CREB-deficient Mice Research Articles

CREB deletion increases resilience to stress and downregulates inflammatory gene expression in the hippocampus

The transcription factor CREB (cyclic AMP response element (CRE)-binding protein) is implicated in the pathophysiology and treatment of depression. Structural and functional studies in both animals and humans suggest that abnormalities of the hippocampus may play a role in depression. CREB regulates thousands of genes, yet to date, only a handful that mediate depression or antidepressant response have been identified as relevant CREB targets. In order to comprehensively identify genes regulated by CREB in the hippocampus, we employed translating ribosome affinity purification (TRAP) to detect actively translating mRNAs in wild type and CREB-deficient mice. Using CrebloxP/loxP; RosaLSL-GFP-L10a mice, we conducted whole genome sequencing to identify transcripts only in cells that lack CREB, as introduction of Cre-recombinase simultaneously deleted CREB and expressed GFP-tagged L10a ribosomes that enabled TRAP. We identified over 200 downregulated genes predominantly associated with inflammation and the immune system, including toll-like receptor 1 (TLR1). To determine if baseline disruption in gene expression in the hippocampus of CREB-deficient mice can modulate behavior, we used unpredictable chronic mild stress (UCMS) to produce a set of behavioral alterations with strong validity for depression.We found that CREB-deficient mice demonstrated resilience to the physiological effects of UCMS and also showed changes in affective behaviors specifically in the presence of stress. TLR1 expression was increased following UCMS in control but not in CREB-deficient mice. The results suggest that CREB-mediated regulation of immune system and inflammatory factors may provide additional targets for the treatment of depression.

A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction

Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD(+)-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes.

Stress-Induced Potentiation of Cocaine Reward: A Role for CRFR1 and CREB

Both clinical and preclinical research have shown that stress can potentiate drug use; however, the underlying mechanisms of this interaction are unknown. Previously, we have shown that a single exposure to forced swim (FS) reinstates extinguished conditioned place preference (CPP) to cocaine and that cAMP response element binding protein (CREB) is necessary for this response. CREB can be activated by corticotropin releasing factor (CRF) receptor type 1 (CRF(R1)) binding, which mediates neuroendocrine and behavioral responses to stress as well as to drugs of abuse. The present experiments investigate whether changes in cocaine reward elicited by previous exposure to stress are mediated by CREB and/or CRF(R1). Chronic exposure to FS in advance of conditioning enhances cocaine CPP in wild-type mice, but this is blocked in CREB-deficient mice. In addition, pretreatment with the CRF(R1) antagonist, antalarmin, before FS exposure blocks this stress-induced enhancement of cocaine CPP. Furthermore, FS-induced increase in phosphorylated CREB (pCREB), specifically in the lateral septum (LS) and nucleus accumbens (NAc) is also blocked by antalarmin. Taken together, these studies suggest that both CREB and CRF(R1) activation are necessary for stress-induced potentiation of drug reward.

CREB, neurogenesis and depression

The transcription factor CREB has been implicated in signalling pathways relevant for pathogenesis and therapy of depression. CREB is upregulated and activated in the hippocampus by chronic antidepressant treatment, similarly as neurogenesis. Surprisingly, a recent study using CREB-deficient mice also demonstrates an upregulation of neurogenesis correlating with an antidepressant behavioral phenotype.1 Interestingly, CREB-deficient mice show a rapid behavioral response to antidepressants, while wild-type mice do not. This minireview tries to reconcile these new findings with established concepts on CREB, neurogenesis and depression. It also outlines some crucial experiments and lines of future research that could clarify some of the pending questions.

CAMP Response Element-Binding Protein Deficiency Allows for Increased Neurogenesis and a Rapid Onset of Antidepressant Response

cAMP response element-binding protein (CREB) has been implicated in the molecular and cellular mechanisms of chronic antidepressant (AD) treatment, although its role in the behavioral response is unclear. CREB-deficient (CREB(alpha delta) mutant) mice demonstrate an antidepressant phenotype in the tail suspension test (TST) and forced-swim test. Here, we show that, at baseline, CREB(alpha delta) mutant mice exhibited increased hippocampal cell proliferation and neurogenesis compared with wild-type (WT) controls, effects similar to those observed in WT mice after chronic desipramine (DMI) administration. Neurogenesis was not further augmented by chronic DMI treatment in CREB(alpha delta) mutant mice. Serotonin depletion decreased neurogenesis in CREB(alpha delta) mutant mice to WT levels, which correlated with a reversal of the antidepressant phenotype in the TST. This effect was specific for the reversal of the antidepressant phenotype in these mice, because serotonin depletion did not alter a baseline anxiety-like behavior in CREB(alpha delta) mutant mice. The response to chronic AD treatment in the novelty-induced hypophagia (NIH) test may rely on neurogenesis. Therefore, we used this paradigm to evaluate chronic AD treatment in CREB(alpha delta) mutant mice to determine whether the increased neurogenesis in these mice alters their response in the NIH paradigm. Whereas both WT and CREB(alpha delta) mutant mice responded to chronic AD treatment in the NIH paradigm, only CREB(alpha delta) mutant mice responded to acute AD treatment. However, in the elevated zero maze, DMI did not reverse anxiety behavior in mutant mice. Together, these data show that increased hippocampal neurogenesis allows for an antidepressant phenotype as well as a rapid onset of behavioral responses to AD treatment.

Cardiomyocyte‐specific inactivation of transcription factor CREB in mice

The transcription factor cAMP response element (CRE)-binding protein (CREB, Creb1) plays a critical role in regulating gene expression in response to activation of the cAMP-dependent signaling pathway, which is implicated in the pathophysiology of heart failure. Using the Cre-loxP system, we generated mice with a cardiomyocyte-specific inactivation of CREB and studied in this model whether CREB is critical for cardiac function. CREB-deficient mice were viable and displayed neither changes in cardiac morphology nor alterations of basal or isoproterenol-stimulated left ventricular function in vivo or of important cardiac regulatory proteins. Since CREB was proposed as a negative regulator of cardiomyocyte apoptosis by enhancing the expression of the antiapoptotic protein Bcl-2, we analyzed the fragmentation of DNA, the activity of caspases 3/7 and the expression of Bcl-2 and did not observe any differences between CREB-deficient and CREB-normal hearts. Our results suggest that the presence of CREB is not critical for normal cardiac function in mice.

Alterations in morphine-induced reward, locomotor activity, and thermoregulation in CREB-deficient mice

Previous studies in our lab have shown a robust decrease in the rewarding properties of morphine in CREB αΔ mutant mice. To determine whether the genetic effects of the global CREB αΔ mutation are specific to reward or generalizable, we examined a variety of morphine-induced behaviors regulated by different neural circuitry. At low doses of morphine (5 and 10 mg/kg), CREB αΔ mutant mice show a reduction in reward yet similar locomotor activity in response to morphine compared to wild type littermates. However, at a high dose (20 mg/kg), CREB αΔ mutant mice show an increase in reward and locomotor activity. Morphine-induced thermoregulation is attenuated in CREB αΔ mutant mice at high doses of morphine compared to wild type animals. The behavioral differences in response to morphine seen in CREB αΔ mutant mice are not due to changes in mu opioid receptor (MOR) mRNA expression, as the CREB deletion has no effect on baseline MOR mRNA in three of the brain regions involved in mediating these behaviors: the ventral tegmental area (VTA), nucleus accumbens (NAc), and hypothalamus. These data demonstrate that at low doses, deficits in morphine-induced changes in CREB deficient mice are limited to reward and thermoregulation. However, at higher doses, CREB mutant mice actually find morphine more rewarding and exhibit increased locomotor activity compared to their wild type littermates. Together, these results indicate that the role of CREB in dose-dependent changes in behaviors induced by morphine is different depending on the brain regions involved in mediating the behavior.

Partial deletion of the cAMP response element-binding protein gene promotes alcohol-drinking behaviors.

The cAMP response element-binding protein (CREB) gene transcription factor has been shown to play a role in the synaptic plasticity associated with drug addictive behaviors; however, the causal role of the CREB gene in alcohol-drinking behaviors is unknown. The present investigation evaluated alcohol-drinking behaviors in mice that are haplodeficient in CREB as a result of targeted CREB (alpha and Delta) gene disruption. It was found that CREB-haplodeficient (+/-) mice have higher preference for ethanol but not for sucrose solution than wild-type (+/+) littermates. The functional aspects of the CREB gene transcription factor were also investigated by measuring the protein levels of phosphorylated CREB (p-CREB) and the expression of cAMP-inducible genes such as neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF). Deletion of the CREB (alpha and Delta) gene significantly decreases total CREB, p-CREB levels and the expression of NPY and BDNF in the brain structures of CREB-deficient (+/-) mice. It was also found that CREB-deficient (+/-) mice displayed more anxiety-like behaviors and that acute ethanol exposure produced anxiolytic effects and significantly increased protein levels of p-CREB and NPY in the central and medial but not in the basolateral amygdala of wild-type mice, but these effects are attenuated in CREB-deficient mice compared with wild-type mice. These results provide the first direct evidence that a haplodeficiency of the CREB gene is associated with increased alcohol-drinking behaviors. Furthermore, alcohol drinking and anxiety-like behaviors in CREB-haplodeficient mice may possibly be related to decreased expression of NPY and BDNF in the brains of these mice.

Consolidation of CS and US representations in associative fear conditioning.

