Progress in Neurobiology 92 (2010) 1–18

Contents lists available at ScienceDirect

Progress in Neurobiology
journal homepage: www.elsevier.com/locate/pneurobio

Biological metals and Alzheimer’s disease: Implications for therapeutics and

diagnostics
James A. Duce a,b, Ashley I. Bush a,c,*
a
The Mental Health Research Institute, Parkville, Victoria 3052, Australia
b
Centre for Neuroscience, The University of Melbourne, Victoria 3010, Australia
c
The Department of Pathology, The University of Melbourne, Victoria 3010, Australia

A R T I C L E I N F O A B S T R A C T

Article history: The equilibrium of metal ions is critical for many physiological functions, particularly in the central
Received 9 February 2010 nervous system, where metals are essential for development and maintenance of enzymatic activities,
Received in revised form 14 April 2010 mitochondrial function, myelination, neurotransmission as well as learning and memory. Due to their
Accepted 16 April 2010
importance, cells have evolved complex machinery for controlling metal-ion homeostasis. However,
disruption of these mechanisms, or absorption of detrimental metals with no known biological function,
Keywords: alter the ionic balance and can result in a disease state, including several neurodegenerative disorders
Alzheimer’s disease
such as Alzheimer’s disease. Understanding the complex structural and functional interactions of metal
Ageing
Copper
ions with the various intracellular and extracellular components of the central nervous system, under
Zinc normal conditions and during neurodegeneration, is essential for the development of effective therapies.
Iron Accordingly, assisting the balance of metal ions back to homeostatic levels has been proposed as a
Metal-protein attenuating compounds disease-modifying therapeutic strategy for Alzheimer’s disease as well as other neurodegenerative
diseases.
ß 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
2. Metals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
2.1. Toxicological metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
2.1.1. Lead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
2.1.2. Aluminium . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
2.2. Biochemically functional metals (biometals). . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
2.2.1. Copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
2.2.2. Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
2.2.3. Iron. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
3. Metal involvement with AD related proteins . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
3.1. b-amyloid precursor protein . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
3.1.1. APP binding to metal. . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
3.1.2. Regulation of APP by metal. . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
3.1.3. Metal involvement in secretase processing of APP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Abbreviations: Ab, b-amyloid; AD, Alzheimer’s disease; ADAM, cell surface metalloproteinase family of disintegrin and metalloprotease; APP, b-amyloid precursor protein;
ApoE, apolipoprotein E; ATP7a, ATPase, Cu2+ transporting, alpha polypeptide (associated with Menkes syndrome); ATP7b, ATPase, Cu2+ transporting, beta polypeptide
(associated with Wilson syndrome); BACE, b-APP-site cleaving enzyme; BBB, blood brain barrier; CCS1, copper chaperone of superoxide dismutase 1; CNS, central nervous
system; CQ, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline).; CSF, cerebral spinal fluid; Ctr1, high affinity copper uptake protein 1; Cu2+, oxidized copper; Cu+, reduced
copper; DFO, desferrioxamine; ERK, extracellular signal-regulated kinase; Fe3+, oxidized iron; Fe2+, reduced iron; GSK-3, glycogen synthase kinase 3; HFE, hemochromatosis
gene; HNE, 4-hydroxy-2-nonenal; H2O2, hydrogen peroxide; IDE, insulin-degrading enzyme; IRE, iron responsive element; JNK, c-Jun NH2-terminal kinase; MMP, matrix
metalloproteinase; MPAC, metal-protein attenuating compound; MT, metallothionein; NEP, neprilysin; NF, neurofilaments; NFT, neurofibrillary tangle; NMDA, N-methyl-D-
aspartic acid; PI3K, phosphoinositol-3-kinase; SOD1, superoxide dismutase 1; TACE, tumor necrosis factor-alpha converting enzyme (also called ADAM-17); ZIP, zinc-
importing protein; ZnT, zinc transporter.
* Corresponding author at: The Mental Health Research Institute, Parkville, Victoria 3052, Australia.
E-mail address: abush@mhri.edu.au (A.I. Bush).

0301-0082/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pneurobio.2010.04.003
2 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18

3.2. b-amyloid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
3.2.1. Ab binding to metal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
3.2.2. Ab production modulated by metal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
3.2.3. Metal modulation of Ab degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
3.2.4. Metal mediated production of reactive oxygen species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
3.3. Tau. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
3.3.1. Tau binding to metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
3.3.2. Metal modulation through kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
3.4. Apolipoprotein E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
4. Therapeutics and diagnostic targeting of metal ions in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
4.1. Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
4.2. Metal chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
4.3. Metal complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
4.4. Metal-protein attenuating compounds (MPACs) or Ionophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

1. Introduction 2.1.1. Lead

Since Alzheimer’s disease (AD) was first described over 100
years ago, it is now thought to affect 2% of the industrial world’s
population and is the third leading cause of death in these
countries (Mattson, 2004). As such, AD represents a huge
socioeconomic problem that requires better diagnostic tools,
management and effective therapies to ease the burden of this
disease. AD brains contain the characteristic pathological
markers of senile plaques and neurofibrillary tangles. Extracel-
lular senile plaques predominantly contain aggregated b-
amyloid (Ab), a small cleaved product from the b-amyloid
precursor protein (APP) (Iwatsubo et al., 1994), while neurofi-
brillary tangles consist of insoluble intra-neuronal inclusions
formed from hyperphosphorylated tau (Clark et al., 1998). The
major risk factor known for AD is age; 95% of all AD cases have no
clear pattern of inheritance and it is believed that interactions
with both genetic and environmental factors contribute to the
etiology of AD (Migliore and Coppede, 2009). However, since it
was first proposed decades ago (Crapper et al., 1973), there has
been an expanding interest in both biochemically functional and
toxicological metals as contributing to the etiology and
pathogenesis of AD.
2. Metals
Lead, seems to particularly affect the developing CNS.
Consequently, children are at a greater risk than adults in suffering
from the neurotoxic effects of lead. One of the basic mechanisms
proposed for leads neuronal toxicity is its substitution for calcium
in intra-cellular signal transduction. This substitution can affect
calcium influx, which is instrumental in the presynaptic release of
neurotransmitters. As early as the second century B.C., mankind
has related the use of lead with behavioural and CNS dysfunction.
However, with the inclusion of lead in paints and fuel, more
modern studies have directly associated lead with intellectual
impairment (Canfield et al., 2003).
Less well studied is the involvement of lead in AD. Recent
rodent and primate animal studies, however, have implicated a
LEARn (latent early-life associated regulation) alteration causing
an elevation of APP and Ab in old age (Basha et al., 2005b; Wu et al.,
2008) as well as increased oxidative stress (Wu et al., 2008). This
elevation is not related to the secretases as neither expression
profiles of the secretases were altered during the animals life post
infant exposure to lead, nor did lead have an effect on the
proteolytic activity of these enzymes on brain extracts. However,
this metal was still able to promote the formation of Ab aggregates
at nanomolar concentrations (Basha et al., 2005a) as well as cause
the accumulation of intracellular Ab and increased dense-core
plaques in the primate model (Wu et al., 2008).

Metals are widely distributed in nature and in biological 2.1.2. Aluminium

systems they can be broadly classified as either toxicological or
biochemically functional. As with many proteins, the mis-
metabolism of any metal ion within the body will result in
obtaining levels outside the normal physiological range and can
result in biological damage (Frausto da Silva and Williams, 2001). It
is also evident that metals play a crucial role in functioning of the
central nervous system (CNS).
2.1. Toxicological metals
Toxicological metals, such as mercury, cadmium, lead and
aluminium, have no known normal biological function and are
detrimental to any organism when absorbed. Interestingly, the
brain appears to be the primary target organ for such metals. While
biochemically functional metals are tightly homeostatically
regulated, non-essential toxicological metals may be able to freely
gain access to an organism by virtue of the fact that they share
properties such as ionic charge or other characteristics with their
biochemically functional counterparts.
Aluminium is the most widely distributed metal in the
environment and is extensively used in daily life, thus providing
easy exposure to human beings. The toxicity of aluminium is
directly linked to its bioavailability. In biological fluids, this
trivalent cation is rarely present as an ion because it complexes
extensively with biologically available ligands such as phosphate,
hydroxide and citrate. Because of its high affinity, aluminium was
used as a phosphate-binding gel in patients with chronic renal
failure to control hyperphosphotemia. It was in this population of
individuals that signs of aluminium neurotoxicity were first
recorded (Alfrey et al., 1976).
One of the first reports linking aluminium to AD demonstrated
that there is an elevated level of the metal in necropsy and biopsy
brain samples from histopathologically confirmed AD patients
(Crapper et al., 1973). However, the linkage between aluminium
and AD has remained controversial since, as a relationship
between increased aluminium exposure and AD has been found
in some studies (Xu et al., 1992) but not confirmed in others
(McDermott et al., 1979). Further suggestion that aluminium may
 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
play a role in the etiology of AD comes from it’s ability to increase;
oxidative stress that is induced by iron and other metals (Xie et al.,
1996), Ab production (Clauberg and Joshi, 1993), plaque deposi-
tion (Kawahara et al., 1994), b-pleated sheet formation (Vyas and
Duffy, 1995), as well as polymerization and aggregation of Ab
protein (Paik et al., 1997). But again controversy lies with many of
these studies as aluminium concentrations used are above those
that are physiologically relevant (for fuller review see (Gupta et al.,
2005; Kawahara, 2005)).
2.2. Biochemically functional metals (biometals)
The biochemically functional metal ion content of the brain is
stringently regulated and there is no passive flux of metals from
the circulation to the brain: movement of metals across the blood
brain barrier (BBB) is highly regulated. While iron, copper and zinc
are being increasingly implicated in interactions with the major
protein components of neurodegenerative disease, this is not
merely due to increased (e.g., toxicological) exposure to these
metals, but rather due to a breakdown in the homeostatic
mechanisms that compartmentalize and regulate metals.
