Methamphetamine Blocks Adenosine A2A Receptor Activation via Sigma 1 and Cannabinoid CB1 Receptors

Abstract

Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A_2A receptor (A_2AR). First, we noticed that methamphetamine does not affect A_2A functionality if the receptor is expressed in a heterologous system. However, A_2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB₁ receptor (CB₁R) and the sigma 1 receptor (σ₁R). Signaling via both adenosine A_2AR and cannabinoid CB₁R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A_2AR-CB₁R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A_2AR- and the CB₁R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σ₁R, alters the expression and function of two interacting receptors, A_2AR, which is a therapeutic target for neuroprotection, and CB₁R, which is the most abundant G protein-coupled receptor (GPCR) in the brain.

Keywords: G protein-coupled receptor GPCR; drug of abuse; heteromer; neuroprotection; striatal neurons.

Figure 1

The effect of methamphetamine on A_2AR–CB₁R colocalization and receptor–receptor interaction. Confocal microscopy images were obtained in HEK-293T cells transfected with 0.5 μg cDNA coding for CB₁R-YFP (A,D), with 0.5 μg cDNA for A_2AR-Rluc (B,E) or with 0.5 μg cDNA for both CB₁R-YFP and A_2AR-Rluc (C,F). Cells were treated with 1 μM methamphetamine (D–F) or vehicle (A–C) for 1 h. Receptors fused to Rluc (red) were identified by immunocytochemistry and receptors fused to YFP (green) were identified by their own fluorescence. Colocalization is shown in yellow. Nuclei were stained with Hoechst (blue). Images are taken near the surface of the slide to observe a higher portion of the plasma membrane. Scale bars: 20 μm. (G): Bioluminescence Resonance Energy Transfer (BRET) assays were performed in HEK-293T cells transfected with a constant amount of the cDNA coding for A_2AR fused to Rluc (1 μg) or the cDNA coding for D₁R fused to Rluc (0.5 μg to 4.5 μg) (red line) and increasing amounts of the cDNA coding for CB₁R fused to YFP (0.5 μg to 4.5 μg). Transfected cells were treated (green dots) or not (blue dots) with 1 μM methamphetamine. BRET is expressed as milli BRET units (mBU) and is given as the mean ± SEM of 8 different experiments grouped by amount of BRET acceptor.

Figure 2

Methamphetamine effect on A_2A–CB₁Het functionality: cAMP and ERK/Akt phosphorylation. HEK-293T cells co-transfected with cDNAs for A_2AR (0.5 µg) and for CB₁R (0.6 µg) were treated (30 min) with vehicle (A,C,E) or 1 µM methamphetamine (B,D,F) and subsequently treated with 500 nM of the A_2AR antagonist SCH 58261 (SCH) or with 500 nM of the CB₁R antagonist SR 141716A (SR). Receptors were activated using 100 nM of the A_2AR agonist CGS 21680 (CGS), with 200 nM of the CB₁R agonist, ACEA or both, before adding 0.5 µM forskolin (FK) or vehicle. The cytosolic cAMP levels (A,B) and the extracellular signal-regulated (ERK) 1/2 (C,D) or Akt (E,F) phosphorylation signals were determined. Results are expressed as percentage over basal and are the mean ± SEM of six experiments performed in triplicates. A one-way ANOVA followed by Bonferroni multiple comparison post hoc test showed a significant effect (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. basal condition; ### p < 0.001 vs. FK condition). In (C–F) a representative Western blot is shown (bottom). p-ERK indicates phosphorylated ERKs, t-ERK indicates total ERKs, p-AKT indicates phosphorylated AKTs.

Figure 3

Methamphetamine effect on A_2A–CB₁Het functionality: ß-arrestin-2 recruitment, dynamic mass redistribution (DMR) and Ca²⁺ mobilization. HEK-293T cells transfected with cDNAs corresponding to A_2AR-YFP (0.4 µg), CB₁R (0.3 µg) and ß-arrestin-2-Rluc (0.5 µg) (A,B) or with A_2AR (0.5 µg), CB₁R (0.6 µg) (C,D) and 6GCaMP calcium sensor (1.5 µg) (E,F) were treated (30 min) with vehicle (A,C,E) or with 1 µM methamphetamine (B,D,F) and subsequently treated with 500 nM of the A_2AR antagonist SCH 58261 (SCH) or with 500 nM of the CB₁R antagonist SR 141716A (SR). Receptors were activated using 100 nM of the A_2AR agonist CGS 21680 (CGS), with 200 nM of the CB₁R agonist ACEA or both. In (E,F), a portion of the cells were stimulated with 1 µM ionomycin as a positive control. The BRET data resulting from ß-arrestin-2 recruitment (A,B), time-dependent DMR representative traces (C,D) and time-dependent representative traces of cytoplasmic Ca²⁺ levels (E,F) are shown. In (A,B), BRET is expressed as milli BRET units (mBU) and is given as the mean ± SEM of six different experiments grouped by amount of BRET acceptor. A one-way ANOVA followed by Bonferroni multiple comparison post hoc test showed a significant effect over 100% (& p < 0.05, &&& p < 0.001 versus ACEA condition; %% p < 0.01 versus CGS condition).

