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Antidepressants increase adult hippocampal neurogenesis and promote neuronal differentiation in rodents, but the underlying molecular mechanisms are unknown. Here we treated human embryonic hippocampal progenitor cells with the antidepressant, sertraline, and investigated changes in cell proliferation and neuronal differentiation. We find that antidepressants increase neuronal differentiation and decrease cell proliferation by activating the glucocorticoid receptor (GR), and by increasing GR-mediated gene transcription of the cell cycle inhibitors p27 Kip1 and p57 Kip2 . Chronic stress and depression are associated with elevated levels of glucocorticoid hormones and with decreased hippocampal neurogenesis. (Gould et al.,1992; David et al., 2009; Boldrini et al., 2009). Antidepressants increase adult hippocampal neurogenesis, and thereby possibly contribute to the resolution of some of the behavioural deficits in depression (Santarelli et al., 2003; David et al., 2009). Glucocorticoids and antidepressants both activate the glucocorticoid receptor (GR). The GR is a nuclear transcription factor, and GR– transactivation (GR binding to DNA) induces transcription of the cyclin- dependent kinase 2 (CDK2)-inhibitors p27 Kip1 and p57 Kip2 , which have been implicated in early neuronal development (Pariante et al., 1997; Shin et al., 2009; Ye et al., 2009). Antidepressants modulate human hippocampal neurogenesis by activating the glucocorticoid receptor Christoph Anacker , Patricia A. Zunszain, Annamaria Cattaneo, Livia A. Carvalho, Sandrine Thuret, Jack Price, Carmine M. Pariante Centre for the Cellular Basis of Behaviour, Institute of Psychiatry, King’s College London, United Kingdom email.: [email protected] BACKGROUND ABSTRACT • Adult neurogenesis & Depression MAP2+, NeuN+, tuj1+ Dcx+, PSA- NCAM+ Antidepressants enhance neuronal differentiation of hippocampal progenitor cells (Wang et al., 2008) Antidepressants increase progenitor cell proliferation in depressed patients or in mice which are co- treated with glucocorticoids (Boldrini et al., 2009; David et al., 2009). HYPOTHESIS Antidepressants enhance neuronal differentiation, but increase progenitor cell proliferation only in the presence of glucocorticoids The effect of antidepressants on neurogenesis is dependent on the glucocorticoid receptor (GR) GR-dependent expression of the CDK2-inhibtiors p27 Kip1 and p57 Kip2 mediates the effect of antidepressants on proliferation and neuronal differentiation RESULTS • Antidepressants modulate hippocampal progenitor cell proliferation by activating the GR Figure 1. BrdU immunocytochemistry (a). Sertraline (SERT 1uM, red bars) and the glucocorticoid dexamethasone (DEX 1uM, black bars) decrease cell proliferation. Only DEX and SERT co-treatment increases cell proliferation (green bars). These effects are abolished by the GR-antagonist RU486 (50nM) (b). *p<0.05, **p<0.01, n=5 a. -30 -20 -10 0 10 20 RU486 (50nM) + + + _ _ SERT 1uM DEX 1uM DEX 1uM + SERT 1uM * _ ** * ** * * BrdU+ cells (% change from veh) b. • Antidepressants induce GR transactivation and decrease GR expression Figure 2. Sertraline induces GR-transactivation (a) and decreases GR mRNA (b) and protein (c) after 12h of cell proliferation. *p<0.05, **p<0.01, ***p0.001; n=3 a. b. c. GRtotal ACTB veh DEX SERT D+S Dcx & MAP2 Immunostaining & Cell counting BrdU Immunostaining GR-DNA binding assay Quantitative Real-time PCR Western Blot METHODS • Proliferation assay • Differentiation assay 7 days Differentiation 1. 2. 3. 3 days Proliferation Treatment Treatment Treatment 4hrs BrdU Treatment (DEX, antidepressants, DEX & antidepressants) 3days Proliferation Human embryonic hippocampal progenitor cell line HPC03A/07 (ReNeuron, UK) CONCLUSIONS • Antidepressants enhance neuronal differentiation and promote neuronal maturation by activating the GR Hoechst Dcx Hoechst MAP2 Figure 3. Sertraline induces differentiation into Dcx+ cells (1) and promotes neuronal maturation into MAP2+ cells (2). This effect is absent if sertraline is not present during the proliferation phase (3). *p<0.05, **p<0.01, ***p0.001; n=5 Sertraline decreases progenitor cell proliferation via a GR-dependent effect; cell proliferation is only increased in the presence of glucocorticoids (Fig 1) Sertraline induces GR transactivation, decreases GR expression, and increases the CDK2-inhibitors p27 Kip1 and p57 Kip2 during cell proliferation (Fig 2) Sertraline increases neuronal differentiation and maturation via a GR- dependent effect, but only if already present during the proliferation phase (Fig 3) ACKNOWLEDGEMENTS This project was funded by the European Union Framework7, the Medical Research Council UK and an NIHR BRC studentship in Biomedical and Mental Health to C. Anacker. • Antidepressants induce expression of p27 Kip1 and p57 Kip2 2. 3. 1. Treatment Lie et al. 2004 BrdU Hoechst RU486 (50nM) + 0.0 0.5 1.0 1.5 2.0 2.5 SERT 1uM veh fold change from control 0.0 0.5 1.0 1.5 2.0 2.5 * fold change from control * p57 Kip2 expression 6h 12h 24h 48h 72h 0.0 0.5 1.5 2.0 2.5 SERT 1uM DEX 1uM DEX 1uM + SERT 1uM * fold change from control p57 Kip2 expression p27 Kip1 expression 6h 12h 24h 48h 72h 0.0 0.5 1.5 2.0 2.5 SERT 1uM DEX 1uM DEX 1uM+ SERT 1uM * fold change from control p27 Kip1 expression RU486 (50nM) SERT1uM veh * ** *** GR protein GR mRNA 6h 12h 24h 48h 72h 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 SERT 1uM DEX 1uM DEX 1uM+ SERT 1uM * * fold change VEH DEX SERT D+S 0.00 0.25 0.50 0.75 1.00 Dex 1uM SERT 1uM Dex+SERT fold induction 1h 6h 12h 24h 72h -30 -20 -10 0 10 20 30 GR transactivation * SERT 1uM % change in GR-DNA binding _ _ + _ _ -45 -35 -25 -15 -5 5 15 25 * SERT DEX + Dcx+ neuroblasts SERT+ DEX _ *** + _ + _ RU486 (50nM) Dcx+ cells (% change) -45 -35 -25 -15 -5 5 15 25 RU486 (50nM) + + _ _ + MAP2+ neurons _ SERT DEX SERT+ DEX ** Dcx+ cells (% change) Dcx+ neuroblasts -30 -20 -10 0 10 RU486 (50nM) + + _ _ + *** _ SERT DEX SERT+ DEX Dcx+ cells (% change) MAP2+ neurons -50 -25 0 25 50 RU486 (50nM) + + _ _ + ** ** SERT DEX SERT+ DEX _ MAP2+ cells (% change) -45 -35 -25 -15 -5 5 15 25 SERT SERT+ DEX Dcx+ neuroblasts DEX Dcx+ cells (% change) -45 -35 -25 -15 -5 5 15 25 SERT SERT+ DEX MAP2+ neurons DEX MAP2+ cells (% change) CONFLICT OF INTEREST J. Price acts as a consultant for ReNeuron Group
