Mitofusin 1 overexpression rescues the abnormal mitochondrial dynamics caused by the Mitofusin 2 <scp>K357T</scp> mutation in vitro
Filippos Stavropoulos, Elena Georgiou, Natasa Schiza, Shaughn Bell, Robert H. Baloh, Kleopas A. Kleopa, Irene Sargiannidou
Abstract
Abstract Background and aims 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 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. Methods Constructs expressing either MFN2 K357T , MFN2 WT , or MFN1 WT 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 WT , MFN2 WT , or MFN2 K357T , as well as double transfected with MFN2 K357T / MFN2 WT or MFN2 K357T / MFN1 WT . Results SH‐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. Interpretation These 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.