cAMP Response Element-binding Protein Levels Research Articles

A decrease in phosphorylation of cAMP-response element-binding protein (CREBP) promotes retinal degeneration

Excitotoxicity, induced either by N-Methyl- d-aspartate (NMDA) or kainic acid (KA), promotes irreversible loss of retinal ganglion cells (RGCs). Although the intracellular signaling mechanisms underlying excitotoxic cell death are still unclear, recent studies on the retina indicate that NMDA promotes RGC death by increasing phosphorylation of cyclic AMP (cAMP) response element (CRE)-binding protein (CREBP), while studies on the central nervous system indicate that KA promotes neuronal cell death by decreasing phosphorylation of CREBP, suggesting that CREBP can elicit dual responses depending on the excitotoxic-agent. Interestingly, the role of CREBP in KA-mediated death of RGCs has not been investigated. Therefore, by using an animal model of excitotoxicity, the aim of this study was to investigate whether excitotoxicity induces RGC death by decreasing Ser 133-CREBP in the retina. Death of RGCs was induced in CD-1 mice by an intravitreal injection of 20 nmoles of kainic acid (KA). Decrease in CREBP levels was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility gel shift assays (EMSAs). Immunohistochemical analysis indicated that CREBP was constitutively expressed in the nuclei of cells both in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) of CD-1 mice. At 6 h after KA injection, nuclear localization of Ser 133-CREBP was decreased in the GCL. At 24 h after KA injection, Ser 133-CREBP was decreased further in GCL and the INL, and a decrease in Ser 133-CREBP correlated with apoptotic death of RGCs and amacrine cells. Western blot analysis indicated that KA decreased Ser 133-CREBP levels in retinal protein extracts. EMSA assays indicated that KA also reduced the binding of Ser 133-CREBP to CRE consensus oligonucleotides. In contrast, intravitreal injection of CNQX, a non-NMDA glutamate receptor antagonist, restored the KA-induced decrease in Ser 133-CREBP both in the GCL and INL, and inhibited loss of RGCs and amacrine cells. These results, for the first time, suggest that KA promotes retinal degeneration by reducing phosphorylation of Ser 133-CREBP in the retina.

Reward or its denial during the neonatal period affects adult spatial memory and hippocampal phosphorylated cAMP response element-binding protein levels of both the neonatal and adult rat

Early life experiences, particularly mother–infant interactions, have been shown to influence adult coping and learning abilities via gene-environment interactions. We have developed a paradigm, in which mother contact is used as either a positive or a negative reinforcer in a T-maze, during postnatal days 10–13. In both neonates receiving (RER) or denied (DER) the expected reward, exposure to the memory test in the absence of the mother resulted in a remarkable increase in the number of pCREB immunopositive cells, when compared to their corresponding levels 2 h after the completion of the training process, but also to the levels of naïve animals. In the CA3 area, the pattern of pCREB immunoreactivity, when evaluated 2 h after the completion of the training on postnatal day 13 seemed to distinguish between the two different neonatal experiences in the T-maze, with the DER pups showing higher levels of pCREB immunopositive cells than the RER. Exposure to the Morris Water Maze (MWM) during adulthood revealed a memory advantage of the DER animals compared to the RER and the animals not exposed to the neonatal experience. Relevantly, in the DER animals an increased number of pCREB immunopositive cells was observed in the CA3 area even 24 h after the end of MWM training. When also measured after exposure to the probe trial, the number of pCREB immunopositive cells was again higher in the DER compared to the RER animals. In conclusion, we show that a learning experience involving discrepancy during the particularly plastic neonatal period is able to induce long-term effects, which result in enhanced adult hippocampal dependent spatial memory. Furthermore, our data document a role of plasticity molecules like pCREB in mediating hippocampal dependent learning and detection of novelty not only in adulthood, but also more importantly in the neonatal period of the rat.

Oligomeric Amyloid-β Inhibits the Proteolytic Conversion of Brain-derived Neurotrophic Factor (BDNF), AMPA Receptor Trafficking, and Classical Conditioning

Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

Gene Expression and Transcription Factor Profiling Reveal Inhibition of Transcription Factor cAMP-response Element-binding Protein by γ-Herpesvirus Replication and Transcription Activator

Herpesvirus replication involves the expression of over 80 viral genes in a well ordered sequence, leading to the production of new virions. Viral genes expressed during the earliest phases of replication often regulate both viral and cellular genes. Therefore, they have the potential to bring about dramatic functional changes within the cell. Replication and transcription activator (RTA) is a potent immediate early transcription activator of the gamma-herpesvirus family. This family includes Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus, human pathogens associated with malignancy. Here we combine gene array technology with transcription factor profiling to identify the earliest DNA promoter and cellular transcription factor targets of RTA in the cellular genome. We find that expression of RTA leads to both activation and inhibition of distinct groups of cellular genes. The identity of the target genes suggests that RTA rapidly changes the cellular environment to counteract cell death pathways, support growth factor signaling, and also promote immune evasion of the infected cell. Transcription factor profiling of the target gene promoters highlighted distinct pathways involved in gene activation at specific time points. Most notable throughout was the high level of cAMP-response element-binding protein (CREB)-response elements in RTA target genes. We find that RTA can function as either an activator or an inhibitor of CREB-response genes, depending on the promoter context. The association with CREB also highlights a novel connection and coordination between viral and cellular "immediate early" responses.

Ultrastructural and transcriptional profiling of neuropathological misregulation of CREB function

We compare here the neurodegenerative processes observed in the hippocampus of bitransgenic mice with chronically altered levels of cAMP-response element-binding protein (CREB) function. The combination of genome-wide transcriptional profiling of degenerating hippocampal tissue with microscopy analyses reveals that the sustained inhibition of CREB function in A-CREB mice is associated with dark neuron degeneration, whereas its strong chronic activation in VP16-CREB mice primarily causes excitotoxic cell death and inflammation. Furthermore, the meta-analysis with gene expression profiles available in public databases identifies relevant common markers to other neurodegenerative processes and highlights the importance of the immune response in neurodegeneration. Overall, these analyses define the ultrastructural and transcriptional signatures associated with these two forms of hippocampal neurodegeneration, confirm the importance of fine-tuned regulation of CREB-dependent gene expression for CA1 neuron survival and function, and provide novel insight into the function of CREB in the etiology of neurodegenerative processes.

Connexin31.1 deficiency in the mouse impairs object memory and modulates open-field exploration, acetylcholine esterase levels in the striatum, and cAMP response element-binding protein levels in the striatum and piriform cortex

Neuronal gap junctions in the brain, providing intercellular electrotonic signal transfer, have been implicated in physiological and behavioral correlates of learning and memory. In connexin31.1 (Cx31.1) knockout (KO) mice the coding region of the Cx31.1 gene was replaced by a LacZ reporter gene. We investigated the impact of Cx31.1 deficiency on open-field exploration, the behavioral response to an odor, non-selective attention, learning and memory performance, and the levels of memory-related proteins in the hippocampus, striatum and the piriform cortex. In terms of behavior, the deletion of the Cx31.1 coding DNA in the mouse led to increased exploratory behaviors in a novel environment, and impaired one-trial object recognition at all delays tested. Despite strong Cx31.1 expression in the peripheral and central olfactory system, Cx31.1 KO mice exhibited normal behavioral responses to an odor. We found increased levels of acetylcholine esterase (AChE) and cAMP response element-binding protein (CREB) in the striatum of Cx31.1 KO mice. In the piriform cortex the Cx31.1 KO mice had an increased heterogeneity of CREB expression among neurons. In conclusion, gap-junctions featuring the Cx31.1 protein might be involved in open-field exploration as well as object memory and modulate levels of AChE and CREB in the striatum and piriform cortex.

Phospho-CREB and other phospho-proteins: Improved recovery from brain tissue

During attempts to quantify levels of phosphorylated cAMP response element binding protein (CREB-P) in guinea pig brain stem auditory nuclei by Western blotting, we compared the decay of CREB-P levels when tissues were homogenized in traditional Lysis buffer containing detergents or in 50 mM Tris–HCl buffer containing 0.32 M sucrose. The decay of CREB-P levels was retarded considerably in the Tris–Sucrose medium as compared to the Lysis buffer. Similarly, the levels of two other phospho-proteins, extracellular regulated kinases (ERK1/2-P) and stress activated protein/Jun-N-terminal kinase (SAP/JNK-P), were better preserved by the Tris–Sucrose medium. These findings imply that the detergents typically present in the Lysis buffer may disrupt organelles and increase the exposure of soluble phospho-proteins to hydrolyzing enzymes. In contrast, such exposure was probably minimized in the Tris–Sucrose medium, which is thought to preserve organelle integrity.

Detection of novelty, but not memory of spatial habituation, is associated with an increase in phosphorylated cAMP response element-binding protein levels in the hippocampus.

There is a growing body of evidence showing that the formation of associative memories is associated with an increase in phosphorylated cAMP response element-binding protein (pCREB) levels. We recently reported increased pCREB levels in the rat hippocampus after an exploration to a novel environment. In the present work, we studied whether this increment in CREB activation is associated with the formation of memory of habituation to a novel environment or with the detection of novelty. Rats were submitted to consecutive open field sessions at 3-h intervals. Measurement of the hippocampal pCREB level, carried out 1 h after each training session, showed that (1) it did not increase when rats explored a familiar environment; (2) it did not increase after a reexposure that improves the memory of habituation; (3) it increased after a brief novel exploration unable to form memory of habituation; and (4) it increased in amnesic rats for spatial habituation. Taken as a whole, our results suggest that the elevated pCREB level after a single open field exploration is not associated with the memory formation of habituation. It is indeed associated with the detection of a novel environment.

The decreased phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein in the central amygdala acts as a molecular substrate for anxiety related to ethanol withdrawal in rats.

Several lines of evidence indicate a high comorbidity between anxiety and alcohol abuse. This study investigated the molecular mechanisms in the amygdaloid neurocircuitry governing anxiety related to ethanol withdrawal and also the phenomenon of alcohol preference. Male Sprague Dawley(R) rats were treated with ethanol or control diet for 15 days, and ethanol-fed rats were withdrawn for 0 and 24 hr. Ethanol-withdrawn or control diet-fed rats were bilaterally infused into central or basolateral amygdala with artificial cerebrospinal fluid or protein kinase A (PKA) activator or inhibitor. These rats were used to measure anxiety levels by the elevated plus-maze test. Protein levels of various signaling molecules related to cyclic adenosine monophosphate (cAMP)-response element binding (CREB) protein signaling in amygdaloid structures were determined by gold immunolabeling procedure. The messenger RNA levels of neuropeptide Y were determined by in situ polymerase chain reaction procedure. Ethanol withdrawal (24 hr) after chronic exposure (15 days) produced anxiety in rats as measured by elevated plus-maze test. Ethanol withdrawal but not treatment significantly decreased the phosphorylation of CREB protein and protein levels of Ca2+/calmodulin-dependent protein kinase IV without modulating the protein levels of total CREB and alpha-catalytic subunit of protein kinase A (PKA-Calpha) in the central and medial amygdala. However, these changes were not observed in the basolateral amygdala. We also investigated the effects of manipulation of the phosphorylation status of CREB in the central amygdala by infusion of the PKA activator (Sp-cAMPS) or inhibitor (Rp-cAMPS) on anxiety levels in rats during ethanol withdrawal. When Sp-cAMPS is specifically infused into the central amygdala, it dose-dependently normalizes the decrease in CREB phosphorylation and prevents the development of anxiety in rats during ethanol withdrawal. On the other hand, Rp-cAMPS infusions into the central or basolateral amygdala decrease CREB phosphorylation, but only infusion into the central amygdala provokes anxiety and increases alcohol preference in normal rats. We also found that alcohol preference provoked by decreased CREB phosphorylation is related to decreased expression of the neuropeptide Y gene in the central amygdala. These novel results suggest the possibility that decreased CREB phosphorylation in the central amygdala acts as a common molecular correlate for anxiety and alcohol-drinking behaviors and also is correlated with anxiety related to ethanol withdrawal.

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways.

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 microM Rp-cAMP) or PKC (with 5-20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses.

Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145

A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (−508 to −501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and competable protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.