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:

Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones

Nature Neuroscience volume 9, pages 1257–1264 (2006)Cite this article

Abstract

Growth cones at the tips of nascent and regenerating axons direct axon elongation. Netrin-1, a secreted molecule that promotes axon outgrowth and regulates axon pathfinding, elevates cyclic AMP (cAMP) levels in growth cones and regulates growth cone morphology and axonal outgrowth. These morphological effects depend on the intracellular levels of cAMP. However, the specific pathways that regulate cAMP levels in response to netrin-1 signaling are unclear. Here we show that 'soluble' adenylyl cyclase (sAC), an atypical calcium-regulated cAMP-generating enzyme previously implicated in sperm maturation, is expressed in developing rat axons and generates cAMP in response to netrin-1. Overexpression of sAC results in axonal outgrowth and growth cone elaboration, whereas inhibition of sAC blocks netrin-1–induced axon outgrowth and growth cone elaboration. Taken together, these results indicate that netrin-1 signals through sAC-generated cAMP, and identify a fundamental role for sAC in axonal development.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Expression of sAC in rat neurons.
Figure 2: sAC overexpression induces axonal outgrowth and growth cone elaboration.
Figure 3: Netrin-1 induction of cAMP is blocked by sAC inhibitors.
Figure 4: Netrin-1–induced growth cone elaboration is blocked by sAC inhibitors.
Figure 5: siRNA-mediated knockdown of sAC reduces netrin-1–dependent growth cone elaboration.
Figure 6: Netrin-1–mediated growth cone elaboration requires cAMP and PKA.
Figure 7: Differentiation of the roles of tmAC and sAC in growth cone signaling.
Figure 8: Netrin-1–induced axonal outgrowth is blocked by sAC inhibitors.

Similar content being viewed by others

References

  1. Song, H. & Poo, M. The cell biology of neuronal navigation. Nat. Cell Biol. 3, E81–E88 (2001).

    Article  CAS  Google Scholar 

  2. Serafini, T. et al. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78, 409–424 (1994).

    Article  CAS  Google Scholar 

  3. Kennedy, T.E., Serafini, T., de la Torre, J.R. & Tessier-Lavigne, M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78, 425–435 (1994).

    Article  CAS  Google Scholar 

  4. Tucker, K.L., Meyer, M. & Barde, Y.A. Neurotrophins are required for nerve growth during development. Nat. Neurosci. 4, 29–37 (2001).

    Article  CAS  Google Scholar 

  5. Nishiyama, M. et al. Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 423, 990–995 (2003).

    Article  CAS  Google Scholar 

  6. Hopker, V.H., Shewan, D., Tessier-Lavigne, M., Poo, M. & Holt, C. Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1. Nature 401, 69–73 (1999).

    Article  CAS  Google Scholar 

  7. Hong, K. et al. A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97, 927–941 (1999).

    Article  CAS  Google Scholar 

  8. Metin, C., Deleglise, D., Serafini, T., Kennedy, T.E. & Tessier-Lavigne, M. A role for netrin-1 in the guidance of cortical efferents. Development 124, 5063–5074 (1997).

    CAS  PubMed  Google Scholar 

  9. Serafini, T. et al. Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87, 1001–1014 (1996).

    Article  CAS  Google Scholar 

  10. Seaman, C., Anderson, R., Emery, B. & Cooper, H.M. Localization of the netrin guidance receptor, DCC, in the developing peripheral and enteric nervous systems. Mech. Dev. 103, 173–175 (2001).

    Article  CAS  Google Scholar 

  11. Keino-Masu, K. et al. Deleted in colorectal cancer (DCC) encodes a netrin receptor. Cell 87, 175–185 (1996).

    Article  CAS  Google Scholar 

  12. Shekarabi, M. & Kennedy, T.E. The netrin-1 receptor DCC promotes filopodia formation and cell spreading by activating Cdc42 and Rac1. Mol. Cell. Neurosci. 19, 1–17 (2002).

    Article  CAS  Google Scholar 

  13. Shekarabi, M. et al. Deleted in colorectal cancer binding netrin-1 mediates cell substrate adhesion and recruits Cdc42, Rac1, Pak1, and N-WASP into an intracellular signaling complex that promotes growth cone expansion. J. Neurosci. 25, 3132–3141 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Lohof, A.M., Quillan, M., Dan, Y. & Poo, M.M. Asymmetric modulation of cytosolic cAMP activity induces growth cone turning. J. Neurosci. 12, 1253–1261 (1992).

