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The neuroprotective effects of Er-Zhi-Wan (EZW), a well-known traditional Chinese formulation, in MPTP-induced Parkinson’s disease (PD) models are poorly understood and require evaluation. A model of PD induced by MPTP was used to evaluate the neuroprotective effects of EZW in mice. The underlying pharmacological mechanisms of EZW for the prevention and treatment of PD were then explored using a combination of multilevel databases, network pharmacology, biological experiments, and LCMS/MS. In vivo data showed that pretreatment with EZW can be neuroprotective against MPTP-induced motor dysfunction and can effectively rescue dopaminergic neurons from MPTP-induced degeneration in mice. Furthermore, data from combined multilevel databases and network pharmacology analysis strategies suggested that the neuroprotective activity of EZW in the treatment of PD is mediated by a complicated multicomponent, multitarget network. Genes such as Grm2, Grm5, Drd2, and Grik2 were identified as important therapeutic targets. Subsequent experimental validation showed that EZW can broadly regulate the mRNA levels of these receptor genes as well as BDNF, and consequently increase the phosphorylation levels of CREB to stimulate CREB signaling. These targets and signaling systems may be responsible for the reversal of neuronal death by EZW after MPTP exposure. The LC-MS/MS results also identified a wide range of chemical components of EZW, including at least 53 precise compounds, further demonstrating the complexity of the network in which EZW exerts its neuroprotective activity. Our work provides evidence for the mechanism of EZW in MPTP-PD models and supports the neuroprotective function of EZW in neurodegenerative diseases.

Despite being banned from production for decades, polychlorinated biphenyls (PCBs) continue to pose a significant risk to human health. This is due to not only the continued release of legacy PCBs from PCB-containing equipment and materials manufactured prior to the ban on PCB production, but also the inadvertent production of PCBs as byproducts of contemporary pigment and dye production. Evidence from human and animal studies clearly identifies developmental neurotoxicity as a primary endpoint of concern associated with PCB exposures. However, the relative role(s) of specific PCB congeners in mediating the adverse effects of PCBs on the developing nervous system, and the mechanism(s) by which PCBs disrupt typical neurodevelopment remain outstanding questions. New questions are also emerging regarding the potential developmental neurotoxicity of lower chlorinated PCBs that were not present in the legacy commercial PCB mixtures, but constitute a significant proportion of contemporary human PCB exposures. Here, we review behavioral and mechanistic data obtained from experimental models as well as recent epidemiological studies that suggest the non-dioxin-like (NDL) PCBs are primarily responsible for the developmental neurotoxicity associated with PCBs. We also discuss emerging data demonstrating the potential for non-legacy, lower chlorinated PCBs to cause adverse neurodevelopmental outcomes. Molecular targets, the relevance of PCB interactions with these targets to neurodevelopmental disorders, and critical data gaps are addressed as well.

Introduction: The incidence of endometrial cancer (EC) has been increasing worldwide. However, because there are limited chemotherapeutic options for the treatment of EC, the prognosis of advanced-stage EC is poor. Methods: Gene expression profile datasets for EC cases registered in The Cancer Genome Atlas (TCGA) was reanalyzed. Highly expressed genes in advancedstage EC (110 cases) compared with early-stage EC (255 cases) were extracted and Gene Ontology (GO) enrichment analysis was performed. Among the enriched genes, Kaplan-Meier (KM) plotter analysis was performed. Candidate genes expression was analyzed in HEC50B cells and Ishikawa cells by RT-qPCR. In HEC50B cells, LIM homeobox1 (LIM1) was knocked down (KD) and cell proliferation, migration, and invasion ability of the cells were evaluated. Xenografts were generated using LIM1-KD cells and tumor growth was evaluated. Ingenuity Pathway Analysis (IPA) of RNA-seq data using LIM-KD cells was performed. Expression of phospho-CREB and CREB-related proteins were evaluated in LIM1-KD cells by western blotting and in xenograft tissue by immunofluorescent staining. Two different CREB inhibitors were treated in HEC50B and cell proliferation was evaluated by MTT assay. Results: Reanalysis of TCGA followed by GO enrichment analysis revealed that homeobox genes were highly expressed in advanced-stage EC. Among the identified genes, KM plotter analysis showed that high LIM1 expression was associated with a significantly poorer prognosis in EC. Additionally, LIM1 expression was significantly higher in high-grade EC cell lines, HEC50B cells than Ishikawa cells. Knockdown of LIM1 showed reduced cell proliferation, migration and invasion in HEC50B cells. Xenograft experiments revealed that tumor growth was significantly suppressed in LIM1-KD cells. IPA of RNA-seq data using LIM-KD cells predicted that the mRNA expression of CREB signalingrelated genes was suppressed. Indeed, phosphorylation of CREB was decreased in LIM1-KD cells and LIM1-KD cells derived tumors. HEC50B cells treated by CREB inhibitors showed suppression of cell proliferation. Conclusion and discussion: Collectively, these results suggested that high LIM1 expression contributed to tumor growth via CREB signaling in EC. Inhibition of LIM1 or its downstream molecules would be new therapeutic strategies for EC. [ABSTRACT FROM AUTHOR]

