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Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.

Forster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.

SUMMARYThe dentate gyrus (DG) of the hippocampus plays a key role in memory formation and it is known to be modulated by septal projections. By performing electrophysiology and optogenetics we evaluated the role of cholinergic modulation in the processing of afferent inputs in the DG. We showed that mature granule cells (GCs), but not adult-born immature neurons, have increased responses to afferent perforant path stimuli upon cholinergic modulation. This is due to a highly precise reconfiguration of inhibitory circuits, differentially affecting Parvalbumin and Somatostatin interneurons, resulting in a nicotinic-dependent perisomatic disinhibition of GCs. This circuit reorganization provides a mechanism by which mature GCs could escape the strong inhibition they receive, creating a window of opportunity for plasticity. Indeed, coincident activation of perforant path inputs with optogenetic release of acetylcholine produced a long-term potentiated response in GCs, essential for memory formation.

Fluorescence nanoscopy imaging permits the observation of periodic supramolecular protein structures in their natural environment, as well as the unveiling of previously unknown protein periodic structures. Deciphering the biological functions of such protein nanostructures requires systematic and quantitative analysis of large number of images under different experimental conditions and specific stimuli. Here we present a method and an open source software for the automated quantification of protein periodic structures in super-resolved images. Its performance is demonstrated by analyzing the abundance and regularity of the spectrin membrane-associated periodic skeleton (MPS) in hippocampal neurons of 2 to 40 days in vitro, imaged by STED and STORM nanoscopy. The automated analysis reveals that both the abundance and the regularity of the MPS increase over time and reach maximum plateau values after 14 DIV. A detailed analysis of the distributions of correlation coefficients provides indication of dynamical assembly and disassembly of the MPS. Fil: Barabas, Federico Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Bordenave, Martín Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Giusti, Sebastian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Unsain, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina

Post-translational modifications, like phosphorylation, ubiquitylation, and sumoylation, have been shown to impact on synaptic neurotransmission by modifying pre- and postsynaptic proteins and therefore alter protein stability, localization, or protein-protein interactions. Previous studies showed that post-translational modifications are essential during the induction of synaptic plasticity, defined by a major reorganization of synaptic proteins. We demonstrated before that neddylation, a post-translational modification that covalently binds Nedd8 to lysine-residues, strongly affects neuronal maturation and spine stability. We now analysed the consequences of inhibiting neddylation on excitatory synaptic transmission and plasticity, which will help to narrow down possible targets, to make educated guesses, and test specific candidates. Here, we show that acute inhibition of neddylation impacts on synaptic neurotransmission before morphological changes occur. Our data indicate that pre- and postsynaptic proteins are neddylated since the inhibition of neddylation impacts on presynaptic release probability and postsynaptic receptor stabilization. In addition, blocking neddylation during the induction of long-term potentiation and long-term inhibition abolished both forms of synaptic plasticity. Therefore, this study shows the importance of identifying synaptic targets of the neddylation pathway to understand the regulation of synaptic transmission and plasticity. Fil: Brockmann, Marisa M.. Universitat Bonn; Alemania. Max Planck Institute Of Psychiatry; Alemania Fil: Döngi, Michael. Universitat Bonn; Alemania Fil: Einsfelder, Ulf. Universitat Bonn; Alemania Fil: Körber, Nils. Universitat Bonn; Alemania Fil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Stein, Valentin. Universitat Bonn; Alemania

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision., Achieving high axial resolution is challenging in single-molecule localization microscopy. Here, the authors present a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope without hardware modification, and show nearly isotropic nanometric resolution.

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An extensive microglial-astrocyte-monocyte-neuronal cross talk seems to be crucial for normal brain function, development, and recovery. However, under certain conditions neuroinflammatory interactions between brain cells and neuroimmune cells influence disease outcome and brain pathology. Microglial cells express a range of functional states with dynamically pleomorphic profiles from a surveilling status of synaptic transmission to an active player in major events of development such as synaptic elimination, regeneration, and repair. Also, inflammation mediates a series of neurotoxic roles in neuropsychiatric conditions and neurodegenerative diseases. The present review discusses data on the involvement of neuroinflammatory conditions that alter neuroimmune interactions in four different pathologies. In the first section of this review, we discuss the ability of the early developing brain to respond to a focal lesion with a rapid compensatory plasticity of intact axons and the role of microglial activation and proinflammatory cytokines in brain repair. In the second section, we present data of neuroinflammation and neurodegenerative disorders and discuss the role of reactive astrocytes in motor neuron toxicity and the progression of amyotrophic lateral sclerosis. In the third section, we discuss major depressive disorders as the consequence of dysfunctional interactions between neural and immune signals that result in increased peripheral immune responses and increase proinflammatory cytokines. In the last section, we discuss autism spectrum disorders and altered brain circuitries that emerge from abnormal long-term responses of innate inflammatory cytokines and microglial phenotypic dysfunctions.

