Ethical statement
Eight to ten week-old CD1 mice were employed for laser capture microdissection (LCM) and transcriptional profiling, and for electrophysiological recordings. C57BL/6 mice expressing cytosolic GFP under the plp promoter [26] were kindly provided by Dr. W.B. Macklin (Aurora, Colorado), with the help of Dr. T. Misgeld (Munchen, Germany), and used for imaging. The proteolipid protein (plp) gene promoter drives the expression of one of the major components of myelin predominantly in oligodendrocytes, SCs and the enteric glia of the gut, both in embryonic and postnatal tissues. Originally created to label oligodendrocytes in the central nervous system, the plp-GFP mice employed in the present study express cytoplasmic GFP in both myelinating and non-myelinating SCs of the peripheral nerves [26]. Mice expressing the Tomato protein specifically in ChAT (choline acetyltransferase)-positive neurons were obtained by crossing the C57BL/6 ChAT-Cre knock-in mice line with C57BL/6 Rosa26.tdTomato mice (Jackson Laboratories). C57BL/6 mice (8–12 weeks) were also used. Mice were maintained under a 12-h light/12-h dark cycle, and kept under constant temperature. Water and food were available ad libitum, and mice were fed with regular chow. All surgical procedures were performed under general anaesthesia (a cocktail of xilazine (48 mg/kg) and zoletil (16 mg/kg) via intraperitoneal injections or via isoflurane as reported in [27]). Paralysis was restricted to one hind limb, and did not impair food or water intake.
All experimental procedures involving animals and their care comply with the ARRIVE guidelines. Procedures carried out in Italy were approved by the ethical committee and by the animal welfare coordinator of the OPBA from the University of Padua. All procedures are specified in the projects approved by the Italian Ministry of Health, Ufficio VI (authorisation numbers: 359/2015 PR; 81/2017 PR; 521/2018 PR; 439/2019 PR) and were conducted in accordance with National laws and policies (D.L. n. 26, March 14, 2014), following the guidelines established by the European Community Council Directive (2010/63/EU) for the care and use of animals for scientific purposes. Animals were handled by specialised personnel under the control of inspectors from the Veterinary Service of the Local Sanitary Service (ASL 16-Padua), who are the local officers of the Ministry of Health.
Animal experiments in the United Kingdom were conducted in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC), under license from the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986, and were approved by the UCL Institute of Neurology Ethical Review Committee.
Toxins and antibodies
α-LTx (LSP-130) and µ-conotoxin GIIIB (C-270) were purchased from Alomone. α-LTx purity was checked by SDS–PAGE, and its neurotoxicity by ex vivo mouse nerve-hemidiaphragm preparations, as previously described [28]. BoNT/A (Xeomin) was from Merz. All other reagents were from Sigma unless stated otherwise.
Antibodies and fluorescent conjugates with relative dilutions: α-BTx AlexaFluor555 (B35451 Thermo Fisher, 1:200), anti-Ctgf neutralizing antibody (70R-CR023 Fitzgerald, 2 µg/40 µl), anti-Ctgf for immunostaining (ab6992 Abcam, 1:200), anti-S100 (Z0311 Dako, 1:400), anti-GAP43 (ab75810 Abcam, 1:200), anti-NF (ab4680 Abcam, 1:800), anti-VAMP1 [29], anti-SNAP25 (ab24737 Abcam, 1:200), anti-SNAP-25 BoNT/A-cleaved [30], anti-syntaxin 1A/1B [31], anti-YAP (13008S, Cell Signaling, 1:200). Secondary AlexaFluor-conjugated antibodies (1:200) were from Thermo Fisher. A list of antibodies and the relative description is provided as Additional file 9.
Sample preparation and RNA extraction
Upon isoflurane anaesthetization, CD1 male mice of ≈ 20 g were injected in the hind limb with α-LTx (5 µg/kg in 15 µl physiological saline, 0.9% NaCl + 0.2% gelatine) or vehicle (15 µl saline). A few minutes before muscle collection, a local injection of fluorescent α-BTx was performed to visualize NMJs. At different time points (0, 4, 16, 72 and 168 h), treated mice (5 mice/group) were sacrificed by anaesthetic overdose (a cocktail of xilazine (48 mg/kg) and zoletil (16 mg/kg) via intraperitoneal injections) followed by cervical dislocation; soleus muscles were then rapidly collected, and frozen in liquid nitrogen-cooled isopentane. Cryo-sections (7–10 µm thick) were transferred to UV-treated microscope glass slides. Microdissection was performed with a PALM RoboMover automatic laser microdissector (Carl Zeiss, Oberkochen, Germany). One hundred NMJs/sample were collected by LCM within 30 min, to preserve RNA integrity, and pooled. Total RNA was isolated by incubation with 50 µl lysis buffer PKD (Qiagen) and 10 µl proteinase K (Promega) at 55 °C overnight, with the sample upside down. The next day, samples were centrifuged for 10 min at 10,000 rpm, and RNA extracted using the Maxwell® 16 LEV RNA FFPE Purification Kit with automated system Maxwell 16 (Promega). RNA extraction was performed following the manufacturer’s protocol starting from the DNase treatment step.
Library preparation
cDNA was obtained using the SMARTer Universal Low Input RNA kit (Clontech Laboratories), according to manufacturer’s instructions. Libraries for RNA-Seq were obtained using the Nextera XT kit (Illumina) according to the manufacturer’s guidelines. RNA sequencing was performed with NextSeq 500 (Illumina), loading a maximum of six, pooling libraries for each cartridge NextSeq High Output (300 cycles).
Computational analysis of sequencing data
One hundred bp paired-end reads were processed by removing Illumina Nextera adapters using Trimmomatic (v0.36). Processed reads were aligned to the mouse genome (GRCm38.p6) with STAR (v020201), using the Gencode M17 gene annotation, based on ENSEMBL release 92. Read duplicates were removed with Picard Tools MarkDuplicates (v2.18.9). All programs were used with default settings unless otherwise specified.
Multidimensional scaling (MDS) was performed with the cmdscale function of the "stats" package in R [32]. The analysis was based on the scaled expression values of 10,956 genes, determined at each time point, averaging the signals from replicates. The Euclidean distance was used as the distance metric in the analysis in Fig. 1B.
Gene expression levels were normalized among replicates using the TMM method implemented in the edgeR Bioconductor package [33]. Differentially expressed genes were detected using edgeR with a double threshold on the log2-fold change (absolute value > 0.75), and the correspondent statistical significance (P < 0.05). Functional annotation of gene lists and enrichment analysis with Gene Ontology terms and KEGG pathways were performed with the clusterProfiler Bioconductor package.
Differentially-expressed genes resulting from H2O2 treatment in R. norvegicus were from [23]. Paralogous M. musculus genes were retrieved from ENSEMBL (release 92). Common up- and down-regulated genes in the two data sets were defined as the intersection of genes up- or down-regulated in at least one of the four time points of α-LTx treatment (4, 16, 72 and 168 h), and in at least one of the two time points upon H2O2 treatment (20 and 40 min).
Droplet digital PCR
Droplet digital PCR (ddPCR) was carried out using the ddPCRTM Supermix for Probes (No dUTP), the QX200TM Droplet Generator, the QX200 Droplet Reader, the C1000 TouchTM Thermal Cycler and the PX1TM PCR Plate Sealer (BIO-RAD, Hercules, California, USA) following the manufacturer’s instructions. Reactions were performed in triplicate in a 96 well plate using 10 μL/reaction of 2 × ddPCR Supermix for Probes (No dUTP), 1 μL/reaction of 20 × target primers/probe (FAM or HEX, BIO-RAD), 1 μL/reaction 20x reference primers/probe (FAM or HEX, BIO-RAD), 3 μL cDNA and 5 μL H2O. Detection of Ctgf and Gapdh by ddPCR was performed using the following PrimePCR™ ddPCR™ Expression Probe Assay designed by BIO-RAD: CTGF-FAM (ID: qMmuCEP0053713) and GAPDH-HEX (ID: dMmuCPE5195283, BIO-RAD). All steps used a ramp rate of 2˚C/s. Results were analyzed in the QX200 Droplet Reader, the RNA targets were quantified using the QuantaSoftTM Software (BIO-RAD), and results were normalized to the control.
