Coordination Chemistry Reviews 256 (2012) 2129–2141

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Coordination Chemistry Reviews

journal homepage: www.elsevier.com/locate/ccr

Review

Copper, zinc and iron in neurodegenerative diseases

(Alzheimer’s, Parkinson’s and prion diseases)
Henryk Kozlowski a,∗ , Marek Luczkowski a , Maurizio Remelli b , Daniela Valensin c
a
Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland
b
Dipartimento di Chimica, Università di Ferrara, Ferrara, Italy
c
Dipartimento di Chimica, Università di Siena, Siena, Italy

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2129
2. Brain metal homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2130
2.1. Copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2130
2.2. Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2131
2.3. Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2132
3. Neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2132
3.1. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2133
3.2. Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2133
3.3. Prion diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2133
4. Metal binding to neurodegenerative proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2134
4.1. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2134
4.2. Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2135
4.3. Prion diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2135
4.4. Metal–protein affinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2136
5. Protein misfolding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2136
6. Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2138
7. Metal based therapeutic strategies for neurogenerative disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2138
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2139
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2139

a r t i c l e i n f o a b s t r a c t

Article history: The basic role of metal ions including copper, zinc and iron in neurological pathologies is generally
Received 27 December 2011 accepted. The relationship between the development of disease and particular metal ions is very compli-
Received in revised form 20 February 2012 cated and complex. Thus, comprehension of metal homeostasis, details of transport and interactions with
Accepted 9 March 2012
biomolecules is essential for understanding the normal and pathological processes occurring in the living
Available online 30 March 2012
system. Homeostasis of metal ions usually involves a huge set of proteins which regulate the proper metal
biology. Disorder in metal homeostasis may result in serious pathologies including neurodegenerative
Keywords:
diseases. Metal ions, especially copper, zinc and iron play very important roles in neurodegeneration
Brain metal homeostasis
Neurodegenerative diseases
having impact on both protein structure (misfolding) and oxidative stress. Metal ion binding to proteins
Metal binding to neurodegenerative involved in neurodegeneration is therefore an important factor for whole brain damage processes. All
proteins these aspects are discussed in the review.
Protein misfolding © 2012 Elsevier B.V. All rights reserved.
Oxidative stress

1. Introduction

The fundamentals of homeostasis and the basic roles of copper,

zinc and iron in biological functioning and neurological disorders
∗ Corresponding author. Tel.: +48 713757251; fax: +48 713757251.
in the brain will be covered in this review. All three metals play
E-mail addresses: henryk.kozlowski@chem.uni.wroc.pl, henrykoz@wchuwr.pl
(H. Kozlowski), marek.luczkowski@chem.uni.wroc.pl (M. Luczkowski), critical roles in the brain biology being present in some regions
rmm@unife.it (M. Remelli), daniela.valensin@unisi.it (D. Valensin). of the brain in millimolar concentrations. Iron and copper are

0010-8545/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.ccr.2012.03.013
2130 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
also effective catalysts for production of oxygen radicals so they
are major cause of the oxidative stress. Furthermore, the metal
ions mentioned above may have a basic impact on the protein
misfolding and progression of the neurodegenerative processes.
All these aspects are discussed below.
2. Brain metal homeostasis
Metal ions are required to sustain a huge number of cellular pro-
cesses, including neuronal ones. Transition metal ions like Fe, Zn,
and Cu play an important role in regulating neuronal activity within
the synapses and they are essential for the biological functions of
several metallo-proteins such as Cu/Zn superoxide dismutase and
cytochrome c oxidase. In the central nervous system (CNS) sev-
eral transition metal ions like iron, copper and zinc are required for
many enzymatic activities, mitochondrial function, neurotransmis-
sion, learning and memory. For example Zn2+ stored in presynaptic
vesicles is involved in neuron signaling process, it is released in
brief pulses into the synaptic cleft, and acts on recognition sites in
the postsynaptic membrane [1].
Even if these transition metal ions are vital to ensure many cel-
lular processes, their concentrations within the cell must be finely
tuned since any aberrations may lead to cell death and severe
diseases. Metal crossing through the blood–brain barrier (BBB) is
strictly regulated; many transporters, enzymes and chaperones
tightly regulate the metal ion content within the brain controlling
metal uptake and delivery to specific domains and preventing a pas-
sive flux of metals from the circulation to the brain [2]. In a healthy
state the concentration of free metal ions is maintained at a very
low level, each physiological metal ion is selectively delivered to
its own site of action in order to tightly control metal reactivity (i.e.
redox activity).
In order to maintain metal-ion homeostasis cells have devel-
oped a well refined machinery whose failure might lead to several
disease state, among which are neurodegenerative disorders [3–8].
It is well accepted that transition metal ions are involved in
Alzheimer’s (AD), Parkinson’s (PD) and prion diseases (TSEs). High
content of copper and zinc is present in amyloid plaques from AD
brains (25.0 ± 7.8 and 69.0 ± 18.4 ␮g/g respectively) [9] and in the
cerebrospinal fluid (CSF) of patients affected by both AD and PD
[10]. In particular Cu and Zn concentration in CSF are almost two
or three fold higher than control levels (10.2 and 5.3 ng/g for cop-
per and zinc respectively). Accumulation of iron is also frequently
observed in brain areas linked to both Parkinson’s and Alzheimer’s
diseases [11].
Proteins associated with neurodegenerative diseases (amyloid-
␤, A␤; ␣-synuclein, ␣S; prion protein, PrP) also bind several
transition metal ions and metal binding affects the aggregation
process of A␤ and ␣S (vide infra Sections 4 and 5). In both PD and AD
copper and iron are associated with reactive oxygen species (ROS)
generation. The Fenton reactions (Cu+ + H2 O2 → Cu2+ + OH• + OH−
or Fe2+ + H2 O2 → Fe3+ + OH• + OH− ) lead to the formation of the
highly reactive hydroxyl radical (OH• ) which induces oxidative
stress in substantia nigra pars compacta dopamine neurons by
lipid membrane peroxidation, DNA damage and protein oxidation
or misfolding (vide infra Section 6). As for PrP, strong evidence
indicates that it has a role in the regulation of brain copper
metabolism. It has been suggested that PrP–copper interactions
might modulate protection against Cu2+ mediated oxidative stress,
copper transport and copper dependent cellular signaling [12].
The possible beneficial or detrimental role of copper in PrP disease
is still a matter of debate [13].
The involvement of iron, copper and zinc in many neurode-
generative diseases is not simply correlated with increased and
toxicological exposure to these metals, but rather it depends on a
breakdown of the metal homeostatic mechanisms. It is, therefore,
crucial to understand the structural and functional interactions
between metals and the various intracellular and extracellular
components of the CNS, both in healthy and in pathological
conditions.
