Investigation of Myelinogenesis in Vitro: Transient Expression of 3,5,3’-Triiodothyronine Nuclear Receptors in Secondary Cultures of Pure Rat Oligodendrocytes

Sarliève, Louis L.; Besnard, François; Labourdette, G.; Yusta, Bernardo; Pascual, Ángel; Aranda, Ana; Luo, Min; Puymirat, Jack; Dussalut, J. H.

doi:10.1007/978-1-4613-0765-5_11

Cited by 12 publications

(13 citation statements)

References 58 publications

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“…The results reported here demonstrate the colocalization of GS and of CNP, a well-known oligodendroglial and myelin marker in the brain (Braun et al, 1988;Vogel et al, 1988). Moreover, secondary cultures of rat oligodendrocytes that contain 90-95% oligodendrocytes (Yusta et al, 1988;Besnard et al, 1989a;Sarlieve et al, 1989;Fressinaud et al, 1990) also form "myelin" membranes (Fressinaud et al, 1990). In such cultures, about 50% of the cells were GS-positive.…”

Section: Discussionmentioning

confidence: 51%

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

Fressinaud¹,

Weinrauder²,

Delaunoy³

et al. 1991

Journal Cellular Physiology

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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.

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Section: Discussionmentioning

confidence: 51%

“…Thus, GS of "pure" oligodendrocyte cultures responds similarly to astrocytes. Both glial cell types express 3,5,3'-triiodothyronine nuclear receptors in vivo (Puymirat et al, 1991) and in vitro (Yusta et al, 1988;Sarlieve et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

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

Fressinaud¹,

Weinrauder²,

Delaunoy³

et al. 1991

Journal Cellular Physiology

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“…The unlabeled cells had the morphology of OL precursors (Labourdette et al, 1979). In 26-day-old OL cultures, more than 90% of the cells expressed GC as previously described (Besnard et al, 1989b;Sarlieve et al, 1989).…”

Section: Resultsmentioning

confidence: 87%

Regulation of cerebroside sulfotransferase activity in cultured oligodendrocytes: Effect of growth factors and insulin

Fressinaud

Sarliève

Labourdette

1989

Journal Cellular Physiology

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Cerebroside sulfotransferase (EC 2.8.2.11, CST) specific activity has been determined in oligodendrocyte (OL)-enriched glial cell cultures from newborn rat brain grown in serum supplemented medium. This activity is detectable at 5 days in vitro (DIV) and reaches its maximum value at 12 DIV. This period corresponds to that of oligodendrocyte precursor proliferation in these cultures. The activity decreases thereafter and remains nearly constant after 24 DIV. The developmental curve of CST activity is parallel in pure oligodendrocyte subcultures but twice higher than in primary cultures. These data confirm that CST is highly enriched in OL. Basic fibroblast growth factor (bFGF) (15 ng/ml) and platelet derived growth factor (PDGF) (0.75 U/ml) both enhance CST activity by 90% and 72%, respectively. This increase is in the same range than that of DNA content in treated cultures, whereas protein increase is smaller (50% and 22%, respectively). In contrast, transforming growth factor beta 1 (TGF beta 1, 0.5 and 5 ng/ml) does not significantly enhance CST activity nor DNA content of OL cultures. Insulin at high concentrations (5 micrograms/ml) also enhances CST activity but has no effect at physiological concentrations (20 ng/ml). These results show that CST activity can be controlled by growth factors. They suggest that CST activity is more closely related to OL and OL precursor proliferation than to myelination itself since its maximal activity preceeds myelination in vitro.

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“…Furthermore, NT,Rs have been localized in rat astrocyte cultures by immunocytochemistry ) using a monoclonal antibody (2B3) raised against the purified liver NT,Rs, characterized by Luo et al (1988). Finally, in rat secondary cultures containing 85 -90% oligodendrocytes (Besnard et al, 1987), we have unequivocally demonstrated the presence of nuclear T, binding capacity in such oligodendrocytes and shown that its level is two to three times higher than in astrocytes (Yusta et al, 1988;Sarlikve et al, 1989).The 2B3 monoclonal antibody against purified rat liver NT,Rs recognized two nuclear proteins with the same molecular weight as the c-erbA gene products p l and cwl: 52 Kd and 45 Kd, respectively, which both bind T, and a non-T, binding protein (NT,Ra,) (Sap et al, 1986;Weinberger et al, 1986;Mitsuhashi et al, 1988; Lazaret al, 1988;Hodin et al, 1989;Strait et al, 1990;Puymirat et al, 1991). It bound to NT,Rs in solution and immunoprecipitated T,-binding proteins in nuclear extracts (Luo et al, 1988;Puymirat et al, 1991; BarakatWalter et al, 1992; for a review, see Puymirat, 1992).…”

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confidence: 84%

Transient expression of 3,5,3′‐triiodothyronine nuclear receptors in rat oligodendrocytes: In vivo and in vitro immunocytochemical studies

Besnard¹,

Luo²,

Miehe³

et al. 1994

J of Neuroscience Research

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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.

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Investigation of Myelinogenesis in Vitro: Transient Expression of 3,5,3’-Triiodothyronine Nuclear Receptors in Secondary Cultures of Pure Rat Oligodendrocytes

Cited by 12 publications

References 58 publications

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

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

Regulation of cerebroside sulfotransferase activity in cultured oligodendrocytes: Effect of growth factors and insulin

Transient expression of 3,5,3′‐triiodothyronine nuclear receptors in rat oligodendrocytes: In vivo and in vitro immunocytochemical studies

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