Seizures regulate the cation-Cl- cotransporter NKCC1 in a hamster model of epilepsy: implications for GABA neurotransmission.

Background: The balance between the activity of the Na+/K+/Cl- cotransporter (NKCC1) that introduces Cl- into the cell and the K+/Cl- cotransporter (KCC2) that transports Cl- outside the cell is critical in determining the inhibitory or excitatory outcome of GABA release. Mounting evidence suggests that the impairment of GABAergic inhibitory neurotransmission plays a crucial role in the pathophysiology of epilepsy, both in patients and animal models. Previous studies indicate that decreased KCC2 expression is linked to audiogenic seizures in GASH/Sal hamsters, highlighting that Cl- imbalance can cause neuronal hyperexcitability. In this study, we aimed to investigate whether the Na+/K+/Cl- cotransporter NKCC1 is also affected by audiogenic seizures and could, therefore, play a role in neuronal hyperexcitability within the GASH/Sal epilepsy model. Methods: NKCC1 protein expression in both the GASH/Sal strain and wild type hamsters was analyzed by immunohistochemistry and Western blotting techniques. Brain regions examined included cortex, hippocampus, hypothalamus, inferior colliculus and pons-medulla oblongata, which were evaluated both at rest and after sound-inducing seizures in GASH/Sal hamsters. A complementary analysis of NKCC1 gene slc12a2 expression was conducted by real-time PCR. Finally, protein and mRNA levels of glutamate decarboxylase GAD67 were measured as an indicator of GABA release. Results: The induction of seizures caused significant changes in NKCC1 expression in epileptic GASH/Sal hamsters, despite the similar brain expression pattern of NKCC1 in GASH/Sal and wild type hamsters in the absence of seizures. Interestingly, the regulation of brain NKCC1 by seizures demonstrated regional specificity, as protein levels exclusively increased in the hippocampus and hypothalamus. Complementary real-time PCR analysis revealed that NKCC1 regulation was post-transcriptional only in the hypothalamus. In addition, seizures also modulated GAD67 mRNA levels in a brain region-specific manner. The increased GAD67 expression in the hippocampus and hypothalamus of the epileptic hamster brain suggests that NKCC1 upregulation overlaps with GABA release in these regions during seizures. Conclusions: Our results indicate that seizure induction causes dysregulation of NKCC1 expression in GASH/Sal animals, which overlaps with changes in GABA release. These observations provide evidence for the critical role of NKCC1 in how seizures affect neuronal excitability, and support NKCC1 contribution to the development of secondary foci of epileptogenic activity.

GSK3ß Inhibition by Phosphorylation at Ser389 Controls Neuroinflammation.

The inhibition of Glycogen Synthase Kinase 3 ß (GSK3ß) by Ser9 phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3ß inhibition by alternative Ser389 phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3ß Ser389 knock-in (KI) mice, in which the phosphorylation of Ser389 GSK3ß is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3ß by Ser389 phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3ß Ser389 KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3ß Ser389 KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3ß regulatory pathway. Overall, our findings indicate that GSK3ß inactivation by Ser389 phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies.

Regulation of GSK3ß by Ser389 Phosphorylation During Neural Development.

GSK3ß is a constitutively active kinase that promotes cell death, which requires strict regulatory mechanisms. Although Akt-mediated phosphorylation at Ser9 is the default mechanism to inactivate GSK3ß, phosphorylation of GSK3ß at Ser389 by p38 MAPK has emerged as an alternative inhibitory pathway that provides cell protection and repair in response to DNA damage. Phosphorylation of Ser389 GSK3ß has been detected in adult brain, where it has been related to neuronal survival and behavior. However, the use of this pathway to regulate GSK3ß in the neonatal developing brain is unknown. In this study, we show that phosphorylation of GSK3ß at Ser389 in the brain is developmentally regulated, with the highest levels corresponding to the first 2 weeks of age. Moreover, we found that the phosphorylation of GSK3ß at Ser389 is the preferential mechanism for inactivating brain GSK3ß in 2-week-old mice. Importantly, we show that phospho-Ser389 GSK3ß expression is predominant in neuronal cell cultures from neonatal brain relative to other cell populations. However, phospho-Ser389 GSK3ß is triggered by DNA double-strand breaks in all developing neural cell types examined. Thus, the phosphorylation of GSK3ß on Ser389 could be a central regulatory mechanism to restrain GSK3ß during neurogenesis early in life.

Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry.

The technical difficulty to isolate microglia, astrocytes and infiltrating immune cells from mouse brain is nowadays a limiting factor in the study of neuroinflammation. Brain isolation requirements are cell-type and animal-age dependent, but current brain dissociation procedures are poorly standardized. This lack of comprehensive studies hampers the selection of optimized methodologies. Thus, we present here a comparative analysis of dissociation methods and Percoll-based separation to identify the most efficient procedure for the combined isolation of healthy microglia, astrocytes and infiltrated leukocytes; distinguishing neonatal and adult mouse brain. Gentle mechanical dissociation and DNase I incubation was supplemented with papain or collagenase II. Dispase II digestion was also used alone or in combination. In addition, cell separation efficiency of 30 % and 30-70 % Percoll gradients was compared. In these experiments, cell yield and integrity of freshly dissociated cells was measured by flow cytometry. We found that papain digestion in combination with dispase II followed by 30 % Percoll separation is the most balanced method to obtain a mixture of microglia, astrocytes and infiltrated immune cells; while addition of dispase II was not an advantage for neonatal brain. These dissociation conditions allowed flow cytometry detection of a slight glial activation triggered by sublethal LPS injection. In conclusion, the enzymes and Percoll density gradients tested here affected differently resting microglia, activated microglia/macrophages, astrocytes and infiltrated lymphocytes. Also, newborn and adult brain showed contrasting reactions to digestion. Our study highlights the strength of flow cytometry for the simultaneous analysis of neuroimmune cell populations once extraction is optimized.

Absence of Notch1 in murine myeloid cells attenuates the development of experimental autoimmune encephalomyelitis by affecting Th1 and Th17 priming.

Inhibition of Notch signalling in T cells attenuates the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Growing evidence indicates that myeloid cells are also key players in autoimmune processes. Thus, the present study evaluates the role of the Notch1 receptor in myeloid cells on the progression of myelin oligodendrocyte glycoprotein (MOG)35-55 -induced EAE, using mice with a myeloid-specific deletion of the Notch1 gene (MyeNotch1KO). We found that EAE progression was less severe in the absence of Notch1 in myeloid cells. Thus, histopathological analysis revealed reduced pathology in the spinal cord of MyeNotch1KO mice, with decreased microglia/astrocyte activation, demyelination and infiltration of CD4+ T cells. Moreover, these mice showed lower Th1 and Th17 cell infiltration and expression of IFN-γ and IL-17 mRNA in the spinal cord. Accordingly, splenocytes from MyeNotch1KO mice reactivated in vitro presented reduced Th1 and Th17 activation, and lower expression of IL-12, IL-23, TNF-α, IL-6, and CD86. Moreover, reactivated wild-type splenocytes showed increased Notch1 expression, arguing for a specific involvement of this receptor in autoimmune T cell activation in secondary lymphoid tissues. In summary, our results reveal a key role of the Notch1 receptor in myeloid cells for the initiation and progression of EAE.

Antimicrobial evaluation of quaternary ammonium polyethyleneimine nanoparticles against clinical isolates of pathogenic bacteria.

Peritonitis is a disease caused by bacterial strains that have become increasingly resistant to many antibiotics. The development of alternative therapeutic compounds is the focus of extensive research, so novel nanoparticles (NPs) with activity against antibiotic-resistant bacteria should be developed. In this study, the antibacterial activity of quaternary ammonium polyethyleneimine (QA-PEI) NPs was evaluated against Streptococcus viridans, Stenotrophomonas maltophilia and Escherichia coli. To appraise the antibacterial activity, minimal inhibitory concentration (MIC), minimal bactericidal concentration and bactericidal assays were utilised with different concentrations (1.56-100 µg/ml) of QA-PEI NPs. Moreover, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and annexin V/propidium iodide toxicity assays were performed in cell cultures. MICs for S. maltophilia and E. coli isolates were 12.5 and 25 µg/ml, respectively, whereas the MIC for S. viridans was 100 µg/ml. Furthermore, the growth curve assays revealed that these QA-PEI NPs at a concentration of 12.5 µg/ml significantly inhibited bacterial growth for the bacterial isolates studied. On the other hand, QA-PEI NPs lacked significant toxicity for cells when used at concentrations up to 50 µg/ml for 48 h. The present findings reveal the potential therapeutic value of this QA-PEI NPs as alternative antibacterial agents for peritonitis, especially against Gram-negative bacteria.

NFAT transcription factors regulate survival, proliferation, migration, and differentiation of neural precursor cells.

The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

NFATc3 promotes Ca(2+) -dependent MMP3 expression in astroglial cells.

Increase in intracellular calcium ([Ca(2+) ]i ) is a key mediator of astrocyte signaling, important for activation of the calcineurin (CN)/nuclear factor of activated T cells (NFAT) pathway, a central mediator of inflammatory events. We analyzed the expression of matrix metalloproteinase 3 (Mmp3) in response to increases in [Ca(2+) ]i and the role of the CN/NFAT pathway in this regulation. Astrocyte Mmp3 expression was induced by overexpression of a constitutively active form of NFATc3, whereas other MMPs and tissue inhibitor of metalloproteinases (TIMP) were unaffected. Mmp3 mRNA and protein expression was also induced by calcium ionophore (Io) and 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and Mmp3 upregulation was prevented by the CN inhibitor cyclosporin A (CsA). Ca(2+) -dependent astrocyte Mmp3 expression was also inhibited by actinomycin D, and a Mmp3 promoter luciferase reporter was efficiently activated by increased [Ca(2+) ]i , indicating regulation at the transcriptional level. Furthermore, Ca(2+) /CN/NFAT dependent Mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (Ast-NSC), demonstrating that the induced Mmp3 expression occurs in astrocytes, and not microglial cells. In an in vivo stab-wound model of brain injury, MMP3 expression was detected in NFATc3-positive scar-forming astrocytes. Because [Ca(2+) ]i increase is an early event in most brain injuries, these data support an important role for Ca(2+) /CN/NFAT-induced astrocyte MMP3 expression in the early neuroinflammatory response. Understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain.

Selenoprotein S expression in reactive astrocytes following brain injury.

Selenoprotein S (SelS) is an endoplasmic reticulum (ER)-resident protein involved in the unfolded protein response. Besides reducing ER-stress, SelS attenuates inflammation by decreasing pro-inflammatory cytokines. We have recently shown that SelS is responsive to ischemia in cultured astrocytes. To check the possible association of SelS with astrocyte activation, here we investigate the expression of SelS in two models of brain injury: kainic acid (KA) induced excitotoxicity and cortical mechanical lesion. The regulation of SelS and its functional consequences for neuroinflammation, ER-stress, and cell survival were further analyzed using cultured astrocytes from mouse and human. According to our immunofluorescence analysis, SelS expression is prominent in neurons and hardly detectable in astrocytes from control mice. However, brain injury intensely upregulates SelS, specifically in reactive astrocytes. SelS induction by KA was evident at 12 h and faded out after reaching maximum levels at 3-4 days. Analysis of mRNA and protein expression in cultured astrocytes showed SelS upregulation by inflammatory stimuli as well as ER-stress inducers. In turn, siRNA-mediated SelS silencing combined with adenoviral overexpression assays demonstrated that SelS reduces ER-stress markers CHOP and spliced XBP-1, as well as inflammatory cytokines IL-1ß and IL-6 in stimulated astrocytes. SelS overexpression increased astrocyte resistance to ER-stress and inflammatory stimuli. Conversely, SelS suppression compromised astrocyte viability. In summary, our results reveal the upregulation of SelS expression in reactive astrocytes, as well as a new protective role for SelS against inflammation and ER-stress that can be relevant to astrocyte function in the context of inflammatory neuropathologies.

Response of transcription factor NFATc3 to excitotoxic and traumatic brain insults: identification of a subpopulation of reactive astrocytes.

Astrocytes react to brain injury triggering neuroinflammatory processes that determine the degree of neuronal damage. However, the signaling events associated to astrocyte activation remain largely undefined. The nuclear factor of activated T-cells (NFAT) is a transcription factor family implicated in activation of immune cells. We previously characterized the expression of NFAT isoforms in cultured astrocytes, and NFAT activation in response to mechanical lesion. Here we analyze NFATc3 in two mouse models of inflammatory brain damage: hippocampal excitotoxicity induced by intracerebral kainic acid (KA) injection and cortical mechanical lesion. Immunofluorescence results demonstrated that NFATc3 is specifically induced in a subset of reactive astrocytes, and not in microglia or neurons. In KA-treated brains, NFATc3 expression is transient and NFATc3-positive astrocytes concentrate around damaged neurons in areas CA3 and CA1. Complementary Western blot and RT-PCR analysis revealed an NFAT-dependent induction of RCAN1-4 and COX-2 in hippocampus as soon as 6 h after KA exposure, indicating that NFAT activation precedes NFATc3 over-expression. Moreover, activation of NFAT by ATP increased NFATc3 mRNA levels in astrocyte cultures, suggesting that NFATc3 expression is controlled through an auto-regulatory loop. Meanwhile, stab wound enhanced NFATc3 expression specifically in a subclass of reactive astrocytes confined within the proximal layer of the glial scar, and GFAP immunoreactivity was attenuated in NFATc3-expressing astrocytes. In conclusion, our work establishes NFATc3 as a marker of activation for a specific population of astrocytes in response to brain damage, which may have consequences for neuronal survival.

A proteomic analysis of PKCε targets in astrocytes: implications for astrogliosis.

Astrocytes are glial cells in the central nervous system (CNS) that play key roles in brain physiology, controlling processes, such as neurogenesis, brain energy metabolism and synaptic transmission. Recently, immune functions have also been demonstrated in astrocytes, influencing neuronal survival in the course of neuroinflammatory pathologies. In this regard, PKCepsilon (PKCε) is a protein kinase with an outstanding role in inflammation. Our previous findings indicating that PKCε regulates voltage-dependent calcium channels as well as morphological stellation imply that this kinase controls multiple signalling pathways within astrocytes, including those implicated in activation of immune functions. The present study applies proteomics to investigate new protein targets of PKCε in astrocytes. Primary astrocyte cultures infected with an adenovirus that expresses constitutively active PKCε were compared with infection controls. Two-dimensional gel electrophoresis clearly detected 549 spots in cultured astrocytes, and analysis of differential protein expression revealed 18 spots regulated by PKCε. Protein identification by mass spectrometry (nano-LC-ESI-MS/MS) showed that PKCε targets molecules with heterogeneous functions, including chaperones, cytoskeletal components and proteins implicated in metabolism and signalling. These results support the notion that PKCε is involved in astrocyte activation; also suggesting that multiple astrocyte-dependent processes are regulated by PKCε, including those associated to neuroinflammation.

Caspase-11 mediates ischemia-induced astrocyte death: involvement of endoplasmic reticulum stress and C/EBP homologous protein.

Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress-dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase-11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress-dependent astrocyte death in ischemic conditions. According to our results, caspase-11 is up-regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER-stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase-11 mRNA levels and luciferase activity driven by a caspase-11 promoter, indicating that caspase-11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress-associated CHOP in caspase-11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase-11. In turn, caspase-11 suppression with siRNA rescued astrocytes from OGD- and ER stress-induced death, supporting the idea that caspase-11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase-1 and caspase-3 significantly reduced astrocyte death, which indicates that these proteases act as death effectors of caspase-11. In conclusion, our work contributes to clarifying the pathways leading to astrocyte death in response to ischemia by defining caspase-11 as a key mediator of the ER stress response acting downstream of CHOP.

mTOR/S6 kinase pathway contributes to astrocyte survival during ischemia.

Neurons are highly dependent on astrocyte survival during brain damage. To identify genes involved in astrocyte function during ischemia, we performed mRNA differential display in astrocytes after oxygen and glucose deprivation (OGD). We detected a robust down-regulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein levels and activity. OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes, as well as by treatment with rapamycin that inhibits S6K1 activity by acting on the upstream regulator mTOR (mammalian target of rapamycin). Astrocytes lacking S6K1 and S6K2 (S6K1;S6K2-/-) displayed a defect in BAD phosphorylation and in the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL. Furthermore reactive oxygen species were increased while translation recovery was impaired in S6K-deficient astrocytes following OGD. Rescue of either S6K1 or S6K2 expression by adenoviral infection revealed that protective functions were specifically mediated by S6K1, because this isoform selectively promoted resistance to OGD and reduction of ROS levels. Finally, "in vivo" effects of S6K suppression were analyzed in the permanent middle cerebral artery occlusion model of ischemia, in which absence of S6K expression increased mortality and infarct volume. In summary, this article uncovers a protective role for astrocyte S6K1 against brain ischemia, indicating a functional pathway that senses nutrient and oxygen levels and may be beneficial for neuronal survival.

SEPS1 gene is activated during astrocyte ischemia and shows prominent antiapoptotic effects.

Contrarily to neurons, astrocytes can survive short periods of ischemia. We have searched for genes implicated in astrocyte resistance to ischemia using oxygen and glucose deprivation (OGD) as a stroke model. A RNA differential display approach uncovered the OGD induction of selenoprotein-S-encoding gene SEPS1. This endoplasmic reticulum (ER) resident protein is known to promote cell survival regulating the ER stress as well as inflammation. We found that suppression of SEPS1 by small interfering RNA severely increases astrocyte injure caused by OGD, suggesting that selenoprotein S protects astrocytes against ischemia. Our data also support that modulation of ER stress is implicated in this effect.

Mechanical lesion activates newly identified NFATc1 in primary astrocytes: implication of ATP and purinergic receptors.

Ca2+-dependent calcineurin is upregulated in reactive astrocytes in neuroinflammatory models. Therefore, the fact that the nuclear factor of activated T cells (NFAT) is activated in response to calcineurin qualifies this family of transcription factors with immune functions as candidates to mediate astrogliosis. Brain trauma induces a neuroinflammatory state in which ATP is released from astrocytes, stimulating calcium signalling. Our goal here is to characterize NFATc1 and NFATc2 in mouse primary astrocyte cultures, also exploring the implication of NFAT in astrocyte activation by mechanical lesion. Quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis and immunofluorescence microscopy identified NFATc1 in astrocytes, but not NFATc2. Moreover, NFATc1 was expressed in the cytosol of resting astrocytes, whereas activation of the Ca2+-calcineurin pathway by ionomycin translocated NFATc1 to the nucleus, which is a requirement for activation. The implication of astrocytic NFAT in brain trauma was analysed using an in vitro scratch lesion model. Mechanical lesion caused a rapid NFATc1 translocation that progressed throughout the culture as a gradient and was maintained for at least 4 h. We also demonstrate that ATP, released by lesion, is a potent inducer of NFATc1 translocation and activation. Moreover, the use of P2Y receptor modulators showed that such ATP action is mediated by stimulation of several G(q)-protein-coupled P2Y purinergic receptors, among which P2Y(1) and P2Y(6) are included. In conclusion, this work provides evidence that newly identified NFATc1 is translocated in astrocytes in response to lesion following a pathway that involves ATP release and activation of metabotropic purinergic receptors.

Glitazones induce astroglioma cell death by releasing reactive oxygen species from mitochondria: modulation of cytotoxicity by nitric oxide.

The glitazones (or thiazolidinediones) are synthetic compounds used in type-2 diabetes, but they also have broad antiproliferative and anti-inflammatory properties still not well understood. We described previously the apoptotic effects of glitazones on astroglioma cells ( J Biol Chem 279: 8976-8985, 2004 ). At certain concentrations, we found a selective lethality on glioma cells versus astrocytes that was dependent on a rapid production of reactive oxygen species (ROS) and seemed unrelated to the receptor peroxisome proliferator activated receptor-gamma. The present study was aimed at characterizing the oxygen derivatives induced by ciglitazone, rosiglitazone, and pioglitazone in C6 glioma cells and to investigate their intracellular source. We examined the interaction of ROS with nitric oxide (NO) and its consequences for glioma cell survival. Fluorescence microscopy and flow cytometry showed that glitazones induced superoxide anion, peroxynitrite, and hydrogen peroxide, with ciglitazone being the most active. ROS production was completely prevented by uncoupling of the electron transport chain and by removal of glucose as an energy substrate, whereas it was unaffected by inhibition of NADPH-oxidase and xanthine-oxidase. Moreover, glitazones inhibited state 3 respiration in permeabilized cells, and experiments with mitochondrial inhibitors suggested that complex I was the likely target of glitazones. Therefore, these results point to the mitochondrial electron transport chain as the source of glitazone-induced ROS in C6 cells. Glitazones also depolarized mitochondria and reduced mitochondrial pH. NO synthase inhibitors revealed that superoxide anion combines with NO to yield peroxynitrite and that the latter contributes to the cytotoxicity of glitazones in astroglioma cells. Future antitumoral strategies may take advantage of these findings.

PKCepsilon induces astrocyte stellation by modulating multiple cytoskeletal proteins and interacting with Rho A signalling pathways: implications for neuroinflammation.

Despite the importance of stellation to maintain astrocyte functionality, the intracellular signals controlling morphology in these cells are poorly characterized. Our goal was to examine the implication of protein kinase C epsilon (PKCepsilon) in astrocyte stellation. We found that the morphological transformation of astrocytes induced by exposure to the pro-inflammatory agent lipopolysaccharide is enhanced by adenoviral expression of wild-type PKCepsilon, and that activation of PKCepsilon is sufficient to trigger a dramatic stellation. Such an effect is mediated by the rearrangement of microtubules and filaments of glial fibrillary acidic protein, disorganization of stress fibres, and formation of new actin filaments within growing cellular processes. Furthermore, PKCepsilon regulates actin-interacting elements such as non-muscle myosin and proteins of the ezrin/radixin/moesin family. We also observed that at least part of the actions of PKCepsilon depend on its catalytic activity. Finally, stellation by PKCepsilon could be blocked by the expression of a constitutively active form of Rho A implicated in the stability of the flat astrocytic morphology. In summary, PKCepsilon stands out as a key intracellular regulator of morphological plasticity in astrocytes, affecting a large range of cytoskeletal elements and inactivating Rho A-dependent pathways. These morphological effects of PKCepsilon may play essential roles during the course of neuroinflammation.

CHOP plays a pivotal role in the astrocyte death induced by oxygen and glucose deprivation.

Ischemia has different consequences on the survival of astrocytes and neurons. Thus, astrocytes show a remarkable resistance to short periods of ischemia that are well known to cause neuronal death. We have used a cell culture model of stroke, oxygen, and glucose deprivation (OGD), to clarify the mechanisms responsible for the exclusive resistance of astrocytes to ischemia. The expression of genes implicated in both ischemia-induced astrocyte death and post-ischemic survival was analysed by the RNA differential display technique. Our study revealed that the expression of the CEBP homologous protein (CHOP)-coding gene is promptly an intensely upregulated following astrocyte oxygen and glucose deprivation. CHOP mRNA induction was accompanied by the activation of other genes (grp78, grp95) that, alike CHOP, are involved in the endoplasmic reticulum (ER) stress response. In addition, drugs that cause ER calcium depletion or protein N-glycosylation inhibition mimicked the effects of OGD on astrocyte survival, further supporting the involvement of ER in the astrocyte responses to OGD. Our experiments also demonstrated that upregulation of CHOP during the ER stress response is required for ischemia to cause astrocyte death. Not only the levels of CHOP mRNA and protein correlate perfectly with the degree of OGD-triggered cell injury, but also astrocyte death induced by OGD is significantly overcome by CHOP antisense oligonucleotide treatment. Nevertheless, we observed that astrocytes undergo apoptosis only when CHOP is permanently upregulated, and not when CHOP increases are transient. Finally, we found that the extent of CHOP induction is determined by the length of the ischemic stimulus. Taken together, our results indicate that permanent upregulation of CHOP is decisive for the induction of astrocyte death by OGD.

Glitazones differentially regulate primary astrocyte and glioma cell survival. Involvement of reactive oxygen species and peroxisome proliferator-activated receptor-gamma.

The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5-20 microM) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 microM was toxic for both cell types. Ciglitazone at 10 microM was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARgamma and production of reactive oxygen species (ROS). PPARgamma does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARgamma. In addition, GW9662, an inhibitor of PPARgamma, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.