Sex differences in CRF1, CRF, and CRFBP expression in C57BL/6J mouse brain across the lifespan and in response to acute stress.

Signaling pathways mediated by corticotropin-releasing factor and its receptor 1 (CRF1) play a central role in stress responses. Dysfunction of the CRF system has been associated with neuropsychiatric disorders. However, dynamic changes in the CRF system during brain development and aging are not well investigated. In this study, we characterized CRF1, CRF, and corticotropin-releasing factor binding protein (CRFBP) expression in different brain regions in both male and female C57BL/6J mice from 1 to 18 months of age under basal conditions as well as after an acute 2-hr-restraint stress. We found that CRF and CRF1 levels tended to increase in the hippocampus and hypothalamus, and to decrease in the prefrontal cortex with aging, especially at 18 months of age, whereas CRFBP expression followed an opposite direction in these brain areas. We also observed area-specific sex differences in the expression of these three proteins. For example, CRF expression was lower in females than in males in all the brain regions examined except the prefrontal cortex. After acute stress, CRF and CRF1 were up-regulated at 1, 6, and 12 months of age, and down-regulated at 18 months of age. Females showed more robust changes compared to males of the same age. CRFBP expression either decreased or remained unchanged in most of the brain areas following acute stress. Our findings suggest that brain CRF1, CRF, and CRFBP expression changes dynamically across the lifespan and under stress condition in a sex- and regional-specific manner. Sex differences in the CRF system in response to stress may contribute to the etiology of stress-related neuropsychiatric disorders.

Enhanced CRFR1-Dependent Regulation of a Ventral Tegmental Area to Prelimbic Cortex Projection Establishes Susceptibility to Stress-Induced Cocaine Seeking.

The ability of stress to trigger cocaine seeking in humans and rodents is variable and is determined by the amount and pattern of prior drug use. This study examined the role of a corticotropin releasing factor (CRF)-regulated dopaminergic projection from the ventral tegmental area (VTA) to the prelimbic cortex in shock-induced cocaine seeking and its recruitment under self-administration conditions that establish relapse vulnerability. Male rats with a history of daily long-access (LgA; 14 × 6 h/d) but not short-access (ShA; 14 × 2 h/d) self-administration showed robust shock-induced cocaine seeking. This was associated with a heightened shock-induced prelimbic cortex Fos response and activation of cholera toxin b retro-labeled VTA neurons that project to the prelimbic cortex. Chemogenetic inhibition of this pathway using a dual virus intersectional hM4Di DREADD (designer receptor exclusively activated by designer drug) based approach prevented shock-induced cocaine seeking. Both shock-induced reinstatement and the prelimbic cortex Fos response were prevented by bilateral intra-VTA injections of the CRF receptor 1 (CRFR1) antagonist, antalarmin. Moreover, pharmacological disconnection of the CRF-regulated dopaminergic projection to the prelimbic cortex by injection of antalarmin into the VTA in one hemisphere and the D1 receptor antagonist, SCH23390, into the prelimbic cortex of the contralateral hemisphere prevented shock-induced cocaine seeking. Finally, LgA, but not ShA, cocaine self-administration resulted in increased VTA CRFR1 mRNA levels as measured using in situ hybridization. Altogether, these findings suggest that excessive cocaine use may establish susceptibility to stress-induced relapse by recruiting CRF regulation of a stressor-responsive mesocortical dopaminergic pathway.SIGNIFICANCE STATEMENT Understanding the neural pathways and mechanisms through which stress triggers relapse to cocaine use is critical for the development of more effective treatment approaches. Prior work has demonstrated a critical role for the neuropeptide corticotropin releasing factor (CRF) in stress-induced cocaine seeking. Here we provide evidence that stress-induced reinstatement in a rat model of relapse is mediated by a CRF-regulated dopaminergic projection from the ventral tegmental area (VTA) that activates dopamine D1 receptors in the prelimbic cortex. Moreover, we report that this pathway may be recruited as a result of daily cocaine self-administration under conditions of extended drug access/heightened drug intake, likely as a result of increased CRFR1 expression in the VTA, thereby promoting susceptibility to stress-induced cocaine seeking.

The Effect of Acute and Repeated Stress on CRH-R1 and CRH-R2 mRNA Expression in Pituitaries of Wild Type and CRH Knock-Out Mice.

The activation of the HPA axis is the endocrine measure of stress responsiveness that is initiated by corticotropin-releasing hormone (CRH). CRH exerts its effects via CRHR1 and CRH-R2 receptors coupled to the cAMP signaling system and this process involves transcription factor cAMP-responsive element-binding protein (CREB).This study investigated the role of CRH and the possible involvement of CREB in gene regulation of CRH receptor, under basal conditions and after stress application in the pituitary. We used wild type (wt +/+) controls and CRH knock-out (CRH-KO -/-) male mice. Using CRH-deficient mice, we were able to investigate the consequences of the lack of the CRH on the expression of CRH receptors and transcriptional regulation mediated by CREB. We estimated the effect of acute (IMO 1×) and repeated (IMO 7×) restraint stressors lasting 30 and 120 min on the expression of mRNA CREB, CRH-R1, and CRH-R2 by qPCR. We found very significant difference in the expression of these peptides under the effect of single and repeated stress in control and CRH-KO mice. Our results indicate that both CRH receptors and CREB might be involved in the regulation of stress response in the pituitary of mice. We propose that regulation of the stress response may be better understood if more were known about the mechanisms of CRH receptor signal transduction and involvement of CREB system.

Urocortins and CRF receptor type 2 variants in the male rat colon: gene expression and regulation by endotoxin and anti-inflammatory effect.

Urocortins (Ucns) 1, 2, and 3 and corticotropin-releasing factor receptor 2 (CRF2) mRNA are prominently expressed in various layers of the upper gut. We tested whether Ucns and CRF2 variants are also expressed in the different layers of the rat colon, regulated by LPS (100 µg/kg ip) and play a modulatory role in the colonic immune response to LPS. Transcripts of Ucns and CRF2b, the most common isoform in the periphery, were detected in all laser microdissected layers, including myenteric neurons. LPS increased the mRNA level of Ucn 1, Ucn 2, and Ucn 3 and decreased that of CRF2b in both the colonic mucosa and submucosa + muscle (S+M) layers at 2, 6, and 9 h after injection with a return to basal at 24 h. In addition, CRF2a, another variant more prominent in the brain, and a novel truncated splice variant CRF2a-3 mRNA were detected in all segments of the large intestine. LPS reciprocally regulated the colonic expression of these CRF2 variants by decreasing both CRF2a and CRF2b, while increasing CRF2a-3 in the mucosa and S+M. The CRF2 antagonist astressin2-B further enhanced LPS-induced increase of mRNA level of interleukin (IL)-1ß, TNF-α, and inducible nitric oxide synthase in S+M layers and IL-1ß in the mucosa and evoked TNF-α expression in the mucosa. These data indicate that Ucns/CRF2 variants are widely expressed in all colonic layers and reciprocally regulated by LPS. CRF2 signaling dampens the CD14/TLR4-mediated acute inflammatory response to Gram-negative bacteria in the colon.

Binge Drinking Decreases Corticotropin-Releasing Factor-Binding Protein Expression in the Medial Prefrontal Cortex of Mice.

