CREB Function Research Articles

Activation of CREB in the nucleus accumbens shell produces anhedonia and resistance to extinction of fear in rats.

Stress triggers psychiatric conditions including depressive and anxiety disorders. The mechanisms by which stress produces persistent changes in behavior are not fully understood. Here we show in rats that stress (footshock) activates the transcription factor cAMP response element binding protein (CREB) within the nucleus accumbens shell (NAS), a brain area involved in encoding reward and aversion. To examine the behavioral significance of altered CREB function in the NAS, we used viral vectors to elevate or disrupt CREB in this region. Elevated CREB produced increases in intracranial self-stimulation thresholds, a depressive-like sign reflecting anhedonia (decreased sensitivity to reward), whereas disruption of CREB function by expression of a dominant-negative CREB had the opposite effect. To determine whether neuroadaptations that produce anhedonia subsequently affect vulnerability to stress-induced behavioral adaptations, we subjected rats with altered CREB function in the NAS to fear conditioning. Although neither elevation nor disruption of CREB function altered the development of conditioned fear, elevation of CREB impaired extinction of conditioned fear. To mimic downstream effects of CREB activation on expression of the opioid peptide dynorphin, we microinjected the κ-opioid receptor (KOR) agonist U50,488 directly into the NAS. KOR stimulation produced anhedonia but had no effect on expression or extinction of conditioned fear. These findings demonstrate that activation of CREB in the NAS produces multiple behavioral signs (anhedonia, impaired extinction) characteristic of experience-dependent psychiatric conditions such as posttraumatic stress disorder. Although CREB activation is a common trigger, expression of these individual signs appears to involve divergent downstream mechanisms.

CREB and the CRTC co-activators: sensors for hormonal and metabolic signals

The cyclic AMP-responsive element-binding protein (CREB) is phosphorylated in response to a wide variety of signals, yet target gene transcription is only increased in a subset of cases. Recent studies indicate that CREB functions in concert with a family of latent cytoplasmic co-activators called cAMP-regulated transcriptional co-activators (CRTCs), which are activated through dephosphorylation. A dual requirement for CREB phosphorylation and CRTC dephosphorylation is likely to explain how these activator-co-activator cognates discriminate between different stimuli. Following their activation, CREB and CRTCs mediate the effects of fasting and feeding signals on the expression of metabolic programmes in insulin-sensitive tissues.

SIK2 Is a Key Regulator for Neuronal Survival after Ischemia via TORC1-CREB

The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.

CREB in long‐term potentiation in hippocampus: Role of post‐translational modifications‐studies In silico

The multifunctionality of proteins is dictated by post-translational modifications (PTMs) which involve the attachment of small functional groups such as phosphate and acetate, as well as carbohydrate moieties. These functional groups make the protein perform various functions in different environments. PTMs play a crucial role in memory and learning. Phosphorylation of synaptic proteins and transcription factors regulate the generation and storage of memory. Among these is the cAMP-regulated element binding protein CREB that regulates CRE containing genes like c-fos. Both phosphorylation and acetylation control the function of CREB as a transcription factor. CREB is also susceptible to O-GlcNAc modification, which inhibits its activity. O-GlcNAc modification occurs on the same or neighboring Ser/Thr residues akin to phosphorylation. An interplay between these modifications was shown to operate in nuclear and cytoplasmic proteins. In this study computational methods were utilized to predict different modification sites in CREB. These in silico results suggest that phosphorylation, O-GlcNAc modification and acetylation modulate the transcriptional activity of CREB and thus dictate its contribution to synaptic plasticity.

CBP gene transfer increases BDNF levels and ameliorates learning and memory deficits in a mouse model of Alzheimer's disease

Cognitive dysfunction and memory loss are common features of Alzheimer's disease (AD). Abnormalities in the expression profile of immediate early genes that play a critical role in memory formation, such as the cAMP-response element binding protein (CREB), have been reported in the brains of AD patients. Here we show that amyloid-β (Aβ) accumulation, which plays a primary role in the cognitive deficits of AD, interferes with CREB activity. We further show that restoring CREB function via brain viral delivery of the CREB-binding protein (CBP) improves learning and memory deficits in an animal model of AD. Notably, such improvements occur without changes in Aβ and tau pathology, and instead are linked to an increased level of brain-derived neurotrophic factor. The resulting data suggest that Aβ-induced learning and memory deficits are mediated by alterations in CREB function, based on the finding that restoring CREB activity by directly modulating CBP levels in the brains of adult mice is sufficient to ameliorate learning and memory. Therefore, increasing CBP expression in adult brains may be a valid therapeutic approach not only for AD, but also for various brain disorders characterized by alterations in immediate early genes, further supporting the concept that viral vector delivery may be a viable therapeutic approach in neurodegenerative diseases.

