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Presenilin-1-deficient neurons are nitric oxide-dependently killed by hydrogen peroxide in vitro
Neuroscience 125 (2004) 563–568
PRESENILIN-1-DEFICIENT NEURONS ARE NITRIC OXIDEDEPENDENTLY KILLED BY HYDROGEN PEROXIDE IN VITRO M. NAKAJIMA AND T. SHIRASAWA*
mechanisms via the genetic mutations still need to be defined but the most reliable hypothesis is as follows. The production of the longer form and/or the shorter form of amyloid -protein (A) is promoted by the mutations of APP, PS1, or PS2. These As enhance the formation of amyloid plaques and exhibit neurotoxicity in the forms of insoluble fibrils or soluble oligomers (Hardy and Selkoe, 2002). Most cases of AD are sporadic, but not inherited. However, the hypothesis described for FAD, the amyloid cascade hypothesis, is accepted for the pathogenic mechanisms of the sporadic AD too. On the other hand, a distinctive risk factor is known for AD, aging (Bachman et al., 1993). Oxidative damage accumulates with age. Thus the hypothesis has been proposed that cellular events involving oxidative stress may play a central role in the pathogenic cascade of AD (Benzi and Moretti, 1995), although the oxidative stress itself would not be the primary event. In fact, the involvement of reactive oxygen species with the pathological features of AD including neurofibrillary tangles, senile plaques, mitochondrial dysfunction, and microglial activation has been reported (Smith et al., 1996; Blass et al., 1990; Colton and Gilbert, 1987). Oxidative stress may be involved in selective neuronal death in AD, in concert with the amyloid cascade. PS1 is suggested to mediate the processing of proteins such as APP, Notch-1, ErbB4, and E-cadherin (Ebinu and Yankner, 2002; Marambaud et al., 2002). In addition, PS1 has been reported for the involvement in membrane protein trafficking, unfolded protein response, and capacitative calcium entry (Naruse et al., 1998; Niwa et al., 1999; Yoo et al., 2000). Developmental functions of PS1 are also suggested in neurogenesis, somatogenesis, and angiogenesis (Shen et al., 1997; Handler et al., 2000; Yuasa et al., 2002; Wong et al., 1997; Koizumi et al., 2001; Nakajima et al., 2003). Although PS1 is a causative gene for FAD, the possible involvement of PS1 in sporadic AD has been examined pathologically: Immunohistochemical analysis of sporadic AD brains for PS1 suggested that PS1 may have a neuroprotective role and that in AD low cellular expression of PS1 protein may be associated with increased neuronal loss (Giannakopoulos et al., 1997). Consistent with this finding, we found in the previous study that CNS neurons cultured from PS1-deficient mice exhibited increased vulnerability to oxidative stress in calciumdependent manners (Nakajima et al., 2001). In the present study, we further investigated the underlying mechanisms of the increased vulnerability of PS1-deficient neurons to oxidative stress and found that the
Department of Molecular Gerontology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan
Abstract—Presenilin-1 (PS1) is the gene responsible for the development of early-onset familial Alzheimer’s disease. To probe the functions of PS1 on neuronal resistance to oxidative stress, we pharmacologically examined the death signals in PS1-deficient neurons induced by oxidative stress. Because the death of primarily cultured neurons lacking PS1 is caused by hydrogen peroxide in calciumdependent manners in vitro [J Neurochem 78 (2001) 807], we tested the neuronal survival-promoting ability of inhibitors against calcium-dependent/cell death-related signaling molecules, such as ERKs, JNK, p38 MAP kinase, calcineurin, calpain, and nitric oxide synthase (NOS). All inhibitors tested failed to rescue the PS1-deficient neurons from the death with the exception of an inhibitor of NOS, NG-nitro-L-arginine methyl ester. Hemoglobin, a nitric oxide (NO) scavenger, also prevented the death of the mutant neurons. NADPH-diaphorase staining, which accounts for NOS activity, was enhanced in the mutant neurons. These results suggest that PS1 has a role for NOS activation in neurons and confers oxidative stress-resistance on neurons in calcium/ NO-dependent manners. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hemoglobin, NADPH-diaphorase, nitric oxide synthase, L-NAME, JNK, p38.
