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Transgenic murine cortical neurons expressing human Bcl-2 exhibit increased resistance to amyloid β-peptide neurotoxicity
Pergamon PII:
Neuroscience Vol. 92, No. 4, pp. 1455–1463, 1999 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(99)00089-5
TRANSGENIC MURINE CORTICAL NEURONS EXPRESSING HUMAN Bcl-2 EXHIBIT INCREASED RESISTANCE TO AMYLOID b-PEPTIDE NEUROTOXICITY C. SAILLE´,* P. MARIN,† J.-C. MARTINOU,‡ A. NICOLE,* J. LONDON* and I. CEBALLOS-PICOT*k *CNRS UMR 8602, CHU Necker—Enfants Malades, 156 Rue de Vaugirard, 75743 Paris Cedex 15, France †INSERM U 114, Colle`ge de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France ‡Serono Pharmaceutical Research Institute, 14 Chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland
Abstract—The generation of reactive oxygen species has been implicated in the neurotoxicity of amyloid b-peptide, the main constituent of the senile plaques that accumulates in the brain of Alzheimer’s disease victims. In this study, we have compared the toxicity of amyloid b-peptide on cultured cortical neurons from control mice and transgenic mice expressing either human copper–zinc superoxide dismutase or human Bcl-2, two proteins that protect cells against oxidative damage. Copper–zinc superoxide dismutase overexpression failed to protect cortical neurons against the toxicity of amyloid b-peptide(25–35) [the minimal cytotoxic fragment of amyloid b-peptide(1–42)] as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide reduction and an enzyme-linked immunoabsorbent assay using an antibody directed against microtubule-associated protein-2 (a specific neuronal protein), ruling out a role for superoxide anion and peroxynitrite in amyloid b-peptide-evoked neurotoxicity. On the contrary, cortical neurons expressing human copper–zinc superoxide dismutase exhibited increased apoptotic nuclei in both untreated and amyloid b-peptide(25–35)-exposed neurons. Transgenic neurons expressing human Bcl-2 were partially protected against amyloid b-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid b-peptide(25–35) and amyloid b-peptide(1–42). This study may be relevant for developing neuroprotective gene therapy to inhibit neuronal apoptosis in Alzheimer’s disease. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: amyloid b-peptide, Alzheimer’s disease, apoptosis, copper–zinc superoxide dismutase, Bcl-2, neuroprotection.
The pathological hallmark of Alzheimer’s disease (AD) is the accumulation of numerous senile plaques associated with degenerating neurons. Amyloid b-peptide (Ab) is a 40–42-amino-acid peptide that is the main constituent of senile plaques. Ab is derived from a larger membrane-spanning glycoprotein called amyloid precursor protein (APP), as a result of proteolytic processing. 21 Several observations suggest that Ab is involved in the pathogenesis of AD: Ab is toxic to neurons in vitro 25,33,41 and in vivo; 32 mutations in the APP locus, which have been linked to early-onset familial AD, lead to overproduction of Ab, 8,13 and transgenic mice expressing these mutations display many of the kTo whom correspondence should be addressed. Abbreviations: Ab, amyloid b-peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; CuZnSOD, copper–zinc superoxide dismutase; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; h-Bcl-2, human Bcl-2; h-CuZnSOD, human CuZnSOD; HEPES, N2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid; MAP-2, microtubule-associated protein-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphatebuffered saline; ROS, reactive oxygen species.
pathological features of AD; 18 mutations of the presenilin-1 gene, which are responsible for the majority of cases of inherited early-onset familial AD, also lead to an elevated production of Ab, 6 and Ab neurotoxicity is exacerbated greatly in cells expressing mutated presenilin-1. 20 Several lines of evidence suggest that the overproduction of reactive oxygen species (ROS) is implicated in Ab neurotoxicity: (i) exposure of cultured neurons or neuronal cell lines to Ab increases the intracellular levels of ROS, leading to the activation of nuclear factor-kB, a redoxmodulated transcription factor involved in Abevoked neurotoxicity; 4,28,56 (ii) the neurotoxicity of Ab is attenuated by the prevention of intracellular glutathione depletion, 36 antioxidants 2,4 and/or free radical scavengers; 22 (iii) antioxidants protect cells against the adverse consequences of presenilin-1 mutations; 20 (iv) in AD brain, regional heterogeneity in the efficiency of free radical defence mechanisms appears to account for the regional heterogeneity of cell vulnerability. 12 Several enzymes have been shown to contribute to cell defence against the deleterious consequences of ROS overproduction.
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Among these enzymes, increased copper–zinc superoxide dismutase (CuZnSOD) activity protects neurons and other cell types against oxidative stress and nerve growth factor deprivation. 19,27,45 By contrast, CuZnSOD down-regulation accelerates spontaneous cell death. 53 In addition to these enzymatic processes, protective effects of the oncogenic gene product Bcl-2 have been reported in various neuronal death paradigms implicating overproduction of ROS. Indeed, overexpression of Bcl-2 in neuronal cells prevents apoptosis induced by oxidative stress associated with glutathione depletion 29 or by 4-hydroxynonenal (an aldehydic product of lipid peroxidation), 31 suggesting that Bcl-2 could act as an antioxidant that suppresses the formation or action of ROS. The present study was undertaken to investigate the neuroprotective effect of CuZnSOD and Bcl-2 against the toxicity evoked by Ab. For that purpose, the decrease in cell viability and the apoptosis induced by Ab were compared on cultured cortical neurons from control mice, and transgenic mice expressing either human CuZnSOD (h-CuZnSOD) or human bcl-2 (h-bcl-2) genes. EXPERIMENTAL PROCEDURES
Materials FVB/N and C57BL/6 × DBA/2 (B6D2) mice were from Iffa–Credo (Lyon, France); heterozygous transgenic mice overexpressing h-CuZnSOD (h-CuZnSOD transgenic) or h-bcl-2 (h-Bcl-2 transgenic) were described previously. 14,38 Culture media were from Life Technology (Cergy-Pontoise, France), fetal calf serum (FCS) from Dutcher (Brumath, France), 96- and four-well culture dishes from Nunc (Denmark), mouse monoclonal anti-microtubule-associated protein-2 (anti-MAP-2) antibody from Biomakor (Rehovot, Israel), monoclonal anti-human Bcl-2 antibody from DAKO (clone 124), peroxidase-coupled goat anti-mouse or antirabbit immunoglobulin G from Biosys (Compie`gne, France), Ab(1–42), Ab(25–35) and Ab(35–25) from Bachem (France), Hoechst 33258 from Molecular Probe (Eugene, OR, U.S.A.) and all other chemicals from Sigma (Saint Quentin Fallavier, France). Primary culture of cortical neurons All studies were conducted according to the EC legislation concerning animal care. Primary neuronal cultures were prepared according to Weiss et al. 55 Cortices were removed from 15–16-day-old FVB/N, B6D2, h-CuZnSOD transgenic or h-Bcl-2 transgenic mouse embryos and dissociated mechanically with a Pasteur pipette in phosphate-buffered saline (PBS) supplemented with glucose (33 mM). Cells were plated on to either 96-well culture dishes (1 × 10 5 cells per well), glass coverslips in four-well plates (3 × 10 5 cells per well) or six-well culture dishes (3 × 10 6 cells per well), successively coated with poly-l-ornithine (mol. wt 40,000, 15 mg/ml) and FCS (10% v/v in culture medium). Serum-free medium consists of a mixture (1:1, v/v) of Dulbecco’s modified essential medium and Ham’s F12 nutrient, supplemented with glucose (33 mM), glutamine (2 mM), NaHCO3 (13 mM), HEPES buffer (5 mM, pH 7.4), penicillin and streptomycin (5 IU/ml and 5 mg/ ml, respectively), and a mixture of salt and hormones containing insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM) and sodium
selenite (Na2SeO3; 30 nM). Cells were cultured at 378C in a humidified atmosphere of 92% air/8% CO2. Cultures, used six days after seeding, were shown to be highly enriched in neurons by immunocytochemistry for MAP-2 and devoid of any detectable glial elements, as assessed by immunocytochemistry for glial fibrillary acidic protein (data not shown). Treatments with amyloid b -peptides Ab(25–35) and Ab(35–25) stock solutions (700 mM in water), and Ab(1–42) stock solution (1 mM in 8.75% acetonitrile/0.25% trifluoroacetic acid) were diluted in serumfree medium (without Ham’s F12 nutrient) just before use, to obtain a ×10 concentrated solution. Concentrated peptides were added to the culture medium for 24 h to reach the appropriate concentrations. Neuronal viability measurement 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Twenty-four hours after the onset of Ab treatment, neurons grown in 96-well culture dishes were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 0.5 mg/ml in PBS containing 33 mM glucose) at 378C for 1 h. The formazan product was dissolved in 100 ml dimethyl sulfoxide and quantified by measuring the optical density at 560 nm. Enzyme-linked immunosorbent assay for microtubuleassociated protein-2. Twenty-four hours after the onset of Ab treatment, neurons grown in 96-well culture dishes were fixed in 100 ml paraformaldehyde (4% w/v in PBS) at 48C for 1 h, washed twice with PBS containing glycine (0.1 M) and PBS/Tween-20 (0.1%, v/v). They were incubated for 1 h at 378C successively with PBS/Tween-20 supplemented with FCS (10%, v/v), a mouse monoclonal anti-MAP-2 antibody (immunoglobulin G1, 1:4000, v/v, in PBS/Tween-20 containing 10% FCS) and peroxidasecoupled goat anti-mouse immunoglobulin G (1:3000, v/v, in PBS/Tween-20 containing 10% FCS). Cells were then incubated with 0.04% (w/v) o-phenylenediamine and 0.012% (v/v) H2O2, diluted in citrate buffer (pH 5), for 5 min. The reaction was stopped by adding H2SO4 (final concentration 0.15 M). MAP-2 was quantified by reading the optical density at 490 nm. Detection and measurement of apoptosis Nuclear condensation and fragmentation (some classical hallmarks of apoptosis) were detected by staining the nuclei with the fluorescent probe Hoechst 33258. Cortical neurons, grown on glass coverslips, were treated with either 10 mM Ab(25–35) or 20 mM Ab(1–42) for 24 h. Cells were fixed in methanol and stained with Hoechst 33258 (1 mg/ml in methanol) for 15 min at 378C. After washing, coverslips were mounted on glass slides in a medium for immunofluorescence (Fluoprep, BioMerieux). Fluorescence microscopy was performed using a Leitz Laborlux S microscope (lex 345 nm, lem 478 nm). Photomicrographs were taken for apoptotic cell counting. Dense fluorescent and fragmented nuclei were considered as apoptotic. Detection of transgene expression Copper–zinc superoxide dismutase activity determination. Neurons grown on six-well culture dishes were scraped off in ice-cold PBS and centrifuged at 200 × g for 5 min. The pellets were frozen and kept in liquid nitrogen until use. The pellets were homogenized in a sodium phosphate buffer (50 mM, pH 7.4) and samples were centrifuged at 20,000 × g for 30 min at 48C. Supernatants were used for protein measurement and determination of CuZnSOD activity, as described previously. 11