Often termed as trace metals, these ions are neither trace in
concentration or irrelevant to functional biology. One estimate is
that 30% of enzymes use metals, however this percentage may be
greater as this estimate has not been evaluated systematically
since the publishing of complete genomes (Guengerich, 2009).
These ions participate in many essential activities and deficiencies
can be lethal. In general, these ions are bound to ligands (e.g.
transferrin or ceruloplasmin) and were for some time thought not
to be found as free species (Rae et al., 1999). However with the
advent of modern cellular and molecular techniques, ‘‘free’’ ionic
metals are now recognized in specific cellular compartments.
While it is unlikely that any ionic metal is truly free, this term is
used in this manuscript to explain the pool of labile, exchangeable
or loosely ligated metals that are accessible by chelator binding.
Here, we will focus on the three major biological transition
metals; copper, zinc and iron that make up a substantial
endogenous pool of transition metals within the brain. Both free
zinc and copper have been reported to act as secondary
messengers within the synaptic cleft and the intracellular pool
of labile iron has been well established to modulate the expression
of various proteins.
2.2.1. Copper
Copper is a redox-active metal that is predominantly used by
organisms living in oxygen-rich environments and exists either in
the oxidized (Cu2+) and reduced (Cu+) valence states (Ridge et al.,
2008). With these changes in redox state, copper can coordinate
with a range of ligands that include carboxylate oxygen, imidazole
nitrogen, cysteine thiolate, and methionine thioether groups and
engages in cation-interactions (Bertini, 2007; Franz, 2008). Copper
may also bind to amides from the backbone (e.g. prion) (Viles et al.,
1999) and side chains (e.g. glutamine in stellacyanin) (Hart et al.,
1996) of proteins. Many enzymes harness the changes in bound
copper oxidation state, in the presence of oxygen, to catalyze redox
chemistry for a wide range of chemical transformations that are
central to biology. Because the free forms of the metal are
potentially damaging, absorption, distribution and excretion are
tightly controlled, and orchestrated by a variety of proteins. In the
plasma, the major copper-binding proteins are ceruloplasmin,
albumin, and transcuprein, which transport the absorbed copper to
all tissues. Transport across cellular membranes is predominantly
through the copper transporters Ctr1 for import and ATP7a
(Menkes) or ATP7b (Wilson) for export. Once inside cells, cytosolic
copper chaperones deliver the metal to specific molecules (e.g.
copper chaperone of superoxide dismutase 1, CCS1, delivers to
3
superoxide dismutase 1, SOD1) (Tapiero et al., 2003). Significant
phenotypes are known to result from a diminution in these
enzymatic activities.
Recently the presence of the ionic species has been reported in
the synapse at micromolar concentrations. In this environment
postsynaptic N-methyl-D-aspartic acid (NMDA) neurites release
ionic copper upon NMDA activation (Schlief et al., 2005). Activation
of synaptic NMDA receptors in the hippocampal neurons results in
trafficking of ATP7a and an associated efflux of free copper (15 mM)
into the synaptic cleft (Fig. 1A) (Schlief et al., 2005). At present, this
is the only known microenvironment where free copper is known.
Catalytic amounts of copper can function as electron acceptors
promoting the reaction of nitric oxide with thiols, and potentially
the release of this metal could function as a molecular switch to
control extracellular S-nitrosylaton of the NMDA receptor, a post-
translational mechanism shown to be crucial for modulating
receptor function (Schlief et al., 2005). Copper has also been
reported by the same group to specifically protect against NMDA-
mediated excitotoxic cell death in primary hippocampal neurons,
and that this protective effect of copper depends on endogenous
nitric oxide production in hippocampal neurons (Schlief et al.,
2006). ATP7a expression is developmentally regulated, peaking
during synaptogenesis, and plays a role in facilitating copper
transport into the brain through the endothelial cells that comprise
the BBB (El Meskini et al., 2007; Niciu et al., 2007; Qian et al., 1998).
While the copper uptake protein, Ctr1, is present in neurons, the
specific mechanism involved in reuptake of copper released into
the synapses is still not yet known but is likely to be energy-
dependent (Giese et al., 2005). This is of relevance as mitochondrial
energy failure is a feature of ageing and AD and may lead to a
pooling to synaptic free copper.
2.2.1.1. Changes in copper homeostasis with ageing. As mentioned,
the dominant risk factor associated with all neurodegenerative
diseases is increasing age. Levels of copper have been reported to
change with age in plasma, cerebral spinal fluid (CSF) and brain
tissue. Levels of plasma copper are low at birth and increase
sharply after postnatal day 10, concomitantly with the predomi-
nant copper storage protein ceruloplasmin (Milne and Johnson,
1993). In subjects greater than 75 years of age copper has
repeatedly been reported as elevated in serum (Madaric et al.,
1994) and plasma (Ekmekcioglu, 2001; Iskra et al., 1993; McMaster
et al., 1992; Menditto et al., 1993; Milne and Johnson, 1993).
Conversely, several animal and human studies have reported a
rise in levels of brain copper from youth to adulthood. Analyses of
normal mice (BL6/SJL) have demonstrated a 46% increase in copper
content (Maynard et al., 2006, 2002). Interestingly, once in
adulthood a marked drop in levels from middle age onwards
occurs. However it is important to note that these gross changes in
copper may be regional specific as electron paramagnetic imaging
has demonstrated an age-related increase in clusters of copper
within the brain (Wender et al., 1992).
2.2.1.2. Changes in copper homeostasis in Alzheimer’s disease. Ho-
meostatic alterations in brain copper levels have been implicated
in the pathogenesis of several neurological disorders including
Alzheimer’s, Parkinson’s and prion diseases (Brown and Kozlowski,
2004; Mercer, 2001; Torsdottir et al., 1999). The serum and CSF
levels of copper are significantly higher in patients with AD
compared to age match controls (Basun et al., 1991; Squitti et al.,
2002a), and potentially correlates with ceruloplasmin protein
expression even though the excess pool of the metal is not bound
to this carrier protein (Squitti et al., 2005), however these findings
await replication. This increased serum copper was reported to
correlate well with higher levels of serum peroxides in AD patients
(Squitti et al., 2002b). Incidentally, copper mediates Low-density
[(Fig._1)TD$IG]
4 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
Fig. 1. Models of zinc and copper in the glutamatergic synapse in health and
Alzheimer’s disease. (A) The healthy synapse. The vesicular zinc transporter ZnT3
transfers zinc into synaptic vesicles. Upon stimulated release zinc concentrations
can achieve 300 mM within the synaptic cleft. Copper is released post-synaptically
following NMDA-induced activation, which causes the translocation of ATP7a and
its associated copper-laden vesicles to the synaptic cleft. Copper concentrations
may reach 15 mM in the cleft. Both copper and zinc are able to inhibit the NMDA
receptor response, which may feedback to prevent further copper from being
released into the cleft. Upon cleavage of APP, Ab is also constitutively released into
the synaptic cleft upon neuronal activity and may normally act as a ‘zinc sponge’ to
lower synaptic levels. Ab would typically be cleared by movement into the
periphery or degradation by extracellular proteases such as neprilysin and insulin
degrading enzyme (IDE). Despite high peak concentrations upon neuronal
stimulation, the average concentrations of free synaptic copper and zinc is kept
low over time by a variety of other ways including putative energy-dependent
reuptake mechanisms as well as buffering by metallothioneins (e.g. MT3) from
neighbouring astrocytes. (B) Alzheimer’s disease. Decreased mitochondrial energy
leads to reduced metal reuptake, which causes the average concentration of metals
to rise over time. This allows copper and zinc to react with Ab released into the
synaptic cleft to form oxidized, cross-linked soluble oligomers and precipitated
amyloid. Ab can bind up to 2.5 moles of metal ions, but becomes more densely
lipoprotein (LDL) oxidation by homocysteine (Nakano et al., 2004)
and plasma homocysteine levels are another risk factor for AD
(Seshadri et al., 2002).
Within the brain, copper has been shown to be associated with
senile plaques, significantly increasing copper content from 79 mM
(in the normal age-matched neuropil) to 390 mM within these
plaques (Lovell et al., 1998). Lovell and colleagues also went on to
report an increase in parenchymal copper levels (Lovell et al.,
1998), however their analysis was confined to the amygdala and
contradicts a larger collection of studies reporting a decrease in
bulk tissue copper levels in AD-affected cortical regions (Deibel
et al., 1996; Loeffler et al., 1996). Considering the increased copper
levels in senile plaques with the overall reduction in copper levels,
a complex picture emerges where copper seems to be abnormally
redistributed in AD and collects outside the cell, leaving the tissue
relatively deficient.
Interestingly, this abnormal redistribution of copper in the
brain may have a lipid component, as rabbits maintained on an
elevated cholesterol and copper diet demonstrated accelerated
plaque formation through aggregation of Ab and promotion of
oxidative events in the brain (Sparks and Schreurs, 2003). This is
supported by the observation that copper in combination with a
high fat diet increases the risk for AD (Morris et al., 2006).
