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

ALS/FTD 中 ESCRT-III 核监测受损的机制

• 批准号: 10705390
• 负责人: Alyssa Coyne
• 金额: $ 57.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705390
• 关键词: Amyotrophic Lateral Sclerosis; Autopsy; Biochemical; Biological; C9ORF72; Cell division; Cell physiology; Cells; Cytoplasm; Data; Defect; Disease; Environment; Event; Excision; Frontotemporal Dementia; Functional disorder; Gene Expression; Genetic; Genome; Health; Homeostasis; Human; Immunoprecipitation; Impairment; Individual; Induced pluripotent stem cell derived neurons; Injury; Libraries; Maintenance; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Molecular; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Envelope; Nuclear Export; Nuclear Inner Membrane; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nuclear Protein; Pathogenicity; Pathologic; Pathway interactions; Patients; Physiological; Plasmids; Point Mutation; Process; Protein Import; Proteins; RNA-Binding Proteins; Role; Series; Small Interfering RNA; Structure; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Tissues; Variant; Yeasts; age related neurodegeneration; cell type; confocal imaging; frontotemporal lobar dementia amyotrophic lateral sclerosis; imaging modality; insight; knock-down; live cell imaging; mutant; novel; nucleocytoplasmic transport; protein TDP-43; protein complex; protein degradation; protein protein interaction; receptor; recruit; seal; sporadic amyotrophic lateral sclerosis; superresolution imaging; therapeutic target; ultra high resolution

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.

肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD) 是两种与年龄相关的疾病 具有共同遗传和病理基础的神经退行性疾病。近期，受干扰 核孔复合体（NPC）的组成和功能已被确定为突出的早期 神经退行性疾病（包括 ALS、AD/FTD 和 HD）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 核细胞质转运 (NCT)、基因表达和基因组组织、NPC 和 NE 稳态 对于神经元功能和生存至关重要。使用诱导多能干细胞 (iPSC) 衍生的神经元 （iPSN）和死后人类中枢神经系统组织，我们最近发现了 NPC 的严重损伤 其特点是人类神经元 NPC 中特定 Nups 的减少，最终导致受损 核蛋白输入并导致 RNA 结合蛋白 TDP- 的功能障碍和错误定位 43，神经变性中的一个突出的病理事件。这种 NPC 伤害级联是由异常的 CHMP7 在 sALS 和 C9orf72 ALS/FTD 神经元中的核积累。 CHMP7 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 介导的细胞质中发挥作用 核监视事件。 CHMP7 的细胞生物学事件和分子机制 人们开始了解酵母和非酵母对 NPC 和 NE 监测和稳态的贡献 神经元哺乳动物细胞在细胞分裂、NE 分解和 NPC 插入过程中。然而了解甚少 关于 CHMP7 在维持长寿、不分裂的人类神经元中 NPC 和 NE 稳态中的作用 其中 NPC 组件很少交换，NE 故障也不会经常发生。我们的 初步数据强调了这样一个事实：调节核定位和激活的分子事件 CHMP7 介导的神经元核监视与最近发现的并不完全一致 在酵母和分裂的哺乳动物细胞中，表明产生 CHMP7 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，我们将使用大量的 iPSN sALS 和 C9orf72 ALS/FTD 患者和适当的对照，并利用基于候选的组合 siRNA 敲低、生化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，以及新生成的 CHMP7 变体质粒库的表达。 总的来说，这些研究将确定促进核进入的分子机制（目标 1）和 CHMP7 的核输出（目标 2）以及 CHMP7 介导的 NPC 损伤（目标 3）的启动，从而产生 ALS/FTD 中的 NPC 损伤级联。因此，这些研究将提供对细胞的基本了解。 促进 CHMP7 介导的人类神经元 NPC 损伤的生物和分子事件，并提供 深入了解缓解 ALS/FTD 中这种早期且重要的致病级联反应的治疗策略。