Gérard Labourdette

Rat oligodendrocytes express the vitamin D3 receptor and respond to 1,25-dihydroxyvitamin D3

The present study investigates the presence of vitamin D receptor (VDR) in cells of the rat oligodendrocyte (OL) lineage. VDR transcripts were detected by in situ hybridization in a fraction of rat OL in secondary cultures. The VDR protein was shown to be co‐localized in cells that are also recognized by an anti‐myelin basic protein (MBP) antibody. Likewise, in vivo, VDR‐positive cells were found in the brain white matter, such as the internal capsule of the striatum or the corpus callosum but also in the spinal cord. At least part of these positive cells in vivo correspond to OL, since they were co‐stained by an anti‐carbonic anhydrase II antiserum. Northern blot analyses of the CG‐4 OL cell line demonstrated that the VDR transcripts are already found in the O‐2A precursors. There was a two‐fold increase in the relative abundance of these transcripts in differentiated OL or in type‐2 astrocytes. 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3] increased the pool of transcripts encoding its own receptor, the VDR. The hormone also enhanced the abundance of the mRNA of the nerve growth factor (NGF) and of its low‐affinity receptor, the p75NTR protein. By contrast, the hormone had no effect on the levels of MBP or proteolipid protein (PLP) mRNA. This finding suggests that unlike retinoic acid (RA) or thyroid hormone, 1,25‐(OH)2D3 has no regulatory action on the synthesis of myelin proteins. GLIA 31:59–68, 2000.

Basic fibroblast growth factor (bFGF) injection activates the glial reaction in the injured adult rat brain

Reactive gliosis is a reaction of glial cells to trauma which is characterized by a phenotypic modification of astrocytes, as well as by a proliferation and a migration of some of these cells to form a glial scar. This scar is currently considered as a physical impediment to neuronal regrowth but it may also be involved in wound healing since the astrocytes beside microglia play a phagocytic role in the clearance of post-traumatic debris. Growth factors are released in the area of the injury and at least some of them could be involved in gliosis. In order to test directly this possibility, we have injected one of them, the basic fibroblast growth factor (bFGF), into several brain areas (cortex, striatum, hippocampus or corpus callosum) of adult 2-month-old rats in the absence of lesion. A glial reaction was observed after 3 days and was maximum after 7 days. It was characterized by an increase in astrocyte proliferation and in glial fibrillary acidic protein (GFAP) expression, resulting in a higher number of GFAP-positive cells per surface unit, and by an increase in the size and branching of the astroglial processes. The GFAP mRNA levels were also strongly increased following the bFGF injection. These effects resemble the reactive gliosis observed after lesion and suggest that bFGF is actually involved in the triggering of glial reactions which follow brain injury. In further experiments, bFGF was injected in the site of electrolytic lesions made in the same various parts of the brain. These injections did not increase significantly the normal reactive gliosis induced by the lesion alone, but it accelerated some of the effects. It also resulted in a higher labeling index and GFAP mRNA levels were strongly enhanced after a 3-day-post-operative delay. This last observation strengthens the idea that one of the main factors driving the astrogliosis is the bFGF normally released in and around the site of the lesion.

Transplantation of oligodendrocyte precursors in the adult demyelinated spinal cord: Migration and remyelination

A demyelinating lesion induced by an injection of lysolecithin into the spinal cord can be partly repaired by oligodendrocyte precursors transplanted at a distance of 6–8 mm from the lesion. Using a non‐toxic fluorescent dye (Hoechst 33342) as a cell marker, we demonstrate that transplanted oligodendrocyte precursors from different origins (periventricular zone fragments from newborn mouse and cultured rat oligodendrocyte progenitor cells) can migrate along specific pathways (i.e. white matter fasciculi, ependymal wall, meninges and blood vessels). These cells can be attracted when passing at the vicinity of the lesion as well as differentiate and remyelinate axons with the lesion. Myelin repair thus appears to be the result of distinct successive events: migration, specific attraction, differentiation and myelination. This can occur in both shiverer and normal adult hosts.

Characterization of oligodendrocytes in primary cultures from brain hemispheres of newborn rats

Characterization of putative oligodendrocytes obtained in primary cultures of brain hemispheres from newborn rats is reported. Most of the oligodendrocytes are scattered in the culture dish until around 20 days after seeding, the time at which they start to form aggregates made up of one to three layers of cells upon the astrocytes. At the electron microscopic level the oligodendrocytes ultrastructure appears undifferentiated but very different from that of the underlying astrocytes. These oligodendrocytes do not react to W1 Wolfgram protein and myelin basic proteins antisera until the sixth day after seeding. On Day 8, a few oligodendrocytes give a positive reaction; after 4 weeks most of them react. These results represent a further step in the identification of oligodendrocytes in culture and in the characterization of their development in vitro.

Isolation of a glial maturation factor from beef brain

Soluble brain extracts from chick embryos and from newborn rats stimulate the morphological maturation of chick neuronal and glial cells in mixed cultures [ 1,2]. Chick brain extracts induce morphological changes in pure astroglial cells in culture derived from brains of newborn rats, with an increase of the level of the glial-specific protein SlOO [3]. In these rat glial structures, brain extracts prepared from postnatal rats and adult beef elicit the same morphological and biochemical stimulatory effects [4] as well as enhance glial cell proliferation. Similar changes induced by brain extracts from rats and pigs in different astroglial cultures have been reported [5-71. It is not known whether the different effects, i.e., morphological changes, enhanced proliferation and biochemical maturation are induced by a single active factor or by several different factors. Therefore, we purified a crude fraction from beef brain extract and could demonstrate that the isolated factor induces the three events simultaneously.

