeutic target; tool; transcriptome sequencing

 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.

 描述（由申请人提供）：C9orf72 非编码区中的六核苷酸重复扩增最近被确定为肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD) 的最常见遗传原因。该疾病机制的主要假设是从扩增的重复序列中获得毒性，具有两种不相互排斥的机制：1）由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) 由重复相关的非 ATG 翻译（RAN 翻译）产生的二肽重复蛋白（DPR）的积累。此外，重复序列在有义和反义方向上转录。这些不同的产品到底如何或在多大程度上导致疾病尚不清楚。在该项目中，建议结合新的基因组、生物化学以及细胞和小鼠建模工具来确定疾病发病机制的分子机制并确定治疗开发的候选靶标。我将在源自大量患者成纤维细胞的人类神经元中定义 C9orf72 重复扩增依赖性 RNA 特征，包括使用全基因组测序方法的 RNA 表达和选择性剪接的改变。我将确定通过反义寡核苷酸 (ASO) 治疗减少含有有义和/或反义重复的转录物是否会逆转 C9orf72 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单个潜在有毒产物来破译含有重复的 RNA 和 RAN 翻译编码的聚二肽的功能贡献。我将使用体内 RNA 标记系统识别与完整细胞中有义或反义六核苷酸重复相关的特定 RNA 结合蛋白，并确定其中任何一个的“功能丧失”是否会导致 C9orf72 神经元中的 RNA 特征。然后，我将通过操纵候选基因变化、扰动途径和 RNA 结合蛋白来破译神经元中的破坏途径。最后，我将通过结合小鼠模型来鉴定由体内 C9orf72 重复扩增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 iPSC 分化神经元与星形胶质细胞或少突胶质细胞的共培养，确定神经胶质细胞中的重复扩增是否通过非细胞自主机制对神经元产生毒性作用。总的来说，我相信我的建议有可能测试关于 C9orf72 介导的 ALS/FTD 发病机制的几个关键假设，并确定能够制定治疗策略的疾病依赖性分子特征。