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.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix/ Per-Arnt-Sim transcription factor superfamily (reviewed by Rowlands and Gustafsson 1997;Hahn 1998 ] i , intracellular calcium concentration; AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; BME, basal medium Eagles; CaMK, calcium/calmodulindependent protein kinase; CBP, cAMP-responsive-element binding protein; ChIP, chromatin immunoprecipitation; CYP1A1, cytochrome P450 1A1; DCF, 2,7-dichlorodihydrofluorescein; DIV, days in vitro; DRE, dioxin-responsive elements; FBS, fetal bovine serum; fura-2AM, fura-2 acetoxymethyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMDA-R, NMDA receptor; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SD, Sprague-Dawley; siAhR, AhR small interfering RNA; siRNA, small interfering RNA; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TTX, tetrodotoxin. AbstractThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor activated by dioxin and polyaromatic hydrocarbons. Recent studies have revealed that AhR activity in central neurons depends on the NMDA receptor. In this study, we investigated how the neuronal activity influence AhR-mediated dioxin-responsive gene expression and neurotoxicity. Our results show that activation of AhR by the selective agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin induced dioxin-responsive gene expression and calcium entry, which were attenuated by AhR small interfering RNA, the NMDA receptor channel blocker MK801, and the action potential blocker tetrodotoxin (TTX). In addition, AhR-mediated gene expression was enhanced in neurons during synaptogenesis (10 days in vitro) compared with younger neurons (4 days in vitro), as was sensitivity to TTX and MK801. Furthermore, TTX and MK801 differentially affected the association of AhR and its transcriptional co-activator cAMP-responsive-element binding protein with the cytochrome P450 1A1 (cyp1A1) gene enhancer. Calcium/calmodulin-dependent protein kinase IV, the cAMP-responsive-element binding protein activating enzyme, was also activated by 2,3,7,8-tetrachlorodibenzop-dioxin in an activity-dependent manner. Finally, we found that neuronal susceptibility to dioxin insult was also maturation and activity-dependent. Together, the results suggest that neuronal activity may facilitate AhR-mediated calcium signaling, which in turn enhances AhR-mediated gene regulation and mediated maturation-dependent dioxin neurotoxicity. Keywords: aryl hydrocarbon receptor, calcium/calmodulindependent protein kinase IV, CREB binding protein, neuronal activity, neuronal survival, NMDA receptor.

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.