Microglia-glioblastoma interactions: New role for Wnt signaling.

Glioblastoma, the most aggressive and fatal type of brain tumor, is capable of interacting with brain immune cells such as microglia, which contributes to the growth of these tumors. Various molecules, including growth factors and cytokines, have been identified as regulators of microglia-glioblastoma interaction. Recent studies suggest that the Wnt family of lipoglycoproteins plays an important role, not only in biological events during development, but also in cancer progression, and can be part of microglia recruitment to glioblastoma as well as of tumor growth and invasion. Here, we discuss recent interesting findings that support a role for Wnt signaling pathways in the microglia-glioblastoma crosstalk.

Metabolomics as a promising tool for early osteoarthritis diagnosis

Osteoarthritis (OA) is the main cause of disability worldwide, due to progressive articular cartilage loss and degeneration. According to recent research, OA is more than just a degenerative disease due to some metabolic components associated to its pathogenesis. However, no biomarker has been identified to detect this disease at early stages or to track its development. Metabolomics is an emerging field and has the potential to detect many metabolites in a single spectrum using high resolution nuclear magnetic resonance (NMR) techniques or mass spectrometry (MS). NMR is a reproducible and reliable non-destructive analytical method. On the other hand, MS has a lower detection limit and is more destructive, but it is more sensitive. NMR and MS are useful for biological fluids, such as urine, blood plasma, serum, or synovial fluid, and have been used for metabolic profiling in dogs, mice, sheep, and humans. Thus, many metabolites have been listed as possibly associated to OA pathogenesis. The goal of this review is to provide an overview of the studies in animal models and humans, regarding the use of metabolomics as a tool for early osteoarthritis diagnosis. The concept of osteoarthritis as a metabolic disease and the importance of detecting a biomarker for its early diagnosis are highlighted. Then, some studies in plasma and synovial tissues are shown, and finally the application of metabolomics in the evaluation of synovial fluid is described.

Malnutrition increases NO production and induces changes in inflammatory and oxidative status in the distal colon of lactating rats.

BACKGROUND: Epidemiological studies have indicated the lack of breast feeding as a risk factor associated with later development of inflammatory bowel disease. Nevertheless, the repercussion of little feeding during suckling on large intestine inflammatory response and anti-oxidant resources has not yet been completely understood. This study hypothesized that unfavorable lactation is able to induce oxidative stress and release of inflammatory mediators modifying the integrity of the colon epithelium in weanling rats. METHODS: Wistar rats were reared under different early nutritional conditions according to litter size in two groups: N6 (6 pups/dam) and N15 (15 pups/dam) until the 25th postnatal day. The distal colon was removed and processed for biochemical, morphometric, and immunohistochemical analyzes. Lipoperoxidation, nitric oxide (NO), reduced (GSH) and oxidized (GSSG) glutathione, tumor necrosis factor-alpha (TNF-α), interleukins-1ß, 4 and 10 (IL-1ß; IL-4; IL-10) levels, and total superoxide dismutase (tSOD), and catalase (CAT) activities were assessed. Morphometric analysis was carried out using paraffin sections and wholemount myenteric plexus preparations. KEY RESULTS: Increased lipoperoxidation, NO, TNF-α and IL-1b levels, reduced tSOD and increased CAT activities were found in the N15 compared to N6 group. No intergroup difference was detected for IL-10, while lower levels of IL-4, GSH and GSSG and lower neuronal size and density were induced by undernutrition. CONCLUSIONS & INFERENCES: Reduced feeding during suckling changed the inflammatory response and oxidative status in the colon of weanling rats. These data suggest potential mechanisms by which malnutrition early in life may increase the vulnerability of the large intestine to insults.

Glioblastoma cells inhibit astrocytic p53-expression favoring cancer malignancy.

