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Induction of myelin-associated glycoprotein expression through neuron–oligodendrocyte contact
Developmental Brain Research 100 Ž1997. 110–116
Research report
Induction of myelin-associated glycoprotein expression through neuron–oligodendrocyte contact Yoshihiro Matsuda ) , Hisami Koito, Hiroshi Yamamoto Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawa-higashi, Kodaira, Tokyo 187, Japan Accepted 11 February 1997
Abstract The role of neurons on expression of myelin-associated glycoprotein ŽMAG. in oligodendrocytes and oligodendroglial differentiation was examined. Primary cultures of oligodendrocytes prepared from neonatal mouse brains were co-cultured with neuronal cells derived from embryonal carcinoma P19 cells. The levels of MAG mRNAs following this co-culture were determined by reverse transcription ŽRT.-PCR. In oligodendrocytes co-cultured in direct contact with P19-derived neurons, the levels of MAG mRNAs, particularly that of the L-type isoform, were markedly higher than those in cultures without any neuronal cells. On the other hand, when the P19-derived neurons were present, but not in direct contact, no significant induction of MAG expression was found, though oligodendrocytes appeared to mature morphologically. The L-MAG expression was also stimulated when just the neuronal cell membrane fraction was added, which implies that there might be some effecterŽs. in the cell membrane which are possibly exerting a signal transduction for myelin formation. These results suggest that morphological differentiation and functional maturation of oligodendrocytes are due to independent factors. The former is caused by some humoral factorŽs. liberated from neuronal cells, while the latter resulted from cellular contact with neuronal cells. Keywords: Mouse; Development; Myelin-associated glycoprotein; Oligodendrocyte; Neural co-culture; Embryonal carcinoma P19 cell; RT-PCR
1. Introduction It is well established that the oligodendroglial cell lineage is regulated by a variety of environmental factors in the brain before differentiating into myelin-forming cells w7,20,28x. However, little is known about the molecular mechanism of myelinogenesis. Myelin-associated glycoprotein ŽMAG. is one of the myelin-specific proteins expressed by myelin-forming oligodendroglia w25x, specifically at the point of contact where they wrap around axons w29x. This implies its involvement in the process whereby the oligodendroglial cell recognizes the axon to sheath. In rodent brains MAG has been reported to appear at around a week after birth and then immediately increase for the period corresponding to active myelinogenesis. Of the two isoforms, L-MAG is first expressed at the onset of myelin
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Corresponding author. Fax: q81 Ž423. 461753.
formation and slowly decreases thereafter, while S-MAG is synthesized later to become a major component replacing L-MAG in adult brains w12,17x. L-MAG has also been found to be involved in the activation of Fyn, a protein tyrosine kinase of the src family, 4 days after birth, which suggests that the tyrosine phosphorylation cascade might be implicated in signal transduction for myelinogenesis w31x. These findings support the idea that MAG, particularly L-MAG might play a critical role in the initial stage of myelin formation. However, the induction mechanism of MAG expression in oligodendroglia is not understood well yet. In the present study we investigated the contribution of neurons to oligodendroglial maturation and MAG induction in primary cultures of oligodendrocytes. Using embryonal carcinoma P19 cell-derived neuronal cells, we examined the effects of neuron-oligodendrocyte co-cultures, and found that direct contact of neurons with oligodendrocytes was essential for the increase of MAG expression.
0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 0 3 9 - 4
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2. Materials and methods 2.1. Primary culture of oligodendrocytes Primary cultures of mouse brain oligodendrocytes were obtained by the method of Knapps et al. w16x and Bottenstein w2x with some modifications. Briefly, the cerebral cortices removed from neonatal BALBrc mice were enzymatically dissociated in a solution of 0.25% trypsin and 0.001% DNase in isotonic buffer described by Raizada et al. w27x at 378C for 15 min. After being washed with Dulbecco’s modified Eagle’s medium ŽDMEM. containing 10% fetal calf serum ŽFCS., the dissociated cells were sieved through 70 m m pore-sized nylon mesh, and seeded on poly-L-lysine coated culture dishes by 5 = 10 6 cellsr60 mm dishes. After 2 days, the medium was exchanged for a serum-free defined medium consisting of DMEMrHam’s F12 Ž1:1. supplemented with insulin Ž5 m grml., sodium selenite Ž30 nM., transferrin Ž50 m grml., biotin Ž5 m grml., hydrocortisone Ž10 nM., triiodothyronine Ž30 nM., and glucose Ž6 grl.. 2.2. Neural co-culture Mouse embryonal carcinoma P19 cells, provided by Dr. Hamada, Tokyo Metropolitan Institute of Medical Sciences, were maintained in a-minimum essential medium Ž a-MEM. containing 10% FCS, and subcultured every other day, as described by McCarrick and Andrews w22x. The P19 EC cells were differentiated into the cells of neuronal phenotype by culture in aggregation under exposure to low doses of retinoic acid. Briefly, the P19 cells were dispersed with 0.025% trypsin and 1 mM EDTA, and seeded in bacteriological grade Petri dishes at a density of 10 5 cellsrml in medium containing 0.7 m M retinoic acid. After 48 h, the cell aggregates were collected and resuspended in the fresh medium containing 0.7 m M retinoic acid again. After an additional 48 h culture in bacteriological grade Petri dishes, the aggregates were dispersed by treatment with trypsin-EDTA, and added onto the oligodendroglial cultures described above.
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defined medium. The membrane fraction of undifferentiating P19 cells was prepared from the cells cultured in a-MEM containing 10% FCS, which had not treated with retinoic acid. 2.4. Determination of mRNAs by RT-PCR Cultured cells were harvested with cell scrapers, and collected by centrifugation Ž500 = g, 5 min.. Total cellular RNA was extracted with RNAzol B ŽBiotecx Lab., USA. according to the supplier’s instruction. cDNA was prepared with random hexadeoxynucleotide primers and Moloney murine leukemia virus reverse transcriptase ŽGIBCO-BRL, USA.. Thirty cycles of PCR were carried out with Ampli-Taq DNA polymerase ŽPerkin Elmer Corp., USA. in a programmable heat block; the cycle profile was denaturation at 938C for 1 min, annealing at 658C for 1 min, and elongation at 728C for 2 min. The following synthetic oligonucleotides were used as primers, which resulted in two different cDNAs amplified for MAG isoforms Ž239 bp for S-MAG and 194 bp for L-MAG.: 5X -GCCCCGAATTCAGAATCTCTGG-3X and 5X TTCTGCATACTCAGCCAGC-3X . The PCR products were fractionated by electrophoresis on 6% polyacrylamide gel, stained with 1 m grml ethidium bromide, and analyzed quantitatively by densitometry. The amount of total RNA used in this RT-PCR assay was fixed to give linear dosedependence of cDNAs amplified for MAG mRNAs. Typically, 10 ng total RNA was used in the present study. Proteolipid protein ŽPLP. mRNAs were also determined by RT-PCR with the following synthetic oligonucleotides for PCR primers: 5X-ACTACAAGACCACCATCTGCG-3X and
2.3. Preparation of membrane fraction of P19 cells After the P19 cells were cultured in aggregation in the presence of retinoic acid for 4 days, as described above, they were dispersed, suspended in DMEMrF12 Ž1:1. containing 5% FCS, and plated on poly-L-lysine-coated culture dishes by 6 = 10 6 cellsr100 mm diameter dish. After 2 days the medium containing 10 m M cytosine arabinoside was added, and then the cells were collected 2–3 days later. The collected cells were suspended in the serum-free defined medium Ž0.6 mlrcell pellet obtained from 100 mm diameter culture dish., and disrupted by freezing-and-thawing. The homogenate was centrifuged at 12,000 rpm for 15 min, and the pellet was resuspended in the serum-free
Fig. 1. Effects of neural co-culture on expression of MAG mRNAs in primary cultures of oligodendrocytes. On day 5 of the oligodendroglial primary culture, 2=10 6 of P19 cells which had been treated with retinoic acid were added per 60 mm diameter dish, and co-culture was carried out for indicated periods. The expression of L-MAG and S-MAG mRNAs in oligodendrocytes with and without P19-derived neuronal cells was determined by RT-PCR. Results are expressed in relative terms, and represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG mRNA in the cells of 2 days in the absence of P19 neurons was defined as 100%.
