Charcot-Marie-Tooth Type2A Mfn2 Domain-Specific Mutants Deferentially Alter Mitochondrial Fusion Dynamics and Motility

Abstract

Mitochondrial function relays on the balance between fusion and fission events that determine mitochondrial communication. Mutations of outer mitochondrial membrane (OMM) fusion protein Mfn2, cause motor neuron degeneration disease Charcot-Marie-Tooth Type 2 (CMT2). Also, Mfn2 plays a controversial role in the ER-mitochondrial communication. Thus, it is unknown if Mfn2 CMT2-associated domain-specific mutations alter mitochondrial fusion dynamics, ER/mitochondria communication, or both. Here we study mitochondrial fusion dynamics in human fibroblasts isolated from skin samples of control and CMT2-confirmed patients from the Centre for Neuromuscular Diseases Biobank, Newcastle. The cells were transfected mitochondrial matrix-targeted mtDsRed and photo-switchable protein mtPA-GFP. By means of 408 nm mediated photo-conversion and time series confocal imaging, we studied mitochondrial continuity and fusion events (f.e.) frequency. We evaluated two domain-specific Mfn2 mutants: L248H (GTPase domain) and M376V (GTPase to HR1 connector). Mfn2-L248H carrying fibroblasts display hyperfused mitochondria and f.e. frequency inhibition 0.42 f.e./min as opposed to 1.2 f.e./min in control fibroblasts. Moreover, Mfn2-M378V fibroblasts showed inhibition of both mitochondrial continuity and f.e. frequency (0.45 f.e./min). In addition, the ultrastructure of control and Mfn2-M378V fibroblasts, evaluated by Transmission Electron Microscopy, showed two populations of mutant mitochondria: high frequency of small round-shaped organelles, consistent with inhibition of mitochondrial fusion, and a group of enlarged mitochondria displaying partially empty cristae. Finally, mitochondrial motility was significantly elevated in Mfn2-M378V fibroblasts, suggesting that Mfn2 GTPase to HR1 connector may have a role in the interaction with the mitochondrial motility machinery. Thus, CMT2A-causing Mfn2 specific-domain mutations display differential defects in human mitochondrial ultrastructure and fusion dynamics. Funding FONDECYT 1150677 to VE.