Influence of meningeal cells on the proliferation and maturation of rat neuroblasts in culture

SummaryNeuronal cells were obtained by dissociating cells from the cerebral hemispheres of rat embryos (10 to 17-day-old), either cleaned entirely or only partially of their meningeal membranes. These cells were seeded on poly-lysine-coated Petri dishes in serum-containing medium. The cultures most enriched in neuronal cells were obtained from brains of 13- to 15-day-old embryos and after 2 h, the culture medium was switched to Dulbeccos modified Eagles medium, without serum, supplemented with the N1 supplements as described by Bottenstein et al. (1980). The proliferation of neuroblasts from 13-day-old embryos in the presence or absence of meningeal cells was studied by using a combination of tritiated thymidine autoradiography and immuno-staining against neurofilament proteins. The neuroblasts seem to proliferate during the first 3 days. The proliferative activity was further enhanced in the presence of meningeal cells. The glioblasts multiply only after a period of one week in culture conditions as observed here. The subsequent development of the neuroblasts was followed over a period of 4 weeks and the ultrastructural appearance of these cells was investigated at 2 and 3 weeks. In the presence of meningeal cells, many neurons, intensely stained for neurofilament proteins, survived for 21 days, while in control cultures they underwent massive degeneration after 2 weeks. Synapses with numerous clear vesicles were abundant in cultures grown under the influence of meningeal cells; they were rare and possessed few vesicles in control cultures. The data indicate that meningeal cells affect the proliferation and maturation of rat neuroblasts in culture.

Effect of norepinephrine and dibutyryl cyclic AMP on S-100 protein level in C6 glioma cells

Abstract S-100 Protein level was determined in C6 glioma cells after treatments by norepinephrine. In growing cells norepinephrine induces an important increase of S-100 protein level continuing during the stationary phase to reach a level higher than in untreated quiescent cells. In quiescent, low density, thymidine blocked cells, S-100 protein level is also enhanced by norepinephrine. In high density, contact inhibited cells, S-100 protein level is not modified although cAMP level is much more stimulated by norepinephrine than is low density cells. Exogenous addition of dibutyryl cyclic AMP mimics the effects of norepinephrine. Our results suggest that cyclic AMP level can mudulate S-100 protein level in C6 cells but that in density inhibited cells, a subsequent step involved in the regulation is no more operative.

Neurons modulate oxytocin receptor expression in rat cultured astrocytes: Involvement of TGF‐β and membrane components

We examined the effect of neurons on oxytocin (OT) receptors (OTR) and OTR gene expression in cultured astrocytes. The addition of neuron‐conditioned medium induced an increase of both OTR binding and OTR mRNA level. This effect was enhanced after the medium was boiled or acidified. As it is known that transforming growth factor‐β (TGF‐β) can be released from carrier proteins by acid or heat, TGF‐β1 and 2 were tested and found to induce an increase of OTR binding. Furthermore, TGF‐β antibody abolished the stimulatory effect of normal or acidified neuron‐conditioned medium. Neurons added to cultured astrocytes without contact mimicked the stimulatory effect of the conditioned medium. In contrast, neurons added with contact, induced a decrease in OTR binding and an increase of mRNA level, whereas neuronal membranes induced a decrease of both OTR binding and mRNA levels. In conclusion, the present data demonstrate that in vitro, neurons are able to modulate astrocytic OTR expression at the level of both protein and mRNA. They stimulate this expression through their release of TGF‐β and inhibit it by the action of unknown membrane components. GLIA 37:169–177, 2002.

ATAD 3A and ATAD 3B are distal 1p-located genes differentially expressed in human glioma cell lines and present in vitro anti-oncogenic and chemoresistant properties.

Human oligodendrogliomas are chemosensitive gliomas usually characterized by a loss of heterozygosity in the large distal regions of the short arm of chromosome 1 (1p LOH). Chemoresistant astrocytomas do not have this genetic signature, suggesting that the 1p arms may contain anti-oncogene and/or genes enabling chemoresistance. We have focused here on two human 1p-distal genes, ATAD 3A and ATAD 3B (1p36-33), and analyzed their gene products in normal human cell lines and tissues and in glioma-derived human cell lines. Using specific anti-peptide antibodies, we have found that ATAD 3A is ubiquitously expressed, whereas ATAD 3B is expressed in embryonic tissues, adult germinative zone and in astrocytoma cell lines but it is not expressed in oligodendroglioma cell lines or in the adult cortex. Furthermore, we have found that human glioma cell lines overexpressing or underexpressing ATAD 3A and ATAD 3B, show modified cell growth, anchorage-independent growth, and chemoresistance to doxorubicin and other genotoxic drugs. These results demonstrate the potential for ATAD 3B as a putative marker in discriminating astrocytomas from oligodendrogliomas. We also have shown that the loss of ATAD 3A/3B may be involved in the transformation pathway and the chemosensitivity of oligodendrogliomas.

S-100 protein in monolayer cultures of glial cells: Basal level in primary and secondary cultures

Abstract Monolayer cultures of astrocytes from newborn rat brain hemispheres have been analysed for the glial-specific protein S-100, during their growth cycle. In primary cultures S-100 protein level increases with a pattern close to that observed with rat brain hemispheres in vivo . This finding suggests that some biochemical maturation of the astrocytes occurs in vitro . In secondary cultures the level of S-100 protein decreases and then increases at the end of the proliferation phase. This modulation, similar to that observed in a clonal culture of tumor cells from rat brain (C6) provides a model to study the relationship between gene expression and the phase of growth of the cells and will allow parallel investigations in normal and tumor cells.