alz disease wiley.pdf

REVIEW ARTICLE

Alzheimers disease: pathological mechanisms and the beneficialrole of melatonin

Introduction

Alzheimers disease (AD) is a devastating disorder affectingaround 35 million people worldwide [1]. Ten years ago,there were 4.5 million persons with AD in the US popu-

lation alone; the number has increased to 5.3 million peoplein 2010, according to the Alzheimers Association [2]. It isestimated there will be 13.2 million people with thisneurodegenerative disorder by 2050 [3].

Alzheimers disease is a primary, progressive neurologicaldisease, which is of unknown etiology in more than 90% ofthe cases. Some characteristic neuropathological and neu-

rochemical features lead to irreversible loss of neurons.Owing to the nature of the primarily affected neuronalcircuits, the clinical hallmarks of AD are progressive

impairment in memory, judgment, decision making, orien-tation to physical surroundings, and distorted language.This illness is the leading cause of dementia in older people[2].

There currently is no cure for AD. A recent meta-analysisof functional outcomes for commercially available acetyl-cholinesterase inhibitors and memantine in the treatment of

patients with AD, the only FDA-approved drugs for AD,revealed only a modest trend favoring active treatment overplacebo [4, 5]. Anti-inflammatory agents may reduce therisk of developing AD [6], but, on the contrary, according

to results obtained from an elderly communitybasedcohort study, anti-inflammatory agents could even bedangerous for cognitive abilities [7]. It is also possible that

anti-inflammatory drugs have no influence at all with theexemption of their well-known collateral effects [8, 9].Vitamin E, estrogens, omega-3 fatty acids, and Ginkgo

biloba have been tested in different studies, and they yieldedcontradictory results. And there is a long list of experi-mental therapies targeting different protagonists in thepathology of AD, such as tau protein, amyloid-b (Abtargets: formation, aggregation, or toxicity), Ab receptorsor N-methyl-D-aspartate (NMDA) antagonists, serotonin

Abstract: Alzheimers disease (AD) is a highly complex neurodegenerativedisorder of the aged that has multiple factors which contribute to its etiology

in terms of initiation and progression. This review summarizes these diverse

aspects of this form of dementia. Several hypotheses, often with overlapping

features, have been formulated to explain this debilitating condition. Perhaps

the best-known hypothesis to explain AD is that which involves the role of

the accumulation of amyloid-b peptide in the brain. Other theories that havebeen invoked to explain AD and summarized in this review include the

cholinergic hypothesis, the role of neuroinflammation, the calcium

hypothesis, the insulin resistance hypothesis, and the association of AD with

peroxidation of brain lipids. In addition to summarizing each of the theories

that have been used to explain the structural neural changes and the

pathophysiology of AD, the potential role of melatonin in influencing each

of the theoretical processes involved is discussed. Melatonin is an

endogenously produced and multifunctioning molecule that could

theoretically intervene at any of a number of sites to abate the changes

associated with the development of AD. Production of this indoleamine

diminishes with increasing age, coincident with the onset of AD. In addition

to its potent antioxidant and anti-inflammatory activities, melatonin has a

multitude of other functions that could assist in explaining each of the

hypotheses summarized above. The intent of this review is to stimulate

interest in melatonin as a potentially useful agent in attenuating and/or

delaying AD.

Sergio A. Rosales-Corral1,2, DarioAcuna-Castroviejo3, Ana Coto-Montes2, Jose A. Boga2, LucienC. Manchester2, Lorena Fuentes-Broto2, Ahmet Korkmaz2, ShuranMa2, Dun- Xian Tan2 and RusselJ. Reiter2

1Centro de Investigacion Biomedica de

Occidente del Instituto Mexicano del Seguro

Social, Guadalajara, Jalisco, Mexico;2Department of Cellular and Structural Biology,

University of Texas Health Science Center at

San Antonio, San Antonio, TX, USA;3Departamento de Fisiologa, Instituto de

Biotecnologa, Universidad de Granada,

Granada, Spain

Key words: Alzheimers disease, amyloid-bpeptide, calcium, cholinergic

neurotransmission, free radicals,

inflammation, insulin resistance, melatonin,

oxidative stress

Address reprint requests to Sergio A. Rosales-

Corral, Centro de Investigacion Biomedica de

Occidente del Instituto Mexicano del Seguro

Social, Sierra Mojada 800 colonia Indepen-

dencia, Guadalajara, Jalisco, CP 45150,

Mexico.

E-mail: [email protected]

Received September 30, 2011;

Accepted October 4, 2011.

J. Pineal Res. 2012; 52:167202Doi:10.1111/j.1600-079X.2011.00937.x

2011 John Wiley & Sons A/SJournal of Pineal Research

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receptors, loss of acetylcholine neurons, cholesterol, andantiaging drugs (reviewed in [10]; Fig. 1).Likewise, there is insufficient clinical evidence to support

the effectiveness of melatonin by itself in managing thecognitive and noncognitive sequelae of people with demen-tia [11]. However, there are molecular and physiological

bases that are worth analyzing, because melatonin mayhave an effective influence on several of the above-mentioned AD protagonists and the most prominenthypotheses to explain the cause of this disease, as reviewed

below. There is also a growing body of evidence indicatingthe potential role of melatonin as an effective adjuvant inAD management [1217]. On the contrary, there is one

report indicating essentially negative results after usingmelatonin in patients with AD [18]. Even worse, there is asingle recent publication which claims that melatonin may

aggravate this neurodegenerative disorder [19].The authors of the current review declare no conflict of

interest related to this paper or financial relationships with

commercial entities. The aim is to put together evidence onmelatonins role in the best-known hypotheses that cur-rently attempt to explain AD pathogenic mechanisms,starting with the fact that AD-related changes begin at the

age when melatonin levels fall significantly. However, it isalso clear from the beginning that more than 100 yr afterthe first clinical report of a case of AD, there is not yet a

satisfactory hypothesis or a model capable of explaining orreproducing the pathogenic mechanisms of this devastatingdisease. Thus, all the proposed treatments for AD are

groping for optimal experimental outcomes in regard toobviously incomplete hypotheses. This incompleteness mayexplain, at least in part, how different models yield widelydifferent results. For example, long-term oral administra-

tion of melatonin in an amyloid precursor protein(APP) + PS1 double transgenic mice model of AD pro-tects against cognitive deficits and markers of neurode-

generation [20], while it fails to protect animals expressingthe Swedish AD mutant gene (Tg2576 mice) exposed toaluminum [21].

Melatonin levels in AD

Cerebral spinal fluid (CSF) melatonin levels are reportedlysignificantly decreased in aged individuals with earlyneuropathological AD-related changes in the temporal

cortex [22]. In aged patients, melatonin levels in CSF havebeen found to be one-half those in young control subjects,but in patients with AD, the CSF melatonin levels are onlyone-fifth those in young subjects [23]. In fact, it is possible

to replicate hippocampal CA1 and CA3 pyramidal neuronloss in rats by merely removing the pineal gland (whichlowers melatonin levels) with this effect being reversed by

melatonin replacement in the drinking water [24]. Also,constant light exposure, which decreases serum melatonin,is enough to cause Alzheimer-like damage, such as memory

deficits, tau hyperphosphorylation at multiple sites, activa-tion of glycogen synthase kinase-3 and protein kinase A, aswell as suppression of protein phosphatase-1 and promi-nent oxidative stress [25].

Melatonin, mechanisms of action

Melatonin is derived from the aminoacid tryptophan in amultistep process involving the synthesis of serotonin,which is subsequently N-acetylated and O-methylated [26].

Melatonin is 5-methoxy-N-acetyltryptamine produced bythe functional elements of the pineal gland; once released, itacts both as an endocrine product and as an antioxidant.

Several precursors of melatonin including tryptophan andserotonin are reduced by aging, and their reduction may belinked to AD appearance [27, 28] (Fig. 2). Tryptophandeficiency is related to an accelerated degradation attrib-

uted, as reviewed below, to homocysteine, a risk factor fordementia and AD [29]. Serotonin deficiency, on the otherhand, is linked to severe psychiatric symptoms in AD [28],

although serotonin dysfunction may appear long beforepsychiatric symptoms; these symptoms are associated withaltered brain serotonin transporter and glucose metabolism

as identified using in vivo molecular imaging [30].

Fig. 1. Major targets for Alzheimersdisease therapy.

Rosales-Corral et al.

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Besides the pineal gland, melatonin is also presumablyproduced in gastrointestinal tract, airway epithelium, pan-creas, adrenal glands, thyroid gland, thymus, urogenital

tract, placenta, and other organs [31]. Even nonendocrinecells, such as mast cells, natural killer cells, eosinophilicleukocytes, platelets, and endothelial cells, may produce

melatonin [32]; this wide synthesis underlines its diversephysiological activities: from the control of biologicalrhythms [33, 34], metabolism of free radicals [3539],immune responsiveness [4042], monitoring of mood and

sleep [4345], cell proliferation and differentiation [46, 47],and control of seasonal reproduction [48, 49]. Importantly,melatonin production declines with age [5053] because of

dysfunction of the sympathetic regulation of pineal mela-tonin synthesis by the suprachiasmatic nucleus (SCN), acondition probably linked to early AD stages, once that the

reactivation of the circadian system using light therapy andmelatonin has shown promising positive results [54].

Firmly established as the key mediator controllingcircadian rhythms [33, 34, 55], it has been discovered that

melatonin, a small, lipophilic molecule, also had thecapacity to directly scavenge the hydroxyl radical (OH)[56, 57]. Almost immediately, a link between melatoninshydroxyl radical-scavenging activity and aging was envi-sioned [58] as well as realizing that aging and Ab-inducedoxidative stress play a key role in AD as well.

