Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking

Abstract

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) has an important function in cell regulation both as a precursor of second messenger molecules and by means of its direct interactions with cytosolic and membrane proteins. Biochemical studies have suggested a role for PtdIns(4,5)P2 in clathrin coat dynamics, and defects in its dephosphorylation at the synapse produce an accumulation of coated endocytic intermediates. However, the involvement of PtdIns(4,5)P2 in synaptic vesicle exocytosis remains unclear. Here, we show that decreased levels of PtdIns(4,5)P2 in the brain and an impairment of its depolarization-dependent synthesis in nerve terminals lead to early postnatal lethality and synaptic defects in mice. These include decreased frequency of miniature currents, enhanced synaptic depression, a smaller readily releasable pool of vesicles, delayed endocytosis and slower recycling kinetics. Our results demonstrate a critical role for PtdIns(4,5)P2 synthesis in the regulation of multiple steps of the synaptic vesicle cycle.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Early postnatal lethality in PI PK1γ-deficient mice.
Figure 2: Alteration of PtdInsP2 metabolism in PIPK1γ-/- mice.
Figure 3: Synaptic transmission in PIPK1γ-/- neurons grown in culture.
Figure 4: Slower rates of endocytosis and recycling in PIPK1γ-/- neurons.
Figure 5: Defects in membrane recycling in PIPK1γ-deficient nerve terminals as revealed by electron microscopy.

Similar content being viewed by others

References

  1. De Camilli, P., Emr, S. D., McPherson, P. S. & Novick, P. Phosphoinositides as regulators in membrane traffic. Science 271, 1533–1539 (1996)

    Article  ADS  CAS  Google Scholar 

  2. Hilgemann, D. W., Feng, S. & Nasuhoglu, C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci. STKE 2001, RE19 (2001)

    CAS  Google Scholar 

  3. Martin, T. F. PI(4,5)P(2) regulation of surface membrane traffic. Curr. Opin. Cell Biol. 13, 493–499 (2001)

    Article  CAS  Google Scholar 

  4. Czech, M. P. Dynamics of phosphoinositides in membrane retrieval and insertion. Annu. Rev. Physiol. 65, 791–815 (2003)

    Article  CAS  Google Scholar 

  5. Valtorta, F. & Meldolesi, J. The presynaptic compartment: signals and targets. Semin. Cell Biol. 5, 211–219 (1994)

    Article  CAS  Google Scholar 

  6. Wang, S. S. & Augustine, G. J. Confocal imaging and local photolysis of caged compounds: dual probes of synaptic function. Neuron 15, 755–760 (1995)

    Article  CAS  Google Scholar 

  7. Wiedemann, C., Schafer, T., Burger, M. M. & Sihra, T. S. An essential role for a small synaptic vesicle-associated phosphatidylinositol 4-kinase in neurotransmitter release. J. Neurosci. 18, 5594–5602 (1998)

    Article  CAS  Google Scholar 

  8. Cremona, O. et al. Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99, 179–188 (1999)

    Article  CAS  Google Scholar 

  9. Lackner, M. R., Nurrish, S. J. & Kaplan, J. M. Facilitation of synaptic transmission by EGL-30 Gqα and EGL-8 PLCβ: DAG binding to UNC-13 is required to stimulate acetylcholine release. Neuron 24, 335–346 (1999)

    Article  CAS  Google Scholar 

  10. Gad, H. et al. Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 27, 301–312 (2000)

    Article  CAS  Google Scholar 

  11. Harris, T. W., Hartwieg, E., Horvitz, H. R. & Jorgensen, E. M. Mutations in synaptojanin disrupt synaptic vesicle recycling. J. Cell Biol. 150, 589–600 (2000)

    Article  CAS  Google Scholar 

  12. Rhee, J. S. et al. Beta phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs. Cell 108, 121–133 (2002)

    Article  CAS  Google Scholar 

  13. Micheva, K. D., Buchanan, J., Holz, R. W. & Smith, S. J. Retrograde regulation of synaptic vesicle endocytosis and recycling. Nature Neurosci. 6, 925–932 (2003)