Electrophysiological recordings of evoked junctional potentials (EJPs)
EJPs were intracellularly recorded in vitro from single soleus muscle fibers following NMJ poisoning by α-LTx. Anaesthetized mice were locally injected with α-LTx (5 μg/kg in 15 μL of 0.9% NaCl, 0.2% gelatin) in the hind limb [20, 21]. In a group of animals, after the local injection of the toxin, 2 µg of anti-Ctgf neutralizing antibody (in 40 µl physiological solution plus 0.2% gelatine) were intraperitoneally injected to induce the biochemical knockout of the molecule [21, 34], which begins rapidly after antibody injection and lasts for a long time, as the half-life of murine IgG antibodies exceeds 11 days [35]. 96 h later, when 50% neurotransmission blockade by the toxin is achieved (a value very suitable for assessing the impact of a treatment on nerve recovery of function), mice were sacrificed by cervical dislocation, and soleus muscles quickly excised and pinned to a Sylgard-coated petri dish (Sylgard 184, Down Corning USA). Recordings were performed in oxygenated Krebs–Ringer solution using intracellular glass microelectrodes (1.5 mm outer diameter, 1.0 mm inner diameter, 15–20 MΩ tip resistance; GB150TF, Science Products GmbH Germany), filled with a 1:2 solution of 3 M KCl and 3 M CH3COOK. Evoked neurotransmitter release was recorded in current-clamp mode, and resting membrane potential was adjusted with current injection to -70 mV. EJPs were elicited by supramaximal nerve stimulation at 0.5 Hz using a suction glass microelectrode (GB150TF, Science Products GmbH Germany) connected to a S88 stimulator (Grass, USA). Muscle fiber contraction during intracellular recordings was blocked by adding 1 μM μ-conotoxin. Intracellularly recorded signals were amplified with an intracellular amplifier (BA-01X, NPI, Germany), digitized using a digital A/C interface (NI PCI-6221, National Instruments, USA), and then fed to a computer for both on-line visualization and off-line analysis using appropriate software (WinEDR, Strathclyde University; pClamp, Axon, USA). Stored data were analyzed off-line using the software pClamp (Axon, USA).
Sciatic nerve compression/transection
The sciatic nerve was exposed at the level of the sciatic notch under general anaesthesia without damaging the gluteal musculature. Nerve compression (crush) was performed using haemostatic forceps, pre-dipped in powdered charcoal, to mark the crush site, by pinching the nerve 0.5 cm from the hip insertion for 20 s at the 3rd click of the haemostatic forceps. Transection of the sciatic nerve was performed using surgical scissor, leaving the edge juxtaposed [36]. The gluteal musculature was re-opposed and the skin sutured using 6–0 braided silk, non-absorbable sutures (ETHLCON2 biological instruments, 8697).
After surgery, mice were intraperitoneally injected once a week with anti-Ctgf antibodies (2 µg in 40 µl physiological solution plus 0.2% gelatine) or vehicle [34, 37], and electrophysiological measurements (in the case of crush) and immunofluorescence (crush and cut) were performed at different time points after injury.
In selected experiments, after sciatic nerve exposure, but before nerve compression, catalase (C1345, Sigma Aldrich) was intra-sciatically injected (1 mM in physiological solution, 2 μl injection volume) using a pulled graduated glass micropipette, gently inserted in the medial area of the sciatic nerve under the perineurium [38].
Electrophysiological recordings of compound muscle action potentials (CMAPs)
CMAPs were recorded from mice 18 or 28 days following sciatic nerve crush [37, 39, 40]. The 28 d time-point corresponds to 50–60% neurotransmission recovery following nerve compression, a value that is very suitable for assessing the impact of a treatment on nerve recovery of function [37]. Upon general anaesthesia, the sciatic nerve was exposed at the sciatic notch, and a small piece of parafilm was slid under the nerve, which was kept moist with phosphate buffered saline (PBS). A pair of stimulating needle electrodes (Grass, USA) were then advanced until gently touching the exposed sciatic nerve, above the site of crush lesion, using a mechanical micromanipulator (MM33, FST, Germany). A pair of electromyography needle electrodes (Grass, USA) were used for electromyographic recording of gastrocnemius muscle fibre activity. The recording needle electrode was inserted halfway into the gastrocnemius muscle, while the indifferent needle electrode was inserted in the distal tendon of the muscle. CMAPs were recorded following supramaximal stimulation of the sciatic nerve at 0.5 Hz (0.4 ms stimulus duration) using a stimulator (S88, Grass, USA) via a stimulus isolation unit (SIU5, Grass, USA) in a capacitance coupling mode. To reach supramaximal stimuli (5–15 mV for controls, up to 50 mV after nerve damage), the sciatic nerve was stimulated with increasingly intense stimuli until the CMAP value ceased to increase. Recorded signals were amplified by an extracellular amplifier (P6 Grass, USA), digitized using a digital A/C interface (National Instruments, USA), and then fed to a computer for both on-line visualization and off-line analysis using appropriate software (WinEDR, Strathclyde University; pClamp, Axon, USA). Stored data were analyzed off-line using pClamp software (Axon, USA).
