Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer’s patients

Free Radical Biology & Medicine, Vol. 33, No. 10, pp. 1372–1379, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter

PII S0891-5849(02)01049-3

Original Contribution OXIDATIVE STRESS AND REDUCED ANTIOXIDANT DEFENSES IN PERIPHERAL CELLS FROM FAMILIAL ALZHEIMER’S PATIENTS CRISTINA CECCHI,* CLAUDIA FIORILLO,* SANDRO SORBI,† STEFANIA LATORRACA,† BENEDETTA NACMIAS,† SILVIA BAGNOLI,† PAOLO NASSI,* and GIANFRANCO LIGURI* *Department of Biochemical Sciences; †Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy (Received 20 March 2002; Revised 23 July 2002; Accepted 1 August 2002)

Abstract—We have measured the levels of typical end products of the processes of lipid peroxidation, protein oxidation, and total antioxidant capacity (TAC) in skin fibroblasts and lymphoblasts taken from patients with familial Alzheimer’s disease (FAD), sporadic Alzheimer’s disease (AD), and age-matched healthy controls. Compared to controls, the fibroblasts and lymphoblasts carrying amyloid precursor protein (APP) and presenilin-1 (PS-1) gene mutations showed a clear increase in lipoperoxidation products, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). In contrast, the antioxidant defenses of cells from FAD patients were lower than those from normal subjects. Lipoperoxidation and antioxidant capacity in lymphoblasts from patients affected by sporadic AD were virtually indistinguishable from the basal values of normal controls. An oxidative attack on protein gave rise to greater protein carbonyl content in FAD patients than in age-matched controls. Furthermore, ADP ribosylation levels of poly(ADPribose) polymerase (PARP) nuclear substrates were significantly raised, whereas the PARP content did not differ significantly between fibroblasts carrying gene mutations and control cells. These results indicate that peripheral cells carrying APP and PS-1 gene mutations show altered levels of oxidative markers even though they are not directly involved in the neurodegenerative process of AD. These results support the hypothesis that oxidative damage to lipid, protein, and DNA is an important early event in the pathogenesis of AD. © 2002 Elsevier Science Inc. Keywords—Familial Alzheimer’s disease, Oxidative stress, Antioxidant capacity, Lipid peroxidation, PARP, Amyloid ␤-peptide, Presenilin gene, Fibroblasts, Lymphoblasts, Free radicals

INTRODUCTION

cases [1]. Several recent studies suggest that presenilin (PS) mutations sensitize cultured neuronal cells to apoptosis [2], as a result of altered calcium efflux from endoplasmic reticulum stores and increased levels of oxidative stress [3,4]. There is strong experimental evidence that oxidative stress is involved in the pathogenesis of Alzheimer’s disease (AD) [5–7], although it is not yet clear whether the resulting alterations act as a causative agent of neuronal degeneration. Some brain regions of AD patients show increased sensitivity to oxygen free radicals; this could be due to a reduction in free radical defenses, an increase in free radical formation, or both [8]. Accumulation of reactive oxygen species (ROS) results in damage to major macromolecules in cells, including lipids, proteins, and nucleic acids [9 –11]. Free radical-induced oxidative alterations to neuronal lipids, proteins, and DNA are particularly extensive in AD brain areas where

Peripheral tissues that express a genetic defect are a valuable tool for studying the primary pathophysiological mechanism by which neurodegenerative disorders develop. Autosomal dominant forms of early onset familial Alzheimer’s disease (EOFAD) are in many cases determined by specific mutations in the genes located on chromosome 21 that encode for the amyloid precursor protein (APP), or in presenilin-1 (PS-1) and presenilin-2 (PS-2), which are mapped on chromosomes 14 and 1, respectively. The PS-1 and APP gene mutations account for 30 –50% of EOFAD (up to 5% of all AD cases); PS-2 gene mutations have been reported as the cause in fewer Address correspondence to: Dr. Gianfranco Liguri, University of Florence, Department of Biochemical Sciences, viale Morgagni 50, 50134 Florence, Italy; Tel: ⫹39 (55) 413765; Fax: ⫹39 (55) 4222725; E-Mail: [email protected]. 1372

Impairment of cellular redox status in Alzheimer’s patients

amyloid ␤-peptide (A␤) is abundant [12]. However, increased A␤ deposition in AD brain regions at late stages of neurodegeneration is associated with a decrease in 8-hydroxyguanosine and nitrotyrosine, which are markers of RNA and protein oxidative damage [13]. According to several studies, increased lipid peroxidation in the brain of AD patients is the main cause of depletion of membrane phospholipids. The lipoperoxidation process may influence the pathogenesis of the disease. In fact, 4-hydroxynonenal (4-HNE), which is increased in AD patients, appears to be toxic and induces neuronal death by altering the ATPases involved in calcium homeostasis [6]. A healthy brain is protected from oxidative damage by antioxidant defenses. These include antioxidant enzymes and free radical scavengers such as ascorbate, vitamin E, and protein sulfhydryls. There is increasing experimental evidence that impairment in cellular total antioxidant capacity (TAC) plays a central role in AD. The activities of the antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase (CAT) are significantly reduced in the frontal and temporal cortex of AD patients [8]. Oxidative stress-mediated neuronal loss may be initiated by a decline in glutathione (GSH), which acts as a scavenger of free radicals and is the most abundant thiol-reducing agent in mammalian tissues [14]. GSH levels are changed in specific regions of the central nervous system of AD patients [15]. Recently, we found a significant reduction in GSH content in lymphoblasts carrying APP and PS-1 and PS-2 gene mutations compared to controls [16]. Oxidation of mitochondrial and nuclear DNA has been observed in some brain regions of AD patients [17]. This can lead to mutations, pathologies, and death. The role of neuronal apoptosis has been extensively investigated in AD [18 –20]. Free radicals and other reactive species may cause strand breaks and other DNA damage, leading to the activation of poly(ADP-ribose) polymerase (PARP, E.C.2.4.2.30), a 116 kDa nuclear enzyme strictly associated to chromatin [21]. PARP catalyzes the covalent modification of histones and several chromatin proteins—including itself— by adding poly(ADP-ribose), using NAD⫹ as a substrate and converting it into nicotinamide [22]. Modified histones can dissociate from DNA, allowing the repair enzymes to act, while the poly(ADP-ribosylation) of other proteins (topoisomerase I and II, DNA ligase II) can trigger signaling pathways like those involved in the arrest of cell cycle [23]. Excessive PARP activation may deplete the intracellular NAD⫹ and ATP, leading to cell injury and death. Though there is strong evidence of oxidative damage in the AD brain, many biochemical alterations are also known to occur in peripheral tissues. Biochemical anomalies, including modifications in glucose metabolism,

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abnormal A␤ processing, altered mitochondrial function, and changes in calcium dynamics, have been observed in fibroblasts from AD patients [24 –26]. Cultured skin fibroblasts and lymphoblasts from FAD patients provide a useful model for investigating dynamic processes, including oxidative metabolism in living cells bearing the genetic drawback present in those tissues marked by AD lesions. The present study is a preliminary assessment of the extent of oxidative stress and the status of antioxidant defenses in AD peripheral cells. To study the effect of APP and PS-1 mutations on antioxidative capacity, the fibroblasts and lymphoblasts of patients with familial and sporadic AD were assayed for oxidative damage to lipids and proteins and for antioxidant capacity. ADP ribosylation of the nuclear enzyme PARP and its protein levels were also determined. Fibroblasts and lymphoblasts from age-matched healthy controls were examined as reference samples. MATERIALS AND METHODS

Materials All reagents were of analytical grade or the highest purity available. Unless otherwise stated, chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cell lines and treatment In this study, we looked at eight FAD patients from two Italian families bearing the APP Val717Ile mutation and from three other Italian families linked to PS-1 gene mutations (two families bear the PS-1 Met146Leu mutation; the other family the PS-1 Leu392Val missense mutation). Clinical assessment of patients took place according to published guidelines [27], and the AD diagnosis fulfilled the Diagnostic and Statistical Manual of Mental Disorders criteria (DSM-IV) [28]. Four subjects affected by sporadic AD and four age-matched healthy controls were also analyzed. Younger subjects were included in these two groups to preclude the influence of aging on the studied markers. The local ethical committee approved the protocol of the study, and written consent was obtained from all subjects or their caregivers. All control subjects were tested and none carried APP or PS-1 mutations. These control subjects also underwent a rigorous diagnostic evaluation in order to exclude other neurological disorders. The main parameters of the study group are shown in Table 1. Skin biopsies of 3 mm punch were obtained from the volar side of the upper arm of the FAD patients and controls. Two explants were performed from each biopsy and plated in 25 cm2 flasks. The cells were grown in

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C. CECCHI et al. Table 1. Main Parameters of the Subjects Studied

Group

Mutation

APP Val717Ile PS-1 Met146Leu / Leu392Val ADa Controlsa a

Number of Sex Age (years) patients (m/f) (mean ⫾ SD) 4 4 4 4

2/2 2/2 2/2 2/2

55.2 ⫾ 4.5 47.5 ⫾ 9.9 56.7 ⫾ 2.7 50.7 ⫾ 9.2

All subjects were tested and none carried APP or PS-1 mutations.

Dulbecco modified Eagle’s medium, supplemented with 10% fetal bovine serum, and were harvested at confluence in T-25 flasks 7 d after the previous subculture. All fibroblast lines were subjected to an equal number of passages (ranging from 10 to 15) and were analyzed in three different experiments before confluence with the control lines. Lymphocytes were isolated from peripheral blood samples and were transformed by the Epstein-Barr virus. Cells were grown in RPMI/HAMS F-12 medium supplemented with 10% fetal bovine serum in T-25 flasks. All lymphoblasts were assayed twice in three distinct experiments. Purification of cytosolic and nuclear fraction Cellular samples (fibroblasts or lymphoblasts) were washed twice in phosphate-buffered saline (PBS), and were harvested in 50 mM Tris-HCl (pH 7.2) containing 0.1 mM phenylmethylsulfonylfluoride (PMSF), 10 ␮g/ml leupeptine, and 10 ␮g/ml aprotinin prior to storage at ⫺80°C until use. Rupture of the plasma membrane was achieved by three freeze-thaw cycles followed by centrifuging at 750 ⫻ g for 10 min at 4°C. Cytosolic fraction was used for estimation of lipoperoxidation, protein carbonyl content, and total antioxidant capacity (TAC). The pellet, which contains the nuclear fraction, was resuspended in 20 mM HEPES (pH 7.4), 250 mM sucrose, 2 mM EGTA, 1 mM EDTA, and finally sonicated twice for 5 s on ice. The protein content was measured in cytosolic and nuclear fractions according to the method of Bradford [29]. Measurement of lipid peroxidation products To assess the rate of lipid peroxidation, we determined the levels of typical end products of the process: malondialdehyde (MDA) plus 4-hydroxy-2-alkenals, exemplified by 4-hydroxy-2-nonenal. These compounds were determined in the cytosolic fraction of the cellular samples prepared as above. Measurements were made using a colorimetric method at 586 nm, according to the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA and 4-HNE in the presence of methanesulfonic acid at 45°C [30].

Measurement of protein carbonyl content Cytosolic fraction was used for the carbonyl content analysis according to the method of Levine et al. [31]. Aliquots of the supernatant were first dried under vacuum and resuspended in 2M HCl containing 10 mM dinitrophenylhydrazine. Proteins obtained by 20% TCA precipitation were then redissolved in guanidine solution (6 M guanidine, 20 mM potassium phosphate, pH 2.3) at 37°C. After removing any insoluble material by centrifuging, spectrophotometric measurement was made at 375 nm, taking the molar extinction coefficient to be 22,000 M⫺1cm⫺1. Total antioxidant capacity (TAC) assay TAC was assayed (Total Antioxidant Status, Randox, UK) in cytosolic fractions using a spectrophotometric method. In outline, 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) is incubated with a peroxidase (metmyoglobin) and H2O2 to generate the radical cation ABTS•⫹. This has a relatively stable blue-green color, which is measured at 600 nm. Antioxidants in the added sample suppress this color production in proportion to their concentration. The results are calibrated using a reference curve based on the soluble antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as a standard. PARP protein levels An aliquot of sonicated nuclear suspension, obtained as described above, was diluted in Laemmly’s sample buffer and boiled for 5 min. Ten micrograms protein were separated on 7.5% SDS-PAGE. After blotting, completeness was checked by suitable staining and the nitrocellulose membranes were blocked in 5% bovine serum albumin. PARP protein levels were determined using the C2-10 anti-PARP monoclonal antibody at 1:10,000 dilution (Oncogene Research Products, Boston, MA, USA). Incubation then took place with the HRPconjugated secondary antibody and was followed by the ECL procedure. The band densities were quantified as densitometric units/␮g protein, using the program Quantity One for image analysis and densitometry (Biorad, Hercules, CA, USA). PARP activity measurement PARP activity was assessed by an immunodot blot method that detects poly(ADP-ribosylated) proteins [32]. In summary, an aliquot of nuclear suspension was diluted in 0.4M NaOH containing 10 mM EDTA and was loaded on to a Hybond N⫹ nylon membrane (Amersham Biosciences, Piscataway, NJ, USA) that had been rinsed with water. The membrane was then washed once with 0.4 M NaOH and saturated in PBS-MT (PBS, pH 7.4,

Impairment of cellular redox status in Alzheimer’s patients

Fig. 1. Lipoperoxidation markers and antioxidant defenses in lymphoblasts from FAD patients carrying APP or PS-1 gene mutation and from age-matched controls (C). Values are means ⫾ SD of three independent experiments, each performed in duplicate. **p ⬍ .01 or *p ⬍ .05 compared to controls.

containing 5% nonfat dried milk and 0.1% Tween 20). It was next incubated overnight with the first antibody, LP96-10 (Alexis Corp., San Diego, CA, USA). The membrane was then washed with PBS-MT and incubated for 30 min with peroxidase-conjugated antirabbit IgG (Amersham Biosciences). The blot was again washed with PBS-MT, followed by washes in PBS prior to analysis by the ECL procedure. Image analysis of the dot blot was performed using the program Quantity One (Biorad). Statistical analysis Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s Test. A p value .05 was taken to be statistically significant. RESULTS

The main characteristics of our subjects are shown in Table 1. Subjects were matched by sex and age. Each fibroblast and lymphoblast line analyzed here was obtained from the same AD patient (or control subject). In particular, fibroblasts were isolated from skin biopsies and lymphocytes from peripheral blood sample, transformed by the Epstein-Barr virus.

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As shown in Fig. 1, MDA plus 4-HNE levels were markedly higher in lymphoblasts from APP (5.38 ⫾ 0.63 nmol/mg protein; p ⬍ .05) and PS-1 (4.98 ⫾ 0.37 nmol/mg protein) patients than in controls (3.94 ⫾ 0.71 nmol/mg protein). 4-HNE, which is one of the most reactive end products of lipid peroxidation, is believed to be the major mediator of oxidative stress [33]. In contrast, measurement of lymphoblast TAC revealed a significantly lower level in APP (0.20 ⫾ 0.01 nmol/mg protein; p ⬍ .01) and PS-1 (0.24 ⫾ 0.02 nmol/mg protein; p ⬍ .05) than in controls (0.29 ⫾ 0.03 nmol/mg protein). Lipoperoxidation and antioxidant capacity in lymphoblasts from sporadic AD were virtually indistinguishable from the basal values of normal controls (data not shown). Alterations in oxidative stress markers were clearer in lymphoblasts carrying APP than PS-1 gene mutation. A similar pattern was observed in fibroblast cell lines (Table 2). These observations of a common behavior in different cellular lines derived from the same subject support the hypothesis that genetic mutations, rather than cell-type features, are involved in abnormal oxidative processes. Moreover, we found a marked increase in protein carbonyl content in FAD patient fibroblasts compared to controls (Table 2). As an index of DNA damage, PARP protein expression and catalytic activity were measured in fibroblasts from FAD patients and age-matched controls. No statistically significant differences in PARP content were found (Fig. 2). On the other hand, ADP-ribosylation levels of PARP nuclear substrates were significantly higher in FAD patients than in healthy controls (Fig. 3). DISCUSSION

Our results confirm that overall lipid peroxidation takes place in fibroblasts and in lymphoblasts from AD patients carrying APP or PS-1 gene mutation, in contrast to age-matched controls. The increase in lipid peroxides could follow from an attack by free radicals on polyunsaturated fatty acids in cell-membrane phospholipids. Several works provide evidence for excess lipoperoxidation within the AD brain [11,34]. However, studies of autopsied brains can not settle whether abnormal oxida-

Table 2. Markers of Oxidative Stress in Familial Alzheimer’s Disease (FAD) Patients and Control Fibroblasts

MDA and 4-HNE (nmol/mg protein) Protein carbonyls (nmol/mg protein) TAC (pmol/mg protein)

APP

PS-1

Control

22.04 ⫾ 2.71** 25.87 ⫾ 3.92 68.94 ⫾ 40.21*

18.34 ⫾ 1.65** 32.10 ⫾ 8.66* 77.33 ⫾ 26.47*

8.90 ⫾ 2.10 18.45 ⫾ 4.35 143.62 ⫾ 28.64

Data are means ⫾ SD of three independent experiments, each performed in duplicate. ** p ⬍ .01 or * p ⬍ .05 compared to controls. MDA ⫽ malondialdehyde; 4-HNE ⫽ 4-hydroxy-2-nonenal; TAC ⫽ total antioxidant capacity.

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Fig. 2. Representative Western blot of PARP protein levels in fibroblasts from control and FAD patients. Densitometric analysis performed by the Quantity One program did not find any significant difference among control subjects (C), APP, and PS-1 patients. Each bar represents the mean ⫾ SD of three experiments performed on all subjects studied.

tive processes are inherent properties of AD cells or are secondary to neurodegeneration. Our study of peripheral cells bearing mutant AD genes supports the involvement of ROS in the pathophysiology of AD. A recent report has suggested that A␤ accumulates faster in membranes containing oxidatively damaged phospholipids than in membranes containing only unoxidized or saturated phospholipids [35]. These results suggest that oxidatively damaged phospholipid membranes promote the transition of A␤ to the ␤-sheet conformation, which has a strong tendency to form fibrillar aggregates in the brain as dense plaques. These findings, therefore, suggest involvement of lipid peroxidation in the pathogenesis of AD. There is published evidence of a significant increase in lipid peroxidation end products in both AD brain regions and ventricular fluid, which are not correlated with the extent of neuritic plaques or neurofibrillary tangles [36,37]. Not only do oxidative processes appear to induce A␤ aggregation, but A␤ itself produces free radicals that are involved in neurodegenerative events [38]. A␤ can fragment and generate free radical peptides [39] with potent lipoperoxidizing effects on the synaptosomal membranes in the neocortex [40]. It has been shown that the addition of A␤ to PC12 cells causes increased ROS production, mitochondrial dysfunction, apoptosis, and cell death [41,42]. 4-HNE, which is generated in response to oxidative insults by A␤, can directly induce neuronal apoptosis at pathophysiological concentrations [43]. How-

ever, another study indicates that, although free radicals and lipid peroxidation may participate in neurodegeneration, the mechanism of A␤-induced neurotoxicity does not involve ROS [33]. Protein carbonyl content is increased in the AD-demented hippocampus and inferior parietal lobule regions compared to age-matched controls [44]. An earlier study found no significant increase in protein oxidation in the serum of AD patients compared to control subjects [45]. In the present study, the occurrence of oxidative stress in FAD patients is also confirmed by the increased level of protein carbonyls found in FAD fibroblasts compared to controls. The extent of the overall increase in protein carbonyl was lower than the observed alteration in lipoperoxidation markers in both groups of FAD patients. The levels of antioxidants are comparable in the PS-1 and APP groups, yet fibroblasts carrying the former gene mutation showed a greater change in protein carbonyl levels. These differences can probably be ascribed to differing activity of the enzymes involved specifically in the detoxification of lipid products like glutathione transferase. Continuing observations of increased oxidative stress (lipoperoxidation, protein carbonyls) in non-neuronal tissues, such as FAD lymphoblasts and fibroblasts, suggest that AD-related abnormalities in ROS are not simply a reflection of neurodegeneration [46]. Thus, these cellular cultures may be well suited to further study because they possess the genetic background in which the human disease is expressed.

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Fig. 3. Representative immunodot blot of ADP-ribosylation levels of PARP nuclear substrates in fibroblasts from control subjects (C), APP, and PS-1 patients. Quantitative data for each group are reported as the mean ⫾ SD of the densitometric analysis performed on all subjects studied (see Material and Methods). **p ⬍ .01 or *p ⬍ .05 compared to controls.

Our previous report demonstrated reduced GSH content in lymphoblasts from patients carrying APP or PS mutations, suggesting that a modified redox status is a common feature of cells carrying these genetic lesions [16]. In the present study, lymphoblasts and fibroblasts with APP and PS-1 gene mutations showed significant impairment in TAC compared to healthy controls. However, no significant difference appeared in the antioxidant status of sporadic AD lymphoblasts compared to basal values. This accords with our previous findings of similar levels of GSH in subjects with sporadic AD and in control cells [16]. The absence of significant alterations in TAC and lipid peroxidation products in sporadic AD supports the hypothesis that the observed changes are strictly correlated with the presence of genetic lesions in the peripheral cells from FAD. The clear signs of oxidative stress in the FAD patients analyzed here could be ascribed to increased reactive oxygen metabolite production or to weakening of antioxidant defenses. Our findings suggest that FAD fibroblasts are more sensitive than lymphoblasts to oxidative stress, possibly due to lower levels of antioxidant defenses. It is well known that every cell type differs in its ability to handle oxidative stress because of varying levels of antioxidant capacity.

Oxidation of mitochondrial and nuclear DNA is a common feature of the AD brain [17]. It was therefore useful to determine the PARP ADP-ribosylation level, since this is activated in response to free radical-mediated injury to DNA. ADP-ribosylation levels of PARP nuclear substrates are significantly higher in FAD patients than in healthy controls. This observation is consistent with the increased DNA damage in FAD patients. In turn, these findings are in accord with experimental studies showing an accumulation of poly(ADP-ribose) in AD neurons [47]. On the other hand, the PARP protein level is unchanged in FAD compared to control cells. Correspondingly, PARP is constitutively expressed at high levels, so that increases in its activity are likely to reflect enhanced catalytic activity rather than a rise in protein content [48]. Although the mechanism by which ROS damages cells remains to be settled, our results show that increased oxidative stress and reduced antioxidant defenses are inherent properties of AD cells from these APP and PS-1 kindred. These findings imply a systemic abnormality in FAD that could prove diagnostically important. Further studies on FAD peripheral cells could clarify the role of oxidative stress in the pathogenic mechanisms of AD.

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Acknowledgements — This work was supported by the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST), by Telethon Italia Fondazione ONLUS (grant no E.0980), and by European Union grants QLK-6-CT-1999-02178 and QLK-6-CT-1999-02112.

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A␤—amyloid ␤-peptide ABTS—2,2'-azino-di-3-ethylbenzthiazoline sulphonate AD—Alzheimer’s disease APP—amyloid precursor protein CAT— catalase ECL— enhanced chemiluminescence EDTA— ethylenediaminetetraacetic acid EGTA— ethylene glycol-bis-␤aminoethyl ether EOFAD— early onset familial Alzheimer’s disease FAD—familial Alzheimer’s disease 4-HNE— 4-hydroxy-2-nonenal GSH— glutathione HEPES—N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid MDA—malondialdehyde PARP—poly(ADP-ribose) polymerase PBS—phosphate-buffered saline PMSF—phenylmethylsulphonylfluoride PS—presenilin ROS—reactive oxygen species SOD—superoxide dismutase TAC—total antioxidant capacity