Alzheimer Metale

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Alzheimers disease, metal ions andmetal homeostatic therapy

Paolo Zatta1

, Denise Drago1

, Silvia Bolognin1

and Stefano L. Sensi2,3

1 CNR-Institute for Biomedical Technologies, Padua Metalloproteins Unit, Department of Biology, University of Padua, Viale G.

Colombo 3-35121 Padua, Italy2 Department of Basic and Applied Medical Science, Molecular Neurology Unit, CeSI-Center for Excellence on Aging,

University G. dAnnunzio, Chieti, 66013, Italy3 Department of Neurology, University of California, Irvine, Irvine, CA, 92697-4292, USA

Mounting evidences support the idea that endogenous

biometals, such as copper, iron, zinc and exogenous

ones such as aluminum, can be involved as factors or

cofactors in the etiopathogenesis of a variety of neuro-

degenerative diseases. Alzheimers disease (AD) is a

multifactorial neurodegenerative condition associatedwith pathological accumulation of amyloid plaques

and with the appearance of deposit of neurofibrillary

tangles. In AD, the process of b-amyloid (Ab) misfolding

and plaque aggregation is greatly influenced by altera-

tions in the homeostasis of the aforementioned metal

ions. Here, we discuss the most recent evidences that

associate metal ion dyshomeostasis with the develop-

ment of AD. As for aluminum, a role for this ion in AD

pathogenesis is still controversial. Thus, here, we also

critically review new findings that argue for and against

the aluminum hypothesis. Finally, it is discussed how

therapeutic strategies aimed at restoring metal homeo-

stasis can delay and modify the progression of AD-

related neurodegeneration.

Introduction

Alzheimers disease (AD) is a devastating neurological

condition with no disease-modifying therapy available so

far. The pathological hallmarks of AD are brain deposition

ofb-amyloid (Ab) in senile plaques (SPs) and the appear-

ance of neurofibrillary tangles (NFTs) made of hyperpho-

sphorylated tau protein (Box 1).

Metal ion dyshomeostasis is a well-recognized cofactor

in several neurodegenerative disorders[1,2]. Metals are

essential for life and have a central role in many bio-

chemical pathways. Genetic dysfunction, environmental

exposure, ageing, inadequate dietary intake and druginteraction can all induce an alteration in their homeosta-

sis leading to deleterious effects and neurotoxicity

(Figure 1).

Metal dyshomeostasis, especially in the case endogen-

ous metal ions such as copper (Cu), iron (Fe), zinc (Zn) or

the exogenous contaminant aluminum (Al), has attracted

the interest of researchers investigating the etiology of a

variety of neurodegenerative conditions and the pathogen-

esis of AD in particular [1,2]. As for AD, the misfolding

process, associated with Ab aggregation, is greatly

influenced by the metal ions (i.e. Al, Cu, Fe and Zn) that

are found in both the core and rim of the AD senile plaques

[38](Table 1).

In recent years, the interest for the role of metal dysho-

meostasis as a pathogenic factor for AD has been strongly

revived after the publication of several key reports indi-cating that therapeutic strategies that restore metal ion

homeostasis in the brain of both AD patients and AD

transgenic mice are able to reverse Ab aggregation, dis-

solve amyloid plaques and delay the AD-related cognitive

impairment[911].

Here, we review the most recent evidences linking metal

ion imbalance and Ab aggregation. We also examine the

participation of the AlAb complex as a cofactor in the

pathogenesis of AD. A critical review of the recent findings

on the deleterious effects of this complex might provide

new arguments for a debate that has animated the AD field

for years. Finally, we discuss new potential approaches for

the treatment of AD that are based on restoration of metal

homeostasis in the brain.

Role of metal homeostasis in AD

The proactive role of metal ions in stimulating Ab aggre-

gation, in addition to their interaction modalities with the

Ab peptide, has been widely investigated in vitro (reviewed

by Refs[12,13]).