Currently, melatonin is recognized both as a free radicalscavenger and as an antioxidant [59]. Thanks to its electron-rich aromatic indole ring, melatonin directly donates an

electron to free radicals at a potential of 715 mV and avoidsredox recycling (reviewed in [60]), while it scavenges, withvarying degrees of efficiency, the hydroxyl radical [56, 60

64], hydrogen peroxide [65], hypochlorous acid [66], singletoxygen [67], superoxide anion radical [68], nitric oxide [69],and the peroxynitrite anion [70] (Fig. 3). Radicals may alsobe added in the C3 amide side chain of melatonin, which

possesses an NC=O functional group [71]. The rateconstant for the scavenging of the OH by melatonin iscalculated to be on the order of 2.7 1010/M/s [72].The free radical-scavenging capacity of melatonin also

extends to its secondary, tertiary, and quaternary meta-bolites in a free radical-scavenging cascade that prolongs

its useful life [7375]. Thus, the interaction of melatoninwith free radicals produces the oxidative pyrrole-ringcleavage, giving N1-acetyl-N2-formyl-5-methoxykynur-amine (AFMK), and this substituted kynuramine may

donate two electrons at different potentials (456 and668 mV, respectively) to function as a reductive moleculecapable to destroy reactive species and to protect macro-

molecules against oxidative damage [76]. Thus, via theAFMK pathway, a single melatonin molecule may scav-enge up to 10 reactive oxygen and reactive nitrogen species

Fig. 2. Melatonin and its precursors,tryptophan and serotonin (*), appearreduced in aging, which is particularlysignificant in Alzheimers disease brain.

Fig. 3. Major oxidant pathways and therole of melatonin as an antioxidant(dashed blue arrows), promoting theactivity of antioxidant enzymes. Derivedfrom metabolic activity, particularlyfrom mitochondria in aging, melatoninplays an important role as a free radicalscavenger (blue balloons).

Alzheimers disease and melatonin

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(ROS/RNS) [73]. Further AFMK deformylation by theaction of arylamineformamidase or hemoperoxidase en-zymes produces N1-acetyl-5-methoxykynuramine (AMK)

[77], which, in addition to its ability to react with variousoxidizing and nitrosating free radical species, particularlysinglet oxygen and nitrogen species, also may destroycarbonate and peroxyl radicals (reviewed in [78]) and

function as an antioxidant [79]. AFMK and AMK metab-olism may lead to other oxidation products, such as 3-indolinone, cinnolinone, and quinazoline compounds, for

which no specific functions have been identified to date [78].Interestingly, the parallel orientation of b-sheets, such astau and Ab filaments, generates channels extending alongthe length of the filament to which aromatic small mole-cules such as indolinones can bind via pp interactions,stacked arrangement of aromatic molecules [80]. Usingfluorescence spectroscopy, atomic force microscopy, and

electron microscopy to screen 29 indole derivatives, Cohenet al. [81] identified three potent inhibitors of amyloid fibrilformation and cytotoxicity, and the indole-3-carbinol was

among them. The interaction of melatonin with Ab will bereviewed below. Additionally, the 3-substituted indolinoneshave been identified as kinase inhibitors [82], which could

be related to the anti-inflammatory actions of melatoninand its metabolites [83].Melatonin may also prevent abnormal elevation of

reactive nitrogen species, stimulate other antioxidant sys-tems, and/or inhibit some pro-oxidant enzymes; theseindirect actions of melatonin contribute to its potentantioxidant activity [8486]. An evaluation in human

diabetic skin fibroblasts demonstrated that melatoninincreases the activity of superoxide dismutase (SOD),catalase (CAT), and glutathione peroxidase (GPx) and

the level of glutathione (GSH) [87] (Fig. 3). Similar resultshave been obtained in fetal rat brain [88], in experimentalbrain trauma [89], as well as in cultured dopaminergic cells[90] and, of course, in AD transgenic mouse brain [91, 92].

These observations allow for the conclusion that melatoninexerts an antioxidant action by increasing the mRNA levelsof the antioxidant enzymes SOD, CAT, and the GSH

system, but it may depend somewhat on the model systeminvestigated.Finally, melatonin exhibits a lower-affinity binding site to

a cytosolic quinine oxidoreductase 2 or QR2, also known asMT3 (reviewed in [93]). This enzyme, as any other quinone,has the ability to transform its substrates into more highlyreactive compounds that are able to cause cellular damage.

Once melatonin binds to its large active site, the enzymeproduces fewer hydrogen bonds and hydrophobic contacts,which diminishes their reactivity [94] (Fig. 4).

Melatonins best-known receptors, MT1 and MT2, aretransmembrane G-protein-coupled heterodimers whosesignaling pathways lead to downstream effects on Ca2+

channels, Ca2+ signaling, and changes in MAP and ERKkinases and/or PI3K/Akt pathways [95] (Fig. 4). Thismeans a broad spectrum of possibilities for melatonin is

one factor that gives the indoleamine a pleiotropic nature[96, 97].Once again, it is worth noting that the pathogenic

mechanisms in AD are not well understood, and there are

Fig. 4. Receptor-mediated or acting directly on its substrates, melatonin exhibits a broad diversity of effects to reduce neurodegenerativechanges in the central nervous system. It is a pleiotropic indoleamine, actually. All the protagonists in neurodegenerative diseases expressmelatonin receptors; when and how cells or their molecular effectors become activated or inhibited according to the expression of theirmelatonin receptors remains unclear. The scheme shows some of the published observations to date, all related to central nervous systemand/or neurodegeneration. Thanks to its ability to transfer electrons, melatonin may repair damaged biomolecules derived from DNAoxidation, such as guanosine. LTP, long-term potentiation; MT, melatonin receptor (G-protein-coupled receptors); CaM, calmodulin; CRT,calreticulin; APP, amyloid b precursor protein; 5-LOX, 5-lipoxygenase; COX-2, cyclooxygenase; iNOS, inducible nitric oxide synthase;PLA2, phospholipase A2.


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many hypotheses regarding this major cause of dementia.The three major hypotheses as well as their derivatives willbe the common thread throughout this review. What has

been published regarding melatonin and its potential role ineach proposed mechanism will be added herein, whereappropriate.

Pathogeny of AD

There is a failure of the intercommunication between

neuronal circuits in Alzheimers disease resulting fromsynaptic loss and the destruction of neurons. As a conse-quence, working memory is not transferred through the

hippocampus to long-term memory circuits. The discon-nection progresses over time and affects some otherfunctions in addition to memory, so that behavior, exec-utive functioning, judgment, movement coordination, and

pattern recognition may become eventually affected.Three major hypotheses have been primarily explored in

an attempt to explain AD: (i) cholinergic hypothesis, (ii)

amyloid cascade hypothesis, and (iii) mitochondrial cascadehypothesis. Even though the amyloid cascade is the mostextended hypothesis [98], the pathogenic role for Ab isunder debate because of reports showing a poor relation-ship between Ab accumulation and cell death in the brain,in addition to other results demonstrating a weak correla-

tion between Ab and cognitive decline [99]. Furthermore,people with Ab deposits do not necessarily suffer AD [100].Even more importantly, there are some published datademonstrating that Ab may be protective in brain disease[101, 102]. Thus, the pathogenic role of Ab in AD deservesfurther scrutiny because all of the hypotheses mentionedinclude a pathological role of Ab in AD.

Amyloid-b processing

Although far from conclusive, Ab is the most studied factorrelated to pathogenic mechanisms of AD. Ab is derivedfrom the catalytic cleavage of an integral membraneprotein, the APP, a ubiquitously expressed type I trans-

membrane protein whose primary function is not wellknown. This is the first obstacle in Alzheimers research,actually. It is known that the lack of APP in hippocampal

neurons enhances neuritic growth, which influences onlythe synapse number at an early age but not in adult animals[103]. The conserved APP intracellular domain, genetically

uncoupled from APP processing and Ab pathogenesis, hasa key role in survival, proper high-affinity choline trans-porter (ChT) targeting, and neuromuscular synapse devel-

opment [104]. ChT is responsible for choline uptake fromthe synaptic cleft, a rate-limiting step in acetylcholinesynthesis [105]. An extensive review [106] tackles in detailthe possible role of APP in synaptic transmission and

neural plasticity, with its respective implications for learn-ing and memory.

Closely associated with lipid rafts in membranes, com-

posed mostly of sphingolipids and cholesterol, the enzymesin charge of APP cleavage are proteases generating solubleisoforms of membrane proteins, a process first related to

the secretion of angiotensin-converting enzyme in thekidney [107]. Thus, the APP chain of 695, 751, or 770

amino acids may suffer consecutive cleavage events. Thelarge extramembranous N-terminal region may undergoproteolysis by the a-secretase that cleaves the moleculebetween Lys687 and Leu688, releasing a large (105125 kDa), soluble ectodomain known as sAPPa [108]. ThissAPPa carries a portion of the Ab sequence [109] thatnormally includes 28 amino acids of the extracellular and

1215 residues of the membrane-spanning region of APP;thus, the subsequent formation of amyloidogenic peptidesmay be precluded. This a-secretase may be modulated bymetal ions and metalloprotease inhibitors and three relateddisintegrin and metalloprotease enzymes, ADAM9 [110],ADAM10 [108], and ADAM17 [111], which seem to exert

an a-secretase activity. It has been speculated that theactivation of these proteases, representing a nonamyloido-genic pathway, may offer a therapeutic method in AD [112,113]. As a matter of fact, it is remarkable that levels of a-secretase ADAM-10 and sAPPa are reduced in the CSF ofpatients with AD compared to that of controls [114].The amyloidogenic pathway is established by the con-

certed action of two secretases, the b-secretase, whichcleaves the APP-N terminus, and the c-secretase, whichcleaves the APP-C terminus in the secondary transmem-

brane region. b-secretase is an aspartyl protease of 501amino acids with two aspartic protease active site motifs,which is known as the b-site APP cleaving enzyme I orBACE-1 [115] and considered a prime drug target forlowering cerebral Ab levels in the treatment for and/orprevention of AD. It is the initiating and rate-limitingenzyme in Ab generation. BACE-1 activity on APP isrelated to the accessibility of APP within a lipid raft zone ofthe membrane [116, 117]. Once APP escapes from process-ing at the a-site, it is cleaved at the luminal domain,resulting in a 12 COOH-terminal fragment (C99), whichremains membrane bound, and the soluble APPb NH2-terminal fragment (sAPPb) [118, 119]. Then, C99 is cleavedfrom the membrane by the c-secretase, a multisubunitprotease complex composed of a presenilin catalytic sub-unit in addition to nicastrin, the anterior pharynx-defective

