V. Moura Neto

Glial fibrillary acidic protein (GFAP): modulation by growth factors and its implication in astrocyte differentiation

Intermediate filament (IF) proteins constitute an extremely large multigene family of developmentally and tissue-regulated cytoskeleton proteins abundant in most vertebrate cell types. Astrocyte precursors of the CNS usually express vimentin as the major IF. Astrocyte maturation is followed by a switch between vimentin and glial fibrillary acidic protein (GFAP) expression, with the latter being recognized as an astrocyte maturation marker. Levels of GFAP are regulated under developmental and pathological conditions. Upregulation of GFAP expression is one of the main characteristics of the astrocytic reaction commonly observed after CNS lesion. In this way, studies on GFAP regulation have been shown to be useful to understand not only brain physiology but also neurological disease. Modulators of GFAP expression include several hormones such as thyroid hormone, glucocorticoids and several growth factors such as FGF, CNTF and TGF beta, among others. Studies of the GFAP gene have already identified several putative growth factor binding domains in its promoter region. Data obtained from transgenic and knockout mice have provided new insights into IF protein functions. This review highlights the most recent studies on the regulation of IF function by growth factors and hormones.

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.

Regulation of the trehalose-6-phosphate synthase complex in Saccharomyces

SummaryTrehalose-6-phosphate synthase is another example of an enzyme of carbohydrate metabolism, in Saccharomyces, which could be regulated by interconversion of forms. Deactivation was mediated both in vivo and in vitro by a cyclic AMP-dependent protein kinase. Reversibility of this process was obtained by a phosphatase treatment leading to an increase in activity. The phosphorylated, less active form of the enzyme proved to be more susceptible to activation by ATP.Mg. Mutants with well defined lesions in the cyclic AMP-dependent protein kinase system were used to corroborate our findings of a possible regulatory mechanism of trehalose-6-phosphate synthase activity by interconversion of forms.

Rearrangement of intermediate filament network of BHK-21 cells infected with vaccinia virus

SummaryAssociation between vaccinia virus (VV) structures and intermediate filaments in specific areas of the cytoplasm of infected cells (virus “factories”) suggests that VV infection interferes with the cellular architecture by modifying the intermediate filament network. To analyse this question, we examined the array of intermediate filaments of BHK-21 cells infected with VV by laser scanning confocal microscopy using an anti-vimentin mouse monoclonal antibody. We observed a marked reorganization of intermediate filaments around the nucleus of infected cells. Bidimensional analysis of32PO4-labeled intermediate filament proteins revealed that the acidic isoform of vimentin and two isoforms of desmin have increased phosphorylation levels in infected cells. Our results suggest that the reorganization of intermediate filaments observed during VV infection could be promoted by an increase in the phosphorylation level of the intermediate filament proteins, vimentin and desmin.

Neuroproteomics: an insight into ALS

Abstract 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.

Microheterogeneity of desmin in the electric organ and dorsal muscle of the electric eel Electrophorus electricus

Abstract Two-dimensional gel electrophoresis of purified desmin from the electric organ of Electrophorus electricus exhibited five isoforms as opposed to four from the dorsal muscle of the eel. Differences in isoforms may indicate different degrees of desmin phosphorylation in these tissues. The similarities detected in the two less acidic isodesmin from the electric organ and from the dorsal muscle of Electrophorus electricus suggest that the electric organ has conserved some characteristics seen in the dorsal muscle, favoring previous suggestions that considered the electric tissue as a differentiated form of striated muscle.

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.

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.

Desmin filaments in the electrocytes of the electric organ of the electric eel Electrophorus electricus

Abstract.Desmin protein is an abundant constituent of the intermediate filaments in the electrocytes of the electric organ of the electric eel Electrophorus electricus. Polyclonal antibodies were raised against purified desmin from the electric organ and used for immunolabeling of the protein in reconstituted filaments. In thick sections of the main electric organ that has been stained with fluorescein-labeled desmin-specific antibodies, light microscope revealed a diffuse meshwork of desmin filaments dispersed in the cytoplasm of electrocytes. In the region under the membrane, the immunostaining was slightly more intense than elsewhere. The meshwork of intermediate filaments composed of desmin was examined by electron microscopy of the main electric organ. Immuno-gold labeling demonstrated a widespread meshwork of desmin filaments in the cytoplasm and in close association with the plasma membrane. These observations suggest that intermediate filaments play a role in the maintenance of the morphology of electrocytes and, as an intracellular meshwork spanning the width of the cell, they may contribute to the organization of the intracellular compartments.