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Immunofluorescence detection of the human Bcl-2 protein. Cortical neurons grown on glass coverslips were fixed in paraformaldehyde (4% in PBS) for 1 h at 48C. After a 1-h preincubation at 378C with PBS containing 5% normal goat serum (solution A), cells were incubated with a mouse anti-h-Bcl-2 antibody (DAKO; 1:20, v/v, in solution A) for 1 h at room temperature. Coverslips were intensively rinsed out before incubation with a rabbit anti-mouse immunoglobulin G conjugated with fluorescein (1:50, v/v, in solution A) for 1 h at room temperature. Human Bcl-2 was detected by fluorescence microscopy, as described above. Western blot analysis. Cortical neurons grown on sixwell culture dishes for six days were harvested in 200 ml sodium dodecyl sulfate (2%). Protein concentration was determined using the bicinchoninic acid method. Proteins (50 mg per lane) were resolved on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred on to polyvinylidene fluoride membranes (Immobilon, Millipore). Membranes were blocked by incubation for 1 h at room temperature in Tris-buffered saline containing 0.05% Tween-20 plus 5% non-fat milk. They were then incubated with either a monoclonal anti-h-Bcl-2 antibody (1:200 dilution) or a polyclonal anti-h-CuZnSOD antibody (1:1000 dilution), 10 followed, respectively, by either an anti-mouse immunoglobulin G or an anti-rabbit immunoglobulin G peroxidase-conjugated antibody (1:1000 dilution). Bcl-2 and CuZnSOD proteins were detected using enhanced chemiluminescence with the ECL kit (Amersham, France) and Fuji films were exposed to the blots. Statistical analysis Statistical analysis was performed by ANOVA followed by a Scheffe´ F-test, unless indicated otherwise. RESULTS
Neurotoxicity of amyloid b -peptide on murine cortical neurons from FVB/N and B6D2 mice A 24-h Ab(25–35) treatment induced a concentration-dependent decrease in MTT reduction in cortical neurons from both genetic backgrounds (Figs 1c, 2c). On FVB/N genetic background control neurons, 1 mM Ab(25–35) had little effect, while 10 and 20 mM decreased MTT reduction to 55.5 ^ 3.6% and 41.0 ^ 8.5%, respectively, compared with control values (mean ^ S.D. of data obtained in four independent experiments performed in triplicate; Fig. 1c). The inverted sequence Ab(35–25) (20 mM) had no effect on cell viability (data not shown). Ab(25–35) (1 mM) induced a slight but non-significant decrease in MTT reduction in neurons from B6D2 mice (Fig. 2c), while 10 and 20 mM Ab(25–35) decreased MTT reduction by 42.5 ^ 4.5% and 69.0 ^ 2.8%, respectively (n 4). Since the inhibition of MTT reduction by Ab(25– 35) could be independent of cellular loss, 49 the amount of neurons was quantified by an MAP-2 enzyme-linked immunosorbent assay (ELISA; a specific marker of neurons) after Ab peptide treatments. On B6D2 genetic background neurons, increasing concentrations of Ab(25–35) (1– 20 mM) induced a decrease in the amount of MAP-2 (Fig. 2d) similar to the magnitude of the
Fig. 1. Ab-induced neuronal death is not modified by CuZnSOD overexpression. (a) The specific CuZnSOD enzymatic activity evaluated in wild-type (WT) and h-CuZnSOD Tg neurons after six days in culture. Data are the means ^ S.D. of results obtained in eight separate cultures. (b) A representative western blot experiment showing human and mouse CuZnSOD expression in transgenic cortical neurons compared with wildtype. (c, d) Cortical neurons were treated for 24 h with increasing concentrations of Ab(25–35). Cell viability, evaluated by MTT reduction (c), and cell number, measured by MAP-2 ELISA (d), are expressed as a percentage of control values and represent the means ^ S.D. of results obtained in four experiments performed on different cultures. **P , 0.01, Ab(25–35) vs vehicle (ANOVA followed by Scheffe´ F-test). ***P , 0.001, h-CuZnSOD transgenic vs wild-type (Student’s t-test).
decrease in MTT reduction (Fig. 2c). Nevertheless, on FVB/N genetic background neurons, the decrease in cell number evaluated by MAP-2 quantification (Fig. 1d) was higher than the magnitude of MTT reduction evoked by the same concentrations of Ab(25–35) (Fig. 1c). Taken together, these results indicate that the decrease in MTT reduction following Ab peptide treatments is related to cell loss and that a 24-h treatment with 10–20 mM Ab(25–35) induces marked neuronal death on cultured neurons from both genetic backgrounds.
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Fig. 2. Bcl-2 overexpression partially protects neurons from Ab-induced cell death. (a, b) Photomicrographs of cortical neurons from wild-type (WT) mice (a) and transgenic mice expressing h-Bcl-2 (h-Bcl-2 Tg; b) immunostained with the anti-h-Bcl-2 antibody. The data illustrated are representative of three experiments performed on different cultures. Omitting the anti-h-Bcl-2 antibody yielded a staining identical to that obtained on wild-type neurons (data not shown). Inset in (a) illustrates a representative western blot showing h-Bcl-2 expression in hBcl-2 Tg cortical neurons compared with wild-type. (c, d) Cell viability, evaluated by MTT reduction (c), and cell number, measured by MAP-2 ELISA (d), are expressed as a percentage of control values and represent the means ^ S.D. of results obtained in four experiments performed on different cultures. **P , 0.01, Ab vs vehicle. $$ P , 0.01, h-Bcl-2 Tg vs wild-type. Scale bar 50 mm. MW, protein molecular weight markers.
Overexpression of copper–zinc superoxide dismutase does not protect cortical neurons against amyloid b -peptide-induced neuronal death CuZnSOD activity quantified in transgenic neurons from mice overexpressing CuZnSOD was increased two-fold (57.1 ^ 4.3 IU/mg protein, mean ^ S.D. of eight determinations performed on different cultures) compared with that measured in control cells (29.3 ^ 4.7 IU/mg protein, n 8) (Fig. 1a). This increase in CuZnSOD activity is probably the consequence of the strong expression of h-CuZnSOD in transgenic neurons, as assessed by western blot experiments performed with an antibody that recognizes both human and mouse CuZnSOD (Fig. 1b). Viability of control and transgenic neurons, estimated by either MTT reduction or MAP-2 quantification, was identical after seven days in culture (data not shown). Moreover, the neurotoxicity evoked by increasing concentrations of Ab (1–20 mM) was similar in control neurons and neurons overexpressing CuZnSOD (Fig. 1c, d). Bcl-2 overexpression reduces amyloid b -peptideinduced neuronal death Immunostaining experiments performed with the anti-h-Bcl-2 antibody showed a strong expression of
h-Bcl-2 in transgenic neurons (Fig. 2b). Human Bcl2 was localized in cell bodies as well as in neuronal processes (Fig. 2b). The strong expression of h-Bcl2 in cultured transgenic neurons was confirmed by western blotting (Fig. 2a, inset). Human Bcl-2 overexpression did not alter neuronal viability evaluated after seven days in culture (data not shown). However, h-bcl-2 transgene expression protected neurons against the toxicity induced by 10 and 20 mM Ab(25–35) (about 30% and 50% of protection as assessed by the MTT assay and the MAP-2 ELISA, respectively; Fig. 2c, d). Inhibition of amyloid b -peptide-caused apoptosis by human bcl-2 but not by human copper–zinc superoxide dismutase transgene expression Experiments were performed to look for the consequences of CuZnSOD and Bcl-2 overexpression on Ab(25–35)- and Ab(1–42)-induced apoptosis. After seven days in culture, about 10–15% of untreated neurons exhibited apoptotic nuclei in both mouse strains (Figs 3e, 4e). The concentration of Ab(25–35) providing a decrease in cell viability of about 50% (10 mM) increased the percentage of apoptotic nuclei to about 25% in both cell strains (Figs 3e, 4e), indicating that both apoptotic and
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Fig. 3. Effect of CuZnSOD overexpression on spontaneous and Ab-induced apoptosis. Neurons, treated for 24 h with 10 mM of Ab(25–35), were fixed in methanol and stained with Hoechst 33258. Fluorescence photomicrographs representative of three experiments performed on different cultures are illustrated. (a) Wild-type (WT) neurons treated with vehicle. (b) Wild-type neurons treated with 10 mM of Ab(25–35). (c) Human CuZnSOD transgenic neurons (h-CuZnSOD Tg) treated with vehicle. (d) h-CuZnSOD Tg neurons treated with 10 mM of Ab(25–35). (e) The quantification of apoptotic nuclei. Dense and fragmented nuclei (arrow and arrowhead, respectively) were counted as apoptotic. Data, expressed as a percentage of total nuclei, represent the mean ^ S.D. of values obtained in three to five fields from at least three different cultures (35–100 nuclei per field). **P , 0.01, Ab(25–35) vs vehicle. $$P , 0.01, h-CuZnSOD transgenic vs wild-type. Scale bar 20 mm.
necrotic pathways occurred in Ab(25–35)-treated neurons. As expected, the number of apoptotic nuclei was similarly increased by a 24-h exposure to Ab(1–42) (20 mM; Fig. 4f). Expression of h-CuZnSOD transgene in cortical neurons did not protect them against Ab(25–35)induced apoptosis. On the contrary, it should be noted that CuZnSOD overexpression slightly but significantly increased the level of apoptotic nuclei in both untreated and Ab-exposed neurons (Fig. 3e). In contrast to what was observed in neurons overexpressing CuZnSOD, Bcl-2 overexpression, which did not alter spontaneous apoptosis (Fig. 4e), suppressed the increase in the percentage of apoptotic nuclei evoked by both Ab(25–35) (Fig. 4e) and Ab(1–42) (Fig. 4f).
