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

项目摘要 C9 orf 72基因中微卫星GGGGCC重复序列的扩增与家族性和 肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的散发形式。同时分子 这种疾病（C9-ALS/FTD）的基础在很大程度上仍然未知，提出的疾病机制包括C9 orf 72 由于单倍不足导致的功能丧失，RNA功能获得（GOF）导致蛋白质螯合， 重复相关的非ATG（RAN）翻译，导致产生毒性C9-RAN二肽重复 proteins.基于我们先前对其他微卫星扩展疾病的研究，该建议旨在 测试我们的隔离失败假设，它整合了RNA和RAN获得功能机制。 根据这一假设，双向正义和反义C9 orf 72转录导致募集 细胞因子重复扩增RNA以产生有义和反义RNA焦点，从而隔离这些RNA 细胞核中的有毒RNA。体细胞重复扩增和/或年龄相关的细胞应激导致 GGGGCC和GGCCCC RNA结合蛋白，随后这些RNA的核质输出， 细胞质中高毒性C9-RAN蛋白的翻译导致神经变性。我们有 产生了C9-ALS/FTD的BAC转基因模型，这将使我们能够检验这一假设。这个鼠标 发展分子（RNA灶，C9-RAN蛋白）和病理生理（神经元丢失，麻痹， C9-ALS/FTD的存活率降低）特征。在这个提议中，我们将首先检验RNA 通过进行RNA-FISH、转录组分析和 在不同发育时期和不同脑和脊髓区域的免疫测定， 无症状、症状前和有症状的C9-BAC小鼠。这些信息将与 用组织病理学和电生理学分析测试C9-RAN蛋白积累 引发神经退行性疾病和急性疾病阶段。应激途径调节RAN的可能性 翻译也将受到考验。最后，我们将测试反义寡核苷酸（阿索）缺口体介导的 有义、反义或有义和反义C9 orf 72转录物的敲低阻断了 我们的C9-BAC转基因小鼠中的RNA和RNA毒性。总的来说，本研究的目的是确定 C9-ALS/FTD疾病发展和进展的致病机制，并提供了一个 用于治疗开发的可获得且充分表征的小鼠模型。

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.