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J Neural Transm (2000) 107: 203–231

An assessment of the antioxidant and the antiamyloidogenic

properties of melatonin: implications for Alzheimer’s disease

M. A. Pappolla1, Y.-J. Chyan1, B. Poeggeler1, B. Frangione1, G. Wilson1,J. Ghiso1, and R. J. Reiter2

1 Department of Pathology, University of South Alabama, School of Medicine,Mobile, AL, and

2 Department of Cellular and Structural Biology, The University of Texas HealthScience Center, San Antonio, TX, U.S.A.

Received June 28, 1999, accepted August 12, 1999

Summary. This review summarizes recent advancements in our understandingof the potential role of the amyloid protein in Alzheimer’s disease. It alsodiscusses the significance of amyloid in initiating the generation of partiallyreduced oxygen species and points out their role in damaging essential macro-molecules in the CNS which leads to neuronal dysfunction and loss. Recentlyacquired experimental data links these destructive oxidative processes withsome neurodegenerative aspects of Alzheimer’s disease. The experimental

findings related to the free radical scavenging and antioxidative properties of melatonin are tabulated and its efficacy and the likely mechanisms involvedin its ability to reduce neuronal damage mediated by oxygen-based reac-tive species in experimental models of Alzheimer’s disease are summarized.Besides the direct scavenging properties and indirect antioxidant actionsof melatonin, its ability to protect neurons probably also stems from itsantiamyloidogenic properties. Melatonin is also unique because of the easewith which it passes through the blood-brain barrier.

Keywords: Alzheimer’s disease, amyloid peptide, neurofibrillary tangles,oxidative stress, free radicals, antioxidant properties of melatonin,neurodegeneration.

Introduction

Alzheimer’s disease (AD) is the most frequent form of intellectual deteriora-tion in elderly individuals and the fourth leading cause of death in developednations. Although the cause of AD remains unknown, progress in this disor-der has accompanied the revolutionary momentum that characterized theDecade of the Brain. Many exciting developments followed the identificationof the amyloid beta peptide and of the microtubule associated protein tau (τ)



204 M. A. Pappolla et al.

as the principal components of the two main pathologic markers of AD, senileplaques and neurofibrillary tangles, respectively.

During the last few years several laboratories have presented compellingevidence indicating that brains with AD are subject to a pervasive level of

oxidative stress. One important set of data comes from in vitro models andsuggests that the amyloid peptide, the main constituent of senile plaques,causes extensive degeneration and death of neurons by mechanisms thatinvolve reactive oxygen intermediates and free radicals. Another independentand equally important body of evidence demonstrates that numerous markersof oxidative stress are accentuated around areas of amyloid deposition inhuman brains and in AD transgenic mice. This review focuses on the role of oxygen free radicals in the pathogenesis of this disease and in the potentialtherapeutic role of the pineal secretory product melatonin. Melatonin exhibitsa unique combination of antioxidant and antiamyloidogenic features whichmay be relevant to the design of preventive and therapeutic strategies in AD.A brief introductory discussion on the biology and neuropathology of the

disorder is presented followed by a consideration of the relationship betweenfree radicals and the pathogenesis of AD and a summary of studies that haveutilized melatonin as an experimental treatment.

Amyloid beta protein and the neuropathology of AD

Many of the recent advances in AD stem from the study of a 40–42 amino acidpeptide called the amyloid beta protein (A), as the essential pathologicmarker of the disorder (Glenner and Wong, 1984; Masters et al., 1985).Deposits of A in the form of amyloid fibrils are widespread in AD, mostlywithin senile plaques (Fig. 1) and in cerebral and meningeal blood vessels

(Abraham et al., 1988; Dickson, 1997; Esiri et al., 1997; Pappolla et al., 1999).The other conspicuous features of AD are intracytoplasmic neuronalinclusions called neurofibrillary tangles (NFT) (Esiri et al., 1997) (Fig. 2).Filamentous bundles of hyperphosphorylated τ proteins are, among othercytoskeletal proteins, the principal components of these lesions (Grundke-Iqbal et al., 1986; Kosik, 1993). Less strident, but nonetheless important, is theextensive neuronal and synaptic loss that affects these brains; these deficitshave been recently implicated as the most significant substrata for the pa-tients’ symptoms (Masliah, 1995; Hyman et al., 1995). Remarkably, senileplaques and neurofibrillary tangles are also present (in substantially fewernumbers) in most intellectually normal individuals fortunate to reach anadvanced age (Pappolla, 1999). In addition, these lesions are consistentlyobserved in the majority of older patients with Down syndrome (Esiri et al.,1997). Most cases of AD are sporadic but approximately 5% have a familialpattern of inheritance. The neuropathology is identical in both forms (Esiriet al., 1997).

Isolation and sequencing of A from meningovascular amyloid was firstaccomplished by Glenner and Wong (1984) whose work has led directly to thecloning of the gene for the much larger amyloid precursor protein (APP)(Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Kitaguchi et al.,



AD, oxidative stress, and melatonin 205

Fig. 1. The “classic” senile plaque, an abundant lesion in AD, is a complex and roughly

spherical focus of neuropil degeneration containing a central core of amyloid (arrow)surrounded by astrocytes, microglia and degenerating (dystrophic) neuritic processes(arrowheads) (reviewed in Pappolla et al., 1992b; Dickson, 1997). A large number of substances have been detected in senile plaques, including serum amyloid P, various acutephase proteins, complement factors, proteoglycans, apolipoproteins, cytokines, and anuncharacterized plaque core protein termed NAC. The neuritic plaque is a particularlyimportant subtype because the density of this lesion has in some studies been correlated

with the severity of the symptoms

Fig. 2. The neuron shown here contains neurofibrillary tangle which is immunoreactivewith antibodies against ubiquitin. This is a filamentous inclusion present within the

cytoplasm of the affected neurons

1988; Ponte et al., 1988; Tanzi et al., 1988). Several isoforms of APP wereidentified (Goldgaber et al., 1987; Robakis et al., 1987; Kitaguchi et al., 1988;Ponte et al., 1988; Tanzi et al., 1988; Weidemann et al., 1989; Golde et al.,1990; Buxbaum et al., 1992, 1993) which derive from alternative splicing of a



single gene in chromosome 21 (Goldgaber et al., 1987). APP is a type Iintegral membrane glycoprotein having a large extracytoplasmic portion, asmaller intracytoplasmic region and a single transmembranous domain(Fig. 3). APP is highly conserved in evolution and has several postulated

functions including growth promoting properties (Ponte et al., 1988), receptorfunction (Kang et al., 1987) and cell adhesion (Schubert et al., 1989; Breen etal., 1991). It is widely expressed in tissues (Weidemann et al., 1989) and is apart-time chondroitin sulfate proteoglycan in glial cells (Shioi et al., 1992).None of the proposed roles of APP have definitely been confirmed in vivo.APP knock out mice fail to show reduced survival although their brainsexhibit reactive gliosis in adult life (Zheng et al., 1995). The processing of APPis the subject of a large and continuously growing body of literature and acomprehensive review on APP processing has recently been published(Selkoe, 1998).

Understanding the pathogenesis of amyloid accumulation in AD is per-haps the major challenge in the Alzheimer’s field. Currently, misprocessing of APP and/or inadequate clearance of A are favored mechanisms (Neve andRobakis, 1998; Selkoe, 1998). Although small amounts of A are generatedby normal cellular metabolism (Haass et al., 1992; Seubert et al., 1992), itis not yet known whether the A peptides that become incorporated intoamyloid fibrils derive from the normally produced pool or from abnormalamyloidogenic processing of APP. It is generally accepted, however, that fibrilformation requires important conformational changes in the secondary struc-ture of A, such as increases in -sheets (Soto et al., 1995). In AD, the basis

Fig. 3. Schematic representation of the main APP domains and of A with the main

secretase cleavage sites indicated by Greek letters. The cysteine rich domain containssites for heparin, copper and zinc. The Kunitz protease inhibitor domain (KPI) and theOX-2 domain correspond to exons 7 and 8, respectively, which are alternatively spliced togive rise at least two to three main isoforms. APP undergoes glycosylation in the regionindicated by CH. Splicing of exon 15 creates the attachment site of a chondroitin sulfate

proteoglycan side-chain, an isoform expressed mainly by glial cells




of these alterations remains speculative, although they can be brought aboutin vitro from interactions between A and a host of potential chaperonproteins (Frangione et al., 1996; Zlokovic et al., 1998; Ghiso et al., 1993;Matsubara et al., 1995), or by metal-catalyzed oxidation (Dyrks et al., 1992).

The characteristics and kinetics of A aggregation are currently the focusof intense scrutiny (for representative studies see references Barrow andZagorski, 1991; Hilbich et al., 1991; Burdick et al., 1992; Mullen et al., 1992;Pike et al., 1993; Naiki and Nakakuki, 1996; Teplow, 1998).

Is amyloid the cause of neuronal degeneration in AD?

The relatively poor morphologic and clinical correlation between amyloidload on one hand and cell loss, neurofibrillary tangles or degree of dementiaon the other, has been the source of controversy regarding the role of amyloidin AD (Neve and Robakis, 1998). In recent years, however, several lines of evidence have significantly strengthened the hypothesis that A causes neu-

ronal degeneration and death. The first piece of evidence comes from geneticstudies which have identified several point mutations within the APP gene.These mutations segregate with a subgroup of patients afflicted with a familialform of AD (FAD), strongly suggesting a relationship between the APP geneand AD (Chartier-Harlin et al., 1991; Goate et al., 1991; Murrell et al., 1991;Naruse et al., 1991; Mullan et al., 1992; Kennedy et al., 1993). Second, it hasbeen demonstrated that A is neurotoxic (Schubert et al., 1989; Yankner etal., 1990; Breen et al., 1991; Shioi et al., 1992; Zhao et al., 1993; Behl et al.,1994a,b; Hensley et al., 1994; Shearman et al., 1994; Zhang et al., 1994;Schubert et al., 1995; Harris et al., 1995), a feature that supports the role of Ain the disease. Third, it has been observed that amyloid deposition (generally)precedes the development of neurofibrillary changes (Tagliavini et al., 1988;Pappolla and Robakis, 1995). Based on these observations, it is thereforelikely that A contributes, directly or indirectly, to neuronal degeneration. Itis also possible that the potential for amyloid to cause AD depends greatly onindividual susceptibilities to A-mediated toxicity.

Is oxidative stress involved in AD?

Involvement of semi-reduced oxygen species in aging has been a topic of interest since Harman’s (1956) seminal paper. Because of the close associa-tion between aging and AD and the qualitative similarities in the neuropa-thology of both conditions, it has been proposed by several investigators thatoxidative stress may play a role in the pathogenesis of AD (Volicer and Crino,1990; Smith et al., 1991; Pappolla et al., 1992a; Reiter, 1995; Munch et al., 1998;Retz et al., 1998).