BACKGROUND: Dysregulation of the corticotropin-releasing factor (CRF) system has been observed in rodent models of binge drinking, with a large focus on CRF receptor 1 (CRF-R1). The role of CRF-binding protein (CRF-BP), a key regulator of CRF activity, in binge drinking is less well understood. In humans, single-nucleotide polymorphisms in CRHBP are associated with alcohol use disorder and stress-induced alcohol craving, suggesting a role for CRF-BP in vulnerability to alcohol addiction. METHODS: The role and regulation of CRF-BP in binge drinking were examined in mice exposed to the drinking in the dark (DID) paradigm. Using in situ hybridization, the regulation of CRF-BP, CRF-R1, and CRF mRNA expression was determined in the stress and reward systems of C57BL/6J mice after repeated cycles of DID. To determine the functional role of CRF-BP in binge drinking, CRF-BP knockout (CRF-BP KO) mice were exposed to 6 cycles of DID, during which alcohol consumption was measured and compared to wild-type mice. RESULTS: CRF-BP mRNA expression was significantly decreased in the prelimbic (PL) and infralimbic medial prefrontal cortex (mPFC) of C57BL/6J mice after 3 cycles and in the PL mPFC after 6 cycles of DID. No significant changes in CRF or CRF-R1 mRNA levels were observed in mPFC, ventral tegmental area, bed nucleus of the stria terminalis, or amygdala after 3 cycles of DID. CRF-BP KO mice do not show significant alterations in drinking compared to wild-type mice across 6 cycles of DID. CONCLUSIONS: These results reveal that repeated cycles of binge drinking alter CRF-BP mRNA expression in the mPFC, a region responsible for executive function and regulation of emotion and behavior, including responses to stress. We observed a persistent decrease in CRF-BP mRNA expression in the mPFC after 3 and 6 DID cycles, which may allow for increased CRF signaling at CRF-R1 and contribute to excessive binge-like ethanol consumption.

PKR deficiency alters E. coli-induced sickness behaviors but does not exacerbate neuroimmune responses or bacterial load.

BACKGROUND: Systemic inflammation induces neuroimmune activation, ultimately leading to sickness (e.g., fever, anorexia, motor impairments, exploratory deficits, and social withdrawal). In this study, we evaluated the role of protein kinase R (PKR), a serine-threonine kinase that can control systemic inflammation, on neuroimmune responses and sickness. METHODS: Wild-type (WT) PKR+/+ mice and PKR-/- mice were subcutaneously injected with live Escherichia coli (E. coli) or vehicle. Food consumption, rotarod test performance, burrowing, open field activity, object investigation, and social interaction were monitored. Plasma TNF-α and corticosterone were measured by ELISA. The percentage of neutrophils in blood was deduced from blood smears. Inflammatory gene expression (IL-1ß, TNF-α, IL-6, cyclooxygenase (COX)-2, iNOS) in the liver and the brain (hypothalamus and hippocampus) were quantified by real-time PCR. Blood and lavage fluid (injection site) were collected for microbiological plate count and for real-time PCR of bacterial 16S ribosomal DNA (rDNA). Corticotrophin-releasing hormone (CRH) expression in the hypothalamus was also determined by real-time PCR. RESULTS: Deficiency of PKR diminished peripheral inflammatory responses following E. coli challenge. However, while the core components of sickness (anorexia and motor impairments) were similar between both strains of mice, the behavioral components of sickness (reduced burrowing, exploratory activity deficits, and social withdrawal) were only observable in PKR-/- mice but not in WT mice. Such alteration of behavioral components was unlikely to be caused by exaggerated neuroimmune activation, by an impaired host defense to the infection, or due to a dysregulated corticosterone response, because both strains of mice displayed similar neuroimmune responses, bacterial titers, and plasma corticosterone profiles throughout the course of infection. Nevertheless, the induction of hypothalamic corticotrophin-releasing hormone (CRH) by E. coli was delayed in PKR-/- mice relative to WT mice, suggesting that PKR deficiency may postpone the CRH response during systemic inflammation. CONCLUSIONS: Taken together, our findings show that (1) loss of PKR could alter E. coli-induced sickness behaviors and (2) this was unlikely to be due to exacerbated neuroimmune activation, (3) elevated bacterial load, or (4) dysregulation in the corticosterone response. Further studies can address the role of PKR in the CRH response together with its consequence on sickness.

ß-Endorphin neuronal transplantation into the hypothalamus alters anxiety-like behaviors in prenatal alcohol-exposed rats and alcohol-non-preferring and alcohol-preferring rats.

