Involvement of Oxidative Stress on the Impairment of Energy Metabolism Induced by Aβ Peptides on PC12 Cells: Protection by Antioxidants

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Neurobiology of Disease 6, 209–219 (1999)A

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nvolvement of Oxidative Stress on the Impairmentf Energy Metabolism Induced by A b Peptidesn PC12 Cells: Protection by Antioxidants

laudia Pereira,* Maria Sancha Santos,† and Catarina Oliveira*Faculty of Medicine and †Department of Zoology and Center for Neuroscience of Coimbra,niversity of Coimbra, 3000 Coimbra, Portugal

eceived January 4, 1999; revised February 25, 1999; accepted for publication March 1, 1999

Alzheimer’s disease is widely held to be associated with oxidative stress due, in part, to the membraneaction of amyloid b-peptide (A b) aggregates. In this study, the involvement of oxidative stress onAb-induced energy metabolism dysfunction was evaluated on PC12 cells. It was shown that A bpeptides (A b25–35 and A b1–40) induce a concentration-dependent accumulation of reactive oxygenspecies (ROS), decrease the cellular redox activity, and lead to the depletion of ATP levels. Theobserved inhibition by A b of mitochondrial function and of glycolysis is blocked by the antioxidantsvitamin E, idebenone, and GSH ethyl ester. Taken together, these data suggest that exposure of PC12

rticle ID nbdi.1999.0241, available online at http://www.idealibrary.com on

cells to A b results in an impairment of energy metabolism, leading to a deficit in ATP levels and to thecompromise of cellular viability. Furthermore, the generation of ROS seems to be a crucial event

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NTRODUCTION

Alzheimer’s disease (AD) is a progressive neurode-enerative disorder characterized by the gradual im-airment of memory function and accumulation ofeurofibrillary tangles and neuritic plaques in brainegions subserving cognitive functions (Selkoe, 1993).

consistent feature of AD patients, detected by brainmaging methods, is the impairment of glucose metabo-ism in brain regions that exhibit neuritic plaques andhat may occur early in the disease process prior toeuronal degeneration (Kalaria & Harik, 1989; Sims,990; Hoyer et al., 1991; Jagust et al., 1991; Kennedy etl., 1995). High levels of oxidative stress (Smith et al.,995; Yankner, 1996; Mark et al., 1996) and increasedevels of protein oxidation (Smith et al., 1991) and lipideroxidation (Lovell et al., 1995) occur in vulnerableegions of AD brain, and histological analyses suggestncreased protein oxidation and glycation in tangle-earing neurons (Smith et al., 1995).Several factors that may contribute to oxidative

tress and neurofibrillary degeneration in AD, includ-ng age-related decreases in energy availability and

itochondrial function (Bowling & Beal, 1995), disrup-

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969-9961/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

d by A b. r 1999 Academic Press

ion of cellular calcium homeostasis (Mattson et al.,993), excitotoxicity (Greenamyre & Young, 1989), andccumulation of amyloid b-peptide (Ab) (Yankner,996), have been described. The amyloid b-peptide,he major component of neuritic plaques, is a 40- to2-amino-acid proteolytic fragment of the amyloidrecursor protein (Kang et al., 1987). Incubation of Ab

n vitro results in a concentration- and time-dependentormation of neurotoxic fibrils (Pike et al., 1993), simi-ar to those characteristic of amyloid-containing plaquesresent in the brains of AD patients. The sequenceorresponding to amino acids 25–35 of Ab (Ab25–35)orms fibrils and is neurotoxic by a mechanism similaro that of Ab1–40/42 (Yankner et al., 1990; Pike et al.,993). The involvement of oxidative stress in Abeurotoxicity has been suggested: Ab accumulation isssociated with protein oxidation (Smith et al., 1991),ipid peroxidation (Lovell et al., 1995), and advancedlycation end products (Smith et al., 1994) in AD brain;he aggregation of Ab in vitro is catalyzed by oxidationnd is inhibited by antioxidants (Dyrks et al., 1992); Ab
eurotoxicity is attenuated by a number of antioxi-ants (Behl et al., 1992; Bruce et al., 1996); Ab itself
nduces reactive oxygen species (ROS) generation (Behl

