Nuclear pore complex quality control in ALS/FTD

项目摘要/摘要 肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)是第二种最常见的 痴呆症包括一系列致命的神经退行性疾病。内含子GGGGCC(G4C2) C9orf72基因中的六核苷酸重复序列扩展(HRE)与ALS和FTD有关，尽管 临床上有两种截然不同的疾病。C9orf72 HRE是家族性和散发性ALS的最常见原因 分别占患者的40%和8%。总体而言，约10%的肌萎缩侧索硬化症患者有家族史 剩下的90%是零星的。疾病发病机制的分子机制仍然不清楚。 明白了。核胞质转运和核孔复合体缺陷是近年来研究的热点 作为包括C9orf72在内的多种神经退行性疾病的重要致病机制 ALS/FTD，散发性ALS的亚群，阿尔茨海默病和亨廷顿病。然而，人们对此知之甚少 关于对NPC及其个别核孔素成分本身的伤害的性质。使用诱导式 多能干细胞来源的脊髓神经元(IPSN)和死后的人类组织，我们已经积累了数据 NPC中跨膜核孔蛋白POM121的丢失启动了一系列病理性级联反应 鼻咽癌的组成、功能和下游细胞存活。值得注意的是，POM121的损失是通过 病理上G4C2重复RNA和非二肽重复多肽或C9ORF72蛋白丢失。考虑到 POM121蛋白没有错位，POM121 RNA代谢没有改变，我们假设 POM121和随后改变的核孔蛋白在C9orf72的早期阶段被异常降解 ALS/FTD的发病机制。最近在酵母和非神经元哺乳动物细胞中的研究表明，核 CHMP7“激活”ESCRT-III介导的核孔复合体和核膜降解 在核孔监测和动态平衡期间的成分。我们新的初步数据表明， POM121从核质和NPC中的丢失是由CHMP7的核积累开始的。 从机制上讲，核CHMP7的增加似乎是G4C2重复RNA介导的损伤的结果 核出口。因此，我们的数据强烈暗示CHMP7降解途径在疾病发病机制中的作用。 有趣的是，敲除CHMP7可以减轻C9orf72 iPSN中的鼻咽癌损伤，使CHMP7成为一个有吸引力的 神经退行性变的治疗靶点。在这项提案中，我们将全面研究这一新途径 包括：1)确定CHMP7介导核孔素去除的降解途径 2)研究病理性G4C2重复RNA启动CHMP7的机制 介导鼻咽癌损伤。最后，3)使用大量单个患者的IPSN脊髓神经元细胞系， 评价CHMP7反义寡核苷酸缓解C9orf72 ALS/FTD和散发性ALS的能力 核孔复合体、核质运输和下游敏感性的介导性改变 IPSN中的压力源。

PROJECT SUMMARY/ABSTRACT Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), the second most common form of dementia, comprise a spectrum of fatal neurodegenerative diseases. An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9orf72 gene, have been linked to ALS and FTD, although clinically two distinct diseases. The C9orf72 HRE is the most common cause of both familial and sporadic ALS accounting for ~40% and ~8% of patients respectively. Overall, about 10% of ALS cases are familial with the remaining 90% being sporadic. The molecular mechanisms underlying disease pathogenesis remain poorly understood. Defects in nucleocytoplasmic transport (NCT) and the nuclear pore complex (NPC) have recently emerged as a prominent pathomechanism underlying multiple neurodegenerative diseases including C9orf72 ALS/FTD, subsets of sporadic ALS, Alzheimer’s Disease, and Huntington’s Disease. However, little is known about the nature of the injury to the NPC and its individual nucleoporin components themselves. Using induced pluripotent stem cell derived spinal neurons (iPSNs) and postmortem human tissue, we have amassed data that loss of the transmembrane nucleoporin POM121 from NPCs initiates a pathological cascade impacting NPC composition, function and downstream cellular survival. Notably, loss of POM121 is mediated by pathologic G4C2 repeat RNA and not dipeptide repeat poly peptides or loss of C9ORF72 protein. Given that POM121 protein is not mislocalized and POM121 RNA metabolism is unaltered, we hypothesized that POM121 and subsequently altered nucleoporin proteins are aberrantly degraded in the early stages of C9orf72 ALS/FTD pathogenesis. Recent work in yeast and non-neuronal mammalian cells has shown that nuclear CHMP7 “activates” ESCRT-III mediated degradation of nuclear pore complexes and nuclear envelope components during nuclear pore surveillance and homeostasis. Our new preliminary data suggests that the loss of POM121 from the nucleoplasm and NPCs is initiated by nuclear accumulation of CHMP7. Mechanistically, increased nuclear CHMP7 appears to be the result of G4C2 repeat RNA mediated impaired nuclear export. Thus, our data strongly implicate a CHMP7 degradative pathway in disease pathogenesis. Intriguingly, knockdown of CHMP7 mitigates NPC injury in C9orf72 iPSNs making CHMP7 an attractive therapeutic target in neurodegeneration. In this proposal we will comprehensively investigate this new pathway including studies to 1) Determine the degradative pathway by which CHMP7 mediates nucleoporin removal from NPCs in iPSNs, 2) Investigate the mechanism by which pathologic G4C2 repeat RNA initiates CHMP7 mediated NPC injury. And finally, 3) using a large battery of individual patient iPSN spinal neuron cell lines, evaluate the ability of CHMP7 antisense oligonucleotides to mitigate C9orf72 ALS/FTD and sporadic ALS mediated alterations in the nuclear pore complex and nucleocytoplasmic transport and downstream sensitivity to stressors in iPSNs.