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Involvement of cystatin C in oxidative stress-induced apoptosis of cultured rat CNS neurons
Brain Research 873 (2000) 252–262 www.elsevier.com / locate / bres
Research report
Involvement of cystatin C in oxidative stress-induced apoptosis of cultured rat CNS neurons 1
Chika Nishio, Kiyomi Yoshida , Keiji Nishiyama, Hiroshi Hatanaka*, Masashi Yamada Institute for Protein Research, Osaka University, 3 -2 Yamadaoka, Suita, Osaka 565 -0871, Japan Accepted 23 May 2000
Abstract Oxidative stress is involved in neuronal degeneration in cerebrovascular injury, neuropathology and aging. When rat CNS neurons were cultured in a high (50%) oxygen atmosphere, the neurons died. This high oxygen-induced cell death showed features of apoptotic cell death, characterized by DNA fragmentation, and was blocked by inhibitor of protein synthesis. We found that cystatin C and HuC mRNA, the products of which are an inhibitor of cysteine proteases and an RNA binding protein, respectively, were up-regulated in neurons cultured in the high oxygen atmosphere. In the present study, we focused on cystatin C. Cystatin C protein levels were also increased in neurons cultured in the high oxygen atmosphere. In situ hybridization with an RNA probe for rat cystatin C and immunocytochemistry with anti-human cystatin C antibody showed that microtubule-associated protein 2 (MAP2)-positive neurons expressed cystatin C mRNA and protein, respectively, in the high oxygen atmosphere. These results indicated that oxidative stress stimulates an increase in cystatin C expression in cultured neurons, and that cystatin C might have important roles in regulation of apoptosis elicited by oxidative stress. 2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Neuronal death Keywords: Cathepsin; PCR subtraction; Death; Survival; Reactive oxygen
1. Introduction Apoptosis is the best studied type of cell death, and has been shown to be mediated by an intrinsic death program [16]. Apoptosis is blocked by protein or RNA synthesis inhibitors [27,32], and is characterized by chromatin condensation and fragmentation of chromosomal DNA into multimers of about 180 bp [46]. This form of cell death
Abbreviations: DF medium, 1:1 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media; DIG, digoxygenin; GFAP, glial fibrillary acidic protein; FITC, fluorescein isothiocyanate; MAP2, microtubuleassociated protein 2; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling *Corresponding author. Tel.: 181-6-6879-8624; fax: 181-6-68798626. E-mail address: [email protected] (H. Hatanaka). 1 Present address: Department of Geriatric Research, The National Institute for Longevity Science, 36-3 Gengo, Morioka-Cho, Ohbu-Shi, Aichi, 474-8522, Japan.
occurs at specific stages during the development of embryonic neurons including those of the sympathetic ganglion, retina, spinal cord, hippocampus and cerebral cortex [11,13,33]. In addition, apoptosis may be related to the neuronal degeneration that occurs during Alzheimer’s and Parkinson’s diseases, and in Down’s syndrome or with aging [8,14,22,26]. Therefore, it is important to study the mechanisms by which apoptosis of neurons is induced. Neuronal death induced by oxidative stress resulting in excessive production of reactive oxygen species, is related to a variety of neuronal degenerative disorders caused by cerebrovascular injury, neuropathology and aging [2,9,39]. Oxidative damage is closely associated with Parkinson’s and Alzheimer’s diseases, familial amyotrophic lateral sclerosis (ALS) and Down’s syndrome [5,8,15,36,38]. In addition, reactive oxygen species have been reported to be related to naturally occurring cell death [19]. To examine the neuronal death caused by oxidative stress in vitro, we cultured CNS neurons under a high oxygen atmosphere (50% O 2 ) [17,23,47]. As we reported
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previously, embryonic rat hippocampal neurons cultured under a 50% oxygen atmosphere died by apoptosis. The hippocampal neurons cultured under a 50% oxygen atmosphere showed nucleosomal DNA fragmentation, nuclear condensation and cytoplasmic compaction [18]. In addition, the oxygen-induced death of cultured hippocampal neurons was blocked by RNA or protein synthesis inhibitors. In this study, we showed that the death of rat brain neurons cultured under a 50% oxygen atmosphere displayed apoptotic features. We focused on the observation that the oxygen-induced death of these cultured neurons was prevented by RNA or protein synthesis inhibitors. This suggested that the oxygen toxicity activates an intracellular death cascade and stimulates up-regulation of some species of mRNA and proteins involved in regulation of neuronal apoptosis. To examine what kinds of RNA were up-regulated by the high oxygen stimulus in the cultured neurons of rat brains, we performed PCR subtraction screening. Cystatin C showed increased expression at both mRNA and protein levels in the neurons cultured in the high oxygen atmosphere. Cystatin C is an inhibitor of cysteine proteases including cathepsin B, H and L [1,3,4,6], and is secreted from various types of cells [12,41]. These results indicated that cystatin C is related to processes of apoptosis stimulated by oxidative stress through regulation of cysteine protease activities in cultured neurons of the rat brain.
2. Materials and methods
2.1. Cell culture Primary cultures of dissociated neurons of rat total brains without the cerebellum were prepared using embryonic day E20–21 rats (Wistar ST, both sexes) as described previously [17]. Briefly, the cells were cultured in a medium consisting of 5% precolostrum newborn calf serum (PNCS, Mitsubishi Kagaku), 5% heat-inactivated (568C, 30 min) horse serum (HS, Gibco) and 90% (v / v) DF medium consisting of a 1:1 mixture of Dulbecco’s modified Eagle’s (DME, Gibco) and Ham’s F12 media (Gibco) containing 15 mM HEPES buffer, pH 7.4, 30 nM selenium and 1.9 mg / ml sodium bicarbonate at a final cell density of 5310 5 cells / cm 2 on a polyethyleneimine-coated culture plates. After incubation for 2 h in a CO 2 incubator under a 20% (v / v) oxygen atmosphere, the medium was changed to DF medium containing 5 mg / ml human transferrin, 5 mg / ml bovine insulin (Colab. Res.), 20 nM progesterone and 30 nM selenium. One day after the medium change, the culture plates were transferred to a 20 or 50% (v / v) O 2 and a constant 5% (v / v) CO 2 atmosphere in a N 2 –O 2 –CO 2 gas incubator (Tabai BNP-110M). The oxygen tension in the 50% O 2 culture medium increased rapidly and reached a plateau level of about 300 mmHg
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within 2–3 h. The tension value of CO 2 and the pH value were stable during the entire culture period in both the 20 and 50% O 2 cultures, as described previously [17]. Cycloheximide (Sigma) was added just before the culture plates were transferred to the O 2 -regulated chambers. For the preparation of RNA from the neurons, the neurons were seeded in polyethyleneimine-coated Falcon 6-well plates. For immunostaining with anti-MAP2 antiserum or anti-cystatin C polyclonal antibody, TUNEL staining and the preparation of lysates from the neurons, polyethyleneimine-coated Nunc multidish 4-well plates were used. For the in situ hybridization and the immunofluorescence analysis, polyethyleneimine-coated Lab-Tek 8-chamber slide plates were used.
2.2. Antibodies, factors and reagents The anti-microtubule-associated protein 2 (MAP2) antiserum was a gift from Dr. H. Murofushi (The University of Tokyo). The anti-human cystatin C polyclonal antibody was purchased from DAKO, the anti-MAP2 monoclonal antibody was from Boeheringer Mannheim, and the antiglial fibrillary acidic protein (GFAP) monoclonal antibody was from Amersham.
2.3. Immunocytochemistry The cells were fixed in 4% paraformaldehyde at room temperature for 20 min. For immunostaining with antiMAP2 antiserum, the anti-MAP2 monoclonal antibody or the anti-cystatin C polyclonal antibody, the cells were blocked with phosphate-buffered saline (PBS) containing 5% (v / v) normal goat serum and 0.1% (v / v) Triton X-100 for 2 h at room temperature, then incubated at 48C overnight with the anti-MAP2 antiserum diluted to 1:5000, with the anti-MAP2 monoclonal antibody diluted to 1:1000, or the anti-cystatin C polyclonal antibody diluted to 1:10,000, with the blocking buffer. For immunostaining with the anti-GFAP antibody, the cells were fixed in 100% methanol, and were blocked with PBS containing 5% (v / v) normal goat serum and 0.3% (v / v) Triton X-100 for 2 h at room temperature, then incubated with the anti-GFAP monoclonal antibody diluted to 1:10,000 with the blocking buffer, overnight at 48C. Staining was performed using a Vectastain ABC kit (Vector Lab.) and 0.02% (w / v) 3,39diaminobenzidine 4-HCl and 0.1% (w / v) (NH 4 ) 2 Ni(SO 4 ) 2 dissolved in 0.05 M Tris–HCl buffer, pH 7.6, containing 0.01% (v / v) H 2 O 2 . For immunofluorescence analysis, rhodamine-conjugated anti-rabbit or mouse antibody and FITC-conjugated anti-rabbit antibody were used for the detection of MAP2 and cystatin C, respectively. The cells were visualized using fluorescence microscopy (Zeiss). The number of immunoreactive cells was determined by examining microphotographs. The number of immunoreactive cells in more than four randomly selected micro-
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scopic fields per well was counted, and that in four wells was averaged.
2.4. TUNEL staining The neurons were cultured under 20 or 50% oxygen atmospheres in the absence or presence of 1 mM cycloheximide for 48 h. The neurons were fixed in 4% paraformaldehyde at room temperature for 20 min, then washed 3 times with PBS. The fixed cells were treated on ice for 2 min with a solution containing 0.1% (w / v) sodium citrate and 0.1% Triton X-100, followed by washing with PBS. TUNEL staining was performed using an In Situ Cell Death Detection kit, POD (Boehringer Mannheim). Staining was performed by incubation with 0.02% (w / v) 3,39diaminobenzidine 4-HCl and 0.1% (w / v) (NH 4 ) 2 Ni(SO 4 ) 2 dissolved in 0.05 M Tris–HCl buffer, pH 7.6, containing 0.01% (v / v) H 2 O 2 .
