Effective therapeutic strategies in a preclinical mouse model of Charcot-Marie-Tooth disease

†Janet Hoenicka, http://orcid.org/0000-0002-6790-6988 ‡Francesc Palau, http://orcid.org/0000-0002-8635-5421 ¶José M. Cuezva, http://orcid.org/0000-0003-1118-248X Received: June 16, 2021. Revised: July 14, 2021. Accepted: July 14, 2021 © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]. 2441 Human Molecular Genetics, 2021, Vol. 30, No. 24 2441–2455 https://doi.org/10.1093/hmg/ddab207 Advance Access Publication Date: 19 July 2021 General Article GENERAL ARTICLE Effective therapeutic strategies in a preclinical mouse model of Charcot–Marie–Tooth disease Cristina Nuevo-Tapioles1,2,3,4, Fulvio Santacatterina1,2,3,4, Brenda Sánchez-Garrido1,2,3,4, Cristina Núñez de Arenas1,2,3,4, Adrián Robledo-Bérgamo1, Paula Martínez-Valero1,2, Lara Cantarero3,5, Beatriz Pardo1,2, Janet Hoenicka3,5,†, Michael P. Murphy6, Jorgina Satrústegui1,2, Francesc Palau3,5,7,8,9,‡and José M. Cuezva1,2,3,4,*,¶ 1Departamento de Biología Molecular, Universidad Autónoma de Madrid (UAM), 28049 Madrid, Spain, 2Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), 28049 Madrid, Spain, 3Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, 28029 Madrid, Spain, 4Instituto de Investigación Hospital 12 de Octubre, 28041 Madrid, Spain, 5Laboratorio de Neurogenética y Medicina Molecular - IPER, Institut de Recerca Sant Joan de Déu, 08950 Barcelona, Spain, 6Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, University of Cambridge, Cambridge CB2 0XY, UK, 7Department of Genetic and Molecular Medicine - IPER, Hospital Sant Joan de Déu, 08950 Barcelona, Spain, 8Clinic Institute of Medicine and Dermatology (ICMiD), Hospital Clínic, 09036 Barcelona, Spain and 9Division of Pediatrics, School of Medicine and Health Sciences, University of Barcelona, 08036 Barcelona, Spain *To whom correspondence should be addressed at: José M. Cuezva, Centro de Biología Molecular “Severo Ochoa,” Universidad Autónoma de Madrid, Nicolás Cabrera, 1, Cantoblanco, 28049 Madrid, Spain. Tel: +34 911964618; Fax: +34 911964420; Email: jmcuezv[email protected] Abstract Charcot–Marie–Tooth (CMT) disease is a neuropathy that lacks effective therapy. CMT patients show degeneration of peripheral nerves, leading to muscle weakness and loss of proprioception. Loss of mitochondrial oxidative phosphorylation proteins and enzymes of the antioxidant response accompany degeneration of nerves in skin biopsies of CMT patients. Herein, we followed a drug-repurposing approach to find drugs in a Food and Drug Administration-approved library that could prevent development of CMT disease in the Gdap1-null mouse model. We found that the antibiotic florfenicol is a mitochondrial uncoupler that prevents the production of reactive oxygen species and activates respiration in human GDAP1-knockdown neuroblastoma cells and in dorsal root ganglion neurons of Gdap1-null mice. Treatment of CMT-affected Gdap1-null mice with florfenicol has no beneficial effect in the course of the disease. However, administration of florfenicol, or the antioxidant MitoQ, to pre-symptomatic GDAP1-null mice prevented weight gain and ameliorated the motor coordination deficiencies that developed in the Gdap1-null mice. Interestingly, both florfenicol and MitoQ halted the decay in mitochondrial and redox proteins in sciatic nerves of Gdap1-null mice, supporting that oxidative damage is implicated in 2442 Human Molecular Genetics, 2021, Vol. 30, No. 24 the etiology of the neuropathy. These findings support the development of clinical trials for translation of these drugs for treatment of CMT patients. Graphical Abstract Introduction Drug repurposing is a strategy to overcome the millionaire costs and decade-long tasks of drug discovery needed to reach a safe treatment for the patients (1). This alternative is especially relevant in the case of rare diseases (RDs) where pharmaceutical investments are scant. Charcot–Marie–Tooth (CMT) disease is a RD and the most common hereditary condition of the peripheral nervous system with an estimated overall population prevalence of 10–28/100 000 in Europe (2). CMT is a heterogeneous genetic disease, which onsets usually in the first two decades of life (3). The