Figure 4

σ₁R involvement in methamphetamine effects on A_2A and CB₁ receptors. HEK-293T cells transfected with cDNAs coding for A_2AR (0.5 µg) (A,B) or for CB₁R (0.6 µg) (C–F) in the absence (A–C,E) or presence (D,F) of siRNA for σ₁R (3 μg) to silence σ₁R were stimulated with 1 µM methamphetamine (Met, white bars) or vehicle (black bars) for 30 min. Cells were treated with 100 nM of the A_2AR agonist CGS 21680 (CGS) (A,B) or with 200 nM of the CB₁R agonist, ACEA (C–F). 0.5 μM of forskolin (FK) was used to determine Gi coupling to the CB₁R. Then, cAMP levels (A,C,D) and ERK1/2 phosphorylation (B,E,F) were determined and expressed as percentage over basal or, in the case of cAMP in CB₂R-expressing cells, over forskolin treatment. Values are the mean ± SEM (n = 5, in triplicates). One-way ANOVA followed by Bonferroni multiple comparison post hoc test showed a significant effect (* p < 0.05, *** p < 0.001 vs. basal condition, ### p < 0.001 versus FK condition). In (B,E,F), a representative Western blot is shown (bottom). In (G), FRET experiments were performed in HEK-293T cells transfected with 0.25 μg of cDNA for σ₁R-YFP and increasing amounts of cDNA for CB₁R-RFP (0.2 μg to 2 μg) or for A_2AR-RFP (0.1 μg to 1.5 μg). When indicated cells were pretreated with 1 µM methamphetamine. Results were grouped by amount of FRET acceptor. p-ERK indicates phosphorylated ERKs, t-ERK indicates total ERKs.

Figure 5

σ₁R involvement in methamphetamine effects on A_2A–CB₁Hets. HEK-293T cells were co-transfected with cDNAs coding for A_2AR (0.5 μg) and for CB₁R (0.6 μg) (A–F). When indicated, σ₁R (B,E) or calneuron-1 (Caln-1) (C,F) were silenced using 3 μg specific siRNA. Cells were pretreated with 1 μM methamphetamine (Met, white bars) or vehicle (black bars) for 30 min. GPCRs were activated either using 100 nM of the A_2AR agonist, CGS 21680 in naïve cells (CGS, (A–C)) or 200 nM of the CB₁R agonist, ACEA, in cells treated with 0.5 μM of forskolin (FK) (D–F). Then, cAMP levels (A–F) were determined and expressed as a percentage over basal (A_2AR) or forskolin (CB₁R). Values are the mean ± SEM of five different experiments performed in triplicates. One-way ANOVA followed by Bonferroni multiple comparison post hoc test showed significant effects (* p < 0.05, *** p < 0.001 vs. basal condition or ### p < 0.001 vs. FK condition). In (G), SRET experiments were performed in HEK-293T cells transfected with constant amounts of cDNAs for σ₁R-Rluc (0.5 μg) or D₁R-Rluc (0.33 μg) and for CB₁R-YFP (1 μg) and increasing amounts of cDNA for A_2AR-RFP (0.25 μg to 2.5 μg). Results were grouped by amount of SRET acceptor.

Figure 6

Methamphetamine effect on A_2A–CB₁Het expression and signaling in striatal neurons. (A,B) Proximity ligation assays (PLA) were performed in striatal neurons treated with 1 µM methamphetamine for two hours (acute, green bars), for one week (chronic, red bars) or with vehicle (control, blue bars) using primary antibodies specific for the A_2A, σ₁ and CB₁ receptors. As a negative control (NC), neurons were only treated with the primary antibody against CB₁R. See Methods for details on the steps in the PLA procedure. Confocal microscopy images (stacks of 4 consecutives planes) show heteroreceptor complexes as red clusters over Hoechst-stained nuclei (blue). Scale bar 20 µm (A). The bar graph (B) shows the number of red dots/neuron and the number above the bars indicates the percentage of neurons presenting red dots. Values are the mean ± SEM (n = 8–10). A two-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis (** p < 0.01, *** p < 0.001). cAMP levels (C) and ERK 1/2 phosphorylation (D) were determined in striatal neurons treated with methamphetamine 1 µM for two hours (acute, green bars) or for one week (chronic, blue bars) or vehicle (control, blue bars). When indicated cells were incubated with 500 nM of the A_2AR antagonist SCH 58261 (SCH) or with 500 nM of the CB₁R antagonist SR 141716A (SR). Receptors activated using 100 nM of the A_2AR agonist CGS 21680 (CGS) or with 200 nM of the CB₁R agonist ACEA. Forskolin (FK) was used to determine Gi coupling to the CB₁ receptor (C). The values are the mean ± SEM of six experiments performed in triplicates. A two-way ANOVA followed by Bonferroni’s multiple comparison post hoc test were used for statistical analysis (* p < 0.05, ** p < 0.01, *** p < 0.001 versus basal condition; ## p < 0.01 versus FK condition).