Glucocorticoid-related molecular signaling pathways regulating hippocampal neurogenesis

Abstract

Stress and glucocorticoid hormones regulate hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. We, therefore, investigated the molecular signaling pathways mediating the effects of cortisol on proliferation, neuronal differentiation, and astrogliogenesis, in an immortalized human hippocampal progenitor cell line. In addition, we examined the molecular signaling pathways activated in the hippocampus of prenatally stressed rats, characterized by persistently elevated glucocorticoid levels in adulthood. In human hippocampal progenitor cells, we found that low concentrations of cortisol (100 nM) increased proliferation (+16%), decreased neurogenesis into microtubule-associated protein 2 (MAP2)-positive neurons (-24%) and doublecortin (Dcx)-positive neuroblasts (-21%), and increased differentiation into S100β-positive astrocytes (+23%). These effects were dependent on the mineralocorticoid receptor (MR) as they were abolished by the MR antagonist, spironolactone, and mimicked by the MR-agonist, aldosterone. In contrast, high concentrations of cortisol (100 μM) decreased proliferation (-17%) and neuronal differentiation into MAP2-positive neurons (-22%) and into Dcx-positive neuroblasts (-27%), without regulating astrogliogenesis. These effects were dependent on the glucocorticoid receptor (GR), blocked by the GR antagonist RU486, and mimicked by the GR-agonist, dexamethasone. Gene expression microarray and pathway analysis showed that the low concentration of cortisol enhances Notch/Hes-signaling, the high concentration inhibits TGFβ-SMAD2/3-signaling, and both concentrations inhibit Hedgehog signaling. Mechanistically, we show that reduced Hedgehog signaling indeed critically contributes to the cortisol-induced reduction in neuronal differentiation. Accordingly, TGFβ-SMAD2/3 and Hedgehog signaling were also inhibited in the hippocampus of adult prenatally stressed rats with high glucocorticoid levels. In conclusion, our data demonstrate novel molecular signaling pathways that are regulated by glucocorticoids in vitro, in human hippocampal progenitor cells, and by stress in vivo, in the rat hippocampus.

Figure 1

Differential effects of mineralocorticoid receptor (MR)- and glucocorticoid receptor (GR)-activation on human hippocampal progenitor cell proliferation. 5′-bromodeoxyuridine (BrdU, 10 μℳ) incorporation and immunocytochemistry was used to assess proliferation of HPC03A/07 cells (a). Cortisol exerts bimodal, dose-dependent effects on proliferation (b). Spironolactone (1 μℳ) blocks the increase in cell proliferation upon treatment with low concentrations of cortisol (c). RU486 (50 nM) blocks the decrease in cell proliferation upon treatment with high concentrations of cortisol (100 μℳ) (d). The MR-agonist, aldosterone (1 nM–1 μℳ), increases cell proliferation (e), while the GR-agonist, dexamethasone (10 nℳ–5 μℳ), decreases cell proliferation (f). GR transactivation is dose-dependently increased by cortisol concentrations (10 μℳ–1 mℳ) (g). Three to four independent experiments were conducted on independent cultures (indicated as n). In proliferation experiments, four wells were analyzed per treatment condition in each experiment and three random, non-overlapping pictures were analyzed for each well. All data are mean±s.e.m. *P<0.05, **P<0.01; ***P<0.001 compared with the vehicle-treated control condition.

Figure 2

Low and high concentrations of cortisol exert differential MR- and GR-dependent effects on neurogenesis and astrogliogenesis. Immunocytochemistry for microtubule-associated protein 2 (MAP2) and S100 calcium-binding protein β (S100ß) (a). If treated only during the proliferation phase, the low cortisol concentration (100 nM) and aldosterone (1 μℳ) decrease the number of MAP2-positive neurons. Both effects are counteracted by spironolactone (1 μℳ) (b). The high cortisol concentration (100 μℳ) and dexamethasone (1 μℳ) also decrease the number of MAP2-positive neurons. These effects are both counteracted by RU486 (50 nℳ) (c). No effects on the number of MAP2-positive neurons are observed when cells are treated only during the differentiation phase (d, e). the low cortisol concentration (100 nM) and aldosterone (1 μM) increase the number of S100β-positive astrocytes. Both effects are counteracted by spironolactone (1 μM) (f). The high cortisol concentration (100 μM) and dexamethasone (1 μℳ) do not significantly alter the number of S100β-positive astrocytes (g). No effects are observed on the number of S100β-positive astrocytes when cells are treated only during the differentiation phase (h, i). Three independent experiments were conducted on independent cultures (n=3). Four wells were analyzed per treatment condition in each experiment and three random, non-overlapping pictures were analyzed for each well. All data are mean±s.e.m. *P<0.05, **P<0.01, compared with the vehicle-treated control condition.

Figure 3

Effects of purmorphamine on neurogenesis. Purmorphamine increases neuronal differentiation into microtubule-associated protein 2 (MAP2)-positive neurons (a) and into doublecortin (Dcx)-positive neuroblasts (b). The decrease in MAP2- and Dcx-positive cells upon treatment with the low cortisol concentration (100 nℳ) is counteracted by purmorphamine (10 nℳ) (c, d). The decrease in MAP2- and Dcx-positive cells upon treatment with the high cortisol concentration (100 μM) is also counteracted by purmorphamine (10 nM) (e, f). Three independent experiments were conducted on independent cultures (n=3). Four wells were analyzed per treatment condition in each experiment and three random, non-overlapping pictures were analyzed for each well. All data are mean±s.e.m. *P<0.05, compared with the vehicle-treated control condition or as indicated.