Retinal Neurons in Primary Cell Culture

Inhibition of Apoptosis by Pigment Epithelial Derived Factor (PEDF)
  • J. F. McGinnis
  • W. Chen
  • J. Tombran-Tink
  • D. Mrazek
  • V. Lerious
  • W. Cao


We have been interested in cellular regulatory mechanisms operating in mammalian retinal neurons, especially those determining sensitivity to factors which either promote or inhibit cell death. The production of reactive oxygen species through oxidative stress is believed to be an important mediator of neuronal cell death although the precise mechanism by which this occurs is unknown. Pigment epithelia derived factor (PEDF) has been shown to promotes neurotrophic differentiation and the survival of neurons of the central nervous system. Using a cell culture system, we demonstrate that multiple populations of retinal neurons can differentiate and be maintained in a chemically defined environment and that cell death induced by reactive oxygen species can be quantitated. The data also show that this induced death is dose dependent and occurs by an apoptotic mechanism which can be inhibited by PEDF. The mechanism by which PEDF provides this protection may be very important for inhibiting the apoptotic death of retinal neurons which occurs in retinitis pigmentosa, macular degeneration, glaucoma or other neurodegenerative diseases. These cultured neurons will also be useful for analyzing apoptotic responses of each of the retinal neuron cell types to a variety of “cytotoxic” agents and to the rescue effect of a number of “factors” either alone or in combination.


Retinal Pigment Epithelial Cell Retinitis Pigmentosa Photoreceptor Cell Primary Cell Culture Retinal Degeneration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Barber, A.J.: Lieth, E.; Khin, S.A.; Antonetti, D.A.; Buchanan, A.G.; Gardner, T.W., and Penn State Retina Res Grp, 1998 Neural apoptosis in the retina during experimental and human diabetes—Early onset and effect of insulin. J. Clin. Invest. 102:783–791.PubMedCrossRefGoogle Scholar
  2. 2.
    Lolley, R.N.; Rong, H., and Craft, CM., 1994 Linkage of photoreceptor degeneration by apoptosis with inherited defect in phototransduction. Invest. Ophthalmol. Vis. Sci. 35:358–362.PubMedGoogle Scholar
  3. 3.
    Papermaster, D.S. and Windle, J., 1995 Death at an early age: Apoptosis in inherited retinal degenerations. Invest. Ophthalmol. Vis. Sci. 36:977–983.PubMedGoogle Scholar
  4. 4.
    Wong, P. 1994 Apoptosis, retinitis pigmentosa, and degeneration. Biochem.Cell Biol. 72:489–498.PubMedGoogle Scholar
  5. 5.
    Davis, A.A.; Bernstein, P.S.; Bok, D.; Turner, J.; Nachtigal, M. and Hunt, R.C., 1995 A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture. Invest. Ophthalmol. Vis. Sci. 36:955–964.PubMedGoogle Scholar
  6. 6.
    Anderson, G.P., 1997 Bcl-2 related proteins, apoptosis, and disease. Trends Pharmacol.Sci. 18:51.PubMedGoogle Scholar
  7. 7.
    Tombran-Tink, J. and Johnson, L.V. 1989 Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells. Invest. Ophthalmol. Vis. Sci. 30:1700–1707.PubMedGoogle Scholar
  8. 8.
    Steele, F.R.; Chader, G.J.; Johnson, L.V., and Tombran-Tink, J., 1993 Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc. Natl. Acad. Sci. U.S.A. 90:1526–1530.PubMedCrossRefGoogle Scholar
  9. 9.
    Taniwaki, T.; Becerra, S.R; Chader, G.J., and Schwartz, J.P., 1995 Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture. J. Neurockem. 6:2509–2517.Google Scholar
  10. 10.
    Taniwaki, T.; Hirashima, N.; Becerra, S.P.; Chader, G.J.; Etcheberrigaray, R., and Schwartz, J.P, 1997 Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity. J. Neurochem. 68:26–32.PubMedCrossRefGoogle Scholar
  11. 11.
    Seigel, G.M.; Tombran-Tink, J.; Becerra, S.P.; Chader, G.J.; Diloreto, D.A.J.; del, C.C.; Lazar, E.S., and del, C.M., 1994 Differentiation of Y79 retinoblastoma cells with pigment epithelial-derived factor and interphotoreceptor matrix wash: effects on tumorigenicity. Growth Factors. 10:289–297.PubMedGoogle Scholar
  12. 12.
    Breen, G.A.; McGinnis, J.F., and de Vellis, J., 1978 Modulation of the hydrocortisone induction of glycerol phosphate dehydrogenase by N6,O2’-dibutyryl cyclic AMP, norepinephrine, and isobutylmethylxanthine in rat brain cell cultures. J. Biol. Chem. 253:2554–2562.PubMedGoogle Scholar
  13. 13.
    McGinnis, J.F.; Stepanik, P.; Chen, W.; Elias, R.; Cao, W., and Lerious, Y, 1999 Unique retina cell phenotypes revealed by immunological analysis of recoverin expression in rat retina cells. J. Neurosci. Res. In Press.Google Scholar
  14. 14.
    Bottenstein, J.E. and Sato, G.H., 1979 Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl. Acad. Sci. U.S.A. 76:514–517.PubMedCrossRefGoogle Scholar
  15. 15.
    Lillien, L. and Cepko, C., 1992 Control of proliferation in the retina: temporal changes in r esponsiveness to FGF and TGF alpha. Development 115:253–266.PubMedGoogle Scholar
  16. 16.
    Reid. D.M.; Laird, D.W., and Molday, R.S., 1992 Characterization and application of an in vitro detection system for studying the binding and phagocytosis of rod outer segments by retinal pigment epithelial cells. Exp. Eye Res. 54:775–783.PubMedCrossRefGoogle Scholar
  17. 17.
    Hsu, Y.-X; Wong, S.Y.C.; Connell, G.J., and Molday, R.S., 1993 Structural and functional properties of rhodopsin from rod outer segment disk and plasma membrane. Biochim. Biophys. Acta Bio-Membr. 1145:85–92.CrossRefGoogle Scholar
  18. 18.
    Donoso, L.A.; Merryman, C.F.; Edelberg, E.; Naids, R., and Lalsow, C., 1985 S-antigen in the developing retina and pineal gland: a monoclonal antibody study. Invest. Ophthalmol. Vis. Sci. 26:561–567.PubMedGoogle Scholar
  19. 19.
    Tombran-Tink, J.; Chader, G.G., and Johnson, L.V., 1991 PEDF: a pigment epithelium-derived factor with potent neuronal differentiative activity [letter]. Exp. Eye Res. 53:411–414.PubMedCrossRefGoogle Scholar
  20. 20.
    Gavrieli, Y.; Sherman, Y. and Ben-Sasson, S.A., 1992 Identificati;on of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493–501.PubMedCrossRefGoogle Scholar
  21. 21.
    Mosmann, T., 1983 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55–63.PubMedCrossRefGoogle Scholar
  22. 22.
    Chang, G.Q.; Hao, Y., and Wong, F., 1993 Apoptosis: final common pathway of photoreceptor death in rd. rds, and rhodopsin mutant mice. Neuron 11:595–605.PubMedCrossRefGoogle Scholar
  23. 23.
    Portera-Cailliau, C.; Sung, C.H.; Nathans, J., and Adler, R., 1994 Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 91:974–978.PubMedCrossRefGoogle Scholar
  24. 24.
    Tso. M.O.M.; Zhang, C.; Abler, A.S.; Chang, C.-J.; Wong, F.; Chang, G.-Q., and Lam. T.T., 1994 Apoptosis leads to photoreceptor degeneration in inherited retinal dystrophy of RCS rats. Invest. Ophthalmol. Vis. Sci. 35:2693–2699.PubMedGoogle Scholar
  25. 25.
    Wong, P., 1994 Apoptosis, retinitis pigmentosa, and degeneration. Biochem.Cell Biol. 72:489–498.PubMedGoogle Scholar
  26. 26.
    Cook, B.; Lewis. G.P.; Fisher, S.K., and Adler, R., 1995 Apoptotic photoreceptor degeneration in experimental retinal detachment. Invest. Ophthalmol. Vis. Sci. 36:990–996.PubMedGoogle Scholar
  27. 27.
    Wong, P.; Kutty, R.K.; Darrow, R.M.; Shivaram, S.; Kutty, G.; Fletcher, R.T.; Wiggert, B.; Chader, G., and Organisciak, D.T., 1994 Changes in clusterin expression associated with light-induced retinal damage in rats. Biochem. Cell Biol. 72:499–503.PubMedCrossRefGoogle Scholar
  28. 28.
    Rosenbaum, D.M.; Rosenbaum, P.S.; Gupta, A.; Michaelson, M.D.; Hall, D.H., and Kessler, J.A., 1997 Retinal ischemia leads to apoptosis which is ameliorated by aurintricarboxylic acid. Vision Res. 37, 3445–3451.PubMedCrossRefGoogle Scholar
  29. 29.
    Garcia-Valenzuela, E.; Shareef, S.; Walsh, J., and Sharma, S.C., 1995 Programmed cell death of retinal ganglion cells during experimental glaucoma. Exp. Eye Res. 61:33-44.Google Scholar
  30. 30.
    Kerrigan, L.A.; Zack, D.J.; Quigley, H.A.; Smith, S.D., and Pease, M.E., 1997 TUNEL-positive ganglion cells in human primary open-angle glaucoma. Arch. Ophthalmol. 115:1031–1035.PubMedGoogle Scholar
  31. 31.
    Steinberg, R.H., 1994 Survival factors in retinal degenerations. Curr.Opin.Neurobiol. 4:515–524.PubMedCrossRefGoogle Scholar
  32. 32.
    Lem, J. and Makino, C.L., 1996 Phototransduction in transgenic mice. Curr. Opin. Neurobiol. 6:453–458.PubMedCrossRefGoogle Scholar
  33. 33.
    Polans, A.S.; Witkowska, D.; Haley, T.L.; Amundson, D.; Baizer, L., and Adamus, G., 1995 Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy. Proc. Natl. Acad. Sci. U.S.A. 92:9176–9180.PubMedCrossRefGoogle Scholar
  34. 34.
    Thirkill, C.E.; Tait, R.C.; Tyler, N.K.; Roth, A.M., and Keltner, J.L., 1992 The cancer-associated retinopathy antigen is a recoverin-like protein. Invest. Ophthalmol. Vis. Sci. 33:2768–2772.PubMedGoogle Scholar

Copyright information

© Kluwer Academic / Plenum Publishers 1999

Authors and Affiliations

  • J. F. McGinnis
    • 1
  • W. Chen
    • 1
  • J. Tombran-Tink
    • 2
  • D. Mrazek
    • 1
  • V. Lerious
    • 1
  • W. Cao
    • 1
  1. 1.Department of Ophthalmology Dean McGee Eye InstituteUniversity of Oklahoma Health Science CenterOklahoma City
  2. 2.Center for Neuroscience ResearchChildren’s National Medical CenterN.W. Washington, DC

Personalised recommendations