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

项目总结/摘要 C9 ORF 72（C9）中的GGGGCC六核苷酸重复扩增（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ALS/FTD重叠综合征。C9 HRE被认为是通过一种有毒的 由扩增的重复RNA和/或二肽重复蛋白（DPR）介导的功能获得， HRE的异常翻译。我们的实验室和其他人最近发现，C9 HRE损害 跨多个物种和模型系统的核质转运，强烈暗示了这一基本原理。 C9介导的神经变性的细胞通路。我们最近未发表的数据表明， C9-ALS/FTD的核转运受损机制涉及核孔蛋白亚群的破坏 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排列在 核孔复合物（NPC），在转运特异性和渗透性中起关键作用， 对于核运输和细胞存活功能上至关重要。目前，对FG的生物学知之甚少- 哺乳动物细胞，特别是中枢神经系统（CNS）中的Nups，对哺乳动物细胞的生长构成了主要障碍。 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FG-Nup在ALS/FTD易感的CNS细胞中的表达和功能， 作为进一步研究C9毒性的框架。我们将使用INTACT转基因小鼠（分离 标记在特定细胞类型中的细胞核），以从限定的神经元和神经胶质细胞群中分离细胞核， 通过质谱法和免疫EM检测FG-Nups的表达和定位，并使用siRNA敲低， 确定哪些FG-NUP对核运输和细胞存活至关重要。随后，我们将展开调查 C9介导的FG-Nup破坏的两种潜在机制：（1）表达改变，和（2）细胞质 错误定位和聚集，这可能是由蛋白质之间的异常蛋白质-蛋白质相互作用引发的。 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的神经元（iPSN）减弱核转运缺陷， 神经毒性总之，这些研究将提供FG-Nup的首次全面评估 生物学在ALS/FTD脆弱的中枢神经系统细胞，阐明机制，其中C9破坏这些必要的 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 Our more recent, unpublished data suggest that the mechanism of nuclear transport impairment in C9-ALS/FTD involves disruption of a subset of nucleoporin proteins (Nups) with low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.