Nucleocytoplasmic transport and nuclear pore disruption in ALS/FTD

项目摘要 C9 ORF 72中的GGGGCC六核苷酸重复扩增（HRE）是最常见的遗传原因。 家族性肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD），虽然潜在的疾病 机制不明确。多项研究，包括我们自己的研究，支持一种功能获得机制， HRE介导的神经毒性。扩增的重复序列可能产生毒性RNA， proteins.它们也可以通过重复相关非ATG翻译（RANT）翻译成毒性二肽 重复蛋白（DPRs）。假设HRE RNA和DPR均介导C9-ALS/FTD的神经毒性。 我们实验室和其他实验室最近的多项研究表明，核孔和/或 核质转运是酵母、果蝇和诱导多能细胞神经变性的主要原因 (iPS)C9- ALS/FTD模型。此外，我们最近的研究，使用C9-ALS/FTD果蝇模型，人类 来源于C9-ALS患者和人C9-ALS CNS组织的iPS神经元表明，核质 转运缺陷可能是ALS/FTD发病机制的基本途径，适合治疗。 该提案将全面研究C9 ORf 72 HRE干扰 核质运输和核孔利用几个互补的模型，包括C9-ALS苍蝇 和小鼠模型以及来自C9 ALS/FTD患者的iPS神经元和脑组织，并研究 调节核质转运可能是ALS/FTD的治疗策略。(1)我们将确定 运动神经元和神经胶质细胞核孔复合体（NPC）的形态学和生化组成， 并在果蝇、iPS、小鼠模型和人脑中表征C9-ALS/FTD中的NPC病理学。知之甚少 关于中枢神经系统NPC，包括细胞类型之间的差异，以及最终如何NPC成分， 核孔蛋白在C9-ALS/FTD模型中失调。因此，了解基本特征， 在CNS和疾病模型中，NPC和核质转运对于 剖析病理学的本质(2)然后我们将研究核质转运的机制 C9-ALS/FTD的破坏。我们假设，NPC和/或核质转运功能的破坏 由于核输出序列的核丢失和/或细胞质积累导致神经变性 (NES)包含C9-ALS的苍蝇、小鼠和iPS模型中的货物。(3)因此，我们将确定 C9-ALS/FTD模型中核质转运中断的后果。(4)最后，我们将确定 如果核质转运的恢复通过采用一系列的方法来挽救C9-ALS/FTD的神经变性， 可能对人类有用的新型化合物。

PROJECT SUMMARY A GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), though the underlying disease mechanism is poorly defined. Multiple studies, including our own, support a gain-of-function mechanism of neurotoxicity mediated by the HRE. Expanded repeats may generate toxic RNAs that sequester RNA-binding proteins. They may also be translated via Repeat-Associated Non-ATG Translation (RANT) into toxic dipeptide repeat proteins (DPRs). Both HRE RNA and DPRs are hypothesized to mediate neurotoxicity in C9-ALS/FTD. Multiple recent studies from our lab and others suggest that disruption of the nuclear pore and/or nucleocytoplasmic transport is a primary cause of neurodegeneration in yeast, fly, and induced pluripotent cell (iPS) models of C9- ALS/FTD. In addition, our recent studies, using a C9-ALS/FTD Drosophila model, human iPS neurons derived from C9-ALS patients, and human C9-ALS CNS tissues, suggest that nucleocytoplasmic transport defects may be a fundamental pathway for ALS/FTD pathogenesis amenable to therapy. This proposal will comprehensively investigate the mechanism by which the C9ORf72 HRE disrupts nucleocytoplasmic transport and nuclear pores utilizing several complementary models including C9-ALS fly and mouse models and iPS neurons and brain tissue from C9 ALS/FTD patients, and investigate whether modulation of nucleocytoplasmic transport may be a therapeutic strategy for ALS/FTD. (1) We will determine the morphological and biochemical composition of the nuclear pore complex (NPC) in motor neurons and glia, and characterize NPC pathology in C9-ALS/FTD in fly, iPS, mouse models and human brain. Little is known about CNS NPCs including differences between cell types and ultimately how the NPC constituents, nucleoporins, are dysregulated in C9-ALS/FTD models. Therefore, understanding the basic characteristics of the NPC and nucleocytoplasmic transport in the CNS and in disease models is fundamentally important to dissecting the nature of pathology. (2) We will then investigate the mechanism of nucleocytoplasmic transport disruption in C9-ALS/FTD. We hypothesize that disrupted NPC and/or nucleocytoplasmic transport function causes neurodegeneration due to nuclear loss and/or cytoplasmic accumulation of nuclear export sequence (NES) containing cargo in fly, mouse, and iPS models of C9-ALS. (3) Therefore, we will determine the consequences of nucleocytoplasmic transport disruption in C9-ALS/FTD models. (4) Finally, we will determine if restoration of nucleocytoplasmic transport rescues neurodegeneration in C9-ALS/FTD by employing a series of novel compounds that may have human utility.