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A protein kinase A–dependent molecular switch in synapsins regulates neurite outgrowth

Nature Neuroscience volume 5, pages 431437 (2002) | Download Citation

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Abstract

Cyclic AMP (cAMP) promotes neurite outgrowth in a variety of neuronal cell lines through the activation of protein kinase A (PKA). We show here, using both Xenopus laevis embryonic neuronal culture and intact X. laevis embryos, that the nerve growth–promoting action of cAMP/PKA is mediated in part by the phosphorylation of synapsins at a single amino acid residue. Expression of a mutated form of synapsin that prevents phosphorylation at this site, or introduction of phospho-specific antibodies directed against this site, decreased basal and dibutyryl cAMP–stimulated neurite outgrowth. Expression of a mutation mimicking constitutive phosphorylation at this site increased neurite outgrowth, both under basal conditions and in the presence of a PKA inhibitor. These results provide a potential molecular approach for stimulating neuron regeneration, after injury and in neurodegenerative diseases.

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References

  1. 1.

    & Signal transduction underlying growth cone guidance by diffusible factors. Curr. Opinion Neurobiol. 9, 355–363 (1999).

  2. 2.

    , & Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells. Nature 306, 265–267 (1983).

  3. 3.

    & cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc. Natl. Acad. Sci. USA 85, 1257–1261 (1988).

  4. 4.

    , , & Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 15, 805–819 (1995).

  5. 5.

    , & cAMP-induced switching in turning direction of nerve growth cones. Nature 388, 275–279 (1997).

  6. 6.

    et al. cAMP-dependent growth cone guidance by netrin-1. Neuron 19, 1225–1235 (1997).

  7. 7.

    et al. Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515–1518 (1998).

  8. 8.

    , , , & Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron 22, 89–101 (1999).

  9. 9.

    & The Neuron (Oxford University Press, New York, NY, 1997).

  10. 10.

    , , & Adenosine-3′,5-monophosphate–dependent phosphorylation of a specific protein in synaptic membrane fractions from rat cerebrum. J. Biol. Chem. 247, 5650–5652 (1972).

  11. 11.

    et al. Synapsins as regulators of neurotransmitter release. Phil. Trans. R. Soc. Lond. B Biol. Sci. 354, 269–279 (1999).

  12. 12.

    et al. Expression of synapsin I correlates with maturation of the neuromuscular synapse. Neuroscience 74, 1087–1097 (1996).

  13. 13.

    , , , & Induction of formation of presynaptic terminals in neuroblastoma cells by synapsin IIb. Nature 349, 697–700 (1991).

  14. 14.

    , , & Overexpression of rat synapsins in NG108-15 neuronal cells enhances functional synapse formation with myotubes. Neurosci. Lett. 260, 93–96 (1999).

  15. 15.

    , & Exogenous synapsin I promotes functional maturation of developing neuromuscular synapses. Neuron 8, 521–529 (1992).

  16. 16.

    , , & Synapsin IIa accelerates functional development of neuromuscular synapses. Proc. Natl. Acad. Sci. USA 91, 3882–3886 (1994).

  17. 17.

    , , & Suppression of synapsin II inhibits the formation and maintenance of synapses in hippocampal culture. Proc. Natl. Acad. Sci. USA 92, 9225–9229 (1995).

  18. 18.

    , , , & Impairment of axonal development and of synaptogenesis in hippocampal neurons of synapsin I–deficient mice. Proc. Natl. Acad. Sci. USA 92, 9230–9234 (1995).

  19. 19.

    , , , & Synapsin III: developmental expression, subcellular localization, and role in axon formation. J. Neurosci. 20, 3736–3744 (2000).

  20. 20.

    et al. Molecular evolution of the synapsin gene family. J. Expt. Zool. (Mol. Dev. Evol.) 285, 360–377 (1999).

  21. 21.

    , & Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I. Proc. Natl. Acad. Sci. USA 84, 7518–7522 (1987).

  22. 22.

    , & Calcium/calmodulin-dependent protein kinase I. cDNA cloning and identification of autophosphorylation site. J. Biol. Chem. 268, 26512–26521 (1993).

  23. 23.

    et al. Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins. Science 245, 1474–1480 (1989).

  24. 24.

    , & Characterization of transcripts from the synapsin III gene locus. J. Neurochem. 73, 2266–2271 (1999).

  25. 25.

    & Embryonic and regenerating Xenopus retinal fibers are intrinsically different. Developmental Biology (Orlando) 114, 475–491 (1986).

