Statistical analyses
For each data set presented, experiments were performed at least three times independently or with at least three animals and p values were calculated in Microsoft Excel (Mac version 16.34) using two-tailed (black asterisks) or one-tailed (gray asterisks) Student’s t-tests. P values: *<0.05, **<0.01, ***<0.001, data are presented as mean values ± SEM. For data sets obtained using animals or their tissues, three to seven animals were used per group and tissues of each animal were processed independently. For data sets obtained using cells, three to ten independent experiments were conducted and analysed. Sample size was determined by the minimal number of animals or individual experiment required to obtain statistically significant results and increased in some cases to improve confidence in the results obtained. No animal or data point was excluded from the analysis.
Animals
Mice homozygous for floxed Hdac8 allele (EMMA, Strain # EM:05717) were crossed with P0-CreERT2 or Plp-CreERT2 mice, in which the expression of the Cre recombinase (Cre) is tamoxifen-inducible and controlled by the Schwann cell-specific P0 promoter or the Schwann cell and oligodendrocyte-specific Plp promoter61. Mice homozygous for floxed Hif1a allele (The Jackson Laboratory, Strain # 007561), were crossed with Plp-CreERT2 mice. To ablate HDAC8 or HIF1a in adult SCs, three- to four-month-old adult mice received daily injections of 2 mg tamoxifen (Sigma) for five consecutive days. As control mice, Cre-negative littermates of HDAC8 or HIF1a knockout mice were used. Genotypes were determined by PCR on genomic DNA.
Stratified random allocation in blocks with gender and age as strata was used. Analysis of the raw data was done at least twice by different researchers, independently. All mice used in this study were from mixed strains backcrossed to C57BL/6 J. Mice were housed in a standard mouse facility with controlled temperature (22 ± 2°C) and humidity (45 ± 10%) in individually ventilated cages containing sawdust bedding, a plastic cylinder and 2 paper tissues on the floor, food pellet and water ad libitum. Light cycle: 12:12 h. This study complies with all relevant ethical regulations concerning animal use, which was approved by the Veterinary office of the Canton of Fribourg, Switzerland, the Veterinary office (Landesuntersuchungsamt) of Rheinland-Pfalz, Germany, and the Veterinary office (Landesdirektion) in Sachsen, Germany.
Surgical procedures
For all surgical procedures, isoflurane (3% for induction, 1.5-2% for narcosis during the operation, Isofluoran, CP Pharma) was used for anesthesia. For analgesia, 0.1 mg/kg body weight buprenorphine (Temgesic; Essex Chemie) was administered by i.p. injection 1 h before surgery and every four hours until the evening on the day of surgery. The following day, 1 ml agarose gel containing 0.027 mg/ml buprenorphine was given twice (morning and evening). The mice were placed on a heat pad during the entire procedure until waking up from anaesthesia. To prevent dehydration of the eyes, a carbomer liquid eye gel (Lacryvisc, Alcon, or Viscotears, Novartis) was used preoperatively. Mice were shaved at the height of the hip for sciatic nerve crush lesion and the field of operation was cleaned and disinfected. Sciatic nerve crush lesions were carried out on three- to four-month-old adult mice (males and females) as follows (by a procedure that we have previously described in ref.10): An incision was made at the height of the hip and the sciatic nerve was exposed on one side. The nerve was crushed (5 times for 10 sec with crush forceps: Ref. FST 00632 − 11). The wound was closed using Histoacryl Tissue Glue (BBraun). After the operation, mice were wrapped in paper towels until recovery from anaesthesia.
Functional recovery experiments
Recovery of motor function was tested at 12, 19 and 30 dpl using the Rotarod test and the inverted grid test. For the Rotarod test, mice were placed four times on the Rotarod apparatus at a fixed speed of 15 revolutions per minute to test balance and motor coordination, at 12, 19 and 30 dpl. The duration of each trial was limited to 1200 sec. Latency to fall from the rotating beam was recorded and the average of the four trials was used for quantification. For the inverted grid test, mice were placed on a cage grid, that was slowly inverted and observed for 30 sec. The test was stopped if the animal fell. For each animal, the average count of grabs and missed steps as well as the average time the animals stayed attached to the inverted grid among the four trials were calculated as described62,63. All trials were video recorded for accurate analysis.
