Mice and treatments

All studies were approved by the State Agency for Nature, Environment and Consumer Protection (LANUV) NRW, Recklinghausen, Germany, # 81–02.04.2017.A084, # 81–02.04.2019.A211, # 81–02.04.2018.A413, and 81–02.2019.A003 and the local IACUC.

In the present study, we used female C57BL/6 mice wild-type mice and inducible neutral sphingomyelinase 2 (Smpd3) on a C57BL/6 background at an age of 8–12 weeks.

Glucocorticosterone (Sigma, Deisenhofen, Germany) was administered via the drinking water at 100 mg/L for 7 days. In the chronic unpredictable stress model, the mice were challenged for 7 days with unpredictable environmental stress, i.e., a reversal of the light/dark cycle, 3 h of 45° tilting of the cage twice each week, shaking at 125 rpm for 45 min, food deprivation for 14 h, predator sounds for 15 min, or wet cages for 1 h with two forms of stress per day in a randomized (unpredictable) order. Mice were sacrificed at day 7 after initiation of the stress. Blood was collected from the heart into heparin-coated needles and tubes immediately after the mice were sacrificed by cervical dislocation, centrifuged at 1300 × g for 5 min at 4 °C in an Eppendorf centrifuge; the plasma was removed and shock frozen in liquid nitrogen.

Embryonic stem cells allowing inducible deletion of neutral sphingomyelinase 2 (Smpd3) were obtained from the European Mouse Mutant Cell Repository (Helmholtz Center Munich, Germany). The gene symbol of the mice is Smpd3tm1a(EUCOMM)Hmg. Transgenic mice were generated, the Flp site was deleted resulting in a mouse strain in which exon 2 of Smpd3 is flanked by two lox sites. The mice were crossed with CreEr mice (Taconic, Cologne, Germany) and deletion of exon 2 was induced by 3 intraperitoneal injections (every 2nd day) of 100 μg/25 g body weight tamoxifen. Floxed mice (littermates) that were injected with the solvent, corn oil, only were used as controls.

Exosomes

To isolate exosomes from blood plasma, we centrifuged samples at 16,500 × g for 20 min. The supernatant was then passed through a 0.22-μm filter, and exosomes were harvested by centrifugation twice at 100,000 × g for 70 min. The final pellet was resuspended in phosphate-buffered saline (PBS). Exosomes were pelleted by centrifugation at 100,000 × g for 70 min, the supernatant was discarded, and the exosomes were resuspended and injected i.v. into healthy mice.

Exosomes in the blood plasma were quantified after ultracentrifugation using an ELISA kit against CD63 following exactly the instructions of the vendor (SBI System Biosciences, Exosome Antibodies and Elisa Kits).

Ceramide in exosomes was in vitro neutralized by incubation of the exosomes that were purified from 150 μL blood plasma as above with 0.5 μg purified IgM anti-ceramide antibody clones S58-9 or 0.5 μg control IgM or 1 μg recombinant neutral ceramidase for 45 min at 37 °C. Exosomes were again purified, the supernatants containing unbound anti-ceramide antibodies or ceramidase removed, exosomes were resuspended in PBS, and i.v. injected.

Measurement of ceramide by kinase assay

Exosomes were purified from 50 μL plasma, resuspended in 200 μL H2O and extracted it in 600 μL CHCl3:CH3OH:1 N HCl (100:100:1, v/v/v). The lower phase was collected, dried, and resuspended in 20 μL of a detergent solution (7.5% [w/v] n-octyl glucopyranoside and 5 mM cardiolipin in 1 mM diethylenetriamine-pentaacetic acid [DTPA]), bath sonicated for 10 min to obtain micelles and the kinase reaction was started by addition of 70 μL of a reaction mixture containing 10 μL diacylglycerol (DAG) kinase (GE Healthcare Europe, Munich, Germany), 0.1 M imidazole/HCl (pH 6.6), 0.2 mM DTPA, 70 mM NaCl, 17 mM MgCl2, 1.4 mM ethylene glycol tetraacetic acid, 2 mM dithiothreitol, 1 µM adenosine triphosphate (ATP), and 5 μCi [32P]γATP. The kinase reaction was performed for 60 min at room temperature under shaking at 300 rpm. The reaction was terminated by the addition of 1 mL CHCl3:CH3OH:1 N HCl (100:100:1, v/v/v), 170 μL buffered saline solution (135 mM NaCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 10 mM HEPES; pH 7.2), and 30 μL of a 100 mM ethylenediaminetetraacetic acid (EDTA) solution. The samples were vortexed, phases were separated, and the lower phase was collected, dried, and separated on Silica G60 thin-layer chromatography (TLC) plates with chloroform/acetone/methanol/acetic acid/H2O (50:20:15:10:5, v/v/v/v/v) and developed with a Fuji phosphorimager. Ceramide levels were determined by comparison with a standard curve; C16/18 and C22/24 ceramides were used as substrates.

