Role of oxidative stress on β-amyloid neurotoxicity elicited during impairment of energy metabolism...

00 (2006) 496–508www.elsevier.com/locate/yexnr

Experimental Neurology 2

Role of oxidative stress on β-amyloid neurotoxicity elicitedduring impairment of energy metabolism in the hippocampus:

Protection by antioxidants

Teresa Montiel a, Ricardo Quiroz-Baez b, Lourdes Massieu a, Clorinda Arias b,⁎

a Departamento de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México AP 70-253, México DF 04510, Méxicob Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas,

Universidad Nacional Autónoma de México, México DF 04510, México

Received 8 July 2005; revised 30 January 2006; accepted 7 February 2006Available online 19 April 2006

Abstract

Age-associated oxidative stress has been implicated in neuronal damage linked with Alzheimer's disease (AD). In addition to the role of β-amyloid peptide (Aβ) in the pathogenesis of AD, reduced glucose oxidative metabolism and decreased mitochondrial activity have been suggestedas associated factors. However, the relationship between Aβ toxicity, metabolic impairment, and oxidative stress is far from being understood. Invivo neurotoxicity of Aβ25–35 peptide has been conflicting. However, in previous studies, we have shown that Aβ25–35 consistently inducessynaptic toxicity and neuronal death in the hippocampus in vivo, when administered during moderate glycolytic or mitochondrial inhibition. In thepresent study, we have investigated whether enhancement of Aβ neurotoxicity during these conditions involves oxidative stress. Results showincreased lipoperoxidation (LPO) when Aβ is administered in the hippocampus of rats previously treated with the glycolysis inhibitor,iodoacetate. Neuronal damage and LPO are efficiently prevented by vitamin E, while the spin trapper, α-phenyl-N-tert-butyl nitrone, shows partialprotection. Aβ stimulates LPO in synaptosomes, but toxicity is only observed in the presence of metabolic inhibitors. Damage and LPO areefficiently prevented by vitamin E. The present results suggest an interaction between oxidative stress and metabolic impairment in the Aβneurotoxic cascade.© 2006 Elsevier Inc. All rights reserved.

Keywords: Alzheimer's disease; Hippocampal damage; Lipoperoxidation; Metabolic inhibition; Neuroprotection; Synaptic protection; Vitamin E

Introduction

Increased brain accumulation of amyloid-β protein (Aβ)associated with Alzheimer's disease (AD) appears to be relatedto a gradual synaptic loss and neuronal death finally leading tocognitive impairment (Cotman et al., 1992; Hisiao et al., 1996;Holcomb et al., 1998; Hsia et al., 1999). Most Aβ toxicitystudies have used synthetic peptides Aβ1–40 or Aβ1–42,analogous to peptides found in neuritic plaques in AD patients.However, the 11-amino-acid fragment Aβ25–35 has beenfound to be the functional domain responsible for Aβneurotoxic properties (Yankner et al., 1990; Pike et al., 1993)and Aβ-induced functional disturbances in vivo (Maurice et al.,

⁎ Corresponding author. Fax: +55 56 22 38 97.E-mail address: [email protected] (C. Arias).

0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.expneurol.2006.02.126

1996; Stepanichev et al., 2003). Moreover, Aβ25–35 containsmethionine 35, which has been shown to play a critical role inthe pro-oxidant and neurotoxic properties exhibited by Aβ1–42(Butterfield and Bush, 2004).

In addition to the well-documented role of Aβ in AD,extensive data from patients and in vivo animal models indicatethat impaired brain metabolism is one of the cardinal andessentially most frequent factors associated with this neurode-generative dementia. In particular, reduced glucose oxidationand mitochondrial activity have been documented in ADpatients (Meier-Ruge and Bertoni-Freddari, 1996; Parker et al.,1994; Sadowski et al., 2004; Sims et al., 1987; Slosman et al.,2001; Tohgi et al., 1998). Although a connection betweenmetabolic impairment and the amyloid neurotoxic cascade hasnot been clearly established, increased free radical generationhas been associated with aging and appears to be involved in

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http://dx.doi.org/10.1016/j.expneurol.2006.02.126

497T. Montiel et al. / Experimental Neurology 200 (2006) 496–508

Aβ neurotoxicity (Lorenzo and Yankner, 1994; Shigenaga etal., 1994; Yatin et al., 1999). Likewise, a decline in the activityof mitochondrial complexes and ATP generation has beenobserved in the aged brain (Bowling et al., 1993; Shigenaga etal., 1994).

Several investigations have revealed the presence ofoxidation products of proteins, lipids, and DNA in postmortemtissue from AD patients, which is indicative of increasedoxidative stress (Butterfield and Boyd-Kimball, 2004; Butter-field and Lauderback, 2002; Smith et al., 1997; Subbarao et al.,1990). In addition, in vitro studies suggest that Aβ promotesoxidative stress and lipid peroxidation in synaptosomes andneuronal cultures (Butterfield et al., 1994; Mark et al., 1997;Keller et al., 1997; Yatin et al., 1999). Consistently, increasedlipoperoxidation has been observed in an animal model ofAlzheimer amyloidosis (Pratico et al., 2001). Altogether, thesestudies favor the suggestion that Aβ plays a central role in thepathogenesis of AD as a mediator of oxidative stress.

However, the induction of Aβ neurotoxicity in vivo after itsintracerebral administration has been intricate. While adminis-tration of Aβ peptides in the hippocampus has yieldedconflicting results (Podlisny et al., 1993; Winkler et al., 1994;Clemens and Stephenson, 1992), toxicity is clearly observedwhen a combination of high doses of Aβ peptides (Aβ1–40 andAβ1–43) is injected into the hippocampus, or when Aβ25–35is coinjected with excitotoxins or the pro-inflammatory factorTNFα (Morimoto et al., 1998; Stéphan et al., 2001; Stepanichevet al., 2003).