The tumor microenvironment has a dynamic and usually cancer-promoting function during all tumorigenic steps. Glioblastoma (GBM) is a fatal tumor of the central nervous system, in which a substantial number of non-tumoral infiltrated cells can be found. Astrocytes neighboring these tumor cells have a particular reactive phenotype and can enhance GBM malignancy by inducing aberrant cell proliferation and invasion. The tumor suppressor p53 has a potential non-cell autonomous function by modulating the expression of secreted proteins that influence neighbor cells. In this work, we investigated the role of p53 on the crosstalk between GBM cells and astrocytes. We show that extracellular matrix (ECM) from p53(+/-) astrocytes is richer in laminin and fibronectin, compared with ECM from p53(+/+) astrocytes. In addition, ECM from p53(+/-) astrocytes increases the survival and the expression of mesenchymal markers in GBM cells, which suggests haploinsufficient phenotype of the p53(+/-) microenvironment. Importantly, conditioned medium from GBM cells blocks the expression of p53 in p53(+/+) astrocytes, even when DNA was damaged. These results suggest that GBM cells create a dysfunctional microenvironment based on the impairment of p53 expression that in turns exacerbates tumor endurance.

Retinoblastoma protein regulates the crosstalk between autophagy and apoptosis, and favors glioblastoma resistance to etoposide.

Glioblastomas (GBMs) are devastating tumors of the central nervous system, with a poor prognosis of 1-year survival. This results from a high resistance of GBM tumor cells to current therapeutic options, including etoposide (VP-16). Understanding resistance mechanisms may thus open new therapeutic avenues. VP-16 is a topoisomerase inhibitor that causes replication fork stalling and, ultimately, the formation of DNA double-strand breaks and apoptotic cell death. Autophagy has been identified as a VP-16 treatment resistance mechanism in tumor cells. Retinoblastoma protein (RB) is a classical tumor suppressor owing to its role in G1/S cell cycle checkpoint, but recent data have shown RB participation in many other cellular functions, including, counterintuitively, negative regulation of apoptosis. As GBMs usually display an amplification of the EGFR signaling involving the RB protein pathway, we questioned whether RB might be involved in mechanisms of resistance of GBM cells to VP-16. We observed that RB silencing increased VP-16-induced DNA double-strand breaks and p53 activation. Moreover, RB knockdown increased VP-16-induced apoptosis in GBM cell lines and cancer stem cells, the latter being now recognized essential to resistance to treatments and recurrence. We also showed that VP-16 treatment induced autophagy, and that RB silencing impaired this process by inhibiting the fusion of autophagosomes with lysosomes. Taken together, our data suggest that RB silencing causes a blockage on the VP-16-induced autophagic flux, which is followed by apoptosis in GBM cell lines and in cancer stem cells. Therefore, we show here, for the first time, that RB represents a molecular link between autophagy and apoptosis, and a resistance marker in GBM, a discovery with potential importance for anticancer treatment.

Neuroproteomics: an insight into ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown aetiology. Diagnosis is made through physical examination, electrophysiological findings, and by excluding other conditions. There is not a single biomarker that concludes the diagnosis. The aim of this study was to investigate differentially expressed proteins in cerebrospinal fluid (CSF) of ALS patients compared to control subjects, with the purpose to identify a panel of possible biomarkers for the disease. The differentially expressed spots/proteins were submitted to two-dimensional (2D) electrophoresis and recognized with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Parkin-like and many iron and zinc binding were some of the proteins found in ALS CSF. Parkin is a ligase involved in ubiquitin-proteasome pathway and mutations in the parkin gene are the most common cause of recessive familial Parkinson's disease. Iron and zinc are involved with many important metabolic processes and are related to neurodegenerative disease. Common features of ALS comprise failure of the ubiquitin-proteasome system and increased levels of metal ions in the brain. Therefore, the identification of these proteins can be a significant step in ALS research. These and other identified proteins are discussed in this study.

Microglial stress inducible protein 1 promotes proliferation and migration in human glioblastoma cells.

Microglial activation is a key event in the progression and infiltration of tumors. We have previously demonstrated that the co-chaperone stress inducible protein 1 (STI1), a cellular prion protein (PrP(C)) ligand, promotes glioblastoma (GBM) proliferation. In the present study, we examined the influence of microglial STI1 in the growth and invasion of the human glioblastoma cell line GBM95. We demonstrated that soluble factors secreted by microglia into the culture medium (microglia conditioned medium; MG CM) caused a two-fold increase in the proliferation of GBM95 cells. This effect was reversed when STI1 was removed from the MG CM. In this context, we have shown that microglial cells synthesize and secrete STI1. Interestingly, no difference was observed in proliferation rates when GBM cells were maintained in MG CM or MG CM containing an anti-PrP(C) neutralizing antibody. Moreover, rec STI1 and rec STI1(Δ230-245), which lack the PrP(C) binding site, both promoted similar levels of GBM95 proliferation. In the migration assays, MG CM favored the migration of GBM95 cells, but migration failed when STI1 was removed from the MG CM. We detected metalloproteinase 9 (MMP-9) activity in the MG CM, and when cultured microglia were treated with an anti-STI1 antibody, MMP-9 activity decreased. Our results suggest that STI1 is secreted by microglia and favors tumor growth and invasion through the participation of MMP-9 in a PrP(C)-independent manner.

