8630 • The Journal of Neuroscience, June 18, 2014 • 34(25):8630–8645 Development/Plasticity/Repair Modulation of GABAA Receptor Signaling Increases Neurogenesis and Suppresses Anxiety through NFATc4 Giorgia Quadrato,1* Mohamed Y. Elnaggar,1,2* Ceren Duman,1,2 Andrea Sabino,1 Kirsi Forsberg,1 and Simone Di Giovanni1,3 1Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 72076 Tuebingen, Germany, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, 72074 Tuebingen, Germany, and 3Molecular Neuroregeneration, Division of Brain Sciences, Department of Medicine, Imperial College London, London W12 ONN, United Kingdom Correlative evidence suggests that GABAergic signaling plays an important role in the regulation of activity-dependent hippocampal neurogenesis and emotional behavior in adult mice. However, whether these are causally linked at the molecular level remains elusive. Nuclear factor of activated T cell (NFAT) proteins are activity-dependent transcription factors that respond to environmental stimuli in different cell types, including hippocampal newborn neurons. Here, we identify NFATc4 as a key activity-dependent transcriptional regulatorofGABAsignalinginhippocampalprogenitorcellsviaanunbiasedhigh-throughputgenome-widestudy.Next,wedemonstrate that GABAA receptor (GABAAR) signaling modulates hippocampal neurogenesis through NFATc4 activity, which in turn regulates GABRA2 and GABRA4 subunit expression via binding to specific promoter responsive elements, as assessed by ChIP and luciferase assays. Furthermore, we show that selective pharmacological enhancement of GABAAR activity promotes hippocampal neurogenesis via the calcineurin/NFATc4 axis. Importantly, the NFATc4-dependent increase in hippocampal neurogenesis after GABAAR stimulation is required for the suppression of the anxiety response in mice. Together, these data provide a novel molecular insight into the regulation of the anxiety response in mice, suggesting that the GABAAR/NFATc4 axis is a druggable target for the therapy of emotional disorders. Key words: adult hippocampal neurogenesis; anxiety; GABAergic signaling; NFATc4 Introduction finding that adult neurogenesis occurs in the adult human hip- Functional and neuroimaging studies have correlated hippocam- pocampus (Spalding et al., 2013) at rates comparable to those pal activity, integrity, and size with emotional behavior in both described in mice (Kempermann et al., 1997) suggests that it humans and animals (Gilbertson et al., 2002; Campbell et al., might as well have a functional role in the regulation of cognitive 2004; Kitayama et al., 2005; Irle et al., 2010; Kheirbek et al., 2013). and emotional human behavior (Kheirbek and Hen, 2013). Specifically, it has been shown that impaired adult hippocampal Therefore, the elucidation of the molecular link between adult neurogenesis is implicated in the pathogenesis of a number of hippocampal neurogenesis and emotional responses in mice cognitive and psychiatric disorders (Snyder et al., 2011; Kheirbek might be critical for the development of more effective treat- et al., 2012; Petrik et al., 2012; Sahay et al., 2011) and that the ments for clinical anxiety disorders. GABA signaling, which is effective treatment of anxiety/depression-related behavior in ro- one of the most important regulator of activity-dependent hip- dents requires intact hippocampal neurogenesis (Santarelli et al., pocampal neurogenesis during adulthood (Ge et al., 2007), is 2003; David et al., 2009; Surget et al., 2011). Importantly, the known to play a central role in the pathophysiology of anxiety disorders (Lydiard, 2003; Nemeroff, 2003). However, it is not Received Jan. 6, 2014; revised April 11, 2014; accepted May 9, 2014. clear whether GABAergic modulation of adult neurogenesis af- Author contributions: G.Q. and S.D.G. designed research; G.Q., M.Y.E., C.D., and A.S. performed research; K.F. fects anxiety levels in mice and which molecular players causally contributed unpublished reagents/analytic tools; G.Q., M.Y.E., and C.D. analyzed data; G.Q. and S.D.G. wrote the regulate these two events. It has been shown that GABAergic paper. excitation shapes neural maturation and differentiation specifi- This work was supported by the Hertie Foundation and the Deutsche Forschungsgemeinschaft (S.D.G.) and the Fortune Program of the University of Tu¨bingen (G.Q.). We thank Tuan Nguyen for important conceptual feedback, cally by the enhancement of GABAA receptor (GABAAR) expres- AlexandraLepierforthedesignandtheproductionoftheretrovirusesused,MariaMinafortechnicalhelp,JefferyD. sion and activity in neural progenitor cells (Tozuka et al., 2005; Molkentin for providing the NFATc4-null mice, and Verdon Taylor for