Serial Review: Oxidatively Modified Proteins in Aging and DiseaseGuest Editor: Earl Stadtman

IS OXIDATIVE DAMAGE THE FUNDAMENTAL PATHOGENIC MECHANISMOF ALZHEIMER’S AND OTHER NEURODEGENERATIVE DISEASES?

GEORGE PERRY,* A KIHIKO NUNOMURA,*† KEISUKE HIRAI,*‡ XIONGWEI ZHU,* M AR PEREZ,§ JESUS AVILA ,§

RUDOLPH J. CASTELLANI,* CRAIG S. ATWOOD,* GJUMRAKCH ALIEV,¶ LAWRENCE M. SAYRE,�

ATSUSHI TAKEDA,*# and MARK A. SMITH**Institute of Pathology, Case Western Reserve University, Cleveland, OH, USA;†Department of Psychiatry and Neurology,

Asahikawa Medical College, Asahikawa, Japan;‡Pharmaceutical Research Laboratories I, Takeda Chemical Industries Ltd., Osaka,Japan;§Centro de Biologia Molecular, Universidad Autonoma de Madrid, Madrid, Spain; Departments of¶Anatomy; �Chemistry,Case Western Reserve University, Cleveland, OH, USA; and#Tohoku University School of Medicine, Department of Neurology,

Sendai, Japan

(Received 12 February 2002;Revised 26 August 2002;Accepted 3 September 2002)

Abstract—In less than a decade, beginning with the demonstration by Floyd, Stadtman, Markesbery et al. [1] ofincreased reactive carbonyls in the brains of patients with Alzheimer’s disease (AD), oxidative damage has beenestablished as a feature of the disease. Here, we review the types of oxidative damage seen in AD, sites involved,possible origin, relationship to lesions, and compensatory changes, and we also consider other neurodegenerativediseases where oxidative stress has been implicated. Although much data remain to be collected, the broad spectrum ofchanges found in AD are only seen, albeit to a lesser extent, in normal aging with other neurodegenerative diseasesshowing distinct spectrums of change. © 2002 Elsevier Science Inc.

Keywords—Alzheimer’s disease, Amyloid-�, Antioxidants, Homeostasis, Neurofibrillary tangles, Oxidative stress,Redox balance, Senile plaque,�, Free radicals

OXIDATIVE DAMAGE IN ALZHEIMER’S DISEASE

Increased reactive carbonyls were the first form of oxi-dative damage identified in AD [1]. Within two years, asuccession of articles showed carbonyl-based damagewas apparent in both senile plaques [2,3], neurofibrillarytangles (NFT) [3,4], and the primary component of thelatter,� (� protein) [4,5]. The significance of these find-ings was initially questioned by suggestions that thelesions of AD, much as vessel walls [6,7], accumulatedamage through low protein turnover [8]. What wasmissing from this criticism was not the accumulativenature of carbonyl modification, but that the productsfirst identified, advanced glycation end products (AGE),

are “active modifications,” meaning they are the result ofmetal-catalyzed redox chemistry and are continuing sitesof redox chemistry [9]. Also, in more recent studies, wehave demonstrated that the lesions are not only sites ofAGE accumulation but also continuing sites of glycation,since the initial Amadori product is closely associatedwith NFT [10].

Early reports of oxidative modifications were fol-lowed in close succession by the identification in NFT ofreactive carbonyls [11,12] and protein adducts of thelipid peroxidation product, hydroxynonenal [13,14].What was remarkable in using these different markers,either resulting from carbonyl adduction or, in the case ofreactive carbonyls, from direct protein oxidation, is thatwhile highly stable modifications, involving cross-linkedproteins, are predominantly associated with the lesions,metastable modifications are more commonly associatedwith the neuronal cytoplasm. Specifically, populations ofneurons involved in AD, and not others, show thischange. This led us to the hypothesis that the most active

This article is part of a series of reviews on “Oxidatively ModifiedProteins in Aging and Disease.” The full list of papers may be found onthe homepage of the journal.

Address correspondence to: Dr. Mark A. Smith, Case Western Re-serve University, Institute of Pathology, 2085 Adelbert Road, Cleve-land, OH 44106, USA; Tel: (216) 368-3670; Fax: (216) 368-8964;E-Mail: mas21@po.cwru.edu.

Free Radical Biology & Medicine, Vol. 33, No. 11, pp. 1475–1479, 2002Copyright © 2002 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/02/$–see front matter

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site of oxidative damage is the neuronal cytoplasm. Toaddress this directly, we focused on oxidative damagethat does not make biological molecules refractory todegradation.