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t al., 1994; Goodman & Mattson, 1994). Although theolecular mechanisms through which Ab induces

euronal death are still controversial, recent reportsuggest the involvement of Ab-induced oxidative stress,resumably initiated through intracellular productionf ROS (Behl et al., 1994; Schubert et al., 1995; Mattsont al., 1995). Membrane lipid peroxidation (Behl et al.,994; Butterfield et al., 1994) and the disruption of ionomeostasis (Mattson et al., 1992, 1993) have beenhown to play an important role in Ab-induced neuro-al death.The aldehyde 4-hydroxynonenal (HNE) has been

eported to be the most cytotoxic product of lipideroxidation and to contribute to membrane damagend cell death at concentrations reached when cells arexposed to various oxidative insults (Esterbauer et al.,991). Recently it was shown that HNE generatedollowing exposure of neurons to Ab binds to mem-rane ion-motive ATPases and impairs their function,uggesting that HNE is a mediator of Ab-inducedisruption of neuronal ion homeostasis and cell death

Mark et al., 1997; Kruman et al., 1997). Furthermore, itas been shown that HNE and HNE-derived advanced

ipid peroxidation end products are increased in theD brain (Markesbery & Lovell, 1998).Recently, we have shown that Ab leads to the

nhibition of mitochondrial respiration on PC12 cellsPereira et al., 1998). The present study was performedn order to evaluate whether Ab also leads to thenhibition of glycolysis and to test the hypothesis thatb peptide disrupts energy metabolism by a mecha-ism involving generation of ROS. In particular, theim of this work was to demonstrate whether Ab: (1)ncreases ROS generation, (2) is responsible for ATPepletion and altered redox status of the cells, and (3)

nhibits the glycolytic pathway. Furthermore, by usingeveral antioxidants, namely vitamin E, idebenone,nd GSH ethyl ester, the involvement of oxidativetress on the inhibition of glycolysis and of enzymaticitochondrial respiratory chain complexes activityas analyzed.

XPERIMENTAL PROCEDURES

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Amyloid b-peptide fragments (Ab25–35 and Ab1–

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0) were obtained from Bachem AG (Bubendorf, Ger-any). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-

olium bromide (MTT), vitamin E (a-tocopherol

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opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

uccinate), GSH ethyl ester, and ubiquinone-1 wereurchased from Sigma Chemical Co. (St. Louis, MO).etal calf serum was obtained from Biokrom KGBerlin, Germany) and horse serum from Gibco (Pais-ey, UK). 28,78-dichlorofluorescin diacetate was pur-hased from Molecular Probes (Eugene, OR). All othereagents were of analytical grade. The kits for lactatend pyruvate measurement were purchased from Boeh-inger Mannheim Co. (Indianapolis, IN) and Sigmahemical Co., respectively. Idebenone was a generousift from Seber (Portugal).

ell Culture

Stock cultures of PC12 cells, purchased from ATCCAmerican Type Culture Collection), were grown rou-inely in 75-cm2 tissue culture flasks in RPMI 1640upplemented with 10% heat-inactivated horse serumnd 5% heat-inactivated fetal calf serum, to whichenicillin (50 U/ml) and streptomycin (50 µg/ml)ere added, and maintained at 37°C in a humidified

ncubator containing 95% air and 5% CO2 (Greene &ischler, 1976). Cells were plated on poly-L-lysine-oated (10 µg/ml) 24-well dishes (0.5 ml/well) foroxicity experiments and on 12-well dishes (1 ml/well)or ATP measurements. Cells were seeded on poly-L-ysine-coated 100-mm dishes (10 ml/dish) for lactatend pyruvate determinations and for evaluation of thenzymatic activity of mitochondrial respiratory chainomplexes. Finally, for the fluorimetric determinationssing the probe 28,78-dichlorofluorescin diacetate (DCF-A), cells were seeded on poly-L-lysine-coated cover-

lips plated in 12-well dishes (2 ml/well).

reatments and Cytotoxicity Analysis

Twenty-four hours after seeding, the medium wasenewed by fresh medium containing the desiredoncentration of the amyloid b-peptide fragments,dded from a 1 mM stock prepared in sterile water.efore use, the Ab1–40 stock was diluted in PBS and

ncubated at 37°C for 24 h, in order to induce peptidereaggregation. When antioxidants were tested, cellsere simultaneously incubated for 24 h in the presence

f amyloid fragments and 1 µM idebenone, 10 µMitamin E (a-tocopherol succinate), or 1 mM GSH ethyl

Pereira, Santos, and Oliveira

ster, after preincubation for approximately 24 h withhe antioxidants. After an additional 24-h incubationeriod, the cells were analyzed.