Alzheimer’s disease (AD) is characterized by progressive neuronal cell loss, the presence of senile plaques and neurofibrillary tangles in the brain. Approximately 10 –17% of AD cases are inherited in autosomal dominant manner (Cruts and Van Broeckhoven, 1998). Three genes were identified so far as causative genes for these familial AD (FAD); the amyloid precursor protein (APP), the presenilin-1 (PS1), and the presenilin-2 (PS2; Chartier et al., 1991; Goate et al., 1991; Alzheimer Disease Collaborative Group, 1995; Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995; Selkoe, 1996). FAD is caused dominantly by mutations in these three genes. Mutations in PS1 are the most common cases of FAD. Pathogenic *Corresponding author. Tel: ⫹813-3964-3241x3025; fax: ⫹813-3579-4776. E-mail address: [email protected] (T. Shirasawa). Abbreviations: A, amyloid -protein; AD, Alzheimer’s disease; APP, amyloid precursor protein; eNOS, endothelial nitric oxide synthase; FAD, familial Alzheimer’s disease; iNOS, inducible nitric oxide synthase; L-NAME, NG-nitro-L-arginine methyl ester; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NO, nitric oxide; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase; PBS, Mg2⫹-, Ca2⫹-free phosphate-buffered saline; PS1, presenilin-1; PS2, presenilin-2; TBS, Tris-buffered saline.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.01.016
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increased neuronal vulnerability was mediated by nitric oxide (NO).
EXPERIMENTAL PROCEDURES Animals The generation of PS1 knockout mice has been described in the report of Yuasa et al. (2002). The stages of mouse embryos subjected to analyses were given as embryonic day where the day of vaginal plug was designated 0.5.
Neuronal cultures Neuronal cultures were prepared as described previously (Nakajima et al., 2001). Briefly, primary dissociated cultures were prepared from neocortices and hippocampi of mouse embryos at embryonic day 15.5. The dissociated cells were plated at an initial density of 8⫻104 cells/0.1 ml/well (0.35 cm2) onto plastic 96-well plates pretreated with poly-L-ornithine. The time of cell seeding was designated culture day 0. Experiments were performed on culture days 7– 8. Ninety to one hundred twenty minutes before hydrogen peroxidetreatment (100 M), culture medium was switched to Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 10 mM glucose, 5 mM HEPES buffer, pH 7.2) (Keller et al., 1998) containing drugs such as signaling molecule inhibitors. Then hydrogen peroxide was added to the cultures. Twenty-four hours later, cell viability was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay as described previously (Mosmann, 1983). The MTT reducing activity was expressed as a percentage of the control. Hydrogen peroxide was purchased from WAKO (Osaka, Japan). U0126, SB203580, FTI-277, cyclosporin A (CsA), and Calpain inhibitor II were from Calbiochem-Novabiochem (La Jolla, CA, USA). NGnitro-L-arginine methyl ester (L-NAME) and hemoglobin were from Sigma (St. Louis, MO, USA). SP600125 was obtained from Biomol (Plymouth Meeting, PA, USA), Microcystin-LR from Alexis Biochemicals (San Diego, CA, USA), and Recombinant murine TNF-␣ from R&D systems (Minneapolis, MN, USA).
NADPH-diaphorase staining Cells were washed three times with Mg2⫹-, Ca2⫹-free phosphatebuffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C. The fixative was washed away with Trisbuffered saline (TBS) containing 50 mM Tris–HCl pH 7.4 and 1.5% NaCl. The reaction solution containing 1 mM NADPH, 0.2 mM nitroblue tetrazolium, 0.2% Triton X-100, 1.2 mM sodium azide, and 100 mM Tris–HCl pH 7.2 was applied to the fixed cells for 1 h at 37 °C. The reaction was terminated by washing the reaction solution with TBS (Dawson et al., 1993).
RESULTS MAP kinase signalings do not mediate the death of PS1-deficient neurons induced by hydrogen peroxide To probe the functions of PS1 on neuronal resistance to oxidative stress, we examined the intracellular signalings in cells prepared from wild-type or PS1-deficient mouse embryos. Because PS1-deficient neurons die of calcium-dependent intracellular events (Nakajima et al., 2001) and the involvement of JNK and p38 activation on cell death have been reported in a variety of contexts (Kawasaki et al., 1997; Tobiume et al., 2001; Iwama et al., 2001; Kim et al., 2001; Crossthwaite et al., 2002), we pharmacologically tested whether the activation of calcium-responsible and cell death-
related MAP kinases such as ERK, JNK and p38 is critical for the PS1-deficient neuronal death by oxidative stress. None of inhibitors of a JNK specific inhibitor SP600125, a p38 specific inhibitor SB203580 and a MEK1/2 inhibitor U0126 prevented the death of PS1-deficient neurons induced by hydrogen peroxide (Fig. 1A–C). As Ras and Raf are known to be activated following the increase of intracellular calcium concentration and to lead to the activation of three MAP kinases of ERK, JNK and p38 (Suzuki et al., 1998; Lerner et al., 1995), a selective farnesyltransferase inhibitor FTI-277 that blocks the Ras/Raf signaling was tested for the ability reducing the mutant neuron death. However, this drug also failed to rescue the PS1-deficient neurons from the death by hydrogen peroxide (Fig. 1D). Next, we examined whether TNF-␣ that activates JNK and p38 signalings (Takeda et al., 2003) led the PS1deficient neurons to death, but this factor did not affect the viability of the mutant neurons (Fig. 1E). Thus the activation of MAP kinases was suggested to be irrelevant for the vulnerability of PS1-deficient neurons to oxidative stress. NO signaling mediates PS1-deficient neuron death by hydrogen peroxide We further investigated the involvement of other calciumdependent, cell death-related cellular machineries. As the activation of a protein phosphatase calcineurin or a family of thiol proteases calpains is calcium-dependent and responsible for a variety of cell death (Ankarcrona et al., 1996; Friberg et al., 1998; Choi et al., 2001; Lee et al., 2000), we tested the cell survival-promoting ability of inhibitors against these cellular molecules. An inhibitor of calcineurin CsA at the range of 0.1–1 M did not prevent the PS1-deficient neuron death caused by hydrogen peroxide (data not shown), and neither did an inhibitor of calpain Calpain inhibitor II at 2 M even in the presence (1 M) or absence of a broad range phosphatase inhibitor Microcystin LR, which supports the cell survivalpromoting effect of Carpain inhibitor II for hydrogen peroxidetreated cerebellar granule neurons (See and Loeffler, 2001; data not shown). Then another cellular machinery was examined as a possible candidate of the key players on the mechanism of the PS1-deficient neuron death. NO synthase (NOS) produces NO in calcium-dependent manners and exerts cytotoxic effects in some conditions (Hwang et al., 2002; Dawson et al., 1991, 1993; Jones et al., 1998). A NOS inhibitor L-NAME rescued the PS1-deficient neurons from the death by hydrogen peroxide (Fig. 2A). Furthermore, an NO scavenger hemoglobin also prevented the mutant neuron death (Fig. 2B). L-NAME and hemoglobin did not show any synergistic effect (Fig. 2C), suggesting that NO is a responsible molecule for the death of PS1-deficient neurons by hydrogen peroxide. NADPH-diaphorase activity is enhanced in PS1-deficient neurons To verify the involvement of NO on the PS1-deficient neuron death by oxidative stress, we performed the NADPHdiaphorase assay that can detect the NOS activity in cells (Dawson et al., 1991). As shown in Fig. 3, a strong activity of NADPH-diaphorase was detected around the perinu-
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Fig. 1. MAP kinase signaling does not mediate PS1-deficient neuron death induced by oxidative stress. (A–D) Neuronal cultures prepared from PS1⫹/⫹, PS1⫾ or PS1⫺/⫺ mice were pretreated with Locke’s buffer containing U0126, SP600125, SB203580 or FTI-277. Then the cultures were exposed to 100 M hydrogen peroxide. (E) Neuronal cultures were treated with TNF-␣ without hydrogen peroxide. Values are the mean⫾S.D. of triplicate wells. Similar results were obtained in at least three independent experiments.
clear regions of cultured PS1-deficient neurons, while the activity in wild-type neurons was weak.
DISCUSSION In the previous study, we have reported that PS1-deficient neurons show a vulnerability to oxidative stress and the death mechanism is dependent on calcium influx. In this study, we revealed a process in the death mechanism: an inhibitor of NOS L-NAME rescued the PS1-deficient neurons from the death by hydrogen peroxide. An NO scavenger hemoglobin also prevented the death of the mutant neurons. The PS1-deficient neurons exhibited the augmented NADPH-diaphorase activity, suggesting that PS1
has a role for NO signaling in neurons and confers oxidative stress-resistance on neurons in calcium- and NOdependent manners. Although the enhanced activation of JNK in the PS1deficient embryonic cells by hydrogen peroxide has been reported previously by others (Kim et al., 2001), the vulnerability of the PS1-deficient neurons to oxidative stress was not referred to the activation of JNK. In addition to the specific JNK inhibitor (SP600125), the inhibitor of Ras/Raf signaling that is an upstream signaling to the MAP kinase (FTI-277) failed to rescue the mutant neurons from the death. Furthermore, the viability of the PS1-deficient neurons was not affected by TNF-␣ treatment that activates JNK and p38 signalings (Takeda et al., 2003). These evi-
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Fig. 3. Enhanced NADPH-diaphorase activity in PS1-deficient neurons. Neuronal cultures from wild-type (A) or PS1-deficient (B) mice were subjected to NADPH-diaphorase staining. Note the localization of NADPH-diaphorase activities in the perinuclear region of PS1deficient neurons, as indicated by arrows (B).
Fig. 2. NO signaling triggers off the death of PS1-deficient neurons by oxidative stress. Neuronal cultures prepared from PS1⫹/⫹, PS1⫾ or PS1⫺/⫺ mice were pretreated with Locke’s buffer containing L-NAME and/or hemoglobin. Then the cultures were exposed to hydrogen peroxide (100 M). Values are the mean⫾S.D. of triplicate wells. Similar results were obtained in at least three independent experiments.
dences have prompted us to the conclusion that the enhanced activation of JNK is irrelevant for the vulnerability of the PS1-deficient neurons to oxidative stress. NOS exists in three different isoforms, namely, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). While iNOS is inducible and calciumindependent, nNOS and eNOS are constitutive and calcium-dependent (Nathan and Xie, 1994). Because the hydrogen peroxide-induced death in PS1-deficient neurons is calcium-dependent and is protected by L-NAME that inhibit the activity of nNOS and eNOS, the involvement of nNOS and/or eNOS is suggested for the PS1deficient neuron death by oxidative stress. To examine
which kinds of NOSs were involved in the neuronal death, we performed Western blotting. Anti-nNOS antibody detected nNOS at the similar levels between the neuronal cultures from wild-type/heterozygous mice and homozygous mice and anti-eNOS antibody (BD Transduction Laboratories N30020 monoclonal, San Diego, CA, USA; Fagan et al., 2001) failed to detect eNOS in both cultures (data not shown). However, we obtained the evidences suggesting the involvement of NOS, i.e. the rescue of the PS1-mutant neurons from the hydrogen peroxide-induced death by NO scavenger (hemoglobin) as well as L-NAME, and the enhanced NADPHdiaphorase activity in the PS1-mutant culture. One possible explanation for this discrepancy is that nNOS and/or eNOS might be modulated to active forms by post-translational control such as phosphorylation or acylation (myristoylation and palmitoylation) in the PS1deficient culture (Matsubara et al., 2003; Nathan and Xie, 1994). Since at least eNOS has been reported to interact with various proteins such as caveolin, NOSIP, hsp90 as well as calmodulin (Fulton et al., 2001), NOSs could be modulated by the changes of these molecules in the mutant neurons. Alternatively, eNOS may exist more in amount in the PS1-deficient culture than the control culture at the lower levels than that of our detection limit on Western blotting, which may induce the enhanced production of NO and the neuronal death. The majority of cells cultured from wild-type or PS1deficient mouse brains are immunopositive for a neuronal marker MAP-2, while a few percent of cells are immunostained with a glial cell marker GFAP (Nakajima et al., 2001). NADPH-diaphorase activity was detected in neuron-like cells showing round-shaped soma (Fig. 3), which were killed by hydrogen peroxide. Because NO shows very short half-life in physiological conditions (Mordvintcev et al., 1991; Rossaint et al., 1993), the cells affected by NO are supposed to be restricted to the cells that produce NO by itself and/or that are located in the neighborhood of the NO-producing cells. Thus NO produced in the PS1-deficient cultures would come from
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the neuronal cells rather than the contaminating non-neuronal cells. Giannakopoulos et al. (1997) reported that late-onset sporadic AD brains showed a marked neuronal loss in the CA1 field of the hippocampus and hilus of the dentate gyrus, subiculum, and entorhinal cortex. In these brain areas, the fraction of neurons showing PS1 immunoreactivity was increased compared with that in the nondemented control brains. In contrast, cortical areas, which displayed no neuronal loss, did not show increase in the fraction of PS1-positive neurons. Our data showing that PS1-lacking neurons are vulnerable to oxidative stress, which accumulates with age, are consistent with the hypothesis of Giannakopoulos et al. (1997) that PS1 expression at low levels in specific areas in brains makes neurons vulnerable. PS1 would have a neuroprotective role. Acknowledgements—We thank Drs. T. Shimizu, M. Ogawara, M. Takahashi and E. Moriizumi for their helpful discussions.
