Neurochemistry International 62 (2013) 540555

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Review

Metal dyshomeostasis and oxidative stress in Alzheimers disease

Mark A. Greenough a,b, James Camakaris b, Ashley I. Bush a,
a
b

The Mental Health Research Institute, The University of Melbourne, Victoria 3010, Australia
Department of Genetics, The University of Melbourne, Victoria 3010, Australia

a r t i c l e

i n f o

Article history:
Available online 8 September 2012
Keywords:
Alzheimers disease
Amyloid
Copper
Zinc
Iron
Homeostasis
Oxidative stress
Neurodegeneration

a b s t r a c t
Alzheimers disease is the leading cause of dementia in the elderly and is dened by two pathological
hallmarks; the accumulation of aggregated amyloid beta and excessively phosphorylated Tau proteins.
The etiology of Alzheimers disease progression is still debated, however, increased oxidative stress is
an early and sustained event that underlies much of the neurotoxicity and consequent neuronal loss.
Amyloid beta is a metal binding protein and copper, zinc and iron promote amyloid beta oligomer formation. Additionally, copper and iron are redox active and can generate reactive oxygen species via Fenton
(and Fenton-like chemistry) and the HaberWeiss reaction. Copper, zinc and iron are naturally abundant
in the brain but Alzheimers disease brain contains elevated concentrations of these metals in areas of
amyloid plaque pathology. Amyloid beta can become pro-oxidant and when complexed to copper or iron
it can generate hydrogen peroxide. Accumulating evidence suggests that copper, zinc, and iron homeostasis may become perturbed in Alzheimers disease and could underlie an increased oxidative stress burden. In this review we discuss oxidative/nitrosative stress in Alzheimers disease with a focus on the role
that metals play in this process. Recent studies have started to elucidate molecular links with oxidative/
nitrosative stress and Alzheimers disease. Finally, we discuss metal binding compounds that are
designed to cross the blood brain barrier and restore metal homeostasis as potential Alzheimers disease
therapeutics.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Copper, zinc and iron are the three most abundant biometals in
mammals, and play catalytic and structural roles in many enzymatic processes. The redox potential of copper and iron are what
facilitate their catalytic activity. However, this potential can also
cause oxidative stress mainly via Fenton (and Fenton-like) chemistry (Jomova and Valko, 2011; Stohs and Bagchi, 1995). Conversely,
zinc is redox inert and usually plays a structural role in protein
folding and stability and coordinating proteinprotein interactions
(Eide, 2011). Zinc also has unique antioxidant properties that enable it to modulate oxidative stress via protection of protein sulfhydryl groups (Powell, 2000). Regulation of copper, zinc and iron
are intimately linked at both an organismal and cellular level and
metal dyshomeostasis is featured in numerous human diseases.
Much of the current knowledge about regulation of copper, zinc
and iron has come from our understanding of genetic disorders
such as Menkes and Wilson diseases (Wang et al., 2011), acrodermatitis enteropathica (Andrews, 2008) and aceruloplasminemia
(Vassiliev et al., 2005). However, in the past decade there has been
Corresponding author. Address: The Mental Health Research Institute, Melbourne Brain Centre, The University of Melbourne, Genetics Lane, Parkville, Victoria
3010, Australia. Tel.: +61 390356532; fax: +61 390353107.
E-mail address: ashleyib@unimelb.edu.au (A.I. Bush).
0197-0186/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuint.2012.08.014

considerable interest in the metal dyshomeostasis that is a feature

of several neurodegenerative conditions. In the case of Alzheimers
disease (AD) there are disturbances in the levels of copper, zinc and
iron in localized regions of the brain associated with disease
pathology. Although the interaction of extraneuronal copper, zinc
and iron with cytotoxic amyloid beta (Ab) had been known for
1520 years, in the last few years much progress has been made
in understanding the associated cellular changes to metal-protein
trafcking and signaling cascades. Further elucidation of these
pathways will not only help to understand what role metals play
in oxidative stress, a feature of AD, but also rationalize development of potential therapeutic compounds targeted to restore metal
balance in the brain. Several such compounds, in particular 8hydroxyquinolines, have shown some efcacy in both animal and
human trials and will be discussed in this review.

2. Alzheimers disease
AD is a progressive neurodegenerative disorder that involves
progressive cortical and hippocampal neuron loss and corresponding cognitive decline. A complex interaction of multiple genetic
and environmental risk factors is thought to underlie the etiology
of AD but age is still considered the number one risk factor for sporadic cases, which make up >95% of all cases. The remaining cases

M.A. Greenough et al. / Neurochemistry International 62 (2013) 540555

have a clear genetic inheritance. Termed familial Alzheimers disease (FAD) they are caused by mutations in the amyloid precursor
protein gene (APP), presenilin 1 (PSEN1) or presenilin 2 (PSEN2)
respectively (Bertram et al., 2010). Two pathological hallmarks of
AD are aggregation of Ab, the major constituent of extraneuronal
senile plaques, and hyper-phosphorylation of Tau, which forms
intracellular neurobrillary tangles (NFTs). Ab peptides are released from APP by the sequential protease activity of b-secretase
(BACE1) (Cole and Vassar, 2008) and c-secretase, a multiprotein
complex that contains either PSEN1 or PSEN2 as its catalytic constituent (Steiner et al., 2008). The length of naturally occurring
Ab can vary but is typically a peptide of 3942 amino acids. Of
the 160 + mutations that have been identied in FAD cases the
majority cause an increase in the prevalence of a longer form;
Ab42, which is considered particularly neurotoxic and more prone
to self-aggregation than shorter peptides. The ratio of Ab42 to
Ab40, is considered a surrogate plasma biomarker and may be a
useful early predictor of AD disease progression (Koyama et al.,
2012).
Senile plaques are composed primarily of extraneuronal aggregated Ab but also contain relatively high amounts of Cu and Zn,
which are known to precipitate Ab in vitro (Bush et al., 1994a,c).
Many researchers now believe that soluble oligomers are the most
toxic form of Ab and that these may start to form early in AD disease progression, long before brillary and plaque pathology becomes evident (Larson and Lesne, 2012). Alarmingly, PET imaging
of Ab, combined with cerebrospinal uid (CSF) measurements of
Ab and Tau, suggest that AD may start some decades before it becomes symptomatic and plaques begin to accumulate (Weiner
et al., 2012). Indeed, plaques may actually be a last ditch cellular
attempt to wall off potentially toxic Ab oligomers. There is also
considerable evidence to indicate perturbations to Ab-degradation
pathways are associated with AD (Wang et al., 2006; Kurz and
Perneczky, 2011). Therefore, an imbalance between Ab production
and Ab clearance is likely to underlie the disease process. Tau plays
an important physiological role in cytoskeletal stability and axonal
transport in neurons as it controls microtubule assembly.
Perturbed microtubule formation and accumulation of phosphorylated tau in NFTs is a contributing factor to neuronal dysfunction
and cell death in AD (Lovestone and Reynolds, 1997). There is substantial evidence to demonstrate that Ab and Tau may act synergistically to potentiate neuronal dysfunction in AD (Huang and Jiang,
2009). Furthermore, molecular evidence suggests tau hyperphosphorylation is the result of perturbed cellular signaling cascades that may, to some extent, be mediated by Ab (Hernandez
et al., 2010).

3. Metal dyshomeostasis and AD

An imbalance of metal homeostasis in the brain is thought to
play an important role in the pathogenesis of AD (Bush, 2012).
The mammalian brain contains an intrinsically high concentration
of Cu, Zn and Fe ions compared to other tissues, which is reective
of its high requirement for these metals in numerous metaldependent enzyme and metabolic processes (Popescu and Nichol,
2011). The concentrations of Cu, Zn and Fe in the brain are tightly
regulated at the level of the blood brain barrier (BBB) (Bobilya
et al., 2008; Bradbury, 1997; Zheng and Monnot, 2012). In AD
brains large net increases in Cu, Zn and Fe have been reported
compared to healthy age-matched controls Cu: 390 lM vs.
70 lM, Zn: 1055 vs. 350 lM, Fe: 940 lM vs. 340 lM respectively
(Lovell et al., 1998). It must be noted that there is some debate in
the literature regarding these values. In particular, Schrag and
colleagues performed a meta-analysis of the existing literature
and concluded that total brain Fe and Cu levels are not signicantly

541

altered in AD brain vs. age-matched controls (Schrag et al., 2011).