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different cDNAs simultaneously amplified for the isoforms, PLP and DM20 were analyzed on 6% polyacrylamide gel electrophoresis Ž286 bp for PLP and 181 bp for DM20.. As internal standard cDNA of b-actin was amplified by 20 cycles of PCR.
3. Results
Fig. 2. Effects of neural co-culture on expression of PLP mRNAs in primary cultures of oligodendrocytes. The cells were prepared, as described in Fig. 1. The expression of PLP and DM20 mRNAs was determined by RT-PCR. Results are expressed in relative terms, and represent the means"S.E.M. of 4 culture dishes. The amount of PLP mRNA in the cells of 2 days in the absence of P19 neurons was defined as 100%.
5X-CCTATACTGGCAGAGGTCTTGC-3X . The conditions of PCR were the same as that for MAG, except that amplification was carried out with 25 reaction cycles. Two
Fig. 1 shows the effect of co-culturing oligodendrocytes with neuronal cells on the expression of MAG mRNAs. Oligodendrocytes in the primary culture had low levels of MAG mRNAs, and increased the expression slowly with the culture period. On the other hand, the cells showed significant increases of MAG mRNAs when cultured with P19-derived neuronal cells. After being co-cultured for 4 days and 7 days, expressions of L-MAG were about 3 and 4.5 times greater, respectively, as much as those in oligodendrocytes alone. There was no difference between the co-culture and the single culture on day 2. It seems likely that the P19 cells were not able to affect oligodendrocytes at that early stage of 2 days, since they were not morpho-
Fig. 3. Effect of neural co-culture on oligodendroglial differentiation. A: the primary culture of oligodendrocytes of 11 DIV Ždays in vitro. without co-culture. B: oligodendrocytes co-cultured for 7 days separately with P19-derived neuronal cells which were grown on a cell culture insert. The total culture period was 11 days, since the co-culture started on day 5. Scale bar in ŽB., 50 m m.
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logically differentiated enough. The P19 cells did not express detectable MAG mRNAs during these culture periods. Proteolipid protein ŽPLP., another myelin-specific protein, was also induced in the co-culture. As shown in Fig. 2, the PLP expression in the primary cultures of oligodendrocytes slowly increased with the culture period, and the increases were markedly magnified when cultured with P19-derived neuronal cells. Neither PLP nor DM20 mRNA was found in the P19 cells during these culture periods. To elucidate whether it is the contact between oligodendrocytes and neuronal cells or some humoral factorŽs. liberated from neuronal cells which is required for the induction of MAG expression, the effect of separate co-culture was investigated using Falcon cell culture inserts ŽBecton Dickinson, USA.. Fig. 3 shows a morphological change under the co-culture condition. In the primary culture of 11 days, oligodendrocytes in the single culture were still immature; most of the cells were bipolar or extended just a few dendrites ŽFig. 3A.. After co-culture for 7 days following 4 days single culture, on the other hand, lots of morphologically matured cells with many refined dendrites were observed ŽFig. 3B., suggesting that neuronal cells stimulated differentiation of oligodendrocytes by liberating some soluble factorŽs. into the culture medium. However, as shown in Fig. 4, oligodendrocytes in the separate co-culture did not show any significant changes in the MAG expression despite their morphologically differentiated appearance. Under the contact condition, on the other hand, a marked increase of MAG mRNAs was confirmed. This suggests that morphological differentiation was not necessarily associated with functional maturation of oligodendrocytes as myelin-forming cells, and that the cellular contact exerted some signal transduction resulting
Fig. 4. Effects of co-culture conditions on MAG induction in primary cultures of oligodendrocytes. The expression of MAG mRNAs in oligodendrocytes without co-culture and with co-culture for 4 days under the separate and contact conditions was determined by RT-PCR. Results are expressed in relative terms, and represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG in the cell preparations of oligodendrocytes Žoligo.. alone was defined as 100%.
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Fig. 5. Dose dependence of neuronal cells on MAG induction in oligodendrocytes. Indicated numbers of P19 cells were added on the primary cultures of oligodendrocytes on day 5, and co-cultured for 4 days. The relative amounts of MAG mRNAs were determined by RT-PCR. Data represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG in the cell preparations with no added P19 cells was defined as 100%.
in induction of the MAG expression. In order to see whether efficient cellular contact can cause a large induction of MAG, next examined was the dose dependence of P19 cells in co-culture. As shown in Fig. 5, the more neuronal cells were added onto oligodendrocytes, the more gene expression was observed. Although the number of oligodendrocytes could not be determined in the primary culture, the number of P19 cells added was always greater throughout the experimental conditions. However, when 2 = 10 5 P19 cells were added, their distribution was so sparse that contact with oligodendrocytes appeared to be rare. In addition, neuronal cells increased their axon extensions to make dense neuronal networks, when the P19 cells were plated in larger numbers. Assuming that the contact of oligodendrocytes with neuronal axons was important, it also appears that the more the P19 cells, the greater the effect. MAG was also induced by adding the membrane fraction of P19-derived neuronal cells. As shown in Fig. 6, the expression of L-MAG markedly increased when the membranous fraction of neurons was added. The soluble fraction gave no significant effects. Furthermore, the P19 cells of non-neuronal phenotype, which had not been treated with retinoic acid, did not have any effects at all. These results highlight how important direct contact between oligodendroglia and neurons is for MAG expression. Interestingly, in this experiment using the membranous fraction, MAG induction occurred in 2 days. In the case of co-culture, on the other hand, no significant change was found in 2 days ŽFig. 1.. This suggests that there are some effector moleculeŽs. on the cell membranes of neurons, which give a signal to induce MAG expression in oligodendrocytes, and that it takes more than 4 days for P19 cells to express them on their cell surfaces including
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Fig. 6. Effect of the membrane fraction of P19 cells on MAG expression. The P19 cells of neuronal phenotype and of undifferentiation were collected, and disrupted by freezing-and-thawing. After centrifugation at 12,000 rpm for 15 min, the pellets and supernatants were used as the membranous and soluble fractions, respectively. The cell pellets resuspended in serum-free defined medium and the supernatants containing 2 mg proteins each were added to oligodendroglial cultures of 5 DIV in 35-mm-diameter dishes, and the cells were harvested 2 days later. The relative amounts of MAG mRNAs were determined by RT-PCR. Data represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG in the cell preparations of oligodendrocytes Žoligo.. alone was defined as 100%.