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(Accepted 21 January 2004)
PRESENILIN-1-DEFICIENT NEURONS ARE NITRIC OXIDEDEPENDENTLY KILLED BY HYDROGEN PEROXIDE IN VITRO M. NAKAJIMA AND T. SHIRASAWA*
mechanisms via the genetic mutations still need to be defined but the most reliable hypothesis is as follows. The production of the longer form and/or the shorter form of amyloid -protein (A) is promoted by the mutations of APP, PS1, or PS2. These As enhance the formation of amyloid plaques and exhibit neurotoxicity in the forms of insoluble fibrils or soluble oligomers (Hardy and Selkoe, 2002). Most cases of AD are sporadic, but not inherited. However, the hypothesis described for FAD, the amyloid cascade hypothesis, is accepted for the pathogenic mechanisms of the sporadic AD too. On the other hand, a distinctive risk factor is known for AD, aging (Bachman et al., 1993). Oxidative damage accumulates with age. Thus the hypothesis has been proposed that cellular events involving oxidative stress may play a central role in the pathogenic cascade of AD (Benzi and Moretti, 1995), although the oxidative stress itself would not be the primary event. In fact, the involvement of reactive oxygen species with the pathological features of AD including neurofibrillary tangles, senile plaques, mitochondrial dysfunction, and microglial activation has been reported (Smith et al., 1996; Blass et al., 1990; Colton and Gilbert, 1987). Oxidative stress may be involved in selective neuronal death in AD, in concert with the amyloid cascade. PS1 is suggested to mediate the processing of proteins such as APP, Notch-1, ErbB4, and E-cadherin (Ebinu and Yankner, 2002; Marambaud et al., 2002). In addition, PS1 has been reported for the involvement in membrane protein trafficking, unfolded protein response, and capacitative calcium entry (Naruse et al., 1998; Niwa et al., 1999; Yoo et al., 2000). Developmental functions of PS1 are also suggested in neurogenesis, somatogenesis, and angiogenesis (Shen et al., 1997; Handler et al., 2000; Yuasa et al., 2002; Wong et al., 1997; Koizumi et al., 2001; Nakajima et al., 2003). Although PS1 is a causative gene for FAD, the possible involvement of PS1 in sporadic AD has been examined pathologically: Immunohistochemical analysis of sporadic AD brains for PS1 suggested that PS1 may have a neuroprotective role and that in AD low cellular expression of PS1 protein may be associated with increased neuronal loss (Giannakopoulos et al., 1997). Consistent with this finding, we found in the previous study that CNS neurons cultured from PS1-deficient mice exhibited increased vulnerability to oxidative stress in calciumdependent manners (Nakajima et al., 2001). In the present study, we further investigated the underlying mechanisms of the increased vulnerability of PS1-deficient neurons to oxidative stress and found that the
Department of Molecular Gerontology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan
Abstract—Presenilin-1 (PS1) is the gene responsible for the development of early-onset familial Alzheimer’s disease. To probe the functions of PS1 on neuronal resistance to oxidative stress, we pharmacologically examined the death signals in PS1-deficient neurons induced by oxidative stress. Because the death of primarily cultured neurons lacking PS1 is caused by hydrogen peroxide in calciumdependent manners in vitro [J Neurochem 78 (2001) 807], we tested the neuronal survival-promoting ability of inhibitors against calcium-dependent/cell death-related signaling molecules, such as ERKs, JNK, p38 MAP kinase, calcineurin, calpain, and nitric oxide synthase (NOS). All inhibitors tested failed to rescue the PS1-deficient neurons from the death with the exception of an inhibitor of NOS, NG-nitro-L-arginine methyl ester. Hemoglobin, a nitric oxide (NO) scavenger, also prevented the death of the mutant neurons. NADPH-diaphorase staining, which accounts for NOS activity, was enhanced in the mutant neurons. These results suggest that PS1 has a role for NOS activation in neurons and confers oxidative stress-resistance on neurons in calcium/ NO-dependent manners. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hemoglobin, NADPH-diaphorase, nitric oxide synthase, L-NAME, JNK, p38.