BACKGROUND: Alcohol exposure has adverse effects on stress physiology and behavioral reactivity. This is suggested to be due, in part, to the effect of alcohol on ß-endorphin (ß-EP)-producing neurons in the hypothalamus. In response to stress, ß-EP normally provides negative feedback to the hypothalamic-pituitary-adrenal axis and interacts with other neurotransmitter systems in the amygdala to regulate behavior. We examined whether ß-EP neuronal function in the hypothalamus reduces the corticosterone response to acute stress, attenuates anxiety-like behaviors, and modulates alcohol drinking in rats. METHODS: To determine whether ß-EP neuronal transplants modulate the stress response, anxiety behavior, and alcohol drinking, we implanted differentiated ß-EP neurons into the paraventricular nucleus (PVN) of the hypothalamus of normal, prenatal alcohol-exposed, and alcohol-preferring (P) and alcohol-non-preferring (NP) rats. We then assessed corticosterone levels in response to acute restraint stress and other markers of stress response in the brain and anxiety-like behaviors in the elevated plus maze and open-field assays. RESULTS: We showed that ß-EP neuronal transplants into the PVN reduced the peripheral corticosterone response to acute stress and attenuated anxiety-like behaviors. Similar transplants completely reduced the hypercorticosterone response and elevated anxiety behaviors in prenatal alcohol-exposed adult rats. Moreover, we showed that ß-EP reduced anxiety behavior in P rats with minimal effects on alcohol drinking during and following restraint stress. CONCLUSIONS: These data further establish a role of ß-EP neurons in the hypothalamus for regulating physiological stress response and anxiety behavior and resemble a potential novel therapy for treating stress-related psychiatric disorders in prenatal alcohol-exposed children and those genetically predisposed to increased alcohol consumption.

Dynamics of appetite-mediated gene expression in daidzein-fed female rats in the meal-feeding method.

We previously found that daidzein decreased food intake in female rats. The present study aimed to elucidate the relationship between dynamics of appetite-mediated neuropeptides and the anorectic effect of daidzein. We examined appetite-mediated gene expression in the hypothalamus and small intestine during the 3 meals per day feeding method. Daidzein had an anorectic effect specifically at the second feeding. Neuropeptide-Y (NPY) and galanin mRNA levels in the hypothalamus were significantly higher after feeding in the control but not in the daidzein group, suggesting that daidzein attenuated the postprandial increase in NPY and galanin expression. The daidzein group had higher corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamus after feeding, and increased cholelcystokinin (CCK) mRNA levels in the small intestine, suggesting that CCK is involved in the hypothalamic regulation of this　anorectic effect. Therefore, daidzein may induce anorexia by suppressing expression of NPY and galanin and increasing expression of CRH in the hypothalamus.

Noise Stress-Induced Changes in mRNA Levels of Corticotropin-Releasing Hormone Family Molecules and Glucocorticoid Receptors in the Rat Brain.

Noise is a widespread stress resource that may lead to detrimental effects on the health. However, the molecular basis of the stress response caused by noise remains elusive. We have studied the effects of acute and chronic noise stress on stress-related molecules in the hypothalamus and hippocampus and also corticosterone responses. Sprague Dawley rats were randomized into control, acute and chronic noise stress groups. While the chronic noise stress group animals were exposed to 100 dB white noise for 4 h/a day during 30 days, the acute noise stress group of animals was exposed to the same level of stress once for 4 h. The expression profiles of corticotropin releasing hormone (CRH), CRH1, CRH2 receptors and glucocorticoid receptor (GR) mRNAs were analysed by RT-PCR. Chronic noise stress upregulated CRH mRNA levels in the hypothalamus. Both acute and chronic noise increased CRH-R1 mRNA in the hypothalamus but decreased it in the hippocampus. GR mRNA levels were decreased by chronic noise stress in the hippocampus. The present results suggest that while corticosterone responses have habituated to continuous noise stress, the involvement of CRH family molecules and glucocorticoid receptors in the noise stress responses are different and structure specific.

Semisynthesis and optimization of G protein-coupled receptor mimics.

We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs.

Catalpol regulates function of hypothalamic-pituitary-adrenocortical-axis in an Alzheimer's disease rat model.

AIMS: To investigate the regulating effects of catalpol on the hypothalamic-pituitary- adrenocortical-axis (HPA) in an Alzheimer's disease (AD) rat model. METHODS: Healthy male Wistar Rats were selected. The AD model was generated by orthotopic injection of beta-amyloid 25-35 (Abeta25-35) into the right lateral ventricle. The animals were divided into five study groups: Catalpol at low dose (5 mg/kg), Catalpol at high dose (10 mg/kg), model control group and sham surgery control group, n = 9 respectively. The serum concentration of hydrocortisone (HYD), adrenocorticotropin (ACTH) and corticotropin releasing hormone (CRH) determined by Enzyme-Linked Immunosorbent Assay (ELISA). Structural alterations of the hypothalamus were examined by H&E stain and electron microscope. The CRH receptor 1 (CRHR1) positive neurons were detected with immunohistochemistry. RESULTS: Serum HYD level was significantly increased (p < 0.01), and both ACTH and CRH were dramatically decreased (p < 0.01) in the AD model group rats compared with normal control rats at day 7. Catalpol treatment was able to improve the hormone secretion disorder in AD model group rats compared with the model group (p < 0.01 or p < 0.05) in particular at 21 days. Structure damage of hypothalamus in the AD rat as evidenced less CRHR1 positive neurons, rough endoplasmic reticulum dilation and degranulation, and mitochondrial swelling under electron microscope. Catalpol treatment at both high and low doses was able to alleviate the structure damage of the hypothalamus in the AD rats. CONCLUSIONS: Catalpol could improve the endocrine function of the HPA and alleviate the structural damage of hypothalamus in AD rats.

Susceptibility to PTSD-like behavior is mediated by corticotropin-releasing factor receptor type 2 levels in the bed nucleus of the stria terminalis.

Posttraumatic stress disorder (PTSD) is a debilitating disease, which affects 8-10% of the population exposed to traumatic events. The factors that make certain individuals susceptible to PTSD and others resilient are currently unknown. Corticotropin-releasing factor receptor type 2 (CRFR2) has been implicated in mediating stress coping mechanisms. Here, we use a physiological PTSD-like animal model and an in-depth battery of tests that reflect the symptomology of PTSD to separate mice into subpopulations of "PTSD-like" and "Resilient" phenotypes. PTSD-like mice are hypervigilant, hyperalert, insomniac, have impaired attention and risk assessment, as well as accompanying attenuated corticosterone levels. Intriguingly, PTSD-like mice show long-term robust upregulation of BNST-CRFR2 mRNA levels, and BNST-CRFR2-specific lentiviral knockdown reduces susceptibility to PTSD-like behavior. Additionally, using a BNST mRNA expression array, PTSD-like mice exhibit a general transcriptional attenuation profile, which was associated with upregulation of the BNST-deacetylation enzyme, HDAC5. We suggest PTSD to be a disease of maladaptive coping.

Stress-induced changes in gene expression of urocortin 2 and other CRH peptides in rat adrenal medulla: involvement of glucocorticoids.

The corticotropin-releasing hormone (CRH) family regulates the endocrine stress response. Here, we examined the effect of immobilization stress (IMO) on gene expression of adrenomedullary CRH family members. Urocortin 2 (Ucn2) has the highest basal gene expression and is increased by > 30-fold in response to single IMO and about 10-fold after six daily repeated IMO. IMO also induced a smaller rise in CRH (six-fold) and CRH receptor type 1 (CRHR1; two-fold with single IMO). The influence of glucocorticoids was examined. Dexamethasone (DEX) or corticosterone greatly increased Ucn2 mRNA levels in PC12 cells in a dose-dependent manner. The DEX elicited rise in Ucn2 was abolished by actinomycin D pre-treatment, indicating a transcriptionally mediated response. DEX also triggered a rise in CRHR1 and lowered CRH mRNA levels. In CRH-knockout mice, where the IMO-induced rise in corticosterone was attenuated, the response of IMO on Ucn2, as well as CRHR2 mRNAs was absent. Overall, the results suggest that the stress-triggered rise in glucocorticoids is involved in the large induction of Ucn2 mRNA levels by IMO, which may allow Ucn2 to act in an autocrine/paracrine fashion to modulate adrenomedullary function, or act as an endocrine hormone.

