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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.

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.

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

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.

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

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

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

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

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.

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

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.

RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.