    Article  CAS  Google Scholar 

  16. Spencer, T. & Filbin, M.T. A role for cAMP in regeneration of the adult mammalian CNS. J. Anat. 204, 49–55 (2004).

    Article  CAS  Google Scholar 

  17. Forte, L.R., Bylund, D.B. & Zahler, W.L. Forskolin does not activate sperm adenylate cyclase. Mol. Pharmacol. 24, 42–47 (1983).

    CAS  PubMed  Google Scholar 

  18. Buck, J., Sinclair, M.L., Schapal, L., Cann, M.J. & Levin, L.R. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc. Natl. Acad. Sci. USA 96, 79–84 (1999).

    Article  CAS  Google Scholar 

  19. Sinclair, M.L. et al. Specific expression of soluble adenylyl cyclase in male germ cells. Mol. Reprod. Dev. 56, 6–11 (2000).

    Article  CAS  Google Scholar 

  20. Jaiswal, B.S. & Conti, M. Identification and functional analysis of splice variants of the germ cell soluble adenylyl cyclase. J. Biol. Chem. 276, 31698–31708 (2001).

    Article  CAS  Google Scholar 

  21. Esposito, G. et al. Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect. Proc. Natl. Acad. Sci. USA 101, 2993–2998 (2004).

    Article  CAS  Google Scholar 

  22. Hess, K.C. et al. The “soluble” adenylyl cyclase in sperm mediates multiple signaling events required for fertilization. Dev. Cell 9, 249–259 (2005).

    Article  CAS  Google Scholar 

  23. Zippin, J.H. et al. Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains. FASEB J. 17, 82–84 (2003).

    Article  CAS  Google Scholar 

  24. Braun, T. & Dods, R.F. Development of a Mn2+-sensitive, “soluble” adenylate cyclase in rat testis. Proc. Natl. Acad. Sci. USA 72, 1097–1101 (1975).

    Article  CAS  Google Scholar 

  25. Jaiswal, B.S. & Conti, M. Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa. Proc. Natl. Acad. Sci. USA 100, 10676–10681 (2003).

    Article  CAS  Google Scholar 

  26. Litvin, T.N., Kamenetsky, M., Zarifyan, A., Buck, J. & Levin, L.R. Kinetic properties of “soluble” adenylyl cyclase. Synergism between calcium and bicarbonate. J. Biol. Chem. 278, 15922–15926 (2003).

    Article  CAS  Google Scholar 

  27. Jackman, A. & Fitzgerald, M. Development of peripheral hindlimb and central spinal cord innervation by subpopulations of dorsal root ganglion cells in the embryonic rat. J. Comp. Neurol. 418, 281–298 (2000).

    Article  CAS  Google Scholar 

  28. Tessier-Lavigne, M., Placzek, M., Lumsden, A.G., Dodd, J. & Jessell, T.M. Chemotropic guidance of developing axons in the mammalian central nervous system. Nature 336, 775–778 (1988).

    Article  CAS  Google Scholar 

  29. Stessin, A.M. et al. Soluble adenylyl cyclase mediates nerve growth factor-induced activation of Rap1. J. Biol. Chem. 281, 17253–17258 (2006).

    Article  CAS  Google Scholar 

  30. Su, A.I. et al. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl. Acad. Sci. USA 101, 6062–6067 (2004).

    Article  CAS  Google Scholar 

  31. Pastor-Soler, N. et al. Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J. Biol. Chem. 278, 49523–49529 (2003).

    Article  CAS  Google Scholar 

  32. Corset, V. et al. Netrin-1-mediated axon outgrowth and cAMP production requires interaction with adenosine A2b receptor. Nature 407, 747–750 (2000).

    Article  CAS  Google Scholar 

  33. Higuchi, H., Yamashita, T., Yoshikawa, H. & Tohyama, M. Functional inhibition of the p75 receptor using a small interfering RNA. Biochem. Biophys. Res. Commun. 301, 804–809 (2003).

    Article  CAS  Google Scholar 

  34. Wu, K.Y. et al. Local translation of RhoA regulates growth cone collapse. Nature 436, 1020–1024 (2005).

    Article  CAS  Google Scholar 

  35. Waschek, J.A. Multiple actions of pituitary adenylyl cyclase activating peptide in nervous system development and regeneration. Dev. Neurosci. 24, 14–23 (2002).

    Article  CAS  Google Scholar 

  36. Guirland, C., Buck, K.B., Gibney, J.A., DiCicco-Bloom, E. & Zheng, J.Q. Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide. J. Neurosci. 23, 2274–2283 (2003).

    Article  CAS  Google Scholar 

  37. Johnson, R.A. et al. Isozyme-dependent sensitivity of adenylyl cyclases to P-site-mediated inhibition by adenine nucleosides and nucleoside 3′-polyphosphates. J. Biol. Chem. 272, 8962–8966 (1997).

    Article  CAS  Google Scholar 

  38. Gille, A. et al. Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides. J. Biol. Chem. 279, 19955–19969 (2004).

    Article  CAS  Google Scholar 

  39. Chen, Y. et al. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289, 625–628 (2000).

    Article  CAS  Google Scholar 

  40. Nobes, C.D. & Hall, A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53–62 (1995).

    Article  CAS  Google Scholar 

  41. Stein, E., Zou, Y., Poo, M. & Tessier-Lavigne, M. Binding of DCC by netrin-1 to mediate axon guidance independent of adenosine A2B receptor activation. Science 291, 1976–1982 (2001).

    Article  CAS  Google Scholar 

  42. Moore, S.W. & Kennedy, T.E. Protein kinase A regulates the sensitivity of spinal commissural axon turning to netrin-1 but does not switch between chemoattraction and chemorepulsion. J. Neurosci. 26, 2419–2423 (2006).

    Article  CAS  Google Scholar 

  43. Roelofs, J. & Van Haastert, P.J. Deducing the origin of soluble adenylyl cyclase, a gene lost in multiple lineages. Mol. Biol. Evol. 19, 2239–2246 (2002).

    Article  CAS  Google Scholar 

  44. Geng, W. et al. Cloning and characterization of the human soluble adenylyl cyclase. Am. J. Physiol. Cell Physiol. 288, C1305–C1316 (2005).

    Article  CAS  Google Scholar 

  45. Wang, G.X. & Poo, M.M. Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434, 898–904 (2005).

    Article  CAS  Google Scholar 

  46. Banker, G. & Goslin, K. Culturing Nerve Cells 2nd edn. (MIT Press, Cambridge, MA, 1998).

    Google Scholar 

  47. de la Torre, J.R. et al. Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 19, 1211–1224 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L.J. Cox for his comments and suggestions. Supported by the US National Institutes of Health (NIH) MH066204 and the Christopher Reeve Paralysis Foundation (S.R.J.); NIH GM07739 and the Barbara and Stephen Friedman Fellowship Endowment (J.H.Z.); NIH 5T32 CA062948 (D.R.H.); NIH 2T32 DA07274 (M.K.); NIH HD38722, the American Diabetes Association and the Hirschl Weill-Caulier Trust (L.R.L.); and NIH HD42060, GM62328 and the Ellison Medical Foundation (J.B.).

Author information

Authors and Affiliations

  1. Department of Pharmacology, Weill Medical College, Cornell University, New York, 10021, New York, USA

    Karen Y Wu, Jonathan H Zippin, David R Huron, Margarita Kamenetsky, Ulrich Hengst, Jochen Buck, Lonny R Levin & Samie R Jaffrey

Authors
  1. Karen Y Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jonathan H Zippin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David R Huron
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Margarita Kamenetsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ulrich Hengst
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jochen Buck
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lonny R Levin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Samie R Jaffrey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Samie R Jaffrey.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Expression of sAC in rat neurons. (PDF 144 kb)

Supplementary Fig. 2

Netrin-1 mediates growth cone elaboration through sAC. (PDF 60 kb)

Supplementary Fig. 3

DRG neurons are susceptible to highly efficient siRNA-mediated mRNA knockdown. (PDF 67 kb)

Supplementary Fig. 4

Netrin-1-mediated growth cone elaboration requires cAMP and PKA. (PDF 48 kb)

Supplementary Fig. 5

Differentiation of the roles of tmAC and sAC in growth cone signaling. (PDF 52 kb)

Supplementary Fig. 6

Netrin-1 mediates axon outgrowth through DCC. (PDF 34 kb)

Supplementary Fig. 7

Netrin-1 mediates growth cone elaboration in a dose-dependent manner. (PDF 31 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, K., Zippin, J., Huron, D. et al. Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones. Nat Neurosci 9, 1257–1264 (2006). https://doi.org/10.1038/nn1767

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn1767

This article is cited by

Search

Advanced 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