ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

Immunosuppressants that target the serine/threonine phosphatase calcineurin are commonly administered following organ transplantation. Their chronic use is associated with reduced insulin secretion and new onset diabetes in a subset of patients, suggestive of pancreatic β cell dysfunction. Calcineurin plays a critical role in the activation of CREB, a key transcription factor required for β cell function and survival. CREB activity in the islet is activated by glucose and cAMP, in large part due to activation of Crtc2, a critical coactivator for CREB. Previous studies have demonstrated that Crtc2 activation is dependent on dephosphorylation regulated by calcineurin. In this study, we sought to evaluate the impact of calcineurin-inhibiting immunosuppressants on Crtc2-CREB activation in the primary β cell. In addition, we further characterized the role and regulation of Crtc2 in the β cell. We demonstrate that Crtc2 is required for glucose dependent up-regulation of CREB target genes. The phosphatase calcineurin and kinase regulation by LKB1 contribute to the phosphorylation status of Crtc2 in the β cell. CsA and FK506 block glucose-dependent dephosphorylation and nuclear translocation of Crtc2. Overexpression of a constitutively active mutant of Crtc2 that cannot be phosphorylated at Ser171 and Ser275 enables CREB activity under conditions of calcineurin inhibition. Furthermore, β cells lacking Crtc2 display impaired glucose-stimulated insulin secretion and cell survival. Together, these results demonstrate that phosphorylation of Crtc2 plays a critical role in regulating CREB activity and contributes to β cell dysfunction and death caused by chronic immunosuppression.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G1-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho β-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of β-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of β-catenin and CREB pathways.

Preclinical results support the use of N-methyl D-aspartate receptor (NMDAR) modulators for cognition enhancement therapeutics. Pregnenolone sulfate (PregS) is a neuroactive steroid derived from cholesterol that augments long term potentiation (LTP) in hippocampal slices and improves memory performance in rats and mice. At micromolar concentrations, PregS is a subtype selective positive allosteric modulator of NMDARs at NR2A and NR2B containing receptors, and at concentrations ranging from pM - nM induces NMDAR-dependent dopamine release in the striatum and from striatal synaptosomes. In this report, we observe that micromolar [PregS] induces an increase in levels of neuronal intracellular calcium ([Ca^2+]i) and surface NMDARs in cortical neurons. Moreover, our results show that PregS stimulated upregulation of surface NR1 subunits in cortical neurons is dependent on NMDARs but independent of channel activity. As PregS has been detected in brain at bulk concentrations of 0.1 nM to 5 nM, we asked whether low, picomolar concentrations of PregS might alter [Ca^2+] levels. We report here that PregS increases [Ca^2+]i signal in cortical neurons in a voltage-gated Na^+ channel and NMDAR-NR2B dependent manner with an EC50 of ~2 pM, at least 6 orders of magnitude higher affinity than its rapid potentiating effect upon the NMDAR-mediated ionotropic response, and within the range of PregS detected in bulk brain tissue. Additionally, calcium (Ca^2+) activation of cyclic AMP response element binding protein (CREB) is critical to the protein synthesis-dependent component of LTP and important in associated behavioral measures of learning and memory. Increased [Ca^2+]i levels are known to induce CREB activation and we now show that 50 pM PregS induces a 44 ± 13% increase in the ratio of pCREB to total CREB that is dependent upon ERK signaling and canonical excitatory synaptic transmission: this includes voltage gated Na+ channels, NMDARs, and voltage-gated Ca^2+ channel activation. The results taken together indicate that PregS may be a useful platform for the development of high-affinity positive modulators of NMDAR-signaling that can be used as cognitive enhancers to treat a variety of neurological disorders: such as Alzheimer's disease, Parkinson's disease, and schizophrenia.

Activation of AMP-activated protein kinase (AMPK) results in the upregulation of several intracellular systems which help to prepare a cell for a high energy challenge. The magnitude of the AMPK response to a 10 min bout of exercise has been found to decrease in red quadriceps (RQ) following training, while putative AMPK roles seem to be maintained; specifically, the biogenesis of mitochondria and higher levels of hexokinase II and glucose transporter 4 (GLUT4). If the AMPK response to exercise is responsible in part for these adaptations, how can they be maintained if the AMPK response is attenuated? The purpose of this study was to determine whether phosphorylation of AMPK in RQ increases during 2-hr training bouts after rats have trained for 8 wks. Male Sprague-Dawley rats ran up to 30 m/min up a 15% grade, 2 hr/day for 8 wks. On the final bout of exercise, trained rats ran for 0 (TRC), 30 (TR1), or 120 min (TR2) up a 15% grade at 30 m/min. Red quadriceps (RQ), soleus, and white quadriceps (WQ) were immediately collected and frozen for analysis. Citrate synthase activity increased in RQ (79 ± 3 vs. 37 ± 4 µmol/g/min) and soleus (64 ± 4 vs. 35 ± 2 µmol/g/min) but not in WQ compared to non-trained controls. In trained rats, maximal increases in T-172 phosphorylation of AMPK occurred after 30 min of exercise (relative values = 1.29 ± 0.06 vs. 1.00 ± 0.06). AMPK phosphorylation did not change significantly in trained rats that ran for 2 hrs (1.31 ± 0.09) compared to rats that ran for 30 min. Similarly, maximal increases in AMPK activity in trained rats occurred after 30 min of exercise (pmoles/min/mg = 2.67 ± .05 vs. 1.09 ± .41) and AMPK activity did not change significantly in trained rats that ran for 2 hrs (2.79 ± .17) compared to rats that ran for 30 min. Previous studies demonstrated a 2−3 fold increase in AMPK activity in non-trained rats after 30 min of exercise at lower work rates. These results demonstrate that the AMPK response to exercise is attenuated even after two-hr bouts of exercise. This implies that the increase in mitochondrial oxidative enzymes, GLUT4, and hexokinase II may be maintained by signals other than the AMPK signaling system. The CREB signaling pathway is one such system. Western analysis of phospho-CREB (Ser133) showed a statistically significant increase in phospho-CREB content in trained rats relative to control. No change in phospho-CREB protein expression was observed between TRC, TR1, and TR2 rats. Significant increases of muscle phospho-CREB content in TRC relative to untrained rats suggest that CREB remains phosphorylated in trained rats even after 24 hrs of rest. Accordingly, chronically increased phospho-CREB in muscle of trained rats relative to controls may explain in part how increased levels of mitochondria are maintained in the face of reduced AMPK response. Alternatively, the attenuated AMPK response may still be above the threshold required for inducing adaptations to endurance training.

In this two-part study, I investigated whether the cyclic-adenosine monophosphate response element-binding (CREB) protein has the potential to be clinically modulated as a therapeutic target for the treatment of neurodegenerative diseases. Part one consisted of a systematic review that was conducted on select articles gathered through a stepwise method to explore (1) the relationship between diseased, neurodegenerative brains and levels of active, phosphorylated CREB (pCREB), (2) increased activation of CREB as a treatment for neurodegenerative symptoms, and (3) a potential therapeutic drug for neurodegenerative diseases that can target CREB signaling. The results of the systematic review showed evidence that suggested excitotoxic concentrations of N-methyl-D-aspartate (NMDA) results in decreased pCREB levels, while decreased pCREB levels were associated with impaired cognition and behavior, increased cell death, as well as decreased CRE-gene transcription and long-term potentiation (LTP). Part two consisted of a systematic review and meta-analysis on clinical trials that used the phosphodiesterase type IV inhibitor, roflumilast, on healthy and schizophrenic patients. It was found that 100 µM roflumilast was able to improve verbal learning in healthy and schizophrenic subjects (ES = 64). Initial evidence indicates that future research on neurodegenerative diseases should further investigate CREB’s potential to be clinically modulated and research investigating PDE4 inhibitor drug therapy for the treatment of neurodegeneration should be expanded upon further in subsequent studies.

Approximately 1 in 5 Americans smoke despite the widely known negative health consequences of the habit. One factor that contributes to the high rates of nicotine addiction and its continued use is the ability of the drug to alter long-term memory. Learning in the presence of nicotine results in changes to the cellular and molecular processes that support the formation and storage of long-term memories. The consolidation of long-term memory requires a number of mechanisms, such as gene transcription. Previous work has found that learning a contextual fear conditioning task in the presence of nicotine results in the upregulation of the c-jun N-terminal kinase (JNK1) gene in the hippocampus and that JNK protein activation is necessary for the nicotine induced enhancement of contextual conditioning. The present study examines the transcription factors involved in the transcriptional regulation of jnk1 in the hippocampi of mice following learning in the presence of nicotine. The hypothesis that cAMP response element binding protein (CREB) regulates jnk1 transcription was examined. Further, a protein/DNA transcription factor array was used as an unbiased examination of changes in transcription factor activity following learning in the presence of nicotine. Using chromatin immunoprecipitation (ChIP), transcription factors identified from the array and CREB were examined for changes in their binding to the jnk1 promoter following fear conditioning in the presence of nicotine. An increase in the binding of phosphorylated CREB was found in the jnk1 promoter of mice trained in the presence of nicotine. This implicates CREB activation in the increase of jnk1 transcription following learning in the presence of nicotine. Additionally, data from the transcription factor array suggest other factors such as PARP, TR, USF-1 and E2F-1 as potentially playing a role in the cognitive effects of nicotine. These findings are discussed with respect to how they inform our understanding of the signaling cascades and genetics involved in the memory enhancing effects of this addictive drug. / Psychology

Obesity-induced chronic inflammation plays a key role in the pathogenesis of insulin resistance and the metabolic syndrome. Proinflammatory cytokines can cause insulin resistance in adipose tissue, skeletal muscle and liver by inhibiting insulin signaling transduction. Interleukin-1 receptor-associated kinase-1 (IRAK-1) is a serine/threonine kinase functioning in Toll-like Receptor signaling pathways, and plays an important role in inflammation and immune response. In our studies, we demonstrated that IRAK-1 is involved with the negative regulation of PI3K-Akt dependent signaling pathway induced by insulin and TLR 2&4 agonists. Out data also indicate that IRAK-1 can interact with IRS-1 protein both in vivo and in vitro. The binding sites for the IRAK1-IRS1 biochemical interaction are IRS-1's PH domain and IRAK-1's proline-rich LWPPPP motif. Our studies also indicate that IRAK-1 is involved with the negative regulation of glycogen synthesis through inhibiting PI3K-Akt signaling pathway and thus releasing GSK3β's inhibitory effect on glycogen synthase. Moreover, our studies also suggest that IRAK-1 is involved in the activation of transcription factors CREB and ATF-1 by stimulating CREB-Ser133 and ATF-1 phosphorylation. CREB transcription factor family induces genes involved in cellular metabolism, gene transcription, cell cycle regulation, cell survival, as well as growth factor and cytokine genes. That may partially explain our finding that IRAK-1 may be also involved with cell proliferation and survival pathway. / Master of Science

Ο α2 –αδρενεργικός υποδοχέας διακρίνεται σε 3 γνωστούς υποτύπους (α2Α, α2Β, α2C) και μετά αλληλεπίδραση με G-πρωτεΐνες (GPCRs), διαμεσολαβεί μέρος των δράσεων των ορμονών- νευρομεταβιβαστών, επινεφρίνη και νορεπινεφρίνη σε πολλά όργανα, συμπεριλαμβανομένου και του νευρικού συστήματος. Πρότυπο μελέτης του νευρικού συστήματος in vitro, αποτελεί η κυτταρική σειρά PC12, που περιλαμβάνει κύτταρα από φαιοχρωμοκύττωμα αρουραίου, τα οποία υπό την επίδραση του Nerve Growth Factor (NGF) διαφοροποιούνται σε συμπαθητικούς νευρώνες. Μετά από διαμόλυνση, αυτά τα κύτταρα εκφράζουν τους υποτύπους των α2-αδρενεργικών υποδοχέων και βάσει δεδομένων από προηγούμενη εμπειρία του εργαστηρίου μας, μπορούν μετά από ενεργοποίηση με επινεφρίνη να οδηγήσουν στην ενεργοποίηση ενός καταρράκτη μεταγωγής σήματος, που περιλαμβάνει τις κινάσες Akt και ERK1/2. Δεδομένου ότι τα μόρια αυτά συμβάλλουν στην ενεργοποίηση του μεταγραφικού παράγοντα CREB (cAMP response element binding protein) θελήσαμε στην παρούσα εργασία να διερευνήσουμε κατά πόσο η ενεργοποίηση των α2-αδρενεργικών υποδοχέων προκαλεί την CREB φωσφορυλίωση. Βάσει προηγούμενων αποτελεσμάτων, που αποδείκνυαν την απελευθέρωση αραχιδονικού οξέος και διαφόρων μεταβολιτών του μετά από ενεργοποίηση των α2 –αδρενεργικών υποτύπων, μελετήσαμε εάν αυτή η απελευθέρωση αραχιδονικού οξέος, μπορούσε να προκαλέσει ενεργοποίηση μέσω φωσφορυλίωσης του CREB και μέσω ποιών μεταβολικών μονοπατιών μπορεί αυτό να πραγματοποιηθεί. Επιπλέον μελετήσαμε εάν αυτή η ενεργοποίηση του CREB ήταν παρούσα και στους 3 υποτύπους και εάν παρουσίαζε υποτυποειδικότητα. Χρησιμοποιήσαμε την τεχνική Western Blotting , σε εκχυλίσματα PC12 κυττάρων, κατάλληλα επεξεργασμένων με επινεφρίνη, παρουσία διαφόρων αναστολέων των μεταβολικών μονοπατιών του αραχιδονικού οξέος. Τα αποτελέσματά μας δείχνουν ότι η επινεφρίνη επάγει τη φωσφορυλίωση του CREB και στους 3 υποτύπους των α2-αδρενεργικών υποδοχέων σε PC12 κύτταρα. Επίσης η απελευθέρωση αραχιδονικού οξέος και η επακόλουθη φωσφορυλίωση του CREB διαμεσολαβείται από την PLC (φωσφολιπάση C) και την εποξυγενάση του κυτοχρώματος P450, αφού έχουμε αναστολή από τους ειδικούς αναστολείς U73122 και κετοκοναζόλη αντίστοιχα. Τα επίπεδα φωσφορυλίωσης ήταν ίδια στους α2A- και α2C-υποτύπους και σημαντικά μεγαλύτερα από τον α2Β-υπότυπο, αποδεικνύοντας ότι παρουσιάζεται σημαντική υποτυποειδικότητα. / α2-adrenergic receptor is divided into 3 known subtypes (α2A, α2Β, α2C) and after interaction with G-proteins (GPCRs) mediates part of actions of hormones-neurotransmitters, epinephrine and nor epinephrine in many organs, including Central Nervous System. Cell line PC12, which origins from cells of rats’ pheochromocytoma , consist a study model of nervous system in vitro and under the influence of Nerve Growth Factor (NGF) is differentiated into sympathetic neurons. After transfection, these cells express the subtypes of α2-adrenergic receptors and based on data from previous experience, after activation with epinephrine, they activate a cascade of signal transduction , which includes kinases Akt and ERK1/2. Based on the fact that these molecules contribute to the activation of transcription factor CREB (cAMP response element binding protein) we study whether the activation of α2-adrenergic receptors can cause direct CREB phosphorylation. Based on previous results, which prove release of arachidonic acid and its metabolites after activation of α2-adrenergic subtypes, we study if the release of arachidonic acid could cause activation, through phosphorylation, of CREB and via which metabolic pathways this happens. Furthermore, we studied if the activation was present in all 3 subtypes and if it presented sub-specificity. We performed the Western Blotting technique in PC12 cells properly pro-incubated with epinephrine and addition of enzymic inhibitors of arachidonic acid metabolism. Our results figure that epinephrine induce CREB phosphorylation in all 3 subtypes of α2-adrenergic receptors in PC12 cells. The release of arachidonic acid and the following phosphorylation of CREB is mediated from phospholipase C (PLC) and cytochrome P450-dependent epoxygenase, as proved by inhibition with the specific inhibitors U73122 and ketokonazole, respectively. The levels of CREB phosphorylation were comparable between α2Α - and α2C- subtypes and higher than the α2Β- subtype, proving that this is an action which presents sub-specificity.

Notre étude à pour but de déterminer l’implication de la corticostérone centrale sur l’activité du réseau hippocampe-cortex préfrontal (HPC-CPF) ainsi que son rôle dans l’émergence et le maintien d’altération de la mémoire de travail (MDT) durant l’alcoolisation chronique (12% durant 6 mois), ou bien après un sevrage aigu (1semaine) ou prolongé (6 semaines). Les effets du sevrage ont également été comparés à ceux résultant d’un stress chronique léger imprédictible (SCLI) modélisant la dépression. Nos données montrent que le sevrage et le SCLI, mais non l’alcoolisation, induisent des troubles de MDT, un déficit d’activation de pCREB (CPF et HPC) ainsi qu’une augmentation excessive des taux de corticostérone spécifiquement dans le CPF après un sevrage. Sur le plan pharmacologique, l’inhibition de la synthèse de la corticostérone restaure la MDT et l’activité de pCREB dans le CPF, chez les souris sevrées et SCLI. Nos résultats montrent également que l’augmentation de pCREB ou le blocage des récepteurs aux minéralocorticoïdes, dans le CPF, mais non dans l’HPC, restaure la MDT des souris sevrées. Ces résultats démontrent que la perturbation de taux de corticostérone dans le CPF joue un rôle clé dans l’émergence des troubles cognitifs et neuronaux après un sevrage. Nous avons également montré qu’un traitement chronique au Diazépam atténue ces altérations transitoirement. Notre étude suggère que des composés agissant sur l'activité de l’axe corticotrope peuvent constituer des stratégies alternatives pour prévenir l'émergence et le maintien des troubles cognitifs induits par le sevrage. / Our study was aimed to determine the involvement of central corticosterone on the activity of hippocampalprefrontal cortex (HPC-PFC) network and its role in the emergence of working memory (WM) alterations during chronic alcohol consumption (12% for 6 months), or after a short (1 week) or a prolonged (6 weeks) withdrawal periods. The alcohol-withdrawal effects were compared to those resulting from an unpredictable mild chronic stress (UCMS), modeling depression. Our data showed that withdrawal and UCMS, but not alcohol, induced WM disorders and deficits of CREB activation in both the PFC and HPC, and an excessive corticosterone increase specifically in the PFC of withdrawn animals. Pharmacological experiments showed that the inhibition of corticosterone synthesis restored pCREB activity in the PFC of both withdrawn and UCMS mice and improved WM. Furthermore, in withdrawn mice, the increase of pCREB or the blockade of the mineralo-corticoid receptor in the PFC, but not in the HPC, restored WM performance. These results demonstrated that corticosterone dysfunction into the PFC plays a key role in the long-lasting cognitive and neural activity disorders of alcohol-withdrawn mice. We also showed that chronic administration of diazepam reduced such alterations only transitorily. Thus, overall, our study suggests that compounds acting on the GCs activity may constitute alternative strategies to prevent the emergence and maintenance of cognitive disorders induced by alcohol withdrawal.