To determine ceramide in the hippocampus and the prefrontal cortex, the brain was prepared and the hippocampus and the prefrontal cortex were carefully dissected. The tissues were homogenized in H2O and extracted and analyzed as above.

Ceramide quantification by liquid chromatography tandem-mass spectrometry (LC–MS/MS)

Samples were subjected to lipid extraction with 1.5 mL methanol/chloroform (2:1, v/v) that contained d18:1/17:0 ceramide (C17 Cer; Avanti Polar Lipids, Alabaster, USA) as internal standard. Extraction was facilitated by incubation at 48 °C with gentle shaking (120 rpm) overnight. To reduce interference from plasma glycerolipids, samples were saponified with 150 µL 1 M methanolic KOH for 2 h at 37 °C with gentle shaking (120 rpm) followed by neutralization with 12 µL glacial acetic acid. After centrifugation at 2200 g for 10 min at 4 °C, organic supernatants were evaporated to dryness using a Savant SpeedVac concentrator (Thermo Fisher Scientific, Dreieich, Germany). Dried residues were reconstituted in 200 µL acetonitrile/methanol/water (47.5:47.5:5 (v:v:v), 0.1% formic acid) and subjected to LC–MS/MS ceramide quantification applying the multiple reaction monitoring (MRM) approach. Chromatographic separation was achieved on a 1260 Infinity HPLC (Agilent Technologies, Waldbronn, Germany) equipped with a Poroshell 120 EC-C8 column (3.0 × 150 mm, 2.7 µm; Agilent Technologies) guarded by a pre-column (3.0 × 5 mm, 2.7 µm) of identical material. MS/MS analyses were carried out using a 6490 triple-quadrupole mass spectrometer (Agilent Technologies) operating in the positive electrospray ionization mode (ESI +). Chromatographic conditions and settings of the ESI source and MS/MS detector have been published elsewhere [53]. The following mass transitions were recorded (qualifier product ions in parentheses): m/z 520.5 → 264.3 (282.3) for C16 Cer, m/z 534.5 → 264.3 (282.3) for C17 Cer, m/z 548.5 → 264.3 (282.3) for C18 Cer, m/z 576.6 → 264.3 (282.3) for C20 Cer, m/z 604.6 → 264.3 (282.3) for C22 Cer, m/z 630.6 → 264.3 (282.3) for C24:1 Cer, and m/z 632.6 → 264.3 (282.3) for C24 Cer. Peak areas of Cer subspecies, as determined with MassHunter software (Agilent Technologies), were normalized to those of the internal standard (C17 Cer) followed by external calibration in the range of 1 fmol to 50 pmol on column. Ceramide quantities were normalized to the actual protein content used for lipid extraction. Ceramide levels are expressed as individual subspecies C16, C18, C20, C22, C24, and C24:1 or as the sum of these.

Loading of exosomes with ceramide

Exosomes isolated from 200 μL blood plasma as above were loaded with C22, C24, and C24:1 ceramide (Avanti Polar Lipids, USA) according to the mean of the amounts of the ceramides in pmol per mg protein in exosomes as determined for each ceramide species by mass spectrometry. We subtracted the amount of each ceramide species (in pmol/mg) present in exosomes from untreated mice from the corresponding values for glucocorticosterone- or CUS-treated mice, respectively, to obtain the amounts for loading the exosomes. The values were as follows: Mean ± SD of C22 ceramide from untreated mice was 12.5 ± 3 pmol/mg protein, C22 ceramide from glucocorticosterone-treated mice was 43.3 ± 9.8 pmol/mg, and C22 from CUS-treated mice was 33.1 ± 13.7 pmol/mg. The mean ± SD of C24 ceramide from untreated mice was 39.3 ± 10.8 pmol/mg protein, C24 ceramide from glucocorticosterone-treated mice was 284.3 ± 25.9 pmol/mg, and C24 from CUS-treated mice was 102.4 ± 14.3 pmol/mg. The mean ± SD of C24:1 ceramide from untreated mice was 17.6 ± 7.6 pmol/mg protein, C24:1 ceramide from glucocorticosterone-treated mice was 74.9 ± 14.3 pmol/mg, and C24:1 from CUS-treated mice was 53.4 ± 6.9 pmol/mg. We determined the protein concentration in an aliquot (25% of the total samples) of the exosomes. We then incubated the remaining exosomes (corresponding to 150 μL blood plasma) with 31 pmol/mg protein C22 ceramide + 145 pmol/mg C24 ceramide + 57 pmol/protein C24:1 ceramide to obtain exosomes that mimic exosomes released after treatment with glucocorticosterone and 21 pmol/mg protein C22 ceramide + 63 pmol/mg C24 ceramide + 36 pmol/protein C24:1 ceramide to obtain exosomes that mimic exosomes released after CUS. Ceramides were suspended in 0.9% NaCl and sonicated prior to use to get a homogenous suspension. Incubations of purified exosomes with ceramides were performed in 100 μL PBS for 60 min at 37 °C. Exosomes were centrifuged again at 100,000 × g for 70 min; the supernatant was discarded, resuspended in PBS, and injected i.v. into healthy mice.

Neutral sphingomyelinase activity

Mice were stressed with glucocorticosterone or chronic unpredictable stress or left untreated as above, sacrificed; the lung, liver, spleen, lymph nodes, yellow peritoneal fat, and brown interscapular fat were removed. Tissues were shock frozen, homogenized in 100 mM HEPES (pH 7.4), 5 mM MgCl2, 1% NP40, and each 10 μg/mL aprotinin/leupeptin using a tip sonicator, and aliquots were diluted tenfold in 100 mM HEPES (pH 7.4), 5 mM MgCl2, 0.2% NP40, and each 10 μg/mL aprotinin/leupeptin. The assay was started by addition of 0.05 μCi [14C]sphingomyelin (52 mCi/mmol; Perkin Elmer, #NEC 663010UC) per sample in 30 μL 100 mM HEPES (pH 7.4), 5 mM MgCl2, 0.2% NP40, and each 10 μg/mL aprotinin/leupeptin. The substrate [14C]sphingomyelin was dried for 10 min in a SpeedVac, resuspended in 100 mM HEPES (pH 7.4), 5 mM MgCl2, 0.2% NP40, and each 10 μg/mL aprotinin/leupeptin and sonicated for 10 min in a bath sonicator before it was added to the samples. The samples were incubated for 60 min at 37 °C with shaking at 300 rpm. Samples were then organically extracted in 4 volumes of CHCl3:CH3OH (2:1, v/v), vortexed, the samples were centrifuged, and an aliquot of the upper aqueous phase was scintillation-counted to determine the release of [14C]phosphorylcholine from [14C]sphingomyelin.

Immunohistochemical bromodeoxyuridine staining

Bromodeoxyuridine (BrdU, 2 mg/25 g body weight) was intraperitoneally injected 3-times, once every 2 h, at a dose of 75 mg/kg, starting 16 h before the mice were sacrificed. Mice were euthanized and perfused via the left heart for 2 min with 0.9% NaCl followed by a perfusion with 4% paraformaldehyde (PFA) buffered in PBS (pH 7.3) for 15 min. The brains were removed, fixed for an additional 36 h in 4% buffered PFA in PBS, embedded in paraffin. Paraffin-embedded sections were dewaxed, treated for 20 min with pepsin at 37 °C, washed, incubated for 2 h with 50% formamide in 300 mM NaCl and 30 mM sodium citrate (pH 7.0) at 65 °C, and washed twice in saline sodium citrate buffer. DNA was denatured for 30 min at 37 °C with 2 M HCl, washed, neutralized for 10 min with 0.1 M borate buffer (pH 8.5), washed, and blocked with 0.05% Tween 20 and 5% FCS in PBS (pH 7.4). The samples were then stained for 45 min at 22 °C with 5 μg/mL BrdU-specific antibodies (Roche, Mannheim, Germany, # 111,703,760,001), washed, and stained with Cy3-coupled F(ab)2 anti-mouse IgG antibody fragments (Jackson ImmunoResearch, West Grove, PA).

All sections were analyzed with a LEICA TCS SL fluorescence confocal microscope. Every tenth section of serial sections of the hippocampus was counted by an investigator blinded to the nature of the samples and the number of BrdU positive cells was calculated.

