- Email: [email protected]
Involvement of caspase-1 proteases in hypoxic brain injury. Effects of their inhibitors in developing neurons
Caspase-1 and inhibitors in hypoxic neurons
Pergamon PII: S0306-4522(99)00501-1
Neuroscience Vol. 95, No. 4, pp. 1157–1165,1157 2000 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
INVOLVEMENT OF CASPASE-1 PROTEASES IN HYPOXIC BRAIN INJURY. EFFECTS OF THEIR INHIBITORS IN DEVELOPING NEURONS C. BOSSENMEYER-POURIE´, V. KOZIEL and J. L. DAVAL* JE 2164, Universite´ Henri Poincare´, 54013 Nancy, Cedex, France
Abstract—To further explore the contribution of caspase-1/interleukin-1b-converting enzyme in the consequences of hypoxia in developing brain neurons, its temporal expression profile was analysed by immunohistochemistry and western blotting in cultured neurons from the embryonic rat forebrain subjected to a hypoxic stress (95% N2/5% CO2 for 6 h), and proteolytic activity of caspase-1 was monitored as a function of time by measuring the degradation of a selective colorimetric substrate (N-acetyl-TyrVal-Ala-Asp-p-nitroanilide). In addition, the influence of pre- and posthypoxic treatments by caspase-1 inhibitors (N-acetyl-TyrVal-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone) was tested on cell outcome. Hypoxia led to delayed apoptotic neuronal death, with an elevation of the expression of both pro-caspase-1 and caspase-1 active cleavage product (ICE p20) for up to 96 h after cell reoxygenation. As reflected by cleavage of the specific substrate, caspase-1 activity progressively increased between 24 h and 96 h posthypoxia, and was blocked by inhibitors in a dose-dependent fashion. The inhibitory compounds, including when given 24 h after hypoxia, prevented neuronal death, reduced apoptosis hallmarks and also increased the number of mitotic neurons, suggesting they might promote neurogenesis. Similar observations were made when neurons were exposed to a sublethal hypoxia (i.e. 3 h). These data emphasize the participation of caspase-1 in neuronal injury consecutive to oxygen deprivation, and provide new insight into the possible cellular mechanisms by which caspase inhibitors may protect developing brain neurons. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: oxygen deprivation, apoptosis, neurogenesis, cell culture, rat forebrain.
apoptosis, 25 suggesting a potential beneficial role for their derivatives in some stressful circumstances. 6,7 However, it is likely that requirement for the activation of specific caspases may vary according to the death-inducing stimulus as well as to the type of target cells. 41 Although ICE has been unequivocally shown to be expressed in the CNS, 22 its effective participation in hypoxia (and/or ischemia)-induced neuronal injury remains unclear. Using primary cultured neurons from the embryonic rat forebrain as a model, we have previously reported that hypoxia for 6 h, without concomitant glucose deprivation, induces delayed cell death presenting morphological and biochemical characteristics of apoptosis. 3,8 In contrast to the deleterious effects of such a long-lasting insult, hypoxia for 3 h has been shown to promote neuronal division, leading to a significant increase in the final number of living neurons. 4 Indeed, apoptosis may be considered as the result of an inappropriate and finally aborted re-entry into the cell cycle, 29,33 and our observations support the conclusion that some neurons exposed to sublethal hypoxia may dodge apoptotic death by fully achieving the cell cycle. To further delineate the participation of ICE-like caspases as well as the consequences of their inhibitors in the neuronal response to transient oxygen deprivation in the developing mammalian brain, we analysed ICE activation and explored the effects of selective peptide inhibitors on cultured neurons exposed to lethal hypoxia. Inhibitors were added to the culture medium either concomitantly to the hypoxic insult or 24 h later. Cell viability and apoptosis-related morphological features were monitored as a function of time after hypoxia/ reoxygenation, and ICE-like specific protease activity was monitored by measuring the hydrolysis of a selective synthetic substrate. 34
In the perinatal period, cerebral hypoxia is known to induce brain injury and remains a major cause of neurodevelopmental impairment. 16 Hypoxic-ischemic cell death was initially attributed to tissue necrosis, but, based upon both in vivo and in vitro findings, selective neuronal damage observed after transient oxygen deprivation progressively appeared to involve apoptosis, 2,3,8,12,35,38 a process implicated in physiological cell elimination during development. 32 Through the activation of an intrinsic, constitutive death programme, apoptosis leads to typical morphological alterations, such as cell shrinkage, chromatin condensation followed by DNA fragmentation. 27 Among the numerous molecular mechanisms involved in the apoptotic cascade, activation of cysteine proteases that belong to the caspase superfamily certainly plays a critical role in the cell outcome. 1,9,10,21 Caspases correspond to the mammalian homologs of the Caenorhabditis elegans death gene ced3, 42 and include the prototypic interleukin-1b-converting enzyme (ICE, capase-1). During apoptosis, caspases cleave their selective substrates at aspartate residues, whereas their inhibitors such as the cowpox virus CrmA protein or the baculovirus p35 protein have been shown to inhibit *To whom correspondence should be addressed. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DEVD-CHO, Nacetyl-Asp-Glu-Val-Asp-aldehyde; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetra-acetate; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; GFAP, glial fibrillary acidic protein; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid; ICE, interleukin-1b-converting enzyme; MTT, 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide; NSE, neuron-specific enolase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; YVAD-CHO, N-acetyl-Tyr-Val-Ala-Asp-aldehyde; YVAD-CMK, N-acetyl-Tyr-ValAla-Asp-chloromethylketone; YVAD-pNA, N-acetyl-Tyr-Val-Ala-Aspp-nitroanilide. 1157
C. Bossenmeyer-Pourie´ et al.
1158 EXPERIMENTAL PROCEDURES
Neuronal cell cultures Animal experimentation was carried out with the highest standards of animal care and housing, according to the N.I.H. Guide for the Care and Use of Laboratory Animals. Primary cultured neurons were obtained from 14-day-old rat embryo forebrains as previously described. 3,4 When they were in the proestrus period, as shown by the observation of daily vaginal smears, Sprague–Dawley female rats (R. Janvier, Le Genest-St-Isle, France) were housed together with males for 24 h, and pregnant dams were maintained in separate cages for 14 days under standard laboratory conditions on a 12:12 h light:dark cycle, with food and water available ad libitum. Living embryos were excised by Caesarian section performed under anaesthesia with halothane. Whole embryos were placed in culture medium previously equilibrated at 378C and consisting of a mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (50:50, ICN Pharmaceuticals, Costa Mesa, CA, U.S.A.) supplemented with 5% inactivated fetal calf serum (Valbiotech, Paris, France). Forebrains were carefully collected, dissected free of meninges and gently dispersed in culture medium. After centrifugation at 700 g for 10 min, the pellet was redispersed in the same medium and passed through a 46-mm-pore size nylon mesh. Aliquots of the cell suspension were transferred into 35 mm Petri dishes (Falcon, Becton Dickinson, Le Pont-de-Claix, France) precoated with poly-l-lysine in order to obtain a final density of 10 6 cells/dish. Cultures were then placed at 378C in a humidified atmosphere of 95% air/5% CO2. The following day, the culture medium was replaced with a fresh hormonally defined serum-free medium consisting of the DMEM/Ham’s F12 mixture enriched with human transferrin (1 mM), bovine insulin (1 mM), putrescine (0.1 mM), progesterone (10 nM), estradiol (1 pM), Na selenite (30 nM), and also containing fibroblast growth factor (2 ng/ml) and epidermal growth factor (10 ng/ml) (Sigma Chemicals, St Louis, MO, U.S.A.). After two additional days, the culture medium was renewed with serum-free medium in the absence of growth factors. Exposure to hypoxia and treatments by inhibitors Six-day-old neuronal cell cultures were submitted to transient hypoxia for 3 or 6 h by transferring the culture dishes to a humidified incubation chamber thermoregulated at 378C and flushed by 95% N2/5% CO2. Cultures were then returned to standard normoxic atmosphere for the next 96 h. The reduction in oxygen delivery to the neurons was assessed by measuring O2 content in samples of extracellular medium collected just prior to the beginning of hypoxia and immediately at the end of the insult, by means of a gas analyser (Corning, Halstead, U.K.), as described by Sher. 36 The effects of two potent ICE-like inhibitors were tested, i.e. Nacetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO, ICE inhibitor I from Oncogene Research Products, Cambridge, MA, U.S.A.), a cellpermeable reversible inhibitor, 24 and N-acetyl-Tyr-Val-Ala-Aspchloromethylketone (YVAD-CMK, ICE inhibitor II from Oncogene Research Products), a cell-permeable irreversible inhibitor. 13 Peptide inhibitors were added at various concentrations to fresh culture medium, either before starting hypoxia or 24 h after cell reoxygenation. Drugs were then left in the culture dishes until the end of the experimental period, i.e. 96 h post-reoxygenation. Concentrations were carefully chosen in agreement with the manufacturer’s recommendations, according to the known Ki values. 28 Analysis of cell outcome Cell morphology was routinely assessed by phase-contrast microscopic observations by means of a Nikon Diaphot TMD. Cell viability was monitored by a spectrophotometric method using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), according to Carmichael et al. 5 Neurons were incubated for 3 h at 378C with MTT (500 mg/ml), washed twice with ice-cold phosphatebuffered saline (PBS), and lysed in dimethylsulfoxide. Optical density was measured at 519 nm and data were compared to those obtained from sister control cells. Characteristic morphological features of apoptosis, necrosis and mitosis were detected after cell reoxygenation by using the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI), as previously documented. 3,4,17,33 Cultures were fixed for 10 min in a mixture of ethanol: acetic acid (3:1), washed for 1 min in distilled water, air-dried, and subsequently stained with DAPI (0.5 mg/ml) for 10 min. Cells were