  26. 26.

    , & Early Development of Xenopus laevis: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000).

  27. 27.

    , , & The synaptic vesicle protein SV2 is a novel type of transmembrane transporter. Cell 70, 861–867 (1992).

  28. 28.

    et al. in Methods in Enzymology (eds. Hunter, T. & Sefton, B. M.) 264–283 (Academic Press, San Diego, California, 1991).

  29. 29.

    et al. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide–dependent protein kinases. Biochem. Biophys. Res. Comm. 142, 436–440 (1987).

  30. 30.

    , , & Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259, 780–785 (1993).

  31. 31.

    et al. Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I–actin interactions. Proc. Natl. Acad. Sci. USA 93, 3679–3683 (1996).

  32. 32.

    , , , & Synapsins as mediators of BDNF-enhanced neurotransmitter release. Nat. Neurosci. 3, 323–329 (2000).

  33. 33.

    et al. Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions. J. Biol. Chem. 271, 21108–21113 (1996).

  34. 34.

    & Neurofilaments help maintain normal morphologies and support elongation of neurites in Xenopus laevis cultured embryonic spinal cord neurons. J. Neurosci. 15, 8331-8344 (1995).

  35. 35.

    Axonal growth–associated proteins. Annu. Rev. Neurosci. 12, 127–156 (1989).

  36. 36.

    , & The effect of tau antisense oligonucleotides on neurite formation of cultured cerebellar macroneurons. J. Neurosci. 11, 1515–1523 (1991).

  37. 37.

    , & CAMs and axonal growth: a critical evaluation of the role of calcium and the MAPK cascade. Mol. Cell. Neurosci. 16, 283–295 (2000).

  38. 38.

    , , , & The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes Dev. 10, 816–825 (1996).

  39. 39.

    , , & Effects of the neuronal phosphoprotein synapsin I on actin polymerization. II. Analytical interpretation of kinetic curves. J. Biol. Chem. 267, 11289–11299 (1992).

  40. 40.

    et al. Phosphorylation-dependent effects of synapsin IIa on actin polymerization and network formation. Eur. J. Neurosci. 9, 2712–2722 (1997).

  41. 41.

    & Synapsins I and II are ATP-binding proteins with differential Ca2+ regulation. J. Biol. Chem. 273, 1425–1429 (1998).

  42. 42.

    & Synapsin III, a novel synapsin with an unusual regulation by Ca2+. J. Biol. Chem. 273, 13371–13374 (1998).

  43. 43.

    et al. Synapsin I is structurally similar to ATP-utilizing enzymes. EMBO J. 17, 977–984 (1998).

  44. 44.

    Mammalian neural stem cells. Science 287, 1433–1438 (2000).

  45. 45.

    , & in Culturing Nerve Cells (eds. Banker, G. & Goslin, K.) 237–260 (MIT Press, Cambridge, Massachusetts, 1998).

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Acknowledgements

This work was supported by U.S. Public Health Service Grants MH39327 (P.G.), AG15072 (P.G.), R29 NS35941 (V.P.) and NS37831 (M-m.P.). B.P. is a 2000 Katowitz-Raden Investigator of the National Alliance for Research in Schizophrenia and Affective Disorders. The catalytic subunit of PKA was a gift from A. Nairn and A. Horiuchi. We thank P. Allen for reviewing the manuscript, and G. Yiu and G. Chaiken for technical assistance.

*This work was also supported by U.S. Public Health Service Grants HG00008 (to J. Ott) and K25-HG00060-01A1 (J.H.)

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Affiliations

  1. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA

    • Hung-Teh Kao
    • , Barbara Porton
    • , Michael Abraham
    • , Andrew J. Czernik
    •  & Paul Greengard
  2. Department of Psychiatry, New York University Medical Center, 550 First Avenue, New York, New York 10016, USA

    • Hung-Teh Kao
  3. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA

    • Hong-jun Song
  4. Department of Biology, University of California, San Diego, 7500 Gilman Drive, La Jolla, California 92093, USA

    • Guo-li Ming
  5. Laboratory of Statistical Genetics, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA

    • Josephine Hoh
  6. The John B. Pierce Laboratory, Yale University School of Medicine, 290 Congress Avenue, New Haven, Connecticut 06510, USA

    • Vincent A. Pieribone
  7. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

    • Mu-ming Poo

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The authors declare no competing financial interests.

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Correspondence to Hung-Teh Kao.

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https://doi.org/10.1038/nn840

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