26 SMITH ET AL.

© 1999 Wiley-Liss, Inc.

DRUG DEVELOPMENT RESEARCH 46:26–33 (1999)

Research Overview

Mitochondrial Abnormalities: A Primary Basis forOxidative Damage in Alzheimer’s Disease

Mark A. Smith,1* Keisuke Hirai,1 Akihiko Nunomura,1,2 and George Perry1

1Institute of Pathology, Case Western Reserve University, Cleveland, Ohio2Department of Psychiatry and Neurology, Asahikawa Medical College, Asahikawa, Japan

ABSTRACT The most striking feature of Alzheimer’s disease (AD) is the number of abnormalitiesaffecting essentially every aspect of brain homeostasis. Recent work suggests that increased oxidativestress that damages lipids, proteins, and nucleic acids and results in the accumulation of redox-activemetals may be responsible for the diversity of systems involved. Interestingly, all of the genetic factors, β-protein precursor, presenilins, and apolipoprotein E, have been linked to reactive oxygen species produc-tion or apoptosis, a process intimately associated with oxidative stress. This leaves open the question ofwhy oxidative damage is increased in AD. In studies of mitochondria, we demonstrated increased mito-chondrial DNA specifically in vulnerable neurons in cases of AD, suggesting that AD is marked by afundamental abnormality in neuronal metabolism. Oxidative stress, therefore, seems to be the elementlinking the multitude of changes in Alzheimer’s disease to a fundamental metabolic deficiency. Drug Dev.Res. 46:26–33, 1999. © 1999 Wiley-Liss, Inc.

Key words: Alzheimer’s disease; oxidative damage; mitochondrial abnormalities

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INTRODUCTION

Damage by reactive oxygen is not only found inthe proteins of neurofibrillary tangles (NFTs) and se-nile plaques of Alzheimer’s disease (AD) but also inthe cytoplasm of neuronal populations vulnerable todeath during the course of AD [Smith et al., 1994a,b,1995a–c, 1996a,b, 1997a,b; Sayre et al., 1997a]. Tounderstand the significance of reactive oxygen dam-age, investigators have been studying two crucial as-pects: (1) the relationship between oxidative stress andother factors, established by genetic or epidemiologicstudies to play an important role in AD; and (2) themetabolic basis for increased reactive oxygen damage.Here, we present evidence that oxidative damage is acentral process in neurodegeneration in AD that linkstogether the diverse factors important for disease ini-tiation and progression. Indeed, not only does oxida-tive damage occur before formation of the pathologiclesions, thereby making it one of the earliest cyto-pathologic markers of neuronal dysfunction in AD, but

it further serves as a marker to uncover the underlyingorigin of AD, which may be a metabolic disorder.

AD ALTERS ESSENTIALLY EVERY FACET OF BRAINHOMEOSTASIS: AGING AND OXIDATIVE STRESS

The pathologic presentation of AD, the leading causeof senile dementia, involves regionalized neuronal deathand an accumulation of intraneuronal and extracellularlesions termed NFTs and senile plaques, respectively [re-viewed in Smith, 1998]. Several independent hypotheseshave been proposed to link the pathologic lesions and neu-ronal cytopathology with, among others, apolipoprotein Egenotype [Corder et al., 1993; Roses, 1995], hyperpho-sphorylation of cytoskeletal proteins [Trojanowski et al.,

Grant sponsor: National Institutes of Health; Grant number:AG09287; Grant sponsor: American Health Assistance Foundation(AHAF); Grant sponsor: Alzheimer’s Association.

