Valeria Sogos

Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor α (TNFα)-inducible NO synthesis, because by affecting NF-κB activation it inhibits inducible Ca-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNFα-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNFα induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNFα-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNFα-elicited NF-κB activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-κB availability.

Differentiation of human bone marrow stem cells into cells with a neural phenotype: diverse effects of two specific treatments

BackgroundIt has recently been demonstrated that the fate of adult cells is not restricted to their tissues of origin. In particular, it has been shown that bone marrow stem cells can give rise to cells of different tissues, including neural cells, hepatocytes and myocytes, expanding their differentiation potential.ResultsIn order to identify factors able to lead differentiation of stem cells towards cells of neural lineage, we isolated stromal cells from human adult bone marrow (BMSC). Cells were treated with: (1) TPA, forskolin, IBMX, FGF-1 or (2) retinoic acid and 2-mercaptoethanol (BME). Treatment (1) induced differentiation into neuron-like cells within 24 hours, while a longer treatment was required when using retinoic acid and BME. Morphological modifications were more dramatic after treatment (1) compared with treatment (2). In BMSC both treatments induced the expression of neural markers such as NF, GFAP, TUJ-1 and neuron-specific enolase. Moreover, the transcription factor Hes1 increased after both treatments.ConclusionOur study may contribute towards the identification of mechanisms involved in the differentiation of stem cells towards cells of neural lineage.

S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10–5 M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT‐induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT‐induced changes. Tumor necrosis factor‐α mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT‐induced increase in TNF‐α expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11‐7082, an inhibitor of nuclear factor‐κB (NF‐κB) activation, and of PD98059, an inhibitor of MEK‐ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF‐κB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury.

The Gustin (CA6) Gene Polymorphism, rs2274333 (A/G), as a Mechanistic Link between PROP Tasting and Fungiform Taste Papilla Density and Maintenance

Taste sensitivity to PROP varies greatly among individuals and is associated with polymorphisms in the bitter receptor gene TAS2R38, and with differences in fungiform papilla density on the anterior tongue surface. Recently we showed that the PROP non-taster phenotype is strongly associated with the G variant of polymorphism rs2274333 (A/G) of the gene that controls the salivary trophic factor, gustin. The aims of this study were 1) to investigate the role of gustin gene polymorphism rs2274333 (A/G), in PROP sensitivity and fungiform papilla density and morphology, and 2) to investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity. Sixty-four subjects were genotyped for both genes by PCR techniques, their PROP sensitivity was assessed by scaling and threshold methods, and their fungiform papilla density, diameter and morphology were determined. In vitro experiments examined cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. Gustin and TAS2R38 genotypes were associated with PROP threshold (p=0.0001 and p=0.0042), but bitterness intensity was mostly determined by TAS2R38 genotypes (p<0.000001). Fungiform papillae densities were associated with both genotypes (p<0.014) (with a stronger effect for gustin; p=0.0006), but papilla morphology was a function of gustin alone (p<0.0012). Treatment of isolated cells with saliva from individuals with the AA form of gustin or direct application of the active iso-form of gustin protein increased cell proliferation and metabolic activity (p<0.0135). These novel findings suggest that the rs2274333 polymorphism of the gustin gene affects PROP sensitivity by acting on fungiform papilla development and maintenance, and could provide the first mechanistic explanation for why PROP super-tasters are more responsive to a broad range of oral stimuli.

EXPRESSION OF BASIC FIBROBLAST GROWTH FACTOR AND ITS RECEPTORS IN HUMAN FETAL MICROGLIA CELLS

The presence of basic fibroblast growth factor (bFGF) and FGF receptors was investigated in microglia cells derived from human fetal brain long‐term cultures. Production of bFGF was suggested through the capability of microglial extracts to stimulate plasminogen activator (PA) synthesis in endothelial cells. The identity of PA‐stimulating activity with bFGF was confirmed by its high affinity for heparin and its cross‐reactivity with polyclonal antibodies to human recombinant bFGF. These antibodies recognized a cell‐associated Mr 18,000 protein as well as trace amounts of the Mr 24,000 bFGF isoform in Western blot. All microglial cells showed bFGF immunoreactivity in the cytoplasm and, sometimes, in the nucleus.

PLATELET-ACTIVATING FACTOR PRODUCTION BY HUMAN FETAL MICROGLIA: EFFECT OF LIPOPOLYSACCHARIDES AND TUMOR NECROSIS FACTOR-ALPHA

Since platelet-activating factor (PAF) exerts neurotoxic effects on brain cells, we explored the possibility of PAF production by human fetal microglial cells in vitro. PAF content in pure cultures was assayed and characterized in basic conditions, and after stimulation with growth factors and cytokines. Results showed that microglia cells synthesized PAF when challenged with tumor necrosis factor-alpha and lipopolysaccharides, whereas other molecules, such as gamma-interferon or basic fibroblast growth factor, were ineffective. The induced PAF production was concentration- and time-dependent. These results are in line with the hypothesis that microglia can start a cascade of events leading to tissue damage, thus playing a central role in the pathogenesis of several central nervous system diseases.

Inhibition of inducible nitric oxide synthase mRNA expression by basic fibroblast growth factor in human microglial cells

The effect of basic fibroblast growth factor (bFGF) on inducible nitric oxide synthase (iNOS) mRNA expression in human cultured ramified microglial cells was investigated. Using RT-PCR and Southern blot analysis, we found that bFGF prevented the iNOS gene expression as induced by LPS/TNF alpha. Also, bFGF dose-dependently inhibited nitrite levels in treated cell supernatants. That the early presence of bFGF during LPS/TNF alpha induction was essential indicates that iNOS gene expression can be transcriptionally regulated.

Differentiation of human adult CD34+ stem cells into cells with a neural phenotype: Role of astrocytes

It has recently been reported that adult hematopoietic stem cells can differentiate into neural cells, opening new frontiers in therapy for neurodegenerative diseases. In this study, adult human hematopoietic stem cells (HSCs) were isolated via magnetic bead sorting, using a specific CD34 antibody and cultured with human astrocyte culture conditioned medium (ACM). In order to evaluate their differentiation into neurons and/or astrocytes, ACM-treated cultures were probed for the expression of several neural markers. We observed morphological modifications and, after 20 days of treatment, cell morphology displayed extending processes. Immunocytochemistry, Western blotting and RT-PCR showed the expression of neuronal markers such as neurofilaments, neuron specific enolase (NSE) and NeuN in ACM-treated HSCs cultured in poly-L-lysine-coated dishes. On the contrary, when the same ACM-treated cells were grown on a plastic substrate, they expressed high levels of glial fibrillary acidic protein (GFAP), with only weak expression of neuronal markers. Nestin, a neural progenitor cell marker, was present in treated cells, regardless of the substrate. These results demonstrate that astrocytes can generate a suitable microenvironment for inducing HSCs to differentiate into neural cells. Therefore, adult bone marrow may represent a readily accessible source of cells for treating neurodegenerative diseases.

Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: Modulation by FGF‐2

CD137 (ILA, 4‐1BB), a member of the tumor necrosis factor receptor family, and its ligand CD137‐L were assayed by RT‐PCR and immunocytochemistry in cultured human brain cells. Results demonstrated that both neurons and astrocytes expressed specific RNA for CD137 and its protein, which was found both on the plasma membrane and in the cytoplasm. Surprisingly, microglia, which also expressed CD137 mRNA, showed negative immunostaining. CD137‐L‐specific RNA was detected only in astrocytes and neurons. When brain cells were treated with fibroblast growth factor‐2 (FGF‐2), upregulation of CD137 but not of its ligand was observed in neurons and astrocytes. Protein localization was also affected. In microglia, an inhibition of RNA expression was induced by treatment, whereas CD137‐L remained negative. Our data are the first demonstration that human brain cells express a protein found thus far in activated immunocompetent cells and epithelia. Moreover, they suggest not only that CD137 and CD137‐L might play a role in interaction among human brain cells, but also that FGF‐2 might have an immunoregulatory function in brain, modulating interaction of the central nervous system with peripheral immunocompetent cells.

Intracellular cholesterol changes induced by translocator protein (18 kDa) TSPO/PBR ligands

One of the main functions of the translocator protein (18 kDa) or TSPO, previously known as peripheral-type benzodiazepine receptor, is the regulation of cholesterol import into mitochondria for steroid biosynthesis. In this paper we show that TSPO ligands induce changes in the distribution of intracellular cholesterol in astrocytes and fibroblasts. NBD-cholesterol, a fluorescent analog of cholesterol, was rapidly removed from membranes and accumulated into lipid droplets. This change was followed by a block of cholesterol esterification, but not by modification of intracellular cholesterol synthesis. NBD-cholesterol droplets were in part released in the medium, and increased cholesterol efflux was observed in [(3)H]cholesterol-prelabeled cells. TSPO ligands also induced a prominent shrinkage and depolarization of mitochondria and depletion of acidic vesicles with cytoplasmic acidification. Consistent with NBD-cholesterol changes, MTT assay showed enhanced accumulation of formazan into lipid droplets and inhibition of formazan exocytosis after treatment with TSPO ligands. The effects of specific TSPO ligands PK 11195 and Ro5-4864 were reproduced by diazepam, which binds with high affinity both TSPO and central benzodiazepine receptors, but not by clonazepam, which binds exclusively to GABA receptor, and other amphiphilic substances such as DIDS and propranolol. All these effects and the parallel immunocytochemical detection of TSPO in potentially steroidogenic cells (astrocytes) and non-steroidogenic cells (fibroblasts) suggest that TSPO is involved in the regulation and trafficking of intracellular cholesterol by means of mechanisms not necessarily related to steroid biosynthesis.