Copper and Copper Proteins in Parkinson’s Disease
Sergio Montes, Susana Rivera-Mancia, Araceli Diaz-Ruiz,
Luis Tristan-Lopez, and Camilo Rios
Neurochemistry Department, National Institute of Neurology and Neurosurgery “Dr. Manuel Velasco Su´
Correspondence should be addressed to Camilo Rios; firstname.lastname@example.org
Received 12 October 2013; Accepted 9 December 2013; Published 8 January 2014
Academic Editor: Ver´
erez de la Cruz
Copyright © 2014 Sergio Montes et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copper is a transition metal that has been linked to pathological and benecial eects in neurodegenerative diseases. In Parkinson’s
disease, free copper is related to increased oxidative stress, alpha-synuclein oligomerization, and Lewy body formation. Decreased
copper along with increased iron has been found in substantia nigra and caudate nucleus of Parkinson’s disease patients. Copper
inuences iron content in the brain through ferroxidase ceruloplasmin activity; therefore decreased protein-bound copper in brain
may enhance iron accumulation and the associated oxidative stress. e function of other copper-binding proteins such as Cu/Zn-
SOD and metallothioneins is also benecial to prevent neurodegeneration. Copper may regulate neurotransmission since it is
released aer neuronal stimulus and the metal is able to modulate the function of NMDA and GABA A receptors. Some of the
proteins involved in copper transport are the transporters CTR1, ATP7A, and ATP7B and the chaperone ATOX1. ere is limited
information about the role of those biomolecules in the pathophysiology of Parkinson’s disease; for instance, it is known that CTR1 is
decreased in substantia nigra pars compacta in Parkinson’sdis ease andt hata mutation in ATP7B could be associated with Parkinson’s
disease. Regarding copper-related therapies, copper supplementation can represent a plausible alternative, while copper chelation
may even aggravate the pathology.
Parkinson’s disease is an age-associated chronic condition;
it is the second most common neurodegenerative disorder,
aecting an important fraction of world population. It is esti-
years and older will be as high as 80 million . e costs, at
the personal and national health care system levels, continue
to rise . e mean age at the onset of Parkinson’s disease is
55 years , and its prevalence dramatically increases aer
Clinically, Parkinson’s disease is characterized by four car-
dinal symptoms: tremor at rest, muscle rigidity, slowness of
movement (bradykinesia, akinesia), and changes in posture
(instability). Usually, tremor begins unilaterally and then
becomes bilateral . Motor symptoms develop distal, and
thus, although tremors in the hands are frequently the rst
observed, tremors in the face are also common. Walking can
be especially dicult for patients; because of the postural
instability, patients have a tendency to fall. As a whole, this
combination of symptoms leads to disability and dependency.
Parkinson’s disease patients, in the long term, become depen-
dent on others for daily living activities, such as dressing
or feeding, and, therefore, the quality of life of patients suf-
fering from Parkinson’s disease is considerably diminished
. Along with movement alterations, Parkinson’s disease
patients show very important and debilitating nonmotor
symptoms, such as autonomic dysfunction, cognitive abnor-
malities, sleep disorders, mood disorders, pain, and sensory
e main pathological hallmark of Parkinson’s disease is
the loss of dopamine-producing neurons, whose cell bodies
are located in the substantia nigra pars compacta, as well as the
presence of aggregates of misfolded proteins (mainly alpha-
synuclein) and other materials, known as Lewy bodies .
e dopaminergic cells in this region project terminals to the
caudate/putamen nuclei; therefore, as a consequence of cell
death, a decreased dopamine content is observed in the
patients. Although a considerable portion of the cases of this
Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume 2014, Article ID 147251, 15 pages
2Oxidative Medicine and Cellular Longevity
disease are linked to genetic defects , the causes behind
Parkinson’s disease are uncertain in the vast majority of cases,
and the disorder is considered multifactorial. In this regard,
many theories have been developed to explain the cause of
protein aggregation and mechanisms underlying cell loss,
including the overproduction of free radicals associated with
mitochondrial dysfunction (either cause or consequence),
alterations of the ubiquitin-proteasome system, inamma-
tion, and exposure to environmental pollutants [8–10]. Alter-
ations in transition metal storage, transport, and cellular
handling have gained attention in neurodegenerative diseases
, particularly as early postmortem reports showed iron
accumulation and decreased copper levels in brains from
Parkinson’s disease patients [12,13].isndingisespecially
important because both metals are involved in the generation
and propagation of free radicals, as well as in protein precip-
itation, as a result of their redox properties. Copper is a special
case because, in addition to the previously mentioned mech-
such as Cu/Zn-SOD and ceruloplasmin. is duality makes
copper interesting for the study of neurodegenerative dis-
eases. e objective of the present review is to gather and
summarize information concerning copper, copper-related
proteins, and mechanisms of damage and protection related
to Parkinson’s disease pathophysiology.
2. Free Copper as a Cause of Damage
ere is important evidence in the literature concerning
the role of free copper as deleterious for neurodegenerative
diseases; this review focuses on its role in Parkinson’s disease.
e main eects of copper are mediated by its redox capacity
and thus by its ability to initiate, maintain, or even potentiate
the generation of free radicals. In addition, copper has been
involved in the inclusions of proteins (as consequence of
misfolding or brillation) and gains of function in copper
cases, the damaging aspects of copper are present when this
metal is present as free ion or linked to low molecular weight
ligands, is released from copper-containing enzymes due to
the surrounding conditions, or shows evident alterations of
Occupational studies have noted that long-term exposure
(20 years) to copper and manganese increases the risk of
Parkinson’s disease . In agreement with these studies,
other environmentally based studies within urban popula-
tions have shown that the incidence of Parkinson’s disease is
greater in those areas with important emissions of copper or
manganese; in such cases, the relative risk for copper was 1.1,
within a 95% condence interval from 0.94 to 1.31, meaning
that copper exposure barely reached signicance .
A meta-analysis performed by Mariani and cols. con-
sidering copper and iron levels in the serum, plasma, and CSF
showed no dierences between Parkinson’s disease patients
and controls; they found that meta-analysis contributed to an
increased dispersion of data analyzed and, thus, that the dif-
ferences observed in individual studies were diluted. ere-
fore, they performed a replication study with newly recruited
Parkinson’s disease patients and controls, but again, they
found no dierences from the results reported for other Par-
kinson’s disease populations .
Copper is a transition metal that, similar to iron, partici-
pates in the cascade of free radical generation as a catalyst in
Fenton chemistry , which also involves hydrogen perox-
ide (this is especially important in brain areas metabolizing
biological amines such as dopamine because hydrogen per-
oxide is a byproduct of the monoamine oxidase metabolism).
e Fenton reaction, Cu(I) + H2O2↔Cu(II) + OH−+OH
turns the relatively stable hydrogen peroxide into the highly
reactive hydroxyl radical, which is known to react with lipids,
proteins, and nucleic acids . It is important to note that in
order for copper to act as a catalyst for the Fenton reaction, at
least two conditions must be fullled: (1) the oxidation state
In this regard, some groups have reported that copper con-
centrations in CSF are higher in Parkinson’s disease patients
than in controls [19–21] and, furthermore, that free copper in
biochemicalmarkerofthedisease. e increased free
copper in CSF could imply that copper is “leaking” from
out of cells, as will be discussed later. Free copper in CFS may
also have other interpretations, considering that tissue from
Parkinson’s patients is diminished in areas intimately related
to the pathology such as the caudate nucleus or the substantia
nigra [12,22]. erefore, one may consider that the loss of
copper in those areas could be an important event, not only
the maintenance of endogenous antioxidant systems, that is,
CU/Zn-SOD. In addition, copper is related to iron control
metabolism. As will be discussed later, decreased copper
may be linked to iron accumulation through a decreased
ferroxidase activity, eected mainly by ceruloplasmin ;
as diminished in samples from patients with Parkinson’s
disease [20,24,25]. Iron accumulation in the basal ganglia
represents a problem by itself because it is well known that
iron participates in the formation and propagation of reactive
oxygen species . In fact, the direct injection of iron into
the substantia nigra causes tissue damage and decreased
dopamine and metabolites, which is considered a model of
Parkinson’s disease .
In an interesting in vitro study, Spencer et al. showed
that copper ions facilitate the oxidation of dopamine and
other related catechols, such as L-Dopa and 6OH-dopamine.
e complexes resulting from dopamine oxidation products
e ndings in this paper are relevant because they suggest
another mechanism of damage to copper, specically in
Although the eect of copper relies on an oxidative mech-
anism, this eect diers from the catalysis of copper in the
Fenton chemistry mentioned above.
Oxidative Medicine and Cellular Longevity 3
Some studies dealing with the copper load in the organ-
ism have been carried out; from these, a recent study showed
that rats exposed to copper (1 mg/L) in the drinking water for
four weeks presented not only liver damage, measured as
increased serum transaminases, but also an increase of 50%
in the brain metal content. e overload of copper was linked
to oxidative parameters such as diminished GSH and lowered
SOD activity, as well as to increased levels of the lipid oxida-
tion marker malondialdehyde .
e direct injection of copper sulfate into the substantia
nigra of rodents has also been tested, and the authors assayed
several copper doses, nding that noxious copper eects
began at 50 nmoles intranigral, which is vefold greater than
the values of iron necessary to produce damage. is injury
consisted of decreased dopamine, increased oxidative stress,
and apoptosis with a sensitive loss of TH immunoreactivity
. In this paradigm, copper served as a toxin for dopamin-
thus acted as a catalyst for free radical overproduction.
Lewy bodies are intracellular inclusions formed mainly
by alpha-synuclein, a protein of unknown function that is
present close to synaptic terminals; the oligomerization of
this protein is considered a key event in the setup or devel-
opment of the disease . One of the reported copper-dam-
aging mechanisms is the oligomerization of alpha-synuclein
; in fact, it is claimed by some authors that copper is highly
ecient in producing the oligomerization of alpha-synuclein
 and that this metal, and not iron, is selectively able to
brillate alpha-synuclein .atwasalsorelatedtothe
ability of copper to cause oxidative damage because the alpha-
synuclein oligomerization is linked to damage to the mito-
chondria and electron chain transfer.
3. Copper as an Essential Metal
Apart from the information described in the preceding para-
per is considered an essential metal . ere is a complex
system for the absorption, distribution, storage, and handling
of this transition metal; the collection of mechanisms of
transport will be considered in a proper section ahead in this
review. In this section, we discuss the general physiological
roles of copper, while the relationship of specic copper pro-
teins and Parkinson’s disease will be discussed in the follow-
e mutation of genes involved with copper transport
shows two extreme cases regarding brain copper, deciency
and overload. In the case of deciency, the mutation of the
ATP7A transporter causes Menkes disease, which is charac-
terized by severe deciency of copper all over the organism.
Individuals carrying the defective gene die at an early age,
evidencing the pivotal role of copper in development. Menkes
patients show severe seizures and a disruption of the brain
energy metabolism . On the other extreme, Wilson’s dis-
ease results from the mutation of a dierent transporter pro-
tein, ATP7B, which is involved in the transport of copper into
the bile and ultimately in copper excretion . Individuals
suering from Wilson’s disease show excess of brain copper,
basal ganglia degeneration, movement disorders, psychiatric
manifestations, and cirrhosis because of the copper burden
in the body . Again, the clinical manifestations of both
diseases indicate the importance of maintaining appropriate
Copper is required in a myriad of reactions in cell
metabolism , particularly in the brain because this organ
has a high respiratory rate and is prone to oxidative stress. In
this regard, one of the most important physiological functions
of copper-dependent proteins relies on their redox capacity.
Copper not only participates in the quenching of reactive
oxygen species as a cofactor in Cu/Zn-SOD but also con-
which transfers electrons between Complexes III (Coenzyme
Q-Cyt C reductase) and IV (Cyt C oxidase) at the inner
membrane of mitochondria. It also participates in neuro-
transmitter synthesis (dopamine beta-hydroxylase), neuro-
transmitter metabolism (diamine oxidase and monoamine
oxidase), the handling or storage of other metals (metalloth-
ioneins, ceruloplasmin, and haephestin), and extracellular
matrix formation (lysyl oxidase), among other functions .
Some other proteins related to central nervous system pathol-
anity for copper. It is now recognized that the interaction
between copper and proteins could be a key event for neuro-
e participation of copper in the brain physiology is
not limited to its incorporation into redox-sensitive proteins;
for example, Schlief and cols. showedthatincultured
hippocampal neurons, copper is released by the neural stim-
ulation of NMDA-type glutamate receptors. e release of
copper was observed as a process that required a calcium
signal but did not operate by the classic fusion of neurotrans-
mitter vesicles with the membrane. In addition, NMDA
receptor activation induced the copper transporter ATP7A
to relocate into synaptic active zones. is eect is related
to the replenishment of synapsis-related copper, indicating
that the continuous use of the glutamatergic synapsis ensures
the availability of copper to be released. Other studies have
shown that copper is enriched in synaptic vesicles , with
even synaptosomes being able to reuptake copper. e copper
concentration on the synaptic cle ranges from 0.2 to 1.7 𝜇M,
but the intracellular neuronal concentrations can reach up
to 3-fold that value. is indicates that specic systems are
activated to concentrate copper inside the cell and to keep it
for release upon stimulation, rendering hundred micromolar
concentrations in the synaptic space during neuronal activity.
As a whole, this evidence suggests that copper plays a mes-
senger, signaling role in the synaptic space.
Electrophysiological studies have shown that, in low con-
centrations, copper is able to inhibit currents induced by
NMDA agonists , suggesting allosteric modulation in
excitatory glutamate signals. e studies of Vlachov´
 showed that copper occupies a dierent site in the
NMDA than that of glutamate or glycine. e IC50 for copper
inhibition in the patch clamp preparation was 0.27 𝜇M,
implying some potency in the modulation of NMDA recep-
tor. AMPA/Kainate receptors in the cortex are also modied
4Oxidative Medicine and Cellular Longevity
by copper (IC50 = 4.5 𝜇M) . Other studies have character-
copper at a concentration of 50 𝜇Mwasabletodisruptlong-
term potentiation by a mechanism involving presynaptic
AMPA receptors . e eects of copper are complex and
time-dependent. While the acute exposure of the neurons
to copper produced a blockage of neurotransmission, when
cells were preexposed to the metal 3 hours before recording,
copper facilitated the glutamatergic response in an AMPA
receptor-mediated manner . is nding was related to
the recruitment of new receptors in the membrane and the
anchoring of the PSD95 protein. e authors further con-
cluded that the release of copper in the glutamatergic synapse
seems to enhance and maintain communication between
Copper has also shown modulatory properties in GABA
A receptors from cortex membranes, due to the reduction of
Cl−currents in a concentration-dependent fashion . Fur-
ther studies have determined that copper inhibits currents in
GABA receptors with an IC50 = 2.4 𝜇M. Copper seems to
act in the gating system of the GABA receptor channel . A
recent study has shown copper blocks with far more potency
against extrasynaptic GABA receptors than synaptic GABA A
receptors. In such conditions, copper may interfere with the
tonic inhibition elicited by GABA .
Electrophysiological studies in neurons from rat olfactory
synapsis, such as the inhibition of TTX-sensitive sodium
channels, delayed responses in rectifying K+channels, and
the inhibition of voltage-dependent calcium channels .
4. Copper-Binding Proteins in
Several copper-dependent enzymes and copper-binding pro-
but we restrict our review to those involved in Parkinson’s
disease through either protective or damaging mechanisms.
4.1. Alpha-Synuclein. Alpha-synuclein is a protein of
unknown function that is enriched at the presynaptic termi-
nals of many neurons. Alpha-synuclein is strongly implicated
in Parkinson’s disease and other neurodegenerative disorders,
such as dementia with Lewy bodies, multiple system atrophy,
and Alzheimer’s disease (nucleopathies). All of these diseases
are characterized by intracellular aggregations of proteins
called Lewy bodies, which are particularly rich in lamentous
Alpha-synuclein is present in the plasma and cerebro-
spinal uid of healthy subjects and Parkinson’s patients [52,
53]. An important issue to highlight is that oligomer protein
levels are higher in Parkinson’s individuals than in paired
subjects; therefore, the polymerization of alpha-synuclein,
although not exclusive to the disease, is clearly related. It has
been observed that the duplication or triplication of the gene
encoding alpha-synuclein is related to a familiar form of the
disease . Alpha-synuclein is physiologically catabolized
defective in patients with idiopathic forms of the disease
. Alpha-synuclein aggregation causes dysfunction of the
ubiquitin-proteasome system .Basedonthisevidence,
cell loading with alpha-synuclein seems to be another factor
e alpha-synuclein protein binds copper. Although
some studies have suggested dierent quantities of metal per
copy of the protein, the most consistent results show two sites
for copper binding per monomer at nanomolar and micro-
molar concentrations . ese sites implicate a histidine
important sites for metal binding. us, alpha-synuclein has
brillation, a phenomenon that yields further precipitation
of similar proteins and is ultimately thought to represent
the “seeding” that gives rise to Lewy bodies . is is in
accordance with the fact that copper is encountered in Lewy
bodies at relatively high concentrations.
e binding of copper to alpha-synuclein is an important
event for the setup/development of the disease because
several interrelated consequences seem to derive from it. e
rst, as has already been discussed, is the conformational
change of alpha-synuclein that facilitates brillation and
aggregation [32,33]. ere is experimental evidence showing
that the copper-alpha-synuclein complex induces changes in
copper’s redox properties, and, thus, this complex has been
linked to increased H2O2production from ascorbic acid
oxidation and, in turn, the dopamine oxidation by H2O2.e
Cu-alpha-synuclein complex itself is also capable of oxidizing
other endogenous antioxidants, for example, GSH . An
interesting study by Davies and cols. showedthatrecom-
binant alpha-synuclein binds both copper and iron; the load-
ing of copper into the protein produced small changes in the
iron binding kinetics, suggesting dierent binding sites for
both metals. Furthermore, alpha-synuclein showed ferrire-
ductase activity. ese authors linked their ndings with a
physiological need for Fe(II) in dopamine synthesis through
tyrosine hydroxylase and with a pathological scenario,
involving the participation of Fe(II) in the Fenton reaction,
increasing the oxidative stress of Parkinson’s disease.
4.2. Ceruloplasmin. Ceruloplasmin is a multicopper con-
taining glycoprotein that is mainly biosynthesized in the
liver . Copper-bound ceruloplasmin is released to the
blood by the liver, where the newly formed enzyme is bound
to copper early in its synthesis in the secretory pathway,
along with the incorporation of a polysaccharide moiety .
amine oxidase, antioxidant, anti-inammatory, nitric oxide
oxidase, and glutathione peroxidase, among other actions
. e most prominent functions of ceruloplasmin are
the following: (a) plasma copper binding (95% of circulating
copper is bound to ceruloplasmin) and (b) iron homeostasis
by means of its ferroxidase activity, with implications for the
control of free radical production, as discussed below.
e soluble form of ceruloplasmin in the blood is
involved in the oxidation of the iron to be incorporated
Oxidative Medicine and Cellular Longevity 5
into transferrin ; therefore, severe copper deciency is
characterized by diminished ferroxidase activity, leading to
iron retention in the liver and defective iron distribution to
the other organs.
In the brain, ceruloplasmin is synthesized by astrocytes,
to glycosylphosphatidylinositol . e glial-derived ceru-
loplasmin is intimately linked to iron eux from the brain
because, as in the liver, ceruloplasmin oxidizes iron that has
been previously transported by ferroportin to be incorpo-
rated into transferrin . Aceruloplasminemia, a genetic
condition producing a lack of function of circulating cerulo-
plasmin, is characterized by iron accumulation, remarkably
in the brain, where it is associated with neurodegeneration
and extrapyramidal parkinsonian symptoms . In fact,
brain, for example, aceruloplasminemia, ferritinopathy, and
a syndrome of neurodegeneration with brain iron accumula-
tion, are characterized by neuronal death and motor mani-
festations similar to those of Parkinson’s disease .
As it has been continuously mentioned, the basal gan-
glia of Parkinson’s disease patients show iron accumulation
and decreased copper [12,13]; those ndings could be in
agreement with the fact that copper-dependent ferroxidase
activity promotes the oxidation of Fe2+ to Fe3+ sothat
Fe3+ can be removed from the brain. erefore, it is possible
that the decreased content of copper in the substantia nigra
is related to the increased iron in this area, as a result of the
defective ferroxidase activity. Accordingly, copper-dependent
ferroxidase activity has been reported to be diminished in the
plasma and cerebrospinal uid of patients with Parkinson’s
disease [17,20,24]. Iron accumulation seems to be an impor-
ease; for example, it has been found that iron, observed by
magnetic resonance imaging, begins to accumulate in the
substantia nigra beforetheappearanceofparkinsoniansymp-
toms. It has also been proposed that iron signals in the
substantia nigra can be a predictive marker of the disease
. Other studies have reached a similar conclusion; taking
advantage of iron echogenicity and of the temporal bone
window at the mesencephalon level in humans, it is possible
to determine echogenic areas in the substantia nigra by using
transcranial ultrasound . Further studies with post-
mortem tissues from Parkinson’s disease patients have shown
that increased echogenicity is related to iron . e specic
accumulation of iron in the substantia nigra has served to
propose the echogenicity of this region as a characteristic
feature of Parkinson’s disease or even a prognostic index of the
disease’s development. In fact, a negative correlation between
brain iron accumulation and copper-dependent ferroxidase
plasma activity  has been reported. Other studies have
conrmed this phenomenon; using magnetic resonance
imaging, two populations of Parkinson’s disease patients were
apparent iron accumulation. It is worth noting that the rst
group also showed diminished ferroxidase activity .
In a study determining serum ceruloplasmin (protein,
not the ferroxidase activity), a positive correlation between
ceruloplasmin and the age of onset of Parkinson’s disease was
found; the stratication of the sample with a cut-o point of
60 years as the age of onset showed decreased ceruloplasmin
in the serum in earlier onset patients. Serum copper was not
dierent between the early and late onset Parkinson’s disease
in comparison with a control group .
A mechanism of ceruloplasmin dysfunction regarding its
ferroxidase activity was proposed in the study by Olivieri et
al. ; they found that the electrophoretic pattern of cerulo-
plasmin was dierent between CSF samples from Parkinson’s
disease patients and controls and that changes were related to
oxidative modications, for example, protein carbonylation.
e experimental oxidation of ceruloplasmin produced a
similar pattern to those obtained from patient’s CSF. Further-
more, oxidized ceruloplasmin produced an accumulation
of iron in cultured cells, whereas functional ceruloplasmin
prevented the iron load and stimulated the synthesis of
proteins related to iron storage and eux. e authors further
discussed the implications of oxidized ceruloplasmin because
this event would lead not only to alterations in the iron eux
from the brain but also to the possibility of releasing copper
from the enzyme, yielding free copper ions to be available for
the production of even more free radicals.
Further evidence of ceruloplasmin malfunction in
Parkinson’s disease has also been derived from studying the
ceruloplasmin gene; ve variants of ceruloplasmin were
found in a screening study in a cohort of patients who dis-
played increased substantia nigra echogenicity .
In a recent study, Ayton et al.  found that postmortem
substantia nigra from Parkinson’s disease patients showed
increased iron and decreased copper; no dierences were
observed in the ceruloplasmin protein levels. However, they
did nd severely reduced ferroxidase activity. ey also found
that decient ceruloplasmin mice displayed neuronal cell
death in the substantia nigra and that neurodegeneration was
partially reduced by the use of an iron chelator. Finally, they
found that the peripheral administration of ceruloplasmin
(5 mg/kg, i.p.) partially prevented both the increased iron in
the substantia nigra and the dopaminergic cell death induced
by MPTP. Ceruloplasmin, due to its antioxidant properties
and its role as an iron regulator in the brain, remains an
attractive target for new therapeutic strategies in Parkinson’s
4.3. Cu/Zn-SOD. Superoxide dismutases are a group of
enzymes that catalyze the reaction of superoxide to hydrogen
peroxide . e function carried out by those enzymes in
the brain is important because superoxide derives from mul-
tiple sources in the cell metabolism, such as the electron tran-
sport chain as a product of one electron oxygen reduction,
and NADPH oxidase. Particularly for dopaminergic areas,
superoxide anion as a byproduct . e cytoplasmic (type
I) and extracellular (type III) superoxide dismutase isoforms
require copper and zinc as cofactors, whereas the mitochon-
drial type II isoform is Mn-dependent .
It is remarkable to note that, in a wide variety of studies
(either with human tissues or in experimental animals),
6Oxidative Medicine and Cellular Longevity
the overexpression of superoxide dismutase has been con-
stantly found to be neuroprotective; this fact underscores the
importance of SOD by itself.
Experimental evidence shows that the overexpression of
Cu/Zn-SOD in mice provides them with resistance to the
dopaminergic neurotoxin MPTP, with regard to dopamine
depletion . In a study by Nakao et al. , substantia nigra
from embryonic mice overexpressing human Cu/Zn-SOD
immunosuppressed rats. e rats were then injured with the
6-OHDA toxin to model Parkinson’s disease. Gras derived
from transgenic mice overexpressing SOD showed a 4-fold
increase in the survival of TH cells compared to those from
littermates with a regular expression of the enzyme. Cells not
Microglial cells transfected with human Cu/Zn SOD1,
when properly stimulated, are able to release the extracellular
isoform of superoxide dismutase into the medium; this
antioxidant enzyme in turn protects neurons against 6-OH
dopamine-induced cell damage . Accordingly, the expo-
sure of cultured astrocytes to dopamine favored the selective
expression of extracellular Cu/Zn-SOD (not SOD 1 or Mn
SOD), depending on the dopamine concentration itself (not
receptors or metabolism) and nuclear factor kappa-B. e
surrounding cells by expressing extracellular SOD . e
protein DJ-1, which is the product of the familiar gene for
Parkinson’s disease, PARK7, enhances the expression of type I
Cu/Zn-SOD in connection with the Erk1/2-Elk1 cascade. DJ-1
null mice were more susceptible to the injection of the toxin
MPTP due to the failure of SOD upregulation .
Physical exercise has been reported as a protective factor
in Parkinson’s disease and other neurodegenerative diseases
suspected to coincide with oxidative stress . In this regard,
experimental studies suggest that physical activity induces
SOD and other antioxidant enzymes .
Ihara et al.  found that blood from Parkinson’s disease
patients showed an increased concentration of hydroxyl
radicals and diminished Cu/Zn-SOD in red blood cells.
Parkinson’s disease patients with a higher concentration of
hydroxyl radicals in the plasma were related to an earlier
onset of the disease and higher Hoehn and Yahr stages. A
previous report showed no dierences in the SOD activity
between Parkinson’s disease patients and age-matched con-
trols; however, in Parkinson’s patients, the SOD activity
decreased signicantly with the duration of the disease ,
suggesting faster deterioration of the antioxidant ability of
Cu/Zn-SOD in Parkinson’s disease. Studies carried out with
postmortem tissues have conrmed that aging reduces the
capacity of antioxidant enzymes, including SOD, in sub-
stantia nigra only selectively, suggesting that this region is
especially susceptible, as indicated by the progression of
Parkinson’s disease .
4.4. Other Copper-Binding Proteins. Metallothioneins are a
family of low molecular weight proteins composed of a high
number of cysteine residues, conferring them with the ability
to bind metals . In the brain, metallothioneins I and
II are expressed in the glia, whereas metallothionein III is
expressed in neurons and metallothionein IV is expressed in
dierent epithelia . Physiologically, metallothioneins are
linked to zinc homeostasis in the brain. e high anity of
metallothionein for copper and the described release of cop-
per in glutamatergic synapsis suggest that metallothioneins
may buer copper ions in the vicinity of the synapsis. In
fact, metallothionein III binds zinc and copper under phys-
iological conditions . Metallothioneins (I and II) can be
upregulated in dierent situations, such as metal intoxication,
steroid use, and oxidative stress. In most of the cases, metal-
lothioneins exert a neuroprotective role [87,88]. e study of
metallothioneins in Parkinson’s disease is appealing because
also to scavenge directly for free radicals. Studies carried
out with postmortem tissues from patients with Parkinson’s
disease showed increased expression of glial metallothionein
Iinthesubstantia nigra and cortex; the authors considered
that the observed eect could be a compensatory mechanism
to protect glial cells from oxidative stress . It has been
observed in rodents that MPTP-induced extrapyramidal
damage reduces the expression of metallothionein I and
other antioxidant enzymes. Parkinson’s disease may aect
the endogenous antioxidant systems as a mechanism for
disease development. e transgenic mice overexpressing
metallothionein I were shown to be more resistant to the
peroxynitrite-releasing agent SIN-1; these animals were also
more resistant to the MPTP model or Parkinson’s disease
than nontransgenic controls. e eect herein observed was
related to increased coenzyme Q10 synthesis .
copper results not only in protein brillation but also in an
increased oxidative stress. In this regard, Meloni and Vaˇ
 found that the complex alpha-synuclein-Cu(II) is able
to oxidize dopamine to o-quinone in the presence of oxygen;
the process involved the cycling of Cu(II) to Cu(I) and back.
By changing dopamine with ascorbate, the authors found that
the hydroxyl radical was produced. e incubation of these
complexes with metallothionein III showed that the copper
ion originally bound to alpha-synuclein was transferred to
metallothionein, at which point the oxidative eects, dopam-
ine oxidation, and production of hydroxyl radicals were abol-
ished. Because metallothionein III is expressed in neurons,
regulating this protein seems a promising strategy, at least in
experimental Parkinson’s disease paradigms.
Amyloid precursor protein possesses a copper-binding
domain that possesses copper reductase activity . Amy-
loid precursor protein knockout mice exhibit high copper
levels in the cortex and liver. ese ndings were the basis of
a suggestion that APP is a membrane copper transporter .
In turn, animals overexpressing APP show decreased copper
in the brain . Additionally, there are reports providing
evidence of the ability of APP to enable ferroxidase activity.
It is considered that APP could be fullling the action of oxi-
dizing iron and exporting it from cells by an association with
reported eect of ceruloplasmin in astrocytes. Amyloid pre-
cursor protein knockout mice fed with a high iron diet dis-
Oxidative Medicine and Cellular Longevity 7
this eect was in turn related to oxidative stress alterations,
such as reduced glutathione and increased protein carbony-
lation. In the same study, the cortical ferroxidase activity
assigned assigned to APP was reduced only in samples from
Alzheimer’s disease and not from Parkinson’s disease ;
Parkinson’s disease remains to be fully elucidated.
bind copper and mercury . DJ-1 expression causes the
cells to become more resistant to either copper or mercury
because DJ-1 confers protection against copper-induced oxi-
dative stress. However, this protection is lost when oxidized
dopamine is present in the medium. is nding supports
other studies showing that mutant rodents lacking DJ-1 are
In addition to their function of editing amyloid precursor
proteins, presenilins are related to copper transport and
homeostasis. In fact, it is claimed that presenilin ablation may
diminish the activity of copper-dependent SODs because of
the decreased availability of intracellular copper . Further
in the transport of copper and the consequences of limited
copper for the synthesis of copper-dependent antioxidant
e fact that prion proteins may accept a considerable
number of copper ions into the extracellular space of the
synapsis has served to suggest an interesting theory regarding
prion proteins as a buering control for copper at the synapsis
. e binding of copper to prion proteins confers its
SOD activity , giving rise to the hypothesis of the gain
of function for prion proteins as a key event in the misfolding
of the proteins and the propagation of these altered forms.
5. Copper Transport
For the reasons expressed above, maintaining adequate cop-
per levels is essential to preserving normal brain functions.
For example, copper concentration in the cerebrospinal uid
is up to 100-fold lower than values in plasma  when the
cytosolic concentration of unbound copper is very low .
Under physiological conditions, most plasma copper ions
are bound to ceruloplasmin, with a small proportion of cop-
per being carried by albumin, transcuprein, and other amino
acids . However, according to experimental evidence,
neither copper-albumin nor copper-ceruloplasmin uptake
represents a signicant contribution to copper transport,
compared to free copper uptake into brain capillaries, the
choroid plexus, and CSF [105,106]. Copper enters the brain
mainly via the BBB (blood-brain barrier), although the BCB
(blood-cerebrospinal uid barrier) is also able to transport‘it
into the brain to a much lesser ex;4tent [102,107]. It is believed
that epithelial cells from the choroid plexus serve as a reser-
ATOX1 (antioxidant 1 copper chaperone) are all involved in
coppertransportintothebrain. e blood-CSF barrier
seems to maintain copper at a certain level by sequestering
copper from the blood and exporting the excess out of
the CNS and back to the blood , as demonstrated by
Monnot et al. , who found that the main direction for
copper transport is from the apical to the basolateral side
of epithelial cells. At the BCB, copper transport is regulated
by two major copper transporters: CTR1 and divalent metal
transporter-1 (DMT1) . ese transporters, together with
ATP7B together with CTR1 achieves this in the opposite
direction .e signicance of the participation of DMT-1
in brain copper transport is still controversial, but it is known
that, in the epithelial cells of the choroid plexus, there is a
coupling between copper and iron homeostasis that involves
this transporter .
5.1. Copper Transporter 1 (CTR1). CTR1isaplasmamem-
brane protein having three transmembrane domains that
form a homotrimeric pore for copper uptake . CTR1 is
present in the brain capillary endothelial cells of the BBB,
choroid plexus of the BCB, and the brain parenchyma [105,
CTR1 is responsible for transporting copper in the intesti-
nal cells and is profusely expressed in brain capillary endothe-
lial cells, where it is considered the major pathway for copper
transport from the blood into the brain . is transporter
is also expressed abundantly in the choroid plexus and, as
opposed to its function in the BBB, transports copper out of
the brain in the BCB .
CTR1 is concentrated on the apical surface in cells of the
choroid plexus; it is also found in the cytoplasm of neurons
in the visual cortex, anterior cingulate cortex, caudate, and
putamen and in cytoplasm of Bergmann glia in human
tissue. It is distributed around neuromelanin granules in the
substantia nigra , most likely regulating the acquisition of
copper by this pigment .
Studies from Davies et al. suggestthat,inthenormal
human brain with adequate cellular copper, CTR1 exists pri-
marily as an internalized protein pool, rather than as an active
membrane-bound transporter, whereas at high copper levels,
it is internalized into the cell and subsequently degraded .
To our knowledge, there are no reports about any muta-
tions of CTR1 in Parkinson’s disease; only a marked reduction
of neuronal CTR1 immunoreactivity and correlation between
CTR1 and copper levels in the substantia nigra of Parkinson’s
disease postmortem human brains have been described .
neural tissue is particularly sensitive to the loss of CTR1 func-
tion, as indicated by marked cell death in the brain and spinal
cord of zebrash in response to CTR1 downregulation .
is occurrence could be attributable to the copper depletion
caused by a decreased level of its main known transporter
to allow it to enter the brain. More studies are needed to
establish the relevance of CTR1 in Parkinson’s disease.
5.2. Antioxidant 1 Copper Chaperone (ATOX1). Human
ATOX1 is a small cytosolic protein of 68 amino acids .
In solution, ATOX1 exists as a monomer, but, in the presence
of metals, it can form dimers . ATOX1 is expressed
abundantly in the pyramidal neurons of cerebral cortex,
8Oxidative Medicine and Cellular Longevity
hippocampus, and locus coeruleus; moderately in the olfac-
tory bulb; and little in the cerebellum (except for Purkinje
e copper chaperone ATOX1 is involved in the delivery
of copper to ATP7A and ATP7B inside the cells [109,114,123].
ATOX1 levels correlate positively with copper content in the
human brain  and function as an antioxidant against
superoxide and hydrogen peroxide .
ATOX1-mediated copper transfer is accompanied by the
upregulation of the Cu-ATPase’s activity, while apo-ATOX1
can retrieve copper from the ATPases and downregulate their
ing of the entire copper-metabolic network . Cultured
Atox1−/−cells exhibit increased ATP7A levels, whereas
Atox1−/−newborn mice show low activity of several copper-
dependent enzymes [123,126,127].
As far as we know, mutations or changes in the ATOX1
levels in Parkinson’s disease have not been described. is
is an interesting molecule to study because of the functions
5.3. P-Type ATPases ATP7A and ATP7B. ATP7A an d ATP7B
are members of the P1B-subfamily of the P-type ATPases;
they catalyze the translocation of copper across cellular mem-
branes by ATP-dependent cycles of phosphorylation and
dephosphorylation . ey have eight transmembrane
domains that form a path through cell membranes for copper
translocation and a large N-terminus with six metal-binding
domains (MBDs), each comprising approximately 70 amino
acids and the highly conserved metal-binding motif GMx-
CxxC (where x is any amino acid) .
ATP7A is expressed in the brain capillaries, choroid
plexus, astrocytes, and neurons from mice [105,128–130]. In
both the epithelial cells of the choroid plexus and the capillary
endothelial cells of the brain, ATP7A is predominantly
located on the basolateral membrane, while ATP7B concen-
trates at the apical membrane .
ATP7A and ATP7B are expressed in neuronal cell bodies
in some brain regions, and both of them are expressed in the
cytoplasm of neurons in the substantia nigra;onlyATP7Bis
associated with neuromelanin .
ATP7A and ATP7B are able to deliver copper for incorpo-
ration into copper-dependent enzymes and to remove excess
of copper from cells, depending on their subcellular location
. ATP7A is important in the delivery of copper from
endothelial cells to the brain , which has been conrmed
by the fact that mice with a mutated ATP7A gene accumulate
copper in brain capillaries and suer from copper deciency
in the brain.
ATP7B, as opposed to ATP7A, is expressed in brain cap-
illaries more than in the choroid plexus anditispossibly
involved in copper transport from the blood to the CSF .
ATP 7A and ATP 7B level s are not associ ated w ith cop p er
brain levels, but their cellular location changes as copper
levels are modied . At physiological conditions, ATP7A
and ATP7B are mainly located in the trans-Golgi network
to incorporate copper into cuproenzymes; when copper
levels are high, these proteins are redistributed to post-Golgi
vesicles and even to the cellular membrane to facilitate copper
Because ATP7A has faster kinetics of copper transport
in relation to ATP7B, a predominant homeostatic role for
ATP7A and a biosynthetic role for ATP7B have been pro-
posed as mediators of the synthesis of cuproenzymes .
Enzymes such as cytochrome c oxidase, SOD1,DBH (dopam-
ine 𝛽-hydroxylase), PAM (peptidylglycine 𝛼-amidating
monooxygenase), lysyl oxidase, and tyrosinase require
ATP7A for metallation in the trans-Golgi network .
e known information about these ATPases comes
mostly from their study in Menkes disease and Wilson’s dis-
ease. Menkes disease is caused by a mutation in the gene
encoding the copper transporter ATP7A that results in severe
copper deciency in the brain , and Wilson’s disease
is caused by a mutation in the gene encoding the copper
transporter ATP7B, resulting in copper accumulation in the
brain . A specic role for these two transporters in
Parkinson’s disease has not been thoroughly investigated, but
disease to some extent .
While Wilson’s disease is an autosomal recessive disorder
caused by mutations in the ATP7B gene , it has been
hypothesized that a single mutated ATP7B allele may act as
a risk factor for (late-onset) parkinsonism [136,138]. Sechi et
UTR region in
a single allele of ATP7B gene in three sisters with levodopa-
responding parkinsonism; mutations in other Parkinson’s
disease-related genes were not found in any of the sisters.
On the other hand, as Parkinson’s disease is an aging-
the behavior of molecules that have some relationship with
its pathophysiology. In this respect, Lenartowicz et al. 
studied ATP7A expression in the mouse liver from P.05 to
P240. ey found that the expression of ATP7A decreases
during the lifespan; in fact, the ATP7A expression in adult
young animals. Interestingly, the same behavior was observed
for liver copper levels .Itwouldbeinterestingtostudy
the behavior of ATP7A expression in the brain as a function
of age and its implications on copper homeostasis.
ATP7B supplies copper to cuproenzymes such as cerulo-
plasmin . As discussed previously, ceruloplasmin activ-
ity is decreased in Parkinson’s disease; to our knowledge,
there are no studies that show or refute any relationship
between ATP7B dysfunction and decreased ceruloplasmin
activity in Parkinson’s disease.
5.4. DMT1. DMT1, also known as divalent cation transporter
1 (DCT1), transports one proton and one atom of Fe(II) in the
same direction, and it also performs a nonselective transport
for multiple divalent metals, including Mn, Cu, Co, Zn, Cd,
and Pb [141,142]. While the presence of DMT1 in the BBB
remains controversial, there are data supporting its presence
in the BCB [110,142,143], although the experiments of Zheng
et al.  suggest a minimum contribution of DMT-1 in
cellular copper uptake in the BCB.
Oxidative Medicine and Cellular Longevity 9
e upregulation of DMT1 in the substantia nigra of
Parkinson’s disease patients and in the substantia nigra of
mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine (MPTP), a neurotoxin known to induce several features
of Parkinson’s disease, has been demonstrated . Addi-
tionally, the CC haplotype derived from single nucleotide
polymorphisms (SNPs) of DMT1 was found to be a possible
risk factor for Parkinson’s disease in the Han Chinese popu-
lation . ese alterations of DMT1 in Parkinson’s disease
are believed to aect iron transport signicantly but not
6. Copper-Related Therapies
e current therapeutic strategies, such as supplying a
dopamine precursor (L-DOPA), dopamine agonists (e.g.,
pramipexole, bromocriptine), and inhibitors of dopamine
breakdown (e.g., selegiline), similar to surgical ablations or
deep brain stimulation, only provide symptomatic relief of the
motor impairment . ere is still an imperative need to
move from symptom-alleviating to disease-modifying thera-
As discussed before, the role of copper in Parkinson’s
disease is controversial, as some evidence points to a need for
increased copper levels, while other results show the opposite.
ere have been some attempts made to clarify the roles of the
Regarding the possibility of increasing brain copper lev-
els, two main options can be tested as follows: (1) to regulate
copper transporters to increase copper entry into the brain
or (2) to administer copper compounds (or copper-releasing
compounds). Regarding the rst option, further knowledge
of the function of copper transporters in Parkinson’s disease
is needed; regarding the second alternative, some strategies
have been already tested.
Using the rodent model of Parkinson’s disease induced
by MPP+(1-methyl-4-phenylpyridinium) intrastriatal injec-
tion, our group found that the administration of CuSO4
(10 𝜇mol/kg i.p.) as a pretreatment 24 h before the lesion
prevented protein nitration, TH inactivation, and dopamine
depletion and decreased the activity of constitutive nitric
oxidesynthase(cNOS)inthestriatum. It is possible that
copper antagonizes NMDA receptor responses by inhibiting
Ca2+ inux and thus inhibiting Ca2+-dependent NOS acti-
vation, reducing protein nitration . Recently, using the
same paradigm, we found that copper pretreatment increased
ceruloplasmin expression and prevented the MPP+-induced
loss of ceruloplasmin ferroxidase activity and the concomi-
tant increase in lipid peroxidation. Additionally, a slight
decrease in ferrous iron was found in the striatum and mes-
encephalon . We consider that the increased ferroxidase
activity is responsible for the decline in ferrous iron content
and the concomitant prevention of lipid peroxidation. As
such, copper-induced ceruloplasmin expression could be an
experimental strategy against the deleterious eects of iron
deposits in Parkinson’s disease.
e use of the hypoxia imaging agent Cu(II) (atsm) in
four dierent models of Parkinson’s disease has been shown
to be neuroprotective by several mechanisms, including the
inhibition of alpha-synuclein nitration and brillation. e
copper compound also showed the protection of dopamine-
producing neurons by TH immunostaining as well as the
preservation of motor function and reduced cognitive decay
EGb761 (an extract of the Ginkgo biloba tree) pretreat-
ment also blocks the neurotoxic actions of MPP+; some
of the protective actions of this extract can be attributed to
the reversing of the MPP+-induced copper depletion in the
striatum of rats and the regulation of copper homeostasis in
other brain regions .
Taking into account the possibility of increased copper
levels in Parkinson’s disease, it has been suggested that copper
chelation can be useful in the treatment of some neurode-
generative diseases, including Parkinson’s disease [21,152].
However, copper chelation was not protective against MPTP
injury  and even, as in the case of diethyldithiocarba-
mate, enhanced neurotoxicity . It is known that iron is
very harmful in Parkinson’s disease and that copper reduces
Fe uptake, possibly through DMT1 .
ere are some studies suggesting that Fe accumulation
is a consequence of copper deciency. Increased ferroportin
expression is associated with neuronal survival aer Fe over-
load . Copper-decient diets reduce ferroportin expres-
sion in the rat liver , possibly leading to Fe accumulation;
in patients with nonalcoholic fatty liver disease, a low hepatic
copper content is associated with a decreased ferroportin
expression, thus contributing to Fe accumulation . In
rats fed a copper-enriched diet, the inux of Fe into the brain
was signicantly decreased compared to that of rats fed with
the control diet . According to those studies, Fe accu-
mulation may be the consequence of copper deciency. Sup-
porting this hypothesis, Fe accumulates in several tissues
during copper deciency .
On the other hand, in a study of patients with smell dys-
function, Henkin et al.  reported that, aer repetitive
transcranial magnetic stimulation (rTMS), patients had
increased copper concentrations in the plasma, erythrocytes,
homeostasis. rTMS has been used with some success to
treat the clinical manifestations of patients with Parkinson’s
disease, resulting in improved motor performance, elevation
of serum dopamine, and improved smell and taste functions
[158–161]. It is not known whether changes in the copper
levels at a systemic level are a reex of changes in brain levels
of copper or whether the improvement observed in patients
with Parkinson’s disease and other neurological disorders
aer rTMS is due, at least in part, to modications in the
e experimental evidence discussed here shows that a
deciency of copper in Parkinson’s disease is more possible
than an excess and that copper supplementation can be a
plausible alternative to treating Parkinson’s disease. However,
need for research about the distribution of copper in dierent
compartments of the brain and other organs, therapeutic
strategies trying to adjust the copper levels in the brain
must be undertaken with caution. e severe consequences
10 Oxidative Medicine and Cellular Longevity
of copper deciency and overload can be illustrated by
Menkes disease and Wilson’s disease, respectively. Addition-
ally, although copper is required for the oxidation of Fe2+ to
avoid oxidative damage, too much copper is also toxic.
Among the interesting facts regarding copper-binding pro-
teins, we found that alpha-synuclein bound to copper acts as
a ferrireductase, thus increasing the availability of iron for the
generation of free radicals; this could be particularly impor-
tant in the caudate/putamen vulnerable regions of the brain,
because of the presence of the oxidative labile dopamine.
In addition, copper-dependent ferroxidase activity of ceru-
loplasmin has been continuously reported to be reduced in
samples from Parkinson’s disease patients. eoretically, the
decreased function of ceruloplasmin would aggravate the
abovementioned situation of alpha-synuclein. Accumulation
but the proportion in which copper or copper proteins are
responsible for iron dyshomeostasis in Parkinson’s disease
is not known accurately. e role of copper transporters in
Parkinson’s disease is an issue that also deserves further
research. Knowledge about copper compartmentalization in
brain will help to establish promissory therapeutic strategies
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
e present paper was supported by CONACYT Grant 18366
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