For both tests, mice were first trained three times and trials were separated by a 30 min recovery period. Recovery of sensory function was tested at 12, 19 and 30 dpl by toe pinch test: each toe of the rear foot on the right side (lesioned side) was pinched with equal pressure applied by the same experimenter using flat tip forceps. Immediate withdrawal was recorded as functional sensitivity of the pinched toe. In case no toe exhibited sensitivity, the same test was applied to toes of the contralateral side (uninjured side), which always resulted in immediate withdrawal.
Constructs
The c-Jun promoter-luciferase construct was generated by PCR on mouse genomic DNA: a 2.5 kb fragment corresponding to the mouse c-Jun promoter (-1702/+781: 94939678–94942161 Mus musculus strain C57BL/6J chromosome 4, GRCm39) was inserted into the pGL3-Basic vector (Promega) between Mlu I and Bgl II restriction sites. The construct was verified by sequencing.
pCEP4F HDAC8-FLAG expression construct was a kind gift from Dr. Edward Seto. pLentiLox 3.7 and pLenti-C-mGFP were acquired from ATCC and Origene, respectively. pCXN2-Flag-TRAF7 and pCXN2-Flag were a kind gift from Dr. Huanjie Yang.
Primary rat Schwann cell cultures
Purified primary rat SC cultures were derived from P2 Wistar rat sciatic nerves, as previously described in ref.64. SCs were then purified by sequential immunopanning in plastic dishes coated with a Thy1.1 antibody65. Identity and purity were checked for each primary preparation by immunofluorescence of SC-specific markers (Sox10, Oct6, Krox20, P0, MAG). SCs were grown in proliferation medium: DMEM containing 10% FCS (Gibco), 1:500 penicillin/streptomycin (Invitrogen), 4 µg/ml crude GGF (bovine pituitary extract, Bioconcept), and 2 µM forskolin (Sigma), at 37°C and 5% CO2/95% air. SC culture protocol mimicking SC demyelination and conversion into repair cells that occur after a PNS lesion was as follows (previously described in ref.10): SCs were first growth-arrested for 8 to 15 h in differentiation medium (DM): DMEM/F-12 (Gibco) containing 0.5% FBS (Gibco), 1:500 penicillin/streptomycin (Invitrogen), 100 µg/ml human apo-transferrin (Sigma), 60 ng/ml progesterone (Sigma), 1 µg/ml insulin (Sigma), 16 µg/ml putrescin (Sigma), 400 ng/ml l-thyroxin (Sigma), 160 ng/ml selenium (Sigma), 10 ng/ml triiodothyronine (Sigma) and 300 µg/ml bovine serum albumin (Sigma). Then 1 mM dbcAMP (Sigma) was added to induce differentiation and cells were incubated in this medium for another 3 days. The medium was then changed to DM only without dbcAMP, and RSCs were incubated in this medium for 3 days (differentiation mimicking adult SC stage). To mimic SC conversion into the repair phenotype, cells were then changed to proliferation medium and incubated in this medium for 1 day.
To inhibit HDAC8, 1 µM, 10 µM or 100 µM PCI-34051 (HDAC8 inhibitor, Selleckchem), 30 µM or 90 µM 1-Naphthohydroxamic acid (HDAC8 inhibitor, MedChemExpress) or their vehicle were added in conditions mimicking the conversion into the repair phenotype at the time of change to proliferation medium.
To inhibit JNK, 1 µM JNK-IN-8 (JNK inhibitor, MedChemExpress) or its vehicle were added to rat SCs in conditions mimicking the conversion into the repair phenotype the day before and at the time of change to proliferation medium for 24 h.
To mimic hypoxia, 250 µM CoCl2 was added to SCs in conditions mimicking the conversion into the repair phenotype 8h after the change to proliferating medium, and incubated for 16h. To induce hypoxia, RSCs were incubated in a hypoxic incubator (CO2-Incubator C16, Labotect) in conditions mimicking the conversion into the repair phenotype, at the time of change to proliferation medium and for 16 h at 5%CO2 and 1%O2/94%N2.
