Research Overview

Metals and Oxidative Homeostasis in Alzheimer’s DiseaseGeorge Perry,n Adam D. Cash, Ravi Srinivas, and Mark A. Smith

Department of Pathology, Case Western Reserve University, Cleveland, Ohio

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Abstract Oxidative damage to every class of biological macromolecule has been characterized inAlzheimer’s disease. Abnormalities in iron and copper metabolism are also being implicated as playing acrucial role in neurodegenerative disease pathogenesis. Metal homeostasis as it pertains to alterations inbrain function in neurodegenerative diseases is reviewed here with its relationship to oxidative stress.While there is documented evidence for alterations in transition metal homeostasis, redox-activity, andlocalization, it is also important to realize that alterations in specific copper- and iron-containingmetalloenzymes also contribute to the neurodegenerative process. These changes offer the opportunityto identify pathways where modification of the disease process can offer new routes for clinical efficacy,from gene therapy to use of antioxidant and chelating drugs. Drug Dev. Res. 56:293–299, 2002. �c 2002

Wiley-Liss, Inc.

Key words: free radicals; neurodegeneration; mitochondria; iron

INTRODUCTION

Interest in the roles of copper, iron, and othertrace redox-active transition metals has grown expo-nentially in recent years as their critical role in thepathogenesis of Alzheimer’s disease (AD) is beingelucidated. While these transition metals are essentialin many biological reactions, shifts in their homeostasis,redox-activity, and/or sequestration can have profoundcellular consequences, including cytotoxicity. Many ofthese alterations in metal homeostasis result inincreased production of free radicals, most of whichare catalyzed by iron or copper, and recent findings linkthe neurodegenerative process to oxidative stress.Together, these findings indicate a direct cause/effectrelationship between metal abnormalities and theincreased oxidative damage present in these diseases.

All aerobic organisms produce free radicals,predominantly superoxide formed as a side productduring the reduction of molecular oxygen by mitochon-dria, where it is estimated that 1% of respiredmolecular oxygen will form O2

- . Since the average cellutilizes 1013 O2/cell/day, this equates to 1011 free radicalspecies produced per cell per day, and even more so forhighly metabolic neurons. While most of the radicals

are sequestered in the mitochondria, oxidative insult isexacerbated by age, metabolic demand, or diseaseprocesses. With respect to neurodegenerative disease,it is notable that most diseases show increases inprevalence with age and that the brain, although only2–3% of total body mass, utilizes 20% of basal oxygenconsumption. Additionally, H2O2, produced by brain-specific oxidases such as monoamine oxidase cantremendously add to this oxygen radical burden incompartments having less protection from reactiveoxygen than mitochondria.

The hydroxyl radical is the most highly reactivewith all categories of biomacromolecules given thevarious free radicals produced by cells. Most hydroxylradicals arise as a consequence of Fenton chemistrybetween reduced transition metals (usually iron(II) orcopper(I)) and H2O2. Importantly, reduction of theresulting oxidized transition metal ions (iron(III) or

DDR

nCorrespondence to: George Perry, Institute of Pathology,Case Western Reserve University, 2085 Adelbert Road,Cleveland, OH 44106.E-mail: gxp7@po.cwru.edu

Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ddr.10099

DRUG DEVELOPMENT RESEARCH 56:293–299 (2002)

�c 2002 Wiley-Liss, Inc.

copper(II)) by vitamin C or other cellular reductantsregenerates the ‘‘active’’ transition metal and leads tothe catalytic production of free radicals by the processof redox cycling. Notably, cellular reductants are oftendepleted in neurodegenerative diseases, suggestingtheir use in redox cycling as well as autooxidantdefense. An alternative pathway for reactive oxygenproduction is the result of the combination of NO andO2

- to yield peroxynitrite [Smith et al., 1997a]. The roleof this process must be carefully considered as metal-dependent peroxidative nitration [Sampson et al.,1998], making peroxynitrite-dependent processes in-distinguishable from metal-dependent processes.Therefore, critical analysis of claims of metal-indepen-dent oxidative damage must demonstrate that theytruly occur devoid of catalytic transition metals.

Protection against oxidant damage comes througha variety of antioxidantsFboth enzymatic and none-nzymatic. In the former regard, metal-containingenzymes such as cytosolic copper-zinc superoxidedismutase (CuZnSOD) and mitochondrial manganesesuperoxide dismutase (MnSOD) convert superoxide toO2 and H2O2. Since these protective enzymes containmetals, there is an apparent delicate balance in placewithin cells regarding the distribution of both proox-idant and antioxidant metal ions. This delicate balanceis critical to cellular homeostasis. Here, we considerevidence that strongly implicates a pivotal role forredox-active metal ions in the pathogenesis of PD, AD,ALS, and a number of other neurodegenerativediseases.

METAL IMBALANCE IN NEURODEGENERATIVEDISEASE

The redox transitions associated with ‘‘free’’ iron,more than any other transition metal, is thought tomediate the generation of oxygen-free radicals andconsequent tissue oxidative stress in vivo. Indeed,alterations in free iron and iron homeostasis have beenimplicated in a number of neurodegenerative disor-ders, including AD [Smith et al., 1997b, 1998a;Castellani et al., 2000a] and those characterized bynigral degeneration such as PD [Castellani et al.,2000b], multiple system atrophy, and progressivesupranuclear palsy [Odetti et al., 2000].

While cellular iron homeostasis is mainly regu-lated by the transferrin receptor and ferritin, it is alsomediated by the lactotransferrin receptor [Kawamataet al., 1993], melanotransferrin, ceruloplasmin [Cas-tellani et al., 1999], and divalent cation transporter.Changes in any of these proteins could, therefore,contribute to alterations in brain iron metabolism [Qianand Wang, 1998]. Control of these regulatory proteinsand therefore regulation of overall cellular iron

metabolism involves the action of two iron regulatoryproteins, IRP-1 and IRP-2. IRP-1, but not IRP-2, israpidly activated by extracellular H2O2, establishing aregulatory connection between the control of ironmetabolism and response to reactive oxygen. Interest-ingly, IRP proteins show significant alterations in AD[Smith et al., 1998a], paralleling alterations in redox-active iron [Smith et al., 1997b].

Copper transport to essential enzymes withoutengendering cellular toxicity initiating from reactiveoxygen generation by ‘‘free’’ copper has necessitatedevolution of a tightly regulated homeostatic systeminvolving copper transport, storage proteins, andchaperonins [Cizewski et al., 1997], Ceruloplasmin, akey copper storage protein, responds to oxidativestress. However, while ceruloplasmin is increased inbrain tissue and cerebrospinal fluid in AD [Castellaniet al., 1999; Loeffler et al., 1996], neuronal levels ofceruloplasmin remain unchanged [Castellani et al.,1999]. Therefore, while increased ceruloplasmin mayindicate a compensatory response to increased oxida-tive stress in AD, its failure to do so in neurons mayplay an important role in potentiating metal-catalyzedreactive oxygen formation [Castellani et al., 1999].Additionally, specific copper chaperonins [Cizewskiet al., 1997] play tightly regulated roles in deliveringcopper to enzymes which require it for activity such asSOD-1 and cytochrome oxidase.

A primary function of glutathione (GSH) is toprevent the toxic interaction between reactive transi-tion metals and free radicals. Perturbations to GSHmetabolism may play an important role in neurode-generative disorders with conditions linked to abnorm-alities in copper homeostasis [Brown et al.,1997; Wonget al., 2001a,b] and redox balance, such as AD[Castellani et al., 1999; Raina et al., 1999a; Russellet al., 1999b; Kim et al., 2000]. Depletion of cellularGSH and inhibition of the GSH-regenerating enzymeglutathione reductase was reported to result in adramatic potentiation of copper toxicity in primaryneuronal cultures without effect on iron toxicity [Whiteet al., 1999]. These studies suggest that conditionsknown to alter neuronal GSH homeostasis such asamyloid-b (Ab) in AD [Russell et al., 1999; Martinset al., 1986] are likely to manifest in toxicity due toendogenous copper.

METALLOENZYME DYSFUNCTION

The principal metalloenzymes responsible forcellular regulation of reactive oxygen are the Mn andCuZn superoxide dismutases (SOD-2 and SOD-1,respectively) that remove O2

- and the enzymes catalaseand peroxidases that remove H2O2. Usually SOD isconsidered the primary defense against buildup of

294 PERRY ET AL.

reactive oxygen because it removes O2- , the initial form

of metabolically produced reactive oxygen. With theadvent of transgenic mouse ‘‘knockout’’ and over-expression models, it has become possible to directlyassess the extent to which alterations of SOD activitymight affect cell viability. If one or the other of theSOD enzymes serve a singular and noncompensableantioxidant function, then knockout animals shouldexhibit increased oxidative stress parameters. Thisoxidative stress may be global or may be localized tothe cellular compartment normally protected by SOD.In support of this notion, while MnSOD knockout miceare embryonic lethal the heterozygote, suffering a 50%drop in mitochondrial SOD activity, but no reductionof CuZnSOD or GSH peroxidase activity, were foundto exhibit increased oxidative damage to mitochondriaas evidenced by increased mitochondrial proteincarbonyls and 8-hydroxydeoxyguanosine in mitochon-drial protein and DNA, respectively [Williams et al.,1998]. In contrast, no damage to cytosolic proteins or tonuclear DNA was observed, indicating that mitochon-drial oxidative imbalance is localized within theorganelle. Analysis of homozygote knockouts showedmitochondrial degeneration so extensive that theycould only be followed for the 2 weeks that these micelived when metal-centered antioxidants were adminis-tered [Melov et al., 1998]. These results suggest thatdecreases in MnSOD activity in vivo can explainincreased oxidative damage in mitochondria andalterations in essential mitochondrial function but notcytoplasmic changes.

Overexpression of human CuZnSOD in mice,resulting in a 10-fold higher level of SOD proteinand SOD activity in both myocytes and endothelialcells and was able to quench a burst of superoxide(EPR detection) and reduce functional damagefollowing 30 min of global ischemia [Wang et al.,1998]. These results indicate that superoxide is animportant mediator of postischemic injury and it istherefore surprising that CuZnSOD knockout miceunder basal conditions show little if any neurodegen-erative phenotype [Bruijn et al., 1998] but showabnormalities in injury repair [Flood et al., 1999].Nonetheless, it is apparent that decreases in CuZnSODactivity can lead to a disruption of cellular antioxidantdefense mechanisms that increase sensitivity to proox-idant conditions. This is exemplified by the recentfinding that a yeast mutant lacking CuZnSOD becomesa victim of iron-mediated oxidative damage [Corsonet al., 1999].

ALZHEIMER’S DISEASE

AD is pathologically characterized by the pre-sence of neurofibrillary tangles (NFT), Ab-laden senile

plaques, and neuropil threads, with a selective loss ofneurons in hippocampal and neocortices and selectedsubcortical regions. Mutations in the genes encodingthe amyloid-b protein precursor (AbPP) and/or thepresenilins lead to the overproduction of AbPP and/oraltered AbPP proteolytic processing, resulting inincreased Ab. Transgenic mice overproducing theAbPP with human mutations led to the developmentof senile plaques, but may not follow the samemechanism of AD. To date, no animal model for evenfamilial AD exists that is complete, with only limitedneuronal loss reported in one model [Irizarry et al.,1997a,b; Calhoun et al., 1998]. Also, the predominantorigin of AD bears no obvious linkage to genetic factorsand instead shows an age-dependent increase inincidence.

Oxidative stress has been linked as a proximalevent in sporadic AD. Many studies have shownimbalances in trace elements, including aluminum,silicon, lead, mercury, zinc, copper, and iron. Homeo-static disruption of copper and iron is of importanceconsidering the substantial evidence for increases inoxidative damage to lipids, sugars, proteins, and nucleicacids, as well as damage to the proteins of NFT andsenile plaques [Smith et al 1994, 1997a, 1998b; Sayreet al., 1997a; Nunomura et al., 1999a; Takeda et al.,2000].

Microparticle-induced X-ray emission revealedthat zinc(II), iron(III), and copper(II) are significantlyincreased in AD neuropil and are further highlyconcentrated within the core and peripheral areas ofsenile plaque [Lovell et al., 1998]. Using an in situ irondetection method, we found an apparent association ofredox-active iron with both NFT and senile plaques inAD [Smith et al., 1997b]. This association of iron withNFT may, in part, be related to iron binding to t [Perezet al., 1998; Sayre et al., 2000]. Also, whereas the iron-regulatory protein IRP-1 was present at similar levels inboth AD and control brain tissues, IRP-2 colocalizedwith redox-active iron in NFT, senile plaques, andneuropil threads [Smith et al., 1998], suggesting thatalterations in IRP-2 might be directly linked toimpaired iron homeostasis in AD.

