Brain Cell Biology

, Volume 36, Issue 5–6, pp 191–202 | Cite as

O-GlcNAc modification of radial glial vimentin filaments in the developing chick brain



We examined the post-translational modification of intracellular proteins by β-O-linked N-acetylglucosamine (O-GlcNAc) with regard to neurofilament phosphorylation in the developing chick optic tectum. A regulated developmental pattern of O-GlcNAcylation was discovered in the developing brain. Most notably, discernible staining occurs along radial glial filaments but not along neuronal filaments in vivo. Immunohistochemical analyses in sections of progressive stages of development suggest upregulation of O-GlcNAc in the ependyma, tectofugal neuron bodies, and radial glial processes, but not in axons. In contrast, double-label immunostaining of monolayer cultures made from dissociated embryonic day (E) 7 optic tecta revealed O-GlcNAcylation of most axons. Labeling of brain sections together with Western blot analyses showed O-GlcNAc modification of a few discrete proteins throughout development, and suggested vimentin as the protein in radial glia. Immunoprecipitation of vimentin from E9 whole brain lysates confirmed O-GlcNAcylation of vimentin in development. These results indicate a regulated pattern of O-GlcNAc modification of vimentin filaments, which in turn suggests a role for O-GlcNAc-modified intermediate filaments in radial glia, but not in neurons during brain development. The control mechanisms that regulate this pattern in vivo, however, are disrupted when cells are placed in vitro.


  1. Akimoto, Y. Comer, F. I., Cole, R. N., Kudo, A., Kawakami, H., Hirano, H., and Hart, G. W. (2003). Localization of the O-GlcNAc transferase and O-GlcNAc-modified proteins in the rat cerebellar cortex. Brain Research. 966, 194–205.PubMedCrossRefGoogle Scholar
  2. Bennett, G.S. and DiLullo, C. (1985). Slow posttranslational modification of a neurofilament protein. J. Cell Biol., 100(5), 1799–1804.PubMedCrossRefGoogle Scholar
  3. Bennett, G. S., Tapscott, S. J., DiLullo, C., and Holtzer, H. (1984). Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons. Brain Res. 304, 291–302.PubMedCrossRefGoogle Scholar
  4. Comer, F. I., Vosesller, K., Wells, L., Accavitti, M. A., and Hart, G.W. (2001). Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal. Biochem. 293, 169–77.PubMedCrossRefGoogle Scholar
  5. Deng, Y., Li, B., Liu, F., Iqbal, K., Grundke-Iqbal, I., Brandt, R., and Gong, C.-X. (2007). Regulation between O-GlcNAcylation and phosphorylation of neurofilament-M and their dysregulation in Alzheimer disease. FASEB J. (published online August 8, 2007)Google Scholar
  6. Dong, D. L.-Y., Xu, Z. S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993). Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M. J Biol Chem. 268, 6679–87.Google Scholar
  7. Dong, D. L.-Y., Xu, Z. S., Hart, G. W., Cleveland, D. W. (1996). Cytoplasmic O-GlcNAc Modification of the Head Domain and the KSP Repeat Motif of the Neurofilament Protein Neurofilament-H. J. Biol. Chem., 271(34), 20845–20852.PubMedCrossRefGoogle Scholar
  8. Galileo, D.S. (2003). Spatio-temporal gradient of oligodendrocyte differentiation in chick optic tectum requires brain integrity and cell-cell interactions. Glia, 41, 25–37.PubMedCrossRefGoogle Scholar
  9. Galileo, D.S., Majors, J., Horwitz, A.F., and Sanes, J.R. (1992). Retrovirally introduced antisense integrin RNA inhibits neuroblast migration in vivo. Neuron, 9, 1117–1131.PubMedCrossRefGoogle Scholar
  10. Gray, G.E. and Sanes, J.R. (1992). Lineage of radial glia in the chicken optic tectum. Development, 114, 271–283.PubMedGoogle Scholar
  11. Herman, J. P., Victor, J. C., and Sanes, J. R. (1993). Developmentally regulated and spatially restricted antigens of radial glial cells. Dev Dyn. 197, 307–18.PubMedGoogle Scholar
  12. Katsumoto, T., Mitsushima, A., and Kurimura, T. (1990). Biol. Cell 68, 139–146.PubMedCrossRefGoogle Scholar
  13. Khidekel, N., Ficarro, S. B., Peters, E. C., amd Hsieh-Wilson, L. C. (2004). Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain. PNAS. 101, 13132–13137.PubMedCrossRefGoogle Scholar
  14. Kroger, S. and Schwarz, U. (1990). The avian tectobulbar tract: development, explant culture, and effects of antibodies on the pattern of neurite outgrowth. J Neurosci., 10(9), 3118–34.PubMedGoogle Scholar
  15. Kroger, S. and Walter, J. (1991). Molecular mechanisms separating two axonal pathways during embryonic development of the avian optic tectum. Neuron, 6(2):291–303.PubMedCrossRefGoogle Scholar
  16. Lee, M. K., and Cleveland, D. W. (1996). Neuronal intermediate filaments. Annu. Rev. Neurosci. 19, 187–217.PubMedCrossRefGoogle Scholar
  17. Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G. W., and Gong, C. X. (2004). O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc Natl Acad Sci U S A. 101, 10804–10809.PubMedCrossRefGoogle Scholar
  18. Nakamura, Y., Hashimoto, R., Amano, M., Nagata, K., Matsumoto, N., Goto, H., Fukusho, E., Mori, H., Kashiwagi, Y., Kudo T., Inagaki, M., and Takeda, M. (2000). Localized phosphorylation of vimentin by rho-kinase in neuroblastoma N2a cells. Genes Cells 5, 823–837.PubMedCrossRefGoogle Scholar
  19. Nixon, R. A., Paskevich, P. A., Sihag, R. K., and Thayer, C. Y. (1994). Phosphorylationon carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, inter-filament spacing and axonal caliber. J. Cell Biol. 126, 1031–1046.PubMedCrossRefGoogle Scholar
  20. Pant, H. C., and Veeranna. (1995). Neurofilament phosphorylation. Biochem. Cell Biol. 73, 575–592Google Scholar
  21. Rakic, P. (1972). Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neurol., 141, 283–312.CrossRefGoogle Scholar
  22. Rex-Mathes, M., Werner, S., Strutas, D., Griffith, L. S., Viebahn, C., Thelen, K., and Schmitz, B. (2001). O-GlcNAc expression in develoing and ageing mouse brain. Biochimic. 83, 583–590.PubMedCrossRefGoogle Scholar
  23. Sanchez, I., Hassinger, L., Sihag, R. K., Cleveland, D. W., Mohan, P., and Nixon, R. A. (2000). Local control of neurofilament accumulation during radial growth of myelinating axons in vivo: Selective role of site-specific phosphorylation. J. Cell Biol. 151, 1013–1024.PubMedCrossRefGoogle Scholar
  24. Shaw, G. (1998). Neurofilaments. Springer-Verlag, Berlin.Google Scholar
  25. Stettler, E. M. and Galileo, D. S. (2005). Radial glia produce and align the ligand fibronectin during neuronal migration in the developing chick brain. J. Comp. Neurol., 468(3), 441–451.CrossRefGoogle Scholar
  26. Treubert-Zimmermann, U., Heyers, D., and Redies, C. (2002). Targeting axons to specific fiber tracts in vivo by altering cadherin expression. J. Neurosci., 22(17), 7617–7626.PubMedGoogle Scholar
  27. Wang, Z., Pandey, A., and Hart, G. W. (2007). Dynamic interplay between O-GlcNAcylation and GSK-3-dependent phosphorylation. Mol. Cell. Proteomics (Epub ahead of print)Google Scholar
  28. Wu, C. C., Russell, R. M., and Karten, H. J. (2000). Ontogeny of the tectorotundal pathway in chicks (Gallus gallus): birthdating and pathway tracing study. J. Comp. Neurol., 417(1), 115–132.PubMedCrossRefGoogle Scholar
  29. Zachara, Z. E., O’Donnell, N., Cheung, W. D., Mercer, J. J., Marth, J. D., and Hart G. W. (2004). Dynamic O-GlcNAc Modification of Nucleocytoplasmic Proteins in Response to Stress. J. Biol. Chem., 279(29) 30133–30142.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Department of Biological SciencesUniversity of DelawareNewarkUSA
  2. 2.Jefferson Medical CollegePhiladelphiaUSA

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