scholarly journals The microRNA miR-124 suppresses seizure activity and regulates CREB1 activity

2016 ◽  
Vol 18 ◽  
Author(s):  
Wei Wang ◽  
Xuefeng Wang ◽  
Lang Chen ◽  
Yujiao Zhang ◽  
Zucai Xu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Neuronal Excitability ◽  
Surface Expression ◽  
Camp Response Element Binding ◽  
Neuronal Firing ◽  
Luciferase Reporter ◽  
Onset Latency ◽  
Drug Induced ◽  
Rat Models ◽  
Patients With Epilepsy

miR-124, a brain-specific microRNA, was originally considered as a key regulator in neuronal differentiation and the development of the nervous system. Here we showed that miR-124 expression was suppressed in patients with epilepsy and rats after drug induced-seizures. Intrahippocampal administration of a miR-124 duplex led to alleviated seizure severity and prolonged onset latency in two rat models (pentylenetetrazole- and pilocarpine-induced seizures), while miR-124 inhibitor led to shortened onset latency in pilocarpine-induced seizure rat models. Moreover, the result of local field potentials (LFPs) records further demonstrated miR-124 may have anti-epilepsy function. Inhibition of neuronal firing by miR-124 was associated with the suppression of mEPSC, AMPAR- and NMDAR-mediated currents, which were accompanied by decreased surface expression of NMDAR. In addition, miR-124 injection resulted in decreased activity and expression of cAMP-response element-binding protein1 (CREB1). a key regulator in epileptogenesis. A dual-luciferase reporter assay was used to confirm that miR-124 targeted directly the 3′UTR of CREB1 gene and repressed the CREB1 expression in HEK293T cells. Immunoprecipitation studies confirmed that the CREB1 antibody effectively precipitated CREB1 and NMDAR1 but not GLUR1 from rat brain hippocampus. These results revealed a previously unknown function of miR-124 in neuronal excitability and provided a new insight into molecular mechanisms underlying epilepsy.

scholarly journals NR4A1 Knockdown Suppresses Seizure Activity by Regulating Surface Expression of NR2B

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yanke Zhang ◽  
Guojun Chen ◽  
Baobing Gao ◽  
Yunlin Li ◽  
Shuli Liang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Seizure Activity ◽  
Surface Expression ◽  
Biological Processes ◽  
Onset Latency ◽  
Downstream Target ◽  
Group A ◽  
Patients With Epilepsy ◽  
The Relationship ◽  
Nuclear Receptor Subfamily

Abstract Nuclear receptor subfamily 4 group A member 1 (NR4A1), a downstream target of CREB that is a key regulator of epileptogenesis, has been implicated in a variety of biological processes and was previously identified as a seizure-associated molecule. However, the relationship between NR4A1 and epileptogenesis remains unclear. Here, we showed that NR4A1 protein was predominantly expressed in neurons and up-regulated in patients with epilepsy as well as pilocarpine-induced mouse epileptic models. NR4A1 knockdown by lentivirus transfection (lenti-shNR4A1) alleviated seizure severity and prolonged onset latency in mouse models. Moreover, reciprocal coimmunoprecipitation of NR4A1 and NR2B demonstrated their interaction. Furthermore, the expression of p-NR2B (Tyr1472) in epileptic mice and the expression of NR2B in the postsynaptic density (PSD) were significantly reduced in the lenti-shNR4A1 group, indicating that NR4A1 knockdown partly decreased surface NR2B by promoting NR2B internalization. These results are the first to indicate that the expression of NR4A1 in epileptic brain tissues may provide new insights into the molecular mechanisms underlying epilepsy.

scholarly journals Neuritin Up-regulates Kv4.2 α-Subunit of Potassium Channel Expression and Affects Neuronal Excitability by Regulating the Calcium-Calcineurin-NFATc4 Signaling Pathway

2016 ◽  
Vol 291 (33) ◽  
pp. 17369-17381 ◽  
Author(s):  
Jin-jing Yao ◽  
Qian-Ru Zhao ◽  
Dong-Dong Liu ◽  
Chi-Wing Chow ◽  
Yan-Ai Mei
Keyword(s):  
Signaling Pathway ◽  
Neural Development ◽  
Cortical Neurons ◽  
Molecular Mechanisms ◽  
Neuronal Excitability ◽  
Outward Current ◽  
Nuclear Accumulation ◽  
Luciferase Reporter ◽  
Activated T Cells ◽  
Α Subunit

Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca2+/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca2+ and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4−/− mice but not in Nfatc2−/− mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4−/− mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions.

scholarly journals IcarisideII facilitates the differentiation of ADSCs to SCs via let-7i/STAT3 axis to preserve erectile function

2019 ◽  
Vol 52 (1) ◽  
Author(s):  
Pingyu Ge ◽  
Yinxue Guo ◽  
Jun Shen
Keyword(s):  
Cell Proliferation ◽  
Erectile Function ◽  
Molecular Mechanisms ◽  
Therapeutic Option ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Regulatory Relationship ◽  
Rat Models ◽  
Protein Levels ◽  
Dose Dependent

Abstract Background IcarisideII (ICAII) could promote the differentiation of adipose tissue-derived stem cells (ADSCs) to Schwann cells (SCs), leading to improvement of erectile function (EF) and providing a realistic therapeutic option for the treatment of erectile dysfunction (ED). However, the underlying molecular mechanisms of ADSCs and ICAII in this process remain largely unclear. Methods ADSCs were treated with different concentrations of ICAII. Cell proliferation was determined by MTT assay. qRT-PCR and western blot were performed to detect expressions of SCs markers, signal transducer and activator of transcription-3 (STAT3), and microRNA-let-7i (let-7i). Luciferase reporter assay was conducted to verify the regulatory relationship between let-7i and STAT3. The detection of intracavernosal pressure (ICP) and the ratio of ICP/mean arterial pressure (MAP) were used to evaluate the EF in bilateral cavernous nerve injury (BCNI) rat models. Results ICAII promoted cell proliferation of ADSCs in a dose-dependent manner. The mRNA and protein levels of SCs markers were increased by ICAII treatment in a dose-dependent manner in ADSCs. Moreover, let-7i was significantly decreased in ICAII-treated ADSCs and upregulation of let-7i attenuated ICAII-induced promotion of SCs markers. In addition, STAT3 was a direct target of let-7i and upregulated in ICAII-treated ADSCs. Interestingly, overexpression of STAT3 abated the let-7i-mediated inhibition effect on differentiation of ADSCs to SCs and rescued the ICAII-mediated promotion effect on it. Besides, combination treatment of ADSCs and ICAII preserved the EF of BCNI rat models, which was undermined by let-7i overexpression. Conclusion ICAII was effective for preserving EF by promoting the differentiation of ADSCs to SCs via modulating let-7i/STAT3 pathway.

scholarly journals P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

2020 ◽  
Vol 21 (16) ◽  
pp. 5921
Author(s):  
Jia-hua Hu ◽  
Cole Malloy ◽  
Dax A. Hoffman
Keyword(s):  
Kainic Acid ◽  
Molecular Mechanisms ◽  
Neuronal Excitability ◽  
Pyramidal Neurons ◽  
Neuronal Firing ◽  
Dependent Manner ◽  
Membrane Excitability ◽  
Hippocampal Pyramidal Neurons ◽  
K Current

The subthreshold, transient A-type K+ current is a vital regulator of the excitability of neurons throughout the brain. In mammalian hippocampal pyramidal neurons, this current is carried primarily by ion channels comprising Kv4.2 α-subunits. These channels occupy the somatodendritic domains of these principle excitatory neurons and thus regulate membrane voltage relevant to the input–output efficacy of these cells. Owing to their robust control of membrane excitability and ubiquitous expression in the hippocampus, their dysfunction can alter network stability in a manner that manifests in recurrent seizures. Indeed, growing evidence implicates these channels in intractable epilepsies of the temporal lobe, which underscores the importance of determining the molecular mechanisms underlying their regulation and contribution to pathologies. Here, we describe the role of p38 kinase phosphorylation of a C-terminal motif in Kv4.2 in modulating hippocampal neuronal excitability and behavioral seizure strength. Using a combination of biochemical, single-cell electrophysiology, and in vivo seizure techniques, we show that kainic acid-induced seizure induces p38-mediated phosphorylation of Thr607 in Kv4.2 in a time-dependent manner. The pharmacological and genetic disruption of this process reduces neuronal excitability and dampens seizure intensity, illuminating a cellular cascade that may be targeted for therapeutic intervention to mitigate seizure intensity and progression.

Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells

2002 ◽  
Vol 283 (5) ◽  
pp. R1140-R1148 ◽  
Author(s):  
Dan D. Hershko ◽  
Bruce W. Robb ◽  
Guangju Luo ◽  
Per-Olof Hasselgren
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Map Kinase ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Camp Response Element Binding ◽  
P38 Map Kinase ◽  
Luciferase Reporter ◽  
Epithelial Cell Line

Mucosal and enterocyte IL-6 production is increased during sepsis and endotoxemia. Recent studies suggest that cAMP potentiates IL-6 production in endotoxin- or IL-1β-stimulated enterocytes, but the molecular mechanisms are not known. We examined the role of the transcription factors NF-κB, activator protein (AP)-1, CCAAT/enhancer binding protein (C/EBP), and cAMP response element-binding protein (CREB) in cAMP-induced IL-6 production in cultured Caco-2 cells, a human intestinal epithelial cell line. In addition, the role of the protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein (MAP) kinase signaling pathways was examined. Treatment of the cells with IL-1β increased IL-6 production and activated the IL-6 promoter in cells transfected with a luciferase reporter plasmid containing a wild-type IL-6 promoter. These effects of IL-1β were significantly potentiated by cAMP. When the binding sites for the individual transcription factors in the IL-6 promoter were mutated, results indicated that all four transcription factors may be involved in the cAMP-induced activation of the IL-6 gene. Treatment of the Caco-2 cells with cAMP increased the DNA binding activity of CREB, C/EBP, and AP-1, but not NF-κB. By using specific blockers, evidence was found that both PKA and p38 MAP kinase (but not PKC or p42/44 MAP kinase) may be involved in the cAMP-induced potentiation of IL-6 production. The present results suggest that cAMP activates multiple transcription factors involved in the regulation of the IL-6 gene and that the activation of these transcription factors may at least in part explain why cAMP potentiates IL-6 production in stimulated enterocytes.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

scholarly journals TEAD4 modulated LncRNA MNX1-AS1 contributes to gastric cancer progression partly through suppressing BTG2 and activating BCL2

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
You Shuai ◽  
Zhonghua Ma ◽  
Weitao Liu ◽  
Tao Yu ◽  
Changsheng Yan ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Mechanistic Model ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tissue Samples ◽  
Reporter Assays

Abstract Background Gastric cancer (GC) is the third leading cause of cancer-related mortality globally. Long noncoding RNAs (lncRNAs) are dysregulated in obvious malignancies including GC and exploring the regulatory mechanisms underlying their expression is an attractive research area. However, these molecular mechanisms require further clarification, especially upstream mechanisms. Methods LncRNA MNX1-AS1 expression in GC tissue samples was investigated via microarray analysis and further determined in a cohort of GC tissues via quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. Cell proliferation and flow cytometry assays were performed to confirm the roles of MNX1-AS1 in GC proliferation, cell cycle regulation, and apoptosis. The influence of MNX1-AS1 on GC cell migration and invasion was explored with Transwell assays. A xenograft tumour model was established to verify the effects of MNX1-AS1 on in vivo tumourigenesis. The TEAD4-involved upstream regulatory mechanism of MNX1-AS1 was explored through ChIP and luciferase reporter assays. The mechanistic model of MNX1-AS1 in regulating gene expression was further detected by subcellular fractionation, FISH, RIP, ChIP and luciferase reporter assays. Results It was found that MNX1-AS1 displayed obvious upregulation in GC tissue samples and cell lines, and ectopic expression of MNX1-AS1 predicted poor clinical outcomes for patients with GC. Overexpressed MNX1-AS1 expression promoted proliferation, migration and invasion of GC cells markedly, whereas decreased MNX1-AS1 expression elicited the opposite effects. Consistent with the in vitro results, MNX1-AS1 depletion effectively inhibited the growth of xenograft tumour in vivo. Mechanistically, TEAD4 directly bound the promoter region of MNX1-AS1 and stimulated the transcription of MNX1-AS1. Furthermore, MNX1-AS1 can sponge miR-6785-5p to upregulate the expression of BCL2 in GC cells. Meanwhile, MNX1-AS1 suppressed the transcription of BTG2 by recruiting polycomb repressive complex 2 to BTG2 promoter regions. Conclusions Our findings demonstrate that MNX1-AS1 may be able to serve as a prognostic indicator in GC patients and that TEAD4-activatd MNX1-AS1 can promote GC progression through EZH2/BTG2 and miR-6785-5p/BCL2 axes, implicating it as a novel and potent target for the treatment of GC.

scholarly journals Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Wu ◽  
Chengying Li ◽  
Bin Xu ◽  
Ying Xiang ◽  
Xiaoyue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Circular Rna ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Candidate Target ◽  
Paired Control ◽  
Reporter Assays ◽  
And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

scholarly journals Knockdown of Circ_SLC39A8 protects against the progression of osteoarthritis by regulating miR-591/IRAK3 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jizhe Yu ◽  
Yushuang Qin ◽  
Naxin Zhou
Keyword(s):  
Cell Viability ◽  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Oa Chondrocytes ◽  
Polymerase Chain ◽  
Apoptosis And Inflammation ◽  
The Relationship

Abstract Background The dysregulation of circular RNAs (circRNAs) has been identified in various human diseases, including osteoarthritis (OA). The purpose of this study was to identify the role and mechanism of circ_SLC39A8 in regulating the progression of OA. Methods The expression levels of circ_SLC39A8, miR-591, and its potential target gene, interleukin-1-receptor-associated kinase 3 (IRAK3), were identified by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were determined by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The relationship between miR-591 and circ_SLC39A8 or IRAK3 was predicted by bioinformatics tools and verified by dual-luciferase reporter. Results Circ_SLC39A8 and IRAK3 were upregulated and miR-591 was downregulated in OA cartilage tissues. Knockdown of circ_SLC39A8 inhibited apoptosis and inflammation in OA chondrocytes, while these effects were reversed by downregulating miR-591. Promotion cell viability effects of miR-591 were partially reversed by IRAK3 overexpression. Conclusion Our findings indicated that knockdown of circ_SLC39A8 delayed the progression of OA via modulating the miR-591-IRAK3 axis, providing new insight into the molecular mechanisms of OA pathogenesis.

scholarly journals Modeling temperature- and Cav3 subtype-dependent alterations in T-type calcium channel mediated burst firing

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Fernando R. Fernandez ◽  
Mircea C. Iftinca ◽  
Gerald W. Zamponi ◽  
Ray W. Turner
Keyword(s):  
Calcium Channel ◽  
Neuronal Excitability ◽  
Neuronal Firing ◽  
Mammalian Brain ◽  
Peripheral Tissues ◽  
Window Current ◽  
Modeling Data ◽  
The Brain ◽  
Firing Neuron ◽  
Cav3 Channel

AbstractT-type calcium channels are important regulators of neuronal excitability. The mammalian brain expresses three T-type channel isoforms (Cav3.1, Cav3.2 and Cav3.3) with distinct biophysical properties that are critically regulated by temperature. Here, we test the effects of how temperature affects spike output in a reduced firing neuron model expressing specific Cav3 channel isoforms. The modeling data revealed only a minimal effect on baseline spontaneous firing near rest, but a dramatic increase in rebound burst discharge frequency for Cav3.1 compared to Cav3.2 or Cav3.3 due to differences in window current or activation/recovery time constants. The reduced response by Cav3.2 could optimize its activity where it is expressed in peripheral tissues more subject to temperature variations than Cav3.1 or Cav3.3 channels expressed prominently in the brain. These tests thus reveal that aspects of neuronal firing behavior are critically dependent on both temperature and T-type calcium channel subtype.

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