Exposure to particulate matter (PM) has been linked with the severity of various diseases. To date, there is no study on the relationship between PM exposure and tendon healing. Open Achilles tenotomy of 20 rats was performed. The animals were divided into two groups according to exposure to PM: a PM group and a non-PM group. After 6 weeks of PM exposure, the harvest and investigations of lungs, blood samples, and Achilles tendons were performed. Compared to the non-PM group, the white blood cell count and tumor necrosis factor-alpha expression in the PM group were significantly higher. The Achilles tendons in PM group showed significantly increased inflammatory outcomes. A TEM analysis showed reduced collagen fibrils in the PM group. A biomechanical analysis demonstrated that the load to failure value was lower in the PM group. An upregulation of the gene encoding cyclic AMP response element-binding protein (CREB) was detected in the PM group by an integrated analysis of DNA methylation and RNA sequencing data, as confirmed via a Western blot analysis showing significantly elevated levels of phosphorylated CREB. In summary, PM exposure caused a deleterious effect on tendon healing. The molecular data indicate that the action mechanism of PM may be associated with upregulated CREB signaling. [ABSTRACT FROM AUTHOR]

Nuclear Ca2+ has emerged as one of the most potent mediators of the dialogue between neuronal synapses and the nucleus that regulates heterochromatin states, transcription factor activity, nuclearmorphology and neuronal gene expression induced by synaptic activity. Recent studies underline the importance of nuclear Ca2+ signaling in longlasting, activity-induced adaptation and maintenance of proper brain function. Diverse forms of neuroadaptation require transient nuclear Ca2+ signaling and cyclic AMP-responsive element-binding protein (CREB1, referred to here as CREB) as its prime target, which works as a tunable switch to drive andmodulate specific gene expression profiles associated with memory, pain, addiction and neuroprotection. Furthermore, a reduction of nuclear Ca2+ levels has been shown to be neurotoxic and a causal factor driving the progression of neurodegenerative disorders, as well as affecting neuronal autophagy. Because of its central role in the brain, deficits in nuclear Ca2+ signalingmay underlie a continuous loss of neuroprotection in the aging brain, contributing to the pathophysiology of Alzheimer’s disease. In this Review, we discuss the principles of the ‘nuclear calcium hypothesis’ in the context of human brain function and its role in controlling diverse forms of neuroadaptation and neuroprotection. Furthermore, we present the most relevant and promising perspectives for future studies. [ABSTRACT FROM AUTHOR]

Schizophrenia is a psychiatric disorder characterized by positive and negative symptoms and cognitive deficits. The exact cause of schizophrenia is still unknown, but substantial evidence indicates that it has a genetic component. Genome wide association studies demonstrate variants within miR-137 host gene are a risk factor for schizophrenia. However, the direct relationship between the pathophysiology of schizophrenia and the dosage of miR-137 remains unclear. Therefore, in this study, we generated transgenic mice overexpressing miR-137 (miR-137 Tg mice) with the neuron-specific Thy-1 promoter and examined schizophrenia-related phenotypes in these mice. Overexpression of miR-137 was observed in various brain regions of the miR-137 Tg mice, with down-regulation of putative miR-137 targets. MiR-137 Tg mice showed sensory gating deficits in a prepulse inhibition test, social deficits in a sociability and social novelty test, and cognitive deficits in a novel object recognition test. Interestingly, the predicted-altered pathways of the medial prefrontal cortex of miR-137 Tg mice were partially overlapped with those of the dorsolateral prefrontal cortex in postmortem brain of patients who died in equal to or less than 4 years after initial diagnosis of schizophrenia in published data. These results suggest that overexpression of miR-137 in the whole brain induces the several phenotypes that are relevant to aspects of psychiatric disorders, such as schizophrenia. Based on these findings, miR-137 Tg mice may have the potential to become a useful tool in researching the pathophysiology of psychiatric disorders. [ABSTRACT FROM AUTHOR]

