All procedures were conducted in accordance with the ethical standards for experiments on animals established and approved by the Animal Ethics Committee of the KU Leuven. Experiments were conducted under the ethical committee approval n° 104/2017. Mice were kept in filter top cages in controlled environment with a room temperature between 20 and 21 °C, humidity between 50 and 60%, and a light–dark cycle of 12 h.
The C3-PMP22 (Tg(PMP22)C3) mouse model  which was obtained from the Academisch Medisch Centrum (University of Amsterdam, the Netherlands) was maintained in a C57BL6/J genetic background. Genotyping was conducted using digital droplet PCR (ddPCR) (see below) until heterozygous animals were identified, and then these mice were crossbred with wild-type (Wt) mice, resulting in either heterozygous or Wt offspring. The genotyping from these litters was done by standard PCR to distinguish between C3 mice and littermate Wt mice. For PCR analysis, genomic DNA was purified from a small section of mouse ear or tail by lysing the tissue overnight at 55 °C with proteinase K (20 mg/ml; Roche, Basel, Switzerland) in a lysis buffer composed of 0.2% SDS, 200 mM NaCl, 100 mM Tris–HCl (pH 8.5), and 5 mM EDTA. Samples were then centrifuged, and DNA was precipitated using isopropanol and pelleted by centrifugation. Pellets were subsequently washed in 75% ethanol and dissolved in Tris–EDTA buffer containing 0.1 mM EDTA and 10 mM Tris–HCl (pH 7.5). Primers used for genotyping were purchased from IDT solutions (Leuven, Belgium) and were the following:
PMP22 FWD primer: 5′- TGG TGA TGA GAA ACA GT -3′.
PMP22 REV primer: 5′- TGA TTC TCT CTA GCA ATG GA -3′.
IL FWD primer: 5′- CTA GGC CAC AGA ATT GAA AGA TCT -3′.
IL REV primer: 5′- GTA GGT GGA AAT TCT AGC ATC C -3′.
PCR program cycle conditions were as follows: (1) 3 min at 94 °C, (2) 10 s at 94 °C, (3) 10 s at 53.8 °C, (4) 10 s at 72 °C, and (5) 5 min at 72 °C, and then the run was held at 15 °C until the plate was removed from the PCR thermal cycler. Steps 2–4 were set to a cycle of 30 times. The subsequent PCR product was ran on a 2% agarose gel, stained using SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA) and visualized using a transilluminator (UVP Visi-Blue™ Transilluminator, Thermo Fisher Scientific).
Digital Droplet PCR
The concentration of genomic DNA was determined using NanoDrop (Thermo Fisher Scientific). Subsequently, a restriction enzyme digest was made using FastDigest Buffer (10 × ; Thermo Fisher Scientific), FastDigest HAEIII (10 U; Thermo Fisher Scientific), 500 ng genomic DNA, and nuclease-free water in a total of 50 μl and incubated at 30 °C overnight or 37 °C for 60 min. PCR reaction mix was formulated by combining ddPCR supermix for probes (no UTP) 2 × (Bio-Rad, Hercules, USA), the 20 × Taqman assay, 2 ng of diluted cDNA, and nuclease-free water. Samples were then mixed by vortexing in short pulses and centrifuged briefly. Assembled reaction mixtures were loaded into the DG8™ Cartridge (Bio-Rad, #1,864,008) followed by 70 μl of Droplet Generation Oil for Probes (Bio-Rad, #1,863,005). The QX200™ Droplet Generator (Bio-Rad) was used for droplet generation according to the manufacturer’s guidelines and then loaded to ddPCR plates (Bio-Rad) and heat sealed with foil. The PCR digest was run on PCR thermal cycler with the following program: (1) 10 min at 95 °C, (2) 30 s at 95 °C, (3) 1 min at 58 °C, and (4) 10 min at 98 °C, (5) and then the run was held at 12 °C until the plate was removed from the PCR thermal cycler. Steps 2–4 were set to a cycle of 39 times. The subsequent loaded ddPCR plate was read using QX200 Droplet Reader and QuantaSoft Software (Bio-Rad).
The human-specific ddPCR copy number variation assay for PMP22 (dHsaCP2500327; Bio-Rad, #10031240) was used to determine the copy number of human PMP22 in the CMT1A mice. The reference gene used in combination with the human PMP22 assay was the commercially established AP3B1 (dHsaCP1000001; Bio-Rad, #10031245) antibody ddPCR copy number variation assay.
HDAC3 Inhibitor Treatment
For treatment, the selective HDAC3 inhibitor (HDAC3i) RGFP966 (99.5% purity; Asclepia, Destelbergen, Belgium) was used as reported before [18, 21]. RGFP966 has a > 200-fold selectivity towards HDAC3 versus other HDACs; HDAC3 IC50 at 0.08 μM versus a HDAC2 IC50 at 17.7 μM and a HDAC1 IC50 at 28.7 μM  (see Additional File 1, Figure S1). Stock solutions of 5 mg/ml or 10 mg/ml RGFP966 were prepared in DMSO. These solutions were diluted 1/10 in sterile injectable water (Aqua ad iniectabilia, Mini-Plasco connect®) containing 30% hydroxyl-beta-cyclodextrin and 0.1 M acetate (pH 5.4) until a 10% DMSO concentration was reached. RGFP966 (either 5 mg/kg or 10 mg/kg body weight) was administered starting at P6. For control mice, 10% DMSO was used in the subsequent steps. The first 7 days, the compound was administered subcutaneously once daily, after which the administration route was switched to intraperitoneal (I.P.) injections, every other day for 2 weeks (7 injections in total). No side effects were observed in the control treated animals. No inclusion or exclusion criteria were applied to the mice. Males and females were included in the study. As genotype identification was not always possible before postnatal day 6, the treatment of genotypes was blinded at certain times, leading to slight disparities in the sample sizes. During phenotyping, investigators were blinded to genotype and treatment.
Motor phenotyping was conducted 2 days after the treatment was completed, starting from postnatal day 30. Muscle force of the mice was determined using a grip strength meter (Columbus Instruments, Columbus, USA). Overall grip strength was assessed using a metal mesh assembly to assess the combined grip strength of the fore- and hindlimbs. Mice were held by their tail and gently pulled backwards while gripping the metal mesh, until they lost their grip. The average of 5 trials per animal was determined. Motor performance was assessed using a Rotarod (Ugo Basile, Gemonio, Italy), with a constant speed of 15 rpm. The latency to fall was recorded to a maximum of 300 s. A rest period of 5 min was given between each trial. A total of 3 trials was recorded, and the mean of these trials is used in graphs.
Initial electrophysiology follow-up characterization of the C3 phenotype was conducted at 2–6 months of age. An additional CMAP tracing is qualitatively shown as part of the initial follow-up at postnatal day 35 to illustrate earlier electrophysiology abnormalities in the C3 versus Wt littermate mice. For the HDAC3i treatment study, electrophysiology was conducted after motor phenotyping was completed, between postnatal days 35 and 36. For CMAP recordings, mice were anaesthetized using 3% isoflurane under a 2.5 l/min oxygen flow. Once sedated, they were kept under constant flow of 2-3% isoflurane and placed on a 37 ± 0.5 °C heating pad to maintain body temperature. Recordings were performed using 0.4 mm platinum coated sub-dermal needle electrodes (Technomed Europe, Maastricht, Netherlands) and the Natus ultraPro S100 (Natus Medical Incorporated, Pleasanton, USA) as described before [20, 22, 23]. The sciatic nerve was stimulated by placing the anode and cathode proximal of the sciatic notch, 0.5 cm apart from each other. The reference electrode was placed at the ankle of the same leg, and the measuring electrode was positioned parallel with the gastrocnemius muscle, subdermally. The distance between these two electrodes was measured to calculate nerve conduction velocities (NCVs). The ground electrode was positioned at the base of the tail. The stimulating current was increased gradually until a stable supramaximal response was generated. The highest supramaximal responses were taken for each mouse out of 3 recordings for each time-point and used in the graphs. For latency and NCV calculations, a mean of the 3 recordings per mouse was used in the graphs.