DISCUSSION
In this study, we show that overexpression of the antiapoptotic/antioxidant protein Bcl-2 suppresses Ab-induced apoptosis in cultured cortical neurons. By contrast, CuZnSOD overexpression did not reduce the apoptotic effect of Ab. The mode of cell death, apoptosis versus necrosis, induced by Ab is still a matter of debate. Apoptosis is selectively increased in primary cultured neurons exposed to Ab 1,17,33,54 and in the brain of transgenic mice overproducing Ab. 32 Both types of cell death were reported on neuronal cell lines depending on the concentration of Ab used, 31 and finally, Ab only triggered necrosis on PC12 cells. 5 In our study, only half of the cell loss, assessed by MAP-2 ELISA or
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Fig. 4. Inhibition of Ab(25–35)- and Ab(1–42)-induced apoptosis in neurons overexpressing Bcl-2. Fluorescence photomicrographs representative of three experiments performed on different cultures are illustrated. (a) Wild-type (WT) neurons treated with vehicle. (b) Wild-type neurons treated with 10 mM of Ab(25–35). (c) Human Bcl-2 transgenic neurons (h-Bcl-2 Tg) treated with vehicle. (d) h-Bcl-2 Tg neurons treated with 10 mM of Ab(25–35). (e, f) The quantification of apoptotic nuclei of neurons treated with Ab(25–35) and Ab(1–42), respectively. Dense and fragmented nuclei (arrow and arrowhead, respectively) were counted as apoptotic. Data, expressed as a percentage of total nuclei, represent the mean ^ S.D. of values obtained in three to five fields from at least three different cultures (35–100 nuclei per field). *P , 0.05, Ab(25–35) vs vehicle. **P , 0.01, Ab(1– 42) vs vehicle. Scale bar 20 mm.
MTT reduction, could be attributed to apoptosis. This suggests that both apoptotic and necrotic cell death occur in mouse cortical neurons exposed to Ab. Our results indicate that h-Bcl-2 overexpression selectively protects neurons against the apoptosis induced by both Ab(25–35) and Ab(1–42). They largely confirm a previous study showing that Bcl2-transfected PC12 cells are protected against delayed apoptosis induced by a relatively low concentration of Ab (50 mM), whereas the rapid necrotic cell death evoked by a higher concentration (300 mM) remained unaltered. 31 Consistent with a
specific protective role of Bcl-2 on Ab-evoked apoptosis, the necrotic cell death induced by Ab on various rat and human neuroblastoma cell lines was not prevented by Bcl-2 overexpression. 3 The biochemical basis for the potent antiapoptotic action of Bcl-2 is still unknown. One of the earliest mechanisms was its inhibitory effect on the formation or action of ROS, such as lipid peroxidation. 7,23,29,35 Accordingly, inhibition of formation and/or detoxification of 4-hydroxynonenal, a product of lipid peroxidation, may be involved in the antiapoptotic action of Bcl-2. 30,31,34 Alternatively, Bcl-2 may act as a pro-oxidant that
Human Bcl-2 inhibits Ab-induced neuronal apoptosis
induces endogenous cellular antioxidants. 52 Mammalian neural cells expressing Bcl-2 have recently been shown to contain increased concentrations of reduced glutathione. 7,15,29,31 PC12 cells overexpressing Bcl-2 had a two-fold increase in CuZnSOD and catalase activities. 7,15 Similarly, a slight increase (about 30%) in antioxidant enzyme activities (CuZnSOD, manganese superoxide dismutase and glutathione peroxidase) was measured in brains from h-Bcl-2 transgenic mice (unpublished results). These data suggest that overexpression of Bcl-2 shifts the cellular redox state to a more reduced state by promoting antioxidant defences. This could account partly for the protective action of Bcl-2 against Ab toxicity. Alteration of the cellular redox state could also modify the activity of transcription factors such as nuclear factor-kB 48 that are involved in the apoptotic process induced by Ab. 4,28,57 However, additional functions of Bcl-2 have emerged following studies performed under low oxygen pressure, which drastically decreases the net formation of ROS. 26,51 Bcl-2 has been implicated in the regulation of calcium fluxes and the blockade of the release of apoptogenic protease activators (including cytochrome c) from mitochondria (see Ref. 46 for review). Owing to the mitochondrial localization of Bcl-2, it could also prevent some mitochondrial deficits induced by Ab that contribute to Ab-induced neuronal death, such as inactivation of pyruvate dehydrogenase or cytochrome c oxidase. 24,50 Whatever the mechanisms involved in the protective effect of Bcl-2, one can speculate that its rapid and sustained down-regulation in human cultured neurons exposed to Ab 37 contributes, at least in part, to the apoptotic neuronal death evoked by this peptide. Although ROS, including superoxide, have been implicated in Ab(25–35) toxicity, 4 CuZnSOD overexpression (a two-fold increase) failed to protect cortical transgenic neurons against Ab(25–35)induced necrosis and/or apoptosis. These results suggest that neither superoxide nor peroxynitrite, resulting from the reaction of superoxide with nitric
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oxide, is involved in the neurotoxicity of Ab(25–35) in vitro. This also suggests that superoxide detoxification does not explain the protection against apoptosis conferred by Bcl-2 overexpression. Our results are in agreement with those of Prehn et al., 42 demonstrating that both acute and chronic treatments of rat hippocampal neurons with Ab(25–35) do not increase the production of superoxide anions and that overexpression of CuZnSOD does not protect them against Ab(25–35) toxicity. However, an eight-fold increase in CuZnSOD activity in the brain of transgenic mice protected them against premature death evoked by concomitant overexpression of APP. 9 Thus, the protective action of CuZnSOD towards Ab toxicity may depend on the level of enzyme expression. Surprisingly, CuZnSOD overexpression did not inhibit spontaneous death of cultured cortical neurons as was described for dopaminergic neurons in culture. 44,47 On the contrary, CuZnSOD overexpression increased spontaneous apoptosis of cortical neurons. An enhanced apoptotic cell death in the thymus was also reported in these transgenic mice. 39 Several reports also showed that an increase in CuZnSOD activity may cause oxidative damage and increased lipid peroxidation. 10,11,16 Indeed, elevated CuZnSOD level leads to increased levels of H2O2 and/or HO · in several tissues, including the brain. 40,43,57 CONCLUSIONS
The functional importance of Bcl-2 and Bcl-2related proteins in the control of apoptosis suggests that they could constitute prone targets for neuroprotective pharmacological interventions or gene therapy to promote neuronal survival in AD.
Acknowledgements—We gratefully acknowledge the financial support from the Fondation IPSEN, the Centre National de la Recherche Scientifique, the Faculte´ Necker and the Universite´ Re´ne´ Descartes, and the help of the Rotary Club.
REFERENCES
1. Anderson A. J., Pike C. J. and Cotman C. W. (1995) Differential induction of immediate early gene proteins in cultured neurons by beta-amyloid (Ab): association of c-jun with Ab-induced apoptosis. J. Neurochem. 65, 1487–1498. 2. Behl C., Davis J. B., Cole G. M. and Schubert D. (1992) Vitamin E protects nerve cells from amyloid b protein toxicity. Biochem. biophys. Res. Commun. 186, 2944–2950. 3. Behl C., Hovey L., Krajewski S., Schubert D. and Reed J. C. (1993) Bcl-2 prevents killing of neuronal cells by glutamate but not by amyloid beta protein. Biochem. biophys. Res. Commun. 197, 949–955. 4. Behl C., Davis J., Lesley R. and Schubert D. (1994) Hydrogen peroxide mediates amyloid b protein toxicity. Cell 77, 1–20. 5. Behl C., Davis J. B., Klier F. G. R. and Schubert D. (1994) Amyloid b protein induces necrosis rather than apoptosis. Brain Res. 645, 253–264. 6. Borchelt D. R., Thinakaran G., Eckman C. B., Lee M. K., Davenport F., Ratovitsky T., Prada C.-M., Kim G., Seekins S., Yager D., Slunt H. H., Wang R., Seeger M., Levey A. I., Gandy S. E., Copeland N. G., Jenkins N. A., Price D. L., Younkin S. G. and Sisodia S. S. (1996) Familial Alzheimer’s disease-linked presenilin-1 variants elevate Ab 1–42/1–40 ratio in vitro and in vivo. Neuron 17, 1005–1013. 7. Bruce-Keller A. J., Begley J. G., Weiming F., Butterfield D. A., Bredesen D. E., Hutchins J. B., Hensley K. and Mattson M. P. (1998) Bcl-2 protects isolated plasma and mitochondrial membranes against lipid peroxidation induced by hydrogen peroxide and amyloid b-peptide. J. Neurochem. 70, 31–39.