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The evaluation of Ab-peptide-induced cytotoxicityas performed by measuring the MTT reduction

bility of PC12 cells, according to the method ofosmann (1983). In brief, MTT was dissolved in PBS

t 5 mg/ml and was 10-fold diluted in serum-freePMI 1640 medium. After incubation of the cells with

he compounds to be tested, the medium was aspi-ated and 0.5 ml of MTT-containing medium wasdded. After an additional 2- to 3-h incubation at 37°C,.5 ml isopropanol/HCl was added to each well andhe absorbance at 570 nm, of solubilized MTT forma-an products, was measured.

easurement of Intracellular Reactive Oxygenpecies Formation

Formation of intracellular peroxides was detectedy fluorescence spectroscopy using a nonfluorescentompound, 28,78-dichlorofluorescin diacetate (DCFH2-A), which is deesterified within cells by endogenous

sterases to the ionized free acid, 28,78-dichlorofluores-in DCFH2 (Bass et al., 1983). This is trapped withinells and thus accumulated 28,78-dichlorofluorescin isapable of being oxidized to fluorescent 28,78-dichloro-uorescein (DCF) by hydroperoxides, although otherechanisms of oxidation cannot be ruled out (Cathcart

t al., 1983). After exposure to b-amyloid, the cells werencubated in 2 ml of 140 mM NaCl, 5 mM KCl, 1 mM

gCl2, 5.6 mM glucose, 1.5 mM CaCl2, and 20 mMepes–Na, pH 7.4, and allowed to take up 5 µMCFH2-DA (added from a 1 mM stock solution inimethyl sulfoxide) at 37°C for 20 min in an atmo-phere of 95% air and 5% CO2. After being loaded withCFH2-DA, cells were washed with the same buffer

nd fluorescence was measured at 502-nm excitationnd 550-nm emission.

nalysis of Adenine Nucleotides

After the incubation period, the medium was re-oved and PC12 cells were extracted, in ice, with 0.3

perchloric acid. The cells were scraped from theells and centrifuged at 15,800g for 5 min. The pelletsere solubilized with 1 M NaOH and analyzed for

otal protein content by the Sedmak method (SedmakGrossberg, 1977), using bovine serum albumin as

tandard. The supernatants were neutralized with 10

b-Induced Oxidative Stress and Metabolic Dysfunction

KOH in 5 M Tris and centrifuged at 15,800g for 5in. The resulting supernatants were assayed for

denine nucleotides by separation in a reverse-phasecK

PLC, as described by Stocchi et al. (1985). Thehromatographic apparatus used was a Beckman Sys-em Gold, consisting of a 126 binary pump model and166 variable UV detector, controlled by a computer.he column used was a Lichrospher 100 RP-18 (5 µm)

rom Merck (Germany). An isocratic elution with 100M KH2PO4 buffer, at pH 7.4, and 1% methanol was

erformed at a flow rate of 1.2 ml/min. The adenineucleotides were detected at 254 nm, for 6 min.

-[3H]Deoxy- D-glucose Uptake Studies

2-[3H]Deoxy-D-glucose ([3H]2DG) uptake was deter-ined as described by Singh et al. (1997). After incuba-

ion with Ab fragments, cells were washed twice witha1 medium (140 mM NaCl, 5 mM KCl, 5 mM MgCl2,mM KH2PO4, 20 mM Hepes, 1.5 mM CaCl2, pH 7.4)

nd then incubated in 2 ml Na1 medium containing25 µM unlabeled 2-deoxy-D-glucose and 1 µCi

3H]2DG (11.4 Ci/mmol), equivalent to 44 nM for 5in at 37°C. The incubation was stopped by rapidlyashing the monolayers twice with ice-cold Na1

edium, followed by addition of 0.1 M NaOH. Ali-uots of the cell digests were used for liquid scintilla-ion counting and for protein determination (Sedmak

Grossberg, 1977). Nonspecific [3H]2DG uptake wasstimated in the presence of 200 mM glucose andubtracted from total uptake.

