Compounds. EHP-101 is a lipidic formulation of VCE-004.8 [(1′R,6′R)-3-(Benzylamine)-6-hydroxy-3′-methyl-4-pentyl-6′-(prop-1-en-2-yl) [1,1′bi(cyclohexane)]-2′,3,6-triene-2,5-dione)] . The chromatographic purity of VCE-004.8 in EHP-101 was 97.6%.
Animals. All experiments were performed in strict accordance with European Union (EU) and governmental regulations. Handling of animals was performed in compliance with the guidelines of animal care set by the EU guidelines 86/609/EEC, and the Ethics Committees on Animal Experimentation at the Cajal Institute (CSIC, Madrid) and the University of Córdoba (UCO, Córdoba, Spain) approved all the procedures described in this study (for EAE at Cajal Institute protocol number: 96 2013/03 CEEA-IC and for cuprizone model at UCO protocol number: 2018PI/02 (UCO). Measures to improve welfare assistance and clinical status as well as endpoint criteria were established to minimize suffering and ensure animal welfare. Briefly, wet food pellets are placed on the bed-cage when the animals begin to develop clinical signs to facilitate access to food and hydration. For the EAE model, female C57BL/6 mice were purchased from Harlan (Barcelona, Spain) and for the cuprizone model, male C56BL/6 mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). All animals were housed in the animal facilities under the following controlled conditions: 12 h light/dark cycle; temperature 20°C (±2°C) and 40-50% relative humidity with free access to standard food and water.
Induction and assessment of EAE. EAE was induced in C57BL/6 female mice at 6-8 weeks of age by subcutaneous immunization with Myelin Oligodendrocyte Glycoprotein Peptide Fragment 35-55 (MOG35–55) (300 µg: peptide synthesis section, CBM, CSIC, Madrid, Spain) and 200 µg of Mycobacterium tuberculosis (H37Ra Difco, Franklin Lakes, NJ, USA) in a 1∶1 mix with incomplete Freund's adjuvant (CFA, Sigma). On the same day and 2 days later, mice were injected intraperitoneally with 200 ng of pertussis toxin (Sigma) in 0.1 ml Phosphate buffer saline (PBS). Control animals (CFA) were inoculated with the same emulsion without MOG and they did not receive pertussis toxin. Treatment started at day 8 post-immunization when animals showed the first symptoms of the disease and consisted of daily oral EHP-101 administration (1, 5, 10 and 20 mg/kg) for the following 21 days. The mice were examined daily for clinical signs of EAE and disease scores were measured as follows: 0, no disease; 1, limb tail; 2, limb tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb and front limb paralysis; 5, moribund and death. All animals were sacrificed at 28 days for further analysis.
Cuprizone-induced demyelination. To induce demyelination, 8-week old C57BL/6 male mice were fed with 0.2% cuprizone (CPZ) TD.140800 diet (Envigo, Barcelona, Spain) for six weeks. Control group (no demyelination) was fed with control mouse TD.00217 diet (Envigo, Barcelona, Spain) for the entire period. To study the effect on remyelination, EHP-101 was administered once daily by oral gavage at 20 mg/kg from week six. For comparison, animals in the CPZ control group (maximal demyelination) received the same volume of vehicle by gavage. To study the dynamic effect of EHP-101 on remyelination, animals in each group were sacrificed at weeks 6, 7 (6+1 W), 8 (6+2 W) for further analysis.
Tissue processing. Mice were anesthetized by i.p. administration of a ketamine-xylazine or pentobarbital solution and they were transcardially perfused with saline 0.9%. The spinal cord was obtained by extrusion with saline. Brain and cervical spinal cord were immediately frozen and kept at -80ºC for RT-PCR analysis. The remaining brain and spinal cord were fixed in 4% paraformaldehyde in 0.1 M PBS, washed in 0.1 M PBS, cryoprotected with a 15% and then a 30% solution of sucrose in 0.1 M PBS, and frozen at -80°C. Free-floating brain and thoracic spinal cord sections (50 µm thick: Leica Microsystems CM1900 cryostat, Barcelona, Spain) were then processed for immunohistochemistry or immunofluorescence. In the case of cuprizone model whole brains were fixed, cryoprotected and frozen at -80ºC for further analysis.