2.2.2. Zinc
Zinc is another transition metal abundantly present in all
animal tissue. Diverse classes of proteins require bound zinc for
normal function, these include; zinc metalloenzymes (e.g. SOD1),
transcription factors containing zinc-binding motifs such as zinc
fingers (e.g. p53 and GAL4) (Vallee et al., 1991), signaling (e.g.
protein kinase C) (Szallasi et al., 1996) and storage to buffer
cytosolic zinc pools (e.g. metallothioneins) (Ebadi et al., 1994). Of
all organs, the brain probably contains the highest levels of zinc,
with a possible exception of pancreatic b islets (Frederickson,
1989). In its free ionic form zinc in the brain is highly enriched in
many of the glutamatergic nerve terminals (10–15%), where it is
released upon neuronal activation (Fig. 1A). Recent studies have
investigated in depth the mechanism involved in maintaining
intraneuronal zinc concentrations (for review see (Sensi et al.,
2009)). Although cytosolic free zinc is typically in the picomolar
range (Frederickson et al., 2005), this is substantially increased
within the synaptic bouton to micromolar concentrations. Upon
neuronal activity or membrane depolarization zinc is released and
the ionic concentration is further amplified to millimolar
concentrations within the synaptic cleft. As with copper (Section
2.2.1), synaptically released zinc interacts with neuronal receptors
such as NMDA (Smart et al., 2004) as well as various neuronal ion
channels and transporters. A multitude of zinc transporters (ZnTs),
aggregated as it becomes loaded with zinc. While the soluble Ab monomers are
constitutively degraded, zinc-loaded Ab oligomers are resistant to degradation.
MT3 is also decreased in AD, so promoting abnormal metal-Ab interaction and
sequestered metal ions by Ab allows unopposed glutamate activation of the NMDA
receptor, which could lead to the increased release of post-synaptic copper. (C)
Ionophore intervention. The metal-free ligands CQ and PBT2 enter the brain and are
attracted to pools of extracellular metals, a feature unique to AD. By binding to
copper and zinc and removing these metals from Ab, CQ and PBT2 facilitate the
dissolution of Ab to monomers. The dissociated metal ions may be in a ternary
complex with the drug itself or in a complex with dissociated Ab. These complexes
are taken up by neighbouring cells, where the elements are separated. While Ab
may be degraded by intracellular proteolysis and CQ/PBT2 is returned to the
extracellular space, the metal ions (Cu or Zn) activate PI3K and sets up a series of
kinase-mediated events. This leads to the phosphorylation and inhibition of GSK-3
resulting in decreased tau phosphorylation as well as the activation of MMP2 and 3,
which in turn facilitates the extracellular break down and clearance of Ab. In
addition, the ionophoric properties of CQ/PBT2 lower average synaptic metal
concentrations and so prevent Ab precipitation, covalent Ab oligomer formation,
and toxic redox activity.
 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
zinc-importing proteins (ZIPs) and buffering proteins such as the
metallothioneins bind cytosolic zinc to prevent free zinc becoming
toxic (Sensi et al., 2009). The zinc transporter ZnT3 has been shown
to be essential for loading zinc into synaptic vesicles (Cole et al.,
1999; Linkous et al., 2008) and is likely to represent the major,
possibly sole, synaptic vesicle zinc transporter. ZnT3 is primarily
co-localized with zinc in glutamatergic synapses (Palmiter et al.,
1996) present in the hippocampus and neocortex. ZnT3 is likely to
govern the downstream effects of synaptic zinc on a variety of
signaling pathways that mediate higher cognitive function.
2.2.2.1. Changes in zinc homeostasis with ageing. In contrast to
copper, zinc levels in rodent and human plasma has been reported
to be highest at birth and steadily decrease with age (Bunker et al.,
1987; Martinez Lista et al., 1993; Monget et al., 1996; Munro et al.,
1987; Ravaglia et al., 2000). One report on healthy men aged 8–89
years indicated that plasma zinc levels tended to remain constant
throughout life until the age of 75 where zinc levels suddenly
decreased (Madaric et al., 1994).
While these changes have not reflected an age-related
alternation in the global zinc levels within the brain (Maynard
et al., 2002), specific areas known to have high concentrations of
zinc such as the hippocampus, are known to decrease with age
(Adlard et al., 2010) In accordance, ZnT3 is also decreased with age
in the same area (Adlard et al., 2010).
2.2.2.2. Changes in zinc homeostasis in Alzheimer’s disease. The
presence of zinc ions in both plasma and CSF have been reported to
further decrease in AD compared to age-matched control patients
(Basun et al., 1991; Baum et al., 2010; Molina et al., 1998). While
nutritional deficiency is common in advanced age, this further
decrease in AD zinc levels may in turn affect brain levels of the ion
and exaggerate amyloid pathology, through it’s entrapment within
the plaque. As previously described with copper, zinc is highly
enriched within AD plaques (1055 mM) compared to normal age-
matched neuropil (350 mM) (Lovell et al., 1998). Histochemically
reactive zinc deposits are also found specifically localized to
cerebral amyloid angiopathy deposits and neurofibrillary tangle
(NFT)-bearing neurons (Friedlich et al., 2004; Suh et al., 2000).
Dyshomeostasis of zinc in the AD brain may arise from
inhibition of zinc export by 4-hydroxynonenal, a peroxidation
product of Ab:copper redox activity (see Section 3.2.4) that is
elevated in AD tissue (Smith et al., 2006b). In addition, rodent
studies have indicated that systemic zinc deficiency increases
brain zinc retention (Takeda et al., 2001) by suppressing levels of
the intracellular zinc export promoter ZnT1 (Chowanadisai et al.,
2005). Indeed, levels of ZnT1, ZnT4 and ZnT6 (Lyubartseva et al.,
2009) as well as ZnT3 (Adlard et al., 2010), are all altered in AD
brain tissue. Interestingly, estrogen can also modulate levels of
ZnT3 and thus synaptic zinc (Lee et al., 2004b), this is of particular
significance as sex is another major risk factor for AD.
2.2.3. Iron
With the exception of a few bacterial species that use
manganese, all other forms of life are iron dependent for a variety
of essential biochemical processes. It is a vital component of the
heme in hemoglobin, myoglobin, and cytochromes and is also an
essential cofactor for non-heme enzymes such as ribonucleotide
reductase, the limiting enzyme for DNA synthesis. When in excess,
iron is toxic because it generates superoxide anions and hydroxyl
radicals that react readily with biological molecules, including
proteins, lipids, and DNA. As a result, humans possess elegant
control mechanisms to maintain iron homeostasis by coordinately
regulating iron absorption, iron recycling, and mobilization of
stored iron. Disruption of these processes causes either iron-
deficient anemia or iron overload disorder (Zhang and Enns, 2009).
5
An average adult brain has approximately 60 mg of non-heme
iron, with some cerebral regions such as the substantia nigra,
globus palladus, caudate nucleus and putamen retaining the
highest levels (Bartzokis et al., 1997; Martin et al., 1998). In the
brain iron is not only important for oxygen transport and electron
transfer but also neurotransmitter synthesis and myelin produc-
tion (Takeda, 2004). Iron transport into the brain across the BBB is a
complex process still not fully understood, but requires the iron
carrier transferrin. Circulating iron, once oxidized to its ferric state
using a ferroxidase (e.g. ceruloplasmin) cannot simply cross the
BBB (Morris et al., 1992) but requires an iron complexed with
transferrin to bind the transferrin receptor on the lumen side of
endothelial cells lining the cerebrovasculature. The transferrin
complex then enters the cells via endocytosis and is transported
further into the brain to be distributed to various cell types that
require the ion. This process is highly regulated by the abundance
or the deficiency in both transferrin (with or without iron
incorporated) and its receptor. Translational regulation of these
proteins as well as other iron binding proteins (e.g. ferritin) is
controlled by iron regulatory proteins that bind to iron responsive
elements (IRE) on RNA to alter the expression levels (Caughman
et al., 1988; Kikinis et al., 1995).
2.2.3.1. Changes in iron homeostasis with ageing. In the healthy
state, there should never be an over-accumulation of free iron.
Unfortunately, although humans have an intricate mechanism for
controlling intestinal absorption of iron, they lack a mechanism
(other than bleeding) for elimination of grossly excessive
quantities. In the developed world, accumulation of excess iron
in a number of tissues from males can begin in early adulthood and
then increase almost linearly with age. Females delay over-
accumulation through menstruation and/or pregnancy, but
postmenopausal women attain parity with men in iron burden
within a few decades. In contrast, plasma levels of iron are reduced
with age and likewise, so are iron-containing proteins such as
hemoglobin (Ahluwalia et al., 2000).
Age-related increases in brain iron have been well documented
in all species examined, including humans (Bartzokis et al., 1994;
Hallgren and Sourander, 1958), non-human primates (Hardy et al.,
2005) and rodents (Maynard et al., 2002; Roskams and Connor,
1994; Sohal et al., 1999; Suh et al., 2005). This accumulated iron is
also evident with increased ferritin expression; thought to occur as
an attempt to store surplus intracellular free iron (Connor et al.,
1990, 1995; Zecca et al., 2001). As with copper, these changes may
be regional as electron paramagnetic imaging has shown that age-
related increases in iron are clustered (Wender et al., 1992).
2.2.3.2. Changes in iron homeostasis in Alzheimer’s disease. The
increase in iron with AD associated senile plaques was first
described by Goodman in 1953 (Goodman, 1953). Since then his
observation has been repeatedly reinforced by others and
validated with the use of various specific and sensitive techniques
(Bishop et al., 2002; Collingwood and Dobson, 2006; Collingwood
et al., 2005; Connor et al., 1992a,b). Within AD plaques, iron
accumulates to nearly 3 times that of normal neuropil levels
(940 mM compared to 340 mM) (Lovell et al., 1998). Deregulation
of brain iron metabolism is multifactorial and comprises non-
genetic and genetic factors. It may also occur at multiple levels,
including iron uptake and release, storage, intracellular metabo-
lism and regulation (Ke and Qian, 2007; Zhu et al., 2007).
The increased iron is found primarily complexed with ferritin in
the plaque-associated neuritic processes (Grundke-Iqbal et al.,
1990) and within neurons and neurofibrillar tangles (Bouras et al.,
1997; LeVine, 1997; Morris et al., 1994). While ferritin levels are
increased in AD, transferrin (involved in the physiological
transport and utilization of iron concentration) is decreased
6 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
(Connor et al., 1992b). Oxidative stress, potentially involving iron
redox state, is one of the earliest events in AD and seems to be
involved in the onset, progression and pathogenesis of the disease
(Castellani et al., 2007; Zhu et al., 2007). Disruption in brain iron
homeostasis through alterations of iron regulatory proteins can
increase the vulnerability of cells to oxidative stress (Connor et al.,
1992b; Pinero et al., 2000). Changes in superoxide levels due to
alteration of SOD1 activity also affect iron metabolism in glial and
neuronal cells (Culotta et al., 2006; Danzeisen et al., 2006).
Lactoferrin, upregulated in AD, exerts an anti-inflammatory
function via its inhibitory effect on hydroxyl radical formation
and, through its anti-oxidative properties, prevents DNA damage
(Sacharczuk et al., 2005).
Several genetic studies on increased risk factors of AD
demonstrate involvement in the genotype of the hemochromatosis
gene (HFE), (known to cause iron overload in hemochromatosis),
and the transferrin C2 allele (Bertram et al., 2007; Robson et al.,
2004). The combination of the C2 variant of the transferrin gene
(TF-C2) and the C282Y allele of HFE gene (HFE C282Y), or the
combination of HFE C282Y and HFE H63D are risk factors for
developing AD. Carriers of such combination were at five times
greater risk for AD. The addition of the apolipoprotein E epsilon4
allele further increases the risk of AD (Connor and Lee, 2006;
Lehmann et al., 2006; Namekata et al., 1997). Brain ferritin iron
may also influence age- and gender-related risk of neurodegenera-
tion as there is a high correlation between brain ferritin iron levels,
measured in vivo by MRI, and age (Bartzokis et al., 2007). Jefferies
and colleagues showed that the transferrin homologue; melano-
transferrin (p97), that binds iron through a high affinity iron-
binding site, could be used as a potential early detection biomarker
for AD (Jefferies et al., 2001; Ujiie et al., 2002).
Interestingly, as with copper, elevated levels of cholesterol and
iron in combination have been observed to promote an AD-like
phenotype in transgenic animals (Ghribi et al., 2006) and to be a
risk factor in humans (Mainous et al., 2005). In a set of 6558 US
adults, followed from 1974 to 1992, an elevated risk of AD occurred
if both dietary cholesterol and iron were above normal (Mainous
et al., 2005).
3. Metal involvement with AD related proteins
3.1. b-amyloid precursor protein
3.1.1. APP binding to metal
There are putative metal binding sites for copper (Atwood et al.,
2000; Barnham et al., 2003; Hesse et al., 1994; Simons et al., 2002;
Valensin et al., 2004) and zinc (Bush et al., 1993, 1994a,b,c) in the
APP sequence. Copper binding sites are located at the amino-
terminal ectodomain of APP and also within the Ab sequence of
APP (described further in Section 3.2.1). The structure of the APP
copper binding domain consist of four ligands (His-147, His-
151,Tyr-168 and Met-170 (Barnham et al., 2003), which show
structural homology to other copper chaperones.
The binding of zinc to APP involves a conserved region of amino
acids between position 170 and 188 of APP (Bush et al., 1993,
1994a). This domain consists of two key cysteine ligands at
position 186 and 187 which are crucial to the binding, as well as
other potential ligands (e.g. Cys-174, Met-170, Asp-177 and Glu-
184) (Ciuculescu et al., 2005). The binding of zinc may have an
important functional role in the dimerization of APP (Ciuculescu
et al., 2005), which could occur in vivo (Scheuermann et al., 2001).
3.1.2. Regulation of APP by metal
APP is a ubiquitously expressed protein that potentially
functions to participate in metal ion homeostasis. There are a
number of in vivo studies that highlight the sensitivity of APP to
copper levels. Modulation of copper levels in fibroblasts from mice
with either chronic copper overload (caused by an over expression
of a copper efflux protein in ‘Mobr’ and ‘Modap’ mice) or copper
deficiency (caused by a mutation in the copper transporter, ATP7a),
can both up- and down-regulate APP mRNA expression (Armen-
dariz et al., 2004; Bellingham et al., 2004b). A study of healthy
postmenopausal women (n = 25) that consumed either a low
(1 mg/d) or a high (3 mg/d) copper content diet (also supplemen-
ted with zinc) confirm that a low copper diet was associated with a
significant decrease in APP expression in platelets (Davis et al.,
2000).
Conversely, both in vivo and in vitro studies in APP and b-
amyloid precursor-like protein 2 knockout mice have demonstrat-
ed that there are significant increases in copper levels in brain and
liver tissue as well as cortical neuron and fibroblast primary
cultures derived from these animals (Bellingham et al., 2004a;
White et al., 1999). Furthermore, the over expression of APP in
various transgenic mouse lines carrying a mutated APP gene
decreased copper levels both in vivo and in vitro at ages varying
from 2.8 to 18 months of age (Bayer et al., 2003; Maynard et al.,
2002; Phinney et al., 2003).
In addition to regulation by copper, APP translation is also
mediated by the IRE type II present in its 50 untranslated region
(Venti et al., 2004). The APP mRNA IRE is located immediately
upstream of an interleukin-1 responsive acute box domain (+101
to +146). APP 50 -UTR conferred translation was selectively down
regulated in response to intracellular metal chelation and this is
reversed by iron influx (Rogers et al., 2002). Iron regulation of APP
mRNA through the APP 50 -UTR points to a role for iron in the
metabolism of APP.
3.1.3. Metal involvement in secretase processing of APP
Metals may also indirectly affect APP by altering APP
processing. The processing of APP is well documented and involves
a number of activities by alpha-, beta- and gamma-secretase (for
review see (Gandy, 2005; Ling et al., 2003; Suh and Checler, 2002)).
Candidate alpha-secretases, tumor necrosis factor-alpha convert-
ing enzyme (TACE or ADAM-17) and ADAM-10 (Buxbaum et al.,
1998), are members of the cell surface metalloproteinase family of
disintegrin and metalloprotease (ADAM) proteins. Beta-secretase
has been identified as a membrane anchored aspartyl protease
termed b-APP-site cleaving enzyme or BACE (Cai et al., 2001; Luo
et al., 2001; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999)
while the high molecular weight active gamma-secretase complex
is comprised of at least four components, presenilin, APH-1, PEN-2
and nicastrin (De Strooper, 2003). Beta- and gamma-secretase
result in a hierarchical and site-specific cleavage of APP to generate
Ab peptides of varying length. All three secretases are now known
to have interactions with different metal species.
The alpha-secretase, TACE, is a multidomain protein that
contains a zinc ion within its catalytic domain (Cross et al., 2002).
Following TACE synthesis, it is maintained in an inactive state
mediated by an intramolecular bond between a cysteine in the
prodomain and a zinc atom in the catalytic site (referred to as the
‘‘cysteine-switch’’ motif) of the protein. It has subsequently been
shown, however, that TACE enzymatic activity can be mediated
independently of this classic cysteine-switch motif, such that other
regions of the prodomain can interact with the catalytic domain
(and the zinc ion) to inhibit enzymatic activity (Buckley et al.,
2005; Gonzales et al., 2004). Similarly, the activity of ADAM-10 can
be inhibited by a dominant negative form of this candidate alpha-
secretase that has a point mutation in its zinc-binding site
(Lammich et al., 1999). Iron may also alter APP cleavage by
modulating furin, a proconvertase involved in the regulation of
alpha-secretase-dependent processing (Silvestri and Camaschella,
2008).
 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
BACE1 binds copper in its C-terminal domain (via cysteine
linkages) and co-immunoprecipitates in brain homogenate with
the copper chaperone CCS (Angeletti et al., 2005). BACE1 interacts
with domain I of CCS (the domain that mediates copper binding)
and may directly compete with SOD1, as BACE1 expression reduces
the activity of SOD1. Overexpression of CCS in cells already
overexpressing BACE1 will restore SOD1 activity (Angeletti et al.,
2005). Accordingly, both BACE1 and CCS are transported together
through the axon (Angeletti et al., 2005). Endocytosis of APP to
BACE1-rich endosomes is required to initiate b-cleavage of APP
(Vassar et al., 1999), and this event is sensitive to flotillin-2 and
cholesterol depletion; both of which are known to be enriched
within lipid rafts along with a subset of cellular APP (Ehehalt et al.,
2003; Schneider et al., 2008). It has recently been observed that
elevated copper reduced flotillin-2 association with lipid rafts and
thereby reduced the endocytosis of APP via lipid rafts (Hung et al.,
2009).
Presenilin, a subunit of the gamma-secretase complex, is also
sensitive to metal levels. Neonatal mouse cortical cultures have
shown that exogenous zinc administration (but not copper or iron)
increases the C-terminal fragments of presenilin 1 by enhancing de
novo synthesis of the protein (Park et al., 2001). More recent data
suggests that zinc chloride causes the oligomerisation of an APP
gamma-secretase substrate and inhibits its cleavage in vitro,
supporting a role for zinc dysregulation in abnormal Ab processing
(Hoke et al., 2005).
A recent report determined APP from human fibroblasts to be
processed simultaneously by both alpha- and beta-secretase, as
copper-deficient fibroblasts secrete beta-secretase cleaved soluble
APP N-termini but also produce alpha-secretase cleaved APP C-
termini (C83) (Cater et al., 2008). Copper deficiency in these cells
also markedly reduced the steady-state level of APP mRNA while
APP protein level remained constant, indicating that copper
deficiency may accelerate APP translation (Cater et al., 2008).
This may be cell-type specific though as copper deficiency in
human neuroblastoma cells did not affect the cleavage of APP
(Cater et al., 2008).
