RNA foci in C9FTD/ALS

描述（由申请人提供）：额颞叶变性伴tdp -43阳性包涵体（FTLD-TDP）和肌萎缩侧索硬化症（ALS）是具有显著临床和神经病理重叠的破坏性神经退行性疾病。最近的一项突破性研究现在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的问题：C9ORF72非编码区扩增的重复序列如何参与疾病的发病机制？我们假设RNA介导的毒性可能是一种机制，因为含有扩增GGGGCC重复序列的转录本在C9ORF72突变携带者的额叶皮层和脊髓中作为核RNA灶积累。RNA灶的形成以及随后的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病灶形成是否是c9FTD/ALS的主要神经毒性机制，需要建立新的C9ORF72六核苷酸重复扩增的体外和体内模型。为此，我们创建了两个组成表达结构来驱动含有18个GGGGCC重复序列（18R）或51个GGGGCC重复序列（51R）的RNA的表达，后者模拟六核苷酸重复扩增。值得注意的是，51R转录物的表达，而不是18R转录物的表达，导致HeLa和COS1细胞系形成独特的核内RNA灶，与C9FTD/ALS病例中观察到的灶相似。我们还发现病灶形成与细胞毒性增强以及rna结合蛋白SAM68和hnRNP-K的隔离有关。这些发现不仅表明我们可以模拟C9FTD/ALS中观察到的RNA灶形成，而且表明我们已经产生了一个有价值的工具来研究它们发挥毒性的机制。在这些令人兴奋的发现之后，本项目的目标是：1)产生和表征过表达非致病性（非灶形成）和致病性（灶形成）C9ORF72重复扩增的转基因小鼠；2)在我们的局灶形成（GGGGCC）51小鼠模型中，评估RNA局灶形成是否会导致行为缺陷和神经变性；3)鉴定被病灶隔离的rna结合蛋白；4)并评估在我们的新细胞和动物模型中，以及在C9FTD/ALS病例的脑组织和成纤维细胞中，隔离蛋白的RNA靶点是否发生改变。总的来说，我们相信这些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。

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.