Cultured Rat Brain Astrocytes Research Articles

A dynamic study of VEGF-A siDOX-EVs trafficking through the in-vitro insert co-culture blood-brain barrier model by digital holographic microscopy.

Modeling the blood-brain barrier has long been a challenge for pharmacological studies. Up to the present, numerous attempts have been devoted to recapitulating the endothelial barrier in vitro to assess drug delivery vehicles' efficiency for brain disorders. In the current work, we presented a new approach for analyzing the morphometric parameters of the cells of an insert co-culture blood-brain barrier model using rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes. This analytical approach could aid in getting further information on drug trafficking through the blood-brain barrier and its impact on the brain indirectly. In the current work, we cultured rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes and then used an insert well to culture the cells in contact with each other to model the blood-brain barrier. Then, the morphometric parameters of the porous membrane of the insert well, as well as each cell type were imaged by digital holographic microscopy before and after cell seeding. At last, we performed folate conjugation on the surface of the EVs we have previously tested for glioma therapy in our previous work called VEGF-A siDOX-EVs and checked how the trafficking of EVs improves after folate conjugation as a clathrin-mediated delivery setup. the trafficking and passage of EVs were assessed by flowcytometry and morphometric analysis of the digital holographic microscopy holograms. Our results indicated that EVs successfully entered through the proposed endothelial barrier assessed by flow cytometry analysis and furthermore, folate conjugation significantly improved EV passage through the blood-brain barrier. Moreover, our results indicated that the VEGF-A siDOX-EVs insert cytotoxic impact on the cells of the bottom of the culture plate. folate-conjugation on the surface of EVs improves their trafficking through the blood-brain barrier and by using digital holographic microscopy analysis, we could directly assess the morphometric changes of the blood-brain barrier cells for pharmacological purposes as an easy, label-free, and real-time analysis.

All-trans Retinoic Acid Increased Transglutaminase 2 Expressions in BV-2 Cells and Cultured Astrocytes.

Activation of microglia and astrocytes has been observed in Alzheimer's disease (AD). Transglutaminase 2 (TG2) is reported to be activated in AD and involved in cell proliferation, differentiation, and inflammation. Moreover, amyloid β (Aβ) aggregation is detected as a characteristic pathology in the AD brain, and is known to be a substrate of TG2. All-trans retinoic acid (ATRA) can modify cell proliferation and differentiation, and is reported to have therapeutic effects on AD pathology. We aimed to assess the effects of ATRA in microglia and astrocytes on TG2 expression and glial functions. After treatment with ATRA, TG2 expression and TG activity were assayed in both murine microglia BV-2 cells and cultured rat brain astrocytes. Endocytosis activity in BV-2 cells and Aβ aggregation by astrocytes conditioned medium were also assessed. In both BV-2 cells and cultured astrocytes, ATRA increased TG2 expression and TG activity. The increase was blocked by AGN194310, an RA receptor antagonist. ATRA enhanced the endocytosis activity in BV-2 cells, and the addition of AGN194310 reversed it. The addition of cystamine, a competitive TG inhibitor, also reduced ATRA-enhanced endocytosis activity. On the other hand, Aβ aggregation was potentiated by ATRA-treated astrocytes conditioned medium compared to control astrocytes conditioned medium. These results suggest that ATRA increased TG2 expression and TG activity via RA receptor in microglia and astrocytes. ATRA-enhanced TGs might be involved in phagocytosis and Aβ aggregation. Adequate control of TGs expression and function in microglia and astrocytes can be an important factor in AD pathology.

Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes

Brain astrocytes are considered as glycolytic cell type, but these cells also produce ATP via mitochondrial oxidative phosphorylation. Exposure of cultured primary astrocytes in a glucose-free medium to extracellular substrates that are known to be metabolised by mitochondrial pathways, including pyruvate, lactate, beta-hydroxybutyrate, alanine and acetate, revealed that among the substrates investigated extracellular pyruvate was most efficiently consumed by astrocytes. Extracellular pyruvate was consumed by the cells almost proportional to time over hours in a concentration-dependent manner with apparent Michaelis–Menten kinetics [Km = 0.6 ± 0.1 mM, Vmax = 5.1 ± 0.8 nmol/(min × mg protein)]. The astrocytic consumption of pyruvate was strongly impaired in the presence of the monocarboxylate transporter 1 (MCT1) inhibitor AR-C155858 or by application of a 10-times excess of the MCT1 substrates lactate or beta-hydroxybutyrate. Pyruvate consumption by viable astrocytes was inhibited in the presence of UK5099, an inhibitor of the mitochondrial pyruvate carrier, or after application of the respiratory chain inhibitor antimycin A. In contrast, the mitochondrial uncoupler BAM15 strongly accelerated cellular pyruvate consumption. Lactate and alanine accounted after 3 h of incubation with pyruvate for around 60% and 10%, respectively, of the pyruvate consumed by the cells. These results demonstrate that consumption of extracellular pyruvate by astrocytes involves uptake via MCT1 and that the velocity of pyruvate consumption is strongly modified by substances that affect the entry of pyruvate into mitochondria or the activity of mitochondrial respiration.

Vitamin D3 Inhibits Phagocytic Activity of Rat Brain Astrocytes in Primary Culture

Vitamin D3 Inhibits Phagocytic Activity of Rat Brain Astrocytes in Primary Culture

ВЛИЯНИЕ ОКИСЛИТЕЛЬНОГО СТРЕССА НА ПРОЦЕСС ФАГОЦИТОЗA АПОПТОТИЧЕСКИХ СУБСТРАТОВ АСТРОЦИТАМИ МОЗГА КРЫС

Brain astrocytes are commonly believed to be relatively tolerant to oxidative stress. In this work, we studied the effect of oxidative stress on the ability of rat brain astrocytes in primary culture to ingest and phagocytose apoptotic substrates. Using fluorescent microscopy, we demonstrated that the ingestion of rat retinal photoreceptor outer segments (POS), used here as a phagocytic substrate, dramatically decreased in astrocytes preincubated with various hydroperoxides. The ability of primary astrocytes to ingest POS also depended on the substrate oxidative status. The ingestion of “oxidized” POS preparations preincubated with hydrogen peroxide was sharply reduced compared to “non-oxidized” POS. We also showed that natural and synthetic antioxidants ( α -tocopherol, Trolox) efficiently protected the phagocytic function of astrocytes from oxidative damage. These data suggest that the phagocytic function of rat brain primary astrocytes is extremely vulnerable to oxidative stress, while oxidative stress tolerance characterizes not all functions of these glial cells.

Rat astrocytes during anoxia: Secretome profile of cytokines and chemokines.

IntroductionThe precise mechanisms of the inflammatory responses after cerebral ischemia in vivo are difficult to elucidate because of the complex nature of multiple series of interactions between cells and molecules. This study explored temporal patterns of secretion of 30 cytokines and chemokines from Sprague Dawley rat astrocytes in primary culture in order to elucidate signaling pathways that are triggered by astrocytes during anoxia.MethodsPrimary cultures of rat brain astrocytes were incubated for periods of 2–24 hr in the absence of oxygen (anoxia) or under normal partial pressure of oxygen (controls). Simultaneous detection of 29 cytokines and chemokines in the samples was performed using a rat cytokine array panel, while the temporal pattern of angiopoietin‐1 (Ang‐1) secretion was determined separately using ELISA. Wilcoxon–Mann–Whitney test was used to compare normoxic and anoxic samples and the Hodge–Lehman estimator with exact 95% confidence intervals was computed to assess the size of differences in cytokine secretion. The obtained data were imported into the Core Analysis tool of Ingenuity Pathways Analysis software in order to relate changes in secretion of cytokines and chemokines from astrocytes during anoxia to potential molecular signal networks.ResultsWith the exception of Ang‐1, concentrations of all cytokines/chemokines in samples collected after anoxia exposure were either the same, or higher, than in control groups. No clear pattern of changes could be established for groups of cytokines with similar effects (i.e., pro‐ or anti‐inflammatory cytokines). The pattern of changes in cytokine secretion during anoxia was associated with the HIF‐1α‐mediated response, as well as cytokines IL‐1β and cathepsin S pathways, which are related to initiation of inflammation and antigen presentation, respectively, and to ciliary neurotrophic factor.ConclusionsThese in vitro findings suggest that astrocytes may play a role in triggering inflammation during anoxia/ischemia of the brain.

Expression of CYP 4A ω-hydroxylase and formation of 20-hydroxyeicosatetreanoic acid (20-HETE) in cultured rat brain astrocytes

Astrocytes secrete vasodilator and vasoconstrictor factors via end feet processes, altering blood flow to meet neuronal metabolic demand. Compared to what is known about the ability of astrocytes to release factors that dilate local cerebral vasculature, very little is known regarding the source and identity of astrocyte derived constricting factors. The present study investigated if astrocytes express CYP 4A ω-hydroxylase and metabolize arachidonic acid (AA) to 20-hydroxyeicotetraenoic acid (20-HETE) that regulates KCa channel activity in astrocytes and cerebral arterial myocyte contractility. Here we report that cultured astrocytes express CYP 4A2/3 ω-hydroxylase mRNA and CYP 4A protein and produce 20-HETE and the CYP epoxygenase metabolites epoxyeicosatrienoic acids (EETs) when incubated with AA. The production of 20-HETE and EETs was enhanced following stimulation of metabotropic glutamate receptors (mGluR) on the astrocytes. Exogenous application of 20-HETE attenuated, whereas inhibition of 20-HETE production with HET-0016 increased the open state probabilities (NPo) of 71pS and 161pS KCa single-channel currents recorded from astrocytes. Exposure of isolated cerebral arterial myocytes to conditioned media from cultured astrocytes caused shortening of the length of freshly isolated cerebral arterial myocytes that was not evident following inhibition of astrocyte 20-HETE synthesis and action. These findings suggest that astrocytes not only release vasodilator EETs in response to mGluR stimulation but also synthetize and release the cerebral arterial myocyte constrictor 20-HETE that also functions as an endogenous inhibitor of the activity of two types of KCa channel currents found in astrocytes.

The chemically synthesized ageladine A-derivative LysoGlow84 stains lysosomes in viable mammalian brain cells and specific structures in the marine flatworm Macrostomum lignano.

Based on the chemical structure and the known chemical synthesis of the marine sponge alkaloid ageladine A, we synthesized the ageladine A-derivative 4-(naphthalene-2-yl)-1H-imidazo[4,5-c]pyridine trifluoroacetate (LysoGlow84). The two-step synthesis started with the Pictet-Spengler reaction of histamine and naphthalene-2-carbaldehyde to a tetrahydropyridine intermediate, which was dehydrogenated with activated manganese (IV) oxide to LysoGlow84. Structure and purity of the synthesized LysoGlow84 were confirmed by NMR spectroscopy and mass spectrometry. The fluorescence intensity emitted by LysoGlow84 depended strongly on the pH of the solvent with highest fluorescence intensity recorded at pH 4. The fluorescence maximum (at 315 nm excitation) was observed at 440 nm. Biocompatibility of LysoGlow84 was investigated using cultured rat brain astrocytes and the marine flatworm Macrostomum lignano. Exposure of the astrocytes for up to 6 h to micromolar concentrations of LysoGlow84 did not compromise cell viability, as demonstrated by several viability assays, but revealed a promising property of this compound for staining of cellular vesicles. Conventional fluorescence microscopy as well as confocal scanning microscopy of LysoGlow84-treated astrocytes revealed co-localization of LysoGlow84 fluorescence with that of LysoTracker® Red DND-99. LysoGlow84 stained unclear structures in Macrostomum lignano, which were identified as lysosomes by co-staining with LysoTracker. Strong fluorescence staining by LysoGlow84 was further observed around the worms’ anterior gut and the female genital pore which were not counterstained by LysoTracker Red. Thus, LysoGlow84 is a new promising dye that stains lysosomes and other acidic compartments in cultured cells and in worms.

Electrophysiology and pharmacology of tandem domain potassium channel TREK-1 related BDNF synthesis in rat astrocytes

In the present study, the functional properties and pharmacology of two-pore domain potassium channel (K2P) TREK-1 in primary cultured rat brain astrocytes were investigated. Western blot, patch clamping techniques, and ELISA were used to detect the distribution and function of TREK-1 as well as the expression of brain-derived neurotrophic factor (BDNF) on the primary cultured astrocytes. It was shown that TREK-1 protein expressed in astrocytes was 2.4-fold higher than it was expressed in microglia. Single channel recording via patch clamping showed that the TREK-1 outward currents in astrocytes could be activated by arachidonic acid (AA) or chloroform with the conductance of 113 ± 14 and 120 ± 13pS, respectively. The current was also sensitive to mechanical stretch and intracellular acidification. Negative pressure (-30cm H2O) and acidification of intracellular solution (pH6.8 or 6.3) both enhanced TREK-1 channel open probability significantly. Further pharmacological studies showed that TREK-1 antagonist penfluridol inhibited AA-induced currents, and both penfluridol and methionine (TREK-1 blockers) significantly increased BDNF level in astrocytes by 50%. These results indicated that TREK-1 channel current was a major component of K2P currents in astrocytes. TREK-1 channels might play important roles in regulating the function of astrocytes and might be used as a drug target for neuroprotection.

Reporter Dyes Demonstrate Functional Expression of Multidrug Resistance Proteins in the Marine Flatworm Macrostomum lignano: The Sponge-Derived Dye Ageladine A Is Not a Substrate of These Transporters

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.

Imipramine Induces Brain-Derived Neurotrophic Factor mRNA Expression in Cultured Astrocytes

Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.

Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.

Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H₂O₂. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, in the presence of horseradish peroxidase, H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials.

NOV/CCN3 upregulates CCL2 and CXCL1 expression in astrocytes through β1 and β5 integrins

Increasing evidence suggests that CCN matricellular proteins play important roles in inflammation. One of the major cell types that handle inflammation in the brain is the astrocyte, which, upon activation, dramatically increases its production of cytokines and chemokines. Here, we report that NOV/CCN3, added to primary cultured rat brain astrocytes, markedly increased the expression of CCL2 and CXCL1 chemokines, as indicated by ELISA and RT-qPCR assays. This effect was selective, as the production of thirteen other cytokines and chemokines was not affected by NOV. NOV expression by astrocytes was demonstrated by immunocytochemistry and Western blot analysis, and astrocyte transfection with NOV small interfering RNA (siRNA) markedly decreased CXCL1 and CCL2 production, indicating that endogenous NOV played a major role in the control of astrocytic chemokine synthesis. NOV was shown to mediate several of its actions through integrins. Here, we observed that siRNAs against integrins beta1 and beta5 decreased basal and abrogated NOV-stimulated astrocyte expression of CCL2 and CXCL1, respectively. Using a panel of kinase inhibitors, we demonstrated that NOV action on CCL2 and CXCL1 production involved a Rho/ROCK/JNK/NF-kappaB and a Rho/qROCK/p38/NF-kappaB pathway, respectively. Thus, distinct integrins and signaling mechanisms are involved in NOV-induced production of CCL2 and CXCL1 in astrocytes. Finally, astrocytic expression of NOV was detected in rat brain tissue sections, and NOV intracerebral injection increased CCL2 and CXCL1 brain levels in vivo. Altogether, our data shed light on the signaling pathways operated by NOV and strongly suggest that NOV mediates astrocyte activation and, therefore, might play a role in neuroinflammation.

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed.

Visualization of Melatonin's Multiple Mitochondrial Levels of Protection Against Mitochondrial Ca2+-Mediated Permeability Transition and Beyond in Rat Brain Astrocytes

Melatonin protects cells against oxidative stress-induced apoptosis due primarily to its ability to effectively scavenge pathological condition-augmented generation of mitochondrial reactive oxygen species (mROS). Once produced, mROS in addition to indiscriminately damage mitochondrial components they crucially activate directly the mitochondrial permeability transition (MPT), one of the critical mechanisms for initiating post mitochondrial apoptotic signaling. Whether or not melatonin targets directly the MPT, however, remains inconclusive, particularly during oxidative stress. Thus, we investigated this possibility of an “oxidation free Ca2+ stress” in the presence of vitamin E after ionomycin exposure as a sole Ca2+-mediated MPT in order to exclude melatonin's primary antioxidative effects as well as Ca2+-mediated oxidative stress. With the application of laser scanning fluorescence imaging microscopy, we visualized for the first time multiple mitochondrial protections provided by melatonin during Ca2+ stress in cultured rat brain astrocytes RBA-1. Melatonin, due to its primary antioxidative actions, completely prevented mCa2+-induced mROS formation for a reduced mROS-activated MPT during ionomycin exposure. In the presence of vitamin E, melatonin, significantly reduced cyclosporin A (CsA) sensitive mitochondrial depolarization and MPT during ionomycin exposure suggesting its direct targeting of the MPT. Moreover, when the MPT was inhibited by CsA, melatonin reduced further MPT-independent mitochondrial depolarization and apoptosis suggesting its targeting beyond the MPT. As astrocytes play active role in regulating neuronal pathophysiology, these multiple mitochondrial protections provided by melatonin against mCa2+- and/or mROS-mediated apoptosis may thus be crucial for the future therapeutic prevention and treatment of astrocyte-mediated neurodegeneration in the CNS.

Visualization of melatonin’s multiple mitochondrial levels of protection against mitochondrial Ca2+‐mediated permeability transition and beyond in rat brain astrocytes

Melatonin protects cells against various types of oxidative stress-induced apoptosis due primarily to its ability to effectively scavenge pathological and disease condition-augmented generation of mitochondrial reactive oxygen species (mROS). Once produced, mROS indiscriminately damage mitochondrial components and more importantly they crucially activate directly the mitochondrial permeability transition (MPT), one of the critical mechanisms for initiating post mitochondrial apoptotic signaling. Whether or not melatonin targets directly the MPT, however, remains inconclusive, particularly during oxidative stress. This study, thus, investigated this possibility of an 'oxidation free Ca(2+) stress' in the presence of vitamin E after ionomycin exposure as a sole Ca(2+)-mediated MPT in order to exclude melatonin's primary antioxidative effects as well as Ca(2+)-mediated oxidative stress. The studies were carried out using cultured rat brain astrocytes RBA-1. With the application of laser scanning multiple fluorescence imaging microscopy, we visualized for the first time multiple mitochondrial protective effects provided by melatonin during Ca(2+) stress. First, melatonin, due to its primary antioxidative actions, completely prevented mCa(2+)-induced mROS formation during ionomycin exposure. Secondly, when melatonin(')s antioxidative effects were prevented due to the addition of vitamin E, melatonin significantly prevented mCa(2+)-mediated MPT and apoptosis suggesting its direct targeting of the MPT. Surprisingly, in the presence of cyclosporin A, a MPT inhibitor, melatonin reduced further mCa(2+)-mediated apoptosis during ionomycin exposure also suggesting its targeting beyond the MPT. As astrocytes are actively involve in regulating synaptic transmission and neurovascular coupling in the CNS, these multiple mitochondrial layers of protection provided by melatonin against mCa(2+)-and/or mROS-mediated apoptosis in astrocytes may be crucial for future therapeutic prevention and treatment of astrocyte-mediated neurodegenerative diseases in the CNS.

Differential SUMOylation of LXRα and LXRβ Mediates Transrepression of STAT1 Inflammatory Signaling in IFN-γ-Stimulated Brain Astrocytes

To unravel the roles of LXRs in inflammation and immunity, we examined the function of LXRs in development of IFN-gamma-mediated inflammation using cultured rat brain astrocytes. LXR ligands inhibit neither STAT1 phosphorylation nor STAT1 translocation to the nucleus but, rather, inhibit STAT1 binding to promoters and the expression of IRF1, TNFalpha, and IL-6, downstream effectors of STAT1 action. Immunoprecipitation data revealed that LXRbeta formed a trimer with PIAS1-pSTAT1, whereas LXRalpha formed a trimer with HDAC4-pSTAT1, mediated by direct ligand binding to the LXR proteins. In line with the fact that both PIAS1 and HDAC4 belong to the SUMO E3 ligase family, LXRbeta and LXRalpha were SUMO-conjugated by PIAS1 or HDAC4, respectively, and SUMOylation was blocked by transient transfection of appropriate individual siRNAs, reversing LXR-induced suppression of IRF1 and TNFalpha expression. Together, our data show that SUMOylation is required for the suppression of STAT1-dependent inflammatory responses by LXRs in IFN-gamma-stimulated brain astrocytes.

Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: Involvement of oxidative stress and mitogen-activated protein kinase cascades

Alzheimer's disease (AD) is a progressive neurodegenerative disorder primarily characterized by excessive deposition of amyloid-β (Aβ) peptides in the brain. One of the earliest neuropathological changes in AD is the presence of a high number of reactive astrocytes at sites of Aβ deposition. Disturbance of glutamatergic neurotransmission and consequent excitotoxicity is also believed as implicated in the progression of this dementia. Therefore, the study of astrocyte responses to Aβ, the main cellular type involved in the maintenance of synaptic glutamate concentrations, is crucial for understanding the pathogenesis of AD. This study aims to investigate the effect of Aβ on the astrocytic glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate–aspartate transporter (GLAST), and their relative participation to glutamate clearance. In addition we have also investigated the involvement of mitogen-activated protein (MAP) kinases in the modulation of GLT-1 and GLAST levels and activity and the putative contribution of oxidative stress induced by Aβ to the astrocytic glutamate transport function. Therefore, we used primary cultures of rat brain astrocytes exposed to Aβ synthetic peptides. The data obtained show that Aβ 1-40 peptide decreased astroglial glutamate uptake capacity in a non-competitive mode of inhibition, assessed in terms of tritium radiolabeled d-aspartate ( d-[ 3H]aspartate) transport. The activity of GLT-1 seemed to be more affected than that of GLAST, and the levels of both transporters were decreased in Aβ 1-40-treated astrocytes. We demonstrated that MAP kinases, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase, were activated in an early phase of Aβ 1-40 treatment and the whole pathways differentially modulated the glutamate transporters activity/levels. Moreover it was shown that oxidative stress induced by Aβ 1-40 may lead to the glutamate uptake impairment observed. Taken together, our results suggest that Aβ peptide downregulates the astrocytic glutamate uptake capacity and this effect may be in part mediated by oxidative stress and the differential activity and complex balance between the MAP kinase signaling pathways.

Activation of P2X7 Receptors Stimulates the Expression of P2Y2 Receptor mRNA in Astrocytes Cultured from Rat Brain

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X(7) ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X(7) receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y(2) in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y(2) expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y(2) sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y(2) mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X(7)-antagonists oATP and BBG. The BzATP-evoked P2Y(2) receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y(2)siRNA, thus supporting evidence of increased P2Y(2) activity. To further investigate the mechanisms by which P2X(7) receptors mediated the P2Y(2) mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y(2) mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X(7)-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X(7) receptors in astrocytes enhances P2Y(2) mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways.

P2X7Receptor Activation in Rat Brain Cultured Astrocytes Increases the Biosynthetic Release of Cysteinyl Leukotrienes

Astrocytes have been recognized as important elements in controlling inflammatory as well as immune processes in the central nervous system (CNS). Recently, glial cells have been shown to produce cysteinyl leukotrienes (CysLTs) which are known lipid mediators of inflammation and whose extracellular concentrations rise under different pathological conditions in the brain. In the same conditions also extracellular concentrations of ATP dramatically increase reaching levels able to activate P2X7 ionotropic receptors for which an emerging role in neuroinflammation and neurodegeneration has been claimed. RTPCR analysis showed that primary cultures of rat brain astrocytes express P2X7 receptors. Application of the selective P2X7 agonist benzoyl benzoly ATP (BzATP) markedly increased [Ca2+]i which was mediated by a calcium influx from the extracellular milieu. The P2X7 antagonist, oATP, suppressed the BzATP-induced calcium increase. Consistent with the evidence that increased calcium levels activate the leukotriene biosynthetic pathway, challenge of astrocytes with either the calcium ionophore A23187 or BzATP significantly increased CysLT production and the cell pre-treatment with EGTA abolished these effects. Again the P2X7 antagonist prevented the BzATP-mediated CysLT efflux, whereas the astrocyte pretreatment with MK-571, a CysLT1 receptor antagonist, was ineffective. The astrocyte pre-treatment with a cocktail of inhibitors of ATP binding cassette (ABC) proteins reduced the BzATP-mediated CysLT production confirming that ABC transporters are involved in the release of CysLTs. The astrocyte P2X7- evoked rise of CysLT efflux was abolished in the presence of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP) whose expression, along with that of 5-lipoxygenase (5-LO) was reported by Northern Blot analysis. The stimulation of P2X7 induced an up-regulation of FLAPmRNA that was reduced by the antagonist oATP. These data suggest that in rat brain cultured astrocytes P2X7ATP receptors may participate in the control of CysLT release thus further supporting a role for extracellular ATP as an integral component of the inflammatory brain response.