Alzheimer’s disease (AD) is characterized by progressive neuronal cell loss, the presence of senile plaques and neurofibrillary tangles in the brain. Approximately 10 –17% of AD cases are inherited in autosomal dominant manner (Cruts and Van Broeckhoven, 1998). Three genes were identified so far as causative genes for these familial AD (FAD); the amyloid precursor protein (APP), the presenilin-1 (PS1), and the presenilin-2 (PS2; Chartier et al., 1991; Goate et al., 1991; Alzheimer Disease Collaborative Group, 1995; Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995; Selkoe, 1996). FAD is caused dominantly by mutations in these three genes. Mutations in PS1 are the most common cases of FAD. Pathogenic *Corresponding author. Tel: ⫹813-3964-3241x3025; fax: ⫹813-3579-4776. E-mail address: [email protected] (T. Shirasawa). Abbreviations: A, amyloid -protein; AD, Alzheimer’s disease; APP, amyloid precursor protein; eNOS, endothelial nitric oxide synthase; FAD, familial Alzheimer’s disease; iNOS, inducible nitric oxide synthase; L-NAME, NG-nitro-L-arginine methyl ester; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NO, nitric oxide; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase; PBS, Mg2⫹-, Ca2⫹-free phosphate-buffered saline; PS1, presenilin-1; PS2, presenilin-2; TBS, Tris-buffered saline.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.01.016
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increased neuronal vulnerability was mediated by nitric oxide (NO).
EXPERIMENTAL PROCEDURES Animals The generation of PS1 knockout mice has been described in the report of Yuasa et al. (2002). The stages of mouse embryos subjected to analyses were given as embryonic day where the day of vaginal plug was designated 0.5.
Neuronal cultures Neuronal cultures were prepared as described previously (Nakajima et al., 2001). Briefly, primary dissociated cultures were prepared from neocortices and hippocampi of mouse embryos at embryonic day 15.5. The dissociated cells were plated at an initial density of 8⫻104 cells/0.1 ml/well (0.35 cm2) onto plastic 96-well plates pretreated with poly-L-ornithine. The time of cell seeding was designated culture day 0. Experiments were performed on culture days 7– 8. Ninety to one hundred twenty minutes before hydrogen peroxidetreatment (100 M), culture medium was switched to Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 10 mM glucose, 5 mM HEPES buffer, pH 7.2) (Keller et al., 1998) containing drugs such as signaling molecule inhibitors. Then hydrogen peroxide was added to the cultures. Twenty-four hours later, cell viability was determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay as described previously (Mosmann, 1983). The MTT reducing activity was expressed as a percentage of the control. Hydrogen peroxide was purchased from WAKO (Osaka, Japan). U0126, SB203580, FTI-277, cyclosporin A (CsA), and Calpain inhibitor II were from Calbiochem-Novabiochem (La Jolla, CA, USA). NGnitro-L-arginine methyl ester (L-NAME) and hemoglobin were from Sigma (St. Louis, MO, USA). SP600125 was obtained from Biomol (Plymouth Meeting, PA, USA), Microcystin-LR from Alexis Biochemicals (San Diego, CA, USA), and Recombinant murine TNF-␣ from R&D systems (Minneapolis, MN, USA).
NADPH-diaphorase staining Cells were washed three times with Mg2⫹-, Ca2⫹-free phosphatebuffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C. The fixative was washed away with Trisbuffered saline (TBS) containing 50 mM Tris–HCl pH 7.4 and 1.5% NaCl. The reaction solution containing 1 mM NADPH, 0.2 mM nitroblue tetrazolium, 0.2% Triton X-100, 1.2 mM sodium azide, and 100 mM Tris–HCl pH 7.2 was applied to the fixed cells for 1 h at 37 °C. The reaction was terminated by washing the reaction solution with TBS (Dawson et al., 1993).
