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

Figures

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  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)

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  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…

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  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

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  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)

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  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…

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  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)