Julie A. Blendy

Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein

The cAMP-responsive element-binding protein (CREB) has been implicated in the activation of protein synthesis required for long-term facilitation, a cellular model of memory in Aplysia. Our studies with fear conditioning and with the water maze show that mice with a targeted disruption of the alpha and delta isoforms of CREB are profoundly deficient in long-term memory. In contrast, short-term memory, lasting between 30 and 60 min, is normal. Consistent with models claiming a role for long-term potentiation (LTP) in memory, LTP in hippocampal slices from CREB mutants decayed to baseline 90 min after tetanic stimulation. However, paired-pulse facilitation and posttetanic potentiation are normal. These results implicate CREB-dependent transcription in mammalian long-term memory.

Spaced training induces normal long-term memory in CREB mutant mice

BACKGROUNDnThe cAMP responsive element binding protein (CREB) is a transcription factor the activity of which is modulated by increases in the intracellular levels of cAMP and calcium. Results from studies with Aplysia, Drosophila and mice indicate that CREB-activated transcription is required for long-term memory. Furthermore, a recent study found that long-term memory for olfactory conditioning can be induced with a single trial in transgenic Drosophila expressing a CREB activator, whereas in normal flies, with presumably lower CREB-mediated transcription levels, conditioning requires multiple spaced trials. This suggests that CREB-mediated transcription is important in determining the type of training required for long-term memory of olfactory conditioning in Drosophila. Interestingly, studies with cultured Aplysia neurons indicated that removing a CREB repressor promoted the formation of long-term facilitation, a cellular model of non-associative memory.nnnRESULTSnHere, we have confirmed that mice lacking the alpha and Delta CREB proteins (CREBalphaDelta-) have abnormal long-term, but not short-term, memory, as tested in an ethologically meaningful task. Importantly, additional spaced training can overcome the profound memory deficits of CREBalphaDelta- mutants. Increasing the intertrial interval from 1 to 60 minutes overcame the memory deficits of the CREBalphaDelta- mice in three distinct behavioral tasks: contextual fear conditioning, spatial learning and socially transmitted food preferences.nnnCONCLUSIONSnPrevious findings and results presented here demonstrate that CREB mutant mice have profound long-term memory deficits. Importantly, our findings indicate that manipulations of CREB function can affect the number of trials and the intertrial interval required for committing information to long-term memory. Remarkably, this effect of CREB function is not restricted to simple conditioning tasks, but also affects complex behaviours such as spatial memory and memory for socially transmitted food preferences.

Reduction of Morphine Abstinence in Mice with a Mutation in the Gene Encoding CREB

Chronic morphine administration induces an up-regulation of several components of the cyclic adenosine 5′-monophosphate (cAMP) signal transduction cascade. The behavioral and biochemical consequences of opiate withdrawal were investigated in mice with a genetic disruption of the α and Δ isoforms of the cAMP-responsive element-binding protein (CREB). In CREBαΔ mutant mice the main symptoms of morphine withdrawal were strongly attenuated. No change in opioid binding sites or in morphine-induced analgesia was observed in these mutant mice, and the increase of adenylyl cyclase activity and immediate early gene expression after morphine withdrawal was normal. Thus, CREB-dependent gene transcription is a factor in the onset of behavioral manifestations of opiate dependence.

TARGETING OF THE CREB GENE LEADS TO UP-REGULATION OF A NOVEL CREB MRNA ISOFORM

To define the role of cAMP signaling in gene control, we have generated mice with a mutation in the cAMP response element binding protein (CREB) gene. Mice carrying this mutation are viable but show an impairment in memory consolidation. In further analysis of these mice, we have found an up‐regulation of a CREB isoform that has not been described previously . The new isoform, termed CREB beta, has nearly the same transactivation potential as the other CREB isoforms and is expressed ubiquitously. The up‐regulation appears to be due to an increase in alternative splicing or mRNA stability, but not to an increase in transcriptional rate. Due to the relatively low levels of expression in all tissues, the role of this isoform is likely to be minor in the wild‐type mouse. However, its dramatic up‐regulation in the mutant mouse, together with the specific deficiencies recently observed in these mice, suggest that it has a very specific role in compensating for CREB alpha and delta in some, but not all, areas where CREB function has been implicated. Together with the up‐regulation of the cAMP response element modulator protein (CREM) mRNA and protein levels demonstrated previously in CREB mutant mice, we suggest that the up‐regulation of CREB beta may also contribute to compensation within the CREB/ATF family of transcription factors, when CREB delta and CREB alpha are absent.

Activating Transcription Factor 1 and CREB Are Important for Cell Survival during Early Mouse Development

ABSTRACT 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.

Molecular genetic analysis of glucocorticoid signaling during mouse development

Glucocorticoids are important in a number of developmental processes in mammals around birth. The pathway of gluconeogenesis is activated in liver shortly after birth due to the combined effects of glucocorticoids and glucagon. We have defined the essential cis-regulatory elements directing hormone-dependent liver-specific expression of the gene for tyrosine aminotransferase, a key gluconeogenic enzyme. The hormone response elements synergize with cell-type specific elements. In the case of glucocorticoids, the glucocorticoid-dependent enhancer is composed of the glucocorticoid response element and binding sites for liver cell-enriched transcription factors, in particular hepatocyte nuclear factor-3. The dependence of the respective enhancer motifs on each other restricts the hormonal activation of the tyrosine aminotransferase gene in liver in response to a hormonal signal. To further understand the role of glucocorticoid signaling via the type II glucocorticoid receptor (GR) in the perinatal period and earlier during development, we have studied the expression of the mouse GR gene. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected as early as day 9.5 of mouse development. To specifically address the role of glucocorticoid signaling via the GR during development, we have disrupted the GR gene by homologous recombination in mouse embryonic stem cells. The majority of GR mutants die shortly after birth and analysis so far has revealed defects in lung, liver, and adrenal function.

Molecular genetic analysis of glucocorticoid signalling in development

A null mutation of the glucocorticoid receptor was generated by homologous recombination. Mutant newborn mice showed impaired lung development, hypertrophy of the adrenal cortex and a strongly reduced size of the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT) was undetectable in the adrenals of the mutant mice. Serum levels of corticosterone were moderately and ACTH levels were strongly elevated in the mutants. A weaker but significant increase of corticosterone and ACTH was observed already in heterozygous animals. This points to a dysregulation of the HPA axis due to defective feedback regulation via the glucocorticoid receptor. Liver gluconeogenetic enzymes were reduced to a variable degree. Whereas survival of heterozygous mutants was not affected, most of the homozygous mutant mice died during the perinatal period.

Expression of the mouse glucocorticoid receptor and its role during development

Genes encoding enzymes involved in gluconeogenesis are activated in liver shortly after birth by the synergistic effect of glucagon and glucocorticoids. This induction is achieved by the synergistic action of hormone responsive and liver-specific enhancer elements. In the case of glucocorticoids, this enhancer is composed of a glucocorticoid-response element (GRE) and a number of cell-specific hepatocyte nuclear factor 3 (HNF-3) binding sites. The GRE binds the ligand-activated glucocorticoid receptor (GR) which is ubiquitously expressed and the HNF-3 element binds a cell-specific protein factor. To further understand the role of cell-specific glucocorticoid signalling in the perinatal period and earlier during development we have studied the expression of the mouse GR gene. The gene has been cloned and fully characterized. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected back to day 9.5 of mouse development. The role of GR during mouse development has been further analysed by disruption of the GR gene in vivo by homologous recombination in mouse embryonic stem cells.

Effects of kainic acid induced seizures on immediate early gene expression in mice with a targeted mutation of the CREB gene

The present study examined the response of immediate early genes following kainic acid induced seizures in mice lacking the alpha and delta isoforms of CREB. mRNA levels for c-fos, c-jun, and Krox-24 were measured following limbic seizure activity and were found to be induced in wild type as well as CREB mutant mice. This effect was also seen for these three mRNAs at the protein level as well as for FOS-B. Furthermore the time course of expression of FOS, JUN, KROX-24, and FOS-B proteins were essentially the same in CREB mutant mice as compared to wild-type controls. These data suggest that CREB alpha and delta are not required for the induction of immediate early genes following pharmacologically induced seizures.

Analysis of the cAMP response on liver-specific gene expression in transgenic mice

Summary— Expression of many genes is modulated by intracellular variations of cyclic AMP (cAMP) levels in response to different signals from the environment. This regulation is mediated via a cAMP‐response element (CRE). This report addresses the role of cAMP in the physiological activation of a subset of liver‐specific genes which are perinatally activated. The tyrosine aminotransferase (TAT) gene and other genes such as phosphoenolpyruvate carboxyquinase (PEPCK) and glucose‐6‐phosphatase, involved in gluconeogenesis, belong to this category. CRE elements derived from the rat TAT −3.6 kb enhancer have been positioned in chimeric constructs, such that the activity of the reporter gene LacZ is dependent on cAMP. The tissue‐specificity of these constructs is guaranteed by the presence of the liver‐specific enhancers of the alpha fetoprotein gene. These constructs have been tested in cells and transgenic mice demonstrating cAMP regulation, liver‐specific expression and perinatal activation of the reporter gene. The CRE is recognized by a number of related proteins of which the cAMP‐response element‐binding factor (CREB) has been best studied. To assess the role of CREB in the in vivo transduction of cAMP signalling, mice deficient in CREB protein have been generated by homologous recombination in embryonic stem (ES) cells. Homozygous mutant mice, although recovering at a lower ratio than expected, do not display impairment of growth or development. The cAMP‐dependent LacZ transgenic mice in a CREB mutant genetic background also show perinatal activation of the reporter gene. Compensation with other members of the family of CRE‐binding proteins, such as cAMP‐response element modulator (CREM) or activating transcription factor‐1 (ATF‐1), may explain the lack of an obvious phenotype in CREB mutant mice.