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.
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References
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 1013:117–124
Alnemri ES, Livingston DJ, Nicholson DW et al (1996) Human ICE/CED-3 protease nomenclature. Cell 87:171
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 48:5404–5407
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 46:4055–4065
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 49:1613–1623
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 118:4058–4066
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 115:125–136
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 32:1207–1216
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516
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 360:654–666
Garcia-Calvo M, Peterson EP, Rasper DM et al (1999) Purification and catalytic properties of human caspase family members. Cell Death Differ 6:362–369
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 78:419–430
Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776
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 283:1111–1117
Jacobson MD, Weil M, Raff MC (1997) Programmed cell death in animal development. Cell 88:347–354
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 40:86–93
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 542:69–76
Lakhani SA, Masud A, Kuida K et al (2006) Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311:847–851
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 12:3655–3671
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 48:526–540
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 24:117–121
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 267:331–335
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 280:11578–11582
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 119:480–486
Love S, Barber R, Srinivasan A, Wilcock GK (2000) Activation of caspase-3 in permanent and transient brain ischaemia in man. Neuroreport 11:2495–2499
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 99:11860–11865
Nicholson DW, Ali A, Thornberry NA et al (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37–43
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 103:7488–7493
Ortega A, Moran J (2011) Role of cytoskeleton proteins in the morphological changes during apoptotic cell death of cerebellar granule neurons. Neurochem Res 36:93–102
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 104:69–77
Petrovsky A, Schellenberger E, Josephson L, Weissleder R, Bogdanov A Jr (2003) Near-infrared fluorescent imaging of tumor apoptosis. Cancer Res 63:1936–1942
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–S98
Rotonda J, Nicholson DW, Fazil KM et al (1996) The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat Struct Biol 3:619–625
Saliba E (2005) Non-invasive techniques to investigate the newborn brain. Arch Pediatr 12:737–740
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 265:111–121
Stennicke HR, Salvesen GS (1997) Biochemical characteristics of caspases-3, −6, −7, and −8. J Biol Chem 272:25719–25723
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 32:1336–1342
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 84:89–100
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 33:3934–3940
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 97:13390–13395
Vaudry D, Pamantung TF, Basille M et al (2002) PACAP protects cerebellar granule neurons against oxidative stress-induced apoptosis. Eur J Neurosci 15:1451–1460
Vaudry D, Falluel-Morel A, Leuillet S, Vaudry H, Gonzalez BJ (2003) Regulators of cerebellar granule cell development act through specific signaling pathways. Science 300:1532–1534
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 61:283–357
Weissleder R, Ntziachristos V (2003) Shedding light onto live molecular targets. Nat Med 9:123–128
Weissleder R, Pittet MJ (2008) Imaging in the era of molecular oncology. Nature 452:580–589
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 6:416–427
Zheng Z, Zhao H, Steinberg GK, Yenari MA (2003) Cellular and molecular events underlying ischemia-induced neuronal apoptosis. Drug News Perspect 16:497–503
Zhu S, Li M, Figueroa BE et al (2004) Prophylactic creatine administration mediates neuroprotection in cerebral ischemia in mice. J Neurosci 24:5909–5912
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.
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Jolivel, V., Arthaud, S., Botia, B. et al. Biochemical Characterization of a Caspase-3 Far-red Fluorescent Probe for Non-invasive Optical Imaging of Neuronal Apoptosis. J Mol Neurosci 54, 451–462 (2014). https://doi.org/10.1007/s12031-014-0325-4
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DOI: https://doi.org/10.1007/s12031-014-0325-4