Determining How Defective Nucleo-Cytoplasmic Trafficking Leads To Neurodegeneration In C9orf72-Related ALS And FTD

摘要 肌萎缩侧索硬化症(ALS)和额颞部痴呆(FTD)最常见的遗传原因是 C9orf72(C9)基因第一内含子中的六核苷酸(G4C2)n重复扩展(HRE)。RNA和 已经显示了分别从C9-HRE转录和翻译的二肽重复序列(Dprs) 有神经毒性。在苍蝇和酵母菌的一系列基因筛查中，几个小组最近表明，两者都 RNA重复和dprs损害核质运输。然而，RNA和蛋白质的同一性 受此缺陷影响的底物，突变的C9运动神经元(MN)，这些特定的下游影响 变化，以及它们对神经毒性的贡献仍不清楚。同样难以捉摸的是 这一机制与散发性肌萎缩侧索硬化症更广泛的相关性，尽管核蛋白在细胞质内积累 如TDP43是几乎所有ALS和FTD患者的神经病理标志。在我们自己的预赛中 我们在C9-HRE细胞模型中进行了大规模的亚细胞蛋白质组分析，并 鉴定并验证了包括PRMT1在内的一些错误定位的候选蛋白。在目前的研究中，我们 将使用患者来源的神经元、患者的中枢神经系统组织和体内的果蝇模型来检验这一假设 ALS/FTD相关的神经毒性是由特定的核/胞浆(N/C)分布中断引起的 核糖核酸和蛋白质的类别。在目标1中，我们将使用患者特定的IPSC来源的MN并使用分子 以及精确的生化亚细胞分级与RNA-Seq和基于MS的定量蛋白质组学相结合。 我们将使用多个C9和控制IPSC，以及一条等基因控制IPSC生产线，在该生产线中，我们有 通过CRISPR/Cas9基因编辑纠正了HRE。鉴定缺失的mRNA和蛋白质- 对患者MNS进行分区是阐明缺陷之间联系的必要的第一步 核质转运与神经毒性。在目标2中，我们将使用细胞模型、患者组织和体内 果蝇C9-HRE毒性模型以系统验证这些分子扰动并评估其 对ALS/FTD相关神经变性的贡献。在目标3中，我们将确定细胞质如何 PRMT1是一种重要的精氨酸甲基转移酶，它的积聚影响着MN的功能和生存。已被占用 总之，我们提出的目标将阐明因异常情况而受损的细胞机制。 核胞质mRNA/蛋白在患者体内的分布，并可能揭示C9和C9的治疗靶点 可能是散发性的肌萎缩侧索硬化症/功能性肌萎缩症。

ABSTRACT The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide (G4C2)n repeat expansion (HRE) in the first intron of the C9orf72 (C9) gene. RNA and dipeptide repeats (DPRs) that are transcribed and translated from the C9-HRE respectively, have been shown to be neurotoxic. In a series of genetic screens in the fly and yeast, several groups recently showed that both RNA repeats and DPRs impair nucleocytoplasmic transport. However, the identities of the RNA and protein substrates affected by this defect in mutant C9 motor neurons (MNs), the specific downstream effects of these changes, and their contribution towards neurotoxicity remain unknown. What also remains elusive is the broader relevance of this mechanism for sporadic ALS, although cytoplasmic accumulation of nuclear proteins such as TDP43 is a neuropathological hallmark in almost all ALS and FTD patients. In our own preliminary work we have conducted large-scale sub-cellular proteomic analysis in a C9-HRE cellular model and have identified and validated a number of mislocalized candidate proteins including PRMT1. In the present study we will use patient-derived neurons, patient CNS tissue, and in vivo Drosophila models to test the hypothesis that ALS/FTD-related neurotoxicity is caused by a disruption the nucleus/cytoplasmic (N/C) distribution of specific classes of mRNAs and proteins. In Aim 1, we will use patient-specific iPSC-derived MNs and employ molecular and precise biochemical subcellular fractionation coupled to RNA-Seq and MS-based quantitative proteomics. We will use multiple C9 and control iPSCs, as well as an isogenic control iPSC line, in which we have corrected the HRE though CRISPR/Cas9 gene editing. Identifying the mRNAs and proteins that are miss- compartmentalized in patient MNs is an essential first step towards elucidating the link between defective nucleocytoplasmic transport and neurotoxicity. In Aim 2, we will use cellular models, patient tissue and in vivo Drosophila models of C9-HRE toxicity to systematically validate these molecular perturbations and assess their contribution towards ALS/FTD-related neurodegeneration. In Aim 3, we will determine how cytoplasmic accumulation of PRMT1, an essential arginine methyltransferase, impacts MN function and survival. Taken together, our proposed aims will shed light into the cellular mechanisms that are compromised by abnormal nucleocytoplasmic mRNA/protein distribution in patients and will likely uncover therapeutic targets for C9 and potentially sporadic ALS/FTD.