The interplay between corticotropin-releasing hormone (CRH) and the dopaminergic system has predominantly been studied in addiction and reward, while CRH-dopamine interactions in anxiety are scarcely understood. We describe a new population of CRH-expressing, GABAergic, long-range-projecting neurons in the extended amygdala that innervate the ventral tegmental area and alter anxiety following chronic CRH depletion. These neurons are part of a distinct CRH circuit that acts anxiolytically by positively modulating dopamine release. Fil: Dedic, Nina. Max Planck Institute Of Psychiatry; Alemania Fil: Kühne, Claudia. Max Planck Institute Of Psychiatry; Alemania Fil: Jakovcevski, Mira. Max Planck Institute Of Psychiatry; Alemania Fil: Hartmann, Jakob. Max Planck Institute Of Psychiatry; Alemania Fil: Genewsky, Andreas J.. Max Planck Institut Of Psychiatry; Alemania Fil: Gomes, Karina S.. Max Planck Institute Of Psychiatry; Alemania Fil: Anderzhanova, Elmira. Max Planck Institute Of Psychiatry; Alemania Fil: Pöhlmann, Max L.. Max Planck Institute Of Psychiatry; Alemania Fil: Chang, Simon. Max Planck Institute Of Psychiatry; Alemania Fil: Kolarz, Adam. Max Planck Institute Of Psychiatry; Alemania Fil: Vogl, Annette M.. Max Planck Institute Of Psychiatry; Alemania Fil: Dine, Julien. Max Planck Institute Of Psychiatry; Alemania Fil: Metzger, Michael W.. Max Planck Institute of Psychiatry; Armenia Fil: Schmid, Bianca. Max Planck Institute Of Psychiatry; Alemania Fil: Almada, Rafael C.. Max Planck Institute Of Psychiatry; Alemania Fil: Ressler, Kerry J.. Harvard Medical School; Estados Unidos Fil: Wotjak, Carsten T.. Max Planck Institute Of Psychiatry; Alemania Fil: Grinevich, Valery. University of Heidelberg; Alemania Fil: Chen, Alon. Max Planck Institute Of Psychiatry; Alemania Fil: Schmidt, Mathias V.. Institute Of Developmental Genetics, Helmholtz Zentrum; Alemania Fil: Wurst, Wolfgang. German Center for Neurodegenerative Diseases; Alemania Fil: Refojo, Damian. Max Planck Institute Of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Deussing, Jan M.. Max Planck Institute Of Psychiatry; Alemania

Neddylation is a ubiquitylation-like pathway that controls cell cycle and proliferation by covalently conjugating Nedd8 to specific targets. However, its role in neurons, nonreplicating postmitotic cells, remains unexplored. Here we report that Nedd8 conjugation increased during postnatal brain development and is active in mature synapses, where many proteins are neddylated. We show that neddylation controls spine development during neuronal maturation and spine stability in mature neurons. We found that neddylated PSD-95 was present in spines and that neddylation on Lys202 of PSD-95 is required for the proactive role of the scaffolding protein in spine maturation and synaptic transmission. Finally, we developed Nae1 CamKIIα-CreERT2 mice, in which neddylation is conditionally ablated in adult excitatory forebrain neurons. These mice showed synaptic loss, impaired neurotransmission and severe cognitive deficits. In summary, our results establish neddylation as an active post-translational modification in the synapse regulating the maturation, stability and function of dendritic spines. Fil: Vogl, Annette M.. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Brockmann, Marisa M.. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Giusti, Sebastian Alejandro. Instituto Max Planck de Psiquiatria de Munich; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: MacCarrone, Giuseppina. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Vercelli, Claudia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Bauder, Corinna A.. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Richter, Julia S.. Instituto Max Planck de Psiquiatria de Munich; Alemania. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Roselli, Francesco. Universita Degli Studi Di Bari; Italia Fil: Hafner, Anne-Sophie. Universite de Bordeaux; Francia Fil: Dedic, Nina. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Wotjak, Carsten T.. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Vogt Weisenhorn, Daniela M.. Institute Of Developmental Genetics; Alemania Fil: Choquet, Daniel. Universite de Bordeaux; Francia Fil: Turck, Christoph W.. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Stein, Valentin. Universitaet Bonn; Alemania Fil: Deussing, Jan M.. Instituto Max Planck de Psiquiatria de Munich; Alemania Fil: Refojo, Damian. Instituto Max Planck de Psiquiatria de Munich; Alemania

Motor neurons in the vertebrate spinal cord are stereotypically organized along the rostro-caudal axis in discrete columns that specifically innervate peripheral muscle domains. Originating from the same progenitor domain, the generation of spinal motor neurons is orchestrated by a spatially and temporally tightly regulated set of secreted molecules and transcription factors such as retinoic acid and the Lim homeodomain transcription factors Isl1 and Lhx1. However, the molecular interactions between these factors remained unclear. In this study we examined the role of the microRNA 9 (miR-9) in the specification of spinal motor neurons and identified Onecut1 (OC1) as one of its targets. miR-9 and OC1 are expressed in mutually exclusive patterns in the developing chick spinal cord, with high OC1 levels in early-born motor neurons and high miR-9 levels in late-born motor neurons. miR-9 efficiently represses OC1 expression in vitro and in vivo. Overexpression of miR-9 leads to an increase in late-born neurons, while miR-9 loss-of-function induces additional OC1(+) motor neurons that display a transcriptional profile typical of early-born neurons. These results demonstrate that regulation of OC1 by miR-9 is a crucial step in the specification of spinal motor neurons and support a model in which miR-9 expression in late-born LMC1 neurons downregulates Isl1 expression through inhibition of OC1. In conclusion, our study contributes essential factors to the molecular network specifying spinal motor neurons and emphasizes the importance of microRNAs as key players in the generation of neuronal diversity. (C) 2014 The Authors. Published by Elsevier Inc. All rights reserved.

