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Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis

Nature Neuroscience volume 5, pages 1926 (2002) | Download Citation

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Abstract

Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated–soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.

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References

  1. 1.

    The Release of Neural Transmitter Substances (Liverpool University Press, Liverpool, 1969).

  2. 2.

    & Spontaneous release of transmitter from growth cones of embryonic neurones. Nature 305, 634–637 (1983).

  3. 3.

    , , & Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis. J. Cell Biol. 144, 507–518 (1999).

  4. 4.

    et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324 (1993).

  5. 5.

    Structural insights into the molecular mechanism of Ca2+-dependent exocytosis. Curr. Opin. Neurobiol. 10, 293–302 (2000).

  6. 6.

    et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

  7. 7.

    , & Neurotoxins affecting neuroexocytosis. Physiol. Rev. 80, 717–766 (2000).

  8. 8.

    et al. A conserved domain is present in different families of vesicular fusion proteins: a new superfamily. Proc. Natl. Acad. Sci. USA 94, 3046–3051 (1997).

  9. 9.

    et al. Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin. J. Neurosci. 18, 2028–2939 (1998).

  10. 10.

    et al. Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron 15, 663–673 (1995).

  11. 11.

    , , , & Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14, 341–351 (1995).

  12. 12.

    , , & Different VAMP/synaptobrevin complexes for spontaneous and evoked transmitter release at the crayfish neuromuscular junction. J. Neurophysiol. 80, 3233–3246 (1998).

  13. 13.

    , , , & Ca2+ or Sr2+ partially rescues synaptic transmission in hippocampal cultures treated with botulinum toxin A and C, but not tetanus toxin. J. Neurosci. 17, 7190–7202 (1997).

  14. 14.

    et al. Tetanus toxin blocks the exocytosis of synaptic vesicles clustered at synapses but not of synaptic vesicles in isolated axons. J. Neurosci. 19, 6723–6732 (1999).

  15. 15.

    et al. Inhibition of axon growth by SNAP-25 antisense oligonucleotides in vitro and in vivo. Nature 364, 445–448 (1993).

  16. 16.

    et al. Common and distinct fusion proteins in axonal growth and transmitter release. J. Comp. Neurol. 222–234 (1996).

  17. 17.

    et al. Distribution of synaptosomal-associated protein 25 in nerve growth cones and reduction of neurite outgrowth by botulinum neurotoxin A without altering growth cone morphology in dorsal root ganglion neurons and PC-12 cells. Neuroscience 91, 695–706 (1999).

  18. 18.

    et al. Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment. J. Neurosci. 19, 9803–9812 (1999).

  19. 19.

    Structure of the chicken gene for SNAP-25 reveals duplicated exons encoding distinct isoforms of the protein. J. Mol. Biol. 233, 67–76 (1993).

  20. 20.

    , , & SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils. J. Biol. Chem. 269, 27427–27432 (1994).

  21. 21.

    , , & Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395, 347–353 (1998).

  22. 22.

    , , & The 25 kDa synaptosomal-associated protein SNAP-25 is the major methionine-rich polypeptide in rapid axonal transport and a major substrate for palmitoylation in adult CNS. J. Neurosci. 12, 4634–4641 (1992).

  23. 23.

    , & Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25. FEBS Lett. 385, 119–123 (1996).

  24. 24.

    & Characterization of the palmitoylation domain of SNAP-25. J. Neurochem. 69, 1864–1869 (1997).

  25. 25.

    et al. The cysteines of synaptosome-associated protein of 25 kDa (SNAP-25) are required for SNARE disassembly and exocytosis, not for membrane targeting. Biochem. J. 357, 625–634 (2001).

  26. 26.

    , & Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues. J. Biol. Chem. 271, 13300–13303 (1996).

  27. 27.

    , , & Organization of the secretory machinery in the rodent brain: distribution of t-SNAREs SNAP-25 and SNAP-23. Brain Res. 831, 11–24 (1999).

  28. 28.

    et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864–869 (2000).

