Cortisol + RU486 (50nM) -5 0 5 10 15 20 25 n=3 BrdU+ cells (% change from veh) MAP2+, NeuN+, tuj1+ Dcx+, PSA- NCAM+ Lie et al. 2004 MR activation mediates the effects of low cortisol concentrations, while high concentrations of cortisol activate the GR in human hippocampal progenitor cells. Cortisol decreases human hippocampal neurogenesis via GR-dependent upregulation of SGK1. SGK1 regulates GR function by phosphorylation. Cortisol - 1nM 10nM 100nM 1μM 10μM 100μM Cortisol + Spironolactone (1μM) -25 -20 -15 -10 -5 0 5 10 15 n=3 BrdU+ cells (% change from veh) * Cortisol - 1nM 10nM 100nM 1μM 10μM 100μM * CORTISOL DECREASES HIPPOCAMPAL NEUROGENESIS BY REGULATING THE ENZYME SGK1 C. Anacker 1 , P.A. Zunszain 1 , A. Cattaneo 2 , K. Musaelyan 1 , S. Thuret 1 , J. Price 1 , C.M. Pariante 1 . 1 Centre for the Cellular Basis of Behaviour, Institute of Psychiatry, King’s College London, United Kingdom 2 IRCCS San Giovanni di Dio, Genetics Unit, Brescia, Italy. BACKGROUND • Glucocorticoids and adult hippocampal neurogenesis in depression Glucocorticoids (cortisol in humans) can activate two intracellular receptors: Mineralocorticoid Receptor (MR), high affinity Glucocorticoid Receptor (GR), low affinity HYPOTHESIS RESULTS • Bimodal effects of cortisol on cell proliferation CONCLUSIONS MR activation by low concentrations of cortisol increases proliferation, while GR activation by high concentrations decreases proliferation GR-dependent activation of SGK1 expression mediates the cortisol-induced reduction in neurogenesis Cortisol-induced SGK1 expression activates the GR by phosphorylation at the GR serine residues S203 and S211 DISCLOSURE: This poster is financially supported by the European Union Framework7, the Medical Research Council UK and an NIHR BRC studentship in Biomedical and Mental Health to C. Anacker. J. Price acts as a consultant for ReNeuron, UK. • Cortisol decreases neuronal differentiation The enzyme, serum- and glucocorticoid-regulated kinase 1 (SGK1) mediates some effects of glucocorticoids on working memory, oligodendrocyte morphology and glucocorticoid responsiveness. (Yuen et al., 2011; Miyata et al., 2011, Luca et al., 2009) GR function is critically regulated by phosphorylation at the serine residues S203, S211 and S226. a. b. Figure 2. (a) The MR-antagonist, spironolactone, blocks the increase in proliferation. (b) The GR- antagonist, RU486, blocks the decrease in proliferation. *p<0.05 • GR activation by cortisol increases SGK1 expression Figure 4. (a) High cortisol concentrations increase SGK1 expression. (b) The effect at 12hrs is blocked by RU486 (50nM). *p<0.05, **p<0.01 • SGK1 mediates the cortisol-induced reduction in neurogenesis -20 -15 -10 -5 0 5 10 15 n=3 BrdU+cells (% change from veh) Cortisol - 1nM 10nM 100nM 1uM 10uM 100uM ** ** Hoechst BrdU MR GR SGK1 inhibitor SGK1 inhibitor -30 -20 -10 0 10 n=3 100μM Cortisol 100μM 100nM _ MAP2+ neurons (% change) -20 -15 -10 -5 0 5 10 15 n=4 _ 10nM 50nM 100nM Cortisol 100μM 100μM 100μM 100μM BrdU+ cells (% change) ** * 1h 3h 12h 72h 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 Cort100μM * ** * n=6 Cort100nM fold change gene expression 0.0 0.5 1.0 1.5 2.0 RU486 (50nM) Spironolactone (1μM) _ _ _ _ _ _ + + Cort 100nM Cort 100μM ** * n=3 fold change gene expression Dcx & MAP2 Immunostaining & Cell counting BrdU Immunostaining Quantitative Real-Time PCR Western Blot METHODS • Proliferation assay • Differentiation assay 7 days Differentiation 1. 2. 3 days Proliferation Treatment Treatment 4hrs BrdU Treatment (Cortisol, Spironolactone 1µM [MR antagonist], RU486 50nM [GR antagonist], GSK650394 [SGK1 inhibitor]) 3days Proliferation Human embryonic hippocampal progenitor cell line HPC03A/07 (ReNeuron, UK) Treatment Adult hippocampal neurogenesis has recently been demonstrated to contribute to the development of depressive symptoms in situations of stress (Snyder et al., 2011) Figure 5. The SGK1-inhibitor, GSK650394, counteracts the effects of cortisol (100µM) on (a) proliferation and (b) neuronal differentiation.*p<0.05, **p<0.01 a. b. a. b. • SGK1 phosphorylates the GR 0.0 0.5 1.0 1.5 2.0 _ + + _ _ + + _ n=5 fold induction 0 1 2 3 4 ** _ + + _ _ + + _ * fold induction 0 1 2 3 4 * _ + + _ _ + + _ * Cort (100uM) SGK1 inhibitor (100nM) fold induction S203 GR S211 GR 0 1 2 3 4 * _ + + _ _ + + _ fold induction S226 GR GR ACTB Figure 6. The SGK1-inhibitor, GSK650394, counteracts the cortisol-induced GR phosphorylation at S203 and S211, but not at S226. *p<0.05, **p<0.01 -40 -30 -20 -10 0 10 20 low Cortisol (100nM) RU486 (50nM) Spironolactone (1 μM) _ _ _ _ _ _ + + high Cortisol (100μM) ** n=3 ** MAP2+ cells (% change) -40 -30 -20 -10 0 10 20 low Cortisol (100nM) high Cortisol (100μM) n=3 MAP2+ cells (% change) a. b. Hoechst MAP2 2. 1. Figure 3. (a) Cortisol treatment during proliferation and differentiation (paradigm 1 ) decreases neuronal differentiation at both 100nM and 100µM via MR and GR-dependent effects, respectively. (b) Treatment only during the differentiation phase (paradigm 2 ) has no effect. **p<0.01 Increased levels of glucocorticoid hormones are commonly observed in situations of chronic stress and in depression. High levels of glucocorticoid hormones decrease adult hippocampal neurogenesis Figure 1. Low concentrations of cortisol (100nM) increase proliferation, while high concentrations (100µM) decrease proliferation.**p<0.01 • MR- and GR-dependent effects of cortisol on proliferation