Metals 2015, 5, 2070-2092; doi:10.

3390/met5042070
OPEN ACCESS

metals
ISSN 2075-4701
www.mdpi.com/journal/metals/
Review

Iron, Aging, and Neurodegeneration

Dafina M. Angelova and David R. Brown *

Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK;
E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +44-1225-383133; Fax: +44-1225-386779.

Academic Editor: Grasso Giuseppe

Received: 5 October 2015 / Accepted: 2 November 2015 / Published: 6 November 2015

Abstract: Iron is a trace element of considerable interest to both chemistry and biology.
In a biological context its chemistry is vital to the roles it performs. However, that same
chemistry can contribute to a more deleterious role in a variety of diseases. The brain is
a very sensitive organ due to the irreplaceable nature of neurons. In this regard regulation of
brain iron chemistry is essential to maintaining neuronal viability. During the course of
normal aging, the brain changes the way it deals with iron and this can contribute to its
susceptibility to disease. Additionally, many of the known neurodegenerative diseases have
been shown to be influenced by changes in brain iron. This review examines the role of iron
in the brain and neurodegenerative diseases and the potential role of changes in brain iron
caused by aging.

Keywords: synuclein; amyloid; prion; Alzheimer’s disease; Parkinson’s disease;

transmissible spongiform encephalopathy; ferrireductase; microglia

1. Introduction

While the atomic nature of matter might be an absolute given in the 21st century, the role of metal
atoms in biological systems remains a developing field. In particular, the difficulty of translating between
chemical and biological systems remains central to advancing concepts that could actually lead to a
better understanding of how our minds work. At the fundamental level, we also still need to understand
how the movement of single electrons can have a significant impact on cellular mechanisms that
Metals 2015, 5 2071

influence the way we age. In biological systems the movement of electrons is often dependent on metal
ions and their role in enzyme activities or more fundamentally as co-factors in catalysis of various
reactions. The regulation of such reactions can be both positive and negative as one results in maintaining
cellular activity essential for life while the opposite can result in the generation of harmful reactive
chemical species of oxygen or nitrogen. The production of reactive oxygen species (ROS) or nitrogen
species (RNS) has been linked to changes in the brain associated with normal aging and also to diseases
that can occur during aging such as the neurodegenerative diseases [1,2]. For many neurodegenerative
diseases, especially the most common ones like Alzheimer’s disease and Parkinson’s disease, aging is a
prerequisite for developing them [3,4]. While we can measure changes in various chemicals or reactions
in the brain, the actual mechanism by which growing older makes us more susceptible to
neurodegenerative diseases is a mystery. Yet, it has quite clearly emerged that changes in certain metals,
especially iron, might be very important to both how the brain ages and to neurodegeneration.
Iron is the second most abundant metal on earth and has multiple oxidation states [5]. This makes it
a leading candidate as a co-factor for enzyme-catalyzed reactions that require electron transfer. Iron can
exist as Fe2+ (highly water soluble) or Fe3+ (much less soluble) in biological systems. Higher oxidation
states are generated by some reactions but are much less stable than Fe2+ and Fe3+. The redox potential
of Fe2+/Fe3+ is highly variable in biological systems and depends largely on the way it is coordinated
within the structure of the ligands it binds [6]. This means that its utility is very high. Iron binding
proteins can have different iron centers with specific classes of co-ordination [7]. The main classes are
heme proteins, iron-sulfur proteins, and a class that is neither. This latter group includes iron storage and
iron transport proteins, proteins coordinating a single Fe atom to histidine, glutamate or aspartate, or two
Fe atoms in an oxygen-bridged center. The latter family includes many oxygen-binding proteins as well
as Fe-oxidizing proteins such as ferritins.
Iron enters the body through the diet and is taken up via two main mechanisms [8]. The first (major)
source of iron is through the metabolism of heme, which is absorbed at the intestinal apical membrane
where the iron is released by heme oxygenase. Iron released in this way can directly enter the cellular
free iron pool, where it can either enter storage in ferritin, or be transported outside the intestinal cell.
The second uptake mechanism is at the brush border of the duodenum where it is first reduced to Fe2+
by the reductase Dcytb and then transported into the enterocytes by divalent metal transporter 1
(DMT-1) [9]. Iron taken up by this route enters the same pathway as heme iron. Once iron reaches the
blood it is largely transported by transferrin. The main uptake mechanism involves the interaction of
transferrin with its receptor at the cell surface. Internalization is through clathrin-coated pits, which
directs transferrin to endosomes. There, the transferrin undergoes a conformational change as a result of
the low pH of the endosomes causing iron release. The iron is reduced and transported to the cytosol.
Iron uptake can also occur through DMT-1, which also transports other divalent metals [10].
Within cells, iron is associated with a variety of proteins as a co-factor, but is also stored. The main
storage protein is ferritin, but other storage proteins exist such as hemosiderin [11,12]. Additionally,
cells possess a pool of free iron known as the labile iron pool [13]. This pool is considered transient but
necessary as the source of iron for newly synthesized proteins that require iron as a co-factor. The need
to tightly regulate this pool of Fe2+ is considerable given the potential of free iron to catalyze radical
generating reactions such as the Haber-Weiss reaction [14]. The production of radicals in the form of
ROS and RSN has the potential to result in unwanted protein modifications and lipid peroxidation. These
Metals 2015, 5 2072

modifications of proteins can also lead to misfolding of the proteins. Aggregation of proteins in the brain
can potentially give rise to neurodegenerative diseases. The necessity of iron for many biological
activities and the potential of iron to cause damage to cells must obviously be balanced. These processes
deteriorate as we age. The consequences for the brain are particularly severe in this regard. However,
the exact role of iron in either aging or neurodegeneration is still under investigation and remains
uncertain. The evidence for its role is explored in this review.

2. Iron in the Brain

The brain contains significant amounts of iron. However, the distribution is not uniform.
Regions such as the substantia nigra and the globus pallidus have the highest levels, exceeding that of
the liver, the main site of iron storage in the body [15–17]. According to cell type, oligodendrocytes
have the highest iron content and astrocytes have very low cellular iron [18]. The main storage protein
for iron is ferritin, which is composed of a mixture of H- or L-ferritin monomers (heavy or light chain),
and varies between cell types as to which is expressed predominantly. Neurons express predominantly
H-ferritin while microglia express L-ferritin [19,20]. Some neurons, such as those of the
substantia nigra, express neuromelanin. Neuromelanin is synthesized from L-Dopa in dopaminergic cells
and forms stable complexes with Fe(III). Thus cells expressing neuromelanin are likely to have increased
iron storage [21,22].
Iron storage increases with age in the brain [23]. Again, this is not uniform and some regions show
greater increases than others. Studies of ferritin levels have indicated high increases in the cortex, globus
pallidus, and substantia nigra. In other regions such as the locus coeruleus the iron concentrations remain
low throughout life [24]. The main cell types accumulating iron with age are microglia and astrocytes [23].
These changes are found in many brain regions including the cortex, cerebellum, hippocampus, and
basal ganglia. In contrast, there is little change in iron in oligodendrocytes despite their higher
concentrations of cellular iron. The origin of increased brain iron remains unclear. However, increased
vascularization of the brain may increase the chance of iron exchange between the blood and tissues of
the brain, thus increasing the concentration of iron [25,26]. Whatever the reason, an increase in a
potentially oxidative metal in the brain increases the chances of deleterious reactions. It is probably no
coincidence that the regions associated with changes in iron are also those associated with several
neurodegenerative diseases.

3. Neurodegenerative Diseases Linked to Iron

This review is aimed at understanding the potential role of iron in aging and neurodegeneration.
However, despite a potential involvement in more common diseases, there are instances of quite specific
diseases directly linked to iron and its metabolism. Some of these diseases have successful treatments
that involve the use of iron chelators.
Friedreich’s Ataxia is the most common of the ataxias and affects adolescents [27]. The disease is
caused by mutations in the gene for frataxin, a protein found in the mitochondria that is associated with
the assembly of Fe-S clusters and may act as an iron chaperone [28]. Mitochondria are also considered
to be the main site of iron accumulation in Friedreich’s Ataxia [29]. Recent research has suggested that
frataxin acts as an allosteric mediator of Fe-S cluster assembly [30]. It is found in tissue with high
Metals 2015, 5 2073

metabolism such as the heart and dorsal root ganglia parallel to the spinal cord [31]. The mutation in the
gene involves trinucleotide repeat insertions (GAA) in the first intron. Severity of the disease appears to
be linked to the number of insertions, which decrease the expression level of frataxin [28]. Patients have
severe neurological problems that seem to be linked to problems with excess iron. Treatment of patients
with the iron chelator deferiprone caused marked reduction in iron levels within the dentate nuclei and
alleviated symptoms including manipulative dexterity, speech fluency, reduction in neuropathy, and
ataxia gait [32].
Neuroferritinopathy is a rare, dominantly inherited disease associated with mutations in the gene for
ferritin [33]. The disease has early and late onset forms and is associated with motor symptoms, spasticity
and cognitive deficits [34]. The cause is usually a frameshift mutation in exon 4 for the
L-ferritin gene, which causes a conformational change in the C-terminus of the molecule and alters its
ability to store iron [35,36]. The mutation results in iron/ferritin-rich aggregates forming in cells [37].
Mouse transgenic models show a strong relation between functional changes and abnormal iron
metabolism [38]. However, attempts to treat the disease with chelation therapy have so far been
unsuccessful [39].
Many of the iron-storage diseases are inherited and pantothenate kinase-associated neurodegeneration
(PKAN, formerly known as Hallervorden-Spatz syndrome) is another such disease [40]. It is
characterized by Parkinson’s-like symptoms as well as significant mental abnormalities. It develops in
childhood and is usually fatal. It is associated with mutations in the gene encoding
pantothenate kinase 2 (PANK2) [41]. For this reason the name was changed from Hallervorden-Spatz
syndrome. PKAN, now the more common term for the disease, is considered one of a family of diseases
called NBIA (neurodegeneration with brain iron accumulation) [42,43]. PKAN brains show specific
areas with high levels of iron accumulation, including the globus pallidus and the
substantia nigra [44]. The exact relation of iron accumulation to disease progress is unclear. It has been
suggested that the disease results in coenzyme A deficiency and subsequently increased cysteine, which
is then able to chelate the iron [45]. However, treatment of patients with iron chelation therapy caused
reduced iron accumulation but no change in symptoms. Therefore, the role of iron in the disease is
unclear and may just be a symptom rather than a cause [46].
NBIA diseases also include neuroferritinopathy, aceruloplasminemia, beta-propeller
protein-associated neurodegeneration, and a number of inherited diseases [43,47,48]. The incidence of
NBIA disease continues to increase and clearly shows the importance of understanding iron metabolism
and its potential link to neuronal loss. Many NBIA diseases are childhood diseases and are clearly not
linked to aging. However, they are illustrative of the potential impact that accumulation of iron in the
brain due to natural aging processes has on the vulnerability of neurons to iron-associated cell death.

4. Iron in Alzheimer’s Disease

Alzheimer’s disease (AD) is both the most common dementia and also the most common
neurodegenerative disease. The greatest risk factor for the disease remains aging. The risk of developing
the disease accelerates as we grow older, with those over 85 having almost a 1 in 2 change of developing
it. AD is progressive and irreversible and results in memory loss, cognitive decline, a variety of
psychological changes including anxiety, depression, and aggression, and eventual loss of physiological
Metals 2015, 5 2074

functions due to dementia [49]. Several brain areas are affected including the hippocampus, temporal
lobe, and frontal cortex. Particularly, the cholinergic innervation is severely disrupted [50]. The exact
cause of the disease remains contentious. There are inherited and sporadic forms of the disease. The
inherited forms, although much rarer, point towards the proteins most likely to be at the heart of the
disease [51]. The lead hypothesis of the cause of AD is the amyloid cascade hypothesis [52]. The
formation of aggregated forms of β-amyloid (Aβ) is a common hallmark of the disease. Aβ is formed
from a large precursor called APP (amyloid precursor protein). Inherited mutations linked to AD are
found in the APP gene or in the genes of proteins associated with processing APP to form Aβ. The
formation of Aβ aggregates in the brain is likely to play a significant role in the disease and may be
associated with the neuronal loss observed [52]. The other significant protein in AD is tau, a
microtubule-associated protein [53]. In AD tau becomes hyper-phosphorylated and forms paired-helical
filaments (PHF), also known as tangles. There are suggestions that PHF are the true cause of AD.
However, it could also be that some interplay between Aβ and tau may be important [54].
APP is a transmembrane protein and as the parent protein for Aβ there is significant debate over its
role in the cell and whether altering its metabolism could be the significant causal effect that initiates
AD [55]. A significant consideration in this debate is that APP binds metals [56]. APP contains a classical
type II copper-binding domain in its N-terminus. The residues His147, His151, and Tyr168 were
identified as the copper coordination sphere of the binding region within the E1 region of the
protein [57]. The His-X-His motif is similar to that seen in Cu/Zn superoxide dismutase. APP has the
potential to reduce Cu2+ to Cu+. The E2 domain of APP has also been suggested to have two metal binding
sites [58]. Of these, Zn2+ is thought to be coordinated between His382, His432, and His436. Copper can
also bind this site with the addition of His313 to the three His of the Zn site. A second, low-affinity site
is coordinated by Glu387, Asp429, and His458. There has also been a report that the E2 site interacts
with iron. A suggested site for iron binding around Glu337 and Glu340 is still debated [59]. The idea
that APP could function as a ferroxidase [60] has been dismissed in favor of the suggestion that APP
can regulate iron export from the cell (Figure 1) [59,61].
As well as physical interactions between metals and APP, there has been considerable research on
the cellular implications of those possible interactions. Of considerable interest are those that might alter
or initiate the disease (Figure 2).
Interaction of APP with copper is thought to alter both its dimerization and the rate of cleavage to
form Aβ [62,63]. However, the aggregation of Aβ is also thought to be influenced by its interaction with
copper and zinc [64–66]. Cu, in particular, is considered to accelerate aggregation. This led the research
group of Ashley Bush to investigate whether copper chelators such as clioquinol could inhibit Aβ
aggregate accumulation and possibly be a treatment for AD itself. While clioquinol proved effective in
depletion of Aβ in transgenic mice [67], it unfortunately did not progress in clinical trials [68].
Metals 2015, 5 2075

Figure 1. Several of the key proteins associated with neurodegeneration have been suggested
to have roles associated with iron. This figure summaries these suggestions. α-synuclein is
associated with PD and has been shown to bind iron and reduce it, thus converting Fe3+ to
Fe2+ which is utilized for many cellular activities. α-synuclein, like most ferrireductases, is
associated with the inner face of the cell membrane. There have also been reports that the
prion protein, associated with diseases like CJD, can also act to reduce iron as a
ferrireductase. Lastly, the amyloid precursor protein (APP) the precursor to beta-amyloid
known from AD, has been shown to enhance iron export out of cells. This process requires
the presence of ferroportin, which mediates the iron export process.

While the relation of copper and zinc to AD and the APP protein has proven quite solid, evidence
supporting a link between iron and AD has taken much longer to emerge [69]. Elevated brain iron has
been reported in patients with AD [70,71]. However, as is clear from this review, this is not unique to
AD. Iron accumulation in AD occurs without a parallel increase in ferritin [72]. IRP proteins normally
respond to increased levels of iron by interacting with genes containing the IRE element and causing
regulation of a number of proteins in a coordinated fashion. This usually ensures that the transferrin
receptor and ferritin are regulated together. In AD it appears there is a dysfunction of this system that
leads to increased free iron that would otherwise be stored in proteins such as ferritin [73]. In a recent
clinical study of cerebral spinal fluid (CSF) from AD patients, it was found that ferritin levels were
predictive of cognitive status [74]. While not being distinctly elevated in all cases of AD, declining
cognitive status from mild cognitive impairment (MCI) to AD was associated with altered ferritin levels
and was also greater with younger onset of AD. This would suggest a relationship between high iron
burden in the brain and an early age for AD onset. Additionally, it was found that ferritin levels were
higher in individuals with the APOE protein allele ε4 [74]. This allele is an epigenetic marker for greater
risk of AD. However, the link between APOE and iron metabolism cannot currently be explained.
Metals 2015, 5 2076

Figure 2. While numerous proteins associated with neurodegeneration may have a role in
iron metabolism in the healthy cell, under certain conditions these processes might be
disturbed. This diagram represents a summary of the potential changes that can occur during
aging which might disturb the way iron is either utilized or its interactions with other
proteins. Changes in the protein might result in changes in levels of iron, particularly Fe2+.
The protein may become overexpressed, resulting in the formation of excess Fe2+, or some
kind of damage to the protein might result in lost activity and reduced Fe2+. Additionally,
aging effects might result in the misfolding of the protein, either as a result of an increase or
a loss of interaction with iron or simply through another process. Misfolded protein could
then aggregate and cause toxic damage to cells. In the case of excess generation of Fe2+,
there could be abnormal interaction with other proteins such as neuromelanin or the Fe2+
could result in oxidative stress. In both cases the end result could be the generation of toxic
species that lead to cell death. Lastly, in some cases the lack of Fe2+ might result in a
breakdown of essential cellular process necessary for cell survival.

As mentioned above, Aβ binds metals. While there has been a focus on copper, there is also evidence
for an interaction with iron [72]. While this could mediate toxicity or oxidation events, there has also
been a suggestion that the interaction could be protective. In this case the Aβ would sequester the iron
and prevent it mediating toxic damage. While this may seem paradoxical given the “bad” reputation of
Aβ, it is actually true that free iron is highly toxic and Aβ can accumulate without any apparent cell
Metals 2015, 5 2077

loss [75,76]. This observation, although suggesting a positive role for Aβ in AD, does not then explain
how iron might be toxic.
Mitochondria are the organelles most likely to show damage in AD [77]. As the site of heme synthesis
in the cell, mitochondria are a major site for iron handling. Mitochondria are the major source of oxygen
radical generation in cells. Damage to mitochondrial-specific proteins, which can increase free iron, has
been shown [78]. Studies of mitochondrial DNA suggest that there is a greater turnover of the organelle
in AD [79]. This supports the fact that there is mitochondrial abnormality in AD [80]. There is no direct
evidence that this dysfunction is causal rather than a result of the damage that leads to cell death in AD
or that there is a direct link between mitochondria and iron. Further research may bring into focus the
causal links between these different factors. However, it is clear that iron disturbance is one important
part of trying to decipher the mystery behind AD.

5. Synucleinopathies and Iron Reduction

Synucleinopathies are a family of diseases associated with the deposition of the protein α-synuclein
in an aggregated form in CNS tissue [81]. These diseases include Parkinson’s disease (PD), Multiple
System Atrophy, and Dementia with Lewy Bodies (DLB) [82,83]. Parkinson’s is very well known, being
the most common of the non-dementing neurodegenerative diseases. It is associated with loss of
dopaminergic neurons from the substantia nigra, which results in a movement disorder [84]. PD has
a long association with disturbances to iron metabolism [85]. However, any mechanistic relation
between the disease and iron metabolism has yet to be firmly established. PD, like AD has sporadic and
inherited forms. The inherited forms are related to mutations in the genes of a long list of proteins which
include α-synuclein, parkin, leucine-rich repeat kinase (LRRK), PINK-1, and DJ-1 [86]. The inherited
forms can be either early or late onset (depending on the mutation). The clinical symptoms of PD include
resting tremors, muscle rigidity, and bradykinesia [87]. Treatments are generally focused on restoring
some amount of dopamine to the patient as the loss of dopaminergic neurons is the principal change that
results in the majority of symptoms. These treatments range from supplying the dopamine precursor
(L-Dopa) to altering dopamine transport and breakdown [88].
Changes in PD patient brains include increased levels of Fe(III) and reduced levels of the
Fe(III)-binding protein ferritin [89]. Increased iron deposits in the substantia nigra are associated with
α-synuclein-positive Lewy bodies in PD and neurodegeneration with brain iron accumulation [90,91].
However, there are also changes in other metals in PD such as zinc in the substantia nigra and high levels
of copper in the cerebrospinal fluid [92]. There is also evidence from epidemiological studies that
increased incidence of PD is associated with environmental metal exposure. Individuals with chronic
industrial exposure to copper, manganese, or iron have an increased rate of PD [93]. In experimental
models, alterations in metal homeostasis were observed with toxin-induced animal models of PD. An
accumulation of iron was observed in the substantia nigra of MPTP-treated mice, which is likely a result
of the observed upregulation of transferrin receptor expression and iron uptake [94,95]. Although these
observations may be consequences of the disease progression, experimental studies using FeCl3 injected
directly into the substantia nigra of rats resulted in a 95% reduction in striatal dopamine and altered
behavior, supporting the idea that iron initiates dopaminergic degeneration in PD [96].
Metals 2015, 5 2078

While observation of changes in metal levels in a disease might be interesting, without a mechanistic
link the findings remain at best a marker of change. The increased cellular iron is likely to come about
through changes either as a result of intake or release. However, with regards to the death of
dopaminergic neurons, there is also the possibility that it is not the absolute amounts that are important
but the ratio of Fe(II) to Fe(III). Similarly, alteration in the interactions of iron with various proteins may
also be critical. For example, the substantia nigra is characterized by high levels of neuromelanin, the
dark pigmented protein that gives the substantia nigra its name. Studies of PD patients have shown high
levels of iron associated with neuromelanin granules [97]. This could suggest that neuromelanin traps
redox-active Fe2+, which is then able to initiate the oxidative process [98,99]. However, another study
suggests that the iron leaves the neuromelanin and migrates to the cytosol in PD [100].
Cellular responses to fluctuating iron levels are regulated by iron regulatory proteins (IRPs) that bind
to iron response elements (IRE) in RNA [101]. However, they also respond to increased levels of
oxidative stress and can then alter cellular protein expression to cause an increase in the free iron
pool [102]. The implication is that oxidative stress can cause a dysregulation of iron metabolism. The
read-out for such a change would be increased iron levels without a corresponding change in
iron-binding proteins such as ferritin. This has been observed in PD [103]. As well as potential
interactions of iron with other proteins, iron could also alter dopamine specifically, causing the
generation of toxic dopamine byproducts that then kill dopaminergic cells specifically. One of the
metabolic products of dopamine 3,4-dihydroxyphenylacetaldehyde (DOPAL) can induce aggregation of
α-synuclein in the presence of iron [104]. DOPAL can also generate reactive oxygen species in the
presence of iron [105].
Synucleins have been linked to metals. Firstly, all three synucleins bind copper [106,107].
α-synuclein also binds other metals including iron [108,109]. Exposure of α-synuclein to metals during
extensive shaking can also accelerate its aggregation [110]. This creates a dichotomy with regards to
α-synuclein in terms of both its potential normal cellular activity and its aggregation, which is associated
with pathological states such as in PD. α-synuclein toxicity is mostly associated with
copper [111,112]. During the aggregation process, copper induces the formation of a unique stellate
oligomer that is highly toxic to neuronal cells in culture [111]. Exposure to iron does not have this effect,
although some studies have shown that iron can accelerate aggregation of α-synuclein [113]. The
aggregation of α-synuclein in cells is associated with the increased expression levels of the protein.
However, increased expression of α-synuclein has also been shown to increase cellular iron
concentrations [114].
Recent studies have shown that α-synuclein can bind both copper and iron simultaneously [109].
When α-synuclein binds copper it is able to undergo redox cycling, as shown by cyclic
voltammetry [106]. The implication is that α-synuclein can use copper to move electrons. Further studies
have shown that α-synuclein can cause the reduction of iron (Figure 1) [109]. Kinetic analysis with
purified recombinant protein has shown that this activity is enzymatic, potentially making
α-synuclein a ferrireductase. The implication is that the potential of α-synuclein to generate Fe2+ in cells
may be its normal cellular role. What this means for the pathology of the synucleinopathies currently
remains unclear. However, there are two distinct possibilities. First, increased expression of α-synuclein
could result in excess production of Fe2+, which could then initiate catastrophic oxidative processes in
the cell, interact with other proteins such as neuromelanin, or simply be neurotoxic (Figure 2). The
Metals 2015, 5 2079

alternative is that when α-synuclein is highly expressed in cells and begins to aggregate the protein
becomes functionally inactive but may still bind Fe3+, thus sequestering iron that is needed for normal
cellular activities such as the synthesis of dopamine. Give the potential role of Fe2+ in numerous aberrant
processes, the former possibility is more likely. However, as with the general role of iron in PD, the role of
iron reduction in PD is still unclear and further research is needed to understand what causative role there is,
if any.