axonal membranes, probably because the effectorŽs. are newly synthesized as P19 cells differentiate into neurons. 4. Discussion It has been demonstrated that both soluble factors produced by neurons and specific forms of contact with neurons are necessary for oligodendrocytes to differentiate w9x. However, information on the initiation of myelination is still sparse. In the later stages of brain development, MAG has been reported to be synthesized at a period which is coincident with that of myelinogenesis w12x, and to be localized in the periaxonal portion of myelin sheaths w29x. This suggests that it plays a critical role in myelin formation. However, little is understood about the regulation of MAG expression in oligodendroglia so far. The present study found evidence that the MAG expression in oligodendrocytes can be induced through direct contact with neuronal cells in vitro. It has been proposed that differentiation of oligodendrocytes, such as process extension and myelin protein syntheses, might proceed under their own intrinsic signals in primary culture w15,18,26x. This is most probably true, since in the present study some of the cells showed morphological differentiation in 1–2 weeks, but the rate for the total progenitor cells was not large. In addition, the expression of myelin-specific proteins in the culture cells was much lower than that in vivo. The level of L-MAG mRNA in the culture cells of 6 days in vitro ŽDIV. was similar to that in the forebrains of mice of post-natal day ŽPND. 7, but the level in the culture cells
of 11 DIV was less than a tenth that of the brains of PND 12, the period that myelination actively proceeds Ždata not shown.. On the other hand, the levels of S-MAG in the culture cells of 6, 8 and 11 DIV were comparable to those in the brains of PND 7, 9 and 12, respectively Ždata not shown.. This suggests that some environmental factorŽs., such as neuron–glia interactions, might be required for the induction of L-MAG expression during the period of myelinogenesis. Ellison et al. w5x recently reported that neurons expressed platelet-derived growth factor ŽPDGF. to support oligodendroglial development, and suggested that by liberating PDGF, neurons regulated the generation of appropriate numbers of differentiated oligodendrocytes needed to myelinate axons during cerebral cortex development. It was also observed in the present study that neuronal cells appeared to liberate some soluble factorŽs. stimulating process extension of oligodendrocytes. However, despite the distinct change in the morphological phenotype the levels of MAG mRNAs were not significantly increased in the co-culture with neuronal cells under the separate condition ŽFigs. 3 and 4.. Only when oligodendrocytes could come into contact with neurons, was the MAG expression up-regulated. These findings suggest that neurons might influence oligodendrocytes on morphological differentiation and functional maturation by different mechanisms. From the time course of increase of MAG mRNA in co-culture, it can be seen that it takes 4 days or more for P19 cells to give signalŽs. for MAG induction to oligodendrocytes ŽFig. 1.. On the other hand, when the membrane fraction of P19-derived neuronal cells was added, the increase of MAG expression became significant in just 2 days ŽFig. 6.. In addition, co-culture with nonneuronal P19 cells which had not been treated with retinoic acid did not alter the MAG expression in oligodendrocytes. These results suggest that some effector moleculeŽs. might be newly synthesized in fully differentiated neurons. In the process of differentiation into the neuronal phenotype to make neuronal networks, the effector moleculeŽs. may be expressed on the cell surface including axonal membranes. It has been reported that MAG has two isoforms resulting from alternative splicing of mRNA w17x, and that L-MAG is selectively up-regulated in the period of myelinogenesis in the developing brain w6,13x. In the present study, the expression of L-MAG was preferentially induced, but SMAG was also increased in co-culture. On the other hand, only L-MAG was increased when the membrane fraction was added. This suggests that the membrane fraction simply contained effector moleculeŽs. which induce the LMAG expression for myelination. On the other hand, neurons in co-culture may have complex effects on oligodendroglia. Various modulatory functions have been attributed to neurons on oligodendroglial lineage w8,10,11,19x; they enhance proliferation, migration, and differentiation of oligodendroglial progenitors as well as stimulating process extension and myelin protein synthesis in mature oligodendroglia. It was also observed that the
Y. Matsuda et al.r DeÕelopmental Brain Research 100 (1997) 110–116
brains of mouse embryos ŽE15–20. and neonates had chiefly S-MAG mRNA ŽKoito et al., unpublished data., suggesting that S-MAG might be preferentially expressed in immature oligodendroglia. Accordingly, in co-culture, the number of immature oligodendrocytes containing SMAG might be increased under the influence of neuronal cells. In the peripheral nervous system, the axon has been shown to be the major regulator of myelin gene expression. The Schwann cells primarily required contact with appropriate axons for significant expression of myelin genes w3,32x, and the myelin components were rapidly down-regulated when the ensheathed axon degenerated w24,30x. In contrast, it has been demonstrated that in oligodendrocytes, synthesis of myelin components occurs in the absence of axons, indicating that myelin gene expression is not necessarily dependent on axonal contact w1,4x. By axotomy, however, the influences of axons on myelin gene expression in oligodendroglia have been evidenced in vivo w14,23x; following optic nerve transection, the myelin protein mRNA levels declined in transected nerves, though significant levels of the mRNAs were found even after 40 days. These findings suggest that axons might have modulatory effects on myelin gene expression in the central nervous system as well. On the other hand, Macklin et al. w21x examined expression of myelin proteolipid protein ŽPLP. and basic protein ŽMBP. mRNAs in cultured cells by dot hybridization analysis, and showed that by co-culturing rat oligodendrocytes with chick spinal cord neurons the levels of these mRNAs were elevated 4-fold. Although their experimental conditions were different from ours in that they used enriched oligodendroglial cell populations, cultured the cells for longer period Ž20 days in total including 11 days of co-culture., and co-cultured oligodendrocytes and neuronal cells from different animal species, their results are in agreement with our present data on MAG. In the present study, to imitate the developmental environment of oligodendrocytes, we studied primary cultures of mouse brain, though the content of oligodendrocytes was not very high. In addition, we clarified the effects of neurons; morphological change was brought about by soluble factorŽs. liberated from neuronal cells, and MAG expression was induced by direct contact with the neuronal cell membrane. We also examined the expression of PLP isoform mRNAs, and found that PLP markedly increased in the co-culture but DM20 did not increase much. These findings support the idea that contact with neurons is required for functional maturation of oligodendroglia for myelination. Identification of the molecules involved in the cellular contact awaits further investigation.
5. Summary Myelin-associated glycoprotein ŽMAG. is considered to be a cell adhesion molecule linking myelin to axon mem-
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branes. Its expression by oligodendrocytes is thought to be critical in the initial stage of myelin formation, but little is known about the mechanism. Although in developing mouse brains in vivo, oligodendroglia express increased quantities of MAG during the process of myelinogenesis, in vitro primary cultures of oligodendrocytes prepared from neonatal brains show no such increase. The effects of neuronal cells on oligodendroglial differentation and MAG expression were examined by co-culturing oligodendrocytes with neuronal cells derived from embryonal carcinoma P19 cells. When oligodendrocytes were co-cultured in direct contact with these neuronal cells, they showed increased expression of MAG mRNA, whereas no such increase was found when the cells were co-cultured without direct contact. However, even when co-cultured without direct contact, most oligodendrocytes developed a morphologically differentiated phenotype with many refined dendrites. These results suggest that neuronal cells might be producing some humoral factors which induce oligodendroglial progenitors to differentiate morphologically. However, for functional maturation with myelin formation, oligodendrocytes require direct contact with neuronal cells.
