Potentiation of cannabinoid signaling in microglia by adenosine A_2A receptor antagonists

2.1 Reagents

Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were purchased from SigmaAldrich (St. Louis, MO), CGS21680, SCH58621, JWH133, SR144528, and forskolin from Tocris Bioscience (Bristol, UK) and tetrahydrocannabinol (Δ⁹-THC) was provided by Phytoplant SL (Córdoba, Spain).

2.2 Expression vectors

Plasmids encoding for A_2ARRluc, A_2ARnRluc8, A_2ARcRluc8, CB₂RYFP, CB₂RnYFP, CB₂RcYFP, and GABA_BRluc and dopamine D₄YFP fusion proteins were used. The human version of adenosine A_2A receptor cDNA without its stop codon was obtained by PCR and subcloned to Rluc-containing vector (pRLuc-N1; PerkinElmer, Wellesley, MA) and nRluc8 and cRluc8-containing vectors (nRluc-pcDNA3.1 and cRluc-pcDNA3.1, previously created in the laboratory) using sense and antisense primers harboring unique restriction sites for HindIII and BamHI. The human version of cannabinoid CB₂ receptor and ghrelin GHS-R1a receptor cDNA without their stop codons were obtained by PCR and subcloned to Rluc-containing vector (pRLuc-N1; PerkinElmer, Wellesley, MA) using sense and antisense primers harboring unique restriction sites for HindIII and BamHI or subcloned to pEYFP-containing vector (pEYFP-N1; Clontech, Heidelberg, Germany) and nRluc8 and cRluc8-containing vectors (nRluc-pcDNA3.1 and cRluc-pcDNA3.1) using sense and antisense primers harboring unique restriction sites for BamHI and KpnI.

2.3 APP transgenic mouse model of Alzheimer's disease (AD)

APP_Sw,Ind transgenic mice (line J9; C57BL/6 background) expressing human APP695 harboring the FAD-linked Swedish (K670N/M671L) and Indiana (V717F) mutations under the PDGFβ promoter were obtained by crossing APP_Sw,Ind to nontransgenic (WT) mice (Mucke et al., 2000).

2.4 Cell culture and transient transfection

HEK-293T cells were grown in DMEM medium (Gibco, Paisley, Scotland, UK) supplemented with 2 mM l-glutamine, 100 U/mL penicillin/streptomycin, MEM nonessential amino acids solution (1/100) and 5% (v/v) heat inactivated fetal bovine serum (FBS) (Invitrogen, Paisley, Scotland, UK). Cells were maintained in a humid atmosphere of 5% CO₂ at 37°C. Cells were transiently transfected with the PEI (PolyEthylenImine, SigmaAldrich) method as previously described (Navarro, Borroto-Escuela, Fuxe, & Franco, 2015). To prepare mice striatal primary microglial cultures, brain was removed from C57/BL6 mice between 2 and 4 days. Microglia cells were isolated as described in (Newell et al., 2015) and grown in DMEM medium supplemented with 2 mM l-glutamine, 100 U/mL penicillin/streptomycin, and 5% (v/v) heat inactivated FBS (Invitrogen).

2.5 Immunocytochemistry

HEK-293T cells or primary microglial culture cells seeded in coverslips were treated with vehicle, with 100 nM A_2AR agonist CGS21680, or with 100 nM CB₂R agonist JWH133 for 3 days. On day 2, 1 μM LPS plus 200 U/mL IFN-γ treatment or vehicle were added. On day 4, cells were fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 20 mM glycine before permeabilization with PBS-glycine containing 0.2% Triton X-100 (5 min incubation). Cells were treated for 1 hr with PBS containing 1% bovine serum albumin. HEK-293T cells were labeled with mouse anti-Rluc antibody (1/100; Millipore, Darmstadt, Germany) and subsequently treated with Cy3 anti-mouse (1/200; Jackson ImmunoResearch [red]) IgG (1 hr each). Microglial cells were labeled with a goat anti-Iba1 (1/100; ab107159; Abcam), a mouse anti-iNOS (1/100; NOS2 [C-11]: sc-7,271; SCB) or a mouse anti-arginase I (1/100; 610708; BD Biosciences) antibody, and subsequently treated with Cy3-conjugated anti-goat (1/200; 705-165-147; Jackson ImmunoResearch [red]) or anti-mouse (1/200; 715–166-150; Jackson ImmunoResearch [red]) IgG secondary antibodies (1 hr each). Nuclei were stained with Hoechst (1/100; SigmaAldrich). Samples were washed several times and mounted with 30% Mowiol (Calbiochem). Samples were observed in a Leica SP2 confocal microscope (Leica Microsystems).

