Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Nerve growth factor (NGF) is a neurotrophic factor secreted by cells that are the targets of innervation of sympathetic and some sensory neurons. However, the mechanism by which the NGF signal is propagated from the axon terminal to the cell body, which can be more than 1 meter away, to influence biochemical events critical for growth and survival of neurons has remained unclear. An NGF-mediated signal transmitted from the terminals and distal axons of cultured rat sympathetic neurons to their nuclei regulated phosphorylation of the transcription factor CREB (cyclic adenosine monophosphate response element – binding protein). Internalization of NGF and its receptor tyrosine kinase TrkA, and their transport to the cell body, were required for transmission of this signal. The tyrosine kinase activity of TrkA was required to maintain it in an autophosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus, an NGF-TrkA complex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.

Bibliography

Riccio, A., Pierchala, B. A., Ciarallo, C. L., & Ginty, D. D. (1997). An NGF-TrkA-Mediated Retrograde Signal to Transcription Factor CREB in Sympathetic Neurons. Science, 277(5329), 1097–1100.

Authors 4
  1. Antonella Riccio (first)
  2. Brian A. Pierchala (additional)
  3. Christopher L. Ciarallo (additional)
  4. David D. Ginty (additional)
References 38 Referenced 350
  1. 10.1126/science.3306916
  2. Barde Y. A., Neuron 2, 1525 (1989); (10.1016/0896-6273(89)90040-8) / Neuron by Barde Y. A. (1989)
  3. Oppenheim R. W., Annu. Rev. Neurosci. 14, 453 (1991) ; (10.1146/annurev.ne.14.030191.002321) / Annu. Rev. Neurosci. by Oppenheim R. W. (1991)
  4. Klein R., FASEB J. 8, 738 (1994). (10.1096/fasebj.8.10.8050673) / FASEB J. by Klein R. (1994)
  5. Crowley C., et al., Cell 76, 1001 (1994); (10.1016/0092-8674(94)90378-6) / Cell by Crowley C. (1994)
  6. Mayo R. A., Zettler C., Pharmacol. Ther. 65, 93 (1993); / Pharmacol. Ther. by Mayo R. A. (1993)
  7. 10.1146/annurev.ne.18.030195.001255
  8. Barbacid M., Curr. Opin. Cell Biol. 7, 148 (1995). (10.1016/0955-0674(95)80022-0) / Curr. Opin. Cell Biol. by Barbacid M. (1995)
  9. Halegoua S., Armstrong R. C., Kremer N. E., Curr. Top. Microbiol. Immunol. 165, 119 (1990). / Curr. Top. Microbiol. Immunol. by Halegoua S. (1990)
  10. Kaplan D. R., Stephens R. M., J. Neurobiol. 25, 1404 (1994); (10.1002/neu.480251108) / J. Neurobiol. by Kaplan D. R. (1994)
  11. Segal R. A., Greenberg M. E., Annu. Rev. Neurosci. 19, 463 (1996); (10.1146/annurev.ne.19.030196.002335) / Annu. Rev. Neurosci. by Segal R. A. (1996)
  12. Blenis J., Proc. Natl. Acad. Sci. U.S.A. 90, 5889 (1993); (10.1073/pnas.90.13.5889) / Proc. Natl. Acad. Sci. U.S.A. by Blenis J. (1993)
  13. Segar R., Krebs E. G., FASEB J. 9, 726 (1995) . (10.1096/fasebj.9.9.7601337) / FASEB J. by Segar R. (1995)
  14. Ginty D. D., Bonni A., Greenberg M. E., Cell 77, 713 (1994); (10.1016/0092-8674(94)90055-8) / Cell by Ginty D. D. (1994)
  15. 10.1006/mcne.1995.1015
  16. 10.1016/0006-8993(74)90536-8
  17. 10.1523/JNEUROSCI.02-04-00431.1982
  18. 10.1016/0304-3940(83)90155-6
  19. Campenot R. B., J. Neurobiol. 25, 599 (1994). (10.1002/neu.480250603) / J. Neurobiol. by Campenot R. B. (1994)
  20. ___, Proc. Natl. Acad. Sci. U.S.A. 74, 4516 (1977); (10.1073/pnas.74.10.4516) / Proc. Natl. Acad. Sci. U.S.A. by ___ (1977)
  21. ; Dev. Biol. 93 1 (1982).
  22. 10.1126/science.8097062
  23. Sympathetic neurons were isolated from superior cervical ganglia of neonatal rats (21). Cells were plated in compartmentalized chambers (Tyler Research Products) as described (8) and grown in growth medium [Dulbecco's modified Eagle's medium containing fetal bovine serum (10%) 5 μM arabinosylcytosine (Ara-C) and NGF (200 ng/ml)]. Medium was replaced every 3 days. After 4 to 7 days medium within the chamber containing cell bodies was replaced with medium lacking NGF. This procedure resulted in the death of those neurons that had not extended processes into adjacent compartments. Neurons grown in center-plated chambers were used 2 to 3 weeks after plating. Neurons grown in side-plated chambers required a longer time to project through two barriers and were used 3 to 4 weeks after plating. For P-CREB immunocytochemistry experiments medium was changed to contain a low concentration of NGF (2 ng/ml) for 48 hours before stimulation with NGF. Neurons were treated with NGF (200 ng/ml) and then fixed with acetone:methanol (1:1) for 3 min. Fixed cells were rinsed with phosphate-buffered saline (PBS) and permeabilized with PBS containing Triton X-100 (0.1%). After blocking with PBS solution containing horse serum (3%) and bovine serum albumin (BSA 3%) at room temperature for 2 hours cells were incubated with anti–P-CREB (1:1000 dilution) in the above solution at 4°C for 18 hours. Immune complexes were detected with an avidin biotin detection system (Vector Laboratories).
  24. Ure D. R., Campenot R. B., J. Neurosci. 17, 1282 (1997). (10.1523/JNEUROSCI.17-04-01282.1997) / J. Neurosci. by Ure D. R. (1997)
  25. NGF was covalently coupled to 1 μm–diameter microspheres by means of a carbodiimide cross-linking method. Amine-modified FluoSpheres (2% solids; Molecular Probes) were washed three times with solution 1 [2( N -morpholino)ethanesulfonic acid (0.1 M pH 6.0)] using centrifugation and gentle resuspension. The FluoSpheres were then resuspended in solution 1 containing NGF (100 μg/ml) to a final concentration of 1% microspheres. EDAC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; Molecular Probes] which was freshly dissolved in solution 1 was then added to this suspension and the mixture was rotated at room temperature for 2 hours. The cross-linking reaction was quenched by the addition of glycine (0.1 M pH 6.0). Beads were then washed four times with a high-salt buffer (10 mM sodium phosphate 1.8 mM potassium phosphate 1 M sodium chloride and 2.6 mM potassium chloride pH 4.0) for 30 min per wash. The beads were next incubated overnight in the high-salt buffer (pH 7.4) washed four times the next day with high-salt buffer (pH 10.0) to remove adsorbed NGF and then resuspended in PBS (pH 7.4) at a final concentration of 0.25% solids. To ensure that all adsorbed NGF was removed from the FluoSpheres we subjected a set of FluoSpheres (control beads) to a similar procedure except EDAC was left out of the cross-linking step. The bioactivity of NGF-coupled FluoSpheres was found to depend on the concentration of NGF and EDAC used. At the concentration of NGF used we found that 2 to 4 μM EDAC was optimal for obtaining bioactive NGF-coupled beads; we also found that 4 μl/ml of the bead solution was sufficient to cover all cell surfaces.
  26. TrkA was immunoprecipitated with anti-panTrk (22) from PC12 cells (7 × 10 6 cells per plate) treated with control medium control beads (4 μl/ml) NGF (100 ng/ml) or NGF-coupled beads (4 μl/ml) (13) and phosphotyrosine protein immunoblotting was done as described (22). Similar results were obtained by immunoprecipitating TrkA from primary cultures of sympathetic neurons (14).
  27. A. Riccio B. A. Pierchala C. L. Ciarallo D. D. Ginty unpublished data.
  28. 10.1523/JNEUROSCI.16-24-07950.1996
  29. Sympathetic neurons grown in center-plated chambers were incubated with medium without NGF for 12 hours then treated with medium containing NGF (200 ng/ml) as described in Fig. 1. Cells were then fixed with PBS containing 4% paraformaldehyde permeabilized with PBS containing Triton X-100 (0.1%) washed with PBS containing glycine (10 mM) and incubated in blocking solution [PBS containing BSA (3%) and normal goat serum (3%)] for 2 hours at room temperature. Cells were then incubated in blocking solution containing anti–P-Trk (1:250 dilution; New England Biolabs). This antibody specifically recognizes TrkA only when it is phosphorylated on Tyr 674 and Tyr 67 5 . The immune complexes were visualized with an avidin biotin detection system (Vector Laboratories).
  30. Ehlers M. D., Kaplan D. R., Price D. L., Koliatsos V. E., J. Cell Biol. 130, 149 (1995). (10.1083/jcb.130.1.149) / J. Cell Biol. by Ehlers M. D. (1995)
  31. Johanson S. O., Crouch M. F., Hendry I. A., Brain Res. 690, 55 (1995). (10.1016/0006-8993(95)00587-G) / Brain Res. by Johanson S. O. (1995)
  32. Berg M. M., Sternberg D. W., Parada L. F., Chao M. V., J. Biol. Chem. 267, 13 (1992); (10.1016/S0021-9258(18)48447-5) / J. Biol. Chem. by Berg M. M. (1992)
  33. Tapley P., Lamballe F., Barbacid M., Oncogene 7, 371 (1992) ; / Oncogene by Tapley P. (1992)
  34. Ohmichi M., Decker S. J., Pang L., Saltiel A. R., Biochemistry 31, 4034 (1992). (10.1021/bi00131a019) / Biochemistry by Ohmichi M. (1992)
  35. K-252a appeared to selectively block TrkA and not other protein kinases necessary for CREB phosphorylation because it completely blocked CREB phosphorylation induced by NGF but not that induced by epidermal growth factor or forskolin (14).
  36. Mains R. E., Patterson P. H., J. Cell Biol. 59, 329 (1973). (10.1083/jcb.59.2.329) / J. Cell Biol. by Mains R. E. (1973)
  37. 10.1016/0896-6273(92)90241-5
  38. We thank A. Ghosh A. Kolodkin D. Linden and members of the Ginty laboratory for discussions and comments on this manuscript and R. Adler D. Anderson R. Mains A. Pieper and C. Tsui for helpful suggestions. We also thank D. Kaplan for providing anti-panTrk (203) and H. Ruan for anti–P-Trk. Supported by a Pew Scholars Award a Klingenstein Award in Neuroscience an Alfred Sloan Research Fellowship and an American Cancer Society Junior Faculty Research Award (D.D.G.); a fellowship from Ministero della Sanità of Italy (A.R.); March of Dimes Birth Defects Foundation research grant 5-FY95-1114; and the Alzheimer's Association–Charles Evans Pilot Research Award.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:44 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 10:23 p.m.)
Indexed 59 minutes ago (Aug. 29, 2025, 6:27 a.m.)
Issued 28 years ago (Aug. 22, 1997)
Published 28 years ago (Aug. 22, 1997)
Published Print 28 years ago (Aug. 22, 1997)
Funders 0

None

@article{Riccio_1997, title={An NGF-TrkA-Mediated Retrograde Signal to Transcription Factor CREB in Sympathetic Neurons}, volume={277}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.277.5329.1097}, DOI={10.1126/science.277.5329.1097}, number={5329}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Riccio, Antonella and Pierchala, Brian A. and Ciarallo, Christopher L. and Ginty, David D.}, year={1997}, month=aug, pages={1097–1100} }