Protein cross-linking, part of the spectrum of reactivecarbonyl-related modifications, not only renders themodified protein more resistant to degradation but alsocompetitively inhibits the proteosome [15]. While theseproperties may underlie the accumulation of ubiquitinconjugates in neurons in AD [16,17], they make use ofcrosslink-related modifications problematic for the eval-uation of recent oxidative damage. Protein nitration is anon-crosslink-related oxidative modification of proteinresulting from either peroxynitrite attack or peroxidativenitration. In investigating the distribution of nitrotyrosinein AD, we found that the major site of nitrotyrosine wasin the cytoplasm of non-NFT-containing neurons [18]and that neurons containing NFT actually showed lowerlevels of nitrotyrosine than similar neurons lacking NFT.These relationships were confirmed when we examinedRNA, a cellular component with a relatively rapid turn-over rate. A major oxidation product of RNA, 8-hydrox-yguanosine (8OHG), has a distribution similar to nitro-tyrosine, except that it is absent from NFT and reduced inthe surrounding cytoplasm [19] even though NFT con-tain associated RNA [20]. The concurrence of RNA andprotein damage suggests that the major site of oxidativedamage in AD is in the neuronal cytoplasm. Therefore, itappears that the association of some oxidation markerswith NFT is, in part, due to their accumulation of dam-age, as discussed earlier, and, as outlined later, the sus-ceptibility of component proteins to regulated oxidativeadduction.

SOURCE OF REACTIVE OXYGEN SPECIES

One of the issues critical to evaluating the mecha-nisms underlying the damage is its source. Both locationand type of damage are important to understand. First,the location of damage, which involves every category ofbiomacromolecules, is restricted to neurons. Classically,nitrotyrosine is considered the product of peroxynitriteattack of tyrosine and 8OHG the product of •OH attack ofguanosine. However, the separation is not simple; nitro-tyrosine can be formed from peroxidative nitration bynitrite and H2O2 and peroxynitrite has •OH-like activity,meaning it can yield 8OHG. In the case of peroxidativenitration, treating tissue sections with nitrite and H2O2

yields increased nitrotyrosine of the same distributionfound during the disease in AD, but not control, cases(Perry and Smith, unpublished observation). An issuewith peroxynitrite is diffusibility, being the result of the

fusion of NO and O2�; it can diffuse several cell diam-

eters from its source to attack vulnerable target proteins[21]. In AD, one of the most striking findings is therestriction of damage to the cell bodies of vulnerableneurons. Although amyloid-� deposits and NFT containredox-active iron, like oxidative damage, the most con-spicuous changes in iron are within the cytoplasm ofvulnerable neurons [22,23]. Significantly, cytoplasmicredox-active iron is barely detectable in controls. Redox-active iron is the critical element for Fenton chemistrygeneration of •OH from H2O2. Ultrastructural localiza-tion of iron shows it is diffusely associated with thecytoplasm primary in the endoplasmic reticulum, butalso in granules identified as lipofuscin as well as theirassociated vacuoles.

Lipofuscin is thought to represent the terminal phaseof autophagic lysosomes that involve iron-rich mito-chondria [24]. Therefore, the increased redox-active ironin such lysosomes in AD lends credence to the notion ofmitochondrial abnormalities in AD. To examine thisissue further, we performed in situ hybridization formitochondrial DNA (mtDNA). mtDNA, as well as theprotein cytochrome oxidase-1, was increased several-fold in vulnerable neurons in AD. Ultrastructural exam-ination showed both markers were in mitochondria, and,in the case of mtDNA, in vacuoles associated with lipo-fuscin—the same sites showing increased redox-activeiron. It is tempting to suggest that electron-dense lipo-fuscin may play a role in modulating metal release frommitochondrial turnover. Ultrastructural examination ofthe site of oxidative damage points to mitochondria. Wenote that the majority is in the endoplasmic reticulum,suggesting that a role for mitochondria is probably not todirectly supply •OH but instead to supply its precursors,H2O2 and redox-active metals. While the proposedmechanism is distinct from nonmetal-catalyzed per-oxynitrite formation, it does not discount an importantrole for nitric oxide (NO). Neurons in AD show activa-tion of nitric oxide synthetase and its modulator, di-methylargininase [25]. NO has strong antioxidant activ-ity (see below) and inhibitor activity for cytochromes.The latter could play a role in the hypometabolism con-sistently found in AD [26] and the altered mitochondrialdynamics noted here.