2.5. Subtractive PCR screening Total RNA was prepared from the neurons cultured under a 20% oxygen atmosphere and from those cultured for 3 h under a 50% oxygen atmosphere, using TRIzol reagent (Gibco BRL). Then, mRNA was purified from the total RNA with Oligotex-dT30 (Super) (Takara). Subtractive PCR screening was performed using a PCR-Select cDNA Subtraction Kit (Clontech) according to manufacturer’s protocol. Briefly, double-stranded cDNA was synthesized from the mRNA, then digested with Rsa I. To subtract mRNA prepared from the cells cultured under a 20% oxygen atmosphere from that of those cultured under 50% oxygen, cDNA from the latter cultures was ligated with one of two adaptors (adaptor 1 or 2). The cDNA from the cells cultured under 20% oxygen was not ligated with the adaptors. cDNAs ligated with adaptor 1 or 2 were separately hybridized with excess amounts of the cDNA from the cells cultured under the 20% oxygen atmosphere. The two samples from the hybridization were mixed together, then fresh denatured cDNA from the 20% oxygen culture was added. The mixture was subjected to second hybridization, then to PCR using the sequences of the adaptors as primers, followed by nested PCR. The PCR products were cloned in the pCR 2.1 vector using an Original TA Cloning Kit (Invitrogen). The clones were sequenced using a sequencer (Li-Cor, model 4000L).
2.6. Nuclease protection assay The neurons were not cultured or were cultured in a 50% oxygen atmosphere for 3 or 5 h, then total RNA was prepared from the neurons as described above. Radiolabeled RNA probes were synthesized from the pCR 2.1 clones in the presence of [a- 32 P] UTP (Amersham) using MAXIscript kits (Ambion) in according to the manufacture’s protocol. RNA protection assay was performed with
a High-Speed Hybridization Ribonuclease Protection Assay Kit (Ambion) in accordance with the instruction manual. The protected bands were visualized, and the intensities of bands were quantified using an image analyzer (BAS 2000, Fuji Film).
2.7. Immunoblotting The neurons were not cultured or were cultured for 24 h under a 20% oxygen atmosphere or for 6, 12, 18, 24 or 36 h under a 50% oxygen atmosphere. Then, the neurons were washed once with ice-cold PBS, and lysed in a buffer containing 1% SDS, 5 mM EDTA, 10 mM Tris–HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride and 5 mg / ml aprotinin. Lysates were boiled for 5 min at 1008C, then centrifuged at 100,000 3g for 30 min. The protein concentration of the clarified lysate was determined by the BCA protein assay. The lysates (10 mg) were resolved by electrophoresis on 10–30% gradient SDS–polyacrylamide gels according to the method of Laemmli [24]. Proteins were transferred onto polyvinylidene difluoride membranes (Millipore) in 0.1 M Tris base, 0.192 M glycine and 20% (v / v) methanol using a semi-dry electrophoretic transfer system. The membranes were blocked with 0.1% (w / v) Tween 20 / TBS (T-TBS) containing 10% (w / v) nonfat dried milk at room temperature for at least 1 h, then incubated with 1.4 mg / ml anti-human cystatin C antibody in T-TBS containing 10% (w / v) nonfat dried milk at room temperature for 1 h. After three washes with T-TBS, the membranes were incubated with peroxidase-coupled goat anti-rabbit IgG secondary antibody (Jackson ImmunoRes. Lab. Inc.) diluted 1:1000 with T-TBS at room temperature for 2 h. The membranes were then washed four times with T-TBS, and visualized using the ECL chemiluminescence system (Amersham).
2.8. In situ hybridization Digoxygenin (DIG)-labeled anti-sense or sense RNA probe of cystatin C was synthesized from pCR 2.1-cystatin C plasmid using a DIG RNA Labeling kit (Boehringer Mannheim). The cells were fixed in 4% paraformaldehyde at room temperature for 20 min, then washed twice with PBS. The cells were washed with H 2 O, incubated with a solution consisting of 0.25% (v / v) acetic acid and 0.1 M triethanolamine for 10 min, then washed with PBS. The cells were dehydrated by treatment with 70% (v / v), 95% (v / v) and 100% (v / v) ethanol each for 2 min, with chloroform for 10 min, with 100% (v / v) and 95% (v / v) ethanol each for 2 min, and were then air-dried. The cells were pre-hybridized at 508C for 1 h with a hybridization solution consisting of 50% (v / v) formamide, 20 mM Tris– HCl (pH 8.0), 5 mM EDTA (pH 8.0), 0.3 M NaCl, 10 mM sodium phosphate buffer (pH8.0), 13Denhardt’s solution, 10% (w / v) dextran sulfate, 0.2% (v / v) sarkosyl, 500 mg / ml yeast tRNA, and 200 mg / ml salmon sperm DNA. The
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cells were hybridized with DIG-labeled anti-sense or sense RNA probe in the hybridization solution at 508C overnight. The cells were washed at 608C for 20 min with 53SSC, at 608C for 30 min with 23SSC containing 50% (v / v) formamide and 0.1 M dithiothreitol, then at 378C for 30 min with RNase buffer consisting of 10 mM Tris–HCl (pH8.0), 1 mM EDTA and 0.5 M NaCl. The cells were incubated at 378C for 30 min with 1 mg / ml RNase A in the RNase buffer, washed at 378C for 30 min with the RNase buffer, then washed at 608C for 30 min with 23SSC containing 50% (v / v) formamide and 0.1 M dithiothreitol. After washing with 0.1 M Tris–HCl (pH8.0) containing 0.15 M NaCl, the cells were stained using alkaline phosphatase-conjugated anti-DIG Fab fragment (Boehringer Mannheim), followed by reaction with xphosphate / 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT).
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3. Results
effect of the protein synthesis inhibition on the oxygeninduced death of cultured rat CNS neurons. Cycloheximide was added at various time points to the cultures under the high oxygen atmosphere. The cultured cells were immunostained 48 h after exposure to the high oxygen atmosphere using anti-MAP2 polyclonal antiserum, then the number of MAP2-positive neurons was counted (Fig. 2). Cycloheximide added to the culture at later time points showed a lesser protective effect against the death induced by oxygen toxicity. When added just before, 2 h or 3 h after transfer to a 50% oxygen atmosphere, cycloheximide effectively prevented the oxygen-induced cell death. On the other hand, almost all of the neurons exposed to cycloheximide 5 h after transfer to a 50% oxygen atmosphere died. In addition, we obtained similar result in MTT assay as that in the counting the number of MAP2-positive cells (data not shown). These results indicated that proteins involved in induction of the death of cultured neurons were expressed between 3 and 5 h after exposure to the high oxygen atmosphere.
3.1. Apoptotic cell death induced by oxygen toxicity in cultured rat CNS neurons
3.2. Subtractive PCR screening to identify genes upregulated by oxidative stress
We have reported that basal forebrain, hippocampal and cerebral cortical neurons cultured under a high oxygen atmosphere show apoptotic cell death characterized by nuclear DNA fragmentation, which can be prevented by protein or RNA synthesis inhibitors [18,47]. In the present study, we used cultured neurons of rat whole brains without cerebellum and screened for genes involved in the high oxygen-induced death of CNS neurons. To determine whether the oxygen-induced death of the cultured rat CNS neurons was also apoptotic, we examined the effects of a protein synthesis inhibitor. Neurons from the embryonic rat (embryonic day 20–21) whole brain without the cerebellum were cultured in 20% or 50% oxygen in the absence or presence of cycloheximide for 48 h, then immunostained with anti-microtubule-associated protein 2 (MAP2) antiserum. Cycloheximide markedly prevented neuronal cell death caused by the high oxygen stimulus (Fig. 1A–C). Apoptotic cell death is characterized by DNA fragmentation [46]. To examine whether high levels of oxygen induce DNA fragmentation, the rat CNS neurons cultured under an atmosphere of 20% or 50% oxygen for 48 h were stained by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) method. Most of the cells cultured under a 50% oxygen atmosphere were TUNEL-positive, although those cultured under a 20% oxygen atmosphere or in the presence of cycloheximide under a 50% oxygen atmosphere were scarcely TUNELpositive (Fig. 1D–F). These results indicated that the high level of oxygen induces apoptotic cell death in the cultured rat CNS neurons. In addition, we examined the commitment time of the
Up-regulation of proteins by high oxygen stimulus might result in the death of the cultured rat brain neurons under the high oxygen atmosphere. To examine what kinds of proteins show oxidative stress-induced increases in their expression, we performed subtractive PCR screening to isolate up-regulated RNA in the neurons cultured under the high oxygen atmosphere. We isolated 85 clones, which were then subjected to sequencing analysis. Of these clones, 45 were unknown, and 40 has been identified previously. To confirm the increases in the levels of mRNA expression of these 85 clones, all clones were analyzed by RNA protection assay using total RNA from the neurons cultured under a 20% oxygen atmosphere and from those cultured for 3 h under a 50% oxygen atmosphere. In result, only two of the clones showed significant increases in the mRNA levels in the neurons cultured under the high oxygen atmosphere as compared with those exposed to normal oxygen level. One ([ 168; 337 bp) of the positive clones had the same sequence as rat cystatin C [12], except for one nucleotide difference (data not shown). The other one was HuC, an RNA binding protein (data not shown) [28]. As shown in Fig. 3A, cystatin C (clone [168) mRNA level increased in the neurons cultured for 3 h under the high oxygen atmosphere. In three independent cultures, the amounts of cystatin C mRNA in the neurons exposed to the high oxygen atmosphere for 3 h were 1.84-, 1.33- and 1.21-times larger than those in the neurons not exposed to the high oxygen atmosphere. In addition, HuC mRNA showed about 1.5-fold increase in the neurons cultured for 3 h under the high oxygen atmosphere (data not shown). In the present study, we focused on cystatin C, because some reports suggest that cathepsins, activities of
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Fig. 1. Inhibitory effect of cycloheximide on apoptosis of cultured CNS neurons under a high-oxygen atmosphere. (A–C) The neurons were cultured under 20 (A) or 50% (B, C) oxygen atmospheres in the absence (A, B) or presence of 1 mM cycloheximide (C) for 48 h. The neurons were immunostained with anti-MAP2 antiserum. Bar represents 100 mm. (D–E) The neurons were cultured in 20 (D) or 50% (E, F) oxygen atmospheres in the absence (D, E) or presence of 1 mM cycloheximide (F) for 24 h. The neurons were stained by the TUNEL method. Bar represents 100 mm.
which are inhibited by cystatin C, are involved in regulation of neuronal cell death [31,37].
3.3. Up-regulation of cystatin C protein in rat CNS neurons cultured under a high oxygen atmosphere To confirm the increased expression of cystatin C in response to oxidative stress, we examined the changes in amounts of cystatin C protein in rat CNS neurons cultured for various periods under a 20% or 50% oxygen atmosphere by Western blotting analysis using anti-human cystatin C antibody. The neurons cultured under a 50% oxygen atmosphere showed gradual increases in the amounts of cystatin C protein until at least 36 h, while those cultured under 20% oxygen showed only small
increases (Fig. 3B). These results suggested that oxidative stress stimulates cystatin C expression in cultured rat CNS neurons.
3.4. Immunocytochemistry with anti-cystatin C antibody in cultured rat CNS neurons To refine the expression of cystatin C in the rat CNS neurons cultured under a 50% atmosphere, the neurons cultured for various periods after exposure to high oxygen were immunostained with anti-human cystatin C antibody (Fig. 4). The cystatin C-positive cells gradually increased, and most of the cells were cystatin C-positive at 48 h after exposure to the high oxygen atmosphere. On the other hand, we observed very few cystatin C-positive cells in the
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signals on immunostaining with both the anti-MAP2 monoclonal and the anti-cystatin C polyclonal antibodies (Fig. 5D–F). These results indicated that the cultured MAP2-positive neurons express cystatin C mRNA and protein in response to high oxygen stimulus.
4. Discussion
Fig. 2. Commitment time of survival-promoting effect of cycloheximide, a protein synthesis inhibitor, on oxygen-induced death of cultured rat CNS neurons. The medium was changed 2 h after the neurons were seeded. 1 mM cycloheximide was added to the culture just before (0 h), and 2, 3, 4, 5 and 6 h after the cultures were transferred to 20% and 50% oxygen atmospheres. The cells were cultured for 48 h after the transfer, and were then immunostained with anti-MAP2 antiserum. Then, the number of MAP2-positive neurons was counted. The values of the cultured neurons in a 50% oxygen atmosphere are expressed as the percentage of those measured in the corresponding culture in a 20% oxygen atmosphere. The values represent the means6S.D. of four individual cultures.
cultures incubated for 24 h under a 20% oxygen atmosphere. In addition, most of the cystatin C-positive cells seemed to be neurons. Because, the cells were cultured in serum-free medium. In addition, when the cultured cells were immunostained using the anti-glial fibrillary acidic protein (GFAP) monoclonal antibody, few GFAP-positive astroglial cells were observed (less than 1% of the corresponding MAP2-positive neurons) (data not shown). These results indicated that the number of neurons that show increased expression of cystatin C gets to increase with incubation under the high oxygen atmosphere.
3.5. MAP2 -positive neurons expressed cystatin C mRNA and protein in high oxygen atmosphere To confirm that the cells expressing cystatin C under the high oxygen atmosphere were neurons, we examined whether MAP2-positive neurons were positive by in situ hybridization analysis using an RNA probe for cystatin C mRNA and immunostaining using anti-cystatin C antibodies. A number of cells cultured for 6 h under a 50% oxygen atmosphere showed positive signals both on immunostaining with anti-MAP2 antiserum and on in situ hybridization analysis with the cystatin C RNA probe (Fig. 5A–C). In addition, some of the cells cultured for 16 h under the high oxygen atmosphere displayed positive
We showed that expression of cystatin C, an inhibitor of cysteine proteases, is up-regulated in response to high oxygen stimulus. Cystatin C is an alkaline protein with a low molecular mass (13.5 kDa), and is a member of Family 2 of the cystatin superfamily [1,3,4,6]. Cystatin C is widely expressed in almost all tissues and is secreted into various biological fluids including urine, blood, seminal fluid, saliva and cerebrospinal fluid [12,41]. In the CNS, large amounts of cystatin C mRNA are present in brain tissue and choroid plexus [12,42]. On immunohistochemical analysis, cystatin C is detected in astrocytes, choroid plexus cells and a small number of neurons [48]. In culture, astrocytes and microglial cells express and secrete cystatin C [44,49]. In transient forebrain ischemia, it has been reported that CA1 pyramidal cells and reactive astrocytes of rat hippocampus show delayed expression of cystatin C, and that immunoreactivity with anti-cystatin C is localized in morphologically degenerative neurons [34]. In addition, immunohistochemical studies with anticystatin C antibody in human brain indicate that intensely immunoreactive neurons are abundant in the cerebral cortex in some aged human subjects and in some Alzheimer’s disease patients [48]. These studies suggested that cystatin C expression may be related to neuronal degeneration in brain diseases. In our study, cultured rat CNS neurons scarcely expressed cystatin C under normal conditions. We found that cystatin C expression was increased in the cultured CNS neurons by exposure to a high oxygen atmosphere, which induced neuronal cell death. Oxidative stress is involved in neuronal degeneration caused by cerebrovascular injury, aging and Alzheimer’s disease [2,5,9,15,39]. Therefore, cystatin C may have important roles in neuronal death caused by oxidative stress in the CNS. Oxidative stress-stimulated up-regulation of cystatin C expression may be mediated by reactive oxygen species including superoxide anion, hydrogen peroxide, hydroxyradical and peroxynitrite, although it remains unclear how reactive oxygen species triggers the cystatin C expression. We cannot exclude the possibility that the oxidative stress stimulates the increase in cystatin C expression through undetermined modifications by oxygen rather than through reactive oxygen species. In addition, we observed that the high oxygen-stimulated increase in expression level of cystatin C mRNA was the highest at 3 h after the exposure to high oxygen, although that of cystatin C protein got to be observed between 6 and 12 h. The time lag of upregulation between mRNA and protein of cystatin C
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Fig. 3. Oxidative stress stimulates increases in expression of cystatin C mRNA and protein in cultured CNS neurons. (A) The neurons were not cultured (0 h) or were cultured in a 50% oxygen atmosphere for 3 or 5 h, then total RNA was prepared. The total RNA was subjected to RNA protection assay using an RNA probe for cystatin C or actin. Intensities of the protected bands were quantified using an imaging analyzer. (B) The neurons were not cultured (0 h) or were cultured for 24 h under a 20% oxygen atmosphere or for 6, 12, 18, 24 or 36 h under a 50% oxygen atmosphere, and were then lysed. The lysates were subjected to Western blotting analysis using anti-cystatin C antibody. Molecular weights are shown on the left. The expression levels of cystatin C protein were quantified by measuring densities of the protein bands.
indicate that there are post-transcriptional regulation and accumulation mechanisms (for examples, increased stability and attenuated exocytosis of cystatin C protein). The
expression level of cystatin C mRNA may increase again after it peaked at 3 h after the exposure to the high oxygen atmosphere.
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Fig. 4. Immunocytochemical staining with anti-cystatin C antibody of cultured CNS neurons under a high oxygen atmosphere. The neurons were not cultured (A) or were cultured for 6 (B), 12 (C), 24 (D) or 48 h (E) under a 50% oxygen atmosphere, or for 24 h (F) under a 20% oxygen atmosphere. The neurons were immunostained with anti-cystatin C antibody. Bar represents 100 mm.
In transient ischemia, delayed death of CA1 pyramidal neurons shows apoptotic features. In this case, immunoreactivities of cathepsin B, H and L, activities of which are inhibited by cystatin C, are increased in the CA1 pyramidal neurons [31]. In addition, in rat hepatocyte and hepatoma cell lines, cathepsin B contributes to bile salt-induced apoptosis [37]. These cathepsins are mainly present in lysosomes, however cathepsins may be released from cells. It is possible that the cathepsins were leaked from dead neurons. Alternatively, the cathepsins may have been released from cells through exocytosis. Cathepsin B is detected in the secretory vesicles from atrial and juxtaglomerular endocrine cells [30,45], and cathepsin H is found in secretory granules from corticotrophic and melanot-
rophic cells of the pancreas [43]. Numerous invasive carcinomas and ras-transformed fibroblasts have been reported to show increased synthesis and secretion of cathepsins [10,40]. Detection of cathepsin L in trophoblastconditioned medium indicates the secretion of cathepsin L from the mouse trophoblast [21]. In addition, cathepsin B and H are able to degrade components of extracellular matrix including laminin, collagen IV and fibronectin [7,20,25,29]. Secreted cystatin C may block the activities of cathepsin released from cells, to protect cell-surface proteins. However, we cannot exclude the possibility that cystatin C may act on intracellular cysteine proteases including cathepsin, to regulate the neuronal cell death. A study using mice lacking cystatin B, a member of the
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Fig. 5. MAP2-positive neurons expressed cystatin C mRNA and protein in CNS neurons cultured under a high oxygen atmosphere. (A–C) The neurons were cultured for 6 h under a 50% oxygen atmosphere, and were then fixed. The fixed neurons were subjected to in situ hybridization with an anti-sense RNA probe for cystatin C (A), and were then immunostained with anti-MAP2 antiserum (B). (C) shows the superimposed images of (A) and (B). The images shown are of the same field. Bar represents 50 mm. (D–F) The neurons were cultured for 16 h under a 50% oxygen atmosphere, and were then fixed. The fixed neurons were immunostained with anti-cystatin C polyclonal antibody (D) and with anti-MAP2 monoclonal antibody (E). (F) shows the superimposed images of (D) and (E). The images shown are of the same field. Bar represents 50 mm.
cystatin superfamily, indicated that cystatin B has a role in preventing apoptosis of cerebellar granule neurons [35]. Therefore, we suggest that cystatins are involved in regulation of apoptosis in neurons.
Acknowledgements We thank Dr. H. Murofushi (The University of Tokyo) for the kind gift of anti-MAP2 antiserum, and Dr. Y.
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Uchiyama and Dr. S. Kametaka (Osaka University) for valuable discussion and kind support in this study. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, by a Research Grant for Neurons and Mental Disorders from the Ministry of Health and Welfare of Japan.
