An innovative approach for a rare disease: Charcot-Marie-Tooth disease type 2A (CMT2A) is a rare, inherited neurodegenerative condition. Affected individuals develop severe progressive muscle weakness, motor deficits, and peripheral neuropathy. Although defects in the gene encoding mitofusin 2 (MFN2) are known to cause CMT2A, the disease remains incurable. Rocha et al. identified specific MFN2 residues contributing to the disease and developed a class of MFN2-agonist drugs. The small molecules restored mitochondrial fusion and activity in the sciatic nerves of mice; they may also help in other diseases linked to mitochondrial trafficking. Science , this issue p. 336

Charcot–Marie-Tooth disease type 2A (CMT2A) is an untreatable childhood peripheral neuropathy caused by mutations of the mitochondrial fusion protein, mitofusin (MFN) 2. Here, pharmacological activation of endogenous normal mitofusins overcame dominant inhibitory effects of CMT2A mutants in reprogrammed human patient motor neurons, reversing hallmark mitochondrial stasis and fragmentation independent of causal MFN2 mutation. In mice expressing human MFN2 T105M, intermittent mitofusin activation with a small molecule, MiM111, normalized CMT2A neuromuscular dysfunction, reversed pre-treatment axon and skeletal myocyte atrophy, and enhanced axon regrowth by increasing mitochondrial transport within peripheral axons and promoting in vivo mitochondrial localization to neuromuscular junctional synapses. MiM111-treated MFN2 T105M mouse neurons exhibited accelerated primary outgrowth and greater post-axotomy regrowth, linked to enhanced mitochondrial motility. MiM111 is the first pre-clinical candidate for CMT2A.

Dynamic regulation of the mitochondrial network by mitofusins (MFNs) modulates energy production, cell survival, and many intracellular signaling events including calcium handling. However, the relative importance of specific mitochondrial functions and their dependence on MFNs vary greatly among cell types. Osteoclasts have many mitochondria, and increased mitochondrial biogenesis and oxidative phosphorylation enhance bone resorption, but little is known about the mitochondrial network or MFNs in osteoclasts. Because expression of each MFN isoform increases with osteoclastogenesis, we conditionally deleted both MFN1 and -2 (dcKO) in murine osteoclast precursors, finding that this increased bone mass in young female mice, and abolished osteoclast precursor differentiation into mature osteoclasts, in vitro. Defective osteoclastogenesis was reversed by overexpression of MFN2 but not MFN1; therefore we generated mice lacking only MFN2 in osteoclasts. MFN2-deficient female mice had increased bone mass at 1 year and resistance to RANKL-induced osteolysis at 8 weeks. To explore whether MFN-mediated tethering or mitophagy is important for osteoclastogenesis, we overexpressed MFN2 variants defective in either function in MFN1/2 dcKO precursors and found that although mitophagy was dispensable for differentiation, tethering was required. Since the master osteoclastogenic transcriptional regulator nuclear factor of activated T cells 1 (NFATC1) is calcium-regulated, we assessed calcium release from the endoplasmic reticulum (ER) and store-operated calcium entry and found that the latter was blunted in dcKO cells. Restored osteoclast differentiation by expression of intact MFN2 or the mitophagy-defective variant was associated with normalization of store-operated calcium entry and Nfatc1 levels, indicating that MFN2 controls mitochondria-ER tethering in osteoclasts.

Cardiac muscle has the highest mitochondrial density of any human tissue, but mitochondrial dysfunction is not a recognized cause of isolated cardiomyopathy. Here, we determined that the rare mitofusin (MFN) 2 R400Q mutation is 15–20× over-represented in clinical cardiomyopathy, whereas this specific mutation is not reported as a cause of MFN2 mutant-induced peripheral neuropathy, Charcot–Marie–Tooth disease type 2A (CMT2A). Accordingly, we interrogated the enzymatic, biophysical, and functional characteristics of MFN2 Q400 versus wild-type and CMT2A-causing MFN2 mutants. All MFN2 mutants had impaired mitochondrial fusion, the canonical MFN2 function. Compared to MFN2 T105M that lacked catalytic GTPase activity and exhibited normal activation-induced changes in conformation, MFN2 R400Q and M376A had normal GTPase activity with impaired conformational shifting. MFN2 R400Q did not suppress mitochondrial motility, provoke mitochondrial depolarization, or dominantly suppress mitochondrial respiration like MFN2 T105M. By contrast to MFN2 T105M and M376A, MFN2 R400Q was uniquely defective in recruiting Parkin to mitochondria. CRISPR editing of the R400Q mutation into the mouse Mfn2 gene induced perinatal cardiomyopathy with no other organ involvement; knock-in of Mfn2 T105M or M376V did not affect the heart. RNA sequencing and metabolomics of cardiomyopathic Mfn2 Q/Q400 hearts revealed signature abnormalities recapitulating experimental mitophagic cardiomyopathy. Indeed, cultured cardiomyoblasts and in vivo cardiomyocytes expressing MFN2 Q400 had mitophagy defects with increased sensitivity to doxorubicin. MFN2 R400Q is the first known natural mitophagy-defective MFN2 mutant. Its unique profile of dysfunction evokes mitophagic cardiomyopathy, suggesting a mechanism for enrichment in clinical cardiomyopathy.

Aims The effects of GRK2 inhibition on myocardial metabolism in heart failure (HF) are unchartered. In this work, we evaluated the impact of pharmacological inhibition of GRK2 by a cyclic peptide, C7, on metabolic, biochemical, and functional phenotypes in experimental HF. Methods and results C7 was initially tested on adult mice ventricular myocyte from wild type and GRK2 myocardial deficient mice (GRK2‐cKO), to assess the selectivity on GRK2 inhibition. Then, chronic infusion of 2 mg/kg/day of C7 was performed in HF mice with cryogenic myocardial infarction. Cardiac function in vivo was assessed by echocardiography and cardiac catheterization. Histological, biochemical, and metabolic studies were performed on heart samples at time points. C7 induces a significant increase of contractility in wild type but not in adult ventricle myocytes from GRK2‐cKO mice, thus confirming C7 selectivity for GRK2. In HF mice, 4 weeks of treatment with C7 improved metabolic features, including mitochondrial organization and function, and restored the biochemical and contractile responses. Conclusions GRK2 is a critical molecule in the physiological regulation of cardiac metabolism. Its alterations in the failing heart can be pharmacologically targeted, leading to the correction of metabolic and functional abnormalities observed in HF.

Phenotypic variations in Charcot-Marie-Tooth disease type 2A (CMT2A) result from the many mutations in the mitochondrial fusion protein, mitofusin 2 (MFN2). While the GTPase domain mutations of MFN2 lack the ability to hydrolyze GTP and complete mitochondrial fusion, the mechanism of dysfunction in HR1 domain mutations has yet to be explored. Using Mfn1/Mfn2 double null cells and Mfn2 knock out (KO) fibroblasts, we measured the ability of this variant protein to change conformations and hydrolyze GTP. We found that a mutation in the HR1 domain (M376A) of MFN2 results in conformational change dysfunction while maintaining GTPase ability. Prolonged exposure to mitofusin agonist MiM 111 reverses mitochondrial fusion dysfunction in the HR1 mutant through encouraging an open conformation, resulting in a potential therapeutic model in this variant. Herein, we describe a novel mechanism of dysfunction in MFN2 variants through exploring domain-specific mitochondrial characteristics leading to CMT2A.