1462 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
C. Saille´ et al. Cai X.-D., Golde T. and Younkin S. (1993) Release of excess amyloid beta-protein from a mutant amyloid beta-protein precursor. Science 259, 514–516. Carlson G. A., Borchelt D. R., Dake A., Turner S., Danielson V., Cofin J. D., Eckman C., Meiners J., Nilsen S. P., Younkin S. G. and Hsiao K. K. (1997) Genetic modification of the phenotypes produced by amyloid precursor protein overexpression in transgenic mice. Hum. molec. Genet. 6, 1951–1959. Ceballos-Picot I., Nicole A., Briand P., Grimber G., Delacourte A., Desfossez A., Javoy-Agid F., Lafon M. and Sinet P. M. (1991) Neuronal-specific expression of human copper–zinc superoxide dismutase gene in transgenic mice: animal model of gene dosage effect in Down’s syndrome. Brain Res. 552, 198–214. Ceballos-Picot I., Nicole A., Clement M., Bourre J. M. and Sinet P. M. (1992) Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper–zinc superoxide dismutase. Mutation Res. 275, 281–293. Ceballos-Picot I. (1997) Oxidative stress in neuronal death and apoptosis. In The Role of Oxidative Stress in Neuronal Death (ed. Landes R. G.), pp. 39–68. Landes Bioscience, Austin, TX. Citron M., Oltersdorf T., Haass C., McConlogue L., Hung A. Y., Seubert P., Vigo-Pelfrey C., Lieberburg I. and Selkoe D. J. (1992) Mutation of the beta-amyloid precursor protein in familial Alzheimer’s disease increases beta-protein production. Nature 360, 672–674. Dubois-Dauphin M., Frankowski H., Tsujimoto Y., Huarte J. and Martinou J. C. (1994) Neonatal motoneurons overexpressing the bcl-2 protooncogene in transgenic mice are protected from axotomy-induced cell death. Proc. natn. Acad. Sci. U.S.A. 91, 3309–3313. Ellerby L. M., Ellerby H. M., Park S. M., Holleran A. L., Murphy A. N., Fiskum G., Kane D. J., Testa M. P., Kayalar C. and Bredesen D. E. (1996) Shift of the cellular oxidation–reduction potential in neural cells expressing Bcl-2. J. Neurochem. 67, 1259–1267. Elroy-Stein O. and Groner Y. (1988) Impaired neurotransmitter uptake in PC12 cells overexpressing human CuZn superoxide dismutase: implications for gene dosage effect in Down’s syndrome. Cell 52, 259–267. Forloni G., Chiesa R., Smiroldo S., Verga L., Salmona M., Tagliavini F. and Angeretti N. (1993) Apoptosis mediated neurotoxicity induced by chronic application of b-amyloid fragment 25–35. NeuroReport 4, 523–526. Games D., Adams D., Alessandrini R., Barbour R., Berthelette P., Blackwell C., Carr T., Clemens J., Donaldson T. and Gillespie F., et al. (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F b-amyloid precursor protein. Nature 373, 523–527. Greenlund L. J. S., Deckwerth T. L. and Johnson E. M. Jr (1995) Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 14, 303–315. Guo Q., Sopher B. L., Furukawa K., Pham D. G., Robinson N., Martin G. M. and Mattson M. P. (1997) Alzheimer’s presenilin mutations sensitize neural cells to apoptosis induced by trophic factor withdrawal and amyloid beta-peptide. Involvement of calcium and oxyradicals. J. Neurosci. 17, 4212–4222. Haass C. and Selkoe D. J. (1993) Cellular processing of b-amyloid precursor protein and the genesis of amyloid b-peptide. Cell 75, 1039–1042. Hensley K., Carney J. M., Mattson M. P., Aksenova M., Harris M., Wu J. F., Floyd R. A. and Butterfield D. A. (1994) A model for b-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer’s disease. Proc. natn. Acad. Sci. U.S.A. 91, 3270–3274. Hockenbery D. M., Oltvai Z. N., Yin C. L., Milliman C. L. and Korsmeyer S. J. (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241–251. Hoshi M., Takashima A., Noguchi K., Murayama M., Sato M., Kondo S., Saitoh Y., Ishiguro K., Hoshino T. and Imahori K. (1996) Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3b in brain. Proc. natn. Acad. Sci. U.S.A. 93, 2719–2723. Iversen L. L., Mortishire-Smith R. J., Pollak S. J. and Shearman M. S. (1995) The toxicity in vitro of b-amyloid protein. Biochem. J. 311, 1–16. Jacobson M. D. and Raff M. C. (1995) Programmed cell death and Bcl-2 protection in very low oxygen. Nature 374, 814–816. Jordan J., Ghadge G. D., Phren J. H. M., Poth P. T., Ross R. P. and Miller R. J. (1995) Expression of human copper–zinc superoxide dismutase inhibits the death of rat sympathetic neurons caused by withdrawal of nerve growth factor. Molec. Pharmac. 47, 1095–1100. Kaltschmidt B., Uherek M., Volk B., Bauerle P. A. and Kaltschmidt C. (1997) Transcription factor NF-kappa-B is activated in primary neurons by amyloid peptides and in neurons surrounding early plaques from patients with Alzheimer disease. Proc. natn. Acad. Sci. U.S.A. 94, 2642–2647. Kane D. J., Sarafian T. A., Anton R., Hahn H., Gralla E. B., Valentine J. S., Ord T. and Bredesen D. E. (1993) Bcl-2 inhibition of neural cell death: decreased generation of reactive oxygen species. Science 262, 1274–1277. Keller J. N., Pang Z., Geddes J. W., Begley J. G., Germeyer A., Waeg G. and Mattson M. P. (1997) Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfunction in synaptosomes by amyloid b-peptide: role of the lipid peroxidation product 4-hydroxynonenal. J. Neurochem. 69, 273–284. Kruman I., Bruce-Keller A. J., Bredesen D., Waeg G. and Mattson M. P. (1997) Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 5089–5100. Laferla F. M., Tinkle B. T., Bieberich C. J., Haudenschild C. C. and Jay G. (1995) The Alzheimer’s Ab peptide induces neurodegeneration and apoptotic cell death in transgenic mice. Nat. Genet. 9, 215–220. Loo T., Copani A., Pike C. J., Whittemore E. R., Walencewicz A. J. and Cotman C. W. (1993) Apoptosis is induced by bamyloid in cultured central nervous system neurons. Proc. natn. Acad. Sci. U.S.A. 90, 7951–7955. Mark R. J., Lowell M. A., Markesbery W. R., Uchida K. and Mattson R. P. (1997) A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid Ab(25–35) peptide. J. Neurochem. 68, 255–264. Martinou J.-C., Dubois-Dauphin M., Staple J. K., Rodriguez I., Frankowski H., Missoten M., Albertini P., Talabot D., Catsicas S. and Pietra C., et al. (1994) Overexpression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron 13, 1017–1030. Mu¨ller W. E. G., Romero F. J., Perovic S., Pergande G. and Pialoglou P. (1997) Protection of flupirtine on b-amyloidinduced apoptosis in neuronal cells in vitro: prevention of amyloid-induced glutathione depletion. J. Neurochem. 68, 2371–2377.
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37. Paradis E., Douillard H., Koutroumatis M., Goodyer C. and Le Blanc A. (1996) Amyloid b peptide of Alzheimer’s disease downregulates Bcl-2 and upregulates Bax expression in human neurons. J. Neurosci. 16, 7533–7539. 38. Paris D., Toyama K., Me´garbane´ A., Casanova M., Sinet P.-M. and London J. (1996) Rapid fluorescence in situ hybridization on interphasic nuclei to discriminate between homozygous and heterozygous transgenic mice. Transgen. Res. 5, 397–403. 39. Peled-Kamar M., Lotem J., Okon E., Sachs L. and Groner Y. (1995) Thymic abnormalities and enhanced apoptosis of thymocytes and bone marrow cells in transgenic mice overexpressing CuZn superoxide dismutase: implications for Down syndrome. Eur. molec. Biol. Org. J. 14, 4985–4993. 40. Peled-Kamar M., Lotem J., Wirguin I., Weiner L., Hermalin A. and Groner Y. (1997) Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type CuZn superoxide dismutase. Proc. natn. Acad. Sci. U.S.A. 94, 3883–3887. 41. Pike C. J., Burdick D., Walencewicz A. J., Glabe C. G. and Cotman C. W. (1993) Neurodegeneration induced by b-amyloid peptides in vitro: the role of peptide assembly state. J. Neurosci. 13, 1676–1687. 42. Prehn J. H., Bindokas V. P., Jordan J., Galindo M. F., Ghadge G. D., Roos R. P., Boise L. H., Thompson C. B., Krajewski S., Reed J. C. and Miller R. J. (1996) Protective effect of transforming growth factor-beta1 on beta-amyloid neurotoxicity in rat hippocampal neurons. Molec. Pharmac. 49, 319–328. 43. Przedborski S., Jackson-Lewis V., Kostic V., Carlson E., Epstein C. J. and Cadet J. L. (1992) Superoxide dismutase, catalase and glutathione peroxidase activities in copper/zinc superoxide dismutase transgenic mice. J. Neurochem. 58, 1760–1767. 44. Przedborski S., Khan U., Kostic V., Carlson E., Epstein C. J. and Sulzer D. (1996) Increased superoxide dismutase activity improves survival of cultured postnatal midbrain neurons. J. Neurochem. 67, 1383–1392. 45. Rabizadeh S., Gralla E. B., Borchelt D. R., Gwinn R., Valentine J. S., Sisodia S., Wong P., Lee M., Hahn H. and Bredesen D. E. (1995) Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: studies in yeast and neural cells. Proc. natn. Acad. Sci. U.S.A. 92, 3024–3028. 46. Reed J. C. (1997) Cytochrome C: can’t live with it—can’t live without it. Cell 91, 559–562. 47. Sanchez-Ramos J. R., Song S., Facca A., Basit A. and Epstein C. J. (1997) Transgenic murine dopaminergic neurons expressing human CuZn superoxide dismutase exhibit increased density in culture, but no resistance to methylphenylpyridinium-induced degeneration. J. Neurochem. 68, 58–67. 48. Sen C. K. and Packer L. (1996) Antioxidant and redox regulation of gene transcription. Fedn Proc. Fedn Am. Socs exp. Biol. 10, 709–720. 49. Shearman M., Hawtin S. and Taylor V. (1995) The intracellular component of cellular 3-(4:5-dimethylthiazol-2-yl-2:5diphenyltetrazolium bromide (MTT) reduction is specifically inhibited by b-amyloid peptides. J. Neurochem. 65, 218–227. 50. Sheehan J. P., Swerdlow R. H., Miller S. W., Davis R. E., Parks J. K., Parker W. D. and Tuttle J. B. (1997) Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer’s disease. J. Neurosci. 17, 4612–4622. 51. Shimizu S., Eguchi Y., Kosaka H., Kamiike W., Matsuda H. and Tsujimoto Y. (1995) Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature 374, 811–813. 52. Steinman H. M. (1995) The Bcl-2 oncoprotein functions as a prooxidant. J. biol. Chem. 270, 3487–3490. 53. Troy C. M. and Shelanski M. L. (1994) Down regulation of copper–zinc superoxide dismutase causes apoptotic death in PC12 neuronal cells. Proc. natn. Acad. Sci. U.S.A. 91, 6384–6387. 54. Watt J. A., Pike C. J., Walencewics-Wasserman A. J. and Cotman C. W. (1994) Ultrastructural analysis of b-amyloid-induced apoptosis in cultured hippocampal neurons. Brain Res. 661, 147–156. 55. Weiss S., Pin J. P., Sebben M., Kemp D. E., Sladeczek F., Gabrion J. and Bockaert J. (1986) Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study. Proc. natn. Acad. Sci. U.S.A. 83, 2238–2242. 56. Yan S. D., Chen X. and Fu J., et al. (1996) RAGE and amyloid-b peptide neurotoxicity in Alzheimer’s disease. Nature 382, 685–691. 57. Yim M. B., Chock P. B. and Stadtman E. R. (1993) Enzyme function of copper–zinc superoxide dismutase as a free radical generator. J. biol. Chem. 268, 4099–4105. (Accepted 29 January 1999)
Neuroscience Vol. 92, No. 4, pp. 1455–1463, 1999 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(99)00089-5
TRANSGENIC MURINE CORTICAL NEURONS EXPRESSING HUMAN Bcl-2 EXHIBIT INCREASED RESISTANCE TO AMYLOID b-PEPTIDE NEUROTOXICITY C. SAILLE´,* P. MARIN,† J.-C. MARTINOU,‡ A. NICOLE,* J. LONDON* and I. CEBALLOS-PICOT*k *CNRS UMR 8602, CHU Necker—Enfants Malades, 156 Rue de Vaugirard, 75743 Paris Cedex 15, France †INSERM U 114, Colle`ge de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France ‡Serono Pharmaceutical Research Institute, 14 Chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland
Abstract—The generation of reactive oxygen species has been implicated in the neurotoxicity of amyloid b-peptide, the main constituent of the senile plaques that accumulates in the brain of Alzheimer’s disease victims. In this study, we have compared the toxicity of amyloid b-peptide on cultured cortical neurons from control mice and transgenic mice expressing either human copper–zinc superoxide dismutase or human Bcl-2, two proteins that protect cells against oxidative damage. Copper–zinc superoxide dismutase overexpression failed to protect cortical neurons against the toxicity of amyloid b-peptide(25–35) [the minimal cytotoxic fragment of amyloid b-peptide(1–42)] as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide reduction and an enzyme-linked immunoabsorbent assay using an antibody directed against microtubule-associated protein-2 (a specific neuronal protein), ruling out a role for superoxide anion and peroxynitrite in amyloid b-peptide-evoked neurotoxicity. On the contrary, cortical neurons expressing human copper–zinc superoxide dismutase exhibited increased apoptotic nuclei in both untreated and amyloid b-peptide(25–35)-exposed neurons. Transgenic neurons expressing human Bcl-2 were partially protected against amyloid b-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid b-peptide(25–35) and amyloid b-peptide(1–42). This study may be relevant for developing neuroprotective gene therapy to inhibit neuronal apoptosis in Alzheimer’s disease. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: amyloid b-peptide, Alzheimer’s disease, apoptosis, copper–zinc superoxide dismutase, Bcl-2, neuroprotection.
