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

项目摘要/摘要 C9ORF72(C9)中的GGGGCC六核苷酸重复序列扩展(HRE)是最常见的已知原因 肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)，包括家族性和散发性形式 以及肌萎缩侧索硬化症/FTD重叠综合征。C9HRE被认为是由一种有毒的 功能的获得，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 跨多个物种和模式系统的核质运输，强烈地暗示了这一基本原理 C9介导的神经退行性变的细胞途径。我们最近未公布的数据表明， C9-ALS/FTD中核转运受损的机制涉及破坏核孔素的一个子集 具有低复杂性苯丙氨酸-甘氨酸结构域的蛋白质(NUP)。在酵母中，FG-NUPS排列在 核孔复合体(NPC)在转运特异性和通透性方面起着关键作用，其亚群是 在功能上对核运输和细胞生存至关重要。目前，人们对纤维蛋白原的生物学知之甚少。 NUPS在哺乳动物细胞中，特别是在中枢神经系统(CNS)中，构成了 了解C9-ALS/FTD中FG-Nup中断的后果。在建议的研究中，我们的目标是 综合评价FG-Nup在ALS/FTD易感中枢神经系统细胞中的表达和功能，为 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(分离 标记在特定细胞类型中的核)，以从定义的神经元和胶质细胞群中分离出核，分析 用质谱仪和免疫-EM法检测FG-nups的表达和定位，并利用siRNA敲除技术 确定哪些FG-NUP对于核运输和细胞生存是必不可少的。随后，我们将调查 C9介导的FG-Nup破坏的两种可能机制：(1)改变表达和(2)细胞质 错误定位和聚集，这可能是由异常的蛋白质-蛋白质相互作用触发的 DPR和FG-低复杂度域。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神经元(IPSN)减轻核转运缺陷并防止 神经毒性。综上所述，这些研究将提供FG-Nup的第一个全面评估 中枢神经系统ALS/FTD易损细胞的生物学，阐明C9扰乱这些必需细胞的机制 FG-nups，并确定治疗干预的新靶点。

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 Our more recent, unpublished data suggest that the mechanism of nuclear transport impairment in C9-ALS/FTD involves disruption of a subset of nucleoporin proteins (Nups) with low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.