Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes.

The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance.

Novel series of tacrine-tianeptine hybrids: Synthesis, cholinesterase inhibitory activity, S100B secretion and a molecular modeling approach.

Tianeptine was linked to various 9-aminoalkylamino-1,2,3,4-tetrahydroacridines using EDC·HCl/HOBt to afford a series of tacrine-tianeptine hybrids. The hybrids were tested for their ability to inhibit AChE and BuChE and IC50 values in the nanomolar concentration scale were obtained. AChE molecular modeling studies of these hybrids indicated that tacrine moiety interacts in the bottom of the gorge with the catalytic active site (CAS) while tianeptine binds to peripheral anionic site (PAS). Furthermore, the compounds 2g and 2e were able to reduce the in vitro basal secretion of S100B, suggesting its therapeutic action in some cases or stages of Alzheimer's disease.

Congenital hypothyroidism alters the oxidative status, enzyme activities and morphological parameters in the hippocampus of developing rats.

Congenital hypothyroidism is associated with delay in cell migration and proliferation in brain tissue, impairment of synapse formation, misregulation of neurotransmitters, hypomyelination and mental retardation. However, the mechanisms underlying the neuropsychological deficits observed in congenital hypothyroidism are not completely understood. In the present study we proposed a mechanism by which hypothyroidism leads to hippocampal neurotoxicity. Congenital hypothyroidism induces c-Jun-N-terminal kinase (JNK) pathway activation leading to hyperphosphorylation of the glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits from hippocampal astrocytes and neurons, respectively. Moreover, hyperphosphorylation of the cytoskeletal proteins was not reversed by T3 and poorly reversed by T4. In addition, congenital hypothyroidism is associated with downregulation of astrocyte glutamate transporters (GLAST and GLT-1) leading to decreased glutamate uptake and subsequent influx of Ca(2+) through N-methyl-D-aspartate (NMDA) receptors. The Na(+)-coupled (14)C-α-methyl-amino-isobutyric acid ((14)C-MeAIB) accumulation into hippocampal cells also might cause an increase in the intracellular Ca(2+) concentration by opening voltage-dependent calcium channels (VDCC). The excessive influx of Ca(2+) through NMDA receptors and VDCCs might lead to an overload of Ca(2+) within the cells, which set off glutamate excitotoxicity and oxidative stress. The inhibited acetylcholinesterase (AChE) activity might also induce Ca(2+) influx. The inhibited glucose-6-phosphate dehydrogenase (G6PD) and gamma-glutamyl transferase (GGT) activities, associated with altered glutamate and neutral amino acids uptake could somehow affect the GSH turnover, the antioxidant defense system, as well as the glutamate-glutamine cycle. Reduced levels of S100B and glial fibrillary acidic protein (GFAP) take part of the hypothyroid condition, suggesting a compromised astroglial/neuronal neurometabolic coupling which is probably related to the neurotoxic damage in hypothyroid brain.

Brain changes in BDNF and S100B induced by ketogenic diets in Wistar rats.

AIMS: We investigated the effects of ketogenic diet (KD) on levels of tumor necrosis factor alpha (TNF-α, a classical pro-inflammatory cytokine), BDNF (brain-derived neurotrophic factor, commonly associated with synaptic plasticity), and S100B, an astrocyte neurotrophic cytokine involved in metabolism regulation. MAIN METHODS: Young Wistar rats were fed during 8weeks with control diet or two KD, containing different proportions of omega 6 and omega 3 polyunsaturated fatty acids. Contents of TNF-α, BDNF and S100B were measured by ELISA in two brain regions (hippocampus and striatum) as well as blood serum and cerebrospinal fluid. KEY FINDINGS: Our data suggest that KD was able to reduce the levels of BDNF in the striatum (but not in hippocampus) and S100B in the cerebrospinal fluid of rats. These alterations were not affected by the proportion of polyunsaturated fatty acids offered. No changes in S100B content were observed in serum or analyzed brain regions. Basal TNF-α content was not affected by KD. SIGNIFICANCE: These findings reinforce the importance of this diet as an inductor of alterations in the brain, and such changes might contribute to the understanding of the effects (and side effects) of KD in brain disorders.

Interleukin-6-induced S100B secretion is inhibited by haloperidol and risperidone.