RELATIONSHIP TO LESIONS

At the same time oxidative damage was established inAD, the putative source of the reactive oxygen wassupposed to be the lesions. Amyloid-� by itself wasproposed to generate reactive oxygen [27]. This mecha-nism has fallen into question for both chemical andbiological issues [28]. Nevertheless, amyloid-�, undersome circumstances, can bind iron and promote catalytic

1476 G. PERRY et al.

redox cycling yielding reactive oxygen [29]. Therefore,it was a surprise when we noted that in vivo oxidativedamage is inversely correlated to amyloid-� load, indi-cating that, rather than being a source of the reactiveoxygen, amyloid-� may be a modulator that can eitherincrease or decrease reactive oxygen production [30–32]. Furthermore, the relative paucity of short-lived ox-idative changes [19], rather than those that accumulate inlong-lived proteins [3], surrounding amyloid-� depositsalso puts into question the idea that reactive oxygenresulting from inflammation is an important mechanismfor oxidative damage. In fact, while inflammation in ADis well established [33], it appears to be a secondaryresponse to the underlying pathological changes. Alter-natively, NO resulting from microglia may be playing anantioxidant role [25].

COMPENSATORY CHANGES

Accumulation of � in neurofibrillary tangles is asso-ciated with the induction of heme oxygenase-1, a potentantioxidant that is the rate-limiting step to convert heme(pro-oxidant) to bilirubin (antioxidant). Heme oxygenasecould play a critical role in metabolizing the heme re-leased from mitochondrial turnover and, as such, reduceoxidative damage. However, there is further complexityof the system, since the � accumulations in AD are alsooxidatively damaged. In studies, performed with normalhuman �, we found phosphorylated (but not nonphospho-rylated) � is susceptible to oxidative damage by hy-droxynonenal [34]. Reaction with hydroxynonenal yieldsthe molecular rearrangement in �, defined by recognitionby the monoclonal antibody Alz50 that is the precursorof neurofibrillary tangle formation [34]. The activation ofthe entire stress-activated phosphorylation pathway [35–38] in neurons prior to neurofibrillary tangle formationgives plausibility to the suggestion that � phosphoryla-tion and heme oxygenase-1 activation are regulated fea-tures of the neuronal reaction to stress that may haveadaptive value [39]. Seen in this light, the amyloid-�deposits and � accumulations may be important adaptiveresponses.

The regulation by phosphorylation over both � [34]and neurofilament [40] oxidative adduction suggests thatthe broad spectrum of damage we have found in AD mayalso be regulated. Critically, neurons in AD are not deador even dying in the immediate sense [41]. It is temptingto view the oxidative damage as well as the supposedpathological lesions [30] as regulators that protect criti-cal systems from failure [42,43]. Certainly, more work isnecessary to understand the fine control over oxidativehomeostasis.

ARE THE FINDINGS OF ALZHEIMER’S DISEASE

GENERAL?

The widespread nature of oxidative damage in ADcan lead one to think that most degenerative diseases willshow the same spectrum of changes. From our ownobservations, nothing could be farther from reality. In noother condition, with the exception of normal aging, havewe found the full spectrum of damage to neuronal mac-romolecules that occurs in AD. This includes analysis ofamyotrophic lateral sclerosis, prion disease, progressivesupranuclear palsy, and Parkinson’s disease [44–50].These observations have led us to consider that eachdisease leads to a different homeostatic balance, one thatis finely tuned to minimize cell death. Since the under-lying pathophysiology is distinct for each of these con-ditions, the rules for each will be distinct. AD, as thearchetype age-related degenerative disease, differs fromthe others in having an exponentially increasing inci-dence with age [51]. Clearly, more studies are necessaryto understand the role of oxidative stress in each disease.

Acknowledgements — Work in the authors’ laboratories is supportedby funding from the National Institutes of Health (NS38648, AG19356,AG14249) and the Alzheimer’s Association (IIRG-98-136, ZEN-99-1789, IIRG-00-2163-Stephanie B. Overstreet Scholars, IIRG-98-140,TLL-99-1872).

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ABBREVIATIONS

A�PP—amyloid-� protein precursorAD—Alzheimer’s diseaseAGE—advanced glycation end products8OHG—8-hydroxyguanosineIBM—inclusion body myositismtDNA—mitochondrial DNANFT—neurofibrillary tanglesNO—nitric oxide

1479Oxidative stress and neurodegenerative disease