Here, we will focus on the three major biological transition met-
als involved in neurodegeneration, copper, zinc and iron. For all the
metal ions similar mechanisms of damage were found including:
(a) free radical production (for redox active metals), (b) protein
aggregation and (c) metal transport alteration [3,6,14–17].
2.1. Copper
Copper is a redox-active transition metal occurring in two main
oxidation states, +2 and +1. It coordinates to different biological
ligands such as imidazole nitrogen, amide and amino nitrogen,
carboxylate oxygen, cysteine and methionine sulfur donor atoms
[18–23]. Although the copper redox activity is fundamental for the
biological activity of a huge number of enzymes, it can become
highly dangerous if not accurately regulated. For these reasons,
specific proteins guarantee that no copper ions can freely circulate.
The full comprehension of copper transport within the CNS is
not yet reached, although the proteins involved in copper homeo-
stasis in the brain and their importance in the CNS have been
identified [24]. The proteins which play a key roles in regulating
copper distribution and transport in peripheral tissues are also
present in the brain as described by the schematic representation
shown in Fig. 1. The major copper binding proteins (cuproenzymes
and/or carrier proteins) in extracellular fluids are ceruloplasmin,
albumin, and transcuprein, which transport the absorbed copper
to all tissues. Ctr1 and ATP7A or ATP7B transport copper across cel-
lular membrane transferring copper into (import) or out of (export)
the cytosol, respectively. Once inside cells, cytosolic copper chap-
erones deliver the metal to intracellular protein targets (e.g. copper
chaperone of superoxide dismutase (CCS), delivers copper to super-
oxide dismutase 1, SOD1) [25].
Copper distribution within CNS varies according to species. Each
region of the brain is characterized by specific copper distribu-
tion which increases with aging. The regions showing the highest
copper concentrations might be associated with the regions with
the highest copper metabolic demand. In rat, the areas showing
high copper enrichment are in the (i) medial geniculate nucleus
(the region responsible of the visual information processing), (ii)
Fig. 1. Schematic representation of copper distribution and transport in a cell of
CNS.
Source: Figure reproduced from Ref. [24] with permission.
 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
superior colliculus (a component of the midbrain associated with
motor functions), (iii) periaqueductal gray (the gray matter modu-
lating pain and defensive behavior), (iv) the lateral amygdala (area
involved in memory and emotional reactions) and in the (v) dor-
somedial aspect of the diencephalon (the part of the forebrain
containing thalamus, hypothalamus and the posterior portion of
the pituitary gland). In humans the highest copper concentration
occurs in the hippocampus [26].
As already mentioned, patients with neurological symptoms
related to dementia show increased copper concentrations [10,27].
In Alzheimer’s-like dementia, copper content in the CNS, is 2-fold
higher than in age matched controls [10,28,29].
Copper, released at the synaptic cleft by ATP7A acts as a modula-
tor of neurotransmission. Recent studies indicate that copper yields
biphasic changes in neurotransmission in rat hippocampal neu-
rons by both blocking and enhancing neurotransmission [30]. It has
also been proposed that copper release protects against excitotoxic
cell death by regulating the activities of extra-synaptic N-methyl
d-aspartate (NMDA) receptors [31,32].
2.2. Iron
Like copper, iron is a redox active metal occurring in two main
oxidation states, +2 and +3. During the last ten years the scien-
tific community has made enormous progress to understand iron
homeostasis [33], however it is still obscure how the brain regulates
fluxes and storage of iron into neurons, oligodentrites, astrocytes
and glial cells. In the healthy state the iron level strictly depends
on specific areas of the brain. Higher iron concentrations are usu-
ally found in the substantia nigra and the globus pallidus [34]. The
dentate gyrus, interpeduncular nucleus, thalamus, ventral pallidus,
nucleus basilis and red nucleus also contain relatively high iron
concentrations. Generally the brain regions responsible for motor
Fig. 2. Representation of labile iron pool formation and related generation of oxidative stress mechanisms in Parkinson’s disease.
Source: Figure adapted from Ref. [17] with permission.
2131
functions seem to have more iron than non-motor related regions
explaining why movement disorders are often associated with iron
loading [35]. As for copper, iron concentration in the brain seems
to increase with aging [35].
Different proteins have been identified as iron binding pro-
teins in the brain: (i) H- and L-chain ferritin in oligodendrocytes,
L-ferritin in microglia and neuromelanin in neurons. Since H- and L-
ferritin have different functions their specific locations might point
to distinctive biological roles. In fact, L-ferritin is mainly involved
with iron storage, while, on the contrary H-ferritin is associated
with stress responses catalysing Fe2+ /Fe3+ oxidation via the fer-
roxidase center. Iron transport within the brain is modulated by
pathways including both transferrin and non-transferrin bound
iron. In the brain, iron uptake seems to be controlled by transfer-
rin receptors, expressed exclusively in gray matter, and by ferritin
receptors present only in white matter.
Iron levels in AD and PD are higher than those expected by nor-
mal aging but the mechanisms associated with such accumulation
are still elusive [36]. One hypothesis is that Fe accumulation is
correlated with neuronal death through glutamate receptors [37].
Iron damage can also depend on altered metal transport mech-
anisms [17]. The divalent metal transporter (DMT1) is responsible
for iron transport across the BBB. Iron accumulation, induced
by neurotoxins such as kainate, 6-hydroxydopamine (6-OHDA)
and MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), might
depend either on increased iron uptake or release from DMT1 and
ferritin, respectively. Such improper handling of iron is thought
to form a labile iron pool, also called ionic iron, which involves
iron in the Fenton reaction with hydrogen peroxide, generated
by dopamine metabolism by monoamine oxidase (MAO), auto-
oxidation and other oxidative processes. Ionic iron might also
determine aggregation of ␣S and A␤, which in turn, can initiate
OH• generation, causing oxidative stress (Fig. 2).
2132 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
Fig. 3. Representation of the disruption of synaptic activity by A␤–Zn2+ interaction at the synaptic cleft.
Source: Figure adapted from Ref. [65] with permission.
2.3. Zinc
Zinc is the most abundant trace metal in the brain where it
plays various functions. It interacts with several proteins confer-
ring on them catalytic or structural properties. As with copper, zinc
is released into the synaptic cleft, where it modulates NMDA and
␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors regulating the activity of glutamatergic synapses, sug-
gesting its possible role as a neurotransmitter in its own right
[38–43].
Several different factors, such as balance between the cellular
redox state, the concentrations of other biological chelating agents
and the energy status of the cell are determinant for zinc distribu-
tion within the cell [44].
