Biomedical subjects

Joan Serratosa

Publications and source records attributed to Joan Serratosa.

13 recordsLinked to original sources

PubMed2007

Upregulation of CCAAT/enhancer binding protein beta in activated astrocytes and microglia.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPbeta in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPbeta expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPbeta in untreated mixed glial cultures. C/EBPbeta is upregulated when glial activation is induced by lipopolysaccharide (LPS). The LPS-induced upregulation of glial C/EBPbeta is rapid (2 h at mRNA level, 4 h at protein level). It is elicited by low concentrations of LPS (almost maximal effect at 1 ng/mL) and it is reversed by the protein synthesis inhibitor cycloheximide. C/EBPbeta nuclear levels increase both in astrocytes and microglia after LPS treatment, and the response is more marked in microglia. The LPS-induced increase in microglial C/EBPbeta is prevented by coadministration of the MAP kinase inhibitors SB203580 (p38 inhibitor) + SP600125 (JNK inhibitor) or SB203580 + U0126 (ERK inhibitor). Systemic injection of LPS also increases brain nuclear levels of C/EBPbeta as shown by Western blot, and this increase is localized in microglial cells as shown by double immunofluorescence, in the first report to our knowledge of C/EBPbeta expression in activated glial cells in vivo. These findings support a role for C/EBPbeta in the activation of astrocytes and, particularly, microglia. Given the nature of the C/EBPbeta-regulated genes, we hypothesize that this factor participates in neurotoxic effects associated with glial activation. (c) 2006 Wiley-Liss, Inc.

Animals↗

PubMed2006

Expression of C/EBPalpha and C/EBPbeta in glial cells in vitro after inducing glial activation by different stimuli.

We studied the involvement of two members of the CCAAT/enhancer binding proteins (C/EBPs) family of transcription factors, C/EBPalpha and C/EBPbeta, in glial activation induced by different stimuli in rat cerebellar neuronal-glial cultures. Glial activation was induced by two types of neuronal death--excitotoxic and apoptotic. We had previously reported that these two induction mechanisms resulted in different patterns of glial activation (K. Pérez-Capote, J. Serratosa, C. Solà, Excitotoxic and apoptotic neuronal death induce different patterns of glial activation in vitro, J. Neurochem. 94 (2005) 226-237), only the former involving an inflammatory response. Glial activation was also induced by lipopolysaccharide (LPS) from E. coli, an inflammatory agent with a known direct effect on glial cells. Using immunocytochemical techniques, here we examined whether changes in C/EBPalpha and C/EBPbeta expression are selectively associated with a determinate pattern of glial activation. Excitotoxic neuronal death increased glial C/EBPbeta expression in the absence of alterations in C/EBPalpha expression, while no effect was observed following neuronal apoptosis. LPS treatment decreased C/EBPalpha and increased C/EBPbeta expression in glial cells. These results implicate C/EBPs in glial activation, although these two factors appear to have different roles. C/EBPalpha expression decreases only in response to LPS, while C/EBPbeta expression is increased by both LPS and excitotoxic neuronal death, although not by neuronal apoptosis. These results show that C/EBPbeta plays an active role in glial activation, but only when this involves an inflammatory reaction, suggesting a role for C/EBPbeta in neuroinflammation.

Animals↗

PubMed2005

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia.

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.

Adenosine↗

PubMed2005

Absence of the cell cycle inhibitor p21Cip1 reduces LPS-induced NO release and activation of the transcription factor NF-kappaB in mixed glial cultures.

We have studied possible differences in glial activation between cells from wild-type and p21Cip1-/- mice. We compared the effect of serum mitogenic stimulation on proliferation rate and on the total number of glial cells after 7 days of culture. No differences between wild-type and p21Cip1-/- glial cells were observed. We also compared the effect of lipopolysaccharide (LPS) from Escherichia coli, an agent widely used to induce glial activation. Nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) release, and nuclear factor kappa-B (NF-kappaB) activation were evaluated as indicators of glial activation. We observed an attenuation of NO release and NF-kappaB activation in p21Cip1-/- glial cells when compared with glial cells from wild-type mice. In contrast, TNF-alpha release was enhanced in p21Cip1-/- glia. These results suggest that the cell cycle inhibitor p21Cip1 plays a role in the inflammatory response induced by LPS.