Several lines of investigation have now converged to support this possibil-ity. One line of evidence is the identification of oxidative markers within theneuropathologic lesions of AD. The first documentation that indicators of oxidation co-localize with senile plaques and neurites was reported by ourlaboratory in 1992 (Pappolla et al., 1992b) and has subsequently been con-firmed by many investigators. These markers include antioxidant enzymes




(Pappolla et al., 1992b; Furuta et al., 1995), heme oxygenase I (Smith et al.,1994a), and pathologic increases of protein carbonyls (Smith et al., 1996)and lipid peroxides (Montine et al., 1997). Likewise, tissue homogenates fromAD brains exhibit evidence of oxidative stress including increased lipid

peroxidation (Subbarao et al., 1990), mitochondrial (Mecocci et al., 1994) andnuclear DNA lesions (Cotman, 1998) and neuronal membrane damage (DeKeyser et al., 1990). One caveat about findings in tissue homogenates is thatAD brains also exhibit evidence of vascular pathology and hypoperfusion (DeJong et al., 1997) that can secondarily cause an additional degree of oxidativeinjury. Thus, co-localization and accentuation of oxidative stress indicatorsaround amyloid deposits are paramount to supporting a relationship betweenamyloid and oxidative stress. In addition, it is also important to appreciatethat oxidative injury in AD is not limited to peri-plaque areas and extendsbeyond the immediate vicinity of senile plaques. Our studies in transgenicmice (Pappolla et al., 1998b) suggest that increases in soluble or micro-aggregated amyloid, not yet recognizable as fully developed plaques, may be

sufficient to produce oxidative stress.A second, but equally important, body of data comes from in vitro studies

and suggests that the neurotoxic properties of A are mediated by oxygen-based free radicals. Cells exposed to A generate increased levels of hydrogenperoxide (H2O2) (Behl et al., 1994b) which in the presence of transition metalsgive rise to destructive hydroxyl radicals (·OH) (Imlay et al., 1988; Behl et al.,1994b; Schubert et al., 1995; Reiter, 1998). Induction of free radicals may beinitiated by binding of A to cell surface receptors such as the advancedglycated end products (AGE) (Yan et al., 1996), although involvement of other A-binding proteins (Yan et al., 1999) or receptor independent mecha-nisms may also be implicated (Pappolla et al., 1998c). Neuronal cells exposed

to A develop extensive oxidative lesions of nuclear and mitochondrial DNA(Loo et al., 1993; Bozner et al., 1997; Pappolla et al., 1999b). In addition,impaired Ca2 homeostasis which appears to follow oxidation of Ca2 mem-brane pumps (Mark et al., 1995) has been demonstrated after exposure of cellsto A (Mattson et al., 1992). Among various adverse effects on neurons(Mattson, 1990), increased intracellular Ca2 leads to activation of calmodulin-dependent nitric oxide synthase and increased intracellular nitric oxide (NO)(Morel et al., 1991) which, in turn, reacts with the superoxide anion to formperoxynitrite anion (ONOO). ONOO can form activated transitional formswith reactive potentials comparable to ·OH, or can be decomposed at physi-ologic pH into the nitrogen dioxide radical and ·OH (Beckman and Crow,1993). The recent demonstration of widespread peroxynitration of proteins inbrains affected with AD (Good et al., 1996; Smith et al., 1997) suggests apathologic role of ONOO in this disease. It has also been reported that anadditional source of free radicals could be A itself, as documented by theability of the peptide to generate ·OH in aqueous solution (Hensley et al.,1994). Aside from a direct effect on neurons, deposits of A initiate microglialand astrocytic reactions, characteristic of neuritic plaques. These activatedcells promote a cascade of pro-inflammatory factors that further amplifiestissue damage and oxidative stress (Meda et al., 1995).




Non-enzymatic glycosylation (glycation) is another pathway which canenhance oxidative lesions in brains with AD. It was first hypothesized by us(Pappolla et al., 1990) that “stagnant” cytoskeletal proteins in early NFT mayundergo glycation and cross-linking within the neuronal cytoplasm in a

manner similar to that observed in extracellular long-lived proteins (Monnierand Cerami, 1981). Long-lived extracellular matrix proteins undergoglycation via the Maillard reaction (Monnier and Cerami, 1981) leading toAmadori rearrangements that accumulate during the aging process. Thispathway is accelerated in patients with diabetes mellitus and accounts to agreat extent for the insoluble qualities of many proteins including collagenand elastin during the aging process. In vitro experiments demonstrated thatpolymerized microtubules are rapidly glycated with glucose-6-phosphate andform insoluble paired helical-like profiles (Pappolla et al., 1990) similar tothose found in neurofibrillary tangles. In support of the hypothesis, twogroups of investigators have reported that the neuropathologic lesions in ADcontain advanced glycation end products characteristic of the Maillard reac-

tion (Smith et al., 1994b; Vitek et al., 1994). It was subsequently demonstratedthat glycation can amplify oxidative stress and enhance abnormal τ phospho-rylation and abnormal assembly of cytoskeletal filaments (Smith et al., 1996;Retz et al., 1998).

Although the contribution from each of the potential mentioned sourcesof free radicals/reactive oxygen intermediates to the overall degree of oxida-tive damage is not certain, it is now generally agreed that A causes oxidationof key cellular components such as membranes (Yan et al., 1996), mitochon-drial (Bozner et al., 1998; Pappolla et al., 1999b) and nuclear DNA (Loo et al.,1993) that culminate in profound metabolic changes and death of neurons.Moreover, abnormal phosphorylation and polymerization of τ, one main com-

ponent of NFT (Grundke-Iqbal et al., 1986; Papasozomenos and Su, 1991;Kosik, 1993), have been induced in vivo and in vitro by heat-shock (Kirby etal., 1994) and oxidative stress (Troncoso et al., 1993), respectively. Oxidationof cystein-322 in the repeat domain of τ appears to control the aggregation of τ into paired helical filaments (Schweers et al., 1995). It is noteworthy thatfibrillar A also causes hyperphosphorylation of τ at serine-202, serine-396and serine-404 (Busciglio et al., 1995).

Despite all the cited evidence, inconsistent in vitro results from variouslaboratories and variability among different batches of amyloid, have createdsignificant controversy concerning the neurotoxic role of amyloid. Based onthese inconsistencies, several investigators went on to suggest that A-mediated toxicity might be caused either by a contaminant or by an artifact of culture (Neve and Robakis, 1998). It should be noted, however, that theability of A to induce neurodegeneration is dependent on several additionalfactors such as the -sheet secondary structure of the peptide (Busciglio et al.,1992; Pike et al., 1993), the state of aggregation, time of exposure, osmolarity,pH and concentration (Barrow and Zagorski, 1991; Hilbich et al., 1991;Burdick et al., 1992; Busciglio et al., 1992; Pike et al., 1993; Lorenzo andYankner, 1994; Naiki and Nakakuki, 1996). Most importantly, recent studiesin a transgenic model of AD have virtually excluded the possibility that A-




mediated oxidative stress is the result of a contaminant or a technical artifact.In these animals, the distribution of the oxidative burden was not only accen-tuated around the amyloid deposits but also overlapped with dystrophic neu-ritic elements (Fig. 4) (Pappolla et al., 1998b; Smith et al., 1998). In addition,one recently created APP transgenic mouse model exhibits evidence of neu-ronal loss associated to A deposition (Calhoun et al., 1998). Taken together,the weight of the evidence suggests that A contributes, directly or indirectly,to neuronal degeneration.

Among the most important unresolved issues regarding the role of oxida-tive stress and AD is whether this form of injury is the cause or consequenceof amyloidogenesis. Aside from A causing oxidative stress, it has also beenproposed that free radical-induced injury would exacerbate a vicious cycle inwhich amyloidogenic processing of APP would be further enhanced, generat-ing more A that in turn would cause more oxidative stress (Pappolla et al.,1995; Yan et al., 1995; Zhang et al., 1997). Emerging data from in vitro studiesappear to support such a mechanism, although more information in this areais necessary. Likewise, evidence of oxidation-mediated amyloidogenesis isstill lacking in in vivo paradigms. Therefore, the possibility of whether such a

Fig. 4. Oxidative markers in AD and in transgenic mice. Expression of numerousoxidation markers in vivo provided remarkable parallels between AD brains and ADtransgenic mice confirming previous in vitro studies that implicate A in oxidative neuro-toxicity (Pappolla et al., 1997, 1998a; Smith et al., 1998). The markers of oxidation weremainly co-localized with the amyloid deposits and overlapped topographically with areasshowing increased products of lipid peroxidation and neuronal dystrophy as demon-strated by anti-HNE, anti-ubiquitin and anti-tau antibodies (see Pappolla et al., 1998b;Smith et al., 1998 for further details). Illustrated here are A deposits in human andmurine transgenic brains visualized by an immunolabelling technique and anti-A anti-bodies (4G8). There is clear overexpression of SOD which co-localizes with A depositsin senile plaques (a marker of oxidative stress) and neuritic elements as detected with

anti-ubiquitin antibodies




self-sustaining cycle is involved in the pathogenesis of AD is intriguing

but in need of more supporting evidence. Other possible pathways linkingoxidation to amyloidogenesis include increased aggregation of A by metalcatalyzed oxidation (Dyrks et al., 1992), and increased susceptibility of M1muscarinic receptor bearing neurons to oxidative stress (Joseph et al., 1996).Decrease M1 muscarinic receptor function causes reductions in cholinergic-mediated protein kinase C activity. This pathway is known to be involved innon-amyloidogenic processing of APP (Haring et al., 1998). Figure 5 illus-trates the manner in which oxidative stress may be involved in the pathogen-esis of AD.

Although the previous discussion favors the argument that APP and Aare central to the pathogenesis of AD, the disease is far more complex in thatother genes and loci also have been linked to the disorder (Sherrington et al.,1995; Hardy and Hutton, 1995; Tanzi et al., 1996). Whereas APP-mutationsaccount for only a small fraction of FAD, the roles of these other genes in ADhave not yet been determined. Late-onset FAD and sporadic AD have beenassociated with the presence of the epsilon-4 allele of the Apo E gene(Rebeck et al., 1993; Strittmatter et al., 1993). The mechanisms leading toincreased risk for developing AD are uncertain and are presently being inves-tigated by several laboratories. Binding of Apo E4 to A appears to modulateamyloid fibril formation although the nature of these interactions is contro-

Fig. 5. Proposed roles of oxidative stress in AD pathogenesis. The pathways indicated bysolid arrows are supported by in vitro and in vivo evidence, as referenced in the text.

The mechanisms indicated by open arrows have been suggested only by in vitro data




versial (Wisniewski et al., 1994; Aleshkov et al., 1997; Naiki et al., 1997; Russoet al., 1998). Interestingly, a recent study also shows that 4-hydroxy-2-nonenalimmunoreactivity, a marker of lipid peroxidation, is increased in AD brains of patients homozygous for Apo E4 (Montine et al., 1997).

In 1995, two partly homologous genes were identified and termedpresenilin 1 and 2 (Sherrington et al., 1995; Hardy and Hutton, 1995; Levy-Lahad et al., 1995; Rogaev et al., 1995; Tanzi et al., 1996). Missense mutationsin these genes account for almost two thirds of early-onset forms of FAD. Thefunctions of these widely expressed proteins are not yet known and mayinclude regulation of apoptosis, “cell fate” (Cribbs et al., 1996) and cytoplas-mic transport (Naruse et al., 1998). A group of investigators have recentlyreported a possible increased susceptibility to oxidative damage or toapoptosis in cells transfected with mutated forms of presenilins (Guo et al.,1997; Zhang et al., 1998). In addition, some presenilin mutations appear toaffect APP processing and enhance the rate of aggregation of A by increas-ing the fraction of the more amyloidogenic peptide A1–42 (Duff et al., 1996;

Lemere et al., 1996; Scheuner et al., 1996). Whether this effect is caused bydirect regulation of APP secretases is currently being studied (Selkoe, 1998).Other proteinaceous components of senile plaques have been described in-cluding anti-chymotrypsin (Abraham et al., 1988) and an α-synuclein derivedfragment named NAC (Ueda et al., 1993). The pathogenic significance of these proteins, also, is uncertain.

