Mitofusins (Mfn) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in Mfn2 cause neurodegenerative Charcot Marie Tooth disease type 2A (CMT2A). Here we show that Mfn2 activity can be determined by Met376 and His380 interactions with Asp725 and Leu727 and controlled by PINK1 kinase-mediated phosphorylation of adjacent Mfn2 Ser378. Small molecule mimics of the peptide-peptide interface of Mfn2 disrupted this interaction, allosterically activating Mfn2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants Mfn2 Arg94Gln and Thr105Met, as evidenced by improved mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of Mfn2 Thr105Met mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism/trafficking.

Summary Mitochondria are dynamic organelles, remodeling and exchanging contents during cyclic fusion and fission. Genetic mutations of mitofusin (Mfn) 2 interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition, Charcot Marie Tooth disease type 2A (CMT2A). It has not been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is incompletely understood. Here we show that mitofusins adopt either a fusion-constrained or fusion-permissive molecular conformation directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated by targeting these conformational transitions. Based on this model we engineered a cell-permeant minipeptide to destabilize fusion-constrained mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons harboring CMT2A gene defects. The relationship between mitofusin conformational plasticity and mitochondrial dynamism uncovers a central mechanism regulating mitochondrial fusion whose manipulation can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.

Summary Mitochondrial fusion and fission are critical to heart health; genetically interrupting either is rapidly lethal. To understand whether it is loss of, or the imbalance between, fusion and fission that underlies observed cardiac phenotypes, we engineered mice in which Mfn-mediated fusion and Drp1-mediated fission could be concomitantly abolished. Compared to fusion-defective Mfn1/Mfn2 cardiac knockout or fission-defective Drp1 cardiac knockout mice, Mfn1/Mfn2/Drp1 cardiac triple knockout mice survived longer and manifested a unique pathological form of cardiac hypertrophy. Over time, however, combined abrogation of fission and fusion provoked massive progressive mitochondrial accumulation that severely distorted cardiomyocyte sarcomeric architecture. Mitochondrial biogenesis was not responsible for mitochondrial superabundance, whereas mitophagy was suppressed despite impaired mitochondrial proteostasis. Similar but milder defects were observed in aged hearts. Thus, cardiomyopathies linked to dynamic imbalance between fission and fusion are temporarily mitigated by forced mitochondrial adynamism at the cost of compromising mitochondrial quantity control and accelerating mitochondrial senescence.

Mutations in the mitochondrial fusion protein mitofusin (MFN) 2 cause the chronic neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A), for which there is currently no treatment. Small-molecule activators of MFN1 and MFN2 enhance mitochondrial fusion and offer promise as therapy for this condition, but prototype compounds have poor pharmacokinetic properties. Herein, we describe a rational design of a series of 6-phenylhexanamide derivatives whose pharmacokinetic optimization yielded a 4-hydroxycyclohexyl analogue, 13, with the potency, selectivity, and oral bioavailability of a preclinical candidate. Studies of 13 cis- and trans-4-hydroxycyclohexyl isostereomers unexpectedly revealed functionality and protein engagement exclusively for the trans form, 13B. Preclinical absorption, distribution, metabolism, and excretion (ADME) and in vivo target engagement studies of 13B support further development of 6-phenylhexanamide derivatives as therapeutic agents for human CMT2A.

Figure 1. Impaired growth rate, sensorimotor function, grip strength, and vision in Thy1.2-MFN2 R94Q transgenic mice. (A) Schematic of the Thy1.2-MFN2 R94Q transgenic construct. The expression of human MFN2 R94Q or control MFN2 WT (N terminus tagged with Flag) was driven by the neuron-specific mouse Thy1.2 promoter. (B) Representative image of a nontransgenic (nTg) mouse, a Thy1.2-MFN2 WT mouse, and a Thy1.2-MFN2 R94Q mouse (5 months old). (C) Immunoblot of Flag-MFN2 WT or Flag-MFN2 R94Q transgene expression (14-month-old mice). Red arrow, Flag-MFN2; black arrow, endogenous mouse Mfn2. Expression levels of MFN2 WT or MFN2 R94Q transgenes were identical and slightly below endogenous Mfn2 levels. n = 3 mice/genotype. (D) Immunostaining of Flag-MFN2 WT or Flag-MFN2 R94Q protein expression and localization. Mouse cortex or spinal cord (5-month-old mice). Anti-Flag (red) and DAPI (blue). Scale bars: 50 μM. Punctate mitochondrial staining was observed in both transgenic lines, but only MFN2 R94Q mice showed mitochondrial accumulations in neuronal cytoplasm and proximal axons. n = 3 mice/genotype. (E) Body weight. Data are represented as mean ± SEM. n = 6-52 per genotype per time point. Student's 2-tailed t test (nTg vs. MFN2 R94Q). *P < 0.05. (F) Survival curve. nTg (n = 59), MFN2 WT (n = 29), MFN2 R94Q (n = 124). log-rank test with Bonferroni's correction. nTg vs. MFN2 WT , P > 0.05, not significant. nTg vs. MFN2 R94Q , P < 0.01. MFN2 WT vs. MFN2 R94Q , P < 0.05. (G) Open-field test (total activity) and (H) open-field test (rearing). Total activity was not significantly different between groups. nTg (n = 5), MFN2 WT (n = 5), MFN2 R94Q (n = 6) (3-month-old mice). (I) Rotarod testing. nTg (n = 5), MFN2 WT (n = 5), MFN2 R94Q (n = 6). (J) Grip-strength testing (forelimbs). nTg (n = 8-11), MFN2 WT (n = 5), MFN2 R94Q (n = 6). (K) Visual acuity measured by OKR. nTg (n = 6), MFN2 WT (n = 3), MFN2 R94Q (n = 7). In G-K, data are represented as mean ± SEM. Two-way ANOVA with Tukey's test was used for multiple comparison.