mGluR1-Mediated Excitation of Cerebellar GABAergic Interneurons Requires Both G Protein-Dependent and Src–ERK1/2-Dependent Signaling Pathways

Stimulation of type I metabotropic glutamate receptors (mGluR1/5) in several neuronal types induces slow excitatory responses through activation of transient receptor potential canonical (TRPC) channels. GABAergic cerebellar molecular layer interneurons (MLIs) modulate firing patterns of Purkinje cells (PCs), which play a key role in cerebellar information processing. MLIs express mGluR1, and activation of mGluR1 induces an inward current, but its precise intracellular signaling pathways are unknown. We found that mGluR1 activation facilitated spontaneous firing of mouse cerebellar MLIs through an inward current mediated by TRPC1 channels. This mGluR1-mediated inward current depends on both G protein-dependent and -independent pathways. The nonselective protein tyrosine kinase inhibitors genistein and AG490 as well as the selective extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors PD98059 and SL327 suppressed the mGluR1-mediated current responses. Following G protein blockade, the residual mGluR1-mediated inward current was significantly reduced by the selective Src tyrosine kinase inhibitor PP2. In contrast to cerebellar PCs, GABA_B receptor activation in MLIs did not alter the mGluR1-mediated inward current, suggesting that there is no cross-talk between mGluR1 and GABA_B receptors in MLIs. Thus, activation of mGluR1 facilitates firing of MLIs through the TRPC1-mediated inward current, which depends on not only G protein-dependent but also Src–ERK1/2-dependent signaling pathways, and consequently depresses the excitability of cerebellar PCs.     

c-FLIP Protects Eosinophils from TNF-α-Mediated Cell Death In Vivo

Understanding the signals that regulate eosinophil survival and death is critical to developing new treatments for asthma, atopy, and gastrointestinal disease. Previous studies suggest that TNF-α stimulation protects eosinophils from apoptosis, and this TNF-α-mediated protection is mediated by the upregulation of an unknown protein by NF-κB. Here, we show for the first time that eosinophils express the caspase 8-inhibitory protein c-FLIP, and c-FLIP expression is upregulated upon TNF-α stimulation. Considering that c-FLIP expression is regulated by NF-κB, we hypothesized that c-FLIP might serve as the “molecular switch” that converts TNFRI activation to a pro-survival signal in eosinophils. Indeed, we found that one c-FLIP isoform, c-FLIP_L, is required for mouse eosinophil survival in the presence of TNF-α both in vitro and in vivo. Importantly, our results suggest c-FLIP as a potential therapeutic target for the treatment of eosinophil-mediated disease.     

Caspase-3 is Involved in IFN-γ- and TNF-α-Mediated MIN6 Cells Apoptosis via NF-κB/Bcl-2 Pathway

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the β-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic β-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it’s activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.     

Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

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LncRNA UCA1 Suppresses the Inflammation Via Modulating miR-203-Mediated Regulation of MEF2C/NF-κB Signaling Pathway in Epilepsy

Although many advances have been made in the pathogenesis of epilepsy recently, the pathological mechanisms of epilepsy are still largely unknown. Exploring the pathological mechanisms and developing novel therapeutic strategies for epilepsy are urgently needed. A SD rat model of epilepsy was established with lithium chloride-pilocarpine. Astrocytes were isolated, cultured from 8 to 12 week rats and identified by flow cytometry and immunofluorescence. Immunohistochemical staining was used for MEF2C and NF-κB in paraffin-embedded sections. RT-qPCR and western blot were used to analyze gene expression. ELISA was used to analyze the concentration of IL-6, TNF-α and Cox-2. Cells were transfected with pcDNA-MEFC2, sh-MEFC2, pcDNA-UCA1, sh-UCA1, miR-203 mimic or miR-203 inhibitor. Cell viability was assessed by MTT assay. Dual luciferase assay was used to determine the direct interaction of lncRNA UCA1/miR-203 and miR-203/MEF2C. MEF2C was down-regulated and inhibited NF-κB expression and the secretion of IL-6 and TNF-α in epilepsy. LncRNA UCA1 was also down-regulated in epilepsy. LncRNA UCA1 over-expression increased the expression of MEF2C and its knock-down decreased MEF2C expression. Luciferase activity showed lncRNA UCA1 directly targeted miR-203 and miR-203 directly targeted MEF2C. MiR-203 suppressed the expression of MEF2C, and promoted NF-κB, phosphorylated IκB/IKK and inflammatory effectors, which was reversed by MEF2C knock-down. Moreover, lncRNA UCA1 could increase the expression of MEF2C to inhibit NF-κB, phosphorylated IκB/IKK and inflammatory effectors, which was also reversed by miR-203 mimic transfection. LncRNA UCA1 inhibited the inflammation via regulating miR-203 mediated regulation of MEF2C/NF-κB signaling in epilepsy. Our investigation elucidated novel pathological mechanisms and provided potential therapeutic targets for epilepsy.

     

Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock

Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.     

A Ca2+-Dependent Switch Activates Axonal Casein Kinase 2α Translation and Drives G3BP1 Granule Disassembly for Axon Regeneration

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TREM2 Attenuates Aβ1-42-Mediated Neuroinflammation in BV-2 Cells by Downregulating TLR Signaling

Neurochemical Research - The pathogenesis of late-onset Alzheimer's disease (LOAD) mainly involves abnormal accumulation of extracellular β-amyloid (Aβ) and the consequent neurotoxic...     

Heme Oxygenase-2 Suppress TNF-α and IL6 Expression via TLR4/MyD88-Dependent Signaling Pathway in Mouse Cerebral Vascular Endothelial Cells

Heme oxygenase (HO) represents an intrinsic antiinflammatory system based on its ability to inhibit expression of proinflammatory cytokines. The constitutive isoform heme oxygenase-2 (HO-2) has high expression and activity in cerebral microvascular endothelial cells (CMVEC). This study was undertaken to evaluate the role of HO-2 in regulation of TLR4/MyD88-dependent signaling and to study the effect of HO-2 on the expression and secretion of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and Interleukin-6 (IL6) in CMVEC. HO-2 short hairpin RNA (shRNA) and HO-2 overexpression plasmids were used to observe the effect of HO-2 on proinflammatory cytokines in CMVEC in vitro, and the results showed that the messenger RNA (mRNA) and protein levels of TNF-α and IL6 were increased and decreased, respectively, compared with control groups. LPS-stimulated TNF-α and IL6 mRNA and protein were also reduced in CMVEC treated with an inhibitor of TLR4 signaling, CLI-095, or HO-2 overexpression. CLI-095 and HO-2 overexpression both reduced TLR4 expression in CMVEC, and HO-2 shRNA blocked these effects of CLI-095. CLI-095 and HO-2 overexpression potently suppressed TLR4/MyD88-dependent proinflammatory cytokine expression in CMVEC. These results suggest that HO-2 plays an important role in protecting CMVEC against cytokine-mediated inflammation.     

Notch-1 Signaling Regulates Microglia Activation via NF-κB Pathway after Hypoxic Exposure In Vivo and In Vitro

Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain.     

Platycodin D Inhibits β-Amyloid-Induced Inflammation and Oxidative Stress in BV-2 Cells Via Suppressing TLR4/NF-κB Signaling Pathway and Activating Nrf2/HO-1 Signaling Pathway

Neurochemical Research - Alzheimer’s disease (AD) is a common neurodegenerative disease associated with deposition of β-amyloid peptide (Aβ). Platycodin D (PLD), a...     

Resveratrol inhibits necroptosis by mediating the TNF-α/RIP1/RIP3/MLKL pathway in myocardial hypoxia/reoxygenation injury

Resveratrol (RES) protects myocardial cells from hypoxia/reoxygenation (H/R)-caused injury.However,the mechanism of this effect has not been clarified.Thus,in t...     

The Enterocyte-Associated Intestinal Microbiota of Breast-Fed Infants and Adults Responds Differently to a TNF-α-Mediated Pro-Inflammatory Stimulus

Co-evolved as an integral component of our immune system, the gut microbiota provides specific immunological services at different ages, supporting the immune education during our infancy and sustaining a well-balanced immunological homeostasis during the course of our life. In order to figure out whether this involves differences in the microbial groups primarily interacting with the host immune system, we developed a non-invasive HT29 cell-based minimal model to fingerprint the enterocyte-associated microbiota fraction in infants and adults. After depicting the fecal microbial community of 12 breast-fed infants and 6 adults by 16S rDNA amplicon pools 454 pyrosequencing, their respective HT29 cell-associated gut microbiota fractions were characterized by the universal phylogenetic array platform HTF-Microbi.Array, both in the presence and absence of a tumor necrosis factor-alpha (TNF-α)-mediated pro-inflammatory stimulus. Our data revealed remarkable differences between the enterocyte-associated microbiota fractions in breast-fed infants and adults, being dominated by Bifidobacterium and Enterobacteriaceae the first and Bacteroides-Prevotella and Clostridium clusters IV and XIVa the second. While in adults TNF-α resulted in a profound impairment of the structure of the enterocyte-associated microbiota fraction, in infants it remained unaffected. Differently from the adult-type gut microbial community, the infant-type microbiota is structured to cope with inflammation, being co-evolved to prime the early immune response by means of transient inflammatory signals from gut microorganisms.     

Serotonin 5-HT2A Receptor Activation Blocks TNF-α Mediated Inflammation In Vivo

Tumor necrosis factor alpha (TNF-α) plays a key role in inflammation, and its production and signaling contribute to many inflammatory related diseases. Recently, we discovered that selective activation of serotonin 5-HT_2A receptors with the agonist (R)-DOI produces a super-potent blockade of proinflammatory markers in primary rat aortic smooth muscle cells. Here, we demonstrate that systemic administration of (R)-DOI can block the systemic effects of TNF-α in whole animal, with potent anti-inflammatory effects in the aortic arch and small intestine. This includes blockade of TNF-α-induced expression of pro-inflammatory cell adhesion (Icam-1, Vcam-1), cytokine (Il-6, IL-1b), and chemokine (Mcp-1, Cx3cl1) genes, and expression of VCAM-1 protein in the intestine. Further, systemic (R)-DOI also prevents the TNF-α-induced increase of circulating IL-6. Importantly, utilizing receptor selective antagonists, we have demonstrated that the mechanism underlying the systemic anti-inflammatory effects of (R)-DOI is activation of serotonin 5-HT_2A receptors. Our results highlight a powerful new role for the serotonin 5-HT_2A receptor in inflammatory processes, and indicate that agonism of serotonin receptors may represent an effective and novel approach to develop powerful small molecule therapeutics for inflammatory diseases and conditions such as atherosclerosis and inflammatory bowel disease.