Astroglial S100B Secretion Is Mediated by Ca2+ Mobilization from Endoplasmic Reticulum: A Study Using Forskolin and DMSO as Secretagogues.

S100B, a homodimeric Ca2+-binding protein, is produced and secreted by astrocytes, and its extracellular levels have been used as a glial marker in brain damage and neurodegenerative and psychiatric diseases; however, its mechanism of secretion is elusive. We used primary astrocyte cultures and calcium measurements from real-time fluorescence microscopy to investigate the role of intracellular calcium in S100B secretion. In addition, the dimethyl sulfoxide (DMSO) effect on S100B was investigated in vitro and in vivo using Wistar rats. We found that DMSO, a widely used vehicle in biological assays, is a powerful S100B secretagogue, which caused a biphasic response of Ca2+ mobilization. Our data show that astroglial S100B secretion is triggered by the increase in intracellular Ca2+ and indicate that this increase is due to Ca2+ mobilization from the endoplasmic reticulum. Also, blocking plasma membrane Ca2+ channels involved in the Ca2+ replenishment of internal stores decreased S100B secretion. The DMSO-induced S100B secretion was confirmed in vivo and in ex vivo hippocampal slices. Our data support a nonclassic vesicular export of S100B modulated by Ca2+, and the results might contribute to understanding the mechanism underlying the astroglial release of S100B.

Hyperammonemia compromises glutamate metabolism and reduces BDNF in the rat hippocampus.

Ammonia is putatively the major toxin associated with hepatic encephalopathy (HE), a neuropsychiatric manifestation that results in cognitive impairment, poor concentration and psychomotor alterations. The hippocampus, a brain region involved in cognitive impairment and depressive behavior, has been studied less than neocortical regions. Herein, we investigated hippocampal astrocyte parameters in a hyperammonemic model without hepatic lesion and in acute hippocampal slices exposed to ammonia. We also measured hippocampal BDNF, a neurotrophin commonly related to synaptic plasticity and cognitive deficit, and peripheral S100B protein, used as a marker for brain damage. Hyperammonemia directly impaired astrocyte function, inducing a decrease in glutamate uptake and in the activity of glutamine synthetase, in turn altering the glutamine-glutamate cycle, glutamatergic neurotransmission and ammonia detoxification itself. Hippocampal BDNF was reduced in hyperammonemic rats via a mechanism that may involve astrocyte production, since the same effect was observed in astrocyte cultures exposed to ammonia. Ammonia induced a significant increase in S100B secretion in cultured astrocytes; however, no significant changes were observed in the serum or in cerebrospinal fluid. Data demonstrating hippocampal vulnerability to ammonia toxicity, particularly due to reduced glutamate uptake activity and BDNF content, contribute to our understanding of the neuropsychiatric alterations in HE.

Methylglyoxal can mediate behavioral and neurochemical alterations in rat brain.

Diabetes is associated with loss of cognitive function and increased risk for Alzheimer's disease (AD). Advanced glycation end products (AGEs) are elevated in diabetes and AD and have been suggested to act as mediators of the cognitive decline observed in these pathologies. Methylglyoxal (MG) is an extremely reactive carbonyl compound that propagates glycation reactions and is, therefore, able to generate AGEs. Herein, we evaluated persistent behavioral and biochemical parameters to explore the hypothesis that elevated exogenous MG concentrations, induced by intracerebroventricular (ICV) infusion, lead to cognitive decline in Wistar rats. A high and sustained administration of MG (3µmol/µL; subdivided into 6days) was found to decrease the recognition index of rats, as evaluated by the object-recognition test. However, MG was unable to impair learning-memory processes, as shown by the habituation in the open field (OF) and Y-maze tasks. Moreover, a single high dose of MG induced persistent alterations in anxiety-related behavior, diminishing the anxiety-like parameters evaluated in the OF test. Importantly, MG did not alter locomotion behavior in the different tasks performed. Our biochemical findings support the hypothesis that MG induces persistent alterations in the hippocampus, but not in the cortex, related to glyoxalase 1 activity, AGEs content and glutamate uptake. Glial fibrillary acidic protein and S100B content, as well as S100B secretion (astroglial-related parameters of brain injury), were not altered by ICV MG administration. Taken together, our data suggest that MG interferes directly in brain function and that the time and the levels of exogenous MG determine the different features that can be seen in diabetic patients.

Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death, Upregulation of p53 and Triggers Low Neurotoxicity.

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin's (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin's effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3ß and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.

Alkaloids from psychotria target sirtuins: in silico and in vitro interaction studies.

Epigenetic enzymes such as histone deacetylases play a crucial role in the development of ageing-related diseases. Among the 18 histone deacetylase isoforms found in humans, class III histone deacetylases, also known as sirtuins, seem to be promising targets for treating neurodegenerative conditions. Recently, Psychotria alkaloids, mainly monoterpene indoles, have been reported for their inhibitory properties against central nervous system cholinesterase and monoamine oxidase proteins. Given the multifunctional profile of these alkaloids in the central nervous system, and the fact that the indole scaffold has been previously associated with sirtuin inhibition, we hypothesized that these indole derivatives could also interact with sirtuins. In the present study, alkaloids previously isolated from Psychotria spp. were evaluated for their potential interaction with human sirtuin 1 and sirtuin 2 by molecular docking and molecular dynamics simulation approaches. The in silico results allowed for the selection of five potentially active compounds, namely, prunifoleine, 14-oxoprunifoleine, E-vallesiachotamine, Z-vallesiachotamine, and vallesiachotamine lactone. The sirtuin inhibition of these compounds was confirmed in vitro in a dose-response manner, with preliminary information on their pharmacokinetics properties.

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.

Huperzine A, but not tacrine, stimulates S100B secretion in astrocyte cultures.

AIMS: The loss of cholinergic function in the central nervous system contributes significantly to the cognitive decline associated with advanced age and dementias. Huperzine A (HupA) is a selective inhibitor of acetylcholinesterase (AChE) and has been shown to significantly reduce cognitive impairment in animal models of dementia. Based on the importance of astrocytes in physiological and pathological brain activities, we investigated the effect of HupA and tacrine on S100B secretion in primary astrocyte cultures. S100B is an astrocyte-derived protein that has been proposed to be a marker of brain injury. MAIN METHODS: Primary astrocyte cultures were exposed to HupA, tacrine, cholinergic agonists, and S100B secretion was measured by enzyme-linked immunosorbent assay (ELISA) at 1 and 24h. KEY FINDINGS: HupA, but not tacrine, at 100µM significantly increased S100B secretion in astrocyte cultures. Nicotine (at 100 and 1000µM) was able to stimulate S100B secretion in astrocyte cultures. SIGNIFICANCE: Our data reinforce the idea that AChE inhibitors, particularly HupA, do not act exclusively on the acetylcholine balance. This effect of HupA could contribute to improve the cognitive deficit observed in patients, which are attributed to cholinergic dysfunction. In addition, for the first time, to our knowledge, these data indicate that S100B secretion can be modulated by nicotinic receptors, in addition to glutamate, dopamine and serotonin receptors.

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.

Dual action of chronic ethanol treatment on LPS-induced response in C6 glioma cells.

In this study we investigated the anti-inflammatory effects of chronic ethanol (EtOH) treatment on lipopolysaccharide (LPS)-stimulated C6 glioma cells. The cells were chronically treated with 200mM EtOH; coincubation with LPS and EtOH was obtained upon addition of 2µg/ml LPS to the incubation medium in the last 24h of EtOH exposure. We found that EtOH prevented the LPS-induced production of tumor necrosis factor α (TNFα) without decreasing cell viability. Either LPS treated or EtOH plus LPS treated cells presented upregulated glial fibrillary acidic protein (GFAP) and downregulated vimentin levels characterizing a program of reactive astrogliosis. Also, EtOH plus LPS stimulation greatly increased the oxidative stress generation evaluated by DCF-DA measurement, while either EtOH alone or LPS alone was unable to induce oxidative stress. Western blot analysis indicated that either EtOH, LPS or EtOH plus LPS treatments are unable to affect Akt/GSK3ß signaling pathway. However, LPS alone and EtOH plus LPS co-treatment inhibited Erk phosphorylation. A dramatic loss of stress fibers was found in EtOH exposed cells, evaluated by cytochemistry using phalloidin-fluorescein. However, LPS alone was not able to disrupt actin organization. Furthermore, cells co-incubated with LPS and EtOH presented reversion of the disrupted stress fibers provoked by EtOH. Supporting this action, RhoA and vinculin immunocontent were upregulated in response to EtOH plus LPS. Interestingly, EtOH suppresses the inflammatory cascade (TNFα production) in response to LPS. Concomitantly it sustains Erk inhibition, increases oxidative stress generation and induces reactive astrogliosis in the presence of LPS, conditions associated with neurotoxicity. The effects observed were not supported by actin reorganization. Altogether, these findings suggest that Erk signaling inhibition could play a role in both suppressing TNFα production and inducing oxidative stress generation and astrogliosis, therefore modulating a dual action of EtOH plus LPS in glial cells.

