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

项目摘要 腓骨肌萎缩症（CMT）是一种遗传性周围神经病，由脱髓鞘和 轴突变性许多轴突变性的病例是由线粒体融合蛋白-2（Mfn 2）的突变引起的， 线粒体表面两种类似发动蛋白的蛋白质之一。线粒体融合蛋白主要介导线粒体 外膜融合，但Mfn 2也可以促进线粒体内ER与线粒体的结合 相关膜（MAM）。在与线粒体分裂共定位的斑点中观察到 动力蛋白Drp 1，并提出影响线粒体的轴突运输，提高了CMT是 由Mfn 2的这些其它功能之一的缺陷引起。初步数据显示，Mfn 2促进 当用真菌处理时，Drp 1-/-细胞中的线粒体收缩和Drp 1-Mfn 1 DKO细胞中的分裂 导致线粒体基质释放钙的毒素PXA。钙的释放和 收缩的线粒体与Mfn 2介导的NCLX调节有关（NCLX是一种Ca/Na交换器， 线粒体内膜）。假设PXA激活NCLX，并且Mfn 2为此是必需的 活动还假设NCLX介导的钙释放引起线粒体收缩， Mfn 2对线粒体分裂的影响与轴突运输有关， CMT患者。这些假设将通过调查三个目标进行检验。目标1.调查联系 PXA、NCLX和Mfn 2之间的关系。这些研究将包括全面测试PXA是否触发 通过激活NCLX从线粒体释放钙，并研究Mfn 2对NCLX的控制。目标2. 研究Mfn 2和线粒体分裂之间的联系。Mfn 2和NCLX对 线粒体分裂将用敲除细胞系以及分裂和融合蛋白的转染进行测试 构建体，然后用一系列成像技术进行分析。目标3.调查生理 Mfn 2和NCLX对裂变的贡献。对线粒体转运蛋白的影响将 在培养的神经元和斑马鱼的轴突过程的kymographs检查。替代功能， 也将考虑对代谢的这种影响和在线粒体自噬中的作用。这些实验将 帮助建立NCLX作为PXA的靶点，评估Mfn 2在线粒体分裂中的新作用， 测试可能对运输或线粒体自噬的下游影响。因此，这些实验可能揭示了一种新的 Mfn 2的功能，并揭示了CMT的根本原因。裂变可能产生的下游效应 对轴突运输的研究将改变对CMT根本原因的理解，并可能提示新的 治疗策略。

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.