Transcription Factor cAMP-response Element Binding Research Articles

Protectin conjugates in tissue regeneration 1 alleviates sepsis-induced acute lung injury by inhibiting ferroptosis

BackgroundAcute lung injury (ALI) is a common and serious complication of sepsis with high mortality. Ferroptosis, categorized as programmed cell death, contributes to the development of lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is an endogenous lipid mediator that exerts protective effects against multiorgan injury. However, the role of PCTR1 in the ferroptosis of sepsis-related ALI remains unknown.MethodsA pulmonary epithelial cell line and a mouse model of ALI stimulated with lipopolysaccharide (LPS) were established in vitro and in vivo. Ferroptosis biomarkers, including ferrous (Fe2+), glutathione (GSH), malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE), were assessed by relevant assay kits. Glutathione peroxidase 4 (GPX4) and prostaglandin-endoperoxide synthase 2 (PTGS2) protein levels were determined by western blotting. Lipid peroxides were examined by fluorescence microscopy and flow cytometry. Cell viability was determined by a CCK-8 assay kit. The ultrastructure of mitochondria was observed with transmission electron microscopy. Morphology and inflammatory cytokine levels predicted the severity of lung injury. Afterward, related inhibitors were used to explore the potential mechanism by which PCTR1 regulates ferroptosis.ResultsPCTR1 treatment protected mice from LPS-induced lung injury, which was consistent with the effect of the ferroptosis inhibitor ferrostatin-1. PCTR1 treatment decreased Fe2+, PTGS2 and lipid reactive oxygen species (ROS) contents, increased GSH and GPX4 levels and ameliorated mitochondrial ultrastructural injury. Administration of LPS or the ferroptosis agonist RSL3 resulted in reduced cell viability, which was rescued by PCTR1. Mechanistically, inhibition of the PCTR1 receptor lipoxin A4 (ALX), protein kinase A (PKA) and transcription factor cAMP-response element binding protein (CREB) partly decreased PCTR1 upregulated GPX4 expression and a CREB inhibitor blocked the effects ofPCTR1 on ferroptosis inhibition and lung protection.ConclusionThis study suggests that PCTR1 suppresses LPS-induced ferroptosis via the ALX/PKA/CREB signaling pathway, which may offer promising therapeutic prospects in sepsis-related ALI.

Activation of protein arginine methyltransferase 1 and subsequent extension of moth lifespan is effected by the ROS/JNK/CREB signaling axis

Previous studies have demonstrated that high physiological levels of reactive oxygen species induce pupal diapause and extend lifespan in the moth Helicoverpa armigera. This has been shown to occur via protein arginine methyltransferase 1 (PRMT1) blockade of Akt-mediated phosphorylation of the transcription factor FoxO, after which activated FoxO promotes the initiation of diapause. However, it is unclear how PRMT1 is activated upstream of FoxO activity. Here, we show that high reactive oxygen species levels in the brains of H.armigera diapause-destined pupae activate the expression of c-Jun N-terminal kinase, which subsequently activates the transcription factor cAMP-response element binding protein. We show that cAMP-response element binding protein then directly binds to the PRMT1 promoter and upregulates its expression to prevent Akt-mediated FoxO phosphorylation and downstream FoxO nuclear localization. This novel finding that c-Jun N-terminal kinase promotes FoxO nuclear localization in a PRMT1-dependent manner to regulate pupal diapause reveals a complex regulatory mechanism in extending the healthspan of H.armigera.

CREB Binding at the Zfp189 Promoter Within Medium Spiny Neuron Subtypes Differentially Regulates Behavioral and Physiological Adaptations Over the Course of Cocaine Use

