RESUMEN
Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.
Asunto(s)
Encéfalo , ARN Circular , Transmisión Sináptica , ARN Circular/genética , Animales , Ratones , Encéfalo/metabolismo , Encéfalo/fisiología , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiologíaRESUMEN
Depression and other psychiatric stress-related disorders are leading causes of disability worldwide. Up to date, treatments of mood disorders have limited success, most likely due to the multifactorial etiology of these conditions. Alterations in inflammatory processes have been identified as possible pathophysiological mechanisms in psychiatric conditions. Here, we review the main features of 2 systems involved in the control of these inflammatory pathways: the CRH system as a key regulator of the stress response and the ATP-gated ion-channel P2X7 receptor (P2X7R) involved in the control of immune functions. The pathophysiology of depression as a stress-related psychiatric disorder is depicted in terms of the impact of CRH and P2X7R function on inflammatory pathways in the brain. Understanding pathogenesis of affective disorders will lead to the development of therapies for treatment of depression and other stress-related diseases.
Asunto(s)
Hormona Liberadora de Corticotropina , Trastornos Mentales , Encéfalo/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Depresión , Humanos , Receptores Purinérgicos P2X7RESUMEN
Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.
Asunto(s)
Diferenciación Celular , AMP Cíclico/metabolismo , Células Piramidales/citología , Células Piramidales/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Adenilil Ciclasas/sangre , Adenilil Ciclasas/metabolismo , Animales , Biomarcadores , Proteína de Unión a CREB/metabolismo , Puntos de Control del Ciclo Celular , Supervivencia Celular , Células Cultivadas , Hormona Liberadora de Corticotropina/metabolismo , Humanos , RatonesRESUMEN
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.
Asunto(s)
Polimorfismo de Nucleótido Simple/genética , Receptores Purinérgicos P2X7/fisiología , Western Blotting , Calcio/metabolismo , Calcio/fisiología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Inmunoprecipitación , Técnicas de Placa-Clamp , Polimorfismo de Nucleótido Simple/fisiología , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Conducta/fisiología , Sistema Hipotálamo-Hipofisario/inmunología , Sistema Hipófiso-Suprarrenal/inmunología , Transducción de Señal , Animales , Sistema Hipotálamo-Hipofisario/enzimología , Ratones , Ratones Transgénicos , Especificidad de Órganos , Sistema Hipófiso-Suprarrenal/enzimologíaRESUMEN
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.