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,*,¶ 1 Departamento de Biología Molecular, Universidad Autónoma de Madrid (UAM), 28049 Madrid, Spain, 2 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), 28049 Madrid, Spain, 3 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, 28029 Madrid, Spain, 4 Instituto de Investigación Hospital 12 de Octubre, 28041 Madrid, Spain, 5 Laboratorio de Neurogenética y Medicina Molecular - IPER, Institut de Recerca Sant Joan de Déu, 08950 Barcelona, Spain, 6 Medical Research Council Mitochondrial Biology Unit, Wellcome Trust/MRC Building, University of Cambridge, Cambridge CB2 0XY, UK, 7 Department of Genetic and Molecular Medicine - IPER, Hospital Sant Joan de Déu, 08950 Barcelona, Spain, 8 Clinic Institute of Medicine and Dermatology (ICMiD), Hospital Clínic, 09036 Barcelona, Spain and 9 Division 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: jmcuezva@cbm.csic.es 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 f lorfenicol 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 f lorfenicol has no beneficial effect in the course of the disease. However, administration of f lorfenicol, 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 f lorfenicol and MitoQ halted the decay in mitochondrial and redox proteins in sciatic nerves of Gdap1-null mice, supporting that oxidative damage is implicated in † 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: journals.permissions@oup.com. 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 journals.permissions@oup.com. 2441 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 that members of the GDAP1 family are responsive and pro- and decade-long tasks of drug discovery needed to reach a tect against stress associated with increased levels of oxidized safe treatment for the patients (1). This alternative is especially glutathione (19). Likewise, GDAP1 mutations lead to defective relevant in the case of rare diseases (RDs) where pharmaceutical mitochondrial complex I activity and oxidative stress (20,21). investments are scant. Charcot–Marie–Tooth (CMT) disease is a Consistent with these observations, loss of mitochondrial and RD and the most common hereditary condition of the peripheral antioxidant proteins have been reported in skin biopsies of CMT nervous system with an estimated overall population prevalence patients (22) and in peripheral nerves of the Gdap1 knock-out of 10–28/100 000 in Europe (2). CMT is a heterogeneous genetic mouse (Gdap1-null) (15), further suggesting that mitochondrial disease, which onsets usually in the first two decades of life (3). function and oxidative stress provide potential targets to treat The symptoms include motor and sensory nerve deficits, loss of CMT disease. proprioception and pinprick sensation and are characterized by Herein, we have performed a screening of 1018 FDA-approved distal muscle weakness and atrophy (3,4). Despite the efforts to small compounds searching for activators of mitochondrial res- characterize and treat the disease, CMT lacks an effective drug piration to be repurposed for the treatment of CMT disease. We or advanced therapy to treat the patients (5). found that the antibiotic florfenicol activated mitochondrial res- More than 90 genes have been linked to CMT disease and piration in human neuroblastoma SH-SY5Y and GDAP1-silenced related neuropathies (6). One of them encodes the ganglioside- SH-SY5Y (G4) cells, and in dorsal root ganglion (DRG) neurons of induced differentiation-associated protein 1 (GDAP1) (7,8). Muta- the Gdap1-null mice. Mechanistically, we show that florfenicol is tions in GDAP1 gene cause axonal CMT with both recessive (AR- a mitochondrial uncoupler that prevents the production of reac- CMT2K) (7) and dominant (CMT2K) inheritance (9), and recessive tive oxygen species (ROS) in mitochondria (mtROS) of SH-SY5Y demyelinating CMT4A (10). GDAP1 is an atypical glutathione and GDAP1-silenced SH-SY5Y (G4) cells. To verify the efficacy S-transferase (GST) (11) of the outer mitochondrial membrane of florfenicol in a preclinical model of CMT disease, we treated (12), which is also located in both the mitochondria-associated Gdap1-null mice before and after development of CMT symp- membranes (MAMs) (13) and the mitochondria–lysosome mem- toms and found that florfenicol is an effective treatment only brane contact sites (14). Mutations in GDAP1 have been associ- when administered before development of CMT disease. More- ated with abnormal changes in mitochondrial morphology and over, aside from increasing mitochondrial respiration by the dynamics (13,15) and with a decreased Ca2+ entry through the uncoupling mtROS-preventing activity of florfenicol as a thera- store-operated calcium entry (SOCE), which leads to a failure in peutic strategy for CMT disease, we also ameliorated mtROS pro- the stimulation of mitochondrial respiration and the inhibition duction with the antioxidant MitoQ in pre-symptomatic Gdap1- of mitochondrial ATP production (15,16). The lack of GDAP1 also null mice. In vivo, both florfenicol and MitoQ prevented the induces an inflammatory response in the spinal cord and sci- development of motor deficiencies, the decline in mitochondrial atic nerve (17). Furthermore, GDAP1 participates in the defense and redox proteins and ameliorated oxidative stress in sciatic against oxidative stress (18). In this context, it has been shown 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/mini- Florfenicol is a potent mitochondrial uncoupler mize the development of symptoms in CMT patients. that prevents the generation of ROS Florfenicol is a highly lipophilic broad-spectrum antibiotic used in veterinary medicine with antibacterial effects in both Results Gram-negative and Gram-positive bacteria because it binds the Searching for activators of mitochondrial respiration prokaryotic 50S ribosomal subunit, leading to the inhibition of To identify activators of oxidative phosphorylation (OXPHOS) protein synthesis (25). Florfenicol also binds the mitoribosome that could be employed for the treatment of mitochondria and inhibits mitochondrial protein synthesis with concomitant l disorders, which are usually ascribed to a deficit of cellu- mitochondrial dysfunction at higher concentrations of the drug lar ATP provision, we screened an FDA-approved library of (70–100 μm) (26). At first sight, it seems unexpected that a low 1018 small compounds that in short-term treatment of 3 h dose of an inhibitor of mitochondrial protein synthesis could stimulated mitochondrial respiration in HCT116 colon cancer result in enhanced cellular respiration. Hence, to explain its cells (Fig. 1A). We used the oligomycin-sensitive respiration positive effect in cellular respiration, we focused on the potential (OSR) as reporter of the drugs’ effect because it represents