symptoms include motor and sensory nerve deficits, loss of proprioception and pinprick sensation and are characterized by distal muscle weakness and atrophy (3,4). Despite the efforts to characterize and treat the disease, CMT lacks an effective drug or advanced therapy to treat the patients (5). More than 90 genes have been linked to CMT disease and related neuropathies (6). One of them encodes the gangliosideinduced differentiation-associated protein 1 (GDAP1) (7,8). Mutations in GDAP1 gene cause axonal CMT with both recessive (ARCMT2K) (7) and dominant (CMT2K) inheritance (9), and recessive demyelinating CMT4A (10). GDAP1 is an atypical glutathione S-transferase (GST) (11) of the outer mitochondrial membrane (12), which is also located in both the mitochondria-associated membranes (MAMs) (13) and the mitochondria–lysosome membrane contact sites (14). Mutations in GDAP1 have been associated with abnormal changes in mitochondrial morphology and dynamics (13,15) and with a decreased Ca2+entry through the store-operated calcium entry (SOCE), which leads to a failure in the stimulation of mitochondrial respiration and the inhibition of mitochondrial ATP production (15,16). The lack of GDAP1 also induces an inflammatory response in the spinal cord and sciatic nerve (17). Furthermore, GDAP1 participates in the defense against oxidative stress (18). In this context, it has been shown that members of the GDAP1 family are responsive and protect against stress associated with increased levels of oxidized glutathione (19). Likewise, GDAP1 mutations lead to defective mitochondrial complex I activity and oxidative stress (20,21). Consistent with these observations, loss of mitochondrial and antioxidant proteins have been reported in skin biopsies of CMT patients (22) and in peripheral nerves of the Gdap1 knock-out mouse (Gdap1-null) (15), further suggesting that mitochondrial function and oxidative stress provide potential targets to treat CMT disease. Herein,we have performed a screening of 1018 FDA-approved small compounds searching for activators of mitochondrial respiration to be repurposed for the treatment of CMT disease. We found that the antibiotic florfenicol activated mitochondrial respiration in human neuroblastoma SH-SY5Y and GDAP1-silenced SH-SY5Y (G4) cells, and in dorsal root ganglion (DRG) neurons of the Gdap1-null mice. Mechanistically, we show that florfenicol is a mitochondrial uncoupler that prevents the production of reactive oxygen species (ROS) in mitochondria (mtROS) of SH-SY5Y and GDAP1-silenced SH-SY5Y (G4) cells. To verify the efficacy of florfenicol in a preclinical model of CMT disease, we treated Gdap1-null mice before and after development of CMT symptoms and found that florfenicol is an effective treatment only when administered before development of CMT disease. Moreover, aside from increasing mitochondrial respiration by the uncoupling mtROS-preventing activity of florfenicol as a therapeutic strategy for CMT disease, we also ameliorated mtROS production with the antioxidant MitoQ in pre-symptomatic Gdap1null mice. In vivo, both florfenicol and MitoQ prevented the development of motor deficiencies, the decline in mitochondrial and redox proteins and ameliorated oxidative stress in sciatic nerves of Gdap1-null mice. These results support the use of Human Molecular Genetics, 2021, Vol. 30, No. 24 2443 florfenicol or MitoQ as therapeutic strategies to prevent/minimize the development of symptoms in CMT patients. Results Searching for activators of mitochondrial respiration To identify activators of oxidative phosphorylation (OXPHOS) that could be employed for the treatment of mitochondria l disorders, which are usually ascribed to a deficit of cellular ATP provision, we screened an FDA-approved library of 1018 small compounds that in short-term treatment of 3 h stimulated mitochondrial respiration in HCT116 colon cancer cells (Fig. 1A). We used the oligomycin-sensitive respiration (OSR) as reporter of the drugs’ effect because it represents an estimate of the activity of ATP synthase (23). We identified 139 compounds that enhanced OSR by 20% when compared to cells treated with the vehicle dimethyl sulfoxide (DMSO) (Fig. 1A,Supplementary Material, Table