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

项目摘要 细胞器动力学深刻地影响细胞的生理学，并受与 细胞骨架。细胞器动力学的改变(即细胞器间和细胞骨架的接触、分裂和 移动性)与多种人类疾病有关，特别是神经性疾病。夏科-玛丽-牙科(Charcot-Marie-Tooth，CMT) 疾病是最常见的遗传性神经病变，由至少80个不同基因的突变引起。 尽管导致CMT的突变通常是与细胞器动力学改变有关的基因，但问题是 关于其致病机制，目前尚存争议。线粒体的分裂是由肌动蛋白的聚合介导的。 内质网通过内质网锚定的肌动蛋白聚合蛋白INF2与线粒体接触。显性激活 INF2基因突变导致线粒体分裂增加和线粒体上肌动蛋白过度堆积， 这会降低线粒体的流动性。INF2中类似的突变也会导致CMT。初步数据显示，1) 肌动蛋白聚集在其他细胞器的分裂部位，包括内小体、溶酶体、过氧化物体和 以及2)INF2基因CMT突变导致内小体和溶酶体流动性降低。这导致了 这一提议的中心假设是：细胞器分裂存在一种保守的分子机制。 以及由肌动蛋白细胞骨架蛋白介导的内质网-细胞器接触部位的流动性。此外，还提出了一种新的解决方案。 由于这些细胞的极端长度，细胞器迁移率的降低特别影响到周围神经元 这可能是CMT的一般致病特征。本项目的具体目标如下：目标1 重点介绍INF2在线粒体、内小体和溶酶体分裂中的作用以及这些过程是如何进行的 被导致CMT的INF2突变所改变。这些研究将通过以下方式在原代人类成纤维细胞中进行 活细胞成像，包括使用一种新颖的、创新的探针，专门标记ER相关的肌动蛋白。 评估细胞器功能并直接将肌动蛋白聚合与表型联系起来的实验 还将执行观察。Aim 2将在原代培养的小鼠神经元中进行，以便适当地 评估细胞器动力学和移动性的改变如何影响神经元健康。基于深度学习的图像 恢复将被用来实现细胞器移动性的高时空分辨率成像。AIM 3将包括 与疾病相关的CMT模型中的细胞器和神经元健康分析。具体地说，神经元 来源于CMT患者成纤维细胞和注射AAVs的小鼠神经元直接表达CMT- 将分析突变的INF2、Mfn2或RAB7A。这些目标的完成将提供对 肌动蛋白在细胞器分裂和移动中的作用，这些过程是如何耦合的，并检验新的假设 这种CMT涉及全球范围内多个细胞器的流动性中断。这将进一步加深我们对 CMT和其他神经退行性疾病的致病机制。该项目还将加强 我的科学训练，为我提供了无价的神经生物学和神经退行性变方面的训练，设计 新的成像探针、先进的成像技术、基于干细胞的重新编程和小鼠模型。

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.