Growth-associated protein 43 (GAP43), a protein kinase C (PKC)-activated phosphoprotein, is often implicated in axonal plasticity and regeneration. In this study, we found that GAP43 can be induced by the endotoxin lipopolysaccharide (LPS) in rat brain astrocytes both in vivo and in vitro. The LPS-induced astrocytic GAP43 expression was mediated by Toll-like receptor 4 and nuclear factor-B (NF-B)-and interleukin-6/signal transducer and activator of transcription 3 (STAT3)-dependent transcriptional activation. The overexpression of the PKC phosphorylation-mimicking GAP43 S41D (constitutive active GAP43) in astrocytes mimicked LPS-induced process arborization and elongation, while application of a NF-B inhibitory peptide TAT-NBD or GAP43 S41A (dominant-negative GAP43) or knockdown of GAP43 all inhibited astrogliosis responses. Moreover, GAP43 knockdown aggravated astrogliosis-induced microglial activation and expression of proinflammatory cytokines. We also show that astrogliosis-conditioned medium from GAP43 knock-down astrocytes inhibited GAP43 phosphorylation and axonal growth, and increased neuronal damage in cultured rat cortical neurons. These proneurotoxic effects of astrocytic GAP43 knockdown were accompanied by attenuated glutamate uptake and expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in LPS-treated astrocytes. The regulation of EAAT2 expression involves actin polymerization-dependent activation of the transcriptional coactivator megakaryoblastic leukemia 1 (MKL1), which targets the serum response elements in the promoter of rat Slc1a2 gene encoding EAAT2. In sum, the present study suggests that astrocytic GAP43 mediates glial plasticity during astrogliosis, and provides beneficial effects for neuronal plasticity and survival and attenuation of microglial activation. Key words: astrogliosis; EAAT2; GAP43; microglial activation; MKL1; neurotoxicity Significance StatementAstrogliosis is a complex state in which injury-stimulated astrocytes exert both protective and harmful effects on neuronal survival and plasticity. In this study, we demonstrated for the first time that growth-associated protein 43 (GAP43), a well known growth cone protein that promotes axonal regeneration, can be induced in rat brain astrocytes by the proinflammatory endotoxin lipopolysaccharide via both nuclear factor-B and signal transducer and activator of transcription 3-mediated transcriptional activation. Importantly, LPS-induced GAP43 mediates plastic changes of astrocytes while attenuating astrogliosis-induced microglial activation and neurotoxicity. Hence, astrocytic GAP43 upregulation may serve to indicate beneficial astrogliosis after CNS injury.
The aryl hydrocarbon receptor (AhR) regulates peripheral immunity; but its role in microglia-mediated neuroinflammation in the brain remains unknown. Here, we demonstrate that AhR mediates both anti-inflammatory and proinflammatory effects in lipopolysaccharide (LPS)-activated microglia. Activation of AhR by its ligands, formylindolo[3,2-b]carbazole (FICZ) or 3-methylcholanthrene (3MC), attenuated LPS-induced microglial immune responses. AhR also showed proinflammatory effects, as evidenced by the findings that genetic silence of AhR ameliorated the LPS-induced microglial immune responses and LPS-activated microglia-mediated neurotoxicity. Similarly, LPS-induced expressions of tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were reduced in the cerebral cortex of AhR-deficient mice. Intriguingly, LPS upregulated and activated AhR in the absence of AhR ligands via the MEK1/2 signaling pathway, which effects were associated with a transient inhibition of cytochrome P450 1A1 (CYP1A1). Although AhR ligands synergistically enhance LPS-induced AhR activation, leading to suppression of LPS-induced microglial immune responses, they cannot do so on their own in microglia. Chromatin immunoprecipitation results further revealed that LPS-FICZ co-treatment, but not LPS alone, not only resulted in co-recruitment of both AhR and NFκB onto the κB site of TNFα gene promoter but also reduced LPS-induced AhR binding to the DRE site of iNOS gene promoter. Together, we provide evidence showing that microglial AhR, which can be activated by LPS, exerts bi-directional effects on the regulation of LPS-induced neuroinflammation, depending on the availability of external AhR ligands. These findings confer further insights into the potential link between environmental factors and the inflammatory brain disorders.
NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity‐dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA‐increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor‐mediated spontaneous as well as miniature excitatory post‐synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application– but not synaptic NMDA receptor‐induced brain‐derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA‐induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA‐induced BDNF expression and the binding activity of cAMP‐responsive element binding protein (CREB) and its calcium‐dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor‐mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.