Cell cytoskeleton and tether extraction.

We perform a detailed investigation of the force × deformation curve in tether extraction from 3T3 cells by optical tweezers. Contrary to conventional wisdom about tethers extracted from cells, we find that actin filaments are present within them, so that a revised theory of tether pulling from cells is called for. We also measure steady and maximum tether force values significantly higher than previously published ones for 3T3 cells. Possible explanations for these differences are investigated. Further experimental support of the theory of force barriers for membrane tube extension is obtained. The potential of studies on tether pulling force × deformation for retrieving information on membrane-cytoskeleton interaction is emphasized.

Different expression of synemin isoforms in glia and neurons during nervous system development.

The synemin gene encodes proteins belonging to the intermediate filament family. These proteins confer resistance to mechanical stress and modulate cell shape. Three synemin isoforms, of 180 (H), 150 (M) and 41 (L) kDa, are produced by alternative splicing of the pre-mRNA and are regulated differently during development. The three isoforms differ in their C-terminal tail domains, while their IF rod domains are identical. Synemins H/M occurred together with nestin and vimentin in glial progenitors during the early differentiation of the developing mouse central nervous system. They are later found in GFAP-labeled cells. In contrast, the L isoform appeared only in neurons, together with neurofilaments and betaIII-tubulin in the brain after birth. However, synemin L appeared from E13 in the peripheral nervous system, where it was confined to the neurons of spinal ganglia. In the meantime, the synemin H/M isoforms were found in both the neurons and Schwann cells of the sensorial ganglia from E11. Tissue fractionation and purification of IFs from adult mouse spinal cord revealed that the synemin L isoform binds to neurofilaments associated with the membrane compartment. This report describes the synthesis of the three synemin isoforms by selective cell types, and their temporal and spatial distributions. Mechanisms specific to neurons and glia probably control the splicing of the common synemin mRNA and the synthesis of each synemin isoform.

Sulfated proteoglycans as modulators of neuronal migration and axonal decussation in the developing midbrain

Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions

Sulfated proteoglycans as modulators of neuronal migration and axonal decussation in the developing midbrain.

Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions.

Thyroid hormone induces cerebellar astrocytes and C6 glioma cells to secrete mitogenic growth factors.

In this study, the effect of thyroid hormone (triiodothyronine, T(3)) on the secretion of mitogenic growth factors in astrocytes and C6 glioma cells was examined. The proliferating activity of T(3) could be due, at least in part, to the astrocyte secretion of acidic and basic fibroblast growth factor (aFGF and bFGF), tumor necrosis factor-beta, and transforming growth factor-beta. In contrast, the conditioned medium (CM) of T(3)-treated C6 cells was mitogenic to this cell line only after hyaluronidase digestion, suggesting the impairment of growth factor mitogenic activity by hyaluronic acid. Furthermore, the presence of bFGF was significantly greater in the CM of both T(3)-treated astrocytes and T(3)-treated C6 cells than in the corresponding control CM. These data show that T(3) induces cerebellar astrocytes to secrete mitogenic growth factors, predominantly bFGF, that could influence astrocyte and neuronal proliferation via autocrine and paracrine pathways.

Cross-talk between neurons and glia: highlights on soluble factors

The development of the nervous system is guided by a balanced action between intrinsic factors represented by the genetic program and epigenetic factors characterized by cell-cell interactions which neural cells might perform throughout nervous system morphogenesis. Highly relevant among them are neuron-glia interactions. Several soluble factors secreted by either glial or neuronal cells have been implicated in the mutual influence these cells exert on each other. In this review, we will focus our attention on recent advances in the understanding of the role of glial and neuronal trophic factors in nervous system development. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership

Cross-talk between neurons and glia: highlights on soluble factors.

The development of the nervous system is guided by a balanced action between intrinsic factors represented by the genetic program and epigenetic factors characterized by cell-cell interactions which neural cells might perform throughout nervous system morphogenesis. Highly relevant among them are neuron-glia interactions. Several soluble factors secreted by either glial or neuronal cells have been implicated in the mutual influence these cells exert on each other. In this review, we will focus our attention on recent advances in the understanding of the role of glial and neuronal trophic factors in nervous system development. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership.

Astroglial cells derived from lateral and medial midbrain sectors differ in their synthesis and secretion of sulfated glycosaminoglycans.

Astroglial cells derived from lateral and medial midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in cocultures. These different properties of the two types of cells may be related to the composition of their extracellular matrix. We have studied the synthesis and secretion of sulfated glycosaminoglycans (GAGs) by the two cell types under control conditions and beta-D-xyloside-stimulated conditions, that stimulate the ability to synthesize and release GAGs. We have confirmed that both cell types synthesize and secrete heparan sulfate and chondroitin sulfate. Only slight differences were observed between the proportions of the two GAGs produced by the two types of cells after a 24-h labeling period. However, a marked difference was observed between the GAGs produced by the astroglial cells derived from lateral and medial midbrain sectors. The medial cells, which contain derivatives of the tectal and tegmental midline radial glia, synthesized and secreted approximately 2.3 times more chondroitin sulfate than lateral cells. The synthesis of heparan sulfate was only slightly modified by the addition of beta-D-xyloside. Overall, these results indicate that astroglial cells derived from the two midbrain sectors have marked differences in their capacity to synthesize chondroitin sulfate. Under in vivo conditions or a long period of in vitro culture, they may produce extracellular matrix at concentrations which may differentially affect neuritic growth.

Involvement of histone H4 gene transcription factor 1 in downregulation of vimentin gene expression during skeletal muscle differentiation.

Upon in vitro myogenesis, the intermediate filament protein vimentin is replaced by desmin, the switch in gene expression occurring essentially at the transcriptional level. Trying to elucidate the molecular mechanisms of this genetic control, we show here that the vimentin promoter is specifically recognized and activated by a protein most probably identical to H4TF-1, and that this factor is present in proliferating myoblasts but disappears upon fusion of these cells into multinucleated myotubes. Our results suggest that H4TF-1 is a differentiation stage-specific factor involved in the downregulation of vimentin gene expression during myogenesis.

Astroglial cells derived from lateral and medial midbrain sectors differ in their synthesis and secretion of sulfated glycosaminoglycans

Astroglial cells derived from lateral and medial midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in cocultures. These different properties of the two types of cells may be related to the composition of their extracellular matrix. We have studied the synthesis and secretion of sulfated glycosaminoglycans (GAGs) by the two cell types under control conditions and ß-D-xyloside-stimulated conditions, that stimulate the ability to synthesize and release GAGs. We have confirmed that both cell types synthesize and secrete heparan sulfate and chondroitin sulfate. Only slight differences were observed between the proportions of the two GAGs produced by the two types of cells after a 24-h labeling period. However, a marked difference was observed between the GAGs produced by the astroglial cells derived from lateral and medial midbrain sectors. The medial cells, which contain derivatives of the tectal and tegmental midline radial glia, synthesized and secreted ~2.3 times more chondroitin sulfate than lateral cells. The synthesis of heparan sulfate was only slightly modified by the addition of ß-D-xyloside. Overall, these results indicate that astroglial cells derived from the two midbrain sectors have marked differences in their capacity to synthesize chondroitin sulfate. Under in vivo conditions or a long period of in vitro culture, they may produce extracellular matrix at concentrations which may differentially affect neuritic growth

The cytoskeleton of the electric tissue of Electrophorus electricus, L.

The electric eel Electrophorus electricus is a fresh water teleost showing an electrogenic tissue that produces electric discharges. This electrogenic tissue is distributed in three well-defined electric organs which may be found symmetrically along both sides of the eel. These electric organs develop from muscle and exhibit several biochemical properties and morphological features of the muscle sarcolema. This review examines the contribution of the cytoskeletal meshwork to the maintenance of the polarized organization of the electrocyte, the cell that contains all electric properties of each electric organ. The cytoskeletal filaments display an important role in the establishment and maintenance of the highly specialized membrane model system of the electrocyte. As a muscular tissue, these electric organs expresses actin and desmin. The studies that characterized these cytoskeletal proteins and their implications on the electrophysiology of the electric tissues are revisited.