In a previous study, we have developed an in vivo model ofAβ neurotoxicity induced by the administration of Aβ25–35 inthe hippocampus of rats previously treated with iodoacetate, aninhibitor of the glycolytic enzyme, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), or with the mitochondrial toxin, 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinatedehydrogenase (Arias et al., 2002). We have consistentlyobserved that in energy-deficient animals, Aβ25–35 induces anextensive lesion in the dentate gyrus, while in energy-competentanimals, Aβ25–35 toxicity is erratic. Similarly, in isolatedsynaptic terminals from cortex and hippocampus, Aβ toxicity isenhanced when incubated in the presence of the metabolicinhibitors (Arias et al., 2002). These observations are consistentwith previous in vitro results (Copani et al., 1991).

In the present study, we have investigated whether increasedfree radical generation is involved in the enhancement of Aβneurotoxicity during energy impairment. Thus, we haveanalyzed the presence of lipoperoxidation (LPO) in thehippocampus in vivo and in isolated nerve terminals after Aβadministration, either in the presence or the absence ofmetabolic inhibitors. On other hand, although some evidencedemonstrates neuroprotective actions of several antioxidants invitro and in vivo models reproducing some features of Aβ-induced neurodegeneration, results are inconsistent (Koppal etal., 1998; Lockhart et al., 1994; Berman and Brodaty, 2004;Sung et al., 2004). Moreover, the precise role of LPO in Aβmediated toxicity has not been clearly established in in vivomodels of Aβ neurotoxicity nor in nerve terminals. Thus, in thepresent study, the neuroprotective potential of the antioxidant α-

tocopherol (vitamin E) was tested and compared to that of thefree radical scavenger α-phenyl-N-tert-butyl nitrone (PBN), in arat model of Aβ toxicity involving metabolic inhibition(Arias et al., 2002). Studies were performed both in therat hippocampus in vivo and in cortical and hippocampalsynaptosomes.

Experimental procedures

In vivo studies

Male Wistar rats (250–320 g) were used throughout thestudy. They were handled according to the Rules for Research inHealth Matters (Mexico), and the local Animal Care Committeeapproved all animal treatments. All efforts were made in orderto reduce the number of animals used for this study and avoidtheir suffering. For chronic glycolysis inhibition studies,animals received a daily intraperitoneal (i.p.) injection of15 mg/kg iodoacetate (Sigma, St. Louis MO, USA) dissolved in10 mM phosphate buffer during 3 days. On day 3, 4 h afterreceiving the last iodoacetate administration, they wereintrahippocampally injected in the dentate gyrus with Aβpeptide fragment 25–35 (1 μg/μl, Sigma, St. Louis MO, USA)solubilized in distilled water and left at room temperature during2 h in order to induce its aggregation and facilitate itsneurotoxicity. When the protective effect of PBN was tested,animals were identically treated with the exception that theyreceived a 100 mg/kg PBN i.p. injection 30 min before Aβ. Insome animals, two additional doses of 50 mg/kg PBN wereadministered immediately and 2 h after Aβ injection.

When the neuroprotective effect of vitamin E (Sigma, St.Louis MO, USA) was tested a group of animals received a dailyi.p. injection of 100 mg/kg during 7 days. At day 5, animalswere treated with iodoacetate during the following 3 days. Onday 7, vitamin E was administered 1 h after Aβ intrahippo-campal injection. When 200 mg/kg vitamin E was tested,animals received 2 daily injections (every 8–12 h) during 7 daysand treated identically as described above. Animals weresacrificed 24 h after Aβ injection and prepared for histologicalanalysis (see below).

For mitochondrial inhibition studies, animals received onedaily i.p. injection of 3-NP (15 mg/kg, Sigma, St. Louis MO,USA) during 2 days. 3-NP was dissolved in 10 mM phosphatebuffer, pH adjusted to 7.0–7.5 with NaOH 1M, and 4 h after thesecond administration, they received an intrahippocampalinjection of Aβ fragment 25–35 (1 μg/μl) in the dentategyrus. PBN and vitamin E treatments were identical to thosedescribed for iodoacetate. Twenty-four hours after the intra-hippocampal injection, animals were anesthetized, transcar-dially perfused, and brains were prepared for histologicalanalysis as described below. As a control for the specificity ofAβ25–35 neurotoxicity, one group of 3-NP- and one ofiodoacetate-treated animals received an intrahippocampaladministration of the scrambled sequence of Aβ25–35 peptide(NH2-IMLKGNGASIG-COOH, Macromolecular AnalysisLab., Albert Einstein College of Medicine, Bronx, N.Y.). Thepeptide with the Aβ scrambled sequence was solubilized in the

498 T. Montiel et al. / Experimental Neurology 200 (2006) 496–508

same vehicle solution as Aβ25–35 and left at room temperatureduring 2 h before its intrahippocampal administration.

For intrahippocampal injections, animals were anesthetizedwith 2% halothane in a 95% O2/5% CO2 mixture and placed ona stereotaxic frame as previously described (Arias et al., 2002).Coordinates used were AP −3.6 mm from bregma, L 2.0 frommidline, and V 2.7 mm from dura according to Paxinos andWatson (1986). A 1 μl volume was unilaterally injected at a rateof 0.5 μl/min, and 2 min later, the needle was withdrawn and theskin sutured.

