Gliopreventive effects of guanosine against glucose deprivation in vitro

Guanosine, a guanine-based purine, is recognized as an extracellular signaling molecule that is released from astrocytes and confers neuroprotective effects in several in vivo and in vitro studies. Astrocytes regulate glucose metabolism, glutamate transport, and defense mechanism against oxidative stress. C6 astroglial cells are widely used as an astrocyte-like cell line to study the astrocytic function and signaling pathways. Our previous studies showed that guanosine modulates the glutamate uptake activity, thus avoiding glutamatergic excitotoxicity and protecting neural cells. The goal of this study was to determine the gliopreventive effects of guanosine against glucose deprivation in vitro in cultured C6 cells. Glucose deprivation induced cytotoxicity, an increase in reactive oxygen and nitrogen species (ROS/RNS) levels and lipid peroxidation as well as affected the metabolism of glutamate, which may impair important astrocytic functions. Guanosine prevented glucose deprivation-induced toxicity in C6 cells by modulating oxidative and nitrosative stress and glial responses, such as the glutamate uptake, the glutamine synthetase activity, and the glutathione levels. Glucose deprivation decreased the level of EAAC1, the main glutamate transporter present in C6 cells. Guanosine also prevented this effect, most likely through PKC, PI3K, p38 MAPK, and ERK signaling pathways. Taken together, these results show that guanosine may represent an important mechanism for protection of glial cells against glucose deprivation. Additionally, this study contributes to a more thorough understanding of the glial- and redox-related protective properties of guanosine in astroglial cells.     

Ornithine and Homocitrulline Impair Mitochondrial Function,Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome

Hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide—MTT—reduction and mitochondrial membrane potential—ΔΨ_m), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1β, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨ_m in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease.     

Adenosine receptors as a new target for resveratrol-mediated glioprotection

Resveratrol, a natural polyphenolic compound, has been studied as a neuroprotective molecule. Our group has demonstrated that such effect is closely associated with modulation of glial functionality, but the underlying mechanisms are not fully understood. Because astrocytes actively participate in the brain inflammatory response, and activation of adenosine receptors can attenuate inflammatory processes, the aim of this study was to investigate the role of adenosine receptors as a mechanism for resveratrol glioprotection, particularly regarding to neuroinflammation. Therefore, primary astrocyte cultures were co-incubated with resveratrol and selective antagonists of A₁, A_2A, and A₃ adenosine receptors, as well as with caffeine (a non-selective adenosine receptor antagonist), and then challenged with bacterial inflammogen lipopolysaccharide (LPS). Caffeine and selective adenosine receptor antagonists abolished the anti-inflammatory effect of resveratrol. In accordance with these effects, resveratrol prevented LPS-induced decrease in mRNA levels of adenosine receptors. Resveratrol could also prevent the activation of pro-inflammatory signaling pathways, such as nuclear factor κB (NFκB) and p38 mitogen-activated protein kinase (p38 MAPK) in a mechanism dependent on adenosine receptors. Conversely, trophic factors and protective signaling pathways, including sirtuin 1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and phosphoinositide 3-kinase (PI3K)/Akt were positively modulated by resveratrol in both LPS-stimulated and unstimulated astrocytes, but adenosine receptor antagonism did not abrogate all effects of resveratrol. To our knowledge, our data provide the first evidence that adenosine receptors are involved in the anti-inflammatory activity of resveratrol in astrocytes, thus exerting an important role for resveratrol-mediated glioprotection.     

S100B Secretion in Acute Brain Slices: Modulation by Extracellular Levels of Ca<Superscript>2+</Superscript> and K<Superscript>+</Superscript>

Hippocampal slices have been widely used to investigate electrophysiological and metabolic neuronal parameters, as well as parameters of astroglial activity including protein phosphorylation and glutamate uptake. S100B is an astroglial-derived protein, which extracellularly plays a neurotrophic activity during development and excitotoxic insult. Herein, we characterized S100B secretion in acute hippocampal slices exposed to different concentrations of K⁺ and Ca²⁺ in the extracellular medium. Absence of Ca²⁺ and/or low K⁺ (0.2 mM KCl) caused an increase in S100B secretion, possibly by mobilization of internal stores of Ca²⁺. In contrast, high K⁺ (30 mM KCl) or calcium channel blockers caused a decrease in S100B secretion. This study suggests that exposure of acute hippocampal slices to low- and high-K⁺ could be used as an assay to evaluate astrocyte activity by S100B secretion: positively regulated by low K⁺ (possibly involving mobilization of internal stores of Ca²⁺) and negatively regulated by high-K⁺ (likely secondary to influx of K⁺).     

Carbon Tetrachloride Increases the Pro-inflammatory Cytokines Levels in Different Brain Areas of Wistar Rats: The Protective Effect of Acai Frozen Pulp

Neurochemical Research - Acai offers health benefits associated with its high antioxidante capacity, phytochemical composition, nutritional and sensory value. Therefore, the objective of this study...     