Most of the glutamatergic synapses in the cerebral

cortex, but not all, co-release Zn along with glutamate

[1417]. This cation has been indicated to have a primary

role in AD owing to its efficacy to induce fast precipitation

of Ab together with its capability to build up protease-

resistant non-structured aggregates [18]. Furthermore,

studies on AD animal models have also shown that genetic

ablation of synaptic Zn greatly reduces the amount of

amyloid plaques [19] and several studies indicate that

compounds affecting Zn homeostasis can decrease Ab

deposition in the brain [9,10,20]. Noteworthy in this

regards, a recent paper has shown that the release of

synaptic Zn2+ facilitates the oligomerization of Ab and

its sequestration in the synaptic cleft [21], suggesting a

potential mechanism for the synaptic deficits observed in

AD. Finally, expression levels of Zn transporters, such as

ZnT1, ZnT4 and ZnT6, were discovered to be altered in the

brain of individuals affected by mild cognitive impairment

(MCI) and AD[22].

Opinion

Corresponding authors: Zatta, P. ([email protected]); Sensi, S.L.

([email protected]).

346 0165-6147/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2009.05.002 Available online 17 June 2009
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Box 1. Molecular mechanisms involved in the pathogenesis of Alzheimers disease

Alzheimers disease is characterized by extracellular deposition of the

b-amyloid (Ab) fibrils in the senile plaques (SPs) and by intraneuronal

aggregates of neurofibrillary tangles (NFTs) made of paired helical

filaments (PHFs) of the hyperphosphorylated tau protein (Figure I).

Senile plaques and neurofibrillary tangles are mainly present in

brain regions (entorhinal cortex, hippocampus, basal forebrain and

amygdala) that are involved in learning, memory and emotional

behavior. The discovery of Abin SPs has led to the formulation of the

amyloid cascade hypothesis, which revolves around the concept thatan imbalance in Ab metabolism leads to abnormal aggregation and

deposition of the peptide, a process that causes neuronal death,

synaptic dysfunction and, ultimately, dementia [45,81]. AD is also

associated with tau pathology[82]. The physiological role of tau is to

control the assembly of neuronal microtubules, thereby providing an

essential element for the stabilization of the neuronal cytoskeleton, a

key system to maintain structural integrity and axonal transport. In

AD, the hyperphosphorylation of the tau protein promotes the

derangement of microtubules and the formation of tangles of

aggregated tau, an additional process that triggers neuronal death

[83,84]. Interestingly, Ab and tau dysmetabolism can influence eachother and enhance their relative contribution to the AD-related

neuronal loss[85,86].

Figure 1. Schematic representation of the factors affecting the delicate balance between metal ion accumulation and deficiency.

Figure I. Pathological hallmarks of AD. Extracellular deposition of the b-amyloid fibrils in the senile plaques (SPs) and intraneuronal aggregates of paired helical

filaments (PHFs) of the hyperphosphorylated tau protein in neurofibrillary tangles (NFTs) represent the hallmark pathogenic features of the disease, and their

observation in a postmortem examination is still required for a diagnosis of AD. In accordance with the amyloid cascade hypothesis, it has been proposed that Ab

aggregation follows a sequence by which the accumulation of soluble Abis followed by the appearance of low molecular weight oligomers that rapidly associate in

higher order aggregates and finally precipitate to form senile plaques. Ab aggregation is greatly influenced by all the metal ions (e.g. Al, Cu, Fe and Zn) that are found inboth the core and rim of the AD senile plaques.

Table 1. Metal levels in patients with Alzheimers disease compared with healthy individuals

Location Zinc mg gS1 (mM)a Coppermg gS1 (mM)a Iron mg gS1 (mM)a

Plaque rim 67 (1024)b 23 (357)b 52 (938)b

Plaque core 87 (1327)b 30 (474) 53 (951)b

Total senile plaque 69 (1055)b 25 (393)b 53 (940)b

Alzheimers neuropil 51 (786)c 19 (304) 39 (695)

Control neuropil 23 (346) 4 (69) 19 (338)aNumbers in brackets represent molar concentrations, which were converted with the assumption of a sample density equivalent to 1 g cm3.bP< 0.05 (plaque values compared with neuropils from patients with Alzheimers disease).

cP< 0.05 (neuropils from patients with Alzheimers disease compared with neuropils from control individuals). Reproduced, with permission, from Ref. [17].