an uncoupling effect of the drug because of its lipophilic nature. estimate of the activity of ATP synthase (23). We identified Indeed, incubation of cells with 1 μm florfenicol promoted a 139 compounds that enhanced OSR by 20% when compared sharp decline in mitochondrial membrane potential (m) to cells treated with the vehicle dimethyl sulfoxide (DMSO) in both SH-SY5Y WT and G4 cell lines, similarly as when the (Fig. 1A, Supplementary Material, Table S1). Since our interest cells were incubated with the classic mitochondrial uncoupler at this stage was to find a drug for the treatment of CMT dinitrophenol (DNP) (Fig. 3A). Interestingly, m in G4 cells was disease, further in-depth investigation of the effect of the significantly augmented when compared to the parental SH- activators on cell viability (Fig. 1B) and mitochondrial respiration SY5Y WT cells (Fig. 3A). As a result, G4 cells produced more (Fig. 1C) was carried out using the neuroblastoma SH-SY5Y superoxide radical in mitochondria (mtROS), as assessed with cell line. Only 86 compounds that surpassed the threshold of the MitoSOX probe than SH-SY5Y WT cells (Fig. 3B). Consistent cellular viability after 24-h incubation with the drugs (Fig. 1B, with the uncoupling effect of florfenicol (Fig. 3A), the drug sig- Supplementary Material, Table S1) were selected for further nificantly diminished the production of mtROS in both SH-SY5Y analysis of mitochondrial respiration in the SH-SY5Y cell line WT and G4 cells (Fig. 3B), to a comparable level of the mitochon- (Fig. 1C). drial antioxidant MitoQ (Fig. 3B). Moreover, in contrast with the Florfenicol (Fig. 1D), a fluorinated synthetic analog of positive effect observed with 1 μm florfenicol in mitochondrial thiamphenicol with bacteriostatic activity; methylthiouracil respiration (Figs 1E and 2C and D), incubation of the cells with (Fig. 1D), an antithyroid preparation; and isosorbide (Fig. 1D), a 100 μm florfenicol promoted a sharp inhibition of mitochondrial heterocyclic compound used to induce the relaxation of smooth respiration (Supplementary Material, Fig. S1A and B). These muscle fibers in coronary heart disease, were the three drugs results further suggested that the drug effect on the respiratory that had the strongest stimulatory effect on mitochondrial function of the organelle is independent from its effect on the respiration in the SH-SY5Y cell line (Fig. 1C and E). In fact, synthesis of mitochondrial encoded proteins. Overall, these find- treatment of the cells with any of these compounds significantly ings thus support that the enhanced mitochondrial respiration increased basal, OSR and maximal respiration of the cells promoted by florfenicol stems from its activity as an uncoupling (Fig. 1E), showing florfenicol and methylthiouracil a stronger agent and emphasize the relevance that oxidative stress plays in effect than that of isosorbide (Fig. 1E). the pathophysiology of CMT disease in agreement with previous reports (20,21). Potential drug candidates for CMT disease Florfenicol does not improve motor coordination Mutations in GDAP1 cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of CMT-affected GDAP1-null mice of CMT neuropathy (24). Hence, we next studied the effect of Our first approach to investigate the potency of florfenicol the 87 drugs that activated mitochondrial respiration in GDAP1 as a repurposed drug for the in vivo treatment of CMT knockdown SH-SY5Y cells (G4 cells) (Fig. 2A). The results con- disease was to verify its effect in Gdap1-null mice that firmed that florfenicol, methylthiouracil and isosorbide were have developed the axonal recessive form of CMT disease also the strongest activators from the screen (Fig. 2B). Indeed, (Supplementary Material, Fig. S2A) (15). To this aim, 7-month-old 24-h incubation with the drugs significantly increased basal, OSR WT and Gdap1-null mice were tested for motor and coordination and maximal respiration of the GDAP1 knockdown cells (Fig. 2C), behavior by rotarod test (Supplementary Material, Fig. S2B). revealing marginal differences in potency among the three drugs Mutant mice had significantly reduced latency to fall in the (Fig. 2C). test when compared to WT mice (Supplementary Mate- Postnatal dorsal root ganglia (DRG) neurons from wild-type rial, Fig. S2B), in agreement with previous findings (15). (WT) and Gdap1-null mice, a mouse model of axonal recessive After, Gdap1-null mice were distributed into two groups and form of the GDAP1-related CMT (15), were incubated for 24 h treated (FLORF, 0.84 mg/ml) or not (KO) with florfenicol, with the drugs (Fig. 2D). Only florfenicol stimulated significantly which was administered in the drinking water for 4 months basal, OSR and maximal respiration of DRG neurons from Gdap1- (Supplementary Material, Fig. S2A). A group of WT mice was null mice when compared to its effect in DRG neurons of WT also maintained for comparison. After ending drug treatment, mice (Fig. 2D). In fact, the effect of methylthiouracil (Fig. 2E) and motor and coordination behavior was monitored by rotarod isosorbide (Fig. 2F) in the respiratory profiles of DRG neurons test (Supplementary Material, Fig. S2C). No significant differ- from Gdap1-null mice was marginal. These findings suggested ences were observed between non-treated and florfenicol- that florfenicol could provide a potential drug for the treatment treated Gdap1-null mice (Supplementary Material, Fig. S2C). of CMT disease. 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 indicate that florfenicol is not able to improve CMT symptoms (Supplementary Material, Fig. S2C). These results most likely 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 of patients with GDAP1 dominant mutations show enhanced ROS production (21), as we have observed in knockdown SH- SY5Y 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 maxi- mum 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 Figure 3. Florfenicol is a mitochondrial uncoupler that prevents the generation or MitoQ significantly improved the muscle strength of of mtROS. (A) The m was assessed in TMRM+ -stained SH-SY5Y WT and G4 the forelimbs (Fig. 4D; Supplementary Material, Fig. S3C and cells treated during 24 h with 1 μm florfenicol (gray bar) or left untreated (black Supplementary Material, Videos S1 and S2). Similar findings bar). The mitochondrial uncoupler DNP 0.5 mm (red bar) was added to collapse were obtained in the grip force test (Fig. 4E), supporting that m. (B) Mitochondrial ROS production was assessed using 5 μm MitoSOX in SH- both florfenicol and MitoQ treatment prevented the wasting of SY5Y 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 muscles in the forelimbs of Gdap1-null mice. three biological replicates. ∗∗ P < 0.01 and ##P < 0.01 when compared to CRL and The muscle strength of the four limbs was determined by WT, respectively, by Student’s t-test. See also Supplementary Material, Fig. S1. 