Dysfunction of the RAR/RXR signaling pathway in the forebrain impairs hippocampal memory and synaptic plasticity.

BACKGROUND: Retinoid signaling pathways mediated by retinoic acid receptor (RAR)/retinoid × receptor (RXR)-mediated transcription play critical roles in hippocampal synaptic plasticity. Furthermore, recent studies have shown that treatment with retinoic acid alleviates age-related deficits in hippocampal long-term potentiation (LTP) and memory performance and, furthermore, memory deficits in a transgenic mouse model of Alzheimer's disease. However, the roles of the RAR/RXR signaling pathway in learning and memory at the behavioral level have still not been well characterized in the adult brain. We here show essential roles for RAR/RXR in hippocampus-dependent learning and memory. In the current study, we generated transgenic mice in which the expression of dominant-negative RAR (dnRAR) could be induced in the mature brain using a tetracycline-dependent transcription factor and examined the effects of RAR/RXR loss. RESULTS: The expression of dnRAR in the forebrain down-regulated the expression of RARß, a target gene of RAR/RXR, indicating that dnRAR mice exhibit dysfunction of the RAR/RXR signaling pathway. Similar with previous findings, dnRAR mice displayed impaired LTP and AMPA-mediated synaptic transmission in the hippocampus. More importantly, these mutant mice displayed impaired hippocampus-dependent social recognition and spatial memory. However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively. Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory. CONCLUSIONS: From these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

CBP/p300 is a cell type-specific modulator of CLOCK/BMAL1-mediated transcription.

BACKGROUND: Previous studies have demonstrated tissue-specific regulation of the rhythm of circadian transcription, suggesting that transcription factor complex CLOCK/BMAL1, essential for maintaining circadian rhythm, regulates transcription in a tissue-specific manner. To further elucidate the mechanism of the cell type-specific regulation of transcription by CLOCK/BMAL1 at the molecular level, we investigated roles of CBP/p300 and tissue-specific cofactors in CLOCK/BMAL1-mediated transcription. RESULTS: As shown previously, CBP/p300 stimulates CLOCK/BMAL1-mediated transcription in COS-1 cells. However, CBP/p300 repressed CLOCK/BMAL1-mediated transcription in NIH3T3 cells and knockdown of CBP or p300 expression by siRNA enhanced this transcription. Studies using GAL4-fusion proteins suggested that CBP represses CLOCK/BMAL1-mediated transcription by targeting CLOCK. We further investigated mechanisms of this cell type-specific modulation of CLOCK/BMAL1-mediated transcription by CBP by examining roles of co-repressor HDAC3 and co-activator pCAF, which are highly expressed in NIH3T3 and COS cells, respectively. CBP repressed CLOCK/BMAL1-mediated transcription in COS-1 cells when HDAC3 was overexpressed, but activated it in NIH3T3 cells when pCAF was overexpressed. CBP forms a complex with CLOCK by interacting with HDAC3 or pCAF; however, direct interaction of CBP with CLOCK was not observed. CONCLUSION: Our findings indicate possible mechanisms by which CBP/p300 tissue-specifically acts cooperatively with pCAF and HDAC3 either as a co-activator or co-repressor, respectively, for CLOCK/BMAL1.

Transgenic up-regulation of alpha-CaMKII in forebrain leads to increased anxiety-like behaviors and aggression.

BACKGROUND: Previous studies have demonstrated essential roles for alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) in learning, memory and long-term potentiation (LTP). However, previous studies have also shown that alpha-CaMKII (+/-) heterozygous knockout mice display a dramatic decrease in anxiety-like and fearful behaviors, and an increase in defensive aggression. These findings indicated that alpha-CaMKII is important not only for learning and memory but also for emotional behaviors. In this study, to understand the roles of alpha-CaMKII in emotional behavior, we generated transgenic mice overexpressing alpha-CaMKII in the forebrain and analyzed their behavioral phenotypes. RESULTS: We generated transgenic mice overexpressing alpha-CaMKII in the forebrain under the control of the alpha-CaMKII promoter. In contrast to alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in anxiety-like behaviors in open field, elevated zero maze, light-dark transition and social interaction tests, and a decrease in locomotor activity in their home cages and novel environments; these phenotypes were the opposite to those observed in alpha-CaMKII (+/-) heterozygous knockout mice. In addition, similarly with alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in aggression. However, in contrast to the increase in defensive aggression observed in alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in offensive aggression. CONCLUSION: Up-regulation of alpha-CaMKII expression in the forebrain leads to an increase in anxiety-like behaviors and offensive aggression. From the comparisons with previous findings, we suggest that the expression levels of alpha-CaMKII are associated with the state of emotion; the expression level of alpha-CaMKII positively correlates with the anxiety state and strongly affects aggressive behavior.

Upregulation of calcium/calmodulin-dependent protein kinase IV improves memory formation and rescues memory loss with aging.

Previous studies have suggested that calcium/calmodulin-dependent protein kinase IV (CaMKIV) functions as a positive regulator for memory formation and that age-related memory deficits are the result of dysfunctional signaling pathways mediated by cAMP response element-binding protein (CREB), the downstream transcription factor of CaMKIV. Little is known, however, about the effects of increased CaMKIV levels on the ability to form memory in adult and aged stages. We generated a transgenic mouse overexpressing CaMKIV in the forebrain and showed that the upregulation of CaMKIV led to an increase in learning-induced CREB activity, increased learning-related hippocampal potentiation, and enhanced consolidation of contextual fear and social memories. Importantly, we also observed reduced hippocampal CaMKIV expression with aging and a correlation between CaMKIV expression level and memory performance in aged mice. Genetic overexpression of CaMKIV was able to rescue associated memory deficits in aged mice. Our findings suggest that the level of CaMKIV expression correlates positively with the ability to form long-term memory and implicate the decline of CaMKIV signaling mechanisms in age-related memory deficits.

Characterization of the promoter of the mouse preproorexin gene.

The neuropeptide Orexin is involved in the regulation of the sleep-awake cycle and feeding behavior. We isolated a 22-kb genomic clone containing the 5' flanking region of the mouse Orexin promoter. We determined that the transcription start site (+1) is located 96 nucleotides upstream of the initiation codon. The start site region contained consensus sequences corresponding to the transcription initiator and TATA box. Analysis of promoter activity using PC12 cells showed that regions between -13 and +112 and between -1,868 and -780 contained nerve growth factor (NGF)-responsive positive regulatory element and a negative regulatory element respectively.

Chronic reduction in dietary tryptophan leads to a selective impairment of contextual fear memory in mice.

The depletion of systemic tryptophan is an important tool to study the effects of reduced 5-HT on cognition. Indeed, previous reports indicated that acute depletion of TRP leads to a memory impairment in human subjects and rodents. From the view of nutrition, it is important to investigate the effects of chronic limitation of L-tryptophan (TRP) on learning and memory formation. In this study, we examined the effects of chronic consumption of a low TRP diet on memory formation in mice. Specifically, we assessed the ability to form contextual fear, cued fear, conditioned taste aversion, and spatial memories in mice fed a TRP-limited diet for at least 1 month. TRP-limited mice showed impaired formation of contextual fear memory that is hippocampus-dependent. In contrast, these mice showed normal hippocampus-dependent spatial memory in the Morris water maze test, as well as in cued fear and conditioned taste aversion memories, which are amygdala-dependent memory processes. Thus, dietary TRP restriction appears to result in selective impairments in hippocampus-dependent contextual fear memory formation in mice.

Involvement of cAMP response element-binding protein activation in salivary secretion.

OBJECTIVE: Saliva secretion is mediated by cAMP and the calcium signaling pathway in salivary acinar cells. The PKA signaling pathway plays an important role in protein secretion through the activation of cAMP, in fluid secretion through the elevation of intracellular calcium and in the activation of cAMP response element-binding protein (CREB), which is involved in these signaling cascades. In this study, we investigated whether the activation of CREB plays a part in the salivary secretion in mice. METHODS: We examined CREB activation by assessing phosphorylation at the serine-133 position using Western blotting. RESULTS: Carbachol (a muscarinic acetylcholine agonist) and isoproterenol (a beta-adrenergic agonist) markedly activated CREB in parotid acinar cells. Carbachol and isoproterenol-induced CREB phosphorylation was blocked by atropine (a muscarinic acetylcholine antagonist) and propranolol (a beta-adrenergic antagonist), respectively. The PKA inhibitor H89 inhibited CREB activation, but the PLC inhibitor U73122 did not. Moreover, carbachol- and isoproterenol-stimulated amylase secretion from parotid acinar cells was inhibited by H89 and adenoviral dominant-negative CREB. CONCLUSION: These results indicate that the muscarinic and beta-adrenergic activation of CREB was mediated through the PKA pathway and that CREB is involved in protein secretion from parotid acinar cells.

Chronic reduction in dietary tryptophan leads to changes in the emotional response to stress in mice.

Acute depletion of brain tryptophan (TRP) levels in humans has been used as a biochemical model of depression. In this study, we examined the effects of consumption of a diet low in TRP on emotional behavior in mice. Specifically, we assessed various parameters of emotional behavior in mice fed a TRP-limited diet for at least 1 mo. TRP-limited mice showed increased defensive, but not offensive, aggression in the resident-intruder test. In the social dominance tube test, these mice showed enhanced social dominance. Since defensive aggression is thought to be a reflection of not only aggression but also fear, these changes in the social behavior of TRP-limited mice are thought to reflect changes in their emotional status. TRP-limited mice also showed increased locomotor activity and mobility in the open field and forced swim tests, respectively, suggesting that their stress/emotional responsiveness was enhanced. Importantly, these mice displayed normal levels of anxiety and motor performance as determined by the elevated zero maze and open field tests, and the rotarod test, respectively, suggesting that their hyperactivity was not due to a reduction in anxiety levels or to enhancement of their motor performance. Thus, dietary TRP restriction appears to result in alterations in the emotional response to stress, in mice.

The administration of retinoic acid down-regulates cAMP-responsive element modulator (CREM) mRNA in vitamin A-deficient testes.

Studies using genetics and vitamin A deficiency (VAD) have shown that vitamin A and retinoids play essential roles in spermatogenesis at the pre-meiotic stage. To understand the mechanisms of control in spermatogenesis by retinoic acid, we investigated whether retinoic acids regulate the expression of downstream transcription factors that are essential for spermatogenesis. In this study, we found that administration of all-trans retinoic acid (ATRA) or retinol to VAD rats down-regulates the testicular mRNA levels of the cAMP responsive element modulator (CREM), an essential transcription factor for spermatogenesis. Conversely, depletion of retinoids from the diet leds to an up-regulation of CREM expression in adult testes. In addition, RT-PCR analysis indicated that ATRA specifically represses the expression of the activator spliced variant of CREM (CREMtau). These results suggest that retinoids function as a negative regulator of CREM expression in testes.

PLCgamma2 Activates CREB-dependent Transcription in PC12 Cells Through Phosphorylation of CREB at Serine 133.

The cAMP and Ca(2+) signaling pathways activate the transcription factor CREB through its phosphorylation at Serine 133. Activation of CREB is involved in the regulation of various biological phenomena. To understand further the mechanisms of the regulation of CREB activity in response to activation of the cAMP and Ca(2+) signaling pathways, we examined the roles of PLCgammas in CREB activation in PC12 cells. siRNA-mediated reduction of the expression of PLCgamma2, but not PLCgamma1, inhibited both the phosphorylation of CREB at S133 and the activation of CREB-dependent transcription following treatment of cells with forskolin or ionomycin, which increases the intracellular concentrations of cAMP or Ca(2+), respectively. Importantly, the siRNA targeting PLCgamma2 completely abolished CREB activation by Ca(2+) signaling but not by cAMP signaling. These results suggest that PLCgamma2 functions as an essential signal transducer leading to CREB activation in response to activation of the Ca(2+) signaling pathway and that the cAMP signaling pathway might activate CREB through phosphorylation of CREB by PKA and another signaling pathway mediated by PLCgamma2.

A BMAL1 mutant with arginine 91 substituted with alanine acts as a dominant negative inhibitor.

Basic-Helix-Loop-Helix-Per-Arnt-Sim (bHLH-PAS) transcription factor, Brain-Muscle-Arnt-Like-protein 1 (BMAL1), forms a heterodimer with the CLOCK protein. The BMAL1/CLOCK complex binds to a specific DNA sequence and plays an essential role in the generation of the circadian rhythm. The basic region of BMAL1 contains an E-R-X-R motif that is highly conserved among basic-helix-loop-helix (bHLH) transcription factors that bind to the E-box transcription element, and is thus thought to constitute a structure required for recognition of this DNA sequence. To understand the role of arginine 91 (E-K-R-R) within the basic region of BMAL1, we examined the effect of mutation of this residue on BMAL1 function. Co-immunoprecipitation and electrophoretic mobility shift assays (EMSA) showed that a BMAL1 R91A mutant forms a heterodimer with CLOCK, but is unable to support DNA binding in vitro. Consistent with these observations, plasmids encoding the R91A and R91H mutants of BMAL1 were unable to stimulate transcription from an E-box containing reporter construct in transient co-transfection analyses in NIH 3T3 cells. More importantly, these mutants suppressed the activation of transcription from the reporter construct mediated by wild-type BMAL1, indicating that they possess dominant negative activity in this assay. These results document further the importance of the basic region of BMAL1 in binding to DNA and suggest that this new mutant might be a useful tool for further analysis of BMAL1 function.

Memory reconsolidation and extinction have distinct temporal and biochemical signatures.

Memory retrieval is not a passive phenomenon. Instead, it triggers a number of processes that either reinforce or alter stored information. Retrieval is thought to activate a second memory consolidation cascade (reconsolidation) that requires protein synthesis. Here, we show that the temporal dynamics of memory reconsolidation are dependent on the strength and age of the memory, such that younger and weaker memories are more easily reconsolidated than older and stronger memories. We also report that reconsolidation and extinction, two opposing processes triggered by memory retrieval, have distinct biochemical signatures: pharmacological antagonism of either cannabinoid receptor 1 or L-type voltage-gated calcium channels blocks extinction but not reconsolidation. These studies demonstrate the dynamic nature of memory processing after retrieval and represent a first step toward a molecular dissection of underlying mechanisms.

CREB required for the stability of new and reactivated fear memories.

The cAMP-responsive element binding protein (CREB) family of transcription factors is thought to be critical in memory formation. To define the role of CREB in distinct memory processes, we derived transgenic mice with an inducible and reversible CREB repressor by fusing CREBS133A to a tamoxifen (TAM)-dependent mutant of an estrogen receptor ligand-binding domain (LBD). We found that CREB is crucial for the consolidation of long-term conditioned fear memories, but not for encoding, storage or retrieval of these memories. Our studies also showed that CREB is required for the stability of reactivated or retrieved conditioned fear memories. Although the transcriptional processes necessary for the stability of initial and reactivated memories differ, CREB is required for both. The findings presented here delineate the memory processes that require CREB and demonstrate the power of LBD-inducible transgenic systems in the study of complex cognitive processes.