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

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 ) 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.

Methods: 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 .

Results: 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.

Interpretation: 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.