The CRF/Urocortins family of stress-related neuropeptides
Scientific background: Maintenance of homeostasis in the presence of real or perceived challenges requires numerous adaptive responses involving changes in the central nervous and neuroendocrine systems. The biological system that has been most closely linked to the stress response in mammals is the neuroendocrine limbic-hypotalamic-pituitary-adrenal (LHPA) axis. Perception of physical or psychological stress by an organism is followed by a series of events, which result in changes in emotional and cognitive functions, modulation of autonomic activities and the secretion of glucocorticoids from the adrenal cortex. Both activation and termination of the behavioral, autonomic and adrenocortical stress responses are critical for adaptation and survival. The neuropeptide corticotropin releasing factor (CRF), expressed and secreted from the parvocellular neurons of the paraventricular nucleus (PVN) in the hypothalamus, represent the final common path for the integration of the neuroendocrine stress response in the brain. CRF and, to a lesser extent, arginine vasopressin playing an important and well-established role in the regulation of the HPA axis under basal and stress conditions. In addition to its hypophysiotropic action, CRF is proposed to integrate the autonomic, metabolic and behavioral responses to stressors. CRF and its receptors are implicated in the control of arousal, anxiety, cognitive functions and appetite, however, the brain circuits and downstream signals responsible for these CRF-linked stress related responses are less understood. Dysregulation of the stress response can have severe psychological and physiological consequences. Chronic hyperactivation of the CRF system has been linked to stress-related emotional disorders such as anxiety, anorexia nervosa and depression.
In addition to CRF, the mammalian CRF-peptide family contains Urocortin 1 (Ucn-1), and the more recently identified peptides, Urocortin 2 (Ucn-2), and Urocortin 3 (Ucn-3). The effects of CRF-related peptides are mediated through activation of two known receptors, CRF receptor type 1 (CRFR1), and CRFR2. Evidence from studies employing competitive peptide or small molecule CRFR1 antagonists provide strong support for the hypothesis that the brain CRF/Urocortin systems play a role in mediating behavioral responses to stress. Data in animal models demonstrating anxiogenic-like behavioral effects of CRF administration and anxiolytic-like activity of CRFR1-selective antagonists led to the suggestion that CRF may be involved in anxiety-related disorders. A central role for the CRF/Urocortin systems in major depression was also suggested. Using different animal models, potential antidepressant-like effects of selective CRFR1 antagonists have been demonstrated. An opposite role for CRFR2 in modulating anxiety-like behavior was suggested based on the complementary behavioral phenotypes of CRFR1 and CRFR2 deficient mice. Mice deficient for CRFR1 display decreased anxiety-like behavior and have an impaired HPA-axis stress response, while CRFR2 mutant mice show increased anxiety-like behaviors and an accelerated HPA response to stress. However, the anxiety-related effects of administration of CRFR2 agonists and antagonists into the cerebral ventricles or into specific brain regions have been less consistent, with some evidence for brain site or ligand specificity.
Schematic representation of the mammalian CRF/Urocortin family of peptides, receptors and binding proteins (Kuperman Y & Chen A. Trends Endocrinol Metab. 2008).
The CRF/Urocortin family of ligands and receptors is of seminal importance to three overlapping fields: stress biology, neuroendocrinology and regulatory peptides. Specifying the contributions of the CRF/Urocortin family of ligands and receptors to the maintenance of homeostasis and to stress-linked allostasis may improve our ability to design therapeutic interventions and thus manage affective and other stress-related disorders. The goal of our recent (see below) and ongoing CRF/Urocortins specific projects and the experiments designed to accomplish them will facilitate our understanding of the molecular basis for the roles of CRF/Urocortins in mediating the physiological and behavioral responses to stress and will improve our knowledge on the pathways by which stress is perceived, processed, and transduced into a neuroendocrine and behavioral responses.
