Advances in Experimental Medicine and Biology 1173

Yan-Zhong Chang Editor

Brain Iron
Metabolism and
CNS Diseases
Advances in Experimental Medicine and Biology

Volume 1173

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Yan-Zhong Chang
Editor

Brain Iron Metabolism

and CNS Diseases

123
Editor
Yan-Zhong Chang
College of Life Sciences
Hebei Normal University
Shijiazhuang, Hebei, China

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Contents

1 Brain Iron Metabolism and CNS Diseases . . . . . . . . . . . . . . . . . . . 1

Anand Thirupathi and Yan-Zhong Chang
2 Cellular Iron Metabolism and Regulation . . . . . . . . . . . . . . . . . . . . 21
Guofen Gao, Jie Li, Yating Zhang and Yan-Zhong Chang
3 Brain Iron Metabolism and Regulation . . . . . . . . . . . . . . . . . . . . . . 33
Peng Yu and Yan-Zhong Chang
4 Iron Pathophysiology in Parkinson Diseases . . . . . . . . . . . . . . . . . . 45
Hong Jiang, Ning Song, Qian Jiao, Limin Shi and Xixun Du
5 Iron Pathophysiology in Alzheimer’s Diseases . . . . . . . . . . . . . . . . . 67
Tao Wang, Shuang-Feng Xu, Yong-Gang Fan, Lin-Bo Li
and Chuang Guo
6 Iron Pathophysiology in Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Mohammed M. A. Almutairi, Grace Xu and Honglian Shi
7 Iron Pathophysiology in Friedreich’s Ataxia . . . . . . . . . . . . . . . . . . 125
Kuanyu Li
8 The Role of Iron in Amyotrophic Lateral Sclerosis . . . . . . . . . . . . . 145
Xian-Le Bu, Yang Xiang and Yansu Guo
9 Iron Pathophysiology in Neurodegeneration with Brain Iron
Accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Sonia Levi, Anna Cozzi and Paolo Santambrogio
10 Diagnostics and Treatments of Iron-Related CNS Diseases . . . . . . . 179
Huan Xiong, Qing-zhang Tuo, Yu-jie Guo and Peng Lei

v
Contributors

Mohammed M. A. Almutairi Department of Pharmacology and Toxicology,

School of Pharmacy, University of Kansas, Lawrence, KS, USA;
Department of Pharmacology and Toxicology, School of Pharmacy, King Saud
University, Riyadh, Saudi Arabia
Xian-Le Bu Department of Neurology and Centre for Clinical Neuroscience,
Daping Hospital, Third Military Medical University, Chongqing, China
Yan-Zhong Chang Laboratory of Molecular Iron Metabolism, Key Laboratory of
Animal Physiology, Biochemistry and Molecular Biology of Hebei Province,
College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei, China
Anna Cozzi Division of Neuroscience, San Raffaele Scientific Institute, Milan,
Italy
Xixun Du Department of Physiology, Medical College of Qingdao University,
Qingdao, China
Yong-Gang Fan College of Life and Health Sciences, Northeastern University,
Hunnan District, Shenyang, China
Guofen Gao Laboratory of Molecular Iron Metabolism, College of Life Sciences,
Hebei Normal University, Shijiazhuang, Hebei, China
Chuang Guo College of Life and Health Sciences, Northeastern University,
Hunnan District, Shenyang, China
Yansu Guo Beijing Geriatric Healthcare Center, Xuanwu Hospital, Capital
Medical University, Beijing, China
Yu-jie Guo West China School of Basic Medical Sciences and Forensic Medicine,
Sichuan University, Sichuan, China
Hong Jiang Department of Physiology, Medical College of Qingdao University,
Qingdao, China

vii
viii Contributors

Qian Jiao Department of Physiology, Medical College of Qingdao University,

Qingdao, China
Peng Lei Department of Neurology and State Key Laboratory of Biotherapy,
West China Hospital, Sichuan University, Sichuan, China;
West China School of Basic Medical Sciences and Forensic Medicine, Sichuan
University, Sichuan, China
Sonia Levi Division of Neuroscience, San Raffaele Scientific Institute, Vita-Salute
San Raffaele University, Milan, Italy
Jie Li Laboratory of Molecular Iron Metabolism, College of Life Sciences, Hebei
Normal University, Shijiazhuang, Hebei, China
Kuanyu Li Jiangsu Key Laboratory of Molecular Medicine, Medical School of
Nanjing University, Nanjing, People’s Republic of China
Lin-Bo Li College of Life and Health Sciences, Northeastern University, Hunnan
District, Shenyang, China
Paolo Santambrogio Division of Neuroscience, San Raffaele Scientific Institute,
Milan, Italy
Honglian Shi Department of Pharmacology and Toxicology, School of Pharmacy,
University of Kansas, Lawrence, KS, USA
Limin Shi Department of Physiology, Medical College of Qingdao University,
Qingdao, China
Ning Song Department of Physiology, Medical College of Qingdao University,
Qingdao, China
Anand Thirupathi Laboratory of Molecular Iron Metabolism, Key Laboratory of
Animal Physiology, Biochemistry and Molecular Biology of Hebei Province,
College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei, China
Qing-zhang Tuo Department of Neurology and State Key Laboratory of
Biotherapy, West China Hospital, Sichuan University, Sichuan, China
Tao Wang College of Life and Health Sciences, Northeastern University, Hunnan
District, Shenyang, China
Yang Xiang Department of Neurology, Chengdu Military General Hospital,
Chengdu, China
Huan Xiong Department of Neurology and State Key Laboratory of Biotherapy,
West China Hospital, Sichuan University, Sichuan, China
Grace Xu Department of Anesthesiology, School of Medicine, University of
Kansas, Kansas City, KS, USA
Shuang-Feng Xu College of Life and Health Sciences, Northeastern University,
Hunnan District, Shenyang, China
Contributors ix

