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

 描述（由申请人提供）：ALS的研究最近取得了重大进展。两个独立的研究小组已经确定了C9 ORF 72基因第一内含子的非编码区中GGGGCC重复序列的扩增是迄今为止确定的肌萎缩侧索硬化症（ALS）和FTD（额颞叶痴呆）的最常见遗传原因。在C9 ORF 72 ALS/FTLD患者中，发现含有GGGGCC（正义）或CCCCGG（反义）重复序列的RNA转录物聚集在额叶皮质和脊髓的核灶中。最近，通过GGGGCC或CCCCGG重复扩增的重复相关非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，并且在ALS和ALS/FTD患者的受累和非受累区域检测到DPR聚集体。在几种核苷酸重复疾病（包括C9-ALS/FTD）中已报告了RAN从正义和反义方向的翻译，这使得RAN翻译在这些扩增疾病中已确定发生，并且还暗示了RAN翻译蛋白的致病作用。我们建立了C9-ALS/FTD的细胞模型，使用原代运动和皮质神经元，这是已知的ALS/FTD退化。通过表达荧光标记的均聚C9 RAN蛋白（或DPR），我们能够使用纵向延时活细胞成像、转基因果蝇模型来解读它们各自对神经元活力的影响，并发现脯氨酸-精氨酸二肽（PR）在体外和体内表达时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，小鼠疾病相关神经元群体中PR聚集体的表达将导致与ALS/FTD患者相似的关键表型和病理学的发展。这一假设是基于广泛的证据，获得的PR聚集体在神经元细胞培养模型中的毒性，以及眼变性和致死性表型时，PR蛋白表达在果蝇眼和运动神经元。 拟议研究的基本原理是，一旦该模型得到充分表征，它将有可能成为解开C9-ALS/FTD神经退行性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.