uantification of Lactate and Pyruvate Levels

After incubation with toxic concentrations of Ab25-35r Ab1–40, PC12 cells were washed twice and scrapedrom the dishes with phosphate-buffered saline, pH.4, at 4°C. An aliquot of the cellular suspension wastored at 220°C for subsequent measurement of lactateevels according to kit instructions. For determinationf pyruvate levels, an aliquot of the cell suspensionas diluted with 8% perchloric acid (1:3, v/v) androteins were pelleted by centrifugation. The superna-

ant was stored at 220°C for further analysis ofyruvate content according with kit instructions.

easurement of Mitochondrial Complexes Activity

211

All activities were measured in sucrose mediumontaining (in mM): sucrose 130, KCl 50, MgCl2 5,H2PO4 5, Hepes 5, pH 7.4.

Copyright r 1999 by Academic PressAll rights of reproduction in any form reserved.

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omplex I (NADH-Ubiquinone Oxidoreductase)

The decrease in NADH absorbance was measured at40 nm, at 30°C, in sucrose medium supplementedith 1 mM KCN, 150 µM NADH, 1.25 mg/ml free

atty acid BSA, and PC12 cell homogenates. Theeaction was initiated with 50 µM ubiquinone-1, after areincubation for 5 min (Ragan et al., 1987). Rotenone

nhibited complex I activity.

omplex II/III (Succinate Dehydrogenase 1uccinate–Cytochrome c Reductase)

The activity of complex II/III was determined inucrose medium supplemented with 8 µM rotenone, 1M KCN, 54 µM cytochrome c, and cell homogenates

ccordingly to Tisdale (1967). After preincubation for 5in, the reaction was initiated by addition of 5 mM

uccinate. Antimycin A at 0.1 µM inhibited enzymectivity.

omplex IV (Cytochrome Oxidase)

The activity of complex IV was measured polaro-raphically (Brautigan et al., 1978) at 30°C in homoge-ates of control or Ab-treated cells resuspended inucrose medium, supplemented with 2 µM rotenone,.1 µg antimycin A, 10 µM cytochrome c, and 0.3 mgriton X-100. The reaction was initiated upon additionf 5 mM ascorbate 1 0.25 mM TMPD. Incubation withCN inhibited complex IV activity.

ata Analysis

Throughout the text, data are expressed as means 6EM of triplicate determinations, from at least threendependent experiments. Statistical significance analy-is was determined by using the unpaired two-tailedtudent’s t test or by the one-way analysis of variance forultiple comparison, followed by the Tukey–Kramer post

oc test (a value of P , 0.05 was considered significant).

ESULTS

ose-Dependent Amyloid b-Peptide Toxicity,ccumulation of ROS, and Depletionf Intracellular ATP Levels

12

The dose-dependent amyloid b-peptide toxicity wasvaluated by determining the percentage of MTT reduc-

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ion upon incubation of PC12 cells for 24 h withncreasing concentrations of Ab25–35 or Ab1–40 pep-ides. As shown in Fig. 1, increasing concentrations ofb25–35 or Ab1–40 peptide induced a decrease in

ellular viability in a dose-dependent manner in PC12ells, with a significant decrease of MTT reductionfter treatment of the cells for 24 h with 10 nM amyloidragments.

To clarify the involvement of oxygen radicals in theb-induced toxicity, we measured the accumulation ofOS after exposure of PC12 cells to increasing concen-

rations of Ab25–35 or Ab1–40 peptides (Figs. 1 and 2).OS accumulation, estimated utilizing a converting

eaction of the probe DCFH2-DA to DCF, was increasedn a dose-dependent manner. The increase in DCFuorescence was significant after treatment of cells for4 h with 10 nM amyloid fragments.The endogenous ATP levels of PC12 cells incubated

or 24 h with different concentrations of Ab25–35 orb1–40 peptides were determined (Fig. 2). The intracel-

ular content of ATP decreased, in a dose-dependentanner, with increasing concentrations of Ab25–35 orb1–40 peptides. It was demonstrated that 10 nMeptide Ab25–35 or Ab1–40 was able to significantlyeduce ATP levels.

ffect of A b Fragments on [ 3H]2DG Uptakend on Lactate and Pyruvate Levels:rotection by Antioxidants