Opinion Trends in Pharmacological Sciences Vol.30 No.7

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Like Zn, Cu is synaptically released [23]and acts as a

potent mediator of Ab aggregation under conditions of mildacidosis[24,25]. Compared with Zn, Cu has the additional

property of producing strong extra-mitochondrial oxidative

stress[26]. Several studies have indicated that, because of

their redox-active nature, transition metal ions such as Cu

and Fe can interact with Ab catalyzing the generation of

H2O2 through a reduction process that uses O2 and bioa-

vailable reducing agents such as cholesterol, vitamin C and

catecholamines [27]. Thus, in the absence of sufficient

detoxifying enzymes such as catalase and glutathione

peroxidase, H2O2 can lead to further generation of

hydroxyl radicals through the Fenton reaction (Box 2) with

relevant consequent neurotoxic effects.

Alteration of intracellular [Cu]i homeostasis has beenimplicated in AD[28,29] given that [Cu]i activates phos-

phoinositol-3-kinase-mediated protein kinase pathways,

thereby increasing the secretion of matrix metalloprotei-

nases (MMPs), a class of enzymes that degrades Ab [30].

According to this model, the amyloid precursor protein

(APP) triggers Cu depletion in the AD brain as APP binds

to Cu and act as a Cu chaperone favoring the efflux of the

ion. Such a model is substantiated by findings in APP-

knockout mice, in which Cu levels are foundto be increased

in the cerebral cortex [31], whereas an overexpression

of APP promotes a significant reduction of [Cu]i in the

brain of AD transgenic mice. Although it is intriguing to

consider Cu and Fe as important players in AD pathogen-

esis given their pro-oxidant activity, it should also be

considered that pathological conditions associated with

increased levels of Cu and Fe (i.e. Wilsons disease) do

not show enhanced deposition of Ab plaques indicating

that the metals might be necessary but are certainly not

sufficient to cause a multifactorial pathology like AD (for

more details seeBox 3).

A new role for Al in AD?

In the context of AD-related metal dyshomeostasis, an

interesting albeit controversial angle is offered by the Al

dysmetabolism. A pathogenic role for Al in AD has been

hypothesized since the 70s; however, such a model has

been partially discredited mostly because of the paucity of

reliable studies and data on Al chemical speciation in

addition to an incomplete understanding of the complexity

of Al chemistry in biological systems. Al is known to be the

most abundant metal ion in the Earths crust and there are

few doubts about its high toxicity for humans and animals

[32]. Altered brain levels of Al have been associated with aspecific neurological condition such as fatal dialysis-linked

encephalopathy (DE) that is due to Al-contaminated water

in dialysate solutions [33]. However, such iatrogenic dis-

ease is not associated with AD-like pathology [34].

Although Al contamination of dialysates has been over-

come by the use of deionised water, new cases of subacute

DE are still reported because of Al-containing drugs are

often prescribed to control hyperphosphatemia and gastric

problems in patients with chronic renal failure [34,35].

Overall, the pathological changes found in DE and sub-

acute Al encephalopathy support the idea that Al can be

Box 2. Fenton reaction

Fe2+ + H2O2! Fe3+ + OH + OH. This is the iron-salt-dependent de-

composition of dihydrogen peroxide, generating the highly reactive

hydroxyl radical, possibly via an oxoiron (IV) intermediate. Addition

of a reducing agent, such as ascorbate, leadsto a damaging cyclethat

targets biological molecules.

Box 3. Metal, homeostasis and AD

Intraneuronal Zn homeostasis is controlled by a balance between

influx, buffering and extrusion. Most Zn enters neurons through

voltage-sensitive Ca channels and Ca-permeable ionotropic glutamate

receptors [87]. Sequestration and buffering is largely controlled by

metallothioneins (MTs), mitochondria, zincosomes and lysosomes

[73,8891]. MTs are present in the central nervous system in three

isoforms (MT-1, MT-2 and MT-3). MT1 and MT2 are expressed in

astrocytes, whereas MT-3 is expressed predominantly in neurons[92].