References [1] T. Akopyan, Protein inhibitors of proteinases from brain, Neurochem.Res. 16 (1991) 513–517. [2] B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7915–7922. [3] A.J. Barrett, H. Kirschke, Cathepsin B, cathepsin H, and cathepsin L, Methods Enzymol. 80 (Pt C) (1981) 535–561. [4] A.J. Barrett, The cystatins: a diverse superfamily of cysteine peptidase inhibitors, Biomed. Biochim. Acta 45 (1986) 1363–1374. [5] C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid beta protein toxicity, Cell 77 (1994) 817–827. [6] L.A. Bobek, M.J. Levine, Cystatins — inhibitors of cysteine proteinases, Crit. Rev. Oral Biol. Med. 3 (1992) 307–332, [published erratum appears in Crit. Rev. Oral Biol. Med. 1993;4(2):251]. [7] M.C. Burleigh, A.J. Barrett, G.S. Lazarus, Cathepsin B1. A lysosomal enzyme that degrades native collagen, Biochem. J. 137 (1974) 387–398. [8] J. Busciglio, B.A. Yankner, Apoptosis and increased generation of reactive oxygen species in Down’s syndrome neurons in vitro, Nature 378 (1995) 776–779. [9] W. Cao, J.M. Carney, A. Duchon, R.A. Floyd, M. Chevion, Oxygen free radical involvement in ischemia and reperfusion injury to brain, Neurosci. Lett. 88 (1988) 233–238. [10] A.F. Chambers, R. Colella, D.T. Denhardt, S.M. Wilson, Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells, Mol. Carcinog. 5 (1992) 238–245. [11] P.G. Clarke, Developmental cell death: morphological diversity and multiple mechanisms, Anat. Embryol. Berl. 181 (1990) 195–213. [12] T. Cole, P.W. Dickson, F. Esnard, S. Averill, G.P. Risbridger, F. Gauthier, G. Schreiber, The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for beta 2-microglobulin in rat brain, Eur. J. Biochem. 186 (1989) 35–42. [13] W.M. Cowan, J.W. Fawcett, D.D. O9Leary, B.B. Stanfield, Regressive events in neurogenesis, Science 225 (1984) 1258–1265. [14] B. Dipasquale, A.M. Marini, R.J. Youle, Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons, Biochem. Biophys. Res. Commun. 181 (1991) 1442–1448. [15] T. Dyrks, E. Dyrks, T. Hartmann, C. Masters, K. Beyreuther, Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation, J. Biol. Chem. 267 (1992) 18210–18217. [16] R.E. Ellis, J.Y. Yuan, H.R. Horvitz, Mechanisms and functions of cell death, Annu. Rev. Cell Biol. 7 (1991) 663–698. [17] Y. Enokido, Y. Akaneya, M. Niinobe, K. Mikoshiba, H. Hatanaka, Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture, Brain Res. 599 (1992) 261–271. [18] Y. Enokido, H. Hatanaka, Apoptotic cell death occurs in hippocampal neurons cultured in a high oxygen atmosphere, Neuroscience 57 (1993) 965–972. [19] L.J. Greenlund, T.L. Deckwerth, J. Johnson-EM, Superoxide dis-
[20]
[21]
[22]
[23]
[24] [25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33] [34]
[35]
[36]
[37]
261
mutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death, Neuron 14 (1995) 303–315. N. Guinec, V. Dalet-Fumeron, M. Pagano, ‘In vitro’ study of basement membrane degradation by the cysteine proteinases, cathepsins B, B-like and L. Digestion of collagen IV, laminin, fibronectin, and release of gelatinase activities from basement membrane fibronectin, Biol. Chem. Hoppe Seyler 374 (1993) 1135– 1146. R.T. Hamilton, K.A. Bruns, M.A. Delgado, J.K. Shim, Y. Fang, D.T. Denhardt, H.M. Nilsen, Developmental expression of cathepsin L and c-rasHa in the mouse placenta, Mol. Reprod. Dev. 30 (1991) 285–292. A. Hartley, J.M. Stone, C. Heron, J.M. Cooper, A.H. Schapira, Complex I inhibitors induce dose-dependent apoptosis in PC12 cells: relevance to Parkinson’s disease, J. Neurochem. 63 (1994) 1987–1990. Y. Kushima, H. Tsukui, Y. Enokido, C. Nishio, H. Hatanaka, High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats, Brain Res. 536 (1990) 16–22. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. T.T. Lah, M.R. Buck, K.V. Honn, J.D. Crissman, N.C. Rao, L.A. Liotta, B.F. Sloane, Degradation of laminin by human tumor cathepsin B, Clin. Exp. Metastasis 7 (1989) 461–468. D.T. Loo, A. Copani, C.J. Pike, E.R. Whittemore, A.J. Walencewicz, C.W. Cotman, Apoptosis is induced by beta-amyloid in cultured central nervous system neurons, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7951–7955. D.P. Martin, R.E. Schmidt, P.S. DiStefano, O.H. Lowry, J.G. Carter, J. Johnson-EM, Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation, J. Cell Biol. 106 (1988) 829–844. M.F. Marusich, H.M. Furneaux, P.D. Henion, J.A. Weston, Hu neuronal proteins are expressed in proliferating neurogenic cells, J. Neurobiol. 25 (1994) 143–155. R.W. Mason, D.A. Johnson, A.J. Barrett, H.A. Chapman, Elastinolytic activity of human cathepsin L, Biochem. J. 233 (1986) 925–927. H. Matsuba, T. Watanabe, M. Watanabe, Y. Ishii, S. Waguri, E. Kominami, Y. Uchiyama, Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney, J. Histochem. Cytochem. 37 (1989) 1689–1697. T. Nitatori, N. Sato, S. Waguri, Y. Karasawa, H. Araki, K. Shibanai, E. Kominami, Y. Uchiyama, Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis, J. Neurosci. 15 (1995) 1001–1011. R.W. Oppenheim, D. Prevette, M. Tytell, S. Homma, Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role of cell death genes, Dev. Biol. 138 (1990) 104–113. R.W. Oppenheim, Cell death during development of the nervous system, Annu. Rev. Neurosci. 14 (1991) 453–501. D.E. Palm, N.W. Knuckey, M.J. Primiano, A.G. Spangenberger, C.E. Johanson, Cystatin C, a protease inhibitor, in degenerating rat hippocampal neurons following transient forebrain ischemia, Brain Res. 691 (1995) 1–8. L.A. Pennacchio, D.M. Bouley, K.M. Higgins, M.P. Scott, J.L. Noebels, R.M. Myers, Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice, Nat. Genet. 20 (1999) 251–258. S. Przedborski, V. Kostic, L. Jackson, V.A.B. Naini, S. Simonetti, S. Fahn, E. Carlson, C.J. Epstein, J.L. Cadet, Transgenic mice with increased cu / zn-superoxide dismutase activity are resistant to Nmethyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity, J. Neurosci. 12 (1992) 1658–1667. L.R. Roberts, H. Kurosawa, S.F. Bronk, P.J. Fesmier, L.B. Agellon,
262
[38]
[39]
[40]
[41]
[42]
[43]
C. Nishio et al. / Brain Research 873 (2000) 252 – 262 W.Y. Leung, F. Mao, G.J. Gores, Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes, Gastroenterology 113 (1997) 1714–1726. D.R. Rosen, T. Siddique, D. Patterson, D.A. Figlewicz, P. Sapp, A. Hentati, D. Donaldson, J. Goto, J.P. O’Regan, H.X. Deng et al., Mutations in Cu / Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature 362 (1993) 59–62, [published erratum appears in Nature 1993 Jul 22;364(6435):362]. T. Satoh, Y. Enokido, T. Kubo, M. Yamada, H. Hatanaka, Oxygen toxicity induces apoptosis in neuronal cells, Cell. Mol. Neurobiol. (1998) 649–666. B.F. Sloane, K. Moin, E. Krepela, J. Rozhin, Cathepsin B and its endogenous inhibitors: the role in tumor malignancy, Cancer Metastasis Rev. 9 (1990) 333–352. C. Tavera, D. Prevot, J.P. Girolami, T.J. Leung, A. Colle, Tissue and biological fluid distribution of cysteine proteinases inhibitor: rat cystatin C, Biol. Chem. Hoppe Seyler 371 (Suppl.) (1990) 187–192. G.F. Tu, A.R. Aldred, B.R. Southwell, G. Schreiber, Strong conservation of the expression of cystatin C gene in choroid plexus, Am. J. Physiol. 263 (1992) R195–R200. Y. Uchiyama, M. Nakajima, D. Muno, T. Watanabe, Y. Ishii, S. Waguri, N. Sato, E. Kominami, Immunocytochemical localization of cathepsins B and H in corticotrophs and melanotrophs of rat pituitary gland, J. Histochem. Cytochem. 38 (1990) 633–639.
[44] A.H. Warfel, F.D. Zucker, B. Frangione, J. Ghiso, Constitutive secretion of cystatin C (gamma-trace) by monocytes and macrophages and its downregulation after stimulation, J. Exp. Med. 166 (1987) 1912–1917. [45] T. Watanabe, M. Watanabe, Y. Ishii, H. Matsuba, S. Kimura, T. Fujita, E. Kominami, N. Katunuma, Y. Uchiyama, An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart, J. Histochem. Cytochem. 37 (1989) 347–351. [46] A.H. Wyllie, R.G. Morris, A.L. Smith, D. Dunlop, Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis, J. Pathol. 142 (1984) 67–77. [47] M. Yamada, Y. Enokido, T. Ikeuchi, H. Hatanaka, Epidermal growth factor prevents oxygen-triggered apoptosis and induces sustained signalling in cultured rat cerebral cortical neurons, Eur. J. Neurosci. 7 (1995) 2130–2138. [48] O. Yasuhara, K. Hanai, I. Ohkubo, M. Sasaki, P.L. McGeer, H. Kimura, Expression of cystatin C in rat, monkey and human brains, Brain Res. 628 (1993) 85–92. [49] F.D. Zucker, A. Warfel, G. Grusky, B. Frangione, D. Teitel, Novel monocyte-like properties of microglial / astroglial cells. constitutive secretion of lysozyme and cystatin-C, Lab. Invest. 57 (1987) 176– 185.
