Exposure of Astrocytes to Thrombin Reduces Levels of the Metabotropic Glutamate Receptor mGluR5

Abstract: Thrombin is one of the first regulatory molecules present at sites of CNS trauma or injury. Exposure of neuronal and glial cells to thrombin produces potent morphological as well as cytoprotective and cytotoxic effects, but little is known about how this important modulator affects neurotransmitter signaling. In astrocyte cultures that have been morphologically differentiated by exposure to transforming growth factor-α, addition of thrombin induced a retraction of astrocytic processes and suppressed the stimulation of phosphoinositide hydrolysis by the selective metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid. In addition to the suppression of phosphoinositide hydrolysis, thrombin treatment produced a corresponding reduction in level of mGluR5 mRNA as demonstrated with ribonuclease protection assay and reduced content of mGluR5 receptor protein as seen with western blotting. In contrast, thrombin exposure up-regulated astrocyte β-actin mRNA levels. A synthetic hexapeptide with a sequence corresponding to the amino-terminus of the thrombin receptor's tethered ligand also mimicked the ability of thrombin to suppress mGluR5 levels and to increase β-actin mRNA content, suggesting that these effects of thrombin are mediated by proteolytically activated cell surface thrombin receptors. Thrombin's suppressive effect on mGluR5 was resistant to pretreatment with pertussis toxin or various protein kinase and protein phosphatase inhibitors. However, the serine/threonine protein kinase inhibitor H-7 did prevent thrombin-induced reversal of astrocyte stellation and induction of β-actin mRNA levels, indicating that these effects of thrombin involve a signaling pathway distinct from the one that mediates the suppressive effects of thrombin on mGluR5.     

Beta-amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes

Accumulation of beta-amyloid peptide (Abeta), which is a landmark of Alzheimer's disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Abeta on biosynthesis and release of diazepam-binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT-PCR and specific radioimmunoassay demonstrated that aggregated Abeta(25-35), at concentrations up to 10(-4) m, induced a dose-dependent increase in DBI mRNA expression and DBI-related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Abeta(25-35) was prevented by Congo red. Measurement of the number of living cells revealed that Abeta(25-35) induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Abeta(25-35)-induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Abeta-induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimer's disease.     

Oligodendrocytes damage in Alzheimer's disease: beta amyloid toxicity and inflammation

Research on Alzheimer's disease (AD) focuses mainly on neuronal death and synaptic impairment induced by beta-Amyloid peptide (Abeta), events at least partially mediated by astrocyte and microglia activation. However, substantial white matter damage and its consequences on brain function warrant the study of oligodendrocytes participation in the pathogenesis and progression of AD. Here, we analyze reports on oligodendrocytes' compromise in AD and discuss some experimental data indicative of Abeta toxicity in culture. We observed that 1 microM of fibrilogenic Abeta peptide damages oligodendrocytes in vitro: while pro-inflammatory molecules (1 microg/ ml LPS + 1 ng/ml IFNgamma) or the presence of astrocytes reduced the Abeta-induced damage. This agrees with our previous results showing an astrocyte-mediated protective effect over Abeta-induced damage on hippocampal cells and modulation of the activation of microglial cells in culture. Oligodendrocytes protection by astrocytes could be, either by reduction of Abeta fibrilogenesis/deposition or prevention of oxidative damage. Likewise, the decrease of Abeta-induced damage by proinflammatory molecules could reflect the production of trophic factors by activated oligodendrocytes and/or a metabolic activation as observed during myelination. Considering the association of inflammation with neurodegenerative diseases. oligodendrocytes impairment in AD patients could potentiate cell damage under pathological conditions.     

Possible linkage between glutamate transporter and mitogen-activated protein kinase cascade in cultured rat cortical astrocytes

The mitogen-activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a variety of signalling molecules. In the present study, we investigated possible linkage between glutamate signalling and the MAPK cascade in cultured rat cortical astrocytes. Exposure of the cells to L-glutamate (100-1000 microM) resulted in an increase in phosphorylated p44/42 MAPK (ERK1/2) in a concentration- and time-dependent manner. The glutamate-induced ERK1/2 phosphorylation was blocked by U0126 and PD98059, specific inhibitors of the MAPK-activating enzyme MEK. Furthermore, L-glutamate-induced ERK1/2 phosphorylation was not mimicked by glutamate receptor agonists and was not blocked by glutamate receptor antagonists. In contrast, the effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that the MEK/ERK cascade is activated by a mechanism related to glutamate transporters. We propose that the glutamate transporter functions as a receptor transmitting extracellular glutamate signal to intracellular messengers.     

