Larissa Daniele Bobermin

Resveratrol Protects C6 Astrocyte Cell Line against Hydrogen Peroxide-Induced Oxidative Stress through Heme Oxygenase 1

Resveratrol, a polyphenol presents in grapes and wine, displays antioxidant and anti-inflammatory properties and cytoprotective effect in brain pathologies associated to oxidative stress and neurodegeneration. In previous work, we demonstrated that resveratrol exerts neuroglial modulation, improving glial functions, mainly related to glutamate metabolism. Astrocytes are a major class of glial cells and regulate neurotransmitter systems, synaptic processing, energy metabolism and defense against oxidative stress. This study sought to determine the protective effect of resveratrol against hydrogen peroxide (H2O2)-induced cytotoxicity in C6 astrocyte cell line, an astrocytic lineage, on neurochemical parameters and their cellular and biochemical mechanisms. H2O2 exposure increased oxidative-nitrosative stress, iNOS expression, cytokine proinflammatory release (TNFα levels) and mitochondrial membrane potential dysfunction and decreased antioxidant defenses, such as SOD, CAT and creatine kinase activity. Resveratrol strongly prevented C6 cells from H2O2-induced toxicity by modulating glial, oxidative and inflammatory responses. Resveratrol per se increased heme oxygenase 1 (HO1) expression and extracellular GSH content. In addition, HO1 signaling pathway is involved in the protective effect of resveratrol against H2O2-induced oxidative damage in astroglial cells. Taken together, these results show that resveratrol represents an important mechanism for protection of glial cells against oxidative stress.

Secretion of S100B, an astrocyte-derived neurotrophic protein, is stimulated by fluoxetine via a mechanism independent of serotonin

S100B is a calcium-binding protein, produced and secreted by astrocytes, which has a putative paracrine neurotrophic activity. Clinical studies have suggested that peripheral elevation of this protein is positively correlated with a therapeutic antidepressant response, particularly to selective serotonin reuptake inhibitors (SSRIs); however, the mechanism underlying this response remains unclear. Here, we measured S100B secretion directly in hippocampal astrocyte cultures and hippocampal slices exposed to fluoxetine and observed a significant increment of S100B release in the presence of this SSRI, apparently dependent on protein kinase A (PKA). Moreover, we found that serotonin (possibly via the 5HT1A receptor) reduces S100B secretion and antagonizes the effect of fluoxetine on S100B secretion. These data reinforce the effect of fluoxetine, independently of serotonin and serotonin receptors, suggesting a putative role for S100B in depressive disorders and suggesting that other molecular targets may be relevant for antidepressant activity.

Resveratrol Prevents Ammonia Toxicity in Astroglial Cells

Ammonia is implicated as a neurotoxin in brain metabolic disorders associated with hyperammonemia. Acute ammonia toxicity can be mediated by an excitotoxic mechanism, oxidative stress and nitric oxide (NO) production. Astrocytes interact with neurons, providing metabolic support and protecting against oxidative stress and excitotoxicity. Astrocytes also convert excess ammonia and glutamate into glutamine via glutamine synthetase (GS). Resveratrol, a polyphenol found in grapes and red wines, exhibits antioxidant and anti-inflammatory properties and modulates glial functions, such as glutamate metabolism. We investigated the effect of resveratrol on the production of reactive oxygen species (ROS), GS activity, S100B secretion, TNF-α, IL-1β and IL-6 levels in astroglial cells exposed to ammonia. Ammonia induced oxidative stress, decreased GS activity and increased cytokines release, probably by a mechanism dependent on protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) pathways. Resveratrol prevented ammonia toxicity by modulating oxidative stress, glial and inflammatory responses. The ERK and nuclear factor-κB (NF-κB) are involved in the protective effect of resveratrol on cytokines proinflammatory release. In contrast, other antioxidants (e.g., ascorbic acid and trolox) were not effective against hyperammonemia. Thus, resveratrol could be used to protect against ammonia-induced neurotoxicity.

Oxidative stress mediated by NMDA, AMPA/KA channels in acute hippocampal slices: neuroprotective effect of resveratrol.

