A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons (original) (raw)

Abstract

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.

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Selected References

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  1. Akhtar S., Juliano R. L. Cellular uptake and intracellular fate of antisense oligonucleotides. Trends Cell Biol. 1992 May;2(5):139–144. doi: 10.1016/0962-8924(92)90100-2. [DOI] [PubMed] [Google Scholar]
  2. Ayala J., Touchot N., Zahraoui A., Tavitian A., Prochiantz A. The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro. Neuron. 1990 May;4(5):797–805. doi: 10.1016/0896-6273(90)90206-u. [DOI] [PubMed] [Google Scholar]
  3. Bucci C., Parton R. G., Mather I. H., Stunnenberg H., Simons K., Hoflack B., Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992 Sep 4;70(5):715–728. doi: 10.1016/0092-8674(92)90306-w. [DOI] [PubMed] [Google Scholar]
  4. Bucci C., Wandinger-Ness A., Lütcke A., Chiariello M., Bruni C. B., Zerial M. Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5061–5065. doi: 10.1073/pnas.91.11.5061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chavrier P., Vingron M., Sander C., Simons K., Zerial M. Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line. Mol Cell Biol. 1990 Dec;10(12):6578–6585. doi: 10.1128/mcb.10.12.6578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Dotti C. G., Banker G. A. Experimentally induced alteration in the polarity of developing neurons. Nature. 1987 Nov 19;330(6145):254–256. doi: 10.1038/330254a0. [DOI] [PubMed] [Google Scholar]
  8. Dotti C. G., Sullivan C. A., Banker G. A. The establishment of polarity by hippocampal neurons in culture. J Neurosci. 1988 Apr;8(4):1454–1468. doi: 10.1523/JNEUROSCI.08-04-01454.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elferink L. A., Anzai K., Scheller R. H. rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain. J Biol Chem. 1992 Mar 25;267(9):5768–5775. [PubMed] [Google Scholar]
  10. Fischer von Mollard G., Südhof T. C., Jahn R. A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature. 1991 Jan 3;349(6304):79–81. doi: 10.1038/349079a0. [DOI] [PubMed] [Google Scholar]
  11. Fletcher T. L., Cameron P., De Camilli P., Banker G. The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture. J Neurosci. 1991 Jun;11(6):1617–1626. doi: 10.1523/JNEUROSCI.11-06-01617.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Forscher P., Smith S. J. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J Cell Biol. 1988 Oct;107(4):1505–1516. doi: 10.1083/jcb.107.4.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gallwitz D., Donath C., Sander C. A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product. Nature. 1983 Dec 15;306(5944):704–707. doi: 10.1038/306704a0. [DOI] [PubMed] [Google Scholar]
  14. Goslin K., Schreyer D. J., Skene J. H., Banker G. Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones. Nature. 1988 Dec 15;336(6200):672–674. doi: 10.1038/336672a0. [DOI] [PubMed] [Google Scholar]
  15. Goud B., Salminen A., Walworth N. C., Novick P. J. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  16. Huber L. A., Pimplikar S., Parton R. G., Virta H., Zerial M., Simons K. Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol. 1993 Oct;123(1):35–45. doi: 10.1083/jcb.123.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huber L. A., de Hoop M. J., Dupree P., Zerial M., Simons K., Dotti C. Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p. J Cell Biol. 1993 Oct;123(1):47–55. doi: 10.1083/jcb.123.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Marcus-Sekura C. J. Techniques for using antisense oligodeoxyribonucleotides to study gene expression. Anal Biochem. 1988 Aug 1;172(2):289–295. doi: 10.1016/0003-2697(88)90447-2. [DOI] [PubMed] [Google Scholar]
  20. Mizoguchi A., Kim S., Ueda T., Kikuchi A., Yorifuji H., Hirokawa N., Takai Y. Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain. J Biol Chem. 1990 Jul 15;265(20):11872–11879. [PubMed] [Google Scholar]
  21. Pagano R. E., Martin O. C. A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells. Biochemistry. 1988 Jun 14;27(12):4439–4445. doi: 10.1021/bi00412a034. [DOI] [PubMed] [Google Scholar]
  22. Pagano R. E., Martin O. C., Kang H. C., Haugland R. P. A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J Cell Biol. 1991 Jun;113(6):1267–1279. doi: 10.1083/jcb.113.6.1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Pfeffer S. R. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. doi: 10.1016/0962-8924(92)90161-f. [DOI] [PubMed] [Google Scholar]
  25. Pfenninger K. H., Johnson M. P. Membrane biogenesis in the sprouting neuron. I. Selective transfer of newly synthesized phospholipid into the growing neurite. J Cell Biol. 1983 Oct;97(4):1038–1042. doi: 10.1083/jcb.97.4.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pfenninger K. H., Maylié-Pfenninger M. F. Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion. J Cell Biol. 1981 Jun;89(3):547–559. doi: 10.1083/jcb.89.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  28. Schroer T. A., Sheetz M. P. Functions of microtubule-based motors. Annu Rev Physiol. 1991;53:629–652. doi: 10.1146/annurev.ph.53.030191.003213. [DOI] [PubMed] [Google Scholar]
  29. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  30. Simons K., Zerial M. Rab proteins and the road maps for intracellular transport. Neuron. 1993 Nov;11(5):789–799. doi: 10.1016/0896-6273(93)90109-5. [DOI] [PubMed] [Google Scholar]
  31. de Hoop M. J., Huber L. A., Stenmark H., Williamson E., Zerial M., Parton R. G., Dotti C. G. The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis. Neuron. 1994 Jul;13(1):11–22. doi: 10.1016/0896-6273(94)90456-1. [DOI] [PubMed] [Google Scholar]
  32. van der Sluijs P., Hull M., Webster P., Mâle P., Goud B., Mellman I. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 1992 Sep 4;70(5):729–740. doi: 10.1016/0092-8674(92)90307-x. [DOI] [PubMed] [Google Scholar]