Acetaminophen protects hippocampal neurons and PC12 cultures from amyloid β-peptides induced oxidative stress and reduces NF-κB activation

Neurochemistry International 41 (2002) 43–54

Acetaminophen protects hippocampal neurons and PC12 cultures from amyloid ␤-peptides induced oxidative stress and reduces NF-␬B activation M. Bisaglia a,b , V. Venezia a,b , P. Piccioli a , S. Stanzione a , C. Porcile a , C. Russo a,b , F. Mancini c , C. Milanese c , G. Schettini a,b,∗ a

Pharmacology and Neuroscience, National Cancer Research Institute c/o Advanced Biotechnology Centre, L.go R. Benzi 10, 16132 Genova, Italy b Section of Pharmacology, Department of Oncology, University of Genova, Genova, Italy c ACRAF-Angelini Ricerche, S. Palomba, Pomezia, Rome, Italy Received 19 July 2001; received in revised form 6 November 2001; accepted 23 November 2001

Abstract The present findings show that an atypical non-steroidal anti-inflammatory drug, such as acetaminophen, retains the ability to recover amyloid ␤-peptides driven neuronal apoptosis through the impairment of oxidative stress. Moreover, this compound reduces the increased NF-␬B binding activity, which occurs in these degenerative conditions. Therapeutic interventions aimed at reducing the inflammatory response in Alzheimer’s disease (AD) recently suggested the application of non-steroidal anti-inflammatory drugs. Although the anti-inflammatory properties of acetaminophen are controversial, it emerged that in an amyloid-driven astrocytoma cell degeneration model acetaminophen proved to be effective. On these bases, we analyzed the role of acetaminophen against the toxicity exerted by different A␤-peptides on rat primary hippocampal neurons and on a rat pheochromocytoma cell line. We found a consistent protection from amyloid ␤-fragments 1-40 and 1-42-induced impairment of mitochondrial redox activity on both cell cultures, associated with a marked reduction of apoptotic nuclear fragmentation. An antioxidant component of the protective activity emerged from the analysis of the reduction of phospholipid peroxidation, and also from a significant reduction of cytoplasmic accumulation of peroxides in the pheochromocytoma cell line. Moreover, activation of NF-␬B by amyloid-derived peptides was greatly impaired by acetaminophen pre-treatment in hippocampal cells. This evidence points out antioxidant and anti-transcriptional properties of acetaminophen besides the known capability to interfere with inflammation within the central nervous system, and suggests that it can be exploited as a possible therapeutic approach against AD. © 2002 Published by Elsevier Science Ltd. Keywords: Acetaminophen; Amyloid; Neuronal cultures; Oxidative stress; NF-␬B

1. Introduction The deposition and excessive accumulation of amyloid ␤-peptide (A␤) is the major pathological hallmark of Alzheimer’s disease (AD), and a possible cause of neurodegeneration (Selkoe, 1996; Hardy, 1997). The main features of this pathology are the appearance of neurofibrillary tangles of hyperphosphorilated cytoskeletal protein tau and the presence of amyloid plaques, containing several peptides generated from the cleavage of the ␤-amyloid precursor protein (APP) by ␤- and ␥-secretases (Coulson et al., 2000). The mechanism by which A␤ induces neuronal death is still unclear. Disruption of calcium homeostasis (Hedin et al., ∗

Corresponding author. Tel.: +39-10-5737-254; fax: +39-10-5737-257. E-mail address: [email protected] (G. Schettini).

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2001; Mattson et al., 1992, 1993; Scorziello et al., 1996) and membrane potential (Mark et al., 1995), as well as upregulation of actin polymerization (Furukawa and Mattson, 1995) have been observed following A␤ toxicity. After all, A␤, through interaction with not yet identified targets on the cell surface (Busciglio et al., 1992; Pike et al., 1992), initiates a cascade of intracellular events that culminates in neuronal death (Forloni et al., 1993; Loo et al., 1993; Scorziello et al., 1996). Apoptosis has been associated with the pathophysiology of AD (Smale et al., 1995; Su et al., 1994), and evidence of A␤-induced expression of several immediate early genes, such as c-jun and c-fos, has been reported (Estus et al., 1997). Moreover, many of the genes newly induced in AD are under immediate-early transcriptional control of NF-␬B, both in neurons and in microglia (Kaltschmidt et al., 1994a,b).

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This leads to the expression of the main components of the inflammatory reaction, such as the acute phase and the cellular-mediated response, and also of the cellular antioxidant system, but is also linked to the pathological evolution of amyloid deposition, being thus a crucial regulator of the cellular fate (Kaltschmidt et al., 1997). Recently, oxidative stress showed to play a primary role in the mechanistic evaluation of A␤-induced neuronal death (Markesbery, 1997; Markesbery and Carney, 1999; Butterfield et al., 1999; Smith et al., 1998), being the brains of AD patients under increased oxidative injury. Moreover, NF-␬B is recognized as a redox-sensitive transcription factor, in that it is implicated in the cellular response to oxidative stress (Mercurio and Manning, 1999). Again, a direct relationship between oxidative stress and mitochondrial abnormalities in AD has been demonstrated (Hirai et al., 2001). In fact, AD brain areas with increased oxidative damage showed also a significant increase in mitochondrial DNA and cytochrome oxidase associated with reduced number of mitochondria. Different antioxidants, from propyl gallate (PG) to Vitamin E, have been analyzed but, at present, they do not provide substantial protection against A␤ peptides, being just able to partly attenuate neuronal damage (Pike et al., 1997; Behl et al., 1992, 1994). As already mentioned, inflammation and glial activation by amyloid-derived peptides play a fundamental role in the complex array of events that leads to neurodegeneration in AD (Canning et al., 1993; Martin and O’Callaghan, 1996; Pike et al., 1995). Activated microglia and astrocytes, the increased expression of various cytokines, complement activation products and apolipoprotein E (apoE) isoforms are typically present around amyloid plaques and dystrophic neurites (Cedazo-Minguez et al., 2001; Emmerling et al., 2000), thus suggesting the development of therapeutic strategies based on anti-inflammatory drugs. Many epidemiological studies have shown the capability of indomethacin or ibuprofen to reduce the risk of AD (McGeer et al., 1996; McGeer and McGeer, 1999). Moreover, non-steroidal anti-inflammatory drugs (NSAIDs), unselective cyclooxygenase (COX) inhibitors, and selective COX-2 inhibitors are under investigation, as they could potentially reduce neuronal prostaglandin production, besides acting on astroglial and microglial cells (McGeer, 2000; Flynn and Theesen, 1999; Hull et al., 2000). Among the latest molecules tested, acetaminophen (paracetamol) has shown the capability to modulate the release of inflammatory molecules like PGE2 and IL6 by A␤-stimulated astrocytes (Landolfi et al., 1998). Albeit some authors hypothesize the ability of acetaminophen to stimulate free radical activity as a causative agent of tardive AD (Jones, 2001), nevertheless the antioxidant properties of this atypical anti-inflammatory drug are directly linked to the capability of inhibiting lipid peroxidation (Porubek et al., 1987). This indicates that the drug is able to interfere with, at least, two different cellular pathways that result activated during inflammation, the

