Protective Effect of N-Arachidonoyl Glycine-GPR18 Signaling after Excitotoxical Lesion in Murine Organotypic Hippocampal Slice Cultures

Abstract

N-arachidonoyl glycine (NAGly) is an endocannabinoid involved in the regulation of different immune cells. It was shown to activate the GPR18 receptor, which was postulated to switch macrophages from cytotoxic to reparative. To study GPR18 expression and neuroprotection after NAGly treatment we used excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). The effect of NAGly was also tested in isolated microglia and astrocytes as these cells play a crucial role during neuronal injury. In the present study, the GPR18 receptor was found in OHSC at mRNA level and was downregulated after N-Methyl-D-aspartate (NMDA) treatment at a single time point. Furthermore, treatment with NAGly reduced neuronal damage and this effect was abolished by GPR18 and cannabinoid receptor (CB)₂ receptor antagonists. The activation but not motility of primary microglia and astrocytes was influenced when incubated with NAGly. However, NAGly alone reduced the phosphorylation of Akt but no changes in activation of the p44/42 and p38 MAPK and CREB pathways in BV2 cells could be observed. Given NAGly mediated actions we speculate that GPR18 and its ligand NAGly are modulators of glial and neuronal cells during neuronal damage.

Keywords: N-arachidonoyl glycine; microglia; neuroprotection.

Figure A1

Representative Western blot membrane of Akt (60 kDa) after 30 min, 2 h and 24 h in BV2 cells. For better visualization contrast and brightness were enhanced. Western blots were performed on different membranes; ß-actin was used as a house-keeping protein.

Figure A2

In green IB4-positive microglia are shown in the dentate gyrus of OHSC. CTL: In control medium microglia cells showed in dentate gyrus a ramified morphology with extensive branching of cytoplasmic processes. After NMDA (10 µM) lesion microglial cells become amoeboid and their number increased. Microglia displayed a ramified morphology after incubation with 0.1 µM NAGly alone. Treatment with NMDA and NAGly (0.1 µM, 10 µM) led to amoeboid morphology of microglial cells and an increase in their numbers. Qualitatively no obvious differences in the morphology of microglia was found when NAGly treated groups were compared to those without NAGly. Scale bar = 50 µm.

Figure 1

Murine microglia, astrocytes, organotypic slices cultures and cell lines BV2, neuronal cell line HT22 express mRNA for GPR18. (a) The gpr18 mRNA was detected in BV2 microglia (BV2), primary microglia (MG), astrocytes (Ast), organotypic hippocampal slice cultures (OHSC) and hippocampal neuronal cell line HT22. The relative concentrations of mRNA were determined via qRT-PCR and normalized to ß-actin. Primary hippocampal neurons (n = 3) express gpr18 mRNA, astrocytes (n = 3) express significantly more gpr18 mRNA than microglia (n = 6, p < 0.05). Expression of GPR18 receptor in (b) OHSC and (c) primary cells. (b) OHSC: GPR18 (red) was found to be colocalized with GFAP (green) in murine astrocytes, NeuN (green) in primary hippocampal neurons and IB4 (green) in microglia (arrows). (c) Primary astrocytes, hippocampal neurons and microglia express GPR18 protein. DAPI (blue) was used to stain DNA in nuclei. Scale bar = 20 µm. The asterisk denotes significant results regarding the respective measurement indicated with the bar.

Figure 2

GPR18 expression measured over time. (a) The relative concentrations of mRNA were determined by qRT-PCR at time points 0 h, 30 min, 2 h, 6 h, 12 h, 24 h and 72 h. Cycle thresholds were normalized to ß-actin. The ΔCt of the control group at time 0 was used for quantification. Each value is presented as the mean (±SEM) of at least 3 replicates, each replicate contains 2 to 3 OHSCs (n_CTL0h = 3; n_NMDA0h = 3; n_CTL30′ = 3; n_NMDA30′ = 4; n_CTL2h = 4; n_NMDA2h = 4; n_CTL12h = 4; n_NMDA12h = 3; n_CTL24h = 3; n_NMDA24h = 3; n_CTL72h = 4; n_NMDA72h = 4). After 6 h of excitotoxical lesion gpr18 level decreased significantly (CTL: 1.09 ± 0.11, n_CTL6h = 4; NMDA: 0.6 ± 0.22, n_NMDA6h = 5; NMDA 6 h vs. CTL 6 h p < 0.05). (b) Changes in the expression of the GPR18 protein (arrows) over 48 h after NMDA treatment in OHSC at time points 0 h, 30 min, 1 h, 2 h, 6 h, 12 h, 16 h, 24 h and 48 h assessed in Western blot analysis. No significant changes over time after NMDA treatment were observed (n_CTL0h = 6; n_NMDA0h = 5; n_CTL30′ = 4; n_NMDA30′ = 3; n_CTL1h = 5; n_NMDA1h = 5; n_CTL2h = 4; n_NMDA2h = 6; n_NMDA6h = 7; n_NMDA6h = 10; n_CTL12h = 3; n_NMDA12h = 3; n_CTL24h = 5; n_NMDA24h = 6; n_CTL48h = 5; n_NMDA48h = 5). Furthermore, representative Western blot for all time points performed on different membranes are shown. GAPDH was used as house-keeping protein. The asterisk denotes significant results regarding the respective measurement indicated with the bar.