  29. 29.

    & Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 87, 297–303 (1980).

  30. 30.

    , & Calcium-independent actions of α-latrotoxin on spontaneous and evoked synaptic transmission in the hippocampus. J. Neurophysiol. 76, 149–158 (1996).

  31. 31.

    , , , & Exoendocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. J. Cell. Biol. 117, 849–869 (1992).

  32. 32.

    & Quantal components of the end-plate potential. J. Physiol. (Lond.) 124, 560–573 (1954).

  33. 33.

    & Structural changes after transmitter release at the frog neuromuscular junction. J. Cell Biol. 88, 564–580 (1981).

  34. 34.

    , & SNAP-25 and synaptotagmin involvement in the final Ca+2-dependent triggering of neurotransmitter exocytosis. Proc. Natl. Acad. Sci. USA 93, 10471–10476 (1996).

  35. 35.

    et al. Synaptic vesicle membrane fusion complex: action of clostriridal neurotoxins on assembly. EMBO J. 13, 5051–5061 (1994).

  36. 36.

    , , & Clostridal neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion. EMBO J. 14, 4705–4713 (1995).

  37. 37.

    et al. Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation. Neuron 30, 399–410 (2001).

  38. 38.

    et al. Distinctive roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature 410, 1057–1064 (2001).

  39. 39.

    et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

  40. 40.

    et al. A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18, 4372–4382 (1999).

  41. 41.

    , , & Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses. Proc. Natl. Acad. Sci. USA 94, 997–1001 (1997).

  42. 42.

    , , , & Miniature synaptic events maintain dendritic spines via AMPA receptor activation. Nat. Neurosci. 2, 44–49 (1999).

  43. 43.

    et al. SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294, 1117–1122 (2001).

  44. 44.

    , & Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 (1988).

  45. 45.

    et al. Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85, 525–535 (1996).

  46. 46.

    , & Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in α-bungarotoxin-treated rats. J. Physiol. (Lond.) 458, 487–499 (1992).

  47. 47.

    et al. Preparative methods for brain slices: a discussion. J. Neurosci. Methods. 59, 139–149 (1995).

  48. 48.

    , , , & Acute effects of ethanol on kainate receptors in cultured hippocampal neurons. Alcohol Clin. Exp. Res. 24, 220–225 (2000).

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Acknowledgements

We thank J. Plomp for advice and help in embryonic neuromuscular electrophysiology, S. Nixon for technical assistance, E. Padilla at the UNM-HSC Animal Resource Facility for maintaining the mouse colony and G. Adamson at the UC Davis EM Pathology lab for help with electron microscopy. We also thank P. De Camilli and A. Klip for antisera, and B. Shuttleworth and L. Anna Cunningham for discussions and for reading the manuscript. The work was supported by NIH MH 4-8989 (M.C.W.).

Author information

Author notes

    • Philip Washbourne

    Present address: Center for Neuroscience, University of California, Davis, California 95616, USA

    • Peter M. Thompson

    Present address: Department of Psychiatry, University of Texas Health Sciences Center, San Antonio, Texas 78284, USA

    • Edmar T. Costa

    Present address: Department of Physiology, Federal University of Pará, 66075-900 Belém, Pará, Brazil

    • Philip Washbourne
    •  & Peter M. Thompson

    The first two authors contributed equally to this work

Affiliations

  1. Department of Neurosciences, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, USA

    • Philip Washbourne
    • , Peter M. Thompson
    • , Mario Carta
    • , Edmar T. Costa
    • , James R. Mathews
    • , C. Fernando Valenzuela
    • , L. Donald Partridge
    •  & Michael C. Wilson
  2. Department of Pathology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, USA

    • Mark W. Becher
  3. Department of Human Anatomy and Genetics, University of Oxford, South Parks Road OX1 3QX, UK

    • Guillermina Lopez-Benditó
    •  & Zoltán Molnár

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael C. Wilson.

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DOI

https://doi.org/10.1038/nn783

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