S1). Since our interest at this stage was to find a drug for the treatment of CMT disease, further in-depth investigation of the effect of the activators on cell viability (Fig. 1B)andmitochondrialrespiration (Fig. 1C) was carried out using the neuroblastoma SH-SY5Y cell line. Only 86 compounds that surpassed the threshold of cellular viability after 24-h incubation with the drugs (Fig. 1B, Supplementary Material, Table S1) were selected for further analysis of mitochondrial respiration in the SH-SY5Y cell line (Fig. 1C). Florfenicol (Fig. 1D), a fluorinated synthetic analog of thiamphenicol with bacteriostatic activity; methylthiouracil (Fig. 1D), an antithyroid preparation; and isosorbide (Fig. 1D), a heterocyclic compound used to induce the relaxation of smooth muscle fibers in coronary heart disease, were the three drugs that had the strongest stimulatory effect on mitochondrial respiration in the SH-SY5Y cell line (Fig. 1C and E). In fact, treatment of the cells with any of these compounds significantly increased basal, OSR and maximal respiration of the cells (Fig. 1E), showing florfenicol and methylthiouracil a stronger effect than that of isosorbide (Fig. 1E). Potential drug candidates for CMT disease Mutations in GDAP1 cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of CMT neuropathy (24). Hence, we next studied the effect of the 87 drugs that activated mitochondrial respiration in GDAP1 knockdown SH-SY5Y cells (G4 cells) (Fig. 2A). The results confirmed that florfenicol, methylthiouracil and isosorbide were also the strongest activators from the screen (Fig. 2B). Indeed, 24-h incubation with the drugs significantly increased basal,OSR and maximal respiration of the GDAP1 knockdown cells (Fig. 2C), revealing marginal differences in potency among the three drugs (Fig. 2C). Postnatal dorsal root ganglia (DRG) neurons from wild-type (WT) and Gdap1-null mice, a mouse model of axonal recessive form of the GDAP1-related CMT (15), were incubated for 24 h with the drugs (Fig. 2D). Only florfenicol stimulated significantly basal, OSR and maximal respiration of DRG neurons from Gdap1null mice when compared to its effect in DRG neurons of WT mice (Fig. 2D). In fact, the effect of methylthiouracil (Fig. 2E)and isosorbide (Fig. 2F) in the respiratory profiles of DRG neurons from Gdap1-null mice was marginal. These findings suggested that florfenicol could provide a potential drug for the treatment of CMT disease. Florfenicol is a potent mitochondrial uncoupler that prevents the generation of ROS Florfenicol is a highly lipophilic broad-spectrum antibiotic used in veterinary medicine with antibacterial effects in both Gram-negative and Gram-positive bacteria because it binds the prokaryotic 50S ribosomal subunit, leading to the inhibition of protein synthesis (25). Florfenicol also binds the mitoribosome and inhibits mitochondrial protein synthesis with concomitant mitochondrial dysfunction at higher concentrations of the drug (70–100 μm) (26). At first sight, it seems unexpected that a low dose of an inhibitor of mitochondrial protein synthesis could result in enhanced cellular respiration. Hence, to explain its positive effect in cellular respiration, we focused on the potential uncoupling effect of the drug because of its lipophilic nature. Indeed, incubation of cells with 1 μm florfenicol promoted a sharp decline in mitochondrial membrane potential (m) in both SH-SY5Y WT and G4 cell lines, similarly as when the cells were incubated with the classic mitochondrial uncoupler dinitrophenol (DNP) (Fig. 3A). Interestingly, m in G4 cells was significantly augmented when compared to the parental SHSY5Y WT cells (Fig. 3A). As a result, G4 cells produced more superoxide radical in mitochondria (mtROS), as assessed with the MitoSOX probe than SH-SY5Y WT cells (Fig. 3B). Consistent with the uncoupling effect of florfenicol (Fig. 3A), the drug significantly diminished the production of mtROS in both SH-SY5Y WT and G4 cells (Fig. 3B), to a comparable level of the mitochondrial antioxidant MitoQ (Fig. 3B). Moreover, in contrast with the positive effect observed with 1 μm florfenicol in mitochondrial respiration (Figs 1E and 2C and D), incubation of the cells with 100 μm florfenicol