Apolipoprotein E receptors mediate the effects of beta-amyloid on astrocyte cultures

We have previously shown that beta-amyloid (Abeta) induces astrocyte activation in vitro and that this reaction is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. However, the effects of Abeta on endogenous apoE and apoJ levels and the potential role of apoE receptors in astrocyte activation have not been addressed. Three activating stimuli (lipopolysaccharide, dibutyryl cAMP, and aged Abeta 1-42) were used to induce activation of rat astrocyte cultures, as assessed by changes in morphology and an increase in interleukin-1beta. However, only Abeta also induced approximately 50% reduction in the amount of released apoE and apoJ and an 8-fold increase in the levels of cell-associated apoE and apoJ. Experiments using two concentrations of receptor-associated protein, an inhibitor of apoE receptors with a differential affinity for the low density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), suggest that LRP mediates Abeta-induced astrocyte activation, whereas LDLR mediates the Abeta-induced changes in apoE levels. Receptor-associated protein had no effect on apoJ levels or on activation by either dibutyryl cAMP or lipopolysaccharide. These data suggest that apoE receptors translate the presence of extracellular Abeta into cellular responses, both initiating and modulating the inflammatory response induced by Abeta.     

Gliotoxic action of glutamate on cultured astrocytes

Because of the well-documented importance of glutamate clearance by astrocytes in protecting neurons against excitotoxicity, it was interesting to examine whether L-glutamate exerts a toxic action on cultured astrocytes. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Exposure of astrocyte cultures of the neonatal rat cerebral cortex to L-glutamate resulted in a concentration- and time-dependent increase in the release of LDH. L-Glutamate-induced gliotoxicity appeared to be mediated predominantly by the increase of oxidative stress because the reduced glutathione content and its effects were almost completely blocked by vitamin E and pyrrolidinedithiocarbamate. To support this notion further, the supplementation or depletion of intracellular reduced glutathione content attenuated or worsened L-glutamate toxicity, respectively. Activation of the glutamate transporter mimicked the action of L-glutamate on astrocytes. In addition, degrees of cell damage were not directly correlated to the levels of glutamate uptake. Moreover, the mechanism of this toxicity required energy and macromolecular synthesis. Taken together, brief exposure to L-glutamate resulted in glutamate uptake and cell swelling, whereas sustained exposure injured astrocytes via oxidative stress instead of the excitatory mechanism.     

Regulation of Astrocyte Morphology by RhoA and Lysophosphatidic Acid

Astrocytes in the CNS undergo morphological changes and start to proliferate after breakdown of the blood–brain barrier. In culture, proliferating astrocytes have a flat, polygonal shape. When treated with cAMP-raising agents, astrocytes adopt a stellate, process-bearing morphology resembling theirin vivoappearance. Stellation is accompanied by loss of actin stress fibers and focal adhesions. Lysophosphatidic acid (LPA), a blood-borne mitogen that signals through its cognate G protein-coupled receptor, stimulates DNA synthesis in astrocytes and causes rapid reversal of cAMP-induced stellation. LPA reversal of stellation is initiated by f-actin reassembly and tyrosine phosphorylation of focal adhesion proteins such as paxillin. Botulinum C3 toxin, which inactivates the Rho GTPase, mimics cAMP-raising agents in inducing stellation, f-actin disassembly, paxillin dephosphorylation, and growth arrest. However, unlike cAMP-induced stellation, C3-induced stellation cannot be reversed by LPA. Conversely, astrocytes expressing activated RhoA fail to undergo cAMP-induced stellation. Thus, RhoA controls astrocyte morphology in that active RhoA directs LPA reversal of stellation, while inactivation of RhoA is sufficient to induce stellation.     

Detection of gamma-aminobutyric acid-induced glutamate release in acute mouse hippocampal slices with a patch sensor

gamma-Aminobutyric acid (GABA)-stimulated release of L-glutamate from various neuronal regions of acute mouse hippocampal slices was detected with a patch sensor that responds to L-glutamate at the sub-micromolar level. The response of the patch sensor to L-glutamate was evaluated in terms of an integrated current. The integrated current increased with the concentration of L-glutamate ranging from 0.50 to 5.0 microM. By using the patch sensor, GABA-induced L-glutamate release from acute mouse hippocampal slices was detected. The effect of antagonists for GABA(A) and GABA(B) receptors on the L-glutamate release was also investigated. The GABA (25 microM) stimulation induced the release of L-glutamate via GABA(A) receptor in the CA1 region, but GABA did not induce L-glutamate release in the CA3 region. However, in the presence of the GABA(B) receptor antagonist (3-aminopropyl)(diethoxymethyl)phosphinic acid (CGP-35348), release of L-glutamate in the CA3 region was evoked by GABA stimulation. The glutamate release was completely suppressed when both GABA(A) and GABA(B) receptor were inhibited. The current results show that the glutamate release in the CA3 region occurs via a GABA(A) pathway when GABA(B) receptors are inhibited.     

Reciprocal Modulation of Astrocyte Stellation by Thrombin and Protease Nexin-1

When cultured astroglia are treated with agents that elevate intracellular cyclic AMP, they become process-bearing stellate cells and resemble differentiated astrocytes in vivo. Thrombin rapidly reversed the stellation induced by dibutyryl cyclic AMP, forskolin, or isoproterenol in cultured rat astrocytes; half-maximal and maximal effects occurred at 0.5 and 8 pM, respectively. The proteolytic activity of thrombin was required for stellation reversal, as thrombin derivatized at its catalytic site serine with a diisopropylphospho group was inactive. Two thrombin inhibitors, protease nexin-1 and hirudin, blocked and reversed the effect of thrombin. The stellation reversal effect of thrombin was specific, as 300-1,000-fold higher concentrations of other serine proteinases, including plasmin, urokinase, trypsin, and T cell serine proteinase-1, were ineffective. Thrombin is a mitogen for astrocytes at concentrations in excess of 30 pM. Thrombin increased both cell number and ornithine decarboxylase activity, an early marker for mitogenic stimulation, in astrocyte cultures. The lowest thrombin concentrations that completely reversed astrocyte stellation, however, did not increase ornithine decarboxylase activity. Moreover, several other mitogens for astrocytes did not reverse dibutyryl cyclic AMP-induced stellation. Thus, the stellation reversal effect of thrombin is distinct from the mitogenic response.     

Beta-amyloid (1-42) affects MTT reduction in astrocytes: implications for vesicular trafficking and cell functionality

Beta-amyloid (Abeta) peptide deposition in the brains of Alzheimer's disease patients results in reactive astrogliosis which may enhance neuronal cell death. Abeta has also been reported to impair important supportive astrocyte functions, such as glutamate uptake in vitro. We studied the effect of amyloid beta-peptide (Abeta) on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, cellular ATP content, lactate release, and proliferation using neonatal rat astrocyte cultures. Abeta(1-42) decreased MTT reduction potently in the absence of cell death, but did not affect cellular ATP levels or lactate release. Moreover, the cells displayed increased proliferation after incubation with Abeta(1-42), confirming that the decreased MTT reduction was not deleterious to cell viability. Abeta(1-42) enhanced transfer of MTT dye to the cell surface leading to cessation of MTT reduction and cell death. Bafilomycin A1, but not brefeldin A, prevented the enhanced trafficking of MTT, suggesting that lysosomes, but not Golgi apparatus, are involved. Our results show that the viability of astrocytes themselves is not diminished by beta-amyloid peptide. However, Abeta alters vesicular trafficking in astrocytes, which may disturb the supportive function of astrocytes in the brains of patients with Alzheimer's disease.     

Astrocyte Heterogeneity: Endogenous Amino Acid Levels and Release Evoked by Non-N-Methyl-D-Aspartate Receptor Agonists and by Potassium-Induced Swelling in Type-1 and Type-2 Astrocytes

The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.     

Calcium signals induced by amyloid beta peptide and their consequences in neurons and astrocytes in culture

In Alzheimer's disease, amyloid beta (Abeta) peptide is deposited in neuritic plaques in the brain. The Abeta peptide 1-42 or the fragment 25-35 are neurotoxic. We here review our recent explorations of the mechanisms of Abeta toxicity in hippocampal cultures. Abeta had no effect on intracellular calcium in neurons but caused striking changes in nearby astrocytes. The [Ca(2+)](c) signals started approximately 5-15 min after Abeta application and consisted of sporadic [Ca(2+)](c) pulses. These were entirely dependent on extracellular Ca(2+), independent of ER Ca(2+) stores and resulted from Ca(2+) influx, probably through Abeta-induced membrane channels. The Ca(2+) signals were closely associated with transient, episodic acidification which may reflect displacement of protons from binding sites or Ca(2+)/2H(+) exchange. Abeta caused an increased rate of generation of reactive oxygen species (ROS), also seen in astrocytes and not in neurons. The increased ROS generation was blocked by inhibitors of the NADPH oxidase, strongly suggesting that this enzyme, normally associated with immune cells, is expressed in astrocytes. ROS generation was also Ca(2+)-dependent, suggesting that Abeta activation of the enzyme may be secondary to the increase in [Ca(2+)](c). Abeta caused delayed neuronal death despite the fact that all responses were seen only in astrocytes. Neurons could not be protected by glutamate receptor antagonists, but were rescued by inhibition of the NADPH oxidase, by antioxidants and by increasing glutathione. These data suggest that Abeta causes Ca(2+)-dependent oxidative stress by activating an astrocytic NADPH oxidase, and that neuronal death follows through a failure of antioxidant support.     

N-myc downstream-regulated gene 2 controls astrocyte morphology via Rho-GTPase signaling

Astrocyte undergoes morphology changes that are closely associated with the signaling communications at synapses. N-myc downstream-regulated gene 2 (NDRG2) is specifically expressed in astrocytes and is associated with several important astrocyte functions, but its potential role(s) relating to astrocyte morphological changes remain unknown. Here, primary astrocytes were prepared from neonatal Ndrg2^+/+ and Ndrg2^−/− pups, and the drug Y27632 was used to induce stellation. We then used a variety of methods to measure the levels of NDRG2, α-Actinin4, and glial fibrillary acidic protein (GFAP), and the activity of RhoA, Rac1, and Cdc42 in Y27632-treated astrocytes as well as in Ndrg2^+/+, Ndrg2^−/−, or Ndrg2^−/− + lentivirus (restore NDRG2 expression) astrocytes. We also conducted live-imaging and proteomics studies of the cultured astrocytes. We found that induction of astrocytes stellation (characterized by cytoplasmic retraction and process outgrowth) resulted in increased NDRG2 protein expression and Rac1 activity and in reduced α-Actinin4 protein expression and RhoA activity. Ndrg2 deletion induced astrocyte flattening, whereas the restoration of NDRG2 expression induced stellation. Ndrg2 deletion also significantly increased α-Actinin4 protein expression and RhoA activity yet reduced GFAP protein expression and Rac1 activity, and these trends were reversed by restoration of NDRG2 expression. Collectively, our results showed that Ndrg2 deletion promoted cell proliferation, interrupted stellation capability, and extensively altered the protein expression profiles of proteins that function in Rho-GTPase signaling. These findings suggest that NDRG2 functions to regulate astrocytes morphology via altering the accumulation of the Rho-GTPase signaling pathway components, thereby supporting that NDRG2 should be understood as a regulator of synaptic plasticity and thus neuronal communications.     

Blockage of amyloid beta peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells

Amyloid beta protein (Abeta) alters signal transduction systems, including increases in the cytosolic free calcium ([Ca2+]i) response which have pathophysiological significance in Alzheimer's disease (AD). The purposes of this study were to elucidate the mechanism involved in Abeta's effect on the Ca2+ signal and to evaluate the effect of fullerenol-1, a water-soluble hydroxyl and superoxide radical scavenger, on the Abeta-induced Ca2+ response. Both Abeta and bradykinin (BK) dose-dependently elevated [Ca2+]i in PC12 cells. Fullerenol-1, at a concentration range between 100 nM and 1 microM, dose-dependently reduced the Abeta-induced [Ca2+]i response, but did not alter the subsequent BK-mediated process. Thapsigargin, an inhibitor of Ca2+-ATPase, released Ca2+ from the internal store and diminished the BK-mediated calcium spike but did not affect the Abeta-induced Ca2+ response. In the absence of extracellular calcium, the Abeta-induced, but not BK-induced, calcium spike was completely abolished. The Ca induced by Abeta did not enter through the voltage-dependent calcium channels or ligand gated calcium channels, because the peak of Abeta-evoked Ca2+ was not significantly altered by various Ca2+ channel blockers or a NMDA receptor antagonist MK801. In addition, neither cholera toxin nor pertussis toxin altered the Abeta-induced Ca response. The results demonstrated that Abeta-stimulated [Ca2+]i increase is due to Ca influx from an extracellular source rather than from the intracellular store. Alteration of the membrane lipid structure and permeability by free radicals generated by Abeta may be a major cause of Ca -influx. Furthermore, fullerenol-1, a novel antioxidant, may provide therapeutic benefits in neurodegenerative diseases such as AD.     

Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex

We previously reported that astrocytes cultured for more than 2 days in a defined medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed calcium oscillation in response to glutamate, whereas the response pattern was transient in the absence of the exogenous growth factors. In the present study, we found that astrocytes showed glutamate-induced calcium oscillation, even in growth factor-free medium, if the cells had been cultured for more than 5 days. The calcium oscillation promoted by the prolonged culture period was suppressed by an inhibitor of EGF receptor tyrosine kinase, but not by a neutralizing antibody to bFGF, indicating that the accumulation of an autocrine factor that activates the EGF receptor leads to calcium oscillation. Astrocytes in our culture system expressed EGF, transforming growth factor alpha (TGFalpha), bFGF and acidic fibroblast growth factor (aFGF). Exogenous aFGF, which induced astrocyte immediate early gene expression to the same extent as EGF or bFGF, did not affect calcium oscillation. Exogenous EGF and bFGF promoted astrocyte hypertrophic morphology and proliferation, as well as calcium oscillation. In contrast, these properties did not accompany calcium oscillation induced by the prolonged culture period. These results suggest that astrocytes possess the ability to promote their own calcium oscillation, which is independent of hypertrophic changes to reactive astrocytes.     

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

Mechanism of membrane depolarization caused by the Alzheimer Abeta1-42 peptide

We report a novel observation that the neurotoxic Alzheimer peptide Abeta1-42, when pre-incubated, causes a dramatic and lasting membrane depolarization in differentiated human hNT neuronal cells and in rodent PC12 cells in a concentration-dependent manner. This phenomenon involves activation of the metabotropic glutamate receptor, mGluR(1). Abeta-induced membrane depolarization in PC12 cells is sensitive to mGluR(1) antagonists and to pertussis and cholera toxins, indicating the involvement of particular G-proteins. The effect is different from the known ability of aggregated Abeta1-42 to cause a calcium influx. Since mGluR(1) agonists mimic the Abeta effect, we deduce that in this cell system glutamate can control the membrane potential and thereby the excitability of its target neurons. We propose that Abeta-induced membrane depolarization described here leads in Alzheimer's disease to hyperexcitability of affected neurons and is a crucially important molecular mechanism for beta-amyloid toxicity and cognitive dysfunction in the disease.     

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.     

Glutamate-mediated overexpression of CD38 in astrocytes cultured with neurones

Recently, a new system of astrocyte-neurone glutamatergic signalling has been identified. It is started in astrocytes by ectocellular, CD38-catalysed conversion of NAD(+) to the calcium mobilizer cyclic ADP-ribose (cADPR). This is then pumped by CD38 itself into the cytosol where the resulting free intracellular Ca(2+) concentration [Ca(2+)](i) transients elicit an increased release of glutamate, which can induce an enhanced Ca(2+) response in neighbouring neurones. Here, we demonstrate that co-culture of either cortical or hippocampal astrocytes with neurones results in a significant overexpression of astrocyte CD38 both on the plasma membrane and intracellularly. The causal role of neurone-released glutamate in inducing overexpression of astrocyte CD38 is demonstrated by two observations: first, in the absence of neurones, induction of CD38 in pure astrocyte cultures can be obtained with glutamate and second, it can be prevented in co-cultures by glutamate receptor antagonists. The neuronal glutamate-mediated effect of neurones on astrocyte CD38 expression is paralleled by increased intracellular cADPR and [Ca(2+)](i) levels, both findings indicating functionality of overexpressed CD38. These results reveal a new neurone-to-astrocyte glutamatergic signalling based on the CD38/cADPR system, which affects the [Ca(2+)](i) in both cell types, adding further complexity to the bi-directional patterns of communication between astrocytes and neurones.     

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogen-activated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98+/-0.18- and 4.4+/-1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 microM AG1478 and 0.25 microM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells.