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

摘要 肌萎缩侧索硬化症（ALS）和额颞叶痴呆症最常见的已知遗传原因 (FTD)是C9 orf 72基因第一内含子中的G4 C2六核苷酸重复扩增。这种重复经历了 蛋白质翻译起始的非规范形式，称为重复相关非AUG（RAN）翻译 （RANT）。RANT起始于非AUG密码子，在C9 orf 72相关ALS/FTD（C9 ALS/FTD）中产生 二肽重复蛋白（DPR），其在患者脑中的内含物内聚集。DPR表达分离 从含有潜在毒性重复序列的RNA种类中释放足以诱导细胞死亡并降低细胞活力， 许多模型系统，包括初级哺乳动物神经元。因此，RANT被认为有助于 发病机理尽管有这种核心的重要性，确切的内源性重复含有转录本 DPRs的翻译来源仍然未知。重复序列位于第一内含子内， 内含子作为RNA变体从前mRNA剪接并迅速降解。在C9 ALS/FTD中，重复RNA 在细胞质中观察并进行翻译。最初的研究表明，RANT可能发生于 一种线性mRNA，其中含有重复的内含子被保留或通过来自C9 orf 72的内部核糖体进入 不能降解的皮革。然而，我们的团队最近发现了第三个潜在的源模板， 对于C9 RANT：在第一个内含子本身内重复触发转录起始，产生加帽重复 含有mRNA。该提案将确定在患者神经元中经历C9 RANT的RNA模板 以及影响其产生和稳定性的靶向途径是否能够调节RANT C9 ALS/FTD患者衍生的iNeurons中产生和抑制毒性。使用患者源性神经元，我 将确定含有重复序列的C9 orf 72转录物的分子性质，所述转录物使用 多核糖体分析、qRT-PCR和杂交链反应与共聚焦显微镜联用， 生化分析与此同时，我将开发选择性靶向此的反义寡核苷酸（ASO）。 新的C9 orf 72转录本，并确定调节C9 orf 72剪接和RNA种类产生的影响 对C9 RANT生成和重复毒性的影响。本建议书中描述的培训将使我为在 细胞与分子神经生物学的接口及其在面向病人的神经系统中的应用 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.