Much attention has been paid to the associative processes that are necessary to fuse together representations of the various components of an episodic memory. In the present study, we focus on the processes involved in the formation of lasting representations of the individual components that make up a fear-conditioning episode. In one-trial contextual fear conditioning experiments, weak conditioning to context occurs if the shock is delivered immediately following placement of the animal in a novel conditioning apparatus, a phenomenon known as the immediate shock deficit. We show that the immediate shock deficit in mice may be alleviated by pre-exposure to either the context or shock. In using this approach to temporally dissect a contextual fear-conditioning task into its constituent representational and associative processes, we are able to examine directly the processes that are important for formation of lasting representations of the context conditioned stimulus (CS) or unconditioned stimulus (US). Our data indicate that the formation of a lasting representation of the context or shock engages protein synthesis-dependent processes. Furthermore, genetic disruption of cAMP-responsive element binding protein (CREB), a transcription factor that regulates the synthesis of new proteins required for long-term memory, disrupts the formation of lasting context memories. We go on to show that the stress hormone epinephrine modulates the consolidation of a context memory, and reverses consolidation deficits in the CREB-deficient mice. Finally we show that disrupting either NMDA or calcium/calmodulin-dependent kinase II (CaMKII) function impairs consolidation of context memories. Together, these data suggest that this approach is particularly suited for the characterization of molecular and cellular processes underlying the formation of stimulus representations.

CAMP response element-binding protein is essential for the upregulation of brain-derived neurotrophic factor transcription, but not the behavioral or endocrine responses to antidepressant drugs.

Antidepressant drugs activate the cAMP signal transduction pathway through a variety of monoamine neurotransmitter receptors. Recently, molecular studies have identified a role for cAMP response element-binding protein (CREB) in the mechanism of action of chronically administered antidepressant drugs. However, the function of CREB in the behavioral and endocrine responses to these drugs has not been thoroughly investigated. We have used CREB-deficient mice to study the effects of two antidepressants, desipramine (DMI) and fluoxetine (FLX), in behavioral, endocrine, and molecular analyses. Behaviorally, CREB-deficient mice and wild-type mice respond similarly to DMI and FLX administration in the forced swim test and tail suspension test. Furthermore, the ability of DMI to suppress an acute corticosterone response after swim stress is maintained in CREB-deficient mice. However, upregulation of a molecular target of CREB, BDNF, is abolished in the CREB-deficient mice after chronic administration of DMI. These data are the first to demonstrate that CREB activation is upstream of BDNF mechanistically in response to antidepressant drug treatment. Therefore, although behavioral and endocrine responses to antidepressants may occur by CREB-independent mechanisms, CREB is critical to target gene regulation after chronic drug administration, which may contribute to long-term adaptations of the system to antidepressant drug treatment.

CAMP Response Element-Binding Protein Is Essential for the Upregulation of Brain-Derived Neurotrophic Factor Transcription, But Not the Behavioral or Endocrine Responses to Antidepressant Drugs

Antidepressant drugs activate the cAMP signal transduction pathway through a variety of monoamine neurotransmitter receptors. Recently, molecular studies have identified a role for cAMP response element-binding protein (CREB) in the mechanism of action of chronically administered antidepressant drugs. However, the function of CREB in the behavioral and endocrine responses to these drugs has not been thoroughly investigated. We have used CREB-deficient mice to study the effects of two antidepressants, desipramine (DMI) and fluoxetine (FLX), in behavioral, endocrine, and molecular analyses. Behaviorally, CREB-deficient mice and wild-type mice respond similarly to DMI and FLX administration in the forced swim test and tail suspension test. Furthermore, the ability of DMI to suppress an acute corticosterone response after swim stress is maintained in CREB-deficient mice. However, upregulation of a molecular target of CREB, BDNF, is abolished in the CREB-deficient mice after chronic administration of DMI. These data are the first to demonstrate that CREB activation is upstream of BDNF mechanistically in response to antidepressant drug treatment. Therefore, although behavioral and endocrine responses to antidepressants may occur by CREB-independent mechanisms, CREB is critical to target gene regulation after chronic drug administration, which may contribute to long-term adaptations of the system to antidepressant drug treatment.

Activating transcription factor 1 and CREB are important for cell survival during early mouse development.

Activating transcription factor 1 (ATF1), CREB, and the cyclic AMP (cAMP) response element modulatory protein (CREM), which constitute a subfamily of the basic leucine zipper transcription factors, activate gene expression by binding as homo- or heterodimers to the cAMP response element in regulatory regions of target genes. To investigate the function of ATF1 in vivo, we inactivated the corresponding gene by homologous recombination. In contrast to CREB-deficient mice, which suffer from perinatal lethality, mice lacking ATF1 do not exhibit any discernible phenotypic abnormalities. Since ATF1 and CREB but not CREM are strongly coexpressed during early mouse development, we generated mice deficient for both CREB and ATF1. ATF1(-/-) CREB(-/-) embryos die before implantation due to developmental arrest. ATF1(+/-) CREB(-/-) embryos display a phenotype of embryonic lethality around embryonic day 9.5 due to massive apoptosis. These results indicate that CREB and ATF1 act in concert to mediate signals essential for maintaining cell viability during early embryonic development.

CREB regulates hepatic gluconeogenesis through the coactivator PGC-1.

When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia [corrected] and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.