2.6 Bioluminescence resonance energy transfer (BRET) assays

For BRET assays, HEK-293T cells were transiently co-transfected with a constant amount of cDNA encoding for A_2ARRluc or GHS-R1aRluc and with increasing amounts of cDNA corresponding to CB₂YFP. For BRET with BiFLC (Bimolecular fluorescence and bioluminescence complementation), HEK-293T cells were transiently co-transfected with a constant and equal amount of cDNA encoding for A_2ARnRluc and A_2ARcRluc (A_2ARnRluc and cRluc or nRluc and A_2ARcRluc for negative controls) and with increasing and equal amounts of cDNA corresponding to CB₂nYFP and CB₂cYFP (nYFP and CB₂cYFP or CB₂nYFP and cYFP for negative controls). Forty-eight hours after transfection cells were adjusted to 20 μg of protein using a Bradford assay kit (Bio-Rad, Munich, Germany) using bovine serum albumin for standardization. To quantify protein-YFP expression, fluorescence was read in a Mithras LB 940 equipped with a high-energy xenon flash lamp, using a 10 nm bandwidth excitation filter at 485 nm reading. For BRET and BRET with BiFLC measurements, readings were collected 1 min after the addition of 5 μM coelenterazine h (Molecular Probes, Eugene, OR) using a Mithras LB 940, which allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify protein-Rluc expression, luminescence readings were performed (using a Mithras LB 940) 10 min after 5 μM coelenterazine h addition. The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-Cf, where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the donor construct expressed alone in the same experiment. GraphPad Prism software (San Diego, CA) was used to fit data. BRET is expressed as milli BRET units, mBU (net BRET × 1,000).

2.7 cAMP determination

Two hours before initiating the experiment, HEK-293T cells or microglia primary cell-culture medium was exchanged to serum-starved DMEM medium. Then, cells were detached, suspended in growing medium containing 50 μM zardaverine and plated in 384-well microplates (2,500 cells/well), pretreated (15 min) with the corresponding antagonists (SCH58621 for A_2AR and SR144528 for CB₂R) -or vehicle- and stimulated with agonists (CGS21680 for A_2AR and JWH133, and Δ⁹-THC for CB₂R) (15 min) before adding 0.5 μM forskolin or vehicle. Readings were performed after 1 h incubation at 25°. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA). Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).

2.8 ERK phosphorylation assays

To determine ERK1/2 phosphorylation, 40,000 transfected HEK-293T cells/well or 50,000 microglial cells/well were plated in transparent Deltalab 96-well microplates and kept at the incubator for 48 hr (transfected HEK-293T cells) or 12 days (microglia culture cells). Two to four hours before initiating the experiment, the medium was substituted by serum-starved DMEM medium. Then, cells were pretreated at 25°C for 10 min with the specific antagonists (SCH58621 for A_2AR and SR144528 for CB₂R) or vehicle in serum-starved DMEM medium and stimulated for an additional 7 min with the specific agonists (CGS21680 for A_2AR and JWH133, and Δ⁹-THC for CB₂R) or vehicle. Cells were then washed twice with cold PBS before addition of lysis buffer (20 min treatment in constant agitation). 10 μL of each supernatant were placed in white ProxiPlate 384-well microplates and ERK 1/2 phosphorylation was determined using AlphaScreen®SureFire® kit (Perkin Elmer) following the instructions of the supplier and using an EnSpire® Multimode Plate Reader (PerkinElmer).

