Selective, Reversible Caspase-3 Inhibitor Is Neuroprotective and Reveals Distinct Pathways of Cell Death after Neonatal Hypoxic-ischemic Brain Injury
2002; Elsevier BV; Volume: 277; Issue: 33 Linguagem: Inglês
10.1074/jbc.m202931200
ISSN1083-351X
AutoresByung Hee Han, Daigen Xu, Junjeong Choi, Yongxin Han, Steven Xanthoudakis, Sophie Roy, John Tam, John P. Vaillancourt, John Colucci, Robert Siman, André Giroux, George S. Robertson, Robert Zamboni, Donald W. Nicholson, David M. Holtzman,
Tópico(s)Neonatal and fetal brain pathology
ResumoHypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model. Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model. hypoxia-ischemia postnatal day 7 caspase-3 inhibitor, (R, S)-({(2S)-2-[5-tert-butyl-3-{[(4-methyl-1,2,5-oxadiazol-3-yl)methyl]amino}-2-oxopyrazin-1(2H)-yl]butanoyl}amino)-5-[hexyl(methyl)amino]-4-oxopentanoic acid bis-hydrochloride intracerebroventricular Boc- Asp(OMe)-fluoromethylketone Asp-Glu-Val-Asp-7- amino-4-methyl-coumarin Val-Asp-Val-Ala-Asp-7- amino-4-(trifluoromethyl)coumarin median inhibitory concentration immunoreactivity mouse cerebellar granule neurons mouse cortical neuron enzyme-linked immunosorbent assay Hypoxic-ischemic (H-I)1encephalopathy in the prenatal and perinatal period is a major cause of morbidity and mortality and often results in cognitive impairment, seizures, and motor impairment leading to cerebral palsy (1Vannucci R.C. 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Despite this, early excitotoxic/necrotic cell death associated with calpain activation and cleavage but not activation of caspase-2 was still present in caspase-3 inhibitor-treated animals. Our results suggest that caspase-3 contributes to delayed cell death and that early events associated with calpain activation may be involved in the rapidly occurring excitotoxic/necrotic component of cell death after neonatal H-I. M826 (molecular weight, 648), a selective, reversible caspase-3 inhibitor was synthesized and provided by Merck Frosst, Inc. To determine potency and selectivity of M826 against different caspases, enzymatic caspase assays were performed with active, recombinant caspase 1–10 as described previously (21Garcia-Calvo M. Peterson E.P. Leiting B. Ruel R. Nicholson D.W. Thornberry N.A. J. Biol. Chem. 1998; 273: 32608-32613Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 40Garcia-Calvo M. Peterson E.P. Rasper D.M. Vaillancourt J.P. Zamboni R. Nicholson D.W. 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To test the ability of M826 to inhibit apoptosis in a cell-based assay system, caspase-3 activation was induced by camptothecin (NT2) or etoposide (mouse cerebellar granule neurons (mCGNs) and mouse cortical neurons (mCORTs)) in the presence of various concentrations of M826. Briefly, mCGN cultures were obtained from 7–9-day-old CD-1 mouse pups, and mCORT cultures were obtained from E16-E18 rat fetuses. After trypsin treatment for mCGNs and papain treatment for mCORTs, tissue was triturated, and cells were plated in 96-well microplates coated with poly-d-lysine (VWR) (105 cells/well for mCGNs and 25,000 cells for mCORTs). mCGNs were grown in Eagle's minimal essential medium (Sigma) with 25 mm glucose, 10% fetal bovine serum (Sigma), 2 mm glutamine, 100 μg/ml gentamicin for 6 days, and mCORTs were grown in neurobasal medium and B27 supplement (Invitrogen) for 4–7 days. Etoposide was then added to a final concentration of 50 μg/ml with and without different concentrations of M826. After 20 h, cells were harvested, cell lysates were prepared, and a cell death ELISA was performed with a commercially available kit per the manufacturer's instructions (Roche Molecular Biochemicals). For NT2 cells, 5000 cells were plated/well in 96-well plates. The following day, camptothecin 5 μg/ml plus and minus different concentrations of M826 or other inhibitor were added for 5 h. Cells were then harvested, lysed, and analyzed as above in the dell death ELISA. Newborn Sprague-Dawley rats (dam plus 10 pups per litter) were obtained from Sasco Breeders when the pups were 3–4 days of age. The pups were housed with their dam in the home cage under a 12:12-h light:dark cycle with food and water freely available throughout the study. The neonatal H-I brain injury was performed based on the Levine procedure (1Vannucci R.C. Pediatr Res. 1990; 27: 317-326Crossref PubMed Scopus (387) Google Scholar, 4Levine S. Am. J. Pathol. 