Histology and determination of lesion volume

Twenty-four hours after the surgery, rats were anesthetizedwith sodium pentobarbital and transcardially perfused with200 ml 0.9% saline followed by 200 ml 3.7% formaldehyde in0.1 M phosphate buffer (pH 7.3). Brains were left in fixative for24 h and transferred successively to 20% and 30% sucrose (24 heach). 40-μm coronal sections were cut in a cryostat and stainedwith cresyl violet. Lesion size was calculated by examination ofall brain sections where neuronal damage was evident, in eachone of the experimental animals. Damaged area in each tissuesection was manually delineated and measured with the aid ofan image analyzer (NIH Macintosh image 1.6). The lesionvolume was calculated as previously described (Arias et al.,2002). Results are expressed as means ± SEM of lesion volumeper each animal group.

Succinate dehydrogenase activity

SDH activity was measured histochemically in tissuesections incubated in the presence of succinate, used as theenzyme substrate, and the electron acceptor Nitro BlueTetrazolium (NBT). The optical density of the dark-blue stainproduced by the formazan product was measured as previouslyreported (Brouillet et al., 1998; Massieu et al., 2001). Tomeasure enzyme activity, 3-NP-treated animals were sacrificed4 h after the administration of the toxin, the control animalswere treated with phosphate buffer 10 mM. All animals weretranscardially perfused with 100 ml cold 0.9% saline followedby 100 ml phosphate-buffered saline (PBS) containing 10%glycerol. Brains were removed, frozen in isopentane, andimmediately sectioned at 16 μM. Brain sections were mountedon slides and kept frozen for 2 days at −20°C. Five tissuesections were used for SDH determination. They wereincubated in PBS at 37°C during 10 min, rinsed with PBS,and incubated with 80–100 μl of a mixture containing 0.3 mMNBT, 0.05 M phosphate buffer pH 7.6, and 0.05 M sodiumsuccinate, added at 4°C. The reaction was started by transferringthe slides to a humidified chamber for 30 min at 37°C in thedark. Sections were rinsed with PBS during 5 min, immersed indistilled water at room temperature for a few seconds, and keptat room temperature until next day when the optical density ofthe dark-blue stain formazan was quantified with the aid of animage analyzer (NIH Macintosh Image, 1.6) as describedpreviously (Massieu et al., 2001). Some sections were handledin parallel and incubated without succinate to determine non-

specific staining. These sections were completely transparent.Data are expressed as percent of control values.

Glyceraldehyde 3-phosphate dehydrogenase activity

Rats were treated either with vehicle (phosphate buffer10 mM) or iodoacetate (15 mg/kg/day) during 3 days. Fourhours after the end of the treatment, animals were sacrificed bydecapitation and the hippocampus extracted. GAPDH activitywas determined as previously reported with minor modifica-tions (Ikemoto et al., 2003). Tissue was homogenized in 1:10(weight/vol) Tris–HCl 0.1 mM pH 8.5 and GAPDH activitywas monitored in a reaction mixture (1 ml total vol) containing(in mM) 1.7 sodium arsenate, 20 sodium fluoride, 1.0 NAD+

and 5 KH2PO4. The reaction was initiated by the addition ofglyceraldehyde 3-phosphate (final concentration 1 mM).GAPDH activity was determined by monitoring the amountof NADH formed and detected at 340 nm during the first 30 swhen the reaction is linear. A molar extinction coefficient of2.07 was used to calculate the amount NADH formed inmicromole. Data are expressed as μmol NADH/min/mg protein.Specific activity is expressed as mean ± SEM.

Determination of ATP in brain homogenates

ATP was determined in tissue homogenates from hippocam-pus using the luciferin–luciferase luminometric assay (Molec-ular Probes, Eugene, OR, USA). The reaction was monitored ina Bio-Orbit Luminometer (model 1251 Labsystems, Helsinki,Finland) as previously described (Massieu et al., 2003a).Animals treated with 3-NP or iodoacetate were anesthetizedwith halothane 4% 4 h after the treatment and placed on astereotaxic apparatus, the skull was exposed to liquid nitrogendirectly, the animal stopped breathing (3–5 min), indicating thatthe medulla was frozen (Delaney and Geiger, 1996). Brainswere removed, placed in liquid nitrogen for 10 min and stored at−70°C. After dissection of the hippocampus on dry ice, tissuewas homogenized in 0.8 M perchloric acid (10 μl/mg wetweight), and centrifuged (10,000 rpm for 10 min at 4°C).Supernatants were recovered and neutralized with 2 M K2CO3

(9% of total volume) and recentrifuged (Matthews et al., 1997).Samples were kept at −20°C and ATP determined 24 h later.Supernatants were eight-fold diluted in distilled water, and a200 μl aliquot was placed in polyethylene tubes in theluminometer. The apparatus injected 300 μl of luciferin–luciferase mixture, and ATP was measured during 20 s. Themaximum peak of the reaction was determined (usually 5 s afterthe reaction is initiated). ATP concentrations were calculatedfrom an ATP standard curve (from 6.5–125 pmol). Data areexpressed as pmol/μg of frozen tissue weight.

Preparation of synaptosomes from hippocampus and cerebralcortex

Male Wistar rats (220–250 g) were decapitated, and apurified synaptosomal fraction was obtained by the proceduredescribed by Loscher et al. (1985), slightly modified. In brief,

Table 1Activities of GADPH and SDH, ATP levels and blood glucose concentration incontrol and treated animals with the metabolic inhibitors

Control Iodoacetate 3-NP

GAPDH activity (μmol ofNADH/min/mg protein)

0.128 ± 0.02 0.061 ± 0.01⁎ –

SDH activity (percent ofcontrol)

100.0 ± 6.71 – 43.43 ± 6.29⁎

ATP (pmol/μg wet weight) 2.810 ± 0.26 2.330 ± 0.42 2.790 ± 0.61

Glucose (mg/dl)Before the first injection 113.00 ± 3.93 111.00 ± 4.82 124.60 ± 8.69at 30 min 117.25 ± 7.18 128.80 ± 3.27 120.80 ± 7.57Before the last injection 107.25 ± 3.63 105.20 ± 7.05 106.20 ± 4.04at 30 min 114.75 ± 5.34 113.20 ± 6.50 112.60 ± 5.12