promoted a sharp inhibition of mitochondrial respiration (Supplementary Material, Fig. S1A and B). These results further suggested that the drug effect on the respiratory function of the organelle is independent from its effect on the synthesis of mitochondrial encoded proteins.Overall, these findings thus support that the enhanced mitochondrial respiration promoted by florfenicol stems from its activity as an uncoupling agent and emphasize the relevance that oxidative stress plays in the pathophysiology of CMT disease in agreement with previous reports (20,21). Florfenicol does not improve motor coordination of CMT-affected GDAP1-null mice Our first approach to investigate the potency of florfenicol as a repurposed drug for the in vivo treatment of CMT disease was to verify its effect in Gdap1-null mice that have developed the axonal recessive form of CMT disease (Supplementary Material, Fig. S2A)(15).To this aim,7-month-old WT and Gdap1-null mice were tested for motor and coordination behavior by rotarod test (Supplementary Material, Fig. S2B). Mutant mice had significantly reduced latency to fall in the test when compared to WT mice (Supplementary Material, Fig. S2B), in agreement with previous findings (15). After, Gdap1-null mice were distributed into two groups and treated (FLORF, 0.84 mg/ml) or not (KO) with florfenicol, which was administered in the drinking water for 4 months (Supplementary Material, Fig. S2A). A group of WT mice was also maintained for comparison. After ending drug treatment, motor and coordination behavior was monitored by rotarod test (Supplementary Material, Fig. S2C). No significant differences were observed between non-treated and florfenicoltreated Gdap1-null mice (Supplementary Material, Fig. S2C). Both groups showed significantly reduced latency to fall in 2444 Human Molecular Genetics, 2021, Vol. 30, No. 24 Figure 1. Florfenicol, methylthiouracil and isosorbide activate mitochondrial respiration of SH-SY5Y neuroblastoma cells. Cells were incubated for 24 h with 1 μm solution of the indicated drugs. (A) One hundred and thirty-nine compounds of a 1018 FDA-approved drug library activate (>20%) OSR of HCT116 colon cancer cells in a 1 μm, 3 h short-term treatment. (B) Viability of SH-SY5Y neuroblastoma cancer cells a 1 μm, 24 h treatment. Eighty-six compounds that had no significant effect on viability (≥75%; in yellow) out of the 139 activators identified were selected for further study. (C) Heat map of the respiration parameters of SH-SY5Y cells treated 24h with 1 μm solution of the 86 selected compounds. The map indicates the OSR, basal (BAS) and maximum (MAX) respiration. The calculated z-score is indicated. (D) Molecular formula of top three mitochondrial activators: florfenicol (blue), methylthiouracil (red) and isosorbide (green). (E) SH-SY5Y cells were treated 1 μm, during 24 h with florfenicol (blue lines and bars), methylthiouracil (red lines and bars) or isosorbide (green lines and bars) or left untreated (black lines and bars) and the respiratory profiles (upper panel) recorded in a Seahorse analyzer. The histograms (bottom panel) show the basal, OSR and maximum oxygen consumption rates (OCR). OL, oligomycin; ROT, rotenone; ANT, antimycin A. Bars indicate the mean ±SEM of three biological replicates. ∗P<0.05 when compared to CRL by Student’s t-test. the rotarod test when compared to age-matched WT mice (Supplementary Material, Fig. S2C). These results most likely indicate that florfenicol is not able to improve CMT symptoms after developing the disease. Human Molecular Genetics, 2021, Vol. 30, No. 24 2445 Figure 2. Florfenicol, methylthiouracil and isosorbide activate mitochondrial respiration of GDAP1-knockdown SH-SY5Y cells and Gdap1−/−DRG neurons. (A) Representative western blot of the expression of GDAP1 in WT SH-SY5Y (WT) and GDAP1-knockdown SH-SY5Y (G4) cells. Tubulin is shown as loading control. (B) Heat map of the respiratory parameters of GDAP1-knockdown G4 cells 24 h-treated with 1 μm solution of the 86 compounds. OSR, basal (BAS) and maximum (MAX) respiration. The calculated z-score is indicated. (C)GDAP1-knockdown G4 cells were treated with 1 μm, during 24 h, florfenicol (blue lines and bars), methylthiouracil (red lines