Active Transport, Cell Nucleus↗

PubMed2005

Excitotoxic and apoptotic neuronal death induce different patterns of glial activation in vitro.

We have studied glial activation in rat cerebellar neuronal-glial cultures after inducing neuronal death using various stimuli. Cultures were exposed to 100 microm glutamate for 20 min, which induces excitotoxic neuronal death, or to potassium/serum deprivation, which induces apoptosis of granule neurons. We evaluated alterations in several parameters related to glial activation: nuclear factor-kappaB activation, nitric oxide and tumour necrosis factor-alpha production, which are associated with a pro-inflammatory response, glial proliferation and phagocytic activity. Although the two experimental models of neuronal damage resulted in the death of most neuronal cells within 24 h, differences were observed in the response of the various glial parameters evaluated. While nitric oxide production was not detected in any case, tumour necrosis factor-alpha production, nuclear factor-kappaB activation and glial proliferation were only induced in the presence of excitotoxic neuronal death. However, phagocytosis was induced in both cases, although earlier in the case of apoptotic neuronal death. These results show that glial cells respond to excitotoxic neuronal death with an inflammatory response associated with proliferation and phagocytosis. In contrast, whilst glial cells do not produce pro-inflammatory molecules in the presence of apoptotic neuronal death, phagocytic activity is rapidly induced.

Animals↗

PubMed2004

Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures.

We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells.

Animals↗

PubMed2004

Effects of beta-AP peptides on activation of the transcription factor NF-kappaB and in cell proliferation in glial cell cultures.

The effect of two beta amyloid peptides (Abeta 25/35 and Abeta 1/42) on the activation of the transcription factor kappaB (NF-kappaB) in pure astroglial, pure microglial and mixed glial cell cultures was compared by means of single or double immunofluorescence and Western blot techniques. We also studied the effect of both peptides in cell proliferation in mixed glial cultures and pure astrocytes. The Abeta 1/42 peptide induced the activation of NF-kappaB in all studied cell cultures and its effect was potentiated by interferon-gamma (IFN-gamma). Abeta 25/35 alone did not induce NF-kappaB activation, but Abeta 25/35 plus IFN-gamma induced the activation of the transcription factor in the mixed and pure microglial cultures, although not in pure astroglia. The Abeta 1/42 peptide, but not Abeta 25/35, induced proliferation in pure astroglial and mixed glial cell cultures. The results suggest that the state of peptide aggregation is related to their ability to activate glial cells.

Amyloid beta-Peptides↗

PubMed2003

High-yield isolation of murine microglia by mild trypsinization.

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05-0.12%) in the presence of 0.2-0.5 mM EDTA and 0.5-0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as >98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon gamma, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-kappaB translocation, NO, and tumor necrosis alpha release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.

Animals↗

PubMed2002

Relationship between beta-AP peptide aggregation and microglial activation.

We compared the relationship between the state of aggregation of two peptides (beta-AP 25-35 and beta-AP 1-42) and microglial activation. After 7 days at 37 degrees C beta-AP 25-35 was in an amorphous state and did not activate microglial cells. In the same conditions, aggregated beta-AP 1-42 activated these cells and caused changes in microglial ramification, increasing the proliferation index and inducing tumor necrosis factor alpha (TNF alpha) release. Neither peptide induced a release of nitric oxide (NO). As the toxicity of beta-AP peptides in cell culture is associated with the formation of amyloid fibrils, we also examined the toxicity of both peptides in microglial cell cultures and in PC 12 cell cultures. The results suggest that the two beta-AP fragments studied have similar neurotoxic effects but different pro-inflammatory activities.

Alzheimer Disease↗

PubMed2002

Structural, kinetic and cytotoxicity aspects of 12-28 beta-amyloid protein fragment: a reappraisal.

A chemical, structural and biological study on the beta-amyloid peptide beta12-28 is reported which was carried out in order to assess the feasibility using this peptide fragment as a model of the natural beta-amyloid protein. The aggregation properties of beta12-28 have been investigated by pulse field-gradient NMR spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. The results obtained suggest that beta12-28 behaviour is comparable to that of the natural beta-amyloid protein although kinetically slower. Translational diffusion coefficients obtained by NMR on an aged beta12-28 solution suggest that the soluble peptide fraction is composed of oligomeric intermediates adopting an extended ellipsoidal assembly rather than a spherical one. The beta12-28 peptide proved to be cytotoxic in PC12 cell cultures as monitored by the MTT assay, although a lack of reproducibility was observed in the dose-response experiments.

Amino Acid Sequence↗

PubMed2002

Astrocytes enhance lipopolysaccharide-induced nitric oxide production by microglial cells.

Several stimuli result in glial activation and induce nitric oxide (NO) production in microglial and astroglial cells. The bacterial endotoxin lipopolysaccharide (LPS) has been widely used to achieve glial activation in vitro, and several studies show that both microglial and, to a lesser extent, astroglial cell cultures produce NO after LPS treatment. However, NO production in endotoxin-treated astrocyte cultures is controversial. We characterized NO production in microglial, astroglial and mixed glial cell cultures treated with lipopolysaccharide, measured as nitrite accumulation in the culture media. We also identified the NO-producing cells by immunocytochemistry, using specific markers for the inducible NO synthase (iNOS) isoform, microglial and astroglial cells. Only microglial cells showed iNOS immunoreactivity. Thus, contaminating microglial cells were responsible for NO production in the secondary astrocyte cultures. We then analysed the effect of astrocytes on NO production by microglial cells using microglial-astroglial cocultures, and we observed that this production was clearly enhanced in the presence of astroglial cells. Soluble factors released by astrocytes did not appear to be directly responsible for such an effect, whereas nonsoluble factors present in the cell membrane of LPS-treated astrocytes could account, at least in part, for this enhancement.

Animals↗

PubMed2002

The p21(Cip1) protein, a cyclin inhibitor, regulates the levels and the intracellular localization of CDC25A in mice regenerating livers.

Liver cells from p21(Cip1-/-) mice subjected to partial hepatectomy (PH) progress into DNA synthesis faster than those from wild-type mice. These cells also show a premature induction of cyclin E/cyclin-dependent kinase (CDK) 2 activity. We studied the mechanisms whereby cells lacking p21(Cip1) showed a premature induction of this activity. Whereas the levels of CDK2, cyclin E, and p27(Kip1) were similar in both wild-type and p21(Cip1-/-) mice, those of the activator CDC25A were much higher in p21(Cip1-/-) quiescent and regenerating livers than in wild-type animals. Moreover, p21(Cip1-/-) cells also showed a premature translocation of CDC25A from cytoplasm into the nucleus. The ectopic expression of p21(Cip1) into mice embryo fibroblasts from p21(Cip1-/-) mice decreased the levels of CDC25A and delayed its nuclear translocation. The levels of CDC25A messenger RNA in p21(Cip1-/-) cells were higher than in wild-type cells, suggesting that this increase might be responsible, at least in part, for the high levels of CDC25A protein in these cells. Thus, the results reported here indicate that p21(Cip1) regulates the levels and the intracellular localization of CDC25A. We also found a good correlation between CDC25A nuclear translocation and cyclin E/CDK2 activation. In conclusion, premature translocation of CDC25A to the nucleus might be involved in the advanced induction of cyclin E/CDK2 activity and DNA replication in cells from animals lacking p21(Cip1).

Animals↗

PubMed2002

Concanavalin-A-induced liver injury is severely impaired in mice deficient in P-selectin.

P-selectin (CD62P) is an adhesion molecule that mediates the initial attachment of leukocytes to activated platelets and endothelial cells in damaged tissues. We evaluated the role of P-selectin in concanavalin A (Con A)-induced hepatitis, a model characterized by CD4(+) T cell activation and infiltration of the liver. Con A injection induced transient P-selectin expression on hepatic venules and platelets. Mice lacking P-selectin showed impaired lymphocyte adhesion to liver venules and sinusoids, a striking reduction in intrasinusoidal occlusion, and decreased lymphocyte infiltration of liver parenchyma. The reduction in transaminase levels and the almost complete abolition of necrotic injury demonstrated that liver damage was lower in P-selectin-deficient mice. In wild-type mice, pretreatment with the P-selectin-blocking monoclonal antibody attenuated the sinusoidal occlusion and reduced the rise in transaminases after Con A treatment. These results implicate P-selectin in the development of Con A-induced liver injury and reveal the protective effect of blocking P-selectin in this hepatitis.

Alanine Transaminase↗