providing the Hes5-GFP mice. Ge et al., 2006). GABAARs are hetero-oligomeric complexes com- The authors declare no competing financial interests. posed of five subunits that are expressed and assembled in a cell *G.Q. and M.Y.E. contributed equally to this work. Correspondenceshouldbeaddressedtoeitherofthefollowing:GiorgiaQuadrato,MolecularNeuroregeneration, type- and spatiotemporal-specific fashion in the brain. The com- Division of Brain Sciences, Department of Medicine, Imperial College London, London W12 ONN, United Kingdom, position of the GABAAR subunits determines their functional E-mail:[email protected];orSimoneDiGiovanni,MolecularNeuroregeneration,DivisionofBrain and pharmacological properties. Specifically, the functional ex- Sciences, Department of Medicine, Imperial college London, London W12 ONN, United Kingdom, E-mail: s.di- pression of the ␣2 (GABRA2) and ␣4 (GABRA4) GABA R sub- [email protected]. A DOI:10.1523/JNEUROSCI.0047-14.2014 units in neural progenitor cells controls adult neurogenesis in the Copyright © 2014 the authors 0270-6474/14/348630-16$15.00/0 mouse DG (Duveau et al., 2011). Vertebrate GABAAR subunits • Quadrato, Elnaggar et al. GABAAR/NFATc4 Axis in Anxiety Disorder J. Neurosci., June 18, 2014 • 34(25):8630–8645 • 8631 are organized in several gene clusters localized on different chro- nestin (1:200; Millipore), monoclonal anti-MAP2 (1:200; Abcam), mosomes originated by gene duplication. Interestingly, GABRA2 polyclonal rabbit anti-cleaved caspase-3 (1:500; Cell Signaling Technol- and GABRA4 subunits belong to the same genomic cluster, and ogy), monoclonal mouse anti-SOX2 (1:100; Abcam), polyclonal rabbit this genomic organization might have been conserved to facili- anti-SOX2 (1:400; Abcam), monoclonal mouse anti-SOX2 (1:400; Ab- tate transcriptional coregulation (Joyce, 2007). However the cam), and polyclonal chicken anti-GFP (1:500; Abcam). The secondary antibodies used were as follows: Alexa Fluor goat anti-mouse 488, goat transcriptional machinery involved in the coordinate regulation anti-rabbit 546, goat anti-rat 647, donkey anti-goat 488, donkey anti- of GABAAR subunits in hippocampal neural progenitor cells still rabbit 568, and donkey anti-mouse 647 (1:1500; Invitrogen). Cell nuclei needs to be investigated. were stained with Hoechst staining (1:5000; Invitrogen) and the glass Here, we demonstrate with several lines of evidence that coverslips were mounted using DAKO fluorescence mounting medium. GABAAR signaling operates through NFATc4 activity, an Cells were imaged using an Axiovert 200M (Zeiss) microscope with activity-dependent transcription factor expressed in hippocam- AxioVision software (version 4.8.1; Zeiss). pal adult-born neurons (Quadrato et al., 2012). NFATc4 modu- Fluorescence intensity threshold analysis. Quantification of the number lates hippocampal neurogenesis by direct transcriptional of viable cells with a staining intensity for anti-GABRA2 or anti-GABRA4 coregulation of GABRA2 and GABRA4 subunit expression by antibodies above the “intermode” threshold was made using ImageJ soft- binding to specific responsive elements on their promoters. Fur- ware. Approximately 100 cells were counted for each coverslip. Cells thermore, we show that selective pharmacological enhancement above intensity threshold were divided by the total number of viable cells to calculate their percentage. Viability of cells was determined using of GABAAR activity promotes hippocampal neuronal differenti- DAPI staining. ation via calcineurin/NFATc4, which in turn modulates innate Microarray analysis. Hippocampal NSP cultures derived from WT and anxiety levels in adult mice. These findings provide a causal link NFATc4 Ϫ/Ϫ mice were differentiated for 1 d with BDNF added to the between modulation of hippocampal neurogenesis and changes medium. Total RNA was isolated from the cells using PureLink RNA in anxiety levels by proposing NFATc4 as a key transcriptional micro kit (Invitrogen) according to manufacturer’s instructions. Whole regulator in GABAergic signaling and suggest that NFATc4/ transcriptome analysis was performed using an Affymetrix Mouse Gene GABAergic signaling is a druggable target for the therapy of emo- 1.1 ST array on the Affymetrix GeneTitan System. For this purpose, tional disorders. triplicates of each sample were analyzed. RNA quality was evaluated on Agilent 2100 Bioanalyzer with RNA integrity numbers (RINs) ranging from 8 to 10; all RINs Ͼ8 were con- Materials and Methods sidered optimal for downstream applications. Animals. Wild-type (WT) and nuclear factor of activated T cell-knock- Double-stranded cDNA was synthesized from 100 ng of total RNA and out (NFATc4 Ϫ/Ϫ) mice (Wilkins et al., 2002) were