A single nucleotide polymorphism substitution from glutamine (Gln, Q) to arginine (Arg, R) at codon 460 of the purinergic P2X7 receptor (P2X7R) has repeatedly been associated with mood disorders. The P2X7R-Gln460Arg variant per se is not compromised in its function. However, heterologous expression of P2X7R-Gln460Arg together with wild-type P2X7R has recently been demonstrated to impair receptor function. Here we show that this also applies to humanized mice coexpressing both human P2X7R variants. Primary hippocampal cells derived from heterozygous mice showed an attenuated calcium uptake upon agonist stimulation. While humanized mice were unaffected in their behavioral repertoire under basal housing conditions, mice that harbor both P2X7R variants showed alterations in their sleep quality resembling signs of a prodromal disease stage. Also healthy heterozygous human subjects showed mild changes in sleep parameters. These results indicate that heterozygosity for the wild-type P2X7R and its mood disorder-associated variant P2X7R-Gln460Arg represents a genetic risk factor, which is potentially able to convey susceptibility to mood disorders. Fil: Metzger, Michael W.. Max Planck Institut Fur Psychiatrie; Alemania Fil: Walser, Sandra M.. Max Planck Institut Fur Psychiatrie; Alemania Fil: Dedic, Nina. Max Planck Institut Fur Psychiatrie; Alemania Fil: Aprile García, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Jakubcakova, Vladimira. Max Planck Institut Fur Psychiatrie; Alemania Fil: Adamczyk, Marek. Max Planck Institut Fur Psychiatrie; Alemania Fil: Webb, Katharine J.. Max Planck Institut Fur Psychiatrie; Alemania Fil: Uhr, Manfred. Max Planck Institut Fur Psychiatrie; Alemania Fil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Schmidt, Mathias V.. Max Planck Institut Fur Psychiatrie; Alemania Fil: Friess, Elisabeth. Max Planck Institut Fur Psychiatrie; Alemania Fil: Steiger, Axel. Max Planck Institut Fur Psychiatrie; Alemania Fil: Kimura, Mayumi. Max Planck Institut Fur Psychiatrie; Alemania Fil: Chen, Alon. Max Planck Institut Fur Psychiatrie; Alemania. Weizmann Institute of Science; Israel Fil: Holsboer, Florian. Max Planck Institut Fur Psychiatrie; Alemania Fil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Wurst, Wolfgang. Institute of Developmental Genetics; Alemania. Technische Universität München-Weihenstephan; Alemania. German Center for Neurodegenerative Diseases; Alemania. Universität München; Alemania Fil: Deussing, Jan M.. Max Planck Institut Fur Psychiatrie; Alemania

A classical view of the neuroendocrine-immune network assumes bidirectional interactions where pro-inflammatory cytokines influence hypothalamic-pituitary-adrenal (HPA) axis-derived hormones that subsequently affect cytokines in a permanently servo-controlled circle. Nevertheless, this picture has been continuously evolving over the last years as a result of the discovery of redundant expression and extended functions of many of the molecules implicated. Thus, cytokines are not only expressed in cells of the immune system but also in the central nervous system, and many hormones present at hypothalamic-pituitary level are also functionally expressed in the brain as well as in other peripheral organs, including immune cells. Because of this intermingled network of molecules redundantly expressed, the elucidation of the unique roles of HPA axis-related molecules at every level of complexity is one of the major challenges in the field. Genetic engineering in the mouse offers the most convincing method for dissecting in vivo the specific roles of distinct molecules acting in complex networks. Thus, various immunological, behavioral, and signal transduction studies performed with different HPA axis-related mutant mouse lines to delineate the roles of beta-endorphin, the type 1 receptor of corticotropin-releasing hormone (CRHR1), and its ligand CRH will be discussed here.

Glucocorticoid (GC) immunosuppression and anti-inflammatory action involve the regulation of several transcription factors (TFs). GCs inhibit the acute production of T-helper (Th) 1 and Th2 cytokines but ultimately favor a shift toward Th2 phenotype. GCs inhibit the transcriptional activity of T-bet Th1 TF by a transrepression mechanism. Here we analyze GC regulation of GATA-3, the master driver of Th2 differentiation. We found that GCs inhibit GATA-3 transcriptional activity. We demonstrate that this mechanism does not involve physical interaction between the glucocorticoid receptor (GR) and GATA-3 or reduction of GATA-3 binding to DNA, as described previously for T-bet. Instead, GCs inhibit GATA-3 activity by inhibition of p38 mitogen-activated protein kinase induced GATA-3 phosphorylation. GCs also inhibit GATA-3 mRNA and protein expression. Finally, GATA-3 inhibition affects the interleukin-5 gene, a central Th2 cytokine. The IC(50) of dexamethasone is 10 nM with a maximum effect at 100 nM. All inhibitory actions were blocked by the GR antagonist RU38486 (1 uM), proving the specificity of GR action. In view of the crucial role of GATA-3 in T-cell differentiation and inflammation, we propose that the mechanism of GATA-3 inhibition compared with that in T-bet may have relevant implications in understanding and modulating the anti-inflammatory and Th-regulatory properties of GCs.