BackgroundOver the course of chronic drug use, brain transcriptional neuroadaptation is thought to contribute to a change in drug use behavior over time. The function of the transcription factor CREB (cAMP response element binding protein) within the nucleus accumbens (NAc) has been well documented in opposing the rewarding properties of many classes of drugs, yet the gene targets through which CREB causally manifests these lasting neuroadaptations remain unknown. Here, we identify zinc finger protein 189 (Zfp189) as a CREB target gene that is transcriptionally responsive to acute and chronic cocaine use within the NAc of mice. MethodsTo investigate the role of the CREB-Zfp189 interaction in cocaine use, we virally delivered modified clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 constructs capable of selectively localizing CREB to the Zfp189 gene promoter in the NAc of mice. ResultsWe observed that CREB binding to the Zfp189 promoter increased Zfp189 expression and diminished the reinforcing responses to cocaine. Furthermore, we showed that NAc Zfp189 expression increased within D1 medium spiny neurons in response to acute cocaine but increased in both D1- and D2-expressing medium spiny neurons in response to chronic cocaine. CREB-mediated induction of Zfp189 potentiated electrophysiological activity of D1- and D2-expressing medium spiny neurons, recapitulating the known effect of CREB on these neurons. Finally, targeting CREB to the Zfp189 promoter within NAc Drd2-expressing neurons, but not Drd1-expressing neurons, was sufficient to diminish cocaine-conditioned behaviors. ConclusionsTogether, these findings point to the CREB-Zfp189 interaction within the NAc Drd2+ neurons as a molecular signature of chronic cocaine use that is causal in counteracting the reinforcing effects of cocaine.

Effect of Insulin Receptor on Juvenile Hormone Signal and Fecundity in Spodoptera litura (F.).

Simple SummaryThe tobacco cutworm, Spodoptera litura (F.), exemplifies strong reproductive capacities and damages many agricultural crops. The insulin signaling pathway is known as a key determinant of female reproduction in insects. However, the detailed molecular mechanisms in these processes are poorly studied. Here, we injected bovine insulin into the newly emerged moth, resulting in gene expression changes in the insulin pathway, while knockdown of SlInR caused an inverse gene expression change involved in the insulin pathway. Further studies indicated that the content of JH-III, Vg, total proteins and triacylgycerol could be suppressed by SlInR dsRNA injection. Furthermore, stunted ovaries and lower fecundity were observed by RNAi. Our studies indicated that SlInR plays a key role in JH-III synthesis and the ovarian development in S. litura.Insulin signaling can regulate various physiological functions, such as energy metabolism and reproduction and so on, in many insects, including mosquito and locust. However, the molecular mechanism of this physiological process remains elusive. The tobacco cutworm, Spodoptera litura, is one of the most important pests of agricultural crops around the world. In this study, phosphoinositide 3-kinase (SlPI3K), protein kinase B (SlAKT), target of rapamycin (SlTOR), ribosomal protein S6 kinase (SlS6K) and transcription factor cAMP-response element binding protein (SlCREB) genes, except transcription factor forkhead box class O (SlFoxO), can be activated by bovine insulin injection. Then, we studied the influence of the insulin receptor gene (SlInR) on the reproduction of S. litura using RNA interference technology. qRT-PCR analysis revealed that SlInR was most abundant in the head. The SlPI3K, SlAKT, SlTOR, SlS6K and SlCREB genes were decreased, except SlFoxO, after the SlInR gene knockdown. Further studies revealed that the expression of vitellogenin mRNA and protein, Methoprene-tolerant gene (SlMet), could be down-regulated by the injection of dsRNA of SlInR significantly. Furthermore, a depletion in the insulin receptor by RNAi significantly decreased the content of juvenile hormone III (JH-III), total proteins and triacylgycerol. These changes indicated that a lack of SlInR could impair ovarian development and decrease fecundity in S. litura. Our studies contribute to a comprehensive insight into reproduction, regulated by insulin and the juvenile hormone signaling pathway through nutrition, and a provide theoretical basis for the reproduction process in pest insects.

Toll-like Receptor 4 Signaling is Critical for the Adaptive Cellular Stress Response Effects Induced by Intermittent Fasting in the Mouse Brain

Among different kinds of dietary energy restriction, intermittent fasting (IF) has been considered a dietary regimen which causes a mild stress to the organism. IF can stimulate proteins and signaling pathways related to cell stress that can culminate in the increase of the body resistance to severe stress conditions. Energy intake reduction induced by IF can induce modulation of receptors, kinases, and phosphatases, which in turn can modulate the activation of transcription factors such as NF-E2-related factor 2 (NRF2) and cAMP response element-binding (CREB) which regulate the transcription of genes related to the translation of proteins such as growth factors: brain-derived neurotrophic factor (BDNF), chaperone proteins: heat shock proteins (HSP), and so on. It has been shown that toll-like receptors (TLRs) are important molecules in innate immune response which are present not only in the periphery but also in neurons and glial cells. In central nervous system, TLRs can exert functions related to set up responses to infection, as well as influence neural progenitor cell proliferation and differentiation, being involved in cognitive parameters such as learning and memory. Little is known about the involvement of TLR4 on the beneficial effects induced by IF protocol. The present work investigated the effects of IF on memory and on the signaling mechanisms associated with NRF2 and CREB in Tlr4 knockout mice. The results suggest that TLR4 participates in the modulatory effects of IF on oxidative stress levels, on the transcription factors CREB and NRF2, and on BDNF and HSP90 expressions in hippocampus.

