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Oligodendrocyte-Type-2 Astrocyte (O-2A) Progenitor Cells Express Glutamine Synthetase: Developmental and Cell Type-Specific Regu

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Oligodendrocyte-Type-2 Astrocyte (O-2A) Progenitor Cells Express Glutamine Synthetase: Developmental and Cell Type-Specific Regu Molecular Psychiatry (1998) 3, 356–361 1998 Stockton Press All rights reserved 1359–4184/98 $12.00 ORIGINAL RESEARCH ARTICLE enriched secondary cultures of O-2A progenitors (A2B5+) express GS (Figure 1c and 1d). In nearly all postmitotic secondary cultures of OL, Oligodendrocyte-type-2 we have investigated GS expression by double immunostaining with the GS antibody and several dif- astrocyte (O-2A) progenitor ferentiation oligodendroglial markers: OL-1,8 2Ј,3Ј-cyc- cells express glutamine lic nucleotide 3Ј-phosphodiesterase (CNP), MBP and myelin/oligodendrocyte glycoprotein (MOG) each spe- synthetase: developmental cific for a particular OL stage of maturation. Figure 2 shows that GS expression in OL secondary cultures and cell type-specific was dependent upon OL differentiation stage. In 4-day OL secondary cultures, 10–15% were A2B5+ O-2A regulation cells. Of these, only 5% expressed GS (data not 1 1 2 shown). Moreover, 10% of pre- or immature OL, D Baas , D Dalenc¸on , C Fressinaud , characterised by the monoclonal rat antibody OL-1 spe- 3 1 L Vitkovic and LL Sarlie`ve cific for the unknown sulfatide, were GS+/OL-1+ and + 1 20% of CNP OL expressed GS. Furthermore, 30% of UPR 416 du CNRS, 5 rue Blaise Pascal, 67084 + + + OL were GS /MBP and about 70% of MOG OL Strasbourg Cedex, France; 2Service de Neurologie B, expressed GS (see also Figure 1). Note that if an OL CHU, Angers, France; 3Division of Neuroscience and was CNP positive it was also OL-1 positive; and if an Behavioral Science, NIMH, NIH, Rockville, USA + + + OL was MOG positive it was OL-1 /CNP /MBP . These data indicated that the number of cells immunostained Keywords: O-2A cells; astrocytes; oligodendrocytes; cul- for GS increased with OL differentiation and matu- ture; glutamine synthetase; mRNA; protein; activity ration (Figure 2). Given that the GS protein was synthesized in each Glutamine synthetase (GS), the enzyme that catalyses macroglial cell type tested (Figure 1), we next com- glutamine synthesis from glutamate and ammonia, pared mRNA levels. GS mRNA expression was ana- plays a central role in the detoxification of brain 9 ammonia.1 In the central nervous system (CNS), GS also lysed by Northern blot using the glial cell line CG-4. subserves additional important functions such as reg- This cell line allowed us to obtain three macroglial cell ulating glutamate, GABA and amino acid metabolism.2 types in vitro. The mRNA sizes were 2.8 kb and 1.4 kb, Oligodendrocytes (OL) form the myelin sheath in the respectively and corresponded to the expected sizes of central nervous system (CNS) and are essential for GS7 and cyclophilin10 mRNA (Figure 3a). GS mRNA efficient propagation of nerve impulses. In culture, OL was expressed in CG-4-derived O-2A progenitors arise from bipotential O-2A progenitor cells. These O- (Figure 3a lane 1), CG-4-derived OL (Figure 3a lane 2) 2A cells give rise to type-2 astrocytes in the presence and CG-4-derived type-2 astrocytes (Figure 3a lane 3). 3–7 of serum. GS is expressed in mature glial cells in vivo Only one GS mRNA species (2.8 kb) was expressed in and in vitro, but it is unknown whether GS is present in CG-4-derived O-2A cells, CG-4-derived type-2 astro- glial progenitors. In addition, a comparison of the GS cytes, and CG-4-derived OL, compared to two GS expression level among the various types of glial cells has never been done in vitro. The current study investi- mRNA species (2.8 kb and 1.4 kb) that were expressed gates in vitro GS expression levels in O-2A progenitors, in type-1 astrocytes. Autoradiogram quantification astrocytes and OL. We demonstrate that the GS gene showed that CG-4-derived O-2A progenitors expressed is expressed in O-2A progenitors and is expressed at 20-fold less GS mRNA than CG-4-derived type-2 astro- different levels in each cultured glial cell type. GS also cytes and 30-fold less than CG-4-derived OL (Figure is stimulated during OL developmental maturation. 3b). Thus, CG-4-derived type-2 astrocytes expressed Thus, the GS gene is expressed in O-2A cells and is 1.5-fold less GS mRNA than CG-4 derived OL (Figure 3b). regulated in a developmental and macroglial cell type- To determine whether GS was functional and equally specific manner. efficient in all glial cell types, GS activity was determ- In highly enriched cultures of O-2A progenitors, ined and compared in type-1 astrocyte primary cul- type-2 astrocytes, OL or type-1 astrocytes, we investi- tures, O-2A progenitors, type-2 astrocyte and OL sec- gated GS expression by double immunostaining with ondary cultures (Figure 4). GS activity in O-2A cells glial specific markers and a GS antibody. Double and type-1 astrocytes was similar. This level was half immunolabelling with GS (Figure 1a) and glial fibril- of that measured in type-2 astrocytes and OL which are lary acidic protein (GFAP) (Figure 1b) showed that GS two cell types differentiated from bipotential glial O- was expressed in GFAP positive type-1 astrocytes. The 2A progenitors. GS activity in the latter two cell types GS antibody (Figure 1c, 1e and 1g) double immunolab- was similar. Note that type-2 astrocytes expressed 1.5- elled cells with the A2B5 (Figure 1d), GFAP (Figure 1f), fold less GS mRNA, but demonstrated the same GS spe- or myelin basic protein (MBP) (Figure 1h) antibodies. cific activity as OL (Figure 3b). These results indicated that type-2 astrocytes (Figure Previous studies indicated that GS activity was upre- 1e and 1f) as well as mature OL (MBP+) (Figure 1g and gulated by bFGF in type-1 astrocytes but not in OL sec- 1h) express GS. Double immunolabelling with A2B5 ondary cultures.4 Since O-2A progenitors were main- and GS demonstrated, for the first time, that highly tained in culture medium containing bFGF, it was Glutamine synthetase in glial lineage D Baas et al 357 Figure 1 Oligodendrocyte progenitors and all three mature macroglial cell types express glutamine synthetase. Primary type- 1 astrocyte cultures after 26 days in vitro (a, b), O-2A progenitor secondary cultures after 13 days in vitro (c, d), type-2 astrocyte secondary cultures after 13 days in vitro (e, f) and oligodendrocyte secondary cultures after 26 days in vitro (g, h) were double immunolabelled with anti-GS (a, c, e, g), anti-GFAP (b and f), A2B5 (d), and anti-MBP (h) antibodies. Cells were grown as described in Materials and Methods. Each pair of panels represents micrographs of the same field. Note that all cell types express GS. Scale bars = 20 ␮m. Glutamine synthetase in glial lineage D Baas et al 358 Figure 4 Specific activity of glutamine synthetase in mac- Figure 2 Developmental regulation of glutamine synthetase roglial cells is cell type-specific. GS specific activity was mea- in oligodendrocytes. Ratio of double labelled cells (GS and sured in type-1 astrocyte primary cultures after 26 days. O-2A one of the markers of oligodendrocyte maturation, OL-1, CNP, progenitor secondary cultures after 13 days, type-2 astrocyte MBP or MOG) expressed as a percentage of cells labelled with secondary cultures after 13 days and oligodendrocyte second- the particular maturation marker in OL secondary cultures. ary cultures after 26 days. Cells were grown and GS activity Cells were grown as described in Materials and Methods for measured as described in Materials and Methods. Values rep- 4 days in secondary cultures (26 days total time in culture). resent the means ± s.d. from ten separate experiments. Stat- ± Values represent the means s.d. from three independent istical analysis was performed using Student’s t-test. * and ** 3 counts of 10 cells in each of three independent experiments. Significantly different from O-2A progenitor cells at P Ͻ 0.05 and P Ͻ 0.001, respectively. necessary to determine whether GS activity was depen- dent on bFGF. Therefore, we measured GS activity in secondary cultures highly enriched with O-2A progeni- tors maintained with or without bFGF (2 ng ml−1). GS specific activity was similar in O-2A cells grown, either in the presence of bFGF (0.10 ± 0.03 ␮mol mg protein−1 min−1) or absence of bFGF (0.09 ± 0.04 ␮mol mg protein−1 min−1) (average ± s.d. from 10 separate experiments). These data suggested that bFGF had no effect on GS expression in O-2A progenitor cells. The CG-4 cell line proved to be interesting for this study because it expressed OL differentiation markers. Indeed, in culture, the CG-4 cell line yielded a high percent of each cell type studied. Ninety eight percent were A2B5+/galactocerebroside (GC)− O-2A cells in serum-free medium. Upon growth factor omission, most cells (98%) acquired a typical differentiated OL (GC+) phenotype or, in the presence of 20% FCS, a type-2 astrocytes phenotype.9 Primary and secondary culture of O-2A progenitors, type-2 astrocytes, OL or type-1 astrocytes, contained also a high percent of each studied cell type and less than 1% of microglia as pre- viously described.3 The present results showed that functional GS was expressed in vitro, not only in one macroglial cell type, but in all including O-2A cells. Previously, it was thought that GS was expressed only in mature macro- glial cells and not in O-2A progenitors. Thus, in these Figure 3 Northern blot analysis of glutamine synthetase mRNA in CG-4 cell line. (a) PolyA+ mRNA prepared from the studies, we report the novel finding that GS was CG-4 cell line maintained as O-2A progenitor cells (lane 1), expressed and was highly efficient in O-2A cells.
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