Together these data demonstrate that there may be a direct
interaction of metals on the secretases and, therefore, also on APP
processing. In addition, the zinc binding domain in the Ab region of
the APP sequence spans the alpha-secretase cleavage site and may,
therefore, modulate the cleavage of Ab from APP and also protect
Ab from proteolytic degradation (Bush et al., 1994b).
3.2. b-amyloid
3.2.1. Ab binding to metal
Ab is rapidly precipitated by zinc over a broad pH range (pH
5.5–7.5) (Atwood et al., 1998; Bush et al., 1994b,c; Yoshiike et al.,
2001), including physiological pH (pH 7.4) (Cherny et al., 1999),
whereas aggregation of Ab by copper and iron is only induced
under mildly acidic conditions (pH 6.8–7.0) (Atwood et al., 1998,
2000; Ha et al., 2007). The Ab affinity for copper is well below
physiological concentrations achieved within the brain, and is
reported to be as high as attomolar for aggregated Ab1–42 (Atwood
et al., 2000), although some reports have lower apparent affinities
in the submicromolar range for soluble Ab (Garzon-Rodriguez
et al., 1999; Syme et al., 2004). The differences between reported
affinities are likely due to the different techniques used to
determine these values and to the conditions (e.g. buffer) of the
experiments (this is covered in more detail by (Faller and Hureau,
2009; Rozga and Bal, 2009)). The affinity for zinc is also in the low
micromolar range (Bush et al., 1994c; Huang et al., 1997). These
levels are of particular relevance to neurotransmission as such
concentrations of free ionic zinc and copper are released at the
synapse (Lee et al., 2002; Schlief et al., 2005) (see Sections 2.2.1 and
7
2.2.2) and may account for the synaptic localization of Ab
precipitation (Fig. 1B) (Terry, 1996). Iron will also precipitate Ab
under acidic conditions, although only copper and zinc have been
shown to be specifically associated with senile plaques and to
copurify with Ab extracted from the human brain (Dong et al.,
2003; Opazo et al., 2002). Of note, these affinities for metals are so
low that the concentrations of zinc, copper and iron present in
common laboratory buffers are sufficient to induce nucleation of
Ab, leading to accelerated aggregation of the peptide (Huang et al.,
2004).
There are up to 2.5 metal binding sites on Ab, possibly
coordinated by oligomers (Atwood et al., 2000). Binding of the
oxidized forms of both zinc and copper (Atwood et al., 2000; Bush
et al., 1994c; Danielsson et al., 2007; Karr and Szalai, 2008; Syme
et al., 2004; Syme and Viles, 2006) as well as the reduced form of
copper (Himes et al., 2008; Hureau et al., 2009; Shearer and Szalai,
2008) to Ab in vitro (either in aqueous solution or membrane
mimetic environments) is mediated by nitrogen ligands from the
three histidine residues at positions 6, 13 and 14, along with an
oxygen ligand (Curtain et al., 2001, 2003). Rat/mouse Ab
(containing amino acid substitutions for histidine at position 13,
arginine at position 5 and tyrosine at position 10) have significant
structural changes in the metal ion coordination chemistry
compared to human Ab (Gaggelli et al., 2008). This supports
histidine at position 13 as having a predominant role in metal
binding and may be responsible for the lower aggregation
propensity of rat Ab when complexed to copper or zinc. It may
also explain why these animals are exceptional among mammals
for not forming cerebral Ab deposits with age (Vaughan and Peters,
1981). However, a role for the two other substitutions in the rat
sequence in aggregation cannot yet be excluded.
The oxygen ligand for metal coordination is most likely from the
aspartate at position 1 (Dorlet et al., 2009; Drew et al., 2009b;
Mekmouche et al., 2005) and alanine at position 2 (Dorlet et al.,
2009; Drew et al., 2009a). While other oxygen ligands have been
proposed, such as tyrosine at position 10, glutamate at position 11
or H2O, these have all been contested (Karr and Szalai, 2007; Syme
et al., 2004), particularly as peptides lacking the first one to three
amino acids do not bind copper in the same fashion as the native
peptide (Karr et al., 2005). Recent in vitro analysis of the copper-
binding domain in the 3–42 truncated Ab (Ab3–42) sequence has
demonstrated that it self-aggregates more rapidly than Ab1–42 and
is a more potent initiator of Ab1–40 aggregation, particularly in the
presence of copper (McColl et al., 2009). Clearly, while the
coordination of metal in Ab is generally accepted as a square-
planar configuration, involving three histidine imidazoles, the
fourth ligand is still yet to be fully elucidated. More detailed
reviews of the metal binding chemistry of Ab have been recently
published (Faller and Hureau, 2009; Rauk, 2009; Rozga and Bal,
2009).
Ab binds equimolar amounts of copper and zinc at pH 7.4, but
under the conditions of acidosis that are thought to occur in the AD
brain (pH 6.6), copper displaces zinc from Ab (Atwood et al., 2000).
As copper has been shown to promote the aggregation of Ab in
vitro and Ab is known to bind copper in AD brain (Dong et al., 2003;
Lovell et al., 1998; Opazo et al., 2002) it may seem surprising that
increasing brain copper levels in transgenic models of the disease
would inhibit Ab pathology (see Section 3.2.2). However, this is
consistent with evidence that copper abnormally redistributes in
AD and collects outside of the cell, leaving the tissue relatively
deficient. This would also go someway to explain the deficiency of
copper-dependent enzymatic activities in AD such as cytochrome c
oxidase (Cardoso et al., 2004; Cottrell et al., 2001; Maurer et al.,
2000; Sullivan and Brown, 2005) and SOD1 (De Deyn et al., 1998;
Omar et al., 1999), as well as the reported elevation of serum
copper (Squitti et al., 2002a, 2005).
8 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
3.2.2. Ab production modulated by metal
In order to investigate if metal-mediated modulation of APP
processing could result in the observed physiological change in Ab
generation, a number of studies have been carried out to examine
the effect copper and zinc may have on the brain from APP
transgenic models. One of the simplest ways to do this is by dietary
supplementation. Copper given to APP23 mice (overexpressing
human APP with the AD-related Swedish mutation) (Bayer et al.,
2003) for 3 months was sufficient to significantly increase copper
levels within the brain compared to non-supplemented APP23
littermates and this was complemented with a normalization in
activity of the copper-dependent enzyme; SOD1. Relative to Ab,
the increase in bioavailable copper in these mice resulted in a
lowering of soluble and insoluble Ab (Bayer et al., 2003). Dietary
zinc has also been administered to 2 transgenic models of AD, the
Tg2576 (overexpressing human APP with the AD-related Swedish
mutation) and TgCRND8 (overexpressing human APP with the AD-
related Swedish and Indiana mutations). The provision of a zinc-
enriched diet also reduced amyloid-beta plaque deposits, but
potentiated Alzheimer-like spatial memory impairments (Linkous
et al., 2009). Correspondingly, lowering dietary zinc in another
transgenic model of AD caused a significant increase in plaque
volume (Stoltenberg et al., 2007).
Similar copper related observations were described with the
TgCRND8 when crossed with a transgenic model containing the
‘toxic-milk’ mutation in the gene encoding the copper transport
protein, ATP7b (Phinney et al., 2003). The mutation in ATP7b
causes the inability of copper to be loaded into secretory vesicles
resulting in an accumulation of intracellular copper. TgCRND8
mice with the ATP7b mutation were confirmed to have elevated
copper within the brain and demonstrated a decrease in amyloid
plaque content as well as the soluble and insoluble Ab levels
suggesting the intracellular shift in copper reduces Ab aggregation.
Plasma levels of Ab were also reduced, including the endogenous
murine Ab content, confirmed a decreased extracellular Ab
content.
As previously explained, the highest pool of free zinc is present
in synaptic vesicles that are released during synaptic transmission
of neocortical glutamatergic fibers (Quinta-Ferreira and Matias,
2005). The activity of the zinc transporter ZnT3 is required for the
passage and pooling of zinc within these pre-synaptic vesicles. The
genetic ablation of ZnT3 in the Tg2576 mouse causes a profound
reduction in the cerebral plaque load (Lee et al., 2002). Further-
more, there is a depletion of an exchangeable zinc pool in the
cerebrovascular wall, which is also associated with a significant
reduction in cerebral amyloid angiopathy (Friedlich et al., 2004). It
is the high concentration of synaptically released zinc in the
synaptic cleft that is believed to play an essential role in amyloid
formation in transgenic models for AD (Fig. 1B).
3.2.3. Metal modulation of Ab degradation
The involvement of metal ions in the aggregation of Ab within
the brain only represents one part of the biological equation, as
metal ions may also participate in the clearance/degradation of Ab
(for review on Ab catabolism, see (Carson and Turner, 2002; Ling
et al., 2003)). Two of the primary Ab degrading enzymes identified
are insulin-degrading enzyme (IDE) and neprilysin (NEP), a neutral
endopeptidase. IDE, which degrades a variety of substrates (e.g.
insulin and amylin) including Ab, is ubiquitously expressed and
localized intra- and extracellularly within the CNS. With the use of
mice deficient in either enzyme, Ab catabolism has been shown to
significantly decrease resulting in an increase of endogenous Ab
(Farris et al., 2003; Iwata et al., 2001). It has subsequently been
demonstrated that NEP is capable of degrading both monomeric
and oligomeric forms of Ab1–40 and Ab1–42 (Kanemitsu et al.,
2003). Conversely, the transgenic overexpression of either IDE or
NEP is sufficient to reduce Ab levels, slow or completely prevent
plaque formation and extend lifespan (Leissring et al., 2003).
