Mitofusin 1 overexpression rescues the abnormal mitochondrial dynamics caused by the Mitofusin 2 K357T mutation in vitro

Background and aimsMitofusin 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(WT)) overexpression. In this study, we compared the therapeutic efficiency between MFN1(WT) and MFN2(WT) overexpression in correcting mitochondrial defects induced by the novel MFN2(K357T) mutation located in the highly conserved R3 region. MethodsConstructs expressing either MFN2(K357T), MFN2(WT), or MFN1(WT) under the ubiquitous chicken beta-actin hybrid (CBh) promoter were generated. Flag or myc tag was used for their detection. Differentiated SH-SY5Y cells were single transfected with MFN1(WT), MFN2(WT), or MFN2(K357T), as well as double transfected with MFN2(K357T)/MFN2(WT) or MFN2(K357T)/MFN1(WT). ResultsSH-SY5Y cells transfected with MFN2(K357T) exhibited severe perinuclear mitochondrial clustering with axon-like processes devoid of mitochondria. Single transfection with MFN1(WT) resulted in a more interconnected mitochondrial network than transfection with MFN2(WT), accompanied by mitochondrial clusters. Double transfection of MFN2(K357T) with either MFN1(WT) or MFN2(WT) resolved the mutant-induced mitochondrial clusters and led to detectable mitochondria throughout the axon-like processes. MFN1(WT) showed higher efficacy than MFN2(WT) in rescuing these defects. InterpretationThese results further demonstrate the higher potential of MFN1(WT) over MFN2(WT) overexpression to rescue CMT2A-induced mitochondrial network abnormalities due to mutations outside the GTPase domain. This higher phenotypic rescue conferred by MFN1(WT), possibly due to its higher mitochondrial fusogenic ability, may be applied to different CMT2A cases regardless of the MFN2 mutation type.