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. 2020 Aug 17;17(1):243.
doi: 10.1186/s12974-020-01908-z.

Angiotensin AT1 and AT2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia

Rafael Rivas-Santisteban  1   2 , Ana I Rodriguez-Perez  2   3 , Ana Muñoz  2   3 , Irene Reyes-Resina  1   2   4 , José Luis Labandeira-García  2   3 , Gemma Navarro  5   6 , Rafael Franco  7   8
Affiliations

Angiotensin AT1 and AT2 receptor heteromer expression in the hemilesioned rat model of Parkinson's disease that increases with levodopa-induced dyskinesia

Rafael Rivas-Santisteban et al. J Neuroinflammation. .

Abstract

Background/aims: The renin-angiotensin system (RAS) is altered in Parkinson's disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT1 and AT2 receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (AT1/2Hets), are present in the central nervous system. We assessed the functionality and expression of AT1/2Hets in Parkinson disease (PD).

Methods: Immunocytochemistry was used to analyze the colocalization between angiotensin receptors; bioluminescence resonance energy transfer was used to detect AT1/2Hets. Calcium and cAMP determination, MAPK activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify receptor expression in mouse primary cultures and in rat striatal sections.

Results: We confirmed that AT1 and AT2 receptors form AT1/2Hets that are expressed in cells of the central nervous system. AT1/2Hets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, AT1/2Hets that are expressed in both striatal neurons and microglia make possible that candesartan, the antagonist of AT1, increases the effect of AT2 receptor agonists. In addition, the level of striatal expression increased in the unilateral 6-OH-dopamine lesioned rat PD model and was markedly higher in parkinsonian-like animals that did not become dyskinetic upon levodopa chronic administration if compared with expression in those that became dyskinetic.

Conclusion: The results indicate that boosting the action of neuroprotective AT2 receptors using an AT1 receptor antagonist constitutes a promising therapeutic strategy in PD.

Keywords: Dyskinesia; G-protein-coupled receptor (GPCR); Heteromer; Levodopa; Neuroinflammation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Human AT1 and AT2 receptors interact in a heterologous expression system. a–c Immunocytochemistry assays were performed in HEK-293T cells expressing AT1R-YFP (1 μg cDNA), which was detected by its own yellow fluorescence (green), and AT2R-Rluc (1 μg cDNA), which was detected by a mouse anti-Rluc antibody and a secondary Cy3 anti-mouse antibody (red). Colocalization is shown in yellow. Cell nuclei were stained with Hoechst (blue). Scale bar: 20 μm. d BRET assays were performed in HEK-293T cells transfected with a constant amount of cDNA for AT2R-Rluc (0.9 μg) or σ1R-Rluc (0.75 μg) (as negative control) and increasing amounts of cDNA for AT1R-YFP (0.5 to 4 μg) or AT2R-YFP (0.1 to 4 μg) (as negative control). Values are the mean ± S.E.M. of 8 independent experiments performed in duplicates. e Schematic representation of BRET assay: the occurrence of energy transfer depends on the distance between the BRET donor (Rluc) and the BRET acceptor (YFP)
Fig. 2
Fig. 2
Functional characterization in HEK-293T cells expressing the AT1R-AT2R heteromer. HEK-293T cells were pretreated with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R) a–c Cytosolic calcium detection assay were performed in HEK-293T cells transfected with the cDNAs for an engineered calcium sensor, 6GCaMP (1 μg), AT1R (1 μg), and/or AT2R (1 μg). Values are the mean ± S.E.M. of 5 independent experiments performed in duplicates. d–f Intracellular cAMP levels were determined by TR-FRET as described in Methods. HEK-293T cells were transfected with cDNAs for AT1R (1 μg) and/or AT2R (1 μg). When Gi coupling was assessed, decreases in [cAMP] were determined using 0.5 μM forskolin added 15 min after the agonists stimulation. Values are the mean ± S.E.M. of 6 independent experiments performed in triplicates. In cAMP one-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (*p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment; +++p < 0.001 versus Ang II treatment; &&&p < 0.001 versus CGP-42112A treatment)
Fig. 3
Fig. 3
Functional characterization of AT1R-AT2R heteromer in HEK-293T cells. HEK-293T cells were transfected with cDNAs for AT1R (1 μg) and/or AT2R (1 μg). Cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R receptors) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R receptors). a–c ERK1/2 phosphorylation was analyzed using an AlphaScreen®SureFire® kit (Perkin Elmer). Values are the mean ± S.E.M. of 5 independent experiments performed in duplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables (*p < 0.05, **p < 0.01, ***p < 0.001; versus vehicle treatment (basal)). d–f DMR tracings represent the picometer-shifts of reflected light wavelength over time. Values are the mean ± S.E.M. 8 independent experiments performed in triplicates
Fig. 4
Fig. 4
AT1R-AT2R heteromer functionality in primary cultures of striatal neurons. For cAMP (a) or ERK1/2 phosphorylation (b), cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and/or 300 nM CGP-42112A for AT2R). Values are the mean ± S.E.M. of 5 independent experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (&p < 0.05 versus CGP-42112A treatment; *p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment in cAMP determinations or versus vehicle treatment (basal) in pERK determinations)
Fig. 5
Fig. 5
AT1R-AT2R heteromer functionality in microglial primary cultures treated with LPS and IFN-γ. a–c Expression of AT1R/AT2R heteromers in primary microglial cultures were determined by PLA, which was performed using specific primary antibodies against AT1 and AT2 receptors (confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue)). Scale bar: 20 μm. d Bar graph showing the percentage of red dots/cell respect non-treated cells; mean ± S.E.M of counts in 5–7 different fields (n = 5; **p < 0.01; Student’s t test versus the control condition). e, f Microglial cultures were incubated for 48 h in the absence (left) or in the presence (right) of 1 μM LPS and 200 U/mL IFN-γ. Microglial cells were pretreated (15 min) with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R receptors) and subsequently with the specific agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R receptors). cAMP (e-f) and ERK1/2 phosphorylation (g-h) were subsequently measured. Values are the mean ± S.E.M. of 5 independent experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (+p < 0.05 versus Ang II treatment in pERK determinations; and *p < 0.05, **p < 0.01, ***p < 0.001; versus forskolin treatment in cAMP measurements or versus vehicle treatment (basal) in pERK measurements)
Fig. 6
Fig. 6
AT1R-AT2R heteromer expression in brain striatal sections of Parkinson’s disease (PD) rat model. a–d PLA assays in striatal sections from the 6-OH-dopamine PD rat model, non-lesioned (a), lesioned (b), and lesioned plus chronically treated with l-DOPA and either lacking (c) or displaying (d) dyskinesias. Confocal microscopy images (stacks of 3 consecutive planes) show heteroreceptor complexes as red clusters and Hoechst-stained nuclei (blue). Scale bar: 20 μm. e Bar graph showing the percentage of red dots/cell. Data are the mean S.E.M . of counts in 9–12 different fields per animal (n = 4 per group). One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison tests were used to compare the red dots/cell values. The number of clusters (r) was the dependent variable and the four animal groups treatments were independent variables (***p < 0.001; versus lesioned condition, ++p < 0.01; versus l-DOPA non-dyskinesia condition)
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