Molecular Characterization of ALS/FTD in a novel C9orf72 BAC mouse model.

项目摘要 C9orf72基因中微卫星GGGGCC重复序列的扩大与家族性和家族性 零星形式的肌萎缩侧索硬化症(ALS)和额颞痴呆(FTD)。而分子 这种疾病的基础(C9-ALS/FTD)仍然很大程度上尚不清楚，提出的发病机制包括C9orf72 由于单倍体不足导致的功能丧失，导致蛋白质隔离的RNA功能获得(GOF)和 重复相关的非ATG(RAN)翻译导致有毒的C9-RAN二肽重复的产生 蛋白质。基于我们之前对其他微卫星扩张性疾病的研究，这项建议旨在 测试我们的隔离失败假说，该假说整合了RNA和RAN增益的作用机制。 根据这一假设，双向正义和反义C9orf72转录导致招募 重复扩增RNA以产生隔离它们的正义和反义RNA焦点的细胞因子 细胞核中含有有毒的核糖核酸。体细胞重复扩增和/或年龄相关的细胞应激导致滴定 GGGGCC和GGCCCC RNA结合蛋白，然后这些RNA的核质输出和 高毒性的C9-RAN蛋白在细胞质中的翻译导致神经退化。我们有 生成了C9-ALS/FTD的BAC转基因模型，这将使我们能够验证这一假设。这只鼠标 发展分子(RNA焦点、C9-RAN蛋白)和病理生理学(神经元丢失、瘫痪、 C9-ALS/FTD的特征。在这个提议中，我们将首先测试RNA的假设 GOF效应通过执行RNA-FISH，转录组分析和 不同发育时期及不同脑区和脊髓组织的免疫学检测 无症状、无症状和有症状的C9-BAC小鼠。此信息将结合使用 通过组织病理学和电生理测试验证了C9-RAN蛋白积聚的假说 会引发神经退化和急性疾病阶段。应激途径调节RAN的可能性 翻译也将接受测试。最后，我们将测试反义寡核苷酸(ASO)缺口介导的 敲除正义和反义C9orf72转录本或同时敲除正义和反义C9orf72转录本可阻止 我们的C9-BAC转基因小鼠的RNA和RNA毒性。总体而言，这项研究的目标是界定 C9-ALS/FTD疾病发生和发展的致病机制并提供 用于治疗开发的可获得且具有良好特性的小鼠模型。

Project Summary The expansion of a microsatellite GGGGCC repeat in the C9orf72 gene has been linked to both familial and sporadic forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While the molecular basis of this disease (C9-ALS/FTD) remains largely unknown, proposed disease mechanisms include C9orf72 loss of function due to haploinsufficiency, RNA gain of function (GOF) leading to protein sequestration and repeat-associated non-ATG (RAN) translation resulting in the production of toxic C9-RAN dipeptide repeat proteins. Based on our prior studies on other microsatellite expansion diseases, this proposal is designed to test our sequestration failure hypothesis, which integrates RNA and RAN gain of function mechanisms. According to this hypothesis, bidirectional sense and antisense C9orf72 transcription results in the recruitment of cellular factors to repeat expansion RNAs to produce sense and antisense RNA foci that sequester these toxic RNAs in the nucleus. Somatic repeat expansion and/or age-related cellular stress results in titration of GGGGCC and GGCCCC RNA binding proteins followed by nucleocytoplasmic export of these RNAs and translation of highly toxic C9-RAN proteins in the cytoplasm that lead to neurodegeneration. We have generated a BAC transgenic model of C9-ALS/FTD that will allow us to test this hypothesis. This mouse develops both the molecular (RNA foci, C9-RAN proteins) and pathophysiological (neuronal loss, paralysis, decreased survival) features of C9-ALS/FTD. In this proposal, we will initially test the hypothesis that RNA GOF effects precede RAN protein accumulation by performing RNA-FISH, transcriptome analysis and immunological assays at various developmental periods and in different brain and spinal cord regions on asymptomatic, pre-symptomatic and symptomatic C9-BAC mice. This information will be used in conjunction with histopathological and electrophysiological assays test the hypothesis that C9-RAN protein accumulation triggers neurodegeneration and the acute disease phase. The possibility that stress pathways modulate RAN translation will also be tested. Finally, we will test whether antisense oligonucleotide (ASO) gapmer-mediated knockdowns of sense, antisense or both sense and antisense C9orf72 transcripts blocks the development of RNA and RNA toxicity in our C9-BAC transgenic mice. Overall, the objective of this study is to define pathogenic mechanisms underlying C9-ALS/FTD disease development and progression and provide an accessible and well-characterized mouse model for therapeutic development.