Advertisement

Journal of Molecular Neuroscience

, Volume 54, Issue 3, pp 451–462 | Cite as

Biochemical Characterization of a Caspase-3 Far-red Fluorescent Probe for Non-invasive Optical Imaging of Neuronal Apoptosis

  • Valérie Jolivel
  • Sébastien Arthaud
  • Béatrice Botia
  • Christophe Portal
  • Bruno Delest
  • Guillaume Clavé
  • Jérôme Leprince
  • Anthony Romieu
  • Pierre-Yves Renard
  • Omar Touzani
  • Heidi Ligeret
  • Pauline Noack
  • Marc Massonneau
  • Alain Fournier
  • Hubert Vaudry
  • David Vaudry
Article

Abstract

Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cerebellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.

Keywords

Apoptosis Caspase-3 Stroke Imaging 

Notes

Acknowledgments

The authors thank Drs. M. Bénard and L. Galas from the Cell Imaging Platform of Normandy (PRIMACEN) for excellent technical assistance in microscopy experiments and Dr. N. Thorel for her contribution to cytometry experiments.

References

  1. Aito H, Aalto KT, Raivio KO (2004) Adenine nucleotide metabolism and cell fate after oxidant exposure of rat cortical neurons: effects of inhibition of poly(ADP-ribose) polymerase. Brain Res 1013:117–124PubMedCrossRefGoogle Scholar
  2. Alnemri ES, Livingston DJ, Nicholson DW et al (1996) Human ICE/CED-3 protease nomenclature. Cell 87:171PubMedCrossRefGoogle Scholar
  3. Bullok K, Piwnica-Worms D (2005) Synthesis and characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis. J Med Chem 48:5404–5407PubMedCrossRefGoogle Scholar
  4. Bullok KE, Maxwell D, Kesarwala AH et al (2007) Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis. Biochemistry 46:4055–4065PubMedCrossRefGoogle Scholar
  5. Chen YH, Zhang YH, Zhang HJ et al (2006) Design, synthesis, and biological evaluation of isoquinoline-1,3,4-trione derivatives as potent caspase-3 inhibitors. J Med Chem 49:1613–1623PubMedCrossRefGoogle Scholar
  6. Cortez-Retamozo V, Swirski FK, Waterman P et al (2008) Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation. J Clin Investig 118:4058–4066PubMedCentralPubMedCrossRefGoogle Scholar
  7. Davoli MA, Fourtounis J, Tam J et al (2002) Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat. Neuroscience 115:125–136PubMedCrossRefGoogle Scholar
  8. Dejda A, Seaborn T, Bourgault S et al (2011) PACAP and a novel stable analog protect rat brain from ischemia: Insight into the mechanisms of action. Peptides 32:1207–1216PubMedCrossRefGoogle Scholar
  9. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516PubMedCentralPubMedCrossRefGoogle Scholar
  10. Fang B, Boross PI, Tozser J, Weber IT (2006) Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition. J Mol Biol 360:654–666PubMedCrossRefGoogle Scholar
  11. Garcia-Calvo M, Peterson EP, Rasper DM et al (1999) Purification and catalytic properties of human caspase family members. Cell Death Differ 6:362–369PubMedCrossRefGoogle Scholar
  12. Gonzalez BJ, Basille M, Vaudry D, Fournier A, Vaudry H (1997) Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts. Neuroscience 78:419–430PubMedCrossRefGoogle Scholar
  13. Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776PubMedCrossRefGoogle Scholar
  14. Hentze H, Schwoebel F, Lund S et al (2001) In vivo and in vitro evidence for extracellular caspase activity released from apoptotic cells. Biochem Biophys Res Commun 283:1111–1117PubMedCrossRefGoogle Scholar
  15. Jacobson MD, Weil M, Raff MC (1997) Programmed cell death in animal development. Cell 88:347–354PubMedCrossRefGoogle Scholar
  16. Juan TS, McNiece IK, Argento JM et al (1997) Identification and mapping of Casp7, a cysteine protease resembling CPP32 beta, interleukin-1 beta converting enzyme, and CED-3. Genomics 40:86–93PubMedCrossRefGoogle Scholar