    Article  CAS  Google Scholar 

  14. Murthy, V. N. & De Camilli, P. Cell biology of the presynaptic terminal. Annu. Rev. Neurosci. 26, 701–728 (2003)

    Article  CAS  Google Scholar 

  15. Heuser, J. E. & Reese, T. S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344 (1973)

    Article  CAS  Google Scholar 

  16. Takei, K., Mundigl, O., Daniell, L. & De Camilli, P. The synaptic vesicle cycle: A single vesicle budding step involving clathrin and dynamin. J. Cell Biol. 133, 1237–1250 (1996)

    Article  CAS  Google Scholar 

  17. Fesce, R. & Meldolesi, J. Peeping at the vesicle kiss. Nature Cell Biol. 1, E3–E4 (1999)

    Article  CAS  Google Scholar 

  18. Aravanis, A. M., Pyle, J. L. & Tsien, R. W. Single synaptic vesicles fusing transiently and successively without loss of identity. Nature 423, 643–647 (2003)

    Article  ADS  CAS  Google Scholar 

  19. Gandhi, S. P. & Stevens, C. F. Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423, 607–613 (2003)

    Article  ADS  CAS  Google Scholar 

  20. McPherson, P. S. et al. A presynaptic inositol-5-phosphatase. Nature 379, 353–357 (1996)

    Article  ADS  CAS  Google Scholar 

  21. Kim, W. T. et al. Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice. Proc. Natl Acad. Sci. USA 99, 17143–17148 (2002)

    Article  ADS  CAS  Google Scholar 

  22. Verstreken, P. et al. Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating. Neuron 40, 733–748 (2003)

    Article  CAS  Google Scholar 

  23. Takei, K. et al. Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94, 131–141 (1998)

    Article  CAS  Google Scholar 

  24. Ford, M. G. et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002)

    Article  ADS  CAS  Google Scholar 

  25. Evans, P. R. & Owen, D. J. Endocytosis and vesicle trafficking. Curr. Opin. Struct. Biol. 12, 814–821 (2002)

    Article  CAS  Google Scholar 

  26. Hay, J. C. et al. ATP-dependent inositide phosphorylation required for Ca(2 + )-activated secretion. Nature 374, 173–177 (1995)

    Article  ADS  CAS  Google Scholar 

  27. Holz, R. W. et al. A pleckstrin homology domain specific for phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5–P2 as being important in exocytosis. J. Biol. Chem. 275, 17878–17885 (2000)

    Article  CAS  Google Scholar 

  28. Khvotchev, M. & Sudhof, T. C. Newly synthesized phosphatidylinositol phosphates are required for synaptic norepinephrine but not glutamate or γ-aminobutyric acid (GABA) release. J. Biol. Chem. 273, 21451–21454 (1998)

    Article  CAS  Google Scholar 

  29. Doughman, R. L., Firestone, A. J. & Anderson, R. A. Phosphatidylinositol phosphate kinases put PI4,5P(2) in its place. J. Membr. Biol. 194, 77–89 (2003)

    Article  CAS  Google Scholar 

  30. Wenk, M. R. et al. PIP kinase Iγ is the major PI(4,5)P(2) synthesizing enzyme at the synapse. Neuron 32, 79–88 (2001)

    Article  CAS  Google Scholar 

  31. Di Paolo, G. et al. Recruitment and regulation of phosphatidylinositol phosphate kinase type 1γ by the FERM domain of talin. Nature 420, 85–89 (2002)

    Article  ADS  CAS  Google Scholar 

  32. Ling, K., Doughman, R. L., Firestone, A. J., Bunce, M. W. & Anderson, R. A. Type Iγ phosphatidylinositol phosphate kinase targets and regulates focal adhesions. Nature 420, 89–93 (2002)

    Article  ADS  CAS  Google Scholar 

  33. Krauss, M. et al. ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Iγ. J. Cell Biol. 162, 113–124 (2003)

    Article  CAS  Google Scholar 

  34. Aikawa, Y. & Martin, T. F. ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5) bisphosphate required for regulated exocytosis. J. Cell Biol. 162, 647–659 (2003)

