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. 1986 Feb 1;102(2):384–392. doi: 10.1083/jcb.102.2.384

Emergence of three myelin proteins in oligodendrocytes cultured without neurons

PMCID: PMC2114066  PMID: 2418030

Abstract

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin- associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.

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Selected References

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  1. Abney E. R., Bartlett P. P., Raff M. C. Astrocytes, ependymal cells, and oligodendrocytes develop on schedule in dissociated cell cultures of embryonic rat brain. Dev Biol. 1981 Apr 30;83(2):301–310. doi: 10.1016/0012-1606(81)90476-0. [DOI] [PubMed] [Google Scholar]
  2. Abney E. R., Williams B. P., Raff M. C. Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting, and cell culture. Dev Biol. 1983 Nov;100(1):166–171. doi: 10.1016/0012-1606(83)90207-5. [DOI] [PubMed] [Google Scholar]
  3. Agrawal H. C., Hartman B. K., Shearer W. T., Kalmbach S., Margolis F. L. Purification and immunohistochemical localization of rat brain myelin proteolipid protein. J Neurochem. 1977 Mar;28(3):495–508. doi: 10.1111/j.1471-4159.1977.tb10420.x. [DOI] [PubMed] [Google Scholar]
  4. Arnheiter H., Dubois-Dalcq M., Lazzarini R. A. Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies. Cell. 1984 Nov;39(1):99–109. doi: 10.1016/0092-8674(84)90195-8. [DOI] [PubMed] [Google Scholar]
  5. Barbarese E., Braun P. E., Carson J. H. Identification of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3360–3364. doi: 10.1073/pnas.74.8.3360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barbarese E., Pfeiffer S. E. Developmental regulation of myelin basic protein in dispersed cultures. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1953–1957. doi: 10.1073/pnas.78.3.1953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bologa-Sandru L., Siegrist H. P., Z'Graggen A., Hofmann K., Wiesmann U., Dahl D., Herschkowitz N. Expression of antigenic markers during the development of oligodendrocytes in mouse brain cell cultures. Brain Res. 1981 Apr 6;210(1-2):217–229. doi: 10.1016/0006-8993(81)90895-7. [DOI] [PubMed] [Google Scholar]
  8. Bologa L., Bisconte J. C., Joubert R., Margules S., Herschkowitz N. Proliferative activity and characteristics of immunocytochemically identified oligodendrocytes in embryonic mouse brain cell cultures. Exp Brain Res. 1983;50(1):84–90. doi: 10.1007/BF00238234. [DOI] [PubMed] [Google Scholar]
  9. Bradel E. J., Prince F. P. Cultured neonatal rat oligodendrocytes elaborate myelin membrane in the absence of neurons. J Neurosci Res. 1983;9(4):381–392. doi: 10.1002/jnr.490090404. [DOI] [PubMed] [Google Scholar]
  10. Brockes J. P., Fryxell K. J., Lemke G. E. Studies on cultured Schwann cells: the induction of myelin synthesis, and the control of their proliferation by a new growth factor. J Exp Biol. 1981 Dec;95:215–230. doi: 10.1242/jeb.95.1.215. [DOI] [PubMed] [Google Scholar]
  11. Campagnoni A. T., Carey G. D., Yu Y. T. In vitro synthesis of the myelin basic proteins: subcellular site of synthesis. J Neurochem. 1980 Mar;34(3):677–686. doi: 10.1111/j.1471-4159.1980.tb11197.x. [DOI] [PubMed] [Google Scholar]
  12. Campagnoni A. T., Hunkeler M. J. Synthesis of the myelin proteolipid protein in the developing mouse brain. J Neurobiol. 1980 Jul;11(4):355–364. doi: 10.1002/neu.480110403. [DOI] [PubMed] [Google Scholar]
