Daniela da Silva Goncalves

Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology.

Primary astrocyte cultures prepared from neonatal hippocampus, cerebral cortex and cerebellum were morphologically distinct. Cells from hippocampus and cortex were almost entirely protoplasmic, whereas cerebellar astrocytes had many processes; in the absence of serum these differences were accentuated. We compared the immunocontent and secretion of the mitogenic protein S100B in these cultures. Immunocontent was 2.5 times higher in cerebellar astrocytes than in hippocampal or cortical astrocytes. Cells from all three regions secreted S100B under basal conditions, but the secretion rate was higher in cerebellar astrocytes. Secretion depended on protein synthesis and was increased by incubation with forskolin or lysophosphatidic acid in mechanisms which were additive. The stellate morphology induced by forskolin was reversed by lysophosphatidic acid in hippocampal but not in cerebellar cultures, suggesting that S100B secretion was not associated with a process‐bearing phenotype of astrocytes.

Extracellular S100B protein modulates ERK in astrocyte cultures.

S100B is a calcium binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates the proliferation of astroglial cells and the survival of neurons. Extracellular signal regulated kinases (ERK) are involved in the transduction of proliferating signals in astrocytes. Here we report that S100B significantly increases the activity of ERK in primary cultures of astrocytes, a result which may be related to previous observations of the effect of this protein on glial proliferation. We further confirm that conversion of S100B to its covalent dimer by oxidation of cysteine residues increases its extracellular activity. Although we cannot exclude S100B involvement in other mechanisms of signal transduction, these results suggest that ERK activity in astrocytes is modulated by extracellular S100B.

Glutamate uptake in cultured astrocytes depends on age: a study about the effect of guanosine and the sensitivity to oxidative stress induced by H2O2

Relatively few studies have been conducted to investigate the relationship between glutamate and development and/or aging. Rat cortical astrocyte cultures were used as a model to investigate glutamate uptake during development. The immunocontent of the markers glial fibrillary acidic protein (GFAP) and S100B increased, while basal secretion of S100B decreased, in astrocytes from 10 to 40 days in vitro (DIV). Basal glutamate uptake increased with age. Exposure to hydrogen peroxide decreased glutamate uptake more potently at 40 than 10 DIV. Moreover, 40 DIV astrocytes showed earlier loss of integrity (at 6 h) than 10 DIV astrocytes (at 24 h) after H(2)O(2) exposure. Addition of guanosine stimulated glutamate uptake only in 10 DIV astrocytes. The present work shows that mature astrocytes in culture present some neurochemical alterations also observed in astrocytes of aged animals. These results can contribute to the understanding of some consequences of the excitotoxicity and oxidative stress during brain aging.

Developmental Changes in S100B Content in Brain Tissue, Cerebrospinal Fluid, and Astrocyte Cultures of Rats

Abstract1. We investigated the content of S100B protein by ELISA in three brain regions (hippocampus, cerebral cortex, and cerebellum) and in cerebrospinal fluid of rats during postnatal development as well as the content and secretion of S100B in pre- and postconfluent primary astrocyte cultures.2. An accumulation of S100B occurred in all brain regions with similar ontogenetic pattern between second and fourth postnatal weeks. However, we observed a decrease in the cerebrospinal fluid S100B after the critical period for synaptogenesis in rodents.3. A similar profile of cell accumulation and decrease in basal secretion was also observed during aging of astrocyte cultures.4. These data contribute to the proposal that S100B is an important glial-derived protein during brain development and that changes in extracellular levels of S100B may be related to glial proliferation and synaptogenesis.

Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA.

The present protocol details a procedure to permeabilize astrocytes in cultures with digitonin as well as to discuss some data about factors that interfere in permeabilization, particularly divalent cations and nucleotides. Two methods to assess astrocyte permeabilization are described: trypan blue exclusion and ELISA for S100B, a specific protein expressed by these cells. Digitonin-permeabilization of astrocytes has been used to investigate intracellular pools of Ca(2+), internal stores of metabolites, phosphoinositide hydrolysis, and recently we standardized a procedure to study protein phosphorylation (Brain Res. 853 (2000) 32-40). A short incubation time (10 min) with 30 microM digitonin permeabilized at least 75% of cells. A range of media with different ionic nature can be used in cell permeabilization without affecting significantly the extent of permeabilization, but calcium and ATP of the order of 10(-5) M induced a partial resealing which deserves to be considered in assays of permeabilized preparations of astrocytes.

High glutamate decreases S100B secretion stimulated by serum deprivation in astrocytes.

