Stavropoulos Filippos, Georgiou Elena, Schiza Natasa, Bell Shaughn, Baloh Robert H, Kleopa Kleopas A, Sargiannidou Irene
Department of Neuroscience, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.
J Peripher Nerv Syst. 2023 Sep;28(3):329-340. doi: 10.1111/jns.12564. Epub 2023 Jun 6.
Mitofusin 1 (MFN1) and MFN2 are outer mitochondrial membrane fusogenic proteins regulating mitochondrial network morphology. MFN2 mutations cause Charcot-Marie-Tooth type 2A (CMT2A), an axonal neuropathy characterized by mitochondrial fusion defects, which in the case of a GTPase domain mutant, were rescued following wild-type MFN1/2 (MFN1/2 ) overexpression. In this study, we compared the therapeutic efficiency between MFN1 and MFN2 overexpression in correcting mitochondrial defects induced by the novel MFN2 mutation located in the highly conserved R3 region.
Constructs expressing either MFN2 , MFN2 , or MFN1 under the ubiquitous chicken β-actin hybrid (CBh) promoter were generated. Flag or myc tag was used for their detection. Differentiated SH-SY5Y cells were single transfected with MFN1 , MFN2 , or MFN2 , as well as double transfected with MFN2 /MFN2 or MFN2 /MFN1 .
SH-SY5Y cells transfected with MFN2 exhibited severe perinuclear mitochondrial clustering with axon-like processes devoid of mitochondria. Single transfection with MFN1 resulted in a more interconnected mitochondrial network than transfection with MFN2 , accompanied by mitochondrial clusters. Double transfection of MFN2 with either MFN1 or MFN2 resolved the mutant-induced mitochondrial clusters and led to detectable mitochondria throughout the axon-like processes. MFN1 showed higher efficacy than MFN2 in rescuing these defects.
These results further demonstrate the higher potential of MFN1 over MFN2 overexpression to rescue CMT2A-induced mitochondrial network abnormalities due to mutations outside the GTPase domain. This higher phenotypic rescue conferred by MFN1 , possibly due to its higher mitochondrial fusogenic ability, may be applied to different CMT2A cases regardless of the MFN2 mutation type.
线粒体融合蛋白1（MFN1）和线粒体融合蛋白2（MFN2）是线粒体外膜融合蛋白，可调节线粒体网络形态。MFN2突变会导致2A型夏科-马里-图斯病（CMT2A），这是一种以线粒体融合缺陷为特征的轴索性神经病，在GTP酶结构域突变的情况下，野生型MFN1/2（MFN1/2）过表达后可得到挽救。在本研究中，我们比较了MFN1和MFN2过表达在纠正由位于高度保守的R3区域的新型MFN2突变引起的线粒体缺陷方面的治疗效果。
构建了在普遍存在的鸡β-肌动蛋白杂交（CBh）启动子下表达MFN2、MFN2或MFN1的载体。使用Flag或myc标签进行检测。将分化的SH-SY5Y细胞分别用MFN1、MFN2或MFN2进行单转染，以及用MFN2/MFN2或MFN2/MFN1进行双转染。
用MFN2转染的SH-SY5Y细胞表现出严重的核周线粒体聚集，轴突样突起中没有线粒体。用MFN1单转染导致线粒体网络比用MFN2转染更相互连接，并伴有线粒体簇。MFN2与MFN1或MFN2双转染可解决突变诱导的线粒体簇，并导致在整个轴突样突起中可检测到线粒体。在挽救这些缺陷方面，MFN1显示出比MFN2更高的疗效。
这些结果进一步证明，由于GTP酶结构域以外的突变，MFN1过表达在挽救CMT2A诱导的线粒体网络异常方面比MFN2过表达具有更高的潜力。MFN1赋予的这种更高的表型挽救可能是由于其更高的线粒体融合能力，可应用于不同的CMT2A病例，而不管MFN2突变类型如何。