    Article  CAS  Google Scholar 

  35. Wenk, M. R. et al. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry. Nature Biotechnol. 21, 813–817 (2003)

    Article  CAS  Google Scholar 

  36. Honda, A. et al. Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell 99, 521–532 (1999)

    Article  CAS  Google Scholar 

  37. Audigier, S. M., Wang, J. K. & Greengard, P. Membrane depolarization and carbamoylcholine stimulate phosphatidylinositol turnover in intact nerve terminals. Proc. Natl Acad. Sci. USA 85, 2859–2863 (1988)

    Article  ADS  CAS  Google Scholar 

  38. Luthi, A. et al. Synaptojanin 1 contributes to maintaining the stability of GABAergic transmission in primary cultures of cortical neurons. J. Neurosci. 21, 9101–9111 (2001)

    Article  CAS  Google Scholar 

  39. Stevens, C. F. & Tsujimoto, T. Estimates for the pool size of releasable quanta at a single central synapse and for the time required to refill the pool. Proc. Natl Acad. Sci. USA 92, 846–849 (1995)

    Article  ADS  CAS  Google Scholar 

  40. Rosenmund, C. & Stevens, C. F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996)

    Article  CAS  Google Scholar 

  41. Kashani, A. H., Chen, B. M. & Grinnell, A. D. Hypertonic enhancement of transmitter release from frog motor nerve terminals: Ca2+ independence and role of integrins. J. Physiol. (Lond.) 530, 243–252 (2001)

    Article  CAS  Google Scholar 

  42. Ryan, T. A. Inhibitors of myosin light chain kinase block synaptic vesicle pool mobilization during action potential firing. J. Neurosci. 19, 1317–1323 (1999)

    Article  CAS  Google Scholar 

  43. Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998)

    Article  ADS  CAS  Google Scholar 

  44. Sankaranarayanan, S. & Ryan, T. A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nature Cell Biol. 2, 197–204 (2000)

    Article  CAS  Google Scholar 

  45. Sankaranarayanan, S. & Ryan, T. A. Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nature Neurosci. 4, 129–136 (2001)

    Article  CAS  Google Scholar 

  46. Bai, J., Tucker, W. C. & Chapman, E. R. PIP(2) increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nature Struct. Mol. Biol. 11, 36–44 (2004)

    Article  CAS  Google Scholar 

  47. Neeb, A., Koch, H., Schurmann, A. & Brose, N. Direct interaction between the ARF-specific guanine nucleotide exchange factor msec7–1 and presynaptic Munc13–1. Eur. J. Cell Biol. 78, 533–538 (1999)

    Article  CAS  Google Scholar 

  48. Fitzsimonds, R. M., Song, H. J. & Poo, M. M. Propagation of activity-dependent synaptic depression in simple neural networks. Nature 388, 439–448 (1997)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank L. Liu, L. Lucast, L. Daniell and W. Yan for technical assistance; J. Kunz and R. Jahn for the antibodies; and J. Morgan and O. Cremona for critical reading of the manuscript. This work was supported in part by National Institutes of Health grants to P.D.C, M.W., T.A.R. and R.M.F., and by pilot grants from the Yale Center for Genomic and Proteomics to P.D.C and M.W.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Pietro De Camilli.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Absence of PIPK1g in knockout mice. (JPG 43 kb)

Supplementary Figure 2

Glutamate release defects in cortical synaptosomes from PIPK1g heterozygotes. (JPG 41 kb)

Supplementary Figure 3

The recycling pool size is significantly smaller in nerve terminals from PIPK1g KO neurons. (JPG 27 kb)

Supplementary Figure 4

Vesicle reacidification is not delayed in PIPK1g KO nerve terminals. (JPG 41 kb)

Supplementary Legends

Legends for Supplementary Figures 1–4. (DOC 43 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paolo, G., Moskowitz, H., Gipson, K. et al. Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking. Nature 431, 415–422 (2004). https://doi.org/10.1038/nature02896

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature02896

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing