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Multiple signaling events in amyloid β-induced, oxidative stress-dependent neuronal apoptosis
Free Radical Biology & Medicine, Vol. 35, No. 1, pp. 45–58, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter
doi:10.1016/S0891-5849(03)00244-2
Original Contribution MULTIPLE SIGNALING EVENTS IN AMYLOID -INDUCED, OXIDATIVE STRESS-DEPENDENT NEURONAL APOPTOSIS ELENA TAMAGNO,* MAURIZIO PAROLA,* MICHELA GUGLIELMOTTO,* GIANNI SANTORO,* PAOLA BARDINI,* LAURA MARRA,* MASSIMO TABATON,† and OLIVIERO DANNI* *Department of Experimental Medicine and Oncology, General Pathology Section, University of Turin, Turin, Italy; and † Department of Neurological Sciences and Vision, University of Genoa, Genoa, Italy (Received 30 December 2002; Revised 17 March 2003; Accepted 10 April 2003)
Abstract—Current evidence suggests that amyloid  peptides (A) may play a major role in the pathogenesis of Alzheimer’s disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by A1– 40 and A1– 42 peptides as well as the relationships between apoptosis and oxidative stress. A peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by ␣-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38MAPK, involved early elevation of p53 protein levels. Pretreatment of neurons with ␣-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of A induced apoptosis. These results suggest that oxidative stress - mediated neuronal apoptosis induced by amyloid  operates by eliciting a SAPK– dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2. © 2003 Elsevier Inc. Keywords—Amyloid  peptides, Oxidative stress, p53, JNKs, p38MAPK, Apoptosis, Free radicals
indicate that exposure of neuronal cells to A peptides is followed by apoptotic cell death [3,4]. The precise mechanism by which A induces neuronal apoptosis is still a matter of debate, but current literature suggests a central role for oxidative stress in AD pathogenesis. Reactive oxygen intermediates (ROI), such as superoxide anion (O2•⫺) and hydrogen peroxide (H2O2), as well as 4-hydroxynonenal (HNE), a major aldehydic end product of lipid peroxidation, may mediate A neurotoxicity [5,6]. Aldehydic end products of polyunsaturated fatty acids, such as 4-hydroxynonenal (HNE), colocalize with intraneuronal neurofibrillary tangles and contribute to the cytoskeletal derangement proper of the disease [7]. Protein oxidation, lipid peroxidation, and peroxynitrite formation have been unequivocally reported in either human brain samples or in experimental models of AD [8]. Either ROI or HNE have been shown to trigger apoptosis in a large variety of cultured cells of different origin [9,10], including some cultured neuronal cells [11,12], and to stimulate stress-
INTRODUCTION
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory loss and cognitive impairment [1]. Although the ethiology of AD is not fully understood, an increasing body of evidence suggests the importance of  amyloid (A) in the initiation/progression of the disease. A, a 39 – 43 amino acid peptide, assembles into insoluble aggregates forming plaques [2]. Degeneration of neurons seems to be a fundamental process responsible for clinical manifestations of many different neurological disorders, including AD. It is generally accepted that A peptides may contribute to neuronal and synaptic loss during the course of the disease and “in vitro” data Address correspondence to: Dr. Elena Tamagno, Department of Experimental Medicine and Oncology, General Pathology Section, University of Turin, Corso Raffaello 30, 10125, Turin, Italy; Tel: ⫹39 (11) 670-7763; Fax: ⫹39 (11) 670-7753; E-Mail: elena.tamagno@ unito.it. 45
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activated protein kinases [13–15] such as JNK and p38MAPK. The latter kinases are known to be activated in neuronal cells undergoing apoptosis after survival signal withdrawal [16,17] or exposure to A [18,19]. By investigating the mechanism(s) underlying oxidative stress-dependent, A-induced apoptosis, we have previously reported that this event requires early and simultaneous generation of hydrogen peroxide and HNE that, in turn, are primarily responsible for JNK and p38MAPK activation as well as for apoptosis [20]. In the present study we show that A-induced apoptosis not only requires oxidative stress-mediated activation of SAPKs, but operates through recruitment of classic apoptotic mitochondrial regulatory proteins involving both p53 and Bcl-2. Moreover, all the biological events induced by A are mimicked by oxidative stress-related reactive intermediates, significantly prevented when cells are pretreated with antioxidants and completely abolished by specific SAPKs inhibitors. MATERIALS AND METHODS
Materials All reagents used for cell culture and differentiation were from Sigma Chemical Company (St. Louis, MO, USA). A1– 40 and A1– 42 were from Bachem (Bubendorf, Switzerland). HNE, SB203580 inhibitor of p38MAPK, and ␣-pifithrin were purchased from Calbiochem (La Jolla, CA, USA). Polyclonal antibodies against caspase 3 and PARP were from Santa Cruz Biotechnology (La Jolla, CA, USA). Monoclonal antibody against p53 was from Zymed Laboratories Inc. (San Francisco, CA, USA); polyclonal antibody against Bax and monoclonal anti Bcl-2 were from Oncogene Research Products (Boston, MA, USA); monoclonal antibody against cytochrome c was from BD Pharmingen (San Diego, CA, USA). JNK inhibitor SP600125 was purchased from Biomol Research Laboratories (Plymouth, PA, USA). SK-N-BE differentiation SK-N-BE neuroblastoma cell line used in this study underwent neuronal differentiation by means of chronic treatment with retinoic acid (RA) [21]. Cells were maintained in RPMI 1640 medium containing 2 mmol/l glutamine and supplemented with 100 ml/l fetal bovine serum, 10 ml/l nonessential amino acids, and 10 ml/l antibiotic mixture (penicillin-streptomycin-amphotericin), in a humidified atmosphere at 37°C with 5% CO2. For differentiation, 2 ⫻ 106 cells were plated in 75 cm2 culture flasks (Costar) and exposed to 10 mol/l RA for 10 d. Growth medium was changed three times a week.
Experimental protocol In all experiments differentiated cells were left for 24 h in serum-free RPMI medium and then treated with A1– 40 and A1– 42. A fragments were dissolved in water at 1 mg/ml directly before use or the solution was aliquoted and stored frozen at ⫺20°C. Refreezing was avoided. Before use these peptides were maintained at 37°C for 5 d, and at the beginning of each experiment were diluted to the desired final concentration [22]. A peptides used in this study were usually employed at 100 nmol/l concentration; in one set of experiments, designed to follow apoptosis and cell death at the end of 1 week of treatment, SK-N-BE cells were exposed to 25 as well as 50 nmol/l concentrations of A. In the experiments in which SK-N-BE were exposed only to HNE, H2O2, or both, these reactive intermediates were used at concentrations of 1 and 10 mol/l and 10 mol/l, respectively. Differentiated cells were exposed to A peptides, HNE, and H2O2, as well as to HNE 1 M/H2O2 10 M mixture for 4 h. To evaluate the morphological occurrence of apoptosis, differentiated SK-N-BE cells were incubated in the presence of the mentioned agents for 24 h. When required, the antioxidants ␣-tocopherol succinate and N-acetylcysteine (final concentration 100 mol/l) as well as inhibitors of JNK and p38MAPK (SP600125 20 mol/l, SB203580 10 mol/l, respectively), ␣-pifithrin (200 nmol/l) or the inhibitor of execution caspases z-VAD.fmk (100 mol/l) were added to culture medium 1 h (antioxidants, ␣-pifithrin, and z-VAD.fmk) or 15 min (SAPK inhibitors) before exposure of cells to the various experimental conditions (i.e., A peptides and H2O2/HNE mixture). Oxidative stress determination Thiobarbituric acid reactive substances (TBARS) formation was evaluated following the method described by Esterbauer et al. [23]. HNE was detected by means of an HPLC procedure essentially as previously described [24]. An aliquot of culture media was added, in equal volume, to acetonitrile-acetic acid (96:4,v:v). Samples were then centrifuged at 250 ⫻ g for 20 min at 4°C and the supernatant was directly injected to HPLC (Waters Associated, Milford, MA, USA) using a RP-18 column (Merck, Darmstadt, Germany). The mobile phase used was 42% acetonitrile:bidistilled water (v:v). Authentic 4-hydroxynonenal (Calbiochem, La Jolla, CA, USA) was used as a standard. End products of lipid peroxidation were also evaluated in terms of fluorescent chromolipid adducts. Total lipids were extracted by the method of Folch et al. [25]. Fluorescent intensity of samples was evaluated at 360/ 430 excitation/emission as described by Esterbauer et al.
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Fig. 1. Induction of oxidative stress in differentiated SK-N-BE cells after treatment with A1– 40 100 nM and A1– 42 100 nM. Oxidative stress has been evaluated in terms of release of H2O2 and of HNE in the medium (panels A and B), of TBARS production (C) and of fluorescent chromolipids formation (D). Data have been obtained at the end of 4 h of incubation at 37°C. Values are means ⫾ SD of three experiments performed in duplicate. 夽 ⫽ significantly different from control (p ⬍ .02).
[26], using quinine sulphate (0.1 g/ml in 0.05 H2SO4) as standard. Oxidative stress was finally monitored, also measuring generation of hydrogen peroxide (H2O2), adding horseradish peroxidase and acetylated ferrocytochrome c to cells. H2O2 release was evaluated as the increase of acetylated ferrocytochrome c oxidation rate as described by Zoccarato et al. [27].
Preparation of cell lysates and cytosolic extracts
Cell viability
Three 75 cm2 flasks for each condition at confluence were harvested, treated with 0.1% Na-deoxycholate, and maintained for 15 min to ⫺80°C. The lysed cells were then added to mannitol-sucrose buffer for a final strength of 1⫻ (210 mM mannitol, 70 mM sucrose, 5 mM EDTA, 5 mM Tris, pH 7.5). The ice-cold suspension was then centrifuged for 10 min at 800 ⫻ g to pellet nuclei. The supernatant was saved, the pelleted material was resuspended in 1⫻ mannitol-sucrose buffer, and centrifugation was repeated. This procedure was repeated three
The extent of necrosis was evaluated using lactate dehydrogenase (LDH) release in culture medium. Enzymatic analysis of LDH activity released by necrotic cells in culture medium was performed as previously described [20]. Values for control and treated cells were expressed as a percentage value of the total LDH activity released by untreated cells after exposure to Triton X-100.
Confluent differentiated cells were treated with the appropriate experimental conditions, quickly placed on ice and washed with ice-cold PBS. Cell lysates and cytosolic extracts were obtained by the method of Andrew and Faller as previously described [28]. Preparation of mitochondria
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Fig. 2. Induction of caspase 3 (A) and caspase 3-like activity (B) obtained in lysates of differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). Induction was prevented by 1 h pretreatment with ␣-tocopherol (100 M) and N-acetylcysteine (100 M). Equal protein loading was confirmed by evaluating the levels of  actin. 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02); ⫹ ⫽ significantly different from A peptides or HNE 1 M/H2O2 10 M mixture (p ⬍ .02).
times. The combined supernatants were then centrifuged to pellet any remaining nuclei, and the resulting supernatant was centrifuged at 10,000 ⫻ g to pellet mitochondria. Isolated mitochondria were re-suspended in a buffer containing 20 mM HEPES, pH 7.6, 1 mM EDTA, 5 mM dithiothreitol, 300 mM KCl, and 5% glycerol. This preparation was briefly sonicated on ice and 5 l of protease inhibitor mixture from Sigma (for mammalian extracts, 100 mM AEBSF [4-(2-amynoethyl) benzenesulfonyl fluoride], 4 mM bestatin, 1.4 mM E64, 2.2mM leupeptin, 1.5 mM pepsatin and 80 M aprotinin) were added per milliliter of buffer. The fractions were again centrifuged at 5000 ⫻ g to pellet any remaining cell debris and supernatant proteins were used for Western blot analysis [29]. Western blot analysis Total cell lysates, cytosolic and mitochondrial extracts were subjected to SDS-PAGE on acrylamide gels using the mini-PROTEAN II electrophoresis cell (Bio-
Rad Laboratories, Segrate, Milano, Italy) according to Laemmli [30]. Proteins were transferred electrophoretically to nitrocellulose membranes (Hybond-C extra; Amersham Life Science, Arlington Heights, IL, USA). Unspecific binding was blocked with 50 g/l nonfat dry milk in 50 mmol/l Tris-HCl, pH 7.4, containing 200 mmol/l NaCl and 0.5 ml/l Tween 20 (TBS-Tween). The blots were incubated with the different primary antibodies, followed by incubation with peroxidase-conjugated antimouse or anti-rabbit immunoglobulins in TBS-Tween containing 20 g/l nonfat dry milk. Immunoblots were developed with ECL-plus reagents from Amersham according to manufacturer’s instructions. Comparative analysis of protein levels was performed by means of computer-assisted densitometric analysis of protein bands using an appropriate software program (Multianalyst, Version 1.1, Bio-Rad Laboratories, Segrate Milano); results were normalized to actin levels in the corresponding blots and expressed as fold increase vs. respective control values; in crucial experiments con-
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Fig. 3. Induction of PARP cleavage (A) and of cytosolic cytochrome c release (B) obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). Induction was prevented by 1 h pretreatment with ␣-tocopherol (100 M) and N-acetylcysteine (100 M). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from PARP and cytochrome c with those obtained for  actin.
cerning effects of ␣-pifithrin and SAPKs inhibitors, data were also expressed as arbitrary units of optical density and as means ⫾ SD of three independent experiments.
[11]. All the experiments were repeated three times and the number of stained cells was counted in 10 randomly selected fields.
Caspase 3-like activity
Statistical analysis of data
Activity of caspase 3 was detected by using a colorimetric kit (Sigma Chemical Company). The colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-asp p-nitroanilide (AcDEVD-pNA) by caspase 3, resulting in the release of p-nitroaniline (p-NA) moiety.
Where appropriate, statistical analysis was performed by means of Student’s t-test or ANOVA test followed by the Bonferroni post test [31].
Morphological detection of apoptosis
Induction of oxidative stress by A peptides
The occurrence of apoptosis was evaluated by 4'-6 diamidino-2-phenylindole (DAPI) staining. To identify apoptotic nuclei with DAPI staining, cells were grown on glass coverslips, washed in PBS, fixed and permeabilized with 95% ethanol for 5 min, and then stained with DAPI solution for 30 min at 37°C. After rinsing in PBS, coverslips were mounted on glass slides and observed by fluorescence microscopy (Leitz Dialux 20 Microscope)
A peptides have been reported to initiate multiple membrane alterations including protein and lipid oxidation [5,32–34]. Oxidative stress induced by A peptides resulted in an early and significant increase in HNE and H2O2; 170% and 400%, respectively (Fig. 1, A and B). Accordingly, exposure of differentiated SK-N-BE cells to A peptides was followed by a significant increase in lipid peroxidation, as evaluated
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Fig. 4. Increase of intracellular p53 levels (A) as evaluated by Western blot analysis obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). The increase was prevented by 1 h pretreatment with ␣-tocopherol and N-acetylcysteine. Pretreatment of cells with specific inhibitors of JNKs (SP600125) and p38MAPK (SB203580) or with ␣-pifithrin completely prevented the increase of p53 protein levels (panels B and C). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from p53 with those obtained for  actin.
by determining fluorescent chromolipid adducts (⫹150%) and TBARS production (⫹ 400%) (Fig. 1, C and D). All the parameters studied were significantly increased after 4 h of incubation with A peptides as compared to control cells. Induction of caspase 3 and PARP cleavage by A peptides and oxidative stress-related intermediates Caspase 3 activation was evaluated either by quantifying both 32 kDa proenzyme and 20 kDa active isoform in SK-N-BE cell lysates by Western blot analysis or by evaluating spectrophotometrically caspase 3-related enzymatic activity. A significant increase (90 –100%) in 20 kDa active isoform levels was detected 4 h after treatment with either A peptides or HNE and H2O2 (alone or in combination), suggesting proteolytic activation of caspase-3 in our experimental conditions. This feature was associated with the concomitant decrease in the level of pro-caspase-3 (Fig. 2A). Ac-DEVD-pNa cleavage activity (i.e., a measure of caspase 3-like activity) was
again significantly increased (400 – 600%) after treatment with A peptides or exposure to the combination of HNE and H2O2. Furthermore, cleavage of PARP (an endogenous substrate of caspase 3) to the 86 kDa Cterminal fragment resulted similarly increased (⫹400 – 500%) after treatment with A peptides as well as prooxidant conditions (Fig. 3). The induction of classic apoptotic cell death was confirmed by data obtained monitoring cytochrome c release in the cytosol (Fig. 3B). Results obtained in cells treated with A peptides or with HNE and/or H2O2 are in agreement with those already obtained for the other parameters of apoptosis, indicating that all the experimental conditions used were effective in increasing cytochrome c release (approximately 250%). Pretreatment of differentiated SK-N-BE cells with ␣-tocopherol and N-acetylcysteine significantly prevented activation of caspase 3, release of cytochrome c, and cleavage of PARP (Fig. 2, A and B; Fig. 3, A and B), suggesting a direct relationship between A peptides-
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Fig. 5. Increase of Bax levels (A) by Western blot analysis obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). The increase was prevented by 1 h pretreatment with ␣-tocopherol and N-acetylcysteine. Pretreatment of cells with specific inhibitors of JNKs (SP600125) and p38MAPK (SB203580) or with ␣-pifithrin, completely prevented the increase of proapoptotic Bax protein levels (panels B and C). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from Bax with those obtained for  actin.
mediated activation of classic caspase-dependent executing pathway and oxidative stress. Role of p 53 in the induction of pro-apoptotic pathways by A peptides and oxidative stress Exposure of SK-N-BE differentiated cells to A peptides, HNE, and/or H2O2 resulted in an early and significant increase (approx. ⫹400%) in p53 protein levels, as evaluated by Western blot analysis (Fig. 4A). Pretreatment with antioxidants was again able to significantly prevent this induction. To evaluate the relative role of p53 and of SAPKs in the apoptotic pathway induced by A peptides, cells were pretreated with a mixture of two specific inhibitors for JNKs and p38MAPK (SB203580⫹SP600125) or with a specific inhibitor for p53 (␣-pifithrin). Specific inhibitors for JNKs and p38MAPK were used simultaneously because we found in a previous study [20] that only this procedure was followed by an almost complete prevention of apoptosis induced by A peptides or by oxidative stress intermediates. Treatment of
cells with the single inhibitors resulted in a significant but incomplete (approximately 50%) prevention of apoptosis, suggesting equal causative involvement of both pathways [20]. As shown in Fig. 4B, inhibition of the two kinases resulted in the complete prevention of p53 induction. Moreover, the pretreatment of cells with ␣-pifithrin also is able to inhibit p53 induction (Fig. 4C). Exposure of cells to A peptides and to HNE and H2O2, alone or in association, resulted in a significant increase (⫹300 – 400%) of the protein levels of the proapoptotic effector Bax (Fig. 5); moreover, antioxidants were able to significantly prevent this induction (Fig. 5A). As expected, the induction of Bax was completely prevented by either SAPKs inhibitors or by ␣-pifithrin, suggesting that Bax was under the control of SAPKs that operated through activation of p53. Data reported in Fig. 6 indicate that the same experimental conditions that were able to elicit an increase of Bax levels also resulted in a significant decrease of Bcl-2 (⫺50%) (Fig. 6A). Interestingly, Bcl-2 levels were strongly affected by
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Fig. 6. Decrease of Bcl-2 levels (A) by Western blot analysis obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). The decrease was prevented by 1 h pretreatment with ␣-tocopherol and N-acetylcysteine. Pretreatment of cells with specific inhibitors of JNKs (SP600125) and p38MAPK (SB203580) completely prevented the decrease of antiapoptotic Bcl-2 protein levels (B). Pretreatment with ␣ pifithrin was ineffective (C). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from Bcl-2 with those obtained for  actin.
SAPKs inhibitors but were essentially p53-independent: treatment of cells with SB203580/SP600125 mixture prevented the decrease of Bcl-2 induced by A peptides or by HNE/H2O2 (Fig. 6B), whereas ␣-pifithrin was ineffective. Similarly, pretreatment with SB203580/SP600125 mixture was able to offer a complete prevention in terms of cyt.c release, caspase 3 activation, and PARP cleavage by A peptides and oxidative stress intermediates (Fig. 7, panels A, B, and C). However, ␣-pifithrin pretreatment resulted only in a significant but incomplete prevention of the mentioned parameters. As documented by densitometric analyses, a residual increase in cytochrome c release, caspase 3 activation, and PARP cleavage was detected in the presence of ␣-pifithrin. The antiapoptotic action of SAPKs inhibitors and ␣-pifithrin was confirmed morphologically by means of DAPI staining (Fig. 8) and once again ␣-pifithrin resulted in an approximate 50 – 60% prevention of A peptides and oxidative stress intermediates mixture-induced apoptosis.
A peptides as well as HNE and/or H2O2 do not induce a necrotic type of cell death In order to assess whether A peptides as well as HNE and/or H2O2 may elicit necrotic cell death in SKN-BE cells, the release of lactate dehydrogenase has been evaluated at 24 (Fig. 9, panel A) and 48 h (Fig. 9, panel B) time points in the presence as well as in the absence of the inhibitor of execution caspases z-VAD.fmk (i.e., to inhibit specifically caspase-mediated apoptosis). Our data indicate that no significant change in LDH release was found, suggesting that A- as well as HNE- and/or H2O2-induced apoptosis represents the major phenomenon in our experimental conditions. Chronic exposure of SK-N-BE cells to lower doses of A peptides is again followed by induction of p53 and activation of caspase 3 Because AD is a chronic disease, we exposed SKN-BE cells to lower concentrations of A peptides for
A, oxidative stress and apoptosis
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Fig. 7. Prevention of cytosolic cytochrome c release (A), caspase 3-like activity (B) and PARP cleavage (C) observed by pretreating differentiated SK-N-BE cells with SAPK inhibitors and ␣-pifithrin, before exposure to A peptides or HNE 1 M/H2O2 10 M mixture. Cell lysates were obtained after 4 h of the latter treatment. Equal protein loading was confirmed by evaluating the levels of  actin. Optical density values of cytochrome c and PARP, normalized against  actin, are means ⫾ SD of three independent experiments. 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02); ⫹ ⫽ significantly different from A peptides or HNE 1 M/H2O2 10 M mixture (p ⬍ .02).
as long as 1 week to evaluate in a preliminary way whether the features previously described may still be recognized. In order to do so we evaluated, as relevant parameters, active form of caspase 3, p53 levels, and LDH release. In these “chronic” experimental conditions we found once again that A peptides were still able to induce both activation of caspase 3 (monitored as a significant increase in the 20 KDa active form)
and recruitment of p53 (Fig. 10, panels A and B) without eliciting any significant increase in LDH release (Fig. 10). DISCUSSION
Although recent evidence suggests the importance of A in AD-related neuronal death, the signaling mecha-
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Fig. 8. Prevention of morphological signs of apoptosis induced by A1– 40 and HNE 1 M/H2O2 10 M mixture. Nuclear SK-N-BE morphology has been evaluated in terms of DAPI staining 24 h after treatments with A1– 40 and HNE 1 M/H2O2 10 M mixture. Inhibitors of JNKs (SP600125) and p38MAPK (SB203580) and ␣-pifithrin were added 15 min and 1 h, respectively, before treatment with A1– 40 or mixture. Apoptotic nuclei are indicated by arrows (A). In panel B apoptosis is expressed as the number of apoptotic cells on the coverslips 24 h after treatment. Data shown are the means ⫾ SD of three independent experiments. 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02); ⫹ ⫽ significantly different from A peptides or HNE 1 M/H2O2 10 M mixture (p ⬍ .02).
nisms controlling this event are still unclear and a matter of debate. The present study indicates that A peptides can induce classic apoptosis (i.e., caspase 3-dependent) throughout oxidative stress-mediated activation of SAPKs that, in turn, can affect proapoptotic mitochondrial regulatory pathways by involving independently both p53- and Bcl-2-dependent processes. Moreover, in our experimental conditions, apoptotic cell death is the major feature observed in SK-N-BE cells exposed to A peptides as well as to HNE and H2O2; necrotic cell death does not significantly occur even if the cells are exposed chronically to A peptides.
All the major events induced by A peptides, including induction of Bax, decrease of Bcl-2, recruitment of p53, cytosolic release of cytochrome c as well as of PARP cleavage, can be mimicked by the simultaneous administration of low concentrations of HNE and H2O2 that are known to be generated early after exposure of SK-N-BE to A peptides [20]. Moreover, since all the parameters affected by A peptides are significantly prevented by pretreatment with ␣-tocopherol and NAC, a direct involvement of oxidative stress is strongly supported. Previous data and present findings indicate that stressactivated protein kinases JNKs and p38MAPK are primar-
A, oxidative stress and apoptosis
Fig. 9. Cell death induced by 24 h (A) or 48 h (B) treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX) as evaluated in terms of LDH release. Pretreatment with z-VAD.fmk did not modify the parameter.
ily involved in A-induced neuronal apotosis. Although no clear consensus has been achieved in the literature concerning the role of JNKs and p38MAPK in determining neuronal apoptosis, Shoji et al. [19] have recently shown that intracellular A accumulation can indeed trigger JNK activation leading to neuronal cell death. Moreover, evidence for a sequential activation of ERK, JNK/SAPK, and p38MAPK in brain tissue from subjects with AD has been provided [35]. In this connection, it has been clearly established that in the brain of AD patients both p38MAPK and JNKs are activated [36,37]. Moreover, in another study Zhu et al. [38] have shown evidence for the activation in AD patient’s brain of MKK6, an upstream kinase activator of p38MAPK. More recently, Wei et al. [39] reported that JNK was rapidly activated by A treatment and that this activation appeared to be critical for A-induced neuronal death. However, these authors reported no activation of p38MAPK by A in their model of cultured SH-SY5Y cells.
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A direct link between oxidative stress and SAPKs has been shown demonstrating that JNK and p38MAPK are activated by HNE and H2O2 in various cell types [13,40,41]. In a previous study we reported that the simultaneous addition of exogenous HNE and H2O2, used at the same low concentrations detected within the first 3 h after A exposure, can elicit activation of JNK and p38MAPK as well as apoptosis [20]. The use of specific inhibitors of JNKs and p38MAPK (SP600125 and SB203580) or of the specific inhibitor of p53 ␣-pifithrin, that selectively blocks the ability of p53 to activate target genes in neurons [42], pointed out that activation of SAPKs is crucial since inhibition of JNKs and p38MAPK completely prevented apoptosis induced by A peptides and mediated by oxidative stress intermediates. However, inhibition of p53 function by ␣-pifithrin still resulted in a significant (approximately 50 – 60%), but not complete, prevention of apoptotic neuronal cell death. p53 is known to play a crucial role in the regulation of apoptosis in many cell types by stimulating expression and/or mitochondrial translocation of the death effector protein Bax [43]. Recently, involvement of p53 in neuronal death occurring in Alzheimer’s disease [44], Parkinson’s disease [45], and traumatic brain injury [46] has been detected. Cell culture studies have established strong relationships between p53 expression and neuronal death induced by DNA damaging agents and glutamate [47]. A critical role for p53 in neuronal apoptosis resulting from ischemic and excitotoxic insults has been confirmed by studies performed in p53-deficient mice [48,49]. However, results by Giovanni et al. [50] described that apoptosis by A in cultured embryonic cortical neurons obtained from p53 knockout mice was mainly p53-independent. In our experimental model the involvement of p53 in A-induced apoptotic cell death seems critical since it accounts for approximately 50 – 60% of total cell number, as suggested by data obtained with the specific inhibitor ␣-pifithrin. Accumulating evidence suggests that the regulation of p53’s transcriptional activities depends on the rate of its phosphorylation [51,52]. Stability of p53, a key factor able to determine its ability to mediate multiple activities, is known to be tightly regulated by JNK in a phosphorylation-dependent manner [53]. A phosphoacceptor site of p53 for JNK phosphorylation, tyrosine 81, seems to be crucial for p53 activities [54]. Moreover, phosphorylation by p38MAPK on residues 33 and 46 is required for UV-induced p53-mediated apoptosis [55], and inhibition of p38MAPK has been found to attenuate transcriptional activities of p53 and p53-dependent apoptosis induced by chemotherapeutic agents [56]. Results obtained with SAPKs inhibitors and ␣-pifithrin suggest that in SK-N-BE cells exposed to
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Fig. 10. Induction of caspase 3 (A) and of p53 protein levels (B) obtained in lysates of differentiated SK-N-BE cells after a chronic (1 week) treatment with low concentrations of A peptides (25–50 nM). Equal protein loading was confirmed by evaluating  actin levels. Densitometric analysis of caspase 3 and p53 levels, normalized against  actin, are means ⫾ SD of three independent experiments. Cell death has been evaluated in terms of LDH release (C). 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02).
A, p53 involvement is downstream to SAPKs activation; in this connection, activation of SAPKs, particularly of JNK isoforms, may act as proapoptotic factor also by phosphorylating and inactivating the antiapoptotic protein Bcl-2 [57]. The involvement of this pathway may explain the 40 –50% residual, p53independent apoptosis. Data presented in this study, including negative effects of pretreatment with ␣-pifithrin on bcl-2 levels and complete prevention of A-induced apoptosis by SAPK inhibitors strongly support this view. Other authors have also demonstrated that UV-induced classic apoptosis in mouse
fibroblasts, mediated by cytochrome c release, can be completely abrogated in the absence of JNK signaling [58]. The scheme in Fig. 11 summarizes results obtained in this study suggesting that oxidative stressmediated neuronal apoptosis induced by amyloid  peptides operates by eliciting a SAPK-dependent multiple regulation of proapoptotic mitochondrial pathways involving both p53 and bcl-2. Finally, since the involvement of p53 has been reported in neurodegenerative diseases, including AD, our findings seem to confirm that chemical inhibitors of p53 activation may be efficient in hampering neurodegeneration [42].
A, oxidative stress and apoptosis
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
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Fig. 11. Proposed pro-apoptotic sequence elicited by A peptides throughout the generation of oxidative stress-related intermediates, related activation of SAPK, and recruitment of p53 protein.
[17]
[18] Acknowledgements — This study was supported by Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST, Rome), National Project: Alteration of signaling systems induced by glyco-oxidative stress: potential substrates of Alzheimer’s disease pathogenesis.
[19]
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ABBREVIATIONS
HNE— 4-hydroxynonenal JNK— c-Jun NH2- terminal kinase p38MAPK—p38-mitogen activated protein kinase PARP—poly-ADP rybose polymerase SAPK—stress-activated protein kinase
doi:10.1016/S0891-5849(03)00244-2
Original Contribution MULTIPLE SIGNALING EVENTS IN AMYLOID -INDUCED, OXIDATIVE STRESS-DEPENDENT NEURONAL APOPTOSIS ELENA TAMAGNO,* MAURIZIO PAROLA,* MICHELA GUGLIELMOTTO,* GIANNI SANTORO,* PAOLA BARDINI,* LAURA MARRA,* MASSIMO TABATON,† and OLIVIERO DANNI* *Department of Experimental Medicine and Oncology, General Pathology Section, University of Turin, Turin, Italy; and † Department of Neurological Sciences and Vision, University of Genoa, Genoa, Italy (Received 30 December 2002; Revised 17 March 2003; Accepted 10 April 2003)
Abstract—Current evidence suggests that amyloid  peptides (A) may play a major role in the pathogenesis of Alzheimer’s disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by A1– 40 and A1– 42 peptides as well as the relationships between apoptosis and oxidative stress. A peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by ␣-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38MAPK, involved early elevation of p53 protein levels. Pretreatment of neurons with ␣-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of A induced apoptosis. These results suggest that oxidative stress - mediated neuronal apoptosis induced by amyloid  operates by eliciting a SAPK– dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2. © 2003 Elsevier Inc. Keywords—Amyloid  peptides, Oxidative stress, p53, JNKs, p38MAPK, Apoptosis, Free radicals
indicate that exposure of neuronal cells to A peptides is followed by apoptotic cell death [3,4]. The precise mechanism by which A induces neuronal apoptosis is still a matter of debate, but current literature suggests a central role for oxidative stress in AD pathogenesis. Reactive oxygen intermediates (ROI), such as superoxide anion (O2•⫺) and hydrogen peroxide (H2O2), as well as 4-hydroxynonenal (HNE), a major aldehydic end product of lipid peroxidation, may mediate A neurotoxicity [5,6]. Aldehydic end products of polyunsaturated fatty acids, such as 4-hydroxynonenal (HNE), colocalize with intraneuronal neurofibrillary tangles and contribute to the cytoskeletal derangement proper of the disease [7]. Protein oxidation, lipid peroxidation, and peroxynitrite formation have been unequivocally reported in either human brain samples or in experimental models of AD [8]. Either ROI or HNE have been shown to trigger apoptosis in a large variety of cultured cells of different origin [9,10], including some cultured neuronal cells [11,12], and to stimulate stress-
INTRODUCTION
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory loss and cognitive impairment [1]. Although the ethiology of AD is not fully understood, an increasing body of evidence suggests the importance of  amyloid (A) in the initiation/progression of the disease. A, a 39 – 43 amino acid peptide, assembles into insoluble aggregates forming plaques [2]. Degeneration of neurons seems to be a fundamental process responsible for clinical manifestations of many different neurological disorders, including AD. It is generally accepted that A peptides may contribute to neuronal and synaptic loss during the course of the disease and “in vitro” data Address correspondence to: Dr. Elena Tamagno, Department of Experimental Medicine and Oncology, General Pathology Section, University of Turin, Corso Raffaello 30, 10125, Turin, Italy; Tel: ⫹39 (11) 670-7763; Fax: ⫹39 (11) 670-7753; E-Mail: elena.tamagno@ unito.it. 45
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activated protein kinases [13–15] such as JNK and p38MAPK. The latter kinases are known to be activated in neuronal cells undergoing apoptosis after survival signal withdrawal [16,17] or exposure to A [18,19]. By investigating the mechanism(s) underlying oxidative stress-dependent, A-induced apoptosis, we have previously reported that this event requires early and simultaneous generation of hydrogen peroxide and HNE that, in turn, are primarily responsible for JNK and p38MAPK activation as well as for apoptosis [20]. In the present study we show that A-induced apoptosis not only requires oxidative stress-mediated activation of SAPKs, but operates through recruitment of classic apoptotic mitochondrial regulatory proteins involving both p53 and Bcl-2. Moreover, all the biological events induced by A are mimicked by oxidative stress-related reactive intermediates, significantly prevented when cells are pretreated with antioxidants and completely abolished by specific SAPKs inhibitors. MATERIALS AND METHODS
Materials All reagents used for cell culture and differentiation were from Sigma Chemical Company (St. Louis, MO, USA). A1– 40 and A1– 42 were from Bachem (Bubendorf, Switzerland). HNE, SB203580 inhibitor of p38MAPK, and ␣-pifithrin were purchased from Calbiochem (La Jolla, CA, USA). Polyclonal antibodies against caspase 3 and PARP were from Santa Cruz Biotechnology (La Jolla, CA, USA). Monoclonal antibody against p53 was from Zymed Laboratories Inc. (San Francisco, CA, USA); polyclonal antibody against Bax and monoclonal anti Bcl-2 were from Oncogene Research Products (Boston, MA, USA); monoclonal antibody against cytochrome c was from BD Pharmingen (San Diego, CA, USA). JNK inhibitor SP600125 was purchased from Biomol Research Laboratories (Plymouth, PA, USA). SK-N-BE differentiation SK-N-BE neuroblastoma cell line used in this study underwent neuronal differentiation by means of chronic treatment with retinoic acid (RA) [21]. Cells were maintained in RPMI 1640 medium containing 2 mmol/l glutamine and supplemented with 100 ml/l fetal bovine serum, 10 ml/l nonessential amino acids, and 10 ml/l antibiotic mixture (penicillin-streptomycin-amphotericin), in a humidified atmosphere at 37°C with 5% CO2. For differentiation, 2 ⫻ 106 cells were plated in 75 cm2 culture flasks (Costar) and exposed to 10 mol/l RA for 10 d. Growth medium was changed three times a week.
Experimental protocol In all experiments differentiated cells were left for 24 h in serum-free RPMI medium and then treated with A1– 40 and A1– 42. A fragments were dissolved in water at 1 mg/ml directly before use or the solution was aliquoted and stored frozen at ⫺20°C. Refreezing was avoided. Before use these peptides were maintained at 37°C for 5 d, and at the beginning of each experiment were diluted to the desired final concentration [22]. A peptides used in this study were usually employed at 100 nmol/l concentration; in one set of experiments, designed to follow apoptosis and cell death at the end of 1 week of treatment, SK-N-BE cells were exposed to 25 as well as 50 nmol/l concentrations of A. In the experiments in which SK-N-BE were exposed only to HNE, H2O2, or both, these reactive intermediates were used at concentrations of 1 and 10 mol/l and 10 mol/l, respectively. Differentiated cells were exposed to A peptides, HNE, and H2O2, as well as to HNE 1 M/H2O2 10 M mixture for 4 h. To evaluate the morphological occurrence of apoptosis, differentiated SK-N-BE cells were incubated in the presence of the mentioned agents for 24 h. When required, the antioxidants ␣-tocopherol succinate and N-acetylcysteine (final concentration 100 mol/l) as well as inhibitors of JNK and p38MAPK (SP600125 20 mol/l, SB203580 10 mol/l, respectively), ␣-pifithrin (200 nmol/l) or the inhibitor of execution caspases z-VAD.fmk (100 mol/l) were added to culture medium 1 h (antioxidants, ␣-pifithrin, and z-VAD.fmk) or 15 min (SAPK inhibitors) before exposure of cells to the various experimental conditions (i.e., A peptides and H2O2/HNE mixture). Oxidative stress determination Thiobarbituric acid reactive substances (TBARS) formation was evaluated following the method described by Esterbauer et al. [23]. HNE was detected by means of an HPLC procedure essentially as previously described [24]. An aliquot of culture media was added, in equal volume, to acetonitrile-acetic acid (96:4,v:v). Samples were then centrifuged at 250 ⫻ g for 20 min at 4°C and the supernatant was directly injected to HPLC (Waters Associated, Milford, MA, USA) using a RP-18 column (Merck, Darmstadt, Germany). The mobile phase used was 42% acetonitrile:bidistilled water (v:v). Authentic 4-hydroxynonenal (Calbiochem, La Jolla, CA, USA) was used as a standard. End products of lipid peroxidation were also evaluated in terms of fluorescent chromolipid adducts. Total lipids were extracted by the method of Folch et al. [25]. Fluorescent intensity of samples was evaluated at 360/ 430 excitation/emission as described by Esterbauer et al.
A, oxidative stress and apoptosis
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Fig. 1. Induction of oxidative stress in differentiated SK-N-BE cells after treatment with A1– 40 100 nM and A1– 42 100 nM. Oxidative stress has been evaluated in terms of release of H2O2 and of HNE in the medium (panels A and B), of TBARS production (C) and of fluorescent chromolipids formation (D). Data have been obtained at the end of 4 h of incubation at 37°C. Values are means ⫾ SD of three experiments performed in duplicate. 夽 ⫽ significantly different from control (p ⬍ .02).
[26], using quinine sulphate (0.1 g/ml in 0.05 H2SO4) as standard. Oxidative stress was finally monitored, also measuring generation of hydrogen peroxide (H2O2), adding horseradish peroxidase and acetylated ferrocytochrome c to cells. H2O2 release was evaluated as the increase of acetylated ferrocytochrome c oxidation rate as described by Zoccarato et al. [27].
Preparation of cell lysates and cytosolic extracts
Cell viability
Three 75 cm2 flasks for each condition at confluence were harvested, treated with 0.1% Na-deoxycholate, and maintained for 15 min to ⫺80°C. The lysed cells were then added to mannitol-sucrose buffer for a final strength of 1⫻ (210 mM mannitol, 70 mM sucrose, 5 mM EDTA, 5 mM Tris, pH 7.5). The ice-cold suspension was then centrifuged for 10 min at 800 ⫻ g to pellet nuclei. The supernatant was saved, the pelleted material was resuspended in 1⫻ mannitol-sucrose buffer, and centrifugation was repeated. This procedure was repeated three
The extent of necrosis was evaluated using lactate dehydrogenase (LDH) release in culture medium. Enzymatic analysis of LDH activity released by necrotic cells in culture medium was performed as previously described [20]. Values for control and treated cells were expressed as a percentage value of the total LDH activity released by untreated cells after exposure to Triton X-100.
Confluent differentiated cells were treated with the appropriate experimental conditions, quickly placed on ice and washed with ice-cold PBS. Cell lysates and cytosolic extracts were obtained by the method of Andrew and Faller as previously described [28]. Preparation of mitochondria
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Fig. 2. Induction of caspase 3 (A) and caspase 3-like activity (B) obtained in lysates of differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). Induction was prevented by 1 h pretreatment with ␣-tocopherol (100 M) and N-acetylcysteine (100 M). Equal protein loading was confirmed by evaluating the levels of  actin. 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02); ⫹ ⫽ significantly different from A peptides or HNE 1 M/H2O2 10 M mixture (p ⬍ .02).
times. The combined supernatants were then centrifuged to pellet any remaining nuclei, and the resulting supernatant was centrifuged at 10,000 ⫻ g to pellet mitochondria. Isolated mitochondria were re-suspended in a buffer containing 20 mM HEPES, pH 7.6, 1 mM EDTA, 5 mM dithiothreitol, 300 mM KCl, and 5% glycerol. This preparation was briefly sonicated on ice and 5 l of protease inhibitor mixture from Sigma (for mammalian extracts, 100 mM AEBSF [4-(2-amynoethyl) benzenesulfonyl fluoride], 4 mM bestatin, 1.4 mM E64, 2.2mM leupeptin, 1.5 mM pepsatin and 80 M aprotinin) were added per milliliter of buffer. The fractions were again centrifuged at 5000 ⫻ g to pellet any remaining cell debris and supernatant proteins were used for Western blot analysis [29]. Western blot analysis Total cell lysates, cytosolic and mitochondrial extracts were subjected to SDS-PAGE on acrylamide gels using the mini-PROTEAN II electrophoresis cell (Bio-
Rad Laboratories, Segrate, Milano, Italy) according to Laemmli [30]. Proteins were transferred electrophoretically to nitrocellulose membranes (Hybond-C extra; Amersham Life Science, Arlington Heights, IL, USA). Unspecific binding was blocked with 50 g/l nonfat dry milk in 50 mmol/l Tris-HCl, pH 7.4, containing 200 mmol/l NaCl and 0.5 ml/l Tween 20 (TBS-Tween). The blots were incubated with the different primary antibodies, followed by incubation with peroxidase-conjugated antimouse or anti-rabbit immunoglobulins in TBS-Tween containing 20 g/l nonfat dry milk. Immunoblots were developed with ECL-plus reagents from Amersham according to manufacturer’s instructions. Comparative analysis of protein levels was performed by means of computer-assisted densitometric analysis of protein bands using an appropriate software program (Multianalyst, Version 1.1, Bio-Rad Laboratories, Segrate Milano); results were normalized to actin levels in the corresponding blots and expressed as fold increase vs. respective control values; in crucial experiments con-
A, oxidative stress and apoptosis
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Fig. 3. Induction of PARP cleavage (A) and of cytosolic cytochrome c release (B) obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). Induction was prevented by 1 h pretreatment with ␣-tocopherol (100 M) and N-acetylcysteine (100 M). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from PARP and cytochrome c with those obtained for  actin.
cerning effects of ␣-pifithrin and SAPKs inhibitors, data were also expressed as arbitrary units of optical density and as means ⫾ SD of three independent experiments.
[11]. All the experiments were repeated three times and the number of stained cells was counted in 10 randomly selected fields.
Caspase 3-like activity
Statistical analysis of data
Activity of caspase 3 was detected by using a colorimetric kit (Sigma Chemical Company). The colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-asp p-nitroanilide (AcDEVD-pNA) by caspase 3, resulting in the release of p-nitroaniline (p-NA) moiety.
Where appropriate, statistical analysis was performed by means of Student’s t-test or ANOVA test followed by the Bonferroni post test [31].
Morphological detection of apoptosis
Induction of oxidative stress by A peptides
The occurrence of apoptosis was evaluated by 4'-6 diamidino-2-phenylindole (DAPI) staining. To identify apoptotic nuclei with DAPI staining, cells were grown on glass coverslips, washed in PBS, fixed and permeabilized with 95% ethanol for 5 min, and then stained with DAPI solution for 30 min at 37°C. After rinsing in PBS, coverslips were mounted on glass slides and observed by fluorescence microscopy (Leitz Dialux 20 Microscope)
A peptides have been reported to initiate multiple membrane alterations including protein and lipid oxidation [5,32–34]. Oxidative stress induced by A peptides resulted in an early and significant increase in HNE and H2O2; 170% and 400%, respectively (Fig. 1, A and B). Accordingly, exposure of differentiated SK-N-BE cells to A peptides was followed by a significant increase in lipid peroxidation, as evaluated
RESULTS
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Fig. 4. Increase of intracellular p53 levels (A) as evaluated by Western blot analysis obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). The increase was prevented by 1 h pretreatment with ␣-tocopherol and N-acetylcysteine. Pretreatment of cells with specific inhibitors of JNKs (SP600125) and p38MAPK (SB203580) or with ␣-pifithrin completely prevented the increase of p53 protein levels (panels B and C). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from p53 with those obtained for  actin.
by determining fluorescent chromolipid adducts (⫹150%) and TBARS production (⫹ 400%) (Fig. 1, C and D). All the parameters studied were significantly increased after 4 h of incubation with A peptides as compared to control cells. Induction of caspase 3 and PARP cleavage by A peptides and oxidative stress-related intermediates Caspase 3 activation was evaluated either by quantifying both 32 kDa proenzyme and 20 kDa active isoform in SK-N-BE cell lysates by Western blot analysis or by evaluating spectrophotometrically caspase 3-related enzymatic activity. A significant increase (90 –100%) in 20 kDa active isoform levels was detected 4 h after treatment with either A peptides or HNE and H2O2 (alone or in combination), suggesting proteolytic activation of caspase-3 in our experimental conditions. This feature was associated with the concomitant decrease in the level of pro-caspase-3 (Fig. 2A). Ac-DEVD-pNa cleavage activity (i.e., a measure of caspase 3-like activity) was
again significantly increased (400 – 600%) after treatment with A peptides or exposure to the combination of HNE and H2O2. Furthermore, cleavage of PARP (an endogenous substrate of caspase 3) to the 86 kDa Cterminal fragment resulted similarly increased (⫹400 – 500%) after treatment with A peptides as well as prooxidant conditions (Fig. 3). The induction of classic apoptotic cell death was confirmed by data obtained monitoring cytochrome c release in the cytosol (Fig. 3B). Results obtained in cells treated with A peptides or with HNE and/or H2O2 are in agreement with those already obtained for the other parameters of apoptosis, indicating that all the experimental conditions used were effective in increasing cytochrome c release (approximately 250%). Pretreatment of differentiated SK-N-BE cells with ␣-tocopherol and N-acetylcysteine significantly prevented activation of caspase 3, release of cytochrome c, and cleavage of PARP (Fig. 2, A and B; Fig. 3, A and B), suggesting a direct relationship between A peptides-
A, oxidative stress and apoptosis
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Fig. 5. Increase of Bax levels (A) by Western blot analysis obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). The increase was prevented by 1 h pretreatment with ␣-tocopherol and N-acetylcysteine. Pretreatment of cells with specific inhibitors of JNKs (SP600125) and p38MAPK (SB203580) or with ␣-pifithrin, completely prevented the increase of proapoptotic Bax protein levels (panels B and C). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from Bax with those obtained for  actin.
mediated activation of classic caspase-dependent executing pathway and oxidative stress. Role of p 53 in the induction of pro-apoptotic pathways by A peptides and oxidative stress Exposure of SK-N-BE differentiated cells to A peptides, HNE, and/or H2O2 resulted in an early and significant increase (approx. ⫹400%) in p53 protein levels, as evaluated by Western blot analysis (Fig. 4A). Pretreatment with antioxidants was again able to significantly prevent this induction. To evaluate the relative role of p53 and of SAPKs in the apoptotic pathway induced by A peptides, cells were pretreated with a mixture of two specific inhibitors for JNKs and p38MAPK (SB203580⫹SP600125) or with a specific inhibitor for p53 (␣-pifithrin). Specific inhibitors for JNKs and p38MAPK were used simultaneously because we found in a previous study [20] that only this procedure was followed by an almost complete prevention of apoptosis induced by A peptides or by oxidative stress intermediates. Treatment of
cells with the single inhibitors resulted in a significant but incomplete (approximately 50%) prevention of apoptosis, suggesting equal causative involvement of both pathways [20]. As shown in Fig. 4B, inhibition of the two kinases resulted in the complete prevention of p53 induction. Moreover, the pretreatment of cells with ␣-pifithrin also is able to inhibit p53 induction (Fig. 4C). Exposure of cells to A peptides and to HNE and H2O2, alone or in association, resulted in a significant increase (⫹300 – 400%) of the protein levels of the proapoptotic effector Bax (Fig. 5); moreover, antioxidants were able to significantly prevent this induction (Fig. 5A). As expected, the induction of Bax was completely prevented by either SAPKs inhibitors or by ␣-pifithrin, suggesting that Bax was under the control of SAPKs that operated through activation of p53. Data reported in Fig. 6 indicate that the same experimental conditions that were able to elicit an increase of Bax levels also resulted in a significant decrease of Bcl-2 (⫺50%) (Fig. 6A). Interestingly, Bcl-2 levels were strongly affected by
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Fig. 6. Decrease of Bcl-2 levels (A) by Western blot analysis obtained in differentiated SK-N-BE cells after 4 h of treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX). The decrease was prevented by 1 h pretreatment with ␣-tocopherol and N-acetylcysteine. Pretreatment of cells with specific inhibitors of JNKs (SP600125) and p38MAPK (SB203580) completely prevented the decrease of antiapoptotic Bcl-2 protein levels (B). Pretreatment with ␣ pifithrin was ineffective (C). Equal protein loading was confirmed by evaluating the levels of  actin. Fold increase versus control values has been calculated by normalizing densitometric values obtained from Bcl-2 with those obtained for  actin.
SAPKs inhibitors but were essentially p53-independent: treatment of cells with SB203580/SP600125 mixture prevented the decrease of Bcl-2 induced by A peptides or by HNE/H2O2 (Fig. 6B), whereas ␣-pifithrin was ineffective. Similarly, pretreatment with SB203580/SP600125 mixture was able to offer a complete prevention in terms of cyt.c release, caspase 3 activation, and PARP cleavage by A peptides and oxidative stress intermediates (Fig. 7, panels A, B, and C). However, ␣-pifithrin pretreatment resulted only in a significant but incomplete prevention of the mentioned parameters. As documented by densitometric analyses, a residual increase in cytochrome c release, caspase 3 activation, and PARP cleavage was detected in the presence of ␣-pifithrin. The antiapoptotic action of SAPKs inhibitors and ␣-pifithrin was confirmed morphologically by means of DAPI staining (Fig. 8) and once again ␣-pifithrin resulted in an approximate 50 – 60% prevention of A peptides and oxidative stress intermediates mixture-induced apoptosis.
A peptides as well as HNE and/or H2O2 do not induce a necrotic type of cell death In order to assess whether A peptides as well as HNE and/or H2O2 may elicit necrotic cell death in SKN-BE cells, the release of lactate dehydrogenase has been evaluated at 24 (Fig. 9, panel A) and 48 h (Fig. 9, panel B) time points in the presence as well as in the absence of the inhibitor of execution caspases z-VAD.fmk (i.e., to inhibit specifically caspase-mediated apoptosis). Our data indicate that no significant change in LDH release was found, suggesting that A- as well as HNE- and/or H2O2-induced apoptosis represents the major phenomenon in our experimental conditions. Chronic exposure of SK-N-BE cells to lower doses of A peptides is again followed by induction of p53 and activation of caspase 3 Because AD is a chronic disease, we exposed SKN-BE cells to lower concentrations of A peptides for
A, oxidative stress and apoptosis
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Fig. 7. Prevention of cytosolic cytochrome c release (A), caspase 3-like activity (B) and PARP cleavage (C) observed by pretreating differentiated SK-N-BE cells with SAPK inhibitors and ␣-pifithrin, before exposure to A peptides or HNE 1 M/H2O2 10 M mixture. Cell lysates were obtained after 4 h of the latter treatment. Equal protein loading was confirmed by evaluating the levels of  actin. Optical density values of cytochrome c and PARP, normalized against  actin, are means ⫾ SD of three independent experiments. 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02); ⫹ ⫽ significantly different from A peptides or HNE 1 M/H2O2 10 M mixture (p ⬍ .02).
as long as 1 week to evaluate in a preliminary way whether the features previously described may still be recognized. In order to do so we evaluated, as relevant parameters, active form of caspase 3, p53 levels, and LDH release. In these “chronic” experimental conditions we found once again that A peptides were still able to induce both activation of caspase 3 (monitored as a significant increase in the 20 KDa active form)
and recruitment of p53 (Fig. 10, panels A and B) without eliciting any significant increase in LDH release (Fig. 10). DISCUSSION
Although recent evidence suggests the importance of A in AD-related neuronal death, the signaling mecha-
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Fig. 8. Prevention of morphological signs of apoptosis induced by A1– 40 and HNE 1 M/H2O2 10 M mixture. Nuclear SK-N-BE morphology has been evaluated in terms of DAPI staining 24 h after treatments with A1– 40 and HNE 1 M/H2O2 10 M mixture. Inhibitors of JNKs (SP600125) and p38MAPK (SB203580) and ␣-pifithrin were added 15 min and 1 h, respectively, before treatment with A1– 40 or mixture. Apoptotic nuclei are indicated by arrows (A). In panel B apoptosis is expressed as the number of apoptotic cells on the coverslips 24 h after treatment. Data shown are the means ⫾ SD of three independent experiments. 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02); ⫹ ⫽ significantly different from A peptides or HNE 1 M/H2O2 10 M mixture (p ⬍ .02).
nisms controlling this event are still unclear and a matter of debate. The present study indicates that A peptides can induce classic apoptosis (i.e., caspase 3-dependent) throughout oxidative stress-mediated activation of SAPKs that, in turn, can affect proapoptotic mitochondrial regulatory pathways by involving independently both p53- and Bcl-2-dependent processes. Moreover, in our experimental conditions, apoptotic cell death is the major feature observed in SK-N-BE cells exposed to A peptides as well as to HNE and H2O2; necrotic cell death does not significantly occur even if the cells are exposed chronically to A peptides.
All the major events induced by A peptides, including induction of Bax, decrease of Bcl-2, recruitment of p53, cytosolic release of cytochrome c as well as of PARP cleavage, can be mimicked by the simultaneous administration of low concentrations of HNE and H2O2 that are known to be generated early after exposure of SK-N-BE to A peptides [20]. Moreover, since all the parameters affected by A peptides are significantly prevented by pretreatment with ␣-tocopherol and NAC, a direct involvement of oxidative stress is strongly supported. Previous data and present findings indicate that stressactivated protein kinases JNKs and p38MAPK are primar-
A, oxidative stress and apoptosis
Fig. 9. Cell death induced by 24 h (A) or 48 h (B) treatment with A peptides as well as HNE, H2O2, or HNE 1 M/H2O2 10 M (MIX) as evaluated in terms of LDH release. Pretreatment with z-VAD.fmk did not modify the parameter.
ily involved in A-induced neuronal apotosis. Although no clear consensus has been achieved in the literature concerning the role of JNKs and p38MAPK in determining neuronal apoptosis, Shoji et al. [19] have recently shown that intracellular A accumulation can indeed trigger JNK activation leading to neuronal cell death. Moreover, evidence for a sequential activation of ERK, JNK/SAPK, and p38MAPK in brain tissue from subjects with AD has been provided [35]. In this connection, it has been clearly established that in the brain of AD patients both p38MAPK and JNKs are activated [36,37]. Moreover, in another study Zhu et al. [38] have shown evidence for the activation in AD patient’s brain of MKK6, an upstream kinase activator of p38MAPK. More recently, Wei et al. [39] reported that JNK was rapidly activated by A treatment and that this activation appeared to be critical for A-induced neuronal death. However, these authors reported no activation of p38MAPK by A in their model of cultured SH-SY5Y cells.
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A direct link between oxidative stress and SAPKs has been shown demonstrating that JNK and p38MAPK are activated by HNE and H2O2 in various cell types [13,40,41]. In a previous study we reported that the simultaneous addition of exogenous HNE and H2O2, used at the same low concentrations detected within the first 3 h after A exposure, can elicit activation of JNK and p38MAPK as well as apoptosis [20]. The use of specific inhibitors of JNKs and p38MAPK (SP600125 and SB203580) or of the specific inhibitor of p53 ␣-pifithrin, that selectively blocks the ability of p53 to activate target genes in neurons [42], pointed out that activation of SAPKs is crucial since inhibition of JNKs and p38MAPK completely prevented apoptosis induced by A peptides and mediated by oxidative stress intermediates. However, inhibition of p53 function by ␣-pifithrin still resulted in a significant (approximately 50 – 60%), but not complete, prevention of apoptotic neuronal cell death. p53 is known to play a crucial role in the regulation of apoptosis in many cell types by stimulating expression and/or mitochondrial translocation of the death effector protein Bax [43]. Recently, involvement of p53 in neuronal death occurring in Alzheimer’s disease [44], Parkinson’s disease [45], and traumatic brain injury [46] has been detected. Cell culture studies have established strong relationships between p53 expression and neuronal death induced by DNA damaging agents and glutamate [47]. A critical role for p53 in neuronal apoptosis resulting from ischemic and excitotoxic insults has been confirmed by studies performed in p53-deficient mice [48,49]. However, results by Giovanni et al. [50] described that apoptosis by A in cultured embryonic cortical neurons obtained from p53 knockout mice was mainly p53-independent. In our experimental model the involvement of p53 in A-induced apoptotic cell death seems critical since it accounts for approximately 50 – 60% of total cell number, as suggested by data obtained with the specific inhibitor ␣-pifithrin. Accumulating evidence suggests that the regulation of p53’s transcriptional activities depends on the rate of its phosphorylation [51,52]. Stability of p53, a key factor able to determine its ability to mediate multiple activities, is known to be tightly regulated by JNK in a phosphorylation-dependent manner [53]. A phosphoacceptor site of p53 for JNK phosphorylation, tyrosine 81, seems to be crucial for p53 activities [54]. Moreover, phosphorylation by p38MAPK on residues 33 and 46 is required for UV-induced p53-mediated apoptosis [55], and inhibition of p38MAPK has been found to attenuate transcriptional activities of p53 and p53-dependent apoptosis induced by chemotherapeutic agents [56]. Results obtained with SAPKs inhibitors and ␣-pifithrin suggest that in SK-N-BE cells exposed to
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Fig. 10. Induction of caspase 3 (A) and of p53 protein levels (B) obtained in lysates of differentiated SK-N-BE cells after a chronic (1 week) treatment with low concentrations of A peptides (25–50 nM). Equal protein loading was confirmed by evaluating  actin levels. Densitometric analysis of caspase 3 and p53 levels, normalized against  actin, are means ⫾ SD of three independent experiments. Cell death has been evaluated in terms of LDH release (C). 夹 ⫽ significantly different from control (p ⬍ .05); 夽 ⫽ significantly different from control (p ⬍ .02).
A, p53 involvement is downstream to SAPKs activation; in this connection, activation of SAPKs, particularly of JNK isoforms, may act as proapoptotic factor also by phosphorylating and inactivating the antiapoptotic protein Bcl-2 [57]. The involvement of this pathway may explain the 40 –50% residual, p53independent apoptosis. Data presented in this study, including negative effects of pretreatment with ␣-pifithrin on bcl-2 levels and complete prevention of A-induced apoptosis by SAPK inhibitors strongly support this view. Other authors have also demonstrated that UV-induced classic apoptosis in mouse
fibroblasts, mediated by cytochrome c release, can be completely abrogated in the absence of JNK signaling [58]. The scheme in Fig. 11 summarizes results obtained in this study suggesting that oxidative stressmediated neuronal apoptosis induced by amyloid  peptides operates by eliciting a SAPK-dependent multiple regulation of proapoptotic mitochondrial pathways involving both p53 and bcl-2. Finally, since the involvement of p53 has been reported in neurodegenerative diseases, including AD, our findings seem to confirm that chemical inhibitors of p53 activation may be efficient in hampering neurodegeneration [42].
A, oxidative stress and apoptosis
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Fig. 11. Proposed pro-apoptotic sequence elicited by A peptides throughout the generation of oxidative stress-related intermediates, related activation of SAPK, and recruitment of p53 protein.
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[18] Acknowledgements — This study was supported by Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST, Rome), National Project: Alteration of signaling systems induced by glyco-oxidative stress: potential substrates of Alzheimer’s disease pathogenesis.
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ABBREVIATIONS
HNE— 4-hydroxynonenal JNK— c-Jun NH2- terminal kinase p38MAPK—p38-mitogen activated protein kinase PARP—poly-ADP rybose polymerase SAPK—stress-activated protein kinase