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. 1999 Apr 1;18(7):1761–1771. doi: 10.1093/emboj/18.7.1761

Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes.

M D Ledesma 1, B Brügger 1, C Bünning 1, F T Wieland 1, C G Dotti 1
PMCID: PMC1171262  PMID: 10202140

Abstract

Neuronal maturation is a gradual process; first axons and dendrites are established as distinct morphological entities; next the different intracellular organization of these processes occurs; and finally the specialized plasma membrane domains of these two compartments are formed. Only when this has been accomplished does proper neuronal function take place. In this work we present evidence that the correct distribution of a class of axonal membrane proteins requires a mechanism which involves formation of protein-lipid (sphingomyelin/cholesterol) detergent-insoluble complexes (DIGs). Using biochemistry and immunofluorescence microscopy we now show that in developing neurons the randomly distributed Thy-1 does not interact with lipids into DIGs (in fully developed neurons the formation of such complexes is essential for the correct axonal targeting of this protein). Using lipid mass spectrometry and thin layer chromatography we show that the DIG lipid missing in the developing neurons is sphingomyelin, but not cholesterol or glucosylceramide. Finally, by increasing the intracellular levels of sphingomyelin in the young neurons the formation of Thy-1/DIGs was induced and, consistent with a role in sorting, proper axonal distribution was facilitated. These results emphasize the role of sphingomyelin in axonal, and therefore, neuronal maturation.