Value represent mean of 3–5 data ± SEM.GADPH and SDH activities and ATP levels were determined in thehippocampus of rats sacrificed 4 h after the treatment with iodoacetate or 3-NP. Blood glucose levels were determined 30 min before and 30 min after thefirst and the last administration of the corresponding metabolic inhibitor.⁎ P < 0.05 relative of control.


neocortex and hippocampus from both hemispheres weredissected on ice, homogenized in a solution containing0.32 M sucrose and 5 mM HEPES, pH 7, and centrifuged at3500 rpm for 10 min (4°C). The supernatant was layered onto1 ml of 1.2 M sucrose and centrifuged at 50,000 rpm (4°C) for30 min. The gradient interface was carefully collected anddiluted with 0.32 M sucrose to a final volume of 2 ml. Thediluted suspension was then layered onto 1 ml 0.8 M sucroseand centrifuged for 30 min at 50,000 rpm. This yielded asynaptosomal pellet, which was resuspended in 2 ml of aLocke's solution, containing (in mM) 154 NaCl, 5.6 KCl, 2.3CaCl2, 1.0 MgCl2, 3.6 NaHCO3, 5.0 glucose, and 5.0 HEPES,pH 7.2. Aliquots of 200 μl containing 50 μg synaptosomalprotein were incubated during 3 h at 37°C with Aβ peptidefragment 25–35 (50 μM) in the presence or absence of either 3-NP (10 μM) or iodoacetate (5 μM). In some experiments,250 μM vitamin E or 10 μM PBN was coincubated for 3 h withthe aforementioned drugs. Higher doses or PBN (25 and 50 μM)were toxic to synaptosomes.

Measurement of LPO by the TBARS assay in brainhomogenates

As an indicator of the levels of lipid peroxidation, theproduction of malondialdehyde (MDA) was assayed asthiobarbituric acid reactive substances (TBARS) in tissuehomogenates (Gluck et al., 2000). According to Esterbauerand Cheeseman (1990), this method is greatly specific for freeMDA. Animals were sacrificed by decapitation 24 h after thedifferent treatments (with the exception of 3-NP-treatedrecumbent animals, which were sacrificed 2–3 h afterrecumbency); the hippocampus was dissected and homogenizedin 10 vol (wt/vol) of a solution containing KCl 1.15%/0.4 mMsodium azide. Three to four mg protein/ml was used for theassay. Samples were incubated during 15 min at 37°C, and thesame volume of trichloroacetic acid (20%) was added. Sampleswere centrifuged at 14,000 × g during 10 min at 4°C. Thesupernatant was mixed with the same volume of 0.8%thiobarbituric acid (Merck, Darmstadt, Germany) and incubatedduring 20 min at 50°C. Optical density was read at 532 nm in aspectrophotometer (Beckman, DU 640, USA). Malonaldehydebis-dimethyl acetal was used as a standard. Protein content wasdetermined by the Bradford (1976)'s method, and data areexpressed as nmol TBARS/mg protein.

In the animals used for LPO determination, the contralateralhippocampus not injected with Aβ was used to determine theeffect of the individual treatments with the metabolic inhibitorson LPO.

Evaluation of mitochondrial redox capacity in synaptosomes

The method employed in the present study is similar to thatcommonly used in cultured cells studies in order to evaluatemitochondrial redox activity through the conversion of MTTtetrazolium salt to formazan crystals by mitochondrial respira-tory chain reactions (Mossman, 1983). Briefly, MTT (Sigma,St. Louis MO) was dissolved in PBS at a concentration of 5 mg/

ml and added to synaptosomes (10-fold diluted in the incubationmedium) after 3-h incubation with the different treatments andallowed to incubate for 1 more h. Then synaptosomes werecentrifuged and the pellet solubilized with isopropanol (0.8 ml).The absorbance of each sample was quantified at 570 nm usinga spectrophotometer (Pharmacia Biotech, USA).

Measurement of LPO by the TBARS assay in synaptosomes

The formation of TBARS in synaptosomes was measuredaccording to a method described previously (Rios andSantamaria, 1991). Synaptosomal fraction was preincubatedduring 3 h with Aβ peptide fragment 25–35 (50 μM) in thepresence or absence of either 3-NP (10 μM) or iodoacetate(5 μM). Afterwards, it was mixed with 2 vol TBA reagent(containing 0.375 g TBA, 15 g trichloroacetic acid and 2.5 mlHCl in 100 ml H2O), and final solutions were heated in a boilingwater bath for 30 min and centrifuged at 3000×g for 15 min.Optical density was then measured in supernatants at 532 nm ina spectrophotometer (Pharmacia Biotech, USA). Results areexpressed as percent of TBARS production compared tocontrol.

Statistics

Data were analyzed by one-way ANOVA followed by aFisher's post hoc test for multiple comparisons.

Results

Hippocampal damage after Aβ injection in rats treated withmetabolic toxins. Protective effect of vitamin E

The effect of iodoacetate on brain GADPH activity and ATPlevels was monitored 4 h after the last injection of the inhibitor.According to the results, enzyme activity was decreased in52.3% relative to control animals; however, ATP levels were


only slightly reduced (Table 1). Blood glucose concentrationwas monitored daily 30 min before and 30 min after iodoacetateinjection. No change in glucose levels was detected (Table 1).