Abstract Erythropoietin (EPO), a cytokine molecule, is best-known for its role in erythropoiesis. Preclinical studies have demonstrated that EPO has robust neuroprotective effects that appear to be independent of erythropoiesis. It is also being clinically tested for the treatment of neuropsychiatric illnesses due to its behavioral actions. A major limitation of EPO is that long-term administration results in excessive red blood cell production and increased blood viscosity. A chemical modification of EPO, carbamoylated erythropoietin (CEPO), reproduces the behavioral response of EPO in animal models but does not stimulate erythropoiesis. The molecular mechanisms involved in the behavioral effects of CEPO are not known. To obtain molecular insight we examined CEPO induced gene expression in neuronal cells. PC-12 cells were treated with CEPO followed by genome-wide microarray analysis. We investigated the functional significance of the gene profile by unbiased bioinformatics analysis. The Ingenuity pathway analysis (IPA) software was employed. The results revealed activation of functions such as neuronal number and long-term potentiation. Regulated signaling cascades included categories such as neurotrophin, CREB, NGF and synaptic long-term potentiation signaling. Some of the regulated genes from these pathways are CAMKII, EGR1, FOS, GRIN1, KIF1B, NOTCH1. We also comparatively examined EPO and CEPO-induced gene expression for a subset of genes in the rat dentate gyrus. The CEPO gene profile shows the induction of genes and signaling cascades that have roles in neurogenesis and memory formation, mechanisms that can produce antidepressant and cognitive function enhancing activity. Highlights • CEPO induces a neurotrophic gene profile and signaling cascades. • CEPO and EPO produce comparable gene regulation in the hippocampal dentate gyrus. • Genes regulated by CEPO have been implicated in antidepressant activity and cognitive function. [ABSTRACT FROM AUTHOR]

The neuroprotective effects of Er-Zhi-Wan (EZW), a well-known traditional Chinese formulation, in MPTP-induced Parkinson's disease (PD) models are poorly understood and require evaluation. A model of PD induced by MPTP was used to evaluate the neuroprotective effects of EZW in mice. The underlying pharmacological mechanisms of EZW for the prevention and treatment of PD were then explored using a combination of multilevel databases, network pharmacology, biological experiments, and LC MS/MS. In vivo data showed that pretreatment with EZW can be neuroprotective against MPTP-induced motor dysfunction and can effectively rescue dopaminergic neurons from MPTP-induced degeneration in mice. Furthermore, data from combined multilevel databases and network pharmacology analysis strategies suggested that the neuroprotective activity of EZW in the treatment of PD is mediated by a complicated multicomponent, multitarget network. Genes such as Grm2, Grm5, Drd2, and Grik2 were identified as important therapeutic targets. Subsequent experimental validation showed that EZW can broadly regulate the mRNA levels of these receptor genes as well as BDNF, and consequently increase the phosphorylation levels of CREB to stimulate CREB signaling. These targets and signaling systems may be responsible for the reversal of neuronal death by EZW after MPTP exposure. The LC-MS/MS results also identified a wide range of chemical components of EZW, including at least 53 precise compounds, further demonstrating the complexity of the network in which EZW exerts its neuroprotective activity. Our work provides evidence for the mechanism of EZW in MPTP-PD models and supports the neuroprotective function of EZW in neurodegenerative diseases. [Display omitted] • EZW contains a complex system of at least 53 compounds identified by LC-MS/MS. • EZW attenuated PD-like phenotypes in MPTP-induced PD mice model. • EZW alleviated the dopaminergic neuron damage in MPTP-induced PD mice model. • EZW alters mRNA expression of multiple PD-associated receptors (mGluRs/Drd2/Grik2). • EZW exerts neuroprotective effects via the BDNF/CREB pathway. [ABSTRACT FROM AUTHOR]

Abstract The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non‐coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up‐regulated in the retinas, Müller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro. MALAT1‐CREB binding maintains CREB phosphorylation by inhibiting PP2A‐mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling.

Despite being banned from production for decades, polychlorinated biphenyls (PCBs) continue to pose a significant risk to human health. This is due to not only the continued release of legacy PCBs from PCB-containing equipment and materials manufactured prior to the ban on PCB production, but also the inadvertent production of PCBs as byproducts of contemporary pigment and dye production. Evidence from human and animal studies clearly identifies developmental neurotoxicity as a primary endpoint of concern associated with PCB exposures. However, the relative role(s) of specific PCB congeners in mediating the adverse effects of PCBs on the developing nervous system, and the mechanism(s) by which PCBs disrupt typical neurodevelopment remain outstanding questions. New questions are also emerging regarding the potential developmental neurotoxicity of lower chlorinated PCBs that were not present in the legacy commercial PCB mixtures, but constitute a significant proportion of contemporary human PCB exposures. Here, we review behavioral and mechanistic data obtained from experimental models as well as recent epidemiological studies that suggest the non-dioxin-like (NDL) PCBs are primarily responsible for the developmental neurotoxicity associated with PCBs. We also discuss emerging data demonstrating the potential for non-legacy, lower chlorinated PCBs to cause adverse neurodevelopmental outcomes. Molecular targets, the relevance of PCB interactions with these targets to neurodevelopmental disorders, and critical data gaps are addressed as well.

Background: The exact cause and mechanisms underlying the pathobiology of autism spectrum disorder (ASD) remain unclear. Dysregulation of long interspersed element-1 (LINE-1) has been reported in the brains of ASD-like mutant mice and ASD brain tissues. However, the role and methylation of LINE-1 in individuals with ASD remain unclear. In this study, we aimed to investigate whether LINE-1 insertion is associated with differentially expressed genes (DEGs) and to assess LINE-1 methylation in ASD. Methods: To identify DEGs associated with LINE-1 in ASD, we reanalyzed previously published transcriptome profiles and overlapped them with the list of LINE-1-containing genes from the TranspoGene database. An Ingenuity Pathway Analysis (IPA) of DEGs associated with LINE-1 insertion was conducted. DNA methylation of LINE-1 was assessed via combined bisulfite restriction analysis (COBRA) of lymphoblastoid cell lines from ASD individuals and unaffected individuals, and the methylation levels were correlated with the expression levels of LINE-1 and two LINE-1-inserted DEGs, C1orf27 and ARMC8. Results: We found that LINE-1 insertion was significantly associated with DEGs in ASD. The IPA showed that LINE-1-inserted DEGs were associated with ASD-related mechanisms, including sex hormone receptor signaling and axon guidance signaling. Moreover, we observed that the LINE-1 methylation level was significantly reduced in lymphoblastoid cell lines from ASD individuals with severe language impairment and was inversely correlated with the transcript level. The methylation level of LINE-1 was also correlated with the expression of the LINE-1-inserted DEG C1orf27 but not ARMC8. Conclusions: In ASD individuals with severe language impairment, LINE-1 methylation was reduced and correlated with the expression levels of LINE-1 and the LINE-1-inserted DEG C1orf27. Our findings highlight the association of LINE-1 with DEGs in ASD blood samples and warrant further investigation. The molecular mechanisms of LINE-1 and the effects of its methylation in ASD pathobiology deserve further study. [ABSTRACT FROM AUTHOR]

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinformatic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac β-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species. [ABSTRACT FROM AUTHOR]

Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated N-acetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction. [ABSTRACT FROM AUTHOR]

PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30–60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4–6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here. [ABSTRACT FROM AUTHOR]

Introduction: The incidence of endometrial cancer (EC) has been increasing worldwide. However, because there are limited chemotherapeutic options for the treatment of EC, the prognosis of advanced-stage EC is poor., Methods: Gene expression profile datasets for EC cases registered in The Cancer Genome Atlas (TCGA) was reanalyzed. Highly expressed genes in advanced-stage EC (110 cases) compared with early-stage EC (255 cases) were extracted and Gene Ontology (GO) enrichment analysis was performed. Among the enriched genes, Kaplan-Meier (KM) plotter analysis was performed. Candidate genes expression was analyzed in HEC50B cells and Ishikawa cells by RT-qPCR. In HEC50B cells, LIM homeobox1 (LIM1) was knocked down (KD) and cell proliferation, migration, and invasion ability of the cells were evaluated. Xenografts were generated using LIM1-KD cells and tumor growth was evaluated. Ingenuity Pathway Analysis (IPA) of RNA-seq data using LIM-KD cells was performed. Expression of phospho-CREB and CREB-related proteins were evaluated in LIM1-KD cells by western blotting and in xenograft tissue by immunofluorescent staining. Two different CREB inhibitors were treated in HEC50B and cell proliferation was evaluated by MTT assay., Results: Reanalysis of TCGA followed by GO enrichment analysis revealed that homeobox genes were highly expressed in advanced-stage EC. Among the identified genes, KM plotter analysis showed that high LIM1 expression was associated with a significantly poorer prognosis in EC. Additionally, LIM1 expression was significantly higher in high-grade EC cell lines, HEC50B cells than Ishikawa cells. Knockdown of LIM1 showed reduced cell proliferation, migration and invasion in HEC50B cells. Xenograft experiments revealed that tumor growth was significantly suppressed in LIM1-KD cells. IPA of RNA-seq data using LIM-KD cells predicted that the mRNA expression of CREB signaling-related genes was suppressed. Indeed, phosphorylation of CREB was decreased in LIM1-KD cells and LIM1-KD cells derived tumors. HEC50B cells treated by CREB inhibitors showed suppression of cell proliferation., Conclusion and Discussion: Collectively, these results suggested that high LIM1 expression contributed to tumor growth via CREB signaling in EC. Inhibition of LIM1 or its downstream molecules would be new therapeutic strategies for EC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kato, Saeki, Imai, Onji, Yano, Yoshida, Sakaue, Fujioka, Sugiyama and Imai.)

This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride accumulation by affecting early (days 0–2) and late (days 4–8), but not middle (days 2–4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein β (C/EBPβ; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPβ expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process. [ABSTRACT FROM AUTHOR]

The purpose of this study was to characterize the inhibitory modulation of cocaine- and amphetamine-regulated transcript (CART) peptides, particularly with respect to the function of the D3 dopamine receptor (D3R), which is activated by its interaction with phosphorylated CaMKIIα (pCaMKIIα) in the nucleus accumbens (NAc). After repeated oral administration of caffeine (30 mg/kg) for five days, microinjection of CART peptide (0.08 μM/0.5 μl/hemisphere) into the NAc affected locomotor behavior. The pCaMKIIα-D3R interaction, D3R phosphorylation and cAMP/PKA/phosphorylated CREB (pCREB) signaling pathway activity were measured in NAc tissues, and Ca2+ influx and pCaMKIIα levels were measured in cultured NAc neurons. We found that CART attenuated the caffeine-mediated enhancement of depolarization-induced Ca2+ influx and CaMKIIα phosphorylation in cultured NAc neurons. Repeated microinjection of CART peptides into the NAc decreased the caffeine-induced enhancement of Ca2+ channels activity, pCaMKIIα levels, the pCaMKIIα-D3R interaction, D3R phosphorylation, cAMP levels, PKA activity and pCREB levels in the NAc. Furthermore, behavioral sensitization was observed in rats that received five-day administration of caffeine following microinjection of saline but not in rats that were treated with caffeine following microinjection of CART peptide. These results suggest that caffeine-induced CREB phosphorylation in the NAc was ameliorated by CART peptide due to its inhibition of D3R phosphorylation. These effects of CART peptides may play a compensatory role by inhibiting locomotor behavior in rats. [ABSTRACT FROM AUTHOR]

The cAMP response element binding protein (CREB) is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo. [ABSTRACT FROM AUTHOR]

The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non-coding RNAs (lnc RNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up-regulated in the retinas, Müller cells, and primary retinal ganglion cells ( RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro. MALAT1- CREB binding maintains CREB phosphorylation by inhibiting PP2A-mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling. [ABSTRACT FROM AUTHOR]

The RNA-binding protein Tristetraprolin (TTP, ZFP36) functions as a tumor suppressor that impairs the development and disables the maintenance of MYC-driven lymphoma. In addition, other human cancers expressed reduced levels of TTP, suggesting that it may function as a tumor suppressor in several malignancies. To identify genes that may be associated with TTP tumor suppressor functions in human cancer, we analyzed The Cancer Genome Atlas (TCGA) breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, and colon adenocarcinoma datasets. These analyses defined a signature of 50 genes differentially regulated between high and low TTP-expressing tumors. Notably, patients with low TTP-expressing breast cancer and lung adenocarcinoma had decreased survival rates and more aggressive tumors with increased necrosis. In addition, analysis across non-TCGA tumor gene expression databases identified a broad spectrum of human cancers having similarities with the TTP-low tumor gene signature, including pancreatic, bladder, and prostate cancer. TTP has documented roles in regulating mRNAs encoding inflammatory proteins, and pathway analysis identified several inflammatory pathways that are altered in tumors with low TTP expression. Surprisingly, the TTP-low tumor gene signature includes a core component of 20 under-expressed CREB target genes, suggesting that the regulation of CREB activity may be related to the tumor suppressor function of TTP. Thus, reduced levels of TTP are a potential biomarker for human cancers with poor outcome, and targeting the CREB pathway may be a therapeutic route for treating aggressive TTP-low tumors. [ABSTRACT FROM AUTHOR]

l-Arginine is considered a conditionally essential amino acid and has been shown to enhance wound healing. However, the molecular mechanisms through which arginine stimulates cutaneous wound repair remain unknown. Here, we evaluated the effects of arginine supplementation on fibroblast proliferation, which is a key process required for new tissue formation. We also sought to elucidate the signaling pathways involved in mediating the effects of arginine on fibroblasts by evaluation of extracellular signal-related kinase (ERK) 1/2 activation, which is important for cell growth, survival, and differentiation. Our data demonstrated that addition of 6 mM arginine significantly enhanced fibroblast proliferation, while arginine deprivation increased apoptosis, as observed by enhanced DNA fragmentation. In vitro kinase assays demonstrated that arginine supplementation activated ERK1/2, Akt, PKA and its downstream target, cAMP response element binding protein (CREB). Moreover, knockdown of GPRC6A using siRNA blocked fibroblast proliferation and decreased phosphorylation of ERK1/2, Akt and CREB. The present experiments demonstrated a critical role for the GPRC6A-ERK1/2 and PI3K/Akt signaling pathway in arginine-mediated fibroblast survival. Our findings provide novel mechanistic insights into the positive effects of arginine on wound healing. [ABSTRACT FROM AUTHOR]

The management of neuropathic pain is still a major challenge because of its unresponsiveness to most common treatments. Curcumin has been reported to play an active role in the treatment of various neurological disorders, such as neuropathic pain. Curcumin has long been recognized as a p300/CREB-binding protein (CBP) inhibitor of histone acetyltransferase (HAT) activity. However, this mechanism has never been investigated for the treatment of neuropathic pain with curcumin. The aim of the present study was to investigate the anti-nociceptive role of curcumin in the chronic constriction injury (CCI) rat model of neuropathic pain. Furthermore, with this model we investigated the effect of curcumin on P300/CBP HAT activity-regulated release of the pro-nociceptive molecules, brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (Cox-2). Treatment with 40 and 60 mg/kg body weight curcumin for 7 consecutive days significantly attenuated CCI-induced thermal hyperalgesia and mechanical allodynia, whereas 20 mg/kg curcumin showed no significant analgesic effect. Chromatin immunoprecipitation analysis revealed that curcumin dose-dependently reduced the recruitment of p300/CBP and acetyl-Histone H3/acetyl-Histone H4 to the promoter of BDNF and Cox-2 genes. A similar dose-dependent decrease of BDNF and Cox-2 in the spinal cord was also observed after curcumin treatment. These results indicated that curcumin exerted a therapeutic role in neuropathic pain by down-regulating p300/CBP HAT activity-mediated gene expression of BDNF and Cox-2. [ABSTRACT FROM AUTHOR]

Primary cilia regulate cellular signaling and are involved in both sensing and transducing extracellular stimuli. A recent study of patients with recurrent miscarriage (RM) identified mutations affecting DYNC2H1, which were involved in ciliary biogenesis. However, there has been no study concerning primary cilia in the decidua. We compared the number and the length of primary cilia in the decidua of 15 patients with unexplained RM with those of 7 pregnant controls who underwent an artificial termination of pregnancy. Immunohistochemistry was performed using antibodies against primary cilia, extravillous trophoblasts (EVTs), macrophages, uterine Natural Killer (uNK) cells, decidual stromal cells, and the activation of TGF-β and CREB signaling in the decidua of early pregnancy was studied. The density of decidual stromal cells, but not EVTs, macrophages or uNK cells, was found to be significantly higher in the decidua of patients compared to controls. The percentage of ciliated decidual stromal cells was significantly decreased in patients. There was no difference in the primary ciliary length. Regarding TGF-β signaling, p-Smad2 in these cells was diminished significantly in patients, and most of the TGF-β-activated decidual stromal cells of both patients and controls had primary cilia. No difference in the activation of CREB was found. Abnormal primary cilia on decidual stromal cells may be one of the explanatory factors for unknown RM. The inactivation of TGF-β signaling may lead to abnormal ciliogenesis in the decidua. • Primary cilia exist on decidual stromal cells in normal pregnant women. • The ciliated decidual stromal cells decreased in patients with recurrent miscarriage. • TGF-β signaling was inactivated in the stromal cells of RM decidua. • Abnormal ciliogenesis may be one of the explanatory factors of unknown RM. [ABSTRACT FROM AUTHOR]

Aims The intravenous anesthetic propofol caused episodic memory impairments in human. We hypothesized propofol caused episodic-like spatial memory retention but not acquisition impairments in rats and rescuing c AMP response element-binding protein ( CREB) signaling using selective type IV phosphodiesterase (PDEIV) inhibitor rolipram reversed these effects. Methods Male Sprague- Dawley rats were randomized into four groups: control; propofol (25 mg/kg, intraperitoneal); rolipram; and rolipram + propofol (pretreatment of rolipram 25 min before propofol, 0.3 mg/kg, intraperitoneal). Sedation and motor coordination were evaluated 5, 15, and 25 min after propofol injection. Invisible Morris water maze ( MWM) acquisition and probe test (memory retention) were performed 5 min and 24 h after propofol injection. Visible MWM training was simultaneously performed to resist nonspatial effects. Hippocampal CREB signaling was detected 5 min, 50 min, and 24 h after propofol administration. Results Rolipram did not change propofol-induced anesthetic/sedative states or impair motor skills. No difference was found on the latency to the platform during the visible MWM. Propofol impaired spatial memory retention but not acquisition. Rolipram reversed propofol-induced spatial memory impairments and suppression on c AMP levels, Ca MKIIα and CREB phosphorylation, brain-derived neurotropic factor ( BDNF) and Arc protein expression. Conclusions Propofol caused spatial memory retention impairments but not acquisition inability possibly by inhibiting CREB signaling. [ABSTRACT FROM AUTHOR]

Aims: To investigate the role and the regulation of the long variant of myeloid cell leukemia-1 protein (Mcl-1L) during liver regeneration. Background: Liver regeneration is an important phenomenon after liver injury. The rat partial hepatectomy (PH) model was used to characterize liver regeneration and Mcl-1L expression after PH. Methods: Male Wistar rats were subjected to 70% PH. The expression of mcl-1L mRNA was determined by quantitative RT-PCR, and protein levels were analyzed by Western blot analysis and immunohistochemistry during liver regeneration. Functional evaluations of Mcl-1L were tested using chemical inhibition (flavopiridol), genetic inhibition (siRNA) of Mcl-1L production, and by assaying for annexin V levels and DNA ladder formation. Serum IL-6 levels were determined by enzyme immunoassays; signal transduction of IL-6-regulated Mcl-1L expression was verified by chemical inhibitors and decoy double-stranded oligodeoxynucleotides. Results: High levels of Mcl-1L were observed in remnant tissue at 4 h after PH. Administration of flavopiridol decreased Mcl-1L accumulation and also inhibited liver regeneration. IL-6 administration promoted the accumulation of Mcl-1L in rat hepatocytes, an effect that was impaired by siRNA treatments that reduced Mcl-1L production. Chemical inhibition and decoy oligonucleotide competition demonstrated that IL-6-induced Mcl-1L production required signaling mediated by JAK kinase, phosphoinositide 3-kinase (PI3K), and cAMP response-element-binding (CREB) proteins. Conclusion: Mcl-1L is an anti-apoptotic protein induced during liver regeneration after PH in rats. The expression of Mcl-1L is induced by IL-6 through the JAK/PI3K/Akt/CREB signaling pathway. Chemotherapy drugs that depend on Mcl-1L- or IL-6-related signaling should be considered carefully before use in patients undergoing hepatectomy for malignant tumor resection. [ABSTRACT FROM AUTHOR]

Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein) expression and intracellular cyclic AMP (cAMP), which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA) substrates such as synapsin (p-syn I). Of interest, we found increased expression of phosphodiesterase 10A (PDE10A) in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease. [ABSTRACT FROM AUTHOR]

Cyclic AMP-response element-binding protein (CREB) is a transcription factor implicated in growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms, and synaptic plasticity associated with memory. To study the phenotype of CREB overexpression in vivo, we generated CREB transgenic (TG) mice in which a myeloid specific hMRP8 promoter drives CREB expression. CREB TG mice developed spontaneous skin abscesses more frequently than wild type (WT) mice. To understand the role of CREB in myeloid function and innate immunity, chemokine expression in bone marrow derived macrophages (BMDMs) from CREB TG mice were compared with BMDMs from WT mice. Our results demonstrated decreased Keratinocyte-derived cytokine (KC) in CREB TG BMDMs but not TNFα protein production in response to lipid A (LPA). In addition, mRNA expression of KC and IL-1β (Interleukin)-1β was decreased in CREB TG BMDMs; however, there was no difference in the mRNA expression of TNFα, MCP-1, IL-6 and IL-12p40. The mRNA expression of IL-1RA and IL-10 was decreased in response to LPA. Nuclear factor kappa B (NFκB) expression and a subset of its target genes were upregulated in CREB TG mouse BMDMs. Although neutrophil migration was the same in both CREB TG and WT mice, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was significantly increased in neutrophils from CREB TG mice. Taken together, CREB overexpression in myeloid cells results in increased abscess formation in vivo and aberrant cytokine and chemokine response, and neutrophil function in vitro. [ABSTRACT FROM AUTHOR]

Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and in vivo knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock. [ABSTRACT FROM AUTHOR]

Abstract: As a partner of cyclin-dependent kinase (CDK) 3, Cyclin C controls cellular proliferation and, together with CDK8, represses gene transcription. In this study, we showed that the highly expressed Cyclin C gene is a direct target of the nuclear hormone all-trans retinoic acid (RA) in HEK293 human embryonal kidney cells. The RA receptor (RAR) γ associates with a Cyclin C promoter region containing two RAR binding sites. The Cyclin C gene also directly responds to the cAMP activator Forskolin via the transcription factor CREB1 (cAMP response element-binding protein 1), for which we identified four binding sites within the first 2250 bp of its promoter. RARγ and CREB1 show functional convergence via the corepressor NCoR1, which controls in particular the Forskolin response of Cyclin C. The histone deacetylases 1, 5, 6, 7 and 11 are involved in the basal expression of Cyclin C, but in HEK293 and MCF-7 human breast carcinoma cells the antiproliferative effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) are not mediated by Cyclin C. However, cell cycle progressing effects of all-trans RA and Forskolin are dependent on Cyclin C expression levels. This suggests that the primary regulation of Cyclin C by all-trans RA and Forskolin mediates some of the cell cycle control actions of these compounds. [Copyright &y& Elsevier]

Prostaglandins are ubiquitous metabolites of arachidonic acid, and cyclooxygenase inhibitors prevent their production and secretion. Animals with loss of cyclooxygenase-2 function have reduced reparative bone formation, but the role of prostaglandins during endochondral bone formation is not defined. The role of PGE2 as a regulator of chondrocyte differentiation in chick growth plate chondrocytes (GPCs) was examined. While PGE2, PGD2, PGF2α, and PGJ2 all inhibited colX expression, approximately 80% at 10−6 M, PGE2 was the most potent activator of cAMP response element (CRE)-mediated transcription. PGE2 dose-dependently inhibited the expression of the differentiation-related genes, colX, VEGF, MMP-13, and alkaline phosphatase gene, and enzyme activity with significant effects at concentrations as low as 10−10 M. PGE2 induced cyclic AMP response element binding protein (CREB) phosphorylation and increased c-Fos protein levels by 5 min, and activated transcription at CRE-Luc, AP-1-Luc, and c-Fos promoter constructs. The protein kinase A (PKA) inhibitor, H-89, completely blocked PGE2-mediated induction of CRE-Luc and c-Fos promoter-Luc promoters, and partially inhibited induction of AP-1-Luc, while the protein kinase C (PKC) inhibitor Go-6976 partially inhibited all three promoters, demonstrating substantial cross-talk between these signaling pathways. PGE2 inhibition of colX gene expression was dependent upon both PKA and PKC signaling. These observations demonstrate potent prostaglandin regulatory effects on chondrocyte maturation and show a role for both PKA and PKC signaling in PGE2 regulatory events. [Copyright &y& Elsevier]

Nuclear Ca2+ has emerged as one of the most potent mediators of the dialogue between neuronal synapses and the nucleus that regulates heterochromatin states, transcription factor activity, nuclear morphology and neuronal gene expression induced by synaptic activity. Recent studies underline the importance of nuclear Ca2+ signaling in long-lasting, activity-induced adaptation and maintenance of proper brain function. Diverse forms of neuroadaptation require transient nuclear Ca2+ signaling and cyclic AMP-responsive element-binding protein (CREB1, referred to here as CREB) as its prime target, which works as a tunable switch to drive and modulate specific gene expression profiles associated with memory, pain, addiction and neuroprotection. Furthermore, a reduction of nuclear Ca2+ levels has been shown to be neurotoxic and a causal factor driving the progression of neurodegenerative disorders, as well as affecting neuronal autophagy. Because of its central role in the brain, deficits in nuclear Ca2+ signaling may underlie a continuous loss of neuroprotection in the aging brain, contributing to the pathophysiology of Alzheimer's disease. In this Review, we discuss the principles of the 'nuclear calcium hypothesis' in the context of human brain function and its role in controlling diverse forms of neuroadaptation and neuroprotection. Furthermore, we present the most relevant and promising perspectives for future studies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Genetic variants in phosphatase and actin regulator-1 (Phactr1) are reported to be associated with arteriosclerotic cardiovascular disease (ASCVD). However, the function of Phactr1 in atherosclerosis remains unclear. Patients with acute coronary syndrome (ACS) who underwent coronary angiography and optical coherence tomography (OCT) were enrolled and divided into non-ST segment elevation (NST-ACS) group and ST-ACS group. The expression of Phactr1 on monocytes was higher in NST-ACS and ST-ACS groups as compared with control group. Furthermore, NST-ACS patients who have more vulnerable features including thin-cap fibroatheroma (TCFA) and large lipid area showed higher levels of Phactr1 on monocytes than those with stable plaques. Through mouse models of atherosclerosis, Phactr1-/-Apoe-/- mice (double knockout mice, DKO) developed more severe atherosclerotic plaques, recruiting more macrophages into subendothelium and having elevated levels of proinflammatory cytokines in plaques. Similarly, Apoe knockout mice (Apoe-/-) receiving DKO bone marrow (BM) exhibited elevated plaque burden compared with Apoe-/- mice receiving Apoe-/- BM, indicating the protective effect of Phactr1 in hematopoietic cells. We found that depletion of Phactr1 in BM-derived macrophages (BMDMs) tended to differentiate into M1 phenotype, produced more proatherogenic cytokines and eventually converted into foam cells driven by oxidized low-density lipoprotein (ox-LDL). Mechanistically, Phactr1 activated CREB signaling via directly binding to CREB, up-regulating CREB phosphorylation and inducing KLF4 expression. Finally, overexpression of KLF4 partly rescued the excessive inflammation response and foam cell formation induced by deficiency of Phactr1. In conclusion, our study demonstrates that elevated Phactr1 in monocytes is a promising biomarker for vulnerable plaques, while increased Phactr1 attenuates atherosclerotic development via activation of CREB and M2 macrophage differentiation., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The impact of low-dose ionizing radiation (IR) on the human brain has recently attracted attention due to the increased use of IR for diagnostic purposes. The aim of this study was to investigate low-dose radiation response in the hippocampus. Female B6C3F1 mice were exposed to total body irradiation with 0 (control), 0.063, 0.125, or 0.5 Gy. Quantitative label-free proteomic analysis of the hippocampus was performed after 24 months. CREB signaling and CREB-associated pathways were affected at all doses. The lower doses (0.063 and 0.125 Gy) induced the CREB pathway, whereas the exposure to 0.5 Gy deactivated CREB. Similarly, the lowest dose (0.063 Gy) was anti-inflammatory, reducing the number of activated microglia. In contrast, induction of activated microglia and reactive astroglia was found at 0.5 Gy, suggesting increased inflammation and astrogliosis, respectively. The apoptotic markers BAX and cleaved CASP-3 and oxidative stress markers were increased only at the highest dose. Since the activated CREB pathway plays a central role in learning and memory, these data suggest neuroprotection at the lowest dose (0.063 Gy) but neurodegeneration at 0.5 Gy. The response to 0.5 Gy resembles alterations found in healthy aging and thus may represent radiation-induced accelerated aging of the brain.