CREB Protein Levels Research Articles

GPR39 protects against corticosterone-induced neuronal injury in hippocampal cells through the CREB-BDNF signaling pathway

BackgroundThe release of zinc from glutamatergic terminals in the hippocampal CA3 region can activate postsynaptic GPR39 receptors and regulate cognition and depression. However, the role and mechanism of GPR39 in the stress-induced depression is still poorly understood. MethodsIn this study, hippocampal cells (HT-22) were treated with corticosterone (CORT). Then the effects of stress on the activity, mitochondrial function and apoptosis of HT-22 cells were observed. The effects of GPR39 on CORT-induced stress injury were analyzed by both siRNA and agonist (TC-G-1008). ResultsCompared with the 500 nM CORT group, the cell viability, apoptosis, mitochondrial membrane potential, and expression levels of BCL-2, CREB and BDNF mRNA were significantly decreased in the GPR39 siRNA+500 nM CORT group, while the expression levels of caspase3, caspase9, AIF and BAX mRNA were significantly increased in the GPR39 siRNA+500 nM CORT group. Compared with the 1 μM CORTgroup, the cell viability, apoptosis, mitochondrial membrane potential, and expression levels of BCL-2, CREB and BDNF were significantly increased in the GPR39 agonist+1 μΜ CORT group, while the expression levels of caspase3, caspase9, AIF and BAX mRNA were significantly decreased in the GPR39 siRNA+500 nM CORT group. Compared with the control group, the mRNA and protein levels of GPR39, CREB and BDNF were significantly increased, and the mRNA and protein levels of CREB and BDNF were significantly decreased after 50 μM zinc sulfate treatment for 6 h. ConclusionsGPR39 may play a neuroprotective role in CORT-induced cell injury via the improvement of CREB-BDNF expression, by inhibiting pro-apoptotic proteins and by upregulating anti-apoptotic proteins.

Effects of Aberrant miR-384-5p Expression on Learning and Memory in a Rat Model of Attention Deficit Hyperactivity Disorder.

Attention deficit hyperactivity disorder (ADHD) is a common neuropsychiatric disorder characterized by inattention, hyperactivity, and impulsivity. It may be accompanied by learning difficulties and working memory deficits. Few studies have examined the role of miRNAs in cognitive dysfunction in ADHD. This study investigated the effects of aberrant miR-384-5p expression on learning and memory in a widely used ADHD rat model. Lentiviral vectors were injected into the lateral ventricles of the rats to increase or decrease miR-384-5p level. To determine whether aberrant miR-384-5p expression affects learning and memory, spontaneous activity and cognitive function were assessed with the open field and Morris water maze tests. In the place navigation experiment of the Morris water maze test, time, and total swimming distance to reach the platform decreased compared to the control group when miR-384-5p was overexpressed, whereas down-regulation of miR-384-5p had the opposite effect. There were no obvious changes in brain tissue morphology following miR-384-5p overexpression or inhibition; however, dopamine (DA) receptor D1 (DRD1) level has decreased and increased, respectively, in the prefrontal cortex (PFC). The luciferase activity of the wild-type DRD1 group has decreased in luciferase reporter assay. Cyclic AMP response element-binding protein (CREB) phosphorylation has increased, and DA transporter (DAT) level has decreased in the PFC of spontaneously hypertensive rats (SHR) by miR-384-5p overexpression. On the other hand, miR-384-5p suppression increased DRD1 and decreased DAT and CREB protein levels relative to control rats. These findings suggest that miR-384-5p may play a critical role in learning and memory impairment in ADHD.

The long-chain fatty acid receptors FFA1 and FFA4 are involved in food intake regulation in fish brain.

We hypothesized that the free fatty acid receptors FFA1 and FFA4 might be involved in the anorectic response observed in fish after rising levels of long-chain fatty acids (LCFAs) such as oleate. In one experiment we demonstrated that intracerebroventricular (i.c.v.) treatment of rainbow trout with FFA1 and FFA4 agonists elicited an anorectic response 2, 6 and 24 h after treatment. In a second experiment, the same i.c.v. treatment resulted after 2 h in an enhancement in the mRNA abundance of anorexigenic neuropeptides pomca1 and cartpt and a decrease in the values of orexigenic peptides npy and agrp1 These changes occurred in parallel with those observed in the mRNA abundance and/or protein levels of the transcription factors Creb, Bsx and FoxO1, protein levels and phosphorylation status of Ampkα and Akt, and mRNA abundance of plcb1 and itrp3 Finally, we assessed in a third experiment the response of all these parameters after 2 h of i.c.v. treatment with oleate (the endogenous ligand of both free fatty acid receptors) alone or in the presence of FFA1 and FFA4 antagonists. Most effects of oleate disappeared in the presence of FFA1 and FFA4 antagonists. The evidence obtained supports the involvement of FFA1 and FFA4 in fatty acid sensing in fish brain, and thus involvement in food intake regulation through mechanisms not exactly comparable (differential response of neuropeptides and cellular signalling) to those known in mammals.

Farnesoid X Receptor (FXR) Aggravates Amyloid-β-Triggered Apoptosis by Modulating the cAMP-Response Element-Binding Protein (CREB)/Brain-Derived Neurotrophic Factor (BDNF) Pathway In Vitro.

BackgroundAlzheimer’s disease (AD), which results in cognitive deficits, usually occurs in older people and is mainly caused by amyloid beta (Aβ) deposits and neurofibrillary tangles. The bile acid receptor, farnesoid X receptor (FXR), has been extensively studied in cardiovascular diseases and digestive diseases. However, the role of FXR in AD is not yet understood. The purpose of the present study was to investigate the mechanism of FXR function in AD.Material/MethodsLentivirus infection, flow cytometry, real-time PCR, and western blotting were used to detect the gain or loss of FXR in cell apoptosis induced by Aβ. Co-immunoprecipitation was used to analyze the molecular partners involved in Aβ-induced apoptosis.ResultsWe found that the mRNA and protein expression of FXR was enhanced in Aβ-triggered neuronal apoptosis in differentiated SH-SY5Y cells and in mouse hippocampal neurons. Overexpression of FXR aggravated Aβ-triggered neuronal apoptosis in differentiated SH-SY5Y cells, and this effect was further increased by treatment with the FXR agonist 6ECDCA. Molecular mechanism analysis by co-immunoprecipitation and immunoblotting revealed that FXR interacted with the cAMP-response element-binding protein (CREB), leading to decreased CREB and brain-derived neurotrophic factor (BDNF) protein levels. Low expression of FXR mostly reversed the Aβ-triggered neuronal apoptosis effect and prevented the reduction in CREB and BDNF.ConclusionsThese data suggest that FXR regulates Aβ-induced neuronal apoptosis, which may be dependent on the CREB/BDNF signaling pathway in vitro.

Pharmacological NOS-1 Inhibition Within the Hippocampus Prevented Expression of Cocaine Sensitization: Correlation with Reduced Synaptic Transmission.

Behavioral sensitization to psychostimulants hyperlocomotor effect is a useful model of addiction and craving. Particularly, cocaine sensitization in rats enhanced synaptic plasticity within the hippocampus, an important brain region for the associative learning processes underlying drug addiction. Nitric oxide (NO) is a neurotransmitter involved in both, hippocampal synaptic plasticity and cocaine sensitization. It has been previously demonstrated a key role of NOS-1/NO/sGC/cGMP signaling pathway in the development of cocaine sensitization and in the associated enhancement of hippocampal synaptic plasticity. The aim of the present investigation was to determine whether NOS-1 inhibition after development of cocaine sensitization was able to reverse it, and to characterize the involvement of the hippocampus in this phenomenon. Male Wistar rats were administered only with cocaine (15mg/kg/dayi.p.) for 5days. Then, animals received 7-nitroindazole (NOS-1 inhibitor) either systemically for the next 5days or a single intra-hippocampal administration. Development of sensitization and its expression after withdrawal were tested, as well as threshold for long-term potentiation in hippocampus, NOS-1, and CREB protein levels and gene expression. The results showed that NOS-1 protein levels and gene expression were increased only in sensitized animals as well as CREB gene expression. NOS-1 inhibition after sensitization reversed behavioral expression and the highest level of hippocampal synaptic plasticity. In conclusion, NO signaling within the hippocampus is critical for the development and expression of cocaine sensitization. Therefore, NOS-1 inhibition or NO signaling pathways interferences during short-term withdrawal after repeated cocaine administration may represent plausible pharmacological targets to prevent or reduce susceptibility to relapse.

Lactobacillus rhamnosus GG and Bifidobacterium bifidum TMC3115 Can Affect Development of Hippocampal Neurons Cultured In Vitro in a Strain-Dependent Manner.

This study examined whether Lactobacillus rhamnosus GG (LGG) and Bifidobacterium bifidum TMC3115 (TMC3115) could morphologically or physiologically influence hippocampal neuronal development in vitro. Hippocampal neurons cultured in vitro were exposed to live or heat-inactivated LGG or TMC3115 for either 6 or 24h. Neuronal morphological changes and drebrin (DRB) and synaptophysin (SYP) protein levels were monitored using immunofluorescence. And the levels of DRB, SYP, and brain-derived neurotrophic factor (BDNF), and cAMP-response element binding protein (CREB) mRNA were detected using RT-PCR. The BDNF, CREB, and phosphorylated-CREB (P-CREB) protein levels were detected by extraction-enzyme-linked immunosorbent assay (ELISA) or Western blot assays. Heat-inactivated LGG and TMC3115 could enhance neuron viability, DRB and SYP protein levels, and BDNF mRNA level were significantly altered after exposure to the tested bacteria with 6h or 24h. There were no significant differences in neuronal morphology or DRB, SYP, or CREB mRNA levels among the groups following bacterial exposure. However, following exposure of live TMC3115 for 24h, the neuronal BDNF and P-CREB protein levels were both significantly up-regulated as detected by western blot assays. These results demonstrated that LGG and TMC3115 could affect neuronal viability, along with hippocampal synaptic and functional development, in a strain-dependent manner, which may also be closely associated with the physiological and culture conditions of each strain. Up-regulated P-CREB may be one of the underlying mechanisms by which the bacteria, especially neurons following exposure of live TMC3115 for 24h, are able to regulate neuronal BDNF protein production.

Lactobacillus Rhamnosus GG and Bifidobacterium Bifidum TMC3115 Can Affect the Development of Hippocampal Neurons Cultured in Vitro in a Strain-dependent Manner (P20-009-19)

ObjectivesThis study aimed to examine whether probiotics could morphologically or physiologically influence hippocampal neuron development. MethodsHippocampal neurons cultured in vitro were exposed to live or heat-inactivated Lactobacillus rhamnosus GG (LGG), or live or heat-inactivated Bifidobacterium bifidum TMC3115 (TMC3115), for either 6 or 24 h. Neuron viability was then tested using the methyl thiazolyl tetrazolium assay. Neuronal morphological changes and drebrin (DRB) and synaptophysin (SYP) protein levels were monitored using immunofluorescence. And the levels of DRB, SYP, and brain-derived neurotrophic factor (BDNF), and cAMP-response element binding protein (CREB) mRNA were detected using RT-PCR. The BDNF, CREB and phosphorylated-CREB (P-CREB) protein levels were detected by ELISA or Western blot assays. ResultsWe found exposure to probiotics could enhance neuron viability, although no significant differences were found in neuronal morphology among the groups following exposure to the test bacteria. However, the synapse development-related proteins, DRB and SYP, as well as BDNF and P-CREB protein levels, were significantly altered in this specific culture system. ConclusionsThese results demonstrated that LGG and TMC3115 exposure can affect neuronal viability, along with synaptic and brain function development, in a strain-dependent manner, which may also be closely associated with the physiological and cultural conditions of each strain, The up-regulated P-CREB protein level may be one of the underlying mechanisms by which the tested bacteria, especially live TMC3115 following exposure for 24 h, are able to regulate neuronal BDNF protein production. Further studies are needed to explore other possible effects probiotic exposure may have on hippocampal neurons, as well as the corresponding mechanisms that underlie them. Funding SourcesThis work was funded by the National Natural Science Foundation of China (Grant number 81872606).

Effect of Exercise and Aβ Protein Infusion on Long-Term Memory-Related Signaling Molecules in Hippocampal Areas.

Alzheimer's disease (AD) results from over-production and aggregation of β-amyloid (Aβ) oligopeptides in the brain. The benefits of regular physical exercise are now recognized in a variety of disorders including AD. In order to understand the effect of exercise at the molecular level, we studied the impact of exercise on long-term memory-related signaling molecules in an AD rat model. The rat model of AD (AD rat) was produced by 14-day osmotic pump infusion of i.c.v. 250pmol/day Aβ1-42. The effects of 4weeks of regular rodent treadmill exercise on the protein levels of CREB, CaMKVI, and MAPK-ERK1/2 in this model were determined by immunoblot analysis in the CA1 and dentate gyrus (DG) areas of the hippocampus, which is among the first brain structures impacted by AD. Aβ infusion caused marked reductions in the basal protein levels of CaMKVI and phosphorylated CREB without significantly affecting total CREB levels in both CA1 and DG areas. As predicted, our exercise regimen totally prevented these effects in the brains of exercised AD rats. Surprisingly, however, neither Aβ infusion nor exercise had any significant effect on the levels of phosphorylated or total ERK in the CA1 and DG areas. Additionally, exercise did not increase any of these molecules in healthy normal rats, which indicated a protective effect of exercise. These findings suggest that CaMKIV is likely a major kinase for phosphorylation of CREB. Therefore, regular exercise is highly effective in preventing the effects of AD even at the molecular level in both areas of the hippocampus. Considering the well-known resistance of the DG area to insults relative to area CA1, the present findings revealed similar molecular vulnerability of the two areas to AD pathology.

Social defeat stress before pregnancy induces depressive-like behaviours and cognitive deficits in adult male offspring: correlation with neurobiological changes

BackgroundEpidemiological surveys and studies with animal models have established a relationship between maternal stress and affective disorders in their offspring. However, whether maternal depression before pregnancy influences behaviour and related neurobiological mechanisms in the offspring has not been studied.ResultsA social defeat stress (SDS) maternal rat model was established using the resident-intruder paradigm with female specific pathogen-free Wistar rats and evaluated with behavioural tests. SDS maternal rats showed a significant reduction in sucrose preference and locomotor and exploratory activities after 4 weeks of stress. In the third week of the experiment, a reduction in body weight gain was observed in SDS animals. Sucrose preference, open field, the elevated-plus maze, light–dark box, object recognition, the Morris water maze, and forced swimming tests were performed using the 2-month-old male offspring of the female SDS rats. Offspring subjected to pre-gestational SDS displayed enhanced anxiety-like behaviours, reduced exploratory behaviours, reduced sucrose preference, and atypical despair behaviours. With regard to cognition, the offspring showed significant impairments in the retention phase of the object recognition test, but no effect was observed in the acquisition phase. These animals also showed impairments in recognition memory, as the discrimination index in the Morris water maze test in this group was significantly lower for both 1 h and 24 h memory retention compared to controls. Corticosterone, adrenocorticotropic hormone, and monoamine neurotransmitters levels were determined using enzyme immunoassays or radioimmunoassays in plasma, hypothalamus, left hippocampus, and left prefrontal cortex samples from the offspring of the SDS rats. These markers of hypothalamic–pituitary–adrenal axis responsiveness and the monoaminergic system were significantly altered in pre-gestationally stressed offspring. Brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate response element binding protein (CREB), phosphorylated CREB (pCREB), and serotonin transporter (SERT) protein levels were evaluated using western blotting with right hippocampus and right prefrontal cortex samples. Expression levels of BDNF, pCREB, and SERT in the offspring were also altered in the hippocampus and in the prefrontal cortex; however, there was no effect on CREB.ConclusionWe conclude that SDS before pregnancy might induce depressive-like behaviours, cognitive deficits, and neurobiological alterations in the offspring.

PO-233 The Neuroprotective Effects of Aerobic Exercise and Oral Resveratrol on Hippocampal Neurons in Diabetic Rats

Objective The purpose of this study is to explore the effects of aerobic exercise combined with oral resveratrol on ethology and BDNF and CREB proteins of hippocampus neurons in diabetic rats, in order to provide a theoretical basis for revealing the neuroprotective mechanism of exercise and resveratrol.
 Methods 45 male Sprague Dawley rats, aged 8 weeks, were randomly divided into 5 groups: normal control (NC), diabetes control (DC), diabetes exercise (DE), diabetes resveratrol (DR) and diabetes exercise and resveratrol (DER). Exercise-related groups performed 8-week swimming training (60min/d，5d/week). Morris maze test, 7d. Escape latency time, strategy of finding platform performance, the protein expression of BDNF and CREB from hippocampus neurons were measured.
 Results 1）Compared with DM, DR and RE groups, the escape latency of DRE group was significantly shortened (p<0.01), and the strategy of finding platform performance was remarkably improved (p<0.05). 2) Compared with NC group, the protein expression of BDNF of DM group was obviously decreased (p <0.01), while in DRE group was improved significantly than that in DE group (p< 0.05). 3) The level of CREB expression in DM group clearly lower than in group NC (p<0.01), and the expression of CREB in DER and DE groups were remarkably increased (P <0.01) .
 Conclusions Eight weeks of swimming training and/or oral resveratrol could increase the expression level of BDNF and CREB protein in the hippocampal neurons of diabetic rats, and improve the ability of spatial learning from behavioral study. It is suggested that the aerobic exercise training and the SIRT1 mechanism of resveratrol perhaps improve the situation of high glucose and indirectly stimulate the expression of BDNF and CREB protein. As a result, that leads to improve the impair of learning and memory which caused by diabetes.

Dexmedetomidine Relieves Acute Inflammatory Visceral Pain in Rats through the ERK Pathway, Toll-Like Receptor Signaling, and TRPV1 Channel.

Dexmedetomidine (DEX) is a highly selective α2 adrenergic receptor agonist. In this study, we aimed to characterize the antinociceptive effects of DEX in acute inflammatory visceral pain (AIVP) induced by acetic acid in rats and to evaluate whether antinociception was regulated by the extracellular signal-regulated protein kinase (ERK) pathway, Toll-like receptor (TLR) signaling, and transient receptor potential (TRP) channel. Acetic acid was administered to 30 male rats with or without DEX. Rats were divided into six groups, as follows: control, disease (received no treatment before acetic acid administration), vehicle-treated, low-dose DEX (lDEX), medium-dose DEX (mDEX), and high-dose DEX (hDEX)-treated groups. Thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), and abdominal withdrawal reflex (AWR) were measured to assess pain. We detected electromyographic (EGM) responses in the rectus abdominis muscle and measured the average arterial blood pressure. Levels of interleukin 1 (IL-1), IL-2, and IL-6 in the serum, as well as tumor necrosis factor α (TNF-α) and prostaglandin E2 (PGE2) in the peritoneal fluid, were measured by ELISA. The expression levels of phospho(p)CREB, pERK1/2, pMEK1, and TRP cation channel subfamily V member 1 (TRPV1), as well as the activation state of TLR4, were determined in the spinal cord of rats by real-time polymerase chain reaction and western blot analysis. TWL and MWT scores were elevated (P < 0.05) in the hDEX and mDEX groups, whereas AWR scores decreased (P < 0.01), compared to those in the disease group. The medium and high doses of DEX suppressed IL-1, IL-6, TNF-α, and PGE2 release, and increased IL-2 release. In addition, protein and mRNA levels of MEK, ERK, and CREB were reduced in the mDEX and hDEX groups. Moreover, TLR4 and its downstream target, nuclear factor kappa B, along with calcitonin gene-related peptide release through the TRPV1 channel, were suppressed by mDEX and hDEX treatment. Taken together, our results suggest that DEX might exert an antinociceptive effect in AIVP in rats through the MEK/ERK pathway, TLR signaling, and TRPV1 channel, resulting in suppression of visceral hypersensitivity.

Glucosamine abrogates the stem cell factor + endothelin-1-induced stimulation of melanogenesis via a deficiency in MITF expression due to the proteolytic degradation of CREB in human melanocytes.

We have already reported that glucosamine (GlcN) distinctly abrogates the pigmentation of human epidermal equivalents stimulated by stem cell factor + endothelin-1 (SE). In this study, we characterized the molecular mechanism involved in the anti-melanogenic effects of GlcN using normal human melanocytes (NHMs) in culture. The SE-stimulated gene (12h) and protein (24h) expression levels of melanocyte-specific proteins (at the indicated times post-stimulation) were significantly abrogated by pretreatment with GlcN for 72h. Western blotting analysis of the phosphorylation of intracellular signaling molecules in the MAPK pathway revealed that despite the significantly decreased level of total CREB protein at all times post-stimulation, the SE-stimulated phosphorylation of ERK, CREB and MITF is not attenuated at 15min post-stimulation in GlcN-treated NHMs. However, the SE-stimulated protein expression level of total MITF at 2 and 6h post-stimulation was significantly abrogated by 72h pretreatment with GlcN. Consistently, pretreatment with GlcN for 72h abrogated the stimulated gene and protein expression levels of MITF at 1h and 2h post-stimulation, respectively. Analysis of gene and protein expression levels also demonstrated that pretreatment with GlcN for 72h significantly reduced the protein levels of CREB and MITF without affecting their gene expression levels prior to the SE stimulation. Silencing with a CREB siRNA distinctly abrogated the SE-stimulated expression of MITF (at 2h post-stimulation) and melanocyte-specific proteins (at 24h post-stimulation). Similarly, transfection of MITF siRNA markedly abrogated the SE-stimulated expression of MITF protein and melanocyte-specific proteins at 2 and 24h post-stimulation, respectively. Finally, the decreased levels of CREB and MITF proteins induced by 72h pretreatment with GlcN were abrogated by the co-addition of the proteosomal degradation inhibitor MG132. These findings suggest that the anti-melanogenic effect elicited by GlcN is mediated via the decreased expression of MITF which results from the attenuated transcriptional activity of CREB due to proteolytic degradation.

PM2.5 induced cardiac hypertrophy via CREB/GSK3b/SOS1 pathway and metabolomics alterations.

The particle matter with diameter less 2.5μm (PM2.5) easier to adsorb toxic substance, and interfere with pulmonary gas exchange. In this study, cardioprotective effects of low molecular weight (LMW) fucoidan in cardiac hypertrophy subjects induced by PM2.5 exposure was conducted by measuring QT interval, Blood pressure, cardiac structure, metabolites and proteins expression in different organs. After PM2.5 exposure, increase in blood pressure, abnormal cardiac function (Prolongation of Action Potential Duration and QT Interval), and structral remodeling (cardiac hypertrophy and fibrosis) were recorded. Fucoidan supplement in consecutive 28 days can reduce the damage to myocardial injury caused by PM2.5. Clearance effect of fucoidan in serum, heart, kidney, lung and liver was found due to organic and inorganic compounds reduced SOS1, CREB, GSK3b, and GRB2 protein level were changed under PM2.5 exposure. Whereas, only CREB level was reduced after fucoidan treatment. Metabolic alteration was also determined that PM2.5 severely damage cardiac tissue and compromise its function. After treatment with fucoidan, the cardiac function was significantly recovered. Our finding demonstrated that LMW could enhance the cardiac status of mice with PM2.5 exposures by rescued QT interval prolongation, action potential and cardiac hypertrophy, and cardiac fibrosis decline.

Abstract 903: Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells

Abstract ErbB2/Her2 overexpression is detected in approximately 30% of human breast cancers and is associated with poor prognosis. Lapatinib, a small molecule dual inhibitor targeting ErbB2 and EGFR, is FDA-approved for the treatment of ErbB2-positive (ErbB2+) advanced or metastatic breast cancer. However, lapatinib resistance (LR) is emerging as a critical issue in clinical oncology. To understand the molecular mechanisms of LR, we first developed a lapatinib-resistant cell line from ErbB2-overexpressing BT474 breast cancer cells by prolonged exposure of parental BT474 cells to gradually increasing concentrations of lapatinib (up to 8 µM). We performed microarray analyses on parental and LR cells to identify novel factors/pathways that contribute to LR. Our microarray data indicated that the gene expression profile of the BT474/LR cells is significantly different from the parental BT474 cells. We found that DARPP32/PPP1R1B is one of the most significantly upregulated genes in the BT474/LR cells. To investigate the role of DARPP32 in LR development, we examined the effect of DARPP32 knockdown via lentiviral shRNA on BT474/LR cell proliferation and apoptosis. Results from MTT, clonogenic, cell cycle, and cleaved caspase-3 and PARP analyses indicated that DARPP32 knockdown significantly sensitized BT474/LR cells to lapatinib. We also found that DARPP32 knockdown renders MDA-MB-361 breast cancer cells, which express high endogenous levels of DARPP32, more sensitive to lapatinib. These data demonstrate that DARPP32 overexpression contributes to LR. To understand the mechanism of DARPP32 overexpression-mediated LR, we focused on the regulation of transcription factor cAMP response element-binding protein (CREB) in this process. As such, DARPP32 overexpression was generally associated with CREB upregulation. We also found that CREB-mediated transcription was increased in BT474/LR cells, and DARPP32 knockdown in BT474/LR and MDA-MB-361 cells significantly downregulated CREB protein levels and CREB-mediated transcription, as indicated by luciferase reporter assays. CREB overexpression rendered BT474 cells resistant to lapatinib and reversed DARPP32 knockdown-induced sensitization in BT474/LR cells. In contrast, CREB knockdown sensitized the cells to lapatinib. Further analysis with MG132 blockage suggests that DARPP32 may regulate CREB protein levels through the proteasomal degradation pathway. Taken together, we have identified DARPP32 overexpression as a critical mediator of acquired lapatinib resistance. The underlying mechanisms involve the deregulation of CREB protein and activity levels. These novel findings advance our understanding of the molecular mechanisms of LR and provide fundamental support for testing the DARPP32-CREB axis in clinical settings for the management of LR breast cancers, which is of significant translational value. Citation Format: Zhikun Ma, Erin W. Howard, Amanda B. Parris, Xiaohe Yang. Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 903.

Chronic antidepressant-like effect of EMD386088, a partial 5-HT6 receptor agonist, in olfactory bulbectomy model may be connected with BDNF and/or CREB signalling pathway.

The removal of the olfactory bulbs has been attributed to behavioral changes and neuroplasticity manifesting themselves among others like increases in brain neurotrophin expression and neurogenesis. Earlier data presented that EMD386088, a 5-HT6 receptor partial agonist, exerts antidepressant-like properties after chronic administration in olfactory bulbectomy (OB) model as was it compared with amitriptyline (AMI). The aim of this study was to compare acute and chronic biochemical effects of EMD386088, administered in its antidepressant active (2.5mg/kg) and non-active (1.25mg/kg) doses, found in the open field test in OB rats, with those of AMI (10mg/kg). The levels of 5-HT6 receptor protein and selected neurotrophins in prefrontal cortex (PFC) and hippocampus (Hp) of rats have been examined. 5-HT6 receptor protein and selected neurotrophins: brain-derived neurotrophic factor (BDNF), cAMP-response element binding protein (CREB), the product of the immediate early gene c-fos (cFos) protein levels were assessed using a Western blot analysis in PFC and Hp of bulbectomized rats after acute or chronic (14-day) EMD386088 or AMI intraperitoneal (ip) treatment. The acute treatment with EMD386088 caused significant increases in CREB and BDNF protein levels in PFC, and an increase in BDNF in Hp of OB rats, while AMI injection decreased CREB and did not change BDNF levels. After the chronic administration of EMD386088, the increasing levels of BDNF and CREB were still observed in PFC and Hp. The antidepressant-like effect of EMD386088 may be associated with the neuroplasticity activation in PFC and Hp in rats.

Suppression of Long Non-Coding RNA LINC00652 Restores Sevoflurane-Induced Cardioprotection Against Myocardial Ischemia-Reperfusion Injury by Targeting GLP-1R Through the cAMP/PKA Pathway in Mice

Long non-coding RNA (lncRNA) and glucagon-like peptide 1 receptor (GLP-1R) are crucial for heart development and for adult heart structural maintenance and function. Herein, we performed a study to explore the effect of lncRNA LINC00652 (LINC00652) on myocardial ischemia-reperfusion (I/R) injury by targeting GLP-1R through the cyclic adenosine monophosphate-protein kinase A (cAMP/PKA) pathway. Bioinformatics software was used to screen the long-chain non-coding RNAs associated with myocardial ischemia-reperfusion and to predict target genes. The mRNA and protein levels of LINC00652, GLP-1R and CREB were detected by RT-qPCR and western blotting. In order to identify the interaction between LINC00652 and myocardial I/R injury, the cardiac function, the hemodynamic changes, the pathological changes of the myocardial tissues, the myocardial infarct size, and the apoptosis of myocardial cells of mice were measured. Meanwhile, the levels of serum IL-1β and TNF-α were detected. LINC00652 was overexpressed in the myocardial cells of mice with myocardial I/R injury. GLP-1R is the target gene of LINC00652. We also determined higher levels of LINC00652 and GLP-1R in the I/R modeled mice. Additionally, si-LINC00652 decreased cardiac pathology, infarct size, apoptosis rates of myocardial cells, and levels of IL-1β and TNF-α, and increased GLP-1R expression cardiac function, normal hemodynamic index, and the expression and phosphorylation of GLP-1R and CREB proteins. Taken together, our key findings of the present highlight LINC00652 inhibits the activation of the cAMP/PKA pathway by targeting GLP-1R to reduce the protective effect of sevoflurane on myocardial I/R injury in mice.

LINC01121 Inhibits Cell Apoptosis While Facilitating Proliferation, Migration, and Invasion Though Negative Regulation of the Camp/PKA Signaling Pathway via GLP1R

Background/Aims: Pancreatic cancer is an aggressive malignancy as a result of highly metastatic potential. The current study was carried out to alter the expression of LINC01121 in pancreatic cancer, with the aim of elucidating its effects on the biological processes of cell proliferation, migration, invasion, and apoptosis. We hypothesized that both the GLP1R gene and cAMP/PKA signaling pathway participate in the aforementioned process. Methods: Microarray data (GSE14245, GSE27890 and GSE16515) and annotating probe files linked to pancreatic cancer were downloaded through the GEO database. The Multi Experiment Matrix (MEM) site was used to predict the target gene of lncRNA. Both pancreatic cancer tissues (n = 56) and paracancerous tissues (n = 45) were collected from patients diagnosed with pancreatic cancer. Immunohistochemistry was applied to identify the positive expression rate of GLP1R protein. Isolated pancreatic cancer cells and PANC-1 cells were independently classified into the blank, negative control (NC), LINC01121 vector, siRNA-LINC01121, siRNA-GLP1R and siRNA-LINC01121 + siRNA-GLP1R groups. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were applied to detect the expressions of LINC01121, GLP1R, cAMP, PKA, CREB, Bcl-2, Bad and PCNA. Cell proliferation, migration, invasion, cycle progression, and apoptosis were examined by MTT assay, scratch test, Transwell assay and flow cytometry analyses of Annexin V-FITC/PI staining. Results: Observations were made indicating that LINC01121 was highly expressed, while low expressions of GLP1R in pancreatic cancer were detected based on microarray data, which was largely in consistent with the data collected of LINC01121 and GLP1R within the tissues. The target prediction program and luciferase activity analysis was testament to the notion suggesting that GLP1R was indeed a target of LINC01121. In contrast to the blank and NC groups, the LINC01121 vector group exhibited increased expressions of LINC01121; decreased mRNA and protein levels of GLP1R, Bad, cAMP, and PKA; increased protein levels of CREB, Bcl-2, PCNA, p-PKA and p-CREB; increased cell proliferation, migration and invasion; and decreased cell apoptosis. There was no significant difference detected among the blank, NC, and siRNA-LINC01121 + siRNA-GLP1R groups, except that decreased LINC01121 expression was determined in the siRNA-LINC01121 + siRNA-GLP1R group. Parallel data were observed in the pancreatic cancer cells and PANC-1 cells. Conclusion: The current study presents evidence indicating that LINC01121 might inhibit apoptosis while acting to promote proliferation, migration, and invasion of pancreatic cancer cells, supplementing the stance held that LINC01121 functions as a tumor promoter by means of its involvement in the process of translational repression of the GLP1R and inhibition of the cAMP/PKA signaling pathway.

The regulation of SKF38393 on the signaling pathway of dopamine D1 receptor in hippocampus during chronic sleep deprivation.

Hippocampal functions are sensitive to sleep deficiency. Dopamine D1 receptor (D1R) in hippocampus can regulate the expression of cAMP response element binding protein (CREB) through PKA, MAPK and phosphoinositide pathway, but which pathway plays the major role in hippocampus during Chronic sleep deprivation (CSD) is unclear. The CSD model was created, SKF rats were administered the D1R agonist (SKF38363), and hippocampus from each animal was dissected for following molecular detection. The gene and protein levels of CREB and key molecules in D1R pathways were measured by real-time PCR and western blotting, respectively. Both the gene and protein expression of CREB in hippocampus decreased by CSD and improved significantly by SKF38393 (p<0.05). Both the gene and protein expression of PKA in hippocampus decreased by CSD and improved significantly by SKF38393 (p<0.05). SKF38393 just significantly improved the gene level of CaMK IV and the protein level of p-CaMK IV (p<0.05) in CSD rats, but it cannot improve the protein expression of ERK1/2 and p-ERK1/2. CSD significantly decreased the expression of CREB in hippocampus. As the key molecules, PKA and CaMK IV play an important role during the improvement of hippocampus by the activation of D1R, and this process might be improved during CSD through the PKA and phosphoinositide pathway.

Early-postnatal iron deficiency impacts plasticity in the dorsal and ventral hippocampus in piglets

In this study, we investigated whether alterations in plasticity markers such as brain-derived neurotrophic factor (BDNF), p75 neurotrophin receptor (p75NTR) and tyrosine receptor kinase B (TrkB) are underlying iron deficiency (ID)-induced cognitive impairments in iron depleted piglets. Newborn piglets were either fed an iron-depleted diet (21mg Fe/kg) or an iron-sufficient diet (88mg Fe/kg) for four weeks. Subsequently, eight weeks after iron repletion (190–240mg Fe/kg) we found a significant decrease in mature BDNF (14kDa) and proBDNF (18kDa and 24kDa) protein levels in the ventral hippocampus, whereas we found increases in the dorsal hippocampus. The phosphorylation of cAMP response element binding protein (CREB) follows the mature BDNF protein level pattern. No effects were found on BDNF and CREB protein levels in the prefrontal cortex. The protein levels of the high affinity BDNF receptor, TrkB, was significantly decreased in both dorsal and ventral hippocampus of ID piglets, whereas it was increased in the prefrontal cortex. Together, our data suggest a disrupted hippocampal plasticity upon postnatal ID.

MiR-9 Regulates the Expression of BACE1 in Dementia Induced by Chronic Brain Hypoperfusion in Rats

Background/Aims: MicroRNA-9 (miR-9) plays important roles in nervous system diseases such as glioblastoma and neurodegenerative disorders. However, how miR-9 contributes to dementia requires further study. In this study, we evaluated the role of miR-9 in dementia and the molecular mechanisms underlying its effects. Methods: A rat model of dementia was created by occlusion of the bilateral common carotid artery (2VO) for 8 weeks. Learning and memory were assessed using the Morris Water Maze (MWM). MicroRNA expression profiling was performed according to a protocol provided by LC Sciences, and quantitative real-time PCR (qRT-PCR) was used to detect the level of miR-9. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining were used to assess pathological changes in brain tissue. Western blot and immunofluorescence were employed to detect the expression of β-site APP cleaving enzyme 1 (BACE1) and c-AMP response element-binding protein (CREB). Results: Learning and memory were significantly impaired in 2VO rats, and these changes were accompanied by neuronal loss and glial activation in brain tissues. miR-9 was greatly upregulated in both the hippocampus and cortex of rats following 2VO. Knockdown of endogenous miR-9 via lentiviral vector-mediated delivery of its antisense molecule (lenti-pre-AMO-miR-9) reduced the vulnerability to dementia, reversed the increase in BACE1 expression, and ameliorated the reduction in CREB expression triggered by 2VO. BACE1 protein levels were significantly increased, but CREB protein levels were significantly decreased in the presence of miR-9 in cultured neonatal rat neurons (NRNs). AMO-miR-9 rescued the upregulation of BACE1 and downregulation of CREB elicited by miR-9 in rats. Dual luciferase assay experiments showed that overexpression of miR-9 inhibited the expression of CREB by targeting its 3’UTR domain. CREB protein was downregulated by miR-9 overexpression which was reversed by miR-9 inhibition in cultured NRNs. TEM imaging showed that miR-9 caused damage to NRNs, which was reversed by addition of AMO-miR-9. Conclusion: We conclude that miR-9 plays an important role in regulating the process of dementia induced by 2VO in rats by increasing BACE1 expression via downregulation of CREB.