Cultured Rat Astrocytes Research Articles

The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA) cycle, we incubated primary rat astrocyte cultures with [U-13C]glucose in the absence or the presence of 100 μM T23 for 2 h and analyzed the 13C metabolite pattern. These incubation conditions did not compromise cell viability and confirmed that the presence of T23 doubled glycolytic lactate production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of 13C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly demonstrate that, in addition to glycolysis, also the mitochondrial TCA cycle is strongly accelerated after exposure of astrocytes to T23, suggesting that a protein tyrosine kinase may be involved in the regulation of the TCA cycle in astrocytes.

IL-17 signalling in astrocytes promotes glutamate excitotoxicity: Indications for the link between inflammatory and neurodegenerative events in multiple sclerosis

ObjectiveTh-17 cells have been exclusively referred to inflammatory events in multiple sclerosis (MS), while their importance in the development of glutamate excitotoxicity and the consequent neurodegeneration has been a completely unexplored concept. Accordingly, the objective of our study was to assess IL-17A effect on astrocyte ability to metabolize and release glutamate, considering that astrocytes had the central role in glutamate homeostasis. MethodsBy using primary rat astrocyte cultures, astrocyte ability to uptake glutamate was estimated by the alterations of glutamate transporters (GLAST and GLT-1) expression, whereas changes in glutamine synthetase expression were used to estimate the ability to metabolize glutamate. Gene expression was determined by real time polymerase chain reaction (rtPCR). IL-17A effect on astrocyte ability to produce glutamate was investigated directly, by measuring the level of released glutamate using high performance liquid chromatography (HPLC). ResultsLower concentrations of IL-17A reduced the expressions of both glutamate transporters and glutamine synthetase; however, this effect was lost when IL-17A was applied in a higher dose. IL-17A did not significantly modify glutamate release from astrocyte in basal conditions, but following Ca2+ stimulation, as well as Ca2+ removal from the culture medium, IL-17A stimulated glutamate release in dose-dependent manner. ConclusionTogether, these results support that IL-17A could promote glutamate excitotoxicity by decreasing astrocyte ability to uptake and convert glutamate to non-toxic glutamine, but also by stimulating Ca2+ dependent glutamate release. Such interactions between IL-17A and glutamate excitotoxicity implicate the potential link between inflammation and neurodegeneration during MS pathogenesis, and identify astrocytes as a potential target in achieving neuroprotective effects in MS.

Fast and Efficient Neural Conversion of Human Hematopoietic Cells.

A major challenge in regenerative medicine is the generation of functionally effective target cells to replace or repair damaged tissues. The finding that most somatic cells can be directly converted into cells of another lineage by the expression of specific transcription factors has paved the way to novel applications. Induced neurons (iNs) represent an alternative source of neurons for disease modeling, drug screening, and potentially, for cell replacement therapy. This unit describes methods for the efficient conversion of blood cells into iNs, including protocols to isolate cord blood CD133+ cells, infect them with Sendai virus vectors that express SOX2 and c-MYC, and differentiate the infected cells (PB-MNCs) into mature neurons. A method to reprogram peripheral blood mononuclear cells into iNs is also described. Support protocols describe how to culture rat astrocytes and characterize the electrophysiology of iNs. © 2016 by John Wiley & Sons, Inc.

The expression of glial cell line-derived neurotrophic factor mRNA by antidepressants involves matrix metalloproteinase-9 activation in rat astroglial cells

Neurotrophic/growth factors derived from glial cells, especially astrocytes, have been implicated in mood disorders and the pharmacological effects of antidepressant drugs. Previous studies demonstrated that the release of glial cell line-derived neurotrophic factor (GDNF) induced by the tricyclic antidepressant amitriptyline was significantly inhibited by a broad-spectrum matrix metalloproteinase (MMP) inhibitor in rat C6 astroglial cells (C6 cells). However, it is unknown whether amitriptyline affects MMP enzymatic activity or expression, and the MMP subtype has yet to be identified. The current study measured the effect of antidepressants on MMP activity with gelatin zymography, an in vitro assay for enzymatic activity, in C6 cells and primary cultured rat astrocytes (primary astrocytes). Treatment with amitriptyline increased zymographic MMP-9 activity without changing MMP-9 mRNA expression in C6 cells. Several different classes of antidepressants significantly increased zymographic MMP-9 activity in C6 cells and primary astrocytes, whereas antipsychotic drugs without antidepressant pharmacological activity did not. The amitriptyline-induced expression of GDNF mRNA was completely blocked by selective inhibition of MMP-9 in C6 cells. Treatment of C6 cells and primary astrocytes with exogenous recombinant MMP-9 increased GDNF mRNA expression, similar to that observed with amitriptyline. Inhibiting MMP-3 blocked amitriptyline-induced zymographic MMP-9 activation in C6 cells and primary astrocytes, indicating that MMP-3 is necessary for MMP-9 activity. The current study suggests that MMP-9 activation is indispensable in the amitriptyline-induced expression of GDNF mRNA in astrocytes and further supports a role of astrocytic neurotrophic/growth factors in the pharmacological effect of antidepressants.

Consequences of a Chronic Exposure of Cultured Brain Astrocytes to the Anti-Retroviral Drug Efavirenz and its Primary Metabolite 8-Hydroxy Efavirenz.

Efavirenz is a widely prescribed non-nucleoside reverse transcriptase inhibitor for the treatment of HIV infections. To test for potential long-term consequences of efavirenz on brain cells, cultured primary astrocytes were incubated with this substance or with its primary metabolite 8-hydroxy efavirenz for up to 7days. Both, efavirenz and 8-hydroxy efavirenz caused time- and concentration-dependent cell toxicity and stimulated in subtoxic concentrations the glycolytic flux (glucose consumption and lactate release) in astrocytes. As 8-hydroxy efavirenz was less toxic than efavirenz and stimulated glycolysis in lower concentrations we tested for a potential hydroxylation of efavirenz to 8-hydroxy efavirenz in astrocytes. Analysis of media and cell lysates by HPLC-UV and mass spectrometry revealed that after 3days of incubation viable astrocytes had accumulated about 17 and 7 % of the applied efavirenz and 8-hydroxy efavirenz, respectively. However, in cultures treated with efavirenz neither 8-hydroxy efavirenz nor any other known metabolite of efavirenz was detectable. These data demonstrate that cultured rat astrocytes efficiently accumulate, but not metabolize, efavirenz and 8-hydroxy efavirenz and that the observed chronic stimulation of glycolysis is mediated by both efavirenz and 8-hydroxy efavirenz.

Time-dependent uptake and trafficking of vesicles capturing extracellular S100B in cultured rat astrocytes.

Astrocytes, the most heterogeneous glial cells in the central nervous system, contribute to brain homeostasis, by regulating a myriad of functions, including the clearance of extracellular debris. When cells are damaged, cytoplasmic proteins may exit into the extracellular space. One such protein is S100B, which may exert toxic effects on neighboring cells unless it is removed from the extracellular space, but the mechanisms of this clearance are poorly understood. By using time-lapse confocal microscopy and fluorescently labeled S100B (S100B-Alexa488 ) and fluorescent dextran (Dextran546 ), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B-Alexa488 from extracellular space and monitored trafficking of vesicles that internalized S100B-Alexa488 . Initially, S100B-Alexa488 and Dextran546 internalized with distinct rates into different endocytotic vesicles; S100B-Alexa488 internalized into smaller vesicles than Dextran546 . At a later stage, S100B-Alexa488 -positive vesicles substantially co-localized with Dextran546 -positive endolysosomes and with acidic LysoTracker-positive vesicles. Cell treatment with anti-receptor for advanced glycation end products (RAGE) antibody, which binds to RAGE, a 'scavenger receptor', partially inhibited uptake of S100B-Alexa488 , but not of Dextran546 . The dynamin inhibitor dynole 34-2 inhibited internalization of both fluorescent probes. Directional mobility of S100B-Alexa488 -positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca2+ ]i ). We conclude that astrocytes exhibit RAGE- and dynamin-dependent vesicular mechanism to efficiently remove S100B from the extracellular space. If a similar process occurs invivo, astroglia may mitigate the toxic effects of extracellular S100B by this process under pathophysiologic conditions. This study reveals the vesicular clearance mechanism of extracellular S100B in astrocytes. Initially, fluorescent S100B internalizes into smaller endocytotic vesicles than dextran molecules. At a later stage, both probes co-localize within endolysosomes. S100B internalization is both dynamin- and RAGE-dependent, whereas dextran internalization is dependent on dynamin. Vesicle internalization likely mitigates the toxic effects of extracellular S100B and other waste products.