2.9 ß-arrestin 2 recruitment assays

Arrestin recruitment was determined as previously described (Navarro et al., 2016, 2018b, 2018c). Briefly, BRET experiments were performed in HEK-293T cells 48 hr after transfection with the cDNA corresponding to the A_2AR-YFP or CB₂R-YFP and 1 μg cDNA corresponding to ß-arrestin 2-Rluc in the presence of A_2AR in coexpression experiments. Cells (20 μg protein) were distributed in 96-well microplates (Corning 3600, white plates with white bottom) and were incubated with specific antagonists (SCH58621 for A_2AR and SR144528 for CB₂R) for 15 min and stimulated with agonists (CGS21680 for A_2AR and JWH133, and Δ⁹-THC for CB₂R) for 10 min prior the addition of 5 μM coelenterazine h (Molecular Probes, Eugene, OR). After 1 min of adding coelenterazine H, BRET between ß-arrestin 2-Rluc and receptor-YFP was determined and quantified. The readings were collected using a Mithras LB-940 (Berthold Technologies, Bad Wildbad, GE) that allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify protein-Rluc expression, luminescence readings were also performed 10 min after adding 5 μM coelenterazine h.

2.10 Dynamic mass redistribution (DMR) assays

Cell mass redistribution induced upon receptor activation were detected by illuminating the underside of a biosensor with polychromatic light and measuring the changes in the wavelength of the reflected monochromatic light that is a sensitive function of the index of refraction. The magnitude of this wavelength shift (in picometers) is directly proportional to the amount of DMR. HEK-293T cells and microglial primary cultures were seeded in 384-well sensor microplates to obtain 70–80% confluent monolayers constituted by approximately 10,000 cells per well. Previous to the assay, cells were washed twice with assay buffer (HBSS with 20 mM HEPES, pH 7.15) and incubated 2 hr with assay-buffer containing 0.1% DMSO (24°C, 30 μL/well). Hereafter, the sensor plate was scanned and a baseline optical signature was recorded for 10 min before adding 10 μL of the specific antagonists (SCH58621 for A_2AR and SR144528 for CB₂R) that were recoded for 30 min followed by the addition of 10 μL of specific agonists (CGS21680 for A_2AR and JWH133, and Δ⁹-THC for CB₂R); all test compounds were dissolved in assay buffer. The cell signaling signature was determined using an EnSpire® Multimode Plate Reader (PerkinElmer) by a label-free technology. Then, DMR responses were monitored for at least 5,000 s. Results were analyzed using EnSpire Workstation Software v 4.10.

2.11 In situ proximity ligation (PLA) assays

Microglia primary cultures grown on glass coverslips were fixed in 4% paraformaldehyde for 15 min, washed with PBS containing 20 mM glycine to quench the aldehyde groups and permeabilized with the same buffer containing 0.05% Triton X-100 (5 min treatment). After 1 hr incubation at 37° with blocking solution, cells were treated with specific antibodies against A_2A or CB₂ receptors (mouse anti-A_2AR -1/100- (Millipore, Darmstadt, Germany); or rabbit anti-CB₂R -1/100- (Santa Cruz Technologies)). Cells were processed using the PLA probes detecting mouse and rabbit antibodies (Duolink II PLA probe anti-Rabbit plus and Duolink II PLA probe anti-Mouse minus) and nuclei were stained with Hoechst (1/200; SigmaAldrich). Coverslips were mounted using Mowiol solution. Samples were observed in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an apochromatic 63X oil-immersion objective (N.A. 1.4), and 405 nm and 561 nm laser lines. For each field of view a stack of two channels (one per staining) and 3 to 4 Z stacks with a step size of 1 μm were acquired. Quantification of cells containing one or more red spots versus total cells (blue nucleus) and, in cells containing spots, the ratio r (number of red spots/ cell), were determined by Duolink Image tool software.

2.12 Data analysis

The data in graphs are the mean ± SD GraphPad Prism software version 5 (San Diego, CA) was used for data fitting and statistical analysis. One- or two-way ANOVA followed by post-hoc Bonferroni test were used depending of the number of factors. Two factors were considered in the case of ligand treatments in resting or activated cells (two levels) or in the case of ligand treatments in microglia from control or transgenic mice (two levels). When pair of values were compared, the Student's t test was used. Significant differences were considered when p < .05.