Immunohistochemistry
Soleus muscles were dissected at different time points after local administration of α-LTx, or 21 days after BoNT/A (0,5 U in 25 µl physiological solution plus 0.2% gelatin), w/wo treatment with anti-Ctgf neutralizing antibodies, then fixed in 4% PFA in PBS for 30 min at RT, and quenched in 0.24% NH4Cl PBS for 20 min. After permeabilization and 2 h saturation in blocking solution (15% goat serum, 2% BSA, 0.25% gelatine, 0.20% glycine, 0.5% Triton-X100 in PBS), samples were incubated with primary antibody against syntaxin 1A/1B for 72 h in blocking solution at 4 °C. Muscles were then washed, and incubated with secondary antibodies and α-BTx AlexaFluor-555 to stain post-synaptic acetylcholine receptors (AChRs). Images were collected with a Leica SP5 confocal microscope equipped with a 40 × and 63 × HCX PL APO NA 1.4 oil immersion objective. Laser excitation line, power intensity and emission range were chosen according to each fluorophore in different samples to minimize bleed-through. Orthogonal projection and 3D analysis were performed with ImageJ software (Orthogonal views command in Image-Stacks section and 3D viewer plugin). The orthogonal projection method was used with a stack to display the XZ and YZ planes at a given point in the 3D image.
Pseudocolor image analysis: the 8 bit image format, acquired by confocal microscope, is associated with a signal intensity gradient (LUT) in grayscale, with pixel intensity values ranging from highest to lowest intensity. The grayscale image is converted in pseudocolors by Fiji imaging software, by assigning colors to the gray levels (pseudocolor Fire). The pseudocolor scale is added in the corresponding figure. White corresponds to the highest intensity, black to the lowest. This colored image, when displayed, allows an easier identification of certain features by the observer.
Sciatic nerves were isolated at different time points following crush or cut, w/wo treatment with anti-Ctgf neutralizing antibodies, fixed in 4% PFA in PBS for 30 min, sucrose-cryoprotected overnight, and embedded in OCT. Samples were slowly frozen in isopentane cooled with liquid nitrogen vapours, and cryo-sliced (20 μm thickness) using a Leica CM1520 cryostat. Slices were processed for immunostaining as described in [37]. In some experiments, whole mount staining of the nerve was performed as described in [41].
Imaging of H2O2 in the murine sciatic nerve
Live imaging experiments were performed in C57BL/6 mice following the protocol described in [27]. Briefly, after anaesthesia was initiated and maintained using isoflurane, the fur on the dorsal hind limb (ankle to hip) was shaved off, and mice were placed on a heat-pad for the duration of surgery. An incision was made in the trochanteric region and the skin covering the entire surface was removed, along with the biceps femoris muscle to expose the underlying sciatic nerve. The connective tissue underneath the sciatic nerve was gently disrupted using curved forceps to enable the placement of a small piece of parafilm aiding the subsequent imaging. Once the sciatic nerve was exposed, 1 mM PF6-AM (A14086 AdooQ Bioscience) in 2 μl saline was injected into the nerve. Peroxyfluor-6 acetoxymethyl ester (PF6-AM) is a chemoselective fluorescent indicator for H2O2. The molecule features a boronate chemical switch that allows for selective detection of H2O2 over other ROS, combined with acetoxymethylester (AM) protected phenol and carboxylic acid groups for enhanced cellular retention and sensitivity [42]. The anaesthetised mouse was then transferred to an inverted LSM780 laser scanning microscope (Zeiss) equipped within an environmental chamber pre-warmed and maintained at 37 °C. Using a 10x, Plan-Apochromat 10x/0.3 M27 (Zeiss), sciatic nerves were imaged, before and after injury, with a frame acquisition rate consistent across comparable data sets. Injury was performed by the compression of the sciatic nerve, in the distal part, for 30 s with a flat handle micro jewellers forceps. One millimolar H2O2 was added directly above the sciatic nerve by drops (total volume 20 μl) at the end of the recordings as a positive control. The mitochondria-targeted antioxidant MitoTEMPO (SML0737, Sigma-Aldrich) or the NOX inhibitor VAS2870 (492,000 Calbiochem) were applied (1 mM) in the same way before injury. Imaging was completed within 20 min of anaesthesia. A minimum of three sciatic nerves were imaged per condition. Mean fluorescence was measured by averaging the intensity across the visible sciatic nerve area.