Subanesthetic doses of ketamine stabilize the fusion pore in a narrow flickering state in astrocytes.

Ketamine is an anesthetic that exhibits analgesic, psychotomimetic, and rapid antidepressant effects that are of particular neuropharmacological interest. Recent studies revealed astrocytic Ca(2+) signaling and regulated exocytosis as ketamine-targeted processes. Thus high-resolution cell-attached membrane capacitance measurements were performed to examine the influence of ketamine on individual vesicle interactions with the plasma membrane in cultured rat astrocytes. Ketamine evoked long-lasting bursts of repetitive opening and closing of the fusion pore that were both time- and concentration-dependent. Moreover, acute application and subanesthetic doses of ketamine elicited a significant increase in the occurrence of bursts that were characterized by a decreased fusion pore conductance, indicating that the fusion pore was stabilized in a narrow configuration. The time- and concentration-dependent increase in burst occurrence was correlated with a decrease in full fission events. This study has demonstrated a novel effect of ketamine manifested as stabilization of a fusion pore incapable of transiting to full vesicle fission, suggestive of an inhibitory effect on vesicle retrieval. This until now unrecognized effect of ketamine on the vesicle fusion pore might play a role in astroglial release and (re)uptake of molecules, modulating synaptic activity. This study demonstrates a novel effect of ketamine on the fusion pore. High-resolution cell-attached membrane capacitance measurements revealed that ketamine evokes long-lasting flickering of a narrow fusion pore that is incapable of transiting to full fission. Astrocytic vesicle fusion/retrieval modified by subanesthetic ketamine doses most likely affects gliotransmission and indicates a non-neuronal mechanism of ketamine action that may contribute to its behavioral effects.

Increased Hypothalamic Levels of Endozepines, Endogenous Ligands of Benzodiazepine Receptors, in a Rat Model of Sepsis.

The mechanisms involved in septic anorexia are mainly related to the secretion of inflammatory cytokines. The term endozepines designates a family of neuropeptides, including the octadecaneuropeptide (ODN), originally isolated as endogenous ligands of benzodiazepine receptors. Previous data showed that ODN, produced and released by astrocytes, is a potent anorexigenic peptide. We have studied the effect of sepsis by means of a model of cecal ligation and puncture (CLP) on the hypothalamic expression of endozepines (DBI mRNA and protein levels), as well as on the level of neuropeptides controlling energy homeostasis mRNAs: pro-opiomelanocortin, neuropeptide Y, and corticotropin-releasing hormone. In addition, we have investigated the effects of two inflammatory cytokines, TNF-α and IL-1β, on DBI mRNA levels in cultured rat astrocytes. Studies were performed on Sprague-Dawley male rats and on cultures of rat cortical astrocytes. Sepsis was induced using the CLP method. Sham-operated control animals underwent the same procedure, but the cecum was neither ligated nor incised. Sepsis caused by CLP evoked an increase of DBI mRNA levels in ependymal cells bordering the third ventricle and in tanycytes of the median eminence. CLP-induced sepsis was also associated with stimulated ODN-like immunoreactivity (ODN-LI) in the hypothalamus. In addition, TNF-α, but not IL-1β, induced a dose-dependent increase in DBI mRNA in cultured rat astrocytes. An increase in the mRNA encoding the precursor of the anorexigenic peptide α-melanocyte stimulating hormone, the pro-opiomelanocortin, and the corticotropin-releasing hormone was observed in the hypothalamus. These results suggest that during sepsis, hypothalamic mRNA encoding endozepines, anorexigenic peptide as well as stress hormone could play a role in the anorexia/cachexia associated with inflammation due to sepsis and we suggest that this hypothalamic mRNA expression could involve TNF-α.

Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the glioprotective action of Pituitary adenylate cyclase-activating polypeptide (PACAP) against H2 O2 -induced astrocyte damages and cell apoptosis in cultured rat astrocytes. PACAP, through activation of its receptor, PAC1-R, and the protein kinase A (PKA), protein kinase C (PKC), and MAP-kinases signaling pathways, prevents accumulation of ROS andinhibition of SOD and catalase activities. This allows the preservation of mitochondrial membrane integrity and the reduction in caspase-3 activation induced by H2 O2 . These data may lead to the development of new strategies for cerebral injury treatment. Cat, catalase; Cyt. C, cytochrome C; SOD, superoxide dismutase.

Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes.

Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo experiments.

CHANGE IN ENVIRONMENTAL GLUCOSE CONCENTRATION ALTERS ASTROCYTIC INSULIN AND INSULIN RECEPTOR LEVELS

BACKGROUNDThe actions of insulin in the brain include effects on glucose homeostasis, cell proliferation, cell differentiation, cognition, and memory. Cellular constituents of the neurovascular unit, including the astrocyte, contribute to insulin responses in the brain. Astrocytes are an ideal target cell type for investigation because of their dynamic character, multitude of functions, and their important role in brain homeostasis. We hypothesize that astrocytes respond to environmental glucose changes and could be a source of insulin supply to the microenvironment.OBJECTIVETo investigate astrocyte insulin and insulin receptor response to environmental glucose changes and to determine insulin secretion levels.METHODSNeonatal rat astrocytes in culture were exposed to changes in environmental glucose levels (LOW‐ 0mM, NORMAL‐5.5mM, and HIGH‐25mM) over three passages. Astrocyte whole cell lysates were used to determine message (qPCR) and protein levels (Western blot) for insulin and insulin receptor. Insulin Ultrasensitive ELISA was used to measure NORMAL astrocytic insulin secretion in response to glucose (25mM) and potassium (30mM) stimuli.RESULTSAstrocytes grown in LOW, NORMAL, and HIGH glucose levels resulted in equal cell counts and morphological appearances. Increasing glucose level from LOW, to NORMAL, or to HIGH in astrocytic culture media significantly increased insulin‐1 (exon 1–2) and insulin‐2 (exon 1–2) mRNA levels (One‐way ANOVA, n=4, p<0.05). Increased glucose level also resulted in a positive correlation with increased protein levels of insulin and insulin receptor in astrocytic cell lysate (Pearson Correlation, r=>0.85, p<0.01, n=3, R2=>0.73). Astrocytes secreted measurable levels of insulin (0.02–0.05μg/L) but did not increase significantly with glucose or potassium stimuli.CONCLUSIONSAstrocytes respond to changes in environmental glucose levels with increased insulin and insulin receptor message and protein levels. Astrocytes secrete measurable amounts of insulin but do not respond to stimuli as other insulin secreting cells. We speculate that such insulin responses to glucose levels from the astrocyte may be adaptive for its own homeostasis rather than to affect the microenvironment.Support or Funding InformationFunding from NIH (NHLBI) R01 HL033833 and HL105997 and the Veterans Administration Research Career Scientist Award

Abstract A03: The effects of the cellular microenvironment on medulloblastoma metastasis