3.1 A_2A and CB₂ receptors interact in a heterologous expression system

In preliminary assays we noticed that a potentially negative control for protein–protein interactions in BRET assays turned out to give a positive signal. The partner receptors were A_2AR and CB₂R, for which cDNAs constructs of fusion proteins containing the human full-length version and either Rluc or YFP were obtained. The cDNAs coding for - A_2ARRluc and CB₂R-YFP were transfected into HEK-293T cells and immunocytochemical studies were performed as indicated in Material and Methods. The results showed a high degree of colocalization; images were taken near the surface of the slide to observe a higher portion of cells' plasma membrane (Figure 1a). Confocal microscopy may suggest that receptors are close in the cell but cannot confirm receptor interactions. Accordingly, energy transfer studies were performed to establish a potential direct interaction. To achieve this objective, BRET studies were performed in cells expressing A_2ARRluc and CB₂RYFP. The results led to a saturable BRET curve (BRET_max of 322 ± 3 mBU and BRET₅₀ of 55 ± 4) indicative of direct interaction, while the negative control performed by replacing A_2ARRluc by GABA_BRluc and CB₂RYFP by the dopamine D₄YFP fusion proteins led to a linear unspecific signal (Figure 1b). It should be noted that BRET_max was higher than that obtained for other interacting GPCRs, something that indicates either a high level of interacting proteins or a close apposition of donor (Rluc) and acceptor (YFP) in the quaternary structure of the heteromer. There is evidence that heteromers may arrange into tetramers, and this possibility for adenosine and cannabinoid receptors was assessed by BRET combined with molecular complementation. For this purpose constructs were generated using A_2AR receptors fused to either the N-terminal or the C-terminal half of the Rluc and CB₂R receptors fused to either the N-terminal or the C-terminal half of the YFP. Transfection with the 4 cDNAs led to a positive and saturable BRET signal (BRET_max of 154 ± 15 mBU and BRET₅₀ of 103 ± 9) suggesting that in the HEK-293T-based heterologous system A_2A and CB₂ receptors may form tetramers (Figure 1c). For negative control, assays were performed in cells expressing a combination of receptors and fusion proteins, and no BRET signal was detected in any case (see Figure 1d).

3.2 Functional characterization of A_2AR-CB₂R tetrameric complex in HEK-293T cells

To assess the properties of A _2A-CB ₂Hets, forskolin-induced intracellular cytosolic cAMP determination, β-arrestin 2 recruitment, ERK1/2 phosphorylation and Dynamic Mass Redistribution (DMR) experiments were performed. DMR is a label-free technique used in GPCR research as it measures rearrangement of cell components (leading to mass redistribution) that is mostly mediated by G protein activation (Simon et al., 2016).

Before characterizing A_2A-CB₂Het functionality, the effect of receptor activation was assayed in single-transfected cells (using 0.7 μg cDNA for A_2AR or 1 μg cDNA for CB₂R). As expected due to Gs coupling, activation of A_2AR with a selective agonist, CGS21680, led to a significant increase in [cAMP] (Figure Sup 1A). This effect was counteracted when in the same experiment cells were pretreated with a selective A_2AR antagonist, SCH58621. In A_2AR-expressing cells neither the CB₂R selective agonist, JWH133, or antagonist, SR144528 (alone or in combination with the A_2AR ligands), exerted any significant effect (Figure S1a). Similar results were obtained in MAPK signaling, β-arrestin 2 recruitment and label-free DMR assays (Figure S1b–d). In summary, A_2AR-expressing cells responded only to the receptor agonist, whose effect was not blocked by CB₂R ligands but by the selective A_2AR antagonist.

Experiments performed in cells expressing CB₂R led to similar results that is, the selective cannabinoid receptor agonist induced CB₂R function was not blocked by A_2AR ligands but by the CB₂R antagonist, SR144528 (Figure S1f–h). Therefore, cAMP assays were consistent with canonical coupling of CB₂R to G_i and, therefore, the agonist, JWH133, led to a decrease in the cytosolic levels induced by 0.5 μM forskolin activation. The effect was nullified by preincubation with SR144528 but not by treatment with A_2AR ligands. (Figure S1e). When experiments on ERK1/2 phosphorylation, β-arrestin 2 recruitment and DMR were performed, JWH133 induced a significant signal that was counteracted by the antagonist SR144528 but not by the A_2AR selective ligands.

