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

基本信息

批准号：
10334500
负责人：
Evangelos Kiskinis
金额：
$ 37万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10334500
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

项目摘要

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.

抽象的肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD) 最常见的遗传原因是 C9orf72 (C9) 基因的第一个内含子中的六核苷酸 (G4C2)n 重复扩展 (HRE)。核糖核酸和已显示分别从 C9-HRE 转录和翻译的二肽重复序列 (DPR) 具有神经毒性。在对果蝇和酵母进行的一系列基因筛选中，几个研究小组最近表明， RNA 重复序列和 DPR 会损害核细胞质运输。然而，RNA 和蛋白质的身份受突变 C9 运动神经元 (MN) 缺陷影响的底物，这些底物的特定下游效应变化及其对神经毒性的贡献仍然未知。同样仍然难以捉摸的是尽管核蛋白在细胞质中积累，但该机制与散发性 ALS 具有更广泛的相关性 TDP43 等是几乎所有 ALS 和 FTD 患者的神经病理学标志。在我们自己的初步我们在 C9-HRE 细胞模型中进行了大规模亚细胞蛋白质组分析，并得到了鉴定并验证了许多错误定位的候选蛋白，包括 PRMT1。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： ALS/FTD 相关的神经毒性是由特定物质的细胞核/细胞质 (N/C) 分布破坏引起的。 mRNA 和蛋白质的类别。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，其中我们有通过 CRISPR/Cas9 基因编辑纠正了 HRE。识别缺失的 mRNA 和蛋白质对患者 MN 进行分区是阐明缺陷之间联系的重要第一步核细胞质运输和神经毒性。在目标 2 中，我们将使用细胞模型、患者组织和体内 C9-HRE 毒性的果蝇模型系统地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（一种重要的精氨酸甲基转移酶）的积累会影响 MN 的功能和存活。采取总之，我们提出的目标将揭示异常损害的细胞机制患者核细胞质 mRNA/蛋白质分布，可能会揭示 C9 和 C9 的治疗靶点潜在散发性 ALS/FTD。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Dissecting the Functional Consequences of De Novo DNA Methylation Dynamics in Human Motor Neuron Differentiation and Physiology.

DOI：
10.1016/j.stem.2018.02.012
发表时间：
2018-04-05
期刊：
Cell stem cell
影响因子：
23.9
作者：
Ziller MJ;Ortega JA;Quinlan KA;Santos DP;Gu H;Martin EJ;Galonska C;Pop R;Maidl S;Di Pardo A;Huang M;Meltzer HY;Gnirke A;Heckman CJ;Meissner A;Kiskinis E
通讯作者：
Kiskinis E

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.

DOI：
10.1016/j.stemcr.2022.02.008
发表时间：
2022-04-12
期刊：
STEM CELL REPORTS
影响因子：
5.9
作者：
Simkin, Dina;Papakis, Vasileios;Bustos, Bernabe, I;Ambrosi, Christina M.;Ryan, Steven J.;Baru, Valeriya;Williams, Luis A.;Dempsey, Graham T.;McManus, Owen B.;Landers, John E.;Lubbe, Steven J.;George, Alfred L., Jr.;Kiskinis, Evangelos
通讯作者：
Kiskinis, Evangelos