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 Florfenicol and MitoQ prevent the development Material, Fig. S3D) were significantly reduced in non-treated of CMT symptoms in Gdap1-null mice Gdap1-null when compared to WT mice. Treatment of mice with Despite the discouraging findings described above, we reasoned florfenicol or MitoQ significantly improved the muscle strength that perhaps florfenicol might prevent or delay the appearance of the four limbs (Fig. 4F; Supplementary Material, Fig. S3C and of CMT symptoms. Therefore, florfenicol was administered to Supplementary Material, Videos S3 and S4), further supporting Gdap1-null mice from the time of weaning and up to 10 months that both treatments prevented muscle wasting of the limbs in of age (Fig. 4A). In this experiment, we also included a group Gdap1-null mice. of non-treated WT and Gdap1-null mice (Fig. 4A). The reduction Finally, motor coordination behavior was assessed by of epidermal nerve density is a common neuropathic abnor- rotarod test (Fig. 4G). Consistent with the results of previous mality observed in skin biopsies of CMT patients (27) that is tests, the latency to fall was significantly reduced in Gdap1- accompanied by loss of the mitochondrial proteins involved in null mice (Fig. 4G). Remarkably, treatment of Gdap1-null mice OXPHOS and scavenging ROS (22). Not surprisingly, fibroblasts 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, florfenicol- and 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) Representative image of four 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 n above 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 The quantification of additional mitochondrial proteins in ROS scavenging from early life prevents the appearance of sciatic nerves, such as NADH9 and COXIV of the respiratory the deleterious muscle wasting phenotype of the GDAP1-CMT chain, and of mitochondrial dynamics (MFN1) and structure mouse model. (HSP60) further confirmed the specific deleterious effect of the ablation of the Gdap1 gene in proteins involved in mitochon- drial energy production when compared to proteins involved in other mitochondrial functions (Fig. 6B). In addition, these find- Florfenicol and MitoQ prevent deterioration ings, further confirmed the positive effect of both treatments in of mitochondrial and redox proteomes in sciatic nerves preventing the loss of the enzymes of energy metabolism in of Gdap1-null mice Gdap1-null mice (Fig. 6B). Overall, the findings indicate that both We have already reported that electrophysiological analysis of florfenicol and MitoQ are two drugs that prevent the devel- the nerves in 5-month Gdap1-null mice showed a significant opment of CMT symptoms by ameliorating the dysfunction of reduction of compound muscle action potential (CMAP) energy metabolism and redox homeostasis in peripheral nerves amplitude obtained for distal (at the ankle) and proximal (at of a mouse model of CMT disease. 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 rea- Discussion soned that prevention of CMT symptoms was enough evidence of the electrophysiological functionality of peripheral nerves. Multiple genetic alterations result in the degeneration of the However, we carried out their analysis at the molecular level peripheral nerves and death of neurons that lead to muscle because sciatic nerves of Gdap1-null mice showed a reduction of weakness and atrophy in CMT patients (27,31). These genetic proteins of energy metabolism and of the antioxidant response alterations are expressed at the protein level by the loss of mito- (15), in agreement with the partial loss of these proteins in chondrial enzymes involved in OXPHOS and ROS scavenging skin biopsies of CMT1A patients as a result of the injury of (22). Despite efforts to find targets and drugs for the treatment epidermal nerve terminals (22). Hence, we next explored the of CMT disease to date, and although different mouse models effect of florfenicol and MitoQ in the expression of selected of the disease are already available (15,32), no clinical benefits proteins of energy metabolism and of the oxidative stress have been advanced limiting current treatments to supportive response in tissue extracts derived from sciatic nerves, skeletal care of the patients (5). Since the duplication of the PMP22 gene muscle, liver and cerebellum of Gdap1-null mice (Figs 5 and is causative for the most prevalent form CMT1A (33), most of 6; Supplementary Material, Figs S5–S7). For this purpose, we the strategies developed look for the downregulation of PMP22 used a quantitative high-throughput reverse phase protein gene. In this context, PXT3003 has been shown to be safe and array (RPPA) approach (29,30) (Supplementary Material, Fig. S4). tolerable in a phase II trial in CMT1A patients (34) (NCT01401257). In agreement with previous observations (15), the peripheral Potential treatments for other subtypes of CMT disease are nerves of GDAP1-null mice showed significant loss of the scant, and among them, HDCA6 inhibitors have shown correc- enzymes involved in the oxidation of glucose and mitochondrial tion of the axonal transport deficits in CMT2 (HSPB1) (35) and respiration (LDHA, PDH, IDH1 and CORE2) and of the antioxidant CMT2D (36) mouse models. Moreover, agonists of MFN2 pro- response (GR, SOD1, catalase and SOD2) when compared to mote mitochondrial fusion and ameliorate mitochondrial traf- WT mice (Fig. 5). Consistent with this last observation, the ficking deficits in cultured mouse neurons containing Mfn2R94G oxidative modification of cellular proteins by MDA and 4HNE and Mfn2T105M mutations (37). Therefore, treatments that target was significantly increased in Gdap1-null mice (Fig. 5). Other molecular pathways to stop the onset and progression of the mitochondrial proteins showed no relevant changes (Fig. 5). disease are imperative to overcome CMT symptoms in patients. Remarkably, both florfenicol and MitoQ treatments signif- Drug repurposing is a strategy that could accelerate the trans- icantly prevented and/or increased the expression of a large lation of active molecules for the treatment of CMT disease. number of proteins of energy metabolism (ENO1, LDHA, PDH, Since mitochondrial dysfunction and oxidative stress are two IDH, CORE2, βF1) and of the antioxidant response (GR, catalase hallmarks of CMT disease (22), which are also reproduced in and SOD2) in the sciatic nerves of GDAP1-null mice (Fig. 5). Both the Gdap1-null mouse model (15), we have screened a library treatments partially ameliorated oxidative damage of nerve pro- of small compounds to search for activators of mitochondrial teins of the Gdap1-null mice, although only MitoQ reached sta- respiration to be tested