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:

Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury

Abstract

The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.

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: CD95L neutralization decreases SCI-induced apoptosis.
Figure 2: Extent of injury 4 weeks after SCI.
Figure 3: CD95L neutralization improves functional recovery.
Figure 4: CD95L neutralization promotes axonal regeneration.
Figure 5: Increased expression of neuronal markers GFAP and GAP-43 in mice treated with neutralizing antibodies to CD95L or to CD95L and TNF.
Figure 6: Immunohistochemical detection of MBP, 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and βIII-tubulin 4 weeks after SCI.

Similar content being viewed by others

References

  1. Crowe, M.J., Bresnahan, J.C., Shuman, S.L., Masters, J.N. & Beattie, M.S. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat. Med. 3, 73–76 (1997).

    Article  CAS  PubMed  Google Scholar 

  2. Martin-Villalba, A. et al. Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke. Cell Death Differ. 8, 679–686 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Tartaglia, L.A., Ayres, T.M., Wong, G.H. & Goeddel, D.V. A novel domain within the 55 kD TNF receptor signals cell death. Cell 74, 845–853 (1993).

    Article  CAS  PubMed  Google Scholar 

  4. Kischkel, F.C. et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14, 5579–5588 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Medema, J.P. et al. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16, 2794–2804 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Scaffidi, C. et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17, 1675–1687 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Springer, J.E., Azbill, R.D. & Knapp, P.E. Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury. Nat. Med. 5, 943–946 (1999).

    Article  CAS  PubMed  Google Scholar 

  8. Li, M. et al. Functional role and therapeutic implications of neuronal caspase-1 and -3 in a mouse model of traumatic spinal cord injury. Neuroscience 99, 333–342 (2000).

    Article  CAS  PubMed  Google Scholar 

  9. Ozawa, H., Keane, R.W., Marcillo, A.E., Diaz, P.H. & Dietrich, W.D. Therapeutic strategies targeting caspase inhibition following spinal cord injury in rats. Exp. Neurol. 177, 306–313 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Zurita, M., Vaquero, J. & Zurita, I. Presence and significance of CD-95 (Fas/APO1) expression after spinal cord injury. J. Neurosurg. 94, 257–264 (2001).

    CAS  PubMed  Google Scholar 

  11. Xu, J. et al. Methylprednisolone inhibition of TNF-alpha expression and NF-κB activation after spinal cord injury in rats. Brain Res. Mol. Brain Res. 59, 135–142 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Li, G.L., Farooque, M. & Olsson, Y. Changes of Fas and Fas ligand immunoreactivity after compression trauma to rat spinal cord. Acta Neuropathol. (Berlin) 100, 75–81 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Lee, Y.B. et al. Role of tumor necrosis factor-α in neuronal and glial apoptosis after spinal cord injury. Exp. Neurol. 166, 190–195 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Kim, G.M. et al. Tumor necrosis factor receptor deletion reduces nuclear factor-κB activation, cellular inhibitor of apoptosis protein 2 expression, and functional recovery after traumatic spinal cord injury. J. Neurosci. 21, 6617–6625 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Farooque, M., Isaksson, J. & Olsson, Y. Improved recovery after spinal cord injury in neuronal nitric oxide synthase-deficient mice but not in TNF-α-deficient mice. J. Neurotrauma 18, 105–114 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Casha, S., Yu, W.R. & Fehlings, M.G. Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat. Neuroscience 103, 203–218 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Basso, D.M., Beattie, M.S. & Bresnahan, J.C. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12, 1–21 (1995).

    Article  CAS  PubMed  Google Scholar 

  18. Rossignol, S. & Dubuc, R. Spinal pattern generation. Curr. Opin. Neurobiol. 4, 894–902 (1994).

    Article  CAS  PubMed  Google Scholar 

  19. Metz, G.A., Merkler, D., Dietz, V., Schwab, M.E. & Fouad, K. Efficient testing of motor function in spinal cord injured rats. Brain Res. 883, 165–177 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Woolf, C.J. A new strategy for the treatment of inflammatory pain. Prevention or elimination of central sensitization. Drugs 47, 1–9 (1994).

    Article  PubMed  Google Scholar 

  21. Lindenlaub, T., Teuteberg, P., Hartung, T. & Sommer, C. Effects of neutralizing antibodies to TNF-α on pain-related behavior and nerve regeneration in mice with chronic constriction injury. Brain Res. 866, 15–22 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Lindsey, A.E. et al. An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat. Neurorehabil. Neural Repair 14, 287–300 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Scherbel, U. et al. Differential acute and chronic responses of tumor necrosis factor- deficient mice to experimental brain injury. Proc. Natl. Acad. Sci. USA 96, 8721–8726 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cohen, P.L. & Eisenberg, R.A. The lpr and gld genes in systemic autoimmunity: life and death in the Fas lane. Immunol. Today 13, 427–428 (1992).

    Article  CAS  PubMed  Google Scholar 

  25. Braun, H., Schafer, K. & Hollt, V. βIII tubulin-expressing neurons reveal enhanced neurogenesis in hippocampal and cortical structures after a contusion trauma in rats. J. Neurotrauma 19, 975–983 (2002).

    Article  PubMed  Google Scholar 

  26. Fournier, A.E. & McKerracher, L. Expression of specific tubulin isotypes increases during regeneration of injured CNS neurons, but not after the application of brain-derived neurotrophic factor (BDNF). J. Neurosci. 17, 4623–4632 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shackelford, D.A. & Nelson, K.E. Changes in phosphorylation of tau during ischemia and reperfusion in the rabbit spinal cord. J. Neurochem. 66, 286–295 (1996).

    Article  CAS  PubMed  Google Scholar 

  28. Davies, S.J. et al. Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390, 680–683 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Fernandes, K.J., Fan, D.P., Tsui, B.J., Cassar, S.L. & Tetzlaff, W. Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M. J. Comp. Neurol. 414, 495–510 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Shuman, S.L., Bresnahan, J.C. & Beattie, M.S. Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats. J. Neurosci. Res. 50, 798–808 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Dong, H. et al. Enhanced oligodendrocyte survival after spinal cord injury in Bax-deficient mice and mice with delayed Wallerian degeneration. J. Neurosci. 23, 8682–8691 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. D'Souza, S.D. et al. Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J. Exp. Med. 184, 2361–2370 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Becher, B., D'Souza, S.D., Troutt, A.B. & Antel, J.P. Fas expression on human fetal astrocytes without susceptibility to fas- mediated cytotoxicity. Neuroscience 84, 627–34 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Li, W. et al. Apoptotic death following Fas activation in human oligodendrocyte hybrid cultures. J. Neurosci. Res. 69, 189–196 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Casha, S., Yu, W.R. & Fehlings, M.G. Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat. Neuroscience 103, 203–218 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Desbarats, J. et al. Fas engagement induces neurite growth through ERK activation and p35 upregulation. Nat. Cell Biol. 5, 118–125 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Martin-Villalba, A. et al. CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons. J. Neurosci. 19, 3809–3817 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Raoul, C., Henderson, C.E. & Pettmann, B. Programmed cell death of embryonic motoneurons triggered through the Fas death receptor. J. Cell Biol. 147, 1049–1062 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ugolini, G. et al. Fas/tumor necrosis factor receptor death signaling is required for axotomy-induced death of motoneurons in vivo. J. Neurosci. 23, 8526–8531 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Brosamle, C., Huber, A.B., Fiedler, M., Skerra, A. & Schwab, M.E. Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1 antibody fragment. J. Neurosci. 20, 8061–8068 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. GrandPre, T., Li, S. & Strittmatter, S.M. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 417, 547–551 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Moon, L.D., Asher, R.A., Rhodes, K.E. & Fawcett, J.W. Regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC. Nat. Neurosci. 4, 465–466 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Grill, R.J., Blesch, A. & Tuszynski, M.H. Robust growth of chronically injured spinal cord axons induced by grafts of genetically modified NGF-secreting cells. Exp. Neurol. 148, 444–452 (1997).

    Article  CAS  PubMed  Google Scholar 

  44. Thallmair, M. et al. Neurite growth inhibitors restrict plasticity and functional recovery following corticospinal tract lesions. Nat. Neurosci. 1, 124–131 (1998).

    Article  CAS  PubMed  Google Scholar 

  45. Lehmann, M. et al. Inactivation of Rho signaling pathway promotes CNS axon regeneration. J. Neurosci. 19, 7537–7547 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Neumann, S., Bradke, F., Tessier-Lavigne, M. & Basbaum, A.I. Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron 34, 885–893 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. Qiu, J. et al. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34, 895–903 (2002).

    Article  CAS  PubMed  Google Scholar 

  48. Ramon-Cueto, A., Plant, G.W., Avila, J. & Bunge, M.B. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants. J. Neurosci. 18, 3803–3815 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Shi, R. & Blight, A.R. Differential effects of low and high concentrations of 4-aminopyridine on axonal conduction in normal and injured spinal cord. Neuroscience 77, 553–562 (1997).

    Article  CAS  PubMed  Google Scholar 

  50. Echtenacher, B., Falk, W., Mannel, D.N. & Krammer, P.H. Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis. J. Immunol. 145, 3762–3766 (1990).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Christopher Reeve Paralysis Foundation. We thank A. Forde for critical reading of the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ana Martin-Villalba.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Demjen, D., Klussmann, S., Kleber, S. et al. Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury. Nat Med 10, 389–395 (2004). https://doi.org/10.1038/nm1007

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1007

This article is cited by

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