CREB Influences Timing and Entrainment of the SCN Circadian Clock

The transcriptional feedback circuit, which is at the core of the suprachiasmatic nucleus (SCN) circadian (i.e., 24 h) clock, is tightly coupled to both external entrainment cues, such as light, as well as rhythmic cues that arise on a system-wide level within the SCN. One potential signaling pathway by which these cues are conveyed to the molecular clock is the CREB/CRE transcriptional cascade. In this study, we employed a tetracycline-inducible CREB repressor mouse strain, in which approximately 60% of the SCN neurons express the transgene, to test CREB functionality in the clock and its effects on overt rhythmicity. We show that attenuated CREB signaling in the SCN led to a significant reduction in light-evoked clock entrainment. An examination of circadian timing revealed that CREB repressor mice exhibited normal free-running rhythms in the absence of external lighting cues. However, under conditions of constant light, which typically leads to a lengthening of the circadian period, CREB repressor mice exhibited a dramatic arrhythmic phenotype, which could be reversed with doxycycline. At a cellular level, the repression of CREB led to a significant reduction in both the expression of the circadian clock proteins PERIOD1 and PERIOD2 and the clock output hormones AVP and VIP. Together, these data support the idea that the CRE transcriptional pathway orchestrates transcriptional events that are essential for both the maintenance of SCN timing and light entrainment of the circadian clock.

Aspartate Release and Signalling in the Hippocampus

The Ca(2+)-dependent release of aspartate from hippocampal preparations was first reported 35 years ago, but the functional significance of this process remains uncertain. Aspartate satisfies all the criteria normally required for identification of a CNS transmitter. It is synthesized in nerve terminals, is accumulated and stored in synaptic vesicles, is released by exocytosis upon nerve terminal depolarization, and activates postsynaptic NMDA receptors. Aspartate may be employed as a neuropeptide-like co-transmitter by pathways that release either glutamate or GABA as their principal transmitter. Aspartate mechanisms include vesicular transport by sialin, vesicular content sensitive to glucose concentration, release mainly outside the presynaptic active zones, and selective activation of extrasynaptic NR1-NR2B NMDA receptors. Possible neurobiological functions of aspartate in immature neurons include activation of cAMP-dependent gene transcription and in mature neurons inhibition of CREB function, reduced BDNF expression, and induction of excitotoxic neuronal death. Recent findings suggest new experimental approaches toward resolving the functional significance of aspartate release.

Continuing the search for the engram: examining the mechanism of fear memories

The goal of my research is to gain insight using rodent models into the fundamental molecular, cellular and systems that make up the base of memory formation. My work focuses on fear memories. Aberrant fear and/or anxiety may be at the heart of many psychiatric disorders. In this article, I review the results of my research group; these results show that particular neurons in the lateral amygdala, a brain region important for fear, are specifically involved in particular fear memories. We started by showing that the transcription factor CREB (cAMP/Ca(2+) response element binding protein) plays a key role in the formation of fear memories. Next, we used viral vectors to overexpress CREB in a subset of lateral amygdala neurons. This not only facilitated fear memory formation but also "drove" the memory into the neurons with relatively increased CREB function. Finally, we showed that selective ablation of the neurons overexpressing CREB in the lateral amygdala selectively erased the fear memory. These findings are the first to show disruption of a specific memory by disrupting select neurons within a distributed network.

Regulation of Genotoxic Stress Response by Homeodomain-interacting Protein Kinase 2 through Phosphorylation of Cyclic AMP Response Element-binding Protein at Serine 271

CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.

Dorsal hippocampal CREB is both necessary and sufficient for spatial memory

Although the transcription factor CREB has been widely implicated in memory, whether it is sufficient to produce spatial memory under conditions that do not normally support memory formation in mammals is unknown. We found that locally and acutely increasing CREB levels in the dorsal hippocampus using viral vectors is sufficient to induce robust spatial memory in two conditions that do not normally support spatial memory, weakly trained wild-type (WT) mice and strongly trained mutant mice with a brain-wide disruption of CREB function. Together with previous results, these findings indicate that CREB is both necessary and sufficient for spatial memory formation, and highlight its pivotal role in the hippocampal molecular machinery underlying the formation of spatial memory.

