Other articles by Ronald B. Tjalkens on PubMed

• CI-1010 Induced Opening of the Mitochondrial Permeability Transition Pore Precedes Oxidative Stress and Apoptosis in SY5Y Neuroblastoma Cells Brain Research. Feb, 2003  |   Pubmed ID: 12560110 The hetero-bifunctional nitroimidazole radiosensitizer CI-1010, R-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide, causes selective irreversible apoptotic loss of retinal photoreceptor cells in vivo. The human neuroblastoma cell line, SH-SY5Y, was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in neuroblastoma cells, as determined by histopathological and ultrastructural analysis and by TUNEL technique. CI-1010 causes a dose-dependent decrease in cell viability in SY5Y cells, as measured by the reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Superoxide dismutase reduced loss of cell viability following CI-1010 treatment suggesting an oxidative stress-mediated mechanism of toxicity. The effects of CI-1010 on mitochondrial membrane potential and intracellular levels of reactive oxygen species were assessed in live SY5Y cells by confocal microscopy using the fluorescent dyes, tetramethylrhodamine methyl ester and 5,6-carboxy-2',7'-dihydrodichlorofluorescein diacetate. CI-1010 caused a rapid depolarization of mitochondria in SY5Y cells followed by an increase in ROS. Both CI-1010-induced mitochondrial depolarization and subsequent increases in ROS were prevented by pretreatment with either the permeability transition pore inhibitor, cyclosporin A (CsA), and by the antioxidant, alpha-tocopherol. However, CsA and alpha-tocopherol were unable to prevent apoptosis in CI-1010-treated cells, suggesting the influence of additional mechanism(s) of CI-1010-induced toxicity. This study evaluates intracellular oxidative stress associated with pore opening prior to apoptosis and provides evidence in support of a mitochondrial mechanism of CI-1010-induced neuronal cell death.
• Prenatal Expression of N-acetyltransferases in C57Bl/6 Mice Chemico-biological Interactions. Mar, 2003  |   Pubmed ID: 12606156 Exposure to carcinogens such as 4-aminobiphenyl (4ABP), found in tobacco smoke and other combustion products, results in the formation of detectable levels of 4ABP-hemoglobin adducts in cord blood and 4ABP-DNA adducts in conceptal tissue. The presence of these adducts requires that the parent compound undergo biotransformation. When exposure occurs in utero, the maternal, placental and conceptal tissues are all possible sites for the formation of DNA-reactive products. One step in the activation of 4ABP is catalyzed by N-acetyltransferases (NAT). The expression of NAT was evaluated in gestational day (GD) 10-18 conceptal tissues from C57Bl/6 mice. There was a quantitative increase in NAT1 and NAT2 mRNAs with increasing gestational age that was also reflected in age-related changes in functional protein measured as 4ABP-NAT activity. The ability to acetylate 4ABP increased from GD10 to 18 and was lower in conceptal tissue than in adult liver. The potential toxicologic significance of prenatal NAT expression was assessed by formation of 4ABP-DNA adducts. At GD 15 and 18, 4ABP-DNA adducts were detected by immunohistochemistry 24 h following a single oral dose of 120 mg 4ABP/kg. Based on nuclear fluorescence, conceptual 4ABP-DNA adducts were present at similar levels at GD15 and 18. Levels of 4ABP-DNA adducts were significantly higher in maternal liver compared with the conceptus. Results from this study show that both NAT genes were expressed prenatally and that functional enzymes were present. These data support the possible in situ generation of reactive products by the conceptus. The relative contributions of maternal activation of 4ABP and that by the conceptus remain to be determined.
• Regional Variation in the Activation Threshold for 1,3-DNB-induced Mitochondrial Permeability Transition in Brainstem and Cortical Astrocytes Neurotoxicology. Jun, 2003  |   Pubmed ID: 12782104 1,3-Dinitrobenzene (DNB) produces edematous, glio-vascular lesions in brainstem nuclei with high energy demands. Astrocytes in vulnerable brainstem nuclei appear to be an early and selective target of DNB and other nitroaromatic compounds, though the molecular basis of this susceptibility is poorly understood. It has been postulated that mitochondria are a principal target of DNB in sensitive cell types [Neuropathol. Appl. Neurobiol. 13 (5) (1987) 371], where redox-cycling of DNB increases levels of reactive oxygen species and disrupts cellular energy metabolism. The present study investigates the role of regional differences in activation of the mitochondrial permeability transition pore (mtPTP) by DNB in brainstem and cortical astrocytes and examines the expression of Bcl-2 proteins as potential regulators of mtPTP function. Neonatal rat astrocytes were cultured from both DNB-sensitive (brainstem) and insensitive (cortex) brain regions and evaluated for DNB-induced alterations in cell morphology and mitochondrial function. Exposure to DNB resulted in rapid changes in the morphology of brainstem astrocytes consistent with loss of ion homeostasis and initiation of necrotic cell death. These changes were not observed in cortical astrocytes at corresponding concentrations of DNB and were prevented in brainstem astrocytes by the mtPTP inhibitor, bongkrekic acid, suggesting that mitochondrial dysfunction is involved in DNB-induced morphological changes in brainstem astrocytes. Mitochondrial depolarization in brainstem astrocytes was observed at DNB concentrations as low as 10 microM, whereas no loss of mitochondrial membrane potential (DeltaPsi(mt)) occurred in cortical astrocytes at less than 100 microM DNB. DNB-induced loss of DeltaPsi(mt) followed apparent first-order kinetics, with EC(50)-values for half-maximal rates of mitochondrial depolarization of approximately 23 and approximately 290 microM in brainstem cortical astrocytes, respectively. DNB-induced mitochondrial depolarization was prevented by pretreatment with bongkrekic acid, indicating that loss of DeltaPsi(mt) was mediated by activation of the mtPTP. Inhibition of succinate dehydrogenase (SDH) activity occurred in astrocytes from both brain regions exposed to DNB and was blocked in brainstem, but not cortical, astrocytes by bongkrekic acid. Constitutive expression of Bcl-X(L) was high in cortical tissue and astrocytes, whereas Bax expression was low. However, Bax was highly expressed in brainstem tissue and astrocytes and Bcl-X(L) expression was markedly lower. The expression of Bcl-2 was similar in both brain regions. These data suggest that the selective vulnerability of brainstem astrocytes to DNB is due to a lower threshold for activation of the mtPTP that is be mediated, in part, by distinct expression patterns of Bcl-2 proteins rather than by intrinsic differences in susceptibility of the electron transport chain.
• The Effects of Genetic Variation in N-acetyltransferases on 4-aminobiphenyl Genotoxicity in Mouse Liver Chemico-biological Interactions. Jul, 2003  |   Pubmed ID: 12902152 Inbred, congenic and transgenic strains of mice were characterized for acetylation of p-aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1, the functional equivalent of murine NAT2, was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean+/-S.D.) were 41.1+/-3.6 for C57Bl/6, 37.9+/-1.11 for A/J and 36.3+/-2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.
