Multiple astrocyte responses to lysophosphatidic acids

Lysophosphatidic acid (LPA) and LPA receptors are enriched in the brain. Moreover, the levels of these receptors and ligand are modulated during brain development and injury, respectively, suggesting multiple roles for LPA in the brain. In cultured astrocytes and glioma-derived cells, LPA increases intracellular calcium concentrations and causes morphological changes. LPA also induces glioma cell migration. In normal astrocytes, LPA stimulates reactive oxygen species synthesis, activation of multiple protein kinases and expression of c-fos and c-jun. It is noteworthy that LPA-induced astrocyte responses vary as a function of the specific brain region of origin of the astrocytes. This may be one factor in the finding of LPA-stimulated proliferation in some, but not all, astrocyte studies. The species and/or developmental stage also differed in many of the astrocyte proliferation analyses. Micromolar LPA is required to elicit some astrocyte responses, including the stimulation of cytokine expression and inhibition of glutamate uptake. These events could significantly impact on survival of injured neurons and micromolar LPA concentrations are likely in diverse brain pathologies. There are important aspects of astrocyte LPA responses still to be fully evaluated, including functions in development and activation, synergy between LPA and other biomediators, and astrocyte interactions with other cells.     

Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC-induced death was delayed and apoptotic, and death rate was dose and treatment duration-dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.     

Reciprocal regulation of miR-23a and lysophosphatidic acid receptor signaling in cardiomyocyte hypertrophy

Earlier, our study demonstrated that lysophosphatidic acid (LPA) receptor mediated cardiomyocyte hypertrophy. However, the subtype-specific functions for LPA1 and LPA3 receptors in LPA-induced hypertrophy have not been distinguished. Growing evidence indicates that microRNAs (miRNAs) are involved in the pathogenesis of cardiac hypertrophy by down-regulating target molecules. The present work therefore aimed at elucidating the functions mediated by different subtypes of LPA receptors and investigating the modulatory role of miRNAs during LPA induced hypertrophy. Experiments were done with cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and we showed that knockdown of LPA1 by small interfering RNA (siRNA) enhanced LPA-induced cardiomyocyte hypertrophy, whereas LPA3 silencing repressed hypertrophy. miR-23a, a pro-hypertrophic miRNA, was up-regulated by LPA in cardiomyocytes and its down-regulation reduced LPA-induced cardiomyocyte hypertrophy. Importantly, luciferase reporter assay confirmed LPA1 to be a target of miR-23a, indicating that miR-23a is involved in mediating the LPA-induced cardiomyocyte hypertrophy by targeting LPA1. In addition, knockdown of LPA3, but not LPA1, eliminated miR-23a elevation induced by LPA. And PI3K inhibitor, LY294002, effectively prevented LPA-induced miR-23a expression in cardiomyocytes, suggesting that LPA might induce miR-23a elevation by activating LPA3 and PI3K/AKT pathway. These findings identified opposite subtype-specific functions for LPA1 and LPA3 in mediating cardiomyocyte hypertrophy and indicated LPA1 to be a target of miR-23a, which discloses a link between miR-23a and the LPA receptor signaling in cardiomyocyte hypertrophy.     

L-glutamate suppresses amyloid beta-protein-induced stellation of cultured rat cortical astrocytes

Alzheimer's amyloid beta-protein (Abeta) has been reported to potentiate glutamate toxicity in neurons, but very little is known about interaction between Abeta and glutamate in astrocytes. Therefore, in the present study, we investigated the effects of Abeta and glutamate on morphology of astrocytes. Cultured rat cortical astrocytes exhibited polygonal morphology in the absence of stimulation and differentiated into process-bearing stellate cells following exposure to Abeta (20 microM). L-Glutamate (30-1,000 microM) had no effect on astrocyte morphology in the absence of stimulation but strongly suppressed Abeta-induced stellation. The suppressive effect of L-glutamate on Abeta-induced stellation was not mimicked by glutamate receptor agonists and not blocked by glutamate receptor antagonists. In contrast, the suppressive effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that Abeta-induced astrocyte stellation is suppressed by a mechanism related to glutamate transporters.     

Down-regulation of astrocytic GLAST by microglia-related inflammation is abrogated in dibutyryl cAMP-differentiated cultures

The influence of neuroinflammation on glutamate uptake by glial cells was examined after exposing primary cultures of rat astrocytes to conditioned culture medium from lipopolysaccharide-activated microglia. While such treatment triggered an inflammatory response in astrocytes, as revealed by the induction of cytokine expression, a significant decrease in GLAST expression and activity was observed after 72 h. This regulation of glutamate transporter was not observed with medium from naive microglia, but was mimicked by direct addition of tumor necrosis factor-alpha (TNF-α), a major cytokine released from activated microglia. Hence, on its own, TNF-α also triggered inflammation in astrocyte cultures, highlighting complex cross-talk between astrocytes and microglia in inflammatory conditions. This putatively detrimental regulation of GLAST in response to inflammation was also studied in cells exposed to dibutyryl cAMP, recognized as a model of astrocytes exhibiting a typical differentiated or activated phenotype. In this model, the conditioned culture medium from activated microglia, as well as TNF-α, were found to increase glutamate uptake capacity. Consistently, both of these treatments caused only modest induction of an inflammatory response in dibutyryl cAMP-matured astrocytes as compared to undifferentiated astrocytes. Together, these results suggest that differentiated/activated astrocytes are endowed with the capacity to confront inflammatory insults and that drugs influencing the astrocytes phenotype would deserve further consideration in the treatment of neurological disorders.     

Lysophosphatidic acid receptor-dependent secondary effects via astrocytes promote neuronal differentiation

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system, receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however, an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects, whereby LPA receptor-expressing cells exposed to, or "primed," by LPA may then act on other cells via distinct, yet LPA-initiated, mechanisms. In the present study, we examined cerebral cortical astrocytes as possible indirect mediators of the effects of LPA on developing cortical neurons. Cultured astrocytes express at least four LPA receptor subtypes, known as LPA(1)-LPA(4). Cerebral cortical astrocytes primed by LPA exposure were found to increase neuronal differentiation of cortical progenitor cells. Treatment of unprimed astrocyte-progenitor cocultures with conditioned medium derived from LPA-primed astrocytes yielded similar results, suggesting the involvement of an astrocyte-derived soluble factor induced by LPA. At least two LPA receptor subtypes are involved in LPA priming, since the priming effect was lost in astrocytes derived from LPA receptor double-null mice (LPA(1)((-/-))/LPA(2)((-/-))). Moreover, the loss of LPA-dependent differentiation in receptor double-null astrocytes could be rescued by retrovirally transduced expression of a single deleted receptor. These data demonstrate that receptor-mediated LPA signaling in astrocytes can induce LPA-dependent, indirect effects on neuronal differentiation.     

Astrocytes treated by lysophosphatidic acid induce axonal outgrowth of cortical progenitors through extracellular matrix protein and epidermal growth factor signaling pathway

Lysophosphatidic acid (LPA) plays important roles in many biological processes, such as brain development, oncogenesis and immune functions, via its specific receptors. We previously demonstrated that LPA-primed astrocytes induce neuronal commitment of cerebral cortical progenitors (Spohr et al. 2008). In the present study, we analyzed neurite outgrowth induced by LPA-treated astrocytes and the molecular mechanism underlying this event. LPA-primed astrocytes increase neuronal differentiation, arborization and neurite outgrowth of developing cortical neurons. Treatment of astrocytes with epidermal growth factor (EGF) ligands yielded similar results, suggesting that members of the EGF family might mediate LPA-induced neuritogenesis. Furthermore, treatment of astrocytes with LPA or EGF ligands led to an increase in the levels of the extracellular matrix molecule, laminin (LN), thus enhancing astrocyte permissiveness to neurite outgrowth. This event was reversed by pharmacological inhibitors of the MAPK signaling pathway and of the EGF receptor. Our data reveal an important role of astrocytes and EGF receptor ligands pathway as mediators of bioactive lipids action in brain development, and implicate the LN and MAPK pathway in this process.     

