Determining the Mechanism of RAN Translation in C9orf72-Associated ALS/FTD

摘要 肌萎缩侧索硬化症(ALS)和额颞叶痴呆最常见的已知遗传原因 (FTD)是位于C9orf72基因第一内含子的G4C2重复序列。这一重复经历了 一种非规范形式的蛋白质翻译启动，称为重复相关非8月(RAN)翻译 (咆哮)。RANT起始于非AUG密码子和C9orf72相关的ALS/FTD(C9ALS/FTD)产生 病人脑内包涵体内聚集的二肽重复蛋白(Dprs)。DPR表达分离 来自潜在有毒的含有重复序列的RNA物种足以诱导细胞死亡并降低细胞活力 许多模型系统，包括原始哺乳动物神经元。因此，咆哮被认为是导致 疾病发病机制。尽管这一点至关重要，但准确的内源性包含重复序列的转录本 从哪里翻译DPR仍然是未知的。重复序列位于第一内含子内，通常， 内含子由前mRNA拼接而成，作为RNA连接物，并迅速降解。在C9ALS/FTD中，重复RNA是 在细胞质中观察并进行翻译。初步研究表明，咆哮可能发生在 含有重复的内含子被保留或通过内部核糖体从C9orf72进入的线状mRNA. 没有经历退化的套索。然而，我们的小组最近确定了第三个潜在的源模板 对于C9RANT：重复触发了第一内含子自身的转录启动，从而产生封顶重复 含有mRNA。这项提议将确定患者神经元中经历C9RANT的RNA模板 以及影响其产量和稳定性的靶向途径是否能够调节RANT C9ALS/FTD患者来源的iNeurons的产生和抑制毒性。使用患者来源的神经元，我 将确定包含C9orf72转录本的重复序列的分子性质，这些转录本使用RANT 多聚体图谱，qRT-PCR和杂交链式反应结合共聚焦显微镜和 生化分析。同时，我将开发反义寡核苷酸(ASO)，选择性地针对这一点 新的C9orf72转录本及其对调控C9orf72剪接和RNA物种产生的影响 关于C9RANT的生成和重复毒性。这份建议书中描述的培训将使我做好在 细胞与分子神经生物学的接口及其面向患者的应用 治疗学。

Abstract The most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a G4C2 hexanucleotide repeat expansion in the first intron of the C9orf72 gene. This repeat undergoes a non-canonical form of protein translation initiation known as repeat associated non-AUG (RAN) translation (RANT). RANT initiates from non-AUG codons and in C9orf72-associated ALS/FTD (C9ALS/FTD) produces dipeptide repeat proteins (DPRs) that aggregate within inclusions in patient brains. DPR expression separate from potentially toxic repeat-containing RNA species is sufficient to induce cell death and reduce cell viability in many model systems, including primary mammalian neurons. As such, RANT is thought to contribute to disease pathogenesis. Despite this central importance, the exact endogenous repeat-containing transcripts from which DPRs are translated remains unknown. The repeat is located within the first intron and typically, introns are spliced from pre-mRNA as RNA lariats and rapidly degraded. In C9ALS/FTD, repeat RNAs are observed in the cytoplasm and undergo translation. Initial studies suggested that RANT could occur from a linear mRNA in which the repeat-containing intron is retained or via internal ribosomal entry from a C9orf72 lariat that fails to undergo degradation. However, our group recently identified a third potential source template for C9RANT: repeat triggered transcriptional initiation within the first intron itself that generates capped repeat containing mRNAs. This proposal will determine the RNA templates that undergo C9RANT in patient neurons and whether targeting pathways which influence their production and stability are capable of modulating RANT production and suppressing toxicity in C9ALS/FTD patient-derived iNeurons. Using patient-derived neurons, I will determine the molecular nature of repeat containing C9orf72 transcripts that undergo RANT using polysome profiling, qRT-PCR, and hybridization chain reaction coupled with confocal microscopy and biochemical assays. In parallel, I will develop antisense oligonucleotides (ASOs) that selectively target this novel C9orf72 transcript and determine the impact of modulating C9orf72 splicing and RNA species production on C9RANT generation and repeat toxicity. The training described in this proposal will prepare me to work at the interface between cellular and molecular neurobiology and its application towards patient-oriented therapeutics.