Peng Yu Laboratory of Molecular Iron Metabolism, Key Laboratory of Animal

Physiology, Biochemistry and Molecular Biology of Hebei Province, College of
Life Sciences, Hebei Normal University, Shijiazhuang, Hebei, China
Yating Zhang Laboratory of Molecular Iron Metabolism, College of Life
Sciences, Hebei Normal University, Shijiazhuang, Hebei, China
Chapter 1
Brain Iron Metabolism and CNS Diseases

Anand Thirupathi and Yan-Zhong Chang

Abstract Iron is the most abundant trace element in the human body. It is well
known that iron is an important component of hemoglobin involved in the transport
of oxygen. As a component of various enzymes, it participates in the tricarboxylic
acid cycle and oxidative phosphorylation. Iron in the nervous system is also involved
in the metabolism of catecholamine neurotransmitters and is involved in the forma-
tion of myelin. Therefore, iron metabolism needs to be strictly regulated. Previous
studies have shown that iron deficiency in the brain during infants and young chil-
dren causes mental retardation, such as delayed development of language and body
balance, and psychomotor disorders. However, if the iron is excessively deposited
in the aged brain, it is closely related to the occurrence of various neurodegenerative
diseases, such as Alzheimer’s disease, Parkinson’s disease, and Friedreich’s ataxia.
Therefore, it is important to fully study and understand the mechanism of brain iron
metabolism and its regulation. On this basis, exploring the relationship between brain
iron regulation and the occurrence of nervous system diseases and discovering new
therapeutic targets related to iron metabolism have important significance for break-
ing through the limitation of prevention and treatment of nervous system diseases.
This review discusses the complete research history of iron and its significant role in
the pathogenesis of the central nervous system (CNS) diseases.

Keywords Iron metabolism · Neurodegenerative diseases · Oxidative stress ·

Alzheimer’s disease · Parkinson’s disease

Iron is an essential player of many biological processes in the brain such as DNA
synthesis, oxygen transport, myelin formation, and mitochondrial functions. It is
necessary to maintain the iron homeostasis for normal physiological actions of the

A. Thirupathi · Y.-Z. Chang (B)

Laboratory of Molecular Iron Metabolism, Key Laboratory of Animal Physiology, Biochemistry
and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University,
20, Nan Er Huan Eastern Road, Shijiazhuang 050024, Hebei Province, China
e-mail: [email protected]
A. Thirupathi
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2019 1

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_1
2 A. Thirupathi and Y.-Z. Chang

brain. However, misregulation of iron homeostasis can cause neurodevelopmental

and neurodegenerative diseases through different mechanisms. Homeostatic mecha-
nism provides the optimum condition to the cells to carry out some essential functions
including maintaining the equilibrium status of the iron concentrations within the
cells and buffering the molecules for stopping the toxic buildup within the cellular
compartments. When the iron level is increased over the iron sequestration capac-
ity, the concentration of the iron can increase, which can cause several damages in
the cellular organelles and cell death in the brain. However, the iron buildup with
neurodegeneration is a primary event or secondary event which is unknown. Here,
we discuss the history of iron research and iron metabolism in the brain. Next, we
discuss the disturbances in the iron homeostasis causing neurodegenerative diseases,
and finally, we discuss the important findings that can overcome the iron accumula-
tion causing diseases and its demerits.

