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

确定缺陷性核细胞质运输如何导致 C9orf72 相关 ALS 和 FTD 中的神经变性

• 批准号: 10112967
• 负责人: Evangelos Kiskinis
• 金额: $ 37.69万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10112967
• 关键词: ALS patients; Affect; Amyotrophic Lateral Sclerosis; Arginine; Biochemical; C9ORF72; CRISPR/Cas technology; Cell Fractionation; Cell Nucleus; Cell model; Cells; Coupled; Cytoplasm; Defect; Dipeptides; Disease; Drosophila genus; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Histone H4; Impairment; In Vitro; Introns; Lead; Light; Link; Mediating; Messenger RNA; Methylation; Modeling; Molecular; Motor Neurons; Mutation; Nerve Degeneration; Neurons; Nuclear; Nuclear Proteins; Pathology; Pathway interactions; Patients; Post-Translational Protein Processing; Production; Protein-Arginine N-Methyltransferase; Proteins; Proteome; Proteomics; RNA; RNA Splicing; RNA Transport; RNA metabolism; Resources; Role; Series; Small Interfering RNA; System; Testing; Therapeutic Intervention; Tissues; Toxic effect; Translating; Translations; Work; Yeasts; arginine methyltransferase; base; experimental study; fly; frontotemporal lobar dementia-amyotrophic lateral sclerosis; gain of function; in vivo; induced pluripotent stem cell; motor neuron degeneration; motor neuron function; mutant; neuropathology; neurotoxic; neurotoxicity; novel; nucleocytoplasmic transport; overexpression; protective effect; protein TDP-43; protein distribution; protein transport; sporadic amyotrophic lateral sclerosis; therapeutic target; trafficking; transcriptome sequencing

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.

抽象的 肌萎缩性外侧硬化症（ALS）和额颞痴呆（FTD）的最常见遗传原因是 C9orf72（C9）基因的第一个内含子中的六核苷酸（G4C2）N重复扩张（HRE）。 RNA和 已显示了分别从C9 hre转录和翻译的二肽重复序列（DPRS） 是神经毒性的。在苍蝇和酵母中的一系列遗传筛选中，最近几个组表明 RNA重复和DPRS会损害核细胞质转运。但是，RNA和蛋白质的身份 突变体C9运动神经元（MN）中受此缺陷影响的底物，这些缺陷的特定下游效应 变化及其对神经毒性的贡献仍然未知。仍然难以捉摸的是 尽管核蛋白的细胞质积累 例如TDP43是几乎所有ALS和FTD患者的神经病理标志。在我们自己的初步 我们已经在C9 HRE细胞模型中进行了大规模的亚细胞蛋白质组学分析，并具有 确定并验证了包括PRMT1在内的许多错误定位的候选蛋白。在本研究中，我们 将使用患者来源的神经元，患者中枢神经系统组织和体内果蝇模型来检验以下假设。 与ALS/FTD相关的神经毒性是由核/细胞质（N/C）特异性分布引起的 mRNA和蛋白质类别。在AIM 1中，我们将使用特异性IPSC衍生的MN并使用分子 并精确的生化亚细胞分馏与RNA-seq和基于MS的定量蛋白质组学结合。 我们将使用多个C9和控制IPSC，以及ISEGENIC CONTROR IPSC系列，其中我们具有 通过CRISPR/CAS9基因编辑纠正了HRE。识别错过的mRNA和蛋白质 在患者MNS中划分是阐明有缺陷之间联系的重要第一步 核质运输和神经毒性。在AIM 2中，我们将使用细胞模型，患者组织和体内 C9 HRE毒性的果蝇模型有系统地验证这些分子扰动并评估其 对ALS/FTD相关的神经变性的贡献。在AIM 3中，我们将确定细胞质如何 PRMT1的积累是一种必需的精氨酸甲基转移酶，会影响MN功能和存活。拍摄 总之，我们提出的目的将把光线放入被异常损害的细胞机制中 患者中的核质mRNA/蛋白质分布，可能会发现C9和C9的治疗靶标 潜在的零星ALS/FTD。