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Selected References

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  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Bittman R., Kasireddy C. R., Mattjus P., Slotte J. P. Interaction of cholesterol with sphingomyelin in monolayers and vesicles. Biochemistry. 1994 Oct 4;33(39):11776–11781. doi: 10.1021/bi00205a013. [DOI] [PubMed] [Google Scholar]
  3. Bradke F., Dotti C. G. Neuronal polarity: vectorial cytoplasmic flow precedes axon formation. Neuron. 1997 Dec;19(6):1175–1186. doi: 10.1016/s0896-6273(00)80410-9. [DOI] [PubMed] [Google Scholar]
  4. Brown D. A., Crise B., Rose J. K. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science. 1989 Sep 29;245(4925):1499–1501. doi: 10.1126/science.2571189. [DOI] [PubMed] [Google Scholar]
  5. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  6. Brügger B., Erben G., Sandhoff R., Wieland F. T., Lehmann W. D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2339–2344. doi: 10.1073/pnas.94.6.2339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caceres A., Banker G., Steward O., Binder L., Payne M. MAP2 is localized to the dendrites of hippocampal neurons which develop in culture. Brain Res. 1984 Apr;315(2):314–318. doi: 10.1016/0165-3806(84)90167-6. [DOI] [PubMed] [Google Scholar]
  8. Caras I. W., Weddell G. N., Williams S. R. Analysis of the signal for attachment of a glycophospholipid membrane anchor. J Cell Biol. 1989 Apr;108(4):1387–1396. doi: 10.1083/jcb.108.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Craig A. M., Banker G. Neuronal polarity. Annu Rev Neurosci. 1994;17:267–310. doi: 10.1146/annurev.ne.17.030194.001411. [DOI] [PubMed] [Google Scholar]
  10. Craig A. M., Blackstone C. D., Huganir R. L., Banker G. Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12373–12377. doi: 10.1073/pnas.91.26.12373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dotti C. G., Parton R. G., Simons K. Polarized sorting of glypiated proteins in hippocampal neurons. Nature. 1991 Jan 10;349(6305):158–161. doi: 10.1038/349158a0. [DOI] [PubMed] [Google Scholar]
  12. Dotti C. G., Simons K. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell. 1990 Jul 13;62(1):63–72. doi: 10.1016/0092-8674(90)90240-f. [DOI] [PubMed] [Google Scholar]
  13. Fiedler K., Kobayashi T., Kurzchalia T. V., Simons K. Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells. Biochemistry. 1993 Jun 29;32(25):6365–6373. doi: 10.1021/bi00076a009. [DOI] [PubMed] [Google Scholar]
  14. Fishman P. H., Pacuszka T., Orlandi P. A. Gangliosides as receptors for bacterial enterotoxins. Adv Lipid Res. 1993;25:165–187. [PubMed] [Google Scholar]
  15. Friedrichson T., Kurzchalia T. V. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature. 1998 Aug 20;394(6695):802–805. doi: 10.1038/29570. [DOI] [PubMed] [Google Scholar]
  16. Fuller S. D., von Bonsdorff C. H., Simons K. Cell surface influenza haemagglutinin can mediate infection by other animal viruses. EMBO J. 1985 Oct;4(10):2475–2485. doi: 10.1002/j.1460-2075.1985.tb03959.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gamble W., Vaughan M., Kruth H. S., Avigan J. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. J Lipid Res. 1978 Nov;19(8):1068–1070. [PubMed] [Google Scholar]
  18. Henke R. C., Hancox K. A., Jeffrey P. L. Characterization of two distinct populations of detergent resistant membrane complexes isolated from chick brain tissues. J Neurosci Res. 1996 Sep 1;45(5):617–630. doi: 10.1002/(SICI)1097-4547(19960901)45:5<617::AID-JNR11>3.0.CO;2-W. [DOI] [PubMed] [Google Scholar]
  19. Horinouchi K., Erlich S., Perl D. P., Ferlinz K., Bisgaier C. L., Sandhoff K., Desnick R. J., Stewart C. L., Schuchman E. H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease. Nat Genet. 1995 Jul;10(3):288–293. doi: 10.1038/ng0795-288. [DOI] [PubMed] [Google Scholar]
  20. Karrenbauer A., Jeckel D., Just W., Birk R., Schmidt R. R., Rothman J. E., Wieland F. T. The rate of bulk flow from the Golgi to the plasma membrane. Cell. 1990 Oct 19;63(2):259–267. doi: 10.1016/0092-8674(90)90159-c. [DOI] [PubMed] [Google Scholar]
  21. Keller P., Simons K. Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol. 1998 Mar 23;140(6):1357–1367. doi: 10.1083/jcb.140.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Killisch I., Dotti C. G., Laurie D. J., Lüddens H., Seeburg P. H. Expression patterns of GABAA receptor subtypes in developing hippocampal neurons. Neuron. 1991 Dec;7(6):927–936. doi: 10.1016/0896-6273(91)90338-z. [DOI] [PubMed] [Google Scholar]
  23. Kobayashi T., Pagano R. E. Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface. J Biol Chem. 1989 Apr 5;264(10):5966–5973. [PubMed] [Google Scholar]
  24. Krämer E. M., Koch T., Niehaus A., Trotter J. Oligodendrocytes direct glycosyl phosphatidylinositol-anchored proteins to the myelin sheath in glycosphingolipid-rich complexes. J Biol Chem. 1997 Apr 4;272(14):8937–8945. doi: 10.1074/jbc.272.14.8937. [DOI] [PubMed] [Google Scholar]
  25. Kuemmel T. A., Schroeder R., Stoffel W. Light and electron microscopic analysis of the central and peripheral nervous systems of acid sphingomyelinase-deficient mice resulting from gene targeting. J Neuropathol Exp Neurol. 1997 Feb;56(2):171–179. doi: 10.1097/00005072-199702000-00008. [DOI] [PubMed] [Google Scholar]