Abstract Medulloblastoma (MB) is the most common pediatric brain tumor, known to be highly malignant and invasive within the brain as well as extraneural locations. The mechanisms inducing MB metastasis and dissemination are not completely understood. Astrocytes, the most abundant cell type in the brain, have been shown in our lab to secrete factors that induce breast cancer brain metastasis (Wang et al., 2013). In addition to astrocytes, many brain cell types have also been implicated in the malignancy of brain tumors, including microglia, pericytes, and endothelial cells as part of the neurovascular unit. The overall goal of this study is to determine the effects of cells in the tumor microenvironment on MB metastasis. Our hypothesis is that astrocytes and/or other cell types in the neurovascular unit and microenvironment secrete factors that bring upon phenotypic changes in MB cells, leading to increased metastasis. To investigate this hypothesis, astrocyte conditioned media (ACM) from neonatal rat astrocyte cultures were used in in vitro assays to determine functional changes to Daoy MB cells. We observed that ACM (both serum free and serum depleted) in the lower chamber of a Boyden chamber Matrigel invasion assay induced invasion of Daoy cells more than 50-fold in comparison to DMEM in the lower well. Brain endothelial cell conditioned media also increased invasion of Daoy cells, which was comparable to ACM. ACM did not significantly affect proliferation or apoptosis of Daoy cells, as tested by BrdU incorporation and Caspase-3/-7 activity, respectively. When cultured appropriately in serum free ACM (+bFGF and EGF), Daoy cells were able to form larger and more proliferative neurospheres in comparison to DMEM, indicative of an increase in neural or cancer stem cell characteristics. To determine the effects of ACM on MB metastasis in vivo, we developed a zebrafish xenotransplant model. Daoy-GFP cells were injected into the hindbrain of 48 hour post fertilization Absolut FLK-mCherry zebrafish embryos. Using time-lapse confocal microscopy, we are able to image the cells and observe their movement within the microenvironment after injection. With this method we are working to determine changes in cell morphology and movement in Daoy-GFP cells that were cultured in ACM prior to injection. In order to further elucidate the mechanism in which ACM increases invasion and neurosphere formation, microarray studies are underway to pin-point a factor(s) or pathway(s) involved. Together, these data indicate that cells comprising the neurovascular unit, namely endothelial cells and astrocytes, contribute in a paracrine manner to MB metastasis and dissemination. Citation Format: Emily Gronseth, Ling Wang, Chris Koceja, Ramani Ramchandran. The effects of the cellular microenvironment on medulloblastoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A03.

Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures

Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

The effect of amyotrophic lateral sclerosis-linked exogenous SOD1-G93A on electrophysiological properties and intracellular calcium in cultured rat astrocytes

Over 150 mutations in the SOD1 gene that encodes Cu/Zn superoxide dismutase (SOD1) cause 20–25% of familial ALS, albeit without a known gain-of-function mechanism. ALS is also non-cell-autonomous, the interactions between motor neurons and their glial neighbours being implicated in disease progression. The aim here was to investigate the biophysical effects of the exogenous human mutant SOD1-G93A on rat astrocytes in culture. Primary cortical astrocyte cultures were treated with recombinant human apo- mSOD1-G93A vs. wild-type control (wtSOD1) and recorded by patch-clamp and calcium imaging. Results showed that exogenous mSOD1 as well as wtSOD1 induced a decrease of membrane resistance, the effect being persistent (up to 13 min) only for the mutant form. Similarly, whole-cell inward currents in astrocytes were augmented by both wt and mSOD1, but the effect was twice larger and only progressed continuously for the latter. Both forms of SOD1 also induced a rise in intracellular Ca2+ activity, the effect being dependent on external Ca2+ and again only persisted with mSOD1, becoming significantly different from wtSOD1 only at longer times (14 min). In conclusion, this study points to membrane permeability and Ca2+ signalling as processes affected by SOD1-G93A that presents the humoral factor triggering the role of astrocytes in ALS pathophysiology.

Simvastatin and atorvastatin facilitates amyloid β-protein degradation in extracellular spaces by increasing neprilysin secretion from astrocytes through activation of MAPK/Erk1/2 pathways.

One of the major neuropathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid β-protein (Aβ) in the brain. Aβ accumulation seems to arise from an imbalance between Aβ production and clearance. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the important Aβ-degrading enzymes in the brain, and deficits in their expression may promote Aβ deposition in patients with sporadic late-onset AD. Statins, which are used clinically for reducing cholesterol levels, can exert beneficial effects on AD. Therefore, we examined whether various statins are associated with Aβ degradation by inducing NEP and IDE expression, and then evaluating the relation between activation of intracellular signaling transduction, inhibition of cholesterol production, and morphological changes to astrocytes. Treating cultured rat astrocytes with simvastatin and atorvastatin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner. The decrease in NEP expression was a result of activation of extracellular signal-regulated kinase (ERK) but not the reduction of cholesterol synthesis pathway. This NEP reduction was achieved by the release to the extracellular space of cultured astrocytes. Furthermore, the cultured medium prepared from simvastatin- and atorvastatin-treated astrocytes significantly induced the degradation of exogenous Aβ. These results suggest that simvastatin and atorvastatin induce the increase of Aβ degradation of NEP on the extracellular of astrocytes by inducing ERK-mediated pathway activity and that these reagents regulate the differential mechanisms between the secretion of NEP, the induction of cholesterol reduction, and the morphological changes in the cultured astrocytes.

Docosahexaenoic acid (DHA) prevents corticosterone-induced changes in astrocyte morphology and function.

The many functions of astrocytes, such as glutamate recycling and morphological plasticity, enable them to stabilize synapses environment and protect neurons. Little is known about how they adapt to glucocorticoid-induced stress, and even less about the influence of dietary factors. We previously showed that omega-3 polyunsaturated fatty acids (ω3PUFA), dietary fats which alleviate stress responses, influence the way astroglia regulate glutamatergic synapses. We have explored the role of docosahexaenoic acid (DHA), the main ω3PUFA, in the astroglial responses to corticosterone, the main stress hormone in rodents to determine whether ω3PUFA help astrocytes resist stress. Cultured rat astrocytes were enriched in DHA or arachidonic acid (AA, the main ω6PUFA) and given 100nM corticosterone for several days. Corticosterone stimulated astrocyte glutamate recycling by increasing glutamate uptake and glutamine synthetase (GS), and altered the astrocyte cytoskeleton. DHA-enriched astrocytes no longer responded to the action of corticosterone on glutamate uptake, had decreased GS, and the cytoskeletal effect of corticosterone was delayed, while AA-enriched cells were unaffected. The DHA-dependent anti-corticosterone effect was related to fewer glucocorticoid receptors, while corticosterone increased DHA incorporation into astrocyte membranes. Thus, DHA helps astrocytes resist the influence of corticosterone, so perhaps promoting a sustainable response by the stressed brain. We show that corticosterone increases the glutamate recycling capacity of rat cortical astrocytes in culture, and alters their morphology, which may be detrimental in the long term. Increasing the membrane incorporation of docosahexaenoic acid (DHA), the main omega-3 in brain, reduces the amount of glucocorticoid receptors (GR) and prevents the effects of corticosterone. This may help the astrocytes maintain a functional phenotype in chronic stress situations.

Ammonia-induced miRNA expression changes in cultured rat astrocytes.

Hepatic encephalopathy is a neuropsychiatric syndrome evolving from cerebral osmotic disturbances and oxidative/nitrosative stress. Ammonia, the main toxin of hepatic encephalopathy, triggers astrocyte senescence in an oxidative stress-dependent way. As miRNAs are critically involved in cell cycle regulation and their expression may be regulated by oxidative stress, we analysed, whether astrocyte senescence is a consequence of ammonia-induced miRNA expression changes. Using a combined miRNA and gene microarray approach, 43 miRNA species which were downregulated and 142 genes which were upregulated by NH4Cl (5 mmol/l, 48 h) in cultured rat astrocytes were found. Ammonia-induced miRNA and gene expression changes were validated by qPCR and 43 potential miRNA target genes, including HO-1, were identified by matching upregulated mRNA species with predicted targets of miRNA species downregulated by ammonia. Inhibition of HO-1 targeting miRNAs which were downregulated by NH4Cl strongly upregulated HO-1 mRNA and protein levels and inhibited astrocyte proliferation in a HO-1-dependent way. Preventing ammonia-induced upregulation of HO-1 by taurine (5 mmol/l) as well as blocking HO-1 activity by tin-protoporphyrine IX fully prevented ammonia-induced proliferation inhibition and senescence. The data suggest that ammonia induces astrocyte senescence through NADPH oxidase-dependent downregulation of HO-1 targeting miRNAs and concomitant upregulation of HO-1 at both mRNA and protein level.

Ketamine Inhibits ATP-Evoked Exocytotic Release of Brain-Derived Neurotrophic Factor from Vesicles in Cultured Rat Astrocytes.

In the brain, astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules, such as brain-derived neurotrophic factor (BDNF). Recent studies uncovered a role of ketamine, an anesthetic and antidepressant, in the regulation of BDNF expression and in the disruption of astrocytic Ca2+ signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca2+ signaling and exocytotic release of BDNF at the single-vesicle level in cultured rat astrocytes. Cells were transfected with a plasmid encoding preproBDNF tagged with the pH-sensitive fluorescent protein superecliptic pHluorin, (BDNF-pHse) to load vesicles and measure the release of BDNF-pHse when the exocytotic fusion pore opens and alkalinizes the luminal pH. In addition, cell-attached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane. Intracellular Ca2+ activity was monitored with Fluo-4 and confocal microscopy, which was also used to immunocytochemically characterize BDNF-pHse-laden vesicles. As revealed by double-fluorescent micrographs, BDNF-pHse localized to vesicles positive for the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, vesicle-associated membrane protein 2 (VAMP2), VAMP3, and synaptotagmin IV. Ketamine treatment decreased the number of ATP-evoked BDNF-pHse fusion/secretion events (P < 0.05), the frequency of ATP-evoked transient (P < 0.001) and full-fusion exocytotic (P < 0.05) events, along with a reduction in the ATP-evoked increase in intracellular Ca2+ activity in astrocytes by ~70% (P < 0.001). The results show that ketamine treatment suppresses ATP-triggered vesicle fusion and BDNF secretion by increasing the probability of a narrow fusion pore open state and/or by reducing astrocytic Ca2+ excitability.

Protective effect of edaravone against manganese-induced toxicity in cultured rat astrocytes

Manganese (Mn), a trace metal, is essential for maintaining the normal regulation of many biochemical and cellular processes. However, accumulation of Mn due to excessive environmental exposure leads to neurological impairment, referred to as manganism. Edaravone (EDA) is a potent free radical scavenger that has been clinically shown to reduce the neuronal injury after cerebral ischemia. In the present study, we aimed to examine the protective effects of EDA against Mn toxicity in astrocyte cultures. Astrocyte cultures were prepared from cerebral cortices of newborn Sprague–Dawley rats. The experiments were performed between 16 and 18 days of cultures. Astrocytes were treated in DMEM medium containing Mn (1–1000μM) for 24h to test Mn toxicity. In order to assess the effect of EDA, cells were pre-treated with different doses of EDA (10, 100 and 1000μM) 6h before Mn treatment. Cell viability (MTT), apoptotic cell death (Hoechst test) and lipid peroxide levels were evaluated in cultures. Our results showed that Mn significantly and dose-dependently reduced cell viability in astrocyte cultures. The apoptotic cell death and lipid peroxides were significantly higher in Mn treated cultures. Treatment of astrocytes with EDA successfully suppressed oxidative stress and cell death due to Mn exposure. The findings of the present study suggest that Mn cytotoxicity is mainly associated with ROS generation and apoptotic cell death. Besides, EDA may have beneficial effects against Mn toxicity. However, further studies are needed to elucidate the molecular mechanisms underlying protective effect of EDA.

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells.

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.