• Manganese Potentiates Lipopolysaccharide-induced Expression of NOS2 in C6 Glioma Cells Through Mitochondrial-dependent Activation of Nuclear Factor KappaB Brain Research. Molecular Brain Research. Mar, 2004  |   Pubmed ID: 15010209 Neuronal injury in manganese neurotoxicity (manganism) is thought to involve activation of astroglial cells and subsequent overproduction of nitric oxide (NO) by inducible nitric oxide synthase (NOS2). Manganese (Mn) enhances the effects of proinflammatory cytokines on expression of NOS2 but the molecular basis for this effect has not been established. It was postulated in the present studies that Mn enhances expression of NOS2 through the cis-acting factor, nuclear factor kappaB (NF-kappaB). Exposure of C6 glioma cells to lipopopolysaccharide (LPS) resulted in increased expression of NOS2 and production of NO that was dramatically potentiated by Mn and was blocked through overexpression of mutant IkappaBalpha (S32/36A). LPS-induced DNA binding of p65/p50 was similarly enhanced by Mn and was decreased by mutant IkappaBalpha. Phosphorylation of IkappaBalpha was potentiated by Mn and LPS and was not blocked by U0126, a selective inhibitor of ERK1/2. Mn decreased mitochondrial membrane potential and increased matrix calcium, associated with a rise in intracellular reactive oxygen species (ROS) that was attenuated by the mitochondrial-specific antioxidant, MitoQ. Blocking mitochondrial ROS also attenuated the enhancing effect of Mn on LPS-induced phosphorylation of IkappaBalpha and expression of NOS2, suggesting a link between Mn-induced mitochondrial dysfunction and activation of NF-kappaB. Overexpression of a dominant-negative mutant of the NF-kappaB-interacting kinase (Nik) prevented enhancement of LPS-induced phosphorylation of IkappaBalpha by Mn. These data indicate that Mn augments LPS-induced expression of NOS2 in C6 cells by increasing mitochondrial ROS and activation of NF-kappaB.
• The Role of Docosahexaenoic Acid in Mediating Mitochondrial Membrane Lipid Oxidation and Apoptosis in Colonocytes Carcinogenesis. Nov, 2005  |   Pubmed ID: 15975958 Docosahexaenoic acid (DHA, 22:6 n-3) from fish oil, and butyrate, a fiber fermentation product, work coordinately to protect against colon tumorigenesis in part by inducing apoptosis. We have recently demonstrated that dietary DHA is incorporated into mitochondrial membrane phospholipids, thereby enhancing oxidative stress induced by butyrate metabolism. In order to elucidate the subcellular origin of oxidation induced by DHA and butyrate, immortalized young adult mouse colonocytes were treated with 0-200 microM DHA or linoleic acid (LA, 18:2 n-6; control) for 72 h with or without 5 mM butyrate for the final 24 h. Cytosolic reactive oxygen species, membrane lipid oxidation, and mitochondrial membrane potential (MP), were measured by live-cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells exhibited a 151% increase in lipid oxidation (P < 0.01), compared with no butyrate treatment, which could be blocked by a mitochondria-specific antioxidant, 10-(6'-ubiquinoyl) decyltriphenylphosphonium bromide (MitoQ) (P < 0.05). Butyrate treatment of LA pretreated cells did not show any significant effect. In the absence of butyrate, DHA treatment, compared with LA, increased resting MP by 120% (P < 0.01). In addition, butyrate-induced mitochondrial membrane potential (MP), dissipation was 21% greater in DHA primed cells as compared with LA at 6 h. This effect was reversed by preincubation with inhibitors of the mitochondrial permeability transition pore, cyclosporin A or bongkrekic acid (1 microM). The functional importance of these events is supported by the demonstration that DHA and butyrate-induced apoptosis is blocked by MitoQ. These data indicate that DHA and butyrate potentiate mitochondrial lipid oxidation and the dissipation of MP which contribute to the induction of apoptosis.
• NF-kappaB-dependent Production of Nitric Oxide by Astrocytes Mediates Apoptosis in Differentiated PC12 Neurons Following Exposure to Manganese and Cytokines Brain Research. Molecular Brain Research. Nov, 2005  |   Pubmed ID: 16168523 Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 1.14.13.39) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IkappaBalpha (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-alpha/IFN-gamma. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-kappaB-dependent expression of NOS2.
• Modulation of Intercellular Calcium Signaling by Melatonin in Avian and Mammalian Astrocytes is Brain Region-specific The Journal of Comparative Neurology. Dec, 2005  |   Pubmed ID: 16261532 Calcium waves among glial cells impact many central nervous system functions, including neural integration and brain metabolism. Here, we characterized the modulatory effects of melatonin, a pineal neurohormone that mediates circadian and seasonal processes, on glial calcium waves derived from different brain regions and species. Diencephalic and telencephalic astrocytes, from both chick and mouse brains, expressed melatonin receptor proteins. Further, using the calcium-sensitive dye Fluo-4, we conducted real-time imaging analyses of calcium waves propagated among mammalian and avian astrocytes. Mouse diencephalic astrocytic calcium waves spread to an area 2-5-fold larger than waves among avian astrocytes and application of 10 nM melatonin caused a 32% increase in the spread of these mammalian calcium waves, similar to the 23% increase observed in chick diencephalic astrocytes. In contrast, melatonin had no effect on calcium waves in either avian or mammalian telencephalic astrocytes. Mouse telencephalic calcium waves radially spread from their initiation site among untreated astrocytes. However, waves meandered among mouse diencephalic astrocytes, taking heterogeneous paths at variable rates of propagation. Brain regional differences in wave propagation were abolished by melatonin, as diencephalic astrocytes acquired more telencephalon-like wave characteristics. Astrocytes cultured from different brain regions, therefore, possess fundamentally disparate mechanisms of calcium wave propagation and responses to melatonin. These results suggest multiple roles for melatonin receptors in the regulation of astroglial function, impacting specific brain regions differentially.