Endothelial cells

bEND3 cells (ATCC; LGC Standards, Wesel, Germany) were cultured in DMEM, supplemented with 10% fetal calf serum, 10 mM penicillin/streptomycin, 1 mM sodium pyruvate, an 2 mM L-glutamine. The cells (50,000 cells per sample) were incubated for 120 min with exosomes isolated from 100 μL blood plasma as above or left untreated. To determine PLD activity in endothelial cells, the medium was removed, the cells washed, homogenized, and the enzyme activity was measured as described below.

PLD activity

Phospholipase D activity was measured employing a commercial colorimetric activity assay (Abcam, #ab183306). To determine PLD in the hippocampus, the hippocampus was removed, homogenized, and incubated in assay buffer on ice for 15 min, insoluble material was removed by 5-min centrifugation at 14,000 rpm at 4 °C, and an aliquot of the lysate was mixed with the PLD substrate, the PLD probe, and the converter enzyme to generate the colored substrate according to the instructions of the vendor. Each sample was measured against a background sample that contained the same amount of lysate and reagents with the omission of PLD substrate. Samples were analyzed at OD 570 nm in a microplate reader (Fluostar Omega, BMG Labtech) in kinetic mode every 2 min. The enzyme activity was calculated and normalized for protein.

Phosphatidic acid analysis

Phosphatidic acid was measured in hippocampus extracts and in endothelial cells extracts using a commercial assay (PromoKine, #PK-CA577-K748) exactly following the protocol of the vendor. Hippocampus was removed, homogenized by tip sonication in 200 μL H2O and extracted in 750 μL CHCl3:CH3OH:12 N HCl (2:4:0.1, v:v:v) and each 250 μL CHCl3 and 1 M NaCl. Samples were vortexed and centrifuged for 5 min at 14,000 rpm to separate phases, the organic phase was dried in a SpeedVac, samples were dissolved in 5% Triton X-100, and the reaction was started by addition of a phosphatidic acid converter. The converter hydrolyzes phosphatidic acid to an intermediate that is converted to a fluorescence substrate in the presence of the provided enzyme mix. Samples were analyzed in a fluorescence reader at excitation/emission of 535/587 nm. The amount of phosphatidic acid was calculated by comparison to a standard curve.

Protein measurements

Protein was measured employing the BioRad Protein Assay Dye (#500,006) from aliquots of the hippocampus homogenates or lysates or exosome preparations to normalize the samples.

Behavioral studies

Behavioral testing was performed between 3:00 p.m. and 6:00 p.m. under diffuse indirect room light. All tests were performed on separate days. If appropriate, animals were tracked with a video camera (Noldus Systems, Worpswede, Germany). For the novelty-suppressed feeding test (latency to feed), mice were fasted for 24 h, placed in a new environment containing one piece of food on a small piece of white paper in the middle of the arena, and the time until mice began eating was recorded. For the light/dark box test, mice were placed in a dark and safe compartment that was connected via a 5-cm × 5-cm rounded-corner aperture to an illuminated, open, and thus aversive area. The time that the mouse spent in each of the separate compartments was measured. In the open-field arena test, the mice were released near the wall of a 50-cm × 50-cm white plastic cage with sidewalls 30 cm high. Animals were observed for 30 min, and the time during which the animal was more than 10 cm away from the wall was recorded. In the coat state test, the appearance of the coat (groomed vs unkempt coat) was scored on the head, neck, back, and ventrum with either 0 for normal status or 1 for unkempt status. For the forced swim test, mice were placed in a cylinder filled with water (21–23 °C) for 15 min. After 24 h, the mice were again placed in a water-filled cylinder for 6 min, and the time of mobility or immobility during the last 4 min of the second trial was recorded. Mice were judged immobile when they moved only to keep their heads above water.

Quantification and statistical analysis

Data are expressed as arithmetic means ± SD. For the comparison of continuous variables from independent groups with one variable (treatment), we used one-way ANOVA followed by post hoc Tukey test for all pairwise comparisons, applying the Bonferroni correction for multiple testing. The P-values for the pairwise comparisons were calculated after Bonferroni correction. We tested all values for normal distribution and similar variances. For the analysis of groups with 2 variables (treatment and genotype), we used one-way ANOVA and post hoc Tukey test for multiple comparison. Statistical significance was set at a P-value of 0.05 or lower (two-tailed). The sample size planning was based on the results of two-sided Wilcoxon-Mann–Whitney tests (free software: G*Power, Version 3.1.7, University of Duesseldorf, Germany).

To avoid bias during analysis, investigators were blinded to the identity results of histological samples and animals. Animals were randomly assigned to cages by a technician who was not involved in the experiments prior to the experiments. Cages were randomly assigned to the various experimental groups.