Primary cell cultures and treatments
Primary cultures of SCs and of spinal cord motor neurons (SCMNs), and the relative co-cultures, were prepared as previously described [20, 43]. Primary SCs were exposed to 50 µM H2O2 in Krebs Ringer Buffer (KRH: HEPES-Na 25 mM at pH 7.4, NaCl 124 mM, KCl 5 mM, MgSO4 1.25 mM, CaCl2 1.25 mM, KH2PO4 1.25 mM, glucose 8 mM) for different amounts of time at 37 °C, and then processed for immunofluorescence or ELISA.
Co-cultures between SCs and SCMNs were kept for 2–3 days in SCMNs medium before treatment, then exposed for different times to α-LTx (0.1 nM) in E4 medium (120 mM NaCl, 3 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES-Na, pH 7.4) at 37 °C, and processed for immunofluorescence.
Immunofluorescence
Samples were exposed to H2O2 with or without the Transcriptional enhancer factor domain (TEAD) inhibitor VT107 (HY-134957 MedChemexpress, 10 μM final concentration). VT107 is a potent inhibitor of TEAD4 palmitoylation [44]. All TEAD homologues require auto-palmitoylation on the sulfydryl of a conserved cysteine to become functional. Samples were then fixed for 15 min in 4% PFA in PBS, quenched (0.24% NH4Cl in PBS), permeabilized with 0.3% Triton X100 in PBS for 5 min at RT, and saturated with 3% goat serum in PBS for 1 h. After incubation with primary antibodies diluted in 3% goat serum in PBS overnight at 4˚C, samples were washed and then incubated with the corresponding AlexaFluor-conjugated secondary antibodies for 1 h at RT. Coverslips were mounted in Mowiol and examined by confocal (Leica SP5) microscopy. Fluorescence intensity was quantified using Fiji Software. Images were acquired using non-saturating settings, and the same imaging parameters were used for all samples.
ELISA
Ctgf was quantified in the supernatant of primary SCs cultured in 24-well plates and exposed for different time periods to 50 µM H2O2. Briefly, the supernatant was collected and centrifuged at 12,000 × g at 4 °C to eliminate cell debris. Aliquots were immediately assayed according to manufacturer’s instructions (Peprotech 900-M317). Ctgf standards were used in the range of 4 to 4000 pg/ml.
In vitro scratch wounding assay
SCs were seeded onto 35 mm Glass Bottom Dishes (Mattek) on a coating of laminin (3 μg/ml) w/wo 100 ng/ml of recombinant human Ctgf (rCtgf, 120–19 Peprotech). Once fully confluent, the monolayers were scratched using a sterile 200 μl pipette tip following the protocol described in [45]. Monolayers were then gently washed twice with DMEM to remove cell debris, and incubated at 37 °C (5% CO2 air atmosphere). Images were acquired 0, 8 and 24 h later. The wound scratch area was measured using ImageJ.
Statistical analysis
Sample size was determined based on data collected in our previously published studies. We used at least N = 4 mice/group for electrophysiological analysis. For imaging and cell cultures studies, at least three independent replicates were performed. Mice were randomly allocated to the different treatment groups by the investigator. We ensured blinded conduct of experiments. For imaging analysis, the quantitation was conducted by an observer who was blind to the experimental groups. No samples or animals were excluded from the analysis. Data displayed in histograms are expressed as means ± SD. GraphPad Prism software was used for all statistical analyses except for RNAseq data, where the glmQLFTest function, which applies a quasi-likelihood F-test, was employed. Statistical significance was evaluated using unpaired Student’s t-test, or paired Student’s t-test for live-imaging experiments. Data were considered statistically different when *p < 0.05, **p < 0.01, ***p < 0.001.