The uptake of glucose, evaluated by the uptake ofritiated deoxyglucose, was not significantly affectedn cells treated for 24 h with toxic concentrations (10M and 1 µM) of Ab25–35 or Ab1–40 peptides (Table 1).The levels of lactate, determined after exposure of

C12 cells to toxic concentrations of Ab fragmentsAb25–35 and Ab1–40), were not significantly differentrom those determined in control cells (Table 2). On thether hand, a significant decline of pyruvate levels wasbserved after incubation of PC12 cells with 10 nM or 1M Ab25–35 or Ab1–40 peptides (Fig. 3). Pyruvate

evels decreased from 8.69 6 0.41 nmol/mg protein inontrol cells to 6.05 6 1.11 and 5.11 6 0.09 nmol/mgrotein in cells treated with 10 nM and 1 µM Ab25–35eptide, respectively (Fig. 3A). A decrease to 4.77 6.77 and 5.25 6 0.48 nmol/mg protein in cells treated,espectively, with 10 nM or 1 µM Ab1–40 peptide was


bserved (Fig. 3B). Pyruvate levels, determined in cellsreated with Ab25–35 or Ab1–40 peptides in theresence of the antioxidants vitamin E (10 µM), idebe-

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one (1 µM), and GSH ethyl ester (1 mM), were similaro those determined in cells incubated in the absence ofhe amyloid fragments (Fig. 3).

rotective Effect of Antioxidants againstb-Induced Inhibition of Complexes I, II–III, and IVf the Mitochondrial Respiratory Chain

After incubation of PC12 cells with toxic concentra-ions of the amyloid b-peptide fragments, the enzy-

atic activity of NADH-ubiquinone oxidoreductasecomplex I) was slightly inhibited (Fig. 4A). Complex Ictivity decreased approximately 20% in cells treatedor 24 h with 1 µM Ab25–35 or Ab1–40, in comparison

ith the activity determined in control cells. Thentioxidants idebenone (1 µM) and GSH ethyl ester (1M) completely prevented the Ab-induced inhibition

f complex I activity, while vitamin E (10 µM), at theoncentration tested, was not able to protect thisomplex of the mitochondrial respiratory chain fromhe deleterious effect of Ab.

When the activity of succinate dehydrogenase 1uccinate–cytochrome c reductase (complexes II–III)

IG. 1. Dose–response curves of the toxic effect of amyloid b-pepells, plated and grown for 24 h in culture medium, were switched tof Ab25–35 (A) or Ab1–40 (B) peptides. The Ab-induced toxicity, everoxides, detected using 28,78-dichlorofluorescin oxidation and fluof control values, are the means 6 SEM of triplicate determinationsifferent compared to control conditions, in the absence of Ab25–35 o


as analyzed, it was observed that the activity of theseomplexes is significantly inhibited by the amyloidragments (Fig. 4B). Complexes II–III activity decreased A

pproximately 60% in cells treated for 24 h with 1 µMb25–35 or Ab1–40 fragments, in comparison with con-

rol cells. The inhibition of complexes II–III was com-letely or partially reverted when cells were incubatedith Ab fragments in the presence of idebenone (1 µM)

nd GSH ethyl ester (1 mM). Vitamin E (10 µM) exertednly a moderate protection against the inhibition of com-lexes II–III induced by Ab1–40 peptide.Cytochrome oxidase activity was also inhibited byb25–35 and Ab1–40 peptide treatment (Fig. 4C). In

omparison with controls, its activity decreased ap-roximately 65% in cells treated for 24 h with 1 µMb25–35 peptide and 35% in cells exposed to 1 µMb1–40 peptide. When cells were exposed to toxic

oncentrations of Ab25–35 or Ab1–40 (1 µM) in theresence of the antioxidants idebenone (1 µM), vita-in E (10 µM), and GSH ethyl ester (1 mM), the

ecrease in the activity of complex IV of the mitochon-rial electron transport chain was completely abol-

shed and reverted to values similar to that determinednder control conditions (Fig. 4).

ISCUSSION

nd of Ab-stimulated intracellular accumulation of peroxides. PC12edium in the absence or in the presence of increasing concentrationsby the reduction of the tetrazolium salt MTT, and the formation of

ce, were assessed 24 h later. The results, expressed as the percentagenct experiments, respectively. **P , 0.01, ***P , 0.001, significantly40 peptides.

213

tides afresh maluated

The goal of this study was to test the hypothesis thatb-induced metabolic dysfunction is a key factor in


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ediating Ab toxicity and that oxidative stress plays aajor role in this process. The present findings demon-

trate that Ab leads to ATP depletion by inhibitingitochondrial function and glycolysis, the two major

nergy-producing cellular pathways, and suggest thatxidative stress may be an important mediator ofnergetic compromise and degeneration induced byb exposure.Treatment of PC12 cells for 24 h with increasing

IG. 2. Dose–response curves of intracellular ATP levels and of intith Ab25–35 (A) or Ab1–40 (B) peptide. PC12 cells, plated and grow

r in the presence of increasing concentrations Ab peptides. The foruorescence, was assessed 24 h later. The ATP levels of control or of cy reverse-phase HPLC with UV detection. Data are the arithmetic m*P , 0.01, ***P , 0.001, significantly different compared to control co

ABLE 1

ffect of Amyloid b-Peptides on [3H]2DG Accumulation byC12 Cells

xperimental condition [Ab peptide][3H]2DG uptake

(% of control)

ontrol 0 100.0 6 1.21 (n 5 4)b25–35 treatment 10 nM 107.9 6 7.92 (n 5 4)

1 µM 100.8 6 2.30 (n 5 4)b1–40 treatment 10 nM 99.03 6 2.50 (n 5 4)

1 µM 98.43 6 2.73 (n 5 4)

Note. [3H]2-Deoxy-D-glucose uptake was determined as describednder Experimental Procedures. After incubation with Ab frag-ents, cells were washed and then incubated in the presence of 125

M unlabeled 2-deoxy-D-glucose and 1 µCi [3H]2DG (11.4 Ci/mol), equivalent to 44 nM, for 5 min at 37°C. Aliquots of the cell

14

igests obtained after addition of NAOH were used for liquidcintillation counting. Nonspecific [3H]2DG uptake was estimated inhe presence of 200 mM glucose and subtracted from total uptake.

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oncentrations of the amyloid b-peptides resulted in aose-dependent decrement of cell survival (Fig. 1),etermined by the MTT assay, in agreement withesults previously published (Shearman et al., 1995;ereira et al., 1998). Concomitant with the decrease inell survival, a significant depletion of ATP levels wasbserved (Figs. 1 and 2), suggesting that the impair-ent of the energy metabolism of PC12 cells is in-

olved in the compromise of cellular viability. Theffect of Ab on energy metabolism was not secondaryo degeneration of PC12 cells, because LDH assays

lar accumulation of peroxides in PC12 cells submitted to treatmenth in culture medium, were switched to fresh medium in the absenceof peroxides, detected using 28,78-dichlorofluorescin oxidation andted with increasing concentrations of Ab peptides were determinedSEM of triplicate determinations of different experiments. *P , 0.05;ns, in the absence of Ab25–35 or Ab1–40 peptides.

ABLE 2

ffect of Amyloid b-Peptides on Intracellular Lactate Levels ofC12 Cells

xperimental condition [Ab peptide]Lactate levels

(nmol/mg protein)

ontrol 0 136.9 6 13.44 (n 5 4)b25–35 treatment 10 nM 148.6 6 13.65 (n 5 4)

1 µM 152.4 6 13.64 (n 5 4)b1–40 treatment 10 nM 159.6 6 17.56 (n 5 4)

1 µM 116.62 6 5.76 (n 5 6)

Note. After incubation with toxic concentrations of Ab25–35 orb1–40, PC12 cells were suspended in phosphate-buffered saline,


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H 7.4, at 4°C. An aliquot of the cellular suspension was stored at20°C for subsequent spectrophotometric measurement of lactate

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evealed no evidence of loss of membrane integrityuring the exposure period examined (data not shown).

