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Neurochemical Research

, Volume 32, Issue 12, pp 2210–2216 | Cite as

Direct Evidence for Calpain Involvement in Apoptotic Death of Neurons in Spinal Cord Injury in Rats and Neuroprotection with Calpain Inhibitor

  • Eric A. Sribnick
  • Denise D. Matzelle
  • Naren L. Banik
  • Swapan K. Ray
Original Paper

Abstract

To demonstrate calpain involvement in neurodegeneration in rat spinal cord injury (SCI), we examined SCI segments for DNA fragmentation, neurons for calpain overexpression, neuronal death, and neuroprotection with calpain inhibitor (E-64-d). After the induction of SCI (40 g cm force) on T12, rats were treated within 15 min with vehicle (DMSO) or E-64-d. Sham animals underwent laminectomy only. Animals were sacrificed at 24 h, and five 1-cm long spinal cord segments were collected: two rostral (S1 and S2), one lesion (S3), and two caudal segments (S4 and S5). Agarose gel electrophoresis of DNA samples isolated from the SCI segments showed both random and internucleosomal DNA fragmentation indicating occurrence of necrosis as well as apoptosis mostly in the lesion, moderately in caudal, and slightly in rostral segments from SCI rats. Treatment of SCI rats with E-64-d (1 mg/kg) reduced DNA fragmentation in all segments. The lesion and adjacent caudal segments (S3 and S4) were further investigated by in situ double-immunofluorescent labelings that showed increase in calpain expression in neurons in SCI rats and decrease in calpain expression in SCI rats treated with E-64-d. In situ combined TUNEL and double-immunofluorescent labelings directly detected co-localization of neuronal death and calpain overexpressin in SCI rats treated with only vehicle while attenuation of neuronal death in SCI rats treated with E-64-d. Previous studies from our laboratory indirectly showed neuroprotective effect of E-64-d in SCI rats. Our current results provide direct in situ evidence for calpain involvement in neuronal death and neuroprotective efficacy of E-64-d in lesion and penumbra in SCI rats.

Keywords

Calpain E-64-d Immunofluorescent labelings Neuronal death SCI 

Notes

Acknowledgments

This work was supported in part by R01 grants (NS-31622, NS-57811, and CA-91460) and a Medical Scientist Training Program (MSTP) grant (GM08716) from the National Institutes of Health, and also a Spinal Cord Injury Research Fund grants (SCIRF-0803 and SCIRF-1205) from the state of South Carolina.

References

  1. 1.
    National Spinal Cord Injury Statistical Center (2006) Spinal cord injury: facts and figures at a glance. National Spinal Cord Injury Statistical Center, Birmingham, ALGoogle Scholar
  2. 2.
    Dumont RJ, Okonkwo DO, Verma S et al (2001) Acute spinal cord injury, part I: pathophysiologic mechanisms. Clin Neuropharmacol 24:254–264CrossRefPubMedGoogle Scholar
  3. 3.
    Ray SK, Matzelle DD, Wilford GG et al (2001) Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra. Ann NY Acad Sci 939:436–449PubMedCrossRefGoogle Scholar
  4. 4.
    Beattie MS, Farooqui AA, Bresnahan JC (2000) Review of current evidence for apoptosis after spinal cord injury. J Neurotrauma 17:915–925PubMedCrossRefGoogle Scholar
  5. 5.
    Dumont RJ, Verma S, Okonkwo DO et al (2001) Acute spinal cord injury, part II: contemporary pharmacotherapy. Clin Neuropharmacol 24:265–279CrossRefPubMedGoogle Scholar
  6. 6.
    Li Y, Field PM, Raisman G (1997) Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells. Science 277:2000–2002CrossRefPubMedGoogle Scholar
  7. 7.
    Nicholls J, Saunders N (1996) Regeneration of immature mammalian spinal cord after injury. Trends Neurosci 19:229–234CrossRefPubMedGoogle Scholar
  8. 8.
    Liu XZ, Xu XM, Hu R et al (1997) Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci 17:5395–5406PubMedGoogle Scholar
  9. 9.
    Springer JE, Azbill RD, Knapp PE (1999) Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury. Nat Med 5:943–946CrossRefPubMedGoogle Scholar
  10. 10.
    Ray SK, Banik NL (2003) Calpain and its involvement in the pathophysiology of CNS injuries and diseases: therapeitic potential of calpain inhibitors for prevention of neurodegeneration. Curr Drug Targets – CNS Neurol Disord 2:173–189CrossRefPubMedGoogle Scholar
  11. 11.
    Ray SK, Hogan EL, Banik NL (2003) Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors. Brain Res Brain Res Rev 42:169–185CrossRefPubMedGoogle Scholar
  12. 12.
    Wyllie AH, Morris RG, Smith AL et al (1984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142:67–77CrossRefPubMedGoogle Scholar
  13. 13.
    Ray SK, Schaecher KE, Shields DC et al (2000) Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease. Brain Res Brain Res Protoc 5:305–311CrossRefPubMedGoogle Scholar
  14. 14.
    Zhivotovsky B, Burgess DH, Vanags DM et al (1997) Involvement of cellular proteolytic machinery in apoptosis. Biochem Biophys Res Commun 230:481–488CrossRefPubMedGoogle Scholar
  15. 15.
    Ray SK, Shields DC, Saido TC et al (1999) Calpain activity and translational expression increased in spinal cord injury. Brain Res 816:375–380CrossRefPubMedGoogle Scholar
  16. 16.
    Banik NL, Chou CH, Deibler GE et al (1994) Peptide bond specificity of calpain: proteolysis of human myelin basic protein. J Neurosci Res 37:489–496CrossRefPubMedGoogle Scholar
  17. 17.
    Jordan J, Galindo MF, Miller RJ (1997) Role of calpain- and interleukin-1β converting enzyme-like proteases in the β-amyloid-induced death of rat hippocampal neurons in culture. J Neurochem 68:1612–1621PubMedCrossRefGoogle Scholar
  18. 18.
    Nath R, Raser KJ, Stafford D et al (1996) Non-erythroid α-spectrin breakdown by calpain and interleukin-1β-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem J 319(Pt 3):683–690PubMedGoogle Scholar
  19. 19.
    McGowan EB, Becker E, Detwiler TC (1989) Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64-d. Biochem Biophys Res Commun 158:432–435CrossRefPubMedGoogle Scholar
  20. 20.
    Perot PL Jr, Lee WA, Hsu CY et al (1987) Therapeutic model for experimental spinal cord injury in the rat: I. Mortality and motor deficit. J Neurotrauma 4:149–159Google Scholar
  21. 21.
    Ray SK, Matzelle DD, Wilford GG et al (2001) Inhibition of calpain-mediated apoptosis by E-64 d-reduced immediate early gene (IEG) expression and reactive astrogliosis in the lesion and penumbra following spinal cord injury in rats. Brain Res 916:115–126CrossRefPubMedGoogle Scholar
  22. 22.
    Chakrabarti AK, Yoshida Y, Powers JM et al (1988) Calcium-activated neutral proteinase in rat brain myelin and subcellular fractions. J Neurosci Res 20:351–358CrossRefPubMedGoogle Scholar
  23. 23.
    Pang Z, Bondada V, Sengoku T et al (2003) Calpain facilitates the neuron death induced by 3-nitropropionic acid and contributes to the necrotic morphology. J Neuropathol Exp Neurol 62:633–643PubMedGoogle Scholar
  24. 24.
    Kim MJ, Jo DG, Hong GS et al (2002) Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death. Proc Natl Acad Sci USA 99:9870–9875CrossRefPubMedGoogle Scholar
  25. 25.
    Wood DE, Thomas A, Devi LA et al (1998) Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene 17:1069–1078CrossRefPubMedGoogle Scholar
  26. 26.
    Blomgren K, Zhu C, Wang X et al (2001) Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of “pathological apoptosis”? J Biol Chem 276:10191–10198CrossRefPubMedGoogle Scholar
  27. 27.
    Nakagawa T, Yuan J (2000) Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol 150:887–894CrossRefPubMedGoogle Scholar
  28. 28.
    Ray SK, Wilford GG, Crosby CV et al (1999) Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells. Brain Res 829:18–27CrossRefPubMedGoogle Scholar
  29. 29.
    Varghese J, Radhika G, Sarin A (2001) The role of calpain in caspase activation during etoposide induced apoptosis in T cells. Eur J Immunol 31:2035–2041CrossRefPubMedGoogle Scholar
  30. 30.
    Gores GJ, Miyoshi H, Botla R et al (1998) Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases. Biochimica Biophys Acta 1366:167–175CrossRefGoogle Scholar
  31. 31.
    Arataki S, Tomizawa K, Moriwaki A et al (2005) Calpain inhibitors prevent neuronal cell death and ameliorate motor disturbances after compression-induced spinal cord injury in rats. J Neurotrauma 22:398–406CrossRefPubMedGoogle Scholar
  32. 32.
    Ray SK, Guyton MK, Sribnick EA et al (2007) Calpain as a target for prevention of neuronal death in injuries and diseases of the central nervous system. In: Lajtha A, Banik N (eds) Handbook of neurochemistry and molecular neurobiology: neural protein metabolism and function. Springer, New York, pp 445–467CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Eric A. Sribnick
    • 1
  • Denise D. Matzelle
    • 1
  • Naren L. Banik
    • 1
  • Swapan K. Ray
    • 1
  1. 1.Department of NeurosciencesMedical University of South CarolinaCharlestonUSA

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