in vivo in the preclinical model of the tistical significance in 4HNE modifications (Fig. 5). These results disease (15). We have discovered that the antibiotic florfenicol is strongly support at the molecular level that the bioenergetic and a mitochondrial uncoupler that prevents mtROS generation and redox compromise that onsets in Gdap1-null mice during devel- increases mitochondrial respiration in GDAP1-silenced SH-SY5Y opment could be prevented by treatment with the antibiotic cells, and DRG neurons of Gdap1-null mice. When florfenicol florfenicol or the mitochondrial-targeted antioxidant MitoQ. is administered from early life to Gdap1-null mice, it prevents It should be noted that except for mitofusin 2 (MFN2) in mus- the development of motor deficiencies and other symptoms of cle (Fig. 6A; Supplementary Material, Fig. S5), ENO1 and SOD1 the CMT neuropathy. Florfenicol also prevents the loss of mito- in liver (Fig. 6A; Supplementary Material, Fig. S6) and none in chondrial proteins involved in metabolic and redox functions of cerebellum (Fig. 6A; Supplementary Material, Fig. S7), ablation of peripheral sciatic nerves. Interestingly, treatment of Gdap1-null the Gdap1 gene had marginal effect in the tissue content of mice with the mitochondrial-targeted antioxidant MitoQ mim- many of the proteins investigated. Likewise, both florfenicol icked at the functional and molecular levels the beneficial effects and MitoQ had negligible impact on the content of the proteins of florfenicol, strongly supporting the implication of oxidative in these tissues (Fig. 6A; Supplementary Material, Figs S5–S7). It damage of metabolic proteins in the etiology of CMT neuropa- should be noted the reduction of SOD2 observed in the liver and thy. Overall, we advance two drugs that by either ameliorating cerebellum of Gdap1-null mice in response to the antioxidant (florfenicol) or scavenging (MitoQ) mtROS production prevent MitoQ (Fig. 6A; Supplementary Material, Figs S5–S7). 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 the n above indicated. ∗ 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. 2450 Human Molecular Genetics, 2021, Vol. 30, No. 24 Figure 6. Muscle, liver and cerebellum of GDAP1 −/− mice show marginal effect in the protein content of metabolic proteins. (A) Scheme summarizing significant changes in protein expression levels [decreased (blue) or increased (red)] in sciatic nerve, muscle, liver and cerebellum of non-treated Gdap1−/− mice (KO) (n = 10) and Gdap1−/− -treated mice with florfenicol (FLORF) (n = 9) or MitoQ (MITOQ) (n = 9) when compared to WT mice (n = 9) as compiled in Fig. 4; Supplementary Material, Figs S4– S6. (B) 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. Additional mitochondrial proteins of OXPHOS (NADH9, COXIV), mitochondrial dynamics (MFN1) and structure (HSP60) are presented. Bars indicate the mean ± SEM of three replicates of the n above indicated. ∗ P < 0.05 when compared to WT mice; #P < 0.05 when compared to Gdap1−/− mice by Student’s t-test. See also Supplementary Material, Figs S4–S7. Human Molecular Genetics, 2021, Vol. 30, No. 24 2451 We support that the two drugs hold great promise to be repur- (42). MitoQ has been shown to decrease blood pressure in old posed for the treatment of these patients. Unfortunately, flor- human subjects (43). Moreover, MitoQ administration reduces fenicol does not revert motor deficiencies when administered oxidative stress and mitochondrial dysfunction in a mouse to already symptomatic CMT mice, which may be due to the model of multiple sclerosis (44) and a phase II clinical trial ROS-mediated progressive accumulation of oxidative damage in multiple sclerosis patients to overcome fatigue is already in neurons. It is likely that the oxidative damage cannot be ongoing (NCT03166800). Since ROS production is a common amended to recover functionality, neither the neurons can be feature in different mitochondrial disorders, including MFN2- replaced because they are post-mitotic cells. In this line, it has related CMT forms, we suggest that florfenicol or MitoQ deserve been suggested that failure of the antioxidant MitoQ in reliev- to be investigated for the treatment of OXPHOS and redox- ing the symptoms of Parkinson’s disease resulted because of related pathologies in appropriate preclinical mouse models the advanced oxidative damage in the patients enrolled in the of these disorders. clinical trial (38). In conclusion, we highlight a drug repurposing strategy to Weight gain is a serious issue in CMT patients because weak- find drugs for the treatment of CMT disease and emphasize the ened muscles make it difficult for them to keep active. In agree- potential clinical relevance of florfenicol and MitoQ for the treat- ment with this, we observed that Gdap1-null mice showed a ment of these patients as positively exemplified at the molecular progressive increase in body weight when compared to WT mice and functional levels in the Gdap1-null mouse model of the dis- due to the accumulation of visceral fat. Interestingly, Gdap1-null ease. We further suggest that several of the plasma metabolites mice treated with florfenicol or MitoQ prevented the body weight previously identified as potential biomarkers of CMT1A disease increase in these animals, presumably because avoiding muscle (22) could help the follow-up of the response to these drugs in weakness allowed the mice to exercise as controls. Indeed, mice future clinical trials to be developed in CMT patients. with a restrained mitochondrial function in skeletal muscle present higher body weight and visceral adipose tissue than WT Materials and Methods mice (39). Of note, weight in these mice can be reestablished by treatment with edaravone, a potent antioxidant and activator Cell lines of mitochondrial respiration (39), supporting the importance of Cells were cultured in an incubator at 37◦ C with a con- preserving mitochondrial and redox function to prevent muscle trolled atmosphere of ambient air 10% CO2 . Human colorectal weakness. carcinoma HCT116 cells were grown in McCoy’s 5A media The doses of florfenicol used to inhibit mitochondrial protein supplemented with 10% fetal bovine serum (FBS). Human synthesis and function (26) are much higher than the ones neuroblastoma WT and GDAP1 knockdown (G4) (13) SH-SY5Y used in this study. Hence, we support that the beneficial effect cells were grown in DMEM-F12 media supplemented with 10% of florfenicol results from its lipophilic nature and uncoupler FBS. For stable control of G4 SH-SY5Y cells, 2 μg/ml puromycin activity that, on the one hand, enhances mitochondrial res- was added to the medium, to maintain the selection. To preserve piration and, on the other, ameliorates mtROS production. In cell lines, aliquots of 5 million cell were gradually frozen (14 h at