production of inflammatory cytokines and the generation of reactive oxygen species (ROS). From these premises, we sought to investigate if acetaminophen can reduce the apoptotic degeneration induced by amyloid derivatives on primary hippocampal neurons and on PC12, a sympathetic-derived cell line, and to analyze the role of the antioxidant properties of the drug in A␤-triggered neuronal apoptosis. Moreover, we studied the capability of the drug to interfere with A␤-induced activation of NF-␬B transcription factor, in order to analyze the role of this drug in neuronal transcriptional activity under A␤ toxicity. 2. Experimental procedures 2.1. Materials [Pyr3]-Amyloid ␤-protein (human, 3-42) was from Peptides International (Louisville, KY), amyloid ␤-protein (1-42) was from Quality Controlled Biochemicals (Hopkinton, MA), amyloid ␤-protein (1-40) was from Biosource International (Camarillo, CA). Acetaminophen was kindly provided by ACRAF S.p. A (Pomezia, Italy). Mouse nerve growth factor (m-NGF) was from Alomone Labs (Tel Aviv, Israel). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), ferrous sulfate (FeSO4 ·7H2 O), propyl gallate and 2-thiobarbituric acid (TBA) were from Sigma (St. Louis, MO), malonaldehyde bis (dimethyl acetal) was from Sigma–Aldrich (Steinheim, Germany). 2 ,7 -Dichloro-dihydrofluoresceindiacetate (H2 -DCFDA) was from Molecular Probes (Poortgebouw, The Netherlands). Polyclonal anti-N3 (pE) antibody was kindly provided by Dr. T. Saido, polyclonal antibody R3659 was a generous gift from Dr. P. Gambetti (Russo et al., 1997). 2.2. Cell cultures PC12 cells were differentiated in DMEM supplemented with NGF (50 ng/ml), 10% fetal bovine serum (FBS), 5% horse serum and antibiotics (100 IU/ml penicillin, 100 ␮g/ml streptomycin) for 7 days. Primary cultures of rat hippocampal neurons were obtained from 18–20-day-old Sprague–Dawley embryos according to the method of Goslin and Banker (1990) with slight modifications. Briefly, hippocampi were excised, trypsinized and resuspended in Neurobasal medium supplemented with 2% B27 (Gibco/Life Technologies, Rockville, MA), 0.5 mM glutamine and antibiotics. Cells were seeded in poly-l-lysine-coated wells and after 8–9 days the cell population was determined to be at least 95% neuronal by immunostaining. All the efforts were made to minimize animal sufferance, in accordance to the present statements on laboratory animal care (UK Animals Act, 1986; European Communities Council Directive 86/609/EEC; NIH Publications no. 80-23, revised 1978).

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2.3. Treatment of cultures Acetaminophen was freshly prepared as 10 mM stock solution in distilled water and applied to the cultures 24 h before peptides challenge. Amyloid ␤-peptides were prepared as 1 mM stock solutions in dimethyl sulfoxide (DMSO), directly administered at the indicated concentrations in the culture medium for the indicated times, and stored at −20 ◦ C for the following treatments. For the oxidative stress experiments, FeSO4 and H2 O2 were added at the indicated concentrations from 100× stock solutions freshly prepared in distilled water. 2.4. Survival assays Mitochondrial function was evaluated by MTT assay (Bisaglia et al., 2000). As a survival test, the “live/dead” assay was used by using a commercial kit (Molecular Probes). Double staining with ethidium homodimer and calcein AM provided evidence of alive versus dead cells by fluorescence microscopy. 2.5. Apoptosis assays PC12 cells were evaluated for apoptotic chromatin condensation and fragmentation using the nuclear staining method by Hoechst 33258, and visualized by fluorescence microscopy. Hippocampal neurons were analyzed for oligonucleosomal DNA fragmentation by a commercial ELISA kit (Roche, Mannheim, Germany), measuring apoptotic fragmentation in a spectrophotometer at 405 nm. 2.6. Confocal microscopy Cytomorphological analyses were carried out on hippocampal neurons seeded on glass coverslips and the day after exposed for 24 h to 1 ␮M concentration of each A␤ peptide. Cells, fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton, were incubated for 20 min with R3659 antibody, that recognizes A␤ N-terminus, and anti-N3 (pE) antibody, specific to pyroglutamate at position 3, at 1:1000 dilutions. Subsequently, secondary antibodies, respectively, FITC- and rhodamine-conjugated, were applied for 40 min. Samples were analyzed on BioRad-MRC 1000 confocal microscope (60× objective, Argon laser ex. = 468 nm, em. = 580 nm), 0.2 ␮m step in z-plane acquisition.

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supernatants were precipitated with 10% trichloroacetic acid (TCA) and centrifuged for 5 min at 4000 rpm at 4 ◦ C. The upper fractions were left to react with the same volume of 0.67% TBA for 15 min at 100 ◦ C, then cooled to RT in the dark and fluorescence was monitored on a Perkin Elmer spectrofluorimeter (ex. = 515 nm; em. = 553 nm). The content of malondialdehyde (MDA) was calculated using a MDA standard curve and related to the protein content, measured apart by Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories). 2.8. Cytoplasmic peroxides measurement Peroxides content in the PC12 cytoplasm was evaluated by using the fluorescent probe H2 -DCFDA. Cells plated onto 96 wells plates were washed twice with Locke’s buffer, then incubated for 10 min with 10 ␮M H2 -DCFDA at 37 ◦ C. After three washes, fluorescence emission was read on a spectrofluorimeter (ex. = 475 nm; em. = 525 nm). 2.9. Electrophoretic mobility shift assay (EMSA) EMSA was performed as previously described (Anrather et al., 1997). Cells were lysed in 400 ␮l of cold buffer A (10 mM Hepes pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol (DTT); 0.5 mM phenylmethylsulfonyl fluoride (PMSF); all reagents from Sigma) and kept on ice for 15 min, after which 25 ␮l of 10% Nonidet NP40 (Sigma) were added. After a brief centrifugation (13,000×g for 2 min), pellets were resuspended in 50 ␮l of cold buffer B (20 mM Hepes pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM PMSF) and shaked for 15 min at 4 ◦ C. The extracts were centrifuged at 4 ◦ C for 5 min at 13,000 × g and, after determination of protein concentration by Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories), supernatant aliquots were frozen at −70 ◦ C. A double-stranded oligonucleotide containing the NF-␬B consensus sequence (M-Medical srl Genenco Life Science) was end-labeled with ␥-32 P-dCTP (3000 Ci/mmol, Amersham Pharmacia Biotech) using Megaprime DNA Labelling System (Amersham). Binding reactions were performed incubating 50,000 cpm of 32 P-labeled probe with 5 ␮g of nuclear proteins in the reaction buffer, containing 1 mg poly dI-dC (Sigma) for 15 min at RT. For competition experiments, a molar excess of cold probe was added to the reaction mixture. After electrophoresis onto 5% non-denaturing polyacrylamide gel at 200 V (4 ◦ C) in 0.5× TBE buffer for 2 h, gels were dried and exposed to X-ray film at −70 ◦ C. Signal intensity was evaluated by densitometry using ImageQuant Utilities (Molecular Dynamics).