Zn2+ is bound to MTs, but this binding can be readily modulated by

changes in the redox state of the two Zn2+/Cys cluster regions and

endogenous (superoxide and peroxynitrites) or exogenous oxidants

promote harmful Zn release from these proteins [72,93,94]. Thus, MT-

3 could be a major source of toxic Zn inside neurons [95].

Mitochondrially sequestered Zn can be re-released into the cytosol

in a Ca-dependent manner, suggesting a potentially injurious inter-play between Zn and Ca dyshomeostasis [91]. Postmortem studies

have shown that the cation is elevated within mature Ab plaques

(Table 1), whereas plaque formation is dramatically reduced in AD

transgenic mice lacking vesicular presynaptic Zn or in AD animals

treated with Zn chelators[9,10,19,20].

Cu homeostasis is controlled by MTs and by the activity of a Cu

transporter ATPase[96]. Ionic Cu is released into the cleft following

postsynaptic stimulation of the N-methyl-D-aspartate (NMDA) recep-

tor [97,98] and is concentrated into postsynaptic vesicles by the

Menkes Cu7aATPase[98].

The amyloid precursor protein, APP, can act as a copper transporter

[99]. APP has two copper binding sites, including one in the Ab

peptide sequence[24,100]and APP overexpression in transgenic mice

has been shown to reduce brain Cu levels [101,102]. Proteins of

similar structure, such as APLP1 and APLP2, also have Cu binding

domains and might similarly promote Cu efflux[31,99,100].

Fe homeostasis is controlled by the Fe transport protein transferrin

(Tf), a serum glycoprotein that binds two atoms of Fe [103], and by

ferritin. Several lines of evidence suggest that Fe dyshomeostasis and

Fe-induced oxidative stress have some role in the pathogenesis of AD.

Abnormal levels of Fe, ferritin and Tf have been reported in the

hippocampus and cerebral cortex of AD brains[104]. Furthermore, in

brain areas more vulnerable to AD-related neurodegeneration, Fe has

been shown to accumulate at a pace that is not matched by similar

levels of ferritin production [105].

Al is largely present in food and beverages such as tea [106]and

in drugs like the antiacids. Despite its ubiquitous presence, only

0.060.1% of the ingested Al is absorbed across the gastrointestinal

tract [107]. Al uptake is limited by the presence of certain dietarycomponents (such as citrate) that complex the ion[108]and by the

competition for uptake exerted by other ions such as Ca, Mg and

Si [109]. To date, it is not completely understood whether Al can

enter the brain and, if it does, by which mechanism. Some authors

h av e s ug ge st ed t ha t A l c an g ai n a cc es s t o t he b r ai n b y

permeating the bloodbrain barrier (BBB) through the activation

of several carriers. In that respect, transferrin-mediated transport of

Al has been suggested [110], whereas other authors have indicated

a possible transporting role for the monocarboxylate transporter

(MTC), a proton co-tra nsporte r that i s l ocated a t both the

luminal and abluminal surfaces of the BBB[111,112]. Finally, recent

findings have suggested that structural and functional pathological

changes of the BBB might promote Al accumulation in the AD brain

[112116].


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neurotoxic whenever the physiological excretion of the

metal is impaired. By contrast, these findings also indicate

that, again, Alper se is not a cause sufficient to promote AD

because the neuropathological and functional hallmarks of

DE and subacute Al encephalopathy in patients with a

history of long term dialysis do not overlap with those

observed in AD patients [3638].

Thus, despite the established neurotoxic activity of the

ion, the aluminum hypothesis for AD continues to be acultural anathema to most neuroscientists. After so many

years of debate we suggest a fresh look at the whole issue

and considering of new evidence that could somehow re-

evaluate the importance of this metal as a cofactor in AD.

