Two Rare Human Mitofusin 2 Mutations Alter Mitochondrial Dynamics and Induce Retinal and Cardiac Pathology in <i>Drosophila</i>

Mitochondrial fusion is essential to organelle homeostasis and organ health. Inexplicably, loss of function mutations of mitofusin 2 (Mfn2) specifically affect neurological tissue, causing Charcot Marie Tooth syndrome (CMT) and atypical optic atrophy. As CMT-linked Mfn2 mutations are predominantly within the GTPase domain, we postulated that Mfn2 mutations in other functional domains might affect non-neurological tissues. Here, we defined in vitro and in vivo consequences of rare human mutations in the poorly characterized Mfn2 HR1 domain. Human exome sequencing data identified 4 rare non-synonymous Mfn2 HR1 domain mutations, two bioinformatically predicted as damaging. Recombinant expression of these (Mfn2 M393I and R400Q) in Mfn2-null murine embryonic fibroblasts (MEFs) revealed incomplete rescue of characteristic mitochondrial fragmentation, compared to wild-type human Mfn2 (hMfn2); Mfn2 400Q uniquely induced mitochondrial fragmentation in normal MEFs. To compare Mfn2 mutation effects in neurological and non-neurological tissues in vivo, hMfn2 and the two mutants were expressed in Drosophila eyes or heart tubes made deficient in endogenous fly mitofusin (dMfn) through organ-specific RNAi expression. The two mutants induced similar Drosophila eye phenotypes: small eyes and an inability to rescue the eye pathology induced by suppression of dMfn. In contrast, Mfn2 400Q induced more severe cardiomyocyte mitochondrial fragmentation and cardiac phenotypes than Mfn2 393I, including heart tube dilation, depressed fractional shortening, and progressively impaired negative geotaxis. These data reveal a central functional role for Mfn2 HR1 domains, describe organ-specific effects of two Mfn2 HR1 mutations, and strongly support prospective studies of Mfn2 400Q in heritable human heart disease of unknown genetic etiology.线粒体融合对于细胞器稳态与器官健康至关重要。令人费解的是，线粒体融合蛋白2（mitofusin 2, Mfn2）的功能丧失突变仅特异性累及神经系统组织，引发夏科-马里-图思综合征（Charcot Marie Tooth syndrome, CMT）与非典型视神经萎缩。由于与CMT相关的Mfn2突变主要集中于GTP酶结构域（GTPase domain），我们推测其他功能结构域内的Mfn2突变可能会影响非神经系统组织。本研究明确了特征尚不充分的Mfn2 HR1结构域（HR1 domain）内罕见人类突变的体外与体内效应。通过人类外显子组测序（human exome sequencing）数据，我们筛选出4个罕见的Mfn2 HR1结构域非同义突变，其中2个经生物信息学预测具有致病性。将这两个突变体（Mfn2 M393I与R400Q）与野生型人源Mfn2（wild-type human Mfn2, hMfn2）分别在Mfn2敲除小鼠胚胎成纤维细胞（Mfn2-null murine embryonic fibroblasts, MEFs）中进行重组表达后发现，相较于野生型hMfn2，二者均未能完全挽救典型的线粒体碎片化表型；其中Mfn2 R400Q可单独在正常MEFs中诱导线粒体碎片化。为在体内比较Mfn2突变在神经系统与非神经系统组织中的效应差异，我们分别在果蝇眼睛或心管中表达hMfn2与上述两个突变体，同时通过器官特异性RNA干扰（RNA interference, RNAi）表达敲除内源性果蝇线粒体融合蛋白（endogenous fly mitofusin, dMfn）。两个突变体均可诱导相似的果蝇眼睛表型：眼睛体积缩小，且无法挽救因dMfn敲除引发的眼部病理损伤。与之形成对比的是，相较于Mfn2 M393I，Mfn2 R400Q可诱导更为严重的心肌细胞线粒体碎片化与心脏表型，包括心管扩张、短轴缩短率降低以及进行性受损的负趋地性运动能力。本研究揭示了Mfn2 HR1结构域的核心功能作用，阐明了2个Mfn2 HR1结构域突变的器官特异性效应，并强烈支持针对未知遗传病因的遗传性人类心脏病开展Mfn2 R400Q相关的前瞻性研究。