2.7. Lipid peroxidation assay 2.10. Statistics Cellular membrane lipid peroxidation was assessed with a TBA reaction according to Zhang and Tang (2000), with minor modifications. After lysis, homogenization and centrifugation of cells for 20 min at 3000 × g at 4 ◦ C, the

Data are expressed as mean ± S.E. values, and statistical significance was assessed by ANOVA followed by Student t-test. Differences were accepted as significant at P < 0.05.

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3. Results 3.1. Acetaminophen rescues neuronal cells from mitochondrial redox impairment A 24 h pre-treatment with 100 ␮M acetaminophen completely rescues differentiated PC12 from mitochondrial impairment by 1 ␮M A␤(1-42) and A␤(1-40) , as assessed by MTT test, and acetaminophen per se does not affect PC12 survival (Fig. 1A). Preliminary experiments on peptides

Fig. 1. Acetaminophen (AAP) protects PC12 and hippocampal neurons from amyloid-induced impairment of mitochondrial function. Panel A: NGF-differentiated PC12 were treated for 24 h with 100 ␮M AAP, then A␤(1-40) and A␤(1-42) were applied at the indicated concentration, and 24 h later mitochondrial function was measured by MTT test. ∗ P < 0.01 vs. control, ∗∗ P < 0.01 vs. A␤(1-42) , ∗∗∗ P < 0.01 vs. A␤(1-40) . Panel B: primary hippocampal neurons were treated for 24 h with 100–500 ␮M AAP, then for 5 days with 25 ␮M A␤(1-40) ; panel C: hippocampal cultures were pre-treated with AAP as in panel B, then 10 ␮M A␤3(pE)-42 was applied for 5 days. Data are mean of three experiments and are expressed as % survival vs. control. ∗ P < 0.01 vs. control, ∗∗ P < 0.05 vs. A␤(1-40) .

Fig. 2. Acetaminophen (AAP) reduces apoptotic chromatin condensation and DNA fragmentation. Panel A: NGF-differentiated PC12 treated for 24 h with 100 M AAP, then for 24 h with 1 ␮M A␤(1-40) , were stained with Hoechst 33258 (left lane), to assess apoptotic nuclear fragmentation, and with ethidium homodimer and calcein AM (right lane), to assess viability in fluorescence microscopy. 1: Control cultures; 2: 1 ␮M A␤(1-40) -treated cultures; 3: A␤(1-40) plus AAP-treated cultures. Panel B: hippocampal neurons were treated for 24 h with 500 ␮M AAP, then 25 ␮M A␤(1-40) was applied and 24 h later DNA fragmentation was assessed by ELISA test. The graph is from one experiment, representative of three repeats; data are mean absorbance values (405 nm), expressed as % DNA fragmentation vs. internal control. ∗ P < 0.005 vs. A␤(1-40) ; ∗∗ P < 0.005 vs. control.

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toxicity on hippocampal cultures were performed to find the concentrations of the peptides which impaired cell survival to a similar extent (data not shown). A␤(1-40) was able to induce a marked neuronal toxicity at 25 ␮M (∼60%, Fig. 1B), while A␤(pE)-42 was effective already at 10 ␮M (Fig. 1C). These concentrations were adopted for the following experiments. Addiction of 100 ␮M acetaminophen is effective against A␤(1-40) toxicity, as it recovers ∼40% of hippocampal cells; however, when acetaminophen concentration is up to 500 ␮M, ∼60% of the cells is recovered. On the other hand, severe toxicity by 10 ␮M A␤3(pE)-42 is not relieved by drug pre-treatment even at high concentrations (Fig. 1C). These results may thus indicate selectivity of action of the drug against the different toxic forms of A␤. 3.2. Acetaminophen reduces apoptotic DNA fragmentation Amyloid toxicity is partially consistent with apoptotic features (Smale et al., 1995), responsible for the onset of a program of progressive cellular suicide. Toxicity by 1 ␮M A␤(1-40) on differentiated PC12 cells triggers chromatin condensation and fragmentation in discrete bodies typical of apoptosis (Fig. 2A). The 24 h pre-treatment with 100 ␮M acetaminophen greatly impairs nuclear fragmentation, as visualized by bisbenzimide nuclear staining, as well as it reduces the number of ethidium homodimer-stained cells,

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as shown in “live/dead” (L/D) test (Fig. 2A, left for bisbenzimide nuclear staining, right for L/D test). Hippocampal DNA fragmentation in histone-associated oligonucleosomes is evident after 25 ␮M A␤(1-40) treatment, and 24 h pretreatment with 500 ␮M acetaminophen significantly reduces nuclear derangement, as measured by ELISA test (Fig. 2B). Interestingly, acetaminophen is antiapoptotic per se, since in these conditions it markedly reduces spontaneous nuclear fragmentation observed in control cells. 3.3. Amyloid fragments bind to membranes and deepen into the cell To focus the attention on the features of A␤ peptides toxicity on cellular membranes, we used confocal microscopy. Fig. 3 shows the interaction of A␤3(pE)-42 with hippocampal neurons, through a z-plane scanning deepened on the 12 ␮m thickness of the cell. This is representative of the marked binding of the different A␤ peptides (notably, A␤(1-40) and A␤(1-42) , data not shown) with membrane components, included the phospholipid bilayer, being extended also to the cytoplasmic environment. Particularly, upper panels (A–B–C) show the widespread diffusion of the parental APP throughout the entire cell volume, while the lower panels (D–E–F) evidence the punctate distribution of A␤3(pE)-42 both on the membranes and in the cytoplasmic region.

Fig. 3. A␤3(pE)-42 binds to neuronal plasma membranes and internal structures. Series of z-plane acquisitions at confocal microscopy of hippocampal neurons exposed for 24 h to 1 mM A␤3(pE)-42 , then incubated with antibodies against amyloid precursor protein (APP) (panels A–B–C) and A␤3(pE)-42 (panels D–E–F) and visualized by secondary antibodies, respectively, FITC and rhodamine-conjugated. Particularly, in panels B–E: thick arrows indicate deposition of the peptides on neuritic and dendritic trees; thin arrows indicate distribution of the peptides on cell body.