Regulation of Kv channel expression and neuronal excitability in rat medial nucleus of the trapezoid body maintained in organotypic culture

Principal neurons of the medial nucleus of the trapezoid body (MNTB) express a spectrum of voltage-dependent K(+) conductances mediated by Kv1-Kv4 channels, which shape action potential (AP) firing and regulate intrinsic excitability. Postsynaptic factors influencing expression of Kv channels were explored using organotypic cultures of brainstem prepared from P9-P12 rats and maintained in either low (5 mm, low-K) or high (25 mm, high-K) [K(+)](o) medium. Whole cell patch-clamp recordings were made after 7-28 days in vitro. MNTB neurons cultured in high-K medium maintained a single AP firing phenotype, while low-K cultures had smaller K(+) currents, enhanced excitability and fired multiple APs. The calyx of Held inputs degenerated within 3 days in culture, having lost their major afferent input; this preparation of calyx-free MNTB neurons allowed the effects of postsynaptic depolarisation to be studied with minimal synaptic activity. The depolarization caused by the high-K aCSF only transiently increased spontaneous AP firing (<2 min) and did not measurably increase synaptic activity. Chronic depolarization in high-K cultures raised basal levels of [Ca(2+)](i), increased Kv3 currents and shortened AP half-widths. These events relied on raised [Ca(2+)](i), mediated by influx through voltage-gated calcium channels (VGCCs) and release from intracellular stores, causing an increase in cAMP-response element binding protein (CREB) phosphorylation. Block of VGCCs or of CREB function suppressed Kv3 currents, increased AP duration, and reduced Kv3.3 and c-fos expression. Real-time PCR revealed higher Kv3.3 and Kv1.1 mRNA in high-K compared to low-K cultures, although the increased Kv1.1 mRNA was mediated by a CREB-independent mechanism. We conclude that Kv channel expression and hence the intrinsic membrane properties of MNTB neurons are homeostatically regulated by [Ca(2+)](i)-dependent mechanisms and influenced by sustained depolarization of the resting membrane potential.

Ultrastructural and transcriptional profiling of neuropathological misregulation of CREB function

We compare here the neurodegenerative processes observed in the hippocampus of bitransgenic mice with chronically altered levels of cAMP-response element-binding protein (CREB) function. The combination of genome-wide transcriptional profiling of degenerating hippocampal tissue with microscopy analyses reveals that the sustained inhibition of CREB function in A-CREB mice is associated with dark neuron degeneration, whereas its strong chronic activation in VP16-CREB mice primarily causes excitotoxic cell death and inflammation. Furthermore, the meta-analysis with gene expression profiles available in public databases identifies relevant common markers to other neurodegenerative processes and highlights the importance of the immune response in neurodegeneration. Overall, these analyses define the ultrastructural and transcriptional signatures associated with these two forms of hippocampal neurodegeneration, confirm the importance of fine-tuned regulation of CREB-dependent gene expression for CA1 neuron survival and function, and provide novel insight into the function of CREB in the etiology of neurodegenerative processes.

Cyclic AMP response element-binding protein is implicated in IL-6 production from arthritic synovial cells

Overproduction of interleukin (IL)-6 from synovial cells is critically involved in the pathogenesis of rheumatoid arthritis (RA). Cyclic adenosine monophosphate (AMP) response element-binding protein (CREB), a leucine zipper transcription factor, is expressed at a high level in synovial cells of patients with RA. Although CREB transactivates IL-6 expression in vascular smooth muscle cells, the relation between CREB expression and IL-6 production from arthritic synovial cells remains unclear. In this study, to determine whether CREB is implicated in IL-6 production from arthritic synovial cells, a dominant negative molecule of activation transcription factor 1 (ATF-1) was transfected into synovial cells obtained from arthritic joints of env-pX rats. These transgenic rats carrying the env-pX gene of human T-cell leukemia virus type-1 develop destructive arthritis with high titers of serum rheumatoid factor and are thus regarded as a suitable model of RA. The dominant negative ATF-1 (ATF-1DN) constitutes a heterodimer with CREB and inhibits CREB function, as CREB/ATF-1DN heterodimers no longer bind to the target sequence of CREB. We showed that transfection of ATF-1DN significantly reduced IL-6 production from arthritic synovial cells. These findings suggest that CREB is implicated in IL-6 production from synovial cells and plays an important role in RA pathogenesis.

Regulation of Energy Stores and Feeding by Neuronal and Peripheral CREB Activity in Drosophila

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), we have examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot with functions of adipose tissue and the liver in flies, on energy balance, stress resistance and feeding behavior. We found that disruption of CREB function in neurons reduced glycogen and lipid stores and increased sensitivity to starvation. Expression of DN-CREB in the fat body also reduced glycogen levels, while it did not affect starvation sensitivity, presumably due to increased lipid levels in these flies. Interestingly, blocking CREB activity in the fat body increased food intake. These flies did not show a significant change in overall body size, suggesting that disruption of CREB activity in the fat body caused an obese-like phenotype. Using a transgenic CRE-luciferase reporter, we further demonstrated that disruption of the adipokinetic hormone receptor, which is functionally related to mammalian glucagon and β-adrenergic signaling, in the fat body reduced CRE-mediated transcription in flies. This study demonstrates that CREB activity in either neuronal or peripheral tissues regulates energy balance in Drosophila, and that the key signaling pathway regulating CREB activity in peripheral tissue is evolutionarily conserved.

Differential effects of acute and repeated citalopram in mouse models of anxiety and depression

Clinically, SSRIs are widely prescribed in the treatment of several anxiety disorders, although very few pre-clinical studies have observed a beneficial effect of this class of drugs in animal models of anxiety. Furthermore, the biphasic pattern observed clinically, an exacerbation of anxiety followed by beneficial effects, is rarely observed in animal studies. In the present study we document this clinical phenomenon in several behavioural paradigms. While a single injection of citalopram induced anxiogenic effects, three administrations of citalopram were sufficient to elicit anxiolytic effects. Congruent with these data, we observed that short-term repeated administration of citalopram was accompanied by increased activation of cAMP response element-binding protein (CREB) in the hippocampus and desensitization of 5-HT1A receptors, two phenomena well associated with chronic rather than acute actions of antidepressants. Moreover, effects of citalopram were abolished in CREBalphaDelta mutant animals in the elevated zero maze (EZM) and tail suspension test (TST), but not in novelty-induced hypophagia (NIH). Further, the desensitization of 5-HT1A receptors elicited by citalopram was not affected by CREB deficiency. The significance of the EZM and TST paradigms in predicting therapeutic efficacy is well known while effects in NIH and 5-HT1A sensitization are less well-established. These data demonstrate that behavioural responses to citalopram are dependent on the frequency of its administration, and that these responses are differentially dependent on CREB function.

ICAM-3 enhances the migratory and invasive potential of human non-small cell lung cancer cells by inducing MMP-2 and MMP-9 via Akt and CREB

We have previously reported that intercellular adhesion molecule-3 (ICAM-3) is associated with an increase of cellular radio-resistance and cancer cell proliferation. In this study, we hypothesized that ICAM-3 has an additional effect on cancer cell migration and invasion because molecules induced by ICAM-3 are known as regulators of cell migration and invasion. To examine this hypothesis, we used NCI-H1299 non-small cell lung cancer (NSCLC) cell line (p53 and PTEN null cell) and constructed an ICAM-3-over-expressing stable transfectant, which exhibited increased cell migration and invasion. The increased migration and invasion resulted from up-regulation of expression and activities of MMP-2 and MMP-9. ICAM-3 also increased Akt phosphorylation, which caused an increase in cellular migration/invasion and MMP activities. Activity of several transcriptional factors located downstream in the Akt pathway was also tested, and constitutive activation of adenosine 3', 5'-monophosphate response element-binding protein (CREB) by ICAM-3 was detected. Blockage of the Akt pathway attenuated CREB activation, and a decrease in CREB expression reduced cellular migration/invasion and activity of MMPs. This result indicates that CREB functions in the signaling pathway between Akt and MMP. We also showed ICAM-3-induced cell migration and invasion in NCI-H460 NSCLC cells (wild-type p53 and PTEN cell) through the same signaling pathway. Taken together, our findings suggest that ICAM-3 stimulates cancer cell migration/invasion via ICAM-3/Akt/CREB/MMP pathway regardless of p53 and PTEN status, and this reflects the possibility that ICAM-3 could be considered as a candidate for anti-cancer drug development and as a cancer diagnostic marker.

Cyclic AMP response element-binding protein is implicated in IL-6 production from arthritic synovial cells

Overproduction of interleukin (IL)-6 from synovial cells is critically involved in the pathogenesis of rheumatoid arthritis (RA). Cyclic adenosine monophosphate (AMP) response element-binding protein (CREB), a leucine zipper transcription factor, is expressed at a high level in synovial cells of patients with RA. Although CREB transactivates IL-6 expression in vascular smooth muscle cells, the relation between CREB expression and IL-6 production from arthritic synovial cells remains unclear. In this study, to determine whether CREB is implicated in IL-6 production from arthritic synovial cells, a dominant negative molecule of activation transcription factor 1 (ATF-1) was transfected into synovial cells obtained from arthritic joints of env-pX rats. These transgenic rats carrying the env-pX gene of human T-cell leukemia virus type-1 develop destructive arthritis with high titers of serum rheumatoid factor and are thus regarded as a suitable model of RA. The dominant negative ATF-1 (ATF-1DN) constitutes a heterodimer with CREB and inhibits CREB function, as CREB/ATF-1DN heterodimers no longer bind to the target sequence of CREB. We showed that transfection of ATF-1DN significantly reduced IL-6 production from arthritic synovial cells. These findings suggest that CREB is implicated in IL-6 production from synovial cells and plays an important role in RA pathogenesis.

Protein kinase A activators and the pan-PPAR agonist tetradecylthioacetic acid elicit synergistic anti-leukaemic effects in AML through CREB

Targeting of signal transduction pathways and transcriptional regulation represents an attractive approach for less toxic anti-leukaemic therapy. We combined protein kinase A (PKA) activation with a pan-peroxisome proliferator-activated receptor (PPAR) activator tetradecylthioacetic acid, resulting in synergistic decrease in viability of AML cell lines. PKA isoform II activation appeared to be involved in inhibition of proliferation but not induction of apoptosis in HL-60 cells. Inhibition of CREB function protected against this anti-leukaemic effect with higher efficiency than enforced Bcl-2 expression. Preclinical studies employing the rat AML model Brown Norwegian Myeloid Leukaemia also indicated anti-leukaemic activity of the combination therapy in vivo. In conclusion, combined PKA and pan-PPAR activation should be explored further to determine its therapeutic potential.

Phosphodiesterase Inhibition Increases CREB Phosphorylation and Restores Orientation Selectivity in a Model of Fetal Alcohol Spectrum Disorders

BackgroundFetal alcohol spectrum disorders (FASD) are the leading cause of mental retardation in the western world and children with FASD present altered somatosensory, auditory and visual processing. There is growing evidence that some of these sensory processing problems may be related to altered cortical maps caused by impaired developmental neuronal plasticity.Methodology/Principal FindingsHere we show that the primary visual cortex of ferrets exposed to alcohol during the third trimester equivalent of human gestation have decreased CREB phosphorylation and poor orientation selectivity revealed by western blotting, optical imaging of intrinsic signals and single-unit extracellular recording techniques. Treating animals several days after the period of alcohol exposure with a phosphodiesterase type 1 inhibitor (Vinpocetine) increased CREB phosphorylation and restored orientation selectivity columns and neuronal orientation tuning.Conclusions/SignificanceThese findings suggest that CREB function is important for the maturation of orientation selectivity and that plasticity enhancement by vinpocetine may play a role in the treatment of sensory problems in FASD.

CREB is a key regulator of striatal vulnerability in chemical and genetic models of Huntington's disease

Evidence of dysregulation of the CREB/CRE transcriptional pathway in animal models of Huntington's disease (HD) suggests that strategies designed to augment CRE-mediated transcription may be of therapeutic value. Here, we investigated the consequences of CREB activation and repression in chemical and transgenic mouse models of HD. In the 3-nitropropionic acid (3-NP) model, CREB phospho-activation in the striatum was potently repressed within the neurotoxic “core” region prior to cell death. Conversely, marked expression of phospho-CREB, as well the CREB-regulated cytoprotective gene Bcl-2, was detected in the “penumbral” region. To examine potential contributory roles for the CREB/CRE transcriptional pathway in striatal degeneration, we used both CREB loss- (A-CREB) and gain- (VP16-CREB) of-function transgenic mouse strains. 3-NP-induced striatal lesion size and motor dysfunction were significantly increased in A-CREB mice compared to controls. Conversely, striatal damage and motor deficits were diminished in VP16-CREB mice. Furthermore, transgenic A-CREB significantly accelerated motor impairment in the YAC128 mouse model of HD. Together, these results indicate that CREB functionality is lost during the early stages of striatal cell stress and that the repression of CREB-mediated transcription contributes to the pathogenic process.