and bars) or isosorbide (green lines and bars) or left untreated (black lines and bars) and the respiratory profiles (upper panel) recorded in a Seahorse analyzer. The histograms (bottom panel) show the basal, OSR and maximum oxygen consumption rates (OCR). OL, oligomycin; ROT, rotenone; ANT, antimycin A. (D)Respiratory profiles (upper panel) and parameters (bottom panel) of DRG neurons from Gdap1−/−mice (blue lines and bars) over DRG neurons of WT mice (black lines and bars) both 24 h-treated with 1 μm florfenicol. (E,F) Respiratory profiles (upper panel) and parameters (bottom panel) of DRG neurons of Gdap1−/−mice were 24 h-treated with 1 μm methylthiouracil (E, red lines and bars) or 1 μm isosorbide (F, green lines and bars) or left untreated (E, F, black lines and bars). Bars indicate the mean ±SEM of three biological replicates. ∗P<0.05 when compared to the respective CRL by Student’s t-test. 2446 Human Molecular Genetics, 2021, Vol. 30, No. 24 Figure 3. Florfenicol is a mitochondrial uncoupler that prevents the generation of mtROS. (A)Them was assessed in TMRM+-stained SH-SY5Y WT and G4 cells treated during 24 h with 1 μm florfenicol (gray bar) or left untreated (black bar). The mitochondrial uncoupler DNP 0.5 mm (red bar) was added to collapse m. (B) Mitochondrial ROS production was assessed using 5 μm MitoSOX in SHSY5Y WT and G4 cells treated during 24 h with 1 μm florfenicol (gray bar), 20 nm MitoQ (yellow bar) or left untreated (black bar). Bars indicate the mean ±SEM of three biological replicates. ∗∗P<0.01 and ##P<0.01 when compared to CRL and WT, respectively, by Student’s t-test. See also Supplementary Material, Fig. S1. Florfenicol and MitoQ prevent the development of CMT symptoms in Gdap1-null mice Despite the discouraging findings described above, we reasoned that perhaps florfenicol might prevent or delay the appearance of CMT symptoms. Therefore, florfenicol was administered to Gdap1-null mice from the time of weaning and up to 10 months of age (Fig. 4A). In this experiment, we also included a group of non-treated WT and Gdap1-null mice (Fig. 4A). The reduction of epidermal nerve density is a common neuropathic abnormality observed in skin biopsies of CMT patients (27)thatis accompanied by loss of the mitochondrial proteins involved in OXPHOS and scavenging ROS (22). Not surprisingly, fibroblasts of patients with GDAP1 dominant mutations show enhanced ROS production (21), as we have observed in knockdown SHSY5Y cells (Fig. 3B). Moreover, the peripheral nervous system of Gdap1-null mice has evidence of oxidative stress (19). Therefore, to investigate the potential implication of ROS in the etiology of CMT, we decided to include an additional group of Gdap1-null mice treated with the mitochondrial-targeted antioxidant MitoQ (Fig. 4A) that ameliorates mtROS production (Fig. 3B)without affecting mitochondrial respiration neither in SH-SY5Y WT nor G4 cells (Supplementary Material, Fig. S1A and B). At 7 months of age, we noted that Gdap1-null mice showed a significant increase in body weight when compared to WT (Supplementary Material, Fig. S3A). In contrast, both florfenicol and MitoQ prevented the increase in body weight of Gdap1-null mice (Supplementary Material, Fig. S3A). Motor coordination of the four groups of mice under study, as assessed with the rotarod at midterm of the experiment (7 months),revealed no significant differences between them (Supplementary Material, Fig. S3B). At 10 months of age, non-treated Gdap1-null mice also showed a relevant increase in body weight when compared to WT mice (Fig. 4B). Both florfenicol and MitoQ treatments significantly diminished the body weight gain of Gdap1-null mice (Fig. 4B). GDAP1-related CMT promotes weakness and wasting of feet and hands leading to pronounced disability of the patients. Hind-limb clasping is a marker of disease progression in a number of mouse models of neurodegeneration (28). Consistent with previous results (15), we observed abnormal hind-limb clasping reflex in non-treated Gdap1-null mice, as assessed by the angle formed between the right hind limb and the body axis when compared to WT mice (Fig. 4C). Remarkably, treatment of mice with florfenicol or MitoQ significantly improved the