FG-nucleoporins and nuclear transport disruption in C9ORF72-ALS/FTD

C9ORF72-ALS/FTD 中的 FG-核孔蛋白和核运输中断

• 批准号: 9431708
• 负责人: Lindsey Renae Hayes
• 金额: $ 19.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9431708
• 关键词: Affinity; Amyotrophic Lateral Sclerosis; Astrocytes; Attenuated; Autopsy; Behavioral; Binding; Binding Proteins; Biological Assay; Biological Models; Biology; C9ORF72; Cell Nucleus; Cell Survival; Cells; Clinical; Data; Defect; Dipeptides; Disease; Enterobacteria phage P1 Cre recombinase; Frontotemporal Dementia; Glycine; Goals; Hippocampus (Brain); Impairment; In Vitro; Injection of therapeutic agent; Investigation; Laboratories; Mammalian Cell; Mass Spectrum Analysis; Mediating; Methods; Microscopy; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Oligodendroglia; Pathologic; Pathway interactions; Patients; Permeability; Phenylalanine; Play; Population; Population Analysis; Proteins; Resolution; Small Interfering RNA; Specificity; Syndrome; Testing; Therapeutic Intervention; Time; Tissues; Toxic effect; Transgenic Mice; Translations; Western Blotting; Yeasts; cell type; gain of function; in vivo; induced pluripotent stem cell; insight; knock-down; neuropathology; neuroprotection; neurotoxicity; new therapeutic target; novel therapeutics; nucleocytoplasmic transport; overexpression; prevent; protein expression; protein protein interaction; therapeutic evaluation; tool

PROJECT SUMMARY/ABSTRACT The GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 (C9) is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including familial and sporadic forms of the disease, as well as the ALS/FTD overlap syndrome. The C9 HRE is thought to cause disease by a toxic gain of function, mediated by expanded repeat RNAs and/or dipeptide repeat proteins (DPRs), produced by aberrant translation of the HRE. Our laboratory and others recently discovered that the C9 HRE impairs nucleocytoplasmic transport across multiple species and model systems, strongly implicating this fundamental cellular pathway in C9-mediated neurodegeneration. Our more recent, unpublished data suggest that the mechanism of nuclear transport impairment in C9-ALS/FTD involves disruption of a subset of nucleoporin proteins (Nups) with low complexity phenylalanine-glycine domains (FG-Nups). In yeast, FG-Nups line the nuclear pore complex (NPC), playing key roles in transport specificity and permeability, and a subset are functionally essential for nuclear transport and cell survival. Currently, little is known about the biology of FG- Nups in mammalian cells, particularly in the central nervous system (CNS), posing a major barrier for understanding the consequences of FG-Nup disruption in C9-ALS/FTD. In the proposed studies, our goal is to comprehensively evaluate FG-Nup expression and function in ALS/FTD-vulnerable cells of the CNS, to serve as a framework for further investigation of C9 toxicity. We will use the INTACT transgenic mouse (isolation of nuclei tagged in specific cell types) to isolate nuclei from defined neuronal and glial populations, analyze the expression and localization of FG-Nups by mass spectrometry and immuno-EM, and use siRNA knockdown to identify which FG-Nups are essential for nuclear transport and cell survival. Subsequently, we will investigate two potential mechanisms of C9-mediated FG-Nup disruption: (1) altered expression, and (2) cytoplasmic mislocalization and aggregation, which may be triggered by aberrant protein-protein interactions between DPRs and the FG-low complexity domain. Finally, we will test whether manipulating these factors in C9 induced pluripotent stem cell-derived neurons (iPSN) attenuates nuclear transport defects and prevents neurotoxicity. Taken together, these studies will provide the first comprehensive assessment of FG-Nup biology in ALS/FTD-vulnerable cells of the CNS, elucidate mechanisms by which C9 disrupts these essential FG-Nups, and identify novel targets for therapeutic intervention.

项目摘要/摘要 C9ORF72（C9）中的GGGGCC六核苷酸重复扩展（HRE）是最常见的原因 肌萎缩性侧面硬化症（ALS）和额颞痴呆（FTD），包括家族和零星形式 该病以及ALS/FTD重叠综合征。 C9 HRE被认为会导致疾病 功能的获取，由扩展的重复RNA和/或二肽重复蛋白（DPRS）介导，由 HRE的异常翻译。我们的实验室和其他人最近发现C9 HRE损害 跨多种物种和模型系统的核细胞质转运，强烈暗示这种基本 C9介导的神经变性中的细胞途径。我们最近未发表的数据表明 C9-ALS/FTD中核转运障碍的机制涉及核孔蛋白的一部分 具有低复杂性苯丙氨酸 - 甘氨酸结构域（FG-NUPS）的蛋白质（NUPS）。在酵母中，FG-Nups列 核孔复合物（NPC），在运输特异性和渗透性中扮演关键作用，并集 对于核运输和细胞存活至关重要。目前，关于FG-的生物学知之甚少 哺乳动物细胞中的NUP，特别是中枢神经系统（CNS）的NUP，为 了解C9-ALS/FTD中FG-NUP破坏的后果。在拟议的研究中，我们的目标是 全面评估CNS的ALS/FTD胶合细胞中的FG-NUP表达和功能，以服务 作为进一步研究C9毒性的框架。我们将使用完整的转基因小鼠（隔离 在特定细胞类型中标记的核）以分离核与定义的神经元和神经胶质种群分析，分析 通过质谱和免疫EM对FG-NUP的表达和定位，并使用siRNA敲低 确定哪些FG-NUP对于核运输和细胞存活至关重要。随后，我们将调查 C9介导的FG-NUP破坏的两种潜在机制：（1）表达改变，（2）细胞质 错误的定位和聚集，这可能是由异常蛋白质 - 蛋白质相互作用引起的 DPRS和FG-LOW复杂性域。最后，我们将测试是否在C9中操纵这些因素 诱导多能干细胞衍生的神经元（IPSN）减轻了核转运缺陷并防止 神经毒性。综上所述，这些研究将对FG-NUP进行首次全面评估 中枢神经系统的ALS/FTD胶合细胞中的生物学，阐明了C9破坏这些必不可少的机制 FG-Nups，并确定治疗干预的新目标。