• Manganese-induced Neurotoxicity: the Role of Astroglial-derived Nitric Oxide in Striatal Interneuron Degeneration Toxicological Sciences : an Official Journal of the Society of Toxicology. Jun, 2006  |   Pubmed ID: 16551646 Chronic exposure to excessive manganese (Mn) is the cause of a neurodegenerative movement disorder, termed manganism, resulting from degeneration of neurons within the basal ganglia. Pathogenic mechanisms underlying this disorder are not fully understood but involve inflammatory activation of glial cells within the basal ganglia. It was postulated in the present studies that reactive astrocytes are involved in neuronal injury from exposure to Mn through increased release of nitric oxide. C57Bl/6 mice subchronically exposed to Mn by intragastric gavage had increased levels of Mn in the striatum and displayed diminutions in both locomotor activity and striatal DA content. Mn exposure resulted in neuronal injury in the striatum and globus pallidus, particularly in regions proximal to the microvasculature, indicated by histochemical staining with fluorojade and cresyl fast violet. Neuropathological assessment revealed marked perivascular edema, with hypertrophic endothelial cells and diffusion of serum albumin into the perivascular space. Immunofluorescence studies employing terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (DUTP)-biotin nick-end labeling revealed the presence of apoptotic neurons expressing neuronal nitric oxide synthase (NOS), choline acetyltransferase, and enkephalin in both the striatum and globus pallidus. In contrast, soma and terminals of dopaminergic neurons were morphologically unaltered in either the substantia nigra or striatum, as indicated by immunohistochemical staining for tyrosine hydroxylase. Regions with evident neuronal injury also displayed increased numbers of reactive astrocytes that coexpressed inducible NOS2 and localized with areas of increased neuronal staining for 3-nitrotyrosine protein adducts, a marker of NO formation. These data suggest a role for astrocyte-derived NO in injury to striatal-pallidal interneurons from Mn intoxication.
• Manganese Suppresses ATP-dependent Intercellular Calcium Waves in Astrocyte Networks Through Alteration of Mitochondrial and Endoplasmic Reticulum Calcium Dynamics Brain Research. Oct, 2006  |   Pubmed ID: 16934782 The neurotoxicity of manganese [Mn] is due in part to glutamate excitotoxicity. Release of ATP by astrocytes is a critical modulator of glutamatergic neurotransmission, which is regulated by calcium (Ca(2+)) waves that propagate through astrocytic networks in response to synaptic activity. It was postulated that Mn alters ATP-dependent intracellular Ca(2+) dynamics in astrocytes, thereby suppressing Ca(2+) wave activity. Confluent primary cultures of cortical astrocytes were loaded with the Ca(2+)-sensitive dye fluo-4 and examined by fluorescence microscopy for Ca(2+) wave activity following micropipet mechanical stimulation of a single cell. Mitochondrial Ca(2+) was evaluated by fluorescence microscopy following addition of ATP using the mitochondrial-specific Ca(2+) dye rhod-2-AM. Imaging studies revealed that pretreatment of astrocytes with 1-10 microM Mn significantly reduced the rate, area, and amplitude of mechanically induced Ca(2+) waves. This attenuation was not a result of inhibited mitochondrial calcium uptake because robust calcium waves were still observed following pretreatment of astrocytes with Ru360, an inhibitor of mitochondrial Ca(2+) uptake, either in coupling or uncoupling conditions. However, determination of endoplasmic reticulum (ER) Ca(2+) levels in cells using the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin indicated that Mn reduced the available pool of releasable ER Ca(2+) at concentrations as low as 1 muM. Examination of ATP-stimulated changes in mitochondrial Ca(2+) indicated that, in cells pretreated with Mn, mitochondria retained high levels of Ca(2+). It is concluded that exposure of astrocytes to low concentrations of Mn(2+) results in sequestration of Ca(2+) within the mitochondria that reduces the available pool of releasable Ca(2+) within the ER, thereby inhibiting calcium wave activity.
• The Peroxisome Proliferator-activated Receptor-gamma Agonist 1,1-bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane Suppresses Manganese-induced Production of Nitric Oxide in Astrocytes and Inhibits Apoptosis in Cocultured PC12 Cells Journal of Neuroscience Research. Feb, 2008  |   Pubmed ID: 18041089 Reactive astrogliosis is a prominent neuropathologic feature of manganism, a neurodegenerative disorder caused by excessive accumulation of manganese (Mn) in the basal ganglia. Activation of astrocytes has been linked to neuronal injury in manganism resulting from overproduction of inflammatory mediators, including tumor necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma), interleukin-1beta (IL-1beta), and nitric oxide (NO), but the signaling mechanisms by which Mn regulates these factors remain poorly understood. We previously reported that Mn enhances production of NO in activated astrocytes that promotes apoptosis in cocultured neuronal cells by a mechanism involving the transcription factor nuclear factor-kappaB (NF-kappaB) (Liu et al., 2005). Because NF-kappaB-dependent expression of inducible nitric oxide synthase (NOS2) can be antagonized by the nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), we postulated that a novel agonist of this receptor, 1,1-bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (cDIM1), would suppress expression of NOS2 in astrocytes and protect cocultured neuronal cells from apoptosis. Submicromolar concentrations of cDIM1 potently suppressed production of NO and expression of NOS2 in cultured astrocytes exposed to Mn and IFNgamma/TNFalpha and prevented apoptosis in cocultures of differentiated PC12 cells, but this neuroprotective effect was lost in the absence of astrocytes. By using fluorescence reporter and chromatin immunoprecipitation (ChIP) assays, we found that cDIM1 prevented activation of NF-kappaB in astrocytes by a mechanism involving stabilization of the nuclear corepressor 2 (NCoR2) on the proximal NF-kappaB binding site of the NOS2 promoter. These data suggest that PPARgamma may be an effective target for limiting inflammatory activation of astrocytes during neurologic injury.
• Manganese Potentiates Nuclear Factor-kappaB-dependent Expression of Nitric Oxide Synthase 2 in Astrocytes by Activating Soluble Guanylate Cyclase and Extracellular Responsive Kinase Signaling Pathways Journal of Neuroscience Research. Jul, 2008  |   Pubmed ID: 18335517 Inflammatory activation of glial cells is associated with neuronal injury in several degenerative movement disorders of the basal ganglia, including manganese neurotoxicity. Manganese (Mn) potentiates the effects of inflammatory cytokines on nuclear factor-kappaB (NF-kappaB)-dependent expression of nitric oxide synthase 2 (NOS2) in astrocytes, but the signaling mechanisms underlying this effect have remained elusive. It was postulated in the present studies that direct stimulation of cGMP synthesis and activation of mitogen-activated protein (MAP) kinase signaling pathways underlies the capacity of Mn to augment NF-kappaB-dependent gene expression in astrocytes. Exposure of primary cortical astrocytes to a low concentration of Mn (10 microM) potentiated expression of NOS2 mRNA and protein along with production of NO in response to interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), which was prevented by overexpression of dominant negative IkappaB alpha. Mn also potentiated IFNgamma- and TNFalpha-induced phosphorylation of extracellular response kinase (ERK), p38, and JNK, as well as cytokine-induced activation of a fluorescent NF-kappaB reporter construct in transgenic astrocytes. Activation of ERK preceded that of NF-kappaB and was required for maximal activation of NO synthesis. Independently of IFNgamma/TNFalpha, Mn-stimulated synthesis of cGMP in astrocytes and inhibition of soluble guanylate cyclase (sGC) abolished the potentiating effect of Mn on MAP kinase phosphorylation, NF-kappaB activation, and production of NO. These data indicate that near-physiological concentrations of Mn potentiate cytokine-induced expression of NOS2 and production of NO in astrocytes via activation of sGC, which promotes ERK-dependent enhancement of NF-kappaB signaling.
• Nuclear Factor Kappa-B Mediates Selective Induction of Neuronal Nitric Oxide Synthase in Astrocytes During Low-level Inflammatory Stimulation with MPTP Brain Research. Jun, 2008  |   Pubmed ID: 18508038 Recent advances in understanding the progression of Parkinson's disease (PD) implicate perturbations in astrocyte function and induction of constitutively expressed neuronal nitric oxide synthase (NOS1) in both human PD and in the MPTP model of the disease. Transcriptional regulation of NOS1 is complex but recent data suggest that nuclear factor kappa-B (NF-kappaB) is an important transcription factor involved in inducible expression of the gene. The data presented here demonstrate that mild activation of primary astrocytes with low or 'sub-optimal' concentrations of MPTP (1 microM) and the inflammatory cytokine tumor necrosis factor alpha (10 pg/ml) and interferon gamma (1 ng/ml) results in selective induction of Nos1 mRNA and protein, increased production of nitric oxide (NO), and a significant elevation in global protein nitration. This mild inflammatory stimulus also resulted in activation and recruitment of p65 to a putative NF-kappaB response element located in the Nos1 promoter region flanking exon 1. A role for NF-kappaB in MPTP-dependent induction of NOS1 was confirmed through overexpression of a mutant IkappaBalpha super repressor of NF-kappaB that prevented induction of NOS1. The data presented here thus demonstrate a role for NF-kappaB in selective induction of NOS1 during early inflammatory activation of astrocytes stimulated by low-dose MPTP and inflammatory cytokines.
• Suppression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Nitric-oxide Synthase 2 Expression in Astrocytes by a Novel Diindolylmethane Analog Protects Striatal Neurons Against Apoptosis Molecular Pharmacology. Jan, 2009  |   Pubmed ID: 18840677 The progressive debilitation of motor functions in Parkinson's disease (PD) results from degeneration of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. Long-term inflammatory activation of microglia and astrocytes plays a central role in the progression of PD and is characterized by activation of the nuclear factor-kappaB (NF-kappaB) signaling cascade and subsequent overproduction of inflammatory cytokines and nitric oxide (NO). Suppression of this neuroinflammatory phenotype has received considerable attention as a potential target for chemotherapy, but there are no currently approved drugs that sufficiently address this problem. The data presented here demonstrate the efficacy of a novel anti-inflammatory diindolylmethane class compound, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in suppressing NF-kappaB-dependent expression of inducible nitric-oxide synthase (NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through a mechanism distinct from that described for the thiazolidinedione-class compound, rosiglitazone. Chromatin immunoprecipitations revealed that micromolar concentrations of DIM-C-pPhtBu prevented association of the p65 subunit of NF-kappaB with enhancer elements in the Nos2 promoter but had little effect on DNA binding of either peroxisome proliferator-activated receptor-gamma (PPAR-gamma) or the nuclear corepressor NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO production and protein nitration in MPTP-activated astrocytes and completely protected cocultured primary striatal neurons from astrocyte-dependent apoptosis. These data demonstrate the efficacy of DIM-C-pPhtBu in preventing the activation of NF-kappaB-dependent inflammatory genes in primary astrocytes and suggest that this class of compounds may be effective neuroprotective anti-inflammatory agents in vivo.
• Analysis of Targeted Mutation in DJ-1 on Cellular Function in Primary Astrocytes Toxicology Letters. Feb, 2009  |   Pubmed ID: 19063952 DJ-1 mutation induces early-onset Parkinson's disease, and conversely over-expression of DJ-1 is associated with cancer in numerous tissues. A gene-trap screening library conducted in embryonic stem cells was utilized for generation of a DJ-1 mutant mouse. Real-time PCR and immunoblotting were utilized to confirm functional mutation of the DJ-1 gene. Normal DJ-1 protein expression in adult mouse tissue was characterized and demonstrates high expression in brain tissue with wide systemic distribution. Primary astrocytes isolated from DJ-1(-/-) mice reveal a decreased nuclear localization of DJ-1 protein in response to rotenone or LPS, with a concomitant increase in mitochondrial localization of DJ-1 found only in the rotenone exposure. Resting mitochondrial membrane potential was significantly lower in DJ-1(-/-) astrocytes, as compared to controls. Our DJ-1 knockout mouse provides an exciting tool for exploring the molecular and physiological roles of DJ-1 to further explicate its functions in neurodegeneration.
• Age-dependent Susceptibility to Manganese-induced Neurological Dysfunction Toxicological Sciences : an Official Journal of the Society of Toxicology. Dec, 2009  |   Pubmed ID: 19812362 Chronic exposure to manganese (Mn) produces a spectrum of cognitive and behavioral deficits associated with a neurodegenerative disorder resembling Parkinson's disease. The effects of high-dose exposure to Mn in occupational cohorts and in adult rodent models of the disease are well described but much less is known about the behavioral and neurochemical effects of Mn in the developing brain. We therefore exposed C57Bl/6 mice to Mn by intragastric gavage as juveniles, adults, or both, postulating that mice exposed as juveniles and then again as adults would exhibit greater neurological and neurochemical dysfunction than mice not preexposed as juveniles. Age- and sex-dependent vulnerability to changes in locomotor function was detected, with juvenile male mice displaying the greatest sensitivity, characterized by a selective increase in novelty-seeking and hyperactive behaviors. Adult male mice preexposed as juveniles had a decrease in total movement and novelty-seeking behavior, and no behavioral changes were detected in female mice. Striatal dopamine levels were increased in juvenile mice but were decreased in adult preexposed as juveniles. Levels of Mn, Fe, and Cu were determined by inductively coupled plasma-mass spectrometry, with the greatest accumulation of Mn detected in juvenile mice in the striatum, substantia nigra (SN), and cortex. Only modest changes in Fe and Cu were detected in Mn-treated mice, primarily in the SN. These results reveal that developing mice are more sensitive to Mn than adult animals and that Mn exposure during development enhances behavioral and neurochemical dysfunction relative to adult animals without juvenile exposure.
• Developmental Exposure to Manganese Increases Adult Susceptibility to Inflammatory Activation of Glia and Neuronal Protein Nitration Toxicological Sciences : an Official Journal of the Society of Toxicology. Dec, 2009  |   Pubmed ID: 19812365 Chronic exposure to manganese (Mn) produces a neurodegenerative disorder affecting the basal ganglia characterized by reactive gliosis and expression of neuroinflammatory genes including inducible nitric oxide synthase (NOS2). Induction of NOS2 in glial cells causes overproduction of nitric oxide (NO) and injury to neurons that is associated with parkinsonian-like motor deficits. Inflammatory activation of glia is believed to be an early event in Mn neurotoxicity, but specific responses of microglia and astrocytes to Mn during development remain poorly understood. In this study, we investigated the effect of juvenile exposure to Mn on the activation of glia and production of NO in C57Bl/6J mice, postulating that developmental Mn exposure would lead to heightened sensitivity to gliosis and increased expression of NOS2 in adult mice exposed again later in life. Immunohistochemical analysis indicated that Mn exposure caused increased activation of both microglia and astrocytes in the striatum (St), globus pallidus (Gp), and substantia nigra pars reticulata (SNpr) of treated mice compared with controls. More robust activation of microglia was observed in juveniles, whereas astrogliosis was more prominent in adult mice preexposed during development. Co-immunofluorescence studies demonstrated increased expression of NOS2 in glia located in the Gp and SNpr. Additionally, greater increases in the level of 3-nitrotyrosine protein adducts were detected in dopamine- and cAMP-regulated phosphoprotein-32-positive neurons of the St of Mn-treated adult mice preexposed as juveniles. These data indicate that subchronic exposure to Mn during development leads to temporally distinct patterns of glial activation that result in elevated nitrosative stress in distinct populations of basal ganglia neurons.
