Interaction between Angiotensin Type 1, Type 2, and Mas Receptors to Regulate Adult Neurogenesis in the Brain Ventricular–Subventricular Zone

Figures

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  Figure 1. Generation of neurospheres from the ventricular-subventricular zone (V-SVZ) of young mice. (A) Photomicrographs showing floating neurospheres obtained from wild-type mice. (B) Representative bands for angiotensinogen (ANG), Ang II type-1 (AT1) and type-2 (AT2) receptors, and β-actin obtained by RT-PCR in neurospheres (NF). Homogenates of striatum (ST) were used as a positive control. (C) ANG was detected in neurosphere culture medium by HPLC and visualized by western blot. ANG (250 μg/mL) was used as a positive control (C+). Bar graphs showing the number (D) and diameter (E) of neurospheres after treatment with angiotensin II (Ang II), AT1 receptor antagonist (ZD7155 or candesartan), and AT2 receptor antagonist (PD123319). Histograms showing the number of neurospheres after treatment with AT2 receptor agonists (CGP42112A or C21) and the AT2 antagonist PD123319 ((F); treatment) or in cultures derived from neurospheres pre-treated with AT2 agonists and reseeded in the absence of any treatment ((G); neurospheres derived from cultures pre-treated with AT2 agonists; pre- = previously treated with). All culture data were obtained from at least three separated experiments. Data are…

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  Figure 2. (A) FACS analysis of neural stem cell (NSC), neural progenitor (NP), and neuroblast (NB) populations in the ventricular–subventricular zone (V-SVZ) within the Lin- population discriminated by levels of CD24 and GLAST. (B) FACS analysis of NSCs within the Lin-/CD24-/low cells discriminated by levels of GLAST and CD9. (C) Histogram showing the quantification of NSCs, NP, and NB FACS analyses 2 h after the last dose of 15 days C21 treatment; the levels of these three populations of cells raised up after 15 days of treatment. (D) Histogram showing FACS analyses quantification of NSC, NP, and NB populations 1 month after C21 treatment showing no differences between experimental groups. Data are means ± standard error of the mean (SEM). * p < 0.05 (Student’s t-test).

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  Figure 3. Bromodeoxyuridine (BrdU (A,B) or doublecortin (DCX)-immunoreactive (-ir) (C,D) cells in the ventricular–subventricular zone (V-SVZ) of wild-type (WT) mice, AT1 receptor-deficient mice (AT1-KO), and AT2-deficient mice (AT2-KO). Number of BrdU (E) and DCX-ir (F) cells in the V-SVZ of WT mice, WT mice treated with the AT1 antagonist candesartan (Cand), WT mice treated with the AT2 agonist C21, and AT2-KO mice untreated or treated with candesartan for 15 days. Data are means ± SEM. * p < 0.05 relative to WT controls (one-way ANOVA and Bonferroni post hoc test); in figures (E,F), $ p < 0.05 relative to AT2-KO group (Student’s t-test). ANOVA = analysis of variance. Scale bar: 200 μm.

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  Figure 4. Expression of AT1 and AT2 receptors in the adult ventricular–subventricular zone (V-SVZ) and neurospheres generated from the V-SVZ of mice. (A) AT1 receptor expression was higher in the microdissected V-SVZ of AT2 receptor deficient (AT2-KO) mice and AT2 receptor expression was lower in the microdissected V-SVZ of AT1 receptor deficient (AT1-KO) mice and wild-type (WT) mice treated with the AT1 antagonist candesartan. (B) AT1 expression was higher in neurospheres treated with the AT2 antagonist PD123319 than in controls, and AT2 expression was lower in neurospheres treated with the AT1 antagonist candesartan than in controls. The number of primary neurospheres obtained from WT, AT1-KO, and AT2-KO mice is shown in (C). (D) Bar graphs showing the number of neurospheres derived from AT1-KO or AT2-KO mice after the corresponding treatments. All culture data were obtained from at least three separate experiments. Data are means ± SEM. * p < 0.05 relative to controls (Student’s t-test or one-way ANOVA and Bonferroni post hoc test). Ang II = angiotensin II; CGP42112A = AT2 receptor agonist; ZD7155 = AT1 receptor antagonist.

