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，并通过与细胞的相互作用来调节。 细胞骨架细胞器动力学的改变（即，细胞器和细胞骨架间的接触，分裂， 移动性）与多种人类疾病，特别是神经病有关。腓骨肌萎缩症（CMT） 疾病是最常见的遗传性神经病，由至少80种不同基因的突变引起。 虽然导致CMT的突变通常发生在与细胞器动力学改变有关的基因中，但问题是， 关于致病机制的问题。线粒体的分裂是由肌动蛋白的聚合介导的， 通过ER锚定的肌动蛋白聚合蛋白INF 2的ER-线粒体接触位点。显性激活 INF 2突变导致线粒体分裂增加和线粒体上肌动蛋白过度积累， 这降低了线粒体的流动性。INF 2中的类似突变也会导致CMT。初步数据显示，（1） 肌动蛋白聚集在其他细胞器的分裂位点，包括核内体、溶酶体、过氧化物酶体， 高尔基体，以及2）INF 2中的CMT突变导致内体和溶酶体移动性降低。这导致 该假说的核心假设是：存在一种保守的分子机制调节细胞器分裂 和流动性介导的肌动蛋白细胞骨架蛋白在ER-细胞器接触网站。此外，建议 细胞器移动性的降低会特别影响外周神经元，因为这些细胞的长度非常长， 因此可能是CMT的一般致病特征。该项目的具体目标如下：目标1 重点关注INF 2在线粒体、内体和溶酶体分裂中的作用，以及这些过程是如何发生的。 由INF 2突变引起CMT。这些研究将在原代人成纤维细胞中进行， 活细胞成像，包括使用一种新的，创新的探针，专门标记ER相关肌动蛋白。 评估细胞器功能并直接暗示表型中肌动蛋白聚合的实验 也将进行观察。目的2将在培养的原代小鼠神经元中进行，以便适当地 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移动性的高时空分辨率成像。目标3将包括 CMT疾病相关模型背景下的细胞器和神经元健康测定。特别是神经元 衍生自CMT患者成纤维细胞和神经元，所述成纤维细胞和神经元来自注射有AAV的小鼠，所述AAV指导CMT的表达。 将分析突变INF 2、MFN 2或RAB 7A。这些目标的完成将提供对以下方面的机械性洞察： 肌动蛋白在细胞器分裂和迁移中的作用，这些过程是如何耦合的，并测试新的假设 CMT涉及多个细胞器的移动性的全局破坏。这将进一步加深我们对 CMT和可能的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，为我提供了宝贵的培训，在神经生物学和神经变性，设计 新型成像探针、先进的成像技术、基于干细胞的重编程和小鼠模型。

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.