Sulforaphane Induces Glioprotection After LPS Challenge

Sulforaphane is a natural compound that presents anti-inflammatory and antioxidant properties, including in the central nervous system (CNS). Astroglial cells are involved in several functions to maintain brain homeostasis, actively participating in the inflammatory response and antioxidant defense systems. We, herein, investigated the potential mechanisms involved in the glioprotective effects of sulforaphane in the C6 astrocyte cell line, when challenged with the inflammogen, lipopolysaccharide (LPS). Sulforaphane prevented the LPS-induced increase in the expression and/or release of pro-inflammatory mediators, possibly due to nuclear factor κB and hypoxia-inducible factor-1α activation. Sulforaphane also modulated the expressions of the Toll-like and adenosine receptors, which often mediate inflammatory processes induced by LPS. Additionally, sulforaphane increased the mRNA levels of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO1), both of which mediate several cytoprotective responses. Sulforaphane also prevented the increase in NADPH oxidase activity and the elevations of superoxide and 3-nitrotyrosine that were stimulated by LPS. In addition, sulforaphane and LPS modulated superoxide dismutase activity and glutathione metabolism. Interestingly, the anti-inflammatory and antioxidant effects of sulforaphane were blocked by HO1 pharmacological inhibition, suggesting its dependence on HO1 activity. Finally, in support of a glioprotective role, sulforaphane prevented the LPS-induced decrease in glutamate uptake, glutamine synthetase activity, and glial-derived neurotrophic factor (GDNF) levels, as well as the augmentations in S100B release and Na⁺, K⁺ ATPase activity. To our knowledge, this is the first study that has comprehensively explored the glioprotective effects of sulforaphane on astroglial cells, reinforcing the beneficial effects of sulforaphane on astroglial functionality.

     

High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.     

Epicatechin gallate increases glutamate uptake and S100B secretion in C6 cell lineage

There is a current interest in dietary compounds, such as green tea polyphenols, that can favor protection against a variety of brain disorders, including Alzheimer’s disease, ischemia, and stroke. The objective of the present study was to investigate the effects of (^_)-epicatechin-3-gallate (ECG), one of three three major green tea antioxidants, on C6 lineage cells. Here, we evaluated cell morphology and integrity and specific astrocyte activities; glutamate uptake and secretion of S100B in the presence of 0.1, 1 and 10 μM ECG. During 6 h of incubation, cell morphology was altered only at 10 μM ECG; however, after 24 h of treatment, cells become stellate in the presence of all concentrations of ECG. Loss of cell integrity was observed after 24 h with 10 μM ECG and represented only 6% of cells, in contrast with 2% observed at basal conditions. ECG (1–10 μM) induced a decrease (about 36%) in glutamate uptake after 1 h of incubation. After 6 h, an opposite effect occurred and ECG induced a sustained increase in glutamate uptake of about 70% from 0.1 μM. In addition, a significant increase in S100B was observed at 1 μM ECG (36%) and 10 μM ECG (69%) after 1 h, in contrast to 6 h of treatment, where all doses of ECG induced a significant increase (about 60%) in S100B secretion. These data demonstrate that ECG induces a significant improvement in glutamate uptake and S100B secretion in C6 cells, indicating that ECG could contribute to the neuroprotective role of astroglial cells.     

Cortical Bilateral Adaptations in Rats Submitted to Focal Cerebral Ischemia: Emphasis on Glial Metabolism

This study was performed to evaluate the bilateral effects of focal permanent ischemia (FPI) on glial metabolism in the cerebral cortex. Two and 9 days after FPI induction, we analyze [¹⁸F]FDG metabolism by micro-PET, astrocyte morphology and reactivity by immunohistochemistry, cytokines and trophic factors by ELISA, glutamate transporters by RT-PCR, monocarboxylate transporters (MCTs) by western blot, and substrate uptake and oxidation by ex vivo slices model. The FPI was induced surgically by thermocoagulation of the blood in the pial vessels of the motor and sensorimotor cortices in adult (90 days old) male Wistar rats. Neurochemical analyses were performed separately on both ipsilateral and contralateral cortical hemispheres. In both cortical hemispheres, we observed an increase in tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and glutamate transporter 1 (GLT-1) mRNA levels; lactate oxidation; and glutamate uptake and a decrease in brain-derived neurotrophic factor (BDNF) after 2 days of FPI. Nine days after FPI, we observed an increase in TNF-α levels and a decrease in BDNF, GLT-1, and glutamate aspartate transporter (GLAST) mRNA levels in both hemispheres. Additionally, most of the unilateral alterations were found only in the ipsilateral hemisphere and persisted until 9 days post-FPI. They include diminished in vivo glucose uptake and GLAST expression, followed by increased glial fibrillary acidic protein (GFAP) gray values, astrocyte reactivity, and glutamate oxidation. Astrocytes presented signs of long-lasting reactivity, showing a radial morphology. In the intact hemisphere, there was a decrease in MCT2 levels, which did not persist. Our study shows the bilateralism of glial modifications following FPI, highlighting the role of energy metabolism adaptations on brain recovery post-ischemia.