1 (APH-1), and the presenilin enhancer 2 (PEN-2) [120,121]. Binding of cholesterol to C99 appears to favor theamyloidogenic pathway in cells by promoting localizationof C99 in lipid rafts [122]. The resultant peptide of 3943

amino acid residues, Ab, is delivered to the extracellularmilieu where it forms insoluble aggregates and becomes themajor component of senile plaques. Ab140 and Ab142 arethe most common Ab isoforms. Aberrant Ab142 accumu-lation within distal neurites and synapses is directlyassociated with subcellular pathology and neurotransmit-

ters [123125], while Ab140 is the predominant form of theAb peptides but less prone to form fibrils [126]. As aconsequence of re-internalization from the extracellularspace [127, 128] or directly by the cleavage of APP in

endosomes generated from the endoplasmic reticulum (ER)or the Golgi apparatus, Ab peptide accumulation alsooccurs inside neurons leading to trafficking problems, early

axonopathy, synaptic loss, and neuron death [129131].What determines which enzyme will gain access to APP,

thus determining the course of events? There are clues

indicating that cholesterol in lipid rafts directly binds theC-terminal transmembrane domain of APP, and this


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interaction may be a determinant in favor of the amyloi-dogenic pathway [122] (Fig. 5). Cholesterol decreases the

secretion of soluble amyloid precursor protein (sAPP) byinterfering with APP maturation and inhibiting glycosyla-tion in the protein secretory pathway, in such a manner that

APP cannot be cleaved by a-secretase [132]. Processing APPat the b-site also requires proper orientation to be accessedby BACE-1 [133], which in turn localizes largely within

cholesterol-rich lipid rafts [117, 134]. It is proposed thatAPP is actually a cholesterol sensor [117]. The AD brainshows significant cholesterol retention and high b- and c-secretase activities as compared to age-matched nonde-mented controls, while cholesterol depletion may be asso-ciated with reduced cellular cholesterol, b-secretase activity,and Ab secretion [116].Another factor influencing the access to APP in cell

membranes seems to be insulin, a significant associationthat links AD to diabetes mellitus. Insulin accelerates APP

trafficking from the trans-Golgi network to the plasmamembrane [135], while the insulin-degrading enzyme (IDE)degrades not only insulin but also Ab and the intracellulardomain of APP [135, 136]. Thus, insulin reduces intracel-lular levels of amyloid and increases amyloid secretion in aprocess that probably involves the activation of the MAPKcascade [137], although it might also use the phosphatidyl

inositol 3 kinase (PI3K)-pathway to release sAPP, the

nonamyloidogenic secreted form of APP [138]. Impor-tantly, this insulinPI3K pathway locates and halts glyco-

gen synthase kinase-3 (GSK-3) to promote glucose storageas glycogen, as part of intermediary metabolism. However,GSK-3 plays another role. Its a-isoform may interactdirectly with presenilins within the c-secretase complex, andit is required for the amyloidogenic APP processing. This isthe reason why GSK-3 has become a target for the

treatment for AD [139]. Insulin deficiency in brain leadsto enhancement of GSK3a/b activation, increases cerebralamyloidosis, and exacerbates behavioral deficits, as dem-

onstrated in APP/PS1 transgenic mouse model of AD byimpairing insulin downstream GSK3 and JNK pathways[140].Not only fibrillar Ab but a variety of Ab oligomers may

cause cellular damage. Soluble oligomers, referred to asamorphous aggregates, micelles, protofibrils, prefibrillaraggregates, amyloid b-derived diffusible legends (ADDLs),Ab*56, globulomers, amylospheroids, toxic soluble Ab,paranuclei, and annular protofibrils [141], appear withinneuronal processes and synapses rather than within the

extracellular space. They are neurotoxic rather than amy-loid fibrils found in amyloid plaques [142] and may inhibitcritical neuronal functions including long-term potentiation[143], a classic experimental paradigm for memory and

synaptic plasticity [143, 144]. Even more, as a consequence

Fig. 5. Several routes to prevent the formation of Ab neurotoxic aggregates are used by melatonin. It directly intervenes, affecting thestability of amyloid b-sheets by disrupting Asp) -His+ salt bridges or affecting the synthesis and maturation of APP, where its ability tosuppress cAMP activity may have a role, because of the cAMP-responsive regions on the APP promoter gene. However, its indirect actionsare significant as well because melatonin may reduce the activity of GSK3 required for the amyloidogenic APP processing, by activatingand/or enhancing the activity of PKC, or by inducing Akt. Both PKC and Akt may turn off GSK-3 through phosphorylation. COX-2,related to APP synthesis in astrocytes, is controlled by melatonin and its metabolites. Finally, melatonin has a key role in cholesterol andfatty acid distribution in membranes, as reviewed later. This is important because, as illustrated, amyloidogenic APP processing seems to befavored by cholesterol/sphingomyelin-enriched lipid rafts. APP, amyloid precursor protein; bs, b secretase; cs, c-secretase; Ab, amyloid b;MT, melatonin receptors; PLC, phospholipase C; DAG, diacyl-glycerol; PKC, protein kinase C; PI3K, phosphatidylinositol-3-kinase; Akt,a serine/threonine protein kinase; PKA, cAMP-dependent protein kinase.


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of inhibition of the proteasome function, soluble oligomersmay cause cell death [124]. Extracellular soluble Ab species,on the other hand, are deposited around neuronal cell

bodies and may interact with the lipid bilayer withindendritic arbors at discrete points, appearing co-localizedwith the postsynaptic density protein 95 (PSD-95) [145],which is related to synapse stabilization and plasticity [146].

Melatonins role on amyloid-b processing

There are some clues indicating an interaction betweenmelatonin and Ab. By using a thioflavin T (Th T)fluorescence assay, which measures the binding abilities of

different compounds with Ab, it is possible to demonstratethat melatonin directly interacts with Ab and prevents itsaggregation [147]. This fact has been well known since 1997,when it was documented using circular dichroism, electron

microscopy, and nuclear magnetic resonance spectroscopy[148]. This phenomenon is not related to the antioxidantproperties of melatonin [149] and involves the disruption of

the His+-Asp) salt bridges in Ab peptide, which aredeterminants for the formation and stabilization of b-sheetstructures [150]. Thus, 24 hr after the incubation with

melatonin, Pappolla et al. [148] showed that originalb-sheet content of Ab was significantly diminished inopposition to the increase in b-sheet content when Abwas incubated alone (Fig. 5).

A direct interaction between the 5-methoxy group onmelatonin and His-13 of Ab may occur as well. Thiseventuality may be attributed to the higher binding energies

in the 5-methoxyindole group, according to single-pointenergy calculations [151]. Further investigations by electro-spray ionization mass spectrometry (ESI-MS), the hydro-

phobic nature of Ab, and melatonin interaction has beenunveiled, and the proteolytic assessment suggests that theinteraction takes place on the 2940 Ab-peptide segment[152]. As compared with some other antiamyloidogenicagents, such as daunomycin or the melatonin analogue 3-indolepropionic acid, melatonin exhibits a moderate degreeof inhibition of aggregation, as evaluated by ESI-MS [153].

It is also possible that melatonin could regulate thesynthesis and full maturation of APP, as it was demon-strated in melatonin-treated PC12 cells, which responded

by decreasing its mRNA encoding b-APP. According toLahiri and Song [154, 155], melatonin accomplishes thiswhile potentiating the nerve growth factormediated

differentiation.Because APP gene promoter contains c-AMP-responsive

regions, it is possible that c-AMP signaling pathways may

induce APP synthesis, and this eventuality could be a linkbetween neuroinflammation and neurodegeneration, asexplored by Lee et al. [156]. They found that prostaglandinsproduced by brain injury or inflammation increases cAMP

formation and stimulates overexpression of APP mRNAand holoprotein in primary cultures of cortical astrocytes.On the other hand, the relationship between melatonin or

its metabolites and neuroinflammation relies importantlyon its ability to inhibit prostaglandins by interfering withthe COX-2/PGE pathway [157], which implies the partic-

ipation of cAMP as a second messenger. In fact, actingthrough its membrane receptors, melatonin may block

cAMP production, protecting white matter against aneonatal excitotoxic challenge. This neuroprotective effectmay be prevented by luzindole, a well-known membrane

melatonin receptor antagonist, or by forskolin, an adenyl-ate cyclase activator [158]. Thus, in a receptor-mediatedmanner and by inhibiting adenylyl cyclase, melatonin mayimpair cAMP signaling, which is probably involved in the

activation of the APP gene promoter. Therefore, melatonincould interfere with APP synthesis (Fig. 5).Acting through its MT2 receptor, melatonin stimulates

phospholipase C (PLC) and, via diacylglycerol (DAG),activates protein kinase C (PKC) [159], which in turnphosphorylates and inactivates GSK-3, whose participation

in APP synthesis is key, as mentioned before. However,PKC is also capable to directly promote a-secretase-mediated cleavage of APP favoring the nonamyloidogenicpathway [160]. Thus, by activating PKC, melatonin might

impair Ab overproduction (Fig. 5). Moreover, actingthrough its membrane receptors, melatonin uses a PI3K-dependent pathway to activate Akt, a serine/threonine

protein kinase, which, besides participating in multiplesurvival pathways, phosphorylates and inactivates GSK-3[161]. PI3K/Akt is the same pathway employed by insulin

and by the insulin growth factor-1 receptor (IGF-1R) tointerrupt GSK-3b activity under oxidative conditions [162](Fig. 5).