Zn homeostasis is controlled by the joint activity of several
Zn transporter families, particularly those involved in zinc ingress
and egress from cells and organelles. In particular Zn2+ homeo-
stasis within neuronal cells results from the balanced control
among (i) influx, (ii) buffering and (iii) extrusion processes. Two
different families of zinc transporters are known: the ZnT fam-
ily, which decreases intracellular zinc concentrations and the ZIP
family, which imports the metal ions from the extracellular space
or organellar lumen into the cytoplasm. The zinc ingress into
neurons takes place through voltage-sensitive Ca channels and
Ca-permeable ionotropic glutamate receptors as well [45]; on the
other hand sequestration and buffering require metallothioneins
(MTs), mitochondria, zincosomes and lysosomes activity [46–50].
The three MTs isoforms expressed in the brain are MT-1, MT-2 and
MT-3 present in astrocytes and in neurons [51,52].
There is a strong indication that Zn2+ is involved in AD pro-
gression only (for review see [53]). Many investigations have been
addressed to monitor changes of zinc levels associated with AD
onset. No consensus on what happens to zinc concentrations and
to ZnT expression has been found. Both increased or decreased
zinc levels are measured in serum [54–56], hippocampal [38] and
CSF [55] of AD patients compared to age-matched controls. Sim-
ilarly, ZnT expression during AD progression, is both increased
or decreased when compared to control [57–59]. High concen-
trations of copper, iron, and zinc are found in amyloid plaques
[9,60,61]. Unlike copper and iron, whose toxicities might depend on
oxidative-stress-type mechanisms [61,62], zinc plays a crucial role
in A␤ aggregation. It can control the rate of self-assembly of A␤ and
regulates the amyloid morphology via specific coordination sites
[63]. Furthermore, zinc can coordinate A␤ via intra-and/or inter-
molecular binding modes, promoting A␤ aggregation [64] and that
synaptic zinc induces the formation of A␤ oligomer with their con-
sequent accumulation at excitatory synapses (Fig. 3) [65].
3. Neurodegenerative diseases
The term neurodegenerative diseases refers to the group of
disorders that share common features and molecular patterns sug-
gesting that their pathology may be directly comparable. They are
characterized by abnormal accumulation of proteins and selective
neuronal degeneration. Depositions of insoluble, fibrous amyloid
proteins are found either extra- or intracellularly in the brain. The
fibrils consist of various amino acid sequences but all share a ␤-
pleated-sheet secondary structure. This sets down the hallmark of
the conformational disorders like Alzheimer’s disease, Parkinson’s
disease and transmissible spongiform encephalopathies where
aggregation of specific proteins has been observed. These proteins
are usually natively partly unfolded but, after misfolding, aggregate
and accumulate as deposits in the brain. A large body of evidence
indicates that transition metal ions like Cu, Zn and Fe have strong
impact on the development of neurodegenerative disorders. As a
matter of fact, metal binding may result in severe conformational
 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
changes of proteins involved in neurodegeneration. The fact that
Cu, Zn and Fe interact with the major protein components is rather
because of the deregulation of the mechanisms that maintain metal
homeostasis [66].
3.1. Alzheimer’s disease
Alzheimer’s disease is the most common form of dementia that
is normally preceded by up to eight years of mild cognitive impair-
ment, the transition stage between the disease and the normal
aging [67]. AD is characterized by the extracellular deposition of
amyloid plaques formed by the aggregated amyloid-␤ peptide that
is cleaved from its integral membrane precursor protein (APP)
and the intracellular deposition of paired helical filaments of the
hyperphosphorylated tau protein in neurofibrillary tangles [68].
The function of the APP is unknown. However, recent evidence
suggests that it plays a role in maintaining copper homeostasis, per-
haps employing its ferroxidase activity [69–71]. The original loci of
the amyloid deposition within the brain have been determined in
the synaptic clefts of glutamatergic synapses of the hippocampus
and cortex. These synapses are involved in long-term potentia-
tion, the fundamental process contributing to memory formation.
The latest studies elucidated them as sites of Cu and Zn release
[72–74]. The pool of vesicular zinc formed as an outcome of ZnT3
protein activity is most likely excreted with glutamate during neu-
rotransmission to deteriorate the response of postsynaptic NDMA
receptors. The Menkes ATPase dependent release of copper from
postsynaptic neurites results in a series of events employing the
metal related S-nitrosylation of NDMA receptors that modulate
effects on the latter one [74]. The increasing number of recently
published papers suggests that glutamatergic synapse might have
unique properties as it may be the site of convergence of chem-
ically exchangeable pools of Zn and Cu [66]. When these metal
ions meet the amyloid-␤, the peptide with its ability to precipi-
tate or crosslink, the resulting series of events engaging interaction
of these metal ions with the peptide might either lead to cop-
per related free radical toxicity accompanied by aggregation or to
inhibition of destructive effects leading to oxidative stress by pep-
tide precipitation induced by Zn binding. This hypothesis might
be supported by the metal swap experiments applying Zn loaded
metallothionein 3 as a factor that can reduce Cu induced toxicity
toward cell cultures [75]. Metallothionein 3 is indeed released by
neighboring astrocytes at the glutametergic synaptic cleft [76].
Zn, Cu and Fe are found in amyloid plaques in relatively high,
even millimolar or submillimolar concentrations (1 mM, 400 ␮M
and 1 mM, respectively) [9]. Zinc and copper collocate within A␤
deposits where they seem to be bound, whereas iron does not
co-purify with the A␤ extracted from plaques and is found pre-
dominantly as a ferritin bound form in neuritic processes associated
with the plaques [77,78]. Interestingly, iron levels are elevated in
the cortex, whereas copper levels are decreased [17,79]. The former
one might be the consequence of atypical distribution of proteins
that regulate iron homeostasis including ceruloplasmin, the cop-
per dependent protein. For that reason, altered iron homeostasis is
rather a secondary than a primary effect [17,80].
The interplay of copper and zinc is not limited only to amyloid-
␤ but spreads through all the key Alzheimer’s disease proteins. To
begin with, Cu+ binds to N-terminal extracellular domain of APP
thus promoting its export outside the neuron [81]. In addition, cel-
lular Cu promotes the expression of APP mRNA [82,83]. C-terminal
domain of ␤-secretase binds Cu+ that allows the protein to interact
with CCS1, the copper chaperone that delivers Cu+ to SOD1. Zn2+
ions, even at low concentration, negatively modulate the activity
of ␥-secretase, another metalloproteinase directly involved in APP
processing. However, physiological implications of the majority of
these interactions are unclear.