The pathological hallmark of Alzheimer’s disease (AD) is the accumulation of numerous senile plaques associated with degenerating neurons. Amyloid b-peptide (Ab) is a 40–42-amino-acid peptide that is the main constituent of senile plaques. Ab is derived from a larger membrane-spanning glycoprotein called amyloid precursor protein (APP), as a result of proteolytic processing. 21 Several observations suggest that Ab is involved in the pathogenesis of AD: Ab is toxic to neurons in vitro 25,33,41 and in vivo; 32 mutations in the APP locus, which have been linked to early-onset familial AD, lead to overproduction of Ab, 8,13 and transgenic mice expressing these mutations display many of the kTo whom correspondence should be addressed. Abbreviations: Ab, amyloid b-peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; CuZnSOD, copper–zinc superoxide dismutase; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; h-Bcl-2, human Bcl-2; h-CuZnSOD, human CuZnSOD; HEPES, N2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid; MAP-2, microtubule-associated protein-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphatebuffered saline; ROS, reactive oxygen species.
pathological features of AD; 18 mutations of the presenilin-1 gene, which are responsible for the majority of cases of inherited early-onset familial AD, also lead to an elevated production of Ab, 6 and Ab neurotoxicity is exacerbated greatly in cells expressing mutated presenilin-1. 20 Several lines of evidence suggest that the overproduction of reactive oxygen species (ROS) is implicated in Ab neurotoxicity: (i) exposure of cultured neurons or neuronal cell lines to Ab increases the intracellular levels of ROS, leading to the activation of nuclear factor-kB, a redoxmodulated transcription factor involved in Abevoked neurotoxicity; 4,28,56 (ii) the neurotoxicity of Ab is attenuated by the prevention of intracellular glutathione depletion, 36 antioxidants 2,4 and/or free radical scavengers; 22 (iii) antioxidants protect cells against the adverse consequences of presenilin-1 mutations; 20 (iv) in AD brain, regional heterogeneity in the efficiency of free radical defence mechanisms appears to account for the regional heterogeneity of cell vulnerability. 12 Several enzymes have been shown to contribute to cell defence against the deleterious consequences of ROS overproduction.
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Among these enzymes, increased copper–zinc superoxide dismutase (CuZnSOD) activity protects neurons and other cell types against oxidative stress and nerve growth factor deprivation. 19,27,45 By contrast, CuZnSOD down-regulation accelerates spontaneous cell death. 53 In addition to these enzymatic processes, protective effects of the oncogenic gene product Bcl-2 have been reported in various neuronal death paradigms implicating overproduction of ROS. Indeed, overexpression of Bcl-2 in neuronal cells prevents apoptosis induced by oxidative stress associated with glutathione depletion 29 or by 4-hydroxynonenal (an aldehydic product of lipid peroxidation), 31 suggesting that Bcl-2 could act as an antioxidant that suppresses the formation or action of ROS. The present study was undertaken to investigate the neuroprotective effect of CuZnSOD and Bcl-2 against the toxicity evoked by Ab. For that purpose, the decrease in cell viability and the apoptosis induced by Ab were compared on cultured cortical neurons from control mice, and transgenic mice expressing either human CuZnSOD (h-CuZnSOD) or human bcl-2 (h-bcl-2) genes. EXPERIMENTAL PROCEDURES
Materials FVB/N and C57BL/6 × DBA/2 (B6D2) mice were from Iffa–Credo (Lyon, France); heterozygous transgenic mice overexpressing h-CuZnSOD (h-CuZnSOD transgenic) or h-bcl-2 (h-Bcl-2 transgenic) were described previously. 14,38 Culture media were from Life Technology (Cergy-Pontoise, France), fetal calf serum (FCS) from Dutcher (Brumath, France), 96- and four-well culture dishes from Nunc (Denmark), mouse monoclonal anti-microtubule-associated protein-2 (anti-MAP-2) antibody from Biomakor (Rehovot, Israel), monoclonal anti-human Bcl-2 antibody from DAKO (clone 124), peroxidase-coupled goat anti-mouse or antirabbit immunoglobulin G from Biosys (Compie`gne, France), Ab(1–42), Ab(25–35) and Ab(35–25) from Bachem (France), Hoechst 33258 from Molecular Probe (Eugene, OR, U.S.A.) and all other chemicals from Sigma (Saint Quentin Fallavier, France). Primary culture of cortical neurons All studies were conducted according to the EC legislation concerning animal care. Primary neuronal cultures were prepared according to Weiss et al. 55 Cortices were removed from 15–16-day-old FVB/N, B6D2, h-CuZnSOD transgenic or h-Bcl-2 transgenic mouse embryos and dissociated mechanically with a Pasteur pipette in phosphate-buffered saline (PBS) supplemented with glucose (33 mM). Cells were plated on to either 96-well culture dishes (1 × 10 5 cells per well), glass coverslips in four-well plates (3 × 10 5 cells per well) or six-well culture dishes (3 × 10 6 cells per well), successively coated with poly-l-ornithine (mol. wt 40,000, 15 mg/ml) and FCS (10% v/v in culture medium). Serum-free medium consists of a mixture (1:1, v/v) of Dulbecco’s modified essential medium and Ham’s F12 nutrient, supplemented with glucose (33 mM), glutamine (2 mM), NaHCO3 (13 mM), HEPES buffer (5 mM, pH 7.4), penicillin and streptomycin (5 IU/ml and 5 mg/ ml, respectively), and a mixture of salt and hormones containing insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM) and sodium
selenite (Na2SeO3; 30 nM). Cells were cultured at 378C in a humidified atmosphere of 92% air/8% CO2. Cultures, used six days after seeding, were shown to be highly enriched in neurons by immunocytochemistry for MAP-2 and devoid of any detectable glial elements, as assessed by immunocytochemistry for glial fibrillary acidic protein (data not shown). Treatments with amyloid b -peptides Ab(25–35) and Ab(35–25) stock solutions (700 mM in water), and Ab(1–42) stock solution (1 mM in 8.75% acetonitrile/0.25% trifluoroacetic acid) were diluted in serumfree medium (without Ham’s F12 nutrient) just before use, to obtain a ×10 concentrated solution. Concentrated peptides were added to the culture medium for 24 h to reach the appropriate concentrations. Neuronal viability measurement 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Twenty-four hours after the onset of Ab treatment, neurons grown in 96-well culture dishes were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 0.5 mg/ml in PBS containing 33 mM glucose) at 378C for 1 h. The formazan product was dissolved in 100 ml dimethyl sulfoxide and quantified by measuring the optical density at 560 nm. Enzyme-linked immunosorbent assay for microtubuleassociated protein-2. Twenty-four hours after the onset of Ab treatment, neurons grown in 96-well culture dishes were fixed in 100 ml paraformaldehyde (4% w/v in PBS) at 48C for 1 h, washed twice with PBS containing glycine (0.1 M) and PBS/Tween-20 (0.1%, v/v). They were incubated for 1 h at 378C successively with PBS/Tween-20 supplemented with FCS (10%, v/v), a mouse monoclonal anti-MAP-2 antibody (immunoglobulin G1, 1:4000, v/v, in PBS/Tween-20 containing 10% FCS) and peroxidasecoupled goat anti-mouse immunoglobulin G (1:3000, v/v, in PBS/Tween-20 containing 10% FCS). Cells were then incubated with 0.04% (w/v) o-phenylenediamine and 0.012% (v/v) H2O2, diluted in citrate buffer (pH 5), for 5 min. The reaction was stopped by adding H2SO4 (final concentration 0.15 M). MAP-2 was quantified by reading the optical density at 490 nm. Detection and measurement of apoptosis Nuclear condensation and fragmentation (some classical hallmarks of apoptosis) were detected by staining the nuclei with the fluorescent probe Hoechst 33258. Cortical neurons, grown on glass coverslips, were treated with either 10 mM Ab(25–35) or 20 mM Ab(1–42) for 24 h. Cells were fixed in methanol and stained with Hoechst 33258 (1 mg/ml in methanol) for 15 min at 378C. After washing, coverslips were mounted on glass slides in a medium for immunofluorescence (Fluoprep, BioMerieux). Fluorescence microscopy was performed using a Leitz Laborlux S microscope (lex 345 nm, lem 478 nm). Photomicrographs were taken for apoptotic cell counting. Dense fluorescent and fragmented nuclei were considered as apoptotic. Detection of transgene expression Copper–zinc superoxide dismutase activity determination. Neurons grown on six-well culture dishes were scraped off in ice-cold PBS and centrifuged at 200 × g for 5 min. The pellets were frozen and kept in liquid nitrogen until use. The pellets were homogenized in a sodium phosphate buffer (50 mM, pH 7.4) and samples were centrifuged at 20,000 × g for 30 min at 48C. Supernatants were used for protein measurement and determination of CuZnSOD activity, as described previously. 11