In our opinion, an unbiased look at the complexity of Al

chemistry in biological systems could reconcile many con-

flicting data reported in the literature [39,40]. One

example of such controversy is given by the issue of Al

deposit in AD brains. Several laboratories have documen-

ted Al accumulation in AD brains by using Laser Microp-

robe Mass Analysis (LAMMA) demonstrating abnormal

high Al concentrations located mostly within the AD neu-

rofibrillary tangles [3,5,8]. However, a recent interestingstudy by Miller and colleagues[41]employed synchrotron-

base infrared and X-ray imaging to investigate metal

deposition in brains of AD patients. They reported that,

whereas Cu, Zn, and Fe can be detected, no Al was found in

the senile plaques. These results are not surprising given

the specific aspects of the technique employed in the study.

In this study, Al was, in fact, part of the coating of the

substrate where biological samples were deposited and the

machine was indeed set to be blind to the analytical

identification of Al. In other words, the system was selec-

tively set to ignore Al.

Although Al is certainly not the only metal involved in

AD pathological features, it is also true that the whole AD

field is still very far from having an exhaustive under-

standing of the molecular determinants involved in the

disorder. Several evidences indicate that Al can contribute

to both tau- and Ab-dependent pathology. Al, a highly

reactive element, can promote tau-dependent pathology

as the ion can easily cross-link hyperphosphorylated

proteins [42]. Moreover, using intracerebral injections of

pair helical tau filaments with and without Al, Lee and

Trojanowski indicated that this metal is shown to co-

localize with several proteins that have a key role in AD

like Ab, ubiquitin, ACT, and ApoE[43,44](Figure 2).

The idea that Al dyshomeostasis might be a cofactor for

AD also fits well with the amyloid cascade hypothesis.

According to this theory, the pathological production of Abpeptide leads to synaptic dysfunction and ultimately

dementia (see also Box 1). More recent studies indicate

that amyloid dysmetabolism synergistically promotes the

development of tau-dependent pathology and the for-

mation of neurofibrillary tangles [39]. Furthermore, new

findings in AD research strongly support the idea that the

severity of AD-related neuronal loss and dementia is

largely mediated by the soluble forms of Ab oligomers,

rather than fibrillar and insoluble Ab deposits [45]. Inboth

AD subjects and AD animal models [46,47], there are

compelling evidences indicating that brain levels of soluble

Abspecies (and hyperphosphorylated tau) correlate better

with cognitive decline rather than plaque density [48].

Furthermore, intraneuronal accumulation of such soluble

Ab oligomers might also have a critical role in AD patho-

genesis (reviewed in Ref. [49]) given that AD-related

synaptic deficits are specifically mediated by these oligo-

mers [50,51]. Studies in AD brain preparations have shown

that the formation of Aboligomers is initiated intracellu-

larly rather than in the extracellular space [52], a phenom-

Figure 2. Al as a possible modulator of tau pathology. Sites of possible interaction

between Al and hyperphosphorylated tau protein that is associated with the

cytoskeletal microfilaments and the neurofibrillary tangles found in AD.

Figure 3. Ab when conjugated with Al undergoes structural changes.

Transmission electron microscopy (TEM) of Ab and Abmetal complexes. Many

short and irregular protofibrillar structures appeared in the Absample. The AbAl

complex triggers the appearance of a large population of small oligomers whereas

the other complexes (AbCu, AbFe, AbZn) form mainly unstructured filaments.


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enon that has been also validated in AD transgenic mice

[53]. Intraneuronal Ab deposition in AD target areas, such

as the hippocampus and the entorhinal cortex, of subjects

with MCI supports the idea that intraneuronal accumu-

lation of Ab acts as an early mediator of synaptic dys-

function and cognitive impairment[54].

Intriguingly, in line with this up-to-date revision of the

amyloid cascade theory, our recent results indicate that

Al, when conjugated with Ab, can have a role in promotinga sort of freezing of the peptide in its oligomeric state,

thereby favoring the formation and assembly of the most

dangerous and neurotoxic amyloid species[55](Figure 3).

Several recent studies have shed some light on novel

pathogenic pathways by which Al dyshomeostasis can

possibly favor AD progression (www.alzforum.com). New

findings have provided a detailed characterization of the in

vitroconformation and aggregation changes stimulated by

Al on different Abfragments and, in particular, on human

Ab [55,56]. Furthermore, when compared with other metal

ions such as Fe, Zn and Cu, Al seems to be very effective in

promoting in vitro structured aggregation of Ab associ-

ated with particularly high neurotoxicity [56]. When con-jugated with Al, Ab undergoes a spontaneous change in the

structural conformation that leads to an increase in its

surface hydrophobicity that is associated with solvent-

exposed hydrophobic patches. Such an AbAl metal com-

plex shows a dramatic reduction in the sequestration in the

brain microcapillaries and (indeed, not surprisingly) an

increased high permeability across the bloodbrain barrier

(BBB), a phenomenon that is leading to intracerebral

accumulation of AbAl [57].

Comparative studies on the aggregation states of both

human (hAb) and rat b amyloid (rAb) in the presence or

absence of Al have shown that AbAl complexes are

capable of increased aggregation when compared with

Ab itself. These studies have also shown that the metal

seems particularly efficient in changing the morphology of

hAbaggregates. This phenomenon is most likely to be due

to the different amino acid sequence of hAb compared with

rAb, a change that seems to be essential for promoting

different levels of toxicity when the two amyloid proteins

are conjugated with Al [58]. Thus, the different aggrega-

tional behavior of rat and human amyloids in the presence

of Al emphasizes the close relationship between the

morphology of Ab aggregates and their cell toxicity [55].

One possible explanation comes from experiments in

human neuroblastoma cells where Al seems to promote

Ab oligomerization and dramatically increase cellulartoxicity.

In a set of microarray and real-time PCR experiments,

in which we investigated the gene expression profile of

human neuroblastoma cells (35 000 genes) treated with

various Abmetal complexes (AbCu, AbZn, AbFe and

AbAl), Ab alone or the metal ions themselves, we

observed that the AbAl complex is able to produce a

selective upregulation of a well known series of AD-related

genes such as the APLP1and APLP2(amyloid precursor-

like protein-1 and -2), MAPT (microtubule-associated

protein tau) and APP genes (Drago et al., unpublished).

Moreover, in functional experiments, we also found that

in neuronal cell cultures exposed to various Ab

metalcomplexes (AbCu, AbZn, AbFe and AbAl) only the

AbAl complex is able to alter glutamate-driven [Ca]irises

[59]. In the same study, AbAl was also found to inhibit the

oxidative respiration in isolated rat brain mitochondria

[59]. These results are in line with a previous study that

indicated that extracellular applications of spherical oli-

gomeric forms of Ab142and several other disease-related

amyloidogenic proteins can cause disruption of [Ca2+]i [60],

whereas equivalent amounts of monomeric and fibrillar

forms of Ab are unable to induce any detectable effect. Our

mitochondrial studies are in agreement with studies from

other laboratories where Ab142 was found capable to

induce a decrease in state 3 respiration, a phenomenon

that is strongly exacerbated when the peptide is conju-

gated with Al[61].

Finally, signs of Al dyshomeostasis were also recently

found in a triple transgenic AD mouse, the 3xTg-AD, that

Figure 4. Homeostatic interplay between brain metals: the domino effect. Bar graph depicts the concentrations of Ca, Cu, Fe, Zn and Al in the brain of CD-1 adult mice fed

for 3 months with a Cu-adequate or a Cu-deficient diet. Note how all the metal levels are strongly affected by the modification of the dietary intake of a single metal (Cu),

highlighting the close interaction between different metal distribution/storage pathways. ** P< 0.01, *P< 0.05 versus control.


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expresses mutant APP, PS1 and tau and is, therefore,

considered to recapitulate the hallmarks of AD pathology

[62]. In that study, experiments employing mass spectrom-

etry indicate that, when compared with the distribution of

other AD-relevant metals (Zn, Cu and Fe), Al is the only

metal ion that is significantly increased in the cortex of 14-

month-old 3xTg-AD mice [59]. Altogether, these data

suggest a potentially intriguing new role for Al in AD

pathogenesis.

Metal homeostatic therapy: a new therapeutic approach

Although metal ion dyshomeostasis is certainly not the only

trigger of the disease, therapeutic interventions aimed at

restoring metal homeostasis remain strong candidates as

disease-modifying strategies for AD treatment. What is

emerging from recent data obtained by several laboratories

is that, more than aiming at chelating strategies, research

should be focused on molecules (called by some authors,

metalprotein attenuation compounds [MPACs]) that are

capable of sequestering Cu and Zn from both amyloid pla-

ques and the synaptic cleft and act as Cu ionophores to

compensate the AD-related [Cu2+]ideficiency[12].

However, it should also be emphasized that pinpointing

a single metal as the major culprit of the disease seems to

be less productive. The jury is out about which metal ion

induces most aggregation and which induces most toxicity,

but it should be understood that a change in the brain levelof a single metal ion can upset the whole metal pool,

resulting in a relevant complex metal imbalance inside

and outside of the central nervous system. Thus, it is likely

that upon such a domino effect, pharmacological and/or

pathological alteration in the level of a single metal can

eventually affect the whole distribution pattern of many

others (Figure 4).

In the past few years, a convergence of pharmacological

studies in AD animal models have made use of metal

Figure 5. Neuroprotective effects of Clioquinol. Clioquinol (CQ) has been recently proposed as disease-modifying drug for AD. CQ seems to have ionophore activity that

favors the entrance into cells of Zn and Cu. Cu entry in particular determines the activation of metalloproteases (MMP) resulting in the degradation of A b. In addition, CQ

could also remove Cu and Zn that is sequestered in senile plaques (SP), thereby reducing the oligomerization of the peptide. Abbreviation: SMON, syndrome subacute

myelo-optico-neuropathy.


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homeostatic strategies and achieved important neuropro-

tective effects. As for AD-related Cu dyshomeostasis, it has

been reported that a small chelating molecule such as apo-

cyclen is able to sequester the synaptic Cu bound to Ab and

also inhibits both Aboligomerization and neuronal death

while actively promoting Ab cleavage[63]. An additional

study has indicated that, as Cu influences the development

of tau-dependent pathology[64], APP/PS1 transgenic mice

treated with the Cu-ligand pyrrolidine dithiocarbamateshowed an increase in brain Cu levels, which activates

Akt-mediated GSK3 phosphorylation, a process that leads

to a substantial decrease in tau phosphorylation and

attenuation of cognitive deficits. Furthermore, Crouch

and colleagues [65] have recently shown that increasing

[Cu2+]i bioavailability in the brain can restore cognitive

function by blocking the accumulation of neurotoxic Ab

trimers and phosphorylated tau. Tetramine derivatives

have also been shown to reduce brain Cu levels in the

brain cortex without affecting the blood levels of the cation

[66]. Moreover, a lipophilic chelator (DP109) that can trap

Ca and Zn at the cell membrane level has also been found

capable reducing brain amyloid deposition in an AD trans-genic model [20].

Finally, a great interest has been generated in the

beneficial effects of 5-chloro-7-iodo-8-hydroxyquinoline

(clioquinol; CQ), a Cu and Zn ionophore that is able to

reduce the size and number of Ab plaques in transgenic AD

mice[10].

PBT2, a second-generation 8-OH quinoline derivative of

CQ has also been shown to promote neuroprotection and

delay cognitive impairment in an AD transgenic model[9].

The proposed mechanism by which CQ can promote neu-

roprotective effects is by enhancing intracellular Cu and Zn

uptake, thereby acting as an ionophore that favors the

clearance of these ions from parenchymal amyloid plaques

and the synaptic space [67]. According to the model, CQ can

also restore intracellular Cu levels and induce the upre-

gulation of matrix metalloproteases MMP-2 and MMP-3,

ultimately promoting the digestion of amyloid oligomers

[68] (Figure 5).

