Soledad Calvo

Glitazones Differentially Regulate Primary Astrocyte and Glioma Cell Survival INVOLVEMENT OF REACTIVE OXYGEN SPECIES AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ

The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor γ (PPARγ). 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 μm) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 μm was toxic for both cell types. Ciglitazone at 10 μm 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 PPARγ and production of reactive oxygen species (ROS). PPARγ 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 PPARγ. In addition, GW9662, an inhibitor of PPARγ, 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.

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.

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.

Veratridine induces apoptotic death in bovine chromaffin cells through superoxide production

The molecular mechanisms involved in veratridine‐induced chromaffin cell death have been explored. We have found that exposure to veratridine (30 μM, 1 h) produces a delayed cellular death that reaches 55% of the cells 24 h after veratridine exposure. This death has the features of apoptosis as DNA fragmentation can be observed. Calcium ions play an important role in veratridine‐induced chromaffin cell death because the cell permeant Ca2+ chelator BAPTA‐AM and extracellular Ca2+ removal completely prevented veratridine‐induced toxicity. Following veratridine treatment, there is a decrease in mitochondrial function and an increase in superoxide anion production. Veratridine‐induced increase in superoxide production was blocked by tetrodotoxin (TTX; 10 μM), extracellular Ca2+ removal and the mitochondrial permeability transition pore blocker cyclosporine A (10 μM). Veratridine‐induced death was prevented by different antioxidant treatments including catalase (100 IU ml−1), N‐acetyl cysteine (100 μM), allopurinol (100 μM) or vitamin E (50 μM). Veratridine‐induced DNA fragmentation was prevented by TTX (10 μM). Veratridine produced a time‐dependent increase in caspase activity that was prevented by Ca2+ removal and TTX (10 μM). In addition, calpain and caspases inhibitors partially prevented veratridine‐induced death. These results indicate that chromaffin cells share with neurons the molecular machinery involved in apoptotic death and might be considered a good model to study neuronal death during neurodegeneration.

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 Gq‐protein‐coupled P2Y purinergic receptors, among which P2Y1 and P2Y6 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.

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.

Minocycline fails to protect cerebellar granular cell cultures against malonate-induced cell death

Experimental and clinical studies support the view that the semisynthetic tetracycline minocycline exhibits neuroprotective roles in several models of neurodegenerative diseases, including ischemia, Huntington, Parkinson diseases, and amyotrophic lateral sclerosis. However, recent evidence indicates that minocycline does not always present beneficial actions. For instance, in an in vivo model of Huntingtons disease, it fails to afford protection after malonate intrastriatal injection. Moreover, it reverses the neuroprotective effect of creatine in nigrostriatal dopaminergic neurons. This apparent contradiction prompted us to analyze the effect of this antibiotic on malonate-induced cell death. We show that, in rat cerebellar granular cells, the succinate dehydrogenase inhibitor malonate induces cell death in a concentration-dependent manner. By using DFCA, monochlorobimane and 10-N-nonyl-Acridin Orange to measure, respectively, H2O2-derived oxidant species and reduced forms of GSH and cardiolipin, we observed that malonate induced reactive oxygen species (ROS) production to an extent that surpasses the antioxidant defense capacity of the cells, resulting in GSH depletion and cardiolipin oxidation. The pre-treatment for 4 h with minocycline (10-100 microM) did not present cytoprotective actions. Moreover, minocycline failed to block ROS production and to abrogate malonate-induced oxidation of GSH and cardiolipin. Additional experiments revealed that minocycline was also unsuccessful to prevent the mitochondrial swelling induced by malonate. Furthermore, malonate did not induce the expression of the iNOS, caspase-3, -8, and -9 genes which have been shown to be up-regulated in several models where minocycline resulted cytoprotective. In addition, malonate-induced down-regulation of the antiapoptotic gene Bcl-2 was not prevented by minocycline, controversially the mechanism previously proposed to explain minocycline protective action. These results suggest that the minocycline protection observed in several neurodegenerative disease models is selective, since it is absent from cultured cerebellar granular cells challenged with malonate.

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.

PKCε upregulates voltage-dependent calcium channels in cultured astrocytes

Astrocytes express voltage‐gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCε), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCε in astrocytes. We found that PKCε potently increases the mRNA levels of two different calcium channel α1 subunits, CaV1.2 (L‐type channel) and CaV2.1 (P/Q‐type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCε activation are functional, since overexpression of constitutively‐active PKCε increased significantly the calcium current density in astrocytes. PKCε raised currents carried by both L‐ and P/Q‐type channels. However, the effect on the P/Q‐type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCε‐induced stellation was significantly reduced by the specific L‐type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCε. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCε‐dependent astrocyte activation.

PKCε 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 (PKCε) 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 PKCε, and that activation of PKCε 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, PKCε 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 PKCε depend on its catalytic activity. Finally, stellation by PKCε 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, PKCε 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 PKCε may play essential roles during the course of neuroinflammation.