Histology was performed at the end of phenotyping (postnatal day 37), when mice were sacrificed. First, mice were anaesthetized by an I.P. injection with sodium pentobarbital (200 mg/kg: Dolethal, Vetoquinol). When the mice were non-responsive to pressure applied to their foot or tail, they were transcardially perfused with PBS. For electron microscopy, mice were additionally perfused with 2.4% glutaraldehyde, 0.1 M Na-cacodylate and 4% paraformaldehyde dissolved in Milli-Q® ultrapure water (Merck, Darmstadt, Germany). Fixation solution was filtered through a 0.2-μm vacuum filter bottle system (Corning®, Lasne, Belgium, #430,771) before use. Isolated sciatic nerves were further post-fixed in Milli-Q® water containing 2.4% glutaraldehyde, 0.1 M Na-cacodylate, and 4% paraformaldehyde overnight at 4 °C. The following day, samples were washed twice with a 0.1 M Na-cacodylate buffer and washed for 30 min. Samples were then transferred to glass vials and post-fixed in 2% osmium tetroxide for 120 min. Samples were subsequently dehydrated as follows: 50% EtOH 15 min (× 2), 70% EtOH 15 min (× 2), 96% EtOH 15 min (× 2), and 100% EtOH 15 min (× 2).
Impregnation was as follows: 20 min propylene oxide (× 2), 60 min at room temperature 1:1 propylene oxide/agar, and then 1:2 propylene oxide/agar overnight at room temperature. Samples were then placed in fresh agar for 6–8 h at room temperature, after which they were placed in a rubber mold containing fresh agar. Samples were oriented so that the most distal region of the sciatic nerve was facing the edge of the molds. To harden the samples, they were placed in a 60 °C oven for 48 h. Samples were trimmed using a Leica EM TRIM (Leica Microsystems), and semi-thin (0.5 μm) transverse sections were cut using a Leica ultra-microtome (Leica Ultracut S, Leica Microsystems). Finally, the sections were stained with 1% toluidine blue at 80 °C for 1 min.
Ultra-thin sections (70 nm) were cut and collected on 75-mesh grids (Van Loenen Instruments, Zaandam, Netherlands) followed by post-staining with 1% uranyl acetate solution and lead citrate before being imaged with a JEOL JEM-1400 Transmission Electron Microscope (Joel Ltd., Akishima, Tokyo, Japan) operated at 80 kV. For statistical analyses, nerve fibers from up to 4 animals per group were used. Each sciatic nerve section was analyzed at × 800 magnification. The ImageJ plugin “GRatio” was used to analyze g-ratios at randomly selected fibers. The investigator was blinded from the genotype and treatment during the analysis. At least 95 fibers were randomly selected per animal at the central region of the sciatic nerve for all animals. For pathological observations, ranges up to × 5000 magnification were used.
For immunohistochemistry of the brachial plexus nerve (specifically, the medial anterior thoracic nerve), animals were transcardially perfused with PBS. Isolated brachial plexus nerves were snap-frozen in isopentane that had been cooled by immersion in liquid nitrogen and placed on dry ice. Samples were then stored at − 80°C until further use. The nerves were later embedded in optimal cutting temperature compound (OCT compound, Fisher Scientific) and stored at − 80 °C until sectioning. Transverse sections of the nerve were cut at a thickness of 20 μm using a cryostat (Cryostar NX70, ThermoFisher Scientific). The average of at least 3 slices was analyzed per animal at an 80 μm depth difference apart from each slice area in each brachial plexus section to avoid analysis of overlapping slices. Sections were collected on a coverslip glass and air-dried for 1–2 h. Sections were then post-fixed in 4% PFA for 15 min at room temperature, followed by immersion in 100% methanol for 6 min at − 20 °C. After fixation, samples were washed twice with PBS and once with PBST (0.3% Triton X-100 (Sigma, St. Louis, USA)), each for 5 min. Samples were then blocked in 5% normal donkey serum (Sigma) and 5% goat serum (Sigma) in PBS for at least 1 h. Sections were then incubated with primary antibodies overnight at 4 °C. Primary antibodies F4/80 (Bio-Rad, #MCA497G) and CD34 (ThermoFisher Scientific, #14–0341-85) were used at a 1:300 and 1:1000 dilution, respectively. After incubation, slides were washed three times for 5 min with PBS, and appropriate secondary antibodies were used at a 1:1000 dilution in blocking buffer for 1 h. Following this, slides were washed three times with PBS for 5 min, after which they were mounted with ProLong® Gold antifade reagent containing DAPI (Invitrogen, Life Technologies, Carlsbad, USA). The amount of F4/80+ cells per brachial plexus nerve section was quantified as the level of macrophages per nerve section, and each time a F4/80+ cell touched a CD34+ cell, this was taken as an interaction.
For immunohistochemistry of gastrocnemius muscle, transverse sections of the muscle were cut at a thickness of 20 μm using a cryostat, allowed to dry for 1–2 h and then post-fixed for 20 min with 4% PFA at room temperature. Sections were then blocked for 1 h and incubated with wheat germ agglutinin primary antibody conjugated to Alexa Fluor-488 (Invitrogen, #W11261). Slides were washed three times for 5 min with PBS before mounting with ProLong® Gold antifade reagent without DAPI (Invitrogen, #P36934). All images were acquired using the Leica SP8 X confocal microscope and analyzed using ImageJ.
Western Blot Analysis
For analysis of whole-protein lysates, snap-frozen whole sciatic nerve tissues were lysed in ice-cold RIPA buffer (50 mM Tris–HCl (pH 7.5), 0.5% SDS, 150 mM NaCl, 0.5% Na-deoxycholic acid, and 1% NP-40), supplemented with complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). Tissues were homogenized using lysing matrix D beads (MP Biomedical, Illkirch Cedex, France) and a tissue homogenizer instrument (FastPrep24-5G- MP Biomedical, USA). For nuclear and cytoplasmic fractionation of sciatic nerve tissue, samples were prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, #78,835) according to the manufacturer’s guidelines. Protein concentration measurements and Western blot preparations were performed as described before [22, 24]. The following primary antibodies were diluted in 5% BSA blocking buffer: myelin basic protein (Millipore, #MAB386) 1:3000, phosphorylated-AKT (Cell Signaling Technology, #9271S) 1:1000, AKT (Cell Signaling Technology, #9272) 1:1000, phosphorylated-ERK1/2 (Cell Signaling Technology, #4370) 1:1000, ERK1/2 (Cell Signaling Technology, #4695) 1/1000, histone H4 (Abcam, #ab10158) 1/1000, CSF-1-R (Abcam, #ab271294) 1/500, and PMP22 (Origene, #TA808964) 1:1000, or acetylated histone H3 (Cell Signaling Technology, #4353S) 1:500. Secondary antibodies conjugated with horseradish peroxidase (Agilent Technologies (Dako); 1:5000, 1 h, RT) were used prior to detection with enhanced chemiluminescence substrate (ECL substrate; ThermoFisher Scientific) and a LAS 4000 Image Analyzer (GE Healthcare, Little Chalfont, UK). Total protein levels were visualized using No-Stain™ Protein Labeling Reagent (ThermoFisher Scientific, #A44717). Luminescent signals were analyzed with ImageQuant TL software (GE Healthcare). The band intensities from the proteins of interest were normalized to the total amount of protein loaded in each lane. For representation in figures, a lane of an equal molecular weight is used.
A priori power analyses were conducted using the software G*Power version 3.1.7. Adequate power (1–β-error) was defined as ≥ 80% with an alpha error of 5%. All other statistical analyses were performed using GraphPad Prism software version 9 (GraphPad software Inc., California, USA) or MS Excel. All data were first checked for normality in GraphPad Prism to select the appropriate statistical test. Additionally, where appropriate, data were checked for outliers using GraphPad Grubbs analysis. The unpaired two-tailed Student’s t test was used for the comparison of two means; one-way ANOVA tests were performed for the multiple group analysis. Data from in vivo studies and Western blot are presented as means ± SD, and data from and histopathological analyses are presented as means ± SEM. Statistical significance was set at the following: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.