Summary of selected projects:
1. Urocortin1 and 2 double-deficient mice show robust anxiolytic phenotype and modified serotonergic activity in anxiety circuits
The urocortin family of neuropeptides is suggested to be involved in homeostatic coping mechanisms of the central stress response, via the activation of corticotropin-releasing factor receptor type 2 (CRFR2). The neuropeptides urocortin-1 and 2 serve as endogenous ligands for the CRFR2, which is highly expressed by the dorsal raphe (DRN) serotonergic neurons and is suggested to be involved in regulating major component of the central stress response. Here, we describe genetically modified mice in which both urocortin 1 and 2 are developmentally deleted. Double knockout mice demonstrated a robust anxiolytic phenotype and altered hypothalamic-pituitary-adrenal axis activity, compared with wild type mice. The significant reduction in anxiety-like behavior observed in these mice was further enhanced following exposure to acute stress, and was correlated with the levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) measured in brain regions associated with anxiety circuits. Thus, we propose that the urocortin/CRFR2 serotonergic system plays an important role in regulating homeostatic equilibrium under challenge conditions. (Neufeld et al., Urocortin1 and 2 double-deficient mice show robust anxiolytic phenotype and modified serotonergic activity in anxiety circuits. Mol Psychiatry 15:426-441, 2010)
Schematic illustration of mouse brain and representative open field exploratory tracks of the double Urocortin1 and Urocortin2 knockout (Ucn1/Ucn2 dKO) mice. Significant reduction in anxiety-like behavior was observed in the Ucn1/Ucn2 dKO mice, which is represented by the open field exploratory behavior of the mutant (blue track) and wild-type (green track) mice. The behavioral data were correlated with the levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) measured in brain regions associated with anxiety circuits. The mouse brain is showing serotonergic projections from the dorsal raphe nucleus (shown in red) to forebrain components of an anxiety-related neuronal system. Ucn1/Ucn2 dKO mice (blue projections) show elevated tissue concentrations of 5-HT and its metabolite 5-HIAA in the CA1 region of the ventral hippocampus, entorhinal cortex, basolateral amygdala, central amygdala and bed nucleus of the stria terminalis compared with WT mice (green projections). (For further information on this figure, please refer to the article by Neufeld-Cohen et al., Molecular Psychiatry 15:426-441, 2010).
2. Prolonged and site-specific over-expression of corticotropin-releasing factor reveals differential roles for extended amygdala nuclei in emotional regulation
Corticotropin releasing factor (CRF) has a key role in the central stress response, and altered levels of this neuropeptide are linked to stress-related psychopathologies such as anxiety and depression. These disorders are associated with the inability to properly regulate the stress response, specifically following exposure to prolonged stressful stimuli. Therefore, the current study assessed the effects of prolonged and site-specific over-expression of CRF, which mimics the state of chronic production, in extended amygdala nuclei that are known to be involved in mediating anxiety-like states. We first constructed and generated lentiviruses that overexpress (OE) CRF in a robust and stable manner, and then generated two male mouse models continuously over-expressing CRF, either at the central nucleus of the amygdala (CeA), or at the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNSTdl). After four months, behavioral assessments were conducted for anxiety and depressive indices on these mice. Surprisingly, prolonged CRF OE at the CeA attenuated stress-induced anxiety-like behaviors, while prolonged CRF OE in the BNSTdl increased depressive-like behaviors, without affecting anxiety levels. These results demonstrate possible differential roles for CRF expressed by distinct loci of the extended amygdala, in mediating stress-induced emotional behaviors. (Regev et al., Prolong and site-specific over-expression of corticotropin releasing factor reveals differential role for extended amygdala nuclei in emotional regulation. Mol. Psychiatry (In Press), 2010)
Design and in-vitro and in-vivo validation of lentiviruses over-expressing CRF. A) In-vitro validation of lentiviruses over-expressing CRF. Schematic representation of the lentiviral construct designed to over-express CRF together with a GFP protein (a). Confirmation of CRF over-expression was performed using western blot analysis (b) and immunocytochemistry (c), in HEK293T cells infected with CRF over-expressing lentiviruses. B) In vivo validation of CRF over-expressing lentiviruses injected into the CeA. Brain sections adapted from the Paxinos & Franklin mouse brain atlas, showing the sites of CeA (a and b). Endogenous mRNA of CRF was detected using in-situ hybridization at the CeA (c) and mice injected with CRF over expressing lentiviruses to the CeA show imunoreactivity to GFP at site of injection (d). (For further information on this figure, please refer to the article by Regev et al., Molecular Psychiatry, In Press, 2010).