1.1 The Evolution of Iron Research

After the bronze era, Iron Age (~1200–1000 BC) began to dominate every creature
in the universe, and it turned into an imperative player of entangled biochemistry,
which means iron is indispensable for life. Iron is one of the most superabundant
metals that contribute to make earth habitable by generating magnetosphere circling
to the planet. Indeed, oxygen-free states of the earth made possible to occur a num-
ber of chemical reactions that initiate the life existence. Importantly, the essential
compound that possibly creates life is a soluble form of iron, hydrogen, and sul-
fide from ores. In the earlier life formation, the soluble iron was rich, but it began
to reduce due to oxygen addition that means life formation is affected slowly but
steadily. Günter Wächtershäuser proposed that chemistry of life occurred in the min-
eral sources [60, 61]. In the sixteenth century, Nicholas Monarde used the iron for
various ailments such as alopecia, acne, wounds, hemorrhoids, and gout, revealed
the biological significance of iron [42].
The underlying biological importance was proposed initially by Lemmery and
Geofgroy in 1713 [57]. Nonetheless, it was nearly required two centuries to compre-
hend iron metabolism in the body. Vincenzo Menghini was discovered the presence
of iron in the blood at the earlier of seventeenth century in the human body [4]. This
group was scorched the blood from various models including birds and humans for
searching iron in the blood. In the eighteenth century, Louis Rene Lecanu discovered
that RBC has a protein called globulin and red coloring matter called hematosine
that contained iron [34, 35]. In the earlier nineteenth century, Fontés and Thivolle
recommended an imperative revelation that a body has unique form of iron in rela-
tion to the hemoglobin in the serum [18]. They additionally found that inadequacy of
iron can affect the serum iron concentration. In the mid of nineteenth century, Holm-
berg and Laurell found the iron-binding proteins called transferrin (Tf) [26]. In the
meantime, Schade and Caroline determined the antibacterial effect of iron-binding
proteins, and they proposed that siderophilin, a bacterial iron-absorbing compound,
1 Brain Iron Metabolism and CNS Diseases 3

which was indistinguishable to Tf proteins [52]. This finding has crucial informa-
tion since most of the drugs that are currently used for chelation therapy belong to
siderophilins. Henry John Horstman Fenton proposed that iron can become toxic in
certain circumstances when the level of iron is surpassing in the cell [14, 15], and the
concept was not acknowledged by the researchers until the disclosure of superoxide
dismutase which converts superoxide into oxygen and hydrogen peroxide.
Studies from 1937 by John Livingood, Fred Fairbrother, and Glenn Seaborg found
an exceptional radioactive labeled iron for analyzing iron homeostasis. Based on
this radioactive iron, McCance and Widdowson in 1938 proposed that iron is not
excreted from the body [44]. Likewise, they theorized that only a small amount of
iron was assimilated from the whole intestinal tract, but the main site of assimilation
occurred at the upper locale of the intestine. Their investigation was accurate with
respect to iron homeostasis except their suggestion that “in hemochromatosis, the
iron deposited in the cells is insoluble and inert, and takes no part in the general
equilibrium. Hence there is a true deficiency of free iron, as evidenced first, by the
anemia, and secondly, by the cure of the anemia by the administration of iron and
for their estimation that the red cell life span was only 30 days.” Ferrokinetic studies
by Pollycove and Mortimer in 1961 were proposed a clear and surmised size of each
iron pool [49].
In 1997, Hediger’s group and Andrews’ group discovered an important protein
called divalent metal protein 1 (DMT1) also known as DCT1 and Nramp2 which
transports the dietary iron using H+ electrochemical gradient as driving force. They
found that the expression of DMT1 in the rat increased the uptake of 55 Fe2+ and Fe2+ .
They also found that the DMT1 expression was reactive to several other divalent metal
ions. Vulpe group discovered a multicopper oxidase protein called hephaestin in the
small intestine in 1999 and Chen group in 2010 identified another type of multicop-
per ferroxidase called zyklopen in the placenta, and the expression of these proteins
is necessary for iron efflux from placental cells and small intestine [6, 59]. The
iron entry into plasma, iron utilization and storage are determined by the interaction
of the peptide hormone called hepcidin and ferroportin (FPN1). FPN1 was identi-
fied by three different groups using different methods. The first group found FPN1
through zebrafish mutant gene [11]. The second group identified FPN1 as a transcript
[39]. This group also identified the Dcytb (duodenal cytochrome b) (also known as
Cybrid1) as an iron-regulated protein using a subtractive cloning strategy [40], and
the third group identified the FPN1 as an iron-responsive element (IRE)-containing
mRNA [55]. The role of hepcidin in relation to iron regulation was identified by Nico-
las group in 2001 through the HAMP1 gene deletion, which encodes hepcidin [45].
All of these discoveries have been greatly promoted to understand iron metabolism
in the body.
4 A. Thirupathi and Y.-Z. Chang

1.2 Iron Metabolism and Cell Iron Homeostasis

1.2.1 Iron Metabolism in the Body

Understanding of iron metabolism begins with dietary iron that can be efficiently
taken to the cells through duodenal enterocytes. This process is firmly managed
by several sensory on and off mechanisms in the epithelium, which is carried out
by a number of proteins such as DMT1, cytochrome b ferrireductase, ferroportin,
TfR1 and hephaestin. Several enigmatic concepts existed in earlier studies about iron
absorption. One of the concepts proposed by Hahn and Whipple is known as “mucosal
block mechanism.” They declared that the first dose of given iron is saturated the
mucosa with ferritin for blocking the absorption of the second dose [21]. In the
1950s and 1960s, some studies had contended against mucosal block mechanism that
anemia can be upregulated by the iron absorption in the iron deficiency condition,
and it was reduced by iron overloading condition. Although studies had given a clear
idea about iron absorption by bowel in the middle of the twentieth century, the bowel
components that direct iron absorption are still elusive. Conrad and Crosby in 1963
demonstrated that the entry of radioactive iron in mucosa sloughed off when the
body needs low iron [8, 9]. They inferred that the iron regulation was determined
by the exchange of iron between mucosal to blood, and the concept is now widely
accepted. However, the mechanism remains elusive.
The intestinal mucosa responds if the body’s iron level is altered and facilitates the
iron absorption accordingly. Iron deficiency condition increases absorption, whereas
iron overload condition decreases the iron absorption. The loss of iron occurs through
desquamation process and an adult man losses 1 mg of iron, and this level varies with
women during their menstruation periods. The healthy population needs 1–2 mg of
iron per day for compensating the loss of iron. Dietary iron absorption takes place
in the duodenum and upper jejunum. Several proteins are involved to carry out this
process such as DMT1, FPN1, and Dcytb. The dietary irons are mostly as inorganic
form, and it is reduced by the Dcytb, a ferrireductase enzyme present in the brush
border of microvilli. Then, DMT1 transports the ferrous form of iron from the api-
cal membrane of the duodenal enterocytes into intestinal epithelial cells where it
can be stored as ferritin or transported into circulation across the basolateral mem-
brane. FPN1 transports the iron from the basolateral membrane to circulation and
non-transported iron is removed from the body through exfoliation of the intesti-
nal epithelium. This epithelium exfoliation may represent the iron excretion because
these cells are expressing TfR1 at the basolateral membrane and iron from the plasma
can enter through endocytosis. For transporting iron through the basolateral mem-
brane, iron must be transported through cell cytoplasm, but this mechanism is not
fully understood. However, this iron transport possibly occurs through cytosolic
chaperons and iron-binding proteins or transcytosis. Iron transport from the basolat-
eral membrane of enterocyte to circulation requires the changes of redox state which
is carried by the hephaestin in the duodenum and ceruloplasmin (CP) in other parts
1 Brain Iron Metabolism and CNS Diseases 5

of the body which converts the intracellular Fe(II) back to extracellular Fe(III) for
transferrin [59].

1.2.2 Cellular Iron Homeostasis

Several researchers are involved to explore the cellular level of iron homeostasis.
We referenced here some of the remarkable researches regarding iron homeostasis.
Finch group first investigated the reticulocytes iron transference in 1949 [17]. In
little later, Jandl and Katz groups proposed the presence of TfR1 for take-up irons
from the reticulocytes [30]. Evan Morgan and his group were evidenced by the cel-
lular level of iron accession mechanism of Tf in the 1970s [2, 43]. Furthermore,
Fineberg and Greenberg provided the molecular level of iron accession mechanism
[16]. They proved that the administration of iron increases the ferritin protein syn-
thesis, and this experiment was further carried out by Drysdale and Munro [12, 63].
Zahringer group detailed that iron administration increased the ferritin mRNA con-
tent in the actinomycin-treated animals. Munro group explained that iron-sensitive
factors called iron-responsive elements (IREs) and iron regulatory proteins (IRPs)
can regulate ferritin mRNA translation. They reported that ferritin availability to iron
was decreased by 5 UTR deletion containing a conserved 28 base sequences from
ferritin mRNA [33]. Some earlier studies reported that TfR1 and ferritin expression
are absolutely inverse. When iron is overabundance, the level of TfR1 is constrained;
whereas the iron is less, the level of TfR1 is high. Therefore, ferritin and TfR1 levels
are precisely altered at the cellular level. Owen and Kuhn evidenced that the sequence
within the 3 UTRs is needed for iron-dependent TfR1 expression, while TfR1’s pro-
moter region is not needed [47]. Further research evidenced that IREs present in
ferritin while TfR1s bind with an IRE-binding proteins called IRPs. There are two
types of IRPs having almost similar function. Higher iron can increase the translation
of ferritin mRNA and decrease the stability of TfR1 mRNA. This crucial mechanism
is orchestrated by the presence of IREs at the 5 UTR of ferritin mRNA and 3 UTR
of the TfR1 mRNA. These IREs of both ferritin and TfR1 can bind with cytosolic
proteins IRP1 and IRP2. Cells acquire iron from the plasma Tf. After loading iron to
Tf, the complex can bind with TfR1 on the cell surface and undergoes endocytosis
through clathrin-coated pits. For triggering the release of iron from the Tf, proton
pump induces the acidification of endosome to pH level 5.5. For redox changing to
transferring iron by DMT1, ferrireductase STEAP3 reduces Fe3+ to Fe2+ , and this
Fe2+ form can be stored in ferritin or utilized by downstream metabolic pathways.
After releasing iron from Tf, the Tf is dissociated from the TfR1 and the apo-Tf is
entered into circulation where it can recapture the Fe3 . Regulation of cellular iron
homeostasis is tightly managed by the post-transcriptional mechanism, and especially
the IRP-IRE system can mediate this post-translational modification for influencing
the synthesis of proteins that are responsible for maintaining the intracellular iron
level. In the iron depletion condition of a cell, IRPs bind with IREs in the 5 UTR or
3 UTR of mRNA for either suppressing the translation of mRNA such as FTH1 and
6 A. Thirupathi and Y.-Z. Chang

FPN1 or by increasing the mRNA translation including TfR1 and DMT1 [24, 25].
Under cellular iron increased conditions, IRPs loses their binding activity to IREs
by acquiring iron–sulfur cluster (4Fe–4S cluster) and proteasomal degradation [20,
51].

1.3 Brain Iron Metabolism and Nervous System Diseases

In the brain, the function of iron in DNA synthesis, gene expression, neurotransmis-
sion, myelination of neurons, and the mitochondrial system can be crucial. There-
fore, the balance of brain iron needs to be strictly regulated. However, iron abnormal
metabolism can lead to misbehave many proteins that are associated with brain
diseases or disorders. The higher iron deposition or iron deficiency may impair the
normal functions of the cells. Over iron accumulation in a specific region of the brain
leads to several neurodegenerative diseases such as Alzheimer’s disease and Parkin-
son’s disease. Dysregulation of iron homeostasis also sets diseases like Friedreich’s
ataxia (FA). Understanding iron metabolism in the CNS is increasing interest. In
addition, numbers of questions are travelling along with scientific discoveries. For
example, what are the characteristics of iron metabolism and its regulatory mecha-
nisms in each cell type of the CNS such as neurons, oligodendrocytes, and microglia
and what is the mechanism of iron transport and regulation across the blood–brain
barrier?

1.3.1 Iron Homeostasis in the Brain

The brain requires iron for its metabolic process like other organs. However, each
type of brain cells such as neurons, oligodendrocytes, and astrocytes possess distinct
iron metabolism. In addition, unequivocal concepts exist that how iron cross the
blood–brain barrier (BBB) or cerebrospinal fluid (CSF) from the circulation and
how it traffics in the brain parenchyma to provide iron to different brain cells. The
distribution of iron in the brain is mostly concentrated in the substantia nigra pars
compacta (SN), circumventricular organs, globus pallidus, and oligodendrocytes. It
is well known that the brain is protected by a special structure called BBB, which
can prevent the pathogen and other macromolecular compounds entry into the brain
and ventricles. Therefore, transporters and receptors can mediate iron transfer across
the luminal membrane of endothelial cells of BBB into the CNS. Nevertheless, these
regulatory molecules are associated with many diseases and are the major baffling
of current research.
The current understanding of iron homeostasis in the brain begins with the Tf/TfR
pathway, may be the major route for iron transport across the luminal membrane of
the endothelium. Transferrin-bound iron then be converted into Fe3+ and translo-
cated across the endosomal membrane by DMT1. Tf will be returned to the luminal
1 Brain Iron Metabolism and CNS Diseases 7

membrane once it releases iron with TfR. Other pathways may also be involved
to transport iron across the BBB such as lactoferrin receptor/lactoferrin and GPI-
anchored melanotransferrin/soluble melanotransferrin. In addition to these pathways,
transferrin-bound iron is transferred across BBB through transcytosis. FPN1 and hep-
haestin might play a key role in transferring iron across the BBB as Fe2+ . Another
possible way for iron transfer for across the BBB is carried out by astrocytes. The
FPN1 released Fe2+ in basolateral surface of endothelial cells. Then, astrocytes oxi-
dize Fe2+ into Fe3+ by CP through their end-feet processes on the capillary endothelia
[38, 46], which are further bound with Tf produced by choroid plexus and oligoden-
drocytes, suggesting that iron not only enters via endothelial cells of BBB but also
enters through epithelial cells of choroid plexus. After entering iron into interstitial
fluid or CSF in ventricles, the iron will binds with Tf and diffuses via CSF and
interstitial fluid of the brain parenchyma, supply iron to the cells expressing TfR
within the CNS. A small amount of transported iron can circulate with citrate and
ATP. Different cell types in the brain are involved to acquire iron using different
pathways. Neurons acquire iron through Tf, astrocytes acquire iron through DMT1,
oligodendrocyte acquires iron from Tf by the TfR1 pathway and from ferritin via
TIM-2 receptors, and microglia acquire iron from ferritin. Although several studies
are well established the iron homeostasis in the CNS, due to BBB, the iron levels in
the peripheral nervous system (PNS) decouple from the brain. However, FPN1 may
play the role to export the iron from the CNS to PNS with the help of ferroxidase.
Furthermore, PNS has little information regarding iron homeostasis. Since BBB has
limited access to iron, it expresses all the iron regulatory proteins as we mentioned
above. However, on the contrary, the peripheral nervous system has little informa-
tion regarding iron regulatory proteins. The individual nerve fiber is protected by
the connective tissue matrix like epineurium, perineurium, and endoneurium in the
PNS. Unlike BBB, the blood–nerve barrier (BNB) permits the proteins and macro-
molecules. Both BBB and BNB shared the common feature in transferring iron, such
as having enzymes, receptors, and transporters. For example, Tf-bound iron is the
main source for CNS, but other sources can also contribute to transfer iron in the
CNS. However, PNS depends on Tf–TfR1 pathway for iron acquisition especially Tf
is accumulated in the Schwann cell (SC) cytoplasm, suggesting a role of Tf-bound
iron in the myelination. Furthermore, SC expresses other iron exporters like FPN1
and ferroxidase CP which associate together to efflux the iron to the axons in the
peripheral nerve. CNS has ferritin as an iron source, but PNS does not have a ferritin-
dependent active mechanism [23]. PNS acquires iron using NTBI through DMT1
[58].

