RNA foci in C9FTD/ALS

C9FTD/ALS 中的 RNA 焦点

• 批准号: 8589205
• 负责人: LEONARD PETRUCELLI
• 金额: $ 23.48万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8589205
• 关键词: Amyotrophic Lateral Sclerosis; Animal Model; Atrophic; Behavioral; Binding; Brain; C9ORF72; COS Cells; Cell Culture Techniques; Cell Line; Cell model; Cells; Cessation of life; Clinical; Complex; Cultured Cells; Cytoplasmic Inclusion; Data; Disease; Fibroblasts; Fluorescent in Situ Hybridization; Frontotemporal Dementia; Frontotemporal Lobar Degenerations; Functional RNA; Genes; Genetic; Genetic Transcription; Goals; Heel; Hela Cells; Heterogeneous-Nuclear Ribonucleoprotein K; In Vitro; Language; Lead; Link; Mediating; Modeling; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear RNA; Pathogenesis; Pathologic Processes; Patients; Prions; Protein Binding; Proteins; RNA; RNA-Binding Proteins; Spinal Cord; Symptoms; Temporal Lobe; Toxic effect; Transcript; Transgenic Mice; Weight; aptamer; base; brain tissue; combat; cytotoxicity; frontal lobe; in vivo; in vivo Model; loss of function; motor impairment; mouse model; mutation carrier; neurotoxic; novel; overexpression; prevent; promoter; protein TDP-43; public health relevance; therapeutic target; tool

DESCRIPTION (provided by applicant): Frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative diseases with significant clinical and neuropathological overlap. A recent groundbreaking study now highlights a genetic link - hexanucleotide (GGGGCC) repeat expansion in C9ORF72 - as the most common cause of ALS and FTLD-TDP. This discovery raises an important question: how do the expanded repeats in a non-coding region of C9ORF72 contribute to disease pathogenesis? We hypothesize that RNA-mediated toxicity is a likely mechanism given that transcripts containing the expanded GGGGCC repeat accumulate as nuclear RNA foci in the frontal cortex and spinal cord of C9ORF72 mutation carriers. RNA foci formation, and the subsequent sequestration and altered activity of RNA-binding proteins by the foci, are emerging as a common pathogenic mechanism in many neurodegenerative diseases caused by non-coding repeat expansions. To determine whether RNA foci formation is a primary neurotoxic mechanism in c9FTD/ALS, novel in vitro and in vivo models of C9ORF72 hexanucleotide repeat expansions are required. To this end, we created two constitutive expression constructs to drive expression of RNA containing 18 GGGGCC repeats (18R) or 51 GGGGCC repeats (51R), the latter to mimic hexanucleotide repeat expansion. Notably, expression of the 51R transcript, but not the 18R transcript, leads to the formation of distinct, intranuclear RNA foci in HeLa and COS1 cell lines reminiscent of the foci observed in C9FTD/ALS cases. We also found foci formation is associated with enhanced cytotoxicity, as well as the sequestration of the RNA-binding proteins SAM68 and hnRNP-K. These findings not only illustrate that we can model the RNA foci formation observed in C9FTD/ALS, but also show that we have generated a valuable tool to study the mechanisms by which they exert toxicity. On the heels of these exciting findings, the goals of this project are: 1) to generate and characterize transgenic mice overexpressing non-pathogenic (non-foci-forming) and pathogenic (foci-forming) C9ORF72 repeat expansions; 2) to evaluate whether RNA foci formation results in behavioral deficits and neurodegeneration in our foci-forming (GGGGCC)51 mouse model; 3) to identify RNA-binding proteins sequestered by the foci; 4) and to evaluate whether RNA targets of the sequestered proteins are altered in our novel cell and animal models, as well as in brain tissue and fibroblasts of C9FTD/ALS cases. Overall, we believe the proposed studies examining RNA foci formation consequences will lead to a better understanding of C9FTD/ALS-related mechanisms and help to uncover promising therapeutic targets.

描述（由申请人提供）：具有 TDP-43 阳性包涵体的额颞叶变性 (FTLD-TDP) 和肌萎缩性脊髓侧索硬化症 (ALS) 是毁灭性的神经退行性疾病，具有显着的临床和神经病理学重叠。最近的一项突破性研究强调了一种遗传联系——C9ORF72 中的六核苷酸 (GGGGCC) 重复扩增——是 ALS 和 FTLD-TDP 的最常见原因。这一发现提出了一个重要问题：C9ORF72 非编码区中的重复序列扩展如何促进疾病发病机制？鉴于包含扩展的 GGGGCC 重复序列的转录物在 C9ORF72 突变携带者的额叶皮层和脊髓中作为核 RNA 聚集点积累，我们假设 RNA 介导的毒性是一种可能的机制。 RNA 病灶的形成，以及随后病灶对 RNA 结合蛋白的隔离和活性改变，正在成为许多由非编码重复扩增引起的神经退行性疾病的常见致病机制。 为了确定 RNA 灶形成是否是 c9FTD/ALS 中的主要神经毒性机制，需要 C9ORF72 六核苷酸重复扩增的新型体外和体内模型。为此，我们创建了两个组成型表达构建体来驱动含有 18 个 GGGGCC 重复序列 (18R) 或 51 个 GGGGCC 重复序列 (51R) 的 RNA 表达，后者模拟六核苷酸重复扩展。值得注意的是，51R 转录本的表达（而非 18R 转录本）导致 HeLa 和 COS1 细胞系中独特的核内 RNA 灶的形成，让人想起在 C9FTD/ALS 病例中观察到的灶。我们还发现病灶形成与增强的细胞毒性以及 RNA 结合蛋白 SAM68 和 hnRNP-K 的隔离有关。这些发现不仅说明我们可以对 C9FTD/ALS 中观察到的 RNA 灶形成进行建模，而且表明我们已经生成了一个有价值的工具来研究它们发挥毒性的机制。继这些令人兴奋的发现之后，该项目的目标是：1) 生成并表征过度表达非致病性（非病灶形成）和致病性（病灶形成）C9ORF72 重复扩增的转基因小鼠； 2) 评估 RNA 病灶形成是否会导致我们的病灶形成 (GGGGCC)51 小鼠模型的行为缺陷和神经变性； 3) 鉴定被病灶隔离的RNA结合蛋白； 4) 并评估隔离蛋白的 RNA 靶点在我们的新型细胞和动物模型以及 C9FTD/ALS 病例的脑组织和成纤维细胞中是否发生改变。 总体而言，我们相信拟议的检查 RNA 灶形成后果的研究将有助于更好地理解 C9FTD/ALS 相关机制，并有助于发现有希望的治疗靶点。