Curcumin has been proposed for treatment of various neuroinflammatory and neurodegenerative conditions, including post-traumatic inflammation during acute spinal cord injury (SCI). In this study, we examined whether curcumin anti-inflammation involves regulation of astrocyte reactivation, with special focus on the injury-induced RANTES (regulated on expression normal T-cell expressed and secreted) from astrocytes in acute SCI. Male Sprague-Dawley (SD) rats were subjected to impact injury of the spinal cord followed by treatment with curcumin (40 mg/kg i.p.). RANTES and inducible nitric oxide synthase expression as well as RANTES-positive astrocytes were all induced by injury accompanied by the elevation of lipid peroxidation, and attenuated by the curcumin treatment. In primary cultured rat astrocytes challenged with lipopolysaccharide (LPS) to mimic astrocyte reactivation following SCI, LPS induces robust increase of RANTES expression and the effect was also reduced by 1 μM curcumin treatment. Furthermore, cortical neurons cultured with astrocyte conditioned medium (ACM) conditioned with both LPS and curcumin (LPS-curcumin/ACM), which characteristically exhibited decreased RANTES expression when compared with ACM from astrocytes treated with LPS alone (LPS/ACM), showed higher level of cell viability and lower level of cell death as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity assay and lactate dehydrogenase release assay, respectively. Knockdown of RANTES expression by siRNA (siRANTES) shows reduced RANTES expression and release from LPS-reactivated astrocytes, and ACM obtained from this condition (LPS-siRANTES/ACM) becomes less cytotoxic as compared with the LPS-ACM. Therefore, curcumin reduction of robust RANTES production in reactivated astrocytes both in vitro and in vivo may contribute to its neuroprotection and potential application in SCI.
Motorcycle exhaust particulates (MEP) contain carcinogenic polycyclic aromatic hydrocarbons including benzo(a)pyrene. This study has determined the ability of MEP to alter the expression of select genes from drug metabolism, cytokine, oncogene, tumor suppressor, and estrogen signaling families of human lung adenocarcinoma CL5 cells. cDNA microarray analyses and confirmation studies were performed using CL5 cells treated with 100 microg/ml MEP extract for 6 h. The results showed that MEP increased the mRNA levels of metabolic enzymes CYP1A1 and CYP1B1, proinflammatory cytokines interleukin (IL)-1alpha, IL-6, and IL-11, fibroblast growth factor (FGF)-6 and FGF-9, vascular endothelial growth factor (VEGF)-D, oncogene fra-1, and tumor suppressor p21. In contrast, MEP decreased tumor suppressor Rb mRNA in CL5 lung epithelial cells. Treatment with 10 microM benzo(a)pyrene for 6 h altered gene expression profiles, in a manner similar to those by MEP. Induction of IL-1alpha, IL-6, IL-11, and FGF-9 mRNA by MEP and benzo(a)pyrene was concentration and time dependent. Cotreatment with 2 mM N-acetylcysteine blocked the MEP- and benzo(a)pyrene-mediated induction. Treatment with MEP or benzo(a)pyrene increased IL-6 and IL-11 releases to CL5 cell medium. Incubation of human lung fibroblast WI-38 with MEP- or benzo(a)pyrene-induced CL5 conditioned medium for 4 days stimulated cell growth of the fibroblasts. Inhalation exposure of rats to 1:10 diluted motorcycle exhaust 2 h daily for 4 weeks increased CYP1A1, FGF-9, and IL-1alpha mRNA in lung. This present study shows that MEP and benzo(a)pyrene can induce metabolic enzyme, inflammatory cytokine, and growth factor gene expression in CL5 cells and stimulate lung epithelium-fibroblast interaction.
g Growth-associated protein 43 (GAP43) is known to regulate axon growth, but whether it also plays a role in synaptogenesis remains unclear. Here, we found that GAP43 regulates the aggregation of gephyrin, a pivotal protein for clustering postsynaptic GABA A receptors (GABA A Rs), in developing cortical neurons. Pharmacological blockade of either protein kinase C (PKC) or neuronal activity increased both GAP43-gephyrin association and gephyrin misfolding-induced aggregation, suggesting the importance of PKC-dependent regulation of GABAergic synapses. Furthermore, we found that PKC phosphorylation-resistant GAP43 S41A , but not PKC phosphorylation-mimicking GAP43 S41D , interacted with cytosolic gephyrin to trigger gephyrin misfolding and its sequestration into aggresomes. In contrast, GAP43 S41D , but not GAP43 S41A , inhibited the physiological aggregation/clustering of gephyrin, reduced surface GABA A Rs under physiological conditions, and attenuated gephyrin misfolding under transient oxygen-glucose deprivation (tOGD) that mimics pathological neonatal hypoxia. Calcineurin-mediated GAP43 dephosphorylation that accompanied tOGD also led to GAP43-gephyrin association and gephyrin misfolding. Thus, PKC-dependent phosphorylation of GAP43 plays a critical role in regulating postsynaptic gephyrin aggregation in developing GABAergic synapses. Proper development of inhibitory GABAergic synapses is critical for establishing an excitatory/inhibitory balance in the neural network (1, 2). The impairment of postsynaptic GABA A receptor (GABA A R) activity is a major cause of neuronal hyperactivity, affecting cognitive development and psychosocial behaviors (3, 4). Postsynaptic surface insertion and clustering of GABA A Rs determine the efficacy of GABAergic synapses (4, 5). Gephyrin, a microtubule-associated protein, is a key scaffolding protein that requires the GABA A R ␥2 subunit for clustering GABA A Rs at the postsynaptic membrane (6, 7). The lack of neuronal gephyrin reduces postsynaptic GABA A R clustering, thereby impairing inhibitory synaptic transmission (8, 9).In central neurons, gephyrin monomers oligomerize to form a hexagonal lattice, also called gephyrin clusters, underneath the cell surface membrane to anchor postsynaptic GABA A Rs (10). However, numerous studies have shown that gephyrin is an aggregation-prone protein that forms large clumps when expressed in nonneural cells or cell-free systems (11, 12). Instead, gephyrin in neurons forms small aggregates/clusters in both the cytosol and submembrane domain for receptor clustering, suggesting a neuronal machinery that regulates gephyrin clustering. To date, a postsynaptic protein, collybistin, a GDP-GTP exchanging factor, is the only gephyrin-interacting protein that can effectively disperse gephyrin clumps into oligomeric clusters in HEK293T cells (13). Gephyrin scaffolding in neurons depends on the dynamic rearrangement of microtubules and actin microfilaments at postsynaptic sites (14,15). Whether cytoskeleton-associated proteins are involved in regulat...
Purpose -The purpose of this paper is to examine how a graduate institute at National Chiayi University (NCYU), by using a model that integrates analytic hierarchy process, cluster analysis and correspondence analysis, can develop effective marketing strategies. Design/methodology/approach -This is primarily a quantitative study aimed at developing a marketing mix for a graduate institute at NCYU in Taiwan. A survey using stratified random sampling was conducted, with 14 universities from four different areas in Taiwan randomly selected for the study. Two questionnaires were conducted: a Likert's five-scale questionnaire regarding school images and an analytic hierarchy process (AHP) questionnaire regarding school selection factors were administered to 640 undergraduate students. Of the total number of questionnaires, 602 (94 percent) valid school image questionnaires and 570 (89 percent) valid school selection factors questionnaires were used. Findings -The results of AHP revealed that the five most important factors for students' school selection were: employability, curriculum, academic reputation, faculty, and research environment. The results of clustering analysis identified five student groups for market segmentation, and they are the Prominence group, the Less aware group, the Pragmatic group, the Austerity group, and the Fastidious group. Finally, the results of correspondence analysis suggested that students of the Pragmatic Group are more likely to be attracted by NCYU, and also, students perceived NCYU to be strongly associated with lower tuition, fewer entrance-exam subjects, lower entrance-exam pass rates, and easier graduation requirements. Research limitations/implications -It would be better to conduct a factor analysis before using AHP. Practical implications -Particularly, NCYU should establish new curricula relevant to internationalization, develop curricula in school finance and educational economics, and form study groups to enhance graduating student employment opportunities. Generally, higher educational institutions may adopt the research model developed in this study to develop their marketing mix for better results. Originality/value -This paper documents research that was the first to integrate AHP, cluster analysis, and correspondence analysis in developing a marketing mix for higher educational institutions.
The emissions from 2- and 4-stroke motorcycles pollute the air of urban areas where motorcycle is a popular means of transportation. This study aimed to determine the endocrine-disrupting activity of motorcycle exhaust particulate (MEP) using MCF-7 human breast cancer cells and immature female Wistar rats treated with organic extracts of MEP. Treatments with 1, 10, and 50 microg/ml MEP extract for 2 and 4 days produced dose-dependent inhibition of thymidine incorporation and cell growth, respectively, in untreated and 1 nM 17beta-estradiol (E2)-treated cells. Treatments of MCF-7 cells with MEP extract replaced [3H]E2 from the estrogen receptor in a time- and concentration-dependent manner. These antiestrogenic and receptor binding properties of MEP extract were blocked by cotreatment of the cells with 2 microM alpha-naphthoflavone, a cytochrome P450 inhibitor and aryl hydrocarbon receptor antagonist. E2 metabolism and HPLC analysis showed that treatment of MCF-7 cells with 50 microg/ml MEP extract for 24 h increased E2 2- and 4-hydroxylation in microsomes. The MEP-mediated increase in E2 2-hydroxylation was inhibited by the addition of 1 microM alpha-naphthoflavone to MCF-7 microsomes. Cotreatment of immature female rats with 10 microg/kg E2 and 10 mg/kg MEP extract intraperitoneally for 3 days decreased the E2-induced uterine weights. MEP extract alone showed no effect on rat uterine weight. The endocrine-disrupting activity of MEP extract was further confirmed in parallel experiments using MCF-7 cells and immature female rats treated with benzo(a)pyrene, an MEP constituent compound. The present findings demonstrate that MEP extract is antiestrogenic in vitro and in vivo and cytochrome P450 induction is an underlying mechanism.
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