Recently, we have found that the redox activity ofAD lesions can be detected directly and be inhibited byinitial exposure of the tissue sections to copper- andiron-selective chelators. Redox activity can be rein-stated following reexposure of the chelator-treatedsections to salts of either copper or iron [Sayre et al.,2000]. These studies suggest that the lesions may playcritical roles in metal homeostasis.

Other studies have dealt with the induciblemitochondrial MnSOD and the constitutive cytoplas-mic CuZnSOD enzymes. The CuZnSOD gene is

METALS AND OXIDATIVE HOMEOSTASIS 295

associated with AD neuropathology and levels ofMnSOD mRNA and CuZnSOD were found to beelevated in AD, whereas the total antioxidant status wasdecreased [De Leo et al., 1998]. Since SOD enzymesare key members of the cellular antioxidant defensesystem, any prooxidant mechanism linked to SOD mustderive from the balance in the local concentrations ofsuperoxide and H2O2, which together produce hydro-xyl radicals via the Haber-Weiss process.

Interest in the factors responsible for aggregationof Ab led to an obvious early focus on the possibleinvolvement of trace metal ions. The first systematicstudy reported that aluminum, iron, and zinc, but notcalcium, cobalt, manganese, copper, magnesium, so-dium, or potassium accelerated aggregation of Ab[Mantyh et al., 1993]. However, more recent studiesindicate that the aggregatory effect of metals dependscritically on the pH. Copper(II) induced aggregation ofAb(1–40) when the pH was lowered from 7.4–6.8, aphenomenon that was not characteristic of other metalstested [Atwood et al., 1998]. A mildly acidic environ-ment together with increased zinc(II) and copper(II)are common features of inflammation and this could bea likely explanation for the rapid deposition of Abfollowing head injury as well as inflammation associatedwith increased oxidative damage due to promotion of�OH-like activity from microglial-derived peroxyni-trite. The association of copper(II), zinc(II), andiron(II) with Ab found in vitro could explain theenrichment of these metals with senile plaques in AD.Evidence that metal ions may actually be involved inpathological aggregation of Ab in vivo is provided bythe recent study reporting that divalent metal ionchelators facilitate solubilization of Ab deposits fromAD tissue, although efficient extraction of Ab alsorequired the presence of magnesium(II) and calciu-m(II) [Cherny et al., 1999]. The finding that divalentmetal ion-induced aggregation of Ab is affected byapolipoprotein E and differentially by the differentapolipoprotein E isoforms, was recently proposed to beconsistent with a role for apolipoprotein E as an in vivochaperone for Ab [Moir et al., 1999].

Although metal-induced aggregation of Ab maynot reflect redox chemistry, the metals bound to solubleor aggregated forms of the peptide do seem to be redoxactive and thus have the capacity to mediate ROSproduction [Sayre et al., 2000; Bondy et al., 1998; Yanget al., 1999; Huang et al., 1999a,b]. Consideredtogether, the studies by us and others indicating thepresence of redox-active iron and copper in ADpathology, suggest that these metal accumulationscould be major contributors to metal homeostasis.

The recent interest in the possible contribution oftransition metals bound to Ab in mediating redox

chemistry and possibly oxidative stress is closely tied tothe ongoing studies directed at elucidating the associa-tion of Ab with free radical production [Yatin et al.,1999]. Since the ‘‘peptide-only shear’’ mechanism,initially proposed to explain radical production, lackedchemical precedent, the consensus has been thatincreased oxidative stress associated with Ab musteither represent peptide interactions with redox-activemetal ions [Dikalov et al., 1999; Rottkamp et al., 2001]or indirect mechanisms triggered by Ab fibrils [Sayreet al., 1997b].

The probability that Ab-associated oxidative stressmight be due at least in part from direct reactiveoxygen production by Ab-bound transition metals, hasbecome the subject of detailed investigation over thepast year. It was reported that Ab binding to copper(II)results in its reduction to copper(I) accompanied bygeneration of H2O2 [Huang et al., 1999a] to a degreethat is most evident for human Ab(1–42). This eventresults in vitro in oxidative stress that follows Ab(1–42)�Ab(1–40)4Ab(40–1)Brat Ab(1–40), the sameorder seen for neurotoxicity associated with thesepeptides. A role for bound copper in possibly mediat-ing Ab peptide neurotoxicity is supported by thefinding that added copper(II) markedly potentiatesAb toxicity in primary neuronal cultures to an extentthat is greatest for Ab(1–42)�Ab(1–40)4Ab(40–1)Brodent Ab(1–40), apparently via H2O2 production[Huang et al., 1999b]. Recent studies with iron showthat Ab toxicity for cells in culture can be potentiatedby iron addition and greatly reduced by the ironchelator deferoxamine [Rottkamp et al., 2001]. Thesefindings suggest a critical role for both iron and copperin Ab activity. Exactly what types of interaction controlthe observed Ab-peptide rank order of biochemicaleffects is still under investigation, but the differencebetween human and rodent Ab(1–40) probably stemsfrom the replacement of the metal-coordinating His-13in the human form by Arg in the rodent form. Inaddition, there is evidence that Met-35 is critical to theradical-generating ability of Ab [Varadarajan et al.,1999].

