pCREB Levels Research Articles

Desipramine improves depression-like behavior and working memory by up-regulating p-CREB in Alzheimer's disease associated mice.

Aggregation of amyloid [Formula: see text] protein (A[Formula: see text] and progressive loss of memory are the main characteristics of Alzheimer's disease (AD). It is noteworthy that approximately 40% of AD patients have depressive symptom. The close correlation between cognitive deficits and mental depression suggests a possibility that antidepression treatment might be beneficial to cognitive improvement in AD. The present study, by using tail-suspension test (TST), forced swimming, alternative electro-stimulus Y maze test and immunohistochemistry, examined the neuroprotective effects of desipramine, a newer generation tricyclic antidepressants (TCA), and investigated its possible molecular mechanism. The results showed that: (1) intra-hippocampal injection of A[Formula: see text] induced depression-like behavior and associative learning deficits in mice, with an increased mean immobility time in tail-suspension and forced swimming test and an increased mean error times in Y maze test; (2) after treatment with desipramine (10[Formula: see text]mg/kg, i.p.), the average immobility time significantly decreased, from [Formula: see text][Formula: see text]s in A[Formula: see text] group to [Formula: see text][Formula: see text]s in A[Formula: see text] plus desipramine group ([Formula: see text]) in TST and from [Formula: see text][Formula: see text]s to [Formula: see text][Formula: see text]s ([Formula: see text] or 9, [Formula: see text]) in forced swimming test, respectively;the mean error times of mice in Y maze test also significantly decreased, from [Formula: see text] in A[Formula: see text] group to [Formula: see text] in A[Formula: see text] plus desipramine group ([Formula: see text], [Formula: see text]); (3) desipramine administration significantly prevented against A[Formula: see text]-induced down-regulation of phosphorylated cAMP response element binding protein (p-CREB) in the hippocampus. These results indicate that A[Formula: see text] could concurrently mimic the depression-like behavior and working memory disorder in mice, while desipramine could effectively reverse both the deficits induced by A[Formula: see text]. The neuroprotection of desipramine may be involved in the up-regulation of p-CREB level in the hippocampus of mice.

Adrenomedullin mediates tumor necrosis factor-α-induced responses in dorsal root ganglia in rats

Adrenomedullin (AM), a member of the calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pain peptide. This study investigated the possible involvement of AM in tumor necrosis factor-alpha (TNF-α)-induced responses contributing to neuronal plasticity in the dorsal root ganglia (DRG). Exposure of the DRG explant cultures to TNF-α (5nM) for 48h upregulated the expression of AM mRNA. The treatment with TNF-α also increased the level of CGRP, CCL-2 and MMP-9 mRNA in the cultured DRG. This increase was attenuated by the co-treatment with the selective AM receptor antagonist AM22-52 (2μM). The blockade of AM receptors inhibited TNF-α-induced increase of the glial fibrillary acidic protein (GFAP), interleukin-1β (IL-1β), phosphorylated cAMP response element binding protein (pCREB) and nuclear factor kappa B (pNF-κB) proteins. On the other hand, the treatment with the AM receptor agonist AM1-50 (10nM) for 96h induced an increase in the level of GFAP, IL-1β, pCREB and pNF-κB proteins. The inhibition of AM activity did not change TNF-α-induced phosphorylation of extracellular signal-related kinase (pERK) while the treatment with AM1-50 still increased the level of pERK in the cultured DRG. Immunofluorescence assay showed the colocalization of AM-like immunoreactivity (IR) with TNF-α-IR in DRG neurons. The present study suggests that the increased AM receptor signaling mediated the many, but not all, TNF-α-induced activities, contributing to peripheral sensitization in neuropathic pain.