  26. Ledesma M. D., Simons K., Dotti C. G. Neuronal polarity: essential role of protein-lipid complexes in axonal sorting. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3966–3971. doi: 10.1073/pnas.95.7.3966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lipsky N. G., Pagano R. E. Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc Natl Acad Sci U S A. 1983 May;80(9):2608–2612. doi: 10.1073/pnas.80.9.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lisanti M. P., Sargiacomo M., Graeve L., Saltiel A. R., Rodriguez-Boulan E. Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9557–9561. doi: 10.1073/pnas.85.24.9557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matlin K. S., Reggio H., Helenius A., Simons K. Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol. 1981 Dec;91(3 Pt 1):601–613. doi: 10.1083/jcb.91.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mays R. W., Siemers K. A., Fritz B. A., Lowe A. W., van Meer G., Nelson W. J. Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells. J Cell Biol. 1995 Sep;130(5):1105–1115. doi: 10.1083/jcb.130.5.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Melkonian K. A., Chu T., Tortorella L. B., Brown D. A. Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells. Biochemistry. 1995 Dec 12;34(49):16161–16170. doi: 10.1021/bi00049a031. [DOI] [PubMed] [Google Scholar]
  32. Moran P., Caras I. W. Proteins containing an uncleaved signal for glycophosphatidylinositol membrane anchor attachment are retained in a post-ER compartment. J Cell Biol. 1992 Nov;119(4):763–772. doi: 10.1083/jcb.119.4.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Parton R. G., Simons K. Digging into caveolae. Science. 1995 Sep 8;269(5229):1398–1399. doi: 10.1126/science.7660120. [DOI] [PubMed] [Google Scholar]
  34. Parton R. G., Simons K., Dotti C. G. Axonal and dendritic endocytic pathways in cultured neurons. J Cell Biol. 1992 Oct;119(1):123–137. doi: 10.1083/jcb.119.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  36. Rao A., Kim E., Sheng M., Craig A. M. Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J Neurosci. 1998 Feb 15;18(4):1217–1229. doi: 10.1523/JNEUROSCI.18-04-01217.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rodgers W., Crise B., Rose J. K. Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction. Mol Cell Biol. 1994 Aug;14(8):5384–5391. doi: 10.1128/mcb.14.8.5384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rouser G., Fkeischer S., Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970 May;5(5):494–496. doi: 10.1007/BF02531316. [DOI] [PubMed] [Google Scholar]
  39. Schroeder R., London E., Brown D. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12130–12134. doi: 10.1073/pnas.91.25.12130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schwarz A., Futerman A. H. Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth. J Neurosci. 1997 May 1;17(9):2929–2938. doi: 10.1523/JNEUROSCI.17-09-02929.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schwarz A., Futerman A. H. The localization of gangliosides in neurons of the central nervous system: the use of anti-ganglioside antibodies. Biochim Biophys Acta. 1996 Oct 29;1286(3):247–267. doi: 10.1016/s0304-4157(96)00011-1. [DOI] [PubMed] [Google Scholar]
  42. Schwarz A., Rapaport E., Hirschberg K., Futerman A. H. A regulatory role for sphingolipids in neuronal growth. Inhibition of sphingolipid synthesis and degradation have opposite effects on axonal branching. J Biol Chem. 1995 May 5;270(18):10990–10998. doi: 10.1074/jbc.270.18.10990. [DOI] [PubMed] [Google Scholar]
  43. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  44. Skibbens J. E., Roth M. G., Matlin K. S. Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts. J Cell Biol. 1989 Mar;108(3):821–832. doi: 10.1083/jcb.108.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sofer A., Futerman A. H. Cationic amphiphilic drugs inhibit the internalization of cholera toxin to the Golgi apparatus and the subsequent elevation of cyclic AMP. J Biol Chem. 1995 May 19;270(20):12117–12122. doi: 10.1074/jbc.270.20.12117. [DOI] [PubMed] [Google Scholar]
  46. Varma R., Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature. 1998 Aug 20;394(6695):798–801. doi: 10.1038/29563. [DOI] [PubMed] [Google Scholar]
  47. Wu G., Ledeen R. W. Stimulation of neurite outgrowth in neuroblastoma cells by neuraminidase: putative role of GM1 ganglioside in differentiation. J Neurochem. 1991 Jan;56(1):95–104. doi: 10.1111/j.1471-4159.1991.tb02567.x. [DOI] [PubMed] [Google Scholar]
  48. Yoshimori T., Keller P., Roth M. G., Simons K. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J Cell Biol. 1996 Apr;133(2):247–256. doi: 10.1083/jcb.133.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Meer G., Stelzer E. H., Wijnaendts-van-Resandt R. W., Simons K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 1987 Oct;105(4):1623–1635. doi: 10.1083/jcb.105.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]

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