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）和额颞叶痴呆症（FTD）最常见的遗传原因是 C9 orf 72（C9）基因第一内含子中的六核苷酸（G4 C2）n重复扩增（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（DPR 神经毒性在对果蝇和酵母进行的一系列基因筛选中，最近有几个研究小组表明， RNA重复序列和DPR损害核质转运。然而，RNA和蛋白质的身份 受此缺陷影响的突变C9运动神经元（MN）的底物，这些特定的下游效应 变化及其对神经毒性的影响尚不清楚。同样难以捉摸的是， 这种机制与散发性ALS有更广泛的相关性，尽管核蛋白在细胞质中的积累 例如TDP 43是几乎所有ALS和FTD患者的神经病理学标志。在我们的初步调查中 我们在C9-HRE细胞模型中进行了大规模的亚细胞蛋白质组学分析， 鉴定并验证了许多错误定位的候选蛋白，包括PRMT 1。在本研究中，我们 将使用患者源性神经元、患者CNS组织和体内果蝇模型来检验以下假设： ALS/FTD相关的神经毒性是由特定的神经细胞核/细胞质（N/C）分布的破坏引起的。 mRNA和蛋白质的种类。在目标1中，我们将使用患者特异性iPSC衍生的MN并采用分子生物学技术。 以及精确的生化亚细胞分级分离与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和对照iPSC，以及同基因对照iPSC系，其中我们具有 通过CRISPR/Cas9基因编辑校正HRE。识别缺失的mRNA和蛋白质- 在患者MN中进行区室化是阐明缺陷性MNS和MNS之间联系的重要第一步。 核质转运和神经毒性。在目标2中，我们将使用细胞模型，患者组织和体内 C9-HRE毒性的果蝇模型，以系统地验证这些分子扰动并评估其 导致ALS/FTD相关的神经变性。在目标3中，我们将确定细胞质如何 PRMT 1（一种必需的精氨酸甲基转移酶）的积累影响MN功能和存活。采取 总之，我们提出的目标将揭示细胞机制，是由异常损害， 患者的核质mRNA/蛋白质分布，并可能揭示C9和 可能是散发性ALS/FTD。

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.