Because the JNK pathway also could be involved inGSK-3 activation [140], we speculate that melatonin, whichprevents JNK activation under oxidative stress conditions[163], may also employ this mechanism to prevent the

activation of GSK-3. It is also feasible that melatonin, in itsrole as antioxidant, might enhance PKC anti-GSK3 activityby avoiding PKC inactivation. This may occur because

PKC is redox sensitive and may be S-glutathiolated andinactivated during oxidative stress in the brain. Oxidativestress is a well-documented phenomenon in Alzheimers[164].Stopping GSK-3 activity could be important not only in

interrupting APP synthesis but also to reduce tau hyper-

phosphorylation, because GSK-3 phosphorylates tau [165,166]. It is worth remembering that the microtubule-associ-ated protein tau is the other key protagonist in ADpathology, being responsible, once hyperphosphorylated,

for paired helical filament (PHF) formation.An indirect interaction between melatonin and Ab

processing has been also proposed involving the hypoxia-

inducible factor-1 (HIF-1), which upregulates BACE-1,facilitating the generation of cytotoxic Ab peptide [167]. Infact, at a pharmacological dose, melatonin may prevent the

generation of Ab peptides by reducing both the BACE-1protein and its mRNA, as demonstrated in a rat modelemployed to evaluate the effect of chronic intermittenthypoxia (CIH) on the Ab generation in the hippocampus.Being a redox-sensitive transcription factor, HIF-1 issusceptible to melatonin redox modulation [164, 168] and,via this indirect manner, melatonin might prevent the

formation of Ab. HIF-1 has been observed to be abundantin AD microvessels where it regulates proinflammatorygene expression [169]. On the contrary, it is also known that

the accumulation of Ab by using a nonhypoxic mechanismmay induce the accumulation and nuclear translocation of


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HIF-1, which in turn mediates a neuroprotective response,presumably by regulating glucose metabolism [170]. HIF-1has a half-life of approximately 5 min in normoxic condi-

tions and less than a minute under hypoxic conditions.Thus, the role of melatonin in these HIF-1 dependentmechanisms is currently only a matter of speculation.Conformational changes in Ab occur in minutes after

addition of melatonin. In fact, the ability of melatonin toinduce conformational changes in Ab has been used toinvestigate the conformation and topology of Ab peptidesinteracting with peptide-tethered planar lipid bilayers [171,172]. Similarly, it has been also demonstrated that lipidcomposition of membrane bilayers plays a dominant role in

mediating conformational changes and in AD pathogeny,as reviewed below.Levels of Ab aggregates in the brain were reduced by

melatonin in aging mice [173], and in 8-month-old APP 695

transgenic [174] mice or in APP + PS1 double-transgenicmice. The latter were supplemented with melatonin from 2to 2.5 months to age 7.5 months [20]. However, in old,

amyloid plaquebearing Tg2576 mice, which started mel-atonin treatment as late as 14 months of age (5 monthslater from the onset of the pathology [175]), melatonin

failed to reproduce its antiamyloid effects (it seems to evenfail to prevent oxidative stress [176]).The melatonin/Ab interaction could be an inconvenience

according to the bioflocculant hypothesis. Even thoughthe investigation related to inhibitors of Ab aggregation asa real promise for many investigators [177], blocking orinhibiting Ab could be a mistake owing to the soluble formsof this peptide, according to this hypothesis. This is becauseAb could have a primordial function: binding to unwantedsolutes in the extracellular fluid, which then precipitates to

build deposits or aggregates. Thus, Ab plaques would be anefficient means of presenting neurotoxins to phagocytes[178].

Extracellular Ab may suppress synaptic plasticity orinhibit long-term potentiation (LTP) [179]. There is, in fact,an odds ratio homocysteine/AD of 4.5 for histopatholog-

ically confirmed AD in a casecontrol study [180], whichalso demonstrated that patients with AD and high homo-cysteine levels showed a more rapid progression over thefollowing 3 yr. Even more so, high homocysteine levels,

considered a risk factor for dementia and AD [29], arerelated to apoptosis [181]. This methionine derivative hasthe ability to induce proapoptotic caspases (caspase 3 and

caspase 9), DNA fragmentation, and the Bcl-2associatedX protein (Bax) while reducing the antiapoptotic Bcl-2protein; these effects may be inhibited in the presence of

melatonin [182].

Cholinergic hypothesis

The cholinergic hypothesis asserts that degeneration ofcholinergic neurons in the basal forebrain and the associ-ated loss of cholinergic neurotransmission in the cerebral

cortex cause deterioration in cognitive function as seen inpatients with AD [183, 184]. This theory was introduced in1971 when it was demonstrated that cholinergic synapses

were modified as a result of learning and that loss ofsensitivity to acetylcholine was related to forgetfulness

[140]. Years later by damaging cholinergic input to theneocortex or hippocampus from the basal forebrain, it waspossible to reproduce a memory impairment as observed in

AD [141]. Based on this old hypothesis, increasing thesynaptic levels of acetylcholine (Ach) with the use ofacetylcholinesterase (AchE) inhibitors has been employedas a treatment and is considered the standard of care for the

treatment for mild-to-moderate AD; meanwhile, the searchfor new AchE inhibitors continues [185]. However,although it remains as a rational approach, nowadays the

real efficacy of this treatment is under debate [142].Both Ab and oxidative stress may reduce Ach synthesis

by reducing choline acetyltransferase activity [186, 187].

However, acetylcholine depletion in the AD brain is alsorelated to free cytosolic ionic calcium and oxidative stress.It is well known that Ab induces elevations of intracellularfree Ca2+ by increasing calcium entry through L-type

voltage-dependent calcium channels [188], and AchErelease is a Ca2+-dependent phenomenon [189]. In thismanner, Ab may elevate AChE activity, as demonstrated inP19 cells [190]. Furthermore, oxidative stress, which is a keyprotagonist in AD, also plays a role in the enhancement ofacetylcholinesterase activity induced by Ab peptide [191].

Melatonins role on the cholinergichypothesis

The Ab-induced AchE activity may be significantly reducedby melatonin, protecting mice from Ab-induced acetylcho-line degradation [147]. Even in LPS injected mice, which

exhibit AchE overactivity, melatonin inhibits this effect asdemonstrated in the neocortical and hippocampal regionsin vivo [192].

Ab selectively interacts with the potentially neurotoxicNMDA receptor via a postsynaptic site [193], leading todysregulation of Ca2+, which is explained because an

intense stimulation of NMDA-type glutamate receptorsresults in a sustained elevation of cytosolic free calcium([Ca2+]c) and its consequential dysregulation [194]. Themechanism of AchE control may be related to the stabil-

ization of Ca2+ levels, because it seems that the increase inAChE expression around amyloid plaques is a consequenceof a disturbance in calcium homeostasis; in fact, intracel-

lular calcium mobilization upregulates AChE expression bymodulating promoter activity and mRNA stability and, onthe contrary, chelation of intracellular Ca2+ may inhibit

acetylcholinesterase expression [195]. Thus, by controlling[Ca2+]c, it is possible to control AchE activity. Melatonincontrols Ca2+ influx through different pathways, and by

these means, it could control acetylcholinesterase expres-sion as reviewed below. Hence, melatonin is important asan acetylcholinesterase and butyrylcholinesterase inhibitor,especially the cyclic 3-hydroxymelatonin analogues, which

exhibit structural similarity to cholinesterase inhibitordrugs [196, 197] (Fig. 6). Melatonin, like insulin and theanticholinesterase drug, donepezil, exhibits an antiamnesic

effect in amnesic mice mediated by enhancement ofcholinergic activity at the expense of decreasing AChEactivity [198].

Considering that oxidative stress and particularly perox-ynitrite (ONOO)) overproduction is significant in AD


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brain, where the latter mediates neurotoxicity of Ab [199], itis possible that S-nitrosylation of metabolic Ach interme-diaries has a significant role. Among the multiple targets ofOONO) in AD brain, choline acetyltransferase (ChAT)

may be a good candidate. ChAT, whose activity has beenshown to decrease in the AD brain [200], may undergoS-nitrosylation followed by lysis and oligomerization, asdemonstrated in cholinergic nerve endings and synaptic

vesicles from Torpedo marmorata electroneurons [186].Even though the mechanism is not fully understood, thehigh-affinity ChT, which provides choline for acetylcholine

synthesis in neurons, seems to be regulated by OONO) aswell [201]. Thus, we speculate that melatonin may promotecholine transport [200, 202] (Fig. 6).

There are some other factors also involving melatonin inthe cholinergic hypothesis even though the mechanism isnot fully understood. For example, ChAT, which bindsacetyl coenzyme A to choline in Ach synthesis, has been

found decreased in the frontal cortex and hippocampus ofAPP 695 transgenic mouse model of AD, and melatonin,chronically administered for 4 months, restored ChAT

levels as observed in the transgenic animals [174]. It ispossible to find ChAT oxidatively modified by the lipidperoxidation product, 4-hydroxy-2-nonenal (HNE) [203], a

diffusible electrophile that covalently binds to proteins viaMichael addition to Cys, His, and Lys residues [164, 204,205]. Thus, owing to its free radical-scavenging activity aswell as its indirect antioxidant actions, melatonin may

reduce ChAT nitrosylation and/or oxidation [186] (Fig. 6).However, in rats infused intracerebroventricularly withamyloid-beta for 14 days, where ChAT activity was signif-

icantly reduced, melatonin was unable to restore theactivity of this enzyme [205].

Oxidative stress and neuroinflammation inthe pathology of AD

It is well known that the accumulation of Ab in plaques aswell as Ab oligomers may produce sequential inflamma-tory/oxidative events and excitotoxicity, causing neurode-generation and cognitive impairment [206]. In one way or

another, all the proposed mechanisms for explaining ADpathogenic mechanisms are connected to oxidative stressand neuroinflammation, widely known hallmarks not only

for AD but in general for all neurodegenerative diseasesand obviously linked to the amyloid cascade [207209].Metabolic signs of oxidative stress in AD are always

evident in neocortex and hippocampus, related to altera-tions in synaptic density. In response to elevated brainperoxide metabolism, AD brains show increased cerebralglucose-6-phosphate dehydrogenase activity [210], which is

the first and rate-limiting enzyme of the pentose phosphatepathway, central to maintenance of the cytosolic pool ofNADPH and thus the cellular redox balance. Even the

brains of preclinical AD individuals, with normal antemor-tem neuropsychological test scores but abundant ADpathology at autopsy, may exhibit increased levels of the

major product of lipid peroxidation, 4-hydroxynonenal,and acrolein, a powerful marker of oxidative damage toprotein [211]. Inferior parietal lobule samples from earlyAD patients compared to age-matched controls have been

examined for proteomic identification of nitrated brainproteins that revealed significant alterations in antioxidantdefense proteins and energy metabolism enzymes, with all

of them being directly or indirectly linked to AD pathology[212].Ab neurotoxic properties depend heavily on free radicals.