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3.4. Acetaminophen reduces lipoperoxidative generation of malondialdehyde Peroxidative disruption of neuronal phospholipid bilayer by 25 ␮M A␤(1-40) generates several products, mostly aldehydes, among which malondialdehyde (MDA) greatly increases by 24 h, as assessed by the thiobarbituric acid (TBA) method (Figs. 4A and 5B). A 24 h pre-treatment with 500 ␮M acetaminophen significantly reduces the amount of the aldehyde in differentiated PC12 (Fig. 4A) and in primary hippocampal neurons (Fig. 5B), suggesting a direct interaction of the drug with the targets of A␤(1-40) toxicity at the membrane level. Interestingly, in hippocampal cells 500 ␮M acetaminophen is in some way lipoperoxidative per se, nevertheless being able to selectively reduce A␤ peptide-induced increase of malondialdehyde. Moreover, as shown in Fig. 5A, a direct comparison of acetaminophen with a specific anti-lipoperoxidant molecule, such as propyl gallate (PG), shows that the amino-phenol derivative is not able to directly react with ROS. In fact, unlike propyl gallate, which specifically reduces the amount of MDA produced by FeSO4 (Pike et al., 1997), the capability of acetaminophen to reduce the rate of MDA generation by two different triggers, such as FeSO4 and H2 O2 , is substantially unmodified. 3.5. Acetaminophen significantly reduces cytoplasmic accumulation of peroxides In neuron-like PC12 cells, formation of cytoplasmic peroxides, notably of hydrogen peroxide, increases of 50% over control after a 24 h challenge with 25 ␮M A␤(1-40) , as detected with the fluorescent probe H2 -DCFDA (Fig. 4B). However, acetaminophen significantly reduces A␤(1-40) triggered accumulation of cytoplasmic hydrogen peroxide to a value comparable to untreated cells. 3.6. Acetaminophen inhibits NF-κB activation induced by Aβ (1-40) A␤ fragments are known to trigger NF-␬B activation, leading to the transcription of several genes responsible for the inflammatory and degenerative reactions that accompany amyloid toxicity. Here we show that in hippocampal neurons 25 ␮M A␤(1-40) is able to rapidly activate nuclear translocation of NF-␬B (Fig. 6), as imaged by electrophoretic mobility shift assay (EMSA). Nuclear levels of NF-␬B increase as early as 30 min after peptide challenge (lane 2), reach a peak at ∼1 h (lane 4) and remain higher than control till 2 h (lane 6). When acetaminophen is added (500 ␮M), it quickly inhibits this activity at 30 min (lane 3), and is still effective after 2 h (lane 7), when the nuclear signal is reduced to a level comparable to control.

Fig. 4. Acetaminophen (AAP) reduces A␤(1-40) -triggered cytoplasmic and membrane-associated oxidative stress in PC12. Neuron-like PC12 were challenged for 24 h with 500 ␮M AAP, then with 25 ␮M A␤(1-40) , and 24 h later oxidative stress was assessed. Panel A: the amount of malondialdehyde (MDA) was measured by thiobarbituric acid (TBA) test. Data are from one experiment, representative of three repeats, they are calculated as pmol MDA/mg protein and are expressed as % values vs. A␤(1-40) -treated cultures. Panel B: cytoplasmic accumulation of (H2 O2 ) was evaluated by oxidation of 2 ,7 -dihydrodichloro-fluorescein diacetate (H2 -DCFDA). Data are values of fluorescence (525 nm) in arbitrary units, and are expressed as % values vs. A␤(1-40) -treated cultures. They are mean of two experiments ∗ P < 0.01 vs. control; ∗∗ P < 0.005 vs. A␤(1-40) .

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Fig. 5. Acetaminophen (AAP) is effective against three different lipoperoxidative insults in hippocampal cells. Panel A: mature hippocampal cultures were treated for 2.5 h with 500 ␮M AAP and 5 ␮M propyl gallate (PG), then 7 ␮M FeSO4 and 25 ␮M H2 O2 were added, and after 20 h lipid peroxidation was evaluated by TBA test. Data are pmol MDA/␮g protein and are expressed as % values vs. control. Panel B: hippocampal neurons were treated for 24 h with 500 ␮M AAP, then 25 ␮M A␤(1-40) was added and after 24 h TBA test was performed. As a positive control, parallel cultures were challenged with 150 ␮M H2 O2 for 30 min in Locke’s buffer, then cells were shifted to complete medium for 24 h. Data are calculated as for the experiment in panel A, and are expressed as % values vs. H2 O2 -treated cultures. Both graphs show one experiment (n = 6), representative of three repeats. ∗ P < 0.05 vs. A(1-40) ; ∗∗ P < 0.05 vs. FeSO4 ; ∗∗∗ P < 0.05 vs. H2 O2 .

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Fig. 6. Acetaminophen (AAP) inhibits A␤(1-40) -induced nuclear translocation of NF-␬B in hippocampal cells. The figure shows an autoradiographic image of hippocampal nuclear extracts analyzed by electrophoretic mobility shift assay (EMSA) with a labeled specific ␬B probe. Cells were pre-treated for 30 min with acetaminophen (lanes 3, 5, 7, 8) before addiction of 50 ␮M A␤(1-40) (lanes 2–7). As a positive control for NF-␬B translocation, cells were pulsed with 50 ␮M glutamic acid (Glu) for 15 min, alone (lane 9), or after a 5 min pre-treatment with 5 mM sodium salicilate (NaSal, lane 10). Competition experiments are shown in lane 12. The figure is representative of three experiments.

4. Discussion The present data show that different amyloid ␤-peptides exert a progressive toxicity on both the neuronally-differentiated pheocromocytoma cell line PC12 and on primary hippocampal neurons. This toxicity leads to apoptotic death in both models and is accompanied by overproduction of reactive oxygen intermediates (ROIs). Moreover, in hippocampal neurons, A␤(1-40) readily activates the transcription factor NF-␬B. The application of the atypical non-steroidal anti-inflammatory drug acetaminophen to both neuronal models before peptides challenge is protective, in that it prevents the three features of amyloid toxicity here investigated. Thus, we provide evidence for an unusual neuroprotective action of acetaminophen against amyloid toxicity. Besides its analgesic-antipyretic action, the latter property confers to the drug a wider pharmacological spectrum with promising applications against neurodegenerative diseases. 4.1. Rationale of studying acetaminophen It has been reported the importance of an anti-inflammatory therapy in preventing the occurrence of AD (Stewart et al., 1997; Bour et al., 2000; Ogawa et al., 2000; Hull et al., 2000), and the employment of both NSAIDs and estrogens emerged useful in reducing the risk or delaying the onset of AD (Emilien et al., 2000). Unlike other molecules, such as ibuprophen or indomethacin, which seem to be effective against amyloid-triggered inflammation mainly through the interaction with cyclooxygenase (COX) 1 and 2, acetaminophen is a weak inhibitor of COX (Marshall et al., 1987). Indeed it is more effective against the central nervous system (CNS) isoform of COX, rather than against the peripheral isomers, as well as it does not inhibit neutrophil

activation, as do other NSAIDs (Abramson and Weissman, 1989). However, recent data show that in the human glial cell line T98G acetaminophen is able to reduce PGE2 production following A␤ stimulation (Landolfi et al., 1998), thus providing evidence of inhibition of A␤-triggered glial activation. 4.2. Role of acetaminophen in mitochondrial disfunction induced by different Aβ peptides Our data, first of all, indicate a different pattern of toxicity of A␤(1-40) on neuron-like PC12 and hippocampal neurons, being the former cultures more susceptible to a shorter time of exposure and smaller concentrations of the amyloid fragment, in accordance with previous reports (Pereira et al., 1998). This higher sensitivity to amyloid toxicity may reflect the clonal origin of PC12 cells, although they have been differentiated to a neuronal phenotype, in comparison to the primary culture. Moreover, acetaminophen exerts an appreciable recovery of hippocampal mitochondrial function even after a more prolonged and more toxic insult, demonstrating to interfere with A␤(1-40) -driven toxicity at the mitochondrial level on both neuronal models. This underlines also the role of mitochondria in AD, in accordance with ex vivo studies indicating decreases in cytochrome oxidase activity in AD post-mortem tissue (Kish et al., 1992; Mutisya et al., 1994; Parker and Parks, 1995). Interestingly, acetaminophen fails to recover mitochondrial damage by the pyroglutaminated form of A␤ (A␤3(pE)-42 ), indicating a specificity of action against the different fragments of the amyloid peptide. A␤3(pE)-42 is the most abundant isoform in extracellular A␤ deposits and soluble A␤ peptides extracted from AD brains, likely because of the acquired resistance of the pyroglutamic modification to most aminopeptidases (Saido et al., 1995, 1996; Russo et al., 1997; Tekirian et al., 1998).

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Nevertheless, it is not more toxic than A␤(1-42) in vitro (Tekirian et al., 1999). Here we found that a long exposure of hippocampal neurons to 10 ␮M A␤3(pE)-42 causes the greatest impairment of mitochondrial function, which is not recovered by the drug pre-treatment. The mechanism of A␤3(pE)-42 -induced mitochondrial impairment needs to be better elucidated to explain this finding. 4.3. Effects of acetaminophen on apoptotic hallmarks The positive effect of acetaminophen against A␤(1-40) toxicity is further confirmed by the reduction of the apoptotic degeneration of nuclei, as assessed by fluorescence methods and by the quantification of oligonucleosomal fragmentation in hippocampal cells. Chromatin condensation and nuclear fragmentation, two characteristic changes in chromatin integrity, are visible under the same conditions that cause mitochondrial sufferance, in agreement with several reports (Forloni et al., 1993; Loo et al., 1993; Estus et al., 1997). It could be of interest to verify whether, under A␤ challenge, mitochondrial membrane potential integrity is affected and, if so, its disruption precedes nuclear and plasma membrane changes, perhaps through mitochondrial release of the protein factor (AIF) capable of inducing nuclear apoptotic derangement (Petit et al., 1996). 4.4. Role of acetaminophen in Aβ-driven oxidative stress It is well documented that oxidative stress is one of the main partners in A␤ neurotoxicity, even if its primary role in amyloid-induced neurodegeneration is still on debate (Pike et al., 1997). Our data confirm that A␤(1-40) promotes the increase of intracellular peroxides both at the cytoplasmic level, and at the membrane sites in PC12, and show a significant lipoperoxidative action also in hippocampal neurons. Moreover, we provide evidence of an appreciable antioxidant activity by acetaminophen, in that it significantly reduces A␤-triggered cellular peroxides accumulation on both models. The drug is particularly effective in PC12, where it almost completely abolishes A␤(1-40) -induced accumulation of thiobarbituric acid reactive substances (TBARS) and it significantly reduces A␤-mediated cytoplasmic overproduction of H2 O2 . In the latter case, the amount of cytoplasmic peroxides is also high in basal conditions, presumably as a consequence of long term culture conditions. Indeed, it has been reported that cellular susceptibility to accumulate oxidative species and by-products of basal oxidative metabolism is increased in long term cultures (Brewer and Cotman, 1989; Colton et al., 1995). Hence, in these conditions, the almost 50% decrease of A␤-mediated accumulation of H2 O2 by acetaminophen is of functional relevance. It might reflect either a specific interaction of the molecule with those cellular targets that mediate A␤ promotion of oxidative stress, or a direct interaction of acetaminophen with the peptide. Indeed, it has already been reported that

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a variety of antioxidant agents, such as Vitamin E, Vitamin C and ␤-carotene, can directly inhibit A␤ aggregation and the formation of ␤-sheet A␤ fibrils (Zhang et al., 1996; Tomiyama et al., 1996). Therefore, it cannot be ruled out the possibility that acetaminophen can interfere with the formation and assembling of A␤ fibrils, thus limiting the access or otherwise interrupting A␤ association with cellular targets. The capability of acetaminophen to specifically interfere with A␤(1-40) -triggered elevation of TBARS is also evident in hippocampal neurons, as the drug completely abolishes the quote of TBARS induced by A␤. Perhaps this leads to the hypothesis that acetaminophen does not directly react with membrane peroxides, but rather upstream, interfering with the mechanism of molecular interaction between A␤ and cellular membranes, that promotes their oxidative disruption. The observation that acetaminophen is somehow lipoperoxidative per se may be explained as a dose-related effect, as the highest protective concentration (500 ␮M) of the compound has been tested, or as part of the cellular actions of the drug, as it has been hypothesized (Jones, 2001). Finally, the antioxidant action of acetaminophen seems not to be related to free radical scavenging properties, as assessed by comparing the activity of the drug with that of the free radical scavenger propyl gallate, known to greatly reduce A␤ toxicity (Behl et al., 1992, 1994; Mattson and Goodman, 1995). In fact, while the latter specifically reduces the lipoperoxidative component of FeSO4 -mediated oxidative stress, without appreciably interfering with H2 O2 -triggered lipid peroxidation, acetaminophen reduces TBARS formation by the two triggers at the same rate, thus showing no specific radical trapping activity. 4.5. Effect of acetaminophen on Aβ-triggered transcriptional activity ROS are known to be described as potent mediators of NF-␬B activation, particularly in stress conditions (Mercurio and Manning, 1999). As a pleiotropic regulator of stress-induced gene expression, NF-␬B is activated in response to a myriad of different agents. Particularly, environmental toxins, acute or chronic inflammation, such as that triggered by amyloid, or UV irradiation can promote cellular accumulation of oxygen derivatives, mostly superoxide anion (O2 − ), which, together with H2 O2 , promotes NF-␬B transcriptional activity. This, in turn, takes to the enhanced expression of the main enzymatic antioxidant systems, which then contribute to cellular redox regulation. Several reports show that, in different neuronal models, A␤ peptides induce NF-␬B activation (Kaltschmidt et al., 1997; Guo et al., 1998); moreover, the nuclear import of this transcriptional factor is selectively blocked by antioxidant compounds such as PDTC, thus pointing to a role for ROS in mediating NF-␬B regulation. Moreover, it has been shown by immunohistochemistry that a marked nuclear signal of the transcription factor is present in brains from AD patients, particularly in primitive plaques, where also the