Figure 3

Treatment protocol. Some OHSC were kept in culture medium and served as controls (CTL). A further group was lesioned with NMDA and conduced as a positive control (NMDA). Cannabinoids were applied alone on day 14 to OHSC (NAGly) or following 4 h incubation with NMDA (NMDA + NAGly). Some OHSC were pre-incubated with an antagonist for CB₂ (AM630) or GPR18 (O-1918) for 15 min before adding NAGly. The fixation was performed on Day 17.

Figure 4

NAGly’s mediated neuroprotection. (a) NAGly (0.1 µM, 1 µM, 10 µM) is protective in organotypic hippocampal slice culture after NMDA (10 µM) damage. In the control group few PI (arrows) positive dead neurons were quantified in the region of dentate gyrus. Application of NMDA (4 h) led to excitotoxical damage in OHSC and was prevented by following incubation with NAGly for 72 h. The increase in the number of dead neurons was significantly reversed by incubation with NAGly (n_CTL = 17; n_NAGly = 5; n_NMDA = 28; n_{NMDA+NAGly0.1µM} = 19; n_{NMDA+NAGly1µM} = 6; n_{NMDA+NAGly10µM} = 15). (b) Application of antagonists, O-1918 (30 µM) or AM630 (10 µM) to NMDA lesioned OHSC had no impact on the amount of damaged cells (n_CTL = 17; n_NMDA = 28; n_NMDA+AM630 = 5; n_NMDA+O-1918 = 10). The neuroprotective effects of NAGly (0.1 µM and 10 µM) were effectively blocked using GPR18 antagonist, O-1918 and CB₂ antagonist, AM630 (n_{NMDA+NAGly0.1µM} = 19; n_{NMDA+NAGly10µM} = 15; n_{NMDA+NAGly0.1µM+O-1918} = 10; n_{NMDA+NAGly10µM+O-1918} = 11; n_{NMDA+NAGly0.1µM+AM630} = 10; n_{NMDA+NAGly10µM+AM630} = 6). (c) Representative pictures of OHSC. The damage in dentate gyrus and the effects of cannabinoids were assessed. In the control group very few PI positive cells are observed. In contrast, incubation with NMDA led to massive increase in amount of damaged neurons. NAGly reduced the number of PI positive cells; this effect was attenuated by co-application with AM630 or O-1918. Scale bar = 50 µm. The asterisk denotes significant results regarding the respective measurement indicated with the bar.

Figure 5

Mechanism behind NAGly mediated neuroprotection in primary microglia (a–c) and primary astrocytes (d). (a) Incubation with NAGly (1 µM; n_NAGly0.1µM = 2281) in microglia led to a 15% significant increase in ramification index in comparison to control (n_CTL = 2607). Higher concentration of NAGly (10 µM; n_NAGly10µM = 1253) showed a stronger rise of 46%. After incubation with LPS (n_LPS = 2021) in combination with NAGly (0.1 µM; n_{LPS+NAGly0.1µM} = 1002, 10 µM, _{nLPS+NAGly10µM} = 1628) no significant effects were observed. The ramification index is approximately 1 for amoeboid cells and tends to 0 for strongly ramified cells. (b) The area and (c) speed of primary wild type microglia cells were not altered after treatment with NAGly or ATP or both. The number of analyzed cells is the same for area and speed (n_CTL = 112; n_NAGly0.1µM = 114; n_NAGly10µM = 105, n_ATP = 128; n_{ATP+NAGly0.1µM} = 122; n_{ATP+NAGly10µM} = 90). (d) NAGly (n_0.1µM = 85; n_10µM = 55) alone did not change the level of GFAP expression significantly versus CTL (n_CTL = 87) in primary astrocytes. Treatment with LPS (10 ng/mL; n_LPS = 65) alone did not increase the activation of astrocytes measured as GFAP index. GFAP index was defined as ratio of GFAP positive astrocytes to all astrocytes. Treatment with LPS and NAGly in low concentration (n_{LPS+NAGly0.1µM} = 55) had no effect on the astrocytes activation. NAGly in high concentration (0.26 ± 0.03, n_{LPS+NAGly10µM} = 35; p < 0.05 vs. LPS) significantly reduced the GFAP positive ratio in comparison to LPS alone. (e) Representative pictures of primary microglia stained with IB4. (f) Representative images of astrocytes stained with GFAP. Scale bar = 50 µm. The asterisk denotes significant results regarding the respective measurement indicated with the bar.

Figure 6

NAGly (0.1 µM) and intracellular signaling cascades analyzed using Western blot. The BV2 cells and primary astrocytes (n > 3) were collected after treatment with NAGly and/or LPS (10 ng/mL) for 10 min, 30 min, 2 h and 24 h and screened for activation of signaling cascades (a) Akt, (b) CREB, (c) p38 MAPK or (d) p44/42 MAPK. (a) NAGly led to decrease in pAkt after 30 min in comparison to the control group (CTL 30 min) in BV2 cells (n_0h = 47; n_CTL10′ = 16; n_NAGly10′ = 11; n_CTL30′ = 15; n_NAGly30′ = 11; n_CTL2h = 12; n_NAGly2h = 9; n_CTL6h = 12; n_NAGly6h = 9; n_CTL24h = 14; n_NAGly24h = 10). (b–d) In the BV2 cells or astrocytes pCREB, p-p38 and p-p44/42 were not affected by NAGly. The asterisk denotes significant results regarding the respective measurement indicated with the bar.