Integration of Nutrient Sensing in Fish Hypothalamus

The knowledge regarding hypothalamic integration of metabolic and endocrine signaling resulting in regulation of food intake is scarce in fish. Available studies pointed to a network in which the activation of the nutrient-sensing (glucose, fatty acid, and amino acid) systems would result in AMP-activated protein kinase (AMPK) inhibition and activation of protein kinase B (Akt) and mechanistic target of rapamycin (mTOR). Changes in these signaling pathways would control phosphorylation of transcription factors cAMP response-element binding protein (CREB), forkhead box01 (FoxO1), and brain homeobox transcription factor (BSX) leading to food intake inhibition through changes in the expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opio melanocortin (POMC), and cocaine and amphetamine-related transcript (CART). The present mini-review summarizes information on the topic and identifies gaps for future research.

Voluntary wheel running promotes improvements in biomarkers associated with neurogenic activity in adult male rats

In rodents, hippocampal neurogenesis and synaptogenesis phenomena are affected by exercise. However, the role of exercise parameters such as intensity, duration, and mode on molecular mechanisms involved in these processes has not been elucidated. In this study, we evaluated the effects of different intensities and modes of running on the expression of genes contributing to neuronal differentiation and synapse formation in the hippocampus of adult male rats. Adult male Wistar rats (n = 24) were randomly divided into control, low-intensity running (LIR), high-intensity running (HIR), and the voluntary wheel running (WR) conditions. Changes in the expression of microRNA-124 (miR-124), microRNA-132 (miR-132), and their respective targets, were analyzed using quantitative RT-PCR and Western blotting techniques. Our results showed that WR compared to treadmill running increased miR-124 and miR-132 expression, while reducing the expression of their respective targets, glucocorticoid receptor (GR), SRY-Box 9 (SOX9), and GTP-activated protein P250 (P250GAP). Differences in expression levels were statistically significant (ps < 0.05), except for the expression of GR in HIR (P = 0.09). Moreover, the expression level of gene coding for the transcription factor cAMP-response element binding protein (CREB) was significantly higher in the WR group compared to the treadmill running groups (P = 0.001). Western blotting techniques indicated that the level of the CREB protein was higher in WR compared to the other groups qualitatively. These findings demonstrated a more dramatic effect for voluntary running on biomarkers that are associated with stimulating neurogenesis and synapse formation in the hippocampus of male rats compared with forced treadmill running. In addition, greater positive effects were observed for lower-intensity treadmill running as compared with high-intensity running.

Mitogen- and stress-activated protein kinase-1 activation is involved in melanocortin-induced BDNF expression in Neuro2a neuronal cells.

Melanocortins are neuropeptides exerting versatile functions in the nervous system. Melanocortin 4 receptor (MC4R) is primarily expressed in the brain and is thought to be a major mediator for melanocortin. Brain-derived neurotrophic factor (BDNF) may be a crucial downstream molecule of MC4R activation, to yield neurite outgrowth, neuroregenerative, anorexigenic and other actions. In this study, we stimulated Neuro2a murine neuronal cells with an α-melanocyte stimulating hormone (α-MSH) analog, [Nle(4), D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH). In Neuro2a cells, NDP-MSH promoted neurite outgrowth. Upon NDP-MSH administration, BDNF expression was greatly enhanced. Furthermore, this effect was effectively reversed by the MC4R antagonist, JKC-363. We found that NDP-MSH treatment activated the ERK cascade and its downstream kinase MSK1 (mitogen- and stress-activated protein kinase-1). Antagonism of the MSK1 cascade by a specific inhibitor or overexpression of a defective MSK1 mutant interrupted the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), blocking BDNF upregulation. In addition, MSK1 activation triggered an epigenetic alteration in histone H3 (Ser10), facilitating the expression of the BDNF gene. Taken together, our results showed that MSK1 kinase positively activates MC4R-induced BDNF expression via modulating the phosphorylation of CREB and histone H3 in Neuro2a neuronal cells.

MiR-134-5p inhibition reduces infarct-induced cardiomyocyte apoptosis via Creb1 upregulation

BackgroundFollowing the recent discovery that microRNA-134-5p (miR-134-5p) is elevated in the early stages of acute myocardial infarction (AMI), we examined the specific role of miR-134-5p in cardiomyocytes during AMI. MethodsTo study miR-134-5p's role in the context of AMI, we used a combination of in vitro experiments in H2O2–treated or hypoxic cardiomyocyte cell cultures as well as in vivo experiments in a murine model of AMI. ResultsH2O2- and hypoxia-induced cardiomyocyte injury upregulated miR-134-5p expression. miR-134-5p overexpression increased cardiomyocyte apoptosis, whereas miR-134-5p inhibition reduced cardiomyocyte apoptosis. We discovered that the transcription factor cAMP-responsive element binding protein 1 (Creb1) is a functional target of miR-134-5p responsible for regulating cardiomyocyte apoptosis. In vivo AMI resulted in the upregulation and downregulation of miR-134-5p and Creb1 in the infarct area, respectively. Circulating miR-134-5p levels were also increased at days 1 and 2 post-AMI. Modulation of myocardial miR-124-5p expression by intramyocardial injection of antagomiR-134-5p or agomiR-134-5p significantly affected cardiomyocyte apoptosis, infarct size, and cardiac function in vivo. ConclusionsmiR-134-5p/Creb1 axis dysregulation plays a role in hypoxia- or oxidative stress-induced cardiomyocyte apoptosis as well as AMI. Circulating miR-134-5p may show promise as a biomarker for AMI or post-AMI cardiac dysfunction. Manipulating the miR-134-5p/Creb1 axis through either inhibition of miR-134-5p or overexpression of Creb1 may show promise as a novel therapeutic strategy to attenuate cardiac dysfunction following AMI.

MAPK-directed activation of the whitefly transcription factor CREB leads to P450-mediated imidacloprid resistance

The evolution of insect resistance to pesticides poses a continuing threat to agriculture and human health. While much is known about the proximate molecular and biochemical mechanisms that confer resistance, far less is known about the regulation of the specific genes/gene families involved, particularly by trans-acting factors such as signal-regulated transcription factors. Here we resolve in fine detail the trans-regulation of CYP6CM1, a cytochrome P450 that confers resistance to neonicotinoid insecticides in the whitefly Bemisia tabaci, by the mitogen-activated protein kinase (MAPK)-directed activation of the transcription factor cAMP-response element binding protein (CREB). Reporter gene assays were used to identify the putative promoter of CYP6CM1, but no consistent polymorphisms were observed in the promoter of a resistant strain of B. tabaci (imidacloprid-resistant, IMR), which overexpresses this gene, compared to a susceptible strain (imidacloprid-susceptible, IMS). Investigation of potential trans-acting factors using in vitro and in vivo assays demonstrated that the bZIP transcription factor CREB directly regulates CYP6CM1 expression by binding to a cAMP-response element (CRE)-like site in the promoter of this gene. CREB is overexpressed in the IMR strain, and inhibitor, luciferase, and RNA interference assays revealed that a signaling pathway of MAPKs mediates the activation of CREB, and thus the increased expression of CYP6CM1, by phosphorylation-mediated signal transduction. Collectively, these results provide mechanistic insights into the regulation of xenobiotic responses in insects and implicate both the MAPK-signaling pathway and a transcription factor in the development of pesticide resistance.

Recent developments in transcriptional and translational regulation underlying long-term synaptic plasticity and memory.

Formation of long-term synaptic plasticity that underlies long-term memory requires new protein synthesis. Years of research has elucidated some of the transcriptional and translational mechanisms that contribute to the production of new proteins. Early research on transcription focused on the transcription factor cAMP-responsive element binding protein. Since then, other transcription factors, such as the Nuclear Receptor 4 family of proteins that play a role in memory formation and maintenance have been identified. In addition, several studies have revealed details of epigenetic mechanisms consisting of new types of chemical alterations of DNA such as hydroxymethylation, and various histone modifications in long-term synaptic plasticity and memory. Our understanding of translational control critical for memory formation began with the identification of molecules that impinge on the 5′ and 3′ untranslated regions of mRNAs and continued with the appreciation for local translation near synaptic sites. Lately, a role for noncoding RNAs such as microRNAs in regulating translation factors and other molecules critical for memory has been found. This review describes the past research in brief and mainly focuses on the recent work on molecular mechanisms of transcriptional and translational regulation that form the underpinnings of long-term synaptic plasticity and memory.

Geniposide ameliorated fluoxetine-suppressed neurite outgrowth in Neuro2a neuroblastoma cells

AimFluoxetine (FXT), a selective serotonin reuptake inhibitor (SSRI), is one of the most common psychiatric medications clinically prescribed; while over-produced serotonin may suppress neurite development. The role of major iridoids like geniposide (GPS) and genipin (GNP) from Gardenia jasminoides Ellis fruit (family Rubiaceae) in ameliorating the anti-neurite outgrowth effect of FXT is poorly understood. In this study, the effects of these iridoids on FXT-suppressed neurite outgrowth in Neuro2a neuroblastoma cells were investigated. Main methodsNeuro2a cells were treated with FXT and GPS. The effect of GPS-FXT co-treatment on neurite outgrowth was observed using inverted phase-contrast microscope imaging system, while neurite outgrowth markers — microtubule-associated protein-2 (MAP2) and growth-associated protein 43 (GAP43) were analyzed using RT-PCR, Western blot and immunofluorescence staining. The transcription factor-cAMP response element binding (CREB), and signaling pathways — mitogen-activated protein kinase (MAPK) and protein kinase B/mammalian target of rapamycin (AKT/mTOR) were also analyzed with the help of Western blot. Key findingsThe results showed that FXT decreased the neurite outgrowth in Neuro2a cells and also downregulated gene and protein expression of MAP2 and GAP43. It also downregulated the protein expression of phosphorylated-CREB, MAPK, and AKT/mTOR signaling pathways. In contrast, GPS counteracted the effects of FXT. GPS-FXT co-treatment increased the percentage of neurite-bearing cells by 3.6-fold at 200 μM as compared to FXT treatment only. SignificanceThis study has provided the possible molecular mechanism showing how FXT exerted its detrimental side-effects on the neurite differentiation, and via the same mechanism how GPS attenuated these side effects.

Activation of prostaglandin EP4 receptor attenuates the induction of cyclooxygenase-2 expression by EP2 receptor activation in human amnion fibroblasts: implications for parturition.

Human amnion fibroblasts produce abundant prostaglandin E2 (PGE2), which plays a crucial role in parturition by stimulating not only myometrial contraction and cervical ripening but also the expression of the rate-limiting enzyme in PGE2 synthesis-namely, cyclooxygenase-2 (COX-2). This feed-forward induction of COX-2 expression by PGE2 is mediated via its receptors coupled with the cAMP and PKA pathway and subsequent phosphorylation of the transcription factors cAMP-response element binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3). Although prostaglandin E receptor (EP)-2 and EP4 for PGE2 are coupled with activation of the cAMP and PKA pathway, the exact roles of these 2 receptors in the regulation of COX-2 expression in amnion fibroblasts remain to be determined. Here, we clarify this issue by employing human amnion tissue and fibroblasts with the long-term objective of specific targeting of prostaglandin synthesis in prevention of preterm birth. We find that an EP2 agonist caused long-lasting increases in CREB phosphorylation and COX-2 expression, whereas an EP4 agonist induced only transient increases in CREB phosphorylation and COX-2 expression in amnion fibroblasts. Moreover, only EP2 stimulation increased STAT3 phosphorylation, whereas only EP4 stimulation increased PI3K activity. EP4 antagonist or inhibition of PI3K enhanced the induction of CREB and STAT3 phosphorylation and COX-2 expression by PGE2 or EP2 stimulation, which was attenuated by EP4 overexpression. Of interest, PGE2 and cortisol, both well-demonstrated stimulants of COX-2 expression in amnion fibroblasts, increased EP2 but decreased EP4 receptor expression. Furthermore, increased EP2 but decreased EP4 abundance were observed in amnion tissue at parturition. We conclude that EP2 and EP4 receptors play different roles in the regulation of COX-2 expression in human amnion fibroblasts. EP2 is the dominant PGE2 receptor mediating the induction of COX-2 at parturition, which can be attenuated by simultaneous activation of PI3K coupled to the EP4 receptor.-Lu, J.-W., Wang, W.-S., Zhou, Q., Gan, X.-W., Myatt, L., Sun, K. Activation of prostaglandin EP4 receptor attenuates the induction of cyclooxygenase-2 expression by EP2 receptor activation in human amnion fibroblasts: implications for parturition.

Adolescent alcohol exposure epigenetically regulates CREB signaling in the adult amygdala

Binge alcohol drinking in adolescence leads to increased risk for alcohol use and other psychiatric disorders in adulthood. The transcription factor cAMP-response element binding (CREB) protein is involved in the neuronal response to adult ethanol exposure, but its role in the enduring effects of adolescent alcohol exposure in adulthood is unknown. We exposed male rats to adolescent intermittent ethanol (AIE) or saline (AIS) during post-natal days 28–41 and evaluated the epigenetic regulation of CREB dynamics in the adult amygdala. A subset of these adult rats was exposed to an acute ethanol challenge. AIE decreased CREB, phosphorylated CREB, CREB-binding protein (CBP) and p300 protein levels in adult amygdaloid brain structures. AIE exposure also causes deficits in Creb1, Cbp, and p300 mRNA expression in the amygdala of AIE adult rats which are normalized after acute ethanol exposure. Interestingly, occupancy of acetylated histone H3K9/14 proteins at specific locations in the Creb1, Cbp, and p300 gene promoter regions was decreased in the amygdala of AIE adult rats and was normalized by acute ethanol exposure. These results suggest that AIE exposure epigenetically reduces CREB and other related transcriptional activators in the amygdala in adulthood that may be associated with the behavioral effects of adolescent alcohol exposure.

Histamine elicits glutamate release from cultured astrocytes

Astrocytes play key roles in regulating brain homeostasis and neuronal activity. This is, in part, accomplished by the ability of neurotransmitters in the synaptic cleft to bind astrocyte membrane receptors, activating signalling cascades that regulate concentration of intracellular Ca2+ ([Ca2+]i) and gliotransmitter release, including ATP and glutamate. Gliotransmitters contribute to dendrite formation and synaptic plasticity, and in some cases, exacerbate neurodegeneration. The neurotransmitter histamine participates in several physiological processes, such as the sleep-wake cycle and learning and memory. Previous studies have demonstrated the expression of histamine receptors on astrocytes, but until now, only a few studies have examined the effects of histamine on astrocyte intracellular signalling and gliotransmitter release. Here, we used the human astrocytoma cell line 1321N1 to study the role of histamine in astrocyte intracellular signalling and gliotransmitter release. We found that histamine activated astrocyte signalling through histamine H1 and H2 receptors, leading to distinct cellular responses. Activation of histamine H1 receptors caused concentration-dependent release of [Ca2+]i from internal stores and concentration-dependent increase in glutamate release. Histamine H2 receptor activation increased cyclic adenosine monophosphate (cAMP) levels and phosphorylation of transcription factor cAMP response-element binding protein. Taken together, these data emphasize a role for histamine in neuron-glia communication.

Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures

Background CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging.ResultsDissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity.ConclusionsDetection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

The Role of CREB, SRF, and MEF2 in Activity-Dependent Neuronal Plasticity in the Visual Cortex.

The transcription factors CREB (cAMP response element binding factor), SRF (serum response factor), and MEF2 (myocyte enhancer factor 2) play critical roles in the mechanisms underlying neuronal plasticity. However, the role of the activation of these transcription factors in the different components of plasticity in vivo is not well known. In this study, we tested the role of CREB, SRF, and MEF2 in ocular dominance plasticity (ODP), a paradigm of activity-dependent neuronal plasticity in the visual cortex. These three proteins bind to the synaptic activity response element (SARE), an enhancer sequence found upstream of many plasticity-related genes (Kawashima et al., 2009; Rodríguez-Tornos et al., 2013), and can act cooperatively to express Arc, a gene required for ODP (McCurry et al., 2010). We used viral-mediated gene transfer to block the transcription function of CREB, SRF, and MEF2 in the visual cortex, and measured visually evoked potentials in awake male and female mice before and after a 7 d monocular deprivation, which allowed us to examine both the depression component (Dc-ODP) and potentiation component (Pc-ODP) of plasticity independently. We found that CREB, SRF, and MEF2 are all required for ODP, but have differential effects on Dc-ODP and Pc-ODP. CREB is necessary for both Dc-ODP and Pc-ODP, whereas SRF and MEF2 are only needed for Dc-ODP. This finding supports previous reports implicating SRF and MEF2 in long-term depression (required for Dc-ODP), and CREB in long-term potentiation (required for Pc-ODP).SIGNIFICANCE STATEMENT Activity-dependent neuronal plasticity is the cellular basis for learning and memory, and it is crucial for the refinement of neuronal circuits during development. Identifying the mechanisms of activity-dependent neuronal plasticity is crucial to finding therapeutic interventions in the myriad of disorders where it is disrupted, such as Fragile X syndrome, Rett syndrome, epilepsy, major depressive disorder, and autism spectrum disorder. Transcription factors are essential nuclear proteins that trigger the expression of gene programs required for long-term functional and structural plasticity changes. Our results elucidate the specific role of the transcription factors CREB, SRF, and MEF2 in the depression and potentiation components of ODP in vivo, therefore better informing future attempts to find therapeutic targets for diseases where activity-dependent plasticity is disrupted.

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it’s expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

Vagus nerve stimulation reduces cocaine seeking and alters plasticity in the extinction network.

Drugs of abuse cause changes in the prefrontal cortex (PFC) and associated regions that impair inhibitory control over drug-seeking. Breaking the contingencies between drug-associated cues and the delivery of the reward during extinction learning reduces rates of relapse. Here we used vagus nerve stimulation (VNS) to induce targeted synaptic plasticity to facilitate extinction of appetitive behaviors and to reduce relapse. Rats self-administered cocaine and were given VNS during extinction. Relapse to drug-seeking was assessed in a cued reinstatement session. We used immunohistochemistry to measure changes in the expression of the phosphorylated transcription factor cAMP response-element binding protein (pCREB) in the PFC and the basolateral amygdala (BLA), which regulate cue learning and extinction. In vivo recordings of evoked field potentials measured drug- and VNS-induced changes in metaplasticity in the pathway from the PFC to the BLA. VNS-treated rats showed improved rates of extinction and reduced reinstatement. Following reinstatement, pCREB levels were reduced in the IL and BLA of VNS-treated rats. Evoked responses in the BLA were greatly reduced in VNS-treated rats, and these rats were also resistant to the induction of LTD. Taken together, these results show that VNS facilitates extinction and reduces reinstatement. Changes in the pathway between the PFC and the amygdala may contribute to these beneficial effects.

Kappa-Opioid Antagonists for Psychiatric Disorders: From Bench to Clinical Trials.

Kappa-opioid receptor (KOR) antagonists are currently being considered for the treatment of a variety of neuropsychiatric conditions, including depressive, anxiety, and substance abuse disorders. A general ability to mitigate the effects of stress, which can trigger or exacerbate these conditions, may explain their putative efficacy across such a broad array of conditions. The discovery of their potentially therapeutic effects evolved from preclinical research designed to characterize the molecular mechanisms by which experience causes neuroadaptations in the nucleus accumbens (NAc), a key element of brain reward circuitry. This research established that exposure to drugs of abuse or stress increases the activity of the transcription factor CREB (cAMP response element binding protein) in the NAc, which leads to elevated expression of the opioid peptide dynorphin that in turn causes core signs of depressive- and anxiety-related disorders. Disruption of KORs-the endogenous receptors for dynorphin-produces antidepressant- and anxiolytic-like actions in screening procedures that identify standard drugs of these classes, and reduces stress effects in tests used to study addiction and stress-related disorders. Although interest in this target is high, prototypical KOR antagonists have extraordinarily persistent pharmacodynamic effects that complicate clinical trials. The development of shorter acting KOR antagonists together with more rapid designs for clinical trials may soon provide insight on whether these drugs are efficacious as would be predicted by preclinical work. If successful, KOR antagonists would represent a unique example in psychiatry where the therapeutic mechanism of a drug class is understood before it is shown to be efficacious in humans.