Also, Cu(II) binds to AbPP and appears to bereduced to copper(I) concomitant with production of adisulfide linkage [Multhaup et al., 1998]. Subsequentexposure to H2O2 results in reoxidation of copper(I)and concomitant site-specific cleavage of AbPP. Redoxchemistry associated with AbPP-bound metals couldthus, alongside that mediated by Ab-bound metals,contribute to a perturbation of oxygen radical home-ostasis and resulting ROS-mediated neuronal toxicity inAD. In this regard, a recent study demonstrated thatcopper- (but not iron- or zinc-) dependent toxicity inprimary neuronal cultures is potentiated by AbPP, and

296 PERRY ET AL.

in particular by the copper(II)-binding domain ofAbPP [White et al., 1999b]. These same workersshowed that the levels of copper (but not iron or zinc)in certain brain regions determined by atomic absorp-tion are increased in AbPP-knockout mice as well asknockout mice for the amyloid precursor-like protein 2,also known to bind copper and zinc [White et al.,1999b]. This finding was proposed to suggest thatperturbations to AbPP metabolism could be respon-sible for initiating pathological changes associated withcopper metabolism in AD.

While in cell culture [Rottkamp et al., 2001] andin transgenic mice [Smith et al 1998c], Ab is associatedwith oxidative damage; in AD, there is no suchassociation [Nunomura et al., 1999a]. In fact, Abdeposition is marked by a reduction in the quantity ofoxidative damage in AD, sporadic [Nunomura et al.,1999b, 2000a, 2001] and genetic cases [Nunomuraet al., 2000b], as well as in Down syndrome [Nunomuraet al., 2000c]. These findings suggest a more compli-cated relationship between homeostatic imbalance inmetals, Ab and other metabolic features is critical tothe quantity of oxidative damage observed. One of themost striking of these changes is a shift in redoxbalance signaled by induction of the pentose phosphatepathway, NQO1 induction as well as increasedsulfhydryls [Raina et al., 1999; Russell et al., 1999;Kim et al., 2000]. Increased availability of reducingequivalents will fundamentally alter the relationshipsbetween metals and redox balance and may explainwhy we note prooxidant effects with normal cells, whilein AD Ab is associated with apparent antioxidantactivity.

CONCLUSIONS AND THERAPEUTIC POTENTIAL

This review has brought to light the growing bodyof evidence for the fundamental role that metals play inoxidative balance evident in AD and a variety of otherneurodegenerative diseases. Although most focus hasbeen on the direct roles of the redox transition metalsiron and copper, nonredox-active metal ions, such aszinc and aluminum, can shift free radical homeostasisby displacing iron and/or copper from sites, e.g.,storage proteins, where they are inactive in oxyradicalproduction. Further, dysregulation of metalloenzymesor operation of mutant forms adds to the burden ofneurons in its efforts to maintain redox homeostasis.The stage is now set to critically evaluate theimportance of these basic research findings as theyare translated into therapeutic modalities such asantioxidants and chelating agents that are being usedclinically.

In these studies, it is essential to understand theintricate homeostatic balance between metals, redox

control, and the pathological lesions [Perry et al.,2000a]. We think it is also necessary to understand thecontribution of each before proposing to eradicate the‘‘pathological’’ entity [Perry et al., 2000b], which mayfurther exacerbate the disease process. We areencouraged that use of antioxidants and iron chelatorsare shown to be clinical effective in AD [Perry et al.,1998]. We think the greatest benefits in metal-directedtherapeutics will derive from treatments that sequesterthe select metals responsible for oxidative imbalancewhile leaving those metals that may have adaptive valuein neuronal survival. Ultimately, the most efficacioustherapy will result from directing our efforts to thefundamental abnormalities, displacing neurons fromphysiological homeostasis. Therapeutics based on thelatter will require a detailed mechanistic understandingof the diseases process.