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Immunofluorescence detection of the human Bcl-2 protein. Cortical neurons grown on glass coverslips were fixed in paraformaldehyde (4% in PBS) for 1 h at 48C. After a 1-h preincubation at 378C with PBS containing 5% normal goat serum (solution A), cells were incubated with a mouse anti-h-Bcl-2 antibody (DAKO; 1:20, v/v, in solution A) for 1 h at room temperature. Coverslips were intensively rinsed out before incubation with a rabbit anti-mouse immunoglobulin G conjugated with fluorescein (1:50, v/v, in solution A) for 1 h at room temperature. Human Bcl-2 was detected by fluorescence microscopy, as described above. Western blot analysis. Cortical neurons grown on sixwell culture dishes for six days were harvested in 200 ml sodium dodecyl sulfate (2%). Protein concentration was determined using the bicinchoninic acid method. Proteins (50 mg per lane) were resolved on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred on to polyvinylidene fluoride membranes (Immobilon, Millipore). Membranes were blocked by incubation for 1 h at room temperature in Tris-buffered saline containing 0.05% Tween-20 plus 5% non-fat milk. They were then incubated with either a monoclonal anti-h-Bcl-2 antibody (1:200 dilution) or a polyclonal anti-h-CuZnSOD antibody (1:1000 dilution), 10 followed, respectively, by either an anti-mouse immunoglobulin G or an anti-rabbit immunoglobulin G peroxidase-conjugated antibody (1:1000 dilution). Bcl-2 and CuZnSOD proteins were detected using enhanced chemiluminescence with the ECL kit (Amersham, France) and Fuji films were exposed to the blots. Statistical analysis Statistical analysis was performed by ANOVA followed by a Scheffe´ F-test, unless indicated otherwise. RESULTS
Neurotoxicity of amyloid b -peptide on murine cortical neurons from FVB/N and B6D2 mice A 24-h Ab(25–35) treatment induced a concentration-dependent decrease in MTT reduction in cortical neurons from both genetic backgrounds (Figs 1c, 2c). On FVB/N genetic background control neurons, 1 mM Ab(25–35) had little effect, while 10 and 20 mM decreased MTT reduction to 55.5 ^ 3.6% and 41.0 ^ 8.5%, respectively, compared with control values (mean ^ S.D. of data obtained in four independent experiments performed in triplicate; Fig. 1c). The inverted sequence Ab(35–25) (20 mM) had no effect on cell viability (data not shown). Ab(25–35) (1 mM) induced a slight but non-significant decrease in MTT reduction in neurons from B6D2 mice (Fig. 2c), while 10 and 20 mM Ab(25–35) decreased MTT reduction by 42.5 ^ 4.5% and 69.0 ^ 2.8%, respectively (n 4). Since the inhibition of MTT reduction by Ab(25– 35) could be independent of cellular loss, 49 the amount of neurons was quantified by an MAP-2 enzyme-linked immunosorbent assay (ELISA; a specific marker of neurons) after Ab peptide treatments. On B6D2 genetic background neurons, increasing concentrations of Ab(25–35) (1– 20 mM) induced a decrease in the amount of MAP-2 (Fig. 2d) similar to the magnitude of the
Fig. 1. Ab-induced neuronal death is not modified by CuZnSOD overexpression. (a) The specific CuZnSOD enzymatic activity evaluated in wild-type (WT) and h-CuZnSOD Tg neurons after six days in culture. Data are the means ^ S.D. of results obtained in eight separate cultures. (b) A representative western blot experiment showing human and mouse CuZnSOD expression in transgenic cortical neurons compared with wildtype. (c, d) Cortical neurons were treated for 24 h with increasing concentrations of Ab(25–35). Cell viability, evaluated by MTT reduction (c), and cell number, measured by MAP-2 ELISA (d), are expressed as a percentage of control values and represent the means ^ S.D. of results obtained in four experiments performed on different cultures. **P , 0.01, Ab(25–35) vs vehicle (ANOVA followed by Scheffe´ F-test). ***P , 0.001, h-CuZnSOD transgenic vs wild-type (Student’s t-test).
decrease in MTT reduction (Fig. 2c). Nevertheless, on FVB/N genetic background neurons, the decrease in cell number evaluated by MAP-2 quantification (Fig. 1d) was higher than the magnitude of MTT reduction evoked by the same concentrations of Ab(25–35) (Fig. 1c). Taken together, these results indicate that the decrease in MTT reduction following Ab peptide treatments is related to cell loss and that a 24-h treatment with 10–20 mM Ab(25–35) induces marked neuronal death on cultured neurons from both genetic backgrounds.
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Fig. 2. Bcl-2 overexpression partially protects neurons from Ab-induced cell death. (a, b) Photomicrographs of cortical neurons from wild-type (WT) mice (a) and transgenic mice expressing h-Bcl-2 (h-Bcl-2 Tg; b) immunostained with the anti-h-Bcl-2 antibody. The data illustrated are representative of three experiments performed on different cultures. Omitting the anti-h-Bcl-2 antibody yielded a staining identical to that obtained on wild-type neurons (data not shown). Inset in (a) illustrates a representative western blot showing h-Bcl-2 expression in hBcl-2 Tg cortical neurons compared with wild-type. (c, d) Cell viability, evaluated by MTT reduction (c), and cell number, measured by MAP-2 ELISA (d), are expressed as a percentage of control values and represent the means ^ S.D. of results obtained in four experiments performed on different cultures. **P , 0.01, Ab vs vehicle. $$ P , 0.01, h-Bcl-2 Tg vs wild-type. Scale bar 50 mm. MW, protein molecular weight markers.
Overexpression of copper–zinc superoxide dismutase does not protect cortical neurons against amyloid b -peptide-induced neuronal death CuZnSOD activity quantified in transgenic neurons from mice overexpressing CuZnSOD was increased two-fold (57.1 ^ 4.3 IU/mg protein, mean ^ S.D. of eight determinations performed on different cultures) compared with that measured in control cells (29.3 ^ 4.7 IU/mg protein, n 8) (Fig. 1a). This increase in CuZnSOD activity is probably the consequence of the strong expression of h-CuZnSOD in transgenic neurons, as assessed by western blot experiments performed with an antibody that recognizes both human and mouse CuZnSOD (Fig. 1b). Viability of control and transgenic neurons, estimated by either MTT reduction or MAP-2 quantification, was identical after seven days in culture (data not shown). Moreover, the neurotoxicity evoked by increasing concentrations of Ab (1–20 mM) was similar in control neurons and neurons overexpressing CuZnSOD (Fig. 1c, d). Bcl-2 overexpression reduces amyloid b -peptideinduced neuronal death Immunostaining experiments performed with the anti-h-Bcl-2 antibody showed a strong expression of
h-Bcl-2 in transgenic neurons (Fig. 2b). Human Bcl2 was localized in cell bodies as well as in neuronal processes (Fig. 2b). The strong expression of h-Bcl2 in cultured transgenic neurons was confirmed by western blotting (Fig. 2a, inset). Human Bcl-2 overexpression did not alter neuronal viability evaluated after seven days in culture (data not shown). However, h-bcl-2 transgene expression protected neurons against the toxicity induced by 10 and 20 mM Ab(25–35) (about 30% and 50% of protection as assessed by the MTT assay and the MAP-2 ELISA, respectively; Fig. 2c, d). Inhibition of amyloid b -peptide-caused apoptosis by human bcl-2 but not by human copper–zinc superoxide dismutase transgene expression Experiments were performed to look for the consequences of CuZnSOD and Bcl-2 overexpression on Ab(25–35)- and Ab(1–42)-induced apoptosis. After seven days in culture, about 10–15% of untreated neurons exhibited apoptotic nuclei in both mouse strains (Figs 3e, 4e). The concentration of Ab(25–35) providing a decrease in cell viability of about 50% (10 mM) increased the percentage of apoptotic nuclei to about 25% in both cell strains (Figs 3e, 4e), indicating that both apoptotic and
Human Bcl-2 inhibits Ab-induced neuronal apoptosis
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Fig. 3. Effect of CuZnSOD overexpression on spontaneous and Ab-induced apoptosis. Neurons, treated for 24 h with 10 mM of Ab(25–35), were fixed in methanol and stained with Hoechst 33258. Fluorescence photomicrographs representative of three experiments performed on different cultures are illustrated. (a) Wild-type (WT) neurons treated with vehicle. (b) Wild-type neurons treated with 10 mM of Ab(25–35). (c) Human CuZnSOD transgenic neurons (h-CuZnSOD Tg) treated with vehicle. (d) h-CuZnSOD Tg neurons treated with 10 mM of Ab(25–35). (e) The quantification of apoptotic nuclei. Dense and fragmented nuclei (arrow and arrowhead, respectively) were counted as apoptotic. Data, expressed as a percentage of total nuclei, represent the mean ^ S.D. of values obtained in three to five fields from at least three different cultures (35–100 nuclei per field). **P , 0.01, Ab(25–35) vs vehicle. $$P , 0.01, h-CuZnSOD transgenic vs wild-type. Scale bar 20 mm.
necrotic pathways occurred in Ab(25–35)-treated neurons. As expected, the number of apoptotic nuclei was similarly increased by a 24-h exposure to Ab(1–42) (20 mM; Fig. 4f). Expression of h-CuZnSOD transgene in cortical neurons did not protect them against Ab(25–35)induced apoptosis. On the contrary, it should be noted that CuZnSOD overexpression slightly but significantly increased the level of apoptotic nuclei in both untreated and Ab-exposed neurons (Fig. 3e). In contrast to what was observed in neurons overexpressing CuZnSOD, Bcl-2 overexpression, which did not alter spontaneous apoptosis (Fig. 4e), suppressed the increase in the percentage of apoptotic nuclei evoked by both Ab(25–35) (Fig. 4e) and Ab(1–42) (Fig. 4f).