6. Prion Diseases

Also known as transmissible spongriform encephalopathies, prion diseases are rare [115]. They are
more widely known because of the concurrent outbreaks of both bovine spongiform encephalopathy
(BSE) in cattle and variant Creutzfeldt-Jakob disease (vCJD) in humans [116,117]. The potential
transmissibility of prion diseases remains an ever-present concern. However, the major form of human
prion disease (sporadic form of Creutzfeldt-Jakob disease) is still very rare and is not naturally
transmissible [118]. Prion diseases can either be inherited (through mutations in the Prnp gene), sporadic
(with no known cause) or transmissible. The transmissibility has largely been demonstrated experimentally,
but may be a result of misadventure such as with vCJD or Kuru (a disease spread by ritual cannibalism).
The disease transmissibility is associated with an abnormal isoform of the prion protein [119]. The prion
protein (PrPc) is a cellular copper-binding glycoprotein expressed at the cell surface [120,121]. The
function of PrPc remains controversial, but the strongest evidence supports its role as an antioxidant
protein associated with increased cell viability [122–124]. The protease-resistant isoform of PrPc often
accumulates at high levels in the central nervous tissue of patients and animals with prion diseases.
Therefore, this abnormal isoform (PrPSc) is a hallmark of the disease. It is tightly associated with the
infectious agent and may be a direct cause of the neurodegeneration seen in the disease [125].
Following the discovery of the copper binding capability of PrPc, extensive studies were carried out
to assess the relation of PrPc and prion diseases to metal homeostasis. The strongest associations were
found between prion diseases and copper metabolism but also manganese metabolism [126]. The latter
was somewhat unexpected and remains controversial. While copper has been associated with normal
PrPc activities, the data suggest that manganese binds to PrPc in disease and can cause its conformational
change [127,128]. Manganese binding to PrP also increases its survival in the environment and increases
its ability to cause prion infection in cells [129].
Studies looking at the levels of trace elements in the brains of animals with BSE, sheep scrapie, patients
with CJD or vCJD, and rodents experimentally infected with scrapie all show a similar
trend [130–132]. They indicate reduced levels of copper and increased levels of manganese. Similar
changes have also been observed in the blood. A recent study has shown increased manganese in prion
plaques in experimental hamsters [133]. Other trace elements were also studied and very little difference
was observed for any other metal. These studies included Fe, which showed no changes in any of the
tissues or diseases analyzed.
Despite these studies showing no changes in iron, there are others that suggest a strong role for PrP
in iron metabolism [134]. An initial study suggested increases in both ferritin and iron response proteins
in astrocytes in rodent scrapie models [135]. The research group of Nina Singh has extensively studied
the links between Fe and prion disease. Their findings initially suggested that PrP could influence iron
Metals 2015, 5 2080

uptake [136]. Cells overexpressing PrP showed increased levels of intracellular iron. Further studies
suggested that CJD patients have increased levels of iron, while PrP knockout mice have decreased iron
levels [137,138]. A number of studies have shown that the divalent metal transporter-1 (DMT-1) is
altered in both PrP-knockout and prion disease [139]. This protein is linked to the transport of multiple
divalent metals and not just iron. However, changes in this protein could explain changes in intracellular
iron. Despite the changes in uptake and storage of iron suggested by these studies, it has also been
suggested that PrPc itself is a ferrireductase (Figure 1) [140]. The implication is that PrPc causes
conversion of Fe3+ to Fe2+, which is then more likely to be transported into the cell, thus resulting in
increased cellular iron. However, unlike alpha-synuclein, for which kinetic studies on purified protein
suggested iron reduction is a result of true enzymatic catalysis109], studies on the ferrireductase activity
of PrPc have only been performed on cell extracts [140]. As PrPc has already been suggested to be
associated with redox balance in cells [123], this observation may only be reporting a secondary effect
rather than a direct function of PrPc in iron reduction.
Unlike other diseases where the association with iron metabolism has been robustly established,
the link between iron and prion diseases remains unclear. The majority of the data do not support this
link. Iron binding to PrP is weak and highly pH dependent, implying there is not likely to be a
physiological interaction of PrPc and iron. However, it will be interesting to see if further data confirm a
role of PrPc in iron metabolism.

7. Microglia and Aging

While iron metabolism is of interest to specific diseases, general aging can cause other changes
upstream that also impact on the same diseases. Here again, changes in the way iron is handled by the
brain impact on the cellular environment. An example of the changing environment in the aging brain is
a change in the supporting cells in the brain, including microglia [141]. Microglia are the resident
macrophages of the brain and are its first and main form of active immune defense. Healthy microglia
are very sensitive to their environment, constantly surveying for and phagocytosing any foreign material
or cellular debris that they encounter. Additionally, they are capable of releasing cytotoxic substances
that can kill neurons that are damaged or infected. Such substances include H2O2, nitric oxide, inflammatory
cytokines, proteases, and neurotransmitters. Activated microglia can act as antigen-presenting cells and
activate T-cells. After an infection has been dealt with, microglia can recruit cells involved in neuronal
repair and secrete anti-inflammatory cytokines [142]. The idea of aging microglia stems from
histological observations of healthy aged brains where the cells often develop dystrophic phenotypic
characteristics [143]. Resting microglia have a ramified morphology with many fine processes extending
from the cell body. Dystrophic microglia found in aging brains lose this fine process of ramification
(Figure 3). Dystrophic microglia often develop abnormally shaped processes with spheroidal swellings
and cytoplasmic fragmentation (cytorrhexis) [143]. Dystrophic microglia have also been associated with
increased release of toxic ROS and inflammatory cytokines and impaired phagocytic ability [144,145].
Cytokines are small proteins involved in intercellular signaling. They are released by a multitude of cell
types including immune cells like microglia and bind to cell surface receptors on other cells where,
through signaling cascades, they alter the transcriptional profile of the target cells. The proinflammatory
cytokines found to be released by dystrophic microglia include IL-6, TNF-α, and IL-1β [145].
Metals 2015, 5 2081

Figure 3. Age-related changes in microglia. When microglia age they lose some of their
processes and develop abnormalities in others. Additionally, they often exhibit cytoplasmic
fragmentation. They also store more iron. Their increased release of neurotoxic substances
and reduced ability to phagocytose debris and toxic protein aggregates leaves
neurons vulnerable.

Ferritin, the main iron storage protein in the brain, is highly expressed in microglia [19]. Microglia
are the main cells in the brain that store iron. One way to identify dystrophic microglia, apart from their
morphology, is their higher levels of iron storage, demonstrated by the expression of the iron storage
protein ferritin. Additionally, an outward rectifier K+ channel called Kv1.3 has been found to increase
expression in dystrophic microglia in aged mouse brains [146]. Dystrophic microglia become more
prevalent with human aging and have been found to increase in a variety of diseases including AD [147]
and Huntington’s disease [148]. There is also evidence that the chronic inflammation that accompanies
neurodegeneration leads to local increases in microglia with high iron and ferritin content, possibly due
to iron scavenging [149]. The association between increased iron storage and an altered microglia
phenotype, particularly a dystrophic one, suggests a possible causative role for iron. As iron can damage
cells, dystrophic microglia possibly develop as a direct result of increased iron storage.

8. Microglia and Neurodegenerative Diseases

The presence of healthy glial cells is critically important to neuronal wellbeing. Microglia maintain
homeostasis in the healthy brain and fight infection when it is present through a complicated system of
signaling molecules. The importance of microglia to neurons is supported by a higher incidence of
dystrophic microglia and microglial apoptosis in AD [150]. The inflammation of the nervous system in
neurodegenerative disease was thought to be due to activated microglia. However, dystrophic microglia
also have impaired neuroprotective ability and generate the low but sustained release of inflammatory
factors seen during neurodegeneration.
The effect of dystrophic microglia on the pathogenic changes occurring in Alzheimer’s disease is not
well understood. Over the progression of the disease, microglia seem to change from exerting
a neuroprotective function to being closer to a classically activated state. This change in phenotype may
Metals 2015, 5 2082

result in microglial neurotoxicity or alternatively in dysfunction that prevents the cells from fulfilling
their protective role [150]. Dystrophic microglia have been co-localized with neurofibrillary tangles in
AD brains [151]. Microglia with impaired phagocytic and motility functions have been
co-localized with Aβ deposits in mouse models of Alzheimer’s disease [152]. Healthy microglia have
been shown to take up Aβ and also to release enzymes that degrade it [153]. However, dystrophic
microglia have impaired Aβ phagocytic ability [144]. If healthy microglia are activated with LPS, they
help reduce the Aβ burden in the brains of mice [154]. Pathologically activated microglia release
pro-inflammatory cytokines and reactive oxygen species that can make neurons more sensitive to Aβ
toxicity [155]. Dystrophic microglia have been found to be hyper-responsive to stimulation [145].
Inflammatory cytokines have been shown to increase the expression of APP in neurons, which can result
in increased production of Aβ through also favoring the amyloidogenic APP processing
pathway [156–158]. The microglial p40 subunit of IL-12 and IL-23 has been found to be elevated in AD
brains and to correlate with a worse Aβ pathology [159].

9. Conclusions

Iron is a two-edged sword for biological systems—essential for many cellular activities, but also able
to cause damage to macromolecules or disrupt sensitive processes. In the brain this balance is even more
delicate given the irreplaceable nature of neurons. Research into the role of iron in both disease and
normal activities in the brain will continue. However, the changes and impact of iron during aging and
within neurodegenerative diseases is now well established.

Author Contributions

DMA and DRB wrote the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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