Multifaceted fusion defects converge on altered mitochondrial distribution and increased mutant mitofusin level in CMT2A models

Charcot-Marie-Tooth type 2A neuropathy (CMT2A) is caused by a hundred different missense mutations in the mitofusin MFN2, a mitochondrial fusion factor. It is well established that different CMT2A alleles expressed in mitofusin deficient cells can exhibit varying levels of fusion activity. However, most CMT2A mutations are dominant. Therefore, to picture the complexity of CMT2A pathogenesis, it is crucial to define whether and how different fusion activity levels translate into different dominant properties. To explore the range of dominant properties among CMT2A alleles, we modelled in drosophila fourteen missense mutations reported in patients. We studied alleles from different mutation hotspots identified in MFN2 protein structure, alleles causing severe or mild forms of CMT2A, or alleles with different level of dominance in patients. Mitochondrial architecture analysis revealed a wide spectrum of composite dominant effects of variable expressivity. Fusion-inhibition and aggregation properties were found in mutants affecting catalytic motifs and hydrophobic cores, and exacerbation of mitochondrial fusion in mutants targeting domain interface. In these mutant categories, mitochondrial cytoplasmic distribution was disturbed to varying degrees that strongly correlate with neurotoxicity levels. Inhomogeneity of mitochondrial repartition was indeed associated with decreased endoplasmic reticulum-mitochondrion overlap, reduced mitochondrial content at neuromuscular junctions, higher locomotor deficits in flies and higher disease severity in patients. At the molecular scale, most CMT2A mutant proteins showed reduced ubiquitination and increased protein level, likely amplifying their dominant properties. Thus, CMT2A mutations induce a broad spectrum of fusion alterations converging towards altered mitochondrial distribution and mutant Mfn accumulation.