Mark A. Smith and George Perry

Case Western Reserve University, Institute of Pathology, 2085 Adelbert Road, Cleveland, Ohio 44106, USA

ABSTRACT

Oxidative damage not only occurs to the proteins comprising the lesions of Alzheimer disease, neurofibrillary tangles and senile plaques, but also precedes lesion formation in neurons at risk of death during the disease. While oxidative stress may not be the primary etiology of the disease, it precedes specific cellular and tissue damage that underlies the onset of dementia. Our assertion links Alzheimer disease to normal aging where oxidative stress has been implicated. Further, our idea is supported by epidemiological data linking the prevalence of Alzheimer disease to diet, an established link to longevity. Additionally, the proteins known from genetics to play a role in AD such as beta protein precursor, apolipoprotein E or presenilins either regulate apoptosis or bind transition metals - both important mechanisms related to oxidative stress. The clinical efficacy of reducing oxidative stress in Alzheimer disease is highlighted by the reduction in prevalence and progression by agents such as nonsteroidal anti-inflammatories, estrogen, and vitamin E which all share the one common feature of reducing oxidative stress.

Therefore, we suggest that oxidative stress is a central feature of the pathogenesis of Alzheimer disease and that treatments that specifically target sources of oxygen radicals may have particular therapeutic efficacy. Specifically, we suggest two broad based treatment strategies, based on preventing the generation of or ameliorating the effects of oxidative stress will be particularly effective.

1. Transition Metal Chelation Therapy: For example, deferoxamine or its derivatives.

2. Dietary Modification: Either (i) the development of water- and fat-soluble antioxidant approaches to increase antioxidants or (ii) reduce total calories and calories from fat.

BACKGROUND

Studies over the past five years have established oxidative stress and damage not only in the lesions of Alzheimer disease but also in neurons at risk of death (Smith et al., 1994a,b, 1995a-c, 1996a,b, 1997a,b; Sayre et al., 1997a). Current studies are establishing how oxidative stress is related to other possible causes of Alzheimer disease as well as whether oxidative stress is an initiator or is instead a result of the disease process (Figure 1). The distinction of where oxidative stress lies in this scheme is more than academic since it is essential to know whether reducing oxidative stress will have therapeutic value to protect brain function. Here we present evidence that oxidative damage is the earliest cytopathological marker of neuronal dysfunction in Alzheimer disease and, moreover, impinges on all proposed pathogenic risk factors and mechanisms implicated in Alzheimer disease.

FIGURE 1

The pathological presentation of Alzheimer disease, the leading cause of senile dementia, involves regionalized neuronal death and an accumulation of intraneuronal and extracellular lesions termed neurofibrillary tangles and senile plaques, respectively (reviewed in Smith, 1997). Several independent hypotheses have been proposed to link the pathological lesions and neuronal cytopathology with, among others, apolipoprotein E genotype (Corder et al., 1993; Roses, 1995); hyperphosphorylation of cytoskeletal proteins (Trojanowski et al., 1993a), and amyloid-ß metabolism (Selkoe, 1997). However, not one of these theories alone is sufficient to explain the diversity of biochemical and pathological abnormalities of Alzheimer disease which involves a multitude of cellular and biochemical changes. Furthermore, attempts to mimic the disease by a perturbation of one of these elements using cell or animal models, including transgenic animals, do not result in the same spectrum of pathological alterations. The most striking case is that while amyloid-ß plaques are deposited in some transgenic rodent models overexpressing ß-protein precursor (Hsiao et al., 1996), there is no neuronal loss - a seminal feature of Alzheimer disease.

What many of these theories have failed to incorporate is that Alzheimer disease is a disease of aging (Katzman, 1986). Importantly, this holds true even in individuals with a genetic predisposition, i.e., those individuals with an autosomal dominant inheritance of Alzheimer disease or in individuals with Down’s syndrome who develop the pathology of Alzheimer disease. Therefore, age is a clear contributor in 100% of Alzheimer disease cases, whatever the genetic background. The free radical theory of aging (Harman, 1956), hypothesizes that the aging process is associated with (i) an increase in the adventitious production of oxygen-derived radicals, i.e., reactive oxygen species (ROS), together with (ii) a concurrent decrease in the ability to defend against such ROS that leads to the accumulation of oxidatively-modified macromolecules. The decrease in ROS buffering capacity also leads to a compromised ability to deal with abnormal sources of ROS such as those associated with genetic predisposition and/or disease status.

Oxygen Radical Generation and Oxidative Stress in Alzheimer Disease

ROS production occurs as a ubiquitous byproduct of both oxidative phosphorylation and the myriad of oxidases necessary to support aerobic metabolism. In Alzheimer disease, in addition to this background level of ROS, there are a number of additional contributory sources that are thought to play an important role in the disease process: (1) Iron, in a redox-active state, is increased in neurofibrillary tangles as well as in amyloid-ß deposits (Good et al., 1992; Smith et al, 1997a). Iron catalyzes the formation of •OH from H₂O₂ as well as the formation of advanced glycation end products. Furthermore, aluminum, which also accumulates in neurofibrillary tangle-containing neurons (Good et al., 1992), stimulates iron-induced lipid peroxidation (Oteiza, 1994). (2) Activated microglia, such as those that surround most senile plaques (Cras et al., 1990), are a source of NO and O₂^- (Colton and Gilbert, 1987) which can react to form peroxynitrite, leaving nitrotyrosine as an identifiable marker (Good et al., 1996; Smith et al., 1997b). (3) Amyloid-ß itself has been directly implicated in ROS formation through peptidyl radicals (Butterfield et al., 1994; Hensley et al., 1994; Sayre et al., 1997b). (4) Advanced glycation end products in the presence of transition metals (see above) can undergo redox cycling with consequent ROS production (Baynes, 1991; Yan et al., 1994, 1995). Additionally, advanced glycation end products and amyloid-ß activate specific receptors such as the receptor for advanced glycation end products (RAGE) and the class A scavenger-receptor to increase ROS production (Yan et al., 1996; El Khoury et al., 1996). (5) Abnormalities in mitochondrial metabolism, such as deficiencies in key enzyme function, resulting in part from detection of the mitochondrial genome, suggest the mitochondria may be the major and possible initiating source of ROS (Davis et al., 1997).

An exact determination of the contribution of each source of oxidative stress is complicated if for no other reason than that most sources have positive feedback. Nonetheless, the overall result is damage including advanced glycation end products (Smith et al., 1994a), nitration (Smith et al., 1997b), lipid peroxidation adduction products (Sayre et al., 1997a) as well as carbonyl-modified neurofilament protein and free carbonyls (Smith et al., 1991; Smith et al., 1994a, 1995b, 1996a; Ledesma et al., 1994; Vitek et al., 1994; Yan et al., 1994; Montine et al., 1996a; Sayre et al., 1997a) with the involvement extending beyond the lesions to neurons not displaying obvious degenerative change. Oxidative crosslinking makes proteins not only insoluble (reviewed in Smith et al., 1995a; Smith et al., 1996b) but also resistant to proteolytic removal (Cras et al., 1995) by competitively inhibiting the proteosome (Friguet et al., 1994). Therefore, oxidative crosslinking may be significant in the accumulation of ubiquitin conjugates in neurofibrillary tangles (Perry et al., 1987) in the face of numerous proteolytic activities which are highly active against abnormal proteins (Smith and Perry, 1994). Indeed, it may not be coincidental that fibrillary inclusions found in neurodegenerative diseases other than Alzheimer disease are also extensively ubiquinated, e.g., Lewy/Pick bodies and Rosenthal fibers (Galloway et al., 1988; Manetto et al., 1989) and are also oxidatively modified (Castellani et al., 1995, 1996, 1997).

The cytopathological significance of oxidative damage is seen by the upregulation of the antioxidant enzyme heme oxygenase-1 in neurons with NFT (Smith et al., 1994b; Premkumar et al., 1995). Furthermore, in quantitative immunocytochemical studies, there is a complete overlap between heme oxygenase-1 and Alz50, an early marker of tau abnormalities, indicating that cytoskeletal abnormalities are associated with increased oxidative stress or vice versa (Smith and Perry, unpublished observation).

Is Oxidative Stress the Process that Ties Together the Seemingly Separable Events of Alzheimer Disease?

Age-related increases in oxidative stress may be the major contributor toward all aspects of Alzheimer disease. In other words, oxidative stress may provide a link between all of the currently accepted views regarding disease pathogenesis (Figure 2).

FIGURE 2

Oxidative Stress And Cytoskeletal Phosphorylation: The mechanisms by which normal soluble cytoskeletal elements, such as tau and neurofilaments, are transformed into insoluble paired helical filaments is an important issue (Selkoe et al., 1982; Smith et al., 1996b). Insolubility has been linked to the most well known posttranslational change of tau, abnormal phosphorylation (Goedert et al., 1991; Greenberg et al., 1992) and a number of specific kinases and phosphatases have been implicated (reviewed in Trojanowski et al., 1993a). However, while increased phosphorylation decreases microtubule stability, a salient feature of the pathology of Alzheimer disease (Perry et al., 1991; Alonso et al., 1994, 1996; Iqbal et al., 1994; Praprotnik et al., 1996a,b), NFT insolubility is not mediated by phosphorylation (Smith et al., 1996b). Indeed, in vitro phosphorylation of normal tau or complete dephosphorylation of NFT has no effect on their solubility (Goedert et al., 1991; Greenberg et al., 1992; Gustke et al., 1992; Smith et al., 1996b). Instead, recent studies suggest tau phosphorylation as found in Alzheimer disease may be part of a novel process similar to that seen during mitosis (Pope et al., 1994; Preuss et al., 1995) suggesting that it might be abortively entering the cell cycle (Vincent et al., 1996; McShea et al., 1997).

Phosphorylation is intimately tied to oxidative stress by the mitogen activated phosphorylation (MAP) pathway (Guyton et al., 1996) as well as through activation of transcription factor NFkB (Schreck et al., 1991). While there is controversy concerning the kinases involved in the phosphorylation of tau in Alzheimer disease, the MAP kinase pathway is implicated (Ledesma et al., 1992; Trojanowski et al., 1993b; Hyman et al., 1994). Therefore, abnormal phosphorylation of proteins in Alzheimer disease may be a consequence of oxidative stress and it is perhaps not surprising that while all pyramidal neurons of the hippocampus show increased free carbonyls (Smith et al., 1996a), lipid peroxide adduction (Sayre et al., 1997a) and nitrotyrosine (Smith et al., 1997b) only a subset of neurons displaying overt degeneration also show increased phosphorylation (Sternberger et al., 1985; Grundke-Iqbal et al., 1986). Heme oxygenase-1, an inducible antioxidant enzyme, also shows the same pattern of involvement since it accumulates concurrent with phosphorylated tau (Smith and Perry, unpublished data). Therefore, induction of the kinase and oxidative stress responses appears to require the same critical level of oxidative damage (Premkumar et al., 1995).

Influences of Amyloid-ß and Other Genetic Factors on Oxidative Stress: A number of mechanisms have been invoked for the neurotoxicity of amyloid-ß (Yankner et al., 1990; reviewed in Iversen et al., 1995; Sayre et al., 1997b) including membrane depolarization (Carette et al., 1993), increased sensitivity to excitotoxins (Koh et al., 1990), and alterations in calcium homeostasis (Mattson et al., 1992). However, the leading hypothesis is that neuronal damage by amyloid-ß is mediated by free radicals and, as such, can be attenuated using antioxidants such as vitamin E (Behl et al., 1992, 1994) or catalase (Zhang et al., 1996) although there has been some controversy regarding whether the protection from the latter involves its antioxidant activity (Lockhart et al., 1994). Critically addressing whether amyloid-ß initiates oxidative damage in Alzheimer disease requires careful study of the relationship of amyloid-ß deposition and increased oxidative damage. In this regard, it will be extremely interesting to determine the oxidative status of the recently reported transgenic rodent models of amyloid-ß deposition (Games et al., 1995; Hsiao et al., 1996).

Mutations in ßPP are the cause of Alzheimer disease in a small number of familial cases where the mutations lead to increased amyloid-ß deposition (reviewed in Selkoe, 1996, 1997). Transgenic rodents overexpressing ßPP, which display many of the neuropathological correlates of Alzheimer disease, support the important role of amyloid-ß (Games et al., 1995; Hsiao et al., 1996). It is interesting to consider that ßPP/amyloid-ß may be related to oxidative stress. Indeed, amyloid-ß is reported to spontaneously generate peptidyl radicals (Butterfield et al., 1994; Goodman et al., 1994; Harris et al., 1995; Prehn et al., 1996). Further, mutations in ßPP are associated with increased DNA fragmentation, possibly involving oxidative mechanisms (see below).

Presenilins 1 and 2 (Sherrington et al., 1995; Selkoe, 1997) are genetic factors where the biological mechanism, although not established, may also involve oxidative damage. Increased presenilin 2, expression increases DNA fragmentation and apoptotic changes (Wolozin et al., 1996), both important consequences of oxidative damage. Apolipoprotein E, in brains and cerebrospinal fluid, is found adducted with the highly reactive lipid peroxidation product, hydroxynonenal (Montine et al., 1996b). Furthermore, apolipoprotein E is a strong chelator of copper and iron, important redox-active transition metals (Miyata and Smith, 1996). Finally, interaction of apolipoprotein E with amyloid-ß only occurs in the presence of oxygen (Strittmatter et al., 1993).

In programmed cell death, i.e., apoptosis, cells are digested within their own membrane by proteases and nucleases as well as by increased ROS. However, without the full range of morphological changes, it is unclear whether DNA fragmentation is apoptotic or is, instead, mediated solely by oxidative stress (Berlin and Haseltine, 1981). While the relative contribution of oxidative stress and apoptosis related DNA fragmentation is unresolved, the relative infrequency of apoptosis defined by morphology (Su et al., 1994; Cotman and Su, 1996) and the broad findings of fragmentation in all cells in cases of Alzheimer disease argues for widespread oxidative DNA damage. Certainly, this interpretation is consistent with the oxidative nuclear damage in all cells of the brain in areas affected in Alzheimer disease (Smith et al, 1996a, 1997a).

Therapeutic Value

An important question in discerning whether reducing oxidative stress may have therapeutic value is whether it is a primary or secondary event in disease pathogenesis (Mattson et al., 1995; Smith et al., 1995c) (Figure 2). Recent evidence supports oxidative damage as the earliest cytopathological and biochemical change of Alzheimer disease (Ledesma et al., 1994; Smith et al., 1994a, 1995a, 1996a, 1997b; Vitek et al., 1994; Yan et al., 1994; Sayre et al., 1997a).

Agents that inhibit free radical formation, reduce both the incidence and the progression of Alzheimer disease (McGeer and Rogers, 1992; Rogers et al., 1993; Rich et al., 1995; Stewart et al., 1997). For the latter, several studies have found epidemiological relationships (Breitner et al., 1994; McGeer and Rogers, 1992) that, together with the results of metal chelation treatment (McLachlan et al., 1991), strongly suggest that oxidative stress precedes cell and tissue damage and therefore agents that prevent oxidative damage show promise in the treatment of Alzheimer disease.

SUMMARY

Oxidative stress may underline all of the commonly accepted notions on Alzheimer disease pathogenesis, including hyperphosphorylation, apolipoprotein E genotype, and mutations of the ß-protein precursor. Further studies, to examine the types and extent of oxidative damage, will undoubtedly identify which antioxidant agents will prove most efficacious in the treatment of Alzheimer disease.

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