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-1ß 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.

Lipopolysaccharide modulates astrocytic S100B secretion: a study in cerebrospinal fluid and astrocyte cultures from rats.

BACKGROUND: Inflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calcium-binding protein secreted by astrocytes, has properties of a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B. METHODS: In this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide. RESULTS: Our data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 µg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein. CONCLUSIONS: Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

Adipocytes as an Important Source of Serum S100B and Possible Roles of This Protein in Adipose Tissue.

Adipocytes contain high levels of S100B and in vitro assays indicate a modulated secretion of this protein by hormones that regulate lipolysis, such as glucagon, adrenaline, and insulin. A connection between lipolysis and S100B release has been proposed but definitive evidence is lacking. Although the biological significance of extracellular S100B from adipose tissue is still unclear, it is likely that this tissue might be an important source of serum S100B in situations related, or not, to brain damage. Current knowledge does not preclude the use of this protein in serum as a marker of brain injury or astroglial activation, but caution is recommended when discussing the significance of changes in serum levels where S100B may function as an adipokine, a neurotrophic cytokine, or an alarmin.

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.

A simple, sensitive and widely applicable ELISA for S100B: Methodological features of the measurement of this glial protein.

S100B expression, particularly extracellular S100B, is used as a parameter of glial activation and/or death in several situations of brain injury. Several immunoassays for S100B measurement are available, which differ with regard to specificity, sensitivity, sample application, and, of course, economic costs. We standardized two protocols for S100B measurement (range between 1.9pg and 10ng/mL) in human and rat samples from brain and adipose tissues, blood serum, cerebrospinal fluid, urine and cell culture. Abundance and secretion of this protein in adipose tissue reinforces the caution about its origin in blood serum. Interestingly, S100B recognition was affected by the redox status of the protein. This aspect should be considered in S100B measurement, assuming that oxidized and reduced forms possibly coexist in vivo and the equilibrium can be modified by oxidative stress of physiological or pathological conditions or even by obtaining sample conditions.

Estimativa dos lípides totais no soro baseada nas determinações de colesterol e/ou triglicérides / Total serum lipids estimated by total cholesterol and/or triglycerides

A concentração dos lípides séricos está diretamente relacionada ao níveis de colesterol, triglicérides efotolípides na amostra. Através de análise de regressão, foram obtidas equações que possibilitam estimar os lípides totais através das concentrações séricas de colesterol e/ou triglicérides e desta forma substituir a detrminação química pela sulfofosfovanilina que apresenta elevada variabilidade analítica e utiliza reagentes corrosivos. Com amostras de soro de 112 indivíduos (51,2% do sexo feminino), duas equações foram obtidas para estimar os lípides totais séricos: lípides totais (mg/dl)= 1,145 x triglicérides + 452 (r2 = 0,866) e lípides totais (mg/dl) = 1,0 x (colesterol total + triglicérides) + 287 (r2 = o,876). Quando os valores de lípides totais quantiticados pela sulfofosfovanilina foram comparados aos estimados por aquelas equações, utilizando como elementos previstos os triglicérides ou a soma destes com o colesterol total não foram observadas diferenças significativas pelo teste "t" (p> 0,99) em ambos os casos. O erro na previsão dos lípides totais presentes nas equações apresentadas é menor que a imprecisão na determinação da química pela sulfofosfovanilina. A utilização de um elemento previsto (triglicérides) ou dois (colesterol + triglicérides) mostrou resuiltados semelhantes no presente estudo.