Memory retrieval of inhibitory avoidance requires histamine H1 receptor activation in the hippocampus

Retrieval represents a dynamic process that may require neuromodulatory signaling. Here, we report that the integrity of the brain histaminergic system is necessary for retrieval of inhibitory avoidance (IA) memory, because rats depleted of histamine through lateral ventricle injections of α-fluoromethylhistidine (a-FMHis), a suicide inhibitor of histidine decarboxylase, displayed impaired IA memory when tested 2 d after training. a-FMHis was administered 24 h after training, when IA memory trace was already formed. Infusion of histamine in hippocampal CA1 of brain histamine-depleted rats (hence, amnesic) 10 min before the retention test restored IA memory but was ineffective when given in the basolateral amygdala (BLA) or the ventral medial prefrontal cortex (vmPFC). Intra-CA1 injections of selective H1 and H2 receptor agonists showed that histamine exerted its effect by activating the H1 receptor. Noteworthy, the H1 receptor antagonist pyrilamine disrupted IA memory retrieval in rats, thus strongly supporting an active involvement of endogenous histamine; 90 min after the retention test, c-Fos-positive neurons were significantly fewer in the CA1s of a-FMHis-treated rats that displayed amnesia compared with in the control group. We also found reduced levels of phosphorylated cAMP-responsive element binding protein (pCREB) in the CA1s of a-FMHis-treated animals compared with in controls. Increases in pCREB levels are associated with retrieval of associated memories. Targeting the histaminergic system may modify the retrieval of emotional memory; hence, histaminergic ligands might reduce dysfunctional aversive memories and improve the efficacy of exposure psychotherapies.

Hippocampal 5-HT7 receptors signal phosphorylation of the GluA1 subunit to facilitate AMPA receptor mediated-neurotransmission in vitro and in vivo.

The 5-HT7 receptor is a GPCR that is the target of a broad range of antidepressant and antipsychotic drugs. Various studies have demonstrated an ability of the 5-HT7 receptor to modulate glutamatergic neurotransmission and cognitive processes although the potential impact upon AMPA receptors has not been investigated directly. The purposes of the present study were to investigate a direct modulation of the GluA1 AMPA receptor subunit and determine how this might influence AMPA receptor function. The influence of pharmacological manipulation of the 5-HT7 receptor system upon phosphorylation of GluA1 subunits was assessed by Western blotting of fractionated proteins from hippocampal neurones in culture (or proteins resident at the neurone surface) and the functional impact assessed by electrophysiological recordings in rat hippocampus in vitro and in vivo. 5-HT7 receptor activation increased cAMP and relative pCREB levels in cultures of rat hippocampal neurones along with an increase in phosphorylation (Ser845) of the GluA1 AMPA receptor subunit evident in whole neurone extracts and within the neurone surface compartment. Electrophysiological recordings in rat hippocampus demonstrated a 5-HT7 receptor-mediated increase in AMPA receptor-mediated neurotransmission in vitro and in vivo. The 5-HT7 receptor-mediated phosphorylation of the GluA1 AMPA receptor provides a molecular mechanism consistent with the 5-HT7 receptor-mediated increase in AMPA receptor-mediated neurotransmission.

A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis.

Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia.

Circadian Gene Circuitry Predicts Hyperactive Behavior in a Mood Disorder Mouse Model

Bipolar disorder, also known as manic-depressive illness, causes swings in mood and activity levels at irregular intervals. Such changes are difficult to predict, and their molecular basis remains unknown. Here, we use infradian (longer than a day) cyclic activity levels in αCaMKII (Camk2a) mutant mice as a proxy for such mood-associated changes. We report that gene-expression patterns in the hippocampal dentate gyrus could retrospectively predict whether the mice were in a state of high or low locomotor activity (LA). Expression of a subset of circadian genes, as well as levels of cAMP and pCREB, possible upstream regulators of circadian genes, were correlated with LA states, suggesting that the intrinsic molecular circuitry changes concomitant with infradian oscillatory LA. Taken together, these findings shed light onto the molecular basis of how irregular biological rhythms and behavior are controlled by the brain.

PMC-12, a traditional herbal medicine, enhances learning memory and hippocampal neurogenesis in mice

The beneficial effects of traditional Korean medicine are recognized during the treatment of neurodegenerative conditions, such as, Alzheimer's disease and neurocognitive dysfunction, and recently, hippocampal neurogenesis has been reported to be associated with memory function. In this study, the authors investigated the beneficial effects of polygonum multiflorum Thunberg complex composition-12 (PMC-12), which is a mixture of four medicinal herbs, that is, Polygonum multiflorum, Polygala tenuifolia, Rehmannia glutinosa, and Acorus gramineus, on hippocampal neurogenesis, learning, and memory in mice. PMC-12 was orally administered to male C57BL/6 mice (5 weeks old) at 100 or 500mg/kg daily for 2 weeks. PMC-12 administration significantly was found to increase the proliferation of neural progenitor cells and the survival of newly-generated cells in the dentate gyrus. In the Morris water maze test, the latency times of PMC-12 treated mice (100 or 500mg/kg) were shorter than those of vehicle-control mice. In addition, PMC-12 increased the levels of BDNF, p-CREB, and synaptophysin, which are known to be associated with neural plasticity and hippocampal neurogenesis. These findings suggest PMC-12 enhances hippocampal neurogenesis and neurocognitive function and imply that PMC-12 ameliorates memory impairment and cognitive deficits.

Sweet Dream Liquid Chinese Medicine Ameliorates Learning and Memory Deficit in a Rat Model of Paradoxical Sleep Deprivation through the ERK/CREB Signaling Pathway.

Sweet dream oral liquid (SDOL), a traditional Chinese herbal compound contains 17 traditional Chinese medicines. It has various pharmacological effects, such as improving brain dysfunction and increasing sleeping quality. This study investigated the neuroprotective effect and the underlying mechanisms of SDOL-impaired hippocampus learning and memory-induced paradoxical sleep deprivation (PSD) in rats. Sixty Male Wistar rats were randomly divided into six groups. Before PSD, SDOL treatment group rats were intragastrically administered SDOL for 25 days at dose of 2.1, 4.2, and 8.4 mL/kg body weight per day. Normal control group, large platform control group, and PSD groups were treated with normal saline instead of SDOL. After 25 days treatment, PSD and SDOL groups were deprived of paradoxical sleep for 72 h. Then two behavioral studies were conducted to test the spatial learning and memory ability using the open field test and Morris water maze test. Expression of the c-fos, c-jun, cyclic AMP response element binding protein (CREB), extracellular signal-regulated protein kinase (ERK), mitogen-activated protein kinases (MAPK)/ERK kinase (MEK), and p-CREB, p-ERK, and p-MEK in the hippocampus were also assayed by western blot. In this study, PSD decreased the levels of p-CREB, p-ERK, p-MEK, c-fos, and c-jun. However, SDOL treatment increased expressions of these proteins. Our results showed that SDOL improved 72-h PSD-induced cognitive impairment. These affects may be mediated by increasing the contents of c-fos, c-jun, and p-CREB/ERK signaling.

CB1 Cannabinoid Agonist (WIN55,212-2) Within the Basolateral Amygdala Induced Sensitization to Morphine and Increased the Level of μ-Opioid Receptor and c-fos in the Nucleus Accumbens.

The basolateral amygdala (BLA) is rich of CB1 cannabinoid receptors (CB1R) and has reciprocal connections with the nucleus accumbens (NAc) which is involved in opioid sensitization. In this study, effects of intra-BLA administration of CB1R agonist on sensitization to antinociceptive effect of morphine and changes in the levels of μ-opioid receptor (MOR), p-CREB, and c-fos in the NAc were investigated. Animals received intra-BLA microinjection of CB1R agonist (WIN55,212-2) once daily for 3 days consecutively (sensitization period). After 5 days free of drug, tail-flick test was performed before and after the administration of an ineffective dose of morphine. Afterward, the levels of MOR, p-CREB, and c-fos proteins were measured in the NAc by Western blot analysis. The results indicated that intra-BLA injection of WIN55,212-2 during sensitization period resulted in the induction of antinociceptive responses by ineffective dose of morphine and caused a significant increase in the MOR and c-fos levels but not p-CREB/CREB ratio in the NAc. These finding revealed that CB1 receptor agonist in the BLA induces development of morphine sensitization and increases expression of MOR in the NAc. It seems that c-fos is one of the important factors involved in the induction of sensitization to antinociceptive effect of morphine.

Diminished CRE-Induced Plasticity is Linked to Memory Deficits in Familial Alzheimer's Disease Mice.

The mechanism underlying impaired learning and memory in Alzheimer's disease is not fully elucidated. The phosphorylation of cyclic-AMP response element binding protein (pCREB) in the hippocampus is thought to be a critical initiating step in the formation of long-term memories. Here, we tested CRE-driven gene expression following learning in mice harboring the familial Alzheimer's disease-linked APPswe/PS1ΔE9 mutations using CRE-β galactosidase reporter. We show that young adult APPswe/PS1ΔE9 mice exhibit impaired recognition memory and reduced levels of pCREB, and its cofactors CREB binding protein (CBP) and p-300 following a learning task, compared to their wild type littermate counterparts. Impairments in learning-induced activation of CREB in these mice are manifested by reduced CRE-driven gene transcription. Importantly, expression of the CRE-driven immediate early gene, Egr-1 (Zif268) is decreased in the CA1 region of the hippocampus. These studies implicate defective CREB-dependent plasticity in the mechanism underlying learning and memory deficits in Alzheimer's disease.

Direct Regulation of Aromatase B Expression by 17β-Estradiol and Dopamine D1 Receptor Agonist in Adult Radial Glial Cells.

Aromatase cytochrome P450arom (cyp19) is the only enzyme that has the ability to convert androgens into estrogens. Estrogens, which are produced locally in the vertebrate brain play many fundamental roles in neuroendocrine functions, reproductive functions, socio-sexual behaviors, and neurogenesis. Radial glial cells (RGCs) are neuronal progenitor cells that are abundant in fish brains and are the exclusive site of aromatase B expression and neuroestrogen synthesis. Using a novel in vitro RGC culture preparation we studied the regulation of aromatase B by 17β-estradiol (E2) and dopamine (DA). We have established that activation of the dopamine D1 receptor (D1R) by SKF 38393 up-regulates aromatase B gene expression most likely through the phosphorylation of cyclic AMP response element binding protein (CREB). This up-regulation can be enhanced by low concentration of E2 (100 nM) through increasing the expression of D1R and the level of p-CREB protein. However, a high concentration of E2 (1 μM) and D1R agonist together failed to up-regulate aromatase B, potentially due to attenuation of esr2b expression and p-CREB levels. Furthermore, we found the up-regulation of aromatase B by E2 and DA both requires the involvement of esr1 and esr2a. The combined effect of E2 and DA agonist indicates that aromatase B in the adult teleost brain is under tight control by both steroids and neurotransmitters to precisely regulate neuroestrogen levels.

The amniotic membrane from the human placenta contains different subregions with different morpho-functional features

Human Amniotic Membrane (AM) has been attributed anti-inflammatory, anti-angiogenic, anti-fibrotic and anti-microbial effects (1-2). A number of clinical studies have used AM for applications in regenerative medicine, but it has yet to be elucidated whether the different areas of the AM have the same plasticity and differentiation potential. Thus, the aim of our study was to map the AM from normal human placenta. AMs were obtained from 24 healthy women undergoing caesarean section. Four areas were considered with respect to the umbilical cord: the first area, closer to the umbilical cord, was named the central area; the second, in the middle, was the intermediate area; the third was the peripheral area (all the three areas were part of the chorion frondosum) and the fourth, the reflected area, corresponded to the chorion laeve. Our results demonstrate the presence of a multi-layered epithelium in different areas of AM, except for the intermediate area, with a number of budding or detaching cells as well as of apoptotic cells, especially in the central area. By means of immune-histochemistry and quantitative analysis with MetaMorph analysis software, we evaluated the in situ expression of different proteins observing that the peripheral area has the highest OCT-4, c-KIT and SOX-2 expression, well known indicators of pluripotency, and the highest levels of CREB and p-CREB, well known for its key role in proliferation, differentiation and apoptosis, whereas mainly the central area expresses high levels of α-fetoprotein, suggesting that it could be more prone to hepatic differentiation. Interestingly, amniotic epithelial cells (AECs) from the peripheral area display the highest quantity of granules in the cytoplasm, as shown in electron microscopy preparations. In addition, in vitro cultured AECs derived from the reflected area demonstrate the highest clonogenic capacity, whereas those from the intermediate area show the highest osteogenic potential, when induced to osteogenic differentiation. Our study is the first to propose the morpho-functional mapping of the AM as a useful tool to identify areas with different stemness properties and plasticity. This could increase the efficiency of human AM application within a therapeutic context.This work was supported by funds granted from MIUR to Prof. Roberta Di Pietro and Lucia Centurione and by Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy to Prof. Ornella Parolini.

Supercritical Fluid Extract of Spent Coffee Grounds Attenuates Melanogenesis through Downregulation of the PKA, PI3K/Akt, and MAPK Signaling Pathways.

The mode of action of spent coffee grounds supercritical fluid CO2 extract (SFE) in melanogenesis has never been reported. In the study, the spent coffee grounds were extracted by the supercritical fluid CO2 extraction method; the chemical constituents of the SFE were investigated by gas chromatography-mass spectrometry (GC-MS). The effects of the SFE and its major fatty acid components on melanogenesis were evaluated by mushroom tyrosinase activity assay and determination of intracellular tyrosinase activity and melanin content. The expression level of melanogenesis-related proteins was analyzed by western blotting assay. The results revealed that the SFE of spent coffee grounds (1–10 mg/mL) and its major fatty acids such as linoleic acid and oleic acid (6.25–50 μM) effectively suppressed melanogenesis in the B16F10 murine melanoma cells. Furthermore, the SFE decreased the expression of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). The SFE also decreased the protein expression levels of p-JNK, p-p38, p-ERK, and p-CREB. Our results revealed that the SFE of spent coffee grounds attenuated melanogenesis in B16F10 cells by downregulation of protein kinase A (PKA), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen-activated protein kinases (MAPK) signaling pathways, which may be due to linoleic acid and oleic acid.

CAMP/PKA/CREB/GLT1 signaling involved in the antidepressant-like effects of phosphodiesterase 4D inhibitor (GEBR-7b) in rats.

ObjectivesGEBR-7b, a potential phosphodiesterase 4D inhibitor, has been shown to have memory-enhancing effects in rodents. However, it is still unknown whether GEBR-7b also has the antidepressant-like effects in rats. Herein, we examined the potential of GEBR-7b to attenuate depression-like behaviors in the rat model of depression induced by chronic unpredictable stress (CUS). Next, we also investigated the alterations of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) catalytic subunit (PKAca), cAMP response element-binding (CREB), and glutamate transporter 1 (GLT1) levels produced by GEBR-7b in the rats model of depression.MethodsEffects of GEBR-7b on CUS (35 days)-induced depression-like behaviors were examined by measuring immobility time in the forced swimming test (FST). Hippocampal cAMP levels were examined by enzyme-linked immunosorbent assay, whereas PKAca, phosphorylation of CREB (pCREB), CREB, and GLT1 in the hippocampus of rats were subjected to Western blot analysis.ResultsCUS exposure caused a depression-like behavior evidenced by the increased immobility time in FST. Depression-like behavior induced by CUS was accompanied by a significant increased GLT, decreased cAMP, PKAca, pCREB activities in hippocampus. However, repeated GEBR-7b administration significantly reversed CUS-induced depression-like behavior and changes of cAMP/PKA/CREB/GLT1 signaling. No alteration was observed in locomotor activity in open field test.ConclusionThese findings indicate that GEBR-7b reversed the depression-like behaviors induced by CUS in rats, which is at least in part mediated by modulating cAMP, PKAca, pCREB, and GLT1 levels in the hippocampus of rats, supporting its neuroprotective potential against behavioral and biochemical dysfunctions induced by CUS.

Identification of the G protein-coupled estrogen receptor (GPER) in human prostate: expression site of the estrogen receptor in the benign and neoplastic gland.

Estrogens are involved in growth, differentiation and pathogenesis of human prostate through the mediation of the classical estrogen receptors ERα and ERβ. The G protein-coupled estrogen receptor (GPER) is a 'novel' mediator of estrogen signaling which has been recently recognized in some human reproductive tissues, but its expression in the prostate gland is still unknown. Here, we investigated GPER in benign (from 5 patients) and neoplastic prostatic tissues (from 50 patients) by immunohistochemical analysis and Western blotting. Normal areas of benign prostates revealed a strong GPER immunoreactivity in the basal epithelial cells while luminal epithelial cells were unreactive and stromal cells were weakly immunostained. GPER was also immunolocalized in adenocarcinoma samples but the immunoreactivity of tumoral areas decreased from Gleason pattern 2 to Gleason pattern 4. Furthermore, a strong GPER immunostaining was also revealed in cells of pre-neoplastic lesions (high-grade prostatic intra-epithelial neoplasia). Western blot analysis of benign and tumor protein extracts showed the presence of a ~42 kDa band, consistent with the GPER molecular weight. An increase in both pAkt and p cAMP-response-binding protein (pCREB) levels was also observed in poorly differentiated PCa samples. Finally, this work identified GPER in the epithelial basal cells of benign human prostate, with a different localization with respect to the classical estrogen receptors. Furthermore, the expression of GPER in prostatic adenocarcinoma cells was also observed but with a modulation of the immunoreactivity according to tumor cell arrangements.

Huwe1 interacts with Gadd45b under oxygen-glucose deprivation and reperfusion injury in primary Rat cortical neuronal cells.

BackgroundGrowth arrest and DNA-damage inducible protein 45 beta (Gadd45b) is serving as a neuronal activity sensor. Brain ischemia induces the expression of Gadd45b, which stimulates recovery after stroke and may play a protective role in cerebral ischemia. However, little is known of the molecular mechanisms of how Gadd45b expression regulated and the down-stream targets in brain ischemia. Here, using an oxygen-glucose deprivation and reperfusion (OGD/R) model, we identified Huwe1/Mule/ARF-BP1, a HECT domain containing ubiquitin ligase, involved in the control of Gadd45b protein level. In this study, we also investigated the role of Huwe1-Gadd45b mediated pathway in BDNF methylation.ResultsWe found that the depletion of Huwe1 by lentivirus shRNA mediated interference significantly increased the expression of Gadd45b and BDNF at 24 h after OGD. Moreover, treatment with Cycloheximide (CHX) inhibited endogenous expression of Gadd45b, and promoted expression of Gadd45b after co-treated with lentivirus shRNA-Huwe1. Inhibition of Gadd45b by lentivirus shRNA decreased the expression levels of brain derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding protein (p-CREB) pathway, while inhibition of Huwe1 increased the expression levels of BDNF and p-CREB. Moreover, shRNA-Huwe1 treatment decreased the methylation level of the fifth CpG islands (123 bp apart from BDNF IXa), while shRNA-Gadd45b treatment increased the methylation level of the forth CpG islands (105 bp apart from BDNF IXa).ConclusionsThese findings suggested that Huwe1 involved in the regulation of Gadd45b expression under OGD/R, providing a novel route for neurons following cerebral ischemia-reperfusion injury. It also indicated that the methylation of BDNF IXa was affected by Gadd45b as well as Huwe1 in the OGD/R model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0178-y) contains supplementary material, which is available to authorized users.

Abstract A157: Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia

Abstract Objective: The Pseudomonas exotoxin (PE)-based anti-CD22 recombinant immunotoxin (RIT) Moxetumomab pasudotox (HA22) has a response rate of 86% in hairy cell leukemia (Kreitman et al 2012), but only about 30% of children with multiply relapsed ALL respond to HA22 treatment (Wayne et al AACR 2014). We attempted to improve activity against B-cell malignancies with a modified immunotoxin, LMB-11, which is missing domain II, has an Fab instead of a dsFv, and has 7 mutations in domain III. These modifications have been associated with reduced side effects, prolonged half-life, diminished immunogenicity, and better responses in in a Burkitt lymphoma animal model (Bera et al 2014). Activity of HA22 and LMB-11 on ALL cells varies significantly in vitro and in vivo (Müller et al AACR 2015). With the goal to improve activity against ALL, we combined CD22-targeting RITs with the multi-kinase inhibitor H89. H89 inhibits PKA (135 nM) and other kinases such as S6K1 (80 nM), MSK1 (120 nM), and ROCK2 (270 nM) (Davies et al 2000). Methods: In vitro cytotoxicity (IC50) was determined by WST-8 assay, and cell death by Annexin V-PE/7-AAD flow cytometry. An in vitro screen for H89 targets was performed by Reaction Biology Corp. Results: 10 uM H89 specifically enhanced activity of LMB-11 or HA22 10- fold on KOPN-8; activity of controls were unchanged. H89 also increased RIT activity by 5 to 10-fold on the ALL cell lines REH, SEM, HAL-01, and Nalm-6 and on 4 primary ALL samples. H89 did not change the rates of RIT-internalization or of furin-cleavage in KOPN-8. However, the time to ADP-ribosylate EF2 and arrest protein synthesis was shortened from 10 to 4 h with H89. We tested phosphorylation states of downstream targets in H89 treated KOPN-8 cells. Phosphorylation level of the PKA targets p-CREB or p-ATF2 were not reduced by H89. A high baseline phosphorylation of rpS6 or GSK-3β at the S6K1 target site was decreased to undetectable levels 15 minutes after addition of H89 to KOPN-8 and MCL1 fell by 50% within 15 minutes. In KOPN-8, a low dose of 5 ng/ml LMB-11 or 10 uM H89, respectively lowered MCL1 but did not induce apoptosis; MCL1 was undetectable and PARP-cleavage induced when H89 and LMB-11 were combined. The more specific S6K1 inhibitor PF-4708671 showed only 2-fold enhancement of RIT-induced cytotoxicity. The ROCK2 inhibitor Stemolecule similarly enhanced cytotoxicity 2-fold. MSK-1 inhibition or PKA-inhibition as well as PKA activation did not alter RIT activity. We performed a kinase screen on 359 kinases of which 21 were inhibited more than 90% by H89. None of the additionally identified kinases showed enhancement of RIT activity once blocked with relevant inhibitors. Despite combinations of small molecule inhibitors (e.g. ROCK2 and S6K1; GSK-3β and ROCK2), we were not able to mimic the 10-fold enhancement of RIT-induced cytotoxicity observed with H89. Conclusion: H89 strongly enhances CD22-targeting RITs against ALL. The increase in activity was in part due to inhibition of S6K1 and ROCK2 and was reflected by a faster onset of ADP-ribosylation of EF2 indicating improved trafficking of RITs through the various intracellular compartments. H89 and RIT lowered MCL1 levels by a distinct mechanism and synergistically induced intrinsic apoptosis. Further pre-clinical testing to assess in vivo efficacy of this combination is ongoing. Citation Format: Fabian Mueller, Xiu Fen Liu, Alan S. Wayne, Ira Pastan. Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A157.

Neuroprotective effects of docosahexaenoic acid on hippocampal cell death and learning and memory impairments in a valproic acid-induced rat autism model

Prenatal exposure to valproic acid (VPA) in rat offspring is capable of inducing experimental autism with neurobehavioral aberrations. This study investigated the effect of docosahexaenoic acid (DHA) on hippocampal cell death, learning and memory alteration in an experimental rat autism model. We found that DHA supplementation (75, 150 or 300mg/kg/day, 21 days) rescued the VPA (600mg/kg) induced DHA reduction in plasma and hippocampus in a dose-dependent manner, increased the levels of hippocampal p-CaMKII and p-CREB without affecting total protein level, and altered BDNF-AKT-Bcl-2 signaling pathway, as well as inhibited the activity of caspase-3. DHA also influenced the content of malondialdehyde (MDA) and the activities of antioxidant enzymes in the VPA-treated offspring. Consistent with the previous results, we also observed that 300mg/kg DHA supplementation markedly increased the cell survival, decreased the cell apoptosis, and increased mature neuronal cell in the hippocampus in VPA-treated offspring. Utilizing the Morris water maze test, we found that DHA prevented cognitive impairment in offspring of VPA-treated rats. The data suggested that DHA may play a neuroprotective role in hippocampal neuronal cell and ameliorates dysfunctions in learning and memory in this rat autism model. Thus, DHA could be used as treatment intervention for mitigating behavioral dysfunctions in autism spectrum disorder (ASD).

The Central Analgesic Mechanism of YM-58483 in Attenuating Neuropathic Pain in Rats.

Calcium channel antagonists are commonly used to treat neuropathic pain. Their analgesic effects rely on inhibiting long-term potentiation, and neurotransmitters release in the spinal cord. Store-operated Ca(2+)channels (SOCCs) are highly Ca(2+)-selective cation channels broadly expressed in non-excitable cells and some excitable cells. Recent studies have shown that the potent inhibitor of SOCCs, YM-58483, has analgesic effects on neuropathic pain, but its mechanism is unclear. This experiment performed on spinal nerve ligation (SNL)-induced neuropathic pain model in rats tries to explore the mechanism, whereby YM-58483 attenuates neuropathic pain. The left L5 was ligated to produce the SNL neuropathic pain model in male Sprague-Dawley rats. The withdrawal threshold of rats was measured by the up-down method and Hargreaves' method before and after intrathecal administration of YM-58483 and vehicle. The SOCCs in the spinal dorsal horn were located by immunofluorescence. The expression of phosphorylated ERK and phosphorylated CREB, CD11b, and GFAP proteins in spinal level was tested by Western blot, while the release of proinflammatory cytokines (IL-1β, TNF-α, PGE2) was measured by enzyme-linked immunosorbent assay (ELISA). Intrathecal YM-58483 at the concentration of 300μM (1.5nmol) and 1000μM (10nmol) produced a significant central analgesic effect on the SNL rats, compared with control+vehicle (n=7, P<0.001). However, both could not prevent the development of neuropathic pain, compared with normal+saline (P<0.001). Immunofluorescent staining revealed that Orai1 and STIM1 (the two key components of SOCCs) were located in the spinal dorsal horn neurons. Western blot showed that YM-58483 could decrease the levels of P-ERK and P-CREB (n=10, #P<0.05), without affecting the expression of CD11b and GFAP (n=10, #P>0.05). YM-58483 also inhibited the release of spinal cord IL-1β, TNF-α, and PGE2, compared with control+vehicle (n=5, #P<0.001). The analgesic mechanism of YM-58483 may be via inhibiting central ERK/CREB signaling in the neurons and decreasing central IL-1β, TNF-α, and PGE2 release to reduce neuronal excitability in the spinal dorsal horn of the SNL rats.

Evidence suggesting phosphodiesterase-3B regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons

Hypothalamic neurons expressing neuropeptide Y (NPY) and agouti related-protein (AgRP) are critical regulators of feeding behavior and body weight, and transduce the action of many peripheral signals including leptin and insulin. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. Since phosphodiesterase-3B (PDE3B) mediates the hypothalamic action of leptin and insulin on feeding, and is expressed in NPY/AgRP neurons, PDE3B could play a significant role in regulating NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we examined the effects of gain-of-function or reduced function of PDE3B on NPY/AgRP gene expression in a clonal hypothalamic neuronal cell line, mHypoE-46, which endogenously express NPY, AgRP and PDE3B. Overexpression of PDE3B in mHypoE-46 cells with transfection of pcDNA-3.1-PDE3B expression plasmid significantly decreased NPY and AgRP mRNA levels and p-CREB levels as compared to the control plasmid. For the PDE3B knockdown study, mHypoE-46 cells transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid were selected with puromycin, and stably transfected cells were grown in culture for 48h. Results showed that PDE3BshRNAmir mediated knockdown of PDE3B mRNA and protein levels (∼60–70%) caused an increase in both NPY and AgRP gene expression and in p-CREB levels. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B, and suggest a significant role for PDE3B in the regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.