Development of a mouse model of C9ORF72 ALS/FTD expressing RAN translated peptide

 描述(申请人提供)：肌萎缩侧索硬化症的研究最近取得了重大进展。两个独立的研究小组已经发现，C9ORF72基因第一内含子非编码区GGGGCC重复序列的扩张是迄今为止已确认的肌萎缩侧索硬化症(ALS)和额颞痴呆(FTD)的最常见遗传原因。C9ORF72 ALS/FTLD患者额叶皮质和脊髓核团聚集了含有GGGGCC(正义)或CCGG(反义)重复序列的RNA转录本。最近，通过GGGGCC或CCCCGG重复序列扩增的重复相关非ATG(RAN)翻译产生的二肽重复产物(DPR)的毒性被认为是C9-ALS/FTD的潜在致病机制，在ALS和ALS/FTD患者的患区和非患区都检测到了DPR聚集体。在包括C9-ALS/FTD在内的几种核苷酸重复疾病中，已经有报道从正义和反义方向翻译RAN，这使得RAN翻译在这些扩张性疾病中已经存在，也暗示了RAN翻译蛋白的致病作用。我们使用已知在ALS/FTD中退化的初级运动神经元和皮质神经元建立了C9-ALS/FTD的细胞模型。通过表达荧光标记的同聚c9RAN蛋白(或dprs)，我们能够利用纵向时移活细胞成像、转基因果蝇模型来破译它们对神经元活性的各自影响，并发现当在体外和体内表达时，Pro-Arg二肽(PR)具有强烈的神经毒性。作为我们研究的下一步，我们建议在这里产生表达PR二肽重复序列的转基因小鼠。我们的假设是，PR聚集体在小鼠疾病相关神经元群体中的表达将导致与ALS/FTD患者相似的关键表型和病理的发展。这一假说的提出是基于大量证据表明，PR聚集体在神经细胞培养模型中获得了毒性，以及当PR蛋白在果蝇眼和运动神经元中表达时，眼睛退化和致死表型。拟议研究的基本原理是，一旦该模型完全表征，它将有可能成为解开C9-ALS/FTD神经退行性变过程背后的基本机制的重要工具，并最终用于开发治疗干预措施。

 DESCRIPTION (provided by applicant): Research on ALS has recently been the subject of major advances. Two independent groups have identified an expansion of GGGGCC repeats in the non-coding region of the first intron of the C9ORF72 gene as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and FTD (frontotemporal dementia) identified to date. Accumulation of RNA transcripts containing GGGGCC (sense) or CCCCGG (antisense) repeats were found to aggregate in nuclear foci in frontal cortex and spinal cord in C9ORF72 ALS/FTLD patients. Recently, toxicity of dipeptide repeat products (DPRs) generated via repeat associated non-ATG (RAN) translation of GGGGCC or CCCCGG repeat expansions has been proposed as potential pathogenic mechanism in C9- ALS/FTD, and DPR aggregates have been detected in affected and non-affected regions in ALS and ALS/FTD patients. RAN translation from sense and anti-sense directions has been reported in several nucleotide repeat disorders, including C9-ALS/FTD, making RAN translation an established occurrence in these expansion disorders, and also implicating a pathogenic role for RAN translated proteins. We established cellular models of C9-ALS/FTD using primary motor and cortical neurons, which are known to degenerate in ALS/FTD. By expressing fluorescently tagged homopolymeric C9RAN proteins (or DPRs) we were able to decipher their respective impact on neuronal viability using longitudinal time-lapse live-cell imaging, transgenic Drosophila models and found that Proline-Arginine dipeptides (PR) are robustly neurotoxic when expressed in vitro and in vivo. As next step in our investigation, we propose here to generate transgenic mice that express PR dipeptide repeats. Our hypothesis is that expression of PR aggregates in disease-relevant neuronal populations in mice will result in development of key phenotypes and pathologies that resemble those in ALS/FTD patients. This hypothesis is formulated based on extensive evidence of gained toxicity of PR aggregates in neuronal cell culture models as well as eye degeneration and lethality phenotypes when PR proteins are expressed in Drosophila eye and motor neurons. The rationale of the proposed research is that, once the model is fully characterized, it will have the potential to become a vital tool to unravel the basic mechanisms behind neurodegenerative processes in C9-ALS/FTD and, ultimately, for development of therapeutic interventions.