fact, long-term in vivo treatment with florfenicol prevents the −20◦ C, 24 h at −70◦ C and then they were kept in liquid nitrogen) downregulation of mitochondrial metabolic and redox proteins in 1 ml culture medium with 10% DMSO. The unfreezing in peripheral nerves. Overall, we postulate that the long-term was carried out at 37◦ C immediately diluting them in culture beneficial effect of florfenicol in GDAP1-null mice results from medium. preventing oxidative damage to metabolic and redox proteins in peripheral nerves what results in less deterioration of motor functions. DRG neuronal cultures Deterioration of the cellular and mitochondrial antioxidant DRGs were obtained from newborn (postnatal days 0–2) WT response is a consistent feature observed in different human and Gdap1 knockout (Gdap1−/− ) mice according to (45). Ganglia genetic disorders that affect mitochondrial activity (30). were digested in collagenase (Worthington, USA) and trypsin Consistently, we showed that Gdap1-null mice present a specific (Sigma-Aldrich, St. Louis, MO), dissociated by trituration and deficiency in metabolic and redox proteins in sciatic nerves, plated in DMEM-F12 containing 50 ng/ml NGF (Tebu-Bio, Le- which leads to an increase oxidative damage and development Perray-en-Yvelines, France), 10% FBS and 5 ng/ml of aphidicolin of CMT symptoms. Remarkably, long-term treatment with MitoQ (A.G. Scientific, San Diego, CA). DRG neurons were seeded on prevents the molecular deterioration and oxidative damage poly-l-lysine and laminin (1 μg/ml)-coated (Sigma-Aldrich, St. observed and the development of CMT symptoms in Gdap1- Louis, MO) plates and used for experimentation between 2 and null mice, strongly supporting oxidative damage as a major 4 DIV. contributing factor in the etiology of the disease. MitoQ is a mitochondrial targeted antioxidant that consists of a quinone moiety linked to a triphenylphosphonium by a 10-carbon alkyl Drug library screening chain (40). MitoQ has no toxic effects in long-term treatments The effects of compounds from a 1018 FDA-approved Drug administered to mice in drinking water and is commercialized Library (Selleckchem, Texas, USA) on mitochondrial respiratory as a dietary supplement for humans (41). Hence, we support parameters were determined in XFe96 Seahorse flux analyzer that MitoQ administration early at the stage of diagnosis to CMT (Agilent Technologies, California, USA) as described in (23). In patients might contribute to prevent the loss of mitochondrial primary screenings, the effect was assessed in 35 000 HCT116 and antioxidant proteins observed in peripheral nerves of mildly colon cancer cells after 1 μm short-term incubation (3 h) of the affected CMT1A patients (22). This treatment might contribute drugs on the basal and OSR using 10 mm glucose, 1 mm pyruvate to halt the development of CMT symptoms. In this context, it has and 2 mm glutamine to identify inhibitors and activators of been shown that Coenzyme Q10 treatment—which plays a role mitochondrial respiration. Inhibitors (23) and activators (this in antioxidant responses—partially resolved visual impairment study) were, respectively, defined when the effect of the drugs in hereditary motor sensory neuropathy related to CMT2A promoted a 40% decrease or activated 20% mitochondrial 2452 Human Molecular Genetics, 2021, Vol. 30, No. 24 respiration when compared to the cells incubated only with the each analysis, 10 000 events were recorded. Data were analyzed vehicle. To validate the potential hits in the appropriate cellular in FlowJo software v10.6.2. models, compounds that positively affected respiration were used for secondary screenings in 35 000 cells of SH-SY5Y WT Animals and G4 cell lines. In the secondary screening, the effect of the drugs was assessed in triplicate assays after 1 μm incubation of Mice were housed in the Animal Facility of the CBMSO with a the drugs for 24 h using 10 mm glucose, 1 mm pyruvate and 2 mm 12-h light/12-h dark cycle and temperature of 18–23◦ C with 40– glutamine. The effect on mitochondrial respiration of the three 60% humidity. The Gdap1 knockout (Gdap1−/− ) mice were previ- confirmed hits, florfenicol, isosorbide and methylthiouracil, ously generated and characterized (15). The Ethics Committee of was further assessed in a XF24 Seahorse flux analyzer (Agilent Animal Experimentation (PROEX 233/19 and PROEX 352/15) and Technologies). WT and Gdap1−/− DRGs were seeded at a density the Institutional Review Board of UAM (CEI 52-961) approved the of 1 × 105 cells/well. Bioenergetics characterization of cultured project. Florfenicol (0.84 mg/ml) (47) and MitoQ (0.14 mg/ml) (48) DRGs was determined through sequential addition of 6 μm treatments were administered in drinking water. oligomycin, 0.25 mm 2,4-dinitrophenol (DNP) and 1 μm antimycin A/1 μm rotenone. Determination of the hind-limb clasping reflex To quantify the hind-limb clasping reflex, mice were suspended Protein extraction and western blot analysis from the tip of the tail and the natural position taken by every mouse was recorded taking a picture of the animal. Then, in a Cells were resuspended in lysis buffer containing 25 mm HEPES, blinded analysis, the angle form between the right hind limb and 2.5 mm EDTA, 1% Triton X-100 supplemented with protease the longitudinal body axis of the mouse was determined using a and phosphatase inhibitor cocktails. Lysates were clarified by protractor. centrifugation at 11 000×g for 15 min. The resulting supernatants were fractionated on SDS-PAGE and transferred onto nitrocel- lulose membranes for immunoblot analysis. Protein concentra- Assessment of mouse motor function: rotarod test tions were determined using Bradford reagent (Bio-Rad protein Motor performance and balance were tested using an accelerat- assay). The primary antibodies used were anti-GDAP1 (1:500, ing rotarod. Each mouse underwent for 2 days the same proce- Abnova, Taipei, Taiwan), anti-tubulin (1:1000, Sigma, Darmstadt, dure. The first day was used to train the mice in one session of Germany), anti-ATF4 (1:500, Cell Signaling, Massachussets, USA), 2 min, walking at 4 r.p.m. Mice that fell off after completing 2 min anti-p-EIF2α (1:500, Cell Signaling), anti-eIF2α (1:500, SantaCruz, walking within 10 min were discarded from the experiment. The Texas, USA), anti-HSP70 (1:500, SantaCruz), anti-HSP60 (1:2000, test sessions were run on day 2. During the test, the speed of the Stressgene SPA-807, Canada), anti-GAPDH (1:20 000, Abcam, Mas- rotarod was accelerated from 4, 8, 12, 16, 20, 24, 28 to 32 r.p.m. sachusetts, USA). Peroxidase conjugated anti-mouse or anti- The mice spent 1 min running at each speed. Their latency was rabbit IgGs (Promega, 1:3000, Wisconsin, USA) were diluted in measured. Mice that fell off six times within 60 s were discarded 5% non-fat dried milk in Tris-buffered saline (TBS) with 1% from the experiment. Tween 20 and used as secondary antibodies. The Novex® ECL (Thermo Fisher Scientific, Massachusetts, USA) system was used Grip force/strength test. The grip force test was used to measure to visualize the bands. the maximum strength that could be performed by a mouse with its forelimbs by taking advantage of the animal’s tendency to grasp to surfaces. One mouse at a time was let to grasp the Cell viability metallic bars and then was gently pulled away until its grasp is Cell viability of SH-SY5Y cells after 24-h treatment with 1 μm broken. The tests were run by the same investigator (C.N.-T.) who of the selected compounds was assessed using the calcein placed her elbow on top of the bench, and at the same distance AM probe (Invitrogen, Massachusetts, USA). 10 000 cells were from the apparatus, to exert the minimum force required to seeded in 200 μl medium into a 96-well black plate with clear break the grasp. The pulling was performed sufficiently slowly flat bottom and treated with the compounds. DMSO treatment to permit the mouse to build up a resistance against it. The test was also added as control. The calcein solution was prepared was repeated five times per mouse, with at least 1 min elapsing by dissolving 50 μg of calcein in 125 μl DMSO and adding between each of the five determinations per animal (49). 25 μl–10 ml sterile phosphate-buffered saline (PBS). After 24 h, 100 μl of calcein solution were added to each well Hanging test: two limbs. The forelimbs muscle strength was and, immediately after, Ex/Em = 485/530 nm fluorescence was measured by monitoring the ability of mice to exhibit sustained recorded in a FLUOstar Omega (BMG LABTECH, Ortenberg, limb tension to oppose their weight. Mice were placed in a 2 mm Germany) luminometer. thick metal bar at 35 cm of a padded surface and time until falling was recorded. The test ended after a hanging time of 2 min was achieved or otherwise after three sessions. Longest Mitochondrial membrane potential and ROS production hanging time (s) and minimal holding impulse (body mass × Cells (3 × 105 ) were incubated with 0.5 μm TMRM+ in 300 μl of hang time) were calculated (50). FACS buffer (1 mm EDTA, 2% FBS in PBS) and processed for flow cytometry to determine m with 1 μm florfenicol, 0.5 mm DNP Hanging test: four limbs. The four limbs muscle strength was or left untreated (46). For the determination of the mitochondrial measured by monitoring the ability of mice to exhibit sustained production of superoxide (mtROS), 3 × 105 cells were incubated limb tension to oppose their weight. Mice were placed in a wire with 5 μm MitoSoX, resuspended in 300 μl of FACS buffer and grid at 35 cm of a padded surface and time until falling was processed for flow cytometry with 1 μm florfenicol, 20 nm MitoQ recorded. The test ended after a hanging time of 2 min was or left untreated. Cells were analyzed in a BD FACScan (46). For achieved or otherwise after three sessions. Longest hanging time Human Molecular Genetics, 2021, Vol. 30, No. 24 2453 (s) and minimal holding impulse (body mass × hang time) were the exception of the hind-limb clasping reflex, the results were calculated (50). not evaluated blind to treatment. Protein extraction from mouse tissues Supplementary Material For protein extraction, tissue sections were homogenized in the Supplementary material is available at HMGJ online. OMNI Bead Ruptor 24 bead mill homogenizer (Fisher Scientific, 15515799, Massachusetts, USA) in T-PER Tissue Protein Extrac- tion Reagent (Cat. No. 78510, Thermo Scientific, Ins. Madrid, Author contributions Spain,) in a 1:8 (w/v) ratio and further freeze thawed three times C.N.-T., F.S., J.S., F.P. and J.M.C. designed experiments and inter- in liquid nitrogen. The protein concentration was determined preted results. C.N.-T., F.S., B.S.-G., C.N.d.A., A.R.-B., L.C. and P.M.- with the Bradford reagent (Cat. No. 5000001, Bio-Rad, Inc. Madrid, V. performed experiments. B.P. and J.H. assisted in mouse experi- Spain) using BSA as standard. ments. M.P.M. contributed to the design and supplied MitoQ. J.S., F.P. and J.M.C. secured funding and C.N.-T. and J.M.C. wrote the paper. All authors read, contributed to and approved the final Printing and processing of RPPAs manuscript. Protein extracts from tissue biopsies were diluted with T-PER to a final protein concentration of 2 μg/μl. Before printing, sam- ples were diluted with PBS to a final protein concentration of Acknowledgements 0.5 μg/μl. Serially diluted protein extracts (0–1.5 μg/μl) derived We thank Dolores Lopez for her technical assistance in the from C2C12 mouse myoblast and HCT116 human colocarcinoma mouse motor behavior experiments. cells were also prepared to assess printing quality and the linear response of protein recognition by the antibodies used (30). Conflict of Interest Statement. M.P.M. consults for Antipodean Phar- Standard curves of BSA (0–2 μg/μl) and mouse IgGs (1–30 ng/μl) maceutical Inc., which is commercializing MitoQ and is the were also prepared as internal negative and positive controls, inventor of Patent US 6331532 B1, dated December18, 2001 enti- respectively. tled ‘Mitochondrially targeted antioxidants’. C.N.-T., J.S., F.P. and Approximately, 1.5 nl of each sample was spotted in quin- J.M.C. are inventors of UAM/CIBERER/HSJD Spanish Patents for tuplicated or triplicate onto nitrocellulose-coated glass slides the treatment of CMT disease. The rest of authors declare no (ONCYTE® SuperNOVA 8 pad—Grace Bio-Labs, 705118, Oregon, competing interests. USA) using a iTWO-300P pico system printer (M2-Automation, Inc., Idaho, USA) equipped with a Piezo Driven Micro-Dispenser (PDMD) 30–150 pl at constant chamber humidity (RH 52%) and Funding temperature (16◦ C), and plate temperature (10◦ C). After printing, arrays were allowed to dry and further ACCI (grant ER18TRL713 to F.P., J.S. and J.M.S.) from CIBERER- blocked in Super G Blocking Buffer (Cat. No. 10501, Grace Instituto de Salud Carlos III, Spain; Ministerio de Economía Biolabs, Madrid, Spain). After, the arrays were incubated y Competitividad (MINECO) (SAF2016-75916-R and PID2019- overnight at 4◦ C with the indicated dilutions of the primary 108674RB-100 to J.M.C, and SAF2015-66625-R to F.P.), Spain. antibodies listed in Supplementary Material, Table S2. After incubation, the arrays were washed with PBS-T and further incubated with goat anti-mouse or goat anti-rabbit highly cross- References adsorbed antibodies conjugated with CF™ 647 (Sigma Aldrich, 1. Sleire, L., Forde, H.E., Netland, I.A., Leiss, L., Skeie, B.S. and SAB4600183–SAB4600185; 1:500, Madrid, Spain). Pads incubated Enger, P.O. (2017) Drug repurposing in cancer. Pharmacol. Res., directly with secondary antibodies and with 0.0001% Fast Green 124, 74–91. FCF (Cat. No. F7252, Sigma Aldrich, Madrid, Spain) were used to 2. Pareyson, D., Saveri, P. and Pisciotta, C. (2017) New devel- evaluate potential unspecific binding to non-masked mouse opments in Charcot-Marie-Tooth neuropathy and related IgGs and the total protein amount present in the spotted diseases. Curr. Opin. Neurol., 30, 471–480. samples, respectively. 3. Pareyson, D. and Solari, A. (2009) Charcot-Marie-Tooth dis- Microarrays were scanned using Typhoon 9410 scanner ease type 1A: is ascorbic acid effective? Lancet Neurol., 8, (GE Healthcare, Inc. Madrid, Spain). The mean fluorescent 1075–1077. intensity of the spots was measured using GenePix® Pro 7 4. Rossor, A.M., Liu, C.H., Petzold, A., Malaspina, A., Laura, M., (USA) and normalized relative to the protein amount contained Greensmith, L. and Reilly, M.M. (2016) Plasma neurofilament in the sample obtained from the FCF stained pad. After heavy chain is not a useful biomarker in Charcot-Marie- quantification, the relative fluorescent intensity was converted Tooth disease. Muscle Nerve, 53, 972–975. into arbitrary units of expressed protein/ng of protein in the 5. Boutary, S., Echaniz-Laguna, A., Adams, D., Loisel-Duwattez, extract using as standard the linear plot of the C2C12 cell line J., Schumacher, M., Massaad, C. and Massaad-Massade, L. (Supplementary Material, Fig. S3) (29,51). (2021) Treating PMP22 gene duplication-related Charcot- Marie-Tooth disease: the past, the present and the future. Transl. Res., 227, 100–111. Statistics 6. Rossor, A.M., Tomaselli, P.J. and Reilly, M.M. (2016) Recent The results shown are means ± SEM. Statistical analysis was advances in the genetic neuropathies. Curr. Opin. Neurol., 29, performed by Student’s t-test. Statistical tests were two-sided 537–548. at the 5% level of significance. Statistical analyses were per- 7. Cuesta, A., Pedrola, L., Sevilla, T., Garcia-Planells, J., Chu- formed using Excel Microsoft 365 and GraphPad Prism 7. With millas, M.J., Mayordomo, F., LeGuern, E., Marin, I., Vilchez, 2454 Human Molecular Genetics, 2021, Vol. 30, No. 24 J.J. and Palau, F. (2002) The gene encoding ganglioside- 20. Cassereau, J., Chevrollier, A., Gueguen, N., Malinge, M.C., induced differentiation-associated protein 1 is mutated in Letournel, F., Nicolas, G., Richard, L., Ferre, M., Verny, C., axonal Charcot-Marie-Tooth type 4A disease. Nat. Genet., 30, Dubas, F. et al. (2009) Mitochondrial complex I deficiency in 22–25. GDAP1-related autosomal dominant Charcot-Marie-Tooth 8. Rzepnikowska, W. and Kochanski, A. (2018) A role for the disease (CMT2K). Neurogenetics, 10, 145–150. GDAP1 gene in the molecular pathogenesis of Charcot- 21. Cassereau, J., Chevrollier, A., Codron, P., Goizet, C., Gueguen, Marie-Tooth disease. Acta Neurobiol. Exp. (Wars), 78, 1–13. N., Verny, C., Reynier, P., Bonneau, D., Lenaers, G. and Pro- 9. Claramunt, R., Pedrola, L., Sevilla, T., López de Munain, A., caccio, V. (2020) Oxidative stress contributes differentially Berciano, J., Cuesta, A., Sánchez-Navarro, B., Millán, J.M., Saifi, to the pathophysiology of Charcot-Marie-Tooth disease type G.M., Lupski, J.R. et al. (2005) Genetics of Charcot-Marie- 2K. Exp. Neurol., 323, 113069. Tooth disease type 4A: mutations, inheritance, phenotypic 22. Soldevilla, B., Cuevas-Martin, C., Ibanez, C., Santacatterina, F., variability, and founder effect. J. Med. Genet., 42, 358–365. Alberti, M.A., Simo, C., Casasnovas, C., Marquez-Infante, C., 10. Baxter, R.V., Ben Othmane, K., Rochelle, J.M., Stajich, J.E., Sevilla, T., Pascual, S.I. et al. (2017) Plasma metabolome and Hulette, C., Dew-Knight, S., Hentati, F., Ben Hamida, M., Bel, S., skin proteins in Charcot-Marie-Tooth 1A patients. PLoS One, Stenger, J.E. et al. (2002) Ganglioside-induced differentiation- 12, e0178376. associated protein-1 is mutant in Charcot-Marie-Tooth dis- 23. Nuevo-Tapioles, C., Santacatterina, F., Stamatakis, K., Nunez ease type 4A/8q21. Nat. Genet., 30, 21–22. de Arenas, C., Gomez de Cedron, M., Formentini, L. and 11. Marco, A., Cuesta, A., Pedrola, L., Palau, F. and Marin, I. (2004) Cuezva, J.M. (2020) Coordinate beta-adrenergic inhibition Evolutionary and structural analyses of GDAP1, involved of mitochondrial activity and angiogenesis arrest tumor in Charcot-Marie-Tooth disease, characterize a novel class growth. Nat. Commun., 11, 3606. of glutathione transferase-related genes. Mol. Biol. Evol., 21, 24. Gonzalez-Sanchez, P., Satrustegui, J., Palau, F. and Del Arco, 176–187. A. (2019) Calcium deregulation and mitochondrial bioen- 12. Pedrola, L., Espert, A., Wu, X., Claramunt, R., Shy, M.E. and ergetics in GDAP1-related CMT disease. Int. J. Mol. Sci., 20, Palau, F. (2005) GDAP1, the protein causing Charcot-Marie- 403. Tooth disease type 4A, is expressed in neurons and is asso- 25. Syriopoulou, V.P., Harding, A.L., Goldmann, D.A. and Smith, ciated with mitochondria. Hum. Mol. Genet., 14, 1087–1094. A.L. (1981) In vitro antibacterial activity of fluorinated 13. Pla-Martin, D., Rueda, C.B., Estela, A., Sanchez-Piris, M., analogs of chloramphenicol and thiamphenicol. Antimicrob. Gonzalez-Sanchez, P., Traba, J., de la Fuente, S., Scorrano, Agents Chemother., 19, 294–297. L., Renau-Piqueras, J., Alvarez, J. et al. (2013) Silencing of 26. Hu, D., Cao, S., Zhang, G., Xiao, Y., Liu, S. and Shang, Y. (2017) the Charcot-Marie-Tooth disease-associated gene GDAP1 Florfenicol-induced mitochondrial dysfunction suppresses induces abnormal mitochondrial distribution and affects cell proliferation and autophagy in fibroblasts. Sci. Rep., 7, Ca2+ homeostasis by reducing store-operated Ca2+ entry. 13554. Neurobiol. Dis., 55, 140–151. 27. Pareyson, D. and Marchesi, C. (2009) Natural history and 14. Cantarero, L., Juárez-Escoto, E., Civera-Tregón, A., Rodríguez- treatment of peripheral inherited neuropathies. Adv. Exp. Sanz, M., Roldán, M., Benítez, R., Hoenicka, J. and Palau, F. Med. Biol., 652, 207–224. (2021) Mitochondrial-lysosome membrane contact sites are 28. Guyenet, S.J., Furrer, S.A., Damian, V.M., Baughan, T.D., La defective in GDAP1-related Charcot-Marie-Tooth neuropa- Spada, A.R. and Garden, G.A. (2010) A simple composite thy. Hum. Mol. Genet., 29, 3589–3605. phenotype scoring system for evaluating mouse models of 15. Barneo-Muñoz, M., Juárez, P., Civera-Tregón, A., Yndriago, cerebellar ataxia. J. Vis. Exp., 39, 1787. L., Pla-Martin, D., Zenker, J., Cuevas-Martín, C., Estela, A., 29. Santacatterina, F., Chamorro, M., de Arenas, C., Navarro, Sánchez-Aragó, M., Forteza-Vila, J. et al. (2015) Lack of GDAP1 C., Martín, M.A., Cuezva, J.M. and Sánchez-Aragó, M. (2015) induces neuronal calcium and mitochondrial defects in a Quantitative analysis of proteins of metabolism by reverse knockout mouse model of Charcot-Marie-Tooth neuropathy. phase protein microarrays identifies potential biomarkers of PLoS Genet., 11, e1005115. rare neuromuscular diseases. J. Transl. Med., 13, 65. 