To inhibit the proteasome, 20 µM MG-132 (proteasome inhibitor, Hycultec) or its vehicle were added to rat SCs in conditions mimicking the conversion into the repair phenotype for 4 to 8 h.
HEK293T cells were obtained from ATCC and cultured in DMEM containing 10% FCS (Gibco) and 1:500 penicillin/streptomycin (Invitrogen) on poly-D-lysine (Sigma)-coated dishes.
Mycoplasma contamination was not tested in primary rat SCs or in HEK293T cells, because of the low incidence of mycoplasma contamination in primary cells, and because mycoplasma contamination results in inefficiency of transfection, which we did not observe in our primary rat SCs or in HEK293T cells.
Generation and use of lentiviruses
To produce lentiviral particles, HEK293T cells (ATCC) were co-transfected with each lentiviral construct: HDAC8 shRNA (TRCN00000877999), TRAF7 shRNA (TRCN0000302734), HIF1a shRNA (TRCN0000232221) or control shRNA (SHC001), together with the packaging constructs pLP1, pLP2 and pLP/VSVG (Invitrogen), using Lipofectamine 2000 (Invitrogen), according to the recommendations of the manufacturer (ViraPower Lentiviral Expression Systems Manual). Lentiviruses were added to primary rat SCs in DM and incubated for two days. Then, infected cells were selected by adding 2 µg/ml puromycin (Sigma) in DM for two days, before adding proliferating medium. HDAC8 shRNA, TRAF7 shRNA, HIF1-α shRNA and control shRNA lentiviral constructs were purchased from Sigma-Aldrich.
Transfection
Confluent primary rat SCs were transfected either in differentiating conditions or in conditions mimicking the conversion into the repair phenotype at the time of change to proliferation medium with Fugene 6 (Promega) at 5:1 ratio Fugene 6:DNA, according to the manufacturer’s recommendations, with the following modifications: the DNA was incubated in OptiMEM (Gibco) for 5 min at room temperature (RT) before addition of Fugene 6. The mix Fugene 6:DNA was then incubated for 30 min at RT before being added to the cells.
Luciferase gene reporter assay
Confluent primary rat SCs previously infected in differentiating conditions with HDAC8 shRNA or control shRNA in 12-well plates were transfected in conditions mimicking the conversion into the repair phenotype at the time of change to proliferation medium with Fugene 6 (Promega) at 3:1 ratio Fugene 6:DNA, according to the manufacturer’s recommendations, with the following modifications: the DNA was incubated in OptiMEM (Gibco) for 5 min at RT before addition of Fugene 6. The mix Fugene 6:DNA was then incubated for 30 min at RT before being added to the cells. Eight hours after transfection, 250 µM CoCl2 or vehicle (H2O) were added to the cells for 16 h. Cells were lysed 24 h after transfection in 110 µl Reporter Lysis Buffer (Promega) and assayed for luciferase activity. Forty microliters of lysate were subjected to two consecutive injections of each 25 µl luciferase substrate and values were recorded after an integration time of 2 and 10 sec, respectively. Efficiency of transfection was evaluated by measuring beta-galactosidase activity of a co-transfected beta-gal construct (Promega). Primary rat SCs were co-transfected with 1 µg of the luciferase construct pGL3b-c-Jun promoter or empty pGL3b (Promega), and 300 ng beta-gal construct (Promega). Luciferase activity was first normalized to beta-galactosidase activity and then normalized to the empty pGL3b luciferase control (transfected at the same ratio Fugene 6:DNA). For beta-galactosidase activity, we followed the instructions of the manufacturer (Promega). Briefly, 50 µl Assay Buffer 2x was added to 50 µl lysate, and beta-galactosidase activity was recorded after a 15–20 min incubation time at 37°C. Endogenous beta-galactosidase activity (as determined from cells only transfected with overexpressing construct at the same ratio Fugene 6:DNA) was subtracted.
RT-PCR
Isolation of RNA was carried out using Trizol reagent (Invitrogen) and cDNA was produced using SuperScript II Reverse Transcriptase (Thermo Scientific), according to the manufacturers’ recommendations. Quantitative real-time PCR analyses were performed with an ABI 7000 Sequence Detection System (Applied Biosystems) using FastStart SYBR Green Master (Roche), according to the manufacturer’s recommendations. A dissociation step was added to verify the specificity of the products formed. Primer sequences were as follows:
for rat c-Jun, forward 5´- CACCTCCGAGCCAAGAACTC-3´, reverse 5´ -CTGGACTGGATGATCAGGCG − 3´
for rat Gapdh, forward 5´-GTATCCGTTGTGGATCTGACAT-3´, reverse 5´-GCCTGCTTCACCACCTTCTTGA-3´
Western blot analysis
For all Western blots and immunoprecipitations on mouse sciatic nerve lysates, we collected the injured sciatic nerve from the lesion site to around 12mm distal to the lesion site (region where the nerve splits into the three branches of tibial, sural and common peroneal nerves). We collected the same region of the contralateral nerve as internal control for each animal. After perineurium removal, sciatic nerves were frozen in liquid nitrogen, pulverized with a chilled mortar and pestle, lysed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM NaVO4, 1 mM EDTA, 0.5% w/v sodium deoxycholate, and 0.5% Nonidet P-40) for 30 min on ice, and centrifuged to pellet debris. Supernatants were collected.
Cells were washed once in PBS, lysed in RIPA buffer for 15 min on ice, and centrifuged to pellet debris. Sciatic nerves and cell lysates were submitted to SDS-PAGE and analyzed by Western blotting.
Primary antibodies used:
HDAC8 (rabbit, 1:1000, Santa Cruz Biotechnology, cat. # sc-11405, lot # D0715), HDAC1 (rabbit, 1:1000, GeneTex, cat. # GTX100513, lot # 39471), HDAC2 (mouse, 1:1000, Sigma, cat. # H2663, lot # 092M4824V), HDAC3 (rabbit, 1:1000, GeneTex, cat. # GTX109679, lot # 39974), Oct6 (rabbit, 1:2000, kind gift from Dr. Dies Meijer, University of Edinburgh), Krox20 (rabbit, 1:500, kind gift from Dr. Dies Meijer, University of Edinburgh), Sox10 (mouse, 1:1000, Abcam, cat. # ab216020, lot #GR3272630-2), P0 (chicken, 1:5000, Aves Labs, cat. # PZO, lot # PZO877982), MBP (rat, 1:759, Bio-Rad, cat. # MCA409S, lot # 158446), c-Jun (rabbit, 1:1000, Cell Signaling, cat. #9165, lot # 13), phospho-c-Jun (rabbit, 1:500, Cell Signaling, cat. #3270, lot # 5), TRAF7 (rabbit, 1:1000, Proteintech, cat. # 11780-1-AP, lot # 00047291), TRAF7 (rabbit, 1:1000, ABclonal, cat. # A3095, lot # 5500008204), HIF1-α (rabbit, 1:1000, Novus Biological, cat. # NB100-479, lot # D108267-1), HIF1-α (mouse, 1:500, R&D Systems, cat. # MAB1536, lot # KRK0521051), VHL (rabbit, 1:1000, ABclonal, cat. # A0377, lot # 5500020894), Ubiquitin (P4D1) (mouse, 1:1000, Cell Signaling, cat. # 3936, lot # 19), phospho-JNK1/JNK2 (rabbit, 1:1000, Invitrogen, cat. # 44-682G, lot # 2465206), GAPDH (mouse, 1:5000, GeneTex, cat. # 28245, lot # 822203823), ß-Actin (mouse, 1:5000, Sigma, cat. # A5441, lot # 122M4782), Lamin A/C (mouse, 1:1000, Sigma, cat. # SAB4200236, lot # 055M4822V), eEF1A1 (rabbit, 1:5000, Abcam, cat. # ab157455, lot #GR231741-10).
All secondary antibodies were from Jackson ImmunoResearch: light-chain specific goat anti-mouse-HRP (horse radish peroxidase) and goat anti-rabbit-HRP, heavy-chain-specific goat anti-chicken-HRP.
Immunoprecipitation
For non-denaturing IPs, tissues or cells were processed as described in the Western blot section in the following RIPA buffer: 10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM NaVO4, 1 mM EDTA, 0.5% w/v sodium deoxycholate, 0.5% Nonidet P-40 and protease inhibitor cocktail (Thermo Scientific, cat. # 78443). Lysates were pre-cleared for 1 h at 4°C with 30 µl protein A/G PLUS agarose beads (Santa-Cruz Biotechnology). Antibodies were conjugated to 30 µl of agarose beads for 2 h at 4°C on a rotating platform. Then, 1 mL of cleared lysate was rotated for 2 h at 4°C with immunoprecipitating antibodies conjugated to the beads.
For denaturating IPs, tissues or cells were lysed in 10 mM Tris/HCl, pH 7.4, 1% SDS, boiled for 5 min at 95°C, and mixed with 9 volumes of RIPA buffer without SDS. Lysates were homogenized with a 20-gauge needle attached to a sterile plastic syringe, pre-cleared for 1 h at 4°C with 30 µl of agarose beads, and rotated overnight at 4°C with immunoprecipitating antibodies. The day after, 30 ul of agarose beads were added and samples were further rotated at 4°C for 2 h.
Two to six micrograms of the following antibodies were used per nerve or per 1x107 cells: HIF1α (mouse, R&D Systems, cat. # MAB1536, lot # KRK0522111), HIF1-α (rabbit, Novus Biological, cat. # NB100-479, lot # D108267-1), HDAC8 (sheep, R&D Systems, cat. # AF4359, lot # CAKS0120091), Normal Goat IgG control (goat, R&D Systems, cat. # AB-108-C, lot # ES41160812), Flag (mouse, Sigma, cat. # F1804, lot # SLBM0089V), GFP (rabbit, Abcam, cat. # ab290, lot # GR3431263-1). Rabbit anti-GFP, mouse anti-Flag and goat anti-IgG antibodies were used as control IPs.
Immunoprecipitates were pelleted, washed four times with RIPA buffer, eluted in 40 µl of Laemmli buffer and incubated at 75°C for 10 min. Analysis was done by Western blot.
Subcellular fractionation
Cytoplasmic and nuclear fractions were obtained by following the protocol described in ref.66. For fractionation on sciatic nerve lysates, we collected the injured sciatic nerve from the lesion site to around 12 mm distal to the lesion site (region where the nerve splits into the three branches of tibial, sural, and common peroneal nerves). We collected the same region of the contralateral nerve as internal control for each animal. After perineurium removal, sciatic nerves were pulverized with a chilled mortar and pestle and lysed in hypotonic buffer (20 mM Tris/HCl, pH7.4, 10 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, and 0.5 mM PMSF) for 30 min on ice.
Cells were harvested by trypsination, resuspended in PBS, centrifuged at 500 x g for 3 min at 4°C and resuspended in hypotonic buffer for 3 min on ice. Then, Nonidet P-40 was added to a final concentration of 0.1% and the lysates were further incubated for 3 min and centrifuged at 1000 x g for 3 min at 4°C. The supernatant (cytoplasmic fraction) was centrifuged a second time at 15000 x g for 3 min at 4°C to pellet remaining debris. The pellet (nuclear fraction), was resuspended in isotonic buffer (20 mM Tris/HCl, pH 7.4, 150 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.3% Nonidet P-40, 0.5 mM DTT, and 0.5 mM PMSF) on ice for 10 min, and centrifuged at 1000 x g for 3 min at 4°C. The pellet was then resuspended in RIPA buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM NaVO4, 1 mM EDTA, 1% w/v sodium deoxycholate, 1% Nonidet P-40, 1% SDS) with protease inhibitors (Thermo Scientific, cat. # 78443) for 30 min and centrifuged at 2000 x g for 3 min at 4°C. Supernatants were collected.
Immunofluorescence and imaging
Mouse sciatic nerves were fixed in situ with 4% PFA for 10 min, dissected, embedded in PolyFreeze (Sigma), and frozen at − 80°C. Sciatic nerve cryosections (5-µm thick) were first incubated with acetone for 10 min at − 20°C, washed in PBS, blocked for 30 min at RT in blocking buffer (0.3% Triton X-100/ 10% BSA/ PBS), and incubated with primary antibodies overnight at 4°C in blocking buffer. Sections were then washed 3 times in blocking buffer and secondary antibodies were incubated for 1 h at RT in the dark. Sections were washed again, incubated with DAPI for 5 min at RT, washed and mounted in Citifluor (Agar Scientific).
Cells were washed with PBS, fixed for 10 min with 4% PFA at RT, washed again in PBS, blocked, incubated with antibodies, DAPI, and mounted as described above.
For whole-mount sciatic nerve staining, nerves were processed as described67 with the following modifications: the perineurium was fully removed after 5 h fixation in 4% PFA and each nerve branch was separated from one another.
Primary antibodies: HDAC8 (sheep, 1:200, R&D Systems, cat. # AF4359, lot # CAKS0120091), TRAF7 (rabbit, 1:200, ABclonal, cat. # A3095, lot # 5500008204), c-Jun (rabbit, 1:200, Cell Signaling, cat. #9165, lot # 13), phospho-c-Jun (rabbit, 1:800, Cell Signaling, cat. #3270, lot # 5), F4/80 (rat, 1:200, GeneTex, cat. # GTX26640, lot # 821401311), NF-M (chicken, 1:1000, GeneTex, cat. # GTX85461, lot # 822102296), Stathmin-2 (rabbit, 1:1000, Novus Biologicals, cat. # NBP1-49461, lot # D-1), GAP43 (rabbit, 1:500, Abcam, cat. # ab75810, lot #GR3299539-7). All secondary antibodies were from Jackson ImmunoResearch. Photos were acquired using a Visitron VisiScope spinning disk confocal system CSU-W1. Fiji (version 2.9.0) and Adobe Photoshop (CC 20.0.4 Release) were used to process images. z-series projections (stated in the figure legends) are shown. Axonal regrowth was measured using NeuronJ software (ImageJ plugin freely available online with a user manual: http://www.imagescience.org/meijering/software/neuronj/). Parameters used were the same as described in the method validation article68: Neurite appearance: Bright, Hessian smoothing scale: 2.0, Cost weight factor: 0.7, Snap window size: 9 x 9, Path-search window size: 2,500 x 2,500, Tracing smoothing range: 5, Tracing subsampling factor: 5, Line width: 1.
Electron microscopy
Mice were killed with 150 mg/kg pentobarbital i.p. (Esconarkon; Streuli Pharma AG) and sciatic nerves were fixed in situ with 4% paraformaldehyde (PFA) and 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Fixed tissues were post-fixed in 2% osmium tetroxide, dehydrated through a graded acetone series as described69 and embedded in Spurr’s resin (Electron Microscopy Sciences). Semithin sections were stained with 1% Toluidine blue for analysis at the light microscope, and ultrathin sections (70-nm thick) were cut as described69. All analyses were done at 5 mm distal to the lesion site. No contrasting reagent was applied. Images were acquired using a Philips CM 100 BIOTWIN equipped with a Morada side mounted digital camera (Olympus).
Mass spectrometry analysis
Protein samples eluted from the beads in 1X Laemmli sample buffer were heated for 10 min at 75°C, treated with 1 mM DTT, and alkylated using 5.5 mM iodoacetamide for 10 min at RT. Samples were fractionated on 4–12% gradients gels and proteins were in-gel digested with trypsin (Promega) into four fractions per sample. Tryptic peptides were purified by STAGE tips and LC-MS/MS measurements were performed on a QExactive HF-X mass spectrometer coupled to an EasyLC 1200 nanoflow-HPLC (all Thermo Scientific) as described70. MaxQuant software (version 1.6.2.10)71 was used for analyzing the MS raw files for peak detection, peptide quantification and identification using a full length Uniprot mouse database (version April 2016). Carbamidomethylcysteine was set as fixed modification and oxidation of methionine was set as variable modification. The MS/MS tolerance was set to 20 ppm and four missed cleavages were allowed for Trypsin/P as enzyme specificity. Based on a forward-reverse database, protein and peptide FDRs were set to 0.01, minimum peptide length was set to seven, and at least one unique peptide per protein had to be identified. The match-between run option was set to 0.7 minutes. MaxQuant results were analysed using Perseus software (version 1.6.2.10)72.