The overproduction of free radicals in the pathogeny of ADmay come from the microglial respiratory burst in responseto Ab-induced neuroinflammatory events [213216]. Themicroglial respiratory burst in AD may result from (i) theinteraction of Ab with specialized receptors, (ii) theastrocyte/microglia intercommunication, or (iii) detectionof damage-associated molecular patterns (DAMPs)

through their corresponding receptors, leading to theactivation of the phagocytic nicotinamide adenine dinucle-otide phosphate (NADPH)-oxidase (PHOX). The activa-

tion of the NADPH oxidase probably both in neurons andin glia [217219] links redox control and neuroinflamma-tory signaling pathways [220].

Ab causes microglial proliferation mediated by PHOX,which is demonstrated by a marked translocation of the

Fig. 6. Melatonin may have a role as an acetylcholine enhancer byblocking the Ca2+-dependent release of anticholinesterase enzyme(red cross #1) or allowing the proper reentry of choline (red cross#2) by avoiding ChT oxidation. It is possible that melatonin re-stores ChAT activity under oxidative stress conditions (red cross#3) as observed in APP transgenic mice. However, the role ofmelatonin in cholinergic hypothesis remains to be clarified becauseoxidative stress along with calcium dysregulation, as observed inchronic cellular stress, is relevant in acetylcholine expression and itsmetabolism as well as Ach receptor activity (red cross #4). Mit,mitochondria; ER, endoplasmic reticulum; ChAT, choline acetyltransferase; Ach, acetylcholine; ChT, choline transporter; AchE,acetylcholinesterase; AchR, acetylcholine receptor.


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cytosolic factors p47phox and p67phox to the microglialmembrane in brains of patients with AD, and this iscorrelated with proinflammatory events, such as TNF-aand IL-1b overproduction [221, 222]. The synergy betweenoxidative and nitrosative stress plus neuroinflammationmay increase the overproduction of OONO) by a1,000,000-fold [223]. PHOX is a multicomponent enzyme

system composed of two integral membrane proteins,p22phox and gp91phox, integrated as cytochrome b558,three essential cytosolic components, p47phox, p67phox,

p40phox, and the above-mentioned GTPase Rac1, of theRho family of small G proteins. In general terms, thecomplex begins its integration when the cytosolic p47phox

subunit becomes phosphorylated and transports the totalcytosolic components to the docking site where theyassemble to the flavocytochrome b558 (reviewed in [224]).GTP-bound Rac coordinates the translocation of the

p47phox/p67phox/p40phox complex and its dissociationfrom GTP permits the subsequent inactivation of the

PHOX complex, a crucial step where SOD plays a key roleacting as a stabilizer of Rac [225]. Once integrated, PHOXtransfers electrons from NADPH to molecular oxygen

generating O2 .Because Ab induces oxidative stress that is related to

mitochondrial damage, a mechanism closely linked toapoptosis is established [226229]. Reciprocally, oxidative

stress may induce intracellular accumulation of Ab,enhancing the amyloidogenic pathway [226, 230, 231](Fig. 7).

Additionally, Ab142 may initiate free radical chainreactions by itself. It has a critical methionine residue atposition 35, which is highly hydrophobic and possesses a

sulfur atom sensitive to oxidation (:S: fi O=S: fiO=S=O) [231], or if the lone pair of electrons on the Satom undergoes one-electron oxidation, it produces apositively charged sulfuranyl radical (MetS+) [232]

(Fig. 7). In this manner, SO bonded MetS+ may initiatefree radical chain reactions with allylic H atoms on

Fig. 7. Ab plaques and oligomers are in the middle of a complex set of interactions among astrocytes, microglia, and neurons originating aneuroinflammatory response. This is linked to reactive oxygen species (ROS) and NOS overproduction (gray clouds) culminating inoxidative stress, which in turn feeds back on neuroinflammation. Organelle dysfunction, particularly mitochondria, adds more free radicalsand aggravates the situation. Even worse, oxidative stress and Ab are interdependent phenomena; thus, the more the oxidation, themore the amyloid accumulation. Newly formed Ab contributes to more neuroinflammation and oxidative stress, closing the vicious cycle. Infact, Ab can be an oxidant by itself, as shown. During inflammatory and oxidative stress, communication between cellular protagonists isimportantly mediated by calcium waves (blue waves) apart from cytokines. Melatonin (green) and its major metabolites AFMK andAMK play key roles by scavenging free radicals directly, while they enhance endogenous antioxidant systems, as shown in Fig. 3. AMK isrelevant particularly in mitochondria, where it takes ETC components as electron donors or acceptors. Going further, melatonin and itsmetabolites have a role in neuroinflammation by regulating both proinflammatory signals and oxidative stress mediators, such as COX2and iNOS by avoiding NF-jB full integration. ctk, cytokines; Ab, amyloid-beta; AFMK, N1-acetyl-N2-formyl-5-methoxykynuramine;AMK, N1-acetyl-5-methoxykynuramine; 3-OHM, cyclic 3-hydroxymelatonin; ETC, electron transport chain; NF-jB, nuclear factor kappaB; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase; PGE2, prostaglandin E2.


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unsaturated acyl chains of lipids making the lipid hydro-peroxide and propagating the chain reaction [233].

Ab can also directly trap molecular oxygen, reducing it toH2O2 in the presence of iron (Fenton reaction), as it hasbeen demonstrated by spectrochemistry in AD brain [234].Fe2+ ions are generated via a redox cycling of iron (Fe2+

M Fe3+), and in the presence of a metal chelator, such asclioquinol, this Ab neurotoxicity is reduced [235]. Thematter is relevant because significant alterations in Cu, Zn,and Fe have been found in AD brain in those areas showing

severe histopathologic alterations [236, 237]. In general,drugs that prevent oxidative stress include antioxidants,modifiers of the enzymes involved in ROS generation and

metabolism, metal-chelating agents and agents, such asanti-inflammatory drugs, that can remove the stimulus forROS generation.

H2O2 is a well-known uncoupler of the mitochondrial

respiratory activity, producing a concentration-dependentinhibition of state 3 (ADP-stimulated) respiration andreducing substantially the ADP:O ratio [238]. An evalua-

tion of electron transport chain complexes and Krebs cycleenzymes revealed that a-ketoglutarate dehydrogenase, suc-cinate dehydrogenase, and aconitase are susceptible to

H2O2 inactivation, which is a reversible process [239].Under normal conditions, excessive ROS are neutralized

by the action of endogenous and exogenous antioxidant

defense systems. In addition to the above-mentionedoxidant-generating properties, Ab may bind the peroxidaseenzyme, CAT with high affinity, inhibiting H2O2 break-down [240] and thus worsening redox conditions. However,

all the antioxidant mechanisms play roles in the AD brain.Thus, the overexpression of superoxide dismutase-2 (SOD-2), which is localized to mitochondria, scavenges hippo-

campal superoxide and prevents memory deficits in Tg2576AD mice [241], which carry both mutant APP andpresenilin 1 transgenes [242]. In another AD mouse model,

3xTg-AD, there are significant rises in the activities of SODand GPx, compared with the controls, whereas levels ofreduced GSH are significantly decreased with a concomi-

tant rise in oxidized glutathione (GSSG). This set of eventsimplicates a high oxidative state and depletion of protondonors [243]. The 3xTg-AD mouse harbors PS1M146 V,APPSwe, and tauP301L mutations and progressively develops

extracellular senile plaques and intracellular neurofibrillarytangles (NFTs) as well as cognitive impairments [244].Interestingly, even the exogenous antioxidant systems seem

to fail in AD, which are apparently not related to under-nourishment because, as demonstrated in 79 patients wherethe plasma chain-breaking antioxidants a-carotene,b-carotene, lycopene, vitamin A, vitamin C, and vitaminE were measured by HPLC in addition to a total antiox-idant capacity assay, a tool for measuring the inhibitoryeffect of antioxidants [245]; all of the measured parameters

were below the normal range.

Microglia activation and neuroinflammatoryresponse

A common factor in AD pathogeny is the overactivation of

microglia with the consequent overexpression of proinflam-matory cytokines and a significant increase in ROS [246

248]. ROS, in turn, may come from the innate immuneresponse promoted by danger signals [249, 250] or from thedamaged mitochondria [251, 252].

Ab peptides may activate microglia through (i) Toll-likereceptors 2 (TLR2), (ii) scavenger receptor (SR), (iii)receptor for advanced glycation end products (RAGE),(iv) a cell surface receptor complex, and (v) TNFR1, whose

deletion, as observed in APP23 transgenic mice (APP23/TNFR1()/))), may inhibit Ab generation and diminishes Abplaque formation in the brain [253]. Ab aggregates asforeign protein particles are recognized by TLRs, and thesebecome important Ab innate immune receptors, as dem-onstrated in antisense knockdown of TLR2 or using

functional blocking antibodies against TLR2, which maysuppress Ab-induced expression of proinflammatory mol-ecules and integrin markers in microglia [254]. Even TLR4could also play a role, as demonstrated in mouse models

homozygous for a destructive mutation of TLR4; theseshow significant increases in diffuse and fibrillar Abdeposits [255]. However, it is not clear whether TLR

signaling pathways involve the clearance of Ab deposits inthe brain or they initiate a neuroinflammatory response,responsible for the synaptic impairment observed in AD

pathology [256]. Important receptors, such as the class Bscavenger receptor CD36 and the LPS-binding moleculeCD14, signal through TLR2. CD36 recognizes a variety of

ischemic by-products acting as ligands, including oxidizedlow-density lipoprotein (LDL), long-chain fatty acids,thrombospondin-1, and, again, Ab. In microglia and inother tissue macrophages, Ab initiates a CD36-dependentsignaling cascade involving the Src kinase family members,Lyn and Fyn, as well as the mitogen-activated proteinkinase, p44/42. Ab also causes the blockade of Src kinasesdownstream of CD36 and inhibits macrophage inflamma-tory responses to b-amyloid [257].Another scavenger receptor, the macrophage receptor

with collagenous structure (MARCO) along with the che-motactic G-protein-coupled receptor formyl-peptide-recep-tor-like-1 (FPRL1) has been documented to be essential in

the amyloid b-induced signal transduction in glial cells [258].Neurons, microglia, and endothelial cells, which surroundthe senile plaques in the AD brain, express higher levels ofRAGE, which may trigger oxidative stress and NF-jBactivation [259]. The interaction of Abwith RAGEmay be adirect interaction [216], or it may involve damagedmolecularpatterns, such as the S100B protein. In primary cortical

neurons, the transcription factor Sp1 mediates IL-1b induc-tion by S100Bwithout evidence of a role for NF-jB, whereasin microglia, S100B stimulates NF-jB or AP-1 transcrip-tional activity and upregulates Cox-2, IL-1b, IL-6, andTNF-a expression through RAGE engagement [260, 261]. Finally,a cell surface receptor complex for fibrillar Ab, linked to thesmall GTPase Rac1 and critical in signaling to PHOX, has

been described. This molecular complex mediates microglialactivation through the stimulation of intracellular tyrosinekinasebased signaling cascades, and it is integrated by the