REFERENCES

Atwood CS, Moir RD, Huang X, et al. 1998. Dramatic aggregationof Alzheimer Ab by Cu(II) is induced by conditions representingphysiological acidosis. J Biol Chem 273:12817–12826.

Bondy SC, Guo-Ross SX, Truong AT. 1998. Promotion of transitionmetal-induced reactive oxygen species formation by b-amyloid.Brain Res 799:91–96.

Brown DR, Qin K, Herms JW, et al. 1997. The cellular prion proteinbinds copper in vivo. Nature 390:684–687.

Bruijn LI, Houseweart MK, Kato S, et al. 1998. Aggregation andmotor neuron toxicity of an ALS-linked SOD1 mutant indepen-dent from wild-type SOD1. Science 281:1851–1854.

Calhoun ME, Wiederhold KH, Abramowski D, et al. 1998. Neuronloss in APP transgenic mice. Nature 395:755–756.

Castellani RJ, Smith MA, Nunomura A, et al. 1999. Is increasedredox-active iron in Alzheimer disease a failure of the copper-binding protein ceruloplasmin? Free Radic Biol Med 26:1508–1512.

Castellani RJ, Harris PLR, Lecrosiey A, et al. 2000a. Evidence for anovel heme-binding protein, HasAh, in Alzheimer disease. AntioxRedox Signal 2:137–142.

Castellani RJ, Siedlak SL, Perry G, Smith MA. 2000b. Sequestrationof iron by Lewy bodies in Parkinson’s disease. Acta Neuropathol100:11–1114.

Cherny RA, Legg JT, McLean CA, et al. 1999. Aqueous dissolutionof Alzheimer’s disease Ab amyloid deposits by biometal depletion.J Biol Chem 274:23223–23228.

Cizewski Culotta V, Klomp LWJ, Strain J, Casareno RLB, Krems B,Gitlin JD. 1997. The copper chaperone for superoxide dismutase.J Biol Chem 272:23469–23472.

Corson LN, Folmer J, Strain JJ, et al. 1999. Oxidative stress and ironare implicated in fragmenting vacuoles of Saccharomycescerevisiae lacking Cu,Zn-superoxide dismutase. J Biol Chem274:27590–27596.

De Leo ME, Borrello S, Passantino M, et al. 1998. Oxidative stressand overexpression of manganese superoxide dismutase inpatients with Alzheimer’s disease. Neurosci Lett 250:173–176.

Dikalov SI, Vitek MP, Maples KR, Mason RP. 1999. Amyloid bpeptides do not form peptide-derived free radicals spontaneously,

METALS AND OXIDATIVE HOMEOSTASIS 297

but can enhance metal-catalyzed oxidation of hydroxylamines tonitroxides. J Biol Chem 274:9392–9299.

Flood DG, Reaume AG, Gruner JA, et al. 1999. Hindlimb motorneurons require Cu/Zn superoxide dismutase for maintenance ofneuromuscular junctions. Am J Pathol 155:663–672.

Huang X, Atwood CS, Hartshorn MA, et al. 1999a. The Ab peptideof Alzheimer’s disease directly produces hydrogen peroxidethrough metal ion reduction. Biochemistry 38:7609–7616.

Huang X, Cuajungco MP, Atwood CS, et al. 1999b. Cu(II)potentiation of Alzheimer Ab neurotoxicity. J Biol Chem274:37111–37116.

Irizarry MC, McNamara M, Fedorchak K, et al. 1997a. APPSwtransgenic mice develop age-related A beta deposits and neuropilabnormalities, but no neuronal loss in CA1. J Neuropathol ExpNeurol 56:965–973.

Irizarry MC, Soriano F, McNamara M, et al. 1997b. Ab deposition isassociated with neuropil changes, but not with overt neuronal lossin the human amyloid precursor protein V717F (PDAPP)transgenic mouse. J Neurosci 17:7053–7059.

Kawamata T, Tooyama I, Yamada T, et al. 1993. Lactotransferrinimmunocytochemistry in Alzheimer and normal human brain. AmJ Pathol 142:1574–1585.

Kim HT, Russell RL, Raina AK, et al. 2000. Protein disulfideisomerase in Alzheimer disease. Antiox Redox Signal 2:485–489.