Research report
Involvement of cystatin C in oxidative stress-induced apoptosis of cultured rat CNS neurons 1
Chika Nishio, Kiyomi Yoshida , Keiji Nishiyama, Hiroshi Hatanaka*, Masashi Yamada Institute for Protein Research, Osaka University, 3 -2 Yamadaoka, Suita, Osaka 565 -0871, Japan Accepted 23 May 2000
Abstract Oxidative stress is involved in neuronal degeneration in cerebrovascular injury, neuropathology and aging. When rat CNS neurons were cultured in a high (50%) oxygen atmosphere, the neurons died. This high oxygen-induced cell death showed features of apoptotic cell death, characterized by DNA fragmentation, and was blocked by inhibitor of protein synthesis. We found that cystatin C and HuC mRNA, the products of which are an inhibitor of cysteine proteases and an RNA binding protein, respectively, were up-regulated in neurons cultured in the high oxygen atmosphere. In the present study, we focused on cystatin C. Cystatin C protein levels were also increased in neurons cultured in the high oxygen atmosphere. In situ hybridization with an RNA probe for rat cystatin C and immunocytochemistry with anti-human cystatin C antibody showed that microtubule-associated protein 2 (MAP2)-positive neurons expressed cystatin C mRNA and protein, respectively, in the high oxygen atmosphere. These results indicated that oxidative stress stimulates an increase in cystatin C expression in cultured neurons, and that cystatin C might have important roles in regulation of apoptosis elicited by oxidative stress. 2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Neuronal death Keywords: Cathepsin; PCR subtraction; Death; Survival; Reactive oxygen
1. Introduction Apoptosis is the best studied type of cell death, and has been shown to be mediated by an intrinsic death program [16]. Apoptosis is blocked by protein or RNA synthesis inhibitors [27,32], and is characterized by chromatin condensation and fragmentation of chromosomal DNA into multimers of about 180 bp [46]. This form of cell death
Abbreviations: DF medium, 1:1 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media; DIG, digoxygenin; GFAP, glial fibrillary acidic protein; FITC, fluorescein isothiocyanate; MAP2, microtubuleassociated protein 2; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TBS, Tris-buffered saline; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling *Corresponding author. Tel.: 181-6-6879-8624; fax: 181-6-68798626. E-mail address: [email protected] (H. Hatanaka). 1 Present address: Department of Geriatric Research, The National Institute for Longevity Science, 36-3 Gengo, Morioka-Cho, Ohbu-Shi, Aichi, 474-8522, Japan.
occurs at specific stages during the development of embryonic neurons including those of the sympathetic ganglion, retina, spinal cord, hippocampus and cerebral cortex [11,13,33]. In addition, apoptosis may be related to the neuronal degeneration that occurs during Alzheimer’s and Parkinson’s diseases, and in Down’s syndrome or with aging [8,14,22,26]. Therefore, it is important to study the mechanisms by which apoptosis of neurons is induced. Neuronal death induced by oxidative stress resulting in excessive production of reactive oxygen species, is related to a variety of neuronal degenerative disorders caused by cerebrovascular injury, neuropathology and aging [2,9,39]. Oxidative damage is closely associated with Parkinson’s and Alzheimer’s diseases, familial amyotrophic lateral sclerosis (ALS) and Down’s syndrome [5,8,15,36,38]. In addition, reactive oxygen species have been reported to be related to naturally occurring cell death [19]. To examine the neuronal death caused by oxidative stress in vitro, we cultured CNS neurons under a high oxygen atmosphere (50% O 2 ) [17,23,47]. As we reported
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02540-3
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previously, embryonic rat hippocampal neurons cultured under a 50% oxygen atmosphere died by apoptosis. The hippocampal neurons cultured under a 50% oxygen atmosphere showed nucleosomal DNA fragmentation, nuclear condensation and cytoplasmic compaction [18]. In addition, the oxygen-induced death of cultured hippocampal neurons was blocked by RNA or protein synthesis inhibitors. In this study, we showed that the death of rat brain neurons cultured under a 50% oxygen atmosphere displayed apoptotic features. We focused on the observation that the oxygen-induced death of these cultured neurons was prevented by RNA or protein synthesis inhibitors. This suggested that the oxygen toxicity activates an intracellular death cascade and stimulates up-regulation of some species of mRNA and proteins involved in regulation of neuronal apoptosis. To examine what kinds of RNA were up-regulated by the high oxygen stimulus in the cultured neurons of rat brains, we performed PCR subtraction screening. Cystatin C showed increased expression at both mRNA and protein levels in the neurons cultured in the high oxygen atmosphere. Cystatin C is an inhibitor of cysteine proteases including cathepsin B, H and L [1,3,4,6], and is secreted from various types of cells [12,41]. These results indicated that cystatin C is related to processes of apoptosis stimulated by oxidative stress through regulation of cysteine protease activities in cultured neurons of the rat brain.
2. Materials and methods
2.1. Cell culture Primary cultures of dissociated neurons of rat total brains without the cerebellum were prepared using embryonic day E20–21 rats (Wistar ST, both sexes) as described previously [17]. Briefly, the cells were cultured in a medium consisting of 5% precolostrum newborn calf serum (PNCS, Mitsubishi Kagaku), 5% heat-inactivated (568C, 30 min) horse serum (HS, Gibco) and 90% (v / v) DF medium consisting of a 1:1 mixture of Dulbecco’s modified Eagle’s (DME, Gibco) and Ham’s F12 media (Gibco) containing 15 mM HEPES buffer, pH 7.4, 30 nM selenium and 1.9 mg / ml sodium bicarbonate at a final cell density of 5310 5 cells / cm 2 on a polyethyleneimine-coated culture plates. After incubation for 2 h in a CO 2 incubator under a 20% (v / v) oxygen atmosphere, the medium was changed to DF medium containing 5 mg / ml human transferrin, 5 mg / ml bovine insulin (Colab. Res.), 20 nM progesterone and 30 nM selenium. One day after the medium change, the culture plates were transferred to a 20 or 50% (v / v) O 2 and a constant 5% (v / v) CO 2 atmosphere in a N 2 –O 2 –CO 2 gas incubator (Tabai BNP-110M). The oxygen tension in the 50% O 2 culture medium increased rapidly and reached a plateau level of about 300 mmHg
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within 2–3 h. The tension value of CO 2 and the pH value were stable during the entire culture period in both the 20 and 50% O 2 cultures, as described previously [17]. Cycloheximide (Sigma) was added just before the culture plates were transferred to the O 2 -regulated chambers. For the preparation of RNA from the neurons, the neurons were seeded in polyethyleneimine-coated Falcon 6-well plates. For immunostaining with anti-MAP2 antiserum or anti-cystatin C polyclonal antibody, TUNEL staining and the preparation of lysates from the neurons, polyethyleneimine-coated Nunc multidish 4-well plates were used. For the in situ hybridization and the immunofluorescence analysis, polyethyleneimine-coated Lab-Tek 8-chamber slide plates were used.
2.2. Antibodies, factors and reagents The anti-microtubule-associated protein 2 (MAP2) antiserum was a gift from Dr. H. Murofushi (The University of Tokyo). The anti-human cystatin C polyclonal antibody was purchased from DAKO, the anti-MAP2 monoclonal antibody was from Boeheringer Mannheim, and the antiglial fibrillary acidic protein (GFAP) monoclonal antibody was from Amersham.
2.3. Immunocytochemistry The cells were fixed in 4% paraformaldehyde at room temperature for 20 min. For immunostaining with antiMAP2 antiserum, the anti-MAP2 monoclonal antibody or the anti-cystatin C polyclonal antibody, the cells were blocked with phosphate-buffered saline (PBS) containing 5% (v / v) normal goat serum and 0.1% (v / v) Triton X-100 for 2 h at room temperature, then incubated at 48C overnight with the anti-MAP2 antiserum diluted to 1:5000, with the anti-MAP2 monoclonal antibody diluted to 1:1000, or the anti-cystatin C polyclonal antibody diluted to 1:10,000, with the blocking buffer. For immunostaining with the anti-GFAP antibody, the cells were fixed in 100% methanol, and were blocked with PBS containing 5% (v / v) normal goat serum and 0.3% (v / v) Triton X-100 for 2 h at room temperature, then incubated with the anti-GFAP monoclonal antibody diluted to 1:10,000 with the blocking buffer, overnight at 48C. Staining was performed using a Vectastain ABC kit (Vector Lab.) and 0.02% (w / v) 3,39diaminobenzidine 4-HCl and 0.1% (w / v) (NH 4 ) 2 Ni(SO 4 ) 2 dissolved in 0.05 M Tris–HCl buffer, pH 7.6, containing 0.01% (v / v) H 2 O 2 . For immunofluorescence analysis, rhodamine-conjugated anti-rabbit or mouse antibody and FITC-conjugated anti-rabbit antibody were used for the detection of MAP2 and cystatin C, respectively. The cells were visualized using fluorescence microscopy (Zeiss). The number of immunoreactive cells was determined by examining microphotographs. The number of immunoreactive cells in more than four randomly selected micro-
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scopic fields per well was counted, and that in four wells was averaged.
2.4. TUNEL staining The neurons were cultured under 20 or 50% oxygen atmospheres in the absence or presence of 1 mM cycloheximide for 48 h. The neurons were fixed in 4% paraformaldehyde at room temperature for 20 min, then washed 3 times with PBS. The fixed cells were treated on ice for 2 min with a solution containing 0.1% (w / v) sodium citrate and 0.1% Triton X-100, followed by washing with PBS. TUNEL staining was performed using an In Situ Cell Death Detection kit, POD (Boehringer Mannheim). Staining was performed by incubation with 0.02% (w / v) 3,39diaminobenzidine 4-HCl and 0.1% (w / v) (NH 4 ) 2 Ni(SO 4 ) 2 dissolved in 0.05 M Tris–HCl buffer, pH 7.6, containing 0.01% (v / v) H 2 O 2 .
