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

C9orf72 六核苷酸重复扩增导致 ALS 和 FTD 神经变性的机制

基本信息

批准号：
9014571
负责人：
Shuying Sun
金额：
$ 9.3万
依托单位：
LUDWIG INSTITUTE FOR CANCER RES LTD
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9014571
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

项目摘要

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（由适用提供）：最近确定在C9orf72非编码区域中的六核苷酸重复扩张是肌萎缩性侧向硬化症（ALS）和额叶临时痴呆（FTD）的最常见遗传原因。疾病机制的主要假设是从扩展的重复序列中获得毒性，具有两个非纯正的机制：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处理； 2）由重复相关的非ATG翻译（RAN翻译）产生的二肽重复蛋白（DPRS）的积累。此外，重复序列既是意义上的和反义方向的翻译。这些不同的产品如何确切或在何种程度上有助于疾病。在这个项目中，提出了结合新的基因组，生物化学以及细胞和小鼠建模工具，以确定I将定义C9ORF72重复扩张依赖性依赖性RNA依赖性RNA特征在人类神经元中得出的大量患者成纤维细胞，包括使用基因组范围序列序列的RNA表达和替代性剪接方法的改变，包括改变RNA的变化。我将确定通过反义寡核苷酸（ASO）治疗降低含义和/或反义重复的转录本是否会逆转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具有单个潜在有毒产物的基因组工程控制细胞来解密重复含RNA的功能贡献，并通过基因组工程控制细胞进行翻译编码的多二肽。我将使用体内RNA标记系统在完整细胞中识别出与hense或反义六核苷酸重复序列相关的特定RNA结合蛋白，并确定其中任何一种的“功能丧失”是否有助于C9orf72神经元中的RNA特征。然后，我将通过操纵候选基因变化，干扰途径和RNA结合蛋白来破坏神经元中的破坏性途径。最后，我将通过将小鼠模型结合使用小鼠模型来鉴定细胞类型特异性，年龄和重复长度依赖性的RNA变化，这是由C9ORF72重复在体内重复扩张引起的，以及IPSC分化的神经元与Artrocytes or oligocydrocydroc，是否可以通过非细胞自主机制对神经元的重复扩展对神经元产生毒性影响。总体而言，我认为我的建议有可能检验有关ALS/FTD的C9ORF72介导的发病机理的几个关键假设，并确定依赖疾病的分子特征，以实现治疗策略的发展。