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

项目总结 Charcot-Marie-Tooth病(CMT)是一种遗传性周围神经病，由脱髓鞘和 轴突变性。许多轴突变性是由Mitofusin-2(Mfn2)突变引起的，Mfn2是 线粒体表面的两种动力蛋白之一。丝裂原蛋白主要介导线粒体 外膜融合，但Mfn2也能促进内质网与线粒体之间的联系 结合膜(MAM)。它是在与线粒体分裂共存的斑点上观察到的 Dynamin Drp1和它被认为影响线粒体的轴突运输，增加了CMT是 由MFn2的这些其他功能中的一个中的缺陷引起。初步数据显示，Mfn2促进 真菌对DRp1-/-细胞线粒体收缩和DRp1-Mfn1-DKO细胞分裂的影响 PXA毒素，引起线粒体基质中钙的释放。钙的释放和细胞的能力 线粒体收缩与Mfn2介导的NCLX(钙/钠交换)的调节有关 线粒体内膜)。假设PXA激活NCLX，并且为此需要Mfn2 活动。还假设NCLX介导的钙释放导致线粒体收缩，并且 Mfn2对线粒体分裂的影响与轴突运输有关，这一过程可能会在 CMT患者。这些假设将通过调查三个目标来检验。目标1.调查关系 在PXA、NCLX和Mfn2之间。这些研究将包括综合测试PXA是否会触发 线粒体激活Nclx释放钙及Mfn2调控Nclx的研究。目标2. 研究Mfn2和线粒体分裂之间的联系。Mfn2和NCLX对大鼠血管内皮细胞的影响 线粒体的分裂将用基因敲除的细胞系和转基因的分裂和融合蛋白进行测试。 构建之后，使用一系列成像技术进行分析。目的3.调查生理问题 Mfn2和Nclx对裂变的贡献的后果。对线粒体运输蛋白的影响 用培养神经元和斑马鱼的轴突动图进行检查。可选功能， 这种对新陈代谢的影响和在吞丝分裂中的作用也将被考虑。总而言之，这些实验将 帮助建立NCLX作为PXA的靶点，评估新提出的Mfn2在线粒体分裂中的作用，以及 测试下游对运输或有丝分裂的影响。因此，这些实验可能会揭示一种新的 Mfn2的功能，并为CMT的根本原因提供了新的线索。裂变可能产生的下游影响 关于轴突运输的研究将改变人们对CMT潜在原因的理解，并可能提出新的 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.