16. Gonzalez-Sanchez, P., Pla-Martin, D., Martinez-Valero, P., 30. Santacatterina, F., Torresano, L., Nunez-Salgado, A., Esparza- Rueda, C.B., Calpena, E., Del Arco, A., Palau, F. and Satrustegui, Molto, P.B., Olive, M., Gallardo, E., Garcia-Arumi, E., Blazquez, J. (2017) CMT-linked loss-of-function mutations in GDAP1 A., Gonzalez-Quintana, A., Martin, M.A. et al. (2018) Different impair store-operated Ca(2+) entry-stimulated respiration. mitochondrial genetic defects exhibit the same protein sig- Sci. Rep., 7, 42993. nature of metabolism in skeletal muscle of PEO and MELAS 17. Fernandez-Lizarbe, S., Civera-Tregon, A., Cantarero, L., Her- patients: a role for oxidative stress. Free Radic. Biol. Med., 126, rer, I., Juarez, P., Hoenicka, J. and Palau, F. (2019) Neuroin- 235–248. flammation in the pathogenesis of axonal Charcot-Marie- 31. Pareyson, D. and Marchesi, C. (2009) Diagnosis, natural Tooth disease caused by lack of GDAP1. Exp. Neurol., 320, history, and management of Charcot-Marie-Tooth disease. 113004. Lancet Neurol., 8, 654–667. 18. Noack, R., Frede, S., Albrecht, P., Henke, N., Pfeiffer, A., Knoll, 32. Morena, J., Gupta, A. and Hoyle, J.C. (2019) Charcot-Marie- K., Dehmel, T., Meyer Zu Horste, G., Stettner, M., Kieseier, Tooth: from molecules to therapy. Int. J. Mol. Sci., 20, 3419. B.C. et al. (2012) Charcot-Marie-Tooth disease CMT4A: GDAP1 33. Nelis, E., Van Broeckhoven, C., De Jonghe, P., Lofgren, A., increases cellular glutathione and the mitochondrial mem- Vandenberghe, A., Latour, P., Le Guern, E., Brice, A., Mostac- brane potential. Hum. Mol. Genet., 21, 150–162. ciuolo, M.L., Schiavon, F. et al. (1996) Estimation of the muta- 19. Niemann, A., Huber, N., Wagner, K.M., Somandin, C., Horn, tion frequencies in Charcot-Marie-Tooth disease type 1 and M., Lebrun-Julien, F., Angst, B., Pereira, J.A., Halfter, H., Welzl, hereditary neuropathy with liability to pressure palsies: a H. et al. (2014) The Gdap1 knockout mouse mechanistically European collaborative study. Eur. J. Hum. Genet., 4, 25–33. links redox control to Charcot-Marie-Tooth disease. Brain, 34. Attarian, S., Vallat, J.M., Magy, L., Funalot, B., Gonnaud, P.M., 137, 668–682. Lacour, A., Pereon, Y., Dubourg, O., Pouget, J., Micallef, J. et al. Human Molecular Genetics, 2021, Vol. 30, No. 24 2455 (2016) Erratum to: an exploratory randomised double-blind 43. Rossman, M.J., Santos-Parker, J.R., Steward, C.A.C., Bispham, and placebo-controlled phase 2 study of a combination of N.Z., Cuevas, L.M., Rosenberg, H.L., Woodward, K.A., Chon- baclofen, naltrexone and sorbitol (PXT3003) in patients with chol, M., Gioscia-Ryan, R.A., Murphy, M.P. et al. (2018) Chronic Charcot-Marie-Tooth disease type 1A. Orphanet J. Rare Dis., supplementation with a mitochondrial antioxidant (MitoQ) 11, 92. improves vascular function in healthy older adults. Hyper- 35. d’Ydewalle, C., Krishnan, J., Chiheb, D.M., Van Damme, P., tension, 71, 1056–1063. Irobi, J., Kozikowski, A.P., Vanden Berghe, P., Timmerman, 44. Mao, P., Manczak, M., Shirendeb, U.P. and Reddy, P.H. V., Robberecht, W. and Van Den Bosch, L. (2011) HDAC6 (2013) MitoQ, a mitochondria-targeted antioxidant, delays inhibitors reverse axonal loss in a mouse model of mutant disease progression and alleviates pathogenesis in an HSPB1-induced Charcot-Marie-Tooth disease. Nat. Med., 17, experimental autoimmune encephalomyelitis mouse 968–974. model of multiple sclerosis. Biochim. Biophys. Acta, 1832, 36. Mo, Z., Zhao, X., Liu, H., Hu, Q., Chen, X.Q., Pham, J., Wei, N., 2322–2331. Liu, Z., Zhou, J., Burgess, R.W. et al. (2018) Aberrant GlyRS- 45. Cabrera, J.R., Viejo-Borbolla, A., Martinez-Martin, N., Blanco, HDAC6 interaction linked to axonal transport deficits in S., Wandosell, F. and Alcami, A. (2015) Secreted herpes sim- Charcot-Marie-Tooth neuropathy. Nat. Commun., 9, 1007. plex virus-2 glycoprotein G modifies NGF-TrkA signaling 37. Rocha, A.G., Franco, A., Krezel, A.M., Rumsey, J.M., Alberti, to attract free nerve endings to the site of infection. PLoS J.M., Knight, W.C., Biris, N., Zacharioudakis, E., Janetka, J.W., Pathog., 11, e1004571. Baloh, R.H. et al. (2018) MFN2 agonists reverse mitochon- 46. Formentini, L., Sánchez-Aragó, M., Sánchez-Cenizo, L. and drial defects in preclinical models of Charcot-Marie-Tooth Cuezva, J.M. (2012) The mitochondrial ATPase inhibitory fac- disease type 2A. Science, 360, 336–341. tor 1 (IF1) triggers a ROS-mediated retrograde pro-survival 38. Snow, B.J., Rolfe, F.L., Lockhart, M.M., Frampton, and proliferative response. Mol. Cell, 45, 731–742. C.M., O’Sullivan, J.D., Fung, V., Smith, R.A., Murphy, 47. Shuang, G., Yu, S., Weixiao, G., Dacheng, W., Zhichao, Z., M.P., Taylor, K.M. and Protect Study Group (2010) A Jing, L. and Xuming, D. (2011) Immunosuppressive activity double-blind, placebo-controlled study to assess the of florfenicol on the immune responses in mice. Immunol. mitochondria-targeted antioxidant MitoQ as a disease- Investig., 40, 356–366. modifying therapy in Parkinson’s disease. Mov. Disord., 25, 48. Formentini, L., Santacatterina, F., Nunez de Arenas, C., 1670–1674. Stamatakis, K., Lopez-Martinez, D., Logan, A., Fresno, M., 39. Sanchez-Gonzalez, C., Nuevo-Tapioles, C., Herrero Martin, Smits, R., Murphy, M.P. and Cuezva, J.M. (2017) Mitochondrial J.C., Pereira, M.P., Serrano Sanz, S., Ramirez de Molina, A., ROS production protects the intestine from inflammation Cuezva, J.M. and Formentini, L. (2020) Dysfunctional oxida- through functional M2 macrophage polarization. Cell Rep., tive phosphorylation shunts branched-chain amino acid 19, 1202–1213. catabolism onto lipogenesis in skeletal muscle. EMBO J., 39, 49. Smith, J.P., Hicks, P.S., Ortiz, L.R., Martinez, M.J. and Mandler, e103812. R.N. (1995) Quantitative measurement of muscle strength in 40. Smith, R.A. and Murphy, M.P. (2010) Animal and human the mouse. J. Neurosci. Methods, 62, 15–19. studies with the mitochondria-targeted antioxidant MitoQ. 50. Aartsma-Rus, A. and van Putten, M. (2014) Assessing func- Ann. N. Y. Acad. Sci., 1201, 96–103. tional performance in the mdx mouse model. J. Vis. Exp., 85, 41. Smith, R.A., Porteous, C.M., Gane, A.M. and Murphy, M.P. 51303. (2003) Delivery of bioactive molecules to mitochondria in 51. Santacatterina, F., Sanchez-Arago, M., Catalan-Garcia, M., vivo. Proc. Natl. Acad. Sci. U. S. A., 100, 5407–5412. Garrabou, G., de Arenas, C.N., Grau, J.M., Cardellach, F. and 42. Takahashi, R., Ikeda, T., Hamaguchi, A., Iwasa, K. and Cuezva, J.M. (2017) Pyruvate kinase M2 and the mitochon- Yamada, M. (2012) Coenzyme Q10 therapy in hereditary drial ATPase inhibitory factor 1 provide novel biomarkers of motor sensory neuropathy type VI with novel mitofusin 2 dermatomyositis: a metabolic link to oncogenesis. J. Transl. mutation. Intern. Med., 51, 791–793. Med., 15, 29.