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

确定 C9orf72 相关 ALS/FTD 中 RAN 翻译的机制

基本信息

批准号：
10620180
负责人：
Shannon Lynne Miller
金额：
$ 4.07万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10620180
关键词：
Affect Antisense Oligonucleotides Biochemical Biological Assay Biological Models Brain C9ORF72 Cell Death Induction Cell Nucleus Cell Survival Cells Cellular Neurobiology Codon Nucleotides Confocal Microscopy Coupled Cytoplasm Dipeptides Disease Enzyme-Linked Immunosorbent Assay Enzymes Exons Future Generations Genes Genetic Genetic Transcription Homeostasis Impairment Introns Measures Messenger RNA Molecular Molecular Neurobiology Nature Neurodegenerative Disorders Neurons Pathogenesis Pathogenicity Pathology Pathway interactions Patients Physiological Production Proteins Quantitative Reverse Transcriptase PCR RNA RNA Splicing Reaction Reading Frames Reporter Ribosomes Role Source Techniques Therapeutic Toxic effect Training Transcript Transcription Initiation Translating Translation Initiation Translations Work clinical application design druggable target frontotemporal lobar dementia amyotrophic lateral sclerosis immunocytochemistry insight knock-down lariat debranching enzyme mRNA Precursor neuronal survival novel patient oriented polysome profiling protein aggregation protein expression therapeutic development tool translational neuroscience

项目摘要

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.

抽象的肌萎缩性侧硬化症（ALS）和额颞痴呆的最常见遗传原因（FTD）是C9ORF72基因的第一个内含子中的G4C2六核苷酸重复膨胀。这种重复发生一种非典型的蛋白质翻译起始形式，称为重复相关的非aug（ran）翻译（咆哮）。从非高级密码子和C9ORF72相关的ALS/FTD（C9ALS/FTD）中启动rant启动二肽重复蛋白（DPRS），在患者大脑中聚集。 DPR表达式分开来自潜在毒性重复的RNA物种足以诱导细胞死亡并降低细胞活力许多模型系统，包括原发性哺乳动物神经元。因此，人们认为rant有助于疾病发病机理。尽管具有至关重要的重要性，但确切的内源性重复转录本从中翻译的DPR仍然未知。重复位于第一个内含子内，通常位于内含子是从前MRNA剪接的RNA肾脏，并迅速降解。在C9als/ftd中，重复RNA为在细胞质中观察到并经历翻译。最初的研究表明，可能是从线性mRNA保留了重复内含子或通过C9orf72的内部核糖体入口未能降解的lariat。但是，我们的小组最近确定了第三个潜在的源模板对于c9rant：在第一个内含子本身内重复触发的转录启动包含mRNA。该建议将确定患者神经元中经历C9RANT的RNA模板以及针对影响其生产和稳定性的目标途径是否能够调节咆哮 C9ALS/FTD患者衍生的不发神经元的产生和抑制毒性。使用患者来源的神经元，我将确定重复的分子性质，其中包含使用C9orf72转录本的分子性质多元体分析，QRT-PCR和杂交链反应与共聚焦显微镜和生化测定。同时，我将开发反义寡核苷酸（ASO），有选择地针对这一点新型C9orf72转录本，并确定调节C9orf72剪接和RNA物种产生的影响关于C9Rant的产生和重复毒性。本提案中描述的培训将使我准备工作细胞和分子神经生物学之间的界面及其在以患者为导向的疗法。