2133
3.2. Parkinson’s disease
Parkinson’s disease is a severe, progressive motor disorder
caused by changes in the central nervous system, tightly linked to
degeneration of dopaminergic neurons of basal ganglia known as
substantia nigra. Familiar or inherited forms of disease are linked
to the mutation of genes coding ␣-synuclein, the key PD protein
[84]. The histopathological hallmark of disease is the presence of
the Lewy bodies that contain the fibrous ␣-synuclein deposits.
Although various biochemical studies suggested that the protein
may have an impact on numerous processes in the cell, like the
reassembly of the SNARE complex required neurotransmitter
release [85], so far none has shown that it is absolutely essential
for anything. Hence, the role of protein remains elusive, over
and above the mechanism leading to its conversion to species
tending to aggregate. The likely factors that could be involved in
the latter process are the transition metal ions, in particular Cu
that interacts with both the soluble and membrane-bound helical
form of ␣-synuclein.
Actually, altered metal homeostasis has been reported in PD.
Zinc and iron are augmented, while copper is decreased in substan-
tia nigra. The elevated iron level may contribute to oxidative stress
and deposition of intracellular inclusion bodies, thus facilitating the
progress of pathology. Under normal physiological conditions the
excess iron is sequestered by ferritin and neuromelanin [86,87].
The reduced expression of the former one and the loss of neurome-
lanin cells in substantia nigra coupled with structure alterations of
the neuromelanin itself may compromise the ability of neurons to
sequester the iron and result in elevated level of this redox active
metal. Iron promotes the aggregation of the key PD protein prob-
ably by distraction of ubiquitin dependent proteasomal system of
protein clearing [88]. The protective role of ferritin and iron chelat-
ing agents in animal models supports these hypotheses.
3.3. Prion diseases
Human prion diseases are a group of fatal neurodegenerative
disorders that share a common pathogenic agent, the prion [89].
There are five known human prion diseases: Creutzfeld–Jakob dis-
ease (CJD), Gerstmann Straussler Scheinker disease, kuru, new
variant Creutzfeld–Jakob disease and fatal familial insomnia. These
diseases are unusual because they can be transferred between
individuals or even between the species. Regardless of etiology,
prion diseases are identified by neuronal vacuolation (spongiosis)
and deposition of amyloid-like plaques. The diverse prion diseases
share various isoforms of common pathogenic unit known as PrPSc ,
the abnormal form of host-encoded cellular prion protein (PrPC )
[90]. The only differences between them seem to be the monomer
conformation and the resulting aggregation properties. It has been
proposed that prions are self-propagating amyloid forms of cellular
counterparts that replicate by fiber fragmentation that can act as
templates for monomer recruitment [91]. The exact mechanism by
which PrP can cause neurodegeneration and cell death is elusive.
The cellular form of prion protein is also widely known as a cop-
per binding protein. The binding of copper is possibly necessary
for its normal cellular function [92]. One of the primary manifes-
tations of this metal ion binding is internalization of the protein
from cellular surface. Another consequence of copper binding to
PrP is the plausible neuroprotective role of the holoprotein fulfilled
by indirect rather than direct involvement in antioxidant protec-
tion of cells. These activities are lost when PrPC is converted to
PrPSc during disease [93,94]. Studies on other metal ion binding
to human PrP suggested possible binding of Zn, Ni and Mn [95].
However, low affinity of the protein toward Zn makes these sugges-
tions questionable and biologically irrelevant. The question of Mn
interaction with the protein is still open to discussion. Manganese
2134 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
Fig. 4. Species distribution diagram for the Cu/A␤16 system at [Cu2+ ]tot = [L]tot
= 0.001 M. Eight mono-nuclear, variously protonated complexes are formed in the
pH range 3–11. The concentration of each species is reported as percentage of copper
involved, as a function of pH.
Source: Diagram drawn using data reported in Ref. [101].
bound protease resistant forms similar to PrPSc are isolated from
the brains of patients with CJD [96]. This binding might be the con-
sequence rather than cause of disease. The potential interaction of
prion protein with Fe might be neglected, since the protein’s affin-
ity toward this metal would favor the metal ion sequestration by
other proteins present in the milieu [92].
4. Metal binding to neurodegenerative proteins
4.1. Alzheimer’s disease
The amyloid-␤ peptide (A␤), whose aggregation results in the
formation of plaques typical of AD [97], binds metal ions influencing
their metabolism. It is widely accepted that the hydrophilic frag-
ment containing the first 16 residues of the A␤ peptide (A␤16) is
highly representative of the coordination behavior of the wild pep-
tide toward metal ions, as also reported in some recent reviews
[97–100].
As far as Cu2+ is concerned, solution-equilibria studies on model
peptides [101,102] agree with spectroscopic investigations about
the presence of different species at physiological pH (Fig. 4). The
A␤16 fragment contains several potential donor sites: Asp1 (NH2 ),
Asp1 (CO), Asp1 (COO− ), Ala2 (CO), Glu3 (COO− ), His6 (Im), Asp7
(COO− ), Tyr10 (O− ), Glu11 (COO− ), His13 (Im), His14 (Im) and
amidyl from the peptide backbone; also water molecules can be
taken into consideration. Some of these have been definitively
excluded from coordination, like e.g. Tyr10 [102,103] even though
this residue plays a role in amyloid aggregation. The main complex
structures around physiological pH have been classified as Form
I (prevailing at pH 6.5) and Form II (prevailing at pH higher than
8); both of them are distorted square planar, with at least three
nitrogen donor-atoms in equatorial position, giving 3N1O or 4N
coordination modes [104]. Both the terminal amino group and side
chain imidazoles of His6, His13 and His14 are involved in com-
plexation [105], in many different combinations and in dynamic
equilibrium [106,107]. The involvement of the amidyl nitrogen of
the Asp1 Ala2 peptide bond is likely for Form II [108], possibly in
place of His13 and/or His14, with a consequent change in complex
geometry. The identity of the oxygen equatorial donor eventually
participating in complex-formation is still debated: the best candi-
dates are carboxylates of Asp1 or Asp7, while, in the case of Form
II, the involvement of Ala2 carbonyl has also been suggested [109].
The presence of a second, independent coordination site for Cu2+
have also been put forward, although its estimated affinity con-
stant rules out any possible biological relevance for this site [99];
Fig. 5. Proposed structure of the mixed metal Cu2+ /Zn2+ complex of A␤ peptide.
Only amino acid residues involved in metal complexation are shown for clarity.