Acknowledgements The authors thank Dr. Akira Hamada, Tokyo Metropolitan Institute of Medical Sciences, for providing embryonal carcinoma P19 cells, and are grateful to Dr. Marcus Wenner for critical reading of the manuscript.
References w1x E.R. Abney, P.P. Bartlett, M.C. Raff, Astrocytes, ependymal cells, and oligodendrocytes develop on schedule in dissociated cell cultures of embryonic rat brain, DeÕ. Biol. 83 Ž1981. 301–310. w2x J.E. Bottenstein, Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes, Proc. Natl. Acad. Sci. USA 83 Ž1986. 1955–1959. w3x J.P. Brockes, M.C. Raff, D.J. Nishiguchi, J. Winter, Studies on cultured rat Schwann cells. III. Assay for peripheral myelin proteins, J. Neurocytol. 9 Ž1980. 67–77. w4x M. Dubois-Dalcq, T. Behar, L. Hudson, R.A. Lazzarini, Emergence of three myelin proteins in oligodendrocytes cultured without neurons, J. Cell Biol. 102 Ž1986. 384–392. w5x J.A. Ellison, S.A. Scully, J. de Vellis, Evidence for neuronal regulation of oligodendrocyte development: cellular localization of platelet-derived growth factor a receptor and A-chain mRNA during cerebral cortex development in the rat, J. Neurosci. Res. 45 Ž1996. 28–39. w6x N. Fujita, S. Sato, T. Kurihara, T. Inuzuka, Y. Takahashi, T. Miyatake, Developmentally regulated alternative splicing of brain myelin-associated glycoprotein mRNA is lacking in the quaking mouse, FEBS Lett. 232 Ž1988. 323–327. w7x R. Hardy, R. Reynolds, Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo, DeÕelopment 111 Ž1991. 1061–1080.
116
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w8x R. Hardy, R. Reynolds, Rat cerebral cortical neurons in primary culture release a mitogen specific for early ŽGD3qrO4y. oligodendroglial progenitors, J. Neurosci. Res. 34 Ž1993. 589–600. w9x R. Hardy, R. Reynolds, Neuron-oligodendroglial interactions during central nervous system development, J. Neurosci. Res. 36 Ž1993. 121–126. w10x S.F. Hunter, J.E. Bottenstein, Bipotential glial progenitors are targets of neuronal cell line-derived growth factors, DeÕ. Brain Res. 49 Ž1989. 33–49. w11x S.F. Hunter, J.E. Bottenstein, O-2A glial progenitors from mature brain respond to CNS neuronal cell-derived growth factors, J. Neurosci. Res. 28 Ž1991. 574–582. w12x T. Inuzuka, N. Fujita, S. Sato, H. Baba, R. Nakano, H. Ishiguro, T. Miyatake, Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system, Brain Res. 562 Ž1991. 173–175. w13x H. Ishiguro, S. Sato, N. Fujita, T. Inuzuka, R. Nakano, T. Miyatake, Immunohistochemical localization of myelin-associated glycoprotein isoforms during the development in the mouse brain, Brain Res. 563 Ž1991. 288–292. w14x G.J. Kidd, P.E. Hauer, B.D. Trapp, Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo, J. Neurosci. Res. 26 Ž1990. 409–418. w15x P.E. Knapp, W.P. Bartlett, R.P. Skoff, Cultured oligodendrocytes mimic in vivo phenotypic characteristics: cell shape, expression of myelin-specific antigens, and membrane production, DeÕ. Biol. 120 Ž1987. 356–365. w16x P.E. Knapp, R.P. Skoff, T.J. Sprinkle, Differential expression of galactocerebroside, myelin basic protein, and 2X ,3X-cyclic nucleotide X 3 -phosphohydrolase during development of oligodendrocytes in vitro, J. Neurosci. Res. 21 Ž1988. 249–259. w17x C. Lai, M.A. Brow, K.-A. Nave, A.B. Noronha, R.H. Quarles, F.E. Bloom, R.J. Milner, J.G. Sutcliffe, Two forms of 1B236rmyelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing, Proc. Natl. Acad. Sci. USA 84 Ž1987. 4337–4341. w18x G. Levi, F. Aloisi, G.P. Wilkin, Differentiation of cerebellar bipotential glial precursors into oligodendrocytes in primary culture: developmental profile of surface antigens and mitotic activity, J. Neurosci. Res. 18 Ž1987. 407–417. w19x J.M. Levine, Neuronal influences on glial progenitor cell development, Neuron 3 Ž1989. 103–113. w20x J.M. Levine, J.E. Goldman, Spatial and temporal patterns of oligodendrocyte differentiation in rat cerebrum and cerebellum, J. Comp. Neurol. 277 Ž1988. 441–455.
w21x W.B. Macklin, C.L. Weill, P.L. Deininger, Expression of myelin proteolipid and basic protein mRNAs in cultured cells, J. Neurosci. Res. 16 Ž1986. 203–217. w22x McCarrick, J.W. and Andrews, P.W., Embryonal carcinoma cells and embryonic stem cells as models for neuronal development and function. In J.N. Wood ŽEd.., Neuronal Cell Lines: A Practical Approach, Oxford University Press, Oxford, 1992, pp. 77–104. w23x K. McPhilemy, L.S. Mitchell, I.R. Griffiths, S. Morrison, A.W. Deary, I. Sommer, P.G.E. Kennedy, Effect of optic nerve transection upon myelin protein gene expression by oligodendrocytes: evidence for axonal influences on gene expression, J. Neurocytol. 19 Ž1990. 494–503. w24x M.J. Politis, N. Sternberger, K. Ederle, P.S. Spencer, Studies on the control of myelinogenesis. IV. Neuronal induction of Schwann cell myelin specific protein synthesis during nerve fiber regeneration, J. Neurosci. 2 Ž1982. 1252–1266. w25x R.H. Quarles, J.L. Everly, R.O. Brady, Evidence for the close association of a glycoprotein with myelin in rat brain, J. Neurochem. 21 Ž1973. 1171–1191. w26x M.C. Raff, R.H. Miller, M. Noble, A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on the culture medium, Nature 303 Ž1983. 390–396. w27x M.K. Raizada, J.W. Yang, R.E. Fellows, Binding of w 125 Ixinsulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain, Brain Res. 200 Ž1980. 389–400. w28x R. Reynolds, G.P. Wilkin, Development of macroglial cells in rat cerebellum. II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell, DeÕelopment 102 Ž1988. 409–425. w29x N.H. Sternberger, R.H. Quarles, Y. Itoyama, H. de F. Webster, Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin forming cells of developing rat, Proc. Natl. Acad. Sci. USA 76 Ž1979. 1510–1514. w30x B.D. Trapp, P. Hauer, G. Lemke, Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells, J. Neurosci. 8 Ž1988. 3515–3521. w31x H. Umemori, S. Sato, T. Yagi, S. Aizawa, T. Yamamoto, Initial events of myelination involve Fyn tyrosine kinase signalling, Nature 367 Ž1994. 572–576. w32x H.J. Weinberg, P.S. Spencer, Studies on the control of myelinogenesis. II. Evidence for neuronal regulation of myelin production, Brain Res. 113 Ž1976. 363–378.
Research report
Induction of myelin-associated glycoprotein expression through neuron–oligodendrocyte contact Yoshihiro Matsuda ) , Hisami Koito, Hiroshi Yamamoto Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawa-higashi, Kodaira, Tokyo 187, Japan Accepted 11 February 1997
Abstract The role of neurons on expression of myelin-associated glycoprotein ŽMAG. in oligodendrocytes and oligodendroglial differentiation was examined. Primary cultures of oligodendrocytes prepared from neonatal mouse brains were co-cultured with neuronal cells derived from embryonal carcinoma P19 cells. The levels of MAG mRNAs following this co-culture were determined by reverse transcription ŽRT.-PCR. In oligodendrocytes co-cultured in direct contact with P19-derived neurons, the levels of MAG mRNAs, particularly that of the L-type isoform, were markedly higher than those in cultures without any neuronal cells. On the other hand, when the P19-derived neurons were present, but not in direct contact, no significant induction of MAG expression was found, though oligodendrocytes appeared to mature morphologically. The L-MAG expression was also stimulated when just the neuronal cell membrane fraction was added, which implies that there might be some effecterŽs. in the cell membrane which are possibly exerting a signal transduction for myelin formation. These results suggest that morphological differentiation and functional maturation of oligodendrocytes are due to independent factors. The former is caused by some humoral factorŽs. liberated from neuronal cells, while the latter resulted from cellular contact with neuronal cells. Keywords: Mouse; Development; Myelin-associated glycoprotein; Oligodendrocyte; Neural co-culture; Embryonal carcinoma P19 cell; RT-PCR
1. Introduction It is well established that the oligodendroglial cell lineage is regulated by a variety of environmental factors in the brain before differentiating into myelin-forming cells w7,20,28x. However, little is known about the molecular mechanism of myelinogenesis. Myelin-associated glycoprotein ŽMAG. is one of the myelin-specific proteins expressed by myelin-forming oligodendroglia w25x, specifically at the point of contact where they wrap around axons w29x. This implies its involvement in the process whereby the oligodendroglial cell recognizes the axon to sheath. In rodent brains MAG has been reported to appear at around a week after birth and then immediately increase for the period corresponding to active myelinogenesis. Of the two isoforms, L-MAG is first expressed at the onset of myelin
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Corresponding author. Fax: q81 Ž423. 461753.
formation and slowly decreases thereafter, while S-MAG is synthesized later to become a major component replacing L-MAG in adult brains w12,17x. L-MAG has also been found to be involved in the activation of Fyn, a protein tyrosine kinase of the src family, 4 days after birth, which suggests that the tyrosine phosphorylation cascade might be implicated in signal transduction for myelinogenesis w31x. These findings support the idea that MAG, particularly L-MAG might play a critical role in the initial stage of myelin formation. However, the induction mechanism of MAG expression in oligodendroglia is not understood well yet. In the present study we investigated the contribution of neurons to oligodendroglial maturation and MAG induction in primary cultures of oligodendrocytes. Using embryonal carcinoma P19 cell-derived neuronal cells, we examined the effects of neuron-oligodendrocyte co-cultures, and found that direct contact of neurons with oligodendrocytes was essential for the increase of MAG expression.
0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 0 3 9 - 4
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2. Materials and methods 2.1. Primary culture of oligodendrocytes Primary cultures of mouse brain oligodendrocytes were obtained by the method of Knapps et al. w16x and Bottenstein w2x with some modifications. Briefly, the cerebral cortices removed from neonatal BALBrc mice were enzymatically dissociated in a solution of 0.25% trypsin and 0.001% DNase in isotonic buffer described by Raizada et al. w27x at 378C for 15 min. After being washed with Dulbecco’s modified Eagle’s medium ŽDMEM. containing 10% fetal calf serum ŽFCS., the dissociated cells were sieved through 70 m m pore-sized nylon mesh, and seeded on poly-L-lysine coated culture dishes by 5 = 10 6 cellsr60 mm dishes. After 2 days, the medium was exchanged for a serum-free defined medium consisting of DMEMrHam’s F12 Ž1:1. supplemented with insulin Ž5 m grml., sodium selenite Ž30 nM., transferrin Ž50 m grml., biotin Ž5 m grml., hydrocortisone Ž10 nM., triiodothyronine Ž30 nM., and glucose Ž6 grl.. 2.2. Neural co-culture Mouse embryonal carcinoma P19 cells, provided by Dr. Hamada, Tokyo Metropolitan Institute of Medical Sciences, were maintained in a-minimum essential medium Ž a-MEM. containing 10% FCS, and subcultured every other day, as described by McCarrick and Andrews w22x. The P19 EC cells were differentiated into the cells of neuronal phenotype by culture in aggregation under exposure to low doses of retinoic acid. Briefly, the P19 cells were dispersed with 0.025% trypsin and 1 mM EDTA, and seeded in bacteriological grade Petri dishes at a density of 10 5 cellsrml in medium containing 0.7 m M retinoic acid. After 48 h, the cell aggregates were collected and resuspended in the fresh medium containing 0.7 m M retinoic acid again. After an additional 48 h culture in bacteriological grade Petri dishes, the aggregates were dispersed by treatment with trypsin-EDTA, and added onto the oligodendroglial cultures described above.
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defined medium. The membrane fraction of undifferentiating P19 cells was prepared from the cells cultured in a-MEM containing 10% FCS, which had not treated with retinoic acid. 2.4. Determination of mRNAs by RT-PCR Cultured cells were harvested with cell scrapers, and collected by centrifugation Ž500 = g, 5 min.. Total cellular RNA was extracted with RNAzol B ŽBiotecx Lab., USA. according to the supplier’s instruction. cDNA was prepared with random hexadeoxynucleotide primers and Moloney murine leukemia virus reverse transcriptase ŽGIBCO-BRL, USA.. Thirty cycles of PCR were carried out with Ampli-Taq DNA polymerase ŽPerkin Elmer Corp., USA. in a programmable heat block; the cycle profile was denaturation at 938C for 1 min, annealing at 658C for 1 min, and elongation at 728C for 2 min. The following synthetic oligonucleotides were used as primers, which resulted in two different cDNAs amplified for MAG isoforms Ž239 bp for S-MAG and 194 bp for L-MAG.: 5X -GCCCCGAATTCAGAATCTCTGG-3X and 5X TTCTGCATACTCAGCCAGC-3X . The PCR products were fractionated by electrophoresis on 6% polyacrylamide gel, stained with 1 m grml ethidium bromide, and analyzed quantitatively by densitometry. The amount of total RNA used in this RT-PCR assay was fixed to give linear dosedependence of cDNAs amplified for MAG mRNAs. Typically, 10 ng total RNA was used in the present study. Proteolipid protein ŽPLP. mRNAs were also determined by RT-PCR with the following synthetic oligonucleotides for PCR primers: 5X-ACTACAAGACCACCATCTGCG-3X and
2.3. Preparation of membrane fraction of P19 cells After the P19 cells were cultured in aggregation in the presence of retinoic acid for 4 days, as described above, they were dispersed, suspended in DMEMrF12 Ž1:1. containing 5% FCS, and plated on poly-L-lysine-coated culture dishes by 6 = 10 6 cellsr100 mm diameter dish. After 2 days the medium containing 10 m M cytosine arabinoside was added, and then the cells were collected 2–3 days later. The collected cells were suspended in the serum-free defined medium Ž0.6 mlrcell pellet obtained from 100 mm diameter culture dish., and disrupted by freezing-and-thawing. The homogenate was centrifuged at 12,000 rpm for 15 min, and the pellet was resuspended in the serum-free
Fig. 1. Effects of neural co-culture on expression of MAG mRNAs in primary cultures of oligodendrocytes. On day 5 of the oligodendroglial primary culture, 2=10 6 of P19 cells which had been treated with retinoic acid were added per 60 mm diameter dish, and co-culture was carried out for indicated periods. The expression of L-MAG and S-MAG mRNAs in oligodendrocytes with and without P19-derived neuronal cells was determined by RT-PCR. Results are expressed in relative terms, and represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG mRNA in the cells of 2 days in the absence of P19 neurons was defined as 100%.
