Treatment Of Glial Cells Research Articles

The Effect of Cocaine on C6 Glial Cells and its Modulation of Oxygen Consumption, Growth and Transcription Factors

Cocaine is a widely abused and addictive psychostimulant drug that acts on the central nervous system (CNS). Glial cells may be the first to experience the toxic effects of cocaine in the mammalian brain, since glial cells outnumber neurons by 10–1. Glial cells were grown in 10% FBS (fetal bovine serum) in Dulbecco's modified Eagle medium (DMEM) and penicillin/streptomycin (100 Units/0.1 mg per ml). Treatment of glial cells with cocaine for 24 and 48 hours caused significant loss of cell viability. We investigated the expression of Hypoxia Inducible Factor‐1α (HIF‐1α) and Vascular Endothelial Growth Factor (VEGF) in 1, 2, 3 and 4 mM of cocaine exposure and also in control samples of glial cells. Preliminary data from different studies indicated that hypoxia is a potent inducer of VEGF in vitro partly because of an increased transcription rate, mediated by binding of HIF‐1 α to a hypoxia responsive element, VEGF gene. Thus following cocaine treatment of various concentrations mentioned above and RNA isolation, real time PCR (Polymerase Chain Reaction ) was carried out using the IQ Sybr Green Super mix. Expression of HIF‐1 α followed by VEGF observed in each concentration compared to control and Flow Cytometry and Elisa experiments confirmed the expression. Oxygen consumption study showed significant reduction in higher concentrations of cocaine. We concluded that cocaine may cause hypoxia in rat C6 glial cells via expression of vascular endothelial growth factor (VEGF).Acknowledgment: This research was supported by National Institute on Minority Health Disparities of the National Institute of Health under award number G12MD007582,FAMU RCMI, Pharmaceutical Research Center

Cocaine induces HIF1 (Hypoxia Inducible Factor1) in rat C6 glial cells (LB619)

Investigations with astroglial cells carry more prominence in drug abuse studies. However, not many investigations have been conducted on the toxicity of various abused drugs, like cocaine. The present study was aimed to discern the effect of cocaine on rat astroglioma cells. Glial cells were grown in 10% FBS (Fetal Bovine Serum) in Dulbecco’s modified Eagle medium (DMEM) and penicillin/streptomycin (100 Units/0.1 mg per ml). Treatment of glial cells with cocaine for 24 and 48 hours caused significant loss of cell viability. We also used rat cytokine array membranes to determine the change in rat C6 glial cell cytokine expression upon cocaine exposure (2, 4 and 7 mM). We found after 24 hours cocaine exposure, expression of vascular endothelial growth factor (VEGF) was relatively high in each concentration compared to control and these results were confirmed with western blot analysis. Preliminary data from different studies indicated that hypoxia is a potent inducer of VEGF in vitro partly because of an increased transcription rate, mediated by binding of hypoxia‐inducible factor‐1 (HIF1) to a hypoxia responsive element, VEGF gene. Thus following cocaine treatment of various concentration and RNA isolation, real time PCR (Polymerase Chain Reaction ) was carried out using the IQ Sybr Green Supermix in conjunction with MyiQ software (Bio‐Rad, Hercules, CA). Expression of HIF1 followed by VEGF observed in each concentration compared to control. We concluded that cocaine may be involved in hypoxia in C6 glial cells.Grant Funding Source: This research was supported by National Institute on Minority Health Disparities of the National Institute of Health under award number G12MD007582, FAMU RCMI, Pharmaceutical Research Center

5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) attenuates the expression of LPS- and Aβ peptide-induced inflammatory mediators in astroglia

BackgroundAlzheimer's disease (AD) pathology shows characteristic 'plaques' rich in amyloid beta (Aβ) peptide deposits. Inflammatory process-related proteins such as pro-inflammatory cytokines have been detected in AD brain suggesting that an inflammatory immune reaction also plays a role in the pathogenesis of AD. Glial cells in culture respond to LPS and Aβ stimuli by upregulating the expression of cytokines TNF-α, IL-1β, and IL-6, and also the expression of proinflammatory genes iNOS and COX-2. We have earlier reported that LPS/Aβ stimulation-induced ceramide and ROS generation leads to iNOS expression and nitric oxide production in glial cells. The present study was undertaken to investigate the neuroprotective function of AICAR (a potent activator of AMP-activated protein kinase) in blocking the pro-oxidant/proinflammatory responses induced in primary glial cultures treated with LPS and Aβ peptide.MethodsTo test the anti-inflammatory/anti-oxidant functions of AICAR, we tested its inhibitory potential in blocking the expression of pro-inflammatory cytokines and iNOS, expression of COX-2, generation of ROS, and associated signaling following treatment of glial cells with LPS and Aβ peptide. We also investigated the neuroprotective effects of AICAR against the effects of cytokines and inflammatory mediators (released by the glia), in blocking neurite outgrowth inhibition, and in nerve growth factor-(NGF) induced neurite extension by PC-12 cells.ResultsAICAR blocked LPS/Aβ-induced inflammatory processes by blocking the expression of proinflammatory cytokine, iNOS, COX-2 and MnSOD genes, and by inhibition of ROS generation and depletion of glutathione in astroglial cells. AICAR also inhibited down-stream signaling leading to the regulation of transcriptional factors such as NFκB and C/EBP which are critical for the expression of iNOS, COX-2, MnSOD and cytokines (TNF-α/IL-1β and IL-6). AICAR promoted NGF-induced neurite growth and reduced neurite outgrowth inhibition in PC-12 cells treated with astroglial conditioned medium.ConclusionThe observed anti-inflammatory/anti-oxidant and neuroprotective functions of AICAR suggest it as a viable candidate for use in treatment of Alzheimer's disease.

Adenosine kinase inhibitor attenuates the expression of inducible nitric oxide synthase in glial cells

The present study demonstrates the anti-inflammatory effect of adenosine kinase inhibitor (ADKI) in glial cells. Treatment of glial cells with IC51, an ADKI, stimulated the extracellular adenosine release and reduced the LPS/IFNγ-mediated production of NO, and induction of iNOS and TNF-α gene expression. The recovery of IC51-mediated inhibition of iNOS expression by adenosine transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI), and the inhibition of LPS/IFNγ-induced iNOS gene expression by exogenous adenosine indicate a role for adenosine release in IC51-mediated iNOS expression. The rescue of IC51-mediated inhibition of iNOS expression by adenosine receptor antagonist for A 2A, 8-(3-chlorostyryl)caffeine (CSC) and alloxazine for A 2B, further supports a role for interaction of adenosine and its receptors in anti-inflammatory activity. The IC51-mediated induction of cAMP levels, downstream target of A 2A and A 2B, and inhibition of LPS/IFNγ-induced expression of iNOS by forskolin, a cAMP activator, document a role for cAMP mediated pathway in anti-inflammatory activity of IC51. Taken together, these studies document that IC51-mediated inhibition of iNOS expression is through activation of adenosine receptors, which activates A 2A and A 2B resulting in increased cAMP levels following LPS/IFNγ stimulation. Moreover, the lack of effect of IC51 or adenosine on NFκB DNA binding activity and its transactivity indicates that the inhibition of iNOS expression mediated by IC51 may be through an NFκB independent pathway.

Contrasting roles of endosomal pH and the cytoskeleton in infection of human glial cells by JC virus and simian virus 40.

Infection of eukaryotic cells by pathogens requires the efficient use of host cell endocytic and cytoplasmic transport mechanisms. Understanding how these cellular functions are exploited by microorganisms allows us to better define the basic biology of pathogenesis while providing better insight into normal cellular functions. In this report we compare and contrast intracellular transport and trafficking of the human polyomavirus JC virus (JCV) with that of simian virus 40 (SV40). We have previously shown that infection of human glial cells by JCV requires clathrin-dependent endocytosis. In contrast, infection of cells by SV40 proceeds by caveola-dependent endocytosis. We now examine the roles of endosomal pH and the cellular cytoskeleton during infection of glial cells by both viruses. Our results demonstrate that JCV infection is sensitive to disruption of endosomal pH, whereas SV40 infection is pH independent. Infection by JCV is inhibited by treatment of glial cells with cytochalasin D, nocodazole, and acrylamide, whereas SV40 infection is affected only by nocodazole. These data point to critical differences between JCV and SV40 in terms of endocytosis and intracellular trafficking of their DNA genomes to the nucleus. These data also suggest a unique sequential involvement of cytoskeletal elements during infection of glial cells by JCV.

Neurotrophin Receptor Death Partner

Neurotrophins, such as nerve growth factor (NGF), are most commonly associated with promoting nerve cell survival and differentiation. However, activation of the low-affinity neurotrophin receptor, p75NTR, which has an intracellular, COOH-terminal type-2 death domain, can lead to apoptosis of nerve cells. Mukai et al . have identified the protein NADE (p75NTR-associated cell death executor) in a yeast two-hybrid screen for proteins that interact with the intracellular domain of p75NTR. NADE and p75NTR could be coprecipitated from pheochromocytoma and neuroblastoma cells treated with NGF. When NADE and p75NTR were coexpressed in 293 cells or when NADE alone was transfected into pheochromocytoma cells, NGF-induced activation of caspase-2 and caspase-3 and apoptosis. In oligodendrocytes, a type of glia, NADE expression was induced by treatment of glial cells with NGF and correlated with increased numbers of apoptotic cells. NADE contains a nuclear export sequence and ubiquitination sequences and may represent a regulated component of the cell death pathway mediated by p75NTR. Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M.T., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L.A., and Sato, T.-A. (2000) NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR. J. Biol. Chem. 275 : 17566-17570. [Abstract] [Full Text]

Effect of HIV-1 gp41 peptides on secretion of beta-amyloid precursor protein in human astroglial cell line, T98G.

To understand the mechanism underlying cognitive deficits in AIDS patients, we examined the influence of gp41 peptides on the expression and the secretion of Alzheimer's amyloid precursor protein (APP) in human astroglial cell line T98G. Western blotting analyses demonstrated that treatment of glial cells with a putative immunosuppressive domain (aa 583-599) of gp41 remarkably downregulated the interleukin 1beta- (IL-1beta) induced elevation of the secreted form of APP (sAPP alpha) containing Kunitz-type protease inhibitor (KPI) domain without significant changes of the expression pattern of APP mRNAs as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Recombinant gp41 protein encoding for ectodomain, including aa 583-599 residues, also elicited a similar dose-dependent inhibitory effect, whereas the control peptides resulted in little change. The molecular mechanism underlying this gp41-mediated reduction of sAPP alpha secretion appears not to be owing to the difference in the function of extracellular proteases based on the finding of similar proteolytic activities responsible for APP metabolism in vitro present in the conditioned media from the cultures treated with or without gp41 peptide. However, the known PKC inhibitors such as H-7 or staurosporine, partially inhibited the elevation of sAPP alpha secretion in response to protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PdBu) as well as to IL-1beta, mimicking the immunosuppressive gp41 peptide. These observations implicate that part of the neurodegenerative cascade in AIDS brains may involve the inhibitory effect of gp41 on secretion of sAPP alpha, a potent glial neurotrophic factor, through impaired PKC response.

Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal alpha(2-6)-linked sialic acids.

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49-58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both alpha(2-3)- and alpha(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the alpha(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the alpha(2-3) or the alpha(2-6) linkage revealed that JCV preferentially interacts with alpha(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-alpha-D-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.

Extracellular Signal-Regulated Kinase and p38 Subgroups of Mitogen-Activated Protein Kinases Regulate Inducible Nitric Oxide Synthase and Tumor Necrosis Factor-α Gene Expression in Endotoxin-Stimulated Primary Glial Cultures

Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO), the product of inducible NO synthase (iNOS), mediate inflammatory and immune responses in the CNS under a variety of neuropathological situations. They are produced mainly by "activated" astrocytes and microglia, the two immune regulatory cells of the CNS. In this study we have examined the regulation of TNFalpha and iNOS gene expression in endotoxin-stimulated primary glial cultures, focusing on the role of mitogen-activated protein (MAP) kinase cascades. The bacterial lipopolysaccharide (LPS) was able to activate extracellular signal-regulated kinase (ERK) and p38 kinase subgroups of MAP kinases in microglia and astrocytes. ERK activation was sensitive to PD98059, the kinase inhibitor that is specific for ERK kinase. The activity of p38 kinase was inhibited by SB203580, a member of the novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs), as revealed by blocked activation of the downstream kinase, MAP kinase-activated protein kinase-2. The treatment of glial cells with either LPS alone (microglia) or a combination of LPS and interferon-gamma (astrocytes) resulted in an induced production of NO and TNFalpha. The two kinase inhibitors, at micromolar concentrations, individually suppressed and, in combination, almost completely blocked glial production of NO and the expression of iNOS and TNFalpha, as determined by Western blot analysis. Reverse transcriptase-PCR analysis showed changes in iNOS mRNA levels that paralleled iNOS protein and NO while indicating a lack of effect of either of the kinase inhibitors on TNFalpha mRNA expression. The results demonstrate key roles for ERK and p38 MAP kinase cascades in the transcriptional and post-transcriptional regulation of iNOS and TNFalpha gene expression in endotoxin-activated glial cells.

Expression of chemokine genes in rat glial cells: The effect of myelin basic protein‐reactive encephalitogenic T cells

Chemokine gene expression and chemokine activity appear to be major components of the immunopathological processes of inflammation and autoimmunity. To initiate an investigation of the role of chemokines in the pathogenesis of autoimmune inflammatory demyelination, we examined the expression of mRNA transcripts encoding four prominent chemokines, IP-10, MIP-1α, MCP-1, and RANTES, in encephalitogenic rat MBP-reactive T cells, astrocytes, and microglia. Astrocytes and microglia, whether as lines or as freshly isolated cells, did not constitutively express IP-10 and MCP-1 mRNA but could be induced with LPS to also produce MIP-1α and RANTES. MBP-reactive T cells were induced with MBP to produce abundant levels of MIP-1α, MCP-1, and RANTES mRNA in different temporal profiles but did not express IP-10 mRNA. In an MHC-II restricted fashion, the antigen-activated MBP-reactive T cells also induced glial cells to express all four chemokines, with the chemokine gene expression greatest following T-cell interactions with MHC-compatible glia. Treatment of glial cells with TNF-α and IFN-γ induced only IP-10, indicating that the expression of chemokine genes other than IP-10 requires a combination of different cytokines or direct cell-cell contact between T cells and glia. Quantitative assays revealed that activated astrocytes, the dominant glia of the CNS, express higher levels of chemokine transcripts than transcripts of the major proinflammatory cytokines TNF-α and IFN-γ. These results underscore the prominent but complex expression of chemokines by cellular component of inflammatory demyelinating lesions. J. Neurosci. Res. 48:192–200, 1997. © 1997 Wiley-Liss, Inc.

Expression of chemokine genes in rat glial cells: The effect of myelin basic protein-reactive encephalitogenic T cells

Chemokine gene expression and chemokine activity appear to be major components of the immunopathological processes of inflammation and autoimmunity. To initiate an investigation of the role of chemokines in the pathogenesis of autoimmune inflammatory demyelination, we examined the expression of mRNA transcripts encoding four prominent chemokines, IP-10, MIP-1 alpha, MCP-1, and RANTES, in encephalitogenic rat MBP-reactive T cells, astrocytes, and microglia. Astrocytes and microglia, whether as lines or as freshly isolated cells, did not constitutively express IP-10 and MCP-1 mRNA but could be induced with LPS to also produce MIP-1 alpha and RANTES. MBP-reactive T cells were induced with MBP to produce abundant levels of MIP-1 alpha, MCP-1, and RANTES mRNA in different temporal profiles but did not express IP-10 mRNA. In an MHC-II restricted fashion, the antigen-activated MBP-reactive T cells also induced glial cells to express all four chemokines, with the chemokine gene expression greatest following T-cell interactions with MHC-compatible glia. Treatment of glial cells with TNF-alpha and IFN-gamma induced only IP-10, indicating that the expression of chemokine genes other than IP-10 requires a combination of different cytokines or direct cell-cell contact between T cells and glia. Quantitative assays revealed that activated astrocytes, the dominant glia of the CNS, express higher levels of chemokine transcripts than transcripts of the major proinflammatory cytokines TNF-alpha and IFN-gamma. These results underscore the prominent but complex expression of chemokines by cellular component of inflammatory demyelinating lesions.