scholarly journals Cyclic AMP response element-binding protein is required in excitatory neurons in the forebrain to sustain wakefulness

SLEEP ◽  
2020 ◽  
Author(s):  
Mathieu E Wimmer ◽  
Rosa Cui ◽  
Jennifer M Blackwell ◽  
Ted Abel
Keyword(s):  
Cyclic Amp ◽  
Intracellular Signaling ◽  
Target Genes ◽  
Binding Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Response Element ◽  
Creb Phosphorylation ◽  
Element Binding Protein ◽  
And Control

Abstract The molecular and intracellular signaling processes that control sleep and wake states remain largely unknown. A consistent observation is that the cyclic adenosine monophosphate (AMP) response element-binding protein (CREB), an activity-dependent transcription factor, is differentially activated during sleep and wakefulness. CREB is phosphorylated by the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway as well as other kinases, and phosphorylated CREB promotes the transcription of target genes. Genetic studies in flies and mice suggest that CREB signaling influences sleep/wake states by promoting and stabilizing wakefulness. However, it remains unclear where in the brain CREB is required to drive wakefulness. In rats, CREB phosphorylation increases in the cerebral cortex during wakefulness and decreases during sleep, but it is not known if this change is functionally relevant to the maintenance of wakefulness. Here, we used the Cre/lox system to conditionally delete CREB in the forebrain (FB) and in the locus coeruleus (LC), two regions known to be important for the production of arousal and wakefulness. We used polysomnography to measure sleep/wake levels and sleep architecture in conditional CREB mutant mice and control littermates. We found that FB-specific deletion of CREB decreased wakefulness and increased non-rapid eye movement sleep. Mice lacking CREB in the FB were unable to sustain normal periods of wakefulness. On the other hand, deletion of CREB from LC neurons did not change sleep/wake levels or sleep/wake architecture. Taken together, these results suggest that CREB is required in neurons within the FB but not in the LC to promote and stabilize wakefulness.

scholarly journals A Novel Cell-Based Assay for G-Protein-Coupled Receptor-Mediated Cyclic Adenosine Monophosphate Response Element Binding Protein Phosphorylation

2006 ◽  
Vol 11 (4) ◽  
pp. 351-358 ◽  
Author(s):  
Julie V. Selkirk ◽  
Lisa M. Nottebaum ◽  
Ian C. Ford ◽  
Mark Santos ◽  
Siobhan Malany ◽  
...  
Keyword(s):  
G Protein ◽  
Binding Protein ◽  
Expression System ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
G Protein Coupled Receptor ◽  
Response Element ◽  
Creb Phosphorylation ◽  
Element Binding Protein ◽  
G Protein Coupled

Currently, the most popular means of assessing functional activity of Gs/olf-coupled receptors is via the measurement of intracellular cyclic adenosine monophosphate (cAMP) accumulation. An additional readout is the downstream phosphorylation of cAMP response element binding protein (CREB), which gives an indication of gene transcription, the ultimate response of many G-protein-coupled receptor (GPCR) signals. Current methods of quantifying CREB phosphorylation are low throughput, and so we have designed a novel higher throughput method using the Odyssey™ infrared imaging system. Functional potencies of both agonists and antagonists correlate well with radioligand binding affinities determined using examples of both an endogenous (adenosine2A receptor in PC-12 cells) and a heterologous (human melanocortin 4 receptor in HEK-293 cells) expression system. For example, the antagonist ZM241385 demonstrates 0.23 ± 0.03 nM affinity for the A2A receptor and has a functional potency of 0.26 ± 0.04 nM determined using cAMP and 0.15 ± 0.06 nM using CREB phosphorylation. These data demonstrate that this novel approach for the measurement of CREB phosphorylation is a useful tool for the assessment of GPCR activity in whole cells and is more amenable to the throughput required for the purposes of drug discovery.

scholarly journals CREB is Required in Excitatory Neurons in the Forebrain to Sustain Wakefulness

2020 ◽  
Author(s):  
Mathieu E. Wimmer ◽  
Cui Rosa ◽  
Jennifer M. Blackwell ◽  
Ted Abel
Keyword(s):  
Cyclic Amp ◽  
Intracellular Signaling ◽  
Target Genes ◽  
Nrem Sleep ◽  
Creb Phosphorylation ◽  
Element Binding Protein ◽  
Cyclic Amp Response Element ◽  
Phosphorylated Creb ◽  
And Control ◽  
Activity Dependent

AbstractThe molecular and intracellular signaling processes that control sleep and wake states remain largely unknown. A consistent observation is that the cyclic-AMP response element binding protein (CREB), an activity-dependent transcription factor, is differentially activated during sleep and wakefulness. CREB is phosphorylated by the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway as well as other kinases, and phosphorylated CREB (pCREB) promotes transcription of target genes. Genetic studies in flies and mice suggest that CREB signaling influences sleep/wake states by promoting and stabilizing wakefulness. However, it remains unclear where in the brain CREB is required to drive wakefulness. In rats, CREB phosphorylation increases in the cerebral cortex during wakefulness and decreases during sleep, but it is not known if this change is functionally relevant to the maintenance of wakefulness. Here, we used the cre/lox system to conditionally delete CREB in the forebrain and in the locus coereleus (LC), two regions known to be important for the production of arousal and wakefulness. We used polysomnography to measure sleep/wake levels and sleep architecture in conditional CREB mutant mice and control littermates. We found that forebrain-specific deletion of CREB decreased wakefulness and increased non-rapid eye movement (NREM) sleep. Mice lacking CREB in the forebrain were unable sustain normal periods of wakefulness. On the other hand, deletion of CREB from LC neurons did not change sleep/wake levels or sleep/wake architecture. Taken together, these results suggest that CREB is required in neurons within the forebrain but not in the LC to promote and stabilize wakefulness.

Ceramide and Cyclic Adenosine Monophosphate (cAMP) Induce cAMP Response Element Binding Protein Phosphorylation via Distinct Signaling Pathways While Having Opposite Effects on Myeloid Cell Survival

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 217-225 ◽  
Author(s):  
Michael P. Scheid ◽  
Ian N. Foltz ◽  
Peter R. Young ◽  
John W. Schrader ◽  
Vincent Duronio
Keyword(s):  
P38 Mapk ◽  
Binding Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Phosphorylation ◽  
Element Binding Protein ◽  
Induced Apoptosis

Abstract The role of ceramide as a second messenger is a subject of great interest, particularly since it is implicated in signaling in response to inflammatory cytokines. Ceramide induces apoptosis in both cytokine-dependent MC/9 cells and factor-independent U937 cells. Elevation of cyclic adenosine monophosphate (cAMP) levels inhibits apoptosis induced by ceramide and several other treatments. One target of cAMP-mediated signaling is the transcription factor CREB (cAMP response element binding protein), and recently CREB phosphorylation at an activating site has been shown to also be mediated by a cascade involving p38 mitogen-activated protein kinase (MAPK), one of the stress-activated MAP kinases. Because no role for p38 MAPK in apoptosis has been firmly established, we examined the relationship between p38 MAPK and CREB phosphorylation under various conditions. Ceramide, or sphingomyelinase, like tumor necrosis factor- (TNF-) or the hematopoietic growth factor, interleukin-3 (IL-3), was shown to activate p38 MAPK, which in turn activated MAPKAP kinase-2. Each of these treatments led to phosphorylation of CREB (and the related factor ATF-1). A selective p38 MAPK inhibitor, SB203580, blocked TNF-– or ceramide-induced CREB phosphorylation, but had no effect on the induction of apoptosis mediated by these agents. The protective agents cAMP and IL-3 also led to CREB phosphorylation, but this effect was independent of p38 MAPK, even though IL-3 was shown to activate both p38 MAPK and MAPKAP kinase-2. Therefore, the opposing effects on apoptosis observed with cAMP and IL-3, compared with ceramide and TNF-, could not be explained on the basis of phosphorylation of CREB. In addition, because SB203580 had no effect of TNF- or ceramide-induced apoptosis, our results strongly argue against a role for p38 MAPK in the induction of TNF-– or ceramide-induced apoptosis.

Ceramide and Cyclic Adenosine Monophosphate (cAMP) Induce cAMP Response Element Binding Protein Phosphorylation via Distinct Signaling Pathways While Having Opposite Effects on Myeloid Cell Survival

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 217-225 ◽  
Author(s):  
Michael P. Scheid ◽  
Ian N. Foltz ◽  
Peter R. Young ◽  
John W. Schrader ◽  
Vincent Duronio
Keyword(s):  
P38 Mapk ◽  
Binding Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Phosphorylation ◽  
Element Binding Protein ◽  
Induced Apoptosis

The role of ceramide as a second messenger is a subject of great interest, particularly since it is implicated in signaling in response to inflammatory cytokines. Ceramide induces apoptosis in both cytokine-dependent MC/9 cells and factor-independent U937 cells. Elevation of cyclic adenosine monophosphate (cAMP) levels inhibits apoptosis induced by ceramide and several other treatments. One target of cAMP-mediated signaling is the transcription factor CREB (cAMP response element binding protein), and recently CREB phosphorylation at an activating site has been shown to also be mediated by a cascade involving p38 mitogen-activated protein kinase (MAPK), one of the stress-activated MAP kinases. Because no role for p38 MAPK in apoptosis has been firmly established, we examined the relationship between p38 MAPK and CREB phosphorylation under various conditions. Ceramide, or sphingomyelinase, like tumor necrosis factor- (TNF-) or the hematopoietic growth factor, interleukin-3 (IL-3), was shown to activate p38 MAPK, which in turn activated MAPKAP kinase-2. Each of these treatments led to phosphorylation of CREB (and the related factor ATF-1). A selective p38 MAPK inhibitor, SB203580, blocked TNF-– or ceramide-induced CREB phosphorylation, but had no effect on the induction of apoptosis mediated by these agents. The protective agents cAMP and IL-3 also led to CREB phosphorylation, but this effect was independent of p38 MAPK, even though IL-3 was shown to activate both p38 MAPK and MAPKAP kinase-2. Therefore, the opposing effects on apoptosis observed with cAMP and IL-3, compared with ceramide and TNF-, could not be explained on the basis of phosphorylation of CREB. In addition, because SB203580 had no effect of TNF- or ceramide-induced apoptosis, our results strongly argue against a role for p38 MAPK in the induction of TNF-– or ceramide-induced apoptosis.

Bumetanide, an Inhibitor of Cation-chloride Cotransporter Isoform 1, Inhibits γ-Aminobutyric Acidergic Excitatory Actions and Enhances Sedative Actions of Midazolam in Neonatal Rats

2013 ◽  
Vol 119 (5) ◽  
pp. 1096-1108 ◽  
Author(s):  
Yukihide Koyama ◽  
Tomio Andoh ◽  
Yoshinori Kamiya ◽  
Satoshi Morita ◽  
Tomoyuki Miyazaki ◽  
...  
Keyword(s):  
Binding Protein ◽  
Hippocampal Slices ◽  
Intraperitoneal Administration ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Aminobutyric Acid ◽  
Neonatal Rats ◽  
Response Element ◽  
Sedative Effects ◽  
Element Binding Protein

Abstract Background: It has been shown that γ-aminobutyric acid exerts excitatory actions on the immature brain due to the increased expression of Na+–K+–2Cl− cotransporter isoform 1. The authors sought to clarify whether midazolam, a γ-aminobutyric acid–mimetic hypnotic agent, causes neuronal excitation that can be blocked by bumetanide, a selective inhibitor of Na+–K+–2Cl− cotransporter isoform 1. Furthermore, the authors examined whether bumetanide potentiates the sedative effects of midazolam in neonatal rats. Methods: The authors measured the effects of midazolam with or without bumetanide on the cytosolic Ca2+ concentration ([Ca]2+i) in hippocampal slices (n = 3 in each condition) from rats at postnatal days 4, 7, and 28 (P4, P7, and P28) using fura-2 microfluorometry. Neuronal activity in the hippocampus and thalamus after intraperitoneal administration of midazolam with or without bumetanide was estimated by immunostaining of phosphorylated cyclic adenosine monophosphate–response element–binding protein (n = 12 in each condition). Furthermore, the authors assessed effects of bumetanide on the sedative effect of midazolam by measuring righting reflex latency (n = 6 in each condition). Results: Midazolam significantly increased [Ca]2+i in the CA3 area at P4 and P7 but not at P28. Bumetanide inhibited midazolam-induced increase in [Ca]2+i. Midazolam significantly up-regulated phosphorylated cyclic adenosine monophosphate–response element–binding protein expression in a bumetanide-sensitive manner in the hippocampus at P7 but not P28. Bumetanide enhanced the sedative effects of midazolam in P4 and P7 but not P28 rats. Conclusion: These results suggest that γ-aminobutyric acid A receptor–mediated excitation plays an important role in attenuated sedative effects of midazolam in immature rats.

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