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inflammatory cytokines IL-6 and IL-1, and the intercellular adhesion molecule 1 (ICAM-1) show the same staining pattern (Strauss et al., 1992; Verbeek et al., 1994; Griffin et al., 1995). This strongly suggests that activation of NF-␬B is linked to enhanced expression of these inflammatory genes, which, in turn, regulate the activity of this transcription factor, establishing, together with amyloid precursor protein, several feedback loops for the control of the inflammatory response to amyloid toxicity (Mrak et al., 1995). There is evidence of the capability of some anti-inflammatory drugs, both NSAIDs and steroids, to attenuate NF-␬B translocation in different models of neuronal and glial toxicity (Grilli et al., 1996; Dodel et al., 1999). Thus, it has been demonstrated that different toxic insults, such as glutamate stimulation of primary neuronal cultures or A␤(1-40) and LPS challenge of astroglial cultures, trigger NF-␬B activation, and that the nuclear signal of the transcription factor is strongly reduced or blocked by the anti-inflammatory compounds (McGeer and McGeer, 1999; McGeer, 2000). According to these findings, our data show that also acetaminophen strongly reduces the increased nuclear signal of NF-␬B after A␤(1-40) challenge, and that this drug is neuroprotective. If the blockade of NF-␬B induction is mediated by a reduction of ROS, remains to be elucidated. Still, there is evidence of a strong reduction of ROS enhancement following amyloid challenge (see previous section), thus suggesting a possible relationship between the antioxidant properties and the blockade of the transcription factor. As for others NSAIDs, such as acetyl salicylic acid or sodium salicylate, acetaminophen blocks NF-␬B activation and exerts a neuroprotective activity, suggesting a link between neuroprotection and the nuclear event (Grilli et al., 1996). Finally, the molecular targets of NF-␬B blockade need further elucidation. Particularly, this early action of the drug may contribute, on the one hand, to ultimately reduce the activation of those inflammatory genes that sustain A␤-induced inflammation, and on the other, to activate those cellular defense systems which contribute to delay the progression of A␤-induced apoptosis and to contain the induction of oxidative stress.

Acknowledgements The financial support of ACRAF, MURST PRIN 2000, MISAN Finalizzato 99 “Invecchiamento cerebrale. . . ” and Telethon Grant E1144 to GS is greatly appreciated. References Abramson, S.R., Weissman, G., 1989. The mechanisms of action of nonsteroidal antiinflammatory drugs. Arthritis Rheum 32, 1–9. Anrather, D., Millan, M.T., Palmetshofer, A., Robson, S.C., Geczy, C., Ritchie, A.J., Bach, F.H., Ewenstein, B.M., 1997. Thrombin activates nuclear factor-␬B and potentiates endothelial cell activation by TNF. J. Immunol. 159, 5620–5628.

Behl, C., Davis, J.B., Lesley, R., Schubert, D., 1992. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem. Biophys. Res. Commun. 186, 944–950. Behl, C., Davis, J.B., Lesley, R., Schubert, D., 1994. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77, 817–827. Bisaglia, M., Natalini, B., Pellicciari, R., Straface, E., Malorni, W., Monti, D., Franceschi, C., Schettini, G., 2000. C3-fullero-trismethanodicarboxylic acid protects cerebellar granule cells from apoptosis. J. Neurochem. 74, 1197–1204. Bour, A.M., Westendorp, R.G., Laterveer, J.C., Bollen, E.L., Remarque, E.J., 2000. Interaction of indomethacin with cytokine production in whole blood. Potential mechanism for a brain-protective effect. Exp. Gerontol. 35, 1017–1024. Brewer, G.L., Cotman, C.W., 1989. Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen. Brain Res. 494, 65–74. Busciglio, J., Lorenzo, A., Yankner, B.A., 1992. Methodological variables in the assessment of beta amyloid neurotoxicity. Neurobiol. Aging 13, 609–612. Butterfield, D.A., Howard, B., Yatin, S., Koppal, T., Drake, J., Hensley, K., Aksenov, M., Aksenova, M., Subramaniam, R., Varadarajan, S., Harris-White, M.E., Pedigo Jr., N.W., Carney, J.M., 1999. Elevated oxidative stress in models of normal brain aging and Alzheimer’s disease. Life Sci. 65, 1883–1892. Canning, D.R., Mc Keon, R.J., De Witt, D.A., Perry, G., Wujek, J.R., Frederickson, R.C.A., Silver, J., 1993. ␤-Amyloid of Alzheimer’s disease induces reactive gliosis that inhibits axonal outgrowth. Exp. Neurol. 124, 289–298. Cedazo-Minguez, A., Wiehager, B., Winblad, B., Huttinger, M., Cowburn, R.F., 2001. Effects of apolipoprotein E (apoE) isoforms, ␤-amyloid (Abeta) and apoE/Abeta complexes on protein kinase C-alpha (PKC-alpha) translocation and amyloid precursor protein (APP) processing in human SH-SY5Y neuroblastoma cells and fibroblasts. Neurochem. Int. 38, 615–625. Colton, C.A., Pagan, F., Snell, J., Cummins, A., Gilbert, D.L., 1995. Protection from oxidation enhances the survival of cultured mesencephalic neurons. Exp. Neurol. 132, 54–61. Coulson, E.J., Paliga, K., Beyreuther, K., Masters, C.L., 2000. What the evolution of the amyloid protein precursor supergene family tells us about its function. Neurochem. Int. 36, 175–184. Dodel, R.C., Du, Y., Bales, K.R., Gao, F., Paul, S.M., 1999. Sodium salicylate and 17␤-estradiol attenuate nuclear transcription factor NF-␬B translocation in cultured rat astroglial cultures following exposure to amyloid A ␤(1-40) and lipopolysaccharides. J. Neurochem. 73, 1453–1460. Emilien, G., Beyruther, K., Masters, C.L., Maloteaux, J.M., 2000. Prospects for pharmacological intervention in Alzheimer disease. Arch. Neurol. 57, 454–459. Emmerling, M.R., Watson, M.D., Raby, C.A., Spiegel, K., 2000. The role of complement in Alzheimer’s disease pathology. Biochim. Biophys. Acta 1502, 158–171. Estus, S., Tucker, M., Van Rooyen, C., Wright, S., Brigham, E.F., Wougulis, M., Rydel, R.E., 1997. Aggregated amyloid-beta protein induces cortical neuronal apoptosis and concomitant apoptotic pattern of gene induction. J. Neurosci. 17, 7736–7745. Flynn, B.L., Theesen, K.A., 1999. Pharmacologic management of Alzheimer disease part III: nonsteroidal antiinflammatory drugs—emerging protective evidence. Ann. Pharmacotherapy 33, 840–849. Forloni, G., Chiesa, R., Smiroldo, S., Verga, L., Salmona, M., Tagliavini, F., Angeretti, N., 1993. Apoptosis mediated neurotoxicity induced by chronic application of beta amyloid fragment 25–35. Neuroreport 4, 523–526. Furukawa, K., Mattson, M.P., 1995. Cytochalasins protect hippocampal neurons against amyloid beta-peptide toxicity: evidence that actin depolymerization suppresses Ca2+ influx. J. Neurochem. 65, 1061– 1068.

M. Bisaglia et al. / Neurochemistry International 41 (2002) 43–54 Goslin, K., Banker, G., 1990. Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions. J. Cell Biol. 110, 1319–1331. Griffin, W.S., Sheng, J.G., Roberts, G.W., Mrak, R.E., 1995. Interleukin-1 expression in different plaque types in Alzheimer’s disease: significance in plaque evolution. J. Neuropathol. Exp. Neurol. 54, 276–281. Grilli, M., Pizzi, M., Memo, M., Spano, P.F., 1996. Neuroportection by aspirin and sodium salicilate through blockade of NF-␬B activation. Science 274, 1383–1385. Guo, Q., Robinson, N., Mattson, M.P., 1998. Secreted ␤-amyloid precursor protein counteracts the proapoptotic action of mutant presenilin-1 by activation of NF-␬B and stabilization of calcium homeostasis. J. Biol. Chem. 273, 12341–12351. Hardy, J., 1997. Amyloid, the presenilins and Alzheimer’s disease. Trends Neurosci. 20, 154–159. Hedin, H.L., Erikkson, S., Fowler, C.J., 2001. Human platelet calcium mobilisation in response to ␤-amyloid (25–35): buffer dependency and unchanged response in Alzheimer’s disease. Neurochem. Int. 38, 145– 151. Hirai, K., Aliev, G., Nunomura, A., Fujioka, H., Russell, R.L., Atwood, C.S., Johnson, A.B., Kress, Y., Vinters, H.V., Tabaton, M., Shimohama, S., Cash, A.D., Siedlak, S.L., Harris, P.L., Jones, P.K., Petersen, R.B., Perry, G., Smith, M.A., 2001. Mitochondrial abnormalities in Alzheimer’s disease. J. Neurosci. 21, 3017–3023. Hull, M., Lieb, K., Fiebich, B.L., 2000. Anti-inflammatory drugs: a hope for Alzheimer’s disease. Exp. Opin. Invest. Drugs 9, 671–683. Jones, G.R., 2001. Causes of Alzheimer’s disease: paracetamol (acetaminophen) today? Amphetamines tomorrow. Med. Hypotheses 56, 121–123. Kaltschmidt, C., Kaltschmidt, B., Neumann, H., Wekerle, H., Baeuerle, P.A., 1994a. Constitutive NF-␬B activity in neurons. Mol. Cell. Biol. 14, 3981–3992. Kaltschmidt, C., Kaltschmidt, B., lannes-Vieira, J., Kreutzberg, G.W., Wekerle, H., Baeuerle, P.A., Gehrmann, J., 1994b. Transcription factor NF-␬B is activated in microglia during experimental autoimmune encephalomyelitis. J. Neuroimmunol. 55, 99–106. Kaltschmidt, B., Uherek, M., Volk, B., Baeuerle, P.A., Kaltschmidt, C., 1997. Transcription factor NF-␬B is activated in primary neurons by amyloid ␤ peptides and in neurons surrounding early plaques from patients with Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 94, 2642–2647. Kish, S.J., Bergeron, C., Rajput, A., Dozic, S., Mastrogiacomo, F., Chang, L.J., Wilson, J.M., DiStefano, L.M., Nobrega, J.N., 1992. Brain cytochrome oxidase in Alzheimer’s disease. J. Neurochem. 59, 776– 779. Landolfi, C., Soldo, L., Polenzani, L., Apicella, C., Capezzone de Joannon, A., Coletta, I., Di Cesare, F., Brufani, M., Pinza, M., Milenese, C., 1998. Inflammatory molecule release by ␤-amyloid-treated T98G astrocytoma cells: role of prostaglandins and modulation by paracetamol. Eur. J. Pharmacol. 360, 55–64. Loo, D.T., Copani, A., Pike, C.J., Whittemore, E.R., Walencewicz, A.J., Cotman, C.W., 1993. Apoptosis is induced by ␤-amyloid in cultured central nervous system neurons. Proc. Natl. Acad. Sci. U.S.A. 90, 7951–7955. Mark, R.J., Hensley, K., Butterfield, D.A., Mattson, M.P., 1995. Amyloid beta-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+ homeostasis and cell death. J. Neurosci. 15, 6239–6249. Markesbery, W.R., 1997. Oxidative stress hypothesis in Alzheimer’s disease. Free Rad. Biol. Med. 23, 134–147. Markesbery, W.R., Carney, J.M., 1999. Oxidative alterations in Alzheimer’s disease. Brain Pathol. 9, 133–146. Marshall, P.J., Kulmacz, R.J., Lands, W.E.M., 1987. Constraints on prostaglandin biosynthesis in tissues. J. Biol. Chem. 262, 3510–3517. Martin, P.M., O’Callaghan, J.P., 1996. In: Aschner, M., Kimelbeg, H.K. (Eds.), The Role of Glia in Neurotoxicity. CRC Press, Boca Raton, USA, pp. 285–310.