Fig. 1. Micrographs of representative animals showing the protective effect of vitaminrats chronically treated with iodoacetate. Rats were treated with iodoacetate (15 mg/kgdentate gyrus on day 3. Some animals received a single i.p (100 mg/kg) injection of P(100 or 200 mg/kg/day i.p) during 7 days. (A) Micrograph of a representative animarepresentative animal injected with the scrambled sequence of Aβ25–35 peptide in anAβ injection in a iodoacetate-treated rat receiving an injection of PBN; (E) Aβbar = 200 μm. (F) Graph shows lesion volume (mm3) in the hippocampus of animalaP < 0.05 relative to Aβ; bP < 0.05 relative to iodoacetate + Aβ.

These results suggest that systemic iodoacetate treatment at theconcentrations used does not induce evident systemic effectsand agree with previous data showing no alteration in blood

E and PBN on neuronal damage induced by Aβ peptide, in the hippocampus in) during 3 days and intrahippocampally injected with Aβ peptide (1 μg/μl) in theBN 30 min before Aβ administration, and some were pretreated with vitamin El injected with Aβ peptide in a control non-treated animal; (B) micrograph of aiodoacetate-treated animal; (C) iodoacetate-treated animal injected with Aβ; (D)injection in a iodoacetate-treated rat receiving vitamin E (100 mg/kg). Scales exposed to the different treatments. Means ± SEM of 5–8 animals per group.


pressure and cardiac metabolism after iodoacetate administra-tion (Liang, 1977). Moreover, no brain damage is inducedduring this administration protocol as evidenced by thehistological analysis. From these data, we can conclude that atthe time Aβ peptide is injected, hippocampal glycolyticmetabolism is moderately impaired. As previously reported(Arias et al., 2002), Aβ neurotoxicity in the hippocampus is 5.6-fold enhanced after chronic glycolysis inhibition with iodoace-tate (Fig. 1F).

In rats chronically treated with vitamin E (100 mg/kg/day),neuronal damage induced by Aβ was prevented, and the lesionvolume significantly reduced to 35% (Fig. 1F). The protectiveeffect of vitamin E was improved when 200 mg/kg/day wasadministered resulting in a lesion volume very similar to thatinduced by the administration of Aβ or the scrambled peptide inintact rats (Fig. 1F). However, no statistical difference wasfound between the different doses of vitamin E. A singleadministration of the spin trapper PBN before Aβ alsosignificantly reduced the lesion volume to 53%. Figs. 1A–Eshow micrographs of tissues from representative animalsexposed to the different treatments. As can be observed, Aβ

Fig. 2. Micrographs of representative animals showing the protective effect of vitaminchronically treated with 3-NP. Animals received two i.p. injections of 3-NP (15 mg/kgwith Aβ25–35 (1 μg/μl) in the dentate gyrus. Some animals were pretreated with vitatreated animal injected with the Aβ scrambled peptide; (B) representative animal tranimal injected with Aβ peptide receiving vitamin E. Scale bar = 200 μm. Graph shotreatments. Means ± SEM of 5–10 animals per group. aP < 0.05 relative to Aβ; bP

administration induced a lesion extending mainly along thedorsal blade of the dentate gyrus characterized by pyknosis ofthe granular cell layer. In animals treated with vitamin E, theextension of the lesion was substantially reduced, and in someanimals, it was limited to the sites adjacent to the needle tract(Figs. 1C and E).

The effect of 3-NP on the activity of succinate dehydroge-nase was studied 4 h after the second administration of the toxinin the hippocampus. A 56% inhibition of enzyme activity wasfound in this region (Table 1). According to a previous study,enzyme activity tends to recover 24 h after 3-NP administration,but it is still significantly inhibited by 20% at this time (Massieuet al., 2001). In animals treated with 3-NP, ATP levels were notreduced 4 h after the last administration of the toxin (Table 1). 3-NP at the doses used induced no changes in blood glucose(Table 1), suggesting a lack of evident systemic effects with theadministration protocol employed. This is in agreement withprevious studies demonstrating systemic changes (such ashypotension and protein extravasations) only when animalsbecome recumbent (Hamilton and Gould, 1987a). Histologicalanalysis revealed no brain damage in the present experimental

E on neuronal damage induced by Aβ peptide in the hippocampus in vivo in rats), and 4 h after the second administration, they were intrahippocampally injectedmin E during 7 days (100 mg/kg/day). (A) Micrograph of a representative 3-NP-eated with 3-NP and injected with Aβ peptide; (C) representative 3-NP-treatedws lesion volume (mm3) in the hippocampus of animals exposed to the different< 0.05 relative to Aβ in the presence of 3-NP + Aβ.

Fig. 3. Effect of Aβ intrahippocampal administration in energy deficient animals(treated with iodoacetate or 3-NP) on lipoperoxidation (LPO), as assessed by theTBARS method 24 h after the injection. (A) LPO was enhanced by Aβ in thehippocampus of rats previously treated with iodoacetate, and this effect wasprevented by vitamin E but not by PBN. Treatment with vitamin E and PBN wasas described in legend to Fig. 1. (B) LPO was not enhanced in the hippocampusof rats treated with 3-NP and injected intrahippocampally with Aβ. LPO is onlyinduced in recumbent rats severely altered by 3-NP. Means ± SEM of 6–9animals in A and 4–6 animals in panel B. aP < 0.05 relative to Aβ; bP < 0.05relative to iodoacetate + Aβ, cP < 0.05 relative to systemic iodoacetate only.


conditions. However, 25–30% of 3-NP-treated animals becomerecumbent after the second administration adopting a severedystonic lateral or ventral position (Hamilton and Gould,1987b). These animals show 85% decrease in enzyme activityand 55% reduction in ATP levels 2–4 h after the onset ofrecumbency and a bilateral lesion in the striatum (Massieu et al.,2003b). Animals showing recumbency were used only for LPOdetermination (see below) and were not injected with Aβ. Fromthese results, we can conclude that at the time Aβ is injectedmitochondrial metabolism is partially impaired. As reportedpreviously (Arias et al., 2002), treatment with 3-NP exacerbatedAβ neurotoxicity in the hippocampus inducing lesions 3.8-foldlarger (Fig. 2D).

Vitamin E (100 mg/kg/day) administration during 7 dayspartially prevented Aβ toxicity and significantly reduced lesionvolume to 49% (Fig. 2D). In Fig. 2, we show micrographsfrom representative animals exposed to the different treat-ments. As can be observed, no damage was induced in animalspretreated with 3-NP and intrahippocampally injected with thescrambled Aβ25–35 peptide (Fig. 2A). In contrast, animalsinjected with Aβ showed a lesion extending along the dorsalblade of the dentate gyrus (Fig. 2B). The extension of thelesion was partially reduced in vitamin E-treated animals(Fig. 2C).

Hippocampal increase of LPO after Aβ injection duringmetabolic inhibition. Effect of vitamin E and PBN

In order to assess if oxidative stress is related to Aβ-inducedneuronal damage in animals previously treated with iodoace-tate or 3-NP, the metabolic product of lipid peroxidation, MDAwas quantified in the hippocampus by the TBARS assay, 24 hafter Aβ administration. Aβ did not augment the content ofperoxide lipids 24 h after its administration in control animals(Fig. 3A). In contrast, in iodoacetate-treated animals Aβadministration induced a significant increase in LPO. Iodoa-cetate treatment alone did not change the levels of LPO in thehippocampus. Treatment with the spin trapper, PBN, failed toreduce LPO induced by Aβ in iodoacetate-treated animals andhad no effect on LPO in the contralateral hippocampus (Fig.3A). In animals receiving two additional injections of PBN(50 mg/kg), Aβ-induced LPO was not reduced (not shown). Incontrast to PBN, vitamin E efficiently reduced the effect of Aβon LPO in iodoacetate-treated animals and partially reducedLPO in the hippocampus of animals treated with iodoacetatealone (Fig. 3A).

LPO was not enhanced in the hippocampus (Fig. 3B),striatum, or motor cortex (data not shown) of animalsreceiving two injections of 3-NP. Only those rats that becamerecumbent showed a high level of LPO in the hippocampus(Fig. 3B). Similarly, the striatum and motor cortex showedincreased LPO 2–3 h after the onset of recumbency(control = 0.124 ± 0.025, recumbent = 0.233 ± 0.046 forcortex n = 6, control = 0.133 ± 0.020, recumbent = 0.410 ± 0.043for striatum n = 5). Aβ administration in the hippocampus of3-NP-treated animals did not enhance LPO as evaluated 24 hlater (Fig. 3B). Since no increase in the content of peroxide

lipids was observed in 3-NP-treated animals, the effect ofvitamin E or PBN on this parameter was not studied.

Mitochondrial redox function and LPO in synaptosomes afterAβ administration in the presence of metabolic toxins.Protection by vitamin E

To assess the protective effects of PBN (10 μM) and vitaminE (250 μM) against Aβ-induced toxicity in the presence ofenergy impairment, cortical synaptosomes were incubated withAβ25–35 plus 3-NP (10 μM) or iodoacetate (5 μM) during 3 h.As shown in Fig. 4A, a significant decrease in MTT reductionwas observed when Aβ25–35 was coincubated with 3-NP oriodoacetate (30 and 40%, respectively). Cotreatment withPBN did not restore MTT reduction, contrary to vitamin E,which completely protected cortical synaptosomes from Aβtoxicity (139 and 115% of control for 3-NP and iodoacetate,respectively).

Furthermore, vitamin E improved mitochondrial redox acti-vity in cortical synaptosomes exposed to individual treatments

Fig. 4. Effect of vitamin E and PBN on mitochondrial activity of neocortical (A) and hippocampal (B) synaptosomes exposed during 3 h to Aβ peptide 25–35 (50 μM)in the presence or absence of 3-NP (10 μM) or iodoacetate (5 μM). The protective effects of 250 μM vitamin E and 10 μM PBN were analyzed in each condition. Dataare expressed as the percentage of control values and represent mean ± SEM of 6–8 independent determinations performed in duplicate. aP < 0.05 relative to Aβ;bP < 0.05 relative to synaptosomes incubated with Aβ in the presence of metabolic inhibitors; cP < 0.05 relative to metabolic inhibitors without Aβ.


with 3-NP or iodoacetate, to levels even higher than thoseobserved in controls (Fig. 4A). Therefore, vitamin E preventedthe normal decline of mitochondrial function in controlsynaptosomes increasing by almost 70% the MTT reductioncapacity, relative to naive synaptosomes after 3 h of incubationin normal medium (data not shown).

The effect of PBN or vitamin E on Aβ25–35-inducedtoxicity in hippocampal synaptosomes is shown in Fig. 4B.Similar results were obtained although the toxic effect of Aβ inthe presence of 3-NP and iodoacetate was slightly larger (35 and45% respectively). While no protection of PBN was observed,vitamin E totally reversed the decline in MTT reduction induced

by Aβ25–35 in the presence of 3-NP or iodoacetate (185 and157% of control, respectively).