*Correspondence to: Mark A. Smith, Institute of Pathology,Case Western Reserve University, 2085 Adelbert Road, Cleveland,OH. E-mail: mas21@po.cwry.edu

MITOCHONDRIAL ABNORMALITIES IN ALZHEIMER’S DISEASE 27

1993a], and amyloid-β metabolism [Selkoe, 1997]. How-ever, not one of these theories alone is sufficient to explainthe diversity of abnormalities found in AD that involves amultitude of cellular and biochemical changes. Further-more, attempts to mimic AD by a perturbation of one ofthese elements using cell or animal models, includingtransgenic animals, do not result in the same spectrum ofpathologic alterations. Perhaps the most striking exampleof this is that while amyloid-β plaques are deposited insome transgenic rodent models overexpressing β-proteinprecursor, there is no neuronal loss [Irizarry et al.,1997a,b]—a seminal feature of AD. This diversity ofchanges suggests that AD stems from a shift in neuronalmetabolism, paralleling the key feature of AD, aging.

AD is a disease with a prevalence that increasesexponentially with aging [Katzman, 1986] (Fig. 1). Im-portantly, this holds true even in individuals with a ge-netic predisposition, i.e., those individuals with anautosomal dominant inheritance of AD or in individualswith Down’s syndrome who develop the pathology of ADin middle age. The free radical theory of aging [Harman,1956] hypothesizes that the aging process is associatedwith multisystem failure due to oxidative damage causedby an imbalance between reactive oxygen production andantioxidant defenses. A corollary of this theory is thataging results in compromised ability to deal with patho-logic sources of reactive oxygen. Furthermore, the abil-ity of reactive oxygen to modify lipid proteins and nucleicacids leaves cells open to multiple system failure or, morecorrectly for those surviving cells, multiple compensa-tions to maintain homeostatic balance.

OXIDATIVE DAMAGE AND RESPONSE

An exact determination of the contribution of eachsource of reactive oxygen is complicated, if for no otherreason than that most sources have positive feedback.Nonetheless, the overall result of unchecked oxygen radi-cals is damage. Such damage found in AD includes ad-vanced glycation end-products [Smith et al., 1994a;Ledesma et al., 1994; Vitek et al., 1994; Yan et al., 1994],

nitration [Good et al., 1996; Smith et al., 1997a], lipidperoxidation adduction products [Montine et al., 1996a;Sayre et al., 1997a], as well as carbonyl-modifiedneurofilament protein and free carbonyls [Smith et al.,1991, 1995b, 1996a]. Importantly, this damage involvesall neurons in populations vulnerable to death in AD, notjust those containing NFTs. What is particularly strikingis that the neurons displaying oxidative damage show noovert signs of degeneration.

The cytopathologic significance of oxidative dam-age is seen by the up-regulation of antioxidant enzymessuch as heme oxygenase-1 [Smith et al., 1994b; Schipperet al., 1995; Premkumar et al., 1995] and superoxidedismutase-1 [Pappolla et al., 1992] in neurons with NFTs.Furthermore, neurons with NFTs accumulate dimethyl-argininase [Smith et al., 1998a], a regulator whose enzy-matic activity removes methylarginine-related inhibitionof nitric oxide synthetase, thus increasing nitric oxide pro-duction. Because nitric oxide reacts with superoxide toform peroxynitrite, increased nitric oxide can be seen asa possible response to increased reactive oxygen. Quan-titative immunocytochemical studies of cases of AD showthat there is a complete overlap between neurons up-regulating heme oxygenase-1 and Alz50, an early markerof τ abnormalities, indicating that cytoskeletal abnormali-ties are associated with increased oxidative stress or viceversa [Smith and Perry, unpublished observation].

PHOSPHORYLATION AS A RESPONSE TOOXIDATIVE STRESS

The mechanisms by which normal soluble cyto-skeletal elements such as τ and neurofilaments are trans-formed into insoluble paired helical filaments is animportant issue [Selkoe et al., 1982; Smith et al., 1996b].Insolubility has been linked to abnormal phosphoryla-tion, the most well known posttranslational change of τ[Goedert et al., 1991; Greenberg et al., 1992], and a num-ber of specific kinases and phosphates have been impli-cated [reviewed in Trojanowski et al., 1993a]. However,while increased phosphorylation decreases microtubulestability, a salient feature of the pathology of AD [Perryet al., 1991; Alonso et al., 1994, 1996; Iqbal et al., 1994;Praprotnik et al., 1996a,b], NFT insolubility is not medi-ated by phosphorylation [Smith et al., 1996b]. Indeed, invitro phosphorylation of normal τ or complete dephos-phorylation of NFT has no effect on their solubility[Goedert et al., 1991; Greenberg et al., 1992; Gustke etal., 1992; Smith et al., 1996b]. Moreover, recent studiessuggest the τ phosphorylation found in AD may be partof a novel process similar to that seen during mitosis [Popeet al., 1994; Preuss et al., 1995], suggesting that neuronsaffected in the disease might be abortively entering thecell cycle [Vincent et al., 1996; McShea et al., 1997, 1999].

Phosphorylation is intimately tied to oxidative stressFig. 1. The prevalence of Alzheimer’s disease is strictly age dependent.Based on the 1998 United States Government Accounting Office report.

28 SMITH ET AL.

by the extracellular receptor kinase (ERK) pathway[Guyton et al., 1996] and by activation of the transcrip-tion factor NFκB [Schreck et al., 1991]. Although thereis controversy concerning the kinases involved in thephosphorylation of τ in AD, the ERK pathway is impli-cated [Ledesma et al., 1992; Trojanowski et al., 1993b;Hyman et al., 1994]. In studies with antibodies specificto activated ERK, we found activity in all pyramidal hip-pocampal neurons in cases of AD but none in controls(Fig. 2). Therefore, abnormal phosphorylation of proteinsin AD may not only be a consequence of oxidative stressbut also a protective response that neurons use to evadethe onset of apoptotic death brought on by oxidativestress. The increased oxidative damage in vulnerableneurons in AD brings neurons to a point of decidingwhether to accept oxidative death through apoptosis,marked by the JNK pathway [Gardner and Johnson,1996], or instead, enter the ERK pathway.

REACTIVE OXYGEN LINKS GENETICS TO ADPATHOGENESIS

A number of mechanisms have been suggested forthe neurotoxicity of amyloid-β [Yankner et al., 1990; re-viewed in Iversen et al., 1995; Sayre et al., 1997b], in-cluding membrane depolarization [Carette et al., 1993],increased sensitivity to excitotoxins [Koh et al., 1990], andalterations in calcium homeostasis [Mattson et al., 1992];

however, the influences of amyloid-β and other geneticfactors on AD may be through their effect on oxidativestress (Fig. 3). Indeed, the leading hypothesis is that neu-ronal damage by amyloid-β is mediated by free radicalsand, as such, can be attenuated using antioxidants suchas vitamin E [Behl et al., 1992, 1994] or catalase [Lockhartet al., 1994; Zhang et al., 1996]. Furthermore, amyloid-βis reported to spontaneously generate peptidyl radicals[Butterfield et al., 1994; Goodman et al., 1994; Harris etal., 1995; Prehn et al., 1996], and mutations in β-precur-sor protein are associated with increased DNA fragmen-tation, possibly involving oxidative mechanisms (seebelow). Critically addressing whether amyloid-β initiatesoxidative damage in AD requires careful study of the re-lationship of amyloid-β deposition and increased oxida-tive damage. In this regard, it is extremely interestingthat the recently reported transgenic rodent models ofamyloid-β deposition [Games et al., 1995; Hsiao et al.,1996] show the full spectrum of oxidative changes of AD[Smith et al., 1998b; Pappolla et al., 1998].

Presenilins 1 and 2 [Sherrington et al., 1995; Selkoe,1997] are genetic factors in which the biological mecha-nism, although not established, may also involve oxida-tive damage. Increased presenilin 2 expression increasesDNA fragmentation and apoptotic changes [Wolozin etal., 1996], both important consequences of oxidative dam-age. Apolipoprotein E, in brains and cerebrospinal fluid,is found adducted with the highly reactive lipidperoxidation product, hydroxynonenal [Montine et al.,1996b]. Furthermore, apolipoprotein E is a strong chela-tor of copper and iron, important redox-active transitionmetals [Miyata and Smith, 1996]. Finally, interaction ofapolipoprotein E with amyloid-β only occurs in the pres-ence of oxygen [Strittmatter et al., 1993].

APOPTOSIS

In programmed cell death, i.e., apoptosis, cells aredigested within their own membrane by proteases andnucleases as well as by increased reactive oxygen. How-

Fig. 2. Hypothetical time line of the alterations in neuronal structure inAlzheimer’s disease (AD). With the onset of AD, normal neurons developnumerous forms of oxidative damage, including nitration (nitrotyrosine),protein oxidation (free carbonyls), and lipid peroxide adducts (lipidperoxidation) prior to the formation of neurofibrillary tangles (pre-NFTs)that evolved into intracellular (I-NFT) and, consequenctly, extracellular(E-NFT) NFTs. Coincident with oxidative damage, extracellular receptorkinase (ERK) is activated. These findings suggest that phosphorylation ab-normalities may be a result of oxidative damage.

Fig. 3. The primary epidemiologic and genetic factors implicated inAlzheimer’s disease are directly linked to oxidative stress.

MITOCHONDRIAL ABNORMALITIES IN ALZHEIMER’S DISEASE 29

ever, without the full range of morphologic changes, it isunclear whether DNA fragmentation is apoptotic or is,instead, mediated solely by oxidative damage to DNAand resulting repair [Berlin and Haseltine, 1981]. Therelative contribution of oxidative stress-related versusapoptosis-related DNA fragmentation in AD is unre-solved. Yet, bearing on this issue, the relative infrequencyof apoptosis defined by morphology [Su et al., 1994;Cotman and Su, 1996] and the broad findings of frag-mentation in all cells in cases of AD argues for wide-spread oxidative DNA damage rather than widespreadapoptosis. Certainly, this interpretation is consistent withthe oxidative nuclear damage in all cells of the brain inareas affected in AD [Smith et al., 1996a, 1997b].

WHAT IS THE INITIAL SOURCE OF INCREASEDREACTIVE OXYGEN PRODUCTION IN AD?

Reactive oxygen is a ubiquitous byproduct of bothoxidative phosphorylation and the myriad of oxidases nec-essary to support aerobic metabolism. In AD, in additionto this background level of reactive oxygen, there are anumber of additional contributory sources that are thoughtto play an important role in the disease process: (1) Iron,in a redox-active state, is increased in NFTs as well as inamyloid-β deposits [Good et al., 1992; Smith et al., 1997b].Iron catalyzes the formation of ·OH from H2O2 as well asthe formation of advanced glycation end-products. Fur-thermore, aluminum, which also accumulates in neu-rofibrillary tangle-containing neurons [Good et al., 1992],stimulates iron-induced lipid peroxidation [Oteiza, 1994].(2) Activated microglia, such as those that surround mostsenile plaques [Cras et al., 1990], are a source of NO andO2

–· [Colton and Gilbert, 1987], which can react to formperoxynitrite, leaving nitrotyrosine as an identifiablemarker [Good et al., 1996; Smith et al., 1997a]. (3) Amy-loid-β itself has been directly implicated in reactive oxy-gen formation through peptidyl radicals [Butterfield et al.,1994; Hensley et al., 1994; Sayre et al., 1997b]. (4) Ad-vanced glycation end-products in the presence of transi-tion metals (see above) can undergo redox cycling withconsequent reactive oxygen species production [Baynes,1991; Yan et al., 1994, 1995]. Additionally, advancedglycation end-products, as well as amyloid-β, activate spe-cific receptors such as the receptor for advanced glycationend-products (RAGE) and the class A scavenger-receptorto increase reactive oxygen production [Yan et al., 1996;El Khoury et al., 1996]. (5) Abnormalities in the mitochon-drial genome [Corral-Debrinski et al., 1994; Davis et al.,1997] or deficiencies in key metabolic enzymes [Sorbi etal., 1983; Sheu et al., 1985; Sims et al., 1987; Blass et al.,1990; Parker et al., 1990] suggest that metabolic abnor-malities affecting mitochondria may be the major and pos-sible initiating source of reactive oxygen in AD.

Although there is support for all listed sources of re-

active oxygen, with the exception of the last, mitochondrialabnormalities, they should only be operative after forma-tion of senile plaques and NFTs. Yet as stated earlier, neu-ronal oxidative damage occurs in the absence of NFTs oradjacent senile plaques. In consideration of the low diffu-sion of reactive oxygen through tissue, these observationspoint to a source within neuronal soma, again supportingmitochondria. Although while there has been considerablecontroversy regarding the role of mitochondrial inheritancein AD [Davis et al., 1997; Hirano et al., 1997; Hutchin et al.,1997; Wallace et al., 1997], we took the view that if mito-chondria are important in increased reactive oxygen gen-eration, mitochondria should show abnormalities in AD. Toassess this issue, we performed in situ hybridization witholigonucleotide probes recognizing wild-type as well asdamaged mitochondrial DNA (mtDNA), i.e., with the 4977-kb common deletion. We found that vulnerable neurons,but no other neurons or glia, showed striking increases inmtDNA [Hirai et al., 1998a,b]. These findings support reac-tive oxygen from mitochondria as the initial source of oxida-tive damage in AD and the earliest biochemical abnormality.Whether the biochemical abnormality is reflected cytologi-cally as increased mitochondria requires further ultrastruc-tural study of neurons in AD cases compared withage-matched controls. Unfortunately, all historical studiesof AD compared neurons with NFTs with those lacking themand presumed neurons lacking NFTs were unaffected bythe disease. Our results invalidate that assumption and showthat all vulnerable neurons are affected.

Although we do not know the reason for mtDNAincrease in AD, its restriction to large neurons suggeststhe increase might be linked to the energy demands ofneurons. That the increased mtDNA is only found in casesof AD, where amyloid-β is also increased, and that amy-loid-β affects a decrease in mitochondrial potential [Prehnet al., 1996] leaves open the possibility that amyloid-βbased mitochondrial damage may be at the heart of thematter. Further studies explicitly addressing the relation-ships between increased mtDNA and amyloid-β depos-its in normal aging and AD are clearly required.

THERAPEUTICS

Oxidative damage resulting, totally or in part, frommitochondrial abnormalities is the earliest cytopathologicand biochemical change of AD. Therefore, it is not sur-prising that agents that inhibit free radical formation[Schinetti et al., 1987; Hamburger and McCay, 1990;Jones et al., 1995; Rong et al., 1996; Wu et al., 1996; Behlet al., 1997; Igawa et al., 1997; Neuzil et al., 1997; Rippleet al., 1997; Shoda et al., 1997; Vane and Botting, 1997]reduce both the incidence and the progression of AD(Fig. 4) [McGeer and Rogers, 1992; Rogers et al., 1993;Breitner et al., 1994; Münch et al., 1994, 1997; Rich etal., 1995; Colaco et al., 1996; Kanowski et al., 1996;

30 SMITH ET AL.

Smalheiser and Swanson, 1996; Stoll et al., 1996; Thal etal., 1996; Henderson, 1997; Kawas et al., 1997;Papasozomenos, 1997; Sano et al., 1997; Shoda et al.,1997; Skolnick, 1997; Stewart et al., 1997]. This relation-ship, together with the efficacy of metal chelation treat-ment [McLachlan et al., 1991], strongly suggest thatoxidative stress is a central process in AD and that agentsthat prevent oxidative damage will be particularly effica-cious in the treatment for the disease.

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