Although inflammation may be a physiological defense process, imbalanced neuroinflammation has been associated with the pathophysiology of brain disorders, including major depression and schizophrenia. Activated glia releases a variety of pro-inflammatory cytokines that contribute to neuronal dysfunction. Elevated levels of S100B, a glia derived protein, have been observed in the serum and CSF of schizophrenic patients suggesting a glial role in the disease. We evaluated whether S100B secretion (in C6 glioma cells and hippocampal slices in Wistar rats) could be directly modulated by the main inflammatory cytokines (IL-1ß, TNF-α, IL-6 and IL-8) altered in schizophrenia, as well as the possible involvement of mitogen-activated protein kinase (MAPK) pathways in these responses. We also investigated the effects of typical and atypical antipsychotic drugs on glial cytokine-induced S100B release. Our results suggest that S100B secretion is increased by pro-inflammatory cytokines via MAPK and that oxidative stress may be a component of this modulation. These results reinforce the idea that the S100B protein is involved in the inflammatory response observed in many brain diseases, including schizophrenia. Moreover the antipsychotics, haloperidol and risperidone, were able to inhibit the secretion of S100B following IL-6 stimulation in C6 glioma cells.

Effects of chronic caloric restriction on kidney and heart redox status and antioxidant enzyme activities in Wistar rats.

Caloric restriction (CR) has been associated with health benefits and these effects have been attributed, in part, to modulation of oxidative status by CR; however, data are still controversial. Here, we investigate the effects of seventeen weeks of chronic CR on parameters of oxidative damage/ modification of proteins and on antioxidant enzyme activities in cardiac and kidney tissues. Our results demonstrate that CR induced an increase in protein carbonylation in the heart without changing the content of sulfhydryl groups or the activities of superoxide dismutase and catalase (CAT). Moreover, CR caused an increase in CAT activity in kidney, without changing other parameters. Protein carbonylation has been associated with oxidative damage and functional impairment; however, we cannot exclude the possibility that, under our conditions, this alteration indicates a different functional meaning in the heart tissue. In addition, we reinforce the idea that CR can increase CAT activity in the kidney.

Methylglyoxal alters glucose metabolism and increases AGEs content in C6 glioma cells.

Methylglyoxal is a dicarbonyl compound that is physiologically produced by enzymatic and non-enzymatic reactions. It can lead to cytotoxicity, which is mainly related to Advanced Glycation End Products (AGEs) formation. Methylglyoxal and AGEs are involved in the pathogenesis of Neurodegenerative Diseases (ND) and, in these situations, can cause the impairment of energetic metabolism. Astroglial cells play critical roles in brain metabolism and the appropriate functioning of astrocytes is essential for the survival and function of neurons. However, there are only a few studies evaluating the effect of methylglyoxal on astroglial cells. The aim of this study was to evaluate the effect of methylglyoxal exposure, over short (1 and 3 h) and long term (24 h) periods, on glucose, glycine and lactate metabolism in C6 glioma cells, as well as investigate the glyoxalase system and AGEs formation. Glucose uptake and glucose oxidation to CO(2) increased in 1 h and the conversion of glucose to lipids increased at 3 h. In addition, glycine oxidation to CO(2) and conversion of glycine to lipids increased at 1 h, whereas the incorporation of glycine in proteins decreased at 1 and 3 h. Methylglyoxal decreased glyoxalase I and II activities and increased AGEs content within 24 h. Lactate oxidation and lactate levels were not modified by methylglyoxal exposure. These data provide evidence that methylglyoxal may impair glucose metabolism and can affect glyoxalase activity. In periods of increased methylglyoxal exposure, such alterations could be exacerbated, leading to further increases in intracellular methylglyoxal and AGEs, and therefore triggering and/or worsening ND.

Moderate exercise training and chronic caloric restriction modulate redox status in rat hippocampus.

Physical activity has been related to antioxidant adaptations, which is associated with health benefits, including those to the nervous system. Additionally, available data suggest exercise and a caloric restriction regimen may reduce both the incidence and severity of neurological disorders. Therefore, our aim was to compare hippocampal redox status and glial parameters among sedentary, trained, caloric-restricted sedentary and caloric-restricted trained rats. Forty male adult rats were divided into 4 groups: ad libitum-fed sedentary (AS), ad libitum-fed exercise training (AE), calorie-restricted sedentary (RS) and calorie-restricted exercise training (RE). The caloric restriction (decrease of 30% in food intake) and exercise training (moderate in a treadmill) were carried out for 3 months. Thereafter hippocampus was surgically removed, and then redox and glial parameters were assessed. Increases in reduced glutathione (GSH) levels and total antioxidant reactivity (TAR) were observed in AE, RS and RE. The nitrite/nitrate levels decreased only in RE. We found a decrease in carbonyl content in AE, RS and RE, while no modifications were detected in thiobarbituric acid reactive substances (TBARS). Total reactive antioxidant potential (TRAP), superoxide dismutase (SOD) activity, S100B and glial fibrilary acid protein (GFAP) content did not change, but caloric restriction was able to increase glutamine synthetase (GS) activity in RS and glutamate uptake in RS and RE. Exercise training, caloric restriction and both combined can decrease oxidative damage in the hippocampus, possibly involving modulation of astroglial function, and could be used as a strategy for the prevention of neurodegenerative diseases.

Induction of S100B secretion in C6 astroglial cells by the major metabolites accumulating in glutaric acidemia type I.

Glutaryl-CoA dehydrogenase deficiency or glutaric acidemia type I (GA I) is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of predominantly glutaric (GA) and 3-hydroxyglutaric (3OHGA) acids and clinically by severe neurological symptoms and structural brain abnormalities, manifested as progressive cerebral atrophy and acute striatum degeneration following encephalopathic crises, whose pathophysiology is still in debate. Considering that reactive astrogliosis is a common finding in brain of GA I patients, in the present study we investigated the effects of GA and 3OHGA on glial activity determined by S100B release by rat C6-glioma cells. We also evaluated the effects of these organic acids on some parameters of oxidative stress in these astroglial cells. We observed that GA and 3OHGA significantly increased S100B secretion and thiobarbituric acid-reactive substances (lipid peroxidation), whereas GA markedly decreased reduced glutathione levels in these glioma cells. This is the first report demonstrating that the major metabolites accumulating in GA I activate S100B secretion in astroglial cells, indicating activation of these cells. We also showed that GA and 3OHGA induced oxidative stress in C6 lineage cells, confirming previous findings observed in brain fresh tissue. It is therefore presumed that reactive glial cells and oxidative damage may underlie at least in part the neuropathology of GA I.

The janus face of resveratrol in astroglial cells.

Astroglial cells are key modulators of neuropathology events. Resveratrol, a redox-active compound present in grapes and wine, has a wide range of biological effects. The aim of this study was to investigate whether resveratrol is able to prevent hydrogen peroxide (H(2)O(2))-induced oxidative damage in C6 astroglial cells. We found that following a short oxidative insult (Model I-1 mM H(2)O(2)/30 min), resveratrol increased glutamate uptake (60%), glutamine synthetase (GS) (139%), glutathione (GSH) (120%), and S100B secretion (24%); and attenuated DCFH oxidation (34%) as compared to H(2)O(2) values. Under less intense (0.1 mM H(2)O(2)), but lasting (6 h) insult (Model II), resveratrol had an opposite effect, potentiating the H(2)O(2)-induced decrease in glutamate uptake (from 34 to 63%), in GS (from 22 to 50%), in GSH (from 22 to 54%), and also potentiating DCFH oxidation (from 24 to 38%). The transcription factor, NF-kappaB, was activated in both models. Cell morphology alterations were also observed in the presence of H(2)O(2) with process-bearing cells, accompanied by cell body retraction and actin reorganization. This effect was not prevented by resveratrol, but was prevented by lysophosphatidic acid (LPA), a specific upstream positive regulator of Rho A. In summary, these findings showed that resveratrol, a redox-active compound, was able to modulate important neurotrophic function of astroglial cells under different oxidative conditions.

Immunoassay for glial fibrillary acidic protein: antigen recognition is affected by its phosphorylation state.

Glial fibrillary acid protein (GFAP) is used commonly as a marker of astrogliosis and astrocyte activation in several situations involving brain injury. Its content may be measured by immunocytochemistry, immunoblotting or enzyme-linked immunosorbent assay (ELISA), usually employing commercial antibodies. Two major post-translational modifications in GFAP (phosphorylation and proteolysis) may alter the interpretation of results or for immunoassay standardization. This study using a non-sandwich ELISA aimed to investigate the putative changes in the immunorecognition due to the phosphorylated state of the antigen by a routinely used polyclonal anti-GFAP antibody from DAKO. Results involving in vitro phosphorylation of purified GFAP or biological samples (brain tissue, cell culture and cerebrospinal fluid) mediated by protein kinase dependent on cAMP indicate that GFAP phosphorylation improves the recognition by the used antibody. These results provide support to the understanding of fast changes in the GFAP-immunoreactivity and suggest that caution is necessary in the interpretation of results using this antibody, as well as indicate that the effect of post-translational modifications must be considered during the standardization of immunoassays with other antibodies.