A_2A-CB₂Het functionality was addressed in cells transfected with cDNAs for A_2AR (0.5 μg) and CB₂R (0.75 μg). Selective agonists, CGS21680 and JWH133, in single treatment or in combination, were used for receptor activation and cytosolic cAMP levels, ERK1/2 phosphorylation, β-arrestin 2 recruitment and DMR were determined. cAMP determination assays were consistent with G_s coupling to A_2A and with G_i coupling to CB₂. However, G_i coupling was negligible upon simultaneous treatment using the two agonists, that is, the signal observed was similar to that obtained using CGS21680 (Figure 2a). This result indicates that A_2AR activation blocks CB₂R/G_i-mediated signaling. Next, we investigated how the antagonist of the A_2AR, SCH58621, blocked agonist's effect. SCH58621 indeed blocked the effect of CGS21680 but, remarkably, it did potentiate the G_i-mediated effect elicited by JWH133. What these results suggest is that binding of SCH58621 to the A_2AR potentiates CB₂ receptor signaling in the A_2A-CB₂Het context. When analyzing the effect of CB₂R antagonist SR144528, we observed a blockade of the JWH133-induced signal and no effect on CGS21680 action. Similar results were observed when DMR, that is mostly mediated by G protein-dependent pathways, was measured. Costimulation with both agonists induced an effect similar to that achieved by CGS21680, thus indicating a blockade of A_2AR over CB₂R/G_i-mediated actions. Consistently, the A_2AR antagonist produced a potentiation in CB₂R signaling (Figure 2d). To determine whether this heteromer print also holds for other signaling outputs, ERK1/2 phosphorylation and β-arrestin recruitment were assayed. Interestingly, pretreatment with the A_2AR antagonist blocked the CGS21680 induced effect, but did not potentiate the cannabinoid signaling (Figure 2b,c). Thus, A_2AR antagonists are increasing CB₂R signaling via inhibition the adenylyl cyclase and in DMR, but not in other signal transduction pathways in which a partial cross-antagonism was detected. In both pERK1/2 and β-arrestin 2 recruitment determinations, treatment with both agonists led to a lack of synergism or additive effects (comparing with data obtained in single-agonist treatments).

3.3 Effect of the main psychoactive component of Cannabis sativa, Δ⁹-THC, on A_2A-CB₂Het functionality

The two main endocannabinoids, which act via cannabinoid receptors, are 2-arachidonoyl glycerol (2-AG) and anandamide. We selected the psychoactive component of Cannabis sativa, Δ⁹-THC, to test its effect when it activates the CB₂R in an A_2A-CB₂Het context. Δ⁹-THC at high concentrations interacts with GPR55 but this receptor is not expressed in significant amount in untransfected HEK-293T cells (Balenga et al., 2014; Moreno et al., 2014). The effect of the natural compound was first tested in CB₂R-expressing cells and the results were fully consistent with those previously reported (Navarro, Borroto-Escuela, et al., 2018a). Δ⁹-THC treatment over A_2AR and CB₂R coexpressing cells showed non-significant effect on Gi-adenylyl cyclase signaling (Figure 2a). To demonstrate if treatment with A_2AR antagonists did potentiate the Δ⁹-THC-induced signal, our first attempt was using the label-free DMR technique. Figure 2e shows that A_2A-CB₂Het-expressing cells responded to Δ⁹-THC and that the signal was blocked by CB₂R antagonist but potentiated by the A_2AR antagonist. Moreover, costimulation of cells with Δ⁹-THC and CGS21680 produced a similar effect than that observed in single CGS21680 treatment. A similar approach, but analyzing pERK levels or β-arrestin recruitment, showed that simultaneous treatment with Δ⁹-THC and the A_2AR agonist did not result in any synergic or additive effect and that Δ⁹-THC-induced effect was partially blocked by the A_2AR antagonist (partial cross-antagonism).