1.3.2 Iron Entry and Its Metabolism in the CNS

Iron passage into the brain had been a long mystery. Initially, it was believed that
the iron entry occurred before the blood–brain barrier maturation at the infant stage.
Now, it is known that endothelial TfR1 mediates the brain iron uptake. However, TfR
8 A. Thirupathi and Y.-Z. Chang

expression is regulated by the availability of iron in CNS. In addition, the lactoferrin

receptor (LfR) and lactoferrin, and GPI-anchored melanotransferrin are involved
to play a pivotal role in iron transport across the BBB. Non-transferrin-bound iron
(NTBI) can also present in the BBB [10]. Blood–brain barrier is consisted of a tight
epithelial barrier similar to the duodenum is a place of dietary iron absorption. In
addition, the presence of BBB can relatively explain the independence of brain iron
from the total body iron. Other sites including retina and testis of the mammalian
body which be separated from the systemic circulation, the iron absorption maybe
similar to the brain.
Endothelial junctional complexes of BBB and astrocytes can decide the passage of
iron into the brain. Endothelial cells of brain express TfR1 in the luminal membrane,
and TfR1 complexes with Tf for further iron transferring. Endosomes can internalize
these complexes to reduce the ferric to ferrous form by endosomal reductase. Nev-
ertheless, the mechanism by which the iron transports from Tf to brain interstitial
system is obscure. There are some possibilities that can bolster this mechanism that
is after the iron detachment from the Tf can be siphoned into the cytosol by DMT1.
Then, the iron transport across the abluminal membrane into the brain is mediated
by the FPN1. Alternatively, TfR1 and Tf release iron in the interface between the
endothelial surface and astrocyte end-foot process, which is then oxidized by the CP
into the ferric form of iron. Subsequently, it is attached with Tf of brain interstitial
fluid (Fig. 1.1). Neurons expressing high levels of TfR1 can utilize efficiently irons
from Tf. However, neurons with lesser level of TfR1 expression or failed expression
can lead to iron dysregulation causing neurodegeneration.
On the other hand, ferrous iron can be moved as NBTI by either attached with ATP
or citrate released from the astrocytes. This NBTI is the primary iron source for the
cells which do not express TfR1 such as astrocytes and oligodendrocytes. However,
the amount of TfR1’s expression is varied in each cell type and its iron status. For
example, oligodendrocytes firmly express transferrin, whereas microglia strongly
express ferritin. Most of the brain cells acquire iron from the TfR1 and endosomal
DMT1 and store iron by ferritin and export via FPN1. It has been reported that
endothelium of BBB, neurons, and astrocytes expresses FPN1 [22, 32]. Cerebrospinal
fluid can also be the iron entry route for brain across the choroid plexus [50].
Neurons including motor, sensory, and interneurons are linked to regulate the
iron metabolism in the brain because neurons are ensheathed by glial cells such as
oligodendrocytes, astrocytes, and Schwann cells, and these glial cells are express-
ing iron regulating transporters, receptors, and exporters such as Tf/TfR and FPN1
for restructuring the neuronal process, myelinate axons, and regulating metabolic
functions. Neurons take up iron via Tf/TfR and DMT1. The newly imported iron
can be stored as ferritin or exported through FPN1. Then, the exported iron from
the neuron is oxidized by astrocytic CP into ferric form [1]. However, within these
neurons there are different gene expressions that are linked with iron metabolism,
thus understanding of iron export from these neurons to other parts of the brain needs
profound clarification.
1 Brain Iron Metabolism and CNS Diseases 9

Fig. 1.1 Understanding of iron homeostasis in the brain. TfR1 of endothelial junctional complexes
and Tf can transfer the iron with the endosomal process. Detachment of iron from Tf, can be pumped
into the cytosol by DMT1 where it can be stored as soluble form of iron. Some iron can be pumped
out by FPN1, which is further oxidized by ceruloplasmin into ferric form for Tf. Non-bound iron
can be either attached with ATP or citrate which is released from astrocytes

1.3.3 Brain Iron Accumulation

Although brain cells have tight regulation for iron entry, iron is the primary source
for several cytosolic proteins and enzymes, mitochondria, and nuclear ferroproteins
of brain cells. It has been known that iron accumulation is directly proportional
to aging. However, this situation is not connected with pathogenesis. Some special
regions such as globus pallidus and substantia nigra in the brain can participate to
increase the iron deposition, but factors causing iron deposition in those regions are
unknown. Possibly, the aging condition can synthesize some special type of neu-
rons to increase the expression of iron-storing protein ferritin. For example, globus
pallidus has rich iron level as equivalent as in the hepatocytes. Different scavenger
cells including macrophages can become iron-rich when the brain cells started to
undergo apoptosis. IRP1 and IRP2 may also play important roles in controlling the
cellular iron homeostasis in the brain. Alterations in these IRPs signaling pathways
can contribute to higher iron deposition in the brain. For example, iron deficiency
condition can allow IRP1 to interact IREs for up-regulating the TfR expression in
10 A. Thirupathi and Y.-Z. Chang

the brain endothelial cells, but alterations in these signaling pathways can increase
the brain iron accumulation.
CSF is the major route for iron export from the brain into the blood from the sub-
arachnoid space. Interference in the CSF route can cause the over iron accumulation
in the brain. For example, transferrin is very low in the CSF, thus exporting iron
from the brain to blood is limited. Some other iron-storing proteins like lactoferrin,
ferritin, and non-protein-bound iron can help to export the iron, but these proteins
function is majorly affected by some pathological conditions. In addition, microglia
and other phagocytic cells are involved to export the iron. Mitochondrial proteins
can also involve in exporting iron. For example, iron/sulfur (Fe/S) clusters are acted
as a prosthetic group of respiratory complexes which can contribute to eliminate the
iron from mitochondria. In other way, the storage protein ferritin can reduce the free
from iron in the cytosol by sequestering them, but changes in this export mechanism
can contribute to increase iron accumulation in the brain. In a simple description,
age factors, genetic mutation, and inflammation can cause the iron accumulation in
the brain [56, 62].

1.4 Iron Metabolism and Brain Diseases

Iron deposition in a particular region of the brain can mishandle the iron metabolism
because this region is already rich with iron, and thereby causing various distinct
disorders called neurodegeneration with brain iron accumulation (NBIA). Neurode-
generation with brain iron accumulation is mainly linked with the mutations in the
iron homeostasis genes and is characterized by gait problems, movement dysfunction,
and motor–cognitive problems. Until now, there are fifteen major genetically distinct
disorders which can be discussed in further content. However, other neurodegener-
ative diseases like Parkinson’s disease, Alzheimer’s disease, and Hunting’s disease
can also be believed to be caused by secondary iron accumulation. This is not nec-
essarily the case. It may also be a disease caused by existing iron deposits (such as
age and inflammation).

1.4.1 Iron Metabolism and Parkinson’s Disease

Parkinson’s disease (PD) is one of the most extensive diseases that relate to dys-
regulation of iron metabolism. Although the causes of PD have been very clear, the
initial discovery of PD was written in 1817 by Parkinson [48]. After this, Jean-Martin
Charcot expanded this research internationally [5]. Since then, several concepts are
associated with PD causes. Recent research has received immense interest to find the
role of iron in causing PD. Patients with PD have higher iron accumulation in the
pars compacta area of the substantia nigra. Several MRI studies have also observed
that elevated level of iron is associated with PD. The cause of PD is linked with the
1 Brain Iron Metabolism and CNS Diseases 11

iron-binding ability of the neuromelanin, a dark pigment present in the dopaminergic

neurons, and can sequestrate the reactive irons in the cytosol of neurons. Reduced
binding capacity of neuromelanin to iron can increase the availability of free iron in
the substantia nigra and consequent oxidative damages and mitochondrial dysfunc-
tion, and thus neuronal cell death. Friedman et al. suggested that despite increasing
ferritin-bound iron, free reactive iron can also contribute to set the PD. It has been
observed that Lewy bodies present in the substantia nigra of PD condition, and the
formation of Lewy bodies may be occured with the iron interaction to ubiquitinated
alpha-synuclein. In the earlier stages of PD, alpha-synuclein can be protective, but
the interaction of iron with alpha-synuclein can increase the oxidative damage and
protein aggregation. Studies found that iron can stimulate alpha-synuclein in the post-
mortem brain. Furthermore, iron chelation can partially reverse the alpha-synuclein
aggregation. However, some studies have observed that there is no iron accumu-
lation in the earlier stages of PD, suggesting a puzzling role of iron in the PD. In
addition, loss of neuromelanin binding to iron can cause oxidative stress through
Fenton reaction, resulting in neurodegeneration.