S100B is a calcium-binding protein expressed and secreted by astrocytes, playing a neurotrophic role in neighboring cells. A protective role of the S100B against glutamate-induced excitotoxicity has recently been proposed. We investigated S100B secretion in rat hippocampal astrocytes exposed to high concentrations of glutamate during serum deprivation (stimulated condition) or not (basal condition), for 30 min. Glutamate at 1 mM had no effect on basal secretion of S100B, but it decreased S100B secretion in serum-deprived astrocytes after 1 h. Secretion was inhibited by Rp-cAMPS or H89. In addition, serum deprivation was accompanied by a transitory increase of intracellular content of cAMP. Our results suggest that high levels of glutamate in a serum-deprived condition could impair S100B secretion from hippocampal astrocytes.

Extracellular ATP stimulates an inhibitory pathway towards growth factor-induced cRaf-1 and MEKK activation in astrocyte cultures

ATP, acting via P2Y, G protein‐coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor‐2 (FGF‐2)‐induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF‐2 cotreatment on the main components of the extracellular‐signal regulated protein kinase (ERK) cascade, cRaf‐1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf‐1 by FGF‐2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf‐1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf‐1 by other growth factors, epidermal growth factor and platelet‐derived growth factor, as well as other MEK1 activators stimulated by FGF‐2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF‐2‐induced cRaf‐1 activation, thereby indicating specificity in the ATP‐induced inhibitory cross‐talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor‐induced activation of cRaf‐1 and MEKKs. Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y‐GPCRs.

Ketogenic diet fed rats have low levels of S100B in cerebrospinal fluid

Ketogenic diets have been used to treat seizure disorders of children resistant to conventional anti-epileptic drug treatment. The mechanism of action of this diet, however, is unknown. Gliosis is a very common characteristic in tissues associated with epileptogenesis and glial cytokines may be involved in the pathology of seizure disorders. We investigate herein, whether ketogenic diet fed rats demonstrate changes in the immunocontent of S100B, an astrocyte-derived cytokine elevated in the temporal lobe of refractory epilepsy. Lower levels of S100B were observed in cerebrospinal fluid with no significant changes in S100B and GFAP content in brain tissue. Ketogenic fed rats presented a lower seizure severity induced by pentylenetetrazole and no change in cerebrospinal fluid S100B after pentylenetetrazole administration. These results support the concept that the ketogenic diet is neuroprotective in seizure disorders. Since S100B has an extracellular activity in neuronal excitability and synaptic plasticity, it would be reasonable to conceive that a decrease in the S100B could be involved in the mechanism of action of the ketogenic diet. However, it is not possible to establish a direct link between reduced CSF S100B and decreased severity of PTZ-induced attacks at present moment. Regardless of this, CSF S100B could be proposed as an index of efficacy of ketogenic diet for seizure disorders.

Protein Kinase C-Mediated in vitro Invasion of Human Glioma Cells through Extracellular-Signal-Regulated Kinase and Ornithine Decarboxylase

We investigated the involvement of protein kinase C (PKC) in the in vitro invasiveness of the A-172, U-87 and U-373 human glioma cell lines, as well as the role of ornithine decarboxylase (ODC) and/or extracellular-signal-regulated kinase (ERK) in the actions of PKC. Thus, cells were treated under serum-free conditions with the PKC activator phorbol 12-myristate 13-acetate (PMA), or with the PKC inhibitors bisindolylmaleimide I (GF 109203X) or calphostin C in the absence or presence of the ODC inhibitor D,L-α-difluoromethylornithine (DFMO), and/or the mitogen-activated protein kinase/extracellular-signal-regulated kinase inhibitor 2′-amino-3′-methoxyflavone (PD 098059). Subsequently, cells were assessed for membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA contents, 72-kD latent, and 59/62-kD activated matrix metalloproteinase 2 (MMP-2) in conditioned media, as well as invasiveness. For these purposes, we used Northern blot analysis, gelatine zymography, and an in vitro filter invasion assay, respectively. Data were related to those found with untreated cells. PKC activity was 2- to 3-fold stimulated by PMA (100 nM for 30 min), and about 2-fold inhibited by calphostin C (40 nM for 2 h) or GF 109203X (5 µM for 20 min). This was accompanied by a similar increase or decrease, respectively, in MT1-MMP mRNA expression, 59/62-kD MMP-2 activity, and in vitro invasion. Inhibition of ODC activity (about 2-fold by 24 h DFMO 5 mM), ERK activation (almost completely by 20 min PD 098059 50 µM), or both these enzymes simultaneously led to a reduction by about half in levels of MT1-MMP mRNA, 59/62-kD MMP-2 activity, and invasion in untreated as well as PMA-stimulated cells. The use of these compounds did not significantly alter the inhibitory effects of GF 109203X or calphostin C. Modulation of PKC and/or ERK activity resulted in corresponding changes in ERK and/or ODC activities, but interference with ODC affected neither ERK nor PKC. Our data suggest a regulatory role for PKC, in co-operation with ERK and ODC, in glioma cell invasion, by modulation of MT1-MMP mRNA expression and MMP-2 activation.