hind-limb clasping reflex of the animals (Fig. 4C),suggesting that both treatments improved CMT symptoms. Determination of the forelimbs muscle strength and maximum strength performed by the forelimbs were assessed by the hanging (Fig. 4D;Supplementary Material, Videos S1 and S2)and grip force (Fig. 4E) tests, respectively. Both the longest hanging time (Fig. 4D;Supplementary Material, Videos S1 and S2)and minimal holding impulse (Supplementary Material, Fig. S3C) were significantly reduced in non-treated Gdap1-null when compared to WT mice. Treatment of mice with florfenicol or MitoQ significantly improved the muscle strength of the forelimbs (Fig. 4D;Supplementary Material, Fig. S3C and Supplementary Material, Videos S1 and S2). Similar findings were obtained in the grip force test (Fig. 4E), supporting that both florfenicol and MitoQ treatment prevented the wasting of muscles in the forelimbs of Gdap1-null mice. The muscle strength of the four limbs was determined by the ability of mice to oppose their weight making use of the four limbs (Fig. 4F;Supplementary Material, Videos S3 and S4). Both the longest hanging time (Fig. 4F;Supplementary Material, Videos S3 and S4) and minimal holding impulse (Supplementary Material, Fig. S3D) were significantly reduced in non-treated Gdap1-null when compared to WT mice. Treatment of mice with florfenicol or MitoQ significantly improved the muscle strength of the four limbs (Fig. 4F;Supplementary Material, Fig. S3C and Supplementary Material, Videos S3 and S4), further supporting that both treatments prevented muscle wasting of the limbs in Gdap1-null mice. Finally, motor coordination behavior was assessed by rotarod test (Fig. 4G). Consistent with the results of previous tests, the latency to fall was significantly reduced in Gdap1null mice (Fig. 4G). Remarkably, treatment of Gdap1-null mice with either florfenicol or MitoQ abolished these differences Human Molecular Genetics, 2021, Vol. 30, No. 24 2447 Figure 4. Florfenicol and MitoQ prevent motor function disabilities of Gdap1−/−mice. (A) Graphical scheme showing the course of mice treatment. After weaning (1 month), florfenicol (0.84 mg/ml) and MitoQ (0.14 mg/ml) were administered in the drinking water of mice. Motor function tests were run at 7 and 10 months of age. (B–G) WT mice (n= 9, black line bar and dots), untreated Gdap1−/−mice (KO, n=10, red line bar and dots), florfenicol-treated Gdap1−/−mice (FLORF, n= 9, gray line bar and dots) and MitoQ-treated Gdap1−/−mice (MITOQ, n=9, yellow line bar and dots). (B) Representative images of mice at 10 months of age. Histograms show the quantification of the fold change in mice weight during the treatments. (C) Representative images of 10-month-old mice suspended by the tail. WT, florfenicoland MitoQ-treated Gdap1−/−mice show the characteristic response trying to escape by splaying the hind limbs away from the trunk of its body. In contrast, hind limbs of non-treated Gdap1−/−mice are held closed to the trunk in an abnormal dystonic posture. Histograms show the quantification of the angle form by the right hind limb and the body axis. (D) Representative image of two limbs hanging test. Histograms show the quantification of the longest time that the mice exhibited sustained limb tension. (E) Representative image of grip force test. Histograms show the quantification of mice strength (g) over body weight (g). (F)Representativeimageoffour limbs hanging test. Histograms show the quantification of the longest time that the mice exhibited sustained limb tension. (G) Representative image of rotarod test. The graph shows the quantification of the mice latency to fall (s) at 4, 8, 12, 16, 20, 24, 28 and 32 r.p.m. of speed. Bars indicate the mean±SEM of the nabove indicated. ∗∗P<0.01 when compared to WT mice and ##P<0.01 when compared to KO mice by Student’s t-test. See also Supplementary Material Figs S2 and S3. 