AhR-deficiency as a cause of demyelinating disease and inflammation

The Aryl hydrocarbon Receptor(AhR) is among the most important receptors which bind pollutants; however it also regulates signaling pathways independently of such exposure. We previously demonstrated that AhR is expressed during development of the central nervous system(CNS) and that its deletion leads to the occurrence of a congenital nystagmus. Objectives of the present study are to decipher the origin of these deficits, and to identify the role of the AhR in the development of the CNS. We show that the AhR-knockout phenotype develops during early infancy together with deficits in visual-information-processing which are associated with an altered optic nerve myelin sheath, which exhibits modifications in its lipid composition and in the expression of myelin-associated-glycoprotein(MAG), a cell adhesion molecule involved in myelin-maintenance and glia-axon interaction. In addition, we show that the expression of pro-inflammatory cytokines is increased in the impaired optic nerve and confirm that inflammation is causally related with an AhR-dependent decreased expression of MAG. Overall, our findings demonstrate the role of the AhR as a physiological regulator of myelination and inflammatory processes in the developing CNS. It identifies a mechanism by which environmental pollutants might influence CNS myelination and suggest AhR as a relevant drug target for demyelinating diseases.


Lipidomic analysis of lipid extracts
UPLC-MS analysis: a quality control QC-1/1 was prepared by pooling 25 µL of each resuspended lipid extract and a fraction was diluted to one-third and one-sixth to give QC-1/3 and QC-1/6, respectively. All samples and QCs were placed in an autosampler at 10 °C and analyzed by reversed phase ultra-performance liquid chromatography (RP-UPLC) coupled to a hybrid quadrupole-orthogonal time-of-flight mass spectrometer equipped with an electrospray ionization (ESI) source (ACQUITY UPLC ® and SYNAPT ® G2 High Definition MS™ mass spectrometer, Waters, Manchester, UK). This technique will be referred to UPLC-ESI-MS hereafter. Lipids chromatographic separation was performed on a CSH C18 1.7 μm column (2.1 x 100 mm) sed at 50 °C. Lipids were eluted with 4:6 v/v acetonitrile/water containing 10 mM ammonium acetate (A) and 1:9 v/v acetonitrile/isopropanol containing 10 mM ammonium acetate (B) at a flow rate of 0.40 mL/min. The percentage of phase B increased from 40 % to 100 % (curve 6) over 10 min and was held at 100 % for 2 min before a rapid return to 40 % followed by an equilibration period of 2.5 min. Data were collected separately both in positive (ESI+) and negative (ESI-) ion modes. ESI source parameters were as follows: source temperature 120 °C, desolvation temperature 450 °C, cone gas flow 20 L/h, desolvation gas flow 800 L/h, capillary voltage for ESI+ ion mode 3,000V, for ESI-ion mode 2,400 V, cone voltage 30 V for ESI+ and 45 V for ESI-. The mass spectrometer was operated in the "MS resolution mode" of acquisition for both polarities. Centroided accurate mass spectra were acquired over the m/z range 50-1000 with a scan time of 0.1 s and an interscan delay of 0.01 s using a target mass resolution of 21,500 (FWHM as defined at m/z 500). Mass measurements were corrected during acquisition using an external reference (Lock-Spray™) comprising a 2 ng/µL solution of leucine enkephalin in acetonitrile/water 50:50, with an analyte-to-reference scan ratio of 20:1. Undiluted quality control samples were injected at the beginning of the batch to condition the chromatographic column. Samples were randomly analyzed and injected in triplicate. A mixture of 48 standard lipids belonging to 10 of the main lipid classes (fatty acids FAs, phosphatidic acids PAs, phosphatidylethanolamines PEs, phosphatidylserines PSs, phosphatidylcholines PCs, phosphatidylglycerols PGs, ceramides CERs, sphingomyelins SMs, diacylglycerols DGs and triacylglycerols TGs), at a final individual concentration of 1 µM, was also periodically injected throughout the analytical batch. The above mixture of lipids was used to create an in-house database compiling the retention time (t R ) values against equivalent number of carbon (ECN) for each main class and sub-class of lipids.
Data pre-processing: raw data files acquired on the UPLC-MS platform were converted to .mzData format using the MassWolf script under R. XCMS set up with parameters suitable for high resolution LC-MS data sets in centroid mode was used for feature detection, feature matching and retention time alignment across all mass spectra files. Subsequent to data preprocessing, a matrix listing peak areas associated to a unique m/z and retention time vs samples and QC, was generated. An ONs weight normalization followed by a total intensity normalization in both ionization modes were performed. The table was then filtered by selecting only variables with a coefficient of variation (CV) inferior to 30 % in the QC-1/1 and a correlation to the dilution (r) superior to 0.7 in the QC-1/1, QC-1/3 and QC-1/6. The final table was then exported for statistical analysis.
Univariate data analysis: A comparison between AhR KO and WT were assessed with the Wilcoxon (Mann-Whitney) test. The p-value were adjusted for multiple comparison by controlling the false discovery rate at a 5 % threshold.
Multivariate data analysis: Unsupervised and supervised multivariate analyses were performed using SIMCA-P+ software version 13.0.3 (Umetrics, Umeå, Sweden). A pareto scaling was applied to the variables prior to unsupervised principal component analysis (PCA) and supervised partial least squares-discriminant analysis (PLS-DA). A permutation test with 999 times permutations on the class labels was conducting to measure over-fitting of the model. Moreover, an orthogonal partial least squares-discriminant analysis (OPLS-DA) model was built based on the PLS-DA model. An S-plot was created from the OPLS-DA model to investigate the lipids related to the statistically significant differences between AhR KO and WT mice. A cross-validated analysis of variance (CV-ANOVA) was carried out on each supervised model to assess the statistical significance of group separation. Moreover, R2 and Q2 quality metrics superior to 0.4 traduced an acceptable biological model. Only variables with a VIP>1 and a p(corr)>|0.6| were considered significant.
Lipid structure assignment: The structure assignment of lipids was based on their m/z and their retention time. LIPID MAPS and METLIN data bases were requested using the mass accuracy with a tolerance window of 5 ppm. A linear relationship between retention time and ECN for each lipid class was determined allowing to determine a theoretical retention time for each lipid. A relative difference of the retention time below 15 % was considered acceptable to confirm the lipid annotation.

Complete western blot:
MAG protein (95 kDa) in whole tissue lysates of WT and AhR-KO mice optic nerves detected by Western Blot in 4 different experiments. Total actin (46 kDa) was used as a loading control (n=4/group).