Indicating the complexity of restoring biometal homeo-

stasis, we have found that CQ, after conjugation with Ab

metal complexes (Cu and Zn), is unexpectedly able to

promotein vitroaggregation and fibrillogenesis of human

Abrather than dissolution of the fibrils[69]. By contrast,

other cell culture studies have shown that CQCu com-

plexes can permeate the cells and markedly inhibit Ab

deposition [67]. Finally, recent studies indicate that CQ

(and probably CQ-related compounds) might also favor thebuffering of synaptic Zn and inhibit the deposition of toxic

Ab oligomers in the synaptic cleft[9,21,70].

No matter what the net result, it is encouraging to note

that clinical trials have shown that both CQ and the CQ

derivative PBT2 can be safely used in AD patients and

attenuate some of the AD-related cognitive deficits[70,71].

It is also intriguing to consider that, because Zn-de-

pendent neuronal death is largely due to intracellular

mobilization of the cation from sources such as metallothio-

neins (MTs), mitochondria, and lysosomes, [19,7276], it

should be pointed out that the potential neuroprotective

role of a new class of low affinity and cell-permeable Zn

chelators able to buffer intraneuronal Zn2+ rises, thereby

preventing Zn2+ neurotoxicity and/or the early intraneur-

onal aggregation and oligomerization of toxic Ab species,

remains to be explored.

Conclusions and future perspectives

Undoubtedly, aging remains the most important risk fac-

tor for the development of neurological disorders and AD in

particular, suggesting that these conditions are likely to bethe result of cumulative metabolic impairment occurring

over decades of life. Metal ion dyshomeostasis per se is

probably not the sole or even the primary cause of AD;

however, in the context of an aging brain it might have a

relevant role in the development and progression of the

disease.

Aging, for instance, is associated with increased levels of

oxidative stress[77], a factor that might also perturb the

Cu/Zn homeostasis, given that MTs tend to be overex-

pressed in the aging brain [78]. In rats, the expression

of the gene encoding for the MT neuronal isoform, MT3, is

increased in neurons obtained from the aging hippocampus

compared with young controls [79]. Notably, MTs arepotent antioxidants and protective factors against stress

conditions. MT-increased expression in the aging brain

might simply reflect a protective endogenous response to

a sub-chronic state of inflammatory and/or oxidative

stress. By contrast, it is possible that the neuroprotective

actions of MTs can be overridden by a concomitant increase

in reactive-oxygen-species-driven [Zn]i accumulation. As in

the aging brain (and even more so in the AD brain), there is

an increase in the level of inflammatory cytokines; a

chronic upregulation of MTs can lead to higher availability

of intracellularly releasable Zn2+ in response to oxidative

stress[78,80]. In that respect, it is interesting to note that

neurons obtained from 3xTg-AD mice respond to oxidative

stress with an enhanced mobilization of [Zn2+]i compared

with control mice[75].

In summary, the metal hypothesis of Alzheimers dis-

ease, with the addition of the concept that the neuropatho-

genic effects of Ab in AD are promoted by (and possibly even

dependent on) the conjugation of the peptide with selected

metals, seems to be a very workable hypothesis. Increas-

ingly sophisticated pharmaceutical approaches are now

implemented to attenuate abnormal interactions between

Aband metals without causing a systemic disturbance on

their systemic levels. The whole AD field will be soon in a

position to verify whether addressing metal dyshomeostasis

can have a strong therapeutic potential in AD.

Finally, arguments for and against the possible role ofAl in AD are represented in the field; however, in light of

recent results it seems premature to discard altogether

this ion as, at least, an accomplice in AD.

AcknowledgementsWe are in debt to Marie Evangeline Oberschlake for the editing and

critical revision of the manuscript. This work was supported by FIRB

2003 (P.Z), 2006 (S.L.S.) and PRIN 2008 (P.Z.) grants.

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