Overexpression of Cu,Zn superoxide dismutase attenuates oxidative inhibition of astrocyte glutamate uptake

Glutamate neurotoxicity in brain is normally prevented by rapid uptake of glutamate by astrocytes. Increased expression of Cu,Zn superoxide dismutase (SOD1) can increase resistance to cerebral ischemia and other oxidative insults, but the cellular mechanisms by which this occurs are not well established. Here we examine whether increased SOD1 expression can attenuate inhibition of astrocyte glutamate uptake by reactive oxygen species. Primary cortical astrocyte cultures were prepared from transgenic mice that overexpress human SOD1 and from nontransgenic littermate controls. Glutamate uptake was assessed after exposure of these cultures to xanthine oxidase plus hypoxanthine, an extracellular superoxide generating system, or to menadione, which generates superoxide in the cytosol. These treatments produced dose-dependent reductions in astrocyte glutamate uptake, and the reductions were significantly attenuated in the SOD1 transgenic astrocytes. A specific effect of reactive oxygen species on glutamate transporters was suggested by the much smaller inhibitory effects of xanthine oxidase/hypoxanthine and menadione on GABA uptake than on glutamate uptake. These findings suggest that the cerebroprotective effects of increased SOD1 expression during cerebral ischemia-reperfusion could be mediated in part by astrocyte glutamate transport.     

Lysophosphatidic Acid-Induced Proliferation-Related Signals in Astrocytes

Abstract: Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 n M LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca²⁺]_i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca²⁺]_i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca²⁺]_i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca²⁺]_i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca²⁺]_i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis.     

Specific LPA receptor subtype mediation of LPA-induced hypertrophy of cardiac myocytes and involvement of Akt and NFkappaB signal pathways

Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth.     

Characterization of lpa(2) (Edg4) and lpa(1)/lpa(2) (Edg2/Edg4) lysophosphatidic acid receptor knockout mice: signaling deficits without obvious phenotypic abnormality attributable to lpa(2)

Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa(1)/lp(A1)/Edg-2/Gpcr26, lpa(2)/lp(A2)/Edg-4, and lpa(3)/lp(A3)/Edg-7 mediate the cellular effects of LPA. We have previously shown that deletion of lpa(1) in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa(2) in mice. Homozygous knockout (lpa(2)((-/-))) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa(1)((-/-)) lpa(2)((-/-)) double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa(1)((-/-)) mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa(1)((-/-)) lpa(2)((-/-)) embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca(2+) mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa(1)((-/-)) lpa(2)((-/-)) mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa(1)((-/-)) fibroblasts], these responses were only partially affected in lpa(1)((-/-)) and lpa(2)((-/-)) fibroblasts. Thus, although LPA(2) is not essential for normal mouse development, it does act redundantly with LPA(1) to mediate most LPA responses in fibroblasts.     

RAFTK/Pyk2 mediates LPA-induced PC12 cell migration

The phospholipid lysophosphatidic acid (LPA) is a normal constituent of serum that functions as a lipid growth factor and intracellular signaling molecule. In this report, we have investigated the signaling mechanism and function of the tyrosine kinase RAFTK/Pyk2 in LPA-induced cell migration. Analysis of tyrosine phosphorylation upon LPA stimulation in neuroendocrine PC12 cells revealed 6 major tyrosine-phosphorylated proteins with estimated sizes of 180, 120, 115, 68, 44, and 42 kDa. These proteins were identified as epidermal growth factor receptor (EGFR), focal adhesion kinase, RAFTK/Pyk2, paxillin, Erk 1, and Erk 2, respectively. Using specific pharmacological inhibitors, we found that the tyrosine phosphorylation of RAFTK/Pyk2 was intracellular Ca2+-dependent, but not EGFR-dependent, during LPA stimulation of these cells. Moreover, the cytoskeletal and signal scaffolding protein, paxillin, associated with and was regulated by RAFTK/Pyk2 in a Ca2+-dependent manner. Characterization of LPA receptors showed that LPA1 (Edg2) and LPA2 (Edg4) are major receptors for LPA, while LPA3 receptor (Edg7) expression was limited. Upon using the LPA1/LPA3 receptor-specific antagonist VPC 32179, we observed that inhibition of the LPA1/LPA3 receptors had no effect on the LPA-induced phosphorylation of RAFTK, strongly suggesting that the LPA2 receptor is a key mediator of RAFTK phosphorylation. Furthermore, LPA induced PC12 cell migration, which was subsequently blocked by the dominant-negative form of FAK, FRNK. Expression of a dominant-negative form of the small GTPase Ras also blocked LPA-induced cell migration and RAFTK phosphorylation. Taken together, these results indicate that RAFTK is a key signaling molecule that mediates LPA-induced PC12 cell migration in a Ras-dependent manner.     

Dual regulation of actin rearrangement through lysophosphatidic acid receptor in neuroblast cell lines: actin depolymerization by Ca(2+)-alpha-actinin and polymerization by rho

Lysophosphatidic acid (LPA) is a potent lipid mediator with actions on many cell types. Morphological changes involving actin polymerization are mediated by at least two cognate G protein-coupled receptors, LPA(1)/EDG-2 or LPA(2)/EDG-4. Herein, we show that LPA can also induce actin depolymerization preceding actin polymerization within single TR mouse immortalized neuroblasts. Actin depolymerization resulted in immediate loss of membrane ruffling, whereas actin polymerization resulted in process retraction. Each pathway was found to be independent: depolymerization mediated by intracellular calcium mobilization, and alpha-actinin activity and polymerization mediated by the activation of the small Rho GTPase. alpha-Actinin-mediated depolymerization seems to be involved in growth cone collapse of primary neurons, indicating a physiological significance of LPA-induced actin depolymerization. Further evidence for dual regulation of actin rearrangement was found by heterologous retroviral transduction of either lpa(1) or lpa(2) in B103 cells that neither express LPA receptors nor respond to LPA, to confer both forms of LPA-induced actin rearrangements. These results suggest that diverging intracellular signals from a single type of LPA receptor could regulate actin depolymerization, as well as polymerization, within a single cell. This dual actin rearrangement may play a novel, important role in regulation of the neuronal morphology and motility during brain development.     

Genetics and cell biology of lysophosphatidic acid receptor-mediated signaling during cortical neurogenesis

Lysophosphatidic acid (LPA) is a small lysophospholipid that signals through G-protein coupled receptors (GPCRs) to mediate diverse cellular responses. Two LPA receptors, LPA(1) and LPA(2), show gene expression profiles in mouse embryonic cerebral cortex, suggesting roles for LPA signaling in cerebral cortical development. Here, we review loss-of-function and gain-of-function models that have been used to examine LPA signaling. Genetic deletion of lpa(1) or both lpa(1) and lpa(2) in mice results in 50-65% neonatal lethality, but not obvious cortical phenotypes in survivors, suggesting that compensatory signaling systems exist for regulating cortical development. A gain-of-function model, approached by increasing receptor activation through exogenous delivery of LPA, shows that LPA signaling regulates cerebral cortical growth and anatomy by affecting proliferation, differentiation and cell survival during embryonic development.     

Unmasking of LPA1 receptor-mediated migration response to lysophosphatidic acid by interleukin-1β-induced attenuation of Rho signaling pathways in rat astrocytes

Action mechanism of lipopolysaccharide (LPS), interleukin-1β (IL-1β), and lysophosphatidic acid (LPA) to regulate motility, an important process of astrogliosis, was investigated in rat astrocytes. While LPA exerted no significant effect on the cell migration, the prior treatment of the cells with LPS or IL-1β resulted in the appearance of migration activity in response to LPA. The LPS induction of the migration response to LPA was associated with the production of IL-1β precursor protein and inhibited by the IL-1 receptor antagonist. The IL-1β treatment also allowed LPA to activate Rac1. The LPA-induced Rac1 activation and migration were inhibited by pertussis toxin, a small interfering RNA specific to LPA(1) receptors, and LPA(1) receptor antagonists, including Ki16425. However, the IL-1β treatment had no appreciable effect on LPA(1) receptor mRNA expression and LPA-induced activation of ERK, Akt, and proliferation. The induction of the migration response to LPA by IL-1β was inhibited by a constitutively active RhoA. Moreover, LPA significantly activated RhoA through the LPA(1) receptor in the control cells but not in the IL-1β-treated cells. These results suggest that IL-1β inhibits the LPA(1) receptor-mediated Rho signaling through the IL-1 receptor, thereby disclosing the LPA(1) receptor-mediated G(i) protein/Rac/migration pathway.     

Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.     

Amyloid β Peptide (25–35) Inhibits Na+-Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l -glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l -glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.     

Glutamate/glutamine metabolism coupling between astrocytes and glioma cells: Neuroprotection and inhibition of glioma growth

Glioma glutamate release has been shown to promote the growth of glioma cells and induce neuronal injuries from epilepsy to neuronal death. However, potential counteractions from normal astrocytes against glioma glutamate release have not been fully evaluated. In this study, we investigated the glutamate/glutamine cycling between glioma cells and astrocytes and their impact on neuronal function. Co-cultures of glioma cells with astrocytes (CGA) in direct contact were established under different mix ratio of astrocyte/glioma. Culture medium conditioned in these CGAs were sampled for HPLC measurement, for neuronal ratiometric calcium imaging, and for neuronal survival assay. We found: (1) High levels of glutaminase expression in glioma cells, but not in astrocytes, glutaminase enables glioma cells to release large amount of glutamate in the presence of glutamine. (2) Glutamate levels in CGAs were directly determined by the astrocyte/glioma ratios, indicating a balance between glioma glutamate release and astrocyte glutamate uptake. (3) Culture media from CGAs of higher glioma/astrocyte ratios induced stronger neuronal Ca²⁺ response and more severe neuronal death. (4) Co-culturing with astrocytes significantly reduced the growth rate of glioma cells. These results indicate that normal astrocytes in the brain play pivotal roles in glioma growth inhibition and in reducing neuronal injuries from glioma glutamate release. However, as tumor growth, the protective role of astrocytes gradually succumb to glioma cells.     

Effects of 5-aminolevulinic acid on the glutamatergic neurotransmission

The haem precursor 5-aminolevulinic acid (ALA) has been proposed to be involved in the neurological dysfunctions presented by patients with acute porphyrias. The effects of ALA on the [3H]glutamate and [3H]MK-801 (dizocilpine) binding to rat cortical membranes and on [3H]glutamate uptake by rat astrocyte cultures were evaluated in the present study in order to elucidate the interaction of ALA with the glutamatergic system and its possible contribution to the in vivo excitatory properties of ALA. ALA (0-1mM) did not affect the binding of 100 nM [3H]glutamate, nor the equilibrium binding constants (K(d) and B(max)) of this neurotransmitter in rat or human cortical membranes. The binding of the NMDA-channel blocker, [3H]MK-801, was not affected by ALA (0-10mM) either. ALA (0-3mM) dose-dependently inhibited glutamate uptake by astrocyte cultures. ALA significantly reduced both the K(m) and V(max) of glutamate uptake indicating an uncompetitive inhibition. The inhibitory effect was irreversible and apparently related to the selective inhibition of the GLT-1 (EAAT2) subtype of glutamate transporter. The finding that ALA significantly increased astrocyte lipoperoxidation in astrocytes incubated under these conditions suggests that the inhibitory effect of ALA might be related to an oxidative damage of the transporter. We propose that the inhibition of glutamate uptake may underlie ALA-induced convulsions.