3.4 Occurrence and functionality of A_2A-CB₂Hets in primary cultures of activated microglia

Since microglial activation leads to upregulation of CB₂ and A_2A receptor expression (Carlisle, Marciano-Cabral, Staab, Ludwick, & Cabral, 2002; Concannon, Okine, Finn, & Dowd, 2015, 2016; Correa et al., 2010; Gyoneva et al., 2014; Madeira, Rashid, Ambrósio, Santiago, & Langmann, 2018; Maresz, Carrier, Ponomarev, Hillard, & Dittel, 2005; Mecha, Carrillo-Salinas, Feliú, Mestre, & Guaza, 2016; van der Putten et al., 2009), our next aim was to identify the heteromer in microglia and any differential expression of A_2A-CB₂Hets in resting versus activated microglial cells. In resting microglia the different signal transduction read-outs led to results that were similar to those found in cotransfected HEK-293T cells. By measuring cytosolic cAMP levels (Figure 3a), ERK1/2 phosphorylation (Figure 3b) and DMR (Figure 3c,d) we observed that both A_2AR and CB₂R agonists produced a characteristic signal and a non-additive effect when used in combination; the signal observed was similar to that obtained with CGS21680. The A_2AR antagonist enhanced CB₂R signaling via G_i (cAMP determinations and DMR read-outs), and a partial cross-antagonism was detectable in ERK1/2 phosphorylation. On the other hand, the CB₂R antagonist had no effect over A_2AR signaling when measuring cAMP levels and MAPK phosphorylation but decreased DMR label-free readings. Interestingly, Δ⁹-THC treatment induced a significant decrease in cAMP levels. Taking into account the data obtained in cells only expressing the CB₂R, it is likely that this effect is derived from Δ⁹-THC stimulation of cannabinoid CB₁ receptors present in these cells (Navarro, Borroto-Escuela, et al., 2018a). However, in ERK1/2 phosphorylation and DMR techniques, coestimulation showed a non-additive effect. Moreover, while the A_2AR antagonists potentiated Δ⁹-THC effect on DMR, SCH58621 partially blocked MAPK pathway activation. Off target effects due to expression of proteins able to be affected by THC such as GPR55, GPR18 or peroxisome proliferator-activated receptor gamma cannot be ruled out.

To analyze the A_2A-CB₂ heteroreceptor complex formation and function in activated microglia, primary cultures were treated for 48 hr with 1 μM LPS and 200 U/mL IFN-γ. The phenotype of activated microglia was assessed by immunocytochemical detection of a microglia marker, ionized calcium binding adaptor molecule 1 (Iba-1) and of M1 and M2 markers, respectively, arginase 1 (Arg-1) and inducible nitric oxide synthase (iNOS). The results demonstrated an important increase in iNOS and Arg-1 fluorescence signal when cultures were previously treated with CGS21680, while a small non-significant decrease in Iba-1 fluorescence was observed by JWH133 pretreatment (Figure 4b–e). No differences in cell shape due to the different treatments were observed upon using a maker or membranes (AlexaFluor 488-conjugated wheat germ agglutinin). These results suggest that A_2AR activation is proinflammatory and probably detrimental for microglial survival and, in contrast, that CB₂R agonists exert a protective action. Cell survival was analyzed by estimating cell death in activated cells treated with A_2AR or CB₂R ligands. Cell death due to LPS plus IFN-γ treatment (circa 40%) was slightly increased when cells were treated with the A_2AR agonist and significantly decreased when the CB₂R agonist was used (Figure 4a). These results indicate a protective role of CB₂R activation on survival of activated microglial cells.

Results of cAMP determination obtained in microglia treated with LPS plus IFN-γ, showed increases upon treatment with the A_2AR agonist whereas CB₂R activation by JWH133 engaged G_i in forskolin-treated cells, being the effect higher than that obtained in resting cells (Figure 3e). A_2AR activation blocked CB₂R/G_i signaling since cells treated with both agonists responded as cells treated with CGS21680. The heteromer print was also detectable in activated cells. In fact, pretreatment with the A_2AR antagonist blocked A_2AR-mediated signal while it potentiated CB₂R-mediated signal using JWH133. The CB₂R antagonist blocked CB₂R function but had no effect over A_2AR activation (similar results were obtained in resting cells).