  13. Carey D. J., Eldridge C. F., Cornbrooks C. J., Timpl R., Bunge R. P. Biosynthesis of type IV collagen by cultured rat Schwann cells. J Cell Biol. 1983 Aug;97(2):473–479. doi: 10.1083/jcb.97.2.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Colman D. R., Kreibich G., Frey A. B., Sabatini D. D. Synthesis and incorporation of myelin polypeptides into CNS myelin. J Cell Biol. 1982 Nov;95(2 Pt 1):598–608. doi: 10.1083/jcb.95.2.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eccleston P. A., Silberberg D. H. The differentiation of oligodendrocytes in a serum-free hormone-supplemented medium. Brain Res. 1984 Sep;318(1):1–9. doi: 10.1016/0165-3806(84)90056-7. [DOI] [PubMed] [Google Scholar]
  16. Fritz R. B., Chou C. H. Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies. J Immunol. 1983 May;130(5):2180–2182. [PubMed] [Google Scholar]
  17. Fry J. M., Lisak R. P., Manning M. C., Silberberg D. H. Serological techniques for detection of antibody to galactocerebroside. J Immunol Methods. 1976;11(2):185–193. doi: 10.1016/0022-1759(76)90146-0. [DOI] [PubMed] [Google Scholar]
  18. Guentert-Lauber B., Monnet-Tschudi F., Omlin F. X., Favrod P., Honegger P. Serum-free aggregate cultures of rat CNS glial cells: biochemical, immunocytochemical and morphological characterization. Dev Neurosci. 1985;7(1):33–44. doi: 10.1159/000112274. [DOI] [PubMed] [Google Scholar]
  19. Hartman B. K., Agrawal H. C., Agrawal D., Kalmbach S. Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4217–4220. doi: 10.1073/pnas.79.13.4217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hartman B. K., Agrawal H. C., Kalmbach S., Shearer W. T. A comparative study of the immunohistochemical localization of basic protein to myelin and oligodendrocytes in rat and chicken brain. J Comp Neurol. 1979 Nov 15;188(2):273–290. doi: 10.1002/cne.901880206. [DOI] [PubMed] [Google Scholar]
  21. Ilyas A. A., Quarles R. H., MacIntosh T. D., Dobersen M. J., Trapp B. D., Dalakas M. C., Brady R. O. IgM in a human neuropathy related to paraproteinemia binds to a carbohydrate determinant in the myelin-associated glycoprotein and to a ganglioside. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1225–1229. doi: 10.1073/pnas.81.4.1225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laursen R. A., Samiullah M., Lees M. B. The structure of bovine brain myelin proteolipid and its organization in myelin. Proc Natl Acad Sci U S A. 1984 May;81(9):2912–2916. doi: 10.1073/pnas.81.9.2912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McCarthy K. D., de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 1980 Jun;85(3):890–902. doi: 10.1083/jcb.85.3.890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McMorris F. A. Cyclic AMP induction of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase in rat oligodendrocytes. J Neurochem. 1983 Aug;41(2):506–515. doi: 10.1111/j.1471-4159.1983.tb04768.x. [DOI] [PubMed] [Google Scholar]
  25. Mirsky R., Winter J., Abney E. R., Pruss R. M., Gavrilovic J., Raff M. C. Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture. J Cell Biol. 1980 Mar;84(3):483–494. doi: 10.1083/jcb.84.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mithen F. A., Wood P. M., Agrawal H. C., Bunge R. P. Immunohistochemical study of myelin sheaths formed by oligodendrocytes interacting with dissociated dorsal root ganglion neurons in culture. Brain Res. 1983 Feb 28;262(1):63–69. doi: 10.1016/0006-8993(83)90469-9. [DOI] [PubMed] [Google Scholar]
  27. Norton W. T., Farooq M., Fields K. L., Raine C. S. The long term culture of bulk-isolated bovine oligodendroglia from adult brain. Brain Res. 1983 Jul 4;270(2):295–310. doi: 10.1016/0006-8993(83)90604-2. [DOI] [PubMed] [Google Scholar]
  28. Omlin F. X., Webster H. D., Palkovits C. G., Cohen S. R. Immunocytochemical localization of basic protein in major dense line regions of central and peripheral myelin. J Cell Biol. 1982 Oct;95(1):242–248. doi: 10.1083/jcb.95.1.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pfeiffer S. E., Barbarese E., Bhat S. Noncoordinate regulation of myelinogenic parameters in primary cultures of dissociated fetal rat brain. J Neurosci Res. 1981;6(3):369–380. doi: 10.1002/jnr.490060312. [DOI] [PubMed] [Google Scholar]
