Hypothesis Paper

HOW IMPORTANT IS OXIDATIVE DAMAGE? LESSONS FROMALZHEIMER’S DISEASE

GEORGE PERRY,* A RUN K. RAINA ,* A KIHIKO NUNOMURA,*,‡ TAKAFUMI WATAYA ,*,§ LAWRENCE M. SAYRE,†

and MARK A. SMITH**Institute of Pathology and†Department of Chemistry, Case Western Reserve University, Cleveland, Ohio, USA;‡Department of

Psychiatry and Neurology, Asahikawa Medical College, Asahikawa, Japan and§Department of Neurology, Kyoto University,Kyoto, Japan

(Received24 September1999;Revised14 December1999;Accepted28 December1999)

Keywords—Alzheimer disease, Amyloid-b, Free radical, Glycation, 8-hydroxyguanosine, Lipid peroxidation,Nitration, Redox-active metals

INTRODUCTION

Historically, efforts to understand oxidative status havefocused on the concept of oxidative stress, i.e., thebreaching of oxidant defenses with consequent detri-ment. By this definition, detection of damage resultingfrom reactive oxygen indicates oxidative stress. We hy-pothesize that what this definition fails to consider is theability of living systems to dynamically regulate theirdefense mechanisms in response to oxidants. Therefore,increases in oxidative damage do not necessitate that thecell is succumbing to oxidative stress given that the cellmay have increased its defenses sufficiently to compen-sate for the increased flux of reactive oxygen responsiblefor the damage. This concept is critically outlined byevidence suggesting that cells that fail to compensate foroxidative imbalance (stress) enter apoptosis, which inturn leads to death within hours. Therefore, we suggestthat cells can only experience oxidative “stress” for shortperiods of time without rapidly dying [1,2]. Particularlygermane to the discussion of degenerative diseases thathave a course of years, those cells experiencing increasedoxidative damage by their continued existence testify totheir increased compensatory response to reactive oxy-gen. This is certainly the case for Alzheimer disease(AD), where damage to every category of macromole-cule is associated with increased sulfhydryls, inductionof heme oxygenase-1, and presence of Cu/Zn superoxide

dismutase [3]. Even those aspects of AD thought mostdeleterious, the pathological lesions, senile plaques andneurofibrillary tangles, may play an important aspect indefenses [4]. Quantitative analysis of the extent of oxi-dative damage in AD shows the oxidative damage isactually reduced in those neurons with the most cytopa-thology [4]. This suggests oxidative defenses extendbeyond the classical antioxidant enzymes and low mo-lecular weight reductants. A prominent role must also beplayed by the distinct structural and biochemical changesthat are associated with and considered part of the spec-trum of disease.

PROTEINS AS PROTECTIVE AGENTS

The importance of this aspect is brought to the fore-front if it is considered that protection of critical cellularcomponents from oxidants can be through the incorpo-ration of “damage” to others. At the present time, wegenerally utilize the exhaustion of cellular reductants asa measure of antioxidant potential; however, cellularmacromolecules may share a similar function. Consistentwith this view is the physiological modification of neu-rofilament heavy (NFH) subunit by carbonyls [5]. In-triguingly, although NFH has a long half-life, the sameextent of carbonyl modification is found throughout thenormal aging process as well as along the length of theaxon. NFH may be uniquely adapted as a carbonyl scav-enger due to its high lysine content (the sequence, lysine-serine-proline, is repeated approximately 50 times) onthe sidearm portion of the molecule, a domain that isexposed on the surface of a neurofilament structure by

Address correspondence to: George Perry, Ph.D., Institute of Pathol-ogy, Case Western Reserve University, 2085 Adelbert Road, Cleve-land, OH 44106, USA; Tel: (216) 368-2488; Fax: (216) 368-8964;E-Mail: gxp7@po.cwru.edu.

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extensive phosphorylation. Although more studies arerequired to understand the role of these modifications inneuronal homeostasis, it is tempting to consider them asaugmentations to the neuronal defenses important inprotecting the major site of neurofilaments, the axon,from some of the most toxic products of oxidation,reactive aldehydes. The slow turnover rate of proteins inthe axon, which can take years, may necessitate thisprotection. Maintenance of steady state levels of car-bonyl adduction suggests the existence of a mechanismfor metabolic regeneration of the neurofilament lysinesanalogous to the scavenging of reactive oxygen shownby methionine on the surface of many proteins andsubsequent regeneration of methione catalyzed by me-thionine sulfoxide reductase [6].

NUCLEIC ACIDS AS PROTECTIVE AGENTS

RNA is extensively modified in AD and, althoughclearly damaged, the rapid turnover of RNA may alsoserve a protective function. With the formation of

•

OH,every macromolecule will be susceptible to attack, butthe most critical aspect for the cell is to reduce damageto systems, such as enzyme active sites, whose com-promise will lead to death. Although RNA alterationmay lead to protein sequence anomalies [7], RNAdestruction can more easily be accommodated in cel-lular metabolism than damage to DNA [8,9] or en-zyme active site destruction. The large pool of neuro-nal RNA may even mean that errors in proteinsynthesis resulting from oxidatively modified RNAcan be corrected by metabolic turnover of abnormalprotein. Certainly, renewal of components is a com-mon theme in biology that, although energeticallywasteful, rids the cells of the consequences of damage.

ANALYSIS OF OXIDATIVE STATUS

In sum, we suggest that analyzing the spectrum ofdamage found in AD necessitates that we revise ourconcept of oxidative damage as a deleterious processwhen we view systems. It is imperative that we considerthe overall homeostatic system before we reach a deci-sion on the short-term, as well as long-term, conse-quences of that damage. If cells survive and functionwith extensive damage, it is unlikely to be damage tocritical systems. Therefore, we are advocating that de-termination of oxidative status be considered as the en-tire balance of oxidant and antioxidant function of a cell.Importantly, in the latter category, it is critical to recog-nize that the full extent of protection from oxidants isonly beginning to be appreciated. Therefore, use of asingle marker of oxidative status ignores the extensive

dynamism shown by all life forms when confronted byoxygen radicals.

MARKERS OF OXIDATIVE DAMAGE

Below, we review an array of in situ detection meth-ods that can be used for the qualitative and semiquanti-tative assessment of various indices related to oxidativestress. The importance of in situ methods over bulkanalysis cannot be overstated when considering thestructural and cellular complexity of disease pathology, afeature particularly striking in neurodegenerative dis-eases where the vulnerable neurons are in the minority.Indeed, in situ detection allows for the detection ofspecific cell types affected or specific subcellular local-ization. Therefore, a process affecting only a small per-centage of the tissue or cells can be readily visualized.Consequently, a positive signal in situ indicates reallevels that cannot be masked by unrelated or compensa-tory responses in adjacent cells. Alternatively, damage tolong-lived proteins, e.g., blood vessel basement mem-brane, provides a cumulative record of long-term oxida-tive insult. Yet the same properties that make long-livedstructures or molecules ideal monitors for aging limitstheir sensitivity in detecting disease-specific changes un-less using in situ techniques. In general, when looking atin situ detection methods, it is essential that the modifi-cations analyzed be both stable and not produced as aresult of time-dependent postexperimental changes suchas postmortem interval or sample preparation.

Nitrotyrosine: A marker of protein tyrosine nitration

Because oxidative stress is associated with high localconcentrations of both superoxide and nitric oxide, pro-duced by the inducible isoform of nitric oxide synthase,the product of their combination, peroxynitrite, has be-come an important secondary oxidative stress marker. Inthe presence of buffering bicarbonate, peroxynitriteforms a CO2 adduct, which augments its reactivity. For-mation of 3-nitrotyrosine by this route has become theclassical protein marker specifically for the presence ofperoxynitrite [10]. However, this conclusion must beseen in light of recent findings that point to “peroxida-tive” nitration from NO2

2 and H2O2 in the presence ofsuitable metalloproteins, e.g., myeloperoxidase [11].Controls consist of (i) omitting the primary antibody; (ii)absorption of the antibody with nitrated proteins or pep-tides; and (iii) chemical reduction of nitrotyrosine bysodium hydrosulfite [12] prior to immunostaining. Theseprocedures are performed in parallel with the antisera toknown markers as controls against artifactual inactiva-tion of either primary or secondary antibodies from theuse of sodium hydrosulfite-reduced sections.

832 G. PERRY et al.

Advanced glycation and lipoxidation end products

Oxidative stress can result in oxidized sugar deriva-tives (glycoxidation), which can subsequently modifyproteins through processes such as those involved in theformation of advanced glycation end products via theMaillard reaction. Similarly, and of more general impor-tance, oxidative stress results in lipid peroxidation andthe production of a range of electrophilic and mostlybifunctional aldehydes that modify numerous proteins.Although there are a number of chemical events thatoccur early and rapidly, many of these are reversible andthus of less pathophysiologic significance. Thus, manyworkers in the field have focused their efforts on identi-fying the more stable protein modifications that formover time, referred to as advanced lipoxidation end prod-ucts. Protein modification can result in both noncross-link and cross-link modifications, the latter arising fromthe potential reactivity of bifunctional lipid-derived mod-ifiers such as 4-hydroxy-2-nonenal (HNE) and malondi-aldehyde.

2,4-Dinitrophenylhydrazine reactivity of protein-boundvs. protein-based carbonyls

The 2,4-dinitrophenylhydrazine (DNP-H) method canyield in situ detection of protein-based reactive carbonyls[13,14]. It is important to recognize that protein-boundcarbonyls can represent either oxidized side-chains or theunivalent adduction of a bifunctional glycoxidation orlipoxidation product such that the adduct still contains afree carbonyl group. Modification of unoxidized lysineamino groups by monofunctional lipid- or sugar-derivedcarbonyl compounds will not interfere because DNP-Hwill displace the carbonyl compound into bulk solution.

Chemical and immunochemical controls are used todefine carbonyl-specific binding. Chemical reduction offree carbonyls and Schiff bases is performed by incubat-ing sections with sodium borohydride, whereas selectivereduction of Schiff bases as opposed to free carbonylscan be achieved by incubation with sodium cyanoboro-hydride. Immunochemical specificity is demonstrated byomission of the DNP-H treatment or the antibody toDNP. Immunoabsorption of the antibody to DNP isperformed by incubating the antibody with pyruvate 2,4-dinitrophenylhydrazone and comparing the resulting im-munoreactivity with unabsorbed antibody.

Nucleic acid damage

Oxidative damage to nucleic acids results in basemodification, substitutions, and deletions. Among themost studied modifications, 8-hydroxyguanosine(8OHG) is considered a signature of oxidative damage to

nucleic acid and is prominent in AD. The specificity ofantibodies to 8OHG is confirmed by comparison withsections in which the primary antibody (i) was omitted or(ii) was absorbed with purified 8OHG [15]. Treatmentwith DNase or RNase before incubation with 8OHGantibody can be used to determine the primary nucleicacid target of oxidative damage.

Mitochondrial DNA is also highly susceptible to ox-idative attack as shown by isolation of mtDNA [8] aswell as in in vitro experiments where amyloid-b toxicitywas associated with mtDNA fragmentation [16].

Cellular response factors

Cells are not passive to increased oxygen radicalproduction but rather upregulate protective responses.Although the major features of antioxidant defense aredetermined by the basic building blocks of cells and diet,oxidation leads to the induction of specific changes. Forexample, heme oxygenase-1, an antioxidant, convertsheme, a pro-oxidant, to biliverdin/bilirubin (antioxi-dants) [17,18]. Given the importance of iron as a catalystfor the generation of reactive oxygen species, changes inproteins associated with iron homeostasis can be used asan index of a cellular response. One such class of pro-teins, the iron regulatory proteins (IRP) respond to cel-lular iron concentrations by regulating the translation ofproteins involved in iron uptake, storage, and utilization[19,20]. Therefore, IRPs are viewed as the central controlof cellular iron concentration.

Additionally, cells may increase sulfhydryl reductionas a means of detoxifying reactive intermediates. Thiscompensation can be detected in tissue by treating sec-tions with tagged N-ethylmaleimide [21] to mark reac-tive sulfhydryls. The specificity of the N-ethylmaleimidelabelling can be verified by prior treatment of the tissuewith untagged N-ethylmaleimide.

CONCLUSIONS

In this Hypothesis Paper, we present methods to studyoxidative damage in AD. We discuss their application toother systems and we consider what we have learnedabout oxidative status from their use. Although seem-ingly simple, one must appreciate that most markers ofdamage lie in the context of a balance between oxidantsand antioxidants. Therefore, neuronal cells affected byAD, despite showing increased oxidative damage, mayactually be in homeostatic balance. This means thatdetection of increased oxidative damage, in cells thatsurvive, must be associated with a commensurate in-crease in compensatory mechanisms. Additionally, notonly is it critical to examine antioxidant responses such

833Neurodegenerative diseases

as heme oxygenase-1 and increases in free sulfhydryls, butit is also critical to examine whether some of the “patho-logical” responses are a part of the increase in defense. InAD, we find that the pathological lesions of the disease,senile plaques and neurofibrillary tangles, as well as neuro-filament proteins, appear to play a role in the protection ofneurons from oxidation. In summary, our studies indicatethat the study of single markers of oxidative damage outsidethe context of oxidative balance is probably not sufficient todetermine oxidative status.

Acknowledgements— Work in the authors’ laboratories was funded bythe National Institutes of Health, the Alzheimer’s Association and theAmerican Health Assistance Foundation.

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ABBREVIATIONS

AD—Alzheimer diseaseDNP-H—2,4-dinitrophenylhydrazine8OHG—8-hydroxyguanosineHNE—4-hydroxy-2-nonenalIRP—iron regulatory proteinsNFH—neurofilament heavy subunit

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