DMR results (Figure 3g,h) were similar to those obtained in cAMP determination assays, but a lack of synergy/additive effect was observed upon determination of pERK levels (Figure 3f). Furthermore a partial cross-antagonism was detectable in activated cells; in fact, the A_2AR antagonist partially blocked CB₂R signaling towards the MAPK pathway. The results obtained in activated microglia show an increase in A_2AR and CB₂R signaling compared to resting cells and the occurrence of A _2A-CB ₂Hets as deduced from identification of the heteromer print(s).

3.5 A_2A-CB₂Het expression and function in the APP_Sw,Ind transgenic mouse model of Alzheimer's disease

Alzheimer's disease (AD) pathological hallmarks include β-amyloid plaques and aggregates of aberrantly phosphorylated tau protein (intracellular neurofibrillary tangles). Whereas the role of reactive microglia in the brain of patients is not well understood, it is known that the expression of A_2A and CB₂ receptors is upregulated in the brain of patients. Such upregulation comes mainly from expression in microglial cells. We took advantage of the APP_Sw,Ind transgenic mouse AD model to better understand the role of A_2A-CB₂Hets in activated microglia. A_2A-CB₂Het expression was addressed in hippocampal microglia obtained from two-day-old pups that were individually cultured. Each culture was assigned to either APP_Sw,Ind or control after genotyping. To check for receptor complex expression, an in situ proximity ligation assay (PLA) was developed using specific antibodies against A_2AR and CB₂R. While only 12% of cultured cells from control animals showed red fluorescence dots, which correspond to clusters of heteroreceptor complexes, the number of cells stained in cells from APP_Sw,Ind transgenic animals increased to 58%. Furthermore, the number of red dots/cell significantly increased from 1.8 to 4.3 dots/cell (Figure 5a,b). These results indicate a clear increase in A_2A-CB₂Het expression in the microglia isolated from the APP_Sw,Ind transgenic animals. Consequently, we questioned about the functional implication of this complex in the first AD stages; it should be noted that young APP_Sw,Ind animals do not display any cognitive impairment.

As per cAMP determination assays, A_2A-CB₂Het functionality when CGS21680 was used was similar in the two groups, transgenic (white bars) and controls (black bars). In contrast, the effect of the CB₂R agonist, JWH133, was potentiated in samples from transgenic animals. In agreement with the features of A_2A-CB₂Het, coactivation produced a similar signal to that obtained by only A_2AR activation (Figure 5c–e). When cells were pretreated with the selective antagonists, the heteromer print was detected; in fact, SCH58621 not only inhibited CGS21680-mediated but also potentiated JWH133-mediated signaling. This was evident in cells from control and APP_Sw,Ind animals. In contrast, SR144528, the CB₂R antagonist, blocked JWH133- but not CGS21680-induced signaling (Figure 5c,e). These results were qualitatively similar in cAMP assays when comparing transgenic and control animals. Results concerning DMR were also similar to those here described for cAMP determination assays (Figure 5d–f).

In contrast the comparison of data from cells obtained from both types of animals led to differential finings in pERK1/2 assays. Analysis of MAPK activation in cells from transgenic mice showed a marked effect of JWH133 and a lack of additive effects upon costimulation. Again there was no potentiation of CB₂R function induced by the A_2AR antagonist (Figure 5g–j). Similar results were obtained when the CB₂R was stimulated with Δ⁹-THC instead of using JWH133. Finally, the partial cross-antagonism in the pERK signal was detected in cells from transgenic and from control animals.

To sum up, the results obtained demonstrate a marked increase in CB₂R-mediated effect that correlates with an increase in A_2A-CB₂Het expression in microglia isolated from the transgenic APP_Sw,Ind mice model. In this model the CB₂R signaling in microglia is reduced when A_2AR is activated but, importantly, potentiated via G_i coupling in the presence of A_2AR antagonists.