• Toxicological and Pathophysiological Roles of Reactive Oxygen and Nitrogen Species Toxicology. Oct, 2010  |   Pubmed ID: 20643181 'Oxidative and Nitrative Stress in Toxicology and Disease' was the subject of a symposium held at the EUROTOX meeting in Dresden 15th September 2009. Reactive oxygen (ROS) and reactive nitrogen species (RNS) produced during tissue pathogenesis and in response to viral or chemical toxicants, induce a complex series of downstream adaptive and reparative events driven by the associated oxidative and nitrative stress. As highlighted by all the speakers, ROS and RNS can promote diverse biological responses associated with a spectrum of disorders including neurodegenerative/neuropsychiatric and cardiovascular diseases. Similar pathways are implicated during the process of liver and skin carcinogenesis. Mechanistically, reactive oxygen and nitrogen species drive sustained cell proliferation, cell death including both apoptosis and necrosis, formation of nuclear and mitochondrial DNA mutations, and in some cases stimulation of a pro-angiogenic environment. Here we illustrate the pivotal role played by oxidative and nitrative stress in cell death, inflammation and pain and its consequences for toxicology and disease pathogenesis. Examples are presented from five different perspectives ranging from in vitro model systems through to in vivo animal model systems and clinical outcomes.
• Low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Causes Inflammatory Activation of Astrocytes in Nuclear Factor-κB Reporter Mice Prior to Loss of Dopaminergic Neurons Journal of Neuroscience Research. Mar, 2011  |   Pubmed ID: 21259327 Neuroinflammation is implicated in the progression of numerous disease states of the CNS, but early inflammatory signaling events in glial cells that may predispose neurons to injury are not easily characterized in vivo. To address this question, we exposed transgenic mice expressing a nuclear factor-κB (NF-κB)-driven enhanced green fluorescent protein (EGFP) reporter construct to low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and examined inflammatory activation of astrocytes in relation to neurobehavioral and neuropathological outcomes. The highest dose of MPTP (60 mg/kg total dose) caused a decrease in locomotor activity and a reduction in stride length. No significant loss of dopaminergic neurons in the substantia nigra was apparent at any dose. In contrast, expression of tyrosine hydroxylase in striatal fibers was reduced at 60 mg/kg MPTP, as were levels of dopamine and DOPAC. Colocalized expression of EGFP and inducible nitric oxide synthase (NOS2) occurred in astrocytes at 30 and 60 mg/kg MPTP and was associated with increased protein nitration in nigral dopaminergic neurons. Inhibition of NF-κB in primary astrocytes by expression of mutant IκBα suppressed expression of NOS2 and protected cocultured neurons from astrocyte-mediated apoptosis. These data indicate that inflammatory activation of astrocytes and enhanced nitrosative stress occurs at low doses of MPTP prior to loss of dopaminergic neurons. NF-κB-mediated expression of NOS2 appears to be a sensitive indicator of neuroinflammation that correlates with MPTP-induced neurochemical and neurobehavioral deficits prior to loss of dopaminergic neurons in the subtantia nigra.
• Manganese-induced NF-kappaB Activation and Nitrosative Stress is Decreased by Estrogen in Juvenile Mice Toxicological Sciences : an Official Journal of the Society of Toxicology. Jul, 2011  |   Pubmed ID: 21512103 Manganese toxicity can cause a neurodegenerative disorder affecting cortical and basal ganglia structures with a neurological presentation resembling features of Parkinson's disease. Children are more sensitive to Mn-induced neurological dysfunction than adults, and recent studies from our laboratory revealed a marked sensitivity of male juvenile mice to neuroinflammatory injury from Mn, relative to females. To determine the role of estrogen (E2) in mediating sex-dependent vulnerability to Mn-induced neurotoxicity, we exposed transgenic mice expressing an NF-κB-driven enhanced green fluorescent protein (EGFP) reporter construct (NF-κB-EGFP mice) to Mn, postulating that supplementing male mice with E2 during juvenile development would attenuate neuroinflammatory changes associated with glial activation, including expression of inducible nitric oxide synthase (NOS2) and neuronal protein nitration. Juvenile NF-κB-EGFP mice were separated in groups composed of females, males, and males surgically implanted with Silastic capsules containing 25 μg of 17-β-estradiol (E2) or vehicle control. Mice were then treated with 0 or 100 mg/Kg MnCl(2) by intragastric gavage from postnatal days 21-34. Manganese treatment caused alterations in levels of striatal dopamine, as well as increases in NF-κB reporter activity and NOS2 expression in both microglia and astrocytes that were prevented by supplementation with E2. E2 also decreased neuronal protein nitration in Mn-treated mice and inhibited apoptosis in striatal neurons cocultured with Mn-treated astrocytes in vitro. These data indicate that E2 protects against Mn-induced neuroinflammation in developing mice and that NF-κB is an important regulator of neuroinflammatory gene expression in glia associated with nitrosative stress in the basal ganglia during Mn exposure.
• 1,3-Dinitrobenzene-induced Metabolic Impairment Through Selective Inactivation of the Pyruvate Dehydrogenase Complex Toxicological Sciences : an Official Journal of the Society of Toxicology. Aug, 2011  |   Pubmed ID: 21551353 Prolonged exposure to the chemical intermediate, 1,3-dinitrobenzene (1,3-DNB), produces neuropathology in the central nervous system of rodents analogous to that observed in various conditions of acute energy deprivation including thiamine deficiency and Leigh's necrotizing encephalopathy. Increased production of reactive intermediates in addition to induction of oxidative stress has been implicated in the neurotoxic mechanism of 1,3-DNB, but a clear metabolic target has not been determined. Here we propose that similar to thiamine deficiency, the effects of 1,3-DNB on metabolic status may be due to inhibition of the thiamine-dependent α-ketoacid dehydrogenase complexes. The effects of 1,3-DNB on astroglial metabolic status and α-ketoacid dehydrogenase activity were evaluated using rat C6 glioma cells. Exposure to 1,3-DNB resulted in altered morphology and biochemical dysfunction consistent with disruption of oxidative energy metabolism. Cotreatment with acetyl-carnitine or acetoacetate attenuated morphological and metabolic effects of 1,3-DNB exposure as well as increased cell viability. 1,3-DNB exposure inhibited pyruvate dehydrogenase complex (PDHc) and the inhibition correlated with the loss of lipoic acid (LA) immunoreactivity, suggesting that modification of LA is a potential mechanism of inhibition. Treatment with antioxidants and thiol-containing compounds failed to protect against loss of LA. Alternatively, inhibition of dihydrolipoamide dehydrogenase, the E3 component of the complex attenuated loss of LA. Collectively, these data suggest that 1,3-DNB impairs oxidative energy metabolism through direct inhibition of the PDHc and that this impairment is due to perturbations in the function of protein-bound LA.