1.4.2 Role of Iron in Alzheimer’s Disease

Abnormal iron metabolism has been considered as a vital marker for the earlier
setting of Alzheimer’s disease (AD). However, the link of iron accumulation to the
AD pathology is not well established. Studies have observed that the patients with
AD have elevated level of iron in the cortical, subcortical, and white matters of the
brain. Amyloid (Aβ) plaque aggregation in a specific region of the brain has been
considered as AD pathology, and Aβ is formed by proteolytic cleavage of amyloid
precursor protein (APP). APP is cleaved by α-secretase and β-secretase. Normally,
alpha pathway is not involved in the generation of amyloidogenesis, whereas beta
pathway is involved in the amyloidogenesis. Overproduction of Aβ42 and alloforms
of Aβ can lead the AD pathogenesis, and the connection of APP and Aβ alloforms
with abnormal iron metabolism has been observed in AD patients. Both ferrous and
ferric forms of iron can interact with the Aβ alloforms. However, Aβ42 fragments
can form amyloid more rapidly. Aggregation of Aβ42 can form monomer, dimers,
oligomers, fibrils, and senile fibril plaques. The ferric form of iron can bind with all
forms of Aβ42. However, it has a difficulty to break from the fibrils, but this plaque
formation with ferric form can initially remove the excess-free form of iron in the
brain. However, the ferrous form can also bind with Aβ42 to revert the function
of amyloid formations. The core of matured amyloid plaque has both ferrous and
ferric form of iron, and the core of amyloid plaque is the place of oxidative stress.
In addition, the ferroxidase activity of APP can prevent the FPN1-mediated iron
loading to Tf resulting in iron retention in the neurons and further causing neuronal
death. Alzheimer’s disease brains have increased the accumulation of iron in the
specific region of the brain such as the hippocampus and cerebral cortex. Aβ plaques
and neurofibrillary tangles have reactive iron and are the important site for catalytic
12 A. Thirupathi and Y.-Z. Chang

reactivity. In addition, iron-binding proteins such as transferrin, ferritin, and IRP2

are linked with Alzheimer’s neurodegeneration. Studies have observed that ferritin-
containing microglia present in the Alzheimer brains, thus suggesting a role of ferritin
in the disruption of the iron homeostasis in the Alzheimer brains.

1.4.3 Iron Overloading in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease, is characterized

by gradual breakdown of nerve cells and cell death. Patients with ALS have higher
iron accumulation in the CNS, and several iron regulatory molecules level is altered
with ALS condition [31, 36]. Dysregulation of iron metabolism can activate microglia
and oxidative stress. At the point, when microglia catabolize the cell debris, it can
express a higher level of ferritin in the ALS. A study observed that iron overloaded
in the activated microglia of motor cortex area, particularly upper motor neuron cell
bodies.

1.4.4 Gene Mutation Induced Disease

Genetic analysis of iron overload condition has revealed a number of target proteins
that control iron homeostasis, and the mutation in these proteins disrupts the iron
homeostasis. Molecular level of research has identified the several loops that control
the iron homeostasis and is mounted in the hepcidin–ferroportin interaction. This
regulatory pathway accounts genetic mutation of iron overloading condition and
related disorder. PKAN is a neuronal iron overloading disease linked with the gene
encoding pantothenate kinase 2 (PANK2) mutations, a crucial enzyme present in
the mitochondrial inter-membrane space and synthesis mitochondrial coenzyme A
from pantothenate. PKAN is previously known as Hallervorden–Spatz syndrome.
This disease is characterized by neurological impairments such as childhood onset
dystonia and spasticity. Study has demonstrated that lack of Pank2−/− in the mice
did not have iron overload causing neurological symptoms in the CNS [64]. Patients
with PKAN have reduced cholesterol level and fatty acid synthesis, and also the
Pank2−/− mice have reduced mitochondrial potential and ATP generation. However,
the exact reason of iron overload in the brain regions is not entrenched, particularly,
why PANK2 associated iron overload in the globus pallidus, the possible reason is
that the other homologues such as PANK1 and PANK3 may be too low to compensate
the loss of PANK2. Likewise, the neurons can cause the iron overload before they
die, which results in iron overload neuron loss in PKAN.
PLAN is linked with different mutations in the PLA2G6 gene, and the symptoms
are dystonia, spasticity, and cerebellar atrophy. PLA2G6 gene encodes phospholipase
A2 group VI, a critical regulator of mitochondrial membrane stability and remodel-
ing. MRI result has shown that iron deposition presents in the globus pallidus and
1 Brain Iron Metabolism and CNS Diseases 13

substantia nigra and striatum varies. PLA2G6 gene producing enzymes regulate fatty
acid synthesis in the brain. However, it is believed that acetyl CoA can recompense
the needs of fatty acid in the brain. MPAN is a condition associated with childhood
or early adulthood. MPAