High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.     

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP-selective receptor in cultured rat astrocytes, human brain tumors, and in response to acute intracranial injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse activities in the nervous system. In addition to its more classic role as a neurotransmitter, PACAP functions as a neurotrophic factor. PACAP exerts these activities by binding to PACAP-selective (PAC1) or nonselective (VPAC1, VPAC2) receptors (-R). Glial cells also exhibit PACAP binding, which is associated with the increased proliferation of astrocytes. The present report demonstrates a distinct spatiotemporal regulation of PACAP, PAC1-R, VPAC1-R, and VPAC2-R expression in primary cultured rat astrocytes. To determine the role of PACAP and PAC1-R expression on glial proliferation, two in vivo models were examined--human brain tumors of glial origin and the reactive gliosis induced by a penetrating stab wound to the mature rat brain. Relative to normal human brain, PAC1-R expression is significantly upregulated in glioma, particularly oligodendrogliomas. While similar polymerase chain reaction (PCR) analysis does not detect PACAP expression, in situ hybridization studies reveal PACAP expression in a limited number of cells within the tumor. In sharp contrast, neither PACAP nor PAC1-R expression are upregulated consequent to injury. These results suggest a distinct role for PACAP and PAC1-R in glioma development and nervous system response to injury.     

S100B content and secretion decrease in astrocytes cultured in high-glucose medium

S100B is an astrocyte calcium-binding protein that plays a regulatory role in the cytoskeleton and cell cycle. Moreover, extracellular S100B, a marker of glial activation in several conditions of brain injury, has a trophic or apoptotic effect on neurons, depending on its concentration. Hyperglycemic rats show changes in glial parameters, including S100B expression. Here, we investigated cell density, morphological and biochemical alterations in primary cortical astrocytes from rats and C6 glioma cells cultured in high-glucose medium. Astrocytes and C6 glioma cells have a reduced content of S100B and glial fibrillary acidic protein when cultured in a high-glucose environment, as well as a reduced content of glutathione and cell proliferation rate. Although these cells have been used indistinctly to study S100B secretion, we observed a contrasting profile of S100B secretion in a high-glucose medium: a decrease in primary astrocytes and an increase in C6 glioma cells. Based on the in vitro neurotrophic effects of the S100B protein, our data suggest that chronic elevated glucose levels affect astrocyte activity, reducing extracellular secretion of S100B and that this, in turn, could affect neuronal activity and survival. Such astrocyte alterations could contribute to cognitive deficit and other impairments observed in diabetic patients.     

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite- promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent- extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.     

Chondroitin sulfate of appican, the proteoglycan form of amyloid precursor protein, produced by C6 glioma cells interacts with heparin-binding neuroregulatory factors

Appican produced by rat C6 glioma cells, the chondroitin sulfate (CS) proteoglycan form of the amyloid precursor protein, contains an E disaccharide, -GlcUA-GalNAc(4,6-O-disulfate)-, in its CS chain. In this study, the appican CS chain from rat C6 glioma cells was shown to specifically bind several growth/differentiation factors including midkine (MK) and pleiotrophin (PTN). In contrast, the appican CS from SH-SY5Y neuroblastoma cells contained no E disaccharide and showed no binding to either MK or PTN. These findings indicate that the E motif is essential in the interaction of the appican CS chain with growth/differentiation factors, and suggest that glial appican may mediate the regulation of neuronal cell adhesion and migration and/or neurite outgrowth.     

Proliferation arrest and induction of CDK inhibitors p21 and p27 by depleting the calcium store in cultured C6 glioma cells

C6 glioma - Ca2+ depletion - proliferation arrest morphology change - CDK inhibitor In this study, we investigated the role of the intracellular calcium store in modulating the cellular proliferation and the expression of cell cycle regulatory proteins in cultured C6 glioma cells. By means of microspectrofluorimetry and Ca(2+)-sensitive indicator fura-2, we found that the intracellular Ca2+ pump inhibitors, thapsigargin (TG) irreversibly and 2,5-ditert-butyl-hydroquinone (DBHQ) reversibly depleted the Ca(2+)-store accompanied with the induction of G0/G1 arrest, an increase in glial fibrillary acidic protein (GFAP) expression and morphological changes from a round flat shape to a differentiated spindle-shaped cell. The machinery underlying these changes induced by Ca(2+)-store depletion was investigated. The results indicated that Ca(2+)-store depletion caused an increased expression of p21 and p27 proteins (cyclin-dependent kinase inhibitors), with unchanged mutant p53 protein of C6 cells but reduced amounts of the cell cycle regulators: cyclin-dependent kinase 2 (CDK2), cdc2, cyclin C, cyclin D1, cyclin D3 and proliferating cell nuclear antigen (PCNA) in a time-dependent manner. These findings indicate a new function of the endoplasmic reticulum (ER) Ca2+ store in regulating cellular proliferation rate through altering the expression of p21 and p27 proteins. Moreover, cellular differentiation as revealed by spindle-shaped morphology and induced GFAP expression were also modulated by the ER Ca2+ store. The implication of this finding is that the abnormal growth of cancer cells such as C6 glioma cells may be derived from a signalling of the ER which can be manipulated by depleting the Ca2+ store.     

Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring.     

The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis.     

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.     

Upregulation of the glutamine transporter through transactivation mediated by cAMP/protein kinase A signals toward exacerbation of vulnerability to oxidative stress in rat neocortical astrocytes

In the present study, we have evaluated the possible functionality in astrocytes of the glutamine (Gln) transporter (GlnT) known to predominate in neurons for the neurotransmitter pool of glutamate. Sustained exposure to the adenylyl cyclase activator forskolin for 24 h led to a significant increase in mRNA expression of GlnT among different membrane transporters capable of transporting Gln, with an increase in [(3)H]Gln accumulation sensitive to a system A transporter inhibitor, in cultured rat neocortical astrocytes, but not neurons. Forskolin drastically stimulated GlnT promoter activity in a manner sensitive to a protein kinase A (PKA) inhibitor in rat astrocytic C6 glioma cells, while deletion mutation analysis revealed that the stimulation was mediated by a cAMP responsive element (CRE)/activator protein-1 (AP-1) like site located on GlnT gene promoter. Forskolin drastically stimulated the promoter activity in a fashion sensitive to a PKA inhibitor in C6 glioma cells transfected with a CRE or AP-1 reporter plasmid, in association with the phosphorylation of CRE binding protein on serine133. Transient overexpression of GlnT significantly exacerbated the cytotoxicity of hydrogen peroxide in cultured astrocytes. These results suggest that GlnT expression is upregulated by cAMP/PKA signals for subsequent exacerbation of the vulnerability to oxidative stress in astrocytes.     

Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with nonglial cells in vitro

I have compared central nervous system (CNS) neurite outgrowth on glial and nonglial cells. Monolayers of glial cells (astrocytes and Schwann cells) or nonglial cells (e.g., fibroblasts) were prepared and were shown to be greater than 95% pure as judged by cell type-specific markers. These monolayers were then tested for their ability to support neurite outgrowth from various CNS explants. While CNS neurites grew vigorously on the glial cells, most showed little growth on nonglial cell monolayers. Neurites grew singly or in fine fascicles on the glial cells at rates greater than 0.5 mm/d. The neurite outgrowth on astrocytes was investigated in detail. Scanning and transmission electron microscopy showed that the neurites were closely apposed to the astrocyte surface and that the growth cones were well spread with long filopodia. There was no evidence of significant numbers of explant- derived cells migrating onto the monolayers. Two types of experiments indicated that factors associated with the astrocyte surface were primarily responsible for the vigorous neurite outgrowth seen on these cells: (a) Conditioned media from either astrocytes or fibroblasts had no effect on the pattern of outgrowth on fibroblasts and astrocytes, and conditioned media factors from either cell type did not promote neurite outgrowth when bound to polylysine-coated dishes. (b) When growing CNS neurites encountered a boundary between astrocytes and fibroblasts, they stayed on the astrocytes and did not encroach onto the fibroblasts. These experiments strongly suggest that molecules specific to the surfaces of astrocytes make these cells particularly attractive substrates for CNS neurite outgrowth, and they raise the possibility that similar molecules on embryonic glial cells may play a role in guiding axonal growth during normal CNS development.     

CCN1 Secretion Induced by Cigarette Smoking Extracts Augments IL-8 Release from Bronchial Epithelial Cells

Inflammation involves in many cigarette smoke (CS) related diseases including the chronic obstructive pulmonary disease (COPD). Lung epithelial cell released IL-8 plays a crucial role in CS induced lung inflammation. CS and cigarette smoke extracts (CSE) both induce IL-8 secretion and subsequently, IL-8 recruits inflammatory cells into the lung parenchyma. However, the molecular and cellular mechanisms by which CSE triggers IL-8 release remain not completely understood. In this study, we identified a novel extracellular matrix (ECM) molecule, CCN1, which mediated CSE induced IL-8 secretion by lung epithelial cells. We first found that CS and CSE up-regulated CCN1 expression and secretion in lung epithelial cells in vivo and in vitro. CSE up-regulated CCN1 via induction of reactive oxygen spices (ROS) and endoplasmic reticulum (ER) stress. p38 MAPK and JNK activation were also found to mediate the signal pathways in CSE induced CCN1. CCN1 was secreted into ECM via Golgi and membrane channel receptor aquaporin4. After CSE exposure, elevated ECM CCN1 functioned via an autocrine or paracrine manner. Importantly, CCN1 activated Wnt pathway receptor LRP6, subsequently stimulated Wnt pathway component Dvl2 and triggered beta-catenin translocation from cell membrane to cytosol and nucleus. Treatment of Wnt pathway inhibitor suppressed CCN1 induced IL-8 secretion from lung epithelial cells. Taken together, CSE increased CCN1 expression and secretion in lung epithelial cells via induction of ROS and ER stress. Increased ECM CCN1 resulted in augmented IL-8 release through the activation of Wnt pathway.     

Proteoglycans of reactive rat cortical astrocyte cultures: Abundance of N-unsubstituted glucosamine-enriched heparan sulfate

“Reactive” astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH₂ to GalNH₂, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a > 5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~ 20% and ~ 40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.     

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury: Involvement of PARK2-dependent mitophagy

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2^+/− mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.     

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2^+/? mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.     

Endoplasmic reticulum stress in glomerular epithelial cell injury

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in specific podocyte proteins. This study addresses mediation of GEC injury, focusing on endoplasmic reticulum (ER) stress. We studied signaling in cultured GECs in the presence or absence of the extracellular matrix (ECM). Adhesion to collagen supports cell survival, but adhesion to plastic (loss of contact with ECM) leads to apoptosis. Compared with collagen-adherent cells, GECs on plastic showed increased protein misfolding in the ER, and an adaptive-protective ER stress response, including increased expression of ER chaperones, increased phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), and a reduction in protein synthesis. Activation of these ER stress pathways counteracted apoptosis. However, tunicamycin (a potent stimulator of ER stress) changed the ER stress response from protective to cytotoxic, as tunicamycin induced the proapoptotic ER stress gene, C/EBP homologous protein-10, and exacerbated apoptosis in GECs adherent to plastic, but not collagen. In GECs adherent to plastic, adaptive ER stress was associated with an increase in polyubiquitinated proteins and "choking" of the proteasome. Furthermore, pharmacological inhibition of the proteasome induced ER stress in GECs. Finally, we show that ER stress (induction of ER chaperones and eIF2α phosphorylation) was evident in experimental FSGS in vivo. Thus interactions of GECs with ECM may regulate protein folding and induction of the ER stress response. FSGS is associated with induction of ER stress. Enhancing protective aspects of the ER stress response may reduce apoptosis and possibly glomerulosclerosis.