Cytokine pathway disruption in a mouse model of schizophrenia induced by Munc18-1a overexpression in the brain

Assays were performed on adult (between 8 and 16-weeks-old) male C57BL6/CBA mice (National Institute for Agronomic Research, Madrid, Spain). Munc18-OE mice were generated as previously described by pronuclear microinjection [27]. WT mice (National Institute for Agronomic Research, Madrid, Spain) used as controls displayed the same background. A total of n = 5 animals per group and experiment were used. All animals were housed under a controlled temperature (22 ± 1°C), at a 12 hour light/dark cycle (lights on: 7:30 a.m. to 7:30 p.m.), and humidity (between 65 and 70%) with food and water ad libitum. Mice were killed by cervical dislocation and their brains were quickly frozen at −80°C. The day of the experiment, the whole cerebral cortex and the dorsal striatum of each mouse were dissected at 4°C. All animal procedures were performed in accordance with the European Directive for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (European Union Directive #86/606/EEC) and approved by the UPV/EHU Ethical Board for Animal Welfare (CEBA) and Genetically Modified Organisms (CEIAB). More recently, new regulations for research procedures with animals have been established (Royal Decree 53/2013).

Samples were homogenized on ice using a Sonic Dismembrator 60 (Thermo Fisher Scientific Inc., Massachusetts, United States) in a cytokine extraction buffer containing 0.05% bovine serum albumin (BSA), 0.02 mg/ml aprotinin, 0.02 mg/ml phenylmethanesulfonyl fluoride (PMSF), 0.05 mg/ml benzethonium chloride, 0.5% polyoxyethylene 20 sorbitan monooleate or Tween 20 (purchased from Sigma Aldrich^©, Missouri, United States), 0.37 mg/ml ethylenediaminetetraacetic acid (EDTA) and 0.023 mg/ml sodium chloride (NaCl) (purchased from Merck, Darmstadt, Germany) followed by centrifugation at 10,000 rpm for 10 minutes at 4°C. The cytokine levels were assessed in the supernatants by ELISA, according to the manufacturer’s instructions (eBioscience Inc., California, United States). All samples were assayed at a dilution 1:2 in the buffer and the assays sensitivities were respectively: 0.7 to 100 pg/ml IFN-γ, 8 to 1,000 pg/ml TNF-α, 8 to 1,000 pg/ml IL-1β, 2 to 200 pg/ml IL-2, 4 to 500 pg/ml IL-6 and 15 to 2,000 pg/ml CCL2 chemokine. The Beckman Coulter DTX 880 Multimode detector (Beckman Coulter Inc., Califronia, United States) was used to determine the concentration of cytokines. Sample cytokine levels were determined using standard concentration curves evaluated in duplicate for each plate. Data were normalized to sample protein concentration, as determined by the Pierce BCA Protein Assay Kit (Thermo Scientific Inc., Massachusetts, United States). Values were expressed in pg cytokine/mg protein.

Samples were homogenized on ice using a Sonic Dismembrator 60 in a lysis buffer containing 50 mM Tris-HCl buffer (pH = 7.4) (Sigma Aldrich^©, Missouri, United States), 150 mM NaCl, 5 mM EDTA and 1% Triton (Sigma Aldrich^©, Missouri, United States). The protein concentration was determined with Pierce BCA Protein Assay Kit, using BSA as a standard. Samples were diluted in a loading buffer containing 70 mM Tris-HCl (pH = 6.8), 2% sodium dodecyl sulfate (SDS), 120 mM dithiothreitol, 6% glycerol and 0.0025% bromophenol blue (purchased from Sigma Aldrich^©, Missouri, United States). Samples were denatured at 95°C for 5 minutes and 20 μg of total protein were loaded on each well. Proteins were separated by electrophoresis in SDS polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked for 1 hour at room temperature with Tris-HCl-buffered saline solution containing 5% nonfat dried milk (Sigma Aldrich^©, Missouri, United States) and 0.05% Tween 20. Membranes were then incubated at 4°C overnight with specific primary antibodies: rabbit monoclonal antibody anti-CD11b (NB110-89474, Novus Biologicals, Colorado United States) at a dilution of 1:1,000, rabbit polyclonal antibody anti-glial fibrillary acidic protein (GFAP) (ab7260-50, lot No. 740637, Abcam®, Cambridge, England) at a dilution of 1:50,000, and rabbit polyclonal antibody anti-NF-κB (NB100-78423, lot No. B128550, Novus Biologicals, Colorado, United States) at a dilution of 1:500. As a control for sample loading and protein transfer, the blots were stripped and re-probed with monoclonal antibody anti-β-actin (A5441, lot No. 061 M4808, Sigma Aldrich^©, Missouri, United States) at a dilution of 1:20,000. The membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Scientific, Massachusetts, Unites States) at a dilution of 1:20,000 in blocking solution for 1 hour at room temperature and developed with the chemiluminescence kit Immobilon™ Western HRP Sustrate (Millipore, Massachusetts, United States). Target proteins were quantified by densitometric scanning of specific immunoreactive bands (integrated optical density (IOD) units). The immunoreactivity value of the target proteins in each sample was first corrected with the corresponding value of β-actin and then calculated as a percentage of the corresponding mean value of the WT group, which was used as a reference value (100%).

Data were analyzed with GraphPad Prism™ version 5.0 (GraphPad Software, California, United States) and InVivoStat (InVivoStat Statistical Software, United Kingdom). Results were expressed as mean ± SEM. Data were analyzed by Student’s t-test. All tests were two-tailed. The level of significance was set to P <0.05.

In the brain cortex samples there were no differences between groups in the levels of IFN-γ (Figure 1a) or CCL2 (Figure 1b). However, cortical TNF-α (Figure 1c) and IL-2 levels (Figure 1d) were significantly lower in Munc18-OE mice than WT (t = 4.03, P <0.01; t = 3.62, P <0.01, respectively).

Interestingly, all the evaluated cytokines were significantly increased in the striatum of Munc18-OE mice compared to WT mice (Figure 2): IFN-γ (t = 2.91, P <0.05), CCL2 (t = 8.24, P <0.0001), TNF-α (t = 2.52, P <0.05), IL-2 (t = 8.51, P <0.0001).

The levels of IL-1β and IL-6 were under detection limits (8 pg/ml and 4 pg/ml, respectively) in both the cortex and striatum of Munc18-OE and WT mice (data not shown).

The microglia and macrophage marker CD11b was identified by western blot as a single band of 160 kDa, GFAP as a band of 55 kDa and the NF-κB p65 subunit as a band of 65 kDa.

In the cortical samples, we observed lower CD11b expression (Figure 3a) in Munc18-OE mice than WT mice (t = 3.01, P <0.05), whereas no differences were detected in the levels of GFAP between both groups (Figure 3b). Although no statistically significant changes were obtained in the NF-κB transcription factor p65 subunit between both groups of mice, a trend of lower levels in Munc18-OE mice was observed (Figure 3c).None of these inflammatory parameters protein levels differed in the striatum of Munc18-OE from those quantified in WT mice (Figure 4).