These results are in agreement with those of LPOexperiments. Fig. 5A shows the percent increase in TBARSproduction induced after 3-h incubation with Aβ-25–35, in thepresence of 3-NP or iodoacetate in cortical synaptosomes, aswell as the effect of vitamin E. While exposure to Aβ alone or incombination with 3-NP or iodoacetate increases TBARS up to27.2, 9.6 and 18.7%, respectively. The coincubation withvitamin E completely prevented the Aβ-induced increase inLPO, in the presence or absence of metabolic toxins. Similarresults were obtained for hippocampal synaptosomes. Aβ

Fig. 5. Effect of vitamin E on LPO as assessed by the TBARS method in cortical (A) and hippocampal synaptosomes (B) exposed during 3 h to Aβ in the presence orabsence of iodoacetate or 3-NP. LPO was enhanced in synaptosomes exposed to Aβ, and no further increase was observed by incubation with metabolic inhibitors. Inall conditions, vitamin E reduced LPO under control values. Data are expressed as the percentage of control determinations and represent mean ± SEM of 5determinations in triplicate; aP < 0.05 relative to Aβ; bP < 0.05 compared to synaptosomes incubated with Aβ in the presence of metabolic inhibitors; cP < 0.05 relativeto metabolic inhibitors without Aβ.


induced an increase of 17 and 19% in LPO levels whencoexposed in the presence of iodoacetate and 3-NP, respective-ly. When vitamin E was added, LPO levels were decreased evenbelow control values (Fig. 5B).

Discussion

We have previously reported that metabolic inhibitionpromotes Aβ toxicity in the rat hippocampus as well as incortical and hippocampal synaptosomes (Arias et al., 2002).Here, we report that enhanced Aβ toxicity might involveoxidative stress because augmented LPO was found, particu-larly when Aβ was administered during glycolysis inhibition.Synaptosomal exposure to Aβ peptide induced a significantenhancement of LPO in the hippocampal and neocorticalfractions, as described previously (Butterfield et al., 1994).However, the present results indicate that this increase in LPO isnot sufficient to reduce mitochondrial function in energycompetent synaptosomes. Noteworthy, Aβ toxicity, as assessedby decreased mitochondrial redox capacity, was only observedwhen it was coincubated with the metabolic toxins. This findingfavors the role of synaptic terminals as targets of Aβ toxicity(Coleman et al., 2004; DeKosky et al., 1996; Mungarro-Menchaca et al., 2002) and supports our previous observationssuggesting that metabolic impairment is a condition thatenhances vulnerability of nerve endings to the damaging effectsof Aβ. The metabolic toxin iodoacetate seems to increase morepotently synaptosomal vulnerability to Aβ peptide toxicity

compared to 3-NP. These results are in accordance withprevious data indicating that synaptosomes use glucose as themain energy source, and that iodoacetate effectively reducesATP levels in this preparation (Kauppinen and Nicholls, 1986).

In contrast to the synaptosomal preparation, acute adminis-tration of Aβ peptide into the hippocampus in vivo did not altersignificantly the levels of peroxide lipids, consistent with thelack of neurotoxicity of this peptide when administered into thebrain of intact rats (Podlisny et al., 1993; Stein-Behrens et al.,1992). Animals treated with iodoacetate or 3-NP alone did notshow a significant increase in LPO relative to control animals,suggesting that mild inhibition of energy metabolism by itselfdoes not induce oxidative stress in the present conditions.Increased LPO was only observed in the hippocampus ofrecumbent rats, which show severe impairment of mitochon-drial metabolism. This result agrees with previous in vivostudies suggesting the induction of oxidative stress during acute3-NP treatments, which correlates with the presence of neuronaldamage (Kim and Chan, 2002; Kim et al., 2000). Therefore, thelack of a significant increase in LPO levels in non-recumbentanimals treated with 3-NP might reflect the lack of severemitochondrial impairment. However, in these conditions,vitamin E reduced significantly the extension of the lesionsinduced by Aβ in the hippocampus, suggesting the participationof oxidative stress in Aβ-neurotoxic cascade. Additional studieswould help to elucidate whether oxidative stress is present inthese conditions. Alternatively, protection by vitamin E in 3-NP-treated animals might be related to the improvement of


mitochondrial activity as observed in synaptosomes, or to otheractions of vitamin E, additional to its antioxidant properties(Parks and Traber, 2000).

Similarly to 3-NP, chronic treatment with iodoacetate did notenhance LPO levels in the hippocampus. A previous studyshowed the generation of OH• radical after the acuteadministration of iodoacetate in the striatum (Matthews et al.,1997). Similarly, data from our laboratory indicate enhancedLPO after the intrastriatal administration of iodoacetate(unpublished results). This suggests that oxidative stress beinduced after the administration of high doses of iodoacetatedirectly into the brain. In contrast to 3-NP, Aβ injection in thehippocampus of iodoacetate-treated animals enhanced LPO.The reasons why iodoacetate potentiates more effectively than3-NP Aβ-induced LPO are not completely understood, but itmight be related to the important reduction of brain glucoseoxidation, and the subsequent lack of pyruvate production tomaintain mitochondrial metabolism. In fact, we have previouslydemonstrated that the administration of pyruvate effectivelyprevents lesions induced by Aβ in the hippocampus ofiodoacetate-treated animals (Arias et al., 2002).

A sustained inhibition of glycolytic metabolism might impaircalcium homeostasis due to failure in ATP-dependent calcium-loading and calcium-extruding mechanisms, favoring theproduction of reactive oxygen species. A recent study inhippocampal cultured astrocytes showed that glycolysis ratherthan mitochondrial metabolism is the main source of energy forintracellular calcium storage (Kahlert and Reiser, 2000).Consistent with this hypothesis, we have recently reportedthat neuronal death induced by a relatively low concentration ofiodoacetate (50 μM) in hippocampal cultured neurons is relatedto disturbances in intracellular calcium storage due to thedecline in ATP levels (Hernández-Fonseca and Massieu, 2005).On the other hand, during 3-NP treatment, mitochondrialmetabolism is only partially impaired and pyruvate would bestill oxidized to some extent in mitochondria. In vitro studieshave shown that 3-NP induces an increase in intracellularcalcium at millimolar concentrations (Lee et al., 2002), and thatmitochondrial inhibitors have only minor effects on endoplas-mic reticulum calcium storage, while glycolysis inhibition leadsto the immediate depletion of calcium stores from this organelle(Kahlert and Reiser, 2000). Therefore, the larger effect of Aβ onLPO in iodoacetate-treated animal might be related to thedisruption of the ionic gradients and the intracellular homeo-stasis of calcium. In fact, previous studies suggest that anincrease in the intracellular concentration of calcium mightparticipate in Aβ neurotoxic cascade (Mattson, 1994).