Loeffler DA, LeWitt PA, Juneau PL, et al. 1996. Increased regionalbrain concentrations of ceruloplasmin in neurodegenerativedisorders. Brain Res 738:265–274.

Lovell MA, Robertson JD, Teesdale WJ, et al. 1998. Copper, ironand zinc in Alzheimer’s disease senile plaques. J Neurol Sci158:47–52.

Mantyh PW, Ghilardi JR, Rogers S, et al. 1993. Aluminum, iron, andzinc ions promote aggregation of physiological concentrations ofbeta-amyloid peptide. J Neurochem 61:1171–1174.

Martins RN, Harper CG, Stokes GB, Masters CL. 1986. Increasedcerebral glucose-6-phosphate dehydrogenase activity in Alzhei-mer’s disease may reflect oxidative stress. J Neurochem 46:1042–1045.

Melov S, Schneider JA, Day BJ, et al. 1998. A novel neurologicalphenotype in mice lacking mitochondrial manganese superoxidedismutase. Nat Genet 18:159–163.

Moir RD, Atwood CS, Romano DM, et al. 1999. Differential effectsof apolipoprotein E isoforms on metal-induced aggregation of Abusing physiological concentrations. Biochemistry 38:4595–4603.

Multhaup G, Ruppert T, Schlicksupp A, et al. 1998. Copper-bindingamyloid precursor undergoes a site-specific fragmentation in thereduction of hydrogen peroxide. Biochemistry 37:7224–7230.

Nunomura A, Perry G, Pappolla MA, et al. 1999a. RNA oxidation isa prominent feature of vulnerable neurons in Alzheimer disease.J Neurosci 19:1959–1964.

Nunomura A, Perry G, Smith MA. 1999b. Oxidation of nucleic acidsin neurons precedes amyloid b deposition in Down syndrome.J Neuropathol Exp Neurol 58:511.

Nunomura A, Perry G, Smith MA, Chiba S. 2000a. Oxidativedamage and amyloid b deposition in Alzheimer disease withreference to apolipoprotein E4 (Japanese). Dementia 14:232.

Nunomura A, Chiba S, Lippa C, et al. 2000b. Increasing amyloid b42 deposition is associated with decreasing neuronal RNAoxidation in Down’s syndrome and familial Alzheimer’s disease.Brain Pathol 10:783.

Nunomura A, Perry G, Hirai K, et al. 2000c. Neuronal oxidativestress precedes amyloid-b deposition in Down’s syndrome.J Neuropathol Exp Neurol 59:1011–1017.

Nunomura A, Perry G, Aliev G, et al. 2001. Oxidative damage is theearliest event in Alzheimer disease. J Neuropathol Exp Neurol60:759–767.

Odetti P, Garibaldi S, Norese R, et al. 2000. Lipoperoxidation isselectively involved in progressive supranuclear palsy. J Neuro-pathol Exp Neurol 59:393–397.

Perez M, Valpuesta JM, de Garcini EM, et al. 1998. Ferritin isassociated with the aberrant tau filaments present in progressivesupranuclear palsy. Am J Pathol 152:1531–1539.

Perry G, Castellani RJ, Hirai K, Smith MA. 1998. Reactive oxygenspecies mediate cellular damage in Alzheimer disease.J Alzheimer Dis 1:45–55.

Perry G, Raina AK, Nunomura A, et al. 2000a. How important isoxidative damage? Lessons from Alzheimer disease. Free RadicBiol Med 28:831–834.

Perry G, Nunomura A, Raina AK, Smith MA. 2000b. Amyloid-bjunkies. Lancet 355:757.

Qian ZM, Wang Q. 1998. Expression of iron transport proteins andexcessive iron accumulation in the brain in neurodegenerativedisorders. Brain Res Rev 27:257–267.

Raina AK, Templeton DJ, Deak JC, et al. 1999. Quinone reductase(NQO1), a sensitive redox indicator, is increased in Alzheimer’sdisease. Redox Report 4:23–27.

Rottkamp CA, Raina AK, Zhu X, et al. 2001. Redox-active ironmediates amyloid-b toxicity. Free Radic Biol Med 30:447–450.

Russell RL, Siedlak SL, Raina AK, et al. 1999. Increased neuronalglucose-6-phosphate dehydrogenase and sulfhydryl levels indicatereductive compensation to oxidative stress in Alzheimer disease.Arch Biochem Biophys 370:236–239.