DISCUSSION
In this study, we show that overexpression of the antiapoptotic/antioxidant protein Bcl-2 suppresses Ab-induced apoptosis in cultured cortical neurons. By contrast, CuZnSOD overexpression did not reduce the apoptotic effect of Ab. The mode of cell death, apoptosis versus necrosis, induced by Ab is still a matter of debate. Apoptosis is selectively increased in primary cultured neurons exposed to Ab 1,17,33,54 and in the brain of transgenic mice overproducing Ab. 32 Both types of cell death were reported on neuronal cell lines depending on the concentration of Ab used, 31 and finally, Ab only triggered necrosis on PC12 cells. 5 In our study, only half of the cell loss, assessed by MAP-2 ELISA or
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Fig. 4. Inhibition of Ab(25–35)- and Ab(1–42)-induced apoptosis in neurons overexpressing Bcl-2. Fluorescence photomicrographs representative of three experiments performed on different cultures are illustrated. (a) Wild-type (WT) neurons treated with vehicle. (b) Wild-type neurons treated with 10 mM of Ab(25–35). (c) Human Bcl-2 transgenic neurons (h-Bcl-2 Tg) treated with vehicle. (d) h-Bcl-2 Tg neurons treated with 10 mM of Ab(25–35). (e, f) The quantification of apoptotic nuclei of neurons treated with Ab(25–35) and Ab(1–42), respectively. Dense and fragmented nuclei (arrow and arrowhead, respectively) were counted as apoptotic. Data, expressed as a percentage of total nuclei, represent the mean ^ S.D. of values obtained in three to five fields from at least three different cultures (35–100 nuclei per field). *P , 0.05, Ab(25–35) vs vehicle. **P , 0.01, Ab(1– 42) vs vehicle. Scale bar 20 mm.
MTT reduction, could be attributed to apoptosis. This suggests that both apoptotic and necrotic cell death occur in mouse cortical neurons exposed to Ab. Our results indicate that h-Bcl-2 overexpression selectively protects neurons against the apoptosis induced by both Ab(25–35) and Ab(1–42). They largely confirm a previous study showing that Bcl2-transfected PC12 cells are protected against delayed apoptosis induced by a relatively low concentration of Ab (50 mM), whereas the rapid necrotic cell death evoked by a higher concentration (300 mM) remained unaltered. 31 Consistent with a
specific protective role of Bcl-2 on Ab-evoked apoptosis, the necrotic cell death induced by Ab on various rat and human neuroblastoma cell lines was not prevented by Bcl-2 overexpression. 3 The biochemical basis for the potent antiapoptotic action of Bcl-2 is still unknown. One of the earliest mechanisms was its inhibitory effect on the formation or action of ROS, such as lipid peroxidation. 7,23,29,35 Accordingly, inhibition of formation and/or detoxification of 4-hydroxynonenal, a product of lipid peroxidation, may be involved in the antiapoptotic action of Bcl-2. 30,31,34 Alternatively, Bcl-2 may act as a pro-oxidant that
Human Bcl-2 inhibits Ab-induced neuronal apoptosis
induces endogenous cellular antioxidants. 52 Mammalian neural cells expressing Bcl-2 have recently been shown to contain increased concentrations of reduced glutathione. 7,15,29,31 PC12 cells overexpressing Bcl-2 had a two-fold increase in CuZnSOD and catalase activities. 7,15 Similarly, a slight increase (about 30%) in antioxidant enzyme activities (CuZnSOD, manganese superoxide dismutase and glutathione peroxidase) was measured in brains from h-Bcl-2 transgenic mice (unpublished results). These data suggest that overexpression of Bcl-2 shifts the cellular redox state to a more reduced state by promoting antioxidant defences. This could account partly for the protective action of Bcl-2 against Ab toxicity. Alteration of the cellular redox state could also modify the activity of transcription factors such as nuclear factor-kB 48 that are involved in the apoptotic process induced by Ab. 4,28,57 However, additional functions of Bcl-2 have emerged following studies performed under low oxygen pressure, which drastically decreases the net formation of ROS. 26,51 Bcl-2 has been implicated in the regulation of calcium fluxes and the blockade of the release of apoptogenic protease activators (including cytochrome c) from mitochondria (see Ref. 46 for review). Owing to the mitochondrial localization of Bcl-2, it could also prevent some mitochondrial deficits induced by Ab that contribute to Ab-induced neuronal death, such as inactivation of pyruvate dehydrogenase or cytochrome c oxidase. 24,50 Whatever the mechanisms involved in the protective effect of Bcl-2, one can speculate that its rapid and sustained down-regulation in human cultured neurons exposed to Ab 37 contributes, at least in part, to the apoptotic neuronal death evoked by this peptide. Although ROS, including superoxide, have been implicated in Ab(25–35) toxicity, 4 CuZnSOD overexpression (a two-fold increase) failed to protect cortical transgenic neurons against Ab(25–35)induced necrosis and/or apoptosis. These results suggest that neither superoxide nor peroxynitrite, resulting from the reaction of superoxide with nitric
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oxide, is involved in the neurotoxicity of Ab(25–35) in vitro. This also suggests that superoxide detoxification does not explain the protection against apoptosis conferred by Bcl-2 overexpression. Our results are in agreement with those of Prehn et al., 42 demonstrating that both acute and chronic treatments of rat hippocampal neurons with Ab(25–35) do not increase the production of superoxide anions and that overexpression of CuZnSOD does not protect them against Ab(25–35) toxicity. However, an eight-fold increase in CuZnSOD activity in the brain of transgenic mice protected them against premature death evoked by concomitant overexpression of APP. 9 Thus, the protective action of CuZnSOD towards Ab toxicity may depend on the level of enzyme expression. Surprisingly, CuZnSOD overexpression did not inhibit spontaneous death of cultured cortical neurons as was described for dopaminergic neurons in culture. 44,47 On the contrary, CuZnSOD overexpression increased spontaneous apoptosis of cortical neurons. An enhanced apoptotic cell death in the thymus was also reported in these transgenic mice. 39 Several reports also showed that an increase in CuZnSOD activity may cause oxidative damage and increased lipid peroxidation. 10,11,16 Indeed, elevated CuZnSOD level leads to increased levels of H2O2 and/or HO · in several tissues, including the brain. 40,43,57 CONCLUSIONS
The functional importance of Bcl-2 and Bcl-2related proteins in the control of apoptosis suggests that they could constitute prone targets for neuroprotective pharmacological interventions or gene therapy to promote neuronal survival in AD.
Acknowledgements—We gratefully acknowledge the financial support from the Fondation IPSEN, the Centre National de la Recherche Scientifique, the Faculte´ Necker and the Universite´ Re´ne´ Descartes, and the help of the Rotary Club.
REFERENCES
1. Anderson A. J., Pike C. J. and Cotman C. W. (1995) Differential induction of immediate early gene proteins in cultured neurons by beta-amyloid (Ab): association of c-jun with Ab-induced apoptosis. J. Neurochem. 65, 1487–1498. 2. Behl C., Davis J. B., Cole G. M. and Schubert D. (1992) Vitamin E protects nerve cells from amyloid b protein toxicity. Biochem. biophys. Res. Commun. 186, 2944–2950. 3. Behl C., Hovey L., Krajewski S., Schubert D. and Reed J. C. (1993) Bcl-2 prevents killing of neuronal cells by glutamate but not by amyloid beta protein. Biochem. biophys. Res. Commun. 197, 949–955. 4. Behl C., Davis J., Lesley R. and Schubert D. (1994) Hydrogen peroxide mediates amyloid b protein toxicity. Cell 77, 1–20. 5. Behl C., Davis J. B., Klier F. G. R. and Schubert D. (1994) Amyloid b protein induces necrosis rather than apoptosis. Brain Res. 645, 253–264. 6. Borchelt D. R., Thinakaran G., Eckman C. B., Lee M. K., Davenport F., Ratovitsky T., Prada C.-M., Kim G., Seekins S., Yager D., Slunt H. H., Wang R., Seeger M., Levey A. I., Gandy S. E., Copeland N. G., Jenkins N. A., Price D. L., Younkin S. G. and Sisodia S. S. (1996) Familial Alzheimer’s disease-linked presenilin-1 variants elevate Ab 1–42/1–40 ratio in vitro and in vivo. Neuron 17, 1005–1013. 7. Bruce-Keller A. J., Begley J. G., Weiming F., Butterfield D. A., Bredesen D. E., Hutchins J. B., Hensley K. and Mattson M. P. (1998) Bcl-2 protects isolated plasma and mitochondrial membranes against lipid peroxidation induced by hydrogen peroxide and amyloid b-peptide. J. Neurochem. 70, 31–39.