Altogether these results allow us to conclude that severalfactors may contribute to oxidative stress and neurotoxicity afterexposure of neurons to Aβ, including decreases in energyavailability, calcium dyshomeostasis and mitochondrial dys-function, as has been suggested (Behl, 1999).

Vitamin E showed a prominent protective effect against Aβneurotoxicity both in vivo and in vitro. Moreover, decreasedlevels of LPO were demonstrated in rats treated withiodoacetate and vitamin E, as well as in synaptosomalpreparations exposed to both metabolic inhibitors. These

findings support the hypothesis that oxidative stress plays afunctional role in the neurotoxicity elicited by Aβ during mildenergy impairment, and that isolated nerve terminals could bevulnerable targets for oxidative stress in AD.

It has been suggested that age-associated increases inoxidative stress may play a role in AD (Behl, 1999; Meccocciet al., 1994), and thus, antioxidants may also have a place in thetherapy for this disease. Vitamin E is the most effective lipid-soluble antioxidant present in mammalian cells. Many studieshave conclusively demonstrated that its major function is toprotect tissue against oxidative damage, although otherbiological properties have also been described (Parks andTraber, 2000). Although numerous in vitro studies, as well assome in vivo investigations in transgenic mice have shown thepotentiality of antioxidants, including vitamin E, in theprevention of Aβ neurotoxicity and LPO levels (Pratico et al.,2001; Sung et al., 2004; Frautschy et al., 2001), human studieshave yielded limited results (Laurin et al., 2004). However,other studies showed that the use of vitamin E was associatedwith reduced prevalence and incidence of AD (Sano et al.,1997; Zandi et al., 2004). In this context, the present studyprovides more evidence about the potential benefits of vitaminE supplementation to protect both hippocampal neurons in vivoand isolated nerve endings. Protection of nerve terminals isparticularly important because it has been reported thatsynapses may be sites where the neurodegenerative cascadeinitiates in AD (Coleman et al., 2004; Floyd, 1999; Mungarro-Menchaca et al., 2002). Therefore, synaptic endings mightrepresent important therapeutic targets for protective treatmentsto slow AD progression and preserve cognitive abilities. Thepresent results are in agreement with a previous study showingsome benefit of α-tocopherol against learning and memorydeficits induced by Aβ1–42 peptide in rats (Yamada et al.,1999).

Vitamin E treatment will be probably more effective whenadministered at a critical early time point before the onset ofclinical symptoms of AD, as it is suggested by a previous studyin transgenic animals (Sung et al., 2004) and the present study,where vitamin E pretreatment during 7 days or its coadminis-tration with Aβ, inhibits the progression of the neurodegener-ative cascade initiated or propagated by oxidative stress. Thisconclusion is supported by the fact that in AD patients withmoderately cognitive impairment, treatment with vitamin Eslows the progression of the disease (Sano et al., 1997).Moreover, the present study shows that vitamin E reduced LPOeven to levels below control values both in synaptosomes and inthe hippocampus in vivo and improved mitochondrial redoxcapacity, suggesting its potentiality in reducing age-associatedalterations in brain oxidative state. Hence, the beneficial effectsof vitamin E could be accounted by the prevention of membranelipoperoxidation as well as by the enhancement of mitochon-drial function (Wang and Quinn, 1999).

PBN has been show to exhibit a remarkable neuroprotectiveeffect against ischemia (Folbergrová et al., 1995; Phillis andClough-Helfman, 1990a,b; Zhao et al., 1994), excitotoxicity,and neurotoxicity induced by the administration of mitochon-drial toxins in vivo (Schulz et al., 1995). However, in the


present study, we observed no improvement of mitochondrialfunction by PBN. In fact, the precise role of PBN as anantioxidant compound is still controversial and some data ruleout direct free radical trapping as the mechanistic basis of theneuroprotective action of PBN (for review, see Floyd, 1999).Nevertheless, in the in vivo model we observed partialprotection of Aβ neurotoxicity by a single i.p. injection ofPBN 30 min before Aβ administration. Improvement of itsneuroprotective efficacy by chronic treatment cannot bediscarded.

In conclusion, the present study provides further evidence forthe influence of metabolic impairment on Aβ neurotoxicity andsuggests that enhanced Aβ toxicity is mediated at least in partby the generation of free radicals, which are more difficult toinactivate when energy metabolism is compromised. Results areconsistent with the previously observed effect of aging onneuronal vulnerability to in vivo Aβ toxicity (Geula et al.,1998), which may be associated with diminished energymetabolic competence (Bowling et al., 1993; Shigenaga et al.,1994).

The observed enhancement of mitochondrial activity andreduction of LPO after treatment with vitamin E encourages thefurther study of its therapeutic potential on the incidence andprogression of AD. Vitamin E might reduce the impact exertedby previous episodes of stroke or heart attack, on the occurrenceand evolution of AD.

Acknowledgments

This work was supported by CONACYT 40306-M andPAPIIT IN-222503 grants to L. Massieu and CONACYT36250-M and PAPITT IN205403 grants to C. Arias.

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Role of oxidative stress on β-amyloid neurotoxicity elicited during impairment of energy metabolism...

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