Both these proteases are members of the zinc metallopeptidase
family of proteins that share a common primary structure in their
sequence that is involved in the binding of zinc. This structure
possesses the short consensus sequence (HEXXH) in which the two
histidines act as the ligand for the catalytic zinc. Some family
members may also have an elongated C-terminal motif
(HEXXHXXGXXH/D) in which there is an additional zinc-binding
histidine (or aspartate) (Gomis-Ruth, 2003). This potentially
means that both IDE and NEP activities could be influenced by
abnormal zinc metabolism. In addition, it has recently been
demonstrated that both enzymes are oxidatively modified in the
AD brain (Caccamo et al., 2005; Shinall et al., 2005; Wang et al.,
2003). There is a concentration dependent loss in activity of both
IDE and NEP upon treatment with hydrogen peroxide. More
specific treatment with various reactive oxygen species differen-
tially affected the activity of NEP and IDE. Likewise, treatment with
4-hydroxy-2-nonenal (HNE), which occurs as a function of
oxidative stress and is augmented in the AD brain, also
differentially affected the activity of these metallopeptidases.
These data suggest that the generation of reactive oxygen species,
perhaps as a product of metal interactions with Ab (see Section
3.2.4), may serve to inactivate the principal degradative enzymes
involved in the regulation of Ab.
An alternative potential mediator of Ab catabolism is plasmin.
Activation of plasmin can be induced by Ab through the
upregulation of its precursor, tissue-plasminogen activator (tPA)
(see (Ling et al., 2003)). While plasmin is not a zinc-dependent
enzyme in the same fashion as NEP and IDE, it has been
demonstrated that it too may be regulated by site-specific
oxidation, perhaps via a modification of the histidine residue in
its active site (Lind et al., 1993). Furthermore, inhibition of
substrate cleavage by tissue-plasminogen activator occurred when
co-incubated with copper and ascorbate (Lind et al., 1993). Lastly,
the tissue-plasminogen activator/plasmin system may also be
involved in the activation of matrix metalloproteinases (MMPs),
another family of zinc-dependent enzymes particularly relevant to
the degradation of extracellular Ab. Recent in vitro studies have
shown that MMP2 can produce site specific degradation of Ab that
then becomes inaccessible when the peptide is incubated with
zinc, but not copper, to form amyloid (Crouch et al., 2009b) (also
see Sections 4.3 and 4.4 and (Yong et al., 1998) for review).
Together these studies provide considerable evidence that a
mis-metabolism of metal ion homeostasis is sufficient to
significantly alter the normal course of APP processing and Ab
generation, deposition and degradation in biologically relevant
animal models. These animal studies are supported by the wealth
of in vitro data characterizing the interactions between metal ions
and Ab.
3.2.4. Metal mediated production of reactive oxygen species
Overwhelming evidence in the literature describes oxidation
injury as a major feature of AD. While under certain in vitro
conditions Ab has been shown to act as an antioxidant (Baruch-
Suchodolsky and Fischer, 2009; Nadal et al., 2008; Shearer and
Szalai, 2008) with neurotrophic and neuroprotective effects
(Whitson et al., 1989), compelling evidence has shown that the
redox activity for variants of Ab is greatest for Ab1–42 human > Ab1–
40 human » Ab1–40 mouse  0 (Huang et al., 1999a), corresponding to
the potentiation of Ab toxicity by copper (Huang et al., 1999b;
Opazo et al., 2002) and the prevalence of Ab1–42 over Ab1–40 in
both sporadic and familial AD. Such studies have shown the
coordination of either oxidized copper (Cu2+) or iron (Fe3+) to Ab
result in the reduction of the metal ions and is the subject of a
focused recent review by Hureau and Faller (2009). Ab’s redox
 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
activity leads to the generation of reactive oxygen species (ROS),
when not accompanied by the oxidation of another moiety,
(Puglielli et al., 2005) and the production of H2O2, in the presence
of biological reducing agents used as electron donors (Dikalov
et al., 2004; Haeffner et al., 2005; Murray et al., 2005; Nelson and
Alkon, 2005; Opazo et al., 2002; Puglielli et al., 2005; Smith et al.,
2006a; Tabner et al., 2002). Possible biological reductants include
cholesterol, long-chain fatty acids, 3,4-dihydroxyphenylalanine
(DOPA), dopamine quinone, dihydroxyindol, isodityrosine and
ascorbate (Dong et al., 2003; Haeffner et al., 2005; Huang et al.,
1999a; Jiang et al., 2007; Murray et al., 2005; Nelson and Alkon,
2005; Opazo et al., 2002; Puglielli et al., 2005; Smith et al., 2007;
Yoshimoto et al., 2005). This can lead to a variety of Ab side-chains
being oxidized, and to covalent oligomerisation. One likely
candidate from the copper-mediated redox reaction is the sulphur
atom of methionine-35 becoming methionine sulfoxide (Ali et al.,
2005; Ciccotosto et al., 2004), however this remains in doubt as it
has also been deemed unnecessary for toxicity (da Silva et al.,
2009; Hou and Zagorski, 2006). Other adducts can also be
generated, including aldehyde adducts to the lysine residues
(Chen et al., 2007) and the tyrosine at position 10 (Barnham et al.,
2004; Haeffner et al., 2005).
Tyrosine at position 10 is particularly susceptible to free radical
attack, due to the conjugated aromatic ring, and in the presence of
Cu2+ and H2O2 forms dityrosine cross-linked oligomers (Atwood
et al., 2004). Both dityrosine and 3-nitrotyrosine are elevated
within the neuronal lesions in AD brain (Hensley et al., 1998). The
formation of dityrosine linkage in Ab cannot be proteolytically
degraded (Atwood et al., 1998) and facilitates further peptide
aggregation, leading to the formation of higher order oligomers
(Barnham et al., 2004). In addition, Ab radicals formed after
reduction of copper can form covalent adducts onto other proteins.
Peroxidases like cycloxygenase 2 (COX2) are particularly vulnera-
ble because of the formation of dityrosine bridges, and cyclox-
ygenase 2:Ab covalent complexes are elevated in AD brain
(Nagano et al., 2004). 2-oxo-histidine adducts of Ab have also
been extracted from AD plaques (Metodiewa, 1998) as have N3-
pyroglutamate modified forms of Ab that are the main ligands for
the amyloid positron emission tomography (PET) ligand Pittsburgh
Compound-B (PIB) (Maeda et al., 2007).
The byproduct H2O2 is also freely permeable across all tissue
and cellular boundaries, and can thus further react with reduced
metal ions (Fe2+, Cu+) to generate hydroxyl radicals via Fenton
chemistry. This in turn generates lipid peroxidation products such
as HNE, protein carbonyl modifications, and nucleic acid adducts
(e.g. 8-OH guanosine), all of which typify AD neuropathology
(Haeffner et al., 2005; Jiang et al., 2007; Markesbery and Lovell,
2007; Murray et al., 2007; Nelson and Alkon, 2005; Opazo et al.,
2002; Puglielli et al., 2005; Smith et al., 2006a, 1996, 1997). In AD,
the H2O2 scavenging defenses, e.g., catalase and glutathione
peroxidase, are likely to be taxed by the catalytic generation of
H2O2 from the Ab:metal complexes.
As a whole, metal dysregulation may not only affect the course
of the disease by interaction with an amyloidogenic pathway but
also with a number of other AD-related pathways. There are other
metal-modulated cellular processes and downstream events that
must be considered. These also may interact with and mediate the
ageing process and subsequent disorders such as AD.
3.3. Tau
3.3.1. Tau binding to metals
The microtubule-associated protein tau is the principal
component of the paired helical filaments that comprise neurofi-
brillary tangles (NFTs) in a number of neurodegenerative disorders,
including AD. As yet the literature linking tau to metal
9
neurochemistry is not as developed as APP’s or Ab’s, but there
are a growing number of in vitro reports that indicate that tau may
well participate in the metal-related abnormalities observed in AD.
However, it is still unclear how the relationship between Ab and
tau lesions may be linked by metal interactions. Various repeat
motifs on tau bind copper and iron in a pH- and stoichiometric-
dependent fashion but have yet to have an affinity determined
under in vivo physiological conditions. Even so, the resulting metal
binding has been shown to result in a conformational change in
tau, promoting its phosphorylation (Malm et al., 2007) and
potentially inducing aggregation (Ma et al., 2006; Ma et al.,
2005; Zhou et al., 2007). Only Fe3+ induces aggregation of
hyperphosphorylated tau and this can be reversed by its reduction
back to Fe2+ (Yamamoto et al., 2002). Indeed, the treatment of tau
aggregates from the AD brain with reducing agents result in the
resolubilization of tau and a release of Fe2+, further demonstrating
the potential role of iron in NFT formation (Yamamoto et al., 2002).
Prior to these reports, NFTs were reported as capable of binding
adventitious copper and iron in a redox-competent manner, acting
as a source for reactive oxygen species within the neuron (Sayre
et al., 2000). In support, recent data indicates that tau interaction
with Cu2+ can mediate the generation of hydrogen peroxide in a
manner analogous to Ab (Su et al., 2007).
While tau is the principal component of NFT, there are other
cytoskeletal components present that are also known to interact
with metals. Neurofilaments (NF), for example, show phosphory-
lation dependent alterations very early in the AD cascade and are
also found within the NFT and in association with the dystrophic
neurites surrounding Ab plaques (King et al., 2000). Purification of
this multi-subunit protein demonstrated that it stoichiometrically
binds at least one copper and four zincs (Pierson and Evenson,
1988). The assembly of NFs may be mediated, in part, by metal ions
such as copper, which foster the assembly of the light NF subunit.
3.3.2. Metal modulation through kinases
Many of the kinases that phosphorylate tau (ultimately
resulting in hyperphosphorylation and dissociation of tau from
the microtubules) are induced by metal ions zinc, copper and iron.
Zinc is able to activate rac-beta serine/threonine protein kinase
(PKB), glycogen synthase kinase 3b (GSK-3b), extracellular signal-
regulated kinase (ERK1/2), c-Jun NH2-terminal kinase (JNK), p38
and p70 S6 kinase, that are all consequently involved in tau
hyperphosphorylation in SH-SY5Y and N2a cells (An et al., 2005;
Bjorkdahl et al., 2005). Tangle-bearing neurons from AD brain have
also been observed to fill with zinc (Bjorkdahl et al., 2005; Suh
et al., 2005). NFs are also phosphorylated at various sites by a
number of the tau-kinases induced by zinc (Bjorkdahl et al., 2005).
Phosphoinositol-3-kinase (PI3K) is rapidly activated by copper
with subsequent downstream activation of serine/threonine
protein kinase (Akt) and phosphorylation of GSK3 (Ostrakhovitch
et al., 2002). Copper may also be able to activate membrane
receptors such as the epidermal growth factor receptor (EGFR) and
result in downstream activation of mitogen-activated protein
kinases (MAPK), including ERK1/2 (Wu et al., 1999).
In contrast, the treatment of hippocampal neuron cultures with
iron citrate results in a decrease in tau phosphorylation at AD-
related epitopes, possibly from a decrease in the activity of the
Cdk5/p25 complex, where p25 may be a regulator of the activity of
protein kinase Cdk5 (Egana et al., 2003).
3.4. Apolipoprotein E
Apolipoprotein E (ApoE) allelic status is another major risk
factor for AD (Corder et al., 1993) and modulates zinc and copper
induced Ab precipitation in accordance with the isoforms ability to
bind these ions (Miyata and Smith, 1996; Moir et al., 1999). The
10 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
fourth isoform of ApoE (ApoE4) is the least protective for risk of AD.
Interestingly this isoform differs from the other ApoE alleles by
containing a cysteine to arginine substitution at position 112 for
isoform 3 and 158 for isoform 2. This cysteine is believed to be
involved in transition metal binding and may be related to the
diminished antioxidant effect of the ApoE4 allele (Moir et al.,
1999).
Interestingly, increased tau phosphorylation in apoE4 trans-
genic mice has been associated with ERK activation, already
described to be modified by zinc, suggesting that apoE4 and zinc
may act in concert to contribute to the pathogenesis of AD (Harris
et al., 2004).
4. Therapeutics and diagnostic targeting of metal ions in AD
One logical and increasingly popular theory for the use of
neurotherapeutic small molecules in AD is to target the initiating
event in the generation of free radicals. This may be done by
administering antioxidants or by employing metal complexing
agents to prevent the metal ions from participating in redox
chemistry. This review will cover some of the more promising
agents, however Scott & Orvig provide a more extensive recent
review (Scott and Orvig, 2009).
A common mistaken belief in targeting metal ions for
therapeutics is that chelation, meaning the removal of metal ions
from tissue, is the obvious intervention. While there are several
medical chelators in use, their approved use is confined to genuine
overexposure situations (e.g., Wilson’s disease, or lead toxicity), or
to rheumatoid arthritis. There have been a few placebo-controlled
clinical trials on AD with chelators reported, yet there is still no
evidence that AD will respond to traditional medical chelation and
the greater risk is that the removal of essential metal ions will
result in adverse effects (e.g. iron deficiency anemia). Another
important property of a potential neurodegenerative therapeutic is
its ability to cross the BBB. This has previously limited a large
number of common metal chelators as possibilities due to their
hydrophilic nature.
While these problems can, to some extent, be overcome by
engineering small molecules that target specific compartments or
organelles, complex situations such as the pooling of metals in
plaques and their relative deficiency within neighbouring cells, call
for the development of small molecules with more sophisticated
properties, e.g. ionophores.
4.1. Antioxidants
As a preventative approach antioxidant molecules may be used
for their ability to neutralize free or incorrectly bound metals,
thereby interfering with the ‘down-stream’ generation of reactive
oxygen species and other radicals (Behl and Moosmann, 2002).
Numerous molecules with antioxidant properties, such as oestro-
gen, melatonin, vitamin C and E (L-ascorbate and - topopherol,
respectively), ginkgo bilboa extract, curcumin and flavonoids have
neuroprotective effects against Ab-induced toxicity in cell- based
experiments (Shishodia et al., 2005; Zatta et al., 2003) and animal
models (Cheng et al., 2006; Defeudis, 2002; Feng et al., 2004;
Matsubara et al., 2003; Yang et al., 2005), but have shown
conflicting results in a clinical setting (Oken et al., 1998; Schneider
et al., 2005; Zandi et al., 2004) and may even be detrimental (Lloret
et al., 2009).
4.2. Metal chelators
Metal chelation in an organism depletes the total pool of
bioavailable metals by binding two or more metal ions to form a
cyclic ring that immobilizes the metal ions. Though not a metal ion
binding small-molecule, metallothionein-3 (MT-3) has chelating
properties and is important for copper and zinc homeostasis
within neurons, astrocytes and the extracellular space of the brain
(Vasak and Hasler, 2000). It has been found to be downregulated in
AD (Yu et al., 2001) and was recently used to protect cultured
neurons from Ab toxicity (Meloni et al., 2008). MT-3’s mechanism
appears to consist of reducing and sequestering the copper from
Ab and swapping it for a zinc ion carried within MT-3. While MT-3
may be involved in the mechanism of action for other therapeutic
compounds (see Fig. 1), it has not to date been used as a therapeutic
for AD itself.
Desferrioxamine (DFO), a preferential iron chelator able to also
bind zinc, copper and aluminium (Keberle, 1964), was the first
medicinal small-molecule chelator tested for the treatment of AD.
Crapper-McLachlan and colleagues reported a significant improve-
ment of some cognitive functions in AD patients on a 2-year-long
placebo controlled phase II clinical trial of DFO injected intramus-
cularly (Crapper McLachlan et al., 1991). However, as with most
metal chelators, DFO is a large hydrophilic molecule, which is not
orally bio-available and does not efficiently penetrate the BBB.
Hence, it is not clear whether the beneficial effect seen with DFO
treatment was due to the drug’s interaction with metals, or due to a
different mechanism all together (Cuajungco et al., 2000). A
smaller clinical trial with d-penicillamine also produced largely
inconclusive cognition results due to the high drop-out rate of
patients but did show a decrease in serum oxidative markers after
6 months (Squitti et al., 2002b).
Preclinically a number of metal chelators have been tested on
AD models with varying success. The hexadentate chelator, DP-109
(DPharm, Rehovot, Israel), is a large synthetic pro-drug that
becomes activated following the cleavage of its two long-chain
esters. A 3-month oral administration of DP-109 to Tg2576 mice
reduced levels of aggregated insoluble Ab in amyloid plaques and
cerebral amyloid angiopathy and conversely increased Ab via
resolubilization (Lee et al., 2004a). As with DFO, how DP-109 exerts
its anti-amyloidogenic effect is still unclear, as it is not expected to
easily cross the BBB. Recently, DP-109 and DP-460 (another
lipophilic chelator that preferentially binds calcium, copper and
zinc) were reported to have neuroprotective effects in a model of
amyotrophic lateral sclerosis (a transgenic mouse containing the
G93A mutation in SOD1) (Petri et al., 2007), another neurodegen-
erative disease associated with metal imbalance (Carri et al., 2003;
Watanabe et al., 2007). Other chelating agents have been reported
to have different effects in vitro, including reduced Ab1–42-induced
oxidative stress (Perluigi et al., 2006) as well as the solubilization of
both hypophosphorylated tau (Shin et al., 2003) and Ab from AD
brain (Cherny et al., 2000). Further in vivo studies with these
chelators are required to advance this therapeutic route and to rule
out any systemic effects.
4.3. Metal complexes
Metallo-complexes are emerging as a new alternative thera-
peutic approach for AD. Unlike chelation, the rational is to remove
metals from biologically deleterious sites and potentially deliver
them to areas of deficiency, thereby maintaining overall metal
homeostasis. Currently, metallo-complexes are able to complete
this strategy either by delivering metals to the relevant cellular
compartment using pyrrolidine dithiocarbamate (Metal-PDTC) or
bis(thiosemicarbazone) (Metal-BTSC) or by preventing the harmful
binding of metal to Ab using platinum (Pt2+) complexed to 1,10
phenanthroline derivatives (L-PtCl2).
PDTC’s ability to interact with copper and zinc gives this metallo-
complex anti-inflammatory, antioxidant and anti-apoptotic prop-
erties (Hayakawa et al., 2003; Liu et al., 1999; Schreck et al., 1992). It
inhibits the transcription-factor regulator nuclear factor-kB (NF-kB)
 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
in a neonatal hypoxia-ischaemia model, as well as activating Akt and
inhibiting GSK-3b (Nurmi et al., 2006). It is suggested to have a
similar role in an AD model (APP/PS1 double transgenic mice)
(Zhang et al., 2009). Oral administration of PDTC was able to increase
cerebral copper levels as well as decrease tau-phosphorylation via
down-regulation of the GSK-3 signaling cascade leading to an
improvement in spatial memory (Malm et al., 2007). While oral
administration of this drug led to no effects on amyloid burden, glial
activation or oxidative stress (Malm et al., 2007; Zhang et al., 2009),
daily intaperitoneal injection over 5 months of the same drug
attenuated astrogliosis and markedly increased cerebral Ab1–42
burden (Zhang et al., 2009).
Metal-BTSCs are small, neutral compounds that cross mem-
branes and have been previously used for a range of different
purposes, particularly as cancer imaging agents (Dearling et al.,
2002; Green et al., 1988; West et al., 1993). Metal-BTSCs
successfully cross the BBB and are relatively non-toxic (Dearling
et al., 2002; Green et al., 1988). Glyoxalbis(N(4)-methyl-3-
thiosemicarbazonato)copper(II) (Cu-GTSM) is a metal-BTSC that
binds and transports copper into cells and is engineered to release
the bound copper in response to the normal intracellular reducing
environment (Dearling et al., 2002; Donnelly et al., 2008;
Fujibayashi et al., 1997). The significance of this is that rather
than just increasing cellular copper levels, Cu-GTSM increases
cellular copper bioavailability. Importantly, this allows the
interaction of the copper with cellular components and subse-
quent triggering of cell signal mechanisms. In contrast, alternative
Metal-BTSCs such as diacetylbis(N(4)-methyl-3-thiosemicarbazo-
nato) copper(II) (Cu-ATSM) also increase cellular copper but the
metal is not released from the organic scaffold and therefore is not
bioavailable, except under hypoxic conditions (Dearling et al.,
2002; Donnelly et al., 2008). Treatment of CHO cells over-
expressing APP with Cu-GTSM has shown that copper bioavail-
ability is significantly increased and the levels of secreted Ab are
reduced in a dose-dependent manner that correlated with copper
uptake (Donnelly et al., 2008). This effect was mediated by
activation of PI3K resulting in GSK3 inhibition, up-regulation of
JNK and ERK activity and finally activation of MMPs (White et al.,
2006). In contrast, no effect on cell signaling, the MMPs or Ab levels
were observed in Cu-ATSM treated cells supporting the hypothesis
that the metal must be bioavailable (i.e. released from the organic
scaffold to induce therapeutic effects). Similar results were
reported in a neuronal cell line as well as in an orally administered
AD mouse model with Cu-GTSM mediated inhibition of GSK3b also
leading to inhibited phosphorylation of tau (Crouch et al., 2009a).
Cu-GTSM restored cognitive performance in the APP/PS1 trans-
genic mice to levels expected for cognitively normal animals
(Crouch et al., 2009a).
In an alternative mechanism of treating AD with metallo-
complexes, L-PtCl2 were designed to bind and alkylate the
imidazole side chains on histidine residues 6, 13 and 14 of Ab.
Alkylation of Ab is proposed to prevent the detrimental binding of
copper to the metal binding site leading to inhibition of subsequent
Ab-copper oligomerisation and binding to the cell membrane
(Barnham et al., 2008). Pt(4,7-diphenyl-[1,10] phenanthroline)Cl2
was identified as a compound that bound Ab, changing the peptide
conformation and inhibiting aggregation (Barnham et al., 2008). In
addition, this complex inhibited Ab-related neurotoxicity and
reversed synaptotoxicity caused by Ab-inhibition of long-term
potentiation on mouse hippocampal slices (Barnham et al., 2008).
Future evaluation of the compound’s ability to cross the BBB and
exert beneficial effects in animal models for AD need to be
performed.
An additional theoretical metal complex based therapeutic for
AD would target the increase of brain iron in the disease. Such
drugs may also attempt to decrease APP translation and hence Ab
11
generation (Reznichenko et al., 2006). A further beneficial effect of
iron depletion could arise from inhibition of hypoxia-inducible
factor; prolyl 4-hydroxylases, which is known to be neuroprotec-
tive (Siddiq et al., 2005).
4.4. Metal-protein attenuating compounds (MPACs) or Ionophores
Similar to metal-complexes, MPACs are able to compete with
endogenous ligands for metal ions. They target the harmful
‘upstream’ metal-protein reactions by using their moderate,
reversible, affinity towards metals and restore normal metal
levels in specific cellular compartments (Masters and Beyreuther,
2006). The first-generation molecule was clioquinol (CQ; 5-chloro-
7-iodo-8-hydroxyquinoline). Originally prescribed as an oral anti-
amebic in addition to being used for the treatment of dysentery
and diarrhoea (Flagstad, 1977; Neldner and Hambidge, 1975), this
quinoline compound is absorbed quickly as it is highly lipophilic
and is excreted easily through its conversion to glucuronated and
sulphate metabolites (Bondiolotti et al., 2007; Kotaki et al., 1983;
Liewendahl and Lamberg, 1967). However, its oral preparation was
withdrawn from the market during the 1960s to 1970s, as it was
suspected to be involved in the development of subacute myelo-
optico-neuropathy (SMON) (Osterman, 1971; Shimada et al., 1971;
Tsubaki et al., 1971), possibly confined to iron deficient individuals
who consumed overdoses for prolonged periods (Yassin et al.,
1998; Yassin et al., 2000).
Despite these previous complications with the compound, oral
treatment with CQ in an AD mouse model (Tg2576) resulted in a
reduction of cortical deposition of amyloid (49%), supporting initial
studies showing CQ’s ability to dissolve synthetic Ab:copper/zinc
aggregates and amyloid deposits from post-mortem AD brain
(Cherny et al., 2001). The 9-week trial led to an improvement in the
general health and weight of these mice compared to untreated
littermates (Cherny et al., 2001) and an improved cognitive
performance in the Tg2576 and APP/PS1 mouse models (Adlard
et al., 2008). Results indicated CQ was an ionophore rather than a
chelator as it was also able to efficiently cross the BBB, complex
with zinc-metallated Ab and increase both copper and zinc in the
brains of treated mice (Adlard et al., 2008; Cherny et al., 2001;
Opazo et al., 2006). CQ has only a moderate affinity for copper and
zinc, through a square, planar arrangement (Di Vaira et al., 2004),
which is sufficient to facilitate dissociation of these metal ions
from the low affinity metal binding sites of Ab. While, subsequent
studies have shown CQ to only reverse copper-suppressed, but not
zinc-suppressed, Ab1–40 fibril formation (Raman et al., 2005), CQ is
able to rescue copper-mediated Ab toxicity in neuronal cell culture
(Abramov et al., 2003) and markedly reduce Ab oligomeric
targeting to the synapse (Deshpande et al., 2009). However, other
studies still highlight that CQ increases oxidative neurotoxicity
(Benvenisti-Zarom et al., 2005). A small phase I study showed CQ to
lower CSF tau but caused no change to CSF-Ab1–42 levels (Regland
et al., 2001). In a subsequent pilot Phase II double-blind, placebo
controlled trial, the oral administration of CQ in moderately severe
AD patients for 36 weeks slowed the rate of cognitive decline with
no signs of severe adverse effects (Ritchie et al., 2003). It also
lowered plasma Ab1–42. Ensuing phase II/III studies, however, were
stalled by the difficulties encountered in preventing di-iodo-8-
hydroxy quinoline contamination during the required larger scale
chemical synthesis for such trials.
The second generation 8-hydroxy quinoline derivative of CQ,
PBT2 lacks iodine and is an easier chemical to synthesize. This
compound also has higher solubility and increased BBB perme-
ability than CQ. In the APP/PS1 transgenic AD mouse model, PBT2
decreased soluble interstitial Ab within hours, and improved
cognitive performance to levels equivalent to or greater than wild-
type controls within days of treatment (Adlard et al., 2008). In
12 J.A. Duce, A.I. Bush / Progress in Neurobiology 92 (2010) 1–18
addition, there was a significant decrease in insoluble Ab load and
the phosphorylation of tau, as well as a significant increase in
synaptophysin levels–suggesting that a number of primary indices
used to characterize AD pathology within the brain had been
successfully modulated (Adlard et al., 2008). Following a successful
phase I study, PBT2 has also completed a randomized, double blind,
placebo controlled Phase II clinical trial in 78 subjects with early
AD over a 12 weeks treatment period. This study demonstrated
safety and tolerability at 50 mg and 250 mg daily doses over the
full 12 weeks, and reduced CSF levels of Ab1–42 at the 250 mg dose.
Improved cognitive performance in patients taking PBT2 (com-
pared to placebo) was observed when tested on executive function
in the Neuropsychological Test Battery (NTB) at the end of the 12
week trial (Faux et al., in press; Lannfelt et al., 2008). These results
appear to be the basis for proceeding with further Phase IIb and
Phase III trials of what may be a disease-modifying drug based
upon the modulation of metals.
The exact mechanism of action of CQ and PBT2 is still uncertain.
However, in cell culture, CQ-copper complexes have been shown to
enter cells where they markedly inhibit the secretion of Ab
thought to occur through its degradation via the upregulation of
MMP-2 and MMP-3 (Fig. 1C). Similar to metal-BTSC, CQ-copper
also promotes phosphorylation of GSK-3 and can thus potentiate
activation of JNK and degradation of Ab1–40 (White et al., 2006).
This leads to a current working hypothesis in AD where CQ or PBT2
enters the brain and is attracted to the extracellular pool of metals
that are in a dissociable equilibrium within the amyloid plaque.
The compound then binds zinc and copper in the amyloid, possibly
forming a ternary complex with Ab, and the drug-metal complex
then enters the cell (Deraeve et al., 2006; Rozga et al., 2009). This
activates MMPs and facilitates the clearance of Ab from the
synapse. In parallel, while bound to the drug, the oxidative
oligomerisation and the toxic redox activity of Ab oligomers are
both blocked.
5. Conclusion
This review has attempted to illustrate the potentially
pleiotropic role that metals have in AD, with functional relevance
to a diverse number of mechanisms known to be disrupted within
the disease. In turn, biological metals represent potent targets for
pharmacological manipulation to limit and perhaps even prevent
the onset and progression of AD. As with any therapeutic target,
issues of specificity and selectivity are important factors that
require careful consideration when testing potential drug candi-
dates. While there are promising preclinical and clinical data in the
literature that show some effectiveness in reversing the clinical
syndrome in patients and models of the disease, large-scale clinical
trial testing is still to be awaited. With a greater understanding of
AD pathogenesis and as the multitude of alternative drug trials
currently underway come to completion, Ab: metal -based
therapeutics remains a promising prospect for the treatment of AD.
Acknowledgements
This work was supported by funds from the National Institute
on Aging (1RO1AG12686), the Australian Research Council, the
Australian National Health & Medical Research Council, Opera-
tional Infrastructure Support (OIS) from the Victorian State
Government and the Alzheimer’s Association.
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