Source: Figure reproduced from Ref. [120] with permission.
the capability of A␤ to bind up to four Cu2+ ion, at alkaline pH,
was also reported [102]. The few available investigations on Cu2+
complexation with amyloid fibrils suggest a coordination behavior
similar to that reported in solution [110], with the proved involve-
ment of ␧-nitrogen atoms of His13 and His14, as well as carboxylate
side-chains of Glu3, Glu11 and Glu22 [111], in a high variety of
coordination modes based on both intra- and inter-molecular inter-
actions, due to the high concentration of binding sites at the solid
state. The involvement of the C-terminus was also suggested, while
the structure of hydrophobic regions of A␤40 does not look to be
affected by copper coordination [111]. Ni2+ , often used as a probe
to investigate Cu2+ coordination behavior, forms complex species
with A␤ of a structure very similar to that found for Cu2+ [112].
The coordination chemistry of Cu+ with A␤ is of much inter-
est, since Cu2+ can be reduced in the brain to produce ROS species
through the Fenton reaction. Cu+ coordination is rather different
from Cu2+ , as expected: Cu+ ion is coordinated to A␤ by means of
two imidazole rings of His residues in a linear fashion. All three his-
tidines are involved in binding, with a dynamic exchange between
different ligand combinations [113–115]. The existence of a dra-
matic difference between the coordination mode of Cu+ to A␤
monomers or oligomers has been reported [116]: in the latter case
tetrahedral geometry is preferred.
There is a general consensus that the Zn2+ complex species
formed at neutral pH have 1:1 stoichiometry with all imidazoles
involved in coordination [99]. As further binding groups, termi-
nal amine, Asp1 or Glu11 carboxylates or water molecules have
been suggested [117], while amide nitrogen atoms, Tyr10 and Arg5
side chains have been excluded. The minimal binding site was rec-
ognized as A␤6–14 (HDSGYEVHH), with the region 11–14 playing
the main recognition role [118]. Also in the Zn2+ case, solid-state
binding-geometries in fibrils are very similar to those described in
solution. The possibility of forming mixed-metal Cu2+ /Zn2+ com-
plexes, in the presence of Zn2+ excess, has been recently reported
[119]. In this case, Cu2+ complex geometry would be modified,
being the His13 and His14 sites engaged with Zn2+ , thus reducing
the redox activity of the copper–A␤ complex (Fig. 5).
Iron coordination to A␤ has been very recently investigated,
using NMR spectroscopy [120,121]. The key role in complex-
formation played by the A␤16 fragment has been confirmed for
the Fe2+ ion. Experimental data suggest that Fe2+ is hexacoordi-
nated, most likely involving the following residues: Asp1 (NH2 , CO
and COO− ), Glu3 (COO− ), His6 (Im and CO), His13 (Im), His14(Im),
but not Tyr10 nor Met35. The binding-site geometry is quite sim-
ilar to Form I of copper (see above), but, different from Cu2+ , no
 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
Fig. 6. Coordination geometry of the primary Cu2+ binding site in ␣S.
Source: Figure reproduced from Ref. [122] with permission.
pH dependence was observed around physiological pH. As far as
Fe3+ and Al3+ are concerned, no complex species was detected at
physiological pH [121].
4.2. Parkinson’s disease
␣-Synuclein (␣S) is the copper-binding protein involved in Lewy
bodies formation which are distinctive of PD. It consists of 140
amino acids and its native monomeric form is unstructured. ␣S
is composed of three distinct domains: (i) the N-terminal region
(1–60) containing two strong anchoring sites for the Cu2+ ion at
Met1 (NH2 ) and His50 (Im); (ii) the so-called non-amyloidogenic
component (NAC), spanning the residues 61–95, and (iii) the neg-
atively charged C-terminal domain (96–140), rich in Asp and Glu
residues, potential additional metal-binding sites.
The binding capability of ␣S toward Cu2+ has recently been
reviewed [100]. It is widely accepted that ␣S can bind at least three
Cu2+ ions, two of them with micromolar or lower affinity and the
third one with millimolar affinity [123]. The two stronger bind-
ing sites are located in the N-terminal domain and correspond to
the Met1 (NH2 ) and His50 (Im) anchoring sites [22]; there is no
agreement in the literature on the fact that they are independent
[124,125] or that the Cu2+ ion can form a macrochelate, binding at
the same time both amine and imidazole groups of Met1 and His50
residues, respectively (Fig. 6) [122,123,126]. The third Cu2+ bind-
ing site is located at the C-terminal; it is weak and unspecific [100].
The presence of different species, characterized by different donor
atom sets, has been ascertained at neutral pH, by means of both
spectroscopic [127] and thermodynamic investigations [127–129].
The comparison between ␣S and ␤S behavior confirmed most of
the above observations [130].
Cu+ coordination to ␣S has recently been investigated [131]:
the reduced copper ion can bind ␣S with micromolar affinity, the
donor-set mainly involving Met1 and Met5 residues.
Fig. 7. Inter-repeat (a) and intra-repeat (b) structures of Cu2+ –PrP complexes with the octarepeat domain.
Source: Figure (a) reproduced from Ref. [136] with permission. Figure (b) drawn using crystallographic data of the Cu2+ /Ac-HGGGW-NH2 complex [134], deposited at the
Cambridge Structural Database.
2135
4.3. Prion diseases
TSEs are spongiform encephalopathies with the peculiarity to be
transmissible: the infectious agent is a mutated form of prion pro-
tein (PrPSc ) which, compared to the normal protein (PrPC ), is highly
insoluble in physiological medium and resistant to proteases, thus
accumulating in neurons as plaques. The exact mechanism of PrPC
post-translational modification to PrPSc is not completely under-
stood. On the other hand it is widely demonstrated that PrPC can
bind metal ions, especially Cu2+ and Zn2+ , with high affinities: the
complex-formation imposes structural constraints on the protein
[132], suggesting that its metal-binding capability is the key for
PrPSc formation, if not for the disease development.
PrPC is a 254 amino acid protein: under physiological condi-
tions, its N-terminal domain is unstructured, while the C-terminal
domain contains three ␣-helices and two ␤-sheets. It is widely
accepted that the main Cu2+ binding sites are located in the
N-terminal domain, containing six histidines (61, 69, 77, 85, 96,
111) which can act as metal anchoring sites [133]. A PrPC peculiar-
ity, highly conserved among mammals and found with some differ-
ences also in birds and fish, is the presence of four repetitions of the
same sequence of eight residues (PHGGGWGQ), called “octarepeat”
and located in the fragment 60–91. Each octarepeat contains one
His and can bind copper or zinc. The binding mode highly depends
on both pH and/or metal/ligand ratio. In fact, at acidic pH or in the
presence of (sub-)stoichiometric levels of Cu2+ ions, the so-called
“inter-repeat” binding mode is adopted: up to four histidines of
this region bind the metal at the same time, with the formation of
a 3N or 4N species where imidazole nitrogen atoms act as donor
atoms (Fig. 7a). Increasing pH, the copper ion can displace one or
more protons from the amide nitrogen atoms of peptidic backbone,
thus changing the complex geometry to the “intra-repeat” mode
(Fig. 7b). This coordination mode is identical to that found both in
solution and at the solid state with peptide models like Ac-HGGGW-
NH2 [134] or Ac-PHGGGWGQ-NH2 [135]; the presence of a Pro
residue before the His anchoring site forces the amide coordination
toward the C-terminus, forming a rather unusual seven-membered
chelate ring. Another interesting feature is the stabilizing role of the
Trp indole ring, interacting with the metal ion through an axially
bound water-molecule. At a sufficiently high copper/PrP ratio, up
to four Cu2+ ions can be bound to the PrP octarepeat domain [23].
One or even two additional Cu2+ ions can be tethered to PrP
at the so-called “fifth binding-site”, located in the amyloidogenic
PrP region of (90–126), where two His residues (96 and 111)
are present. Most of the literature agrees that these histidines
behave independently [137] and that the preferred anchoring site,
in humans, is His111 [133]. The most accredited coordination
geometry at physiological pH is [NIm , 2N− , O] for both the sites;
the presence of an equilibrium between different species where
2136 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
the amide coordination is directed toward either the N- or the C-
terminus has been suggested [138]. The involvement of Met109
and/or Met112 residues in complexation at the site centered at
His111 is still a matter of debate [139,140].
The “inter-repeat” binding mode is uniquely available for Zn2+
(with an affinity much lower than copper), since this metal ion
is unable to displace amide protons [100]. However, due to the
high zinc concentration that can be present in the brain, Zn2+ can
influence the Cu2+ distribution in PrP complexes [141,142] in the
presence of low copper levels.
4.4. Metal–protein affinity
The classical way that biochemists use to estimate the affinity
of a ligand (e.g. a metal ion, M) for a substrate (e.g. a protein, P)
is the evaluation of the dissociation constant, Kd , referring to the
equilibrium:
[M][P]
MP  M + P Kd = (1)
[MP]
where charges are omitted for the sake of simplicity. Kd is a
conditional constant, usually obtained by experimentally deter-
mining the free metal concentration, [M], when [P] = [MP], at known
total concentration of P and under physiological conditions [133].
Whatever experimental technique (most often spectroscopic) is
employed to measure [M], this procedure deals with the chem-
ical system, where many complex-formation equilibria are often
active at the same time (e.g. see Fig. 4), as the main (if not the
unique) species forming was the 1:1 complex. Taking into account
this simplification and the differences in the experimental meth-
ods employed, in the matrix (usually containing a buffer and/or a
competitor), in temperature and ionic strength, the high variability
of Kd values reported in the literature for a specific system is not
surprising. In particular, a problem commonly not correctly faced
is the account in which competitor’s action has to be taken; more-
over, if ternary species, containing the competitor, are formed, the
Kd value is unreliable [143]. In addition, in some cases it not possi-
ble to classify the “binding sites” of a substrate but it is much better
to distinguish among different “binding modes” [133,144]. A big
advantage of this method is the possibility of working with very
low amounts of high-molecular-weight substrates, like recombi-
nant proteins.
An alternative way to investigate the metal–protein affinity is
the rigorous determination of the speciation model (stoichiometry
and thermodynamic stability constant of every species present in
solution), through the study of simplified model systems, mimick-
ing the binding site(s). In this case, the experimental conditions and
procedures are strictly defined and highly reproducible, no buffer or
competitor is normally necessary; in addition, the knowledge of the
speciation model allows the evaluation of Kd and the calculation of
distribution and competition diagrams. Details of calculation pro-
cedure requested to estimate Kd from a given speciation model are
described elsewhere [133,144]. A drawback here can derive from
the different behavior between the full-length protein and its low
molecular-weight models, possibly due to the protein secondary or
tertiary structure or the action of residues not included in the pep-
tide model. The former problem can be neglected if the binding site
is located in an unstructured domain of the protein. In summary,
the two methods are complementary and, together, they can give
a complete picture of the system under study.
The affinities of above proteins, involved in the main neurode-
generative disorders, with metal ions, and the resulting biological
consequences, have been recently reviewed [99,122,128,133,144]
and they will be extensively examined in another chapter of this
volume; for these reasons they will not be discussed here.
5. Protein misfolding
On the basis of the growing number of pathological processes
in which folding abnormalities are involved, it has been proposed
that at least half of human diseases are somehow associated with
protein misfolding [145]. These folding abnormalities are either
associated with diseases caused by decreased concentrations of a
specific protein that never achieves its folded functional structure
(lack of function mechanism) or might be due to intra- or extracel-
lular accumulation of insoluble, abnormally aggregated proteins
(gain of function mechanism) [146]. Diseases based on the first
mechanism are distributed over each phase of life and are mostly
associated with genetic diseases, while those induced by protein
aggregation are predominantly associated with aging. Neurode-
generative disorders constitute one of the most common groups
of the latter type of protein misfolding diseases, with exception
of the prion diseases that might be elucidated both by “loss of
function” and “gain of function” mechanisms (vide supra). In most
cases large structural rearrangements between the frequently ␣-
helical or unordered structure of the monomeric native protein and
the aggregated material rich in ␤-sheet conformation is observed
[147]. The initiating event of the mechanism of formation of amy-
loid fibrils is protein misfolding, which results in the generation of
aggregation-prone structures that grow by an autocatalytic mech-
anism. The critical event in that process, known as nucleation
dependent model, generates protein oligomers that act as a seed of
successive propagation of protein misfolding [148–150]. Aggrega-
tion starts when protein exceeds critical concentration. In addition
to mature fibrils, number of other structures has been described
as a meta-stable intermediate of the protein aggregation process,
including soluble oligomers, pores, annular structures, spherical
micelles and protofibrils. These diverse species with progressive
degrees of aggregation are present simultaneously in dynamic
equilibrium between each other [151–153].
The discovery of A␤ in the senile plaques set the principle for
the amyloid cascade hypothesis where the imbalance of A␤ pep-
tide metabolism followed by aggregation and deposition leads to
neuronal death and in consequence to dementia in AD. This idea
has been further implemented to other neurodegenerative disor-
ders. However, the hypothesis that deposited aggregates are toxic
has been challenged by the results of studies indicating that these
plaques were found in people without any clinical symptoms of
AD. Moreover, in some animal models of AD and other neurode-
generative disorders the symptoms have been identified before the
occurrence of protein aggregates has been detected. As a conse-
quence, today the most accepted hypothesis affirms that protein
misfolding and early stages of oligomerization are actual culprits
of neurodegeneration. Perhaps formation of amyloid-like fibrils
is a protective mechanism of sequestration and isolation of toxic
misfolded intermediates [154], unless aggregates and associated
proteins are in dynamic equilibrium with soluble versions of the
protein, thus acting as reservoir of toxic oligomeric species (Fig. 8)
[153,155,156].
All of the proteins involved in the most common neurodegener-
ative disorders, amyloid-␤ peptide and hyperphosphorylated tau
protein for AD, ␣-synuclein for PD and prion protein for TSEs are
affected by metals when it comes to their aggregation.
Focusing on AD, protein misfolding associated with amyloid-␤
aggregation is greatly affected by various biophysical and chemi-
cal factors including metal ions as Cu, Zn, and Fe which have been
found in senile plaques in post-mortem AD brains. However, the
exact role of these metal ions, especially Fe, in ␤-sheet formation
is rather unclear. Among them Zn2+ is very efficient inductor of
fast precipitation of the amyloid peptide leading to formation of
protease-resistant aggregates, whereas Cu and Fe demonstrate lim-
ited ability to induce aggregation in the physiological pH range that
 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
Fig. 8. Two pathways of amyloidogenic protein aggregation.
Source: Figure reproduced from Ref. [157] with permission.
drastically increases in slightly acidic pH [158–162]. The proamy-
loidogenic activity of Zn is neuroprotective rather than neurotoxic
effect, since Zn attenuates A␤ toxicity in cortical cultures. The
possible mechanism of action might be interrelated with compe-
tition with Cu for the peptide binding sites thus reducing the Cu
related Fenton type redox chemistry of amyloid-␤ species. In gen-
eral, the role of Cu depends on the conditions and type of aggregate
[163,164], since some reports suggest the inhibitory effect of Cu on
the amyloidogenic processes [158,165,166].
A number of hypothetical mechanisms of A␤-induced neu-
rotoxicity have been proposed. All of them require the peptide
to aggregate, all of them were toxic, despite what the actual
aggregation state was. Although generally speaking, most of the
aggregation states have been poorly characterized, there is a grow-
ing interest in the scientific community in oligomers that are
particularly toxic. Oxidative stress induced by Cu interaction with
hallmark peptide or the pool of labile Fe may indeed contribute to
the formation of oligomers through the formation of non-native
covalent bonds, e.g. dityrosine [167,168].
The aggregation properties of ␣-synuclein, the key PD protein,
rely on the N-terminal domain of the protein. The key step is the
transformation of the native protein to the partially folded inter-
mediate that gains the ␤-sheet content. The residues 8–16 were
claimed to be critical to the formation of ␤-sheet structure [169].
However, these observations are in contrast to another study that
suggests that residues 64–100 regulate protein aggregation [170].
Current research has suggested that presence of metal ions like Cu
may promote self-oligomerization of the protein [171]. The actual
mechanism leading to self-association relies on induction of con-
formational changes rather than induction of aggregation. Beside
copper, aluminum and iron were assigned the most effective pro-
moters of polymerization [172]. Metal ion concentrations required
to cause the aggregation are well above physiological values sug-
gesting that the metals themselves are not necessary for the process
and promote the aggregation through oxidative stress induction
[173]. On the contrary other reports indicate that even at physio-
logical concentration Cu2+ may accelerate the rate of ␣-synuclein
aggregation [123].
The biological activity of ␣-synuclein is feasibly related to the
control of vesicular transport of neurotransmitters within the
dopaminergic neuron. This requires the protein to interact with
the vesicular membranes that influences its structure and pro-
motes aggregation, where the membrane surface serves as “seed”
for the polymerization [174,175]. The predominantly ␣-helical
2137
membrane bound form is believed to have higher propensity for
the formation of toxic oligomeric species, that form pores in the
membrane of the vesicle [176]. Coordination of Cu2+ could lock
the protein in ␣-helical conformation thus leading to increased
toxicity [177]. Another factor that may stabilize the toxic oligomers
are dopamine-quinones that are the components of the modified
form of neuromelanin [178].
The basic concept of so-called “prion hypothesis” is that mis-
folded prion protein interacts specifically with host PrPC catalyzing
its conversion to the pathogenic form of the protein. The true neu-
rotoxic species, like in AD and PD is almost certainly represented by
the oligomeric intermediate. Available data suggest that neurotoxic
infectious species are represented by the oligomeric intermedi-
ates that act as a seeds to bind PrPC and catalyze its conversion
into the misfolded form by incorporation into the growing polymer
[179,180].
At the same time long PrPSc polymers break into smaller pieces
that cause the increase in the number of effective nuclei to pro-
mote further conversion of PrPC to its pathogenic form [181]. Metal
ions may influence the equilibrium between different forms of PrP
aggregates. Copper inhibits the potential of the protein to aggregate
[182], although in some cases non-specific interactions accelerate
aggregation [183]. The former hypothesis is supported by the obser-
vations that critical metal ion increases prion infectivity in animal
models [184], since it promotes oligomer formation in other sys-
tems [167,168]. Manganese binding to PrP may generate the form
of the protein that may act as a seed catalyzing the aggregation,
with no effect of the metal on the aggregation itself [185].
The nucleation dependent mechanism of aggregation provides
a reasonable explanation for the infectious nature of prions. More-
over, it comprises most reliable model elucidating the mechanism
of other neurodegenerative disorders [149,186]. These observa-
tions suggest that protein misfolding has an intrinsic ability to
be transmissible. This raises the question as to whether or not
neurodegenerative diseases are transmitted by infection through
a prion-like phenomenon [181].
Although it is theoretically possible, biological and pharmacoki-
netic barriers would prevent some amyloid aggregates, e.g. toxic
oligomeric species, from acting like prions [150]. It is likely to be
the problem especially for some of the intracellular aggregates like
Lewy bodies in PD. The unusual resistance of PrPSc to proteases
may be the key in the efficiency of prions as infectious agents [187].
The other reason for non transmissibility of neurodegenerative dis-
eases other than prion diseases is the formation of hyperstable
2138 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141
aggregates that are poor at propagating the misfolding [188]. In
fact fragmentation of aggregates is essential for effective propa-
gation of prion diseases [189,190]. Despite that, although the key
proteins of amyloid-like diseases are less hardy than prion, they
seem to spread in an infection-like manner within regions of the
brain most vulnerable to the disorder [157].
6. Oxidative stress
All the mentioned neurodegenerative diseases, although
exhibiting different symptoms and affecting different parts of the
brain, share many common features such as impaired mitochon-
drial function [66] and increased oxidative damage [191].
Mitochondria play key role in regulating cellular survival and
death [192] and they are an important source of reactive oxygen
species (ROS) within most mammalian cells [193–195] such that
mitochondria damage highly contributes to neurodegeneration.
Free radicals are normally produced in the organism and they
can interact with cellular constituents. Such reaction is strongly
dependent on aging yielding activation of cytosolic stress signaling
pathways. The term oxidative stress is usually referred to the level
of oxidative damage in a cell caused by ROS. They can cause serious
injuries affecting proteins, nucleic acid and lipid membranes. In the
healthy state ROS toxicity is normally handled (ROS are converted
into metabolically nondestructive molecules or are scavenged after
their formation) and any ROS damage is readily repaired. Once
triggered, ROS induced damage is often irreversible and creates a
positive feed-forward process, such that, when mitochondrial func-
tions are impaired by ROS further ROS generation is favored. This
vicious relationship finally yields oxidative stress and accelerated
aging.
The generation of free radicals is strongly related to the involve-
ment of redox-active trace metals. In particular Fe2+ /Fe3+ and
Cu+ /Cu2+ redox couples modulate the redox state of the cell which
should be strictly maintained to physiological limits.
Copper induced oxidative stress can occur by means of two dif-
ferent mechanisms. The first one is related to ROS generation via
a Fenton-like reaction [196,197]. Cu2+ , in the presence of superox-
ide anion radical or biological reductants, can be reduced to Cu+
(Cu2+ + O2 − → Cu+ + O2 ) which in turn, catalyzes the formation of
reactive hydroxyl radicals through the decomposition of hydrogen
peroxide via the Fenton reaction (Cu+ + H2 O2 → Cu2+ + OH• + OH− )
[198–200]. The resulting hydroxyl radical, extremely reactive, can
cause extensive injury to biological molecules resulting in lipid
peroxidation and DNA damage.
Copper modulates oxidative stress by also reducing the levels
of glutathione [201]. Normally glutathione (GSH) concentration
in the cells is in the millimolar range. GSH acts as potent cel-
lular antioxidant and as substrate for several enzymes removing
ROS. Glutathione is involved in intracellular copper metabolism
and it can modulate copper toxicity by directly coordinating the
metal with high affinity [202]. Moreover the stabilization of copper
reduced state, makes it unavailable for redox cycling.
Alteration of copper homeostasis, with the formation of an
extensive pool of copper, might result in reduced glutathione lev-
els [203], which in turn may enhance the cytotoxic effect of ROS
by favoring the copper catalytic activity and as a consequence ROS
production.
Cu+ may catalyze free radical oxidation of A␤ and ␣S via the
generation of free radicals by the Fenton reaction. The neurotoxi-
city of Cu2+ –A␤ interaction is correlated with the metal reduction
to Cu+ and subsequent generation of hydrogen peroxide that
can be catalytically decomposed to form hydroxyl radical [204].
Investigations on electron transfer reactivity of Cu(I)/Cu(II)–A␤
complexes reports conflicting results, recent observations support
the occurrence of preorganization mechanism characterized by
small positive reduction potential (0.3 eV) [205].
As copper, Fe2+ can generate reactive hydroxyl radicals from
the Fenton reaction (Fe2+ + H2 O2 → Fe3+ + OH• + OH− ) [200]. The
hydroxyl radical is highly reactive, its reaction with DNA bases
or deoxyribose backbone might produce damaged bases or strand
breaks yielding to deleterious effects [206].
As a matter of fact, iron can stimulate (i) free radical forma-
tion, (ii) increased protein and DNA oxidation in the Alzheimer’s
and Parkinson’s brain, (iii) enhanced lipid peroxidation, and (iv)
decreased level of cytochrome c oxidase and advanced glycation
end products. In addition the iron-dependent HIF-1 prolyl-4-
hydroxylase might be activated by excess of iron in brain such
to modulate the proteasomal-mediated degradation of HIF. Iron-
chelating drugs stabilize HIF-1, which, in turn, would transactivate
the expression of established protective genes, including vascular
endothelial growth factor (VEGF), erythropoietin, aldolase and p21
[207].
Contrary to copper and iron, zinc is a redox inert metal and
does not take part in oxidation-reduction reactions. However zinc
deficiency has been associated with increased levels of oxidative
damage including increased lipid, protein and DNA oxidation [208].
Zinc can act as an antioxidant by protecting sulfhydryl groups of
proteins against free radical attack and by behaving as antagonists
of redox-active transition metals, such as iron and copper [209].
In the latter case the redox active metal is pushed off its binding
site and it is replaced by an isostructurally similar redox-inactive
metal (e.g. copper replacement by zinc) [210]. Such substitution
allows the redox metal to leave the cells reducing the formation of
hydroxyl radicals.
The beneficial effects of zinc have been also ascribed to the
induction of other species that serves as definitive antioxidants.
To this end, metallothioneins (MTs) seem the most effective pro-
teins [211]. Recent studies have shown that the MTs represent a
connection between cellular zinc and the redox state of the cell
[212]. Under conditions of high oxidative stress, changes in the cel-
lular redox state result in release of zinc from metallothionein as a
result of sulfide/disulfide exchange. Finally it has been shown that
metallothioneins efficiently prevents deleterious redox activity
associated with copper binding to both ␣S and A␤ [213,214,75,215].
7. Metal based therapeutic strategies for neurogenerative
disease
As largely discussed Cu, Fe and Zn play key roles in many
neurodegenerative diseases. Many reports support the use of
compounds that modulate metal binding to neurodegenerative
proteins as promising therapeutic strategy for neurodegeneration,
especially for AD. The use of metal chelators results in regulation
of metal-induced A␤ aggregation and neurotoxicity both in vitro
and in vivo [7,216–218]. These compounds, differentiate from
the high affinity metal depleting chelators traditionally used for
metal overload disease (e.g. hemochromatosis (Fe) and Wilson’s
disease) and they are based on selectively targeting the copper
and zinc bound to A␤. They are usually referred to metal–protein
attenuating compounds (MPACs) [217]. Among these compounds,
clioquinol and 8-hydroxiquinoline improved cognition in phase
II clinical trials [219,220]. Recently, copper and iron chelators
have resulted effective in both inhibiting A␤ fibrils aggregation
and potentially capable to cross the blood–brain barrier (BBB)
[221–226]. Similarly a novel prochelator, HLA20A has been devel-
oped for AD targeting. The effective molecule HLA20, released
after HLA20A binding to acetylcholinesterase (AChE), modulates
APP regulation and A␤ reduction and equilibrate metal ions con-
centration in the brain [227]. Finally, metal based drugs (platinum
and/or ruthenium complexes), inhibiting A␤/metal interactions
 H. Kozlowski et al. / Coordination Chemistry Reviews 256 (2012) 2129–2141 2139

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