Sampson JB, Ye Y, Rosen H, Beckman JS. 1998. Myeloperoxidaseand horseradish peroxidase catalyze tyrosine nitration in proteinsfrom nitrite and hydrogen peroxide. Arch Biochem Biophys356:207–213.

Sayre LM, Zelasko DA, Harris PLR, et al. 1997a. 4-Hydroxynone-nal-derived advanced lipid peroxidation end products areincreased in Alzheimer’s disease. J Neurochem 68:2092–2097.

Sayre LM, Zagorski MG, Surewicz WK, et al. 1997b. Mechanisms ofneurotoxicity associated with amyloid b deposition and the role offree radicals in the pathogenesis of Alzheimer’s disease: a criticalappraisal. Chem Res Toxicol 10:518–526.

Sayre LM, Perry G, Harris PLR, et al. 2000. In situ redox catalysisby neurofibrillary tangles and senile plaques in Alzheimer disease:a central role for bound transition metals. J Neurochem 74:270–279.

Smith MA, Taneda S, Richey PL, et al. 1994. Advanced Maillardreaction end products are associated with Alzheimer diseasepathology. Proc Natl Acad Sci USA 91:5710–5714.

Smith MA, Harris PLR, Sayre LM, et al. 1997a. Widespreadperoxynitrite-mediated damage in Alzheimer’s disease. J Neurosci17:2653–2657.

Smith MA, Harris PLR, Sayre LM, Perry G. 1997b. Ironaccumulation in Alzheimer disease is a source of redox-generatedfree radicals. Proc Natl Acad Sci USA 94:9866–9868.

Smith MA, Wehr K, Harris PLR, et al. 1998a. Abnormal localizationof iron regulatory protein (IRP) in Alzheimer disease. Brain Res788:232–236.

298 PERRY ET AL.

Smith MA, Sayre LM, Anderson VE, et al. 1998b. Cytochemicaldemonstration of oxidative damage in Alzheimer disease byimmunochemical enhancement of the carbonyl reaction withdinitrophenylhydrazine. J Histochem Cytochem 46:731–735.

Smith MA, Hirai K, Hsiao K, et al. 1998c. Amyloid-b deposition inAlzheimer transgenic mice is associated with oxidative stress. JNeurochem 70:2212–2215.

Takeda A, Smith MA, Avila J, et al. 2000. In Alzheimer disease,heme oxygenase is coincident with Alz50, an epitope of tauinduced by 4-hydroxy-2-nonenal modification. J Neurochem75:1234–1241.

Varadarajan S, Yatin S, Kanski J, et al. 1999. Methionine residue 35is important in amyloid b-peptide-associated free radical oxidativestress. Brain Res Bull 50:133–141.

Wang P, Chen H, Qin H, et al. 1998. Overexpression of humancopper, zinc-superoxide dismutase (SOD1) prevents postischemicinjury. Proc Natl Acad Sci USA 95:4556–4560.

White AR, Bush AI, Beyreuther K, et al. 1999a. Exacerbation ofcopper toxicity in primary neuronal cultures depleted of cellularglutathione. J Neurochem 72:2092–2098.

White AR, Reyes R, Mercer JFB, et al. 1999b. Copper levels areincreased in the cerebral cortex and liver of APP and APLP2knockout mice. Brain Res 842:439–444.

Williams MD, Van Remmen H, Conrad CC, et al. 1998. Increasedoxidative damage is correlated to altered mitochondrial functionin heterozygous manganese superoxide dismutase knockout mice.J Biol Chem 273:28510–28515.

Wong B-S, Liu T, Li R, et al. 2001a. Increased levels of oxidativestress markers detected in the brains of mice devoid of prionprotein. J Neurochem 76:565–572.

Wong B-S, Liu T, Paisley D, et al. 2001b. Induction of hemeoxygenase-1 and neuronal nitric oxide synthase in Doppel-expressing mice devoid of prion protein: implications for Doppelfunction. Mol Cell Neurosci 17:768–775.

Yang EY, Guo-Ross SX, Bondy SC. 1999. The stabilization of ferrousiron by a toxic b-amyloid fragment and by an aluminum salt. BrainRes 839:221–226.

Yatin SM, Varadarajan S, Link CD, Butterfield DA. 1999. In vitroand in vivo oxidative stress associated with Alzheimer’s amyloidb-peptide(1-42). Neurobiol Aging 20:325–330.

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