Interferon-γ (IFNG) is a cytokine that exerts potent antiproliferative and tumoricidal effects in a variety of cancers. Moreover, IFNG modulates normal pituitary hormone secretion, and was shown to inhibit the expression of the ACTH precursor POMC in murine ACTH-secreting AtT-2010/21/2008 tumor cells. We have studied the functional role of IFNG on pituitary tumor cells, focusing on the involvement of IFNG in the molecular events leading to the control of POMC transcriptional repression. Herein, it is shown that IFNG inhibits AtT-20 tumor cell proliferation without inducing apoptosis. Unexpectedly, an activated janus kinases–signal transducer and activator of transcription (JAK–STAT1) cascade is required for IFNG inhibitory action on POMC promoter activity. Factor-kappa B (NF-κB) is necessary for the inhibitory action of IFNG on Pomc transcription, since loss of NF-κB activity with IκB super-repressor abolishes this effect. In addition, 1 and 2 IFNG receptor immunoreactivity was detected in human corticotropinoma cells. Interestingly, IFNG inhibits ACTH production from these cells in primary cell culture, without affecting basal ACTH biosynthesis in normal non-tumoral pituitary cells. In conclusion, our data show for the first time that POMC transcription can be negatively regulated by a JAK–STAT1 and NF-κB-dependent pathway.

Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in the pathophysiology of affective disorders. Both, basic and clinical studies suggested that disrupting CRH signaling through CRH type 1 receptors (CRH-R1) can ameliorate stress-related clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicited behavioral and neurochemical changes we created different mouse lines overexpressing CRH in distinct spatially restricted patterns. CRH overexpression in the entire central nervous system, but not when overexpressed in specific forebrain regions, resulted in stress-induced hypersecretion of stress hormones and increased active stress-coping behavior reflected by reduced immobility in the forced swim test and tail suspension test. These changes were related to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonist treatment and recapitulated the effect of stress-induced activation of the endogenous CRH system. Moreover, we identified enhanced noradrenergic activity as potential molecular mechanism underlying increased active stress-coping behavior observed in these animals. Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologies and treatments that target the central CRH system.

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations., PLoS ONE, 11 (3), ISSN:1932-6203

Corticotropin-releasing hormone (CRH) coordinates hormonal and behavioral responses to stress. The mitogen-activated protein kinase extracellular signal-related kinase 1/2 (ERK1/2) mediates several functions in different forebrain structures and recently has been implicated in CRH signaling in cultured cells. To study in vivo CRH-mediated activation of central ERK1/2, we investigated the expression pattern of the phosphorylated ERK1/2( p -ERK1/2) in the mouse brain after intracerebroventricular CRH injections. As shown by immunohistochemistry and confocal microscopy analysis, CRH administration increased p -ERK1/2 levels specifically in the CA3 and CA1 hippocampal subfields and basolateral complex of the amygdala, both structures related to external environmental information processing and behavioral aspects of stress. Other regions such as hypothalamic nuclei and the central nucleus of the amygdala, also related to central CRH system but involved in the processing of the ascending visceral information and neuroendocrine-autonomic response to stress, did not show CRH-mediated ERK1/2 activation. To dissect the involvement of CRH receptor 1 (CRHR1) and CRHR2, we used conditional knockout mice in which Crhr1 is inactivated in the anterior forebrain and limbic structures. The conditional genetic ablation of Crhr1 inhibited the p -ERK1/2 increase, underlining the involvement of CRHR1 in the CRH-mediated activation. These findings underscore the fact that CRH activates p -ERK1/2 through CRHR1 only in selected brain regions, pointing to a specific role of this pathway in mediating behavioral adaptation to stress.

An essential event in immune activation is the increase of cytokines in both plasma and immune tissues. Steroid hormones influence several adaptive responses in both health and disease. Cytokines and steroids have an intimate cross-communication in many systems, making possible a satisfactory adaptive response to environmental changes. The ultimate level of integration of the cytokine-steroids cross-talk is the molecular level. We have demonstrated this in four types of cross-talk mechanisms on different cells in which steroids have major roles: (1) The tumor necrosis factor (TNF)-glucocorticoid receptor (GR) transcriptional interaction in cellular targets of TNF-induced cytotoxicity. TNF potentiates the transactivation activity of GR and the priming with TNF increases the protective action of GR on TNF-induced cytotoxicity. (2) The GR-T cell receptor (TCR) antagonism in GR-TCR-induced T cell apoptosis and its modulation by cAMP. cAMP inhibits the TCR-induced apoptosis through a PKA-CREB-dependent mechanism and potentiates glucocorticoid-induced apoptosis by means of a CREB-independent mechanism. (3) The GR influence on Th1-Th2 cytokine expression and differentiation. Glucocorticoids inhibit the induction of GATA-3 and T-bet transcription factors. (4) The influence of ER/Smad-4 signaling cross-communication on prolactinoma pathogenesis. Physical and functional interactions between Smad-4 and estrogen receptors take place in prolactinoma cells, providing a molecular explanation to link the tumorigenic action of these two important players of prolactinoma pathogenesis. The molecular cross-talk between steroids and transcription factors is the mechanism that provides the basis for the outcome of adaptive responses integrating the systemic information provided by hormones and cytokines.