2.5. Subtractive PCR screening Total RNA was prepared from the neurons cultured under a 20% oxygen atmosphere and from those cultured for 3 h under a 50% oxygen atmosphere, using TRIzol reagent (Gibco BRL). Then, mRNA was purified from the total RNA with Oligotex-dT30 (Super) (Takara). Subtractive PCR screening was performed using a PCR-Select cDNA Subtraction Kit (Clontech) according to manufacturer’s protocol. Briefly, double-stranded cDNA was synthesized from the mRNA, then digested with Rsa I. To subtract mRNA prepared from the cells cultured under a 20% oxygen atmosphere from that of those cultured under 50% oxygen, cDNA from the latter cultures was ligated with one of two adaptors (adaptor 1 or 2). The cDNA from the cells cultured under 20% oxygen was not ligated with the adaptors. cDNAs ligated with adaptor 1 or 2 were separately hybridized with excess amounts of the cDNA from the cells cultured under the 20% oxygen atmosphere. The two samples from the hybridization were mixed together, then fresh denatured cDNA from the 20% oxygen culture was added. The mixture was subjected to second hybridization, then to PCR using the sequences of the adaptors as primers, followed by nested PCR. The PCR products were cloned in the pCR 2.1 vector using an Original TA Cloning Kit (Invitrogen). The clones were sequenced using a sequencer (Li-Cor, model 4000L).
2.6. Nuclease protection assay The neurons were not cultured or were cultured in a 50% oxygen atmosphere for 3 or 5 h, then total RNA was prepared from the neurons as described above. Radiolabeled RNA probes were synthesized from the pCR 2.1 clones in the presence of [a- 32 P] UTP (Amersham) using MAXIscript kits (Ambion) in according to the manufacture’s protocol. RNA protection assay was performed with
a High-Speed Hybridization Ribonuclease Protection Assay Kit (Ambion) in accordance with the instruction manual. The protected bands were visualized, and the intensities of bands were quantified using an image analyzer (BAS 2000, Fuji Film).
2.7. Immunoblotting The neurons were not cultured or were cultured for 24 h under a 20% oxygen atmosphere or for 6, 12, 18, 24 or 36 h under a 50% oxygen atmosphere. Then, the neurons were washed once with ice-cold PBS, and lysed in a buffer containing 1% SDS, 5 mM EDTA, 10 mM Tris–HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride and 5 mg / ml aprotinin. Lysates were boiled for 5 min at 1008C, then centrifuged at 100,000 3g for 30 min. The protein concentration of the clarified lysate was determined by the BCA protein assay. The lysates (10 mg) were resolved by electrophoresis on 10–30% gradient SDS–polyacrylamide gels according to the method of Laemmli [24]. Proteins were transferred onto polyvinylidene difluoride membranes (Millipore) in 0.1 M Tris base, 0.192 M glycine and 20% (v / v) methanol using a semi-dry electrophoretic transfer system. The membranes were blocked with 0.1% (w / v) Tween 20 / TBS (T-TBS) containing 10% (w / v) nonfat dried milk at room temperature for at least 1 h, then incubated with 1.4 mg / ml anti-human cystatin C antibody in T-TBS containing 10% (w / v) nonfat dried milk at room temperature for 1 h. After three washes with T-TBS, the membranes were incubated with peroxidase-coupled goat anti-rabbit IgG secondary antibody (Jackson ImmunoRes. Lab. Inc.) diluted 1:1000 with T-TBS at room temperature for 2 h. The membranes were then washed four times with T-TBS, and visualized using the ECL chemiluminescence system (Amersham).
2.8. In situ hybridization Digoxygenin (DIG)-labeled anti-sense or sense RNA probe of cystatin C was synthesized from pCR 2.1-cystatin C plasmid using a DIG RNA Labeling kit (Boehringer Mannheim). The cells were fixed in 4% paraformaldehyde at room temperature for 20 min, then washed twice with PBS. The cells were washed with H 2 O, incubated with a solution consisting of 0.25% (v / v) acetic acid and 0.1 M triethanolamine for 10 min, then washed with PBS. The cells were dehydrated by treatment with 70% (v / v), 95% (v / v) and 100% (v / v) ethanol each for 2 min, with chloroform for 10 min, with 100% (v / v) and 95% (v / v) ethanol each for 2 min, and were then air-dried. The cells were pre-hybridized at 508C for 1 h with a hybridization solution consisting of 50% (v / v) formamide, 20 mM Tris– HCl (pH 8.0), 5 mM EDTA (pH 8.0), 0.3 M NaCl, 10 mM sodium phosphate buffer (pH8.0), 13Denhardt’s solution, 10% (w / v) dextran sulfate, 0.2% (v / v) sarkosyl, 500 mg / ml yeast tRNA, and 200 mg / ml salmon sperm DNA. The
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cells were hybridized with DIG-labeled anti-sense or sense RNA probe in the hybridization solution at 508C overnight. The cells were washed at 608C for 20 min with 53SSC, at 608C for 30 min with 23SSC containing 50% (v / v) formamide and 0.1 M dithiothreitol, then at 378C for 30 min with RNase buffer consisting of 10 mM Tris–HCl (pH8.0), 1 mM EDTA and 0.5 M NaCl. The cells were incubated at 378C for 30 min with 1 mg / ml RNase A in the RNase buffer, washed at 378C for 30 min with the RNase buffer, then washed at 608C for 30 min with 23SSC containing 50% (v / v) formamide and 0.1 M dithiothreitol. After washing with 0.1 M Tris–HCl (pH8.0) containing 0.15 M NaCl, the cells were stained using alkaline phosphatase-conjugated anti-DIG Fab fragment (Boehringer Mannheim), followed by reaction with xphosphate / 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT).
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3. Results
effect of the protein synthesis inhibition on the oxygeninduced death of cultured rat CNS neurons. Cycloheximide was added at various time points to the cultures under the high oxygen atmosphere. The cultured cells were immunostained 48 h after exposure to the high oxygen atmosphere using anti-MAP2 polyclonal antiserum, then the number of MAP2-positive neurons was counted (Fig. 2). Cycloheximide added to the culture at later time points showed a lesser protective effect against the death induced by oxygen toxicity. When added just before, 2 h or 3 h after transfer to a 50% oxygen atmosphere, cycloheximide effectively prevented the oxygen-induced cell death. On the other hand, almost all of the neurons exposed to cycloheximide 5 h after transfer to a 50% oxygen atmosphere died. In addition, we obtained similar result in MTT assay as that in the counting the number of MAP2-positive cells (data not shown). These results indicated that proteins involved in induction of the death of cultured neurons were expressed between 3 and 5 h after exposure to the high oxygen atmosphere.
3.1. Apoptotic cell death induced by oxygen toxicity in cultured rat CNS neurons
3.2. Subtractive PCR screening to identify genes upregulated by oxidative stress
We have reported that basal forebrain, hippocampal and cerebral cortical neurons cultured under a high oxygen atmosphere show apoptotic cell death characterized by nuclear DNA fragmentation, which can be prevented by protein or RNA synthesis inhibitors [18,47]. In the present study, we used cultured neurons of rat whole brains without cerebellum and screened for genes involved in the high oxygen-induced death of CNS neurons. To determine whether the oxygen-induced death of the cultured rat CNS neurons was also apoptotic, we examined the effects of a protein synthesis inhibitor. Neurons from the embryonic rat (embryonic day 20–21) whole brain without the cerebellum were cultured in 20% or 50% oxygen in the absence or presence of cycloheximide for 48 h, then immunostained with anti-microtubule-associated protein 2 (MAP2) antiserum. Cycloheximide markedly prevented neuronal cell death caused by the high oxygen stimulus (Fig. 1A–C). Apoptotic cell death is characterized by DNA fragmentation [46]. To examine whether high levels of oxygen induce DNA fragmentation, the rat CNS neurons cultured under an atmosphere of 20% or 50% oxygen for 48 h were stained by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) method. Most of the cells cultured under a 50% oxygen atmosphere were TUNEL-positive, although those cultured under a 20% oxygen atmosphere or in the presence of cycloheximide under a 50% oxygen atmosphere were scarcely TUNELpositive (Fig. 1D–F). These results indicated that the high level of oxygen induces apoptotic cell death in the cultured rat CNS neurons. In addition, we examined the commitment time of the
Up-regulation of proteins by high oxygen stimulus might result in the death of the cultured rat brain neurons under the high oxygen atmosphere. To examine what kinds of proteins show oxidative stress-induced increases in their expression, we performed subtractive PCR screening to isolate up-regulated RNA in the neurons cultured under the high oxygen atmosphere. We isolated 85 clones, which were then subjected to sequencing analysis. Of these clones, 45 were unknown, and 40 has been identified previously. To confirm the increases in the levels of mRNA expression of these 85 clones, all clones were analyzed by RNA protection assay using total RNA from the neurons cultured under a 20% oxygen atmosphere and from those cultured for 3 h under a 50% oxygen atmosphere. In result, only two of the clones showed significant increases in the mRNA levels in the neurons cultured under the high oxygen atmosphere as compared with those exposed to normal oxygen level. One ([ 168; 337 bp) of the positive clones had the same sequence as rat cystatin C [12], except for one nucleotide difference (data not shown). The other one was HuC, an RNA binding protein (data not shown) [28]. As shown in Fig. 3A, cystatin C (clone [168) mRNA level increased in the neurons cultured for 3 h under the high oxygen atmosphere. In three independent cultures, the amounts of cystatin C mRNA in the neurons exposed to the high oxygen atmosphere for 3 h were 1.84-, 1.33- and 1.21-times larger than those in the neurons not exposed to the high oxygen atmosphere. In addition, HuC mRNA showed about 1.5-fold increase in the neurons cultured for 3 h under the high oxygen atmosphere (data not shown). In the present study, we focused on cystatin C, because some reports suggest that cathepsins, activities of
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Fig. 1. Inhibitory effect of cycloheximide on apoptosis of cultured CNS neurons under a high-oxygen atmosphere. (A–C) The neurons were cultured under 20 (A) or 50% (B, C) oxygen atmospheres in the absence (A, B) or presence of 1 mM cycloheximide (C) for 48 h. The neurons were immunostained with anti-MAP2 antiserum. Bar represents 100 mm. (D–E) The neurons were cultured in 20 (D) or 50% (E, F) oxygen atmospheres in the absence (D, E) or presence of 1 mM cycloheximide (F) for 24 h. The neurons were stained by the TUNEL method. Bar represents 100 mm.
which are inhibited by cystatin C, are involved in regulation of neuronal cell death [31,37].