2448 Human Molecular Genetics, 2021, Vol. 30, No. 24 (Fig. 4G), strongly supporting that florfenicol or mitochondrial ROS scavenging from early life prevents the appearance of the deleterious muscle wasting phenotype of the GDAP1-CMT mouse model. Florfenicol and MitoQ prevent deterioration of mitochondrial and redox proteomes in sciatic nerves of Gdap1-null mice We have already reported that electrophysiological analysis of the nerves in 5-month Gdap1-null mice showed a significant reduction of compound muscle action potential (CMAP) amplitude obtained for distal (at the ankle) and proximal (at the hip) stimulation, indicative for an axonal neuropathy (15). In morphological studies, we did not detect any significant reduction in the axonal number correlating with the observed reduction in CMAP amplitude in 5-month-old mice (15). We reasoned that prevention of CMT symptoms was enough evidence of the electrophysiological functionality of peripheral nerves. However, we carried out their analysis at the molecular level because sciatic nerves of Gdap1-null mice showed a reduction of proteins of energy metabolism and of the antioxidant response (15), in agreement with the partial loss of these proteins in skin biopsies of CMT1A patients as a result of the injury of epidermal nerve terminals (22). Hence, we next explored the effect of florfenicol and MitoQ in the expression of selected proteins of energy metabolism and of the oxidative stress response in tissue extracts derived from sciatic nerves, skeletal muscle, liver and cerebellum of Gdap1-null mice (Figs 5 and 6;Supplementary Material, Figs S5–S7). For this purpose, we used a quantitative high-throughput reverse phase protein array (RPPA) approach (29,30)(Supplementary Material, Fig. S4). In agreement with previous observations (15), the peripheral nerves of GDAP1-null mice showed significant loss of the enzymes involved in the oxidation of glucose and mitochondrial respiration (LDHA, PDH, IDH1 and CORE2) and of the antioxidant response (GR, SOD1, catalase and SOD2) when compared to WT mice (Fig. 5). Consistent with this last observation, the oxidative modification of cellular proteins by MDA and 4HNE was significantly increased in Gdap1-null mice (Fig. 5). Other mitochondrial proteins showed no relevant changes (Fig. 5). Remarkably, both florfenicol and MitoQ treatments significantly prevented and/or increased the expression of a large number of proteins of energy metabolism (ENO1, LDHA, PDH, IDH, CORE2, βF1) and of the antioxidant response (GR, catalase and SOD2) in the sciatic nerves of GDAP1-null mice (Fig. 5). Both treatments partially ameliorated oxidative damage of nerve proteins of the Gdap1-null mice, although only MitoQ reached statistical significance in 4HNE modifications (Fig. 5). These results strongly support at the molecular level that the bioenergetic and redox compromise that onsets in Gdap1-null mice during development could be prevented by treatment with the antibiotic florfenicol or the mitochondrial-targeted antioxidant MitoQ. It should be noted that except for mitofusin 2 (MFN2) in muscle (Fig. 6A;Supplementary Material, Fig. S5), ENO1 and SOD1 in liver (Fig. 6A;Supplementary Material, Fig. S6) and none in cerebellum (Fig. 6A;Supplementary Material, Fig. S7), ablation of the Gdap1 gene had marginal effect in the tissue content of many of the proteins investigated. Likewise, both florfenicol and MitoQ had negligible impact on the content of the proteins in these tissues (Fig. 6A;Supplementary Material, Figs S5–S7). It should be noted the reduction of SOD2 observed in the liver and cerebellum of Gdap1-null mice in response to the antioxidant MitoQ (Fig. 6A;Supplementary Material, Figs S5–S7). The quantification of additional mitochondrial proteins in sciatic nerves, such as NADH9 and COXIV of the respiratory chain, and of mitochondrial dynamics (MFN1) and structure (HSP60) further confirmed the specific deleterious effect of the ablation of the Gdap1 gene in proteins involved in mitochondrial energy production when compared to proteins involved in other mitochondrial functions (Fig. 6B). In addition, these findings, further confirmed the positive effect of both treatments in preventing the loss of the enzymes of energy metabolism in Gdap1-null mice (Fig. 6B). Overall, the findings indicate that both florfenicol and MitoQ are two drugs that prevent the development of CMT symptoms by ameliorating the dysfunction of energy metabolism and redox homeostasis in peripheral nerves of a mouse model of CMT disease. Discussion Multiple genetic