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different cDNAs simultaneously amplified for the isoforms, PLP and DM20 were analyzed on 6% polyacrylamide gel electrophoresis Ž286 bp for PLP and 181 bp for DM20.. As internal standard cDNA of b-actin was amplified by 20 cycles of PCR.
3. Results
Fig. 2. Effects of neural co-culture on expression of PLP mRNAs in primary cultures of oligodendrocytes. The cells were prepared, as described in Fig. 1. The expression of PLP and DM20 mRNAs was determined by RT-PCR. Results are expressed in relative terms, and represent the means"S.E.M. of 4 culture dishes. The amount of PLP mRNA in the cells of 2 days in the absence of P19 neurons was defined as 100%.
5X-CCTATACTGGCAGAGGTCTTGC-3X . The conditions of PCR were the same as that for MAG, except that amplification was carried out with 25 reaction cycles. Two
Fig. 1 shows the effect of co-culturing oligodendrocytes with neuronal cells on the expression of MAG mRNAs. Oligodendrocytes in the primary culture had low levels of MAG mRNAs, and increased the expression slowly with the culture period. On the other hand, the cells showed significant increases of MAG mRNAs when cultured with P19-derived neuronal cells. After being co-cultured for 4 days and 7 days, expressions of L-MAG were about 3 and 4.5 times greater, respectively, as much as those in oligodendrocytes alone. There was no difference between the co-culture and the single culture on day 2. It seems likely that the P19 cells were not able to affect oligodendrocytes at that early stage of 2 days, since they were not morpho-
Fig. 3. Effect of neural co-culture on oligodendroglial differentiation. A: the primary culture of oligodendrocytes of 11 DIV Ždays in vitro. without co-culture. B: oligodendrocytes co-cultured for 7 days separately with P19-derived neuronal cells which were grown on a cell culture insert. The total culture period was 11 days, since the co-culture started on day 5. Scale bar in ŽB., 50 m m.
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logically differentiated enough. The P19 cells did not express detectable MAG mRNAs during these culture periods. Proteolipid protein ŽPLP., another myelin-specific protein, was also induced in the co-culture. As shown in Fig. 2, the PLP expression in the primary cultures of oligodendrocytes slowly increased with the culture period, and the increases were markedly magnified when cultured with P19-derived neuronal cells. Neither PLP nor DM20 mRNA was found in the P19 cells during these culture periods. To elucidate whether it is the contact between oligodendrocytes and neuronal cells or some humoral factorŽs. liberated from neuronal cells which is required for the induction of MAG expression, the effect of separate co-culture was investigated using Falcon cell culture inserts ŽBecton Dickinson, USA.. Fig. 3 shows a morphological change under the co-culture condition. In the primary culture of 11 days, oligodendrocytes in the single culture were still immature; most of the cells were bipolar or extended just a few dendrites ŽFig. 3A.. After co-culture for 7 days following 4 days single culture, on the other hand, lots of morphologically matured cells with many refined dendrites were observed ŽFig. 3B., suggesting that neuronal cells stimulated differentiation of oligodendrocytes by liberating some soluble factorŽs. into the culture medium. However, as shown in Fig. 4, oligodendrocytes in the separate co-culture did not show any significant changes in the MAG expression despite their morphologically differentiated appearance. Under the contact condition, on the other hand, a marked increase of MAG mRNAs was confirmed. This suggests that morphological differentiation was not necessarily associated with functional maturation of oligodendrocytes as myelin-forming cells, and that the cellular contact exerted some signal transduction resulting
Fig. 4. Effects of co-culture conditions on MAG induction in primary cultures of oligodendrocytes. The expression of MAG mRNAs in oligodendrocytes without co-culture and with co-culture for 4 days under the separate and contact conditions was determined by RT-PCR. Results are expressed in relative terms, and represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG in the cell preparations of oligodendrocytes Žoligo.. alone was defined as 100%.
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Fig. 5. Dose dependence of neuronal cells on MAG induction in oligodendrocytes. Indicated numbers of P19 cells were added on the primary cultures of oligodendrocytes on day 5, and co-cultured for 4 days. The relative amounts of MAG mRNAs were determined by RT-PCR. Data represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG in the cell preparations with no added P19 cells was defined as 100%.
in induction of the MAG expression. In order to see whether efficient cellular contact can cause a large induction of MAG, next examined was the dose dependence of P19 cells in co-culture. As shown in Fig. 5, the more neuronal cells were added onto oligodendrocytes, the more gene expression was observed. Although the number of oligodendrocytes could not be determined in the primary culture, the number of P19 cells added was always greater throughout the experimental conditions. However, when 2 = 10 5 P19 cells were added, their distribution was so sparse that contact with oligodendrocytes appeared to be rare. In addition, neuronal cells increased their axon extensions to make dense neuronal networks, when the P19 cells were plated in larger numbers. Assuming that the contact of oligodendrocytes with neuronal axons was important, it also appears that the more the P19 cells, the greater the effect. MAG was also induced by adding the membrane fraction of P19-derived neuronal cells. As shown in Fig. 6, the expression of L-MAG markedly increased when the membranous fraction of neurons was added. The soluble fraction gave no significant effects. Furthermore, the P19 cells of non-neuronal phenotype, which had not been treated with retinoic acid, did not have any effects at all. These results highlight how important direct contact between oligodendroglia and neurons is for MAG expression. Interestingly, in this experiment using the membranous fraction, MAG induction occurred in 2 days. In the case of co-culture, on the other hand, no significant change was found in 2 days ŽFig. 1.. This suggests that there are some effector moleculeŽs. on the cell membranes of neurons, which give a signal to induce MAG expression in oligodendrocytes, and that it takes more than 4 days for P19 cells to express them on their cell surfaces including
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Fig. 6. Effect of the membrane fraction of P19 cells on MAG expression. The P19 cells of neuronal phenotype and of undifferentiation were collected, and disrupted by freezing-and-thawing. After centrifugation at 12,000 rpm for 15 min, the pellets and supernatants were used as the membranous and soluble fractions, respectively. The cell pellets resuspended in serum-free defined medium and the supernatants containing 2 mg proteins each were added to oligodendroglial cultures of 5 DIV in 35-mm-diameter dishes, and the cells were harvested 2 days later. The relative amounts of MAG mRNAs were determined by RT-PCR. Data represent the means"S.E.M. of 4 culture dishes. The amount of L-MAG in the cell preparations of oligodendrocytes Žoligo.. alone was defined as 100%.
axonal membranes, probably because the effectorŽs. are newly synthesized as P19 cells differentiate into neurons. 4. Discussion It has been demonstrated that both soluble factors produced by neurons and specific forms of contact with neurons are necessary for oligodendrocytes to differentiate w9x. However, information on the initiation of myelination is still sparse. In the later stages of brain development, MAG has been reported to be synthesized at a period which is coincident with that of myelinogenesis w12x, and to be localized in the periaxonal portion of myelin sheaths w29x. This suggests that it plays a critical role in myelin formation. However, little is understood about the regulation of MAG expression in oligodendroglia so far. The present study found evidence that the MAG expression in oligodendrocytes can be induced through direct contact with neuronal cells in vitro. It has been proposed that differentiation of oligodendrocytes, such as process extension and myelin protein syntheses, might proceed under their own intrinsic signals in primary culture w15,18,26x. This is most probably true, since in the present study some of the cells showed morphological differentiation in 1–2 weeks, but the rate for the total progenitor cells was not large. In addition, the expression of myelin-specific proteins in the culture cells was much lower than that in vivo. The level of L-MAG mRNA in the culture cells of 6 days in vitro ŽDIV. was similar to that in the forebrains of mice of post-natal day ŽPND. 7, but the level in the culture cells
of 11 DIV was less than a tenth that of the brains of PND 12, the period that myelination actively proceeds Ždata not shown.. On the other hand, the levels of S-MAG in the culture cells of 6, 8 and 11 DIV were comparable to those in the brains of PND 7, 9 and 12, respectively Ždata not shown.. This suggests that some environmental factorŽs., such as neuron–glia interactions, might be required for the induction of L-MAG expression during the period of myelinogenesis. Ellison et al. w5x recently reported that neurons expressed platelet-derived growth factor ŽPDGF. to support oligodendroglial development, and suggested that by liberating PDGF, neurons regulated the generation of appropriate numbers of differentiated oligodendrocytes needed to myelinate axons during cerebral cortex development. It was also observed in the present study that neuronal cells appeared to liberate some soluble factorŽs. stimulating process extension of oligodendrocytes. However, despite the distinct change in the morphological phenotype the levels of MAG mRNAs were not significantly increased in the co-culture with neuronal cells under the separate condition ŽFigs. 3 and 4.. Only when oligodendrocytes could come into contact with neurons, was the MAG expression up-regulated. These findings suggest that neurons might influence oligodendrocytes on morphological differentiation and functional maturation by different mechanisms. From the time course of increase of MAG mRNA in co-culture, it can be seen that it takes 4 days or more for P19 cells to give signalŽs. for MAG induction to oligodendrocytes ŽFig. 1.. On the other hand, when the membrane fraction of P19-derived neuronal cells was added, the increase of MAG expression became significant in just 2 days ŽFig. 6.. In addition, co-culture with nonneuronal P19 cells which had not been treated with retinoic acid did not alter the MAG expression in oligodendrocytes. These results suggest that some effector moleculeŽs. might be newly synthesized in fully differentiated neurons. In the process of differentiation into the neuronal phenotype to make neuronal networks, the effector moleculeŽs. may be expressed on the cell surface including axonal membranes. It has been reported that MAG has two isoforms resulting from alternative splicing of mRNA w17x, and that L-MAG is selectively up-regulated in the period of myelinogenesis in the developing brain w6,13x. In the present study, the expression of L-MAG was preferentially induced, but SMAG was also increased in co-culture. On the other hand, only L-MAG was increased when the membrane fraction was added. This suggests that the membrane fraction simply contained effector moleculeŽs. which induce the LMAG expression for myelination. On the other hand, neurons in co-culture may have complex effects on oligodendroglia. Various modulatory functions have been attributed to neurons on oligodendroglial lineage w8,10,11,19x; they enhance proliferation, migration, and differentiation of oligodendroglial progenitors as well as stimulating process extension and myelin protein synthesis in mature oligodendroglia. It was also observed that the
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brains of mouse embryos ŽE15–20. and neonates had chiefly S-MAG mRNA ŽKoito et al., unpublished data., suggesting that S-MAG might be preferentially expressed in immature oligodendroglia. Accordingly, in co-culture, the number of immature oligodendrocytes containing SMAG might be increased under the influence of neuronal cells. In the peripheral nervous system, the axon has been shown to be the major regulator of myelin gene expression. The Schwann cells primarily required contact with appropriate axons for significant expression of myelin genes w3,32x, and the myelin components were rapidly down-regulated when the ensheathed axon degenerated w24,30x. In contrast, it has been demonstrated that in oligodendrocytes, synthesis of myelin components occurs in the absence of axons, indicating that myelin gene expression is not necessarily dependent on axonal contact w1,4x. By axotomy, however, the influences of axons on myelin gene expression in oligodendroglia have been evidenced in vivo w14,23x; following optic nerve transection, the myelin protein mRNA levels declined in transected nerves, though significant levels of the mRNAs were found even after 40 days. These findings suggest that axons might have modulatory effects on myelin gene expression in the central nervous system as well. On the other hand, Macklin et al. w21x examined expression of myelin proteolipid protein ŽPLP. and basic protein ŽMBP. mRNAs in cultured cells by dot hybridization analysis, and showed that by co-culturing rat oligodendrocytes with chick spinal cord neurons the levels of these mRNAs were elevated 4-fold. Although their experimental conditions were different from ours in that they used enriched oligodendroglial cell populations, cultured the cells for longer period Ž20 days in total including 11 days of co-culture., and co-cultured oligodendrocytes and neuronal cells from different animal species, their results are in agreement with our present data on MAG. In the present study, to imitate the developmental environment of oligodendrocytes, we studied primary cultures of mouse brain, though the content of oligodendrocytes was not very high. In addition, we clarified the effects of neurons; morphological change was brought about by soluble factorŽs. liberated from neuronal cells, and MAG expression was induced by direct contact with the neuronal cell membrane. We also examined the expression of PLP isoform mRNAs, and found that PLP markedly increased in the co-culture but DM20 did not increase much. These findings support the idea that contact with neurons is required for functional maturation of oligodendroglia for myelination. Identification of the molecules involved in the cellular contact awaits further investigation.
5. Summary Myelin-associated glycoprotein ŽMAG. is considered to be a cell adhesion molecule linking myelin to axon mem-
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branes. Its expression by oligodendrocytes is thought to be critical in the initial stage of myelin formation, but little is known about the mechanism. Although in developing mouse brains in vivo, oligodendroglia express increased quantities of MAG during the process of myelinogenesis, in vitro primary cultures of oligodendrocytes prepared from neonatal brains show no such increase. The effects of neuronal cells on oligodendroglial differentation and MAG expression were examined by co-culturing oligodendrocytes with neuronal cells derived from embryonal carcinoma P19 cells. When oligodendrocytes were co-cultured in direct contact with these neuronal cells, they showed increased expression of MAG mRNA, whereas no such increase was found when the cells were co-cultured without direct contact. However, even when co-cultured without direct contact, most oligodendrocytes developed a morphologically differentiated phenotype with many refined dendrites. These results suggest that neuronal cells might be producing some humoral factors which induce oligodendroglial progenitors to differentiate morphologically. However, for functional maturation with myelin formation, oligodendrocytes require direct contact with neuronal cells.
Acknowledgements The authors thank Dr. Akira Hamada, Tokyo Metropolitan Institute of Medical Sciences, for providing embryonal carcinoma P19 cells, and are grateful to Dr. Marcus Wenner for critical reading of the manuscript.
References w1x E.R. Abney, P.P. Bartlett, M.C. Raff, Astrocytes, ependymal cells, and oligodendrocytes develop on schedule in dissociated cell cultures of embryonic rat brain, DeÕ. Biol. 83 Ž1981. 301–310. w2x J.E. Bottenstein, Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes, Proc. Natl. Acad. Sci. USA 83 Ž1986. 1955–1959. w3x J.P. Brockes, M.C. Raff, D.J. Nishiguchi, J. Winter, Studies on cultured rat Schwann cells. III. Assay for peripheral myelin proteins, J. Neurocytol. 9 Ž1980. 67–77. w4x M. Dubois-Dalcq, T. Behar, L. Hudson, R.A. Lazzarini, Emergence of three myelin proteins in oligodendrocytes cultured without neurons, J. Cell Biol. 102 Ž1986. 384–392. w5x J.A. Ellison, S.A. Scully, J. de Vellis, Evidence for neuronal regulation of oligodendrocyte development: cellular localization of platelet-derived growth factor a receptor and A-chain mRNA during cerebral cortex development in the rat, J. Neurosci. Res. 45 Ž1996. 28–39. w6x N. Fujita, S. Sato, T. Kurihara, T. Inuzuka, Y. Takahashi, T. Miyatake, Developmentally regulated alternative splicing of brain myelin-associated glycoprotein mRNA is lacking in the quaking mouse, FEBS Lett. 232 Ž1988. 323–327. w7x R. Hardy, R. Reynolds, Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo, DeÕelopment 111 Ž1991. 1061–1080.
116
Y. Matsuda et al.r DeÕelopmental Brain Research 100 (1997) 110–116
w8x R. Hardy, R. Reynolds, Rat cerebral cortical neurons in primary culture release a mitogen specific for early ŽGD3qrO4y. oligodendroglial progenitors, J. Neurosci. Res. 34 Ž1993. 589–600. w9x R. Hardy, R. Reynolds, Neuron-oligodendroglial interactions during central nervous system development, J. Neurosci. Res. 36 Ž1993. 121–126. w10x S.F. Hunter, J.E. Bottenstein, Bipotential glial progenitors are targets of neuronal cell line-derived growth factors, DeÕ. Brain Res. 49 Ž1989. 33–49. w11x S.F. Hunter, J.E. Bottenstein, O-2A glial progenitors from mature brain respond to CNS neuronal cell-derived growth factors, J. Neurosci. Res. 28 Ž1991. 574–582. w12x T. Inuzuka, N. Fujita, S. Sato, H. Baba, R. Nakano, H. Ishiguro, T. Miyatake, Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system, Brain Res. 562 Ž1991. 173–175. w13x H. Ishiguro, S. Sato, N. Fujita, T. Inuzuka, R. Nakano, T. Miyatake, Immunohistochemical localization of myelin-associated glycoprotein isoforms during the development in the mouse brain, Brain Res. 563 Ž1991. 288–292. w14x G.J. Kidd, P.E. Hauer, B.D. Trapp, Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo, J. Neurosci. Res. 26 Ž1990. 409–418. w15x P.E. Knapp, W.P. Bartlett, R.P. Skoff, Cultured oligodendrocytes mimic in vivo phenotypic characteristics: cell shape, expression of myelin-specific antigens, and membrane production, DeÕ. Biol. 120 Ž1987. 356–365. w16x P.E. Knapp, R.P. Skoff, T.J. Sprinkle, Differential expression of galactocerebroside, myelin basic protein, and 2X ,3X-cyclic nucleotide X 3 -phosphohydrolase during development of oligodendrocytes in vitro, J. Neurosci. Res. 21 Ž1988. 249–259. w17x C. Lai, M.A. Brow, K.-A. Nave, A.B. Noronha, R.H. Quarles, F.E. Bloom, R.J. Milner, J.G. Sutcliffe, Two forms of 1B236rmyelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing, Proc. Natl. Acad. Sci. USA 84 Ž1987. 4337–4341. w18x G. Levi, F. Aloisi, G.P. Wilkin, Differentiation of cerebellar bipotential glial precursors into oligodendrocytes in primary culture: developmental profile of surface antigens and mitotic activity, J. Neurosci. Res. 18 Ž1987. 407–417. w19x J.M. Levine, Neuronal influences on glial progenitor cell development, Neuron 3 Ž1989. 103–113. w20x J.M. Levine, J.E. Goldman, Spatial and temporal patterns of oligodendrocyte differentiation in rat cerebrum and cerebellum, J. Comp. Neurol. 277 Ž1988. 441–455.
w21x W.B. Macklin, C.L. Weill, P.L. Deininger, Expression of myelin proteolipid and basic protein mRNAs in cultured cells, J. Neurosci. Res. 16 Ž1986. 203–217. w22x McCarrick, J.W. and Andrews, P.W., Embryonal carcinoma cells and embryonic stem cells as models for neuronal development and function. In J.N. Wood ŽEd.., Neuronal Cell Lines: A Practical Approach, Oxford University Press, Oxford, 1992, pp. 77–104. w23x K. McPhilemy, L.S. Mitchell, I.R. Griffiths, S. Morrison, A.W. Deary, I. Sommer, P.G.E. Kennedy, Effect of optic nerve transection upon myelin protein gene expression by oligodendrocytes: evidence for axonal influences on gene expression, J. Neurocytol. 19 Ž1990. 494–503. w24x M.J. Politis, N. Sternberger, K. Ederle, P.S. Spencer, Studies on the control of myelinogenesis. IV. Neuronal induction of Schwann cell myelin specific protein synthesis during nerve fiber regeneration, J. Neurosci. 2 Ž1982. 1252–1266. w25x R.H. Quarles, J.L. Everly, R.O. Brady, Evidence for the close association of a glycoprotein with myelin in rat brain, J. Neurochem. 21 Ž1973. 1171–1191. w26x M.C. Raff, R.H. Miller, M. Noble, A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on the culture medium, Nature 303 Ž1983. 390–396. w27x M.K. Raizada, J.W. Yang, R.E. Fellows, Binding of w 125 Ixinsulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain, Brain Res. 200 Ž1980. 389–400. w28x R. Reynolds, G.P. Wilkin, Development of macroglial cells in rat cerebellum. II. An in situ immunohistochemical study of oligodendroglial lineage from precursor to mature myelinating cell, DeÕelopment 102 Ž1988. 409–425. w29x N.H. Sternberger, R.H. Quarles, Y. Itoyama, H. de F. Webster, Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin forming cells of developing rat, Proc. Natl. Acad. Sci. USA 76 Ž1979. 1510–1514. w30x B.D. Trapp, P. Hauer, G. Lemke, Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells, J. Neurosci. 8 Ž1988. 3515–3521. w31x H. Umemori, S. Sato, T. Yagi, S. Aizawa, T. Yamamoto, Initial events of myelination involve Fyn tyrosine kinase signalling, Nature 367 Ž1994. 572–576. w32x H.J. Weinberg, P.S. Spencer, Studies on the control of myelinogenesis. II. Evidence for neuronal regulation of myelin production, Brain Res. 113 Ž1976. 363–378.