Melatonin as a free radical scavenger and neural antioxidant

A variety of partially reduced toxic oxygen-based molecules (Fig. 6) aregenerated in the brain. The damage that these species inflict is referred to as

oxidative stress (Sies, 1991). Melatonin has proven to be highly protective of the brain against oxidative deterioration (Reiter, 1995a, 1999; Reiter et al.,1996, 1998) for several reasons. First, melatonin has repeatedly been shown,using a wide variety of techniques – e.g., electron spin resonance spectros-copy, pulse radiolysis, etc. – to be a very efficient scavenger of the highly toxic·OH both in vitro (Tan et al., 1993, 1998; Poeggeler et al., 1994, 1995, 1996;Matuszek et al., 1997; Susa et al., 1997; Pähkla et al., 1998; Roberts et al., 1998;Stasica et al., 1998a, 1998b; Brömme et al., 1999; Mahal et al., 1999) as well asin vivo (Li et al., 1997; Tan et al., 1998a). The action of melatonin is notewor-thy because the ·OH is very reactive and indiscriminately damages any mol-ecule in the vicinity of where it is produced. It is estimated that 50% of thetotal oxidative damage that organisms sustain is a consequence of the unceas-ing plundering by the ·OH. Melatonin scavenges this molecular brigand witha calculated rate constant of 2.7 1010 M1 s1 (Matuszek et al., 1997) which isgreater than that of most other antioxidants (Acworth et al., 1997).

Besides detoxifying the ·OH, melatonin also neutralizes other reactiveoxygen intermediates that mutilate essential neuronal molecules (Beckman,1997). Thus, melatonin scavenges the highly reactive ONOO (Gilad et al.,1997; Cuzzocrea et al., 1998) and reportedly scavenges one of its precursormolecules, i.e., nitric oxide (NO·) (Noda et al., 1999). Furthermore, melatonin




has been shown to prevent the toxicity of singlet oxygen (1O2) (Cagnoli et al.,1995; King and Scaiano, 1997; Zang et al., 1998), a reactive oxygen by-productwith high neuronal toxicity.

The brain is highly susceptible to oxidative damage for several reasons.Particularly noteworthy are the high concentrations of polyunsaturated fattyacids (PUFA) in the CNS. These molecules are easily oxidized in the processof lipid peroxidation. The peroxidation of lipids is especially damaging inas-much as one of the products generated during the breakdown of lipids, i.e., theperoxyl radical (LOO·), is sufficiently toxic to re-initiate (propagate) lipidperoxidation (Hall, 1997). Thus, lipid peroxidation more dramatically than

Fig. 6. A summary of the products that are formed when oxygen (O2) undergoes succes-sive one electron reductions within cells. The most reactive and toxic of the productsformed is the hydroxyl radical (·OH). Other species, however, also destroy neuronalelements including the peroxynitrite anion (ONOO), nitric oxide (NO·), singlet oxygen(1O2), and the peroxyl radical (LOO·). Much of the superoxide anion radical (O 2

·)generated in cells undergoes dismutation due to the catalytic action of a family of super-oxide dismutases (SOD). The resulting product, hydrogen peroxide (H2O2) as well ashydroperoxides, can be enzymatically removed from cells by the actions of two enzymes,catalase (CAT, this enzyme is rather deficient in the brain) and glutathione peroxidase(GPx). GPx oxidizes glutathione (GSH) (and utilizes H2O2 and hydroperoxides as sub-strates) to its disulfide form (GSSG) which is recycled back to GSH in the presence of glutathione reductase (GRd). The enzyme required to generate the co-factor (NADPH)for reduction of GSSG is glucose-6-phosphate dehydrogenase (G6PDH). Nitric oxide

synthase (NOS) is potentially a proxidative enzyme since NO· can have inherent toxicityin addition to combining with O2

· to form ONOO. Melatonin’s antioxidative actionsinclude direct scavenging of the ·OH, ONOO, 1O2 and possibly O2

· and the peroxylradical (LOO·). Additionally, melatonin may stimulate important antioxidative enzymes(indicated by ≠) in the brain including SOD, GPx, GRd and G6PDH while inhibiting theproxidative enzyme, NOS (indicated by Ø). Thus, melatonin has a number of means bywhich it reduces oxidative destruction of essential neural elements. Unlike some other

antioxidants, melatonin also readily crosses the blood-brain barrier




other free radical damaging processes is particularly devastating because of the chain reaction that results.

The major chain breaking antioxidant is vitamin E (α-tocopherol). Someevidence suggests that melatonin may function as a chain breaking antioxi-

dant as well due to its ability to detoxify the LOO·. While it has been sug-gested that melatonin is a more efficient scavenger of the LOO· than isvitamin E (Pieri et al., 1994, 1995), this is not supported by all findings(Scaiano, 1995; Marshall et al., 1996; Livrea et al., 1997; Longoni et al., 1997).Melatonin is, however, an effective LOO· but probably not with the highefficiency claimed by Pieri and co-workers (1994, 1995). It does, however,have one distinct advantage over vitamin E in protecting the brain againstlipid peroxidation; thus, melatonin readily crosses the blood-brain barrier(Menendez-Pelaez et al., 1993). This contrasts with vitamin E which traversesthis barrier much less easily.

Besides the evidence that illustrates its ability to directly scavenge reactiveoxygen intermediates, melatonin also acts as an indirect antioxidant in that it

stimulates several important antioxidative enzymes. These enzymes provideprotection against free radical damage by metabolizing radical precursors aswell as reactive oxygen intermediates to non-toxic products. Major anti-oxidative enzymes in the brain include a family of enzymes the superoxidedismutases, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase (Fig. 6). Both pharmacological as well as physi-ological levels of melatonin have been shown to stimulate either mRNA levelsor the activities of these antioxidative agents (Barlow-Walden et al., 1995;Pablos et al., 1995, 1997; Kotler et al., 1998).

Since glutathione peroxidase also functions as a peroxynitrite reductase(Sies et al., 1997), this enzyme not only removes toxic hydroperoxides and

H2O2 from intracellular compartments, it also reduces the highly reactiveONOO. Furthermore, physiological levels of melatonin have also beenshown to inhibit nitric oxide synthase (NOS) which would reduce the forma-tion of NO·, one of the precursors required for ONOO generation (Bettahiet al., 1996; Pozo et al., 1997).

That melatonin is capable of protecting the brain from oxidative damagehas been amply demonstrated (Reiter, 1998). In numerous models of experi-mentally-induced oxidative destruction of the brain, melatonin has proveneffective in significantly reducing the damage. Thus, in in vivo excitotoxicity(Guisti et al., 1996, Manev et al., 1996a, 1996b; Tan et al., 1998b), ischemia-reperfusion (Cho et al., 1997, Guerrero et al., 1997; Kilic et al., 1999), trau-matic brain injury (Mesenge et al., 1998), hyperoxia (Pablos et al., 1997b),porphyric neuropathy (Princ et al., 1997; Carneiro and Reiter, 1998), andmodels of Parkinson’s disease (Acuña-Castroviejo et al., 1997; Jin et al., 1998;Mayo et al., 1998b) melatonin reduced macromolecular damage caused byprocesses which work via free radical mechanisms. Also, melatonin has beenshown effective in reducing amyloid -induced lipid peroxidation in vitro(Pappolla et al., 1997; Daniels et al., 1998). To date there are no publishedreports in which melatonin has failed to protect the CNS from destructiveprocesses which involve free radicals and reactive oxygen intermediates. Fur-




thermore, melatonin reduces apoptosis in neurons subjected to oxidativestress (Lezoualc’h et al., 1996, 1998; Manev et al., 1996a; Mayo et al., 1998a,1999). Apoptosis is one likely mechanism whereby neurons are lost in the ADpatients.

Melatonin as an antioxidant and antiamyloidogenic agent in AD

Since the severity of dementia correlates best with neuronal and/or synapticloss (Hyman et al., 1995; Masliah, 1995), enhancing neuronal function andsurvival has been the primary objectives of most therapeutic strategies. Wehave recently reported that melatonin totally prevents cell death and oxida-tive damage of cultured neuroblastoma cells and primary cell neurons ex-posed to A (Pappolla et al., 1997, 1999b) (Fig. 7). As previously discussed,neurons exposed to A display a spectrum of indicators of cellular and oxida-tive damage including increased lipid peroxidation, increased intracellularfree Ca2 concentration, oxidative damage of mitochondrial DNA and emer-

gence of markers of programmed cell death. Remarkably, when all the men-tioned indicators of neuronal injury were assessed in neurons exposed to Ain combination with melatonin, it was observed that this indoleamine com-pletely protected neurons exposed to the amyloid peptide. There were virtu-ally no differences between A-treated cells and control neurons. Thesestrong neuroprotective effects may, therefore, provide the basis for a newtherapy strategy in AD.

Because of the relationship between oxidative stress and AD and therecently established antioxidant properties of melatonin (Reiter et al., 1993;Tan et al., 1993, 1998), it was initially thought that the observed cytoprotectiveeffects were mostly due to the intracellular antioxidant activities of the

indoleamine. While investigating various mechanisms of action, however, wealso found that melatonin strongly inhibited spontaneous formation of -sheets and amyloid fibrils (Pappolla et al., 1998a). These effects were demon-strated by a number of techniques including circular dichroism, nuclearmagnetic resonance spectroscopy and electron microscopy (Fig. 8). The newproperties provide an additional degree of cytoprotection that is synergisticwith the marked intracellular antioxidant effects of melatonin. Inhibition of -fibrillogenesis is important for the following reasons. First, it has been demon-strated that A-induced neurotoxicity is linked to the -sheet structure butnot to the random conformation of the peptide (Simmons et al., 1994;Harrigan et al., 1995). Second, the soluble -sheet conformation is a precursorof the insoluble -sheet structure in amyloid plaques and can act as a seed fornucleation and fibril formation (Jarret and Lansbury, 1993; Snyder et al.,1994). Lastly, peptide solutions containing higher amounts of -sheet struc-tures are resistant to proteolytic degradation (Soto and Castaño, 1996). Thus,by blocking the formation of -sheets, one could potentially reduce neurotox-icity and facilitate clearance of the peptide via increased proteolytic degrada-tion. Reduction of the amyloid -sheet structures could then represent one of the most important therapeutic targets to interrupt the development of patho-logical changes in AD. It is important to recognize that the antiamyloidogenic




and antioxidant properties of melatonin may go hand-in-hand since it hasbeen proposed that aggregation of amyloid peptides may be driven by oxida-tive mechanisms (Dyrks et al., 1992). The strong antiamyloidogenic propertiesof melatonin, however, are not observed with other antioxidants (B.Poeggeler and M. A. Pappolla, unpublished).

Melatonin has major advantages over other endogenous and syntheticantioxidants. One is the proposed role of melatonin in the aging process(Iguchi et al., 1982; Pierpaoli, 1991; Dori et al., 1994; Reiter, 1995, 1997; Reiteret al., 1996). Decreased secretion of melatonin with aging is well documented(Iguchi et al., 1982; Reiter, 1992; Dori et al., 1994; Reiter et al., 1998) and moreprofound reductions are reported in populations with dementia (Souetreet al., 1989; Mishima et al., 1994; Liu et al., 1999). These intriguing facts are in

Fig. 7. Melatonin protection of primary hippocampal neurons against A neurotoxicity.Cell viability experiments assessed by a sensitive florescent probe (bodipy green) andconfocal laser microscopy in fetal primary rat hippocampal neurons. Panel A showscontrol cells. In Panel B, neurotoxicity of 1µM A1-42 was evidenced by markedlydecreased bodipy green fluorescence of neurons (shown here by a pseudocolor scale). C

Melatonin (1µM) prevented A toxicity as indicated by increased viability of neurons(upper red pseudocolor). D N-acetyl serotonin (1µM, used here as a control indole) hasno neuroprotective activity. This sensitive probe permits assessment of the neurotoxiceffects of A1-42 as well as the neuroprotective properties of melatonin at relatively lowconcentrations of peptide and hormone. The effects illustrated have been extensively

confirmed by eight different methods and across various neuronally-derived cell lines




sharp contrast with conventional antioxidants which, despite their cytopro-tective characteristics, have no comparable correlates with the pathophysiol-ogy of human aging.

Melatonin’s intracellular distribution allows it to provide on-site protec-tion against ·OH-mediated oxidative damage to biomolecules and melatonin’santioxidant activity is not limited to either lipophilic or hydrophilic intracel-lular structures or compartments as occurs with vitamin E and vitamin C,

Fig. 8. Inhibition of amyloid fibril formation by melatonin. Illustrated are typical areasfrom a representative experiment showing of A1-40 incubated for 48 hr either alone (A),with N-acetyl-serotonin (control indole compound) (B), PBN (C), or melatonin (D) (Bar 200ηm). Melatonin inhibited formation of amyloid fibrils. EM grids were extensivelyand carefully examined and a negative result was only documented when fibrils weretotally absent from the grids. No inhibition of fibril formation was seen under the condi-tions tested with NAT or PBN. At the time of this writing these results were reproduced

more than ten times on independent different-day experiments




respectively. Melatonin diffuses widely into cellular compartments and incontrast to vitamin E it penetrates the blood-brain barrier with ease(Menendez-Pelaez et al., 1993; Reiter et al., 1993; Reiter, 1998). In addition tothese properties, there also are important structural (chemical) and mechanis-

tic (functional/scavenging) differences between melatonin and common phe-nolic chain breaking antiooxidants like vitamin E. Vitamin E possesses areactive hydroxyl group, which enables it to donate a hydrogen atom therebyreducing free radicals which in turn, promote radical chain reactions. Becauseof their high reactivity, chain breaking antioxidants such as vitamin Eautooxidize in the presence at transition metals and increase the formation of primary radicals such as the ·OH. In contrast, melatonin is especially efficientin detoxifying ·OH which are the initiators of radical chain reactions. Of alloxygen-derived free radicals, ·OH are the most reactive (Halliwell, 1993).Melatonin is an ·OH scavenger with a rate constant three orders of magnitudehigher than exhibited by chain breaking antioxidants such as vitamin E. Theproduct of the interaction of melatonin with two ·OH has been identified as

cyclic 3-hydroxymelatonin (Tan et al., 1998); this product is excreted in theurine and is an index of in vivo ·OH generation. Perhaps, the most significantdifference that sets melatonin apart from other antioxidant compounds is therelatively weak pro-oxidant potential of melatonin in itself as well as of themolecules generated by melatonin while scavenging reactive species (Reiteret al., 1993; Reiter, 1998). Finally, the reported lack of toxicity of melatoninmakes it a prime candidate for experimental testing against neurodegene-rative diseases (Reiter et al., 1997; Reiter, 1998, 1999).

A major question in the melatonin field is whether physiological levels of the hormone have a role in the total antioxidant and/or neuroprotectivecapacity of the organism. The bulk of the research conducted thus far utilized

what were presumed to be pharmacologic levels of melatonin. This assump-tion was based on the normally low levels of melatonin in blood. Withoutknowledge of melatonin concentrations in specific organs, cells and subcellu-lar compartments, however, it is not possible at this point to know whatconstitutes a physiological or pharmacological level of melatonin. It is known,for example, that concentrations of melatonin in brain cells can be muchhigher than would be predicted on the basis of serum levels of theindoleamine (Menendez-Pelaez et al., 1993). Also regarding oxidative dam-age, data from in vivo mutagenesis studies have emerged suggesting that eventhe apparently miniscule (in the pM range) amount of endogenous melatoninis sufficient to reduce the oxidative damage from mutagen exposure (Tan etal., 1994). Likewise, models of ischemia and excitotoxicity show that theendogenous blood levels of the indoleamine are sufficient for neuroprotectionin vivo (Manev et al., 1996; Kilic et al., 1999). Besides the direct antifibri-llogenic actions on A and the antioxidant effects of melatonin, other syner-gistic cytoprotective effects of this hormone are possible at the cellular level.Such effects may involve signal transduction pathways (melatonin inhibitsphospholipase A2 and cyclooxygenase) (Franchi et al., 1987), 5-lipoxygenase(Benitez-King and Anton-Tay, 1993; Carlberg and Wiesenberg, 1995; Uz etal., 1997), and increases in the intrinsic cellular antioxidant defenses (Reiter et




al., 1997; Reiter, 1998). Considering these findings, it is possible that thereduction in endogenous melatonin levels in advanced age (Iguchi et al., 1992;Touitou et al., 1984; Sack et al., 1986; Reiter, 1992) influences the onset and/or severity of the symptoms of AD.

No controlled, double-blind studies are currently available concern-ing the preventive or therapeutic potential of melatonin in AD. Recent anec-dotal observations on patients with AD appear to support our hypothesisthat melatonin might slow down the rate of cognitive impairment in AD(Pappolla et al., unpublished observations). The authors experience with twoAD patients given melatonin is as follows. An 80-year-old male with a diagno-sis of probable AD, global dementia scale five (Schneider et al., 1997),was treated with 6 mg melatonin daily for more than one year and showedfour points of improvement in the Mini-Mental State Examination a year lateron follow-up reassessment. He is currently stable. An 83-year-old womanwith advanced AD, who had loss spoken communication, recovered threeword communication (yes, no and good) after one year treatment. Although

she later died of possible bronchopneumonia, her improvement in terms of communication is an exceedingly rare event in advanced AD. It must beemphasized that although encouraging, these are uncontrolled isolated obser-vations that must be confirmed in double-blind, randomized placebo-controlled trials.

Concluding remarks

Most recent developments on the AD field resulted from the compositionalanalysis of senile plaques and neurofibrillary tangles that began almost twodecades ago. These research efforts have provided intriguing information

about the role of several factors, including oxidative stress, in the pathogen-esis of this condition. Although many facets of the biology of this diseaseremain to be defined, strides have been made in the development of animalmodels and in the elucidation of unique biological properties of several pro-teins like A, APP, presenilins and apolipoproteins. The value of this infor-mation is centered in the development of strategies either aimed at reducingproduction or aggregation of A or to abrogating the neurotoxicity andinflammation that this peptide appears to cause in brain regions responsiblefor our intellectual functioning. Based on the findings reviewed here in itwould seem that interventions with antioxidants and antiamyloidogenicagents such as melatonin may be beneficial in deferring neurodegenerativeconditions such as AD.

References

Abraham CR, Selkoe DJ, Potter H (1988) Immunochemical identification of the serineprotease inhibitor α1-antichymotrypsin in the brain amyloid deposits of Alzheimer’sdisease. Cell 52: 487–501

Acuña-Castroviejo D, Coto-Montes A, Monti MG, Ortiz GG, Reiter RJ (1997)Melatonin is protective against MPTP-induced striatal and hippocampal lesions. LifeSci 60: PL23–PL29




Acworth IN, McCabe DR, Maher TJ (1997) The analysis of free radicals, their reactionproducts, and antioxidants. In: Baskin SI, Salem H (eds) Oxidants, antioxidants, andfree radicals. Taylor and Francis, Washington, pp 23–77

Aleshkov S, Abraham CR, Zannis VI (1997) Interaction of nascent ApoE2 ApoE3 andApoE4 isoforms expressed in mammalian cells with amyloid peptide beta (1–40).

Relevance to Alzheimer’s disease. Biochemistry 36: 10571–10580Barlow-Walden LR, Reiter RJ, Abe M, Pablos MI, Chen LD, Poeggeler P (1995)Melatonin stimulates brain glutathione peroxidase activity. Neurochem Int 26: 497–502

Barrow CJ, Zagorski MG (1991) Solution structures of peptide and its constituentfragments: relation to amyloid deposition. Science 253: 179–182

Beckman JS (1997) Nitric oxide, superoxide, and peroxynitrite in CNS injury. In: WelchKMA, Caplan LR, Ries DJ, Siesjö BK, Weir B (eds) Primer on cerebrovasculardiseases. Academic Press, New York, pp 209–210

Beckman JS, Crow JP (1993) Pathological implications of nitric oxide superoxide andperoxynitrite formation. Biochem Soc Transact 21: 330–334

Behl C, Davis JB, Klier FG, Schubert D (1994a) Amyloid beta peptide induces necrosisrather than apoptosis. Brain Res 645: 253–264

Behl C, Davis JB, Lesley R, Schubert D (1994b) Hydrogen peroxide mediates amyloid

protein toxicity. Cell 77: 817–827Benitez-King G, Anton-Tay F (1993) Calmodulin mediates melatonin cytoskeletal ef-

fects. Experientia 49: 635–641Bettahi I, Pozo D, Ozuna C, Reiter RJ, Acuña-Castroviejo D, Guerrero JM (1996)

Physological concentrations of melatonin inhibit nitric oxide synthase activity in rathypothalamus. J Pineal Res 20: 205–210

Bozner P, Grishko V, LeDoux SP, Wilson GL, Chyan Y-J, Pappolla MA (1997) Theamyloid protein induces oxidative damage of mitochondrial DNA. J NeuropatholExp Neurol 56: 1356–1362

Breen KC, Bruce M, Anderson BH (1991) Beta amyloid precursor protein mediatesneuronal cell-cell and cell-surface adhesion. J Neurosci Res 28: 90–100

Brömme HJ, Ebelt H, Peschke D, Peschke E (1999) Alloxan acts as a prooxidant onlyunder reducing conditions: influence of melatonin. Cell Mol Life Sci 55: 487–493

Burdick D, Soreghan B, Kwon M, Kosmoski J, Knauer M, Henschen A, Yates J, CotmanCW, Glabe C (1992) Assembly and aggregation properties of synthetic Alzheimer’sA4/ amyloid peptide analogs. J Biol Chem 267: 546–554

Busciglio J, Lorenzo A, Yankner BA (1992) Methodological variables in the assessmentof beta amyloid neurotoxicity. Neurobiol Aging 13: 609–612

Busciglio J, Lorenzo A, Yeh J, Yankner BA (1995) -Amyloid fibrils induce tau phospho-rylation and loss of microtubule binding. Neuron 14: 879–888

Buxbaum JD, Oishi M, Chen HI, Pinkas-Kramarski R, Jaffe EA, Gandy SE, GreengardP (1992) Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer /A4 amyloid protein precursor. Proc Natl Acad Sci USA 89: 10075–10078

Buxbaum JD, Koo EH, Greengard P (1993) Protein phosphorylation inhibits productionof Alzheimer amyloid beta/A4 peptide. Proc Natl Acad Sci USA 90: 9195–9198

Cagnoli CM, Atabay C, Kharlamova E, Manev H (1995) Melatonin protects neuronsfrom singlet oxygen-induced apoptosis. J Pineal Res 18: 222–226

Calhoun ME, Wiederhold KH, Abramowski D, Phiney AL, Probst A, Sturchler-PierratC, Staufenbiel ME, Sommer B, Jucker M (1998) Neuron loss in APP transgenic mice.Nature 395: 755–756

Carlberg C, Wiesenberg I (1995) The orphan receptor family RZR/ROR, melatonin andlipoxygenase: an unexpected relationship. J Pineal Res 18: 171–178

Carneiro RCG, Reiter RJ (1998) Melatonin protects against lipid peroxidation inducedby δ-aminolevulinic acid in rat cerebellum, cortex and hippocampus. Neuroscience82: 293–299




Chartier-Harlin MC, Crawford F, Houlden H, Warren A, Hughes D, Fidani L, Goate A,Rossor M, Roques P, Hardy J, Mullan M (1991) Early-onset Alzheimer’s diseasecaused by mutations at codon 717 of the beta-amyloid precursor protein gene. Nature353: 844–846

Cho S, Joh JH, Baik HH, Didinis C, Volpe BT (1997) Melatonin administration protects

CA1, hippocampal neurons after transient forebrain ischemia in rats. Brain Res 755:335–338Cotman CW (1998) Apoptosis decision cascades and neuronal degeneration in

Alzheimer’s disease. Neurobiol Aging 19 [Suppl 1]: S29–S32Cribbs DH, Chen LS, Cotman CW, LaFerla FM (1996) Injury induces presenilin-1 gene

expression in mouse brain. Neuroreport 7(11): 1773–1776Cuzzocrea S, Zingarelli B, Costantino G, Caputi AP (1998) Protective effect of melatonin

in a non-septic shock model induced by zymosan in the rat. J Pineal Res 25: 24–33

Daniels WMU, van Rensburg SJ, van Zyl JM, Taljaard JJF (1998) Melatonin prevents -amyloid-induced lipid peroxidation. J Pineal Res 24: 78–92

De Jong GI, De Vos RA, Steur EN, Luiten PG (1997) Cerebrovascular hypoperfusion: arisk factor for Alzheimer’s disease? Animal model and postmortem human studies.Ann NY Acad Sci 826: 56–74

De Keyser J, Ebinger G, Vanguelin G (1990) D1-dopamine receptor abnormality infrontal cortex points to a functional alteration of cortical cell membranes inAlzheimer’s disease. Arch Neurol 47: 761–763

Dickson DW (1997) The pathology of senile plaques. J Neuropathol Exp Neurol 56: 321–339

Dori D, Casale G, Solerte SB, Fioravanti M, Migliorati G, Cuzzoni G, Ferrari E (1994)Chrono-neuroendocrinological aspects of physiological aging and senile dementia.Chronobiology 21: 121–126

Duff K, Eckman C, Zehr C, Yu X, Prada C-M, Perez-tur J, Hutton M, Buee L, HarigayaY, Yager D, Morgan D, Gordon MN, Holcomb L, Refolo L, Zenk B, Hardy J,Younkin S (1996) Increased amyloid-42(43) in brains of mice expressing mutantpresenilin 1. Nature 383: 710–713

Dyrks T, Dyrks E, Hartmann T, Masters C, Beyreuther K (1992) Amyloidogeneicity of

beta amyloid and beta A4-forming amyloid protein precursor fragment by metal-catalyzed oxidation. J Biol Chem 267: 18210–18217Esiri MM, Hyman B, Beyreuther K, Masters CL (1997) Aging and dementia, chapter 4.

In: Graham DI, Lantio PL (eds) Greenfield’s neuropathology, 6th edn. Arnold Pubs,London, pp 151–233

Franchi AM, Gimeno MF, Cardinali DP, Vacas MI (1987) Melatonin, 5-methoxytryp-tamine and some of their analogs as cyclooxygenase inhibitors in rat medial basalhypothalamus. Brain Res 405: 384–388

Frangione B, Castaño EM, Wisniewski T, Ghiso J, Prelli F, Vidal R (1996)Apolipoprotein E and amyloidogenesis. Ciba Foundation Symposium 199: 132–141

Furuta A, Price DL, Pardo C, Troncoso JC, XU Z-S, Taniguchi N, Martin LJ (1995)Localization of superoxide dismutases in Alzheimer’s disease and Down syndromeneocortex and hippocampus. Am J Pathol 146: 357–367

Ghiso J, Matsubara E, Koudinov A, Choi-Miura NH, Tomita M, Wisniewski T, FrangioneB (1993) The cerebrospinal-fluid soluble form of Alzheimer’s amyloid beta is com-plexed to SP-40, 40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex. Biochem J 293: 27–30

Gilad E, Cuzzocrea S, Zingarelli B, Salzman AL, Szabo C (1997) Melatonin as aperoxynitrite scavenger. Life Sci 60: PL169–PL170

Giusti P, Lipartiti M, Franceschini D, Shiavo N, Floreani M, Manev H (1996)Neuroprotection by melatonin from kainate-induced excitotoxicity in rats. FASEB J10: 891–896




Glenner GG, Wong CW (1984) Alzheimer’s disease: initial report of the purification andcharacterization of a novel cerebrovascular amyloid protein. Biochem Biophys ResCommun 120: 885–890

Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L, Giuffra L,Haynes A, Irving N, James L, Mant R, Newton P, Rooke K, Roques P, Talbot C,

Pericak-Vance M, Roses A, Williamson R, Rossor M, Owen M, Hardy J (1991)Segregation of a missense mutation in the amyloid precursor protein gene withfamilial Alzheimer’s disease. Nature 349: 704–706

Golde TE, Estus S, Usiak M, Younkin LH, Younkin SG (1990) Expression of betaamyloid protein precursor mRNAs: recognition of a novel alternatively spliced formand quantitation in Alzheimer’s disease using PCR. Neuron 4: 253–267

Goldgaber D, Lerman MI, McBride OW, Saffiotti U, Gadijusek DC (1987) Characteriza-tion and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer’sdisease. Science 235: 887–880

Good PF, Werner P, Hsu A, Olanow CW, Perl DP (1996) Evidence for neuronal oxida-tive damage in Alzheimer’s disease. Am J Pathol 149: 21–28

Grundke-Iqbal I, Iqbal K, Tung YC, Quinlan M, Wiesniewski HM, Binder LI (1986)Abnormal phosphorylation of the microtubule-associated protein τ (tau) inAlzheimer’s cytoskeletal pathology. Proc Natl Acad Sci USA 83: 4913–4917

Guerrero JM, Reiter RJ, Ortiz GG, Pablos MI, Sewerynek E, Chuang JI (1997) Melato-nin prevents increases in nitric oxide and cyclic GMP production after transient brainischemia and reperfusion in the Mongolian gerbil (Meriones unguiculatus). J PinealRes 23: 24–31

Guo Q, Soper BL, Furukawa K, Robinson N, Martin GM, Mattson MP (1997)Alzheimer’s presenilin mutation sensitizes neural cells to apoptosis induced bytrophic factor withdrawal and amyloid beta-peptide: involvement of calcium andoxyradicals. J Neurosci 17: 4212–4222

Haass C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A, Ostaszewski BL,Lieberburg I, Koo EH, Schenk D, Teplow DB, Selkoe DJ (1992) Amyloid -peptideis produced by cultured cells during normal metabolism. Nature 359: 322–324

Hall ED (1997) Lipid peroxidation. In: Welch KMA, Caplan LR, Ries DJ, Siesjö BK,Weir B (eds) Primer on cerebrovascular diseases. Academic Press, New York, pp

200–204Halliwell B (1993) Oxidative DNA damage: meaning and measurement. In: Halliwell B,Auroma OI (eds) DNA and free radicals. Ellis Harwood, London pp 67–79

Hardy J, Hutton M (1995) Two new genes for Alzheimer’s disease. Trends Neurosci 18:436

Haring R, Fisher A, Marciano D, Pittel Z, Kloog Y, Zuckerman A, Eshhar N, HeldmanE (1998) Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to beta-amyloid precursorprotein secretion. J Neurochem 71: 2094–2103

Harman D (1956) Aging: a theory based on free radical and radiation chemistry. JGerontol 11: 298–300

Harrigan MR, Kunkel DD, Nguyen LB, Malouf AT (1995) Amyloid is neurotoxic inhippocampal slice culture. Neurobiol Aging 16: 779–789

Harris ME, Hensley K, Butterfield DA, Leedle RA, Carney JM (1995) Direct evidenceof oxidative injury produced by the Alzheimer’s beta-amyloid peptide (1–40) incultured hippocampal neurons. Exp Neurol 131: 193–202

Hensley K, Carney JM, Mattson MP, Aksenova M, Harris M, Wu JF, Floyd RA,Butterfield DA (1994) A model for beta-amyloid aggregation and neurotoxicitybased on free radical generation by the peptide: relevance to Alzheimer disease. ProcNatl Acad Sci USA 91: 3270–3274

Hilbich C, Kisters-Woike B, Reed J, Masters CL, Beyreuther K (1991) Aggregation andsecondary structure of synthetic amyloid A4 peptides in Alzheimer’s disease. J MolBiol 218: 149–163




Hyman BT, West HL, Gomez-Isla T, Mui S (1995) Quantitative neuropathology inAlzheimer’s disease: neuronal loss in high-order association cortex parallels demen-tia. In: Iqbal K, Mortimer JA, Winblad B, Wiesniewski HM (eds) Research advancesin Alzheimer’s disease and related disorders. John Wiley and Sons, New York

Iguchi H, Kato K, Ibayashi H (1982) Age-dependent reduction in serum melatonin

concentrations in healthy subjects. J Clin Endocrinol Metabol 55: 27–29Imlay JA, Chin SM, Linn S (1988) Toxic DNA damage by hydrogen peroxide through theFenton Reaction in vivo and in vitro. Science 240: 640–642

Jarrett JT, Lansbury PT Jr (1993) Seeding “one-dimensional crystallization” of amyloid:a pathogenic mechanism in Alzheimer’s disease and scrapie? Cell 73: 1055–1058

Jin BK, Shin DY, Jeong MY, Gwag MR (1998) Melatonin protects nigral dopaminergicneurons from 1-methyl-4-phenylpyridinium (MPP) neurotoxicity in rats. NeurosciLett 245: 61–64

Joseph JA, Villalobos-Molina R, Denisova N, Erat S, Jimenez N, Strain J (1996) In-creased sensitivity to oxidative stress and the loss of muscarinic receptor responsive-ness in senescence. Ann NY Acad Sci 786: 112–119

Kang J, Lemaire H-G, Unterbeck A, Salbaum JM, Masters CL, Grzeschik K-H,Multhaup G, Beyreuther K, Muller-Hill B (1987) The precursor of Alzheimer’sdisease amyloid A4 protein resembles a cell-surface receptor. Nature 325: 733–736

Kennedy AM, Newman S, McCaddon A, Ball J, Roques P, Mullan M, Hardy J, Chartier-Harlin MC, Frackowiak RS, Warrington EK (1993) Familial Alzheimer’s disease. Apedigree with a missense mutation in the amyloid precursor protein gene (amyloidprecursor protein 717 valineÆglycine). Brain 116: 309–324

King M, Scaiano JC (1997) The excited states of melatonin. Photochem Photobiol 65:538–542

Kilic E, Ozdemir YG, Bolay H, Kelestimur H, Dalkara T (1999) Pinealectomy aggravatesand melatonin administration attenuates brain damage in focal ischemia. J CerebBlood Flow Metab 19: 511–516

Kirby BA, Merril CR, Ghanbari H, Wallace WC (1994) Heat shock proteins protectagainst stress-related phosphorylation of tau in neuronal PC12 cells that have ac-quired thermotolerance. J Neurosci 14: 5687–5693

Kitaguchi N, Takahashi Y, Tokushima Y, Shiojiri S, Ito H (1988) Novel precursor of

Alzheimer’s disease amyloid protein shows protease inhibitory activity. Nature 331:530–532Kosik KS (1993) The molecular and cellular biology of Tau. Brain Pathol 3: 39–43Kotler M, Rodriquez C, Sainz RM, Antolin I, Menendez Pelaez A (1998) Melatonin

increases gene expression for antioxidant enzymes in rat brain cortex. J Pineal Res 24:83–89

Lemere CA, Lopera F, Kosik KS, Lendon CL, Ossa J, Saido TC, Yamaguchi H, Ruiz A,Martinez A, Madrigal L, Hincapie L, Arango JC, Anthony DC, Koo EH, Goate AM,Selkoe DJ, Arango JC (1996) The E280A presenilin 1 Alzheimer mutation producesincreased A42 deposition and severe cerebellar pathology. Nature Med 2: 1146–1150

Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, Pettingell WH, Yu CE,Jondro PD, Schmidt SD, Wang K (1995) Candidate gene for the chromosome 1familial Alzheimer’s disease locus. Science 269: 973–977

Li XJ, Zhang LM, Gu J, Zhang AZ, Sun FY (1997) Melatonin decreases production of hydroxyl radical during cerebral ischemia-reperfusion. Acta Pharmacol Sinica 18:394–396

Liu R-Y, Zhou J-N, van Heerikhuize J, Hofman MA, Swaab DF (1999) Decreasedmelatonin levels in postmortem cerebrospinal fluid in relation to aging Alzheimer’sdisease and apolipoprotein E-ε4/4 genotype. J Clin Endocrinol Metab 84: 323–327

Livrea MA, Tesoriere L, D’Arpa D, Morreale M (1997) Reaction of melatonin withlipoperoxyl radicals in phospholipid bilayers. Free Radic Biol Med 23: 706–711




Longoni B, Salgo G, Pryor WA, Marchiafava M (1997) Effects of melatonin on lipidperoxidation induced by oxygen radicals. Life Sci 62: 853–859

Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW (1993)Apoptosis is induced by -amyloid in cultured central nervous system neurons. ProcNatl Acad Sci USA 90: 7951–7955

Lorenzo A, Yankner BA (1994) Beta-amyloid neurotoxicity requires fibril formation andis inhibited by Congo red. Proc Natl Acad Sci USA 91: 12243–12247Mahal HS, Sharma HS, Mukherjee T (1999) Antioxidant properties of melatonin: a pulse

radiolysis study. Free Radic Biol Med 26: 557–565Manev H, Uz T, Kharlamov A, Joo JY (1996) Increased brain damage after stroke or

excitotoxic seizures in melatonin-deficient rats. FASEB J 10: 1546–1551Manev H, Uz T, Kharlamov A, Cognali CM, Franseschini D, Giusti D (1996a) In

vivo protection against kainate-induced apoptosis by pineal hormone melatonin:effect of exogenous melatonin and circadian rhythm. Restr Neurol Neurosci 9: 251–256

Mark RJ, Hensley K, Butterfield DA, Mattson MP (1995) Amyloid -peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2 homeostasisand cell death. J Neurosci 15: 6239–6249

Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI, Halliwell BA (1996) Evaluationof the antioxidant activity of melatonin in vitro. Free Radic Biol Med 21: 307–315

Masliah E (1995) Mechanisms of synaptic dysfunction in Alzheimer’s disease. HistolHistopathol 10: 509–519

Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K (1985)Amyloid plaque core protein in Alzheimer disease and Down Syndrome. Proc NatlAcad Sci USA 82: 4245–4249

Matsubara E, Frangione B, Ghiso J (1995) Characterization of apolipoprotein J-Alzheimer’s A beta interaction. J Biol Chem 270: 7563–7567

Mattson MP (1990) Excitatory amino acids growth factors and calcium: a teeter-totter model for neural plasticity and degeneration. Adv Exp Med Biol 268: 211–220

Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg I, Rydel RE (1992) -Amyloid

peptides destabilize calcium homeostasis and render human cortical neurons vulner-able to excitotoxicity. J Neurosci 12: 376–389Matuszek Z, Reszka KJ, Chignell CF (1997) Reaction of melatonin and related indoles

with hydroxyl radicals: EPR and spin trapping investigations. Free Radic Biol Med23: 367–372

Mayo JC, Sainz RM, Uria H, Antolin I, Esteban MM, Rodriquez C (1998a) Melatoninprevents apoptosis induced by 6-hydroxydopamine in neural cells: implications forParkinson’s disease. J Pineal Res 24: 179–192

Mayo JC, Sainz RM, Uria H, Antolin I, Esteban MM, Rodriquez C (1998b) Inhibition of cell proliferation: a mechanism likely to mediate prevention of neuronal cell death bymelatonin. J Pineal Res 24: 179–192

Mayo JC, Sainz RM, Antolin I, Rodriquez C (1999) Ultrastructural confirmation of neuronal protection of melatonin against the neurotoxin 6-hydroxydopamine celldamage. Brain Res 818: 221–227

Mecocci P, MacGarvey U, Beal MF (1994) Oxidative damage to mitochondrial DNA isincreased in Alzheimer’s disease. Ann Neurol 36: 747–751

Meda L, Cassatella MA, Szendrei GI, Otvos L Jr, Baron P, Villalba M, Ferrari D, RossiF (1995) Activation of microglial cells by -amyloid protein and interferon-γ. Nature374: 647–650

Menendez-Pelaez A, Poeggeler B, Reiter RJ, Barlow-Walden L, Pablos MI, Tan DX(1993) Nuclear localization of melatonin in different mammalian species:immunocylochemical and radioimmunoassay evidence. J Cell Biochem 53: 373–382




Mesenge C, Margaill I, Verrecchia C, Allix M, Boulu RG, Plotkine M (1998) Protectiveeffect of melatonin in a model of traumatic brain injury in mice. J Pineal Res 25: 41–46

Mishima K, Okawa M, Hishikawa Y, Hozumi S, Hori H, Takahashi K (1994) Morningbright light therapy for sleep and behavior disorders in elderly patients with demen-

tia. Acta Psychiatr Scand 89: 1–7Monnier VM, Cerami A (1981) Non-enzymatic browning in vivo: possible process foraging of long-lived proteins. Science 211: 491–493

Montine KS, Kim PJ, Olson SJ, Markersbery WR, Montine TJ (1997) 4-Hydroxy-2-nonenal pyrrole adducts in human neurodegenerative disease. J Neuropathol ExpNeurol 56: 866–871

Morel F, Doussiere J, Vignais PV (1991) The superoxide-generating oxidase of phago-cytic cells. Physiological, molecular and pathological aspects. Eur J Biochem 201:523–546

Mullan M, Crawford F, Axelman K, Houlden H, Lilius L, Winblad B, Lannfelt L (1992)A pathogenic mutation for probable Alzheimer’s disease in the APP gene at the N-terminus of beta-amyloid. Nature Genet 1: 345–347

Munch G, Schinzel R, Loske C, Wong A, Durany N, Li JJ, Vlassara H, Smith MA, PerryG, Riederer P (1998) Alzheimer’s disease-synergistic effects of glucose deficit oxida-tive stress and advanced glycation end products. J Neural Transm 105: 439–461

Murrell J, Farlow M, Ghetti B, Benson MD (1991) A mutation in the amyloid precursorprotein associated with hereditary Alzheimer’s disease. Science 254: 97–99

Naiki H, Nakakuki K (1996) First-order kinetic model of Alzheimer’s -amyloid fibrilextension in vitro. Lab Invest 74: 374

Naiki H, Gejyo F, Nakakuki K (1997) Concentration-dependent inhibitory effects of apolipoprotein E on Alzheimer’s beta-amyloid fibril formation in vitro. Biochemistry36: 6243–6250

Naruse S, Igarashi S, Kobayashi H, Aoki K, Inuzuka T, Kaneko K, Shimizu T, Iihara K,Kojima T, Miyatake T, Tsuji S (1991) Mis-sense mutation Val–Ile in exon 17 of amyloid precursor protein gene in Japanese familial Alzheimer’s disease. Lancet Lett337: 978–979

Naruse S, Thinakaran G, Luo JJ, Kusiak JW, Tomita T, Iwatsubo T, Qian X, Ginty DD,

Price DL, Borchelt DR, Wong PC, Sisodia SS (1998) Effects of PS1 deficiency onmembrane protein trafficking in neurons. Neuron 21: 1213–1221Neve RL, Robakis NK (1998) Alzheimer’s disease: a re-examination of the amyloid

hypothesis. Trends Neurosci 21: 15–19Noda Y, Mori A, Liburty R, Parker L (1999) Melatonin and its precursors scavenge nitric

oxide. J Pineal Res 27: 159–163Pablos MI, Agapito MT, Gutierrez R, Recio JM, Reiter RJ (1995) Melatonin stimulates

the activity of the detoxifying enzyme glutathione peroxidase in several tissues of rats.J Pineal Res 19: 111–115

Pablos MI, Guerrero JM, Ortiz GG, Agapito MT, Reiter RJ (1997a) Both melatonin anda putative nuclear melatonin receptor agonist CGP 52608 stimulate glutathione per-oxidase and glutathione reductase activities in mouse brain in vivo. NeuroendocrinolLett 18: 49–58

Pablos MI, Reiter RJ, Chuang JI, Ortiz GG, Guerrero JM, Sewerynek E, Agapito MT,Melchiorri D, Lawrence R, Deneke SM (1997b) Acutely administered melatoninreduces oxidative damage in lung and brain induced by hyperbaric oxygen. J ApplPhysiol 83: 354–358

Pähkla R, Zilmer M, Kullisaar T, Rägo L (1998) Comparison of the antioxidant activityof melatonin and pinoline in vitro. J Pineal Res 24: 96–101

Papasozomenos SC, Su Y (1991) Altered phosphorylation of tau protein in heat-shockedrats and patients with Alzheimer disease. Proc Natl Acad Sci 88: 4543–4547

Pappolla MA (1999) Molecular biology and neuropathology of Alzheimer’s disease. In:Cruz F (ed) Neuropathologia. (in press)




Pappolla MA, Robakis NK (1995) Neuropathology and molecular biology of Alzheimer’sdisease. In: Stein M, Baum M (eds) Perspectives in behavioral medicine: Alzheimer’sdisease. Academic Press, San Diego

Pappolla MA, Alzofon J, McMahon J, Thedoropoulos TJ (1990) Ultrastructural evidencethat insoluble microtubules are components of neurofibrillary tangles. Eur Arch

Psych Neurol Sci 239: 314–319Pappolla MA, Omar RA, Kim KS, Robakis NK (1992a) Immunohistochemical evidenceof oxidative stress in Alzheimer’s disease. Am J Pathol 140: 621–628

Pappolla MA, Omar RA, Sambamurti K, Anderson JP, Robakis NK (1992b) The genesisof the senile plaque. Further evidence in support of its neuronal origin. Am J Pathol141: 1151–1159

Pappolla MA, Sambamurti K, Efthimiopoulos S, Refolo L, Omar RA, RobakisNK (1995) Heat-shock induces abnormalities in the cellular distribution of amy-loid precursor protein (APP) and APP fusion proteins. Neurosci Lett 192: 105–108

Pappolla MA, Sos M, Omar RA, Bick RJ, Hickson-Bick DLM, Reiter RJ,Efthimiopoulos S, Robakis NK (1997) Melatonin prevents death of neuro-blastoma cells exposed to the Alzheimer’s amyloid peptide. J Neurosci 17: 1683–1690

Pappolla MA, Bozner P, Soto C, Shao H, Robakis N, Zagorski M, Frangione B, Ghiso J(1998a) Inhibition of Alzheimer -fibrillogenesis by melatonin. J Biol Chem 273:7185–7189

Pappolla MA, Chyan YJ, Omar RA, Hsiao K, Perry G, Smith MA, Bozner P (1998b)Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a transgenicmouse model of Alzheimer’s disease: a chronic oxidative paradigm for testing anti-oxidant therapies in vivo. Am J Pathol 152: 871–877

Pappolla MA, Chyan Y-J, Bozner P, Martin B, Frangione B, Hsiao K, Omar RA, PerryG, Smith MA, Ghiso J (1998c) NADPH cytochrome P450 reductase mediates oxida-tive stress by the amyloid protein. Society for Neuroscience, 28th Annual Meeting,Los Angeles, CA (Abstract #698.4)

Pappolla MA, Chyan Y-J, Poeggeler B, Bozner P, Ghiso J, LeDoux SP, Wilson GL(1999a) A mediated oxidative damage of mitochondrial DNA: prevention by mela-

tonin. J Pineal Res 27: 226–229Pappolla MA, Chyan Y-J, Bozner P, Soto C, Shao H, Reiter RJ, Brewer G, Robaski NK,Zagorski MG, Frangione B, Ghiso J (1999b) Dual anti-amyloidogenic and anti-oxidant properties of melatonin. A new therapy for Alzheimer’s disease. In: Iqbal K,Mortimer J, Winblad B, Wisniewski HM (eds) Research advance in Alzheimer’sdisease. Wiley and Sons, New York, pp 661–669

Pieri C, Marra M, Moroni F, Recchioni R, Marcheselli F (1994) Melatonin, a peroxylradical scavenger more efficient than vitamin E. Life Sci 55: PL271–PL276

Pieri C, Moroni F, Marra M, Marcheselli F, Recchioni R (1995) Melatonin is an efficientantioxidant. Arch Gerontol Geriatr 20: 159–165

Pierpaoli W (1991) The pineal gland: circadian or seasonal aging clock? Aging (Milano)3: 99–101

Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW (1993) Neurodegenerationinduced by -amyloid peptide in vitro: the role of peptide assembly state. J Neurosci13: 1676–1687

Poeggeler B, Saarela S, Reiter RJ, Tan DX, Chen LD, Manchester LC, Barlow-WaldenLR (1994) Melatonin – a highly potent endogenous radical scavenger and electrondonor: new aspects of the oxidation chemistry of this indole assessed in vitro. Ann NYAcad Sci 738: 419–420

Poeggeler B, Reiter RJ, Hardeland R, Sewerynek E, Melchiorri D, Barlow-Walden LR(1995) Melatonin, a mediator of electron transfer and repair reactions, acts synergis-tically with the chain-breaking antioxidants ascorbate, Trolox and glutathione.Neuroendocrinol Lett 17: 87–92




Poeggeler B, Reiter RJ, Hardeland R, Tan DX, Barlow-Walden LR (1996) Melatoninand structurally-related endogenous indoles act as potent electron donors and radicalscavengers in vitro. Redox Reports 2: 179–184

Ponte P, Gonzalez-DeWhitt Schilling P, Miller J, Hsu D, Greenberg B, Davis K, WallaceW, Lieberburg I, Fuller F (1988) A new A4 amyloid mRNA contains a domain

homologous to serine proteinase inhibitors. Nature 331: 525–527Pozo D, Reiter RJ, Calvo JR, Guerrero JM (1997) Inhibition of cerebellar nitric oxidesynthase and cyclic GMP production by melatonin via complex formation withcalmodulin. J Cell Biochem 65: 430–442

Princ FG, Maxit AG, Cardalda C, Batlle A, Juknat A (1998) In vivo protection bymelatonin against δ-aminolevulinic acid-induced oxidative damage and its antioxi-dant effect on the activity of haem enzymes. J Pineal Res 24: 1–8

Rebeck GW, Reiter JS, Strickland DK, Hyman BT (1993) Apolipoprotein E in sporadicAlzheimer’s disease: allelic variation and receptor interactions. Neuron 11: 575–580

Reiter RJ (1992) The aging pineal gland and its physiological consequences. BioEssays14: 169–175

Reiter RJ (1995a) Oxidative processes and antioxidative defense mechanisms in the agingbrain. FASEB J 9: 526–533

Reiter RJ (1995b) The pineal gland and melatonin in relation to aging: a summary of thetheories and of the data. Exp Gerontol 30: 199–212

Reiter RJ (1997) Aging and oxygen toxicity: relation to changes in melatonin. Age 20:201–213

Reiter RJ (1998) Oxidative damage in the central nervous system: protection by melato-nin. Prog Neurobiol 56: 359–384

Reiter RJ, Poeggeler B, Tan D-X, Chen L-D, Manchester LC (1993) Antioxidant capacityof melatonin: a novel function not requiring a receptor. Neuroendocr Lett 15: 103–116

Reiter RJ, Pablos MI, Agapito MT, Guerrero JM (1996) Melatonin in the context of thefree radical theory of aging. Ann NY Acad Sci 786: 362–368

Reiter RJ, Guerrero JM, Escames G, Pappolla MA, Acuna-Castroviejo D (1997) Prophy-lactic actions of melatonin in oxidative neurotoxicity. Ann NY Acad Sci 825: 70–

78Reiter RJ, Guerrero JM, Garcia JJ, Acuña-Castroviejo D (1998) Reactive oxygen inter-mediates, molecular damage and aging: relation to melatonin. Ann NY Acad Sci 854:410–454

Reiter RJ, Cabrera J, Sainz RM, Mayo JC, Manchester LC, Tan DX (1999) Melatoninas a pharmacological agent against neuronal loss in experimental models of Huntington’s disease, Alzheimer’s disease and Parkinsonism. Ann NY Acad Sci (inpress)

Retz W, Gsell W, Munch G, Rösler M, Riederer P (1998) Free radicals in Alzheimer’sdisease. J Neural Transm [Suppl] 54: 221–236

Robakis NK, Pappolla MA (1990) Oxygen free radicals and amyloidosis in Alzheimer’sdisease: is there a connection? Editorial. Neurobiol Aging 15: 457–459

Robakis NK, Ramakrishna N, Wolfe G, Wisniewski HM (1987) Molecular cloning andcharacterization of a cDNA encoding the cerebrovascular and the neuritic plaqueamyloid peptides. Proc Natl Acad Sci USA 84: 4190–4194

Roberts JE, Hu DN, Wishart JF (1998) Pulse radiolysis studies of melatonin and 6-chloromelatonin. J Photochem Photobiol B: Biol 42: 125–132

Rogaev EI, Sherrington R, Rogaeva EA, Levesque G, Ikeda M, Liang Y, Chi H, Lin C,Holman K, Tsuda T (1995) Familial Alzheimer’s disease in kindreds with missensemutations in a gene on chromosome 1 related to the Alzheimer’s disease type 3 gene.Nature 376: 775–778

Russo C, Angelini G, Dapino D, Piccini A, Piombo G, Schettini G, Chen S, Teller J,Zaccheo D, Gambetti P, Tabaton M (1998) Opposite roles of apolipoprotein E in




normal brains and in Alzheimer’s disease. Proc Natl Acad Sci USA 95: 15598–15602

Sack RL, Lewy AJ, Erb DL, Vollmer WM, Singer CM (1986) Human melatonin produc-tion decreases with age. J Pineal Res 3: 379–388

Scaiano JC (1995) Exploratory laser flash photolysis study of free radical reactions and

magnetic field effects in melatonin chemistry. J Pineal Res 19: 189–195Scheuner D, Eckman C, Jensen M, Song X, Citron M, Suzuki N, Bird TD, Hardy J,Hutton M, Kukull W, Larson E, Levy-Lahad E, Viitanen M, Peskind E, Poorkaj P,Schellenberg G, Tanzi R, Wasco W, Lannfelt L, Selkoe D, Younkin S (1996) Secretedamyloid beta-protein similar to that in the senile plaques of Alzheimer’s disease isincreased in vivo by the presenilin 1 and 2 and APP mutations linked to familialAlzheimer’s disease. Nature Med 2: 864–870

Schneider LS, Olin JT, Doody RS, Clark CM, Morris JC, Reisberg B, Schmitt FA,Grundman M, Thomas RG, Ferris SH (1997) Validity and reliability of theAlzheimer’s Disease Cooperative Study-Clinical Global Impression of Change.Alzheimer Dis Assoc Disord 11 [Suppl 2]: S22–S32

Schubert D, Jin LW, Saitoh T, Cole G (1989) The regulation of amyloid betaprotein precursor secretion and its modulatory role in cell adhesion. Neuron 3: 689–694

Schweers O, Mandelkow EM, Biernat J, Mandelkow E (1995) Oxidation of cysteine-322in the repeat domain of microtubule-associated protein tau controls the in vitroassembly of paired helical filaments. Proc Natl Acad Sci USA 92: 8463–8467

Selkoe DJ (1998) The cell biology of -amyloid precursor protein and presenilin inAlzheimer’s disease. Trends Cell Biol 8: 447–453

Seubert P, Vigo-Pelfrey C, Esch F, Lee M, Dovey H, Davis D, Sinha S, Schlossmacher M,Whaley J, Swindlehurst C, McCormack R, Wolfert R, Selkoe DJ, Lieberburg I,Schenk D (1992) Isolation and quantification of soluble Alzheimer’s -peptide frombiological fluids. Nature 359: 325–327

Shearman MS, Ragan CI, Iversen LL (1994) Inhibition of PC12 cell redox activity is aspecific early indicator of the mechanism of beta-amyloid mediated cell death. ProcNatl Acad Sci USA 91: 1470–1474

Schubert D, Behl C, Lesley R, Brack A, Dargusch R, Sagara Y, Kimura H (1995)

Amyloid peptides are toxic via a common oxidative mechanism. Proc Natl Acad SciUSA 92: 1989–1993Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H,

Lin C, Li G, Holman K, Tsuda T, Mar L, Fonci J-F, Bruni AC, Montesi MP, Sorbi S,Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P,Polinsky RJ, Wasco W, Da Silva HAR, Haines JL, Pericak-Vance MA, Tanzi RE,Roses AD, Fraser PE, Rommens JM, St George-Hyslop PH (1995) Cloning of a genebearing missense mutations in early-onset familial Alzheimer’s disease. Nature 375:734

Sies H (1991) Oxidative stress, oxidants and antioxidants. Academic Press, New York,385 pp

Sies H, Sharov VS, Klutz LO, Brivida K (1997) Glutathione peroxidase protects againstperoxynitrite-mediated reactions. J Biol Chem 272: 27812–27817

Shioi J, Anderson JP, Ripellino JA, Robakis NK (1992) Chondroitin sulfate proteoglycanform of the Alzheimer’s amyloid precursor. J Biol Chem 267: 13819–13822

Simmons LK, May PC, Tomaselli KJ, Rydel RE, Fuson KS, Brigham EF, Wright S,Lieberburg I, Becker GW, Brems DN, Li W (1994) Secondary structure of amyloidbeta peptide correlates with neurotoxic activity in vitro. Mol Pharmacol 45: 373–379

Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd RA,Markesberry WR (1991) Excess brain protein oxidation and enzyme dysfunctionin normal aging and in Alzheimer disease. Proc Natl Acad Sci USA 88: 10540–10543




Smith MA, Kutty RK, Richey PL, Yan SD, Stern D, Chader GJ, Wiggert B, Petersen RB,Perry G (1994a) Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer’s disease. Am J Pathol 145: 42–47

Smith MA, Taneda S, Richey PL, Miyata S, Yan S-D, Stern D, Sayre LM, Monnier VM,Perry G (1994b) Advanced Maillard reaction end products are associated with

Alzheimer disease pathology. Proc Natl Acad Sci USA 91: 5710–5714Smith MA, Tabaton M, Perry G (1996) Early contribution of oxidative glycation inAlzheimer disease. Neurosci Lett 217: 210–211

Smith MA, Harris PLR, Sayre LM, Beckman JS, Perry G (1997) Widespreadperoxynitrite-mediated damage in Alzheimer’s disease. J Neurosci 17: 2653–2657

Smith MA, Hirai K, Hsiao K, Pappolla MA, Harris PL, Siedlak SL, Tabaton M, Perry G(1998) Amyloid-beta deposition in Alzheimer transgenic mice is associated withoxidative stress. J Neurochem 70: 2212–2215

Snyder SW, Ladror US, Wade WS, Wang GT, Barrett LW, Matayoshi ED, Huffaker HJ,Krafft GA, Holzman TF (1994) Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths. Biophys J 67: 1216–1228

Soto C, Castaño M (1996) The conformation of Alzheimer’s peptide determines therate of amyloid formation and its resistance to proteolysis. Biochem J 314: 701–707

Soto C, Castaño EM, Kumar RA, Beavis RC, Frangione B (1995) Fibrillogenesis of synthetic amyloid-beta peptides is dependent on their initial secondary structure.Neurosci Lett 200: 105–108

Souetre E, Salvati E, Krebs B, Belugou JL, Darcourt G (1989) Abnormal melatoninresponse to 5-methoxypsoralen in dementia. Am J Psychiatry 146: 1037–1040

Strittmatter WJ, Weisgraber KH, Huang DY, Dong LM, Salvesen GS, Pericak-Vance M,Schmechel D, Saunders AM, Goldgaber D, Roses AD (1993) Binding of humanapolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects andimplications for late-onset Alzheimer’s disease. Proc Natl Acad Sci USA 90: 80098–80102

Stasica P, Ulanski P, Rosiak JM (1998a) Melatonin as a hydroxyl radical scavenger. JPineal Res 25: 65–66

Stasica P, Ulanski P, Rosiak JM (1998b) Reactions of melatonin with radicals in deoxy-genated solution. J Radioanal Nucl Chem 232: 107–113Subbarao KV, Richardson JS, Ang LC (1990) Autopsy samples of Alzheimer’s cortex

show increased peroxidation in vitro. J Neurochem 55: 342–345Susa N, Ueno S, Furukawa Y, Ueda J, Sugiyama M (1997) Potent protective effect of

melatonin on chromium VI-induced DNA single-strand breaks, cytotoxicity, andlipid peroxidation in primary cultures of rat hepatocytes. Toxicol Appl Pharmacol144: 377–384

Tagliavini F, Giaccone G, Frangione B, Bugiani O (1988) Preamyloid deposits in thecerebral cortex of patients with Alzheimer’s disease and nondemented individuals.Neurosci Lett 93: 191–196

Tan DX, Chen LD, Poeggeler B, Manchester LC, Reiter RJ (1993) Melatonin: a potentendogenous hydroxyl radical scavenger. Endocr J 1: 57–60

Tan DX, Reiter RJ, Chen LD, Poeggeler B, Manchester LC, Barlow-Walden LR (1994)Both physiological and pharmacological levels of melatonin reduce DNA adductformation induced by the carcinogen safrole. Carcinogenesis 15: 215–218

Tan DX, Manchester LC, Reiter RJ, Plummer BF, Hardies LJ, Weintraub ST,Vijayalaxmi, Shepherd AMM (1998a) A novel melatonin metabolite, cyclic 3-hydroxymelatonin: a biomarker of in vivo hydroxyl radical generation. BiochemBiophys Res Commun 253: 614–620

Tan DX, Manchester LC, Reiter RJ, Qi W, Kim SJ, El-Sokkary GH (1998b) Melatoninprotects hippocampal neurons in vivo against kainic acid-induced damage in mice. JNeurosci Res 54: 382–389




Tanzi RE, McClatchey AI, Lamperti ED, Villa-Komaroff L, Gusella JF, Neve RL (1988)Protease inhibitor domain encoded by an amyloid protein precursor mRNA associ-ated with Alzheimer’s disease. Nature 331: 528–530

Tanzi RE, Kovacs DM, Kim TW, Moir RD, Guenette SY, Wasco W (1996) The genedefects responsible for familial Alzheimer’s disease. Neurobiol Dis 3: 159–168

Teplow DB (1998) Structural and kinetic features of amyloid beta-protein fibrillogenesis.Amyloid 5: 121–142Touitou Y, Fevre A, Bogdan A (1984) Patterns of plasma melatonin with aging and

mental condition: stability of nyctoemeral rhythms and differences in season varia-tion. Acta Endocrinol 106: 145–151

Troncoso JC, Costello A, Watson AL Jr, Johnson GVW (1993) In vitro polymerization of oxidated tau into filaments. Brain Res 613: 313–316

Ueda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero DAC, Kondo J,Ihara Y, Saitoh T (1993) Molecular cloning of a cDNA encoding an unrecognizedcomponent of amyloid in Alzheimer disease. Proc Natl Acad Sci USA 90: 11282–11286

Uz T, Longone P, Manev H (1997) Increased hippocampal 5-lipoxygenase mRNAcontent in melatonin-deficient, pinealectomized rats. J Neurochem 69: 2220–2223

Vitek MP, Bhattacharya K, Glendening JM, Stopa E, Vlassara H, Bucala R, Manogue K,Cerami A (1994) Advanced glycosylation end products contribute to amyloidosis inAlzheimer disease. Proc Natl Acad Sci USA 91: 4766–4770

Volicer L, Crino B (1990) Involvement of free radicals in dementia of the Alzheimer’stype: a hypothesis. Neurobiol Aging 11: 567–571

Weidemann A, Konig G, Bunke D, Fischer P, Salbaum JM, Masters CL, Beyreuther K(1989) Identification biogenesis and localization of precursors of Alzheimer’s diseaseA4 amyloid protein. Cell 57: 115–126

Wisniewski T, Castaño EM, Golabek A, Vogel T, Frangione B (1994) Acceleration of Alzheimer’s fibril formation by apolipoprotein E in vitro. Am J Pathol 145: 1030–1035

Yan SD, Yan SF, Chen X, Fu J, Chen M, Kuppusamy P, Smith MA, Perry G, GodmanGC, Nawroth P (1995) Non-enzymatically glycated tau in Alzheimer’s disease in-

duces neuronal oxidant stress resulting in cytokin gene expression and release of amyloid -eptide. Nature Med 1: 693–699Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Nagashima M,

Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM (1996) RAGE and amyloid- peptide neurotoxicity in Alzheimer’s disease. Nature 382: 685–691

Yan SD, Shi Y, Zhu A, Fu J, Zhu H, Zhu Y, Gibson L, Stern E, Collison K, Al-MohannaF, Ogawa S, Roher A, Clarke SG, Stern DM (1999) Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Abeta-induced cytotoxicity. J BiolChem 274: 2145–56

Yanker BA, Duffy LK, Kirschner DA (1990) Neurotrophic and neurotoxic effects of amyloid protein: reversal by tachykinin neuropeptides. Science 270: 279–282

Zang LY, Cosma G, Gardner H, Vallyathan V (1998) Scavenging of reactive oxygenspecies by melatonin. Biochem Biophys Acta 1425: 469–477

Zhang L, Zhao B, Yew DT, Kusiak JW, Roth GS (1997) Processing of Alzheimer’samyloid precursor protein during H2O2-induced apoptosis in human neuronal cells.Biochem. Biophys Res Commun 235: 845–848

Zhang Z, Drzewiecki GJ, Hom JT, May PC, Hyslop PA (1994) Human cortical neuronal(HCN) cell lines: a model for amyloid beta neurotoxicity. Neurosci Lett 177: 162–164

Zhang Z, Hartmenn H, Do, VM, Abramowski D, Stuchler-Pierrat C, Staufenbiel M,Sommer B, de Wetering M, Clevers H, Saftig P, De Strooper B, He X, Yankner BA(1998) Destabilization of -catenin by mutations in presenilin-1 potentiates neuronalapoptosis. Nature 395: 698–702




Zhao X, Valantas JA, Vyas S, Duffy LK (1993) Comparative toxicity of amyloid betapeptide in neuroblastoma cell lines: effects of albumin and physalaemin. CompBiochem Physiol 106: 165–170

Zheng H, Jiang M, Trumbauer ME, Sirinathsinghji DJ, Hopkins R, Smith DW, HeavensRP, Dawson GR, Boyce S, Conner MW et al. (1995) beta-Amyloid precursor protein-

deficient mice show reactive gliosis and decreased locomotor activity. Cell 81: 525–31Zlokovic BV, Frangione B, Ghiso G (1998) Neurovascular interactions of Alzheimer’s

amyloid peptide with apolipoprotein J and E. In: Finch CE (ed) Normal brainfunctions and during neurodegeneration. Plenum, New York, pp 71–87

Authors’ address: Prof. R. J. Reiter, Department of Cellular and Structural Biology,The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX78284-7762, U.S.A., e-mail: [email protected]


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