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Actin-mediated regulation of organelle dynamics in Charcot-Marie-Tooth disease

肌动蛋白介导的夏科-玛丽-图思病细胞器动力学调节

基本信息

批准号：
10327608
负责人：
Cara Rae Schiavon
金额：
$ 6.98万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10327608
关键词：
Actins Affect Biochemical Biological Biological Assay Cancer cell line Cell physiology Cells Cellular biology Charcot-Marie-Tooth Disease Computer software Coupled Cytoskeletal Proteins Cytoskeleton Data Dependovirus Disease Endoplasmic Reticulum Endosomes Environment Fibroblasts Functional disorder Genes Goals Golgi Apparatus Health Human Image Imaging Techniques Impairment Label Length Link Lysosomes Measures Mediating Mitochondria Modeling Molecular Mus Mutation Nerve Degeneration Neurobiology Neurodegenerative Disorders Neurons Neuropathy Organelles Pathogenicity Pathology Patients Peripheral Pharmacology Phenotype Physiological Play Polymers Positioning Attribute Process Proteins Regulation Research Resolution Role Site Small Interfering RNA Testing Training base cell motility cell type deep learning depolymerization design experience experimental study genetic regulatory protein hereditary neuropathy human disease imaging approach imaging probe in vivo innovation insight live cell imaging mouse model mutant nanobodies nerve stem cell novel peroxisome polymerization programs restoration spatiotemporal stem cell technology stem cells

项目摘要

Project Summary Organelle dynamics profoundly affect the physiology of the cell and are regulated by interactions with the cytoskeleton. Alterations in organelle dynamics (i.e., inter-organelle and cytoskeletal contacts, fission, and mobility) are associated with a variety of human diseases, particularly neuropathies. Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neuropathy and is caused by mutations in at least eighty different genes. Although the mutations that cause CMT are often in genes linked to altered organelle dynamics, questions remain regarding the pathogenic mechanism. Mitochondrial fission is mediated by the polymerization of actin at ER-mitochondria contact sites via the ER-anchored, actin polymerizing protein INF2. Dominant activating mutations in INF2 cause increased mitochondrial fission and excessive actin accumulation on mitochondria, which reduces mitochondrial mobility. Similar mutations in INF2 also cause CMT. Preliminary data show that 1) actin accumulates at fission sites of other organelles including endosomes, lysosomes, peroxisomes, and the Golgi, and 2) that CMT mutations in INF2 cause a reduction in endosome and lysosome mobility. This leads to the central hypothesis of this proposal: there is a conserved molecular mechanism regulating organelle fission and mobility mediated by actin cytoskeletal proteins at ER-organelle contact sites. Furthermore, it is proposed that reduction in organelle mobility specifically affects peripheral neurons due to the extreme length of these cells and hence may be a general pathogenic feature of CMT. The Specific Aims of this project are as follows: Aim 1 focuses on the role of INF2 in mitochondrial, endosomal, and lysosomal fission and how these processes are altered by mutations in INF2 that cause CMT. These studies will be carried out in primary human fibroblasts via live-cell imaging, including the use of a novel, innovative probe that specifically labels ER-associated actin. Experiments to assess organelle functions and to directly implicate actin polymerization in the phenotypes observed will also be performed. Aim 2 will be carried out in cultured primary mouse neurons in order to properly assess how alterations in organelle dynamics and mobility affect neuronal health. Deep learning-based image restoration will be used to achieve high spatiotemporal resolution imaging of organelle mobility. Aim 3 will include organelle and neuronal health assays in the context of disease-relevant models of CMT. Specifically, neurons derived from CMT patient fibroblasts and neurons from mice injected with AAVs directing expression of CMT- mutant INF2, MFN2, or RAB7A will be analyzed. Completion of these aims will provide mechanistic insight into the role of actin in organelle fission and mobility, how these processes are coupled, and test the novel hypothesis that CMT involves global disruption of mobility of multiple organelles. This will further our understanding of the pathogenic mechanism of CMT and perhaps other neurodegenerative disorders. The project will also enhance my scientific training by providing me with invaluable training in neurobiology and neurodegeneration, designing novel imaging probes, advanced imaging techniques, stem-cell based reprogramming, and mouse models.

项目概要细胞器动力学深刻影响细胞的生理机能，并受到与细胞相互作用的调节细胞骨架。细胞器动力学的改变（即细胞器间和细胞骨架接触、裂变和流动性）与多种人类疾病，特别是神经病有关。腓骨肌萎缩症 (CMT) 该疾病是最常见的遗传性神经病，由至少 80 个不同基因的突变引起。尽管导致 CMT 的突变通常发生在与细胞器动力学改变相关的基因中，但仍存在疑问关于致病机制仍有待探讨。线粒体裂变是由肌动蛋白聚合介导的内质网-线粒体通过内质网锚定的肌动蛋白聚合蛋白 INF2 接触位点。显性激活 INF2 突变导致线粒体裂变增加和线粒体上肌动蛋白过度积累，这会降低线粒体的流动性。 INF2 的类似突变也会导致 CMT。初步数据表明 1) 肌动蛋白聚集在其他细胞器的裂变位点，包括内体、溶酶体、过氧化物酶体和高尔基体，2) INF2 中的 CMT 突变导致内体和溶酶体流动性降低。这导致该提案的中心假设：存在调节细胞器裂变的保守分子机制以及由内质网细胞器接触位点的肌动蛋白细胞骨架蛋白介导的移动性。此外，建议由于这些细胞的长度极长，细胞器流动性的降低特别影响周围神经元因此可能是 CMT 的一般致病特征。本项目的具体目标如下：目标1 重点关注 INF2 在线粒体、内体和溶酶体裂变中的作用以及这些过程是如何进行的因 INF2 突变而改变，导致 CMT。这些研究将通过以下方法在原代人成纤维细胞中进行活细胞成像，包括使用专门标记 ER 相关肌动蛋白的新型创新探针。评估细胞器功能并直接暗示表型中肌动蛋白聚合的实验观察也将被执行。目标 2 将在培养的原代小鼠神经元中进行，以便正确地评估细胞器动力学和流动性的改变如何影响神经元健康。基于深度学习的图像恢复将用于实现细胞器迁移率的高时空分辨率成像。目标 3 将包括在疾病相关的 CMT 模型中进行细胞器和神经元健康检测。具体来说，神经元源自注射 AAV 的小鼠的 CMT 患者成纤维细胞和神经元，指导 CMT- 的表达将分析突变的 INF2、MFN2 或 RAB7A。完成这些目标将为我们提供机械洞察肌动蛋白在细胞器裂变和移动性中的作用，这些过程如何耦合，并检验新的假设 CMT 涉及多个细胞器的整体流动性破坏。这将加深我们对 CMT 和其他神经退行性疾病的致病机制。该项目还将增强我的科学培训为我提供了神经生物学和神经变性方面的宝贵培训，设计新颖的成像探针、先进的成像技术、基于干细胞的重编程和小鼠模型。