washed twice with distilled water, air-dried, and treated with antifading medium (10 mg/ml p-phenylenediamine in 90% glycerol, pH 9.0). Normal, apoptotic, necrotic and mitotic neurons were scored under fluorescence microscopy (Zeiss Axioscop) at an excitation wavelength of 365 nm by counting concerned cells in three distinct areas of at least 100 neurons per culture dish. Immunohistochemical and western blot analyses In its active form, ICE is an oligomeric enzyme with 20,000 mol. wt (p20) and 10,000 mol. wt (p10) subunits derived from the cleavage of a 45,000 mol. wt proenzyme. 40 Expression of ICE/caspase-1 was studied in neuronal cells by immunohistochemistry and western blotting by using an affinity-purified goat polyclonal antibody (ICE p20/M-19, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) raised against a peptide corresponding to amino acids 276–294 mapping at the carboxyterminus of the p20 subunit, and thus reacts with both the ICE precursor and the active cleavage product ICE p20. Cultures were rinsed twice with PBS, then fixed for 10 min in methanol at 2108C, and rinsed again with PBS. Non-specific binding sites for IgG were blocked by incubating the cells for 20 min with 10% horse serum (Gibco-BRL, Inchinnan, U.K.) in PBS. Thereafter, cultured neurons were incubated for 60 min at room temperature in buffer containing ICE p20 polyclonal antibody diluted at 1:40. Following two washing steps to remove unfixed antibodies, the cells were incubated for 120 min with anti-goat IgG conjugated to rhodamine isothiocyanate (RITC, dilution 1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.). Neuronal cells were finally washed three times with PBS, coverslipped using mounting medium (Aquapolymount w), and kept in the dark until subsequent analysis by means of a Zeiss Axioscop fluorescence microscope. In some experiments, culture preparations were stained with a polyclonal antibody specific for Bcl-2 diluted at 1:100 (goat IgG, Santa Cruz Biotechnology) and revealed in the presence of an anti-goat IgG conjugated to fluorescein isothiocyanate (FITC, dilution 1:100, Jackson ImmunoResearch Laboratories). For western blotting, adhering and non-adhering cultured cells were harvested, and cell extraction was performed by using a buffer solution consisting of 10 mM HEPES (pH 7.5), 5 mM EDTA, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM KCl and 0.1% Triton X-100. Cell lysates were then centrifuged for 20 min at 14,000 g, and the cytosolic supernatant was collected. The nuclear pellet was resuspended in the same buffer, centrifuged at 14,000 g for 20 min, and the final supernatant was pooled with the primary one for subsequent protein separation. Final samples, each containing 30 mg total proteins, were electrophoresed on sodium dodecyl sulfate (SDS)–polyacrylamide gel (5% stacking, 10% running) according to Laemmli, 23 in parallel with molecular weight standards. After separation, proteins were transferred on to a polyvinylidene difluoride membrane (PVDF, New England Nuclear, Boston, MA, U.S.A.) using a Tris–glycine buffer (48 mM Tris base, 39 mM glycine, 0.037% SDS and 20% methanol) by means of a semi-dry electrotransferring unit (Bio-Rad, Ivry-sur Seine, France) at 160 mA for 30 min. Subsequent experimental steps were performed by using a commercially available kit for western blot detection of proteins with peroxidase-labeled secondary antibodies and the chemiluminescent substrate luminol from Boehringer (Mannheim, Germany). Non-specific sites were blocked by incubating the membrane while shaking for 1 h in 1% blocking reagent provided by the supplier. Blots were then probed for 1 h with either anti-ICE p20 goat polyclonal antibody at a 1:500 dilution in 0.5% blocking solution or anti-Bcl-2 goat polyclonal antibody diluted at 1:400. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBS–Tween), the membrane was incubated for 30 min with peroxidase-labeled secondary antibody (40 mU/ml, Boehringer), and washed four times in TBS–Tween. Chemiluminescent detection of specifically-labeled proteins was finally performed according to the supplier’s instructions by using Kodak X-Omat films. Assay for interleukin-1b -converting enzyme activity ICE-like proteolytic activity was measured at various time intervals after cell reoxygenation in crude cell extracts by a spectrophotometric method using the selective colorimetric substrate peptide, N-acetylTyr-Val-Ala-Asp-p-nitroanilide (YVAD-pNA, Biomol Research Laboratories, Plymouth Meeting, PA, U.S.A.), the sequence of which is based on precursor interleukin-1b Asp-116 cleavage site for
Caspase-1 and inhibitors in hypoxic neurons
1159
Fig. 1. Temporal effects of hypoxia on neuronal outcome. Effects of ICE/caspase-1 inhibitors added concomitantly to lethal hypoxia on final cell viability. Cell viability was measured by the conversion of the MTT dye at various time intervals after hypoxia for 3 or 6 h (H3 and H6, respectively) and at 96 h after a 6-h hypoxia in cultured neurons treated by ICE inhibitors used at two different concentrations. Data were obtained from three separate experiments, each using more than 10 dishes for each experimental condition, and are expressed as mean percentages (^S.D.) of changes from matched normoxic controls (*P , 0.05 and **P , 0.01: statistically significant difference from controls, Student’s t-test).
ICE. 34 Upon reactivity with ICE/caspase-1, the chromophore p-nitroaniline (pNA) is released from the substrate and produces a yellow color which is directly proportional to the amount of YVADase activity present in the sample. 39 On completion of the experimental protocol, cell lysates were prepared essentially as previously described, 11,18 with minor modifications. Culture medium from each culture dish was collected and centrifuged at 200 g for 10 min to collect non-adhering cells. Neuronal cells remaining on the bottom of the culture dishes were washed in ice-cold PBS, scraped off, and harvested. After centrifugation at 200 g for 10 min, the two pellets were pooled and then lysed by a 20-min incubation at 48C in a buffer containing 25 mM HEPES (pH 7.5), 5 mM EDTA, 5 mM MgCl2, 5 mM dithiothreitol, 1 mM PMSF and 150 mM NaCl. Cell lysate was then centrifuged at 14,000 g for 20 min at 48C. The supernatant corresponding to cytoplasmic extract was collected for the measurement of total proteins 26 and then for analysis of ICE/ caspase-1 activity. The assay was conducted in a final volume of 500 ml containing 20 mM HEPES (pH 7.5), 0.1 mM NaCl, 5 mM dithiothreitol, 10% sucrose, saturating substrate concentration (100 mM) and cell lysate (100 mg protein). After incubation at 378C for 30 min, time by which the reaction was shown to be linear in preliminary experiments, absorbance was measured at an excitation wavelength of 405 nm, and activity was finally reported as optical density units. RESULTS
Cell viability and nuclear morphology Preliminary cellular characterization by immunohistochemistry using antibodies specifically raised against neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) showed that our preparations were highly enriched in neurons, depicting 92.5 ^ 2.8% of NSE-positive cells in six-day-old cultures. By the same time, the level of contamination by GFAP-positive cells was evaluated to 7.6 ^ 1.6%. When compared to control cultures maintained in normoxia, transient exposure to an anaerobic environment routinely reduced by 78–80% the partial pressure of oxygen (PO2) measured in the culture medium. Following hypoxia for
6 h, morphological alterations could be first noticed by 48 h after reoxygenation, as previously reported. 3 Thereafter, analysis of cell viability depicted a significant 20% reduction of the number of living cells compared to control cultures at 72 h post-reoxygenation, and then a 36% decrease at 96 h (Fig. 1). Conversely, hypoxia for 3 h led to a progressive increase in the number of living cells, as already documented. 4 Although not shown, ICE inhibitors by themselves did not alter neurons, since cell viability remained unaffected following treatments by these compounds in the absence of hypoxia. Addition of inhibitors concomitantly to the hypoxic insult significantly reduced cell death consecutive to a 6-h hypoxia, 15 mM YVAD-CMK even increasing neuronal viability over basal values (Fig. 1). Both inhibitory compounds were also effective in rescuing neuronal cells when they were added 24 h post-reoxygenation (Fig. 2). Again, treatments by ICE inhibitors led to viability rates which were higher than those obtained from control normoxic neurons. In order to correlate these observations with specific changes in nuclear morphology, DAPI staining was performed at the experimental end-point (96 h) in controls, hypoxic cells, and cells treated by the caspase inhibitors at 24 h after hypoxia for 6 h. As illustrated in Figs 3 and 4, our data confirmed the increased proportion of neurons exhibiting typical nuclear hallmarks of apoptosis following a 6-h hypoxia (23.0 ^ 4.1% versus 1.8 ^ 0.3% in controls). The percentage of necrotic cells was also augmented, though to a lower extent (11.0 ^ 0.4% versus 6.4 ^ 0.7% in controls). Treatments by the caspase inhibitors reduced both necrosis and apoptosis after hypoxia, but they also increased significantly the proportion of mitotic neurons in culture preparations (1.9 ^ 0.2% with YVAD-CHO versus 0.3 ^ 0.2% in controls), as did hypoxia for 3 h (6.1 ^ 0.9%).
1160
C. Bossenmeyer-Pourie´ et al.
Fig. 2. Effects of ICE/caspase-1 inhibitors added 24 h after hypoxia on post-insult neuronal outcome. Cell viability was measured in cultured neurons at 96 h after hypoxia for 3 h (H3) or 6 h (H6) and in neurons exposed to hypoxia for 6 h and treated the following day by ICE inhibitors. Data were obtained from three separate experiments, each using more than 10 dishes for each experimental condition, and are expressed as mean percentages (^S.D.) of changes from matched normoxic controls (**P , 0.01: statistically significant difference from controls, Student’s t-test).
Expression of interleukin-1b -converting enzyme in cultured neurons As illustrated in Fig. 5, immunohistochemical staining against ICE p20 revealed enhanced expression of the protease in cultured neurons exposed to hypoxia for 6 h. Although not shown, immunoreactivity was already elevated by 24 h postreoxygenation, and then continued to increase progressively. Conversely, basal immunoreactivity in control normoxic cultures remained very low, and similar low reactivity was observed in cultures exposed to hypoxia for 3 h. The reverse was observed for Bcl-2 expression; the latter protein was repressed after hypoxia for 6 h, whereas its expression was augmented after hypoxia for 3 h (Fig. 5). In good agreement with immunohististochemical analysis, western blotting revealed time-dependent increases in ICE levels. This technique allowed to separate the enzyme precursor and the active cleavage product, leading to the observation of two high-intensity bands, respectively at 45,000 mol. wt and 20,000 mol. wt, in cellular extracts obtained at 96 h from neuronal cells previously exposed to hypoxia for 6 h (Fig. 6). By contrast, immunoreactivity bands were hardly detectable following hypoxia for 3 h, and data were far from reproducible, due to low levels of pro-ICE and ICE p20 in these preparations (not shown). In parallel, Bcl-2 protein, which was shown to act upstream of ICE in the cell response and to inhibit ICE caspase activity, 37 was transiently induced by 48 h to finally decrease under basal values at 96 h posthypoxia. In case of a hypoxic insult lasting for 3 h, Bcl-2 expression peaked at 48 h and remained higher than in controls at 96 h (Fig. 6). Interleukin-1b -converting enzyme proteolytic activity— effects of inhibitors
Fig. 3. Typical morphological consequences of a 6-h hypoxia (H6) followed by reoxygenation for 96 h on nuclear chromatin of cultured neurons, and influence of the ICE/caspase-1 inhibitor YVAD-CHO added at 10 mM 24 h after the hypoxic insult (DAPI staining).
Whereas spectrophotometric analysis of the degradation of the selective substrate for ICE-like proteases (i.e. YVADpNA) showed an increasing basal caspase activity in control cultures during the whole experimental period, transient hypoxia for 6 h stimulated the proteolytic action (Fig. 7). ICE activity progressively increased with time after hypoxia to finally reach 30% over control values at 96 h post-insult.
Caspase-1 and inhibitors in hypoxic neurons
1161
Fig. 4. Consequences of hypoxia on the rates of necrosis, apoptosis and mitosis in cultured neurons. Influence of delayed administration of ICE inhibitors. Numbers of normal, apoptotic, necrotic and mitotic neurons were scored by fluorescence microscopy in cultured neurons stained by DAPI at 96 h after hypoxia for 3 or 6 h (H3 and H6, respectively) and in neurons exposed to hypoxia for 6 h and treated the following day by ICE inhibitors. Nuclei were considered apoptotic when they exhibited marked signs of chromatin condensation or when they fragmented into apoptotic bodies. Data are reported as mean percentage of total neurons (^S.D.) and were obtained from three separate experiments by counting concerned cells in three distinct areas of at least 100 neurons per culture dish (**P , 0.01: statistically significant difference from controls, Student’s t-test).
Figure 8 depicts the effects on caspase final activity (i.e. at 96 h) of the different inhibitors tested, including several nonspecific protease inhibitors, such as PMSF, aprotinin, EDTA, and EGTA. These latter non-specific compounds were used at effective concentrations and were without significant consequences on ICE activity. Moreover, it clearly appeared that
YVAD-CHO and YVAD-CMK are very potent inhibitors of ICE in our culture model. It should be noticed, however, that the known selective inhibitor of CPP32/caspase-3, DEVDCHO (N-acetyl-Asp-Glu-Val-Asp-CHO), was also able to inhibit the degradation of the ICE substrate when it was used at 10 mM.
Fig. 5. Immunohistochemical detection of ICE p20 and Bcl-2 in cultured neurons at 96 h following exposure to hypoxia for 3 h or 6 h and in matched controls. The experiments were repeated three times with similar observations.
C. Bossenmeyer-Pourie´ et al.
1162 DISCUSSION
In our culture model, a hypoxic episode for 6 h leads to neuronal cell death appearing within several days following reoxygenation. Cell injury mainly reflects apoptosis, as previously documented by characteristic nuclear features, time-dependent changes in the rates of RNA and protein synthesis, sensitivity to protein synthesis inhibitors, as well as by induction of specific sets of genes implicated in the apoptotic pathway. 3,4,8 According to the present study, the apoptotic process initiated by lethal hypoxia involves the activation of ICE-like cysteine proteases. Moreover, whereas ICE inhibitors protect neuronal cells from delayed death, these compounds may act, at least partly, by stimulating neurogenesis. Using a selective peptide substrate for ICE, caspase activity was depicted in our control neurons from the developing rat forebrain during their normal life span in vitro. Indeed, a selective fraction of neurons in culture is committed to die by naturally occurring apoptosis, as confirmed by the analysis of nuclear morphology, and cysteine proteases have been implicated in physiological programmed cell death. 10 As a consequence of neuronal exposure to hypoxia at levels which are likely to occur in various pathological situations, the proteolytic action of ICE increased with time, indicating that ICE caspase family may contribute to the resulting cell injury. In good agreement with activity monitoring, transient hypoxia for 6 h resulted in progressive up-regulation of the 45,000 mol. wt ICE proenzyme, and mainly of the 20,000 mol. wt subunit which corresponds to an active protein fragment. 40 As previously documented by our laboratory, 4 a sublethal period of hypoxia (i.e. lasting for 3 h) induces enhanced neuronal rates of DNA synthesis, persistent over-expression of anti-apoptotic proteins, along with induction of proliferating cell nuclear antigen (a cofactor for DNA polymerase). As confirmed in the present study, such biochemical changes are associated with a significant increase (around 15%) in the final number of living neurons compared to normoxic controls. Taken together, these data, coupled to the previous demonstration that the cell cycle inhibitor olomoucine prevented apoptosis consecutive to a 6-h hypoxia and also impaired the stimulatory effects of a 3-h insult, 4 strongly suggest that sensitive neuronal cells enter the cell cycle in response to hypoxia and then, depending on the severity of the insult, at least some of them may dodge apoptotic death by fully achieving cell division. Among the negative regulators of apoptosis which are stimulated in response to moderate hypoxia in our model, Bcl-2 certainly plays a pivotal role. Bcl-2 expression peaks at 48 h after a 3-h hypoxia and remains high at 96 h. Conversely, Bcl-2 levels are markedly lower after a 6-h hypoxia, to finally decline below control values at 96 h, when ICE levels and activity are maximal. These observations are of interest, since it has been shown that Bcl-2 family members are functionally linked to ICE. 37 Bcl-2 would participate upstream of ICE proteases and may contribute to apoptosis inhibition by preventing the post-translational activation of ICE. ICE family members have been proposed to be required in many apoptotic paradigms, 37,42 and their activation was reported as an early event before the appearance of typical nuclear features of apoptosis. The recent discovery
of a caspase-activated DNAse constitutes a linkage between caspase induction and DNA fragmentation. 14 Several lines of evidence suggest that caspase activation may mediate delayed neuronal death after temporary cerebral ischemia in vivo 6,30 as well as hypoxia/hypoglycemiainduced neuronal damage in vitro, 31 and the ICE/caspase-1 family has been implicated in staurosporine-induced apoptosis of cultured rat hippocampal neurons as an upstream initiator of reactive oxygen species. 20 Our data are in favour of the participation of ICE/caspase-1 in hypoxic damage in developing brain neurons, and this conclusion is supported by the beneficial effects of potent and selective ICE inhibitors. Pharmacological treatments by ICE inhibitors during hypoxia significantly improved final cell outcome in our model. Notably, these compounds decreased the rate of apoptotic cells which was markedly elevated within four days after hypoxia. However, a reduction of the percentage of necrotic cells was also recorded, though the rate of necrosis consecutive to hypoxia was much more modest than that of apoptosis. This could potentially be explained by the fact that increased necrosis may partially result from the secondary transformation of apoptotic cells which cannot be eliminated by phagocytosis in vitro. Also, it appeared that neurons are not irreversibly proceeding on to death by the end of the hypoxic insult itself, inasmuch as we showed that delayed application of caspase inhibitors, i.e. 24 h after reoxygenation, can still rescue cultured cells from lethal injury. These data suggest that, in this model, the actual commitment to apoptosis induced by oxygen deprivation occurs late after cell reoxygenation. Since it has been reported that caspase-1 can activate caspase-3-like proteases in various models, it is conceivable that the effects of ICE inhibitors added during the post-reoxygenation period may reflect subsequent impairment of late caspase-3 stimulation by ICE. In this respect, Tamatani et al. 38 reported that prolonged hypoxia up-regulated sequentially caspase-1 and caspase-3 proteases in a model of rat cortical neurons. However, their experimental conditions differed from ours, and these profiles were obtained within 24 h in neurons constantly maintained under low oxygen pressure. During the time we conducted our experiments, several reports showed an in vivo attenuation of hypoxic-ischemic brain damage by various caspase inhibitors administred either before or after the ischemic insult, 6,15,19 including in a rat model of neonatal hypoxia-ischemia, 7 supporting the concept of a prolonged therapeutic window for ischemic brain injury. Still, by monitoring the cleavage of YVAD-pNa in cell lysates from selective brain regions, Chen and colleagues 6 reported a time-dependent increase in ICE activity in areas highly sensitive to oxygen supply, such as the hippocampus, but no change in more resistant areas, such as the cerebral cortex. Our study is in good agreement with such data. Stimulation of caspase-1 activity, in contrast to caspase-3, could not be shown in the developmental study by Cheng et al. 7 Interestingly, the use of ICE inhibitors not only reduced cell mortality associated to hypoxia, but repeatedly led to increased viability measured in cultured neurons. Furthermore, an augmentation of typical mitotic cells was clearly observed by nuclear staining. Similarly to sublethal hypoxia, this certainly corresponds to the division of neurons. Indeed, immunohistochemical characterization repeatedly
Caspase-1 and inhibitors in hypoxic neurons
1163
Fig. 6. Western blot analysis of ICE p20 and Bcl-2 immunoreactivities in cultured neurons previously exposed to hypoxia. For ICE analysis, cellular extracts were prepared from neurons reoxygenated for 72 h (R72) or 96 h (R96) after exposure to hypoxia for 6 h (H6) and from their matched controls (C). Concerning Bcl-2, neurons were taken at 48 h (R48) or 96 h (R96) after exposure to hypoxia for either 3 h (H3) or 6 h (H6). Whereas one representative blot for each protein with the corresponding densitometric analysis are presented for illustration, similar profiles were obtained from three separate experiments using different culture sets.
Fig. 7. Temporal evolution of ICE/caspase-1 protease activity in cultured neurons exposed to hypoxia. ICE activity was evaluated as function of time in cellular extracts from control neurons and from neurons exposed to hypoxia for 6 h (H6) by measuring the cleavage of the ICE selective colorimetric substrate peptide, YVAD-pNA. Data were obtained from three different experiments using separate culture preparations, and are expressed as optical density units at 405 nm (^S.D.) (**P , 0.01: statistically significant difference from controls, Student’s t-test).
1164
C. Bossenmeyer-Pourie´ et al.
Fig. 8. Effects of specific and non-specific inhibitors on hypoxia-induced ICE/caspase-1 activity in cultured neurons. ICE protease activity was evaluated at 96 h after hypoxia for 6 h (H6) and in matched controls by measuring the cleavage of the selective colorimetric substrate YVAD-pNA at 405 nm. When used, inhibitors were added to the cell incubating medium at the concentration indicated (TIU, trypsin inhibitor unit) just before induction of hypoxia, and were left until the end of the experimental period. Data were obtained from three different experiments using separate culture preparations, and are expressed as optical density units at 405 nm (^S.D.) (**P , 0.01: statistically significant difference from basal activity measured in controls constantly maintained under normoxia, Dunnett’s test for multiple comparisons).
showed very high purity of neuronal cultures grown in such chemically-defined conditions, without significant change in final neuron/glia ratio between hypoxic and control sister cultures. 4 Also, at the microscopic level, aspect and size of stained nuclei were quite homogeneous, including for dividing cells. Therefore, it may be postulated that, at least in neurons from the immature brain, caspase inhibitors might
challenge neuronal loss consecutive to severe hypoxia by promoting neurogenesis. Acknowledgements—The authors wish to thank the “Institut National de la Sante´ et de la Recherche Me´dicale” (INSERM), the “Fondation pour la Recherche Me´dicale” and the “Association pour la Recherche contre le Cancer” (ARC) for supporting this work.
REFERENCES
1. Alnemri E. S. (1997) Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J. Cell Biochem. 64, 33–42. 2. Beilharz E. J., Williams C. E., Dragunow M., Sirimanne E. S. and Gluckman P. D. (1995) Mechanisms of delayed cell death following hypoxic-ischemic injury in the immature rat: evidence for apoptosis during selective neuronal loss. Molec. Brain Res. 29, 1–14. 3. Bossenmeyer C., Chihab R., Muller S., Schroeder H. and Daval J. L. (1998) Hypoxia/reoxygenation induces apoptosis through biphasic induction of protein synthesis in central neurons. Brain Res. 787, 107–116. 4. Bossenmeyer-Pourie´ C., Chihab R., Schroeder H. and Daval J. L. (1999) Transient hypoxia may lead to neuronal proliferation in the developing mammalian brain: from apoptosis to cell cycle completion. Neuroscience 91, 221–231. 5. Carmichael J., de Graff W. G., Gazdar A. F., Minna J. D. and Mitchell J. B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936–942. 6. Chen J., Nagayama T., Jin K., Stetler R. A., Zhu R. L., Graham S. H. and Simon R. P. (1998) Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J. Neurosci. 18, 4914–4928. 7. Cheng Y., Deshmukh M., D’Costa A., Demaro J. A., Gidday J. M., Shah A., Sun Y., Jacquin M. F., Johnson E. M. Jr and Holtzman D. M. (1998) Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J. clin. Invest. 101, 1992–1999. 8. Chihab R., Ferry C., Koziel V., Monin P. and Daval J. L. (1998) Sequential activation of activator protein-1-related transcription factors and JNK protein kinases may contribute to apoptotic death induced by transient hypoxia in developing brain neurons. Molec. Brain Res. 63, 105–120. 9. Chinnaiyan A. M. and Dixit V. M. (1996) The cell-death machine. Curr. Biol. 6, 555–562. 10. Cohen G. M. (1997) Caspases; the executioners of apoptosis. Biochem. J. 326, 1–16. 11. Datta R., Kojima H., Banach D., Bump N. J., Talanian R. V., Alnemri E. S., Weichselbaum R. R., Wong W. W. and Kufe D. W. (1997) Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis. J. biol. Chem. 272, 1965–1969. 12. Edwards A. D. and Mehmet H. (1996) Apoptosis in perinatal hypoxic-ischaemic cerebral damage. Neuropath. appl. Neurobiol. 22, 494–498. 13. Enari M., Hug H. and Nagata S. (1995) Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature 375, 78–81. 14. Enari M., Sakahira H., Yokoyama H., Okawa K., Iwamatsu A. and Nagata S. (1998) A caspase-activated DNAse that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50. 15. Fink K., Zhu J., Namura S., Shimizu-Sasamata M., Endres M., Ma J., Dalkara T., Yuan J. and Moskowitz M. A. (1998) Prolonged therapeutic window for ischemic brain damage caused by delayed caspase activation. J. cereb. Blood Flow Metab. 18, 1071–1076. 16. Gray P. H., Tudehope D. I., Masel J. P., Burns Y. R., Mohay H. A., O’Callaghan M. J. and Williams G. M. (1993) Perinatal hypoxic-ischaemic brain injury: prediction of outcome. Devl med. Child Neurol. 35, 965–973. 17. Gschwind M. and Huber G. (1997) Detection of apoptotic or necrotic death in neuronal cells by morphological, biochemical, and molecular analysis. In Neuromethods, Apoptosis Techniques and Protocols (ed. Poirier J.), Vol. 29, pp. 13–31. Humana, Totowa. 18. Harvey N. L., Butt A. J. and Kumar S. (1997) Functional activation of Nedd2/ICH-1 (caspase-2) is an early process in apoptosis. J. biol. Chem. 272, 13,134–13,139. 19. Himi T., Ishizaki Y. and Murota S. (1998) A caspase inhibitor blocks ischaemia-induced delayed neuronal death in the gerbil. Eur. J. Neurosci. 10, 777–781. 20. Krohne A. J., Preis E. and Prehn J. H. M. (1998) Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector. J. Neurosci. 18, 8186–8197. 21. Kumar S. (1995) ICE-like proteases in apoptosis. Trends biochem. Sci. 20, 198–202. 22. Kumar S., Kinoshita M., Noda M., Copeland N. G. and Jenkins N. A. (1994) Induction of apoptosis by mouse Nedd2 gene which encodes a protein similar to the product of Caenorhabditis elegans cell death ge ced-3 and mammalian IL-1b-converting enzyme. Genes Dev. 8, 1613–1626. 23. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Caspase-1 and inhibitors in hypoxic neurons
1165
24. Lin Y. Z., Yao S. Y. and Hawiger J. (1996) Role of the nuclear localization sequence in fibroblast growth factor-1-stimulated mitogenic pathways. J. biol. Chem. 271, 5305–5308. 25. Livingston D. J. (1997) In vitro and in vivo studies of ICE inhibitors. J. Cell Biochem. 64, 19–26. 26. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275. 27. MacManus J. P., Buchan A. M., Hill I. E., Rasquinha I. and Preston E. (1993) Global ischaemia can cause DNA fragmentation indicative of apoptosis in rat brain. Neurosci. Lett. 164, 89–92. 28. Margolin N., Raybuck S. A., Wilson K. P., Chen W., Fox T., Gu Y. and Livingston D. J. (1997) Substrate and inhibitor specificity of interleukin-1-bconverting enzyme and related caspases. J. biol. Chem. 272, 7223–7228. 29. Meikrantz W. and Schlegel R. (1995) Apoptosis and the cell cycle. J. Cell Biochem. 58, 160–174. 30. Namura S., Zhu J., Fink K., Endres M., Srinivasan M., Tomaselli K. J., Yuan J. and Moskowitz M. A. (1998) Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J. Neurosci. 18, 3659–3668. 31. Nath R., Probert A. Jr, McGinnis K. M. and Wang K. K. W. (1998) Evidence for activation of caspase-3-like protease in excitotoxin- and hypoxia/ hypoglycemia-injured neurons. J. Neurochem. 71, 186–195. 32. Oppenheim R. W. (1991) Cell death during development of the nervous system. A. Rev. Neurosci. 14, 453–501. 33. Park D. S., Morris E. J., Greene L. A. and Geller H. M. (1997) G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecininduced neuronal apoptosis. J. Neurosci. 17, 1256–1270. 34. Reiter L. A. (1994) Peptidic p-nitroanilide substrates of interleukin-1 beta-converting enzyme. Int. J. Pept. Protein Res. 43, 87–96. 35. Rosenbaum D. M., Michaelson M., Batter D. K., Doshi P. and Kessler J. A. (1994) Evidence of hypoxia-induced, programmed cell death of cultured neurons. Ann. Neurol. 36, 864–870. 36. Sher P. K. (1990) Chronic hypoxia in neuronal cell culture: metabolic consequences. Brain Dev. 12, 293–300. ¨ rd T., Keane R. W., Bredesen D. E. and Kayalar C. (1996) Bcl-2 expression in neural cells blocks activation of 37. Srinivasan A., Foster L. M., Testa M. P., O ICE/CED-3 family proteases during apoptosis. J. Neurosci. 16, 5654–5660. 38. Tamatani M., Ogawa S. and Tohyama M. (1998) Roles of Bcl-2 and caspases in hypoxia-induced neuronal cell death: a possible neuroprotective mechanism of peptide growth factors. Molec. Brain Res. 58, 27–39. 39. Thornberry N. A. (1994) Interleukin-1 beta converting enzyme. Meth. Enzymol. 244, 615–631. 40. Thornberry N. A., Bull H. G., Calaycay J. R., Chapman K. T., Howard A. D., Kostura M. J., Miller D. K., Molineaux S. M., Weidner J. R., Aunins J., Elliston K. O., Ayala J. M., Casano F. J., Chin J., Ding G. J. F., Egger L. A., Gaffney E. P., Limjuco G., Palyha O. C., Raju S. M., Rolando A. M., Salley J. P., Yamin T. T., Lee T. D., Shively J. E., MacCross M., Mumford R. A., Schmidt J. A. and Tocci M. J. (1992) A novel heterodimeric cysteine protease is required for interleukin-1b processing in monocytes. Nature 356, 768–774. 41. Woo M., Hakem R., Soengas M. S., Duncan G. S., Shahinian A., Ka¨gi D., Hakem A., McCurrach M., Khoo W., Kaufman S. A., Senaldi G., Howard T., Lowe S. W. and Mak T. W. (1998) Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev. 12, 806–819. 42. Yuan J., Shaham S., Ledoux S., Ellis H. M. and Horvitz H. R. (1993) The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1b-converting enzyme. Cell 75, 641–652. (Accepted 4 October 1999)
Pergamon PII: S0306-4522(99)00501-1
Neuroscience Vol. 95, No. 4, pp. 1157–1165,1157 2000 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
INVOLVEMENT OF CASPASE-1 PROTEASES IN HYPOXIC BRAIN INJURY. EFFECTS OF THEIR INHIBITORS IN DEVELOPING NEURONS C. BOSSENMEYER-POURIE´, V. KOZIEL and J. L. DAVAL* JE 2164, Universite´ Henri Poincare´, 54013 Nancy, Cedex, France
Abstract—To further explore the contribution of caspase-1/interleukin-1b-converting enzyme in the consequences of hypoxia in developing brain neurons, its temporal expression profile was analysed by immunohistochemistry and western blotting in cultured neurons from the embryonic rat forebrain subjected to a hypoxic stress (95% N2/5% CO2 for 6 h), and proteolytic activity of caspase-1 was monitored as a function of time by measuring the degradation of a selective colorimetric substrate (N-acetyl-TyrVal-Ala-Asp-p-nitroanilide). In addition, the influence of pre- and posthypoxic treatments by caspase-1 inhibitors (N-acetyl-TyrVal-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone) was tested on cell outcome. Hypoxia led to delayed apoptotic neuronal death, with an elevation of the expression of both pro-caspase-1 and caspase-1 active cleavage product (ICE p20) for up to 96 h after cell reoxygenation. As reflected by cleavage of the specific substrate, caspase-1 activity progressively increased between 24 h and 96 h posthypoxia, and was blocked by inhibitors in a dose-dependent fashion. The inhibitory compounds, including when given 24 h after hypoxia, prevented neuronal death, reduced apoptosis hallmarks and also increased the number of mitotic neurons, suggesting they might promote neurogenesis. Similar observations were made when neurons were exposed to a sublethal hypoxia (i.e. 3 h). These data emphasize the participation of caspase-1 in neuronal injury consecutive to oxygen deprivation, and provide new insight into the possible cellular mechanisms by which caspase inhibitors may protect developing brain neurons. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: oxygen deprivation, apoptosis, neurogenesis, cell culture, rat forebrain.