Glutamate is the major excitatory neurotransmitter in the brain and over-stimulation of the glutamate receptors, NMDA, AMPA and kainate (KA), may cause neuronal death in epilepsy, seizures and neurodegenerative diseases. Mitochondria have critical cellular functions that influence neuronal excitability, such as regulation of Ca(2+) homeostasis and ATP production to maintain Na(+)K(+)-ATPase in the central nervous system (CNS). However, mitochondria are also the primary site of reactive oxygen species (ROS) production, and oxidative stress can induce cellular damage. Resveratrol, a polyphenol found in grapes and wines, presents antioxidant and neuroprotective effects on brain pathologies. This study sought to determine the neuroprotective effect of resveratrol against glutamate toxicity in acute hippocampal slices, using specific inhibitors of glutamate channels, and to investigate the targets of glutamate excitotoxicity, such as mitochondrial membrane potential (ΔΨ(m)), Na(+)K(+)-ATPase and glutamine synthetase (GS) activity. Resveratrol decreases intracellular ROS production, most likely by mechanisms involving NMDA, AMPA/KA, intracellular Ca(2+) and the heme oxygenase 1 (HO1) pathway, and prevents mitochondrial dysfunction and impairments in Na(+)K(+)-ATPase and GS activity after glutamate activation. Taken together, these results show that resveratrol may exhibit an important neuroprotective mechanism against neuropsychiatric disorders, focusing on mitochondrial bioenergetics and oxidative stress, as well as inhibitory effects on ionic channels.

Gap junction inhibitors modulate S100B secretion in astrocyte cultures and acute hippocampal slices

Astrocytes sense, integrate, and respond to stimuli generated by neurons or neural injury; this response involves gap junction (GJ) communication. Neuronal vulnerability to injury increased when cocultures of astrocytes and neurons were exposed to GJ inhibitors. However, GJ uncoupling could limit the extension of a lesion. We investigated a possible link between GJ communication and S100B secretion. S100B is a calcium‐binding protein of 21 kDa that is predominantly expressed and secreted by astrocytes, which has trophic paracrine activity on neurite growth, glial proliferation, and neuronal survival. GJ inhibitors were analyzed in isolated astrocytes in primary cultures from hippocampus, acute hippocampal slices, and C6 glioma cells, which were used as a negative control. Our data indicate that GJ blocking stimulates S100B secretion in astrocyte cultures and acute hippocampal slices. Different assays were used to confirm cell integrity during exposure to GJ inhibitors. S100B secretion was observed with different types of GJ inhibitors; the resulting event was dependent on time, the nature of the inhibitor, its putative molecular target of GJ blocking, and/or the cell preparation used. Only carbenoxolone induced a fast and persistent increase in S100B secretion in both preparations. Endothelin‐1 increased S100B secretion in astrocyte cultures at 1 hr, but a decrease was observed at 6 hr or in acute hippocampal slices. Physiologically, a local GJ closure associated with release of S100B in injury conditions favors the idea of a common mechanism available to limit the extension of lesion and increase the chances of cell survival.

Guanosine protects C6 astroglial cells against azide‐induced oxidative damage: a putative role of heme oxygenase 1

Guanosine, a guanine‐based purine, is an extracellular signaling molecule that is released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies. Our group recently showed that guanosine presents antioxidant properties in C6 astroglial cells. The heme oxygenase 1 signaling pathway is associated with protection against oxidative stress. Azide, an inhibitor of the respiratory chain, is frequently used in experimental models to induce oxidative and nitrosative stress. Thus, the goal of this study was to investigate the effect of guanosine on azide‐induced oxidative damage in C6 astroglial cells. Azide treatment of these cells resulted in several detrimental effects, including induction of cytotoxicity and mitochondrial dysfunction, increased levels of reactive oxygen/nitrogen species, inducible nitric oxide synthase expression and NADPH oxidase, decreased glutamate uptake and EAAC1 glutamate transporter expression, decreased glutathione (GSH) levels, and decreased activities of glutamine synthetase (GS), superoxide dismutase and catalase (CAT). The treatment also increased nuclear factor‐κB activation and the release of proinflammatory cytokines tumor necrosis factor α and IL‐1β. Guanosine strongly prevented these effects, protecting glial cells against azide‐induced cytotoxicity and modulating glial, oxidative and inflammatory responses through the activation of the heme oxygenase 1 pathway. These observations reinforce and support the role of guanosine as an antioxidant molecule against oxidative damage.

The neuroprotective effect of two statins: simvastatin and pravastatin on a streptozotocin-induced model of Alzheimer’s disease in rats

Astrocytes play a fundamental role in glutamate metabolism by regulating the extracellular levels of glutamate and intracellular levels of glutamine. They also participate in antioxidant defenses, due to the synthesis of glutathione, coupled to glutamate metabolism. Although the cause of Alzheimer’s disease (AD) remains elusive, some changes in neurochemical parameters, such as glutamate uptake, glutamine synthetase activity and glutathione have been investigated in this disease. A possible neuroprotective effect of two statins, simvastatin and pravastatin (administered p.o.), was evaluated using a model of dementia, based on the intracerebroventricular (ICV) administration of streptozotocin (STZ), and astrocyte parameters were determined. We confirmed a cognitive deficit in rats submitted to ICV-STZ, and a prevention of this deficit by statin administration. Moreover, both statins were able to prevent the decrease in glutathione content and glutamine synthetase activity in this model of AD. Interestingly, simvastatin increased per se glutamate uptake activity, while both statins increased glutamine synthetase activity per se. These results support the idea that these drugs could be effective for the prevention of alterations observed in the STZ dementia model and may contribute to reduce the cognitive impairment and brain damage observed in AD patients.

Guanosine inhibits LPS-induced pro-inflammatory response and oxidative stress in hippocampal astrocytes through the heme oxygenase-1 pathway

Guanosine, a guanine-based purine, is an extracellular signaling molecule that is released from astrocytes and has been shown to promote central nervous system defenses in several in vivo and in vitro injury models. Our group recently demonstrated that guanosine exhibits glioprotective effects in the C6 astroglial cell line by associating the heme oxygenase-1 (HO-1) signaling pathway with protection against azide-induced oxidative stress. Astrocyte overactivation contributes to the triggering of brain inflammation, a condition that is closely related to the development of many neurological disorders. These cells sense and amplify inflammatory signals from microglia and/or initiate the release of inflammatory mediators that are strictly related to transcriptional factors, such as nuclear factor kappa B (NFκB), that are modulated by HO-1. Astrocytes also express toll-like receptors (TLRs); TLRs specifically recognize lipopolysaccharide (LPS), which has been widely used to experimentally study inflammatory response. This study was designed to understand the glioprotective mechanism of guanosine against the inflammatory and oxidative damage induced by LPS exposure in primary cultures of hippocampal astrocytes. Treatment of astrocytes with LPS resulted in deleterious effects, including the augmentation of pro-inflammatory cytokine levels, NFκB activation, mitochondrial dysfunction, increased levels of oxygen/nitrogen species, and decreased levels of antioxidative defenses. Guanosine was able to prevent these effects, protecting the hippocampal astrocytes against LPS-induced cytotoxicity through activation of the HO-1 pathway. Additionally, the anti-inflammatory effects of guanosine were independent of the adenosinergic system. These results highlight the potential role of guanosine against neuroinflammatory-related diseases.

Treadmill Exercise Induces Hippocampal Astroglial Alterations in Rats

Physical exercise effects on brain health and cognitive performance have been described. Synaptic remodeling in hippocampus induced by physical exercise has been described in animal models, but the underlying mechanisms remain poorly understood. Changes in astrocytes, the glial cells involved in synaptic remodeling, need more characterization. We investigated the effect of moderate treadmill exercise (20 min/day) for 4 weeks on some parameters of astrocytic activity in rat hippocampal slices, namely, glial fibrillary acidic protein (GFAP), glutamate uptake and glutamine synthetase (GS) activities, glutathione content, and S100B protein content and secretion, as well as brain-derived neurotrophic factor (BDNF) levels and glucose uptake activity in this tissue. Results show that moderate treadmill exercise was able to induce a decrease in GFAP content (evaluated by ELISA and immunohistochemistry) and an increase in GS activity. These changes could be mediated by corticosterone, whose levels were elevated in serum. BDNF, another putative mediator, was not altered in hippocampal tissue. Moreover, treadmill exercise caused a decrease in NO content. Our data indicate specific changes in astrocyte markers induced by physical exercise, the importance of studying astrocytes for understanding brain plasticity, as well as reinforce the relevance of physical exercise as a neuroprotective strategy.

Atypical neuroleptic risperidone modulates glial functions in C6 astroglial cells

Risperidone has demonstrated therapeutic advantages over conventional neuroleptics and offers a valuable emerging option for the treatment of social behavior associated with autistic disorder. Considering the putative involvement of astroglial cells in neuropsychiatric disorders, we investigated the effect of risperidone on parameters of astrocyte activity - glutamate uptake, glutamine synthetase (GS) activity and glutathione (GSH) levels. Risperidone was able to induce a significantly increase on glutamate uptake (32%); GS activity (15%); GSH levels (58%). These findings imply the perspectives for further investigations directed on astrocytes from different brain areas. Our present results suggest that risperidone might exert its neuroprotective effects against brain illness at least partially via modulation of astrocyte functions.