53

Mattson, M.P., Goodman, Y., 1995. Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium. Brain Res. 676, 219–224. Mattson, M.P., Cheng, B., Davis, D., Bryant, K., Lieberburg, I., Rydel, R.E., 1992. ␤-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J. Neurosci. 12, 376–389. Mattson, M.P., Barger, S.W., Cheng, B., Lieberburg, I., Smith-Swintosky, V.L., Rydel, R.E., 1993. ␤-Amyloid precursor protein metabolites and loss of neuronal Ca2+ homeostasis in Alzheimer’s disease. Trends Neurosci. 16, 409–414. McGeer, P.L., 2000. Cyclo-oxygenase-2 inhibitors: rationale and therapeutic potential for Alzheimer’s disease. Drugs Aging 17, 1–11. McGeer, P.L., McGeer, E.G., 1999. Inflammation of the brain in Alzheimer’s disease: implications for therapy. J. Leukocyte Biol. 65, 409–415. McGeer, P.L., Schulzer, M., McGeer, E.G., 1996. Arthritis and antiinflammatory agents as possible protective factors for Alzheimer’s disease: a review of 17 epidemiologic studies. Neurology 47, 425–432. Mercurio, F., Manning, A.M., 1999. NF-␬B as a primary regulator of the stress response. Oncogene 18, 6163–6171. Mrak, R.E., Sheng, J.G., Griffin, W.S., 1995. Glial cytokines in Alzheimer’s disease: review and pathogenic implications. Hum. Pathol. 26, 816–823. Mutisya, E.M., Bowling, A.C., Beal, M.F., 1994. Cortical cytochrome oxidase activity is reduced in Alzheimer’s disease. J. Neurochem. 63, 2179–2184. Ogawa, O., Umegaki, H., Sumi, D., Hayashi, T., Nakamura, A., Thakur, N.K., Yoshimura, J., Endo, H., Iguchi, A., 2000. Inhibition of inducible nitric oxide synthase gene expression by indomethacin or ibuprofen in ␤-amyloid protein-stimulated J774 cells. Eur. J. Pharmacol. 408, 137–141. Parker Jr., W.D., Parks, J.K., 1995. Cytochrome c oxidase in Alzheimer’s disease brain: purification and characterization. Neurology 45, 482– 486. Pereira, C., Santos, M.S., Oliveira, C., 1998. Mitochondrial function impairment induced by amyloid ␤-peptide in PC12 cells. Neuroreport 9, 1749–1755. Petit, P.X., Susin, S.A., Zamzami, N., Mignotte, B., Kroemer, G., 1996. Mitochondria and programmed cell death: back to the future. FEBS Lett. 396, 7–13. Pike, C.J., Cummings, B.J., Cotman, C.W., 1992. ␤-Amyloid induces neuritic dystrophy in vitro: similarities with Alzheimer pathology. Neuroreport 3, 769–772. Pike, C.J., Walencewicz-Wasserman, A.J., Kosmoski, J., Cribbs, D.H., Glabe, C.G., Cotman, C.W., 1995. Structure-activity analyses of ␤-amyloid peptides: contributions of the beta 25–35 region to aggregation and neurotoxicity. J. Neurochem. 64, 253–265. Pike, C.J., Ramezam-Arab, N., Cotman, C.W., 1997. ␤-Amyloid neurotoxicity in vitro: evidence of oxidative stress but not protection by antioxidants. J. Neurochem. 69, 1601–1611. Porubek, D.J., Rundgren, M., Harvison, P.J., Nelson, S.D., Moldeus, P., 1987. Investigation of mechanisms of acetaminophen toxicity in isolated rat hepatocytes with the acetaminophen analogues 3,5-dimethylacetaminophen and 2,6-dimethylacetaminophen. Mol. Pharmacol. 31, 647–653. Russo, C., Saido, T.C., De Busk, L., Tabaton, M., Gambetti, P., Teller, J.K., 1997. Heterogeneity of water-soluble amyloid beta-peptide in Alzheimer’s disease and Down’s syndrome brains. FEBS Lett. 409, 411–416. Saido, T.C., Iwatsubo, T., Mann, D.M., Shimada, H., Ihara, Y., Kawashima, S., 1995. Dominant and differential deposition of distinct ␤-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron 14, 457–466. Saido, T.C., Yamao-Harigaya, W., Iwatsubo, T., Kawashima, S., 1996. Amino- and carboxy-terminal heterogeneity of ␤-amyloid peptides deposited in human brain. Neurosci. Lett. 215, 173–176.

54

M. Bisaglia et al. / Neurochemistry International 41 (2002) 43–54

Scorziello, A., Meucci, O., Florio, T., Fattore, M., Forloni, G., Salmona, M., Schettini, G., 1996. Beta 25–35 alters calcium homeostasis and induces neurotoxicity in cerebellar granule cells. J. Neurochem. 66, 1995–2003. Selkoe, D.J., 1996. Amyloid beta-protein and the genetics of Alzheimer’s disease. J. Biol. Chem. 271, 18295–18298. Smale, G., Nichols, N.R., Brady, D.R., 1995. Evidence for apoptotic cell death in Alzheimer’s disease. Exp. Neurol. 133, 225–230. Smith, M.A., Hirai, K., Hsiao, K., Pappolla, M.A., Harris, P.L.R., Siedlak, S.L., Tabaton, M., Perry, G., 1998. Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress. J. Neurochem. 70, 2212–2215. Stewart, W.F., Kawas, C., Corrada, M., Metter, E.J., 1997. Risk of Alzheimer’s disease and duration of NSAID use. Neurology 48, 626– 632. Strauss, S., Bauer, J., Ganter, U., Jonas, U., Berger, M., Volk, B., 1992. Detection of interleukin-6 and alpha 2-macroglobulin immunoreactivity in cortex and hippocampus of Alzheimer’s disease patients. Lab. Invest. 66, 223–230. Su, J.H., Anderson, A.J., Cummings, B., Cotman, C.W., 1994. Immunohistochemical evidence for apoptosis in Alzheimer’s disease. Neuroreport 5, 2529–2533.

Tekirian, T.L., Saido, T.C., Markesbery, W.R., Russel, M.J., Wekstein, D.R., Patel, E., Geddes, J.W., 1998. N-terrminal heterogeneity of parenchymal and cerebrovascular Abeta deposits. J. Neuropath. Exp. Neurol. 57, 76–94. Tekirian, T.L., Yang, A.Y., Glabe, C., Geddes, J.W., 1999. Toxicity of pyroglutaminated amyloid ␤-peptides 3(pE)-40 and -42 is similar to that of A␤1-40 and -42. J. Neurochem. 73, 1584–1589. Tomiyama, T., Shoji, A., Kataoka, K., Suwa, Y., Asano, S., Kaneko, H., Endo, N., 1996. Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger. J. Biol. Chem. 271, 6839–6844. Verbeek, M.M., Otte-Holler, I., Westphal, J.R., Weisseling, P., Ruiter, D.J., Waal, R.M., 1994. Accumulation of intercellular adhesion molecule-1 in senile plaques in brain tissue of patients with Alzheimer’s disease. Am. J. Pathol. 144, 104–116. Zhang, H.Y., Tang, X.C., 2000. Huperzine B, a novel acetylcholinesterase inhibitor, attenuates hydrogen peroxide induced injury in PC12 cell. Neurosci. Lett. 292, 41–44. Zhang, Z., Rydel, R.E., Drzewiecki, G.J., Fuson, K., Wright, S., Wogulis, M., Audia, J.E., May, P.C., Hyslop, P.A., 1996. Amyloid beta-mediated oxidative and metabolic stress in rat cortical neurons: no direct evidence for a role for H2 O2 generation. J. Neurochem. 67, 1595–1606.