• Detection of Nitric Oxide Formation in Primary Neural Cells and Tissues Methods in Molecular Biology (Clifton, N.J.). 2011  |   Pubmed ID: 21815072 Nitric oxide (NO) is a free radical molecule with a short half-life (
• Role of Oxidative Stress and the Mitochondrial Permeability Transition in Methylmercury Cytotoxicity Neurotoxicology. Oct, 2011  |   Pubmed ID: 21871920 Oxidative stress has been implicated in the pathogenesis of methylmercury (MeHg) neurotoxicity. Studies of mature neurons suggest that the mitochondrion may be a major source of MeHg-induced reactive oxygen species and a critical mediator of MeHg-induced neuronal death, likely by activation of apoptotic pathways. It is unclear, however, whether the mitochondria of developing and mature neurons are equally susceptible to MeHg. Murine embryonal carcinoma (EC) cells, which differentiate into neurons following exposure to retinoic acid, were used to compare the differentiation-dependent effects of MeHg on ROS production and mitochondrial depolarization. EC cells and their neuronal derivatives were pre-incubated with the ROS indicator 2',7'-dichlorofluoroscein diacetate or tetramethylrhodamine methyl ester, an indicator of mitochondrial membrane potential, with or without cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore opening, and examined by laser scanning confocal microscopy in the presence of 1.5 μM MeHg. To examine consequences of mitochondrial perturbation, immunohistochemical localization of cytochrome c (cyt c) was determined after incubation of cells in MeHg for 4 h. MeHg treatment induced earlier and significantly higher levels of ROS production and more extensive mitochondrial depolarization in neurons than in undifferentiated EC cells. CsA completely inhibited mitochondrial depolarization by MeHg in EC cells but only delayed this response in the neurons. In contrast, CsA significantly inhibited MeHg-induced neuronal ROS production. Cyt c release was also more extensive in neurons, with less protection afforded by CsA. These data indicate that neuronal differentiation state influences mitochondrial transition pore dynamics and MeHg-stimulated production of ROS.
• Gene Deletion of Nos2 Protects Against Manganese-induced Neurological Dysfunction in Juvenile Mice Toxicological Sciences : an Official Journal of the Society of Toxicology. Mar, 2012  |   Pubmed ID: 22174044 The mechanisms underlying cognitive and neurobehavioral abnormalities associated with childhood exposure to manganese (Mn) are not well understood but may be influenced by neuroinflammatory activation of microglia and astrocytes that results in nitrosative stress due to expression of inducible nitric oxide synthase (iNOS/NOS2). We therefore postulated that gene deletion of NOS2 would protect against the neurotoxic effects of Mn in vivo and in vitro. Juvenile NOS2 knockout (NOS2(-/-)) mice were orally exposed to 50 mg/kg of MnCl₂ by intragastric gavage from days 21 to 34 postnatal. Results indicate that NOS2(-/-) mice exposed to Mn were protected against neurobehavioral alterations, despite histopathological activation of astrocytes and microglia in Mn-treated mice in both genotypes. NOS2(-/-) mice had decreased Mn-induced formation of 3-nitrotyrosine protein adducts within neurons in the basal ganglia that correlated with protection against Mn-induced neurobehavioral defects. Primary striatal astrocytes from wildtype mice caused apoptosis in cocultured striatal neurons following treatment with MnCl₂ and tumor necrosis factor-α, whereas NOS2(-/-) astrocytes failed to cause any increase in markers of apoptosis in striatal neurons. Additionally, scavenging nitric oxide (NO) with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) prevented the ability of Mn- and cytokine-treated wildtype astrocytes to cause apoptosis in cocultured striatal neurons. These data demonstrate that NO plays a crucial role in Mn-induced neurological dysfunction in juvenile mice and that NOS2 expression in activated glia is an important mediator of neuroinflammatory injury during Mn exposure.
• Manganese Inhibits ATP-induced Calcium Entry Through the Transient Receptor Potential Channel TRPC3 in Astrocytes Neurotoxicology. Jan, 2013  |   Pubmed ID: 23131343 Chronic exposure to elevated levels of manganese (Mn(2+)) causes neuronal injury and inflammatory activation of glia. Astrocytes selectively accumulate Mn(2+), which inhibits mitochondrial respiration and increases production of reactive oxygen species. We previously reported that sub-acute exposure to low micromolar levels of Mn(2+) in primary astrocytes inhibited ATP-induced calcium (Ca(2+)) signaling, associated with decreased levels of endoplasmic reticulum Ca(2+) and increased mitochondrial Ca(2+) loads. In the present studies, we postulated that the mechanism underlying the capacity of Mn(2+) to inhibit these purinergic signals in astrocytes could be due to competition with Ca(2+) for entry through a plasma membrane channel. These data demonstrate that acutely applied Mn(2+) rapidly inhibited ATP-induced Ca(2+) waves and transients in primary striatal astrocytes. Mn(2+) also decreased influx of extracellular Ca(2+) induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a direct activator of the transient receptor potential channel, TRPC3. The TRPC3 inhibitor, pyrazole-3, prevented ATP- and OAG-dependent transport of Mn(2+) from extracellular stores, demonstrated by a dramatic reduction in the rate of fluorescence quenching of Fura-2. These data indicate that Mn(2+) can acutely inhibit ATP-dependent Ca(2+) signaling in astrocytes by blocking Ca(2+) entry through the receptor-operated cation channel, TRPC3. Loss of normal astrocytic responses to purinergic signals due to accumulation of Mn(2+) could therefore comprise critical homeostatic functions necessary for metabolic and trophic support of neurons.
• Neuroprotective Efficacy and Pharmacokinetic Behavior of Novel Anti-inflammatory Para-phenyl Substituted Diindolylmethanes in a Mouse Model of Parkinson's Disease The Journal of Pharmacology and Experimental Therapeutics. Apr, 2013  |   Pubmed ID: 23318470 There are currently no registered drugs that slow the progression of neurodegenerative diseases, in part because translation from animal models to the clinic has been hampered by poor distribution to the brain. The present studies examined a selected series of para-phenyl-substituted diindolylmethane (C-DIM) compounds that display anti-inflammatory and neuroprotective efficacy in vitro. We postulated that the pharmacokinetic behavior of C-DIM compounds after oral administration would correlate with neuroprotective efficacy in vivo in a mouse model of Parkinson's disease. Pharmacokinetics and metabolism of 1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (C-DIM5), 1,1-bis(3'-indolyl)-1-(phenyl)methane, 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (C-DIM8), and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) were determined in plasma and brain of C57Bl/6 mice after oral and intravenous administration at 10 and 1 mg/Kg, respectively. Putative metabolites were measured in plasma, liver, and urine. C-DIM compounds given orally displayed the highest area under the curve, Cmax, and Tmax levels, and C-DIM12 exhibited the most favorable pharmacokinetics of the compounds tested. Oral bioavailability of each compound ranged from 6% (C-DIM8) to 42% (C-DIM12). After pharmacokinetic studies, the neuroprotective efficacy of C-DIM5, C-DIM8, and C-DIM12 (50 mg/Kg per oral) was examined in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid for 14 days, a model of progressive neurodegeneration with a strong neuroinflammatory component. C-DIM5 and C-DIM12 given orally once daily after one week of exposure to MPTP and probenecid prevented further loss of dopaminergic neurons in the substantia nigra pars compacta and striatal dopamine terminals, indicating that these compounds could be effective therapeutic agents to prevent neurodegeneration.
• The Atrazine Metabolite Diaminochlorotriazine Suppresses LH Release from Murine LβT2 Cells by Suppressing GnRH-induced Intracellular Calcium Transients Toxicology Research. May, 2013  |   Pubmed ID: 24052811 The primary metabolite of the herbicide atrazine (ATRA), diaminochlorotriazine (DACT), has been suggested to cause disruption in the hypothalamic-pituitary-gonadal axis leading to inhibition of luteinizing hormone (LH) release. DACT is a reactive electrophile known to form covalent protein adducts both in vitro and in vivo following ATRA exposure and maybe targeting proteins involved in GnRH-induced calcium signaling and subsequent LH release. To test this hypothesis, LβT2 pituitary cells were exposed to 300 μM DACT for 24 hrs and examined by fluorescence microscopy for GnRH-induced changes in intracellular calcium and LH release. LβT2 cells exposed to DACT had markedly diminished GnRH-induced intracellular calcium transients and a significant decreased LH release in response to GnRH. DACT appeared to cause a selective decrease in caffeine-sensitive ryanodine receptor-operated calcium stores in LβT2 cells, rather than in thapsigargin-sensitive ER calcium stores. This sensitivity correlated with the formation of covalent protein adducts by DACT, as determined by mass spectrometry. ERp57 was identified by mass spectrometry as a target of DACT adduction in the ER that could potentially mediate the effects of DACT on inhibition of GnRH-induced calcium signaling and inhibition of LH release. Intracellular calcium responses to GnRH and release of LH were restored in DACT-treated cells with the addition of a calcium ionophore (A23187). These data suggest that DACT forms adducts on proteins involved in calcium handling within the ER and that dysfunction in this critical signaling system is associated with loss of normal sensitivity to GnRH and subsequent decreased release of LH.
• Repeated Exposure to Low Doses of Kainic Acid Activates Nuclear Factor Kappa B (NF-κB) Prior to Seizure in Transgenic NF-κB/EGFP Reporter Mice Neurotoxicology. Sep, 2014  |   Pubmed ID: 24813937 Predicting seizurogenic properties of pharmacologically active compounds is difficult due to the complex nature of the mechanisms involved and because of the low sensitivity and high variability associated with current behavioral-based methods. To identify early neuronal signaling events predictive of seizure, we exposed transgenic NF-κB/EGFP reporter mice to multiple low doses of kainic acid (KA), postulating that activation of the stress-responsive NF-κB pathway could be a sensitive indicator of seizurogenic potential. The sub-threshold dose level proximal to the induction of seizure was determined as 2.5mg/kg KA, using video EEG monitoring. Subsequent analysis of reporter expression demonstrated significant increases in NF-κB activation in the CA3 and CA1 regions of the hippocampus 24h after a single dose of 2.5mg/kg KA. This response was primarily observed in pyramidal neurons with little non-neuronal expression. Neuronal NF-κB/EGFP expression was observed in the absence of glial activation, indicating a lack of neurodegeneration-induced neuroinflammation. Protein expression of the immediate-early gene, Nurr1, increased in neurons in parallel to NF-κB activation, supporting that the sub-threshold doses of KA employed directly caused neuronal stress. Lastly, KA also stimulated NF-κB activation in organotypic hippocampal slice cultures established from NF-κB/EGFP reporter mice. Collectively, these data demonstrate the potential advantages of using genetically encoded stress pathway reporter models in the screening of seizurogenic properties of new pharamacologically active compounds.
• Diindolylmethane Analogs Bind NR4A1 and Are NR4A1 Antagonists in Colon Cancer Cells Molecular Endocrinology (Baltimore, Md.). Oct, 2014  |   Pubmed ID: 25099012 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compounds exhibit antineoplastic activity in multiple cancer cell lines and the p-hydroxyphenyl analog (DIM-C-pPhOH) inactivates nuclear receptor 4A1 (NR4A1) in lung and pancreatic cancer cell lines. Using a series of 14 different p-substituted phenyl C-DIMs, we show that several compounds including DIM-C-pPhOH directly interacted with the ligand binding domain of NR4A1. Computational-based molecular modeling studies showed high-affinity interactions of DIM-C-pPhOH and related compounds within the ligand binding pocket of NR4A1, and these same compounds decreased NR4A1-dependent transactivation in colon cancer cells transfected with a construct containing 3 tandem Nur77 binding response elements linked to a luciferase reporter gene. Moreover, we also show that knockdown of NR4A1 by RNA interference (small interfering NR4A1) or treatment with DIM-C-pPhOH and related compounds decreased colon cancer cell growth, induced apoptosis, decreased expression of survivin and other Sp-regulated genes, and inhibited mammalian target of rapamycin signaling. Thus, C-DIMs such as DIM-C-pPhOH directly bind NR4A1 and are NR4A1 antagonists in colon cancer cells, and their antineoplastic activity is due, in part, to their interactions with nuclear NR4A1.
• Domoic Acid-induced Seizures in California Sea Lions (Zalophus Californianus) Are Associated with Neuroinflammatory Brain Injury Aquatic Toxicology (Amsterdam, Netherlands). Nov, 2014  |   Pubmed ID: 25286249 California sea lions (CSLs) exposed to the marine biotoxin domoic acid (DA) develop an acute or chronic toxicosis marked by seizures and act as sentinels of the disease. Experimental evidence suggests that oxidative stress and neuroinflammation are important mechanisms underlying the seizurogenic potential of environmental toxicants but these pathways are relatively unstudied in CSLs. In the current study, we investigated the role of glutamate-glutamine changes and gliosis in DA-exposed CSLs to better understand the neurotoxic mechanisms occurring during DA toxicity. Sections from archived hippocampi from control and CSLs diagnosed with DA toxicosis were immunofluorescently stained for markers of gliosis, oxidative/nitrative stress and changes in glutamine synthetase (GS). Quantitative assessment revealed increasing loss of microtubule associated protein-2 positive neurons with elevations in 4-hydroxynonenal correlating with chronicity of exposure, whereas the pattern of activated glia expressing nitric oxide synthase 2 and tumor necrosis factor followed pathological severity. There was no significant change in the amount of GS positive cells but there was increased 3-nitrotyrosine in GS expressing cells and in neurons, particularly in animals with chronic DA toxicosis. These changes were consistently seen in the dentate gyrus and in the cornu ammonis (CA) sectors CA3, CA4, and CA1. The results of this study indicate that gliosis and resultant changes in GS are likely important mechanisms in DA-induced seizure that need to be further explored as potential therapies in treating exposed wildlife.
• Novel Para-phenyl Substituted Diindolylmethanes Protect Against MPTP Neurotoxicity and Suppress Glial Activation in a Mouse Model of Parkinson's Disease Toxicological Sciences : an Official Journal of the Society of Toxicology. Feb, 2015  |   Pubmed ID: 25406165 The orphan nuclear receptor NR4A2 (Nurr1) constitutively regulates inflammatory gene expression in glial cells by suppressing DNA binding activity of NF-κB. We recently reported that novel 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methane (C-DIM) compounds that activate NR4A family nuclear receptors in cancer lines also suppress inflammatory gene expression in primary astrocytes and prevent loss of dopaminergic neurons in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp). In this study, we postulated that the basis for this neuroprotection involves blockade of glial activation and subsequent expression of NF-κB-regulated inflammatory genes. To examine this mechanism, we treated transgenic NF-κB/EGFP reporter mice with MPTPp for 7 days (MPTPp7d) followed by daily oral gavage with either vehicle (corn oil; MPTPp14d) or C-DIMs containing p-methoxyphenyl (C-DIM5), p-hydroxyphenyl (C-DIM8), or p-chlorophenyl (C-DIM12) groups. Each compound conferred significant protection against progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), even when given after 7 days of dosing with MPTPp. C-DIM12 had the greatest neuroprotective activity in MPTPp-treated mice, and was also the most potent compound in suppressing activation of microglia and astrocytes, expression of cytokines and chemokines in quantitative polymerase chain reaction (qPCR) array studies, and in reducing expression of NF-κB/EGFP in the SN. C-DIM12 prevented nuclear export of Nurr1 in dopaminergic neurons and enhanced expression of the Nurr1-regulated proteins tyrosine hydroxylase and the dopamine transporter. These data indicate that NR4A-active C-DIM compounds protect against loss of dopamine neurons in the MPTPp model of PD by preventing glial-mediated neuronal injury and by supporting a dopaminergic phenotype in TH-positive neurons in the SNpc.
• The Nurr1 Activator 1,1-Bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane Blocks Inflammatory Gene Expression in BV-2 Microglial Cells by Inhibiting Nuclear Factor κB Molecular Pharmacology. Jun, 2015  |   Pubmed ID: 25858541 NR4A family orphan nuclear receptors are an important class of transcription factors for development and homeostasis of dopaminergic neurons that also inhibit expression of inflammatory genes in glial cells. The identification of NR4A2 (Nurr1) as a suppressor of nuclear factor κB (NF-κB)-related neuroinflammatory genes in microglia and astrocytes suggests that this receptor could be a target for pharmacologic intervention in neurologic disease, but compounds that promote this activity are lacking. Selected diindolylmethane compounds (C-DIMs) have been shown to activate or inactivate nuclear receptors, including Nurr1, in cancer cells and also suppress astrocyte inflammatory signaling in vitro. Based upon these data, we postulated that C-DIM12 [1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane] would suppress inflammatory signaling in microglia by a Nurr1-dependent mechanism. C-DIM12 inhibited lipopolysaccharide (LPS)-induced expression of NF-κB-regulated genes in BV-2 microglia including nitric oxide synthase (NOS2), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2), and the effects were attenuated by Nurr1-RNA interference. Additionally, C-DIM12 decreased NF-κB activation in NF-κB-GFP (green fluorescent protein) reporter cells and enhanced nuclear translocation of Nurr1 primary microglia. Chromatin immunoprecipitation assays indicated that C-DIM12 decreased lipopolysaccharide-induced p65 binding to the NOS2 promoter and concurrently enhanced binding of Nurr1 to the p65-binding site. Consistent with these findings, C-DIM12 also stabilized binding of the Corepressor for Repressor Element 1 Silencing Transcription Factor (CoREST) and the Nuclear Receptor Corepressor 2 (NCOR2). Collectively, these data identify C-DIM12 as a modulator of Nurr1 activity that results in inhibition of NF-κB-dependent gene expression in glial cells by stabilizing nuclear corepressor proteins, which reduces binding of p65 to inflammatory gene promoters.
• Nuclear Receptor 4A (NR4A) Family - Orphans No More The Journal of Steroid Biochemistry and Molecular Biology. Apr, 2015  |   Pubmed ID: 25917081 The orphan nuclear receptors NR4A1, NR4A2 and NR4A3 are immediate early genes induced by multiple stressors, and the NR4A receptors play an important role in maintaining cellular homeostasis and disease. There is increasing evidence for the role of these receptors in metabolic, cardiovascular and neurological functions and also in inflammation and inflammatory diseases and in immune functions and cancer. Despite the similarities of NR4A1, NR4A2 and NR4A3 and their interactions with common cis-genomic elements, they exhibit unique activities and cell-/tissue-specific functions. Although endogenous ligands for NR4A receptors have not been identified, there is increasing evidence that structurally-diverse synthetic molecules can directly interact with the ligand binding domain of NR4A1 and act as agonists or antagonists, and ligands for NR4A2 and NR4A3 have also been identified. Since NR4A receptors are key factors in multiple diseases, there are opportunities for the future development of NR4A ligands for clinical applications in treating multiple health problems including metabolic, neurologic and cardiovascular diseases, other inflammatory conditions, and cancer.
• Angiotensin II Regulates Brain (pro)renin Receptor Expression Through Activation of CAMP Response Element-binding Protein American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. May, 2015  |   Pubmed ID: 25994957 We reported that brain (pro)renin receptor (PRR) expression levels are elevated in deoxycorticosterone acetate (DOCA)-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether angiotensin (Ang II) type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplying 0.9% saline as the drinking solution to C57BL/6J mice; SHAM pellet-implanted mice provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly (ICV) infused with the AT1R blocker losartan, angiotensin converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 weeks. ICV infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for Ang II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which Ang II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with Ang II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors as well as CREB knockdown abolished Ang II-induced increases in PRR levels. Ang II also induced PRR up-regulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that Ang II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that Ang II acts via AT1R to up-regulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter.