Control of calcium flux and mitochondrial fission by the Charcot Marie Tooth disease protein Mfn2.

腓骨肌萎缩症蛋白 Mfn2 对钙通量和线粒体裂变的控制。

• 批准号: 10322143
• 负责人: Carla M Koehler
• 金额: $ 38.05万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10322143
• 关键词: Affect; Apoptosis; Axon; Axonal Transport; Binding; Biochemical; Biological Assay; Biological Testing; Calcium; Carrier Proteins; Cell Line; Cells; Charcot-Marie-Tooth Disease; Chimeric Proteins; Complement; Data; Defect; Demyelinations; Disease; Dynamin; Gene Deletion; Gene Mutation; Genes; Imaging Techniques; Inherited; Inner mitochondrial membrane; Knock-out; Lead; Learning; Light; Link; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Metabolism; Microscopy; Mitochondria; Mitochondrial Matrix; Modeling; Mutation; Mycotoxins; Neurons; OPA1 gene; Outcome; Outer Mitochondrial Membrane; Oxidative Phosphorylation; Patients; Peripheral; Peripheral Nervous System Diseases; Physiological; Play; Process; Proteins; Regulation; Respiration; Role; Series; Signal Transduction Pathway; Site; Sodium; Spottings; Surface; Techniques; Testing; Transfection; Zebrafish; axonal degeneration; constriction; design; experimental study; genetic analysis; hereditary neuropathy; human disease; insight; knockout gene; mutant; novel; release of sequestered calcium ion into cytoplasm; treatment strategy

Project summary Charcot-Marie-Tooth disease (CMT) is a hereditary peripheral neuropathy resulting from demyelination and axon degeneration. Many cases of axon degeneration are caused by mutations in Mitofusin-2 (Mfn2), which is one of two dynamin-like proteins on the surface of mitochondria. Mitofusins primarily mediate mitochondrial outer membrane fusion, but Mfn2 can also promote association between ER and mitochondria in mitochondria associated membranes (the MAM). It is observed in spots that colocalize with the mitochondrial fission dynamin Drp1 and it was proposed to affect axonal transport of mitochondria, raising the possibility that CMT is caused by defects in one of these other functions of Mfn2. Preliminary data show that Mfn2 promotes constriction of mitochondria in Drp1–/– cells and fission in Drp1-Mfn1 DKO cells when treated with the fungal toxin PXA that causes the release of calcium from the mitochondrial matrix. Calcium release and the ability to constrict mitochondria was linked to Mfn2-mediated regulation of NCLX (a Ca/Na exchanger in the mitochondrial inner membrane). It is hypothesized that PXA activates NCLX and that Mfn2 is required for this activity. It is also hypothesized that NCLX-mediated calcium release causes mitochondrial constriction and that the effects of Mfn2 on mitochondrial fission are linked to axonal transport, a process that could be disrupted in CMT patients. These hypotheses will be tested by investigating three aims. Aim 1. Investigate connections between PXA, NCLX, and Mfn2. These studies will include comprehensive tests whether PXA triggers calcium release from mitochondria by activating NCLX and investigates of the control of NCLX by Mfn2. Aim 2. Investigate connections between Mfn2 and mitochondrial fission. Effects of Mfn2 and NCLX on mitochondrial fission will be tested with knockout cell lines and transfections of fission and fusion protein constructs followed by analyses with a range of imaging techniques. Aim 3. Investigate the physiological consequences of Mfn2 and NCLX contributions to fission. Effects on mitochondrial transport proteins will be examined with kymographs of axonal processes in cultured neurons and zebrafish. Alternative functions, such effects on metabolism and a role in mitophagy, will also be considered. Together, these experiments will help establish NCLX as the target of PXA, assess the newly proposed role of Mfn2 in mitochondrial fission, and test possible downstream effects on transport or mitophagy. These experiments may therefore reveal a novel function for Mfn2 and shed new light on the underlying causes of CMT. Possible downstream effects of fission on axonal transport will change the understanding of the underlying causes of CMT and may suggest novel treatment strategies.

项目摘要 Charcot-Marie-Tooth病（CMT）是由脱髓鞘和 轴突变性。许多轴突变性的病例是由丝拟拟南诺夫蛋白-2（MFN2）突变引起的，这是 线粒体表面上的两个动力蛋白样蛋白之一。介脂蛋白原发性介导线粒体 外膜融合，但MFN2也可以促进线粒体中ER和线粒体之间的关联 相关机制（MAM）。在与线粒体裂变共定位的斑点中观察到了这一点 Dynamin DRP1并被提议影响线粒体的轴突运输，从而提高了CMT的可能性 由MFN2的其他功能之一中的缺陷引起。初步数据显示MFN2促进 用真菌处理DRP1 - / - 细胞中线粒体的收缩和DRP1-MFN1 DKO细胞的裂变 毒素PXA导致从线粒体基质中释放钙。钙释放和能力 线粒体收缩与MFN2介导的NCLX的调节有关（ca/na交换器 线粒体内膜）。假设PXA激活NCLX，并且需要MFN2 活动。还假设NCLX介导的钙释放会导致线粒体收缩，并且 MFN2对线粒体裂变的影响与轴突运输有关，该过程可能会破坏 CMT患者。这些假设将通过研究三个目标来检验。目标1。调查联系 在PXA，NCLX和MFN2之间。这些研究将包括PXA是否触发的全面测试 通过激活NCLX并研究MFN2对NCLX的控制，从线粒体释放了钙。目标2。 研究MFN2与线粒体裂变之间的联系。 MFN2和NCLX对 线粒体裂变将通过基因敲除细胞系和裂变和融合蛋白的翻译进行测试 结构进行了一系列成像技术的分析。目标3。研究生理 MFN2和NCLX对裂变的贡献的后果。对线粒体转运蛋白的影响将 在培养的神经元和斑马鱼中的轴突过程的基因学检查。替代功能， 也将考虑这种对代谢和线粒体作用的影响。这些实验将在一起 帮助建立NCLX作为PXA的靶标，评估MFN2在线粒体裂变中的新提出的作用，并评估 测试可能对运输或线粒体的下游影响。因此，这些实验可能会揭示出小说 MFN2的功能，并为CMT的根本原因开发了新的灯光。裂变可能的下游效应 关于轴突运输将改变对CMT的根本原因的理解，并可能暗示新颖 治疗策略。