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factors/cytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4/Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17β-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smadl physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type β transforming growth factor family have important roles., Facultad de Ciencias Exactas, Instituto de Investigaciones Bioquímicas de La Plata

Nur factors are critical for proopiomelanocortin (POMC) induction by CRH in corticotrophs, but the pathways linking CRH to Nur are unknown. In this study we show that in AtT-20 corticotrophs CRH and cAMP induce Nur77 and Nurr1 expression and transcription at the NurRE site by protein kinase A (PKA) and calcium-dependent and -independent mechanisms. Calcium pathways depend on calmodulin kinase II (CAMKII) activity, and calcium-independent pathways are accounted for in part by MAPK activation (Rap1/B-Raf/MAPK-ERK kinase/ERK1/2), demonstrated by the use of molecular and pharmacological tools. AtT-20 corticotrophs express B-Raf, as do other cells in which cAMP stimulates MAPK. CRH/cAMP stimulated ERK2 activity and increased transcriptional activity of a Gal4-Elk1 protein, which was blocked by overexpression of dominant negative mutants and kinase inhibitors and stimulated by expression of B-Raf. The MAPK kinase inhibitors did not affect Nur77 and Nurr1 mRNA induction but blocked CRH or cAMP-stimulated Nur transcriptional activity. Moreover, MAPK stimulated phosphorylation and transactivation of Nur77. The functional impact of these pathways was confirmed at the POMC promoter. In conclusion, in AtT-20 corticotrophs the CRH/cAMP signaling that leads to Nur77/Nurr1 mRNA induction and transcriptional activation, and thus POMC expression, is dependent on protein kinase A and involves calcium/calmodulin kinase II (Nur induction/activation) and MAPK calcium-dependent and -independent (Nur phosphorylation-activation) pathways.

Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers. Fil: Rybak Wolf, Agnieszka. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Stottmeister, Christin. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Glažar, Petar. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Jens, Marvin. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Pino, Natalia. Max Planck Institute of Psychiatry; Alemania Fil: Giusti, Sebastian. Max Planck Institute of Psychiatry; Alemania Fil: Hanan, Mor. The Hebrew University of Jerusalem; Israel Fil: Behm, Mikaela. Stockholms Universitet; Suecia Fil: Bartok, Osnat. The Hebrew University of Jerusalem; Israel Fil: Ashwal Fluss, Reut. The Hebrew University of Jerusalem; Israel Fil: Herzog, Margareta. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Schreyer, Luisa. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Papavasileiou, Panagiotis. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Ivanov, Andranik. Max-Delbrück Center for Molecular Medicine; Alemania Fil: Öhman, Marie. Stockholms Universitet; Suecia Fil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Max Planck Institute of Psychiatry; Alemania Fil: Kadener, Sebastian. The Hebrew University of Jerusalem; Israel Fil: Rajewsky, Nikolaus. Max-Delbrück Center for Molecular Medicine; Alemania

Genetic mouse models based on the Cre-loxP system have been extensively used to explore the influence of specific gene deletions on different aspects of behavioral neurobiology. However, the interpretation of the effects attributed to the gene deletion might be obscured by potential side effects secondary to the Cre recombinase transgene insertion or Cre activity, usually neither controlled nor reported. Here, we performed a comprehensive behavioral analysis of endophenotypes of neuropsychiatric disorders in the extensively used NestinCre mouse line, commonly employed to restrict genetic modifications to the CNS. We observed no alterations in locomotion, general exploratory activity, learning and memory, sociability, startle response and sensorimotor gating. Although the overall response to stimuli triggering anxiety-like behaviors remained unaltered in NestinCre mice, a strong impairment in the acquisition of both contextual- and cued-conditioned fear was observed. These results underline the importance of adequately controlling the behavioral performance of the employed Cre-lines per-se in pre-clinical neurobehavioral research. Fil: Giusti, Sebastian Alejandro. Max Planck Institute of Psychiatry; Alemania Fil: Vercelli, Claudia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación En Biomedicina de Buenos Aires; Argentina Fil: Vogl, Annette M.. Max Planck Institute of Psychiatry; Alemania Fil: Kolarz, Adam W.. Max Planck Institute of Psychiatry; Alemania Fil: Pino, Natalia S.. Max Planck Institute of Psychiatry; Alemania Fil: Deussing, Jan M.. Max Planck Institute of Psychiatry; Alemania Fil: Refojo, Damian. Max Planck Institute of Psychiatry; Alemania

We focus on how the IL-1 system, T-helper1 (Th1) or Th2 cytokines and glucocorticoids, converge to give a unified physiological response. Glucocorticoids inhibit IL-1 and IL-1 receptor antagonist (IL-1ra) expression, Th1 cytokines stimulate both and Th2 cytokines stimulate IL-1ra and inhibit IL-1. Thus, during the Th1 response there is a window for IL-1 inflammatory activity, absent during the Th2 response. We also study the interactions among glucocorticoid and cytokine transcriptional activity. Glucocorticoids inhibit cytokine-induced transcription factors (NFkB, AP1) and cytokines enhance glucocorticoid receptor (GR) transcriptional activity, thus reciprocally fine tuning immunological control mechanisms.

