Fig 1: Functionality of OX1R-CB2R heteromer in HEK-293T cells. (A,B,E) HEK-293T cells were transfected with the cDNA for the CB2 receptor (1 µg) (A), for the OX1 receptor (1 µg) (B) or both (E) and were pretreated with selective receptor antagonists (1 µM SB334867 for OX1R or 1 µM SR144528 for CB2R) or vehicle and treated with selective agonists (100 µM Orexin-A for OX1R and/or 100 nM JWH133 for CB2R) followed by 0.5 µM forskolin stimulation (15 min). Values are the mean ± S.E.M. of six independent experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis (** p < 0.01, *** p < 0.001) versus FK condition. (C,D,F) HEK-293T cells were transfected with the cDNA for the GCaMP6 calcium sensor (1 µg) and for the CB2 receptor (1 µg) (C), for the OX1 receptor (1 µg) (D) or both receptors (F). Receptors were activated using selective agonists (100 nM orexin-A for OX1R and/or 100 nM JWH133 for CB2R). When indicated, cells were pretreated with selective antagonists (1 µM SB334867 for OX1R or 1 µM SR144528 for CB2R). Cytosolic calcium readings were collected and data are the mean ± S.E.M. from six independent experiments. (G) ERK1/2 phosphorylation was determined in HEK-293T cells expressing the CB2 receptor (1 µg cDNA) and the OX1 receptor (1 µg cDNA). Results are expressed in percentage with respect to basal condition. Values are the mean ± S.E.M. of five independent experiments performed in triplicates. A resentative Western blot is shown (bottom). p-ERK1/2: phosphorylated ERKs; t-ERK1/2: total ERKs. * p < 0.05, *** p < 0.001. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis * p < 0.05 versus basal condition in ERK1/2 phospohorylation assays or versus FK condition in cAMP determination assays (dashed line).
Fig 2: Computational model of the heteromeric TM4/5 CB2R-OX1R interface. (A,B) The possible modulation (indicated by the black arrows) of an antagonist to a neighbor receptor through a network of aromatic residues connecting both orthosteric sites, viewed from the membrane. (A) the CB2R/JWH133-OX1R/SB334867 complex; The experimental structure of the OX1R antagonist SB334867 revealed the presence of two copies of the ligand in the binding pocket interacting antiparallelly with each other, which resulted in a large ligand volume near TM5. (B) The CB2R/SR144528-OX1R/orexin-A. Similarly, the CB2R antagonist SR144528 puts an aromatic ring at the region where most CB ligands place the alkyl chain (such as in JHW133), also towards TM5. The CB2R receptor is shown in gold and the OX1R receptor in blue. Small-molecule ligands and aromatic side chains connecting the pockets are shown as sticks; orexin-A with a thick green cartoon. Dotted boxes indicate TMs 4 and 5 of both receptors. In (C) detail of the CB2R/SR144528-OX1R/orexin-A complex viewed from the extracellular side superposing different orientations of the extracellular portion of TM5 and proximal region of the second extracellular loop (ECL2) (i) from the OX2R/orexin-B complex (PDB id 7L1U, cylinder and loop in light blue), which would clash sterically with the TM4 of the CB2R; and (ii) from the OX1R/SB334867 complex (PDB id 6TQ7, pink cylinder), which orients in a direction that does not directly face TM4 and better avoids the possible clashes with it TM4. Arrows indicate the movements of TM5 helices towards the TM5 modeled in the heteromer (see Section 4, dark blue) [33]. This complex shows a characteristic signaling where the OX1R selective antagonist potentiates the cannabinoid CB2R function in cAMP signaling while it blocks the indirect MAPK pathway. These interesting results are also observed in resting microglia. However, in activated microglia, the OX1R antagonist blockade became stronger, inducing a higher potentiation of CB2R cannabinoid signaling not only in cAMP but also in MAPK signaling. These results can be explained because in primary microglia from the transgenic AD model, the CB2-OX1 receptor heteromer expression is significantly higher than in equivalent cells from control animals, as shown by PLA. This finding suggests that the heteromer could be a target to combat neurodegeneration. We have previously shown that primary microglia from this AD model have an activated phenotype that is, likely, neuroprotective [38,39,40]. This hypothesis would explain why cognitive deficits do not appear at birth but later in life. Future research should address whether the CB2-OX1 receptor heteromer is a suitable target to skew microglia to the neuroprotective phenotype.
Fig 3: Functionality of CB2R and OX1R heteromer in the microglia from the APPSw,Ind mouse. Primary microglia from APPSw,ind (A,B) or age-matched control animals (B,D) were pre-treated with selective receptor antagonists (1 µM SR144528 for CB2 or 1 µM SB334867 for OX1 receptors) for 15 min and subsequently treated with selective agonists (100 nM JWH133 for CB2 or 100 nM orexin-A for OX1 receptors) in single or combined treatments. cAMP levels (A,B) were detected 15 min after forskolin addition. Values are the mean ± S.E.M. of seven different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test was used for statistical analysis (* p < 0.05 versus forskolin treatment). (C,D) ERK1/2 phosphorylation was analyzed using an AlphaScreen® SureFire® kit (Perkin Elmer). Values are the mean ± S.E.M. of five different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis (** p < 0.01, *** p < 0.001ersus untreated cells). (E) A proximity ligation assay (PLA) was performed in primary microglia from APPSw,Ind transgenic mice or age-matched controls, using specific primary antibodies against CB2R or against OX1R (1/100). Representative images corresponding to stacks of four sequential planes are shown. Cell nuclei were stained with Hoechst (blue) and receptor complexes appear as red dots. The number of red dots/cell was quantified using Andy’s algorithm Fiji’s plug-in (see Section 4). Scale bar: 15 µm. Values are the mean ± S.E.M. of five different experiments performed in duplicates. One-way ANOVA and Bonferroni’s multiple comparison post hoc test were used for statistical analysis (* p < 0.05 versus control).
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