Phosphorylated cAMP Response Element Binding Research Articles

Human Tau may Modify Glucocorticoids-Mediated Regulation of cAMP-dependent Kinase and Phosphorylated cAMP Response Element Binding Protein

Phosphorylation of the cAMP response element binding protein (CREB) by cAMP-dependent kinase (PKA) is critical to memory formation. However, activation of PKA can also increase tau phosphorylation, which may contribute to memory impairment. Therefore, the regulation of PKA may be part of the mechanism by which glucocorticoids (GCs) influence memory. Additionally, the cellular response to GCs may be affected by the presence of human tau. The goal of this paper was to study GCs-mediated regulation of PKA as well as CREB and tau phosphorylation in wild-type HEK293 cells and HEK293 cells stably expressing human tau441 (HEK293/tau441 cells). By using dexamethasone (DEX) as GCs, we found that DEX induced a tau-dependent selective decrease in the level of PKA RIIβ subunit protein. The observed decrease in RIIβ expression was not due to alterations of mRNA levels and was reversed by inhibiting the proteasome with lactacystin. Moreover, the decrease in RIIβ did not diminish the co-localization of the catalytic subunit of PKA with tau and might contribute to the DEX-induced increase in tau phosphorylation at Ser-214. DEX also induced a tau-dependent decrease in CREB phosphorylation that could not be reversed by activating PKA with forskolin. Taken together, these results show that human tau protein may alter the GCs-mediated regulation of PKA activity and CREB phosphorylation.

Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism

The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.

Involvement of transcription factor GATA-4 in regulation of CYP19 gene during folliculogenesis and luteinization in buffalo ovary

CYP19 gene encode aromatase, the key enzyme of estrogen biosynthesis, is regulated in species- and tissue-specific manner by alternate use of different promoters. Previously, we have reported the cloning and characterization of tissue-specific promoter and transcripts in buffalo ovary and placenta. In human and rat ovary, FSH induces the phosphorylation of transcription factor CREB (cAMP response element binding protein) through PKA pathway which binds to cAMP response element like sequence (CLS) in CYP19 gene ovarian promoter. However, in buffalo as well as in bovine, in silico analysis of ovary specific promoter sequence identified a single base pair deletion in CLS site and is designated as CLS-like sequence. To understand if CLS with a point mutation is still a functional cis-element and is involved in FSH stimulated transactivation of CYP19 gene in buffalo ovary, the present study was thus aimed to functionally characterize the role of buffalo CLS in CYP19 gene transactivation. We also studied the involvement of GATA-4, having consensus binding sites in CYP19 gene ovarian promoter in the vicinity of CLS during different stages of the buffalo estrus cycle. Reporter construct analyses and EMSA results showed that CLS is playing no significant role in CYP19 gene regulation in buffalo ovary. Real time absolute quantification of GATA-4 showed the differential expression of GATA-4 mRNA during folliculogenesis and luteinization with significantly higher transcript abundance in large follicle in comparison to other tissues. Western blot analysis of granulosa cells nuclear protein isolated from different stage of follicular development (small and large follicles) and differentiation (corpus luteum) showed that abundance of phosphorylated GATA-4 (Ser261) was significantly higher in granulosa cell nuclear protein of large follicles as compared to small follicles and corpora lutea. Interestingly, binding studies using ChIP showed significantly enhanced binding to the CYP19 gene promoter in large follicle which was seen to be declined in the luteal tissue. Similar results were obtained in the in vitro experiments as well. Finally, RNAi experiments were performed to validate the involvement of GATA-4 in CYP19 gene regulation. Results of RNAi showed that knockdown of GATA-4 mRNA significantly declined CYP19 gene mRNA as well as 17β-estradiol contents. In conclusion, result of the present study indicated that that in the absence of consensus CRE (cAMP response element); GATA-4 could be a downstream effector of cAMP/PKA pathway in regulation of CYP19 gene during folliculogenesis and luteinization.

CAMP/PKA Agonist Restores the Fasting-Induced Down-Regulation of nNOS Expression in the Paraventricular Nucleus.

Gene expression of neuronal nitric oxide synthase (nNOS) changes in the hypothalamic paraventricular nucleus (PVN) depending on feeding conditions, which is decreased during food deprivation and restored by refeeding, and phosphorylated cAMP response element binding protein (pCREB) was suggested to play a role in its regulation. This study was conducted to examine if the fasting-induced down-regulation of the PVN-nNOS expression is restored by activation of cAMP-dependent protein kinase A (cAMP/PKA) pathway. Freely moving rats received intracerebroventricular (icv) injection of cAMP/PKA activator Sp-cAMP (40 nmol) or vehicle (sterilized saline) following 48 h of food deprivation. One hour after drug injections, rats were transcardially perfused with 4% paraformaldehyde, and the PVN tissues were processed for nNOS or pCREB immunohistochemistry. Sp-cAMP significantly increased not only nNOS but also pCREB immunoreactivities in the PVN of food deprived rats. Fasting-induced down-regulation of the PVN-nNOS was restored by 1 h after the icv Sp-cAMP. Results suggest that cAMP/PKA pathway may mediate the regulation of the PVN-nNOS expression depending on different feeding conditions.

Localization of phosphorylated CREB in the honeybee brain

Event Abstract Back to Event Localization of phosphorylated CREB in the honeybee brain Katrin Gehring1*, Karin Heufelder1 and Dorothea Eisenhardt1 1 Freie Universität Berlin, Neurobiology, Germany The transcription factor CREB (cAMP response element binding protein) is involved in regulation of growth, differentiation, neuronal viability and circadian rhythms. It also plays an essential role in the formation of long term memory (LTM). CREB induces gene expression after being phosphorylated by several kinases. A huge body of work analyzed this CREB phosphorylation but little is known about its role in the honeybee (Apis mellifera), a well-known invertebrate model system for learning and memory. Our aim is to characterize Apis mellifera CREB and localize CREB dependent processes in the honeybee brain. We use different antibodies to detect CREB and phosphorylated CREB (pCREB) in bee homogenate and in fixed bee brain slices. Counterstaining of synaptic proteins and DNA were performed to further characterize pCREB containing neurons. pCREB immunoreactivity can be observed in a wide range of cell nuclei including nuclei of neurons known to be involved in processing sensory information, for example intrinsic mushroom body neurons (Kenyon cells). In contrast to the assumed distribution of pCREB, which, as a transcription factor, is thought to be predominantly located in the nucleus, we also find strong immunoreactivity in axonal and dendritic parts of Kenyon cells. We investigated the distribution of pCREB immunoreactivity in the honeybee brain in bees with different age. We compared one day old bees with more experienced bees and found increased pCREB levels in aged bees and, focusing on specific brain neuropils, an ongoing redistribution and sharpening of the pCREB signal in distinct neuronal subsets. Since the antennal lobes and the mushroom bodies are involved in processing sensory information, ongoing experiments focus on changes of pCREB and CREB levels in these neuropils after learning. Keywords: CREB, Honeybee Conference: Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012. Presentation Type: Poster Presentation (see alternatives below as well) Topic: Learning, Memory and Behavioral Plasticity Citation: Gehring K, Heufelder K and Eisenhardt D (2012). Localization of phosphorylated CREB in the honeybee brain. Conference Abstract: Tenth International Congress of Neuroethology. doi: 10.3389/conf.fnbeh.2012.27.00149 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Apr 2012; Published Online: 07 Jul 2012. * Correspondence: Ms. Katrin Gehring, Freie Universität Berlin, Neurobiology, Berlin, Germany, katrin.gehring@fu-berlin.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Katrin Gehring Karin Heufelder Dorothea Eisenhardt Google Katrin Gehring Karin Heufelder Dorothea Eisenhardt Google Scholar Katrin Gehring Karin Heufelder Dorothea Eisenhardt PubMed Katrin Gehring Karin Heufelder Dorothea Eisenhardt Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

CREB involvement in the regulation of striatal prodynorphin by nicotine

The transcription factor cAMP response element binding (CREB) protein plays a pivotal role in drug-dependent neuronal plasticity. CREB phosphorylation at Ser133 is enhanced by drugs of abuse, including nicotine. Dynorphin (Dyn) contributes to the addictive process and its precursor gene prodynorphin (PD) is regulated by CREB. PD mRNA and Dyn synthesis were enhanced in the striatum following acute nicotine, suggesting genomic regulation. These studies investigated PD transcription in mice acutely treated with nicotine, determined the role of CREB, and characterized the receptors involved. Acute nicotine increased adenylyl cyclase activity, cAMP, and pCREB Ser133 levels in striatum and enhanced CREB binding to CRE elements (DynCREs) of the PD promoter, preferentially DynCRE3. DynCRE3 binding was dose dependent with 1mg of nicotine giving a maximal response. Additionally, DynCRE binding was time dependent, rising by 15min, reaching a maximum at 1h, and returning to control by 3h, a temporal pattern similar to that of cAMP and pCREB. Supershift experiments showed that CREB and pCREB Ser133 were the major contributors to DynCRE3 binding complex. The nAChR antagonist mecamylamine and the dopamine D1-like receptor antagonist SCH 23390 prevented the nicotine-induced increase of pCREB and nuclear protein binding to DynCRE3. Our findings suggest that nicotine regulates PD expression in striatum at the transcriptional level and CREB is involved. Dopamine D1 receptor stimulation by nAChR-released dopamine appears to be an underlying mechanism. Altered Dyn synthesis might be relevant for the behavioral actions of nicotine and especially its aversive properties.

Disrupting Effect of Drug-Induced Reward on Spatial But Not Cue-Guided Learning: Implication of the Striatal Protein Kinase A/cAMP Response Element-Binding Protein Pathway

The multiple memory systems hypothesis posits that different neural circuits function in parallel and may compete for information processing and storage. For example, instrumental conditioning would depend on the striatum, whereas spatial memory may be mediated by a circuit centered on the hippocampus. However, the nature of the task itself is not sufficient to select durably one system over the other. In this study, we investigated the effects of natural and pharmacological rewards on the selection of a particular memory system during learning. We compared the effects of food- or drug-induced activation of the reward system on cue-guided versus spatial learning using a Y-maze discrimination task. Drug-induced reward severely impaired the acquisition of a spatial discrimination task but spared the cued version of the task. Immunohistochemical analysis of the phosphorylated form of the cAMP response element binding (CREB) protein and c-Fos expression induced by behavioral testing revealed that the spatial deficit was associated with a decrease of both markers within the hippocampus and the prefrontal cortex. In contrast, drug reward potentiated the cued learning-induced CREB phosphorylation within the dorsal striatum. Administration of the protein kinase A inhibitor 8-Bromo-adenosine-3',5'-cyclic monophosphorothioate Rp isomer (Rp-cAMPS) into the dorsal striatum before training completely reversed the drug-induced spatial deficit and restored CREB phosphorylation levels within the hippocampus and the prefrontal cortex. Therefore, drug-induced striatal hyperactivity may underlie the declarative memory deficit reported here. This mechanism could represent an important early step toward the development of addictive behaviors by promoting conditioning to the detriment of more flexible forms of memory.

Kalopanaxsaponins A and B Isolated from Kalopanax pictus Ameliorate Memory Deficits in Mice

The stem-bark of Kalopanax pictus (KP, family Araliaceae), which contains triterpenoid saponins, has been shown to exhibit anticarcinogenic, antiinflammatory, antirheumatoid and antidiabetic activities. In a preliminary study, a KP methanol extract demonstrated acetylcholinesterase activity in vitro and memory enhancement in scopolamine-treated mice. Therefore, we isolated acetylcholinesterase inhibitors, kalopanaxsaponins A and B, from a KP butanol (BuOH) fraction, measured acetylcholinesterase activity in vitro, and investigated their memory-enhancing effects in a passive avoidance test, Y-maze test and Morris water maze test. These constituents inhibited acetylcholinesterase activity and significantly reversed scopolamine-induced deficits. They also increased brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding (p-CREB) protein expression but reduced TNF-α increased by scopolamine. Based on these findings, kalopanaxsaponins A and B may ameliorate memory deficits by inhibiting acetylcholinesterase activity and inducing BDNF and p-CREB expression.

Decreased CREB levels suppress epilepsy

Epilepsy is a common neurologic disorder yet no treatments aimed at preventing epilepsy have been developed. Several molecules including genes containing cAMP response elements (CREs) in their promoters have been identified that contribute to the development of epilepsy, a process called epileptogenesis. When phosphorylated cAMP response element binding protein (CREB) increases transcription from CRE regulated promoters. CREB phosphorylation is increased in rodent epilepsy models, and in the seizure onset region of humans with medically intractable epilepsy (Rakhade et al., 2005; Lee et al., 2007; Lund et al., 2008). Here we show that mice with decreased CREB levels (CREBα∆ mutants) have a ~50% reduction in spontaneous seizures following pilocarpine induced status epilepticus (SE) and require more stimulation to electrically kindle. Following SE, brain derived neurotrophic factor (BDNF) and inducible cAMP early repressor (ICER) mRNAs are differentially up-regulated in the hippocampus and cortex of the CREBα∆ mutants compared to wild-type mice, which may be contributing to differences in the severity of epilepsy. In contrast, we found no difference in KCC2 mRNA levels between the CREBα∆ and wild-type mice after SE. The mechanism by which BDNF and ICER mRNAs increase specifically in the CREBα∆ compared to wild-type mice following SE is not known. We did, however, find an increase in specific cAMP response element modulator (CREM) mRNA transcripts in the CREBα∆ mutants that might be responsible for the differential regulation of BDNF and ICER after SE. Altering CREB activity following a neurologic insult provides a therapeutic strategy for modifying epileptogenesis.

Repeated Exposure to the κ-Opioid Receptor Agonist Salvinorin A Modulates Extracellular Signal-Regulated Kinase and Reward Sensitivity

Repeated exposure to drugs of abuse and stress increase dynorphin, a κ opioid receptor (KOR) ligand, in the nucleus accumbens (NAc). Acute KOR activation produces dysphoria that might contribute to addictive behavior. How repeated KOR activation modulates reward circuitry is not understood. We used intracranial self-stimulation (ICSS), a method that provides a behavioral index of reward sensitivity, to measure the effects of repeated administration of the KOR agonist salvinorin A (salvA) (2 mg/kg) on the reward-potentiating effects of cocaine (5.0 mg/kg). In separate rats, we measured the effects of salvA on activation of extracellular signal regulated kinase (ERK), cyclic adenosine monophosphate (cAMP) response element binding protein, and c-Fos within the NAc. SalvA had biphasic effects on reward: an immediate effect was to decrease the rewarding impact of ICSS, whereas a delayed effect was to increase the rewarding impact of ICSS. Repeated salvA produced a net decrease in the reward-potentiating effects of cocaine. In the NAc, both acute and repeated salvA administration increased phosphorylated ERK, whereas only acute salvA increased c-Fos and repeated salvA increased phosphorylated cAMP response element binding protein. The KOR antagonist nor-binaltorphimine (20 mg/kg) blocked the immediate and delayed effects of salvA and prolonged the duration of cocaine effects in ICSS. Repeated salvA might trigger opponent processes such that "withdrawal" from the dysphoric effects of KOR activation is rewarding and decreases the net rewarding valence of cocaine. The temporal effects of salvA on ERK signaling suggest KOR-mediated engagement of distinct signaling pathways within the NAc that might contribute to biphasic effects on reward sensitivity.

Essential Role of Store-Operated Calcium Entry in Urotensin-Ii Induced Vascular Smooth Muscle Cells Proliferation

Urotensin-II (U-II) is a vasoactive peptide with many effects in the cardiovascular system. It has been described that U-II promotes vascular smooth muscle cell (SMC) proliferation and migration. However, its signalling pathway remains unclear. The aim of this study is to determine the role of store-operated Ca2+ entry (SOCE) and the activation of cAMP response element-binding (CREB) transcription factor in U-II evoked SMC proliferation. We used 5-bromo-2-deoxyuridine (Brdu), and immunofluorescence assays to determine SMC proliferation. Ca2+ imaging experiments were performed to study Ca2+ entry in cultured SMC isolated from thoracic rat aorta. Our results show that U-II (100 nM) evokes Ca2+ and Mn2+ entry that was significantly reduced by SOCE inhibitors Gadolinium (10 µM) and 2-aminoethyl diphenylborinate (2APB, 75 µM). Moreover, U-II promotes dose dependent SMC proliferation, reaching maximum effect with 100 nM concentration. This event was significantly abolished by SOCE inhibition with 2APB (75 µM). Furthermore, RNA silencing of the new players of SOCE, Stim1 and Orai1, inhibited Ca2+ entry induced by UII in SMC, and effectively prevented SMC proliferation. On the other hand, we have determined that U-II promotes the phosphorylation of CREB and its translocation into cell nucleus. P-CREB activation was also sensitive to SOCE inhibitors. In conclusion, our data demonstrate an essential role of Ca2+ entry through Stim1 and Orai1 dependent SOCE pathway and CREB activation in U-II induced vascular SMC proliferation. Acknowledgements: This study was supported by Red Cardiovascular RECAVA and Instituto Carlos III (RD06-0014-0020; PS09-02287) and from the Government of Andalucía by Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía (P08-CVI-3913; P09-CTS-4715).

Glucagon Receptor Knockout Prevents Insulin-Deficient Type 1 Diabetes in Mice

OBJECTIVETo determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency.RESEARCH DESIGN AND METHODSWe compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr−/−) mice and wild-type (Gcgr+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction.RESULTSGcgr+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower.CONCLUSIONSWe conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice.

Soluble adenylyl cyclase mediates bicarbonate-dependent corneal endothelial cell protection

Cyclic AMP produced from membrane receptor complex bound adenylyl cyclases is protective in corneal endothelial cells (CEC). CEC also express soluble adenylyl cyclase (sAC), which is localized throughout the cytoplasm. When activated by HCO(3)(-), cAMP concentration ([cAMP]) increases by ∼50%. Here we ask if cAMP produced from sAC is also protective. We examined the effects of HCO(3)(-), pH, phosphodiesterase 4 inhibition by rolipram, sAC inhibition by 2HE (2-hydroxyestradiol), and sAC small interfering RNA (siRNA) knockdown on basal and staurosporine-mediated apoptosis. HCO(3)(-) (40 mM) or 50 μM rolipram raised [cAMP] to similar levels and protected endothelial cells by 50% relative to a HCO(3)(-)-free control, whereas 2HE, which decreased [cAMP] by 40%, and H89 (PKA inhibitor) doubled the apoptotic rate. sAC expression was reduced by two-thirds in the absence of HCO(3)(-) and was reduced to 15% of control by sAC siRNA. Protection by HCO(3)(-) was eliminated in siRNA-treated cells. Similarly, caspase-3 activity and cytochrome c release were reduced by HCO(3)(-) and enhanced by 2HE or siRNA. Analysis of percent annexin V+ cells as a function of [cAMP] revealed an inverse, nonlinear relation, suggesting a protective threshold [cAMP] of 10 pmol/mg protein. Relative levels of phosphorylated cAMP response element binding protein and phosphorylated Bcl-2 were decreased in CEC treated with 2HE or siRNA, suggesting that HCO(3)(-)-dependent endogenous sAC activity can mobilize antiapoptotic signal transduction. Overall, our data suggest a new role for sAC in endogenous cellular protection.

Fitting MITF regulation of PDE4D3 into the skin pigmentation puzzle

Fitting MITF regulation of PDE4D3 into the skin pigmentation puzzle

CREB Inhibits AP-2α Expression to Regulate the Malignant Phenotype of Melanoma

BackgroundThe loss of AP-2α and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2α during melanoma progression remains unknown.Methodology/Principal FindingsHerein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2α protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2α. Loss of AP-2α expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2α promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2α expression following CREB silencing increases endogenous p21Waf1 and decreases MCAM/MUC18, both known to be downstream target genes of AP-2α involved in melanoma progression.Conclusions/SignificanceSince AP-2α regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21Waf1, our data identified CREB as a major regulator of the malignant melanoma phenotype.

Intensification of long-term memory deficit by chronic stress and prevention by nicotine in a rat model of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cholinergic dysfunction and deposition of beta-amyloid (Aβ) in regions of the brain associated with learning and memory. The sporadic nature and late onset of most AD cases suggests that aside from biological determinants, environmental factors such as stress may also play a role in the progression of the disease. Behavioral and molecular studies were utilized to evaluate the effects of chronic nicotine treatment in the prevention of impairment of long-term memory. The rat model of AD was induced by i.c.v. osmotic pump infusion of Aβ peptides. Chronic psychosocial stress and chronic nicotine treatment were instituted for 6 weeks. Spatial memory testing in the Radial Arm Water Maze revealed that, although stress, by itself, did not affect long-term memory, the combination of chronic stress and Aβ infusion impaired long-term memory significantly more than Aβ peptides infusion alone. Chronic nicotine treatment completely prevented Aβ- and stress/Aβ combination-induced memory impairment. Furthermore, molecular findings in hippocampal CA1 region of stress/Aβ rats indicated marked reduction in the protein levels of phosphorylated cAMP response element binding (p-CREB) and calcium-calmodulin-dependent protein kinase IV (CaMKIV), with significant increases in the levels of brain-derived neurotrophic factor (BDNF). These disturbances in signaling pathways, which may be the underlying mechanisms of impairment of long-term memory in these rats, were totally prevented by chronic nicotine treatment.

Involvement of cAMP in nerve growth factor-triggered p35/Cdk5 activation and differentiation in PC12 cells

The signaling mechanisms underlying cell differentiation have been extensively studied with the use of rat PC12 cells as a model system. Nerve growth factor (NGF) is a trophic factor inducing PC12 cell differentiation through the activation of the p35/cyclin-dependent kinase 5 (Cdk5) complex. It has been reported that adenylyl cyclase activation and cAMP production may be involved in NGF-dependent actions. Our previous results indicate that cAMP activates the p35/Cdk5 complex in reproductive cells. Therefore, the role of cAMP in NGF-triggered p35/Cdk5 activation and PC12 differentiation was interesting to explore. Our results indicate that roscovitine, a molecular inhibitor of Cdk5, blocks cAMP-triggered PC12 differentiation, which was evaluated by neurite initiation, a decrease in proliferation, and cell cycle G(1) arrest. The following data show that cAMP treatment increased Cdk5 activity through p35 upregulation. cAMP downstream components, protein kinase A (PKA) and phosphorylated cAMP response element binding protein (CREB), are involved in this regulation. The immunocytochemical results indicate that PKA inhibition disrupted cAMP-triggered p35/Cdk5 localization in PC12 cells. In addition, adenylyl cyclase inhibition was found to diminish NGF-induced intracellular cAMP production, CREB phosphorylation, and p35 expression. The cAMP antagonist and the PKA inhibitors reduced NGF-induced p35 expression. Finally, NGF-triggered PC12 differentiation was partially decreased by adenylyl cyclase or PKA inhibitors. In conclusion, these results demonstrate that cAMP may play a role in NGF-p35/Cdk5-dependent PC12 differentiation.

A Rapid Interference between Glucocorticoids and cAMP‐Activated Signalling in Hypothalamic Neurones Prevents Binding of Phosphorylated cAMP Response Element Binding Protein and Glucocorticoid Receptor at the CRE‐Like and Composite GRE Sites of Thyrotrophin‐Releasing Hormone Gene Promoter

Glucocorticoids or cAMP increase, within minutes, thyrotrophin-releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at -101/-94 bp and a composite GRE element (cGRE) at -218/-197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br-cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone-bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP- and glucocorticoid-mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP-1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br-cAMP. ChIP assays revealed phospho-CREB, c-Jun, Sp1, c-Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA-polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br-cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE-2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as a mechanism by which glucocorticoids rapidly suppress cAMP and noradrenaline-stimulated TRH transcription.

FGF but not EGF induces phosphorylation of the cAMP response element binding protein in olfactory mucosa-derived cell cultures

The stem/progenitor cells of the olfactory epithelium are potentially useful cells for autologous cell-based therapy because of their relative accessibility compared to other sources of neural stem cells. However, they have very limited potential to self-renew in vitro under growth factor stimulation compared to central nervous system-derived stem/progenitor cells. Using a sphere-forming assay and immunocytochemistry to identify cells that contained phosphorylated cAMP response element binding protein (pCREB) as an indicator of cell responsiveness to growth factor activation, we found that olfactory-spheres primed with FGF2 responded to FGF2 and EGF stimulation. In contrast, olfactory-spheres primed with EGF failed to respond to FGF2 or EGF stimulation despite the detection of FGFR1 and EGFR and their transcripts. These data demonstrate that FGF2 but not EGF permit the maintenance of a subset of cells responsive to FGF2 and EGF, whereas EGF induces unresponsive to either growth factor possibly via intrinsic mechanisms of regulation.

Neuropeptide Y Signaling in the Central Nucleus of Amygdala Regulates Alcohol‐Drinking and Anxiety‐Like Behaviors of Alcohol‐Preferring Rats

The neuropeptide Y (NPY) system of the central nucleus of amygdala (CeA) has been shown to be involved in anxiety and alcoholism. In this study, we investigated the molecular mechanisms by which NPY in the CeA regulates anxiety and alcohol drinking behaviors using alcohol-preferring (P) rats as an animal model. Alcohol-preferring rats were bilaterally cannulated targeting the CeA and infused with artificial cerebrospinal fluid (aCSF) or NPY. Alcohol drinking and anxiety-like behaviors were assessed by the 2-bottle free-choice paradigm and light/dark box (LDB) exploration test, respectively. The levels of NPY and related signaling proteins were determined by the gold immunolabeling procedure. The mRNA levels of NPY were measured by in situ RT-PCR. Double-immunofluorescence labeling was performed to observe the colocalization of NPY and Ca(2+)/calmodulin-dependent protein kinase IV (CaMK IV). We found that NPY infusion into the CeA produced anxiolytic effects, as measured by the LDB exploration test, and also decreased alcohol intake in P rats. NPY infusion into the CeA significantly increased levels of CaMK IV and phosphorylated cAMP responsive element-binding (pCREB) protein and increased mRNA and protein levels of NPY, but produced no changes in protein levels of CREB or the catalytic alpha-subunit of protein kinase A (PKA-Calpha) in the CeA. We also observed that alcohol intake produced anxiolytic effects in P rats in the LDB test and also increased NPY expression and protein levels of pCREB and PKA-Calpha without modulating protein levels of CREB or CaMK IV, in both the CeA and medial nucleus of amygdala. In addition, we found that CaMK IV-positive cells were co-localized with NPY in amygdaloid structures of P rats. These results suggest that NPY infusion may increase the expression of endogenous NPY in the CeA, which is most likely attributable to an increase in CaMK IV-dependent CREB phosphorylation and this molecular mechanism may be involved in regulating anxiety and alcohol drinking behaviors of P rats.