B-class scavenger receptor CD36, the integrin-associatedprotein/CD47, and the a6b1-integrin [262].NF-jB may be activated from a variety of pathways,

from the canonical pathway where the proinflammatoryTNF-a, IL-1, and LPS exert their action in addition to


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DAMPS, to the noncanonical pathway where CD40 andlymphotoxin receptors activate a p52/relB complex. More-over, there are other atypical pathways, where genotoxic

stress, hypoxia, UV light, H2O2, or the epidermal growthfactor receptor 2, among others, may intervene (reviewed in[263]). The link between NF-jB and neurodegenerativedisorders, particularly AD, is an old one [264, 265].

In rat primary cultures of microglial cells and humanneutrophils and monocytes, Ab activates PHOX, and thiseffect may be potentiated by the proinflammatory stimulus,

such as interferon-gamma or TNF-a, but blocked bytyrosine kinase inhibitors [266]. Mediated by PHOX,oligomeric Ab may induce ROS production, possiblythrough N-methyl-D-aspartate receptors (NMDAR), andthese PHOX-related ROS, in turn, release the prostanoidprecursor arachidonic acid through the activation of ERKs,which phosphorylate cytosolic phospholipase A2a [219].The rate-limiting enzyme, COX-2, can be induced by

multiple cellular factors such as growth factors or theproinflammatory cytokines IL-1b and TNF-a in neurons,astrocytes, and microglia. COX-2 in turn regulates PGE2signaling in neurons [267] and can activate APP transcrip-tion in astrocytes [156], as well as glutamate release from

astrocytes, which is responsible for excitotoxic damage inAD [193, 268, 269]. PGE2 and COX-2 feedback each otherand modulate neuroinflammation, regulating the produc-

tion of multiple inflammatory molecules.

Melatonins role in neuroinflammation andoxidative stress

Importantly, melatonin has a key role in Ab-inducedassembly of PHOX and the subsequent production of

ROS, as demonstrated in cultures of microglia incubated inthe presence of fibrillar Ab. According to Zhou et al. [270],melatonin may impair the assembly of PHOX by inhibiting

the translocation of p47phox and p67phox subunits ofPHOX from the cytosol to the plasma membrane. Thisbecomes feasible owing to blockade of the phosphorylationof p47phox, a PI3K-dependent phenomenon, and conse-

quently impairing the binding of p47phox to gp91phox.This mechanism is related to melatonins capacity to inhibitAkt (protein kinase B) activity in microglia, which is the

Ser/Thr kinase downstream of PI3K in these cells [270]. It isworth mentioning that the activation of the PI3K/Aktpathway may be mediated by H2O2, acting as an intracel-

lular messenger [271]; melatonin is known to directlyscavenge H2O2 [234, 239] (Fig. 7).Melatonin directly detoxifies H2O2 and produces the

biogenic amine AFMK and a potent free radical scavenger,which in turn may suffer deformylation, giving riseAMK.This latter antioxidant and free radical scavenger isparticularly relevant in mitochondria [78, 157, 239] (Fig. 7).

Melatonin and/or its metabolites function as antioxidants[91, 272], free radical scavengers, and antiapoptosis agentsand prevent abnormal nitric oxide (NO) elevation [273] in

the cerebral cortex.Between microglia and astrocytes, a fluid communication

exists. Several astrocyte factors released including trans-

forming growth factor b (TGF-b), macrophage colony-stimulating factor, granulocyte/macrophage colony-stimu-

lating factor, IL-10, IL-b, and ApoE modulate microgliaactivity [274276]. Glial Ca2+ waves can trigger responsesin microglial cells, and the calcium waves arise, in vitro, in

response to Ab administration [277]. Extracellular ATP, inits role as a DAMP and as part of the innate immunereceptor surveillance behind the Ab-induced inflammasomeactivation [278], may also elicit Ca2+ waves and activate a

microglial inflammatory response [279]. As will be ex-plained below, melatonin also has a role in this process.Even though the underlying mechanisms and their scopes

in neuroinflammation remain to be unveiled, it has beensuggested that melatonin could have modulatory effects onATP-dependent gliotransmission or glial calcium waves

derived from different brain regions and species, regulatingastroglial function [280]. Studied in the context of rhythmiccircadian outputs to pervasive neurobehavioral states, afunctional shift in the mode of intercellular communication

between junctional coupling and calcium waves in glial cellswas found to be induced by melatonin [281]. However, it iswell known that melatonin modulates intracellular free

Ca2+, and by this means, melatonin may protect cells fromcalcium-dependent pathways of death, such as calpain andcaspase-3 in cells undergoing excitotoxicity and oxidative

stress, as demonstrated in vitro in rat C6 astroglial cells[282]. By controlling Ca2+ influx, melatonin attenuatesglutamate-mediated excitotoxicity, which is responsible for

NMDAR-mediated damage of neurons. This in turn is oneof the postulated Ab-mechanisms of damage, as mentionedabove [219]. In in vitro experiments with a hippocampal cellline challenged with H2O2, Ab, or glutamate, cell death wasprevented by the melatonin derivative, AFMK, which isformed by the interaction of melatonin with H2O2 or O

_2

[76] (Fig. 7). Additionally, melatonin may inhibit not only

glutamate-induced ion currents but also ion currents fromthe other ionotropic glutamate receptors, kainate, andAMPA [283]. Also, it is possible that melatonin antagonizes

glutamate release, as observed in cortical synaptosomes inold mice and in neurotoxicity induced by KCl [284].We reported in vivo that melatonin significantly reduced

the proinflammatory response, decreasing by nearly 50%the Ab-induced levels of proinflammatory cytokines IL1-b,IL6, and TNF-a [285]. We speculated that melatoninaffected NF-jB DNA binding activity based on a previousreport by Natarajan et al. [286]and Chuang et al. [287],who found that NF-jB DNA binding activity was inhibitedby melatonin and was lower at night when endogenous

melatonin levels are high. Furthermore, 60 min after anintraperitoneal injection of melatonin, a reduction in NF-jB DNA binding activity was replicated. More recently, ithas been demonstrated in Ab-treated brain slices thatmelatonin reduces NF-jB-induced IL-6 in a concentration-dependent manner [288]. By administering melatonin, it isalso possible to reduce Ab-induced impairment in learningand memory in rats along with a significant decrease inpositive glial cells expressing NF-jB-induced IL-1b inaddition to C1q in hippocampus [289], both of which are

involved in glial activation (Fig. 7). The critical comple-ment component C1q, in turn, may induce the translocationof NF-jB p50p50 homodimers, at least as observed inhuman monocytes [290], and it is always related to ADpathology usually linked to fibrillar b-amyloid [291].


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The pleiotropic transcription factor NF-jB, composed ofhomo- and heterodimers of five members of the Rel familyincluding NF-jB1 (p50), NF-jB2 (p52), RelA (p65), RelB,and c-Rel (Rel) plays a key role in inflammatory processesbut also it is a protagonist in plasticity and neuronaldevelopment (reviewed in [292]). Thus, it is complicated topoint out that NF-jB inhibition may be a therapeutic targetin AD. Nonetheless, by using an immunological assay, it ispossible to demonstrate how melatonin prevents the Ab-induced expression of NF-jB [147]; more specifically,according to Deng et al. [293], melatonin inhibits p52NF-jB binding as demonstrated by examining the expres-sion of LPS-induced iNOS and COX-2. The latter action

involves a promiscuous histone acetyltransferase (HAT),within the nuclear cofactor p300, which is essential forCOX-2 transcriptional activation by proinflammatorymediators. By inhibiting p300 HAT activity, melatonin

may suppress p52 acetylation, binding, and transactivation[293]. In this manner, it is possible to block the rate-limitingenzyme COX-2 (Fig. 7). This enzyme is induced by multiple

cellular factors, such as growth factors or the proinflam-matory cytokines IL-1b and TNF-a in neurons, astrocytes,and microglia. COX-2 in turn regulates PGE2 signaling in

neurons [267] and can activate APP transcription inastrocytes [156], as well as glutamate release from astro-cytes, which is responsible for excitotoxic damage in AD

[268, 269, 293]. PGE2 and COX-2 feedback on each otherand modulate neuroinflammation, regulating the produc-tion of multiple inflammatory molecules.

Experiments using transformed lymphatic-derived endo-

thelial cell line demonstrated the ability of melatonin toprevent TNF-a induced phosphorylation of NF-jB p65,although the mechanism is unclear [294]. The administra-

tion of melatonin 1 hr after closed head injury also mayinhibit the activation of NF-jB during the late phase(8 days), an effect attributed to its prolonged antioxidant

effect at the site of injury. However, melatonin did not alterearly phase (24 hr after closed head injury), which implies aselective mechanism of neuroprotection [295]. One could

expect that such an interference with NF-jB would alsoaffect the role of this transcription factor in plasticity andneurogenesis. Thus, even though not linked to these NF-jB-dependent mechanisms, there are reports indicating asignificant depression as well as instability of synaptictransmission in the central nervous system (CNS),although melatonin-dependent fluctuations in synaptic

potentials were apparent only when the involved circuitwas tetanized [296]. Such depressive effects of melatonin insynaptic transmission would be expected to influence

epileptic seizure activity [297]. Nonetheless, other resultsindicate that, instead of depressing synaptic transmission,melatonin modulates neuronal excitability in the hippo-campus, and this modulatory activity depends on its

receptors [298, 299]. In fact, melatonin may modulatespecific forms of plasticity in hippocampal pyramidalneurons, as demonstrated by electrophysiological methods

[300] where neurons exposed to melatonin were found tochange their excitability in response to repetitive stimula-tion, which reveals melatonin as an activity-dependent

modulator of subsequent synaptic plasticity (metaplastic-ity; Fig. 4).

It seems that melatonin may thus regulate neuroinflam-mation through free radical control and modulation ofimportant proinflammatory transcription factors and their

signaling pathways while reducing glutamate excitotoxicity,whether it be by inhibiting glutamate-induced ion currentsor by controlling the glutamate delivery. On the other hand,even though functional cytoplasmic membrane melatonin

receptors have been described in astrocytes derived fromchick brain [301], which could suggest a role for melatoninas a metabolism regulator in astrocytes, these receptors

have not been corroborated in human glia.The nuclear hormone retinoid z receptor/retinoid orphan

receptor (RZR/ROR), from the retinoid-related orphan

receptors family, are likely associated with melatoninsignaling and have been identified in the promoter regionof 5-lipoxygenase (5-LOX), a key protagonist in neuroin-flammation. By repressing the expression of 5-LOX mRNA

in human B lymphocytes, melatonin may reduce theproinflammatory response via nuclear receptor RZR/RORa [302]. Furthermore, the transcriptional activationof RZR/RORa by melatonin is possible even in thenanomolar range [303, 304]. There are no reports confirm-ing this effect of melatonin in the CNS, but 5-LOX is widely

expressed in the brain [305] where it has neuromodulatoryand neuroendocrine functions and plays an important rolein aging and AD, as we will review later. It is worth

mentioning that, in addition to AA-derived leukotrienes,5-LOX also modulates the c-secretase activity in mem-branes, favoring Ab formation [306].Although there is an isolated report indicating that

melatonin is not an important modulator of macrophageand microglia function [307], melatonins role controllingthe primarily microglia-guided neuroinflammatory

response is demonstrated in multiple reports. This is aconsequence of its regulation of the NF-jB overexpression[308], the amount of LPS-induced proinflammatory cyto-

kines [192], or prevention of GSK-3b activation andneuroinflammation in response to Ab, as observed inastrocytes and microglial cells [166]. Meanwhile, a cumu-

lative dose of 10 mg/kg melatonin may attenuate kainicacid-induced neuronal death, lipid peroxidation, and mi-croglial activation, reducing the number of DNA breaks invivo, as demonstrated in adult male SpragueDawley rats

[309].The antioxidant and immunomodulatory effects have

inserted melatonin into the two-hit hypothesis [310], which

states that although either oxidative stress or abnormalitiesin mitotic signaling can, independently, serve as initiators inAD, both processes are necessary to propagate the path-

ological features of the disease.

The mitochondrial cascade hypothesis

According to Swerdlow and Khan [311], the mitochondrialcascade hypothesis asserts that inheritance determinesmitochondrial baseline function and durability, and mito-

chondrial durability influences how mitochondria changewith age. Thus, according to this hypothesis, once athreshold of mitochondrial changes is reached, AD histo-

pathology and symptoms ensue. Even though it wasformulated in 2004 as a formal hypothesis, some strong


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evidence linking AD to mitochondrial damage in braincells had been reported many years earlier. For example,in 1980, by investigating the mechanism for the production

of acetyl-CoA used in acetylcholine synthesis, a small, butsignificant, reduction in the activity of the pyruvatedehydrogenase (besides ATP-citrate lyase and acetoace-tyl-CoA thiolase) was found in postmortem brain tissue

from cases of AD [312]. In 1985, the activity of thepyruvate dehydrogenase complex was reported to bereduced to about 30% of control values in histologically

unaffected occipital cortex as well as in histologicallyaffected frontal cortex of the brains of patients with AD[313]. Likewise, AD as a primary defect in cytochromeoxidase was proposed years later [314]. However, itcurrently is debatable whether Ab is a downstreamproduct of the mitochondrial functional decline or whetherAb-induced mitochondrial damage is an extension of theamyloid cascade hypothesis. The amyloid cascade, pro-posed 20 yr ago, suggested that faulty metabolism of APPwas the initiating event in AD pathogenesis, leading

subsequently to the aggregation of Ab, specifically Ab142 [315, 316]. However, long before the appearance ofextracellular Ab deposits, they are detectable withinmitochondria [317].Beyond hypotheses, some other important features have

been found since those early years. It has been demon-

strated that Ab142 uncouple the mitochondrial respiratorychain, and this event plays a key role in pathology of AD[311]. Structurally, Ab induces swelling of isolated mito-chondria [318, 319] and, functionally, decreases ATP

synthesis and the activity of various mitochondrial en-zymes, as demonstrated in vivo [320] and in vitro incultured neuronal cells or in Ab-exposed astrocytes [319321]. Later, different neurotoxic mechanisms for Ab wereproposed, including disruption of mitochondrial functionvia binding of the Ab-binding alcohol dehydrogenase(ABAD) protein [322]or the formation of ion channelsallowing calcium uptake, which induces neuritic abnormal-ity in a dose- and time-dependent manner [323], or the

opening of the mitochondrial permeability transition porecoupled to inhibition of respiratory complexes [324, 325].We have found (Rosales-Corral et al., unpublished data)that following the intracerebral injection of fibrillar Ab, thepeptide is revealed both intracellularly and intramitoc-hondrially, deep in the cristae, coinciding with other reportswhich demonstrate that Ab progressively accumulates inmitochondria where it is associated with diminished enzy-matic activity of the respiratory chain complexes III and IV[317]. Presence of Ab in mitochondria is related to areduction in the rate of oxygen consumption by the electrontransport chain [317]. The enzymes in charge of importingAb to mitochondria have been identified as a complex oftranslocases, i.e., translocases of the outer membrane

(TOM) and the translocase of the inner membrane (TIM)[326] (Fig. 8).In addition to direct effects caused by Ab in mitochon-

dria, there are severe changes attributed to the Ab-inducedoxidative stress. A disturbance of mitochondrial dynamics,a term that includes fission, fusion, movement, and

mitochondrial architecture, seems to be implicated inAD pathogeny. It has been demonstrated in human brains

of patients with AD where mitochondrial distributiontends to be predominantly perinuclear and fission orfragmentation prevails over fusion, a phenomenon related

to a low metabolic capability [327, 328]. These eventsinvolve large dynamin-related GTPases, such as thedynamin-related protein (Drp1). Localized to mitochon-dria, Drp1 is a key factor in mitochondrial division and

particularly sensitive to redox regulation [328]. It has beenreported that NO overproduced in response to Ab proteincould be responsible for the impairment of Drp1 via

S-nitrosylation [329], and this eventuality may lead to animbalance of fission/fusion in mitochondria, which in turnis correlated with neuronal damage and synaptic loss

[330].Another important feature related to AD pathogeny is

mtDNA damage. Perhaps because it is not protected byhistones, mtDNA with its 37 genes is more susceptible to

oxidative stress-induced deletions and point mutationsthan nuclear DNA. Even though the consequences ofthese alterations remain to be clarified [331], mtDNA

damage is usually linked to dysfunction (decrement ofmitochondrial electron transport chain efficiency) andapoptosis [332, 333].

Also related to Ab-induced oxidative stress, mitochon-drial proteins and lipids become disturbed leading todysfunction. We have found significant alterations in

cholesterol and fatty acids content in mitochondrial mem-branes following the injection of Ab (Rosales-Corral et al.,unpublished data), associated with functional impairment;as a consequence of increased membrane permeability and

changes in lipid polarity owing to oxidative injury,cytochrome c is released from the intermembrane space ofmitochondria [334], behaving as an important intermediate

in apoptosis and associated with impaired mitochondrialrespiration, as observed in brain, platelets, and fibroblastsof patients with AD [335].

An important feature related to the direct interactionbetween Ab and cyclophilin D (CypD) has been found[336]. Ca2+-associated CypD is part of the mitochondrial

permeability transition pore (MtPTP) and translocatesfrom the matrix to the inner membrane where it appearslinked to oxidative stress, and by facilitating the opening ofmPTP, CypD causes mitochondrial swelling with cellular

and synaptic perturbations [337, 338]. The importance ofthe association of Ab/CypD is underlined by the fact that adeficiency in CypD may attenuate Ab-induced mitochon-drial oxidative stress, an effect accompanied by improvedsynaptic function and an improved cognitive performance,as observed in APP transgenic/CypD double-mutant mice

[336] (Fig. 8).As a result of Ab entrance and mitochondrial damage,

energy demands of cells become impaired. We have foundfunctional disorders of F0F1-ATPase in submitochondrial

particles obtained from platelets of patients with Alzhei-mers-type dementia [339], but the impairment of ion-motive ATPases in response to Ab is reproducible inhippocampal neurons in culture [340]. Nonetheless, anotherreport on F0F1-ATPase, searching in isolated mitochon-dria from platelets and postmortem motor cortex and

hippocampus from patients with AD, did not find abnor-malities in F0F1-ATPase functioning [341].


