Angiotensin II type 1/adenosine A 2A receptor oligomers: a novel target for tardive dyskinesia

Abstract

Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells. Interestingly, by protein-protein docking and molecular dynamics simulations, we described that a stable heterotetrameric interaction may exist between AT1R and A2AR bound to antagonists (i.e. losartan and istradefylline, respectively). Accordingly, we subsequently ascertained the existence of AT1R/A2AR heteromers in the striatum by proximity ligation in situ assay. Finally, we took advantage of a TD animal model, namely the reserpine-induced vacuous chewing movement (VCM), to evaluate a novel multimodal pharmacological TD treatment approach based on targeting the AT1R/A2AR complex. Thus, reserpinized mice were co-treated with sub-effective losartan and istradefylline doses, which prompted a synergistic reduction in VCM. Overall, our results demonstrated the existence of striatal AT1R/A2AR oligomers with potential usefulness for the therapeutic management of TD.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

AT1R and A2AR physically interact in HEK-293T cells. (a) Co-distribution of AT1R and A2AR in HEK-293T cells. Transiently transfected HEK-293T cells with AT1RYFP (red) and A2ARCFP (green) were fixed and observed by confocal microscopy. Co-distribution (yellow) is shown in the merge image. Scale bar: 10 µm. (b). Direct interaction between AT1 and A2A receptors. BRET saturation curves in HEK-293T cells expressing A2ARRluc and AT1RYFP (blue) or GABAB2RRluc and AT1RYFP (red). Plotted on the x-axis is the fluorescence value obtained from the YFP, normalized with the luminescence value of Rluc-tagged vectors 10 min after benzyl-coelenterazine incubation. Results are expressed as mean ± SEM (n = 3, in triplicate).

Figure 2

Conformational arrangement of AT1R/A2AR heterotetramer. Model generated by protein-protein docking and 2 μs MD simulation. (a) Top view of tetramer (AT1R in blue, A2AR in green, losartan in purple and istradefylline in brown). (b) Side view of interaction between A2AR and AT1R.

Figure 3

A2AR expression potentiates AT1R functioning. (a) Representative Angiotensin II-mediated intracellular Ca2+ accumulation determined by Fluo4 assay. HEK-293T cells were transiently transfected with A2AR (black trace), AT1R (red trace) and A2AR + AT1R (blue trace). Cells were loaded with Fluo4-NW dye and challenged with Angiotensin II (50nM). The [Ca2+]i dynamics is shown as change in fluorescence of the Fluo4 signal (F) expressed as percentage of the maximal Ca2+ influx elicited by ionomycin (Fi) in each experimental conditions. (b) Quantification of the AT1R-mediated [Ca2+]i accumulation measured by Fluo4. The integrated area under the curve (AUC) of the normalized AT1R-mediated Fluo4 signal (F) is expressed as percentage of the corresponding ionomycin signal (Fi) for each transfection. The data are expressed as the mean ± SEM of three independent experiments performed in triplicate. The asterisk indicates statistically significant differences (**P < 0.01, ***P < 0.001; 1-way ANOVA with a Newman-Keuls post-hoc test).

Figure 4

Detection of AT1R and A2AR proximity in mice striatal sections. (a) Immunohistochemistry detection of AT1R and A2AR in mice striatum. Representative confocal microscopy images of AT1R (red) and A2AR (green) immunoreactivities in the striatum are shown. Lower panels show a magnification of the square area shown in the upper panel. Arrows indicate potential location of medium spiny neurons (MSN) cell bodies. Superimposition of images revealed a high receptor co-distribution in yellow (merge). Scale bars: 350 μm (upper panels) and 10 μm (lower panels). (b) Photomicrographs of dual recognition of AT1R and A2AR with P-LISA. Representative images from wild-type (left) and A2AR-KO (right) mice striatum. (c) Quantification of P-LISA signals for AT1R and A2AR proximity confirmed the significant difference of P-LISA signal density between wild type and A2AR-KO mice (***P < 0.001). Values in the graph correspond to the mean ± SEM (dots/nuclei) of at list three animals and 5 images per animal. Scale bar: 10 μm.

Figure 5

Effect of AT1R and A2AR blocking in the TD animal model. The effect of different doses of losartan (a) or istradefylline (b) on total vacuous chewing movements (VCM) in the reserpine-based animal model of TD in mice was monitored during 10 min. (c) Effects of sub-effective dose co-administration (i.p.) of losartan (0.05 mg/ml) and istradefylline (0.03 mg/ml) in the VCM of TD animal model. (d) Effect of losartan in the VCM of TD animal model performed in A2AR-KO mice. Results are represented as the mean ± SEM (n = 10 animals). *P < 0.05 compared to the vehicle group (one-way ANOVA, followed by Newman-Keuls test).