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: 10154169
• 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/10154169
• 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.

项目概要 腓骨肌萎缩症 (CMT) 是一种由脱髓鞘和神经损伤引起的遗传性周围神经病 许多轴突变性病例是由 Mitofusin-2 (Mfn2) 突变引起的。 线粒体表面的两种动力蛋白之一，主要介导线粒体。 外膜融合，但Mfn2还可以促进线粒体中ER与线粒体的结合 在与线粒体裂变共定位的斑点中观察到相关膜（MAM）。 dynamin Drp1 被认为会影响线粒体的轴突运输，这增加了 CMT 的可能性 由 Mfn2 的这些其他功能之一的缺陷引起的 初步数据表明 Mfn2 促进。 用真菌处理时，Drp1–/– 细胞中线粒体收缩，Drp1-Mfn1 DKO 细胞中线粒体分裂 毒素 PXA 导致钙从线粒体基质中释放并具有释放钙的能力。 收缩线粒体与 Mfn2 介导的 NCLX（线粒体中的 Ca/Na 交换器）调节有关 线粒体内膜）重新认识到 PXA 激活 NCLX，并且 Mfn2 是此过程所必需的。 人们还认为 NCLX 介导的钙释放会导致线粒体收缩。 Mfn2 对线粒体裂变的影响与轴突运输有关，这一过程可能会在 CMT 患者将通过调查三个目标来检验这些假设。 这些研究将包括 PXA 是否触发的全面测试。 通过激活 NCLX 从线粒体释放钙并研究 Mfn2 对 NCLX 的控制。 研究 Mfn2 和线粒体裂变之间的联系。 将通过敲除细胞系以及裂变和融合蛋白的转染来测试线粒体裂变 目的 3. 研究生理学。 Mfn2 和 NCLX 对裂变的影响将影响线粒体转运蛋白。 通过培养神经元和斑马鱼的轴突过程的 kymographs 进行检查， 这些实验也将一起考虑对新陈代谢的影响和线粒体自噬的作用。 帮助建立 NCLX 作为 PXA 的靶标，评估新提出的 Mfn2 在线粒体裂变中的作用，以及 因此，测试对运输或线粒体自噬的可能的下游影响。 Mfn2 的功能并为 CMT 可能的下游影响提供了新的线索。 对轴突运输的研究将改变对 CMT 根本原因的理解，并可能提出新的建议 治疗策略。