  17. Kume T, Taguchi R, Katsuki H et al (2006) Serofendic acid, a neuroprotective substance derived from fetal calf serum, inhibits mitochondrial membrane depolarization and caspase-3 activation. Eur J Pharmacol 542:69–76PubMedCrossRefGoogle Scholar
  18. Lakhani SA, Masud A, Kuida K et al (2006) Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311:847–851PubMedCentralPubMedCrossRefGoogle Scholar
  19. Lapeyre M, Leprince J, Massonneau M et al (2006) Aryldithioethyloxycarbonyl (Ardec): a new family of amine protecting groups removable under mild reducing conditions and their applications to peptide synthesis. Chemistry 12:3655–3671PubMedCrossRefGoogle Scholar
  20. Lazarovici P, Cohen G, Arien-Zakay H et al (2012) Multimodal neuroprotection induced by PACAP38 in oxygen-glucose deprivation and middle cerebral artery occlusion stroke models. J Mol Neurosci 48:526–540PubMedCentralPubMedCrossRefGoogle Scholar
  21. Lin TN, He YY, Wu G, Khan M, Hsu CY (1993) Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 24:117–121PubMedCrossRefGoogle Scholar
  22. Liu Y, Kati W, Chen CM, Tripathi R, Molla A, Kohlbrenner W (1999) Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction. Anal Biochem 267:331–335PubMedCrossRefGoogle Scholar
  23. Liu H, Chang DW, Yang X (2005) Interdimer processing and linearity of procaspase-3 activation. A unifying mechanism for the activation of initiator and effector caspases. J Biol Chem 280:11578–11582PubMedCrossRefGoogle Scholar
  24. Lloyd-Jones D, Adams R, Carnethon M et al (2009) Heart disease and stroke statistics–2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 119:480–486PubMedCrossRefGoogle Scholar
  25. Love S, Barber R, Srinivasan A, Wilcock GK (2000) Activation of caspase-3 in permanent and transient brain ischaemia in man. Neuroreport 11:2495–2499PubMedCrossRefGoogle Scholar
  26. Moretti A, Weig HJ, Ott T et al (2002) Essential myosin light chain as a target for caspase-3 in failing myocardium. Proc Natl Acad Sci U S A 99:11860–11865PubMedCentralPubMedCrossRefGoogle Scholar
  27. Nicholson DW, Ali A, Thornberry NA et al (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37–43PubMedCrossRefGoogle Scholar
  28. Ohtaki H, Nakamachi T, Dohi K et al (2006) Pituitary adenylate cyclase-activating polypeptide (PACAP) decreases ischemic neuronal cell death in association with IL-6. Proc Natl Acad Sci U S A 103:7488–7493PubMedCentralPubMedCrossRefGoogle Scholar
  29. Ortega A, Moran J (2011) Role of cytoskeleton proteins in the morphological changes during apoptotic cell death of cerebellar granule neurons. Neurochem Res 36:93–102PubMedCrossRefGoogle Scholar
  30. Pantos C, Mourouzis I, Saranteas T et al (2009) Thyroid hormone improves postischaemic recovery of function while limiting apoptosis: a new therapeutic approach to support hemodynamics in the setting of ischaemia-reperfusion? Basic Res Cardiol 104:69–77PubMedCrossRefGoogle Scholar
  31. Petrovsky A, Schellenberger E, Josephson L, Weissleder R, Bogdanov A Jr (2003) Near-infrared fluorescent imaging of tumor apoptosis. Cancer Res 63:1936–1942PubMedGoogle Scholar
  32. Raymond SB, Skoch J, Hills ID, Nesterov EE, Swager TM, Bacskai BJ (2008) Smart optical probes for near-infrared fluorescence imaging of Alzheimer’s disease pathology. Eur J Nucl Med Mol Imaging 35(Suppl 1):S93–S98PubMedCrossRefGoogle Scholar
  33. Rotonda J, Nicholson DW, Fazil KM et al (1996) The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat Struct Biol 3:619–625PubMedCrossRefGoogle Scholar
  34. Saliba E (2005) Non-invasive techniques to investigate the newborn brain. Arch Pediatr 12:737–740PubMedCrossRefGoogle Scholar
  35. Smolewski P, Grabarek J, Halicka HD, Darzynkiewicz Z (2002) Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis. J Immunol Methods 265:111–121PubMedCrossRefGoogle Scholar