  30. Poduslo J. F., Berg C. T., Dyck P. J. Schwann cell expression of a major myelin glycoprotein in the absence of myelin assembly. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1864–1866. doi: 10.1073/pnas.81.6.1864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Poduslo S. E., Miller K., Wolinsky J. S. The production of a membrane by purified oligodendroglia maintained in culture. Exp Cell Res. 1982 Jan;137(1):203–215. doi: 10.1016/0014-4827(82)90021-0. [DOI] [PubMed] [Google Scholar]
  32. Poduslo S. E., Miller K., Zoller S. Purification and maintenance in culture of oligodendroglia from human multiple sclerosis brain. J Neurol Sci. 1982 Jun;54(3):395–400. doi: 10.1016/0022-510x(82)90202-7. [DOI] [PubMed] [Google Scholar]
  33. Quarles R. H. Glycoproteins from central and peripheral myelin. Prog Clin Biol Res. 1980;49:55–77. [PubMed] [Google Scholar]
  34. Quarles R. H. Myelin-associated glycoprotein in development and disease. Dev Neurosci. 1983;6(6):285–303. doi: 10.1159/000112356. [DOI] [PubMed] [Google Scholar]
  35. Raff M. C., Fields K. L., Hakomori S. I., Mirsky R., Pruss R. M., Winter J. Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture. Brain Res. 1979 Oct 5;174(2):283–308. doi: 10.1016/0006-8993(79)90851-5. [DOI] [PubMed] [Google Scholar]
  36. Raff M. C., Miller R. H., Noble M. A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature. 1983 Jun 2;303(5916):390–396. doi: 10.1038/303390a0. [DOI] [PubMed] [Google Scholar]
  37. Raff M. C., Mirsky R., Fields K. L., Lisak R. P., Dorfman S. H., Silberberg D. H., Gregson N. A., Leibowitz S., Kennedy M. C. Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture. Nature. 1978 Aug 24;274(5673):813–816. [PubMed] [Google Scholar]
  38. Raff M. C., Williams B. P., Miller R. H. The in vitro differentiation of a bipotential glial progenitor cell. EMBO J. 1984 Aug;3(8):1857–1864. doi: 10.1002/j.1460-2075.1984.tb02059.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ranscht B., Clapshaw P. A., Price J., Noble M., Seifert W. Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2709–2713. doi: 10.1073/pnas.79.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Richardson C. D., Berkovich A., Rozenblatt S., Bellini W. J. Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus. J Virol. 1985 Apr;54(1):186–193. doi: 10.1128/jvi.54.1.186-193.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roussel G., Nussbaum J. L. Comparative localization of Wolfgram W1 and myelin basic proteins in the rat brain during ontogenesis. Histochem J. 1981 Nov;13(6):1029–1047. doi: 10.1007/BF01002642. [DOI] [PubMed] [Google Scholar]
  42. Roussel G., Sensenbrenner M., Labourdette G., Wittendorp-Rechenmann E., Pettmann B., Nussbaum J. L. An immunohistochemical study of two myelin-specific proteins in enriched oligodendroglial cell cultures combined with an autoradiographic investigation using [3H]thymidine. Brain Res. 1983 Jun;284(2-3):193–204. doi: 10.1016/0165-3806(83)90004-4. [DOI] [PubMed] [Google Scholar]
  43. Sarliève L. L., Fabre M., Susz J., Matthieu J. M. Investigations on myelination in vitro: IV. "Myelin-like" or premyelin structures in cultures of dissociated brain cells from 14--15-day-old embryonic mice. J Neurosci Res. 1983;10(2):191–210. doi: 10.1002/jnr.490100208. [DOI] [PubMed] [Google Scholar]
  44. Sobue G., Pleasure D. Schwann cell galactocerebroside induced by derivatives of adenosine 3',5'-monophosphate. Science. 1984 Apr 6;224(4644):72–74. doi: 10.1126/science.6322307. [DOI] [PubMed] [Google Scholar]
  45. Sternberger N. H., Itoyama Y., Kies M. W., Webster H deF Immunocytochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat C.N.S. J Neurocytol. 1978 Apr;7(2):251–263. doi: 10.1007/BF01217922. [DOI] [PubMed] [Google Scholar]
  46. Sternberger N. H., Itoyama Y., Kies M. W., Webster H. D. Myelin basic protein demonstrated immunocytochemically in oligodendroglia prior to myelin sheath formation. Proc Natl Acad Sci U S A. 1978 May;75(5):2521–2524. doi: 10.1073/pnas.75.5.2521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sternberger N. H., Quarles R. H., Itoyama Y., Webster H. D. Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1510–1514. doi: 10.1073/pnas.76.3.1510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stoffel W., Hillen H., Giersiefen H. Structure and molecular arrangement of proteolipid protein of central nervous system myelin. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5012–5016. doi: 10.1073/pnas.81.16.5012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Temple S., Raff M. C. Differentiation of a bipotential glial progenitor cell in a single cell microculture. Nature. 1985 Jan 17;313(5999):223–225. doi: 10.1038/313223a0. [DOI] [PubMed] [Google Scholar]
  50. Tennekoon G. I., Cohen S. R., Price D. L., McKhann G. M. Myelinogenesis in optic nerve. A morphological, autoradiographic, and biochemical analysis. J Cell Biol. 1977 Mar;72(3):604–616. doi: 10.1083/jcb.72.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Townsend L. E., Agrawal D., Benjamins J. A., Agrawal H. C. In vitro acylation of rat brain myelin proteolipid protein. J Biol Chem. 1982 Aug 25;257(16):9745–9750. [PubMed] [Google Scholar]
  52. Trapp B. D., Quarles R. H. Immunocytochemical localization of the myelin-associated glycoprotein. Fact or artifact? J Neuroimmunol. 1984 Jul;6(4):231–249. doi: 10.1016/0165-5728(84)90011-0. [DOI] [PubMed] [Google Scholar]
  53. Trapp B. D., Quarles R. H. Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin. J Cell Biol. 1982 Mar;92(3):877–882. doi: 10.1083/jcb.92.3.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Trapp B. D., Quarles R. H., Suzuki K. Immunocytochemical studies of quaking mice support a role for the myelin-associated glycoprotein in forming and maintaining the periaxonal space and periaxonal cytoplasmic collar of myelinating Schwann cells. J Cell Biol. 1984 Aug;99(2):594–606. doi: 10.1083/jcb.99.2.594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Trapp B. D., Webster H. D., Johnson D., Quarles R. H., Cohen S. R., Murray M. R. Myelin formation in rotation-mediated aggregating cell cultures: immunocytochemical, electron microscopic, and biochemical observations. J Neurosci. 1982 Jul;2(7):986–993. doi: 10.1523/JNEUROSCI.02-07-00986.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wood P. M., Williams A. K. Oligodendrocyte proliferation and CNS myelination in cultures containing dissociated embryonic neuroglia and dorsal root ganglion neurons. Brain Res. 1984 Feb;314(2):225–241. doi: 10.1016/0165-3806(84)90045-2. [DOI] [PubMed] [Google Scholar]
  57. Wood P., Okada E., Bunge R. The use of networks of dissociated rat dorsal root ganglion neurons to induce myelination by oligodencrocytes in culture. Brain Res. 1980 Aug 25;196(1):247–252. doi: 10.1016/0006-8993(80)90732-5. [DOI] [PubMed] [Google Scholar]
  58. Zeller N. K., Behar T. N., Dubois-Dalcq M. E., Lazzarini R. A. The timely expression of myelin basic protein gene in cultured rat brain oligodendrocytes is independent of continuous neuronal influences. J Neurosci. 1985 Nov;5(11):2955–2962. doi: 10.1523/JNEUROSCI.05-11-02955.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zurbriggen A., Vandevelde M., Steck A., Angst B. Myelin-associated glycoprotein is produced before myelin basic protein in cultured oligodendrocytes. J Neuroimmunol. 1984 Feb;6(1):41–49. doi: 10.1016/0165-5728(84)90041-9. [DOI] [PubMed] [Google Scholar]

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