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. 1994 Feb 2;124(4):537–546. doi: 10.1083/jcb.124.4.537

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

PMCID: PMC2119915  PMID: 8106551

Abstract

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.

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Selected References

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  1. Arends M. J., Morris R. G., Wyllie A. H. Apoptosis. The role of the endonuclease. Am J Pathol. 1990 Mar;136(3):593–608. [PMC free article] [PubMed] [Google Scholar]
  2. Batistatou A., Greene L. A. Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. J Cell Biol. 1991 Oct;115(2):461–471. doi: 10.1083/jcb.115.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buckmaster A., Nobes C. D., Edwards S. N., Tolkovsky A. M. Nerve Growth Factor is Required for Induction of c-Fos Immunoreactivity by Serum, Depolarization, Cyclic AMP or Trauma in Cultured Rat Sympathetic Neurons. Eur J Neurosci. 1991;3(7):698–707. doi: 10.1111/j.1460-9568.1991.tb00855.x. [DOI] [PubMed] [Google Scholar]
  4. Bursch W., Kleine L., Tenniswood M. The biochemistry of cell death by apoptosis. Biochem Cell Biol. 1990 Sep;68(9):1071–1074. doi: 10.1139/o90-160. [DOI] [PubMed] [Google Scholar]
  5. Chun L. L., Patterson P. H. Role of nerve growth factor in the development of rat sympathetic neurons in vitro. I. Survival, growth, and differentiation of catecholamine production. J Cell Biol. 1977 Dec;75(3):694–704. doi: 10.1083/jcb.75.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chun L. L., Patterson P. H. Role of nerve growth factor in the development of rat sympathetic neurons in vitro. II. Developmental studies. J Cell Biol. 1977 Dec;75(3):705–711. doi: 10.1083/jcb.75.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clarke P. G. Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl) 1990;181(3):195–213. doi: 10.1007/BF00174615. [DOI] [PubMed] [Google Scholar]
  8. Claude P., Hawrot E., Dunis D. A., Campenot R. B. Binding, internalization, and retrograde transport of 125I-nerve growth factor in cultured rat sympathetic neurons. J Neurosci. 1982 Apr;2(4):431–442. doi: 10.1523/JNEUROSCI.02-04-00431.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen G. M., Sun X. M., Snowden R. T., Dinsdale D., Skilleter D. N. Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J. 1992 Sep 1;286(Pt 2):331–334. doi: 10.1042/bj2860331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen J. J., Duke R. C., Fadok V. A., Sellins K. S. Apoptosis and programmed cell death in immunity. Annu Rev Immunol. 1992;10:267–293. doi: 10.1146/annurev.iy.10.040192.001411. [DOI] [PubMed] [Google Scholar]
  11. Cunningham T. J. Naturally occurring neuron death and its regulation by developing neural pathways. Int Rev Cytol. 1982;74:163–186. doi: 10.1016/s0074-7696(08)61172-9. [DOI] [PubMed] [Google Scholar]
  12. Edwards S. N., Buckmaster A. E., Tolkovsky A. M. The death programme in cultured sympathetic neurones can be suppressed at the posttranslational level by nerve growth factor, cyclic AMP, and depolarization. J Neurochem. 1991 Dec;57(6):2140–2143. doi: 10.1111/j.1471-4159.1991.tb06434.x. [DOI] [PubMed] [Google Scholar]
  13. Hawrot E., Patterson P. H. Long-term culture of dissociated sympathetic neurons. Methods Enzymol. 1979;58:574–584. doi: 10.1016/s0076-6879(79)58174-9. [DOI] [PubMed] [Google Scholar]
  14. Henderson C. E., Camu W., Mettling C., Gouin A., Poulsen K., Karihaloo M., Rullamas J., Evans T., McMahon S. B., Armanini M. P. Neurotrophins promote motor neuron survival and are present in embryonic limb bud. Nature. 1993 May 20;363(6426):266–270. doi: 10.1038/363266a0. [DOI] [PubMed] [Google Scholar]
  15. Holtzman D. M., Li Y., Parada L. F., Kinsman S., Chen C. K., Valletta J. S., Zhou J., Long J. B., Mobley W. C. p140trk mRNA marks NGF-responsive forebrain neurons: evidence that trk gene expression is induced by NGF. Neuron. 1992 Sep;9(3):465–478. doi: 10.1016/0896-6273(92)90184-f. [DOI] [PubMed] [Google Scholar]
  16. Johnson E. M., Jr, Gorin P. D., Brandeis L. D., Pearson J. Dorsal root ganglion neurons are destroyed by exposure in utero to maternal antibody to nerve growth factor. Science. 1980 Nov 21;210(4472):916–918. doi: 10.1126/science.7192014. [DOI] [PubMed] [Google Scholar]
  17. Kerr J. F., Wyllie A. H., Currie A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug;26(4):239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Koike T., Tanaka S. Evidence that nerve growth factor dependence of sympathetic neurons for survival in vitro may be determined by levels of cytoplasmic free Ca2+. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3892–3896. doi: 10.1073/pnas.88.9.3892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levi-Montalcini R., Booker B. DESTRUCTION OF THE SYMPATHETIC GANGLIA IN MAMMALS BY AN ANTISERUM TO A NERVE-GROWTH PROTEIN. Proc Natl Acad Sci U S A. 1960 Mar;46(3):384–391. doi: 10.1073/pnas.46.3.384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Martin D. P., Schmidt R. E., DiStefano P. S., Lowry O. H., Carter J. G., Johnson E. M., Jr Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J Cell Biol. 1988 Mar;106(3):829–844. doi: 10.1083/jcb.106.3.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Martin S. J., Lennon S. V., Bonham A. M., Cotter T. G. Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis. J Immunol. 1990 Sep 15;145(6):1859–1867. [PubMed] [Google Scholar]
  22. Mesner P. W., Winters T. R., Green S. H. Nerve growth factor withdrawal-induced cell death in neuronal PC12 cells resembles that in sympathetic neurons. J Cell Biol. 1992 Dec;119(6):1669–1680. doi: 10.1083/jcb.119.6.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mobley W. C., Schenker A., Shooter E. M. Characterization and isolation of proteolytically modified nerve growth factor. Biochemistry. 1976 Dec 14;15(25):5543–5552. doi: 10.1021/bi00670a019. [DOI] [PubMed] [Google Scholar]
  24. Oberhammer F., Fritsch G., Schmied M., Pavelka M., Printz D., Purchio T., Lassmann H., Schulte-Hermann R. Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease. J Cell Sci. 1993 Feb;104(Pt 2):317–326. doi: 10.1242/jcs.104.2.317. [DOI] [PubMed] [Google Scholar]
  25. Oppenheim R. W. Cell death during development of the nervous system. Annu Rev Neurosci. 1991;14:453–501. doi: 10.1146/annurev.ne.14.030191.002321. [DOI] [PubMed] [Google Scholar]
  26. Peitsch M. C., Polzar B., Stephan H., Crompton T., MacDonald H. R., Mannherz H. G., Tschopp J. Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J. 1993 Jan;12(1):371–377. doi: 10.1002/j.1460-2075.1993.tb05666.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Raff M. C. Social controls on cell survival and cell death. Nature. 1992 Apr 2;356(6368):397–400. doi: 10.1038/356397a0. [DOI] [PubMed] [Google Scholar]
  28. Rukenstein A., Rydel R. E., Greene L. A. Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms. J Neurosci. 1991 Aug;11(8):2552–2563. doi: 10.1523/JNEUROSCI.11-08-02552.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Saadat S., Sendtner M., Rohrer H. Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture. J Cell Biol. 1989 May;108(5):1807–1816. doi: 10.1083/jcb.108.5.1807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schwartz L. M., Smith S. W., Jones M. E., Osborne B. A. Do all programmed cell deaths occur via apoptosis? Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):980–984. doi: 10.1073/pnas.90.3.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scott S. A., Davies A. M. Inhibition of protein synthesis prevents cell death in sensory and parasympathetic neurons deprived of neurotrophic factor in vitro. J Neurobiol. 1990 Jun;21(4):630–638. doi: 10.1002/neu.480210410. [DOI] [PubMed] [Google Scholar]
  32. Thoenen H. The changing scene of neurotrophic factors. Trends Neurosci. 1991 May;14(5):165–170. doi: 10.1016/0166-2236(91)90097-e. [DOI] [PubMed] [Google Scholar]
  33. Tolkovsky A. M., Walker A. E., Murrell R. D., Suidan H. S. Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons. J Cell Biol. 1990 Apr;110(4):1295–1306. doi: 10.1083/jcb.110.4.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ucker D. S., Obermiller P. S., Eckhart W., Apgar J. R., Berger N. A., Meyers J. Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes. Mol Cell Biol. 1992 Jul;12(7):3060–3069. doi: 10.1128/mcb.12.7.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Waring P., Egan M., Braithwaite A., Mullbacher A., Sjaarda A. Apoptosis induced in macrophages and T blasts by the mycotoxin sporidesmin and protection by Zn2+ salts. Int J Immunopharmacol. 1990;12(4):445–457. doi: 10.1016/0192-0561(90)90028-l. [DOI] [PubMed] [Google Scholar]
  36. Wright L. L., Cunningham T. J., Smolen A. J. Developmental neuron death in the rat superior cervical sympathetic ganglion: cell counts and ultrastructure. J Neurocytol. 1983 Oct;12(5):727–738. doi: 10.1007/BF01258147. [DOI] [PubMed] [Google Scholar]
  37. Wyllie A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980 Apr 10;284(5756):555–556. doi: 10.1038/284555a0. [DOI] [PubMed] [Google Scholar]
  38. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]

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