180

Melatonins role in mitochondrialhypothesis

Melatonins role in this context is mostly related to itsability to scavenge free radicals in addition to its indirect

antioxidant properties because it enhances endogenousantioxidant systems in mitochondria [342348]. It does thisbasically by maintaining and regenerating the GSH con-

tent, which is an important antioxidant mechanism inmitochondria [346]. Similarly, melatonin reduces peroxida-tion of lipids in mitochondrial membranes and free radicalleakage from this organelle. Thus, it is possible to repro-

duce in vitro mtDNA damage by adding Ab142 to neuronsin culture, but the addition of melatonin prevents signifi-cantly mtDNA damage [14, 347]. Added to drinking water

and chronically administered, melatonin prevents mito-chondrial impairment, maintaining or even increasing ATPproduction in senescent-prone mice suffering age-dependent

mitochondrial dysfunction accompanied by an importantoxidative/nitrosative stress [348]. It is worth noting that,additionally, melatonin seems to accumulate in the mito-chondria, in such a manner that mitochondrial melatonin

levels could be even 100 times higher than melatonin levelsin plasma [346]; this claim, however, requires confirmation.Indole propionamide, similar to melatonin but with a

longer half-life has been proven to protect against mito-chondrial toxins capable of collapsing the mitochondrialproton potential, causing severe mitochondrial dysfunction

and ATP deprivation. Under those circumstances, thisrecently discovered endogenous indole may act as arecyclable electron and proton carrier, restoring the proton

gradient and mitochondrial ATP synthesis [349].In reference to MtPTP, the antiapoptotic effects of

melatonin have been explained by its ability to inhibit theopening of the protein channels responsible for calcium and

cytochrome c (cyt c) release from mitochondria. Also, itmay function by reducing the loss of the mitochondrialmembrane potential in the presence of glucose deprivation-

related events [350] (Fig. 8).Cardiolipin is an important component (20%) of the

inner mitochondria membrane. Being particularly suscep-

tible to oxidative stress, cardiolipin becomes implicated incyt c release during apoptotic events [351], in part because itsensitizes mitochondria to Ca2+ mPTP [352]. One of the

Fig. 8. Both Ab and abnormal phosphorylated tau play key roles in mitochondrial dysfunction long before amyloid plaques appear.Hyperphosphorylated tau may cause ETC dysfunction by impairing complex I activity, although its major capacity for damaging may comefrom its ability to interact with ANT from MtPTP, which leads to swelling, mitochondrial dysfunction, and cell death, ultimately. Amyloid-b, whose mitochondrial receptors (ABAD and Hsp) have been identified, causes ETC dysfunction by interrupting the activity of complex IIIand complex IV, and possibly it may disrupt ion-motive ATPase. Moreover, Ab impairs energy metabolism by inhibiting directly the activityof the a-ketoglutarate enzyme during the tricarboxylic acid cycle and the pyruvate dehydrogenase before the cycle. Ab also disrupts Ca2+

homeostasis, which overloads mitochondrial matrix and may lead to complex II deficiency, membrane potential loss, ATP reduction, andROS overproduction in addition to MtPTP disturbance. Ab-induced oxidative stress has been related to membrane dysfunction, oxidationof ETC components, free radical leak, and MtDNA damage because MtDNA is particularly vulnerable to these events. The way CytCescapes from mitochondria is not completely clear, but once released, it initiates a chain of events leading to apoptosis. Melatonin(represented as red crosses) tends to accumulate inside mitochondria, where (1) it may reduce oxidative stress and its deleterious conse-quences on MtDNA, proteins, and membrane lipids, such as cardiolipin; (2) it strongly inhibits MtPTP currents and prevents cytochrome crelease in a dose-dependent manner; (3) it may recycle electron carriers, such as NADH; (4) it may prevent apoptosis by impairing Cytcrelease from mitochondria; (5) Ca2+ regulation by melatonin may protect mitochondrial functioning. ROS/RNS, reactive oxygen/nitrogenspecies; Ub, ubiquitin; ABAD, amyloid-b binding alcohol dehydrogenase; Hsp, heat-shock protein; Cytc, cytochrome c; ETC, electrontransport chain; MtPTP, mitochondrial permeability transition pore; MtDNA, mitochondrial DNA; APAF-1, apoptosis-activating factor 1.


181

probable mechanisms of mitochondrial protection bymelatonin relates to the prevention of cardiolipin oxidation,avoiding MtPTP opening and restoring Ca2+ balance (as

reviewed in [353]). However, it is also possible thatmelatonin directly inhibits MtPTP at single-channel andcellular levels, as demonstrated in patch-clamp recordingson the inner mitochondrial membrane [350].

Three other important proapoptotic factors related tomitochondrial functioning and signaling have been dem-onstrated to be modulated by melatonin in brain. The

executory caspase-3, which is known to be directly linked tocyt c release and widely linked to cell death in AD [99, 320,354], can be downregulated by melatonin [92]. On the

contrary, melatonin may enhance bcl-2 expression asdemonstrated in AD transgenic mice [92] and in ischemicbrain [355]. Bcl-2 is recognized as an antiapoptotic protec-tive factor, and its relation to Ab is also widely known,because Ab may deplete bcl-2 as demonstrated in humanprimary neuron cultures [356], in microglia [357], or inhuman neurons from patients with AD [358]. Furthermore,

the proapoptotic bcl-2associated X protein (Bax), whichmoves from the cytosol to the mitochondria, binds to bcl-2,and promotes cyt c release, is increased in the presence of

Ab in human neurons [356]. However, under a variety ofexperimental conditions, melatonin has demonstrated itsutility in diminishing bax [174, 359361]. Thus, melatonin

modulates mitochondrial pathways to apoptosis.Mitochondrial damage is linked not only to energy

dysmetabolism and leakage of free radicals, which in turnfeeds back to induce oxidative stress, but also to increased

leakage of Ca2+ currents, besides the above-mentionedapoptosis-inducing factors.

Calcium hypothesis

Other hypotheses have been proposed, complementing the

amyloid cascade theory. For example, it has been proposedthat a calcium-signaling deficit causes accumulation of APPbecause APP a-processing is a Ca2+-dependent process,and this phenomenon provides excessive substrate for

b- and c-secretases, the enzymes responsible for APPprocessing and Ab overproduction (Fig. 1) [362]. That Abincreases calcium uptake has been demonstrated in PC12

cells [363], in human cortical neurons linked to glutamateexcitotoxicity [364], in AD brain frontal cortex, and inplasma membrane vesicles from both rat and human brain

[365]. This occurs via stimulation of L voltage-sensitivecalcium channels, as demonstrated in cultured neurons[366], but Ab also may increase calcium uptake viapotassium channels, the NMDA receptor, the nicotinicreceptor, or even by its own calcium-conducting pores(reviewed in [367]). Conversely, calcium accelerates Abaggregation, even at physiological concentrations [368], and

it is exacerbated by synthetic calcium ionophores [369]. Atthe same time, Ab-induced calcium waves feed the neuro-inflammatory response, and this increases Ab aggregationand calcium waves, as mentioned previously [277279].During the events leading to oxidative stress and neur-

oinflammation in AD pathogeny, the glutamate-override of

the glutamate/cystine-antiporter system, which controls thelevels of glutamate by exchanging cystine in cells for the

neurotransmitter glutamate, may lead to an excessiveglutamate activity and consequently excessive influx ofcations, Ca2+ in particular [268, 269]. Glutamate receptors,

especially NMDA, are deeply involved in AD pathology[370]by controlling Ca2+ influx. Ionic calcium, in turn,activates a number of enzymes, including phospholipases,endonucleases, xanthine oxidase, neuronal nitric oxide

synthase, as well as proteases, such as the calcium-depen-dent cysteine protease, calpains, among others [190, 269,338, 367, 369]. Thus, the glutamate-induced overestimation

of NMDA receptors becomes neurotoxic; this process iscommon to several neurodegenerative diseases, and it iswell known as glutamate excitotoxicity (reviewed in [269]).

Melatonins role in calcium hypothesis

Melatonin may reduce NMDA-induced high [Ca2+]c levels

in addition to its ability to directly inhibit the mitochondrialpermeability transition pores, a mechanism linked partic-ularly to oxidative stress, as mentioned before [350]. Several

other mechanisms have been postulated to explain theregulation of intracellular Ca2+ by melatonin. As anexample, it is possible that melatonin acting on its MT2

receptor may inhibit adenylyl cyclase, and with this, itdecreases cAMP formation, blocking the cAMP-dependentprotein kinase (PKA), which would activate calcium release

channels [370372]. The reader is reminded that pineal andcortical melatonin receptors, MT1 and MT2, are signifi-cantly decreased in AD brain [373]. Conversely, it has beenknown from many years that calcium influx regulates

melatonin production in the pineal gland [374] (Fig. 9).Melatonin also may inhibit the mobilization of Ca2+

from ER as well as Ca2+ influx through voltage-sensitive

channels [375]. Importantly, melatonin controls theNMDA receptor whose activation comprises multipleregulatory sites controlling Ca2+ influx into the cell. On

the contrary, in the presence of the Ca2+ ionophore A-23187, the inhibitory effect on Ca2+ by melatonin issuppressed [376], returning to a glutamate-derived excit-atory state. The mechanism involved in NMDA-R control

may also imply redox modulation [350, 377]. However, it isalso possible that melatonin increases the concentration ofthe NMDA receptor subunits 2A and 2B, as demonstrated

in rat hippocampus, in a dose-dependent manner [378].Another melatonin mechanism of protection related to

calcium influx control is the multifunctional calcium-

modulated protein (calmodulin, or CaM) [379], whichmediates the calcium requirement for retrograde axonaltransport of AchE [380]. Melatonin interaction with CaM is

so avid that CaM had been considered a receptor formelatonin [381, 382] (Fig. 4). Even though more recentNMR and molecular dynamic studies suggest a loweraffinity [383], it has been demonstrated that melatonin

decreases, in a specific manner, the activity and autophos-phorylation of CaM kinase II, a key protein kinaseinvolved in neurite maturation [384], and causes neurite

enlargement through an increase in tubulin polymerizationderived from its CaM antagonism [385, 386]. In thismanner, apart from its antioxidant and antiapoptotic

effects as well as its anti-AchE actions related to Ca2+

and CaM modulatory effects, melatonin may preclude


182

microfilament and microtubule collapse, as demonstrated in

N1E-115 cells [372]. Even more, by activating the Ca2+-dependent a isoform of PKC [387], melatonin may restoreneurite formation, microtubule enlargement, and microfil-

ament organization in microspikes and growth cones incells damaged with H2O2. On the contrary, the PKCinhibitor, bisindolylmaleimide, blocks neurite formation

and microfilament reorganization elicited by melatonin.Thus, by regulating Ca2+ and CaM, melatonin possessesmodulatory actions on cytoskeletal

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