Mechanisms of impaired ESCRT-III nuclear surveillance in ALS/FTD

肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTD）是两种与年龄有关的疾病， 神经退行性疾病的共同遗传和病理基础。最近， 核孔复合体（NPC）的组成和功能已被确定为一个突出的和早期的 包括ALS、AD/FTD和HD在内的神经退行性疾病的病理机制。作为全国人大和 周围的核膜（NE）环境严格控制基本的细胞过程，包括 核质转运（NCT），基因表达和基因组组织，NPC和NE稳态是 对神经元的功能和存活至关重要。使用诱导多能干细胞（iPSC）衍生的神经元 （iPSNs）和死后的人CNS组织，我们最近发现了NPC的显著损伤 其特征在于来自人神经元NPC的特异性NUP减少， 核蛋白的输入，并有助于功能障碍和错误定位的RNA结合蛋白TDP- 43，一个突出的神经变性病理事件。这种NPC损伤级联反应是由异常的 CHMP 7在sALS和C9 orf 72 ALS/FTD神经元中的核积累。CHMP 7是一种ESCRT-III蛋白， 虽然通常主要定位于细胞质，但被认为主要在ESCRT-III介导的 核监视事件。CHMP 7的细胞生物学事件和分子机制 对NPC和NE监视和稳态的贡献开始在酵母和非酵母中被理解。 在细胞分裂、NE分解和NPC插入期间的哺乳动物神经元细胞。然而， 关于CHMP 7在维持长寿命的非分裂人类神经元中NPC和NE稳态中的作用， 其中NPC组件很少更换，并且NE故障不会经常发生。我们 初步数据强调了这样一个事实，即调节核定位和激活的分子事件 在神经元中CHMP 7介导的核监视与最近鉴定的那些并不完全一致 在酵母和分裂的哺乳动物细胞中，提示产生CHMP 7的不同神经元机制 引发ALS/FTD的致病级联反应。在拟议的研究中，我们将采用大量的iPSN sALS和C9 orf 72 ALS/FTD患者和适当的对照，并利用基于候选物的组合， siRNA敲除、生物化学蛋白质相互作用技术、最近开发和优化的活细胞和 超分辨率成像策略和新产生的CHMP 7变体质粒文库的表达。 总的来说，这些研究将确定促进核进入（Aim 1）的分子机制， CHMP 7的核输出（Aim 2）以及CHMP 7介导的NPC损伤（Aim 3）的起始，从而引起 ALS/FTD中NPC损伤级联。因此，这些研究将提供对细胞的基本了解， 促进人神经元中CHMP 7介导的NPC损伤的生物和分子事件， 深入了解缓解ALS/FTD中这种早期和重要致病级联反应的治疗策略。

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two age-related neurodegenerative diseases that share genetic and pathological underpinnings. Recently, disruptions to the composition and function nuclear pore complex (NPC) have been identified as a prominent and early pathomechanism underlying neurodegenerative diseases including ALS, AD/FTD, and HD. As the NPC and surrounding nuclear envelope (NE) environment critically control fundamental cellular processes including nucleocytoplasmic transport (NCT), gene expression, and genome organization, NPC and NE homeostasis is essential for neuronal function and survival. Using induced pluripotent stem cell (iPSC) derived neurons (iPSNs) and postmortem human CNS tissues, we have recently identified a significant injury to the NPC characterized by the reduction in specific Nups from the human neuronal NPC that culminates in impaired nuclear protein import and contributes to the dysfunction and mislocalization of the RNA binding protein TDP- 43, a prominent pathological event in neurodegeneration. This NPC injury cascade is initiated by the aberrant nuclear accumulation of CHMP7 in sALS and C9orf72 ALS/FTD neurons. CHMP7 is an ESCRT-III protein that while normally localized predominantly to the cytoplasm, is thought to function primarily in ESCRT-III mediated nuclear surveillance events. The cell biological events and molecular mechanisms underlying CHMP7’s contribution to NPC and NE surveillance and homeostasis are beginning to be understood in yeast and non- neuronal mammalian cells during cell division, NE breakdown, and NPC insertion. However, little is understood regarding CHMP7’s role in maintaining NPC and NE homeostasis in long-lived, non-dividing human neurons where NPC components are infrequently exchanged, and NE breakdown does not routinely occur. Our preliminary data highlight the fact that the molecular events that regulate the nuclear localization and activation of CHMP7 mediated nuclear surveillance in neurons are not perfectly concordant with those recently identified in yeast and dividing mammalian cells suggestive of distinct neuronal mechanisms that give rise to CHMP7 initiated pathogenic cascades in ALS/FTD. In the proposed studies, we will employ iPSNs from a large number of sALS and C9orf72 ALS/FTD patients and appropriate controls and utilize a combination of candidate based siRNA knockdown, biochemical protein interaction techniques, recently developed and optimized live cell and super resolution imaging strategies, and expression of a newly generated CHMP7 variant plasmid library. Collectively, these studies will identify the molecular mechanisms that facilitate the nuclear entry (Aim 1) and nuclear export (Aim 2) of CHMP7 as well as the initiation of CHMP7 mediated NPC injury (Aim 3) to give rise to NPC injury cascades in ALS/FTD. Therefore, these studies will provide an essential understanding of the cell biological and molecular events that facilitate CHMP7 mediated NPC injury in human neurons and provide insights into therapeutic strategies for alleviating this early and significant pathogenic cascade in ALS/FTD.