Ontogeny of steroid metabolizing enzymes in rat oligodendrocytes.

Rat oligodendroglial cells were isolated from newborn and developing brains and used immediately after, for quantification of steroid metabolizing activities. Oligodendrocytes (Ol) and their progenitor cells were incubated with [(14)C] testosterone, [(14)C] progesterone, [(14)C] pregnenolone or [(14)C] dehydroepiandrosterone (DHEA). Oligodendrocytes and their progenitor cells expressed different steroid metabolizing enzymes. The main activities were 5 alpha reduction of testosterone and progesterone and 3 beta hydroxy steroid dehydrogenase-isomerase which transformed pregnenolone into progesterone and DHEA into Delta 4 androstenedione. 5 alpha reductase activity increased in male and female rats in parallel with testosterone or progesterone. Contrary to this, 3 beta hydroxysteroid dehydrogenase-isomerase activity was found to be high in the young rat and to decrease when testosterone and progesterone plasma concentration increased.

Adrenal gland SNAP-25 expression is altered in thyroid hormone receptor knock-out mice.

SNAP-25 is a protein in neurons and neuroendocrine cells, which is involved, together with syntaxin and VAMP, in neurotransmitter release and neurite outgrowth. Since the thyroid hormone receptors TR alpha and TR beta are essential for nervous system development, their possible role in regulating the expression of these vesicle trafficking proteins was examined by analysing SNAP-25 levels in TR alpha and TR beta knock-out mice. Immunoblotting and RT-PCR showed that SNAP-25 levels are increased in the adrenal gland, but not in cerebellum, in knock-out mice, while syntaxin-1 and VAMP-2 are unaffected in either tissue. Treatment of the pheochromocytoma-derived cell line PC12 with the thyroid hormone L-3,5,3'-triiodothyronine (T3) decreased SNAP-25 expression. Together, these data suggest that thyroid hormones exert a negative regulatory effect on SNAP-25 in adrenal medullary neuroendocrine cells.

Phospholipid composition in late infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Neuronal ceroid lipofuscinosis (NCL) is a relatively common group of inherited neurodegenerative disorders characterised by the accumulation of autofluorescent lipopigments (ceroid) similar to lipofuscin. Because of this property, studies have concentrated on fatty acid metabolism and lipid peroxidation. METHODS: In the present study, the fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and the molecular species compositions of diacylglycerophosphocholine (diacyl GPC), diacylglycerophosphoethanolamine (diacyl GPE) and alkenylacyl GPE (plasmalogens) were investigated in cultured skin fibroblasts from three patients with a confirmed diagnosis of the late infantile form of the disease (LINCL, CLN2) and three healthy age-matched controls. RESULTS: Relatively minor differences in the fatty acid compositions of PC and PE were observed between patients and controls. However, dimethyl acetals of plasmalogens were found to be 40% higher in the patients compared to in the controls. Control and LINCL fibroblasts displayed only slight differences in the molecular compositions of diacyl GPE and diacyl GPC. In contrast, compared with normal cells, LINCL fibroblasts had higher levels of alkenylacyl GPE species containing both 18 : 1 and polyunsaturated fatty acids, but lower levels of species with 16 : 0 or 18 : 0 in the sn-1 position. CONCLUSION: The molecular composition of PC and PE subclasses in skin fibroblasts of healthy subjects and patients suffering from LINCL is here described for the first time. While few differences are noticeable in the fatty acid composition of PC and PE and the molecular species distribution of diacylGPC and diacylGPE, the alkenylacyl GPE (or ethanolamine plasmalogens) were found to differ significantly between patients and healthy controls.

Rat oligodendrocytes express the vitamin D(3) receptor and respond to 1,25-dihydroxyvitamin D(3).

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 D(3) [1,25-(OH)(2)D(3)] 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 p75(NTR) 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)(2)D(3) has no regulatory action on the synthesis of myelin proteins.

Increased density of oligodendrocytes in childhood ataxia with diffuse central hypomyelination (CACH) syndrome: neuropathological and biochemical study of two cases.

We report neuropathological, biochemical and molecular studies on two patients with childhood ataxia with diffuse central nervous system hypomyelination (CACH) syndrome, a leukodystrophy recently defined according to clinical and radiological criteria. Both had severe cavitating orthochromatic leukodystrophy without atrophy, predominating in hemispheric white matter, whereas U-fibers, internal capsule, corpus callosum, anterior commissure and cerebellar white matter were relatively spared. The severity of white matter lesions contrasted with the rarity of myelin breakdown products and astroglial and microglial reactions. In the white matter, there was an increase in a homogeneous cell population with the morphological features of oligodendrocytes, in many instances presenting an abundant cytoplasm like myelination glia. These cells were negative for glial fibrillary acidic protein and antibodies PGM1 and MIB1. Some were positive for myelin basic protein, proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein, but the majority were positive for human 2'-3' cyclic nucleotide 3' phosphodiesterase and all were positive for carbonic anhydrase II, confirming that they are oligodendrocytes. Myelin protein and lipid content were reduced. The PLP gene, analyzed in one case, was not mutated or duplicated. The increased number of oligodendrocytes without mitotic activity suggests an intrinsic oligodendroglial defect or an abnormal interaction with axons or other glial cells. This neuropathological study supports the notion that CACH syndrome constitutes a specific entity.

Thyroid hormone receptor isoforms are sequentially expressed in oligodendrocyte lineage cells during rat cerebral development.

In the mammalian brain, thyroid hormones regulate myelination. Their actions are mediated by interactions with nuclear receptors that function as ligand-regulated transcription factors. Two genes, alpha and beta, encode different isoforms, of which only the beta and alpha1 isoforms are authentic nuclear triiodothyronine (T3)-receptors (NT3R). In agreement with the important role of T3 on myelination and oligodendrocyte generation, the presence of NT3Rs has been reported in oligodendrocytes and their precursors. We and others have shown that both progenitors and oligodendrocytes in vitro express the alpha1 and alpha2 isoforms, but the expression of the beta1 isoform is confined to differentiated oligodendrocytes, suggesting that they have different functions. To establish if this is the case during development in vivo, we have studied NT3R isoform expression in glial cells isolated by density gradient centrifugation from rat brains of various ages. We report the presence of the alpha1 NT3R and its variant alpha2, but not that of the beta1 isoform, in newborn rat glial progenitors. The pattern of expression of beta1, both at the level of mRNA and protein, parallels the increase in the number of oligodendrocytes. We found a significant change in the kinetic parameters of [125I]-T3 binding to NT3Rs in these cells during the first month of life, consisting of an increase in the binding capacity that peaks with myelination, and a significative decrease in Kd that coincides with the switch from the alpha to the beta1 isoform. Thus, the expression of NT3R isoforms in the rat oligodendrocyte lineage changes radically from the alpha to the beta1 isoform during the period when oligodendrocytes differentiate from progenitors.

Glutamine synthetase expression and activity are regulated by 3,5,3'-triodo-L-thyronine and hydrocortisone in rat oligodendrocyte cultures.

Glutamine synthetase plays a central role in the detoxification of brain ammonia. Previously, we demonstrated that in vitro glutamine synthetase is expressed by all macroglial cell types and is developmentally regulated in oligodendrocyte lineage. Furthermore, glutamine synthetase is increased in secondary cultures of oligodendrocytes following a 72 h treatment with 30 nM 3,5,3'-triodo-L-thyronine [Baas, D., Bourbeau, D., Sarliève, L. L., Ittel, M. E., Dussault, J. H. and Puymirat, J., Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone. Glia, 1997, 19, 324-332]. Hydrocortisone also increases glutamine synthetase activity after 72 h [Fressinaud, C., Weinrauder, H., Delaunoy, J. P., Tholey, G., Labourdette, G. and Sarliève, L. L., Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors. J. Cell. Physiol., 1991, 149, 459-468]; however, it is still unknown whether these increases in glutamine synthetase expression in oligodendrocytes after 3,5,3'-triodo-L-thyronine and hydrocortisone application are dose- and time-dependent. To further investigate this issue, we measured glutamine synthetase levels by Northern analysis, immunostaining and determination of glutamine synthetase activity after 3,5,3'-triodo-L-thyronine or hydrocortisone stimulation. We find that in rat oligodendrocyte secondary cultures, 3,5,3'-triodo-L-thyronine and hydrocortisone cause a dose- and time-dependent increase in glutamine synthetase mRNA, protein and activity. However, these hormones do not exert an additive or synergistic effect. Because purines, pyrimidines, and certain amino acids necessary for the synthesis of myelin components, are, in part, provided by the glutamine synthetase pathway, 3,5,3'-triodo-L-thyronine effect on myelination development and maturation could be mediated in part, through the glutamine synthetase gene regulation.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells express glutamine synthetase: developmental and cell type-specific regulation.

Glutamine synthetase (GS), the enzyme that catalyses glutamine synthesis from glutamate and ammonia, plays a central role in the detoxification of brain ammonia. In the central nervous system (CNS), GS also subserves additional important functions such as regulating glutamate, GABA and amino acid metabolism. Oligodendrocytes (OL) form the myelin sheath in the central nervous system (CNS) and are essential for efficient propagation of nerve impulses. In culture, OL arise from bipotential O-2A progenitor cells. These O-2A cells give rise to type-2 astrocytes in the presence of serum. GS is expressed in mature glial cells in vivo and in vitro, but it is unknown whether GS is present in glial progenitors. In addition, a comparison of the GS expression level among the various types of glial cells has never been done in vitro. The current study investigates in vitro GS expression levels in O-2A progenitors, astrocytes and OL. We demonstrate that the GS gene is expressed in O-2A progenitors and is expressed at different levels in each cultured glial cell type. GS also is stimulated during OL developmental maturation. Thus, the GS gene is expressed in O-2A cells and is regulated in a developmental and macroglial cell type-specific manner.

Posttranscriptional regulation of oligodendroglial thyroid hormone (T3) receptor beta 1 by T3.

3,5,3'-triiodo-L-thyronine interacts with the genome by binding and activating nuclear 3,5,3'-triiodo-L-thyronine receptors. To determine how in secondary oligodendrocyte cultures, exogenous 3,5,3'-triiodo-L-thyronine influences the expression of different 3,5,3'-triiodo-L-thyronine receptor isoforms, we studied the regulation of alpha 1, alpha 2 and beta 1 3,5,3'-triiodo-L-thyronine receptor mRNAs. In culture, we find that beta 1, 3,5,3'-triiodo-L-thyronine receptor mRNA, but not alpha 1 and alpha 2 3,5,3'-triiodo-L-thyronine receptor mRNAs, is up-regulated by 3,5,3'-triiodo-L-thyronine in a time and dose dependent manner. In addition, we present evidence indicating that beta 1 3,5,3'-triiodo-L-thyronine receptor expression is posttranscriptionally regulated by 3,5,3'-triiodo-L-thyronine. Previous studies from our laboratory and others have shown that in the rat oligodendrocyte lineage, 3,5,3'-triiodo-L-thyronine receptors alpha 1 and alpha 2 were expressed in both early progenitor cells and mature oligodendrocytes. In contrast, beta 1 3,5,3'-triiodo-L-thyronine receptor was found to be expressed only in mature oligodendrocytes. This suggests that thyroid hormone may influence oligodendrocyte differentiation and maturation via 3,5,3'-triiodo-L-thyronine receptor beta 1, which is expressed only in oligodendrocytes and not in progenitor cells. We therefore show that this effect is indirect and is mediated by 3,5,3'-triiodo-L-thyronine which acts posttranscriptionally on the 3,5,3'-triiodo-L-thyronine receptor beta 1 gene.

Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone.

The development of oligodendrocyte progenitor cells is regulated by epigenetic factors which control their proliferation and differentiation. When oligodendrocyte progenitor cells, purified on a Percoll centrifugation gradient from neonate rat brain, are cultured in serum-free medium in the presence of platelet-derived-growth factor (PDGF), they divide and their differentiation is delayed. Triiodothyronine (T3) treatment of progenitor cells blocks their proliferation and induces their differentiation into oligodendrocytes. T3 also induces morphological differentiation of oligodendrocytes as indicated by the marked increase in the length of oligodendrocyte processes. To determine whether the effects of T3 on progenitor cell proliferation and oligodendrocyte maturation are causally related, or instead, are independent, we examined the influence of T3 on secondary cultures of postmitotic oligodendrocytes. We show that T3 increases morphological and functional maturation of postmitotic oligodendrocytes as indicated by a well developed network of branched processes and by the expression of myelin/oligodendrocyte glycoprotein (MOG) and glutamine synthetase (GS). T3 increases glutamine synthetase activity and its message level after a lag period of 24-48 h, and these levels increase through a posttranscriptional event. In contrast, no effect of T3 was observed on myelin basic protein (MBP) gene expression as determined by Northern blot analysis. Our results indicate that thyroid hormones participate in the control of the progenitor cell proliferation and differentiation as well as in oligodendrocyte maturation and that these two T3-regulated events are independent.

Expression of alpha and beta thyroid receptors during oligodendrocyte differentiation.

To determine which thyroid receptor (TR) isoforms are expressed during oligodendrocyte differentiation, we studied the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2) and one beta (beta 1) TR isoforms in a bipotential oligodendrocyte-type 2-astrocyte (O-2A) progenitor cell line (CG-4) as well as in rat O-2A progenitor cells and oligodendrocytes. O-2A progenitor cells expressed only TR alpha-mRNAs, whereas oligodendrocytes and type 2-astrocytes also expressed TR beta 1-mRNAs. The differential expression of alpha 1 and beta 1 TRs suggests specific functions for both types of TRs during oligodendrocyte differentiation. We present evidence for a possible role of TR alpha 1 in the effect of thyroid hormones on the proliferation of CG-4 cells maintained as progenitor cells.

High gelsolin content of developing oligodendrocytes.

The actin-binding protein gelsolin that severs and caps the actin microfilaments under the control of the cytoplasmic free calcium and the membranous phosphatidylinositol 4,5-bisphosphate, is essentially restricted to the oligodendroglia in the central nervous system. Immunocytochemistry showed that gelsolin is an early marker of oligodendrocytes, both in vivo, in the rat cerebellum, and in vitro, in oligodendrocyte culture. We report the early appearance of gelsolin in A2B5-positive precursor oligodendrocyte cells and the specific expression of gelsolin in OL-1-, GC-, and MBP-positive oligodendrocytes in culture. The protein was distributed throughout the cell body and in the branched cell processes of cultured oligodendrocytes, but not in the MBP-positive membrane sheets. Gelsolin is thus cytosolic and not a myelin component. The quantitative study demonstrated that that the cerebellar gelsolin content changes significantly with age, with the maximal value at the age of 21 days, confirming that large amounts of gelsolin are transiently synthesized during development, especially from the first events of myelinogenesis. The results are consistent with gelsolin being involved, through its effects on the actin cytoskeleton, in the motile events occurring during the growth of the oligodendroglial processes towards the axons and the wrapping of the myelin sheaths around the axons.

Expression of thyroid hormone receptor isoforms in rat oligodendrocyte cultures. Effect of 3,5,3'-triiodo-L-thyronine.

3,5,3'-Triiodo-L-thyronine (T3) acts at the genomic level by interacting with nuclear T3 receptors (T3Rs). We have used double immunostaining to follow the expression of T3Rs and oligodendrocytes (OL) lineage markers in rat secondary cultures consisting of 85-90% OL. Using antibodies against different synthetic peptides of T3Rs (alpha common: alpha 1 + alpha 2 and beta 1) we find that alpha-T3R is expressed in both O-2A progenitors and in mature OL, while beta 1-T3R is expressed only in mature OL. In cultured OL, beta 1-T3R mRNA is upregulated the most by T3. OL exhibit more numerous and longer processes when treated by T3.

Transient expression of 3,5,3'-triiodothyronine nuclear receptors in rat oligodendrocytes: in vivo and in vitro immunocytochemical studies.

It is generally accepted that the action of thyroid hormones is mediated through specific nuclear receptors. Recent studies have demonstrated the homology of the thyroid receptor with the cellular product of the oncogen v-erbA. So far, two genes have been identified and classified as alpha and beta subtypes. In this study, the expression of nuclear triiodothyronine (T3) receptors (NT3Rs) was examined in secondary cultures containing 85-90% oligodendrocytes (OL) prepared from newborn rat brain primary cultures enriched in OL. These cultures, which are able to produce myelin membranes, were examined by double immunolabelling with a monoclonal antibody (2B3) raised against purified rat liver NT3Rs and with antibodies against two maturation markers of OL: an early marker, galactocerebroside (GC), and myelin basic protein (MBP), which is expressed later than GC. 2B3 recognized three nuclear proteins with the same molecular weights as beta 1, alpha 1, and alpha 2 subtypes with different capacities for binding T3. In 5-day-old OL secondary cultures (25 days, total time in culture), 2B3-NT3R immunoreactivity was located in 77% of morphologically immature OL (GC)+ cells, whereas only 44% of morphologically mature OL were immunoreactive. Only 35% of the MBP+ cells co-expressed NT3Rs. In the corpus callosum of developing rat brain, at all ages studied from 7-60 days postnatal, the total absence of NT3Rs in dark OL (morphologically mature), confirmed by ultrastructural immunocytochemistry, indicates an even more dramatic decrease during maturation. Furthermore, the percentage of medium OL (less mature) stained by 2B3 is reduced by approximately half in 60- compared to 20-day-old rat brain. It is of interest to note that the in vitro observation with maturation markers mirrors the in vivo decrease of NT3R expression during development. It is interesting that NT3Rs are absent in vivo before the critical period of active myelination. These data indicate the presence of a nuclear T3 binding protein in the nuclei of OL at the time of myelination both in vitro and in vivo. The transient expression of these NT3Rs during active myelination argues in favour of a direct effect of thyroid hormones on OL.

Molecular species of choline and ethanolamine glycerophospholipids in rat brain myelin during development.

The composition of the molecular species of various phospholipid subclasses was examined in myelin isolated from brain of 15-, 21- and 90-day-old rats. The molecular species of diacylglycerophosphocholine (PtdCho), diacylglycerophosphoethanolamine (PtdEtn) and plasmenyl-ethanolamine (PlsEtn) were quantified by high-performance liquid chromatography (HPLC) after phospholipase C treatment and dinitrobenzoyl derivatization. In rat brain myelin, each phospholipid subclass showed a specific pattern of molecular species that changed during development. PtdCho contained large amounts of saturated/monounsaturated and disaturated species and low amounts of saturated/polyunsaturated species. During brain development, the levels of saturated/monounsaturated molecular species increased whereas those of the disaturated and saturated/polyunsaturated species decreased. PtdEtn were characterized by their low levels of disaturated species and a high content of saturated/monounsaturated and saturated/polyunsaturated species, of which those containing fatty acids of the n-3 series decreased, whereas those containing fatty acids of the n-6 series did not change during brain development. The levels of saturated/monounsaturated species increased in PtdEtn. No disaturated molecular species could be detected in PlsEtn. This alkenylacyl subclass contained large amounts of saturated/polyunsaturated, saturated/monounsaturated and dimonounsaturated molecular species. During development, the levels of saturated/polyunsaturated molecular species decreased while those of the two others increased. The data indicated that myelin sheaths undergo phospholipid changes during brain development and maturation.

Interleukin-1 binding sites on astrocytes.

Interleukin-1 has been shown to have regulatory effects on glial cell functions. In this study, we examined the capacity of astroglial cells to specifically bind recombinant iodinated human interleukin-1 alpha. This was performed in mouse brain by both in situ and in vitro autoradiography, on areas of gliosis and on astrocytes and microglia primary and secondary cultures respectively. Specific binding was shown in the brain sections over areas of glial proliferation, and in addition, quantitative autoradiography was performed. Analysis of competition experiments by autoradiography led to EC50 values of 5 x 10(-11) M for human interleukin-1 alpha and approximately 10(-9) M for the interleukin-1 receptor antagonist. In cultures, iodinated human interleukin-1 alpha bound specifically to astrocytes but was unable to bind to microglial cells. Competition binding experiments in astrocyte cultures led to EC50 values of 8 x 10(-11) M and 1 x 10(-10) M for human interleukin-1 alpha and mouse interleukin-1 beta respectively, and an EC50 higher than 10(-9) M for the antagonist. The presence of interleukin-1 receptors on astroglial cells provides biochemical support for the various effects of interleukin-1 in the central nervous system, particularly those concerning the formation of scar tissue, possibly by astroglia proliferation after brain injury.

The alpha and beta thyroid receptors are expressed by cultured ependymal cells. Correlation with the effect of L-3,5,3'-triiodothyronine on glutamine synthetase mRNAs.

It is generally accepted that L-3,5,3'-triiodothyronine (L-T3) acts at the genomic level through an interaction with specific nuclear L-T3 receptors (NT3R). Using antibodies raised against different peptides of NT3R, we report here the immunocytochemical localization of the alpha, alpha 2, beta 1 NT3R subtypes in ependymal cell primary cultures. The alpha and beta thyroid hormone receptors are both expressed. While the alpha and alpha 2 subtypes are found in almost all cells, the beta 1 receptors are present in few cells only. The possibility that alpha and beta receptors are colocalized is discussed. We also demonstrate that ependymal cells respond to L-T3 with a marked increase of the expression of the glutamine synthetase messenger RNAs.

The expression of nuclear 3,5,3' triiodothyronine receptors is induced in Schwann cells by nerve transection.

The effects of thyroid hormones on the nervous system are mediated by the presence of nuclear T3 receptors (NT3R). In this study, the expression of NT3R was investigated in spinal cord, dorsal root ganglia (DRG), or sciatic nerve of adult rats after immunostaining with a 2B3-NT3R monoclonal antibody which recognizes both alpha and beta types of NT3R. The specificity of this monoclonal antibody was confirmed by Western blots. The 2B3-NT3R monoclonal antibody recognized one band corresponding to a molecular weight of 57 kDa in extract of spinal cord or DRG. No staining was observed on immunoblot of intact sciatic nerve. In the spinal cord, the nuclei of the neurons and glial cells including both astrocytes and oligodendrocytes exhibited 2B3-NT3R immunoreactivity. While all the nuclei of the DRG sensory neurons expressed the NT3R, all the nuclei of the satellite and Schwann cells were devoid of any immunoreaction. In the sciatic nerve, the nuclei of the Schwann cells also lacked 2B3-NT3R-immunoreactivity. After sciatic nerve transection in vivo, Schwann cell nuclei, which never expressed NT3R in intact nerves of adult rats, displayed a clear 2B3-NT3R immunoreaction in proximal and distal stumps adjacent to the section. Double immunostaining with antibodies raised to 3-sulfogalactosylceramide or S100 confirmed that most of the NT3R containing nuclei belong to Schwann cells. In dissociated cell cultures grown in vitro from sciatic nerves, Schwann cells exhibited 2B3-NT3R immunoreactivity. These data suggest that the inhibition of NT3R expression in Schwann cells ensheathing axons in intact nerve is reversed when the axons are degenerating or lacking.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of cerebroside sulfotransferase and 2',3'-cyclic nucleotide 3'-phosphodiesterase by basic fibroblast growth factor in relation to proliferation in rat oligodendrocyte cultures.

Previous results (Fressinaud, C., Sarliève, L.L., and Labourdette, G. J. J. Cell. Physiol., 141:667-674, 1989b) have shown that cerebroside sulfotransferase (CST; EC 2.8.2.11) is enriched in pure rat oligodendrocyte (OL) cultures and that its activity is increased by factors mitogenic for OL precursors and galactocerebroside (GC) expressing OL, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor, and high insulin concentrations. In contrast, transforming growth factor beta or low insulin concentrations were found to be ineffective in this culture system. As bFGF mainly enhanced the proliferation of OL precursors (GC negative cells) rather than that of differentiated (GC+) cells, a relationship between OL precursor proliferation and CST increase was suggested. This hypothesis was first tested in 20-day-old OL cultures grown in chemically defined medium. The dose-response curve of [125I] Iododeoxyuridine ([125I]dUrd) incorporation toward bFGF was parallel to that of CST specific activity, and maximal stimulation was reached at 5 ng/ml bFGF for both. In contrast, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) specific activity decreased after bFGF treatment. To determine if CST increase was linked to the proliferation of OL precursors induced by bFGF, cell proliferation was blocked by cytosine arabinoside (ARA-C). From 10(-8) to 10(-5) M ARA-C there was a dose-dependent inhibition of cell proliferation and a decrease in CST specific activity, whereas CNP specific activity was enhanced. When the cells were treated with bFGF and 10(-6) M ARA-C together, the proliferation was completely blocked and CST activity decreased by 72% below control values, whereas CNP activity was not significantly decreased. Immunocytochemical studies showed that the number of sulfatide-expressing cells and the number of cycling cells were increased after bFGF treatment, but that there was no overlapping between these two populations. Taken together these results suggest that CST activity and sulfatide expression appear shortly after the arrest of OL precursor division.

Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors.

Glutamine synthetase (GS, EC 6.3.1.2.) has long been considered as a protein specific for astrocytes in the brain, but recently GS immunoreactivity has been reported in oligodendrocytes both in mixed primary glial cell cultures and in vivo. We have investigated its expression and regulation in "pure" oligodendrocyte cultures. "Pure" oligodendrocyte secondary cultures were derived from newborn rat brain primary cultures enriched in oligodendrocytes as described by Besnard et al. (1987) and were grown in chemically defined medium. These cultures contain more than 90% galactocerebroside-positive oligodendrocytes and produce "myelin" membranes (Fressinaud et al., 1990) after 6-10 days in subcultures (30-35 days, total time in culture). The presence of GS in oligodendrocytes from both primary glial cell cultures and "pure" oligodendrocyte cultures was confirmed by double immunostaining with a rabbit antisheep GS and guinea pig antirat brain myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase. In "pure" oligodendrocyte cultures, about half of cells were labeled with anti-GS antibody. Furthermore, on the immunoblot performed with a rabbit antisheep GS, the GS protein in "pure" oligodendrocyte secondary cultures was visualized as a single band with an apparent molecular mass of about 43 kDa. In contrast, two protein bands for GS were observed in cultured astrocytes. On the immunoblot performed with a rabbit antichick GS, two immunopositive protein bands were observed: a major one migrating as the purified adult chick brain GS and a minor one with a lower molecular mass. Two similar immunoreactive bands were also observed in pure rat astrocyte cultures. Compared to pure rat astrocyte cultures, "pure" oligodendrocyte cultures of the same age displayed an unexpectedly high GS specific activity that could not be explained by astrocytic contamination of the cultures (less than 5%). As for cultured astrocytes, treatment of oligodendrocyte cultures with dibutyryl-adenosine 3':5'-cyclic monophosphate, triiodothyronine, or hydrocortisone increased significantly GS specific activity. Interestingly, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor that increase the GS activity in astrocytes do not affect this activity in oligodendrocytes. Thus we confirm the finding of Warringa et al. (1988) that GS is also expressed in oligodendrocytes. We show that its activity is regulated similarly in astrocytes and oligodendrocytes by hormones, but that it is regulated differently by growth factors in these two cell types.