RESULTS MAP kinase signalings do not mediate the death of PS1-deficient neurons induced by hydrogen peroxide To probe the functions of PS1 on neuronal resistance to oxidative stress, we examined the intracellular signalings in cells prepared from wild-type or PS1-deficient mouse embryos. Because PS1-deficient neurons die of calcium-dependent intracellular events (Nakajima et al., 2001) and the involvement of JNK and p38 activation on cell death have been reported in a variety of contexts (Kawasaki et al., 1997; Tobiume et al., 2001; Iwama et al., 2001; Kim et al., 2001; Crossthwaite et al., 2002), we pharmacologically tested whether the activation of calcium-responsible and cell death-
related MAP kinases such as ERK, JNK and p38 is critical for the PS1-deficient neuronal death by oxidative stress. None of inhibitors of a JNK specific inhibitor SP600125, a p38 specific inhibitor SB203580 and a MEK1/2 inhibitor U0126 prevented the death of PS1-deficient neurons induced by hydrogen peroxide (Fig. 1A–C). As Ras and Raf are known to be activated following the increase of intracellular calcium concentration and to lead to the activation of three MAP kinases of ERK, JNK and p38 (Suzuki et al., 1998; Lerner et al., 1995), a selective farnesyltransferase inhibitor FTI-277 that blocks the Ras/Raf signaling was tested for the ability reducing the mutant neuron death. However, this drug also failed to rescue the PS1-deficient neurons from the death by hydrogen peroxide (Fig. 1D). Next, we examined whether TNF-␣ that activates JNK and p38 signalings (Takeda et al., 2003) led the PS1deficient neurons to death, but this factor did not affect the viability of the mutant neurons (Fig. 1E). Thus the activation of MAP kinases was suggested to be irrelevant for the vulnerability of PS1-deficient neurons to oxidative stress. NO signaling mediates PS1-deficient neuron death by hydrogen peroxide We further investigated the involvement of other calciumdependent, cell death-related cellular machineries. As the activation of a protein phosphatase calcineurin or a family of thiol proteases calpains is calcium-dependent and responsible for a variety of cell death (Ankarcrona et al., 1996; Friberg et al., 1998; Choi et al., 2001; Lee et al., 2000), we tested the cell survival-promoting ability of inhibitors against these cellular molecules. An inhibitor of calcineurin CsA at the range of 0.1–1 M did not prevent the PS1-deficient neuron death caused by hydrogen peroxide (data not shown), and neither did an inhibitor of calpain Calpain inhibitor II at 2 M even in the presence (1 M) or absence of a broad range phosphatase inhibitor Microcystin LR, which supports the cell survivalpromoting effect of Carpain inhibitor II for hydrogen peroxidetreated cerebellar granule neurons (See and Loeffler, 2001; data not shown). Then another cellular machinery was examined as a possible candidate of the key players on the mechanism of the PS1-deficient neuron death. NO synthase (NOS) produces NO in calcium-dependent manners and exerts cytotoxic effects in some conditions (Hwang et al., 2002; Dawson et al., 1991, 1993; Jones et al., 1998). A NOS inhibitor L-NAME rescued the PS1-deficient neurons from the death by hydrogen peroxide (Fig. 2A). Furthermore, an NO scavenger hemoglobin also prevented the mutant neuron death (Fig. 2B). L-NAME and hemoglobin did not show any synergistic effect (Fig. 2C), suggesting that NO is a responsible molecule for the death of PS1-deficient neurons by hydrogen peroxide. NADPH-diaphorase activity is enhanced in PS1-deficient neurons To verify the involvement of NO on the PS1-deficient neuron death by oxidative stress, we performed the NADPHdiaphorase assay that can detect the NOS activity in cells (Dawson et al., 1991). As shown in Fig. 3, a strong activity of NADPH-diaphorase was detected around the perinu-
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Fig. 1. MAP kinase signaling does not mediate PS1-deficient neuron death induced by oxidative stress. (A–D) Neuronal cultures prepared from PS1⫹/⫹, PS1⫾ or PS1⫺/⫺ mice were pretreated with Locke’s buffer containing U0126, SP600125, SB203580 or FTI-277. Then the cultures were exposed to 100 M hydrogen peroxide. (E) Neuronal cultures were treated with TNF-␣ without hydrogen peroxide. Values are the mean⫾S.D. of triplicate wells. Similar results were obtained in at least three independent experiments.
clear regions of cultured PS1-deficient neurons, while the activity in wild-type neurons was weak.
DISCUSSION In the previous study, we have reported that PS1-deficient neurons show a vulnerability to oxidative stress and the death mechanism is dependent on calcium influx. In this study, we revealed a process in the death mechanism: an inhibitor of NOS L-NAME rescued the PS1-deficient neurons from the death by hydrogen peroxide. An NO scavenger hemoglobin also prevented the death of the mutant neurons. The PS1-deficient neurons exhibited the augmented NADPH-diaphorase activity, suggesting that PS1
has a role for NO signaling in neurons and confers oxidative stress-resistance on neurons in calcium- and NOdependent manners. Although the enhanced activation of JNK in the PS1deficient embryonic cells by hydrogen peroxide has been reported previously by others (Kim et al., 2001), the vulnerability of the PS1-deficient neurons to oxidative stress was not referred to the activation of JNK. In addition to the specific JNK inhibitor (SP600125), the inhibitor of Ras/Raf signaling that is an upstream signaling to the MAP kinase (FTI-277) failed to rescue the mutant neurons from the death. Furthermore, the viability of the PS1-deficient neurons was not affected by TNF-␣ treatment that activates JNK and p38 signalings (Takeda et al., 2003). These evi-
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Fig. 3. Enhanced NADPH-diaphorase activity in PS1-deficient neurons. Neuronal cultures from wild-type (A) or PS1-deficient (B) mice were subjected to NADPH-diaphorase staining. Note the localization of NADPH-diaphorase activities in the perinuclear region of PS1deficient neurons, as indicated by arrows (B).
Fig. 2. NO signaling triggers off the death of PS1-deficient neurons by oxidative stress. Neuronal cultures prepared from PS1⫹/⫹, PS1⫾ or PS1⫺/⫺ mice were pretreated with Locke’s buffer containing L-NAME and/or hemoglobin. Then the cultures were exposed to hydrogen peroxide (100 M). Values are the mean⫾S.D. of triplicate wells. Similar results were obtained in at least three independent experiments.
dences have prompted us to the conclusion that the enhanced activation of JNK is irrelevant for the vulnerability of the PS1-deficient neurons to oxidative stress. NOS exists in three different isoforms, namely, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). While iNOS is inducible and calciumindependent, nNOS and eNOS are constitutive and calcium-dependent (Nathan and Xie, 1994). Because the hydrogen peroxide-induced death in PS1-deficient neurons is calcium-dependent and is protected by L-NAME that inhibit the activity of nNOS and eNOS, the involvement of nNOS and/or eNOS is suggested for the PS1deficient neuron death by oxidative stress. To examine
which kinds of NOSs were involved in the neuronal death, we performed Western blotting. Anti-nNOS antibody detected nNOS at the similar levels between the neuronal cultures from wild-type/heterozygous mice and homozygous mice and anti-eNOS antibody (BD Transduction Laboratories N30020 monoclonal, San Diego, CA, USA; Fagan et al., 2001) failed to detect eNOS in both cultures (data not shown). However, we obtained the evidences suggesting the involvement of NOS, i.e. the rescue of the PS1-mutant neurons from the hydrogen peroxide-induced death by NO scavenger (hemoglobin) as well as L-NAME, and the enhanced NADPHdiaphorase activity in the PS1-mutant culture. One possible explanation for this discrepancy is that nNOS and/or eNOS might be modulated to active forms by post-translational control such as phosphorylation or acylation (myristoylation and palmitoylation) in the PS1deficient culture (Matsubara et al., 2003; Nathan and Xie, 1994). Since at least eNOS has been reported to interact with various proteins such as caveolin, NOSIP, hsp90 as well as calmodulin (Fulton et al., 2001), NOSs could be modulated by the changes of these molecules in the mutant neurons. Alternatively, eNOS may exist more in amount in the PS1-deficient culture than the control culture at the lower levels than that of our detection limit on Western blotting, which may induce the enhanced production of NO and the neuronal death. The majority of cells cultured from wild-type or PS1deficient mouse brains are immunopositive for a neuronal marker MAP-2, while a few percent of cells are immunostained with a glial cell marker GFAP (Nakajima et al., 2001). NADPH-diaphorase activity was detected in neuron-like cells showing round-shaped soma (Fig. 3), which were killed by hydrogen peroxide. Because NO shows very short half-life in physiological conditions (Mordvintcev et al., 1991; Rossaint et al., 1993), the cells affected by NO are supposed to be restricted to the cells that produce NO by itself and/or that are located in the neighborhood of the NO-producing cells. Thus NO produced in the PS1-deficient cultures would come from
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the neuronal cells rather than the contaminating non-neuronal cells. Giannakopoulos et al. (1997) reported that late-onset sporadic AD brains showed a marked neuronal loss in the CA1 field of the hippocampus and hilus of the dentate gyrus, subiculum, and entorhinal cortex. In these brain areas, the fraction of neurons showing PS1 immunoreactivity was increased compared with that in the nondemented control brains. In contrast, cortical areas, which displayed no neuronal loss, did not show increase in the fraction of PS1-positive neurons. Our data showing that PS1-lacking neurons are vulnerable to oxidative stress, which accumulates with age, are consistent with the hypothesis of Giannakopoulos et al. (1997) that PS1 expression at low levels in specific areas in brains makes neurons vulnerable. PS1 would have a neuroprotective role. Acknowledgements—We thank Drs. T. Shimizu, M. Ogawara, M. Takahashi and E. Moriizumi for their helpful discussions.
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(Accepted 21 January 2004)