Immunohistochemistry analysis. For IHC analysis, free-floating thoracic spinal cord (50 µm) sections were washed with 0.1M Phosphate buffer (PB). Endogenous peroxidase activity was inhibited with 3.3% hydrogen peroxide in methanol. The sections were blocked with 2.5% normal horse serum and then incubated overnight at 4ºC in blocking buffer with a rabbit anti-Teneurin 4 antibody (1:50: Novus Biological, Colorado, USA). Slides were incubated with ImmPRESS reagent (Vector Laboratories; Burlingame, Ca, USA) and then developed with diaminobenzidine chromogen (Merck, Darmstadt, Germany). Samples were photographed, digitalized using a Leica DFC420c camera and analyzed using Image J software. Myelin integrity was analyzed using the Hito CryoMyelinStain™ Kit (Gold phosphate complex Myelin Staining Kit) following manufacturer’s recommendation (Hitobiotech Corp., Kingsport, TN, USA).
Confocal microscopy analysis. For antigen retrieval, spinal cord or brain sections were boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0) or Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) (Sigma-Aldrich, St. Louis, MO, USA). The sections were washed three times in PBS. Nonspecific antibody-binding sites were blocked for 1 h at room temperature with 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA in PBS). Next, the sections were incubated overnight at 4 °C with the following primary antibodies diluted in PBS with 3% BSA: microglia cells were stained with a rabbit anti-ionized calcium binding adaptor molecule 1 (Iba-1) antibody (1∶1,000; Wako Chemical Pure Industry, Osaka, Japan), astrocytes were stained with a mouse anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), myelin basic protein was marked with a rabbit anti-Myelin Basic Protein (MBP) antibody (1:1000; Abcam, Cambridge, UK), oligodendrocytes were marked with a mouse anti-Olig2 (1:100, Santa Cruz, CA, USA) and a rabbit anti-GSTPi (1:250, Abcam, Cambridge, UK) axonal damage was determined with a mouse anti-Neurofilament H (NF-H) Nonphosphorylated antibody (SMI-32) (1:50; Biolegend, CA, USA). After extensive washing in PBS, slides were incubated with secondary antibodies for 1 h at room temperature in the dark. The immunoreactions were revealed using anti-rabbit Texas Red (1:100), anti-mouse/rabbit Alexa 488 (1:100) obtained from Thermo Fischer Scientific, Walthamm, MA, USA. The slides were then mounted using Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, Ca, USA). All images were acquired using a spectral confocal laser-scanning microscope LSM710, (Zeiss, Jena, Germany) with a 20×/0.8 Plan-Apochromat lens and quantified in 9-15 randomly chosen fields using ImageJ software (http://rsbweb.nih.gov/ij/).
RNA-Seq and bioinformatic analysis. Total RNA was isolated from spinal cord tissue using QIAzol lysis reagent (Qiagen, Hilden, Germany) and purified with RNeasy Lipid Tissue Mini kit (Qiagen). Then, samples were processed for high throughput sequencing using poly-A selection with the TruSeq Stranded mRNA Library Prep Kit (Cat. No. RS-122-2101, Illumina, San Diego, CA, USA). In brief, 1 µg of total RNA from each sample was used to construct a cDNA library, followed by sequencing on the Illumina HiSeq 2500 system with single end 50 bp reads and ~40 millions of reads per sample (n=3 per group). FASTQ files were pre-processed with Trimmomatic (v0.36)  and aligned to mouse genome assembly mm10 using HISAT2 (v2.1.0) . Then, counts per gene matrix were obtained with featureCounts (v1.6.1)  using the in-built RefSeq annotation for mm10 genome assembly and the differential expression analysis was carried out using DESeq2 (v1.20.0) , excluding genes with less than 15 counts across all samples. The functional over-representation analyses were performed using EnrichR  and clusterProfiler . All the P values were adjusted to control the false discovery rate (FDR) using the Benjamini and Hochberg approach . RNA-seq data have been deposited in the Gene Expression Omnibus databank (accession no. GSE131854).
Quantitative reverse transcriptase-PCR. Total RNA (1 µg) was retrotranscribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and the cDNA analyzed by real-time PCR using the iQTM SYBR Green Supermix (Bio-Rad) and a CFX96 Real-time PCR Detection System (Bio-Rad). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene was used to standardize mRNA expression in each sample. Gene expression was quantified using the 2-ΔΔCt method and the percentage of relative expression against controls was represented. The primers used in this study are described in Table 1.
Data analysis. All the in vivo data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) followed by the Tukey´s post-hoc test for parametric analysis or Kruskal-Wallis post-hoc test in the case of non-parametric analysis tests were used to determine the statistical significance. The level of significance was set at p˂0.05. Statistical analyses were performed using GraphPad Prism version 8.00 (GraphPad, San Diego, CA, USA).