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  Figure 5. (A) Generation of neurospheres from the ventricular–subventricular zone (V-SVZ) of aged mice (20–22 months old) compared to young mice (3–4 months old). Bar graphs showing the number of spheres quantified after treatment with the AT2 receptor agonist CGP42112A and the AT2 receptor antagonist PD123319 ((B); treatment), or in cultures derived from neurospheres pre-treated with AT2 agonists and reseeded in the absence of any treatment ((C); neurospheres derived from cultures pre-treated with AT2 agonists; pre-: previously treated with). (D) Number of neurospheres after treatment with CGP42112A and the Mas receptor antagonist A779 or the Mas receptor agonist angiotensin 1–7 (Ang 1–7). All culture data were obtained from at least three separate experiments. Data are means ± SEM. * p < 0.05 relative to control group; in figure (B), $ p < 0.05 relative to aged group (one-way ANOVA and Bonferroni post hoc test). SEM = standard error of the mean; ANOVA = analysis of variance.

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  Figure 6. Immunofluorescence labelling for AT1 ((A); red) and AT2 ((B); red) receptors showing their presence in DCX-positive cells (green) and glial fibrillary acidic protein (GFAP)-positive cells (green) in the rat ventricular–subventricular zone (V-SVZ). Nuclei have been labelled with Hoechst-33342 (blue). (C) Representative bands of AT1 and AT2 receptors and β-actin by RT-PCR in rat laser-microdissected V-SVZ. Microdissected cells obtained from the striatum (ST) were used as a positive control. LV = lateral ventricle; DCX = doublecortin. bp = base pairs. Scale bar: 100 μm.

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  Figure 7. Expression of AT1 and AT2 receptors in the adult ventricular–subventricular (V-SVZ) of rats and mice. The level of mRNA of AT1 (A) and AT2 (B) was higher in rats than in mice. However, the ratio of AT1/AT2 receptors in the microdissected V-SVZ was higher in mice than in rats (C). (D) Representative bands of AT1 and AT2 receptors in the mouse V-SVZ detected by western blot. Striatum (ST) was used as a positive control. (E) AT2 receptor expression was higher in rats treated with the AT1 receptor antagonist candesartan (Cand) than in control rats. (F) The ratio of AT1/AT2 was higher in aged than in young rats. Representative photomicrographs of coronal sections immunostained for BrdU (G) and DCX (I) of young and aged rat V-SVZ. The estimated number of BrdU and DCX-ir cells in the V-SVZ of the experimental groups is shown in (H,J). Data are means ± SEM. * p < 0.05 relative to the control group of the same age (Student’s t-test); in figures (H,J), $ p < 0.05 relative to the control young group (Student’s t-test). BrdU = bromodeoxyuridine; DCX = doublecortin; SEM = standard error of the mean. Scale bar: 100 μm.

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  Figure 8. (A) Model of the role of angiotensin II (Ang II) AT1 and AT2 receptor activity on the proliferation of V-SVZ cells. Ang II, via AT1 receptors, set a basal inhibition of proliferation that was antagonized by the AT2 receptors, which set a basal level of Ang II-dependent regulation of neurogenesis. In addition, AT2 receptor activity may exert an additional direct stimulatory effect on proliferation, which was modulated by Mas receptors (MasR). (B) Aging, candesartan (or AT1 deletion), and PD (or AT2 deletion) regulate AT1/AT2 expression ratio. Aging and PD (or AT2 deletion) increased ratio and candesartan (or AT1 deletion) increased ratio in mice (i.e., decreased AT2 expression) and decreased ratio in rats (i.e., increased AT2 expression). Candesartan = AT1 receptor antagonist; PD123319 = AT2 receptor antagonist.