MicroRNAs (miRNAs) are conserved noncoding RNAs that function as posttranscriptional regulators of gene expression. miR-9 is one of the most abundant miRNAs in the brain. Although the function of miR-9 has been well characterized in neural progenitors, its role in dendritic and synaptic development remains largely unknown. In order to target miR-9 in vivo, we developed a transgenic miRNA sponge mouse line allowing conditional inactivation of the miR-9 family in a spatio-temporal-controlled manner. Using this novel approach, we found that miR-9 controls dendritic growth and synaptic transmission in vivo. Furthermore, we demonstrate that miR-9-mediated downregulation of the transcriptional repressor REST is essential for proper dendritic growth. DOI: http://dx.doi.org/10.7554/eLife.02755.001, eLife digest Messages are sent back and forth in our brains by cells called neurons that connect to each other in complex networks. Neurons develop from stem cells in a complicated process that involves a number of different stages. In one of the final stages, tree-like structures called dendrites emerge from the neurons and connect with neighboring neurons via special junctions called synapses. A group of small RNA molecules called microRNAs have roles in controlling the development of neurons. One microRNA, called miR-9, is abundant in the brain and is known to be involved in the early stages of neuron development. However, its role in the formation of dendrites and synapses remains unclear. Giusti et al. studied this microRNA in mice. A length of DNA, coding for an RNA molecule that binds to miR-9 molecules and stops them performing their normal function, was inserted into the mice. These experiments showed that miR-9 is involved in controlling dendrite growth and synaptic function. To enable a neuron to produce dendrites, miR-9 binds to and interferes with the RNA molecules that are needed to make a protein called REST. This protein is a transcription factor that switches off the expression of other genes so, in effect, miR-9 allows a set of genes that are needed for dendrite growth to be switched on. The methodology developed by Giusti et al. could be used to study the functions of other microRNAs. DOI: http://dx.doi.org/10.7554/eLife.02755.002

Immune system disorders are often accompanied by alterations in the reproductive axis. The bacterial endotoxin (lipopolysaccharide, LPS) has inflammatory effects and activates cytokine release in the

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and ß-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders. Fil: Bonfiglio, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina; Fil: Inda, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina; Fil: Senin, Sergio Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina; Fil: Maccarrone, Giuseppina. Max-Planck-Institut Für Psychiatrie; Alemania; Fil: Refojo, Damian. Max-Planck-Institut Für Psychiatrie; Alemania; Fil: Giacomini, Damiana Paula. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Invest.bioquimicas de Bs.as(i); Argentina; Fil: Turck, Christoph W.. Max-Planck-Institut Für Psychiatrie; Alemania; Fil: Holsboer, Florian. Max-Planck-Institut Für Psychiatrie; Alemania; Fil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina; Fil: Silberstein, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Conicet -; Instituto P; Argentina

The corticotropin-releasing hormone receptor 1 (CRHR1) critically controls behavioral adaptation to stress and is causally linked to emotional disorders. Using neurochemical and genetic tools, we determined that CRHR1 is expressed in forebrain glutamatergic and γ-aminobutyric acid-containing (GABAergic) neurons as well as in midbrain dopaminergic neurons. Via specific CRHR1 deletions in glutamatergic, GABAergic, dopaminergic, and serotonergic cells, we found that the lack of CRHR1 in forebrain glutamatergic circuits reduces anxiety and impairs neurotransmission in the amygdala and hippocampus. Selective deletion of CRHR1 in midbrain dopaminergic neurons increases anxiety-like behavior and reduces dopamine release in the prefrontal cortex. These results define a bidirectional model for the role of CRHR1 in anxiety and suggest that an imbalance between CRHR1-controlled anxiogenic glutamatergic and anxiolytic dopaminergic systems might lead to emotional disorders.

Glucocorticoids (GCs) and cAMP-dependent signaling pathways exert diverse and relevant immune regulatory functions, including a tight control of T cell death and homeostasis. Both of these signaling molecules inhibit TCR-induced cell death and FasL expression, but the underlying mechanisms are still poorly understood. Therefore, to address this question, we performed a comprehensive screening of signaling pathways downstream of the TCR, in order to define which of them are targets of cAMP- and GC-mediated inhibition. We found that cAMP inhibited NF-κB and ERK pathways through a PKA-dependent mechanism, while Dexamethasone blocked TCR-induced NF-κB signaling. Although GCs and cAMP inhibited the induction of endogenous FasL mRNA expression triggered by TCR activation, they potentiated TCR-mediated induction of FasL promoter activity in transient transfection assays. However, when the same FasL promoter was stably transfected, the facilitatory effect of GCs and cAMP became inhibitory, thus resembling the effects on endogenous FasL mRNA expression. Hence, the endogenous chromatinization status known to occur in integrated or genomic vs. episomic DNA might be critical for proper regulation of FasL expression by cAMP and GCs. Our results suggest that the chromatinization status of the FasL promoter may function as a molecular switch, controlling cAMP and GC responsiveness and explaining why these agents inhibit FasL expression in T cells but induce FasL in other cell types.

