Life Science Open Day | 2006 | Weizmann Institute of Science

972 8 934 4490

Department of Neurobiology

972 8 934 4131

alon.chen@weizmann.ac.il

All living organisms struggle towards maintaining a dynamic equilibrium called homeostasis. Physiological or psychological stimuli that disrupt the organism’s homeostatic equilibrium are known as stressors. The stressors trigger a series of coordinated physiological and behavioral responses, known as the stress response, aimed to enhance the probability of survival by protecting homeostatic equilibrium. Maintenance of homeostasis in the presence of real or perceived challenges requires numerous adaptive responses involving changes in the central nervous, endocrine and immune systems. When a situation is perceived as stressful the brain activates many neuronal circuits to adapt to the demand. Corticotropin releasing factor (CRF), originally isolated from hypothalamus, plays an important and well established role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis under basal and stress conditions (Fig. 1A). In addition to its hypophysiotropic action, CRF is proposed to integrate the endocrine, autonomic and behavioral responses to stressors (Fig. 1A). CRF and its receptors are implicated in the control of arousal, anxiety, cognitive functions and appetite. 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 melancholic depression. In addition to CRF, the mammalian CRF-peptide family contains urocortin 1, and the recently identified peptides, urocortin 2 and urocortin 3 (Fig. 1B). The effects of CRF-related peptides are mediated through activation of two known receptors, CRF receptor type 1 (CRFR1), and CRFR2 (Fig 1B). CRFR1 mRNA is widely expressed in mammalian brain and pituitary, with high levels in the anterior pituitary, cerebral cortex, cerebellum, amygdala, hippocampus and olfactory bulb. CRFR2 mRNA is expressed in a discrete pattern in the brain with highest densities in the lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, olfactory bulb, mesencephalic raphe nuclei and medial amygdala. Receptor binding and intracellular cAMP accumulation studies in cells stably transfected with CRF receptors have demonstrated that CRFR1 and CRFR2 differ pharmacologically. CRF has relatively lower affinity for CRFR2 compared to its affinity for CRFR1, Ucn 1 has equal affinities for both receptors, and Ucn 2 and Ucn 3 appear to be selective for CRFR2 (Fig. 1B).

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

Homeostatic regulation of neuroendocrine stress by the CRF/Urocortin peptide family and their receptors

Alon chen

Shosh Gil

Yael Kuperman

Limor Regev

Adi Neufeld

Ifat Musseri

Meital Ten-Bosch

Fig. 1 Schematic representation of the CRF actions (A) and members of the CRF family of peptide and receptors (B).

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.

The long term goal of our research is to examine the hypothesis that the CRF/urocortin family of peptides and receptors play important roles in the modulation of neuroendocrine, metabolic and behavioral responses to challenge. The CRF/urocortin family of ligands and cognate receptors is of seminal importance to three overlapping fields: neuroendocrinology, stress biology and regulatory peptides. Specifying the contributions of the CRF 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 disorders.

We are using integrative molecular, biochemical, physiological and behavioral methods, with focus on the generation of mutant mice models as an in vivo tool, to study the central roles of

CRF family of neuropeptides and receptors in coordinating the neuroendocrine, autonomic and behavioral responses to stress (Fig. 2). Genetic manipulation of CRF signaling pathways in the whole animal context will permit us to understand their roles in behavioral and physiological functions. In order to explore the relative importance of CRF family of ligands and receptors in specific brain nuclei, we are currently using the lentivirus-based system to functionally overexpress the relevant genes or alternatively to knockdown their expression using small interfering RNA (siRNA). A site-specific knockdown or overexpression of the CRF family genes in adult mice will provide us with a powerful in vivo model to explore the relative importance of these genes in different brain sites to the stress response and will help to unravel the controversies in this field. Genetically modified mice are subjected to stress-related endocrine, metabolic and behavioral studies, based on the specific brain site targeted (Fig. 2). We aim to probe the physiological roles of CRFR1 and CRFR2-dependent pathways in discrete brain nuclei in the maintenance of homeostasis, which will refine our understanding of the central roles of CRF family of ligands and receptors in stress-related disorders.

Urocortin 2 deficient mice exhibit gender specific alterations in circadian HPA axis and in depressive-like behavior

Urocortin 2 (Ucn 2) is a member of the CRF family and a specific ligand for the type 2 CRF receptor. Ucn 2 is expressed in discrete regions of the rodent central nervous system including stress-related nuclei. To determine the physiological role of Ucn 2, mice null for Ucn 2 were generated and the HPA axis activity, stress-related behaviors and alterations in expression levels of CRF-related ligands and receptors were studied. Ucn 2 mRNA and peptide were undetectable in null mutant mice and the brain expression of CRF family peptides and receptors was altered compared with wild type (WT) littermates. Nocturnal basal ACTH and corticosterone (CORT) levels were higher in female, but not male, mutant mice relative to WT littermates. A 24 hr CORT profile showed elevated levels in females during the interphase light-dark cycle transition. In situ hybridization showed increased hypothalamic vasopressin, but not CRF, expression in female knockout mice, which positively correlated with changes in water intake at the light-dark interphase. Thus, Ucn 2 may play a role in the regulation of basal daily rhythms of ACTH, CORT and fluid intake in females. ACTH and CORT levels did not differ between mutant and WT mice subjected to restraint or swim stress. Female Ucn 2-deficient mice were less immobile than WT littermates in modified forced swim and tail suspension tests, an antidepressant-like phenotype. In contrast, male WT and null mutant mice did not differ in these tests. Ucn 2 null and WT mice did not differ in tests of locomotor activity, anxiety-like behavior, appetitive operant learning or fear conditioning. Gender differences in HPA axis activation and the prevalence of mood disorders are well-documented. The differential phenotype of Ucn 2 deficiency in female mice may imply a role for Ucn 2 in these gender differences.

Selected publicationsChen, A., Kaganovsky, E., Rahimipour, S., Ben-Aroya, N., Okon,

E., and Koch, Y. (2002) Two forms of gonadotropin-releasing hormone (GnRH) are expressed in human breast tissue and overexpressed in breast cancer: a putative mechanism for the antiproliferative effect of GnRH by down-regulation of acidic ribosomal phosphoproteins P1 and P2. Cancer Res. 62, 1036-1044.

Chen, A., Ganor, Y., Rahimipour, S., Ben-Aroya, N., Koch, Y., and Levite, M. (2002) The neuropeptides GnRH-II and GnRH-I are

Fig. 2 Schematic representation of our research methodologies.

produced by human T cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs. Nat. Med. 8, 1421-1426.

Chen, A., Vaughan, J., and Vale, W.W. (2003) Glucocorticoids regulate the expression of the mouse Urocortin II Gene: a putative connection between the corticotropin-releasing factor receptor pathways. Mol. Endocrinol. 17, 1622-1639.

Chen, A., Blount, A., Vaughan, J., and Vale, W.W. (2004) Urocortin II gene is highly expressed in mouse skin and skeletal muscle tissues: localization, basal expression in CRFR1 and CRFR2-null mice and regulation by glucocorticoids. Endocrinology 145, 2445-2457.

Chen, A., Perrin, M., DiGruccio M., Vaughan, J., Brar, B., Arias, C., Lewis, K., Rivier, J., Sawchenko, P., and Vale, W. (2005) A soluble mouse brain splice variant of type 2a corticotropin-releasing factor (CRF) receptor binds ligands and modulates their activity. Proc. Natl. Acad. Sci. U S A. 102, 2620-2625.

Chen, A., Perrin, M., Brar, B., Li, C., Jamieson, P., Digruccio, M., Lewis, K., and Vale, W. (2005) Mouse corticotropin releasing factor receptor type 2{alpha} gene: isolation, distribution, pharmacological characterization and regulation by stress and glucocorticoids. Mol. Endocrinol. 19, 441-458.

Chen, A., Zorrilla, E., Smith, S., Rousso, D., Levy, C., Donaldson, C., Roberts, A., Koob, G., Lee, K.F., and Vale, W. (2006) Urocortin 2 knockout mice exhibit gender specific alterations in circadian HPA axis and in depressive-like behavior (J. Neurosci., In Press).

Internal SupportThe Center for New Scientists, Women’s Health Research Center,

Nella and Leon Benoziyo Center for Neurosciences.