  36. Stennicke HR, Salvesen GS (1997) Biochemical characteristics of caspases-3, −6, −7, and −8. J Biol Chem 272:25719–25723PubMedCrossRefGoogle Scholar
  37. Takadera T, Fujibayashi M, Kaniyu H, Sakota N, Ohyashiki T (2007) Caspase-dependent apoptosis induced by thapsigargin was prevented by glycogen synthase kinase-3 inhibitors in cultured rat cortical neurons. Neurochem Res 32:1336–1342PubMedCrossRefGoogle Scholar
  38. Tanaka M, Sawada M, Miura M, Marunouchi T (1998) Insulin-like growth factor-I analogue prevents apoptosis mediated through an interleukin-1 beta converting enzyme (caspase-1)-like protease of cerebellar external granular layer neurons: developmental stage-specific mechanisms of neuronal cell death. Neuroscience 84:89–100PubMedCrossRefGoogle Scholar
  39. Thornberry NA, Peterson EP, Zhao JJ, Howard AD, Griffin PR, Chapman KT (1994) Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry 33:3934–3940PubMedCrossRefGoogle Scholar
  40. Vaudry D, Gonzalez BJ, Basille M et al (2000) The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32. Proc Natl Acad Sci U S A 97:13390–13395PubMedCentralPubMedCrossRefGoogle Scholar
  41. Vaudry D, Pamantung TF, Basille M et al (2002) PACAP protects cerebellar granule neurons against oxidative stress-induced apoptosis. Eur J Neurosci 15:1451–1460PubMedCrossRefGoogle Scholar
  42. Vaudry D, Falluel-Morel A, Leuillet S, Vaudry H, Gonzalez BJ (2003) Regulators of cerebellar granule cell development act through specific signaling pathways. Science 300:1532–1534PubMedCrossRefGoogle Scholar
  43. Vaudry D, Falluel-Morel A, Bourgault S et al (2009) Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. Pharmacol Rev 61:283–357PubMedCrossRefGoogle Scholar
  44. Weissleder R, Ntziachristos V (2003) Shedding light onto live molecular targets. Nat Med 9:123–128PubMedCrossRefGoogle Scholar
  45. Weissleder R, Pittet MJ (2008) Imaging in the era of molecular oncology. Nature 452:580–589PubMedCentralPubMedCrossRefGoogle Scholar
  46. Zhang Z, Fan J, Cheney PP et al (2009) Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo. Mol Pharm 6:416–427PubMedCrossRefGoogle Scholar
  47. Zheng Z, Zhao H, Steinberg GK, Yenari MA (2003) Cellular and molecular events underlying ischemia-induced neuronal apoptosis. Drug News Perspect 16:497–503PubMedCrossRefGoogle Scholar
  48. Zhu S, Li M, Figueroa BE et al (2004) Prophylactic creatine administration mediates neuroprotection in cerebral ischemia in mice. J Neurosci 24:5909–5912PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Valérie Jolivel
    • 1
    • 2
    • 3
    • 4
  • Sébastien Arthaud
    • 5
  • Béatrice Botia
    • 1
    • 2
    • 4
  • Christophe Portal
    • 3
    • 6
  • Bruno Delest
    • 3
  • Guillaume Clavé
    • 3
    • 6
  • Jérôme Leprince
    • 1
    • 2
    • 4
    • 5
  • Anthony Romieu
    • 6
    • 7
  • Pierre-Yves Renard
    • 6
    • 7
  • Omar Touzani
    • 8
  • Heidi Ligeret
    • 3
  • Pauline Noack
    • 3
  • Marc Massonneau
    • 3
  • Alain Fournier
    • 4
    • 9
  • Hubert Vaudry
    • 1
    • 2
    • 4
    • 5
  • David Vaudry
    • 1
    • 2
    • 4
    • 5
  1. 1.Rouen Institute for Research and Innovation in Biomedicine (IRIB)University of RouenMont-Saint-AignanFrance
  2. 2.INSERM U982, DC2NUniversity of RouenMont-Saint-AignanFrance
  3. 3.QUIDD, Pharmaparc IIVoie de l’InnovationVal de ReuilFrance
  4. 4.International Associated Laboratory Samuel de Champlain
  5. 5.Cell Imaging Platform of Normandy (PRIMACEN)University of RouenMont-Saint-AignanFrance
  6. 6.Equipe de chimie Bio-Organique, COBRA CNRS UMR 6014 & FR 3038IRCOFMont-Saint-AignanFrance
  7. 7.University of Rouen, IRCOF, rue TesnièreMont-Saint-AignanFrance
  8. 8.CERVOxy team, CNRS UMR 6232CaenFrance
  9. 9.INRS–Institut Armand-FrappierUniversity of QuébecLavalCanada

Personalised recommendations