apoptosis, 25 suggesting a potential beneficial role for their derivatives in some stressful circumstances. 6,7 However, it is likely that requirement for the activation of specific caspases may vary according to the death-inducing stimulus as well as to the type of target cells. 41 Although ICE has been unequivocally shown to be expressed in the CNS, 22 its effective participation in hypoxia (and/or ischemia)-induced neuronal injury remains unclear. Using primary cultured neurons from the embryonic rat forebrain as a model, we have previously reported that hypoxia for 6 h, without concomitant glucose deprivation, induces delayed cell death presenting morphological and biochemical characteristics of apoptosis. 3,8 In contrast to the deleterious effects of such a long-lasting insult, hypoxia for 3 h has been shown to promote neuronal division, leading to a significant increase in the final number of living neurons. 4 Indeed, apoptosis may be considered as the result of an inappropriate and finally aborted re-entry into the cell cycle, 29,33 and our observations support the conclusion that some neurons exposed to sublethal hypoxia may dodge apoptotic death by fully achieving the cell cycle. To further delineate the participation of ICE-like caspases as well as the consequences of their inhibitors in the neuronal response to transient oxygen deprivation in the developing mammalian brain, we analysed ICE activation and explored the effects of selective peptide inhibitors on cultured neurons exposed to lethal hypoxia. Inhibitors were added to the culture medium either concomitantly to the hypoxic insult or 24 h later. Cell viability and apoptosis-related morphological features were monitored as a function of time after hypoxia/ reoxygenation, and ICE-like specific protease activity was monitored by measuring the hydrolysis of a selective synthetic substrate. 34
In the perinatal period, cerebral hypoxia is known to induce brain injury and remains a major cause of neurodevelopmental impairment. 16 Hypoxic-ischemic cell death was initially attributed to tissue necrosis, but, based upon both in vivo and in vitro findings, selective neuronal damage observed after transient oxygen deprivation progressively appeared to involve apoptosis, 2,3,8,12,35,38 a process implicated in physiological cell elimination during development. 32 Through the activation of an intrinsic, constitutive death programme, apoptosis leads to typical morphological alterations, such as cell shrinkage, chromatin condensation followed by DNA fragmentation. 27 Among the numerous molecular mechanisms involved in the apoptotic cascade, activation of cysteine proteases that belong to the caspase superfamily certainly plays a critical role in the cell outcome. 1,9,10,21 Caspases correspond to the mammalian homologs of the Caenorhabditis elegans death gene ced3, 42 and include the prototypic interleukin-1b-converting enzyme (ICE, capase-1). During apoptosis, caspases cleave their selective substrates at aspartate residues, whereas their inhibitors such as the cowpox virus CrmA protein or the baculovirus p35 protein have been shown to inhibit *To whom correspondence should be addressed. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DEVD-CHO, Nacetyl-Asp-Glu-Val-Asp-aldehyde; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetra-acetate; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; GFAP, glial fibrillary acidic protein; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid; ICE, interleukin-1b-converting enzyme; MTT, 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide; NSE, neuron-specific enolase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; YVAD-CHO, N-acetyl-Tyr-Val-Ala-Asp-aldehyde; YVAD-CMK, N-acetyl-Tyr-ValAla-Asp-chloromethylketone; YVAD-pNA, N-acetyl-Tyr-Val-Ala-Aspp-nitroanilide. 1157
C. Bossenmeyer-Pourie´ et al.
1158 EXPERIMENTAL PROCEDURES
Neuronal cell cultures Animal experimentation was carried out with the highest standards of animal care and housing, according to the N.I.H. Guide for the Care and Use of Laboratory Animals. Primary cultured neurons were obtained from 14-day-old rat embryo forebrains as previously described. 3,4 When they were in the proestrus period, as shown by the observation of daily vaginal smears, Sprague–Dawley female rats (R. Janvier, Le Genest-St-Isle, France) were housed together with males for 24 h, and pregnant dams were maintained in separate cages for 14 days under standard laboratory conditions on a 12:12 h light:dark cycle, with food and water available ad libitum. Living embryos were excised by Caesarian section performed under anaesthesia with halothane. Whole embryos were placed in culture medium previously equilibrated at 378C and consisting of a mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (50:50, ICN Pharmaceuticals, Costa Mesa, CA, U.S.A.) supplemented with 5% inactivated fetal calf serum (Valbiotech, Paris, France). Forebrains were carefully collected, dissected free of meninges and gently dispersed in culture medium. After centrifugation at 700 g for 10 min, the pellet was redispersed in the same medium and passed through a 46-mm-pore size nylon mesh. Aliquots of the cell suspension were transferred into 35 mm Petri dishes (Falcon, Becton Dickinson, Le Pont-de-Claix, France) precoated with poly-l-lysine in order to obtain a final density of 10 6 cells/dish. Cultures were then placed at 378C in a humidified atmosphere of 95% air/5% CO2. The following day, the culture medium was replaced with a fresh hormonally defined serum-free medium consisting of the DMEM/Ham’s F12 mixture enriched with human transferrin (1 mM), bovine insulin (1 mM), putrescine (0.1 mM), progesterone (10 nM), estradiol (1 pM), Na selenite (30 nM), and also containing fibroblast growth factor (2 ng/ml) and epidermal growth factor (10 ng/ml) (Sigma Chemicals, St Louis, MO, U.S.A.). After two additional days, the culture medium was renewed with serum-free medium in the absence of growth factors. Exposure to hypoxia and treatments by inhibitors Six-day-old neuronal cell cultures were submitted to transient hypoxia for 3 or 6 h by transferring the culture dishes to a humidified incubation chamber thermoregulated at 378C and flushed by 95% N2/5% CO2. Cultures were then returned to standard normoxic atmosphere for the next 96 h. The reduction in oxygen delivery to the neurons was assessed by measuring O2 content in samples of extracellular medium collected just prior to the beginning of hypoxia and immediately at the end of the insult, by means of a gas analyser (Corning, Halstead, U.K.), as described by Sher. 36 The effects of two potent ICE-like inhibitors were tested, i.e. Nacetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO, ICE inhibitor I from Oncogene Research Products, Cambridge, MA, U.S.A.), a cellpermeable reversible inhibitor, 24 and N-acetyl-Tyr-Val-Ala-Aspchloromethylketone (YVAD-CMK, ICE inhibitor II from Oncogene Research Products), a cell-permeable irreversible inhibitor. 13 Peptide inhibitors were added at various concentrations to fresh culture medium, either before starting hypoxia or 24 h after cell reoxygenation. Drugs were then left in the culture dishes until the end of the experimental period, i.e. 96 h post-reoxygenation. Concentrations were carefully chosen in agreement with the manufacturer’s recommendations, according to the known Ki values. 28 Analysis of cell outcome Cell morphology was routinely assessed by phase-contrast microscopic observations by means of a Nikon Diaphot TMD. Cell viability was monitored by a spectrophotometric method using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), according to Carmichael et al. 5 Neurons were incubated for 3 h at 378C with MTT (500 mg/ml), washed twice with ice-cold phosphatebuffered saline (PBS), and lysed in dimethylsulfoxide. Optical density was measured at 519 nm and data were compared to those obtained from sister control cells. Characteristic morphological features of apoptosis, necrosis and mitosis were detected after cell reoxygenation by using the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI), as previously documented. 3,4,17,33 Cultures were fixed for 10 min in a mixture of ethanol: acetic acid (3:1), washed for 1 min in distilled water, air-dried, and subsequently stained with DAPI (0.5 mg/ml) for 10 min. Cells were
washed twice with distilled water, air-dried, and treated with antifading medium (10 mg/ml p-phenylenediamine in 90% glycerol, pH 9.0). Normal, apoptotic, necrotic and mitotic neurons were scored under fluorescence microscopy (Zeiss Axioscop) at an excitation wavelength of 365 nm by counting concerned cells in three distinct areas of at least 100 neurons per culture dish. Immunohistochemical and western blot analyses In its active form, ICE is an oligomeric enzyme with 20,000 mol. wt (p20) and 10,000 mol. wt (p10) subunits derived from the cleavage of a 45,000 mol. wt proenzyme. 40 Expression of ICE/caspase-1 was studied in neuronal cells by immunohistochemistry and western blotting by using an affinity-purified goat polyclonal antibody (ICE p20/M-19, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) raised against a peptide corresponding to amino acids 276–294 mapping at the carboxyterminus of the p20 subunit, and thus reacts with both the ICE precursor and the active cleavage product ICE p20. Cultures were rinsed twice with PBS, then fixed for 10 min in methanol at 2108C, and rinsed again with PBS. Non-specific binding sites for IgG were blocked by incubating the cells for 20 min with 10% horse serum (Gibco-BRL, Inchinnan, U.K.) in PBS. Thereafter, cultured neurons were incubated for 60 min at room temperature in buffer containing ICE p20 polyclonal antibody diluted at 1:40. Following two washing steps to remove unfixed antibodies, the cells were incubated for 120 min with anti-goat IgG conjugated to rhodamine isothiocyanate (RITC, dilution 1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.). Neuronal cells were finally washed three times with PBS, coverslipped using mounting medium (Aquapolymount w), and kept in the dark until subsequent analysis by means of a Zeiss Axioscop fluorescence microscope. In some experiments, culture preparations were stained with a polyclonal antibody specific for Bcl-2 diluted at 1:100 (goat IgG, Santa Cruz Biotechnology) and revealed in the presence of an anti-goat IgG conjugated to fluorescein isothiocyanate (FITC, dilution 1:100, Jackson ImmunoResearch Laboratories). For western blotting, adhering and non-adhering cultured cells were harvested, and cell extraction was performed by using a buffer solution consisting of 10 mM HEPES (pH 7.5), 5 mM EDTA, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM KCl and 0.1% Triton X-100. Cell lysates were then centrifuged for 20 min at 14,000 g, and the cytosolic supernatant was collected. The nuclear pellet was resuspended in the same buffer, centrifuged at 14,000 g for 20 min, and the final supernatant was pooled with the primary one for subsequent protein separation. Final samples, each containing 30 mg total proteins, were electrophoresed on sodium dodecyl sulfate (SDS)–polyacrylamide gel (5% stacking, 10% running) according to Laemmli, 23 in parallel with molecular weight standards. After separation, proteins were transferred on to a polyvinylidene difluoride membrane (PVDF, New England Nuclear, Boston, MA, U.S.A.) using a Tris–glycine buffer (48 mM Tris base, 39 mM glycine, 0.037% SDS and 20% methanol) by means of a semi-dry electrotransferring unit (Bio-Rad, Ivry-sur Seine, France) at 160 mA for 30 min. Subsequent experimental steps were performed by using a commercially available kit for western blot detection of proteins with peroxidase-labeled secondary antibodies and the chemiluminescent substrate luminol from Boehringer (Mannheim, Germany). Non-specific sites were blocked by incubating the membrane while shaking for 1 h in 1% blocking reagent provided by the supplier. Blots were then probed for 1 h with either anti-ICE p20 goat polyclonal antibody at a 1:500 dilution in 0.5% blocking solution or anti-Bcl-2 goat polyclonal antibody diluted at 1:400. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBS–Tween), the membrane was incubated for 30 min with peroxidase-labeled secondary antibody (40 mU/ml, Boehringer), and washed four times in TBS–Tween. Chemiluminescent detection of specifically-labeled proteins was finally performed according to the supplier’s instructions by using Kodak X-Omat films. Assay for interleukin-1b -converting enzyme activity ICE-like proteolytic activity was measured at various time intervals after cell reoxygenation in crude cell extracts by a spectrophotometric method using the selective colorimetric substrate peptide, N-acetylTyr-Val-Ala-Asp-p-nitroanilide (YVAD-pNA, Biomol Research Laboratories, Plymouth Meeting, PA, U.S.A.), the sequence of which is based on precursor interleukin-1b Asp-116 cleavage site for
Caspase-1 and inhibitors in hypoxic neurons
1159
Fig. 1. Temporal effects of hypoxia on neuronal outcome. Effects of ICE/caspase-1 inhibitors added concomitantly to lethal hypoxia on final cell viability. Cell viability was measured by the conversion of the MTT dye at various time intervals after hypoxia for 3 or 6 h (H3 and H6, respectively) and at 96 h after a 6-h hypoxia in cultured neurons treated by ICE inhibitors used at two different concentrations. Data were obtained from three separate experiments, each using more than 10 dishes for each experimental condition, and are expressed as mean percentages (^S.D.) of changes from matched normoxic controls (*P , 0.05 and **P , 0.01: statistically significant difference from controls, Student’s t-test).
ICE. 34 Upon reactivity with ICE/caspase-1, the chromophore p-nitroaniline (pNA) is released from the substrate and produces a yellow color which is directly proportional to the amount of YVADase activity present in the sample. 39 On completion of the experimental protocol, cell lysates were prepared essentially as previously described, 11,18 with minor modifications. Culture medium from each culture dish was collected and centrifuged at 200 g for 10 min to collect non-adhering cells. Neuronal cells remaining on the bottom of the culture dishes were washed in ice-cold PBS, scraped off, and harvested. After centrifugation at 200 g for 10 min, the two pellets were pooled and then lysed by a 20-min incubation at 48C in a buffer containing 25 mM HEPES (pH 7.5), 5 mM EDTA, 5 mM MgCl2, 5 mM dithiothreitol, 1 mM PMSF and 150 mM NaCl. Cell lysate was then centrifuged at 14,000 g for 20 min at 48C. The supernatant corresponding to cytoplasmic extract was collected for the measurement of total proteins 26 and then for analysis of ICE/ caspase-1 activity. The assay was conducted in a final volume of 500 ml containing 20 mM HEPES (pH 7.5), 0.1 mM NaCl, 5 mM dithiothreitol, 10% sucrose, saturating substrate concentration (100 mM) and cell lysate (100 mg protein). After incubation at 378C for 30 min, time by which the reaction was shown to be linear in preliminary experiments, absorbance was measured at an excitation wavelength of 405 nm, and activity was finally reported as optical density units. RESULTS
Cell viability and nuclear morphology Preliminary cellular characterization by immunohistochemistry using antibodies specifically raised against neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) showed that our preparations were highly enriched in neurons, depicting 92.5 ^ 2.8% of NSE-positive cells in six-day-old cultures. By the same time, the level of contamination by GFAP-positive cells was evaluated to 7.6 ^ 1.6%. When compared to control cultures maintained in normoxia, transient exposure to an anaerobic environment routinely reduced by 78–80% the partial pressure of oxygen (PO2) measured in the culture medium. Following hypoxia for
6 h, morphological alterations could be first noticed by 48 h after reoxygenation, as previously reported. 3 Thereafter, analysis of cell viability depicted a significant 20% reduction of the number of living cells compared to control cultures at 72 h post-reoxygenation, and then a 36% decrease at 96 h (Fig. 1). Conversely, hypoxia for 3 h led to a progressive increase in the number of living cells, as already documented. 4 Although not shown, ICE inhibitors by themselves did not alter neurons, since cell viability remained unaffected following treatments by these compounds in the absence of hypoxia. Addition of inhibitors concomitantly to the hypoxic insult significantly reduced cell death consecutive to a 6-h hypoxia, 15 mM YVAD-CMK even increasing neuronal viability over basal values (Fig. 1). Both inhibitory compounds were also effective in rescuing neuronal cells when they were added 24 h post-reoxygenation (Fig. 2). Again, treatments by ICE inhibitors led to viability rates which were higher than those obtained from control normoxic neurons. In order to correlate these observations with specific changes in nuclear morphology, DAPI staining was performed at the experimental end-point (96 h) in controls, hypoxic cells, and cells treated by the caspase inhibitors at 24 h after hypoxia for 6 h. As illustrated in Figs 3 and 4, our data confirmed the increased proportion of neurons exhibiting typical nuclear hallmarks of apoptosis following a 6-h hypoxia (23.0 ^ 4.1% versus 1.8 ^ 0.3% in controls). The percentage of necrotic cells was also augmented, though to a lower extent (11.0 ^ 0.4% versus 6.4 ^ 0.7% in controls). Treatments by the caspase inhibitors reduced both necrosis and apoptosis after hypoxia, but they also increased significantly the proportion of mitotic neurons in culture preparations (1.9 ^ 0.2% with YVAD-CHO versus 0.3 ^ 0.2% in controls), as did hypoxia for 3 h (6.1 ^ 0.9%).
1160
C. Bossenmeyer-Pourie´ et al.
Fig. 2. Effects of ICE/caspase-1 inhibitors added 24 h after hypoxia on post-insult neuronal outcome. Cell viability was measured in cultured neurons at 96 h after hypoxia for 3 h (H3) or 6 h (H6) and in neurons exposed to hypoxia for 6 h and treated the following day by ICE inhibitors. Data were obtained from three separate experiments, each using more than 10 dishes for each experimental condition, and are expressed as mean percentages (^S.D.) of changes from matched normoxic controls (**P , 0.01: statistically significant difference from controls, Student’s t-test).
Expression of interleukin-1b -converting enzyme in cultured neurons As illustrated in Fig. 5, immunohistochemical staining against ICE p20 revealed enhanced expression of the protease in cultured neurons exposed to hypoxia for 6 h. Although not shown, immunoreactivity was already elevated by 24 h postreoxygenation, and then continued to increase progressively. Conversely, basal immunoreactivity in control normoxic cultures remained very low, and similar low reactivity was observed in cultures exposed to hypoxia for 3 h. The reverse was observed for Bcl-2 expression; the latter protein was repressed after hypoxia for 6 h, whereas its expression was augmented after hypoxia for 3 h (Fig. 5). In good agreement with immunohististochemical analysis, western blotting revealed time-dependent increases in ICE levels. This technique allowed to separate the enzyme precursor and the active cleavage product, leading to the observation of two high-intensity bands, respectively at 45,000 mol. wt and 20,000 mol. wt, in cellular extracts obtained at 96 h from neuronal cells previously exposed to hypoxia for 6 h (Fig. 6). By contrast, immunoreactivity bands were hardly detectable following hypoxia for 3 h, and data were far from reproducible, due to low levels of pro-ICE and ICE p20 in these preparations (not shown). In parallel, Bcl-2 protein, which was shown to act upstream of ICE in the cell response and to inhibit ICE caspase activity, 37 was transiently induced by 48 h to finally decrease under basal values at 96 h posthypoxia. In case of a hypoxic insult lasting for 3 h, Bcl-2 expression peaked at 48 h and remained higher than in controls at 96 h (Fig. 6). Interleukin-1b -converting enzyme proteolytic activity— effects of inhibitors
Fig. 3. Typical morphological consequences of a 6-h hypoxia (H6) followed by reoxygenation for 96 h on nuclear chromatin of cultured neurons, and influence of the ICE/caspase-1 inhibitor YVAD-CHO added at 10 mM 24 h after the hypoxic insult (DAPI staining).
Whereas spectrophotometric analysis of the degradation of the selective substrate for ICE-like proteases (i.e. YVADpNA) showed an increasing basal caspase activity in control cultures during the whole experimental period, transient hypoxia for 6 h stimulated the proteolytic action (Fig. 7). ICE activity progressively increased with time after hypoxia to finally reach 30% over control values at 96 h post-insult.
Caspase-1 and inhibitors in hypoxic neurons
1161
Fig. 4. Consequences of hypoxia on the rates of necrosis, apoptosis and mitosis in cultured neurons. Influence of delayed administration of ICE inhibitors. Numbers of normal, apoptotic, necrotic and mitotic neurons were scored by fluorescence microscopy in cultured neurons stained by DAPI at 96 h after hypoxia for 3 or 6 h (H3 and H6, respectively) and in neurons exposed to hypoxia for 6 h and treated the following day by ICE inhibitors. Nuclei were considered apoptotic when they exhibited marked signs of chromatin condensation or when they fragmented into apoptotic bodies. Data are reported as mean percentage of total neurons (^S.D.) and were obtained from three separate experiments by counting concerned cells in three distinct areas of at least 100 neurons per culture dish (**P , 0.01: statistically significant difference from controls, Student’s t-test).
Figure 8 depicts the effects on caspase final activity (i.e. at 96 h) of the different inhibitors tested, including several nonspecific protease inhibitors, such as PMSF, aprotinin, EDTA, and EGTA. These latter non-specific compounds were used at effective concentrations and were without significant consequences on ICE activity. Moreover, it clearly appeared that
YVAD-CHO and YVAD-CMK are very potent inhibitors of ICE in our culture model. It should be noticed, however, that the known selective inhibitor of CPP32/caspase-3, DEVDCHO (N-acetyl-Asp-Glu-Val-Asp-CHO), was also able to inhibit the degradation of the ICE substrate when it was used at 10 mM.
Fig. 5. Immunohistochemical detection of ICE p20 and Bcl-2 in cultured neurons at 96 h following exposure to hypoxia for 3 h or 6 h and in matched controls. The experiments were repeated three times with similar observations.
C. Bossenmeyer-Pourie´ et al.
1162 DISCUSSION
In our culture model, a hypoxic episode for 6 h leads to neuronal cell death appearing within several days following reoxygenation. Cell injury mainly reflects apoptosis, as previously documented by characteristic nuclear features, time-dependent changes in the rates of RNA and protein synthesis, sensitivity to protein synthesis inhibitors, as well as by induction of specific sets of genes implicated in the apoptotic pathway. 3,4,8 According to the present study, the apoptotic process initiated by lethal hypoxia involves the activation of ICE-like cysteine proteases. Moreover, whereas ICE inhibitors protect neuronal cells from delayed death, these compounds may act, at least partly, by stimulating neurogenesis. Using a selective peptide substrate for ICE, caspase activity was depicted in our control neurons from the developing rat forebrain during their normal life span in vitro. Indeed, a selective fraction of neurons in culture is committed to die by naturally occurring apoptosis, as confirmed by the analysis of nuclear morphology, and cysteine proteases have been implicated in physiological programmed cell death. 10 As a consequence of neuronal exposure to hypoxia at levels which are likely to occur in various pathological situations, the proteolytic action of ICE increased with time, indicating that ICE caspase family may contribute to the resulting cell injury. In good agreement with activity monitoring, transient hypoxia for 6 h resulted in progressive up-regulation of the 45,000 mol. wt ICE proenzyme, and mainly of the 20,000 mol. wt subunit which corresponds to an active protein fragment. 40 As previously documented by our laboratory, 4 a sublethal period of hypoxia (i.e. lasting for 3 h) induces enhanced neuronal rates of DNA synthesis, persistent over-expression of anti-apoptotic proteins, along with induction of proliferating cell nuclear antigen (a cofactor for DNA polymerase). As confirmed in the present study, such biochemical changes are associated with a significant increase (around 15%) in the final number of living neurons compared to normoxic controls. Taken together, these data, coupled to the previous demonstration that the cell cycle inhibitor olomoucine prevented apoptosis consecutive to a 6-h hypoxia and also impaired the stimulatory effects of a 3-h insult, 4 strongly suggest that sensitive neuronal cells enter the cell cycle in response to hypoxia and then, depending on the severity of the insult, at least some of them may dodge apoptotic death by fully achieving cell division. Among the negative regulators of apoptosis which are stimulated in response to moderate hypoxia in our model, Bcl-2 certainly plays a pivotal role. Bcl-2 expression peaks at 48 h after a 3-h hypoxia and remains high at 96 h. Conversely, Bcl-2 levels are markedly lower after a 6-h hypoxia, to finally decline below control values at 96 h, when ICE levels and activity are maximal. These observations are of interest, since it has been shown that Bcl-2 family members are functionally linked to ICE. 37 Bcl-2 would participate upstream of ICE proteases and may contribute to apoptosis inhibition by preventing the post-translational activation of ICE. ICE family members have been proposed to be required in many apoptotic paradigms, 37,42 and their activation was reported as an early event before the appearance of typical nuclear features of apoptosis. The recent discovery
of a caspase-activated DNAse constitutes a linkage between caspase induction and DNA fragmentation. 14 Several lines of evidence suggest that caspase activation may mediate delayed neuronal death after temporary cerebral ischemia in vivo 6,30 as well as hypoxia/hypoglycemiainduced neuronal damage in vitro, 31 and the ICE/caspase-1 family has been implicated in staurosporine-induced apoptosis of cultured rat hippocampal neurons as an upstream initiator of reactive oxygen species. 20 Our data are in favour of the participation of ICE/caspase-1 in hypoxic damage in developing brain neurons, and this conclusion is supported by the beneficial effects of potent and selective ICE inhibitors. Pharmacological treatments by ICE inhibitors during hypoxia significantly improved final cell outcome in our model. Notably, these compounds decreased the rate of apoptotic cells which was markedly elevated within four days after hypoxia. However, a reduction of the percentage of necrotic cells was also recorded, though the rate of necrosis consecutive to hypoxia was much more modest than that of apoptosis. This could potentially be explained by the fact that increased necrosis may partially result from the secondary transformation of apoptotic cells which cannot be eliminated by phagocytosis in vitro. Also, it appeared that neurons are not irreversibly proceeding on to death by the end of the hypoxic insult itself, inasmuch as we showed that delayed application of caspase inhibitors, i.e. 24 h after reoxygenation, can still rescue cultured cells from lethal injury. These data suggest that, in this model, the actual commitment to apoptosis induced by oxygen deprivation occurs late after cell reoxygenation. Since it has been reported that caspase-1 can activate caspase-3-like proteases in various models, it is conceivable that the effects of ICE inhibitors added during the post-reoxygenation period may reflect subsequent impairment of late caspase-3 stimulation by ICE. In this respect, Tamatani et al. 38 reported that prolonged hypoxia up-regulated sequentially caspase-1 and caspase-3 proteases in a model of rat cortical neurons. However, their experimental conditions differed from ours, and these profiles were obtained within 24 h in neurons constantly maintained under low oxygen pressure. During the time we conducted our experiments, several reports showed an in vivo attenuation of hypoxic-ischemic brain damage by various caspase inhibitors administred either before or after the ischemic insult, 6,15,19 including in a rat model of neonatal hypoxia-ischemia, 7 supporting the concept of a prolonged therapeutic window for ischemic brain injury. Still, by monitoring the cleavage of YVAD-pNa in cell lysates from selective brain regions, Chen and colleagues 6 reported a time-dependent increase in ICE activity in areas highly sensitive to oxygen supply, such as the hippocampus, but no change in more resistant areas, such as the cerebral cortex. Our study is in good agreement with such data. Stimulation of caspase-1 activity, in contrast to caspase-3, could not be shown in the developmental study by Cheng et al. 7 Interestingly, the use of ICE inhibitors not only reduced cell mortality associated to hypoxia, but repeatedly led to increased viability measured in cultured neurons. Furthermore, an augmentation of typical mitotic cells was clearly observed by nuclear staining. Similarly to sublethal hypoxia, this certainly corresponds to the division of neurons. Indeed, immunohistochemical characterization repeatedly
Caspase-1 and inhibitors in hypoxic neurons
1163
Fig. 6. Western blot analysis of ICE p20 and Bcl-2 immunoreactivities in cultured neurons previously exposed to hypoxia. For ICE analysis, cellular extracts were prepared from neurons reoxygenated for 72 h (R72) or 96 h (R96) after exposure to hypoxia for 6 h (H6) and from their matched controls (C). Concerning Bcl-2, neurons were taken at 48 h (R48) or 96 h (R96) after exposure to hypoxia for either 3 h (H3) or 6 h (H6). Whereas one representative blot for each protein with the corresponding densitometric analysis are presented for illustration, similar profiles were obtained from three separate experiments using different culture sets.
Fig. 7. Temporal evolution of ICE/caspase-1 protease activity in cultured neurons exposed to hypoxia. ICE activity was evaluated as function of time in cellular extracts from control neurons and from neurons exposed to hypoxia for 6 h (H6) by measuring the cleavage of the ICE selective colorimetric substrate peptide, YVAD-pNA. Data were obtained from three different experiments using separate culture preparations, and are expressed as optical density units at 405 nm (^S.D.) (**P , 0.01: statistically significant difference from controls, Student’s t-test).
1164
C. Bossenmeyer-Pourie´ et al.
Fig. 8. Effects of specific and non-specific inhibitors on hypoxia-induced ICE/caspase-1 activity in cultured neurons. ICE protease activity was evaluated at 96 h after hypoxia for 6 h (H6) and in matched controls by measuring the cleavage of the selective colorimetric substrate YVAD-pNA at 405 nm. When used, inhibitors were added to the cell incubating medium at the concentration indicated (TIU, trypsin inhibitor unit) just before induction of hypoxia, and were left until the end of the experimental period. Data were obtained from three different experiments using separate culture preparations, and are expressed as optical density units at 405 nm (^S.D.) (**P , 0.01: statistically significant difference from basal activity measured in controls constantly maintained under normoxia, Dunnett’s test for multiple comparisons).
showed very high purity of neuronal cultures grown in such chemically-defined conditions, without significant change in final neuron/glia ratio between hypoxic and control sister cultures. 4 Also, at the microscopic level, aspect and size of stained nuclei were quite homogeneous, including for dividing cells. Therefore, it may be postulated that, at least in neurons from the immature brain, caspase inhibitors might
challenge neuronal loss consecutive to severe hypoxia by promoting neurogenesis. Acknowledgements—The authors wish to thank the “Institut National de la Sante´ et de la Recherche Me´dicale” (INSERM), the “Fondation pour la Recherche Me´dicale” and the “Association pour la Recherche contre le Cancer” (ARC) for supporting this work.
REFERENCES
1. Alnemri E. S. (1997) Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J. Cell Biochem. 64, 33–42. 2. Beilharz E. J., Williams C. E., Dragunow M., Sirimanne E. S. and Gluckman P. D. (1995) Mechanisms of delayed cell death following hypoxic-ischemic injury in the immature rat: evidence for apoptosis during selective neuronal loss. Molec. Brain Res. 29, 1–14. 3. Bossenmeyer C., Chihab R., Muller S., Schroeder H. and Daval J. L. (1998) Hypoxia/reoxygenation induces apoptosis through biphasic induction of protein synthesis in central neurons. Brain Res. 787, 107–116. 4. Bossenmeyer-Pourie´ C., Chihab R., Schroeder H. and Daval J. L. (1999) Transient hypoxia may lead to neuronal proliferation in the developing mammalian brain: from apoptosis to cell cycle completion. Neuroscience 91, 221–231. 5. Carmichael J., de Graff W. G., Gazdar A. F., Minna J. D. and Mitchell J. B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936–942. 6. Chen J., Nagayama T., Jin K., Stetler R. A., Zhu R. L., Graham S. H. and Simon R. P. (1998) Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J. Neurosci. 18, 4914–4928. 7. Cheng Y., Deshmukh M., D’Costa A., Demaro J. A., Gidday J. M., Shah A., Sun Y., Jacquin M. F., Johnson E. M. Jr and Holtzman D. M. (1998) Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J. clin. Invest. 101, 1992–1999. 8. Chihab R., Ferry C., Koziel V., Monin P. and Daval J. L. (1998) Sequential activation of activator protein-1-related transcription factors and JNK protein kinases may contribute to apoptotic death induced by transient hypoxia in developing brain neurons. Molec. Brain Res. 63, 105–120. 9. Chinnaiyan A. M. and Dixit V. M. (1996) The cell-death machine. Curr. Biol. 6, 555–562. 10. Cohen G. M. (1997) Caspases; the executioners of apoptosis. Biochem. J. 326, 1–16. 11. Datta R., Kojima H., Banach D., Bump N. J., Talanian R. V., Alnemri E. S., Weichselbaum R. R., Wong W. W. and Kufe D. W. (1997) Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis. J. biol. Chem. 272, 1965–1969. 12. Edwards A. D. and Mehmet H. (1996) Apoptosis in perinatal hypoxic-ischaemic cerebral damage. Neuropath. appl. Neurobiol. 22, 494–498. 13. Enari M., Hug H. and Nagata S. (1995) Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature 375, 78–81. 14. Enari M., Sakahira H., Yokoyama H., Okawa K., Iwamatsu A. and Nagata S. (1998) A caspase-activated DNAse that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50. 15. Fink K., Zhu J., Namura S., Shimizu-Sasamata M., Endres M., Ma J., Dalkara T., Yuan J. and Moskowitz M. A. (1998) Prolonged therapeutic window for ischemic brain damage caused by delayed caspase activation. J. cereb. Blood Flow Metab. 18, 1071–1076. 16. Gray P. H., Tudehope D. I., Masel J. P., Burns Y. R., Mohay H. A., O’Callaghan M. J. and Williams G. M. (1993) Perinatal hypoxic-ischaemic brain injury: prediction of outcome. Devl med. Child Neurol. 35, 965–973. 17. Gschwind M. and Huber G. (1997) Detection of apoptotic or necrotic death in neuronal cells by morphological, biochemical, and molecular analysis. In Neuromethods, Apoptosis Techniques and Protocols (ed. Poirier J.), Vol. 29, pp. 13–31. Humana, Totowa. 18. Harvey N. L., Butt A. J. and Kumar S. (1997) Functional activation of Nedd2/ICH-1 (caspase-2) is an early process in apoptosis. J. biol. Chem. 272, 13,134–13,139. 19. Himi T., Ishizaki Y. and Murota S. (1998) A caspase inhibitor blocks ischaemia-induced delayed neuronal death in the gerbil. Eur. J. Neurosci. 10, 777–781. 20. Krohne A. J., Preis E. and Prehn J. H. M. (1998) Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector. J. Neurosci. 18, 8186–8197. 21. Kumar S. (1995) ICE-like proteases in apoptosis. Trends biochem. Sci. 20, 198–202. 22. Kumar S., Kinoshita M., Noda M., Copeland N. G. and Jenkins N. A. (1994) Induction of apoptosis by mouse Nedd2 gene which encodes a protein similar to the product of Caenorhabditis elegans cell death ge ced-3 and mammalian IL-1b-converting enzyme. Genes Dev. 8, 1613–1626. 23. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Caspase-1 and inhibitors in hypoxic neurons
1165
24. Lin Y. Z., Yao S. Y. and Hawiger J. (1996) Role of the nuclear localization sequence in fibroblast growth factor-1-stimulated mitogenic pathways. J. biol. Chem. 271, 5305–5308. 25. Livingston D. J. (1997) In vitro and in vivo studies of ICE inhibitors. J. Cell Biochem. 64, 19–26. 26. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275. 27. MacManus J. P., Buchan A. M., Hill I. E., Rasquinha I. and Preston E. (1993) Global ischaemia can cause DNA fragmentation indicative of apoptosis in rat brain. Neurosci. Lett. 164, 89–92. 28. Margolin N., Raybuck S. A., Wilson K. P., Chen W., Fox T., Gu Y. and Livingston D. J. (1997) Substrate and inhibitor specificity of interleukin-1-bconverting enzyme and related caspases. J. biol. Chem. 272, 7223–7228. 29. Meikrantz W. and Schlegel R. (1995) Apoptosis and the cell cycle. J. Cell Biochem. 58, 160–174. 30. Namura S., Zhu J., Fink K., Endres M., Srinivasan M., Tomaselli K. J., Yuan J. and Moskowitz M. A. (1998) Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J. Neurosci. 18, 3659–3668. 31. Nath R., Probert A. Jr, McGinnis K. M. and Wang K. K. W. (1998) Evidence for activation of caspase-3-like protease in excitotoxin- and hypoxia/ hypoglycemia-injured neurons. J. Neurochem. 71, 186–195. 32. Oppenheim R. W. (1991) Cell death during development of the nervous system. A. Rev. Neurosci. 14, 453–501. 33. Park D. S., Morris E. J., Greene L. A. and Geller H. M. (1997) G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecininduced neuronal apoptosis. J. Neurosci. 17, 1256–1270. 34. Reiter L. A. (1994) Peptidic p-nitroanilide substrates of interleukin-1 beta-converting enzyme. Int. J. Pept. Protein Res. 43, 87–96. 35. Rosenbaum D. M., Michaelson M., Batter D. K., Doshi P. and Kessler J. A. (1994) Evidence of hypoxia-induced, programmed cell death of cultured neurons. Ann. Neurol. 36, 864–870. 36. Sher P. K. (1990) Chronic hypoxia in neuronal cell culture: metabolic consequences. Brain Dev. 12, 293–300. ¨ rd T., Keane R. W., Bredesen D. E. and Kayalar C. (1996) Bcl-2 expression in neural cells blocks activation of 37. Srinivasan A., Foster L. M., Testa M. P., O ICE/CED-3 family proteases during apoptosis. J. Neurosci. 16, 5654–5660. 38. Tamatani M., Ogawa S. and Tohyama M. (1998) Roles of Bcl-2 and caspases in hypoxia-induced neuronal cell death: a possible neuroprotective mechanism of peptide growth factors. Molec. Brain Res. 58, 27–39. 39. Thornberry N. A. (1994) Interleukin-1 beta converting enzyme. Meth. Enzymol. 244, 615–631. 40. Thornberry N. A., Bull H. G., Calaycay J. R., Chapman K. T., Howard A. D., Kostura M. J., Miller D. K., Molineaux S. M., Weidner J. R., Aunins J., Elliston K. O., Ayala J. M., Casano F. J., Chin J., Ding G. J. F., Egger L. A., Gaffney E. P., Limjuco G., Palyha O. C., Raju S. M., Rolando A. M., Salley J. P., Yamin T. T., Lee T. D., Shively J. E., MacCross M., Mumford R. A., Schmidt J. A. and Tocci M. J. (1992) A novel heterodimeric cysteine protease is required for interleukin-1b processing in monocytes. Nature 356, 768–774. 41. Woo M., Hakem R., Soengas M. S., Duncan G. S., Shahinian A., Ka¨gi D., Hakem A., McCurrach M., Khoo W., Kaufman S. A., Senaldi G., Howard T., Lowe S. W. and Mak T. W. (1998) Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev. 12, 806–819. 42. Yuan J., Shaham S., Ledoux S., Ellis H. M. and Horvitz H. R. (1993) The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1b-converting enzyme. Cell 75, 641–652. (Accepted 4 October 1999)