n order to analyze whether the cause of ATP depletionbserved in Ab-treated cells resulted from mitochon-rial impairment and/or glycolysis inhibition, theffect of toxic concentrations of Ab on the activity ofeveral complexes of the mitochondrial respiratoryhain and also on the uptake of glucose and on thentracellular levels of pyruvate and lactate, two majorroducts of glycolysis, was studied. Recently, welearly demonstrated the inhibition of mitochondrialunction by Ab peptides, showing that the activity ofeveral complexes of the mitochondrial respiratoryhain was compromised after Ab treatment (com-lexes I, II–III, and IV). We also demonstrated that theb fragments induce the decrease of mitochondrialxygen consumption and the depolarization of theitochondrial membrane (Pereira et al., 1998). There-

ore, the mitochondrial function impairment observednder our experimental conditions could account, at

east in part, for the depletion of ATP levels. However,ecause PC12 cells are extremely dependent uponlycolysis for the synthesis of ATP (Morelli et al., 1986),he inhibition of glycolysis by toxic concentrations ofb, demonstrated by the decrease of pyruvate levels

Fig. 3), seems to play a major role in the depletion of

IG. 3. Effect of toxic concentrations of Ab25–35 (A) or Ab1–40 (B)he intracellular pyruvate levels of control and Ab-treated PC12 cell

10 µM), idebenone (1 µM), or GSH ethyl ester (1 mM), were determeterminations of different experiments. *P , 0.05, **P , 0.01, significantleptides; 1P , 0.05, 11P , 0.01, 111P , 0.001, significantly different


he intracellular ATP pool. The inhibition of mitochon-rial respiration by Ab (Pereira et al., 1998) couldecrease the utilization of pyruvate by mitochondria

cep

eading to its accumulation and consequent conversiono lactate; however, the maintenance of lactate levels is

ore consistent with the hypothesis that Ab induceshe inhibition of glycolytic enzymes, decreasing pyru-ate production.The decrease in cellular ATP levels by Ab has been

ecently reported (Zhang et al., 1996; Keller et al., 1997a;ark et al., 1997). Mark et al. (1997) and also Blanc et al.

1997) showed that the impairment of glucose trans-ort precedes ATP depletion in cultured rat corticaleurons exposed to Ab. Under our experimental condi-

ions, the uptake of glucose was not significantlyffected, suggesting that the impairment of glucoseetabolism, rather than its transport, may play an

mportant role in Ab-induced energy failure. Ouresults, in agreement with others (Blanc et al., 1997;

ark et al., 1997), support the notion that glucosetilization is reduced by Ab, either as a result of the

mpairment of glucose transport or as a result of thenhibition of glucose utilization by glycolysis. Manytudies have documented reduced glucose availabilityo neuronal tissue and evidence for the energetic stressn the brains of AD patients (Hoyer, 1991). Recenttudies of ‘‘at-risk’’ patients suggest that reduced glucosevailability may precede neuronal degenerationKennedy et al., 1995). Disruption of glucose utilization

es on endogenous pyruvate levels. Protective action of antioxidants.ated in the presence or in the absence of the antioxidants vitamin E

spectrophotometrically. Data are means 6 SEM values of triplicateent compared to control conditions, in the absence ofAb25–35 or Ab1–40red to conditions, in the absence of the antioxidants.

215

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ould contribute to the metabolic deficit in AD, and ourxperimental data suggest a role for Ab in such arocess.


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The inhibition of Ab-induced toxicity was previ-usly shown to be prevented by several antioxidantsBehl et al., 1992, 1997; Pappolla et al., 1997), suggestinghe involvement of oxidative stress on Ab-inducedoxicity. In the present work it was demonstrated thatb25–35 and Ab1–40 peptides increase ROS accumula-

ion in a dose-dependent manner, the increase in ROSroduction being well correlated with the decrease inell survival and the depletion of ATP levels (Figs. 1nd 2). This suggests that oxidative stress is respon-ible for the energetic compromise and toxicity onC12 cells after Ab exposure. This hypothesis wasonfirmed by the protective effect of several antioxi-ants against Ab-induced depletion of pyruvate levels

Fig. 3) and inhibition of the mitochondrial complexes, II–III, and IV (Fig. 4). It has been clearly demon-trated that mitochondrial dysfunction occurs underxidative stress conditions due to the increased ROSeneration, supporting our conclusion that ROS are

nvolved in the impairment of mitochondrial functionnd also in glycolytic dysfunction (Imberti et al., 1993;enzi & Moretti, 1995; Bolanos et al., 1996).Several reports argue in favor of the generation of

eactive oxygen species induced by Ab fragmentsBehl et al., 1994; Hensley et al., 1994; Butterfield et al.,994), which seem to be responsible for the inductionf lipid peroxidation in cell membranes (Mattson et al.,992; Behl et al., 1994; Butterfield et al., 1994; Goodman

