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.

Taurine has potent protective function against glutamate-induced neuronal injury presumably through its function in regulation of intracellular free calcium level, [Ca2+]i. In this communication, we report that taurine exerts its protective function through one or more of the following mechanisms: 1. Inhibition of glutamate-induced calcium influx through L-, N- and P/Q-type voltage-gated calcium channels and NMDA receptor calcium channel; 2. Attenuation of glutamate-induced membrane depolarization; 3. Prevention of glutamate-induced apoptosis via preventing glutamate-mediated down-regulation of Bcl-2; 4. Prevention of cleavage of Bcl-2 by calpain. This action of taurine is due to its inhibition on glutamate induced calpain activation. Based on these observations, we propose that taurine protects neurons against glutamate-induced neurotoxicity in part, by preventing glutamate-induced membrane depolarization, elevation of [Ca2+]i, activation of calpain, reduction of Bcl-2 and apoptosis.

Previously, we reported that L-glutamic acid decarboxylase isoform 65 (GAD65) could be cleaved in vitro to release a stable truncated form which lacks amino acid 1-69 from the N-terminus, GAD65(D1-69). However, whether such a truncated form is also present under certain physiological conditions remains elusive. In the present study, we showed that, upon sustained neuronal stimulation, GAD65 could be cleaved into a truncated form in a rat synaptosomal preparation. This truncated form had similar electrophoretic mobility to purified recombinant human GAD65(D1-69). Furthermore, we demonstrated that this conversion was calcium dependent. Calcium-chelating reagents such as EDTA and 1,2-bis-(oaminphenoxy)-ethane-N,N,N¢,N¢-tetra-acetic acid tetra-acetoxy-methyl ester prevented the cleavage of GAD65. In addition, our data suggested that calpain, a calcium-dependent cysteine protease, is activated upon neuronal stimulation and could be responsible for the conversion of full-length GAD65 to truncated GAD65 in the brain. Moreover, calpain inhibitors such as calpain inhibitor I or calpastatin could block the cleavage. Results of our in vitro cleavage assay using purified calpain and immunopurified rat GAD65 also supported the idea that GAD65 could be directly cleaved by calpain.

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for γ-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that μ-calpain is a good candidate for conversion of fGAD 67 to tGAD 67 . This conclusion is based on the following observations: 1. Purified recombinant GAD 67 is cleaved by μ-calpain at specific sites; 2. In brain synaptosomal preparation, GAD 67 is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. In μ-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when μ-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; 5. μ-calpain is activated by neuronal stimulation and Ca 2+ -influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed. KeywordsCalpain; GABA synthesis; GAD; Proteolytic CleavageIn the central nervous system (CNS), γ-aminobutyric acid (GABA) is synthesized by a single enzymatic reaction catalyzed by L-glutamic decarboxylase (EC 4.1.1.15; GAD) (1,2). Mammalian species express two isoforms of GAD, namely, GAD 65 and GAD 67 , referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Previously, we reported **Address correspondence and reprint requests to Dr. Jang-Yen Wu, Florida Atlantic University, Department of Biomedical Science, 777 Glades Road, Boca Raton, FL 33431-0991, U.S. ; Email: jwu@fau.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the presence of the truncated GAD (tGAD) derived from proteolytic cleavage of the full-length (fGAD) in vivo as well as in vitro (3,4). The presence of smaller forms of GAD was also observed from other laboratories (5-8). However, no information was reported regarding the identity of the endogenous proteases responsible for GAD cleavage and their physiological significance. Recently, we found that recombinant human brain GAD 65 is specifically cleaved at arginine 69 from the N-terminal of fGAD 65 to produce tGAD 65 , which is ∼2-3 fold more active than fGAD 65 (3). In addition, GAD 67 was found to be cleaved at two specific sites, one at arginine 70 and another at arginine 90, to produ...

Prenatal exposure to buprenorphine renders offspring vulnerable to cerebral impairments. In this study, our data demonstrate, for the first time, that prenatal exposure to buprenorphine escalates astrocyte activation concurrent with indications of endoplasmic reticulum (ER) stress in the hippocampi of neonates, and this can be prevented by the coadministration of dextromethorphan with buprenorphine. Furthermore, dextromethorphan can inhibit the accumulation of GPR37 in the hippocampus of newborns caused by buprenorphine and is accompanied by the proapoptotic ER stress response that involves the procaspase-3/CHOP pathway. Primary astrocyte cultures derived from the neonates of the buprenorphine group also displayed aberrant ER calcium mobilization and elevated basal levels of cyclooxygenase-2 (COX-2) at 14 days in vitro while showing sensitivity to lipopolysaccharide-activated expression of COX-2. Similarly, these long-lasting defects in the hippocampus and astrocytes were abolished by dextromethorphan. Our findings suggest that prenatal exposure to buprenorphine might instigate long-lasting effects on hippocampal and astrocytic functions. The beneficial effects of prenatal coadministration of dextromethorphan might be, at least in part, attributed to its properties in attenuating astrocyte activation and hippocampal ER stress in neonates.