1960; 36: 1-17PubMed Google Scholar, 5Rice J.E. Vannucci R.C. Brierley J.B. Ann. Neurol. 1981; 9: 131-141Crossref PubMed Scopus (1980) Google Scholar) as described previously (9Han B.H. D'Costa A. Back S.A. Parsadanian M. Patel S. Shah A.R. Gidday J.M. Srinivasan A. Deshmukh M. Holtzman D.M. Neurobiol. Dis. 2000; 7: 38-53Crossref PubMed Scopus (264) Google Scholar, 12Cheng Y. Deshmukh M. D'Costa A. Demaro J.A. Gidday J.M. Shah A. Sun Y. Jacquin M.F. Johnson E.M. Holtzman D.M. J. Clin. Invest. 1998; 101: 1992-1999Crossref PubMed Scopus (482) Google Scholar, 42Han B.H. Holtzman D.M. J. Neurosci. 2000; 20: 5775-5781Crossref PubMed Google Scholar). M826 was dissolved in phosphate-buffered saline containing 20% dimethyl sulfoxide. For in vivo studies, either vehicle or compound (3 or 30 nmol in 5 μl of vehicle) was intracerebroventricular (ICV)-injected into the left hemisphere of P7 rats as described previously (12Cheng Y. Deshmukh M. D'Costa A. Demaro J.A. Gidday J.M. Shah A. Sun Y. Jacquin M.F. Johnson E.M. Holtzman D.M. J. Clin. Invest. 1998; 101: 1992-1999Crossref PubMed Scopus (482) Google Scholar, 42Han B.H. Holtzman D.M. J. Neurosci. 2000; 20: 5775-5781Crossref PubMed Google Scholar, 43Cheng Y. Gidday J.M. Yan Q. Shah A.R. Holtzman D.M. Ann. Neurol. 1997; 41: 521-529Crossref PubMed Scopus (172) Google Scholar). After H-I in P7 rats, tissues from the hippocampus and cortex, both ipsi- and contra-lateral to the ligation, were dissected and frozen in dry ice. Tissue samples were homogenized in lysis buffer (10 mm Hepes, pH 7.4, 5 mm MgCl2, 1 mm dithiothreitol, 1% Triton X-100, 2 mm EGTA, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture) and centrifuged at 12,000 × g for 10 min at 4 °C. Caspase-3-like activity was determined by DEVD-AMC cleavage assay as described previously (9Han B.H. D'Costa A. Back S.A. Parsadanian M. Patel S. Shah A.R. Gidday J.M. Srinivasan A. Deshmukh M. Holtzman D.M. Neurobiol. Dis. 2000; 7: 38-53Crossref PubMed Scopus (264) Google Scholar, 42Han B.H. Holtzman D.M. J. Neurosci. 2000; 20: 5775-5781Crossref PubMed Google Scholar). Caspase-2-like activity was determined using VDVAD-AFC as a caspase-2 substrate. Tissue lysate (10 μl) was incubated in a 96-well plate with 90 μl of assay buffer (10 mm Hepes, pH 7.4, 42 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, 10% Sucrose) containing 30 μm VDVAD-AFC (Calbiochem). The emitted fluorescence was measured every 15 min for 60 min at 37 °C at an excitation wavelength of 405 nm and an emission wavelength of 500 nm using a microplate fluorescence reader (Bio-Tek Instruments). VDVAD-AFC cleavage activity was obtained from the slope by plotting fluorescence units against time. Ac-AFC (Calbiochem) was used to obtain a standard curve, and the enzyme activity was calculated as the pmol of AFC/mg of protein/min. Brain tissue lysates prepared as described above for the caspase assay were subjected to Western blotting. Proteins (40 μg/lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) as described previously (9Han B.H. D'Costa A. Back S.A. Parsadanian M. Patel S. Shah A.R. Gidday J.M. Srinivasan A. Deshmukh M. Holtzman D.M. Neurobiol. Dis. 2000; 7: 38-53Crossref PubMed Scopus (264) Google Scholar, 42Han B.H. Holtzman D.M. J. Neurosci. 2000; 20: 5775-5781Crossref PubMed Google Scholar). Blots were blocked with 3% dried milk in Tris-buffered saline containing 0.05% Tween 20 overnight. Blots were then incubated for 2–3 h with primary antibody followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG and visualized with enhanced chemiluminescence (Amersham Biosciences). Primary antibodies used were as follows: rabbit anti-procaspase-3 at 1:1000 (Santa Cruz), rabbit anti-active caspase-3 at 1:1000 (Cell Signaling Technology), rabbit anti-cleaved poly(ADP-ribose) polymerase at 1:200 (Cell Signaling Technology), mouse monoclonal anti-α-spectrin at 1:2000 (Chemicon), rabbit anti-p35 at 1:1000 (Santa Cruz), rabbit anti-caspase-2 (C-20) at 1:100 (Santa Cruz), and goat anti-β-actin used at 1:2000 (Santa Cruz). Brain tissues were prepared as described previously (9Han B.H. D'Costa A. Back S.A. Parsadanian M. Patel S. Shah A.R. Gidday J.M. Srinivasan A. Deshmukh M. Holtzman D.M. Neurobiol. Dis. 2000; 7: 38-53Crossref PubMed Scopus (264) Google Scholar, 10Han B.H. DeMattos R.B. Dugan L.L. Kim-Han J.S. Brendza R.P. Fryer J.D. Kierson M. Cirrito J. Quick K. Harmony J.A. Aronow B.J. Holtzman D.M. Nat. Med. 2001; 7: 338-343Crossref PubMed Scopus (179) Google Scholar, 42Han B.H. Holtzman D.M. J. Neurosci. 2000; 20: 5775-5781Crossref PubMed Google Scholar) and subjected to immunofluorescent labeling. Briefly, tissue sections were blocked with Tris-buffered saline containing 1% bovine serum albumin, 0.2% dry milk, and 0.3% Triton X-100 for 30 min and incubated overnight with one of the following primary antibodies: rabbit anti-active caspase-3 at 1:10,000 (Merck Frosst), rabbit anti-caspase-3 cleaved spectrin p120 at 1:20,000 (Merck Frosst), and rabbit anti-calpain-cleaved spectrin Ab38 (44Roberts-Lewis J.M. Savage M.J. Marcy V.R. Pinsker L.R. Siman R. J. Neurosci. 1994; 14: 3934-3944Crossref PubMed Google Scholar) at 1:50,000. Biotinylated secondary antibodies were then applied to the sections, and Cy3 or tetramethylrhodamine was used to visualize staining using TSA™-direct tyramide signal amplification kit (PerkinElmer Life Sciences). For nuclear counterstaining, the sections were incubated with 10 μm4,6-diamidino-2-phenylindole (Sigma) for 2 min followed by washing with Tris-buffered saline. Slides were cover-slipped with Vectashield mounting media (Vector) and examined with a Nikon fluorescence microscope or Zeiss LSM 5 pascal confocal microscope. Forty-micron brain sections were mounted onto glass slides and dried. DNA fragmentation was detected using a terminal dUTP nick-end labeling apoptosis detection kit (Upstate Biotechnology) according to the manufacturer's instruction. To quantify cell death, DNA fragmentation was assayed using a cell death ELISA kit as described (45Salgame P. Varadhachary A.S. Primiano L.L. Fincke J.E. Muller S. Monestier M. Nucleic Acids Res. 1997; 25: 680-681Crossref PubMed Scopus (112) Google Scholar). To assess regional area loss, 1 week after H-I, brain sections were prepared as described above, and damage due to H-I was determined by calculating the amount of surviving tissue in coronal sections as described previously (12Cheng Y. Deshmukh M. D'Costa A. Demaro J.A. Gidday J.M. Shah A. Sun Y. Jacquin M.F. Johnson E.M. Holtzman D.M. J. Clin. Invest. 1998; 101: 1992-1999Crossref PubMed Scopus (482) Google Scholar,42Han B.H. Holtzman D.M. J. Neurosci. 2000; 20: 5775-5781Crossref PubMed Google Scholar). Briefly, coronal sections from the genu of the corpus callosum to the end of the dorsal hippocampus were stained with cresyl violet as previously described (46Holtzman D.M. Sheldon R.A. Jaffe W. Cheng Y. Ferriero D.F. Ann. Neurol. 1996; 39: 114-122Crossref PubMed Scopus (145) Google Scholar). The cross-sectional areas of the striatum, cortex, and hippocampus in each of eight equally spaced reference planes were photo-scanned, and the area of each brain region was calculated using SigmaScan Pro (Jandel Scientific Software). The sections utilized for quantification corresponded approximately to plates 12, 15, 17, 20, 23, 28, 31, and 34 in the rat brain atlas (47Paxinos G. Watson C. The Rat Brain in Stereotaxic Coordinates. 2 Ed. Academic Press Ltd., Sydney, Australia1986Google Scholar). Data are presented as the mean ± S.E. and were analyzed by analysis of variance followed by Dunn's multiple comparison method with significance set at p < 0.05 unless otherwise stated. The structure of M826 (molecular weight, 648) is shown in Fig. 1. Utilizing recombinant, activated caspases, we determined the potency and selectivity of M826 to different caspases (Table I). M826 inhibited caspase-3 activity at a median inhibitory concentration (IC50) value of 5 nm. It also inhibited enzymatic activity of caspase-7 that has similar protein structure and substrate specificity to caspase-3 (21Garcia-Calvo M. Peterson E.P. Leiting B. Ruel R. Nicholson D.W. Thornberry N.A. J. Biol. Chem. 1998; 273: 32608-32613Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 48Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. J. Biol. Chem. 1997; 272: 9677-9682Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 49Hotchkiss R.S. Chang K.C. Swanson P.E. Tinsley K.W. Hui J.J. Klender P. Xanthoudakis S. Roy S. Black C. Grimm E. Aspiotis R. Han Y. Nicholson D.W. Karl I.E. Nat. Immunol. 2000; 1: 496-501Crossref PubMed Scopus (475) Google Scholar). We compared the potency of M826 to that of peptide caspase inhibitors in an enzyme assay system with brain tissue lysates after neonatal H-I that contained active caspase-3. M826 was 50–1000-fold more potent than the peptide-based caspase-3 inhibitors, z-DEVD-fmk, z-VAD-fmk, and Boc-D-fmk (data not shown). We then tested the ability of M826 to inhibit apoptosis in a cell-based assay system in which caspase-3-dependent cell death occurs. M826 potently inhibited DNA fragmentation induced by camptothecin or etoposide in cell cultures with an IC50 of 30–120 nm (Table II). As a comparison, z-VAD-fmk had an IC50 of 1 μm for inhibiting DNA fragmentation induced by camptothecin in NT2 cells. Because we were unable to test the potency of M826 against recombinant activated caspase-9, we assessed whether M826 could block the formation of the large and small subunits of caspase-3 in NT2 cells treated with camptothecin. This cleavage is mediated by activated caspase-9. Despite the ability of M826 to potently inhibit caspase-3 activation, high concentrations of M826 (>1 μm) did not block the cleavage of pro-caspase-3 between the large and small subunit (data not shown).Table ISelective inhibition of caspase-3/7 activity by M826Recombinant caspaseIC5010.052>1030.00540.350.261.770.0182.19NT1017.7 Open table in a new tab Table IIInhibition of DNA fragmentation by M826 in cultured cellsCell typesIC50NT20.03mGCN0.05mCORT0.12 Open table in a new tab To evaluate the effects of the selective caspase-3 inhibitor, M826, on caspase-3 activation and its downstream effects, we first determined the concentration of M826 present in the brain after direct ICV administration. M826 (30 nmol) was injected ICV into the left hemisphere of P7 rats, and concentrations of the compound were analyzed (Fig. 2A). M826 was detectable bilaterally in the cortex with a similar time course profile. The brain concentration 24 h after ICV injection remained greater than 1 μm. To test the ability of M826 to inhibit caspase-3 activity after H-I, P7 rats were ICV-injected with either vehicle or M826 immediately before exposure to hypoxia (2 h after the carotid ligation), and then caspase-3 activity in the brain was assessed 24 h after H-I. Consistent with previous results (9Han B.H. D'Costa A. Back S.A. Parsadanian M. Patel S. Shah A.R. Gidday J.M. Srinivasan A. Deshmukh M. Holtzman D.M. Neurobiol. Dis. 2000; 7: 38-53Crossref PubMed Scopus (264) Google Scholar, 12Cheng Y. Deshmukh M. D'Costa A. Demaro J.A. Gidday J.M. Shah A. Sun Y. Jacquin M.F. Johnson E.M. Holtzman D.M. J. Clin. Invest. 1998; 101: 1992-1999Crossref PubMed Scopus (482) Google Scholar), caspase-3 activity markedly increased unilaterally in the hippocampus and cortex as assessed 24 h after H-I (Fig. 2B). A single ICV injection of M826 completely blocked H-I-induced caspase-3 activation. When given immediately after H-I, M826 (3 or 30 nmol) also blocked most caspase-3 activation at 24 and 48 h post-H-I (Fig.2C). To test whether caspase-3 inhibition by M826 results in neuroprotection against neonatal H-I brain injury, different measures of cell death were assessed (Fig. 3). We first utilized an ELISA assay that quantitatively detects DNA fragmentation (45Salgame P. Varadhachary A.S. Primiano L.L. Fincke J.E. Muller S. Monestier M. Nucleic Acids Res. 1997; 25: 680-681Crossref PubMed Scopus (112) Google Scholar). M826 significantly reduced chromosomal DNA cleavage by ∼30% as compared with vehicle-treated animals (Fig. 3A). We next investigated whether other indicators of tissue injury were affected by M826. Consistent with the ELISA assay, there was a clear decrease in the number of terminal dUTP nick-end labeling-positive cells in M826versus vehicle-treated ani
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