Mattson, 1994, 1996; Mark et al., 1995; Keller et al.,997a). Free radical-induced lipid peroxidation leadso the formation of several aldehydic products, such as

NE (Esterbauer et al., 1991). Recently, it has beenhown that Ab induces a large increase in the levels of-hydroxynonenal, which by itself is capable of induc-ng several of the oxidative stress responses previouslyemonstrated for Ab (Blanc et al., 1997, 1998; Keller etl., 1997a,b; Mark et al., 1997; Kruman et al., 1997).ecause glutathione plays a major role in cellularetabolism/detoxification of HNE (Hartley et al., 1995),

nder conditions of GSH depletion, it would be prob-ble that HNE levels would be increased. Under ourxperimental conditions, following Ab exposure, we

IG. 4. Protective action of antioxidants against inhibition of the actuccinate–cytochrome c reductase (B), and cytochrome c oxidase (Cnzymatic activity of NADH-ubiquinone oxidoreductase (complex I)I–III), and cytochrome c oxidase (complex IV) of control and Ab-trea

(10 µM), idebenone (1 µM), or GSH ethyl ester (1 mM), was meas

16

ercentage of control values, are means 6 SEM values of triplicate determiignificantly different compared to control conditions, in the absence of Aignificantly different compared to conditions, in the absence of the antioxid


ave evidence supporting that, in PC12 cells, Abnduces lipid peroxidation and decreases GSH levelsdata not shown). Therefore, HNE could be respon-ible for the inhibitory effect of Ab fragments on thectivity of the mitochondrial respiratory chain com-lexes (Fig. 4), mediated by its conjugation and inacti-ation of the mitochondrial proteins (Subramaniam etl., 1997) and/or alteration of fluidity of the lipids ofhe mitochondrial inner membrane (Chen & Yu, 1994).urthermore, it has been demonstrated that HNEffects glycolysis, inactivating the rate-limiting en-ymes of the glycolytic pathway in the absence of GSHMiwa et al., 1997). Therefore, the depletion of GSHbserved under our experimental conditions can de-rease the protection of glycolytic enzymes from HNEnactivation, leading to pyruvate depletion (Fig. 3),

hich can then potentiate the ROS-mediated cellamage induced by Ab, because pyruvate has recentlyeen shown to act as a scavenger of hydrogen peroxideDesagher et al., 1997). The antioxidants idebenone1 µM), vitamin E (10 µM), and GSH ethyl ester (1 mM)ttenuate Ab-induced depletion of pyruvate levels andnhibition of mitochondrial respiratory chain com-lexes, suggesting that lipid peroxidation and HNEontribute to the energetic compromise observed afterb treatment (Figs. 3 and 4).Our data clearly show that Ab induces mitochon-

rial dysfunction and impairment of glycolysis, lead-ng to ATP depletion, by a mechanism involving theeneration of ROS, which may contribute to the patho-enesis of Alzheimer’s disease. Antioxidants like idebe-one, vitamin E, and GSH may play an important role

n pretreating neuronal cell death occurring in thiseurodegenerative disorder.

CKNOWLEDGMENTS

The present work was supported by JNICT (Portuguese Researchouncil) and the Human Capital Mobility Program (EU), ProposalRB 4050 PL 932039. C. Pereira is a recipient of Grant PRAXISXI/BD/3889/94. We wish to thank Dr. Teresa Proenca (University

NADH-ubiquinone oxidoreductase (A), succinate dehydrogenase 1

xic concentrations of Ab25–35 or Ab1–40 (10 nM and 1 µM). Theate dehydrogenase 1 succinate–cytochrome c reductase (complexes

12 cells, in the presence or in the absence of the antioxidants vitamins described under Experimental Procedures. Data, expressed as the


ivity of) by to

, succinted PCured a

nations of different experiments. *P , 0.05, **P , 0.01, ***P , 0.001,b25–35 or Ab1–40 peptides; 1P , 0.05, 11P , 0.01, 111P , 0.001,ants.

A
b-Induced Oxidative Stress and Metabolic Dysfunction 217

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ospitals, Coimbra, Portugal) for help in the determination ofyruvate and lactate levels.

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