Corticotropin-releasing hormone (CRH) plays a key role in adjusting the basal and stress-activated hypothalamic-pituitary-adrenal axis (HPA). CRH is also widely distributed in extrahypothalamic circuits, where it acts as a neuroregulator to integrate the complex neuroendocrine, autonomic, and behavioral adaptive response to stress. Hyperactive and/or dysregulated CRH circuits are involved in neuroendocrinological disturbances and stress-related mood disorders such as anxiety and depression. This review describes the main physiological features of the CRH network and summarizes recent relevant information concerning the molecular mechanism of CRH action obtained from signal transduction studies using cells and wild-type and transgenic mice lines. Special focus is placed on the MAPK signaling pathways triggered by CRH through the CRH receptor 1 that plays an essential role in CRH action in pituitary corticotrophs and in specific brain structures. Recent findings underpin the concept of specific CRH-signaling pathways restricted to specific anatomical areas. Understanding CRH action at molecular levels will not only provide insight into the precise CRH mechanism of action, but will also be instrumental in identifying novel targets for pharmacological intervention in neuroendocrine tissues and specific brain areas involved in CRH-related disorders. Fil: Bonfiglio, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Inda, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina Fil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina Fil: Holsboer, Florian. Max Planck Institute of Psychiatry; Alemania Fil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina Fil: Silberstein Cuña, Susana Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina

There is an urgent need to generate new drugs or improve existing ones in the pharmacology of mood disorders. The corticotropin-releasing hormone (CRH) system is closely involved in the development and course of depression, and drugs targeting this system arguably offer hope to improve the current tools for drug treatment of depression. Recent clinical studies in depressed patients showed that CRHR1 antagonists improve clinical symptoms of anxiety and depression and reduce stress hormone release following psychosocial stress. These effects of CRHR1 antagonists were not associated with reduced secretory capacity of corticotrophic cells because of CRH receptor abundance at the pituitary level, which contrasts with CRH receptors in the brain. This is in accordance with previous studies showing that CRH injections into the mouse brain activate MAPK pathways in a brain region-specific manner pointing toward differences in signaling pathways beyond the receptor level. We will highlight this and discuss how these brain area-specific differences may offer opportunities for drug discovery. An additional puzzle in the search of new targets for depression is the lack of bona fide animal models helping to discover the antidepressants that are not monoamine based. We recently developed a conditional mouse model that overexpresses CRH in a spatio-temporal-regulated fashion and permits to dissect precisely the contribution of different brain areas to the CRH-dependent behaviors. Recent findings obtained with this mouse model and its usefulness in the context of the CRH-dependent, region-specific changes in depression will be discussed.

Corticotropin-releasing hormone (CRH) coordinates neuroendocrine and behavioral adaptations to stress. Acute CRH administration in vivo activates extracellular signal-regulated kinase 1/2 (ERK1/2) in limbic brain areas, acting through the CRH receptor type 1 (CRH-R1). In the present study, we used CRH-COE-Cam mice that overexpress CRH in limbic-restricted areas, to analyze the effect of chronic CRH overexpression on ERK1/2 activation. By immunohistochemistry and confocal microscopy analysis we found that pERK1/2 levels in the basolateral amygdala (BLA) were similar in control and CRH overexpressing mice under basal conditions. Acute stress caused comparably increased levels of corticosterone in both control (CRH-COEcon-Cam) and CRH overexpressing (CRH-COEhom-Cam) animals. CRH-COEhom-Cam mice after stress showed reduced pERK1/2 immunoreactivity in the BLA compared to CRH-COEhom-Cam animals under basal conditions. Radioligand binding and in situ hybridization revealed higher density of CRH-R1 in the amygdala of CRH-COEhom mice under basal conditions compared to control littermates. A significant reduction of the receptor levels was observed in this area after acute stress, suggesting that stress may trigger CRH-R1 internalization/downregulation in these CRH overexpressing mice. Chronic CRH overexpression leads to reduced ERK1/2 activation in response to acute stress in the BLA.

Conditional CRH overexpressing mice: an animal model for stress-elicited pathologies and treatments that target the central CRH system

Cushing's disease is a severe clinical condition caused by hypersecretion of corticosteroids due to excessive ACTH secretion from a pituitary adenoma. In the last years there was only few progress elucidating the molecular mechanisms responsible for constitutive and autonomous ACTH secretion of pituitary corticotrophinomas. As a consequence, no effective drug therapy is currently available, particularly if surgical excision is not successful. IFN-γ is a cytokine that exerts potent antiproliferative and tumoricidal effects in a variety of cancers. Moreover, IFN-γ modulates normal pituitary hormone secretion, and has been shown to inhibit the expression of the precursor of ACTH, POMC, in murine ACTH secreting AtT-20 tumor cells. In order to identify molecular targets for the treatment of Cushing's disease we have studied the involvement of IFN-γ in the molecular events leading to the control of POMC transcriptional repression. We demonstrate that IFN-γ inhibits proliferation and POMC transcriptional activity of AtT-20 tumor cells via the activation of the JAK-STAT1 signalling cascade. Furthermore, the transcription factor NF-κB is associated with the transcriptional inhibition of POMC by IFN-γ. In addition, α and β IFN-γ receptor subunits are present at the protein level in human corticotroph tumor cells. Interestingly, IFN-γ inhibits ACTH production from human ACTH secreting tumor cells in primary cell culture without affecting basal ACTH biosynthesis in normal non-tumoral pituitary cells. In conclusion, our data for the first time show that POMC transcription can be negatively regulated by a JAK-STAT1 and NF-κB-dependent signalling pathway. The development of therapeutic agents that target this novel IFN-γ-JAK-STAT1/NF-κB pathway could therefore prove valuable for the effective treatment of Cushing's disease.

Glucocorticoids (GCs) immunosuppression acts via regulation of several transcription factors (TF), including AP-1, NFkB and NFAT. GCs inhibit Th1 cytokines and promote a shift towards Th2 differentiation. Th1 phenotype depends on TF T-bet. In this study we examined GC regulation of T-bet. We found that GCs inhibit T-bet transcriptional activity. We show that glucocorticoid receptor (GR) physically interacts with T-bet both in transfected cell lines and in primary splenocyte cultures with endogenous GR and T-bet. This interaction also blocks GR-dependent transcription. We show both in vitro and in vivo at endogenous binding sites that the mechanism underlying T-bet inhibition further involves reduction of T-bet binding to DNA. Using specific mutations of GR, we demonstrate that the first zinc finger region of GR is required for T-bet inhibition. GCs additionally inhibit T-bet both at mRNA and protein expression levels, revealing another layer of GR action on T-bet. Finally, we examined the functional consequences of GR/T-bet interaction on interferon-gamma, showing that GCs inhibit transcriptional activity of T-bet on its promoter. In view of the crucial role of T-bet in T cell differentiation and inflammation, we propose that GR inhibitory interaction with T-bet may be an important mechanism underlying the immunosuppressive properties of GCs. Fil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Druker, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Toscano, Marta Alicia. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Inmunogenética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Rein, Theo. Max Planck Institut für Psychiatrie; Alemania Fil: Holsboer, Florian. Max Planck Institut für Psychiatrie; Alemania Fil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

The molecular mechanisms governing the pathogenesis of ACTH-secreting pituitary adenomas are still obscure. Furthermore, the pharmacological treatment of these tumors is limited. In this study, we report that bone morphogenetic protein-4 (BMP-4) is expressed in the corticotrophs of human normal adenohypophysis and its expression is reduced in corticotrophinomas obtained from Cushing's patients compared with the normal pituitary. BMP-4 treatment of AtT-20 mouse corticotrophinoma cells has an inhibitory effect on ACTH secretion and cell proliferation. AtT-20 cells stably transfected with a dominant-negative form of the BMP-4 signal cotransducer Smad-4 or the BMP-4 inhibitor noggin have increased tumorigenicity in nude mice, showing that BMP-4 has an inhibitory role on corticotroph tumorigenesis in vivo. Because the activation of the retinoic acid receptor has an inhibitory action on Cushing's disease progression, we analyzed the putative interaction of these two pathways. Indeed, retinoic acid induces both BMP-4 transcription and expression and its antiproliferative action is blocked in Smad-4dn- and noggin-transfected Att-20 cells that do not respond to BMP-4. Therefore, retinoic acid induces BMP-4, which participates in the antiproliferative effects of retinoic acid. This new mechanism is a potential target for therapeutic approaches for Cushing's disease. Fil: Giacomini, Damiana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Páez Pereda, Marcelo. Max Planck Institute of Psychiatry; Alemania. Affectis Pharmaceuticals; Alemania Fil: Theodoropoulou, Marily. Max Planck Institute of Psychiatry; Alemania Fil: Labeur, Marta. Max Planck Institute of Psychiatry; Alemania Fil: Refojo, Damian. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Gerez, Juan Atilio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Chervin, Alberto. Hospital Santa Lucía; Argentina Fil: Berner, Silvia. Hospital Santa Lucía; Argentina Fil: Losa, Marco. Max Planck Institute of Psychiatry; Alemania Fil: Buchfelder, Michael. University of Gottingen Medical School; Alemania Fil: Renner, Ulrich. Max Planck Institute of Psychiatry; Alemania Fil: Stalla, Günter K.. Max Planck Institute of Psychiatry; Alemania Fil: Arzt, Eduardo Simon. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina

Bone morphogenetic protein-4 (BMP-4), a member of the transforming growth factor-Beta(TGF-Beta) family, is overexpressed in different prolactinoma models and induces the development of these lineage adenomas. SMAD proteins activated by growth factors of the TGF-Beta and BMP family interact with estrogen receptors to stimulate the proliferation of prolactin and growth hormone-secreting cells. Furthermore, BMP-4 presents differential expression in normal and adenomatous corticotropes and inhibitory action on corticotropinoma cell proliferation. Moreover, BMP-4 mediates the antiproliferative action of retinoic acid in these cells. The present review highlights not only the crucial and opposite role of BMP-4 in the progression of pituitary adenomas but also that BMP-4 and retinoic acid interaction might serve as a potential new mechanism target for therapeutic approaches for Cushing disease.

The secretion of interleukin 2 (IL-2) is a key event in T cell activation. IL-2 allows T cells to enter into the S phase of the cell cycle and divide. After the activation phase takes place, T lymphocytes proliferate and differentiate to generate effector T cells. Thereby, T helper (Th) precursor cells, which are functionally immature, may become Th1 or Th2 effector cells. These subsets are responsible for cell-mediated immunity and humoral responses, respectively. Both T cell activation and Th differentiation are processes that depend on changes in the pattern of gene expression. The expression and changes in the genes responsible for these events are regulated by transcription factors. This review will focus on both the transcription factors involved in the control of IL-2 as well as those that are key to T helper differentiation.