Histone deacetylase inhibition mediates urocortin-induced antiproliferation and neuronal differentiation in neural stem cells.

During cortical development, cell proliferation and cell cycle exit are carefully regulated to ensure that the appropriate numbers of cells are produced. Urocortin (UCN) is a member of the corticotrophin releasing hormone (CRH) family of neuropeptides that regulates stress responses. UCN is widely distributed in adult rat brain. However, the expression and function of UCN in embryonic brain is, as yet, unclear. Here, we show that UCN is endogenously expressed in proliferative zones of the developing cerebral cortex and its receptors are exhibited in neural stem cells (NSCs), thus implicating the neuropeptide in cell cycle regulation. Treatment of cultured NSCs or organotypic slice cultures with UCN markedly reduced cell proliferation. Furthermore, blocking of endogenous UCN/CRHRs system either by treatment with CRHRs antagonists or by neutralization of secreted UCN with anti-UCN antibody increased NSCs proliferation. Cell cycle kinetics analysis demonstrated that UCN lengthened the total cell cycle duration via increasing the G1 phase and accelerated cell cycle exit. UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological and RNA interference approaches, we further demonstrated that antiproliferative action of UCN appeared to be mediated through a HDAC inhibition-induced p21 upregulation. Moreover, UCN treatment in vitro and in vivo led to an increase in neuronal differentiation of NSCs. These findings suggest that UCN might contribute to regulate NSCs proliferation and differentiation during cortical neurogenesis.

The mouse cochlea expresses a local hypothalamic-pituitary-adrenal equivalent signaling system and requires corticotropin-releasing factor receptor 1 to establish normal hair cell innervation and cochlear sensitivity.

Cells of the inner ear face constant metabolic and structural stress. Exposure to intense sound or certain drugs destroys cochlea hair cells, which in mammals do not regenerate. Thus, an endogenous stress response system may exist within the cochlea to protect it from everyday stressors. We recently described the existence of corticotropin-releasing factor (CRF) in the mouse cochlea. The CRF receptor type 1 (CRFR1) is considered the primary and canonical target of CRF signaling, and systemically it plays an essential role in coordinating the body-wide stress response via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we describe an essential role for CRFR1 in auditory system development and function, and offer the first description of a complete HPA equivalent signaling system resident within the cochlea. To reveal the role of CRFR1 activation in the cochlea, we have used mice carrying a null ablation of the CRFR1 gene. CRFR1(-/-) mice exhibited elevated auditory thresholds at all frequencies tested, indicating reduced sensitivity. Furthermore, our results suggest that CRFR1 has a developmental role affecting inner hair cell morphology and afferent and efferent synapse distribution. Given the role of HPA signaling in maintaining local homeostasis in other tissues, the presence of a cochlear HPA signaling system suggests important roles for CRFR1 activity in setting cochlear sensitivity, perhaps both neural and non-neural mechanisms. These data highlight the complex pleiotropic mechanisms modulated by CRFR1 signaling in the cochlea.

Chronic stress exacerbates tau pathology, neurodegeneration, and cognitive performance through a corticotropin-releasing factor receptor-dependent mechanism in a transgenic mouse model of tauopathy.

Because overactivation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in Alzheimer's disease (AD), dysregulation of stress neuromediators may play a mechanistic role in the pathophysiology of AD. However, the effects of stress on tau phosphorylation are poorly understood, and the relationship between corticosterone and corticotropin-releasing factor (CRF) on both ß-amyloid (Aß) and tau pathology remain unclear. Therefore, we first established a model of chronic stress, which exacerbates Aß accumulation in Tg2576 mice and then extended this stress paradigm to a tau transgenic mouse model with the P301S mutation (PS19) that displays tau hyperphosphorylation, insoluble tau inclusions and neurodegeneration. We show for the first time that both Tg2576 and PS19 mice demonstrate a heightened HPA stress profile in the unstressed state. In Tg2576 mice, 1 month of restraint/isolation (RI) stress increased Aß levels, suppressed microglial activation, and worsened spatial and fear memory compared with nonstressed mice. In PS19 mice, RI stress promoted tau hyperphosphorylation, insoluble tau aggregation, neurodegeneration, and fear-memory impairments. These effects were not mimicked by chronic corticosterone administration but were prevented by pre-stress administration of a CRF receptor type 1 (CRF(1)) antagonist. The role for a CRF(1)-dependent mechanism was further supported by the finding that mice overexpressing CRF had increased hyperphosphorylated tau compared with wild-type littermates. Together, these results implicate HPA dysregulation in AD neuropathogenesis and suggest that prolonged stress may increase Aß and tau hyperphosphorylation. These studies also implicate CRF in AD pathophysiology and suggest that pharmacological manipulation of this neuropeptide may be a potential therapeutic strategy for AD.

Urocortins and CRF type 2 receptor isoforms expression in the rat stomach are regulated by endotoxin: role in the modulation of delayed gastric emptying.

Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF(2)) by urocortin 1, 2, or 3 (Ucns) exerts powerful effects on gastric function; however, little is known about their expression and regulation in the stomach. We investigated the expression of Ucns and CRF(2) isoforms by RT-PCR in the gastric corpus (GC) mucosa and submucosa plus muscle (S+M) or laser captured layers in naive rats, their regulations by lipopolysaccharide (LPS, 100 µg/kg ip) over 24 h, and the effect of the CRF(2) antagonist astresssin(2)-B (100 µg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF(2b,) the most common wild-type CRF(2) isoform in the periphery, were expressed in all layers, including myenteric neurons. LPS increased Ucn mRNA levels significantly in both mucosa and S+M, reaching a maximal response at 6 h postinjection and returning to basal levels at 24 h except for Ucn 1 in S+M. By contrast, CRF(2b) mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF(2a), reportedly only found in the brain, and the novel splice variant CRF(2a-3) were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF(2a) and an increase of CRF(2a-3) in the GC 6 h postinjection. Astressin(2)-B exacerbated LPS-delayed GE (42-73%, P < 0.001). These data indicate that Ucn and CRF(2) isoforms are widely distributed throughout the rat stomach and inversely regulated by immune stress. The CRF(2) signaling system may act to counteract the early gastric motor alterations to endotoxemia.

Natural variation in the molecular stress network correlates with a behavioural syndrome.

In several species, individuals from the same population behave differently from each other. A functional link between variation in personality traits and the stress response has been suggested by studies in artificial selection lines in fish, birds and mammals. The aim of this study was to test whether the expression of genes involved in the stress response co-varies with personality traits in a natural population. Four personality traits, excreted cortisol level and brain expression of six candidate genes (CRF, CRF-R2, POMC1, GR1, GR2, MR) were measured in non-stressed wild-caught threespine sticklebacks (Gasterosteus aculeatus). We found correlations between variation in personality traits and variation in the expression of genes involved in the stress response. Aggressiveness was negatively correlated with cortisol levels. Boldness and aggressiveness formed a behavioural syndrome and were both positively correlated with brain expression of glucocorticoid receptors (GR1 and GR2). Boldness and exploration were positively correlated with expression of POMC1 but not with each other. Our results are compatible with a model that suggests that the aggressiveness-boldness behavioural syndrome could be the consequence of a physiological pleiotropic effect of glucocorticoid receptors, which are involved in the stress response and behaviour variation.

The pseudo signal peptide of the corticotropin-releasing factor receptor type 2a decreases receptor expression and prevents Gi-mediated inhibition of adenylyl cyclase activity.

The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF(2(a))R in comparison with the CRF(1)R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G(s) protein, G(i)-mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF(1)R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.