1462 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
C. Saille´ et al. Cai X.-D., Golde T. and Younkin S. (1993) Release of excess amyloid beta-protein from a mutant amyloid beta-protein precursor. Science 259, 514–516. Carlson G. A., Borchelt D. R., Dake A., Turner S., Danielson V., Cofin J. D., Eckman C., Meiners J., Nilsen S. P., Younkin S. G. and Hsiao K. K. (1997) Genetic modification of the phenotypes produced by amyloid precursor protein overexpression in transgenic mice. Hum. molec. Genet. 6, 1951–1959. Ceballos-Picot I., Nicole A., Briand P., Grimber G., Delacourte A., Desfossez A., Javoy-Agid F., Lafon M. and Sinet P. M. (1991) Neuronal-specific expression of human copper–zinc superoxide dismutase gene in transgenic mice: animal model of gene dosage effect in Down’s syndrome. Brain Res. 552, 198–214. Ceballos-Picot I., Nicole A., Clement M., Bourre J. M. and Sinet P. M. (1992) Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper–zinc superoxide dismutase. Mutation Res. 275, 281–293. Ceballos-Picot I. (1997) Oxidative stress in neuronal death and apoptosis. In The Role of Oxidative Stress in Neuronal Death (ed. Landes R. G.), pp. 39–68. Landes Bioscience, Austin, TX. Citron M., Oltersdorf T., Haass C., McConlogue L., Hung A. Y., Seubert P., Vigo-Pelfrey C., Lieberburg I. and Selkoe D. J. (1992) Mutation of the beta-amyloid precursor protein in familial Alzheimer’s disease increases beta-protein production. Nature 360, 672–674. Dubois-Dauphin M., Frankowski H., Tsujimoto Y., Huarte J. and Martinou J. C. (1994) Neonatal motoneurons overexpressing the bcl-2 protooncogene in transgenic mice are protected from axotomy-induced cell death. Proc. natn. Acad. Sci. U.S.A. 91, 3309–3313. Ellerby L. M., Ellerby H. M., Park S. M., Holleran A. L., Murphy A. N., Fiskum G., Kane D. J., Testa M. P., Kayalar C. and Bredesen D. E. (1996) Shift of the cellular oxidation–reduction potential in neural cells expressing Bcl-2. J. Neurochem. 67, 1259–1267. Elroy-Stein O. and Groner Y. (1988) Impaired neurotransmitter uptake in PC12 cells overexpressing human CuZn superoxide dismutase: implications for gene dosage effect in Down’s syndrome. Cell 52, 259–267. Forloni G., Chiesa R., Smiroldo S., Verga L., Salmona M., Tagliavini F. and Angeretti N. (1993) Apoptosis mediated neurotoxicity induced by chronic application of b-amyloid fragment 25–35. NeuroReport 4, 523–526. Games D., Adams D., Alessandrini R., Barbour R., Berthelette P., Blackwell C., Carr T., Clemens J., Donaldson T. and Gillespie F., et al. (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F b-amyloid precursor protein. Nature 373, 523–527. Greenlund L. J. S., Deckwerth T. L. and Johnson E. M. Jr (1995) Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 14, 303–315. Guo Q., Sopher B. L., Furukawa K., Pham D. G., Robinson N., Martin G. M. and Mattson M. P. (1997) Alzheimer’s presenilin mutations sensitize neural cells to apoptosis induced by trophic factor withdrawal and amyloid beta-peptide. Involvement of calcium and oxyradicals. J. Neurosci. 17, 4212–4222. Haass C. and Selkoe D. J. (1993) Cellular processing of b-amyloid precursor protein and the genesis of amyloid b-peptide. Cell 75, 1039–1042. Hensley K., Carney J. M., Mattson M. P., Aksenova M., Harris M., Wu J. F., Floyd R. A. and Butterfield D. A. (1994) A model for b-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer’s disease. Proc. natn. Acad. Sci. U.S.A. 91, 3270–3274. Hockenbery D. M., Oltvai Z. N., Yin C. L., Milliman C. L. and Korsmeyer S. J. (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241–251. Hoshi M., Takashima A., Noguchi K., Murayama M., Sato M., Kondo S., Saitoh Y., Ishiguro K., Hoshino T. and Imahori K. (1996) Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3b in brain. Proc. natn. Acad. Sci. U.S.A. 93, 2719–2723. Iversen L. L., Mortishire-Smith R. J., Pollak S. J. and Shearman M. S. (1995) The toxicity in vitro of b-amyloid protein. Biochem. J. 311, 1–16. Jacobson M. D. and Raff M. C. (1995) Programmed cell death and Bcl-2 protection in very low oxygen. Nature 374, 814–816. Jordan J., Ghadge G. D., Phren J. H. M., Poth P. T., Ross R. P. and Miller R. J. (1995) Expression of human copper–zinc superoxide dismutase inhibits the death of rat sympathetic neurons caused by withdrawal of nerve growth factor. Molec. Pharmac. 47, 1095–1100. Kaltschmidt B., Uherek M., Volk B., Bauerle P. A. and Kaltschmidt C. (1997) Transcription factor NF-kappa-B is activated in primary neurons by amyloid peptides and in neurons surrounding early plaques from patients with Alzheimer disease. Proc. natn. Acad. Sci. U.S.A. 94, 2642–2647. Kane D. J., Sarafian T. A., Anton R., Hahn H., Gralla E. B., Valentine J. S., Ord T. and Bredesen D. E. (1993) Bcl-2 inhibition of neural cell death: decreased generation of reactive oxygen species. Science 262, 1274–1277. Keller J. N., Pang Z., Geddes J. W., Begley J. G., Germeyer A., Waeg G. and Mattson M. P. (1997) Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfunction in synaptosomes by amyloid b-peptide: role of the lipid peroxidation product 4-hydroxynonenal. J. Neurochem. 69, 273–284. Kruman I., Bruce-Keller A. J., Bredesen D., Waeg G. and Mattson M. P. (1997) Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 5089–5100. Laferla F. M., Tinkle B. T., Bieberich C. J., Haudenschild C. C. and Jay G. (1995) The Alzheimer’s Ab peptide induces neurodegeneration and apoptotic cell death in transgenic mice. Nat. Genet. 9, 215–220. Loo T., Copani A., Pike C. J., Whittemore E. R., Walencewicz A. J. and Cotman C. W. (1993) Apoptosis is induced by bamyloid in cultured central nervous system neurons. Proc. natn. Acad. Sci. U.S.A. 90, 7951–7955. Mark R. J., Lowell M. A., Markesbery W. R., Uchida K. and Mattson R. P. (1997) A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid Ab(25–35) peptide. J. Neurochem. 68, 255–264. Martinou J.-C., Dubois-Dauphin M., Staple J. K., Rodriguez I., Frankowski H., Missoten M., Albertini P., Talabot D., Catsicas S. and Pietra C., et al. (1994) Overexpression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron 13, 1017–1030. Mu¨ller W. E. G., Romero F. J., Perovic S., Pergande G. and Pialoglou P. (1997) Protection of flupirtine on b-amyloidinduced apoptosis in neuronal cells in vitro: prevention of amyloid-induced glutathione depletion. J. Neurochem. 68, 2371–2377.
Human Bcl-2 inhibits Ab-induced neuronal apoptosis
1463
37. Paradis E., Douillard H., Koutroumatis M., Goodyer C. and Le Blanc A. (1996) Amyloid b peptide of Alzheimer’s disease downregulates Bcl-2 and upregulates Bax expression in human neurons. J. Neurosci. 16, 7533–7539. 38. Paris D., Toyama K., Me´garbane´ A., Casanova M., Sinet P.-M. and London J. (1996) Rapid fluorescence in situ hybridization on interphasic nuclei to discriminate between homozygous and heterozygous transgenic mice. Transgen. Res. 5, 397–403. 39. Peled-Kamar M., Lotem J., Okon E., Sachs L. and Groner Y. (1995) Thymic abnormalities and enhanced apoptosis of thymocytes and bone marrow cells in transgenic mice overexpressing CuZn superoxide dismutase: implications for Down syndrome. Eur. molec. Biol. Org. J. 14, 4985–4993. 40. Peled-Kamar M., Lotem J., Wirguin I., Weiner L., Hermalin A. and Groner Y. (1997) Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type CuZn superoxide dismutase. Proc. natn. Acad. Sci. U.S.A. 94, 3883–3887. 41. Pike C. J., Burdick D., Walencewicz A. J., Glabe C. G. and Cotman C. W. (1993) Neurodegeneration induced by b-amyloid peptides in vitro: the role of peptide assembly state. J. Neurosci. 13, 1676–1687. 42. Prehn J. H., Bindokas V. P., Jordan J., Galindo M. F., Ghadge G. D., Roos R. P., Boise L. H., Thompson C. B., Krajewski S., Reed J. C. and Miller R. J. (1996) Protective effect of transforming growth factor-beta1 on beta-amyloid neurotoxicity in rat hippocampal neurons. Molec. Pharmac. 49, 319–328. 43. Przedborski S., Jackson-Lewis V., Kostic V., Carlson E., Epstein C. J. and Cadet J. L. (1992) Superoxide dismutase, catalase and glutathione peroxidase activities in copper/zinc superoxide dismutase transgenic mice. J. Neurochem. 58, 1760–1767. 44. Przedborski S., Khan U., Kostic V., Carlson E., Epstein C. J. and Sulzer D. (1996) Increased superoxide dismutase activity improves survival of cultured postnatal midbrain neurons. J. Neurochem. 67, 1383–1392. 45. Rabizadeh S., Gralla E. B., Borchelt D. R., Gwinn R., Valentine J. S., Sisodia S., Wong P., Lee M., Hahn H. and Bredesen D. E. (1995) Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: studies in yeast and neural cells. Proc. natn. Acad. Sci. U.S.A. 92, 3024–3028. 46. Reed J. C. (1997) Cytochrome C: can’t live with it—can’t live without it. Cell 91, 559–562. 47. Sanchez-Ramos J. R., Song S., Facca A., Basit A. and Epstein C. J. (1997) Transgenic murine dopaminergic neurons expressing human CuZn superoxide dismutase exhibit increased density in culture, but no resistance to methylphenylpyridinium-induced degeneration. J. Neurochem. 68, 58–67. 48. Sen C. K. and Packer L. (1996) Antioxidant and redox regulation of gene transcription. Fedn Proc. Fedn Am. Socs exp. Biol. 10, 709–720. 49. Shearman M., Hawtin S. and Taylor V. (1995) The intracellular component of cellular 3-(4:5-dimethylthiazol-2-yl-2:5diphenyltetrazolium bromide (MTT) reduction is specifically inhibited by b-amyloid peptides. J. Neurochem. 65, 218–227. 50. Sheehan J. P., Swerdlow R. H., Miller S. W., Davis R. E., Parks J. K., Parker W. D. and Tuttle J. B. (1997) Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer’s disease. J. Neurosci. 17, 4612–4622. 51. Shimizu S., Eguchi Y., Kosaka H., Kamiike W., Matsuda H. and Tsujimoto Y. (1995) Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature 374, 811–813. 52. Steinman H. M. (1995) The Bcl-2 oncoprotein functions as a prooxidant. J. biol. Chem. 270, 3487–3490. 53. Troy C. M. and Shelanski M. L. (1994) Down regulation of copper–zinc superoxide dismutase causes apoptotic death in PC12 neuronal cells. Proc. natn. Acad. Sci. U.S.A. 91, 6384–6387. 54. Watt J. A., Pike C. J., Walencewics-Wasserman A. J. and Cotman C. W. (1994) Ultrastructural analysis of b-amyloid-induced apoptosis in cultured hippocampal neurons. Brain Res. 661, 147–156. 55. Weiss S., Pin J. P., Sebben M., Kemp D. E., Sladeczek F., Gabrion J. and Bockaert J. (1986) Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study. Proc. natn. Acad. Sci. U.S.A. 83, 2238–2242. 56. Yan S. D., Chen X. and Fu J., et al. (1996) RAGE and amyloid-b peptide neurotoxicity in Alzheimer’s disease. Nature 382, 685–691. 57. Yim M. B., Chock P. B. and Stadtman E. R. (1993) Enzyme function of copper–zinc superoxide dismutase as a free radical generator. J. biol. Chem. 268, 4099–4105. (Accepted 29 January 1999)