3.3. Up-regulation of cystatin C protein in rat CNS neurons cultured under a high oxygen atmosphere To confirm the increased expression of cystatin C in response to oxidative stress, we examined the changes in amounts of cystatin C protein in rat CNS neurons cultured for various periods under a 20% or 50% oxygen atmosphere by Western blotting analysis using anti-human cystatin C antibody. The neurons cultured under a 50% oxygen atmosphere showed gradual increases in the amounts of cystatin C protein until at least 36 h, while those cultured under 20% oxygen showed only small
increases (Fig. 3B). These results suggested that oxidative stress stimulates cystatin C expression in cultured rat CNS neurons.
3.4. Immunocytochemistry with anti-cystatin C antibody in cultured rat CNS neurons To refine the expression of cystatin C in the rat CNS neurons cultured under a 50% atmosphere, the neurons cultured for various periods after exposure to high oxygen were immunostained with anti-human cystatin C antibody (Fig. 4). The cystatin C-positive cells gradually increased, and most of the cells were cystatin C-positive at 48 h after exposure to the high oxygen atmosphere. On the other hand, we observed very few cystatin C-positive cells in the
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signals on immunostaining with both the anti-MAP2 monoclonal and the anti-cystatin C polyclonal antibodies (Fig. 5D–F). These results indicated that the cultured MAP2-positive neurons express cystatin C mRNA and protein in response to high oxygen stimulus.
4. Discussion
Fig. 2. Commitment time of survival-promoting effect of cycloheximide, a protein synthesis inhibitor, on oxygen-induced death of cultured rat CNS neurons. The medium was changed 2 h after the neurons were seeded. 1 mM cycloheximide was added to the culture just before (0 h), and 2, 3, 4, 5 and 6 h after the cultures were transferred to 20% and 50% oxygen atmospheres. The cells were cultured for 48 h after the transfer, and were then immunostained with anti-MAP2 antiserum. Then, the number of MAP2-positive neurons was counted. The values of the cultured neurons in a 50% oxygen atmosphere are expressed as the percentage of those measured in the corresponding culture in a 20% oxygen atmosphere. The values represent the means6S.D. of four individual cultures.
cultures incubated for 24 h under a 20% oxygen atmosphere. In addition, most of the cystatin C-positive cells seemed to be neurons. Because, the cells were cultured in serum-free medium. In addition, when the cultured cells were immunostained using the anti-glial fibrillary acidic protein (GFAP) monoclonal antibody, few GFAP-positive astroglial cells were observed (less than 1% of the corresponding MAP2-positive neurons) (data not shown). These results indicated that the number of neurons that show increased expression of cystatin C gets to increase with incubation under the high oxygen atmosphere.
3.5. MAP2 -positive neurons expressed cystatin C mRNA and protein in high oxygen atmosphere To confirm that the cells expressing cystatin C under the high oxygen atmosphere were neurons, we examined whether MAP2-positive neurons were positive by in situ hybridization analysis using an RNA probe for cystatin C mRNA and immunostaining using anti-cystatin C antibodies. A number of cells cultured for 6 h under a 50% oxygen atmosphere showed positive signals both on immunostaining with anti-MAP2 antiserum and on in situ hybridization analysis with the cystatin C RNA probe (Fig. 5A–C). In addition, some of the cells cultured for 16 h under the high oxygen atmosphere displayed positive
We showed that expression of cystatin C, an inhibitor of cysteine proteases, is up-regulated in response to high oxygen stimulus. Cystatin C is an alkaline protein with a low molecular mass (13.5 kDa), and is a member of Family 2 of the cystatin superfamily [1,3,4,6]. Cystatin C is widely expressed in almost all tissues and is secreted into various biological fluids including urine, blood, seminal fluid, saliva and cerebrospinal fluid [12,41]. In the CNS, large amounts of cystatin C mRNA are present in brain tissue and choroid plexus [12,42]. On immunohistochemical analysis, cystatin C is detected in astrocytes, choroid plexus cells and a small number of neurons [48]. In culture, astrocytes and microglial cells express and secrete cystatin C [44,49]. In transient forebrain ischemia, it has been reported that CA1 pyramidal cells and reactive astrocytes of rat hippocampus show delayed expression of cystatin C, and that immunoreactivity with anti-cystatin C is localized in morphologically degenerative neurons [34]. In addition, immunohistochemical studies with anticystatin C antibody in human brain indicate that intensely immunoreactive neurons are abundant in the cerebral cortex in some aged human subjects and in some Alzheimer’s disease patients [48]. These studies suggested that cystatin C expression may be related to neuronal degeneration in brain diseases. In our study, cultured rat CNS neurons scarcely expressed cystatin C under normal conditions. We found that cystatin C expression was increased in the cultured CNS neurons by exposure to a high oxygen atmosphere, which induced neuronal cell death. Oxidative stress is involved in neuronal degeneration caused by cerebrovascular injury, aging and Alzheimer’s disease [2,5,9,15,39]. Therefore, cystatin C may have important roles in neuronal death caused by oxidative stress in the CNS. Oxidative stress-stimulated up-regulation of cystatin C expression may be mediated by reactive oxygen species including superoxide anion, hydrogen peroxide, hydroxyradical and peroxynitrite, although it remains unclear how reactive oxygen species triggers the cystatin C expression. We cannot exclude the possibility that the oxidative stress stimulates the increase in cystatin C expression through undetermined modifications by oxygen rather than through reactive oxygen species. In addition, we observed that the high oxygen-stimulated increase in expression level of cystatin C mRNA was the highest at 3 h after the exposure to high oxygen, although that of cystatin C protein got to be observed between 6 and 12 h. The time lag of upregulation between mRNA and protein of cystatin C
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Fig. 3. Oxidative stress stimulates increases in expression of cystatin C mRNA and protein in cultured CNS neurons. (A) The neurons were not cultured (0 h) or were cultured in a 50% oxygen atmosphere for 3 or 5 h, then total RNA was prepared. The total RNA was subjected to RNA protection assay using an RNA probe for cystatin C or actin. Intensities of the protected bands were quantified using an imaging analyzer. (B) The neurons were not cultured (0 h) or were cultured for 24 h under a 20% oxygen atmosphere or for 6, 12, 18, 24 or 36 h under a 50% oxygen atmosphere, and were then lysed. The lysates were subjected to Western blotting analysis using anti-cystatin C antibody. Molecular weights are shown on the left. The expression levels of cystatin C protein were quantified by measuring densities of the protein bands.
indicate that there are post-transcriptional regulation and accumulation mechanisms (for examples, increased stability and attenuated exocytosis of cystatin C protein). The
expression level of cystatin C mRNA may increase again after it peaked at 3 h after the exposure to the high oxygen atmosphere.
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Fig. 4. Immunocytochemical staining with anti-cystatin C antibody of cultured CNS neurons under a high oxygen atmosphere. The neurons were not cultured (A) or were cultured for 6 (B), 12 (C), 24 (D) or 48 h (E) under a 50% oxygen atmosphere, or for 24 h (F) under a 20% oxygen atmosphere. The neurons were immunostained with anti-cystatin C antibody. Bar represents 100 mm.
In transient ischemia, delayed death of CA1 pyramidal neurons shows apoptotic features. In this case, immunoreactivities of cathepsin B, H and L, activities of which are inhibited by cystatin C, are increased in the CA1 pyramidal neurons [31]. In addition, in rat hepatocyte and hepatoma cell lines, cathepsin B contributes to bile salt-induced apoptosis [37]. These cathepsins are mainly present in lysosomes, however cathepsins may be released from cells. It is possible that the cathepsins were leaked from dead neurons. Alternatively, the cathepsins may have been released from cells through exocytosis. Cathepsin B is detected in the secretory vesicles from atrial and juxtaglomerular endocrine cells [30,45], and cathepsin H is found in secretory granules from corticotrophic and melanot-
rophic cells of the pancreas [43]. Numerous invasive carcinomas and ras-transformed fibroblasts have been reported to show increased synthesis and secretion of cathepsins [10,40]. Detection of cathepsin L in trophoblastconditioned medium indicates the secretion of cathepsin L from the mouse trophoblast [21]. In addition, cathepsin B and H are able to degrade components of extracellular matrix including laminin, collagen IV and fibronectin [7,20,25,29]. Secreted cystatin C may block the activities of cathepsin released from cells, to protect cell-surface proteins. However, we cannot exclude the possibility that cystatin C may act on intracellular cysteine proteases including cathepsin, to regulate the neuronal cell death. A study using mice lacking cystatin B, a member of the
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Fig. 5. MAP2-positive neurons expressed cystatin C mRNA and protein in CNS neurons cultured under a high oxygen atmosphere. (A–C) The neurons were cultured for 6 h under a 50% oxygen atmosphere, and were then fixed. The fixed neurons were subjected to in situ hybridization with an anti-sense RNA probe for cystatin C (A), and were then immunostained with anti-MAP2 antiserum (B). (C) shows the superimposed images of (A) and (B). The images shown are of the same field. Bar represents 50 mm. (D–F) The neurons were cultured for 16 h under a 50% oxygen atmosphere, and were then fixed. The fixed neurons were immunostained with anti-cystatin C polyclonal antibody (D) and with anti-MAP2 monoclonal antibody (E). (F) shows the superimposed images of (D) and (E). The images shown are of the same field. Bar represents 50 mm.
cystatin superfamily, indicated that cystatin B has a role in preventing apoptosis of cerebellar granule neurons [35]. Therefore, we suggest that cystatins are involved in regulation of apoptosis in neurons.
Acknowledgements We thank Dr. H. Murofushi (The University of Tokyo) for the kind gift of anti-MAP2 antiserum, and Dr. Y.
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Uchiyama and Dr. S. Kametaka (Osaka University) for valuable discussion and kind support in this study. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, by a Research Grant for Neurons and Mental Disorders from the Ministry of Health and Welfare of Japan.
References [1] T. Akopyan, Protein inhibitors of proteinases from brain, Neurochem.Res. 16 (1991) 513–517. [2] B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7915–7922. [3] A.J. Barrett, H. Kirschke, Cathepsin B, cathepsin H, and cathepsin L, Methods Enzymol. 80 (Pt C) (1981) 535–561. [4] A.J. Barrett, The cystatins: a diverse superfamily of cysteine peptidase inhibitors, Biomed. Biochim. Acta 45 (1986) 1363–1374. [5] C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid beta protein toxicity, Cell 77 (1994) 817–827. [6] L.A. Bobek, M.J. Levine, Cystatins — inhibitors of cysteine proteinases, Crit. Rev. Oral Biol. Med. 3 (1992) 307–332, [published erratum appears in Crit. Rev. Oral Biol. Med. 1993;4(2):251]. [7] M.C. Burleigh, A.J. Barrett, G.S. Lazarus, Cathepsin B1. A lysosomal enzyme that degrades native collagen, Biochem. J. 137 (1974) 387–398. [8] J. Busciglio, B.A. Yankner, Apoptosis and increased generation of reactive oxygen species in Down’s syndrome neurons in vitro, Nature 378 (1995) 776–779. [9] W. Cao, J.M. Carney, A. Duchon, R.A. Floyd, M. Chevion, Oxygen free radical involvement in ischemia and reperfusion injury to brain, Neurosci. Lett. 88 (1988) 233–238. [10] A.F. Chambers, R. Colella, D.T. Denhardt, S.M. Wilson, Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells, Mol. Carcinog. 5 (1992) 238–245. [11] P.G. Clarke, Developmental cell death: morphological diversity and multiple mechanisms, Anat. Embryol. Berl. 181 (1990) 195–213. [12] T. Cole, P.W. Dickson, F. Esnard, S. Averill, G.P. Risbridger, F. Gauthier, G. Schreiber, The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for beta 2-microglobulin in rat brain, Eur. J. Biochem. 186 (1989) 35–42. [13] W.M. Cowan, J.W. Fawcett, D.D. O9Leary, B.B. Stanfield, Regressive events in neurogenesis, Science 225 (1984) 1258–1265. [14] B. Dipasquale, A.M. Marini, R.J. Youle, Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons, Biochem. Biophys. Res. Commun. 181 (1991) 1442–1448. [15] T. Dyrks, E. Dyrks, T. Hartmann, C. Masters, K. Beyreuther, Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation, J. Biol. Chem. 267 (1992) 18210–18217. [16] R.E. Ellis, J.Y. Yuan, H.R. Horvitz, Mechanisms and functions of cell death, Annu. Rev. Cell Biol. 7 (1991) 663–698. [17] Y. Enokido, Y. Akaneya, M. Niinobe, K. Mikoshiba, H. Hatanaka, Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture, Brain Res. 599 (1992) 261–271. [18] Y. Enokido, H. Hatanaka, Apoptotic cell death occurs in hippocampal neurons cultured in a high oxygen atmosphere, Neuroscience 57 (1993) 965–972. [19] L.J. Greenlund, T.L. Deckwerth, J. Johnson-EM, Superoxide dis-
[20]
[21]
[22]
[23]
[24] [25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33] [34]
[35]
[36]
[37]
261
mutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death, Neuron 14 (1995) 303–315. N. Guinec, V. Dalet-Fumeron, M. Pagano, ‘In vitro’ study of basement membrane degradation by the cysteine proteinases, cathepsins B, B-like and L. Digestion of collagen IV, laminin, fibronectin, and release of gelatinase activities from basement membrane fibronectin, Biol. Chem. Hoppe Seyler 374 (1993) 1135– 1146. R.T. Hamilton, K.A. Bruns, M.A. Delgado, J.K. Shim, Y. Fang, D.T. Denhardt, H.M. Nilsen, Developmental expression of cathepsin L and c-rasHa in the mouse placenta, Mol. Reprod. Dev. 30 (1991) 285–292. A. Hartley, J.M. Stone, C. Heron, J.M. Cooper, A.H. Schapira, Complex I inhibitors induce dose-dependent apoptosis in PC12 cells: relevance to Parkinson’s disease, J. Neurochem. 63 (1994) 1987–1990. Y. Kushima, H. Tsukui, Y. Enokido, C. Nishio, H. Hatanaka, High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats, Brain Res. 536 (1990) 16–22. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. T.T. Lah, M.R. Buck, K.V. Honn, J.D. Crissman, N.C. Rao, L.A. Liotta, B.F. Sloane, Degradation of laminin by human tumor cathepsin B, Clin. Exp. Metastasis 7 (1989) 461–468. D.T. Loo, A. Copani, C.J. Pike, E.R. Whittemore, A.J. Walencewicz, C.W. Cotman, Apoptosis is induced by beta-amyloid in cultured central nervous system neurons, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7951–7955. D.P. Martin, R.E. Schmidt, P.S. DiStefano, O.H. Lowry, J.G. Carter, J. Johnson-EM, Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation, J. Cell Biol. 106 (1988) 829–844. M.F. Marusich, H.M. Furneaux, P.D. Henion, J.A. Weston, Hu neuronal proteins are expressed in proliferating neurogenic cells, J. Neurobiol. 25 (1994) 143–155. R.W. Mason, D.A. Johnson, A.J. Barrett, H.A. Chapman, Elastinolytic activity of human cathepsin L, Biochem. J. 233 (1986) 925–927. H. Matsuba, T. Watanabe, M. Watanabe, Y. Ishii, S. Waguri, E. Kominami, Y. Uchiyama, Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney, J. Histochem. Cytochem. 37 (1989) 1689–1697. T. Nitatori, N. Sato, S. Waguri, Y. Karasawa, H. Araki, K. Shibanai, E. Kominami, Y. Uchiyama, Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis, J. Neurosci. 15 (1995) 1001–1011. R.W. Oppenheim, D. Prevette, M. Tytell, S. Homma, Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role of cell death genes, Dev. Biol. 138 (1990) 104–113. R.W. Oppenheim, Cell death during development of the nervous system, Annu. Rev. Neurosci. 14 (1991) 453–501. D.E. Palm, N.W. Knuckey, M.J. Primiano, A.G. Spangenberger, C.E. Johanson, Cystatin C, a protease inhibitor, in degenerating rat hippocampal neurons following transient forebrain ischemia, Brain Res. 691 (1995) 1–8. L.A. Pennacchio, D.M. Bouley, K.M. Higgins, M.P. Scott, J.L. Noebels, R.M. Myers, Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice, Nat. Genet. 20 (1999) 251–258. S. Przedborski, V. Kostic, L. Jackson, V.A.B. Naini, S. Simonetti, S. Fahn, E. Carlson, C.J. Epstein, J.L. Cadet, Transgenic mice with increased cu / zn-superoxide dismutase activity are resistant to Nmethyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity, J. Neurosci. 12 (1992) 1658–1667. L.R. Roberts, H. Kurosawa, S.F. Bronk, P.J. Fesmier, L.B. Agellon,
262
[38]
[39]
[40]
[41]
[42]
[43]
C. Nishio et al. / Brain Research 873 (2000) 252 – 262 W.Y. Leung, F. Mao, G.J. Gores, Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes, Gastroenterology 113 (1997) 1714–1726. D.R. Rosen, T. Siddique, D. Patterson, D.A. Figlewicz, P. Sapp, A. Hentati, D. Donaldson, J. Goto, J.P. O’Regan, H.X. Deng et al., Mutations in Cu / Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature 362 (1993) 59–62, [published erratum appears in Nature 1993 Jul 22;364(6435):362]. T. Satoh, Y. Enokido, T. Kubo, M. Yamada, H. Hatanaka, Oxygen toxicity induces apoptosis in neuronal cells, Cell. Mol. Neurobiol. (1998) 649–666. B.F. Sloane, K. Moin, E. Krepela, J. Rozhin, Cathepsin B and its endogenous inhibitors: the role in tumor malignancy, Cancer Metastasis Rev. 9 (1990) 333–352. C. Tavera, D. Prevot, J.P. Girolami, T.J. Leung, A. Colle, Tissue and biological fluid distribution of cysteine proteinases inhibitor: rat cystatin C, Biol. Chem. Hoppe Seyler 371 (Suppl.) (1990) 187–192. G.F. Tu, A.R. Aldred, B.R. Southwell, G. Schreiber, Strong conservation of the expression of cystatin C gene in choroid plexus, Am. J. Physiol. 263 (1992) R195–R200. Y. Uchiyama, M. Nakajima, D. Muno, T. Watanabe, Y. Ishii, S. Waguri, N. Sato, E. Kominami, Immunocytochemical localization of cathepsins B and H in corticotrophs and melanotrophs of rat pituitary gland, J. Histochem. Cytochem. 38 (1990) 633–639.
[44] A.H. Warfel, F.D. Zucker, B. Frangione, J. Ghiso, Constitutive secretion of cystatin C (gamma-trace) by monocytes and macrophages and its downregulation after stimulation, J. Exp. Med. 166 (1987) 1912–1917. [45] T. Watanabe, M. Watanabe, Y. Ishii, H. Matsuba, S. Kimura, T. Fujita, E. Kominami, N. Katunuma, Y. Uchiyama, An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart, J. Histochem. Cytochem. 37 (1989) 347–351. [46] A.H. Wyllie, R.G. Morris, A.L. Smith, D. Dunlop, Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis, J. Pathol. 142 (1984) 67–77. [47] M. Yamada, Y. Enokido, T. Ikeuchi, H. Hatanaka, Epidermal growth factor prevents oxygen-triggered apoptosis and induces sustained signalling in cultured rat cerebral cortical neurons, Eur. J. Neurosci. 7 (1995) 2130–2138. [48] O. Yasuhara, K. Hanai, I. Ohkubo, M. Sasaki, P.L. McGeer, H. Kimura, Expression of cystatin C in rat, monkey and human brains, Brain Res. 628 (1993) 85–92. [49] F.D. Zucker, A. Warfel, G. Grusky, B. Frangione, D. Teitel, Novel monocyte-like properties of microglial / astroglial cells. constitutive secretion of lysozyme and cystatin-C, Lab. Invest. 57 (1987) 176– 185.