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Circadian genes in the medial accumbal shell (mNAcSh) region regulate binge alcohol consumption. Here, we investigated if antisense-induced knockdown of major circadian genes (Per1, Per2, and NPAS2) in the mNAcSh of mice exposed to intermittent access two-bottle choice (IA2BC) paradigm modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP), key epigenetic modifiers associated with withdrawal-associated behaviors such as anxiety. Adult male C57BL/6J mice (N = 28), surgically implanted with bilateral guide cannulas above the mNAcSh, were chronically (4 weeks) exposed to alcohol (20% v/v) or saccharin (0.03%) via IA2BC paradigm. In the fourth week, a mixture of antisense (AS-ODNs; N = 14/group) or nonsense (NS-ODNs; N = 14/group) oligodeoxynucleotides against circadian genes were bilaterally infused into the mNAcSh. Subsequently, alcohol/saccharin consumption and preference were measured followed by euthanization of animals and verification of microinjection sites by visual inspection and the expression of HDAC-2 and CBP by using RT-PCR along with the verification of antisense-induced downregulation of circadian genes in the mNAcSh. As compared with NS-ODNs, AS-ODNs infusion significantly attenuated the alcohol-induced increase in HDAC-2 and reduction in CBP expression in the mNAcSh along with a significant reduction in alcohol consumption and preference. No significant effect was observed on either saccharin consumption or preference. Our results suggest that circadian genes in the mNAcSh may have a causal to play in mediating epigenetic changes observed after chronic alcohol consumption., (© 2021 International Society for Neurochemistry.)

The statin atorvastatin (ATV) given as a post-treatment has been reported beneficial in stroke, although the mechanisms involved are not well understood so far. Here, we investigated in vitro the effect of post-treatment with ATV and its main bioactive metabolite ortho-hydroxy ATV (o-ATV) on neuroprotection after oxygen and glucose deprivation (OGD), and the role of the pro-survival cAMP response element-binding protein (CREB). Post-OGD treatment of primary cultures of rat cortical neurons with o-ATV, but not ATV, provided neuroprotection to a specific subset of cortical neurons that were large and positive for glutamic acid decarboxylase (large-GAD (+) neurons, GABAergic). Significantly, only these GABAergic neurons showed an increase in phosphorylated CREB (pCREB) early after neuronal cultures were treated post-OGD with o-ATV. We found that o-ATV, but not ATV, increased the neuronal uptake of glutamate from the medium; this provides a rationale for the specific effect of o-ATV on pCREB in large-GABAergic neurons, which have a higher ratio of synaptic (pCREB-promoting) vs extrasynaptic (pCREB-reducing) N-methyl-D-aspartate (NMDA) receptors (NMDAR) than that of small-non-GABAergic neurons. When we pharmacologically increased pCREB levels post-OGD in non-GABAergic neurons, through the selective activation of synaptic NMDAR, we observed as well long-lasting neuronal survival. We propose that the statin metabolite o-ATV given post-OGD boosts the intrinsic pro-survival factor pCREB in large-GABAergic cortical neurons in vitro, this contributing to protect them from OGD., (© 2017 International Society for Neurochemistry.)

Brain-derived neurotrophic factor (BDNF) has been implicated in the potent modulation of synaptic plasticity at both pre-synaptic and post-synaptic sites. However, the molecular mechanism underlying BDNF-mediated pre-synaptic modulation remains incompletely understood. Here, we report that BDNF treatment for over 4 h could significantly enhance the expression of c-Jun NH2-terminal kinase-interacting protein 3 (JIP3) in cultured hippocampal neurons. This enhancement could be blocked by the Trk inhibitor K252a or by a cAMP response element-binding protein (CREB) inhibitor. In addition, chromatin immunoprecipitation (ChIP) assays revealed that CREB could bind with the JIP3 promoter region and the BDNF treatment could increase this binding. Using dual-luciferase assays we further characterized the cAMP response element (CRE) site in the JIP3 promoter. Finally, we found that BDNF-increased JIP3 expression contributes to the BDNF-induced modulation of neurotransmitter release. Together, our studies reveal that in hippocampal neurons BDNF up-regulates JIP3 expression via CREB activation, which contributes to the enhancement of neurotransmitter release; thus, we have identified a novel mechanism that BDNF modulates pre-synaptic transmission., (© 2015 International Society for Neurochemistry.)

It has recently been proposed that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. We hypothesized that inhibition of ERK1/2 activity could prevent audiogenic seizures by altering GABA and glutamate release mechanisms. Krushinsky-Molodkina rats, genetically prone to audiogenic seizure, were recruited in the experiments. Animals were i.p. injected with an inhibitor of ERK1/2 SL 327 at different doses 60 min before audio stimulation. We demonstrated for the first time that inhibition of ERK1/2 activity by SL 327 injections prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. The obtained data also demonstrated unchanged levels of GABA production, and an increase in the level of vesicular glutamate transporter 2. The study of exocytosis protein expression showed that SL 327 treatment leads to downregulation of vesicle-associated membrane protein 2 and synapsin I, and accumulation of synaptosomal-associated protein 25 (SNAP-25). The obtained data indicate that the inhibition of ERK1/2 blocks seizure behavior presumably by altering the exocytosis machinery, and identifies ERK1/2 as a potential target for the development of new strategies for seizure treatment. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. Here we report that inhibition of ERK1/2 by SL 327 prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. Accumulation of VGLUT2 was associated with differential changing of synaptic proteins VAMP2, SNAP-25 and synapsin I. The obtained data indicate that the inhibition of ERK1/2 alters neurotransmitter release by changing the exocytosis machinery, thus preventing seizures., (© 2014 International Society for Neurochemistry.)

NMDA-type glutamate receptors mediate both trophic and excitotoxic signalling in CNS neurons. We have previously shown that blocking NMDAR- post-synaptic density-95 (PSD95) interactions provides significant protection from excitotoxicity and in vivo ischaemia; however, the mechanism of neuroprotection is unclear. Here, we report that blocking PSD-95 interactions with the Tat-NR2B9c peptide enhances a Ca²⁺-dependent protective pathway converging on cAMP Response Element binding protein (CREB) activation. We provide evidence that Tat-NR2B9c neuroprotection from oxygen glucose deprivation and NMDA toxicity occurs in parallel with the activation of calmodulin kinase signalling and is dependent on a sustained phosphorylation of the CREB transcription factor and its activator CaMKIV. Tat-NR2B9c-dependent neuroprotection and CREB phosphorylation are blocked by coapplication of CaM kinase (KN93 and STO-609) or CREB (KG-501) inhibitors, and by siRNA knockdown of CaMKIV. These results are mirrored in vivo in a rat model of permanent focal ischaemia. Tat-NR2B9c application significantly reduces infarct size and causes a selective and sustained elevation in CaMKIV phosphorylation; effects which are blocked by coadministration of KN93. Thus, calcium-dependent nuclear signalling via CaMKIV and CREB is critical for neuroprotection via NMDAR-PSD95 blockade, both in vitro and in vivo. This study highlights the importance of maintaining neuronal function following ischaemic injury. Future stroke research should target neurotrophic and pro-survival signal pathways in the development of novel neuroprotective strategies., (© 2013 International Society for Neurochemistry.)

Vascular endothelial growth factor (VEGF), an angiogenic factor induced by hypoxia, also exerts direct effects on neural tissues. VEGF up-regulation after hypoxia coincides with expression of its two tyrosine kinase receptors Flt-1(VEGFR-1) and Flk-1 (KDR/VEGFR-2), which are the key mediators of physiological angiogenesis. We have recently shown that hypoxic-preconditioning (PC) leading to tolerance to hypoxia-ischemia in neonatal piglet brain resulted in increased expression of VEGF. In this study, we used a hypoxic-preconditioning model of ischemic tolerance to analyze the expression and cellular distribution of VEGF receptors and phosphorylation of cAMP-response element-binding protein (CREB) in newborn piglet brain. The response of Flt-1 and Flk-1 mRNA to PC alone was biphasic with peaks early (6 h) and late (1 week) after PC. The mRNA expression of Flt-1 and Flk-1 in piglets preconditioned 24 h prior to hypoxia-ischemia was significantly higher than non-preconditioned piglets and remained up-regulated up to 7 days. Furthermore, PC prior to hypoxia-ischemia significantly increased the protein levels of Flt-1 and Flk-1 compared with hypoxia-ischemia in a time-dependent manner. Double-immunolabeling indicated that both Flt-1 and Flk-1 are expressed in neurons and endothelial cells with a similar time course of expression following PC and that PC leads to the growth of new vessels. Finally, our data demonstrate that PC significantly phosphorylated and activated cAMP-response element-binding protein in nucleus. These results suggest that mechanism(s) initiated by PC can induce VEGF receptor up-regulation in newborn brain and that VEGF-VEGF receptor-coupled signal transduction pathways could contribute to the establishment of tolerance following hypoxia-ischemia., (© 2012 International Society for Neurochemistry.)

KCNIP3/KChIP3 (voltage-dependent K+ channel interacting protein 3), alias Calsenilin and downstream regulatory element antagonist modulator (DREAM), is a multifunctional protein that modulates A-type potassium channels, affects processing of amyloid precursor protein and regulates transcription. KCNIP3 has been described to negatively influence the activity of CREB (cAMP/Ca(2+)-response element binding protein), an essential factor in neuronal activity-dependent gene expression regulation. However, reports on intracellular localization of KCNIP3 in neurons are diverse and necessitate additional analyses of distribution of KCNIPs in cells to clarify the potential of KCNIP3 to fulfill its functions in different cell compartments. Here, we examined localization of the entire family of highly similar KCNIP proteins in neuronal cells and show that over-expressed isoforms of KCNIP1/KChIP1, KCNIP2/KChIP2, KCNIP3/KChIP3, and KCNIP4/KChIP4 had varied, yet partially overlapping subcellular localization. In addition, although some of the over-expressed KCNIP isoforms localized to the nucleus, endogenous KCNIPs were not detected in nuclei of rat primary cortical neurons. Moreover, we analyzed the role of KCNIP proteins in cAMP/Ca(2+)-response element (CRE)-dependent transcription by luciferase reporter assay and electrophoretic mobility shift assay and report that our results do not support the role for KCNIPs, including DREAM/Calsenilin/KChIP3, in modulation of CREB-mediated transcription in neurons., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

We evaluated whether changes in expression and activity of the three sodium/calcium exchanger isoforms, NCX1, NCX2, and NCX3 occurred in PC12 cells when the extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) were silenced, pharmacologically blocked, or activated with nerve growth factor (NGF). Several findings suggesting that MAPKs control NCX emerged: (1) A decrease in NCX1 and NCX3 basal expression occurred when JNK or MEK1, the extracellular-signal-regulated kinases 1/2 upstream activator, were pharmacologically blocked, respectively; (2) NGF increased cAMP response element-binding 1 (CREB1) and Specificity Protein 1 (Sp1) binding to ncx1 promoter and CREB1 binding to two different sequences close to ncx2 transcription start site on genomic DNA; (3) An up-regulation of NCX1 and NCX3, abrogated upon either MEK1 or p38 blockade, and a down-regulation of NCX2, abolished upon p38 blockade, occurred upon NGF-induced MAPK activation. The NCX1 up-regulation was abolished upon either CREB1 or Sp1 silencing, whereas NCX2 down-regulation was abrogated only by CREB1 silencing. The NCX3 up-regulation was unaffected by CREB1 or Sp1 silencing and abolished upon proteasomal inhibition; (4) Whole-cell Na(+) /Ca(2+) exchange decreased when MEK1 and JNK were blocked and increased when MAPKs were activated by NGF. Collectively, these results demonstrate a MAPK-dependent regulation of NCX expression and activity which could be relevant in mediating some of the effects of MAPKs in neurons., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβ protein. PKIβ played a very specific role in the Ascl1-mediated differentiation process as other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) brain. Our prior studies demonstrated reduced mitochondrial number in susceptible hippocampal neurons in the brain from AD patients and in M17 cells over-expressing familial AD-causing amyloid precursor protein (APP) mutant (APPswe). In the current study, we investigated whether alterations in mitochondrial biogenesis contribute to mitochondrial abnormalities in AD. Mitochondrial biogenesis is regulated by the peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α)-nuclear respiratory factor (NRF)-mitochondrial transcription factor A pathway. Expression levels of PGC-1α, NRF 1, NRF 2, and mitochondrial transcription factor A were significantly decreased in both AD hippocampal tissues and APPswe M17 cells, suggesting a reduced mitochondrial biogenesis. Indeed, APPswe M17 cells demonstrated decreased mitochondrial DNA/nuclear DNA ratio, correlated with reduced ATP content, and decreased cytochrome C oxidase activity. Importantly, over-expression of PGC-1α could completely rescue while knockdown of PGC-1α could exacerbate impaired mitochondrial biogenesis and mitochondrial deficits in APPswe M17 cells, suggesting reduced mitochondrial biogenesis is likely involved in APPswe-induced mitochondrial deficits. We further demonstrated that reduced expression of p-CREB and PGC-1α in APPswe M17 cells could be rescued by cAMP in a dose-dependent manner, which could be inhibited by PKA inhibitor H89, suggesting that the PKA/CREB pathway plays a critical role in the regulation of PGC-1α expression in APPswe M17 cells. Overall, this study demonstrated that impaired mitochondrial biogenesis likely contributes to mitochondrial dysfunction in AD., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Cholesterol is a major component of membrane lipid rafts. It is more abundant in the brain than in other tissues and plays a critical role in maintaining brain function. We report here that a significant enhancement in apoptosis in rat cerebellar granule neurons (CGNs) was observed upon incubation with 5mM K(+) /serum free (LK-S) medium. Cholesterol enrichment further potentiated CGN apoptosis incubated under LK-S medium. On the contrary, cholesterol depletion using methyl-beta-cyclodextrin protected the CGNs from apoptosis induced by LK-S treatment. Cholesterol enrichment, however, did not induce apoptosis in CGNs that have been incubated with 25mM K(+) /serum medium. Mechanistically, increased I(K) currents and DNA fragmentation were found in CGNs incubated in LK-S, which was further potentiated in the presence of cholesterol. Cholesterol-treated CGNs also exhibited increased cAMP levels and up-regulation of Kv2.1 expression. Increased levels of activated form of PKA and phospho-CREB further supported activation of the cAMP/PKA pathway upon treatment of CGNs with cholesterol-containing LK-S medium. Conversely, inhibition of PKA or small G protein Gs abolished the increase in I(K) current and the potentiation of Kv2.1 expression, leading to reduced susceptibility of CGNs to LK-S and cholesterol-induced apoptosis. Our results demonstrate that the elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1. Our data provide new evidence for the role of cholesterol in eliciting neuronal cell death., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Recent evidence suggests that the persistence of cocaine seeking during periods of protracted drug abstinence following chronic cocaine exposure is mediated, in part, by neuroadaptations in the mesolimbic dopamine system. Specifically, incubation of cocaine-seeking behavior coincides with increased brain-derived neurotrophic factor (BDNF) protein expression in the ventral tegmental area (VTA). However, the molecular mechanisms that regulate time-dependent changes in VTA BDNF protein expression during cocaine abstinence are unclear. The goal of these experiments was to determine whether VTA BDNF transcript levels are altered following cocaine abstinence and identify the molecular mechanisms regulating cocaine-induced changes in VTA BDNF transcription. Rats were allowed to self-administer cocaine (0.25 mg/infusion, i.v.) for 14 days on a fixed-ratio schedule of reinforcement followed by 7 days of forced drug abstinence. BDNF protein and exon I-containing transcripts were significantly increased in the VTA of cocaine-experienced rats following 7 days of forced drug abstinence compared to yoked saline controls. Cocaine-induced changes in BDNF mRNA were associated with increased acetylation of histone 3 and binding of CREB-binding protein to exon I-containing promoters in the VTA. Taken together, these results suggest that drug abstinence following cocaine self-administration remodels chromatin in the VTA resulting in increased expression of BDNF, which may contribute to neuroadaptations underlying cocaine craving and relapse., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Regulation of gene expression via brain-derived neurotrophic factor (BDNF) is critical to the development of the nervous system and may well underlie cognitive performance throughout life. We now describe a mechanism by which BDNF can exert its effects on postsynaptic receptor populations that may have relevance to both the normal and diseased brain where BDNF levels either rise or fall in association with changes in excitatory neurotransmission. Increased levels of NMDA receptors (NMDARs) occur in rat cortical neurons via synthesis of new NMDA receptor 1 (NR1) subunits. The majority of synthesis is controlled by binding of cAMP response element binding protein (CREB) and early growth response factor 3 (Egr3) to the core NR1 promoter (NR1-p) region. BDNF-mediated NR1 transcription depends upon induction of the mitogen-activated protein kinase (MAPK) pathway through activation of the TrK-B receptor. Taken together with the fact that NMDAR activation stimulates BDNF synthesis, our results uncover a feed-forward gene regulatory network that may enhance excitatory neurotransmission to change neuronal behavior over time., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Neocortical circuits are most sensitive to sensory experience during a critical period of early development. Previous studies implicate that brain-derived neurotrophic factor (BDNF) and GABAergic inhibition may control the timing of the critical period. By using an in vitro maturation model, we found that neurons at DIV (day in vitro) 7, around a period when functional synapses start to form and GABAergic inhibition emerges, displayed the most dynamic activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB by exogenous BDNF. The BDNF-stimulated transcriptional up-regulation of CREB target genes was also the highest in DIV 7 neurons. The basal level of ERK1/2 and CREB activity, as well as the expression of CREB target genes, increased along with maturation, and neurons at DIV 13 and 22 displayed less dynamic responses to BDNF. Furthermore, we found that the developmentally regulated GABAergic inhibition correlated with the decline of BDNF-mediated signaling during maturation. BDNF stimulation along with suppression of GABAergic inhibition enhanced the activation of ERK1/2-CREB signaling and gene transcription in mature neurons. Conversely, BDNF stimulation along with enhancement of GABAergic inhibition reduced the overall induction of intracellular signaling in younger neurons. We propose that the less dynamic molecular changes may play a certain role in the loss of plasticity during maturation., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

In vivo exposure of rodents to ethanol leads to a long-lasting increase in Fyn kinase activity in the dorsomedial striatum (DMS). In this study, we set out to identify a molecular mechanism that contributes to the enhancement of Fyn activity in response to ethanol in the DMS. Protein tyrosine phosphatase α (PTPα) positively regulates the activity of Fyn, and we found that repeated systemic administration or binge drinking of ethanol results in an increase in the synaptic localization of PTPα in the DMS, the same site where Fyn resides. We also demonstrate that binge drinking of ethanol leads to an increase in Fyn activity and to the co-localization of Fyn and PTPα in lipid rafts in the DMS. Finally, we show that the level of tyrosine phosphorylated (and thus active) PTPα in the synaptic fractions is increased in response to contingent or non-contingent exposure of rats to ethanol. Together, our results suggest that the redistribution of PTPα in the DMS into compartments where Fyn resides is a potential mechanism by which the activity of the kinase is increased upon ethanol exposure. Such neuroadaptations could be part of a mechanism that leads to the development of excessive ethanol consumption., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Following various types of nerve injury, cyclooxygenase 2 and prostaglandin E2 (PGE2) are universally and chronically up-regulated in injured nerves and contribute to the genesis of neuropathic pain. Persistent high levels of PGE2 likely exert chronic effects on nociceptive dorsal root ganglion (DRG) neurons. In the present study, we tested the hypothesis that injured nerve-derived PGE2 contributes to the up-regulation of the pro-inflammatory cytokine interleukin-6 (IL-6) in DRG neurons following partial sciatic nerve ligation. In naive adult rats, IL-6 was expressed in only a few small size DRG neurons which all co-expressed EP4 receptors. Partial sciatic nerve ligation increased and shifted IL-6 expression from small to medium and large size damaged DRG neurons. Perineural injection of a selective cyclooxygenase 2 inhibitor or a selective EP4 receptor antagonist significantly suppressed the up-regulation of IL-6 in DRG, suggesting that injured nerve derived PGE2 contributes to the de novo synthesis of IL-6 in DRG neurons through EP4 receptors. In cultured sensory ganglion explants, a stabilized PGE2 analog increased IL-6 mRNA and protein levels through the activation of EP4, protein kinase A, protein kinase C, extracellular regulated protein kinase/MAPK, cAMP response element binding protein and NFκB signalling pathways. Taken together, these data indicate that facilitating the de novo synthesis of pain-related cytokines in injured medium and large size DRG neurons is a novel mechanism underlying the role of injured nerve derived PGE2 in the genesis of neuropathic pain., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Curcumin, a major active component of Curcuma longa, possesses antioxidant and neuroprotective activities. The present study explores the mechanisms underlying the neuroprotective effect of curcumin against corticosterone and its relation to 5-hydroxy tryptamine (5-HT) receptors. Exposure of cortical neurons to corticosterone results in decreased mRNA levels for three 5-HT receptor subtypes, 5-HT(1A), 5-HT(2A) and 5-HT(4), but 5-HT(1B,) 5-HT(2B), 5-HT(2C), 5-HT(6) and 5-HT(7) receptors remain unchanged. Pre-treatment with curcumin reversed this effect on mRNA for the 5-HT(1A) and 5-HT(4) receptors, but not for the 5-HT(2A) receptor. Moreover, curcumin exerted a neuroprotective effect against corticosterone-induced neuronal death. This observed effect of curcumin was partially blocked by either 5-HT(1A) receptor antagonist p-MPPI or 5-HT(4) receptor antagonist RS 39604 alone; whereas, the simultaneous application of both antagonists completely reversed the effect. Curcumin was also found to regulate corticosterone-induced morphological changes such as increases in soma size, dendritic branching and dendritic spine density, as well as elevate synaptophysin expression in cortical neurons. p-MPPI and RS 39604 reversed the effect of curcumin-induced change in neuronal morphology and synaptophysin expression of corticosterone-treated neurons. In addition, an increase in cyclic adenosine monophosphate (cAMP) level was observed after curcumin treatment, which was further prevented by RS 39604, but not by p-MPPI. However, curcumin-induced elevation in protein kinase A activity and phosphorylation of cAMP response element-binding protein levels were inhibited by both p-MPPI and RS 39604. These findings suggest that the neuroprotection and modulation of neuroplasticity exhibited by curcumin might be mediated, at least in part, via the 5-HT receptor-cAMP-PKA-CREB signal pathway., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine (NE) and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP response element binding protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. NE-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which NE and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how NE and β1-adrenergic receptors may affect striatal physiology., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Hypoxic ischemia (HI) in neonates causes significant neurodevelopmental sequelae. Pharmacological agents designed to target specific transcription factors expressed in neurons and vasculature may provide powerful therapy against HI. Vascular endothelial growth factor-A (VEGF-A) and cAMP response element-binding protein (CREB) both underlie learning and memory, and survival of the nervous system. We examined whether CREB activation is a shared pathway underlying VEGF-A's protection in neurons and cerebral vascular endothelial cells. VEGF-A was used in a HI model of rat pups and in oxygen-glucose-deprivation (OGD) models of immortalized H19-7 neurons and b.End3 cerebral vascular endothelial cells. We found that VEGF-A activated VEGF receptor-2 (VEGFR-2), phosphorylated CREB in neurons and endothelial cells, and protected against HI, and inhibiting VEGFR-2 before VEGF-A reduced the protective effect of VEGF-A in rat pups. VEGF-A also up-regulated VEGFR-2 and phosphorylated CREB, and protected H19-7 neurons and b.End3 endothelial cells against OGD. Inhibiting VEGFR-2 and extracellular signal-regulated kinase (ERK), respectively, reduced VEGF-A-induced CREB phosphorylation and protection of H19-7 and b.End3 cells against OGD. Transfecting H19-7 and b.End3 cells with a serine-133 phosphorylation mutant CREB also inhibited VEGF-A's protection of both types of cells. We conclude that CREB phosphorylation through VEGFR-2/ERK signaling is the shared pathway that underlies VEGF-A's protection of neurons and vascular endothelial cells.

Chronic use of marijuana impairs synaptic plasticity and cognitive function. However, the molecular mechanisms by which marijuana alters long-term synaptic plasticity are largely unknown. Here, we show that repeated in vivo exposures to Delta9-THC for 7 consecutive days significantly impaired hippocampal long-term potentiation (LTP) of excitatory glutamatergic synaptic transmission. The Delta9-THC exposure-induced decrease in LTP was prevented by pharmacological inhibition or deletion of the cannabinoid 1 receptor (CB1R). To determine the molecular mechanisms underlying Delta9-THC-altered LTP, we targeted expression and function of the glutamate receptors (GluR) and phosphorylation status of cAMP-response element-binding protein (CREB). Chronic in vivo exposure to Delta9-THC produced CB1R-dependent decreases in expression of hippocampal GluR1, NR2A, and NR2B, the ratio of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/NMDA receptor-gated currents, and phosphorylation of CREB. Our results suggest that reduced expression and function of the GluR subunits and phosphorylation of CREB may underlie the impaired long-term synaptic plasticity induced by repeated in vivo exposure to Delta9-THC.

Most recent studies aimed at defining the cellular and molecular mechanisms of Pavlovian fear conditioning have focused on protein kinase signaling pathways and the transcription factor cAMP-response element binding protein (CREB) that promote fear memory consolidation in the lateral nucleus of the amygdala (LA). Despite this progress, there still remains a paucity of information regarding the genes downstream of CREB that are required for long-term fear memory formation in the LA. We have adopted a strategy of using microarray technology to initially identify genes induced within the dentate gyrus following in vivo long-term potentiation (LTP) followed by analysis of whether these same genes are also regulated by fear conditioning within the LA. In the present study, we first identified 34 plasticity-associated genes that are induced within 30 min following LTP induction utilizing a combination of DNA microarray, qRT-PCR, and in situ hybridization. To determine whether these genes are also induced in the LA following Pavlovian fear conditioning, we next exposed rats to an auditory fear conditioning protocol or to control conditions that do not support fear learning followed by qRT-PCR on mRNA from microdissected LA samples. Finally, we asked whether identified genes induced by fear learning in the LA are downstream of the extracellular-regulated kinase/mitogen-activated protein kinase signaling cascade. Collectively, our findings reveal a comprehensive list of genes that represent the first wave of transcription following both LTP induction and fear conditioning that largely belong to a class of genes referred to as 'neuronal activity dependent genes' that are likely calcium, extracellular-regulated kinase/mitogen-activated protein kinase, and CREB-dependent.

Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.

Recent evidence showed that neurotransmitters are synthesised in glial cells, such as the Schwann cells, which form myelin sheaths in the PNS. While the presence of GABA type A (GABA-A) receptors has been previously demonstrated in these cells, the evidence of GABA synthesis remained still elusive. In an attempt to demonstrate the presence of GABA in rat Schwann cells, we adopted a strategy, using several integrated neurochemical, molecular as well as immunocytochemical approaches. We first demonstrated the presence of glutamic acid decarboxylase of 67 kDa (GAD67) in Schwann cells, a crucial enzyme of the GABA synthesis mechanism. Second, we demonstrated that GABA is synthesized and localized in Schwann cells. As the third step we showed that allopregnanolone (10 nM), a potent allosteric modulator of GABA-A receptors, stimulates GABA synthesis through increased levels of GAD67 in Schwann cells. Analysis of intracellular signalling mechanisms revealed that the protein kinase A pathway, through enhanced cAMP levels and cAMP response element binding protein phosphorylation, modulates the allosteric action of allopregnanolone at the GABA-A receptor in Schwann cells. Our findings are the first to demonstrate that this GABA mechanism is active in Schwann cells thus establishing new potential therapeutic targets to control Schwann cell biology, which may prove useful in the treatment of several neurodegenerative disorders.

The main function of the rat pineal gland is to transform the circadian rhythm generated in the suprachiasmatic nucleus into a rhythmic signal of circulating melatonin characterized by a large nocturnal increase that closely reflects the duration of night period. This is achieved through the tight coupling between environmental lighting and the expression of arylalkylamine-N-acetyltransferase, the rhythm-controlling enzyme in melatonin synthesis. The initiation of Aanat transcription at night is controlled largely by the norepinephrine-stimulated phosphorylation of cAMP response element-binding protein by protein kinase A. However, to accurately reflect the duration of darkness, additional signaling mechanisms also participate to fine-tune the temporal profile of adrenergic-induced Aanat transcription. Here, we reviewed some of these signaling mechanisms, with emphasis on the more recent findings. These signaling mechanisms can be divided into two groups: those involving modification of constitutively expressed proteins and those requiring synthesis of new proteins. This review highlights the pineal gland as an excellent model system for studying neurotransmitter-regulated rhythmic gene expression.

Tyrosine hydroxylase (TH) promoter activity is induced by 17beta-estradiol (E(2)) in PC12 cells expressing estradiol receptor-alpha (ERalpha) requiring a cAMP/calcium response element (CRE/CaRE) at -45. To examine whether membrane-initiated estradiol signaling is underlying this induction, cells co-transfected with TH reporter construct and ERalpha expression vector were exposed to membrane-impermeant estradiol conjugate (beta-estradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin, E(2)BSA). TH promoter activity was elevated by E(2)BSA in dose- and time-dependent manner. E(2)BSA also elicited rapid phosphorylation of CRE binding protein (CREB) and increased CRE-driven promoter activity. Over-expression of dominant negative forms of CREB, with mutations in DNA binding or phosphorylation site, prevented TH promoter response to E(2)BSA. Pre-treatment with protein kinase A (PKA) and MEK inhibitors reduced E(2) dependent phosphorylation of CREB and ERK, and also decreased induction of TH promoter activity by E(2) or E(2)BSA. Blocking S-palmitoylation of ERalpha with C451A mutation and/or pre-treatment with 2-Bromopalmitate did not prevent but instead enhanced E(2) or E(2)BSA-elicited induction of TH promoter activity. These findings reveal, for the first time, that estradiol induction of TH gene transcription with ERalpha in PC12 cells involves membrane-initiated estradiol signaling, rapid activation of dual PKA/MEK signaling pathways, leading to CREB phosphorylation, acting at CRE/CaRE. The data demonstrate possible mechanism whereby estradiol affects catecholaminergic systems in vivo.

NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity-dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA-increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor-mediated spontaneous as well as miniature excitatory post-synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application- but not synaptic NMDA receptor-induced brain-derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA-induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA-induced BDNF expression and the binding activity of cAMP-responsive element binding protein (CREB) and its calcium-dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor-mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.

Glutamate signaling in the mature retinal tissue is very important for accurate sensory decoding by retinal neurons and orchestrates the fine-tuned output from the retina to higher-order centers at the cerebral cortex. In this study, we show that glutamate induces a rapid extracellular-regulated kinase and cAMP-responsive element binding protein (CREB) phosphorylation in cultured developing retinal neurons. This process is reliant on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors and nitric oxide (NO) signaling and independent of NMDA receptors activation, as it is blocked by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate antagonists as well as inhibiting NO synthase with NG-nitro-L-arginine methyl ester but not by the NMDA channel blocker dizocilpine maleate. The effect of NO on extracellular-regulated kinase and CREB is mediated by the classical NO/soluble guanylyl cyclase/protein kinase G pathways as it is inhibited by the soluble guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor KT5823, respectively. Immunocytochemical data suggest that increased CREB phosphorylation in response to glutamate occurs in glial cell nuclei. We also have supporting evidence suggesting that neuronally produced NO directly reaches the glial cells and stimulates CREB phosphorylation. Hence, the results indicate the importance of neuronal-glial communication and glutamate/NO/CREB linkage during retinal development.

Leptin is an adipose hormone with well characterized roles in regulating food intake and energy balance. A novel neuroprotective role for leptin has recently been discovered; however, the underlying mechanisms are not clearly defined. The purpose of this study was to determine whether leptin protects against delayed neuronal cell death in hippocampal CA1 following transient global cerebral ischemia in rats and to study the signaling mechanism responsible for the neuroprotective effects of leptin. Leptin receptor antagonist, protein kinase inhibitors and western blots were used to assess the molecular signaling events that were altered by leptin after ischemia. The results revealed that intracerebral ventricle infusion of leptin markedly increased the numbers of survival CA1 neurons in a dose-dependent manner. Infusion of a specific leptin antagonist 10 min prior to transient global ischemia abolished the pro-survival effects of leptin, indicating the essential role of leptin receptors in mediating this neuroprotection. Both the Akt and extracellular signal-related kinase 1/2 (ERK1/2) signaling pathways appear to play a critical role in leptin neuroprotection, as leptin infusion increased the phosphorylation of Akt and ERK1/2 in CA1. Furthermore, pharmacological inhibition of either pathway compromised the neuroprotective effects of leptin. Taken together, the results suggest that leptin protects against delayed ischemic neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt and ERK1/2 MAPK signaling pathways.

The netrin-1 receptor Deleted in Colorectal Cancer (DCC) is required for the formation of major axonal projections by embryonic cortical neurons, including the corpus callosum, hippocampal commissure, and cortico-thalamic tracts. The presentation of DCC by axonal growth cones is tightly regulated, but the mechanisms regulating DCC trafficking within neurons are not well understood. Here, we investigated the mechanisms regulating DCC recruitment to the plasma membrane of embryonic cortical neurons. In embryonic spinal commissural neurons, protein kinase A (PKA) activation recruits DCC to the plasma membrane and enhances axon chemoattraction to netrin-1. We demonstrate that PKA activation similarly recruits DCC and increases embryonic cortical neuron axon extension, which, like spinal commissural neurons, respond to netrin-1 as a chemoattractant. We then determined if depolarization might recruit DCC to the plasma membrane. Neither netrin-1 induced axon extension, nor levels of plasma membrane DCC, were altered by depolarizing embryonic spinal commissural neurons with elevated levels of KCl. In contrast, depolarizing embryonic cortical neurons increased the amount of plasma membrane DCC, including at the growth cone, and increased axon outgrowth evoked by netrin-1. Inhibition of PKA, phosphatidylinositol-3-kinase, protein kinase C, or exocytosis blocked the depolarization-induced recruitment of DCC and suppressed axon outgrowth. Inhibiting protein synthesis did not affect DCC recruitment, nor were the distributions of trkB or neural cell adhesion molecule (NCAM) influenced by depolarization, consistent with selective mobilization of DCC. These findings identify a role for membrane depolarization modulating the response of axons to netrin-1 by regulating DCC recruitment to the plasma membrane.

After injury to the CNS, the accumulation of extracellular glutamate induces neuronal excitotoxicity, leading to secondary tissue damage. Astrocytes can reduce excess extracellular glutamate primarily through the astrocytic glutamate transporter-1 and the Na(+)-dependent glutamate/aspartate transporter (GLAST). In this study, we used an in vitro model of cadmium-induced cellular stress and found that glutamate uptake activity of astrocytes was suppressed because of cadmium-induced inhibition of GLAST expression. The blockage of cadmium-triggered Ca(2+) influx by Ca(2+) chelators elevated GLAST transcription and glutamate uptake activity in astrocytes, suggesting that the suppression of GLAST expression in cadmium-treated astrocytes was Ca(2+)-dependent. This was supported by the findings showing the reduction of GLAST mRNA in astrocytes after treatment with Ca(2+)-ionophore A23187. Cadmium reduced human GLAST promoter activity; however, it increased the binding of Ca(2+)-sensitive activator protein-1 (AP-1) and cAMP response element binding protein (CREB) to their specific elements derived from the human GLAST promoter. These results demonstrate that AP-1 and CREB may be coupled with Ca(2+)-dependent pathway triggered by cadmium to mediate the inhibition of GLAST transcription. Our results suggest that Ca(2+) influx into astrocytes after CNS injury could cause the down-regulation of GLAST expression, thus reducing the astrocytic glutamate uptake function, which in turn may exacerbate secondary damage after CNS injury.

Stress can cause damage and atrophy of neurons in the hippocampus by deregulating the expression of neurotrophic factors that promote neuronal plasticity. The endocannabinoid system represents a physiological substrate involved in neuroprotection at both cellular and emotional levels. The lack of CB1 receptor alters neuronal plasticity and originates an anxiety-like phenotype in mice. In the present study, CB1 knockout mice exhibited an augmented response to stress revealed by the increased despair behavior and corticosterone levels showed in the tail suspension test and decreased brain derived neurotrophic factor (BDNF) levels in the hippocampus. Interestingly, local administration of BDNF in the hippocampus reversed the increased despair behavior of CB1 knockout mice, confirming the crucial role played by BDNF on the emotional impairment of these mutants. The neurotrophic deficiency seems to be specific for BDNF as no differences were found in the levels of nerve growth factor and NT-3, two additional neurotrophic factors. Moreover, BDNF impairment is not related to the activity of its specific tyrosine kinase receptor or the activity of the transcription factor cAMP responsive element binding. These results suggest that the lack of CB1 receptor originates an enhanced response to stress and deficiency in neuronal plasticity by decreasing BDNF levels in the hippocampus that lead to impairment in the responses to emotional disturbances.

Basic fibroblast growth factor (bFGF) has proven neuroprotective efficacy in the rodent retina against a diverse array of injurious stimuli. However, there is no consensus to date as to the molecular mechanisms underlying this neuroprotection. The study presented herein demonstrates increased expression of endogenous bFGF in the albino mouse retina in response to acute exposure to sublethal levels of light stress. The increased expression correlates with significant photoreceptor protection from light damage. The neuroprotection is likely to be mediated by bFGF as we demonstrate that a shorter exposure to bright light stress that does not up-regulate bFGF fails to protect photoreceptors from light damage. Furthermore, intravitreal bFGF injection into the retina of mice 3 h prior to light damage affords almost complete photoreceptor protection from light-induced degeneration. In addition, injected bFGF induces the activation of protein kinase B and extracellular signal-regulated kinase 1/2 signalling which correlate directly with the pathways we find to be activated in response to light stress and up-regulated bFGF. Moreover, we demonstrate that both bright light pre-conditioning and intravitreal bFGF injection result in dramatic increases in levels of inactive glycogen synthase kinase 3beta and cyclic AMP response element binding protein phosphorylation indicating a potential mechanism by which bFGF promotes survival of photoreceptors in vivo.

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase at vertebrate neuromuscular junctions (nmjs). Two ColQ transcripts as ColQ-1 and ColQ-1a, driven by two promoters: pColQ-1 and pColQ-1a, were found in mammalian slow- and fast-twitch muscles, respectively, which have distinct expression pattern in different muscle fibers. In this study, we show the differential expression of CoQ in different muscles is triggered by calcitonin gene-related peptide (CGRP), a known motor neuron-derived factor. Application of CGRP, or dibutyryl-cAMP (Bt(2)-cAMP), in cultured myotubes induced the expression of ColQ-1a transcript and promoter activity; however, the expression of ColQ-1 transcript did not respond to CGRP or Bt(2)-cAMP. The CGRP-induced gene activation was blocked by an adenylyl cyclase inhibitor or a dominant negative mutant of cAMP-responsive element (CRE) binding protein (CREB). Two CRE sites were mapped within the ColQ-1a promoter, and mutations of the CRE sites abolished the response of CGRP or Bt(2)-cAMP. In parallel, CGRP receptor complex was dominantly expressed at the nmjs of fast muscle but not of slow muscle. These results suggested that the expression of ColQ-1a at the nmjs of fast-twitch muscle was governed by a CGRP-mediated cAMP signaling mechanism.

Repeated morphine administration leads to molecular alterations of the neural circuitry in the locus coeruleus and nucleus accumbens. These changes include increased activity of several components of the cAMP signaling pathway that are thought to be associated with psychological and somatic signs of opiate withdrawal. The neuropeptide galanin has been shown to attenuate cAMP signaling in multiple cell types. The current study demonstrates that acute galanin treatment blocks the consequences of increased cAMP signaling following chronic opiate administration and withdrawal in Cath.a cells and primary cultures of striatal neurons as measured by phosphorylation of the transcription factor cAMP regulatory element-binding protein (CREB). In addition, galanin-mediated attenuation of CREB phosphorylation is independent of galanin-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in Cath.a cells. These data suggest that galanin receptors may serve as an additional potential therapeutic target for the treatment of opiate withdrawal.

Repeated cocaine administration to rats outside their home cages sensitizes the behavioral effects of the drug, and enhances induction of the immediate early gene product Fos in nucleus accumbens. We hypothesized that the same treatment regimen would also enhance cocaine-induced activation of intracellular signaling kinases that phosphorylate cyclic AMP-regulated element-binding protein (CREB), an important mediator of c-fos transcription. Phosphorylation levels of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), calcium/calmodulin kinases (CaMKs) II and IV, and CREB were used to assess endogenous functional activity of these signaling molecules in rats behaviorally sensitized outside their home cages. Protein kinase A (PKA)-specific phosphorylation of Ser845 in the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR1 was used to assess endogenous functional activity of PKA. Using western blots and immunohistochemistry, we detected cocaine-induced CREB phosphorylation after repeated cocaine administration, but not after repeated saline administration. Using western blots and MAPK activity assays, we found that cocaine-induced phosphorylation and activation of ERK, but not of CaMKs II or IV or GluR1, was augmented in nucleus accumbens of cocaine-sensitized rats. Unilateral infusions of the MAPK kinase inhibitor U0126 into nucleus accumbens attenuated cocaine-induced ERK and CREB phosphorylation in cocaine-sensitized rats. In contrast, unilateral infusions of the PKA inhibitor Rp-isomer of adenosine-3',5'-cyclicmonophosphorothioate (Rp-cAMPs) did not affect cocaine-induced CREB phosphorylation. Therefore, enhanced activation of ERK, but not PKA, enables and mediates cocaine-induced CREB phosphorylation in nucleus accumbens of rats that are sensitized by repeated cocaine administration outside their home cages.

Cyclic AMP response element binding protein (CREB) is a constitutive transcription factor that activates transcription following stimulus-dependent phosphorylation at Ser133, implicated in synaptic plasticity and neuronal survival pathways. The prevailing view that CREB is exclusively nuclear has been questioned by several studies, and, for example, mitochondrial localization has been reported. Using subcellular fractionation of rat brain cortex coupled with western immunoblotting with Ser133-phospho-CREB (pCREB) antibodies, we found a robust pCREB immunoreactivity (IR) in mitochondria-enriched fractions. The pCREB antibodies also stained the mitochondria, in addition to nuclei, of glial cells in primary cortical cultures. However, two CREB antibodies against different epitopes and gel shift assay detected the CREB protein mainly in the nuclear fraction. The two-dimensional electrophoretic mobility of mitochondrial pCREB IR differed markedly from the nuclear CREB/pCREB IR, indicating that the pCREB antibody cross-reacts with another mitochondrial protein. Immunoprecipitation of the mitochondrial pCREB IR produced three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as E2, E1 alpha-subunit, and E1 beta-subunit of pyruvate dehydrogenase complex. The cross-reacting epitope was identified as phospho-Ser300 of the alpha-subunit. In conclusion, this study confirms the presence of pCREB-like IR in brain mitochondria that, after careful scrutiny, turned out to be pyruvate dehydrogenase rather than authentic CREB.