alterations result in the degeneration of the peripheral nerves and death of neurons that lead to muscle weakness and atrophy in CMT patients (27,31). These genetic alterations are expressed at the protein level by the loss of mitochondrial enzymes involved in OXPHOS and ROS scavenging (22). Despite efforts to find targets and drugs for the treatment of CMT disease to date, and although different mouse models of the disease are already available (15,32), no clinical benefits have been advanced limiting current treatments to supportive care of the patients (5). Since the duplication of the PMP22 gene is causative for the most prevalent form CMT1A (33), most of the strategies developed look for the downregulation of PMP22 gene. In this context, PXT3003 has been shown to be safe and tolerable in a phase II trial in CMT1A patients (34) (NCT01401257). Potential treatments for other subtypes of CMT disease are scant, and among them, HDCA6 inhibitors have shown correction of the axonal transport deficits in CMT2 (HSPB1) (35)and CMT2D (36) mouse models. Moreover, agonists of MFN2 promote mitochondrial fusion and ameliorate mitochondrial trafficking deficits in cultured mouse neurons containing Mfn2R94G and Mfn2T105M mutations (37). Therefore, treatments that target molecular pathways to stop the onset and progression of the disease are imperative to overcome CMT symptoms in patients. Drug repurposing is a strategy that could accelerate the translation of active molecules for the treatment of CMT disease. Since mitochondrial dysfunction and oxidative stress are two hallmarks of CMT disease (22), which are also reproduced in the Gdap1-null mouse model (15), we have screened a library of small compounds to search for activators of mitochondrial respiration to be tested in vivo in the preclinical model of the disease (15). We have discovered that the antibiotic florfenicol is a mitochondrial uncoupler that prevents mtROS generation and increases mitochondrial respiration in GDAP1-silenced SH-SY5Y cells, and DRG neurons of Gdap1-null mice. When florfenicol is administered from early life to Gdap1-null mice, it prevents the development of motor deficiencies and other symptoms of the CMT neuropathy. Florfenicol also prevents the loss of mitochondrial proteins involved in metabolic and redox functions of peripheral sciatic nerves. Interestingly, treatment of Gdap1-null mice with the mitochondrial-targeted antioxidant MitoQ mimicked at the functional and molecular levels the beneficial effects of florfenicol, strongly supporting the implication of oxidative damage of metabolic proteins in the etiology of CMT neuropathy. Overall, we advance two drugs that by either ameliorating (florfenicol) or scavenging (MitoQ) mtROS production prevent the development of CMT disease in a preclinical mouse model. Human Molecular Genetics, 2021, Vol. 30, No. 24 2449 Figure 5. Florfenicol and MitoQ prevent the metabolic and redox deficiencies observed in sciatic nerves of GDAP1 −/−mice. Sciatic nerve extracts derived from WT mice (black boxed) (n= 9), non-treated Gdap1−/−mice (KO, red boxed) (n= 10) and Gdap1−/−-treated mice with florfenicol (FLORF, grey boxed) (n= 9) or MitoQ (MITOQ, yellow boxed) (n= 9) were spotted onto the arrays and processed. The arrays were developed with the indicated primary antibodies and the quantity of protein in the extracts expressed in arbitrary units/ng of protein using as standard the linear plot of the C2C12 cell line (Supplementary Material, Fig. S3). Box plots represent 25th–75th percentiles with the median value in the middle line and with all data represented from minimal to maximal values of the expression level of the proteins. Enzymes of glucose metabolism (ENO1, LDHA, PDH), TCA cycle (IDH1, MDH2), fatty acid oxidation (HADHA), OXPHOS (CORE2, β-F1-ATPase), mitochondrial dynamics (MFN2, OPA1), redox homeostasis (G6PDH, GR, SOD1, Catalase, SOD2) and oxidative stress (MDA, 4HNE) are presented. Bars indicate the mean ±SEM of three replicates of thenaboveindicated.∗and ∗∗ indicates a P<0.05 and P<0.001 when compared to WT mice, respectively; # and ##, P<0.05 and P<0.001 when compared to Gdap1−/− mice, respectively, by Student’s t-test. See also Supplementary Material, Fig. S3.