Despite this, numerous methods including proton induced X-ray
emission, immunohistochemistry and synchrotron X-ray uorescence have detected focalized concentrations of Cu, Zn and Fe in
areas of amyloid plaque pathology from AD patients and transgenic
mouse models (Danscher et al., 1997; Friedlich et al., 2004; Lee
et al., 1999, 2002; Lovell et al., 1998; Miller et al., 2006; Stoltenberg
et al., 2007; Suh et al., 2000). Systemic metal dyshomeostasis is
also evident in AD patients with higher than age-matched normal
levels of Cu reported in both the CSF and serum respectively (Basun
et al., 1991; Squitti et al., 2002). In contrast, plasma Zn levels
normally decline with age (Bunker et al., 1987; Monget et al.,
1996; Munro et al., 1987; Ravaglia et al., 2000) but there is
evidence that both plasma and CSF Zn levels are further depleted
in AD patients compared to age-matched control patients (Basun
et al., 1991; Baum et al., 2010; Molina et al., 1998). Hence, global
changes to Cu and Zn are a feature of AD.
4. Oxidative stress and AD
4.1. Markers of oxidative stress
Increased oxidative stress is associated with normal aging but it
is further exacerbated in several neurodegenerative disorders
including AD and Parkinsons disease (Jomova et al., 2010). Mounting evidence implicates oxidative stress as an early event in AD disease progression mediated, in part, by pathological increases in
Ab:metal interactions that facilitate reactive oxygen species
(ROS) production. Markers of oxidative stress that are elevated in
AD brain include lipid peroxidation, DNA oxidation, protein oxidation, advanced glycation end-products (AGEs) and reactive nitrogen species (Buttereld et al., 2011). Homocysteine (Hcy) is a
potential blood biomarker of AD as numerous groups have reported elevated serum or plasma Hcy levels in AD patients compared to age-matched controls (Clarke et al., 1998; Doecke et al.,
2012; Gallucci et al., 2004; Guidi et al., 2005; Joosten et al.,
1997; McCaddon et al., 1998; Quadri et al., 2004; Selley, 2003;
Trojanowski et al., 2010). The mechanism of Hcy induced toxicity
has not been fully elucidated but strong interactions with Fe
(Baggott and Tamura, 2007) and Cu (White et al., 2001), together
with associated increases in protein carbonyls suggest a causal
relationship between Hcy and oxidative damage (Sibrian-Vazquez
et al., 2010). However, circulating Hcy increases normally with age
and there is some doubt in the literature as to whether Hcy levels
are elevated in AD (Ariogul et al., 2005; Luchsinger et al., 2004;
Mizrahi et al., 2003). The generation of Hcy is part of the complex
and synergistic methionine biosynthetic pathway and is affected
by factors such as dietary intake of folate and vitamin B12
(Cito et al., 2010). Population differences and confounding medical
conditions may also account for some of the contradiction in the
literature (Cito et al., 2010). Another factor that contributes to
oxidative stress is a reduced ability to cope with a rise in prooxidants such as Ab and there is evidence that major antioxidant
defense systems are perturbed in AD (see chapter 7).
4.2. Mechanisms of oxidative stress
Redox cycling between Cu1+/Cu2+ and Fe2+/Fe3+ facilitates the
activation of molecular oxygen, a process utilized by numerous
enzymes including cytochrome-c oxidase (CCO), an integral part
of the mitochondrial electron transport chain and ATP production
(Yoshikawa et al., 2011). However, unregulated interaction of Cu
and Fe with molecular oxygen also facilitates the generation of
ROS. Levels of Cu and Fe are intrinsically high in the mitochondria, a highly oxygenated microenvironment that produces

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substantial levels of hydrogen peroxide (H2O2), which make it the

most susceptible site for intracellular ROS generation (Cerqueira
and Kowaltowski, 2012). Mitochondrial dysfunction is a feature
of normal aging and neurodegeneration, potentially due to an
increase in ROS (Beal, 2005). Conversely, redox active metals also
provide catalytic function to antioxidant enzymes such as
superoxide dismutase (SOD). It is important to have a feedback
system to maintain a balance between the roles that divalent
metals play in the generation of ROS and antioxidant defense.
Indeed, these metals are kept within strict physiological limits
by numerous metal-binding proteins that maintain metal
homeostasis by regulated cellular metal uptake, trafcking,
sequestration and efux.
The superoxide anion radical (O2) is considered the primary
ROS in biological systems and is generated primarily via metabolic
processes in mitochondria (Jomova et al., 2010). The generation of
highly reactive O2 is susceptible to conversion to other secondary ROS, rstly via a dismutation reaction catalyzed by SOD enzymes that produce H2O2. In the presence of reduced metals
(Cu1+ and Fe2+) H2O2 can generate highly reactive hydroxyl radicals
(OH). H2O2 is readily diffusible across biological membranes and
H2O2-induced oxidative damage is associated with neurodegeneration. SOD enzymes work in concert with H2O2 removing enzymes
to maintain essential but safe levels of cellular oxidants. In a
normal healthy cellular environment excess H2O2 is usually removed by the action of catalases and glutathione peroxidases.
However, these systems may become overwhelmed during neurodegeneration as levels of pro-oxidants rise. In the case of AD this is
largely mediated by neuropathological increases in Ab that can reduce Cu2+ and Fe3+ to generate H2O2 and downstream highly reactive ROS via Fenton and HaberWeiss chemistry (see Fig. 1)
(Cherny et al., 2001; Dikalov et al., 2004; Hu et al., 2006; Nelson
and Alkon, 2005; Opazo et al., 2002; Tabner et al., 2002). The redox
chemistry of Ab has been comprehensively reviewed elsewhere
(Smith et al., 2007). The presence of high concentrations of Cu
and Fe in the vicinity of Ab-rich plaques (Rajendran et al., 2009),

Fig. 1. Oxidative stress mediated by redox cycling of Cu2+/Cu+ and Fe3+/Fe2+ in the
presence of Ab. Ab/Cu and Ab/Fe complexes are capable of producing hydrogen
peroxide (H2O2). The generated H2O2 may degrade to form unstable ROS via Fenton
Chemistry or a HaberWeiss reaction, leading to oxidative cellular damage and
subsequent neuronal dysfunction and cell loss.

provide the fuel for the generation of ROS that can affect lipid
peroxidation and the formation of deleterious protein and DNA
adducts (Bush and Tanzi, 2008). Although the afnity of Ab to react
with Cu and Fe put it in the pro-oxidant category it may, under
certain conditions, also function as an antioxidant (Kontush,
2001). For example, at physiological (nanomolar) concentrations
Ab has been shown to be neuroprotective and neurotrophic to
cultured cells (Whitson et al., 1990; Whitson et al., 1989; Yankner
et al., 1990). As concentrations of Ab rise however the switch to
pro-oxidant become apparent and the redox activity of Ab variants
is greatest for human Ab42 > human Ab40  mouse/rat Ab40 (Huang
et al., 1999a), corresponding to their relative capacity to reduce
Cu2+ to Cu+ and generate H2O2 in cell free assays (Huang et al.,
1999b).
Lipid peroxidation results in the formation of peroxyl radicals,
which once formed, can then be rearranged via a cyclisation reaction to endoperoxides with the end product of this reaction being
malondialdehyde (MDA) (Jomova and Valko, 2011). Another major
product of lipid peroxidation is the aldehyde 4-hydroxy-2-nonenal
(HNE) and elevated levels of both MDA and HNE have been reported in AD brain and AD transgenic mouse models (Haeffner
et al., 2005; Nelson and Alkon, 2005; Opazo et al., 2002; Puglielli
et al., 2005; Smith et al., 2006; Torres et al., 2011). HNE is highly
reactive and readily forms protein adducts by covalently binding
to cysteine, lysine and histidine residues (Buttereld et al., 2011).
Unlike other protein modications such as phosphorylation this
reaction is irreversible. High levels of redox metals, oxygen and lipids (polyunsaturated fatty acids) make the brain particularly susceptible to lipid peroxidation. In AD the presence of elevated
levels of extracellular Ab at potential sites of lipid peroxidation
only serves to elevate the risk of oxidative damage. Tellingly, areas
where AD pathology is concentrated such as the hippocampus possess higher levels of free HNE in AD patients compared to agematched controls (Markesbery and Lovell, 1998). Increases in brain
levels of HNE in patients with mild cognitive impairment (MCI)
also suggests that oxidative stress is an early event in the pathogenesis of AD (Torres et al., 2011; Williams et al., 2006). Supporting
this notion, markers of oxidative stress, including lipid peroxidation, have been shown to precede (and accompany) Ab pathology
in AD transgenic mouse models (Pratico et al., 2001; Yao et al.,
2004). Recently, a study by Gwon and colleagues provided a potential molecular mechanism to explain how oxidative stress can induce Ab42 production via HNE or Fe2+-mediated modication of
c-secretase activity (Gwon et al., 2012). Using human derived cultured neuroblastoma (SH-SY5Y) cells, they demonstrated that
exogenous addition of HNE or Fe2+ enhanced c-secretase activity
in a luciferase reporter assay with a concomitant rise in the Ab42/
Ab40 ratio (Gwon et al., 2012). Further investigation identied nicastrin, one component of mature c-secretase complexes that liberate Ab from APP, as specically modied by HNE (Gwon et al.,
2012). Additionally, a reduced glutathione (GSH) analogue or the
c-secretase inhibitor L685 458 (GSI) could suppress the increase
in c-secretase activity with the authors reasoning that Ab may amplify amyloidogenic processing of APP via increased HNE activation
of c-secretase activity (Gwon et al., 2012). In other words, a positive feedback system could exist in which Ab not only participates
but also promotes lipid peroxidation, facilitated by increases in
extraneuronal Fe2+.
Further evidence that links oxidative stress and aging with
amyloidogenic processing of APP was recently provided by Mao
and colleagues (Mao et al., 2012). They crossed APP transgenic
(Tg) mice with a strain that was engineered to express the human
form of mitochondria-targeted antioxidant catalase (MCAT). They
found that MCAT expression reduced amyloidogenic processing
of APP in the brain in the APP Tg mice, consistent in this case with
a lowering of BACE1 mRNA and protein expression (Mao et al.,

M.A. Greenough et al. / Neurochemistry International 62 (2013) 540555

2012). MCAT expression also corresponded with increased levels of

several known Ab interacting proteins including the Ab degrading
enzymes neprilysin and insulin-degrading enzyme (Mao et al.,
2012). Taken together, these results suggest that expression of
MCAT may reduce the production and/or clearance of Ab. Although
the mechanism of MCAT modulation of gene expression is not
known, increased lifespan in the MCAT/APP Tg mice compared to
APP Tg mice (Mao et al., 2012) indicates that boosting this intrinsic
mitochondrial antioxidant defense system can offer some level of
protection against AD-related insults.
5. Nitrosative stress and AD
The excessive production of nitric oxide (NO) may also contribute to neuronal cell damage by a process termed nitrosative
stress (Gu et al., 2010). NO is an important signaling molecule but
it is also highly diffusible and in the presence of O2 can form
highly reactive nitrogen species (RNS), including peroxynitrite
(ONOO). Peroxinitrite can then react with vulnerable protein
tyrosine residues in a reaction termed nitration which may contribute to protein misfolding and neuronal injury (Nakamura and
Lipton, 2011). Tellingly, nitration of Tau is thought to inhibit its
ability to self-polymerize and nitrated Tau has been reported in
NFTs and neuritic plaques in AD brain as well as several other
protein misfolding disorders that have associated tauopathies
(Reynolds et al., 2006). In mammals NO is synthesized by three
isozymes: endothelial (eNOS), neuronal (nNOS) and inducible
(iNOS) oxide synthase. All three are expressed in the brain
although, as its name suggests, iNOS is inducible. Inammatory
stimuli can induce iNOS and activated microglia are a source of
neurotoxic amounts of NO characteristic of several neurodegenerative disorders such as AD (Gu et al., 2010). In neurons, excessive
activation of N-methyl-D-aspartate receptors (NMDARs) by glutamate (termed excitotoxicity) drives Ca2+ inux that in turn activates nNOS and can also induce excessive NO production (Gu
et al., 2010).
Another form of NO mediated nitrosative stress that has been
implicated in AD is S-nitrosylation (Lipton et al., 1993). Unlike peroxinitrite-mediated nitration of tyrosine residues to 3-nitrotyrosine on proteins, S-nitrosylation is a reversible modication of
thiol groups of cysteine residues (Lipton et al., 1993; Stamler
et al., 1992). It can regulate a wide range of biological processes
including signal transduction, gene transcription and vesicle
trafcking (Nakamura and Lipton, 2011). Like other forms of
post-translational modication such as phosphorylation it can be
neurotoxic or neuroprotective, dependent on the target protein
(Nakamura and Lipton, 2011). One of the rst demonstrations that
S-nitrosylation could impart neuroprotection was its modulatory
effect on NMDAR activity of cultured rat neurons under conditions
of excitotoxicty, thus preventing the excessive inux of Ca2+
(Lipton et al., 1993). Excessive Ca2+ inux via NMDAR overstimulation can generate ROS and activate cell death pathways
(e.g. caspases) and is thought to mediate neurotoxicity in
numerous neurological and neurodegenerative disorders including
AD (Lipton, 2007). Interestingly, Lipton and colleagues found that
NO binding to NMDAR at a specic cysteine residue (Cys399) on
the NR2A subunit could induce a conformational change that
allows the combined action of glutamate and Zn2+ (which can
now bind more tightly) to desensitize and close NMDAR ion
channels (Lipton et al., 2002; Nakamura and Lipton, 2011).
Glutamate is a well-characterized neurotransmitter but the role
of synaptic Zn2+ in neuromodulation is only just starting to be
elucidated. The implications of lowered levels of bioavailable
synaptic free Zn2+ in the context of pathological levels of extraneuronal Ab:Zn2+ complexes will be discussed later in this review.

543

6. Mechanisms of nitrosative stress in AD

In spite of the wealth of information to support a role for oxidative/nitrosative stress in neurodegeneration very little is known
about the actual molecular mechanisms that govern this process.
For example, Ab is a known pro-oxidant and can cause lipid
peroxidation but how does this relate to mitochondrial oxidative
stress? Much of the focus on Ab-mediated synaptic damage has
been on extraneuronal Ab but the contribution of intraneuronal
Ab is often overlooked. Both Ab and APP have been found in mitochondrial membranes and mitochondrial dysfunction features in
AD with multiple lines of evidence described in the AD brain (Devi
et al., 2006; Gibson et al., 1998; Maurer et al., 2000; Smith et al.,
1996), transgenic AD mice (Caspersen et al., 2005; Eckert et al.,
2008; Lustbader et al., 2004; Manczak et al., 2006; Reddy et al.,
2004), and cell culture models (Matsumoto et al., 2006; Schmidt
et al., 2008). Changes in the expression of mitochondrial genes,
including those that regulate mitochondrial biogenesis, have also
been reported in the AD brain and mouse AD models (Reddy
et al., 2010). A recent study by Guix and colleagues has provided
mechanistic insight that links pathogenic Ab42 production with
NO production and mitochondrial dysfunction using a cultured
murine neuronal cell aging model (Guix et al., 2012). More specifically, they found that as hippocampal neurons in culture age, increases in oxidative stress via the production of ONOO- led to
nitrotyrosination of PSEN1, a modication that caused an increase
in total Ab production and Ab42/Ab40 ratio (Guix et al., 2012). The
increase in ONOO- was consistent with a loss of mitochondrial
SOD2 activity and SOD2 knockout mouse-derived neurons displayed a concomitant increase in the Ab42/Ab40 ratio (Guix et al.,
2012). Furthermore, increased levels of nitrotyrosinated PSEN1
were found in AD brain compared to age-matched controls (Guix
et al., 2012). This mechanistic study builds on previous work by
several groups that have demonstrated that protein nitration can
cause inactivation of manganese-dependent SOD2 (Aoyama et al.,
2000; Ischiropoulos et al., 1992; MacMillan-Crow and Thompson,
1999), which in an APP/PSEN1 double Tg mouse model was mediated by Ab (Anantharaman et al., 2006). The site of SOD2 inactivation by ONOO is the catalytic metal binding site, co-coordinated
by critical tyrosine residues, inducing dityrosine crosslinked
SOD2 (MacMillan-Crow and Thompson, 1999). Increased levels of
various other forms of nitrated proteins have been identied in
AD brain (Castegna et al., 2003; Smith et al., 1997; Sultana et al.,
2006) and an increase in nitrated proteins was also reported in patients with MCI, which is considered a transition state between
cognitively normal and AD disease progression (Buttereld et al.,
2006). Hence, protein nitration is not only an early marker of oxidative stress in neurodegeneration, but it may be exacerbated by
pathological increases in Ab42, which itself may become dityrosine
crosslinked and form neurotoxic stable oligomers in the presence
of Cu2+ and H2O2 (Atwood et al., 2004). Additionally, nitrosative
stress has been associated with pathological perturbations to mitochondrial biogenesis (Nakamura and Lipton, 2010). Neurons have
high energy requirements and as such possess a large number of
mitochondria that are highly mobile and can fuse and divide
(Barsoum et al., 2006; Bereiter-Hahn and Voth, 1994). Mitochondrial ssion/fusion is governed by competing dynamin-related
GTPases and an equilibrium shift in one direction could play a role
in neurodegeneration (Bossy-Wetzel et al., 2003). In mammals the
ssion process is mediated by dynamin-related protein 1 (Drp1)
(Smirnova et al., 2001) and in cultured embryonic cortical rat
neurons Ab oligomers can induce excessive Drp1-dependent
mitochondrial ssion and neuronal damage in a NO dependent
manner (Barsoum et al., 2006). Subsequent studies revealed this
was due to S-nitrosylation of Drp1, which could be induced by

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exogenous addition of the physiological NO donor, N-nitrocysteine,

or Ab oligomers or endogenous overexpression of APP (Cho et al.,
2009). Strikingly, there was a ten-fold increase in S-nitrosylated
Drp1 in AD vs. control brain (Cho et al., 2009) providing pathological relevance to the described mechanism and consistent with
morphological changes in synaptic mitochondria as visualized by
electron microscopy of AD brain (Baloyannis, 2011).
7. Reduced antioxidant defense is a feature of AD
7.1. Glutathione
A large body of evidence implicates compromised antioxidant
defense systems as a contributing factor in AD pathogenesis. Glutathione (c-glutamylcysteinylglycine; GSH) is a ubiquitous and
highly abundant cellular antioxidant that is synthesized metabolically from the precursors glutamate, cysteine and glycine. Thiol reduced GSH normally accounts for the majority (>98%) of total
cellular glutathione but it can also exist as oxidized glutathione
disulde (GSSG) or glutathione adducts (Ballatori et al., 2009). Glutathione peroxidase (GPx) catalyses the oxidation of GSH to GSSG
whilst the reverse reaction is driven by glutathione reductase
(GR) and requires NADPH. Coupled to the oxidation of GSH GPx
can reduce H2O2, highlighting the importance of both GPx and
GR in maintaining the cellular redox state. Indeed, measurement
of erythrocyte levels of GSH, expressed as a ratio of GSH/GSSG, provide a dynamic marker of oxidative stress in vivo (Sohal et al.,
1990). Several groups have independently conrmed reduced
GSH/GSSG ratios in Alzheimers patient erythrocytes compared to
controls (Bermejo et al., 2008; Liu et al., 2005; Torres et al.,
2011; Vina et al., 2004), and in one particular study this correlated
with cognitive status (Vina et al., 2004). There are also reports of
reduced GSH/GSSG levels in MCI patients (Bermejo et al., 2008),
consistent with rising HNE and MDA levels (Torres et al., 2011;
Williams et al., 2006), which may provide an early indicator of possible AD disease progression. Attempts to measure GPx and GR
activity in AD patients have provided conicting results. Some
studies found activity of GPx increased in AD (Martin-Aragon
et al., 2009; Torres et al., 2011; Zafrilla et al., 2006) whereas others
found reduced GPx activity (Bermejo et al., 2008; Casado et al.,
2008; Padurariu et al., 2010; Rinaldi et al., 2003). Similarly, activity
of GR in AD patients has either been reported as decreased (Casado
et al., 2008; Torres et al., 2011; Zafrilla et al., 2006) or no change
(Baldeiras et al., 2008; Martin-Aragon et al., 2009) compared to
controls. These discrepancies indicate that the GSH/GSSG ratio
may be a more reliable marker of oxidative stress in AD. Postmortem detection of GSH levels in AD patient brains have provided
conicting results (Adams et al., 1991; Aksenov and Markesbery,
2001; Gu et al., 1998; Perry et al., 1987), which may be attributable
to factors such as quality of post-mortem tissue.
7.2. Superoxide dismutase
Reduced Cu/Zn-dependent SOD1 activity has been reported in
post-mortem AD patient frontal lobe tissue as well as transgenic
AD mice (Marcus et al., 1998; Murakami et al., 2011). Additionally,
knock-in mice expressing a pathological mutation that causes FAD
in humans were found to have increased ROS production (Guo
et al., 1999), consistent with reduced SOD1 function (Leutner
et al., 2000). Altered localization and expression patterns of both
SOD1 and the Mn-dependent isoform SOD2, has been detected in
the neocortex and hippocampus of post-mortem AD brain tissue
(Furuta et al., 1995). Reduced levels of SOD enzymes and increased
levels of oxidative stress markers such as MDA have also been reported in the bloodstream of patients with MCI and AD compared

to age-matched healthy control groups (Padurariu et al., 2010;

Rinaldi et al., 2003; Torres et al., 2011). Conversely, the Australian
Imaging Biomarker and Lifestyle (AIBL) group recently reported
that SOD levels were signicantly elevated in the plasma of AD patients compared to age-matched controls (Doecke et al., 2012). This
study was carried out on a large cohort of 754 healthy individuals
and 207 AD patients and the results were cross-validated with 58
healthy controls and 112 individuals with AD from the Alzheimer
Disease Neuroimaging Initiative (ADNI) cohort (Doecke et al.,
2012). Despite the discrepancies in reported peripheral levels of
SOD protein, changes to SOD activity may be reective of the
changes in metal homeostasis that occur in AD or due to protein
modication such as tyrosine nitration or cysteine nitrosylation
that would preclude the active site from metal-binding (see
above). In a recently published study by our group SOD1 specic
activity was reduced in cultured embryonic broblast cells and
brain tissue from presenilin knockout mice (Greenough et al.,
2011). We reasoned that this was due to the apparent requirement
for presenilin-mediated copper (and zinc) uptake into these cells
and subsequent lack of metallation of SOD1 in the presenilin null
background. In this particular case protein nitrosylation was excluded, as critical cysteine residues were still reactive. Further
analysis revealed a loss of expression of CCS (the copper chaperone
for SOD1), in presenilin knockout cells and tissue, consistent with a
reduced capacity for copper delivery to SOD1. Why CCS expression
is down-regulated in presenilin null cells and brain tissues that
have a lowered copper status is unclear but may reect a functional, two-way interaction since a recent screen to identify genes
that interact with Drosophila presenilin (Psn) identied CCS via its
ability to suppress a Psn-mediated dominant negative phenotype
(van de Hoef et al., 2009). In light of our ndings, it is notable that
BACE1 binds Cu, interacts with CCS and BACE1 expression reduces
the activity of SOD1 (Angeletti et al., 2005). CCS deciency increases Ab production through BACE1 processing (Gray et al.,
2010). Thus, two critical components of the b-amyloidogenic pathway functionally interact with CCS. Taken together, we may expect
that a decrease in PS activity would lower CCS levels, which would
in turn increase BACE1 processing of APP.
7.3. Metallothioneins
Metallothioneins (MTs) are small cysteine-rich proteins that
have a high capacity to bind metals with varying afnities
(Cu > Cd > Zn) (Toriumi et al., 2005). They are not antioxidants
per se but due to their high metal afnity can buffer potentially
toxic levels of metals via sequestration. Therefore MTs play an indirect role in the modulation of NO and ROS (Datta and Lianos, 2006).
Four isoforms have been identied in humans designated MT1
MT4 (Vasak and Hasler, 2000). Metal transcription factor 1 (MTF1) regulates the expression of MT1 and MT2 genes in mammals,
which are induced by Zn, Cu and other metals (Ghoshal and Jacob,
2001; Tapia et al., 2004). MT1 and MT2 are ubiquitously expressed
and reportedly upregulated in AD, potentially in response to
increased metal mediated oxidative stress (Adlard et al., 1998;
Hidalgo et al., 2006; Zambenedetti et al., 1998). Conversely, MT3,
which is not induced by metals (Palmiter et al., 1992) is enriched
in the CNS (neurons and astrocytes) and is lower in AD brain (Tsuji
et al., 1992; Uchida et al., 1991; Yu et al., 2001). MT3 was formerly
called growth inhibitory factor (or GIF) as it was originally identied for its ability to inhibit neurite outgrowth of cultured rat neurons in the presence of AD brain extracts (Uchida et al., 1991). In
the absence of brain extract however MT3 is neuroprotective for
cultured neurons indicating MT3 alone could not mediate its GIF
activity (Uchida et al., 2002). This function is not shared by other
MTs and although the mechanism is poorly understood it is potentially mediated by a unique TCPCP motif within the MT3 b-domain

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that has been proposed to induce a characteristic conformation

that facilitates MT3 domaindomain interactions (Ding et al.,
2010). Interestingly, El Ghazi and colleagues demonstrated that
mouse MT3 can form part of one or several multiprotein complexes (El Ghazi et al., 2006) and recently proposed a model that
links astrocyte secretion of MT3 containing protein complexes
with regulation of neuronal growth (El Ghazi et al., 2010). Additionally, MT3 can effectively redox silence reactive soluble and
aggregated Ab:Cu2+ complexes in vitro by a Zn/Cu metal swap
mechanism (Meloni et al., 2007, 2008). Recently, this model was
further rened and kinetic analysis revealed that the rapid transfer
of Cu2+ from Ab:Cu2+ to MT3 did not involve a direct Ab:Cu2+:MT3
complex but instead free Cu2+ released from Ab to MT3 (Pedersen
et al., 2012). Associated structural and morphological changes to
Ab were also reported, attributable to Zn-induced aggregation
(Pedersen et al., 2012). Whether this mechanism has physiological
relevance in vivo is yet to be determined although several studies
have implicated a role for synaptic MT3 zinc release and subsequent binding to Ab in neuroprotection (Bell and Vallee, 2009;
Durand et al., 2010).

8. Interactions of metals and AD related proteins

8.1. Ab oligomerization and the role of metals in neurotoxicity
The amyloid cascade hypothesis is the leading model of AD
pathophysiology and initially extracellular Ab brils were thought
to be the primary toxic Ab species due their high abundance in
amyloid plaques of the AD affected brain. However, plaque burden
is a poor correlate of disease severity (Braak and Braak, 1990,
1991), which has caused considerable contention amongst
researchers for many years. Research in the early 1990s identied
that Ab in the soluble portion of AD brain was a better correlate
with synaptic change (Lue et al., 1999) and markers of disease
severity (McLean et al., 1999) than insoluble Ab aggregates. Intermediate stable soluble Ab oligomers are now widely believed to be
a major cause of neurotoxicity in AD (Frackowiak et al., 1994; Kuo
et al., 1996; Podlisny et al., 1995; Roher et al., 1993). Pioneering
work by Lambert and colleagues demonstrated that small diffusible Ab42 oligomers could inhibit long term potentiation (LTP is a
physiological marker of memory and learning) and mediate toxicity in rat hippocampal neurons under conditions that block bril
formation (Lambert et al., 1998). Since then, numerous groups
have tried to identify the dominant toxic Ab oligomer species that
mediates neurotoxicity in AD as these would make attractive targets for Ab immunotherapy. So far this has included cell culture derived dimeric and trimeric Ab (Selkoe, 2008), AD brain derived
dimers (Shankar et al., 2008) and AD brain and synthetically derived dodecamers (Barghorn et al., 2005; Bernstein et al., 2009;
Gong et al., 2003). The inherent transient nature of Ab and propensity to aggregate in vitro together with variance in source material
and experimental conditions is likely to account for different ndings. As described recently by De Strooper and colleagues, a lack of
consistency in experimental conditions makes comparative analysis of soluble Ab oligomer studies virtually impossible (Benilova
et al., 2012). Despite the lack of agreement amongst researchers
several mechanisms have been proposed for Ab oligomer-induced
neurotoxicity including oxidative stress, perturbed Ca2+ and biometal homeostasis, impaired axonal transport, altered membrane
integrity and mitochondrial dysfunction (Crouch et al., 2008). The
relative contribution of any of these mechanisms to neuronal dysfunction and ultimately neuronal death is hotly debated although
deleterious effects are likely to be cumulative. A wealth of evidence
from our group and others over the past two decades has indicated
that biometals, namely Cu and Zn and Fe, not only play a role in Ab

545

aggregation but also facilitate ROS generation via Ab:Cu2+ ligands.

Ab is a metalloprotein that can bind Cu2+ and Zn2+ at high and
low afnity metal binding sites, coordinated by histidine residues
in the N-terminus (Atwood et al., 2000b; Curtain et al., 2001;
Danielsson et al., 2007; Syme and Viles, 2006). Numerous studies
have demonstrated that both Cu and Zn can promote the oligomerization of Ab in vitro. Iron is also in abundance at the periphery of
amyloid plaques and in vitro studies have demonstrated that Ab
may bind Fe with lowered afnity and at mildly acidic pH (6.8
7.0), consistent with the conditions found in AD brain (Bush and
Tanzi, 2008). Furthermore, the micromolar concentrations of Cu
and Zn released from cortical synapses are sufcient to induce
Ab aggregation (Ha et al., 2007; Jun and Saxena, 2007; Stellato
et al., 2006; Tougu et al., 2008). A key Ab amino residue in this
reaction is reported to be tyrosine10 (Y10) (Barnham et al.,
2004). Oxidative modication to Y10 in the presence of Cu2+ and
H2O2 can form dityrosine crosslinked Ab, a highly stable oligomeric
species that displays enhanced toxicity when added to cultured
neurons (Atwood et al., 2004). Once formed dityrosine crosslinked
Ab is resistant to proteolytic degradation (Atwood et al., 1998)
which potentiates further aggregation and the formation of higher
order Ab oligomers (Barnham et al., 2004). Not surprisingly, dityrosine oxidation products are elevated in the AD brain, particularly in
the hippocampus (Hensley et al., 1998). Another Ab residue that is
susceptible to Cu2+-mediated oxidation is the sulphur atom of
methionine-35 (Met35). Raman spectroscopic analysis of AD brain
plaques indicated elevated levels of methionine oxidation (Dong
et al., 2003) and amino acid substitution of Met35 has been shown
to abrogate the neurotoxic effects of Ab peptides to cultured cells
(Ali et al., 2005; Ciccotosto et al., 2004; Varadarajan et al., 2001;
Varadarajan et al., 1999). More recent in vitro analysis of synthetic
Ab peptides using NMR and EPR spectroscopy have shown that
Met35 is not required for Cu2+-binding (da Silva et al., 2009; Hou
and Zagorski, 2006). Despite the lack of consensus regarding
the exact co-ordination and kinetics of Ab:Cu binding, due in part
to differences in experimental conditions such as peptide preparation and pH effects, there is little doubt that Cu2+ potentiates Ab
toxicity via its redox potential and propensity to cause Ab
oligomerization.
8.2. APP and metal regulation
Numerous in vitro and in vivo studies have found interactions
between APP and Cu. Structural studies have revealed that putative
metal binding sites for Cu are located in the amino-terminal ectodomain and Ab sequence of bAPP (Atwood et al., 2000a; Barnham
et al., 2003; Hesse et al., 1994; Simons et al., 2002; Valensin
et al., 2004) with the Cu binding domain of APP coordinated by
His-147, His-151, Tyr-168 and Met-170 residues (Barnham et al.,
2003). APP is ubiquitously expressed and modulation of intracellular Cu levels by overexpression of the Cu exporter ATP7a in transformed human broblasts reduces APP levels (Bellingham et al.,
2004b). Consistent with this observation, adding Cu to ATP7aoverexpressing (and parental) CHO cells increases APP expression
(Borchardt et al., 1999). Copper overload in cultured primary
mouse broblasts by expression of mutant forms of ATP7a (Mobr
and Modap mice) in combination with high Cu exposure also increases APP levels (Armendariz et al., 2004). Regulation was evident at the transcriptional (mRNA) level and is mediated by a
copper regulatory region between 490 and + 104 of the APP promoter region (Bellingham et al., 2004b). Additionally, in vivo and
in vitro studies in APP knockout mice and cultured cortical neurons
and broblasts from these animals suggest APP can modulate Cu
levels (Bellingham et al., 2004a; White et al., 1999). Taken together, these results imply a feedback system exists in which Cu
can regulate APP expression and APP can in turn regulate Cu levels.

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Furthermore, intracellular Cu levels can modulate the processing of

APP. In transformed human broblasts genetically engineered to
be profoundly Cu decient, higher levels of amyloidogenic processing of APP were evident by the secretion of more sAPPb into the
culture media as well as more APP C-termini (C99) (Cater et al.,
2008). Conversely, genetically modied broblasts that have a constitutively high Cu content secreted more non-amyloidogenic sAPPa (Cater et al., 2008). Consistent with a modulatory effect of Cu on
APP processing, the addition of low micromolar concentrations of
Cu to parental or chronically Cu-decient CHO cells (10 or 50 lM
Cu, respectively) reduced Ab generation but increased intracellular
full length APP and secreted sAPP (Borchardt et al., 1999). Recent
studies using cultured human neuroblastoma and primary mouse
cortical neurons cells have reported net increases in APP at the cell
surface in the presence of elevated Cu (Acevedo et al., 2011; Hung
et al., 2009) suggestive of a role for Cu in APP trafcking. Utilizing
cell surface biotinylation, live cell confocal imaging and antibody
endocytosis techniques this observation was attributed to an increase in the rate of APP exocytosis and a concomitant decrease
in endocytosis (Acevedo et al., 2011). Cu-induced retention of
APP at the cell surface was consistent with reduced levels of Ab
in cholesterol-rich lipid rafts, proposed sites of amyloidogenic processing (Hung et al., 2009). Paradoxically, low cellular Cu levels
corresponded with a concentration of Cu, Ab and cholesterol in
lipid rafts (Hung et al., 2009). Hence, in AD brain where localized
tissues are Cu-decient this would increase the likelihood of
pro-oxidant Ab:Cu2+ complexes that could catalytically oxidize
cholesterol and long chain fatty acids, as observed in the brain of
transgenic AD mouse models (Jiang et al., 2007; Nelson and Alkon,
2005; Opazo et al., 2002; Puglielli et al., 2005; Smith et al., 2006;
Yoshimoto et al., 2005).
Brain MRI scans revealed elevated levels of Fe2+ in AD patients
vs. healthy controls (Bartzokis et al., 1994a,b; Bartzokis and Tishler,
2000). Furthermore, increased redox activity in hippocampal slices
isolated from post-mortem AD patients vs. age-matched controls
was attributed to elevated cytoplasmic Fe2+ levels and subsequent
oxidation of ribosomal RNA (Honda et al., 2005). It was noted by
our group that a couple of features of APP suggested a potential
role in iron regulation, most notably, APP mRNA contains an iron
response element (IRE) in the 50 untranslated region and subsequently APP translation is sensitive to Fe levels (Rogers et al.,
2002; Venti et al., 2004), and APP possesses a REXXE ferroxidase
consensus motif (Gutierrez et al., 1997). Ferroxidases oxidize Fe2+
to Fe3+, facilitating the binding of Fe3+ to transferrin that only recognizes ferric iron. Cellular Fe2+ export is mediated via interaction
of a ferroxidase with ferroportin on the cell surface for incorporation of Fe3+ into transferrin (De Domenico et al., 2007). Ceruloplasmin (CP) is a well-characterized member of the ferroxidase family.
In humans, mutations in Cp cause aceruloplasminemia, a condition
that causes pathological brain Fe2+ accumulation in glial cells and
dementia (Harris et al., 1995; Patel et al., 2002). However neurons
do not contain CP, which prompted members of our group to consider whether APP may serve that function in neurons. Exhaustive
in vitro and in vivo analyses using synthetic peptides, APP knockout
mice and cultured cell models revealed that APP indeed possesses
ferroxidase activity, interacts with cell surface ferroportin in an
Fe2+-dependent manner and facilitates export of Fe2+ from cultured
neurons (Duce et al., 2010). Furthermore, free Zn2+ or Zn2+ derived
from Zn2+:Ab42 aggregates potently inhibited APP ferroxidase
activity suggesting that pathological interactions of synaptic Zn2+
(e.g. from Zn2+:Ab oligomers) could inhibit neuronal Fe2+ export
(Duce et al., 2010). Decreased APP ferroxidase activity correlated
strongly with increasing Ab levels specically in AD cortical tissue
providing further support for a physiological role for APP in iron
export (Duce et al., 2010). Recently it was also reported that APP,
like CP, exerts amine oxidase activity that mediates the release of

the catecholamines; dopamine, norepinephrine and epinephrine

(Duce et al., 2012). The site for amine oxidase activity overlaps
the ferroxidase motif and it was suggested that pairing ferroxidation with amine oxidation might be a means of preventing the oxidation of catecholamines by Fe3+ into toxic products (Duce et al.,
2012). It was also recently discovered that Tau plays a role in the
trafcking of APP to the cell surface of cultured cortical neurons
and Tau knockout mice accumulate neurotoxic levels of Fe in the
hippocampus, frontal neocortex and substantia nigra (Lei et al.,
2012).
APP also binds zinc (Bush et al., 1993, 1994a,b,c), coordinated by
two critical cysteine residues Cys-186 and Cys-187 with other
potential ligand sites including Met-170, Cys-174, Asp-177 and
Glu-184 (Ciuculescu et al., 2005). In vitro analysis of this metal
binding region has demonstrated that Zn2+ can facilitate dimerization of the peptide (Ciuculescu et al., 2005), which may have important functional consequences as APP dimers can occur in vivo
(Scheuermann et al., 2001). Hence, a picture has emerged whereby
APP has a complex role in biometal homeostasis. On the one hand
APP expression is sensitive to Cu and Fe levels and in turn APP
can modulate intracellular Cu2+ and Fe2+ levels. Conversely Zn2+ appears to play more of a structural role that could impact its function.
8.3. Presenilins and copper/zinc regulation
We recently demonstrated that presenilins are responsible for a
signicant proportion (up to 50%) of Cu2+ and Zn2+ uptake in cultured mouse embryonic broblasts with organismal Cu and Zn
deciencies in several presenilin knockout mouse tissues including
brain (Greenough et al., 2011). Presenilin mediated Cu2+ and Zn2+
uptake was not dependent on its c-secretase function in that study.
This implied that lowered Cu/Zn status in presenilin null cells was
not due to changes in c-secretase substrate activities such as an
increase in sAPP or loss of Ab, both of which bind Cu and Zn with
high and low afnity (Atwood et al., 2000a,b; Barnham et al.,
2003; Dahms et al., 2012; Giuffrida et al., 2007; Kong et al., 2007,
2008). Numerous studies have implicated presenilins in protein
maturation and post-Golgi trafcking/turnover, including several
cell surface receptors, extensively reviewed elsewhere (Coen and
Annaert, 2010). We hypothesize that presenilins may inuence
copper transport activity at the level of protein trafcking.
Regulation of cellular copper import by copper transporter 1
(CTR1) and copper export by ATP7a, have been shown to be dependent on endocytosis/exocytosis of the respective transporters
(Greenough et al., 2004; Guo et al., 2004; Molloy and Kaplan,
2009; Nose et al., 2010; Pase et al., 2004; Petris et al., 1996,
2003). Tellingly, perturbations of neuronal endocytic pathways
have been shown to be one of the earliest pathological features
in AD patients (Cataldo et al., 2000; Coen and Annaert, 2010). We
are currently investigating a potential link with presenilin and
trafcking of several key metal transporters, including the copper
transporters; CTR1 and ATP7a, and several Zn2+ transporters.
Alternatively, non-selective plasma membrane ion channels may
also modulate biometal uptake. Transient receptor potential
melastatin 7 (TRPM7), a non-selective Ca2+ channel, is Zn2+
permeable (Inoue et al., 2010). Interestingly, Oh and colleagues
demonstrated that TRPM7-mediated Zn2+ (and Ca2+) uptake was
facilitated by expression of wild type PSEN1 but not FADassociated mutant PSEN1 in cultured HEK293 cells (Oh et al.,
2012). Similar to our study, Zn2+ uptake was not sensitive to
pharmacological c-secretase inhibition. Decits in Ca2+ signaling
are well known in AD and previous reports have demonstrated a
role for presenilin in Ca2+ homeostasis (LaFerla, 2002; Mattson
et al., 2001). Presenilin appears to function at the level of capacitative calcium entry (CCE), an intracellular Ca2+ relling mechanism
that couples inositol-1,4,5-triphosphate-mediated release of stored

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ER-calcium with Ca2+ uptake via plasma membrane channels

(Berridge, 2002). Hence, major routes of cellular Zn2+ may overlap
with those for Ca2+ entry and are sensitive to functional presenilin.
This has important implications in terms of the re-uptake of Zn2+ in
AD, whereby defective Zn2+ uptake could conceivably result in
the pooling of synaptic zinc at the sites of amyloid deposition
(see Section 9).
9. The relationship of synaptic Zn2+ release and AD
Zn is released from the presynaptic terminals of glutamatergic
neurons and can reportedly reach concentrations up to 300 lM
upon neurotransmission (Frederickson et al., 2000). The zinc transporter, ZnT3, is responsible for presynaptic release of vesicular Zn2+
and is expressed primarily in the hippocampus and neocortex,
regions associated with higher cognitive function in mammals
(Adlard et al., 2010; Palmiter et al., 1996). Lee and colleagues
demonstrated that genetic ablation of ZnT3 in an AD mouse model
overexpressing Swedish mutant APP (Tg2576) caused a decreased
amyloid plaque load (Lee et al., 2002) and cerebral amyloid angiopathy (Friedlich et al., 2004), providing evidence that synaptic Zn2+
mediates Ab pathology. In 2009, Deshpande and colleagues reported that synaptic Zn2+ potentiates the synaptic targeting of Ab
oligomers added to rat hippocampal slices and cultured human
cortical neurons which then occluded NMDAR2B receptors, affecting downstream signaling pathways (Deshpande et al., 2009). In
2010, Adlard and colleagues described several pertinent ndings;
(1) cortical ZnT3 expression decreases with age in healthy mice
and humans and this is further exacerbated in AD, (2) ZnT3 (KO)
knockout mice display age-dependent decits in multiple memory
and learning tasks that phenocopies those observed in AD APP Tg
mice, and (3) aged ZnT3 KO mice have altered levels of several
pre- and post-synaptic markers, including lowered NMDAR2a/b,
AMPAR, PSD95 and SNAP25 (Adlard et al., 2010). Taken together,
the authors proposed a model whereby loss of trans-synaptic
Zn2+ via normal aging is exacerbated in AD by the trapping of
synaptic Zn2+ by Ab, leading to decreased expression of select
post-synaptic targets and cognitive decline. Lee and colleagues
demonstrated an age-dependent decline in brain ZnT3 expression
and concomitant reduction in synaptic Zn2+ in APP Tg AD mice
compared to WT littermate controls (Lee et al., 2012), consistent
with reduced ZnT3 protein and mRNA expression in postmortem
AD brain (Adlard et al., 2010; Beyer et al., 2009). They also reported
APP Tg AD mice had a distinct pool of Zn2+ and ZnT3 expression in
dystrophic neurites and reactive glial cells at sites of amyloid
deposition, consistent with a previous study that used APP/PSEN1
double-Tg AD mice (Lee et al., 2012; Stoltenberg et al., 2007).
Reduced activation of Erk-dependent signaling pathways has also
been reported in the hippocampal regions of ZnT3 knockout mice,
consistent with memory and learning decits observed in these
animals (Sindreu et al., 2011). In the context of AD, Nakashima
and colleagues have demonstrated gender-specic changes to
Zn-dependent synaptic plasticity in a 3x-Tg AD mouse model
(Nakashima and Dyck, 2010). In addition, several other groups
have reported a role for synaptic Zn2+ in cognition (Daumas
et al., 2004; Frederickson et al., 1990, 2000; Martel et al., 2010)
potentially as a neuromodulator of synaptic plasticity and longterm potentiation (Li et al., 2001; Lu et al., 2000; Pan et al., 2011).
10. Metal ionophores as AD therapeutics
10.1. Introduction current AD therapies
The current FDA-approved treatments for AD are limited to
small number of drugs that are designed to alleviate some of the

547

symptomatic memory-decits and behavioral changes associated

with the disease. Most of these drugs are of the class of acetylcholinesterase inhibitors (AChEIs), designed to increase the availability
of acetylcholine (ACh), a neurotransmitter involved in cholinergic
neurotransmission (Lleo et al., 2006). The only other approved drug
is memantine, an N-methyl-D-aspartate (NMDA)-receptor antagonist that protects against pathological activation of NMDA receptors (NMDAR) by glutamate (Doraiswamy, 2002; Farlow, 2004;
Lipton, 2005). However, the above strategies are not long-term
treatments for AD as their effectiveness diminishes over time and
they do not modify the underlying pathobiology. Exhaustive animal and human trials have tested numerous disease-modifying
drugs, targeted to the aggregation pathology of either Ab or Tau,
but none to date have made it to FDA approval stage, mostly due
to adverse side effects or lack of cognitive improvement in trial
participants (reviewed in (Herrmann et al., 2011). Given that oxidative stress is an early and sustained event in the pathogenesis
of AD it is perhaps not surprising that several types of naturally
occurring antioxidants, including vitamin E, Ginkgo Biloba, curcumin and resveratrol have been trialed in both animals and humans.
However, the limited ability of most of these compounds to cross
the BBB prevents oral administration as an effective therapeutic.

10.2. Clioquinol
In light of the nding that Cu2+ and Zn2+ could provoke Ab oligomerization, targeting pathological metal:Ab interactions was considered as an alternative AD therapeutic strategy. In early testing,
the drug clioquinol (CQ), an 8-hydroxyquinoline (8-OHQ), was considered for its moderate Cu2+ and Zn2+ binding afnities and ability
to cross the BBB. The hypothesis was that if it were possible to
preferentially bind metals in the vicinity of extraneuronal Ab then
it might offer some protection from adverse oxidative reactions
that occur from the interaction of Cu2+ and Zn2+ with Ab, namely
the release of H2O2. The binding afnities of CQ for Cu2+ and Zn2+
are such that it can competitively dissociate these metals from
the lower afnity binding sites of Ab (Opazo et al., 2006). A study
by Cherny and colleagues demonstrated a dramatic reduction in
cortical Ab plaque pathology (49%) from CQ-treated 15 monthold APPTg2576 AD mice vs. sham-treated littermates with
improvements to weight and general health measures (Cherny
et al., 2001). Importantly, there was no depletion in any of the
brain metals measured, including Cu, Zn and Fe, in the CQ-treated
animals (Cherny et al., 2001). This indicated that CQ was not simply acting as a chelator by stripping metals from the brain. CQ was
then assessed in a small phase 2 clinical trial, where oral dosing for
36 weeks slowed the rate of cognitive decline and reduced CSF
Ab42 levels in moderately severe AD patients vs. placebo controls
(Ritchie et al., 2003). Although chelation of Cu and Zn from metallated-Ab species and potential dissolution of Ab plaques was the
intended role for CQ (Bush, 2008), a subsequent study using cultured cells provided important insights into a potential mode of action of CQ via activation of cellular signalling pathways. Using
Chinese hamster ovary (CHO) and mouse neuroblastoma (N2a)
cells it was found CQ could act as a metal ionophore, effectively
increasing the intracellular pool of bioavailable Cu and Zn (White
et al., 2006). The downstream effect of this was a metal-dependent
activation of the PI3KAkt signaling pathway and subsequent neuroprotective phosphorylation of JNK and ERK1/2 kinases (White
et al., 2006). A signicant decrease in secreted Ab and upregulation
of Ab-degrading matrix metalloproteases, MMP1 and MMP2, was
also reported (White et al., 2006). Taken together, these results
suggest that CQ may act by (i) removing Cu and Zn from extracellular Ab, and/or (ii) stimulating Ab degradation pathways by activating intracellular signaling cascades that are metal-dependent.

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10.3. PBT2
PBT2 (Prana Biotechnology Ltd) is a 2nd generation 8-OHQ
derivative of CQ. In vivo testing of AD transgenic mice demonstrated that PBT2 crossed the BBB more effectively than CQ, significantly reduced Ab levels and plaque burden, improved cognitive
performance and rescued Ab-induced impairment of LTP (Adlard
et al., 2008). Follow up studies comparing young (4 months) vs.
old (14 months) AD transgenic mice established that short-term
PBT2 treatment (11 days) could restore dendritic spine density deficits and increase levels of several biochemical markers of synaptic
plasticity and learning, including BDNF, pro-BDNF, CaMKII and
NMDAR1A/2A (Adlard et al., 2011). In a 12-week Phase 2 clinical
trial of 78 patients with mild to moderate AD, PBT2 was safe and
well tolerated at 50 and 250 mg daily doses (Lannfelt et al.,
2008). As a potential disease-modifying drug in humans, the
250 mg/day dose of PBT2 signicantly lowered CSF Ab levels and
improved cognitive performance over baseline in several key executive function tests (Faux et al., 2010; Lannfelt et al., 2008). In addition to increasing intracellular pools of labile Zn2+ and modulating
phospho-Tau levels, studies using cultured human SH-SY5Y neuronal cells established a model mechanism of action for PBT2 (Crouch
et al., 2011). In concert with Zn2+, PBT2 could activate the calcineurin pathway, resulting in phosphorylation of several downstream
targets such as CREB and CaMKII and decrease the levels of
pro-apoptotic caspase-3 (Crouch et al., 2011). Recently, several
8-OHQ derivatives of CQ were shown to rescue TDP-43 proteinopathies in a yeast model (Tardiff et al., 2012).
10.4. BTSCs
Bis(thiosemicarbazonoto) or BTSCs are another class of compounds with metal ionophore characteristics currently being
tested as potential therapeutics for AD. As proof of concept Donnelly and colleagues demonstrated that Cu2+- and Zn2+-BTSC complexes could increase intracellular [copper] or [zinc] and reduce
Ab levels in APP expressing CHO cells, consistent with a reported
induction of PI3 K/JNK signaling responsible for regulation of Abdegrading MMP1/2 activity (Donnelly et al., 2008). Six-month-old
AD transgenic mice treated with Cu2+-gtsm, a copper-BTSC complex, displayed marked improvements in spatial learning and
memory, reduced abundance of trimeric Ab and co-treatment of
hippocampal slices with Cu2+-gtsm restored LTP compared to
treatment with Ab42 alone (Crouch et al., 2009). Importantly, when
Cu2+-gtsm enters the cell Cu2+ is reduced to Cu+, causing its disassociation from the ligand. The increase in bioavailable copper from
Cu2+-gtsm treatment was accompanied by a rise in phosphoGSK3b, Akt and ERK1/2 levels, and concomitant decrease in phospho-Tau in mice and cultured cell models (Crouch et al., 2009).
What is important to establish now, is the dynamics of cellular
transport of both 8-OHQ and metal-BTSC ionophores. How they
enter neuronal cells; what are the dissociation kinetics; what trafcking pathways determine their bioavailability and ultimate efux are all questions that remain largely unanswered. Passive
uptake is likely due to the inherent lipophilic nature of these compounds. However, there is some evidence that Cu2+-gtsm (and
other BTSC analogues) can be taken up by a combined passive/carrier mediated pathway, although the high afnity Cu transporter
CTR1 does not appear to be involved (Price et al., 2011). Accumulation of Cu when Cu2+-gtsm treated neuronal cells were depleted
of ATP suggests an energy dependent carrier mediated Cu2+ efux
system is utilized (Price et al., 2011). The Cu2+-translocating ATPase (ATP7a) seems a likely candidate although its role was not
investigated. These are important questions that need addressing;
especially in light of the nding that glutamate/glycine induced
NMDAR activation can stimulate trafcking of ATP7a to the plasma

membrane and subsequent release of copper from cultured murine

hippocampal neurons (Schlief et al., 2005). The authors of that
study then demonstrated a potential neuroprotective role for
ATP7a mediated release of Cu2+ from NMDA excitotoxicity, dependent on endogenous NO (Schlief and Gitlin, 2006; Schlief et al.,
2006). The proposed model of neuroprotection is based on the
known role of nitrosylation by NO at specic cysteine residues
on NMDARs that modulates their activity (Boehning and Snyder,
2003; Lipton et al., 2002). In this scenario Cu2+ would act as an
electron donor (Ford et al., 2005) (Fig. 2). Two features central to
the pathophysiology of AD are excessive NMDAR activity and
sequestration of extraneuronal Cu2+ with Ab oligomers. Hence, it
is not unreasonable to predict that in the case of AD excessive
Ab:Cu2+ interactions could result in a lack of bioavailable Cu2+
and subsequent lack of modulation of NMDAR activity (Fig. 2).
Reduced levels of intracellular Cu have been reported in cultured cortical neurons derived from AD transgenic mice, consistent
with a loss of bioavailable Cu at the site of oxidative damage and
neuronal loss in AD (Bellingham et al., 2004a). Work by Khosravani
and colleagues demonstrated that cellular prion protein (PrPc)
forms a signaling complex with NMDARs and abrogation of PrPc
in mouse hippocampal neurons enhanced NMDAR currents
(Khosravani et al., 2008). PrPc possesses several octarepeat regions
that bind Cu2+ with a high afnity (Jackson et al., 2001). Moreover,
several other groups have reported pathological interactions of
PrPc with Ab oligomers that can disrupt LTP induction (Barry
et al., 2011; Freir et al., 2011; Gimbel et al., 2010; Lauren et al.,
2009). Recently, You and colleagues proposed a model that links
PrPc with NMDAR activity modulation in a Cu-dependent manner
(You et al., 2012). Using a combination of rodent models; primary
hippocampal neurons from normal rat, transgenic 5XFAD mice and
PrPc knockout mice, they demonstrated that Cu loaded PrPc could
reduce NMDAR afnity for glycine, a co-agonist for NMDAR current
stimulation (You et al., 2012). Depletion of copper, either by direct
chelation or an overabundance of Ab42, phenocopied the effect or
PrPc knockout (You et al., 2012). These ndings indicate that (a)
bioavailable synaptic Cu may be critical for preventing NMDAR
excitotoxicity, and (b) pathological levels Cu-binding Ab oligomers
may act as a Cu sink, increasing glycine-mediated steady-state currents that contribute to neurotoxicity. Copper dependent coimmunoprecipitation of PrPC with the NR1 subunit of NMDAR,
which contains a glycine-binding site, suggests this is a direct
modulatory effect (You et al., 2012).
As a growing number of independent studies are implicating
roles for Cu and Zn in NMDAR kinetics, it is becoming clear that
we need to establish what effect metal-ionophores such as 8-OHQs
and BTSCs have on NMDAR stimulation in both physiological and
pathological situations. For example, PBT2 and Cu2+-gtsm have
been shown to effectively increase intraneuronal [Cu2+] and in
the case of PBT2 the source of Cu2+ can be extracellular synaptic
Ab. Although depletion of extracellular Cu from the synapse might
seem counter to the two proposed models of Cu-dependent neuroprotection via NMDAR modulation presented above, Cu2+ bound to
Ab would not be bioavailable to carry out this role. The usual cellular response to increasing cytoplasmic [Cu2+] is trafcking of
Cu2+-loaded ATP7a from the trans-Golgi network to the PM, where
it is implicated in Cu release (Petris et al., 1996). Hence, we propose
a model whereby PBT2 treatment in AD patients may not only increase intraneuronal [Cu2+] and activate Ab-degradation pathways,
but also promote the release of bioavailable Cu2+ via a stimulation
of the ATP7a trafcking pathway that has been implicated in protection from NMDAR excitotoxicity (Fig. 2).
Accumulating evidence indicates that metal ionophores such as
8-OHQs and BTSCs have the potential to provide therapeutic benet to AD patients. This approach to AD drug therapy is unique
in that the intended target is not a specic pathological target such

M.A. Greenough et al. / Neurochemistry International 62 (2013) 540555

549

Fig. 2. A proposed model of protection against NMDAR excitotoxicity mediated by NO and inhibited by extraneuronal Ab in AD. (A) Antagonism of NMDARs by the addition of
Glutamate/Glycine (Glu/Gly) causes a re-distribution of the intracellular copper exporter ATP7a to the plasma membrane (PM) where it releases Cu2+. Cu2+ then acts as an
electron donor in the conversion of NO to NO2- that can modulate NMDAR-mediated Ca2+ inux by S-nitrosylation at specic cysteine residues on NMDARs. This model is
based on experimental evidence using cultured mouse hippocampal neurons (Schlief and Gitlin, 2006; Schlief et al., 2006) (B) In AD, extraneuronal Ab oligomers can
potentially bind Cu2+, reducing its bioavailability to NO, resulting in excessive inux of Ca2+ by NMDARs. Alternatively, You and colleagues demonstrated that Cu, potentially
bound to PrPc, could reduce NMDAR afnity for glycine, and pathological Ab-Cu interaction could inhibit the proposed modulatory effect of PrPc on NMDAR stimulation (You
et al., 2012).

as reduction of Ab or phospho-Tau. Numerous failures of targeted

pharmaco- or immuno-therapies have highlighted the importance
of considering alternative strategies to combat AD, a disease of
ever-increasing prevalence due to aging populations worldwide.
Instead, it is the ability of metal ionophores to modulate potentially toxic oxidative interactions of Zn2+ and Cu2+ and activating
neuroprotective signaling cascades by restoring metal homeostasis
that makes them a more holistic approach to AD treatment.
11. Conclusion
Disturbances in the distribution of the Cu and Zn are central to
the aggregation pathology of Ab associated with AD. Additionally,
elevated levels of Cu and Fe at sites of Ab oligomer formation can
promote Ab:Cu2+ and Ab:Fe3+ interactions that lead to the generation of H2O2, increased levels of which are a feature of AD. The Metal Hypothesis of Alzheimers Disease has become the basis of
several drug discovery programs that have screened numerous metal-interacting compounds for efcacy in the treatment of AD in human and animal studies as well as cell culture models (Bush and
Tanzi, 2008). Some of these compounds, including PBT2, have been
very effective at reducing Ab pathology, increasing levels of markers of neuroprotection and improving memory and cognition in
AD animal studies. Clinical trials of PBT2 (and CQ) have also shown
promise. However, these trials have been limited to small numbers
of patients within short trial periods. To assess whether PBT2, or indeed any other metal-interacting compound, has the potential to
provide signicant therapeutic benet for the treatment of AD will
require much larger (and more costly) human trials. There has been
much debate over time as to whether AD patients have elevated,
normal or lowered levels of brain Cu, Zn or Fe. However, as described in this review, dysregulated Cu, Zn and Fe distribution is
more important than bulk tissue accumulation, and can contribute

to Ab-mediated oxidative stress and potentially Tau pathology. Further elucidation of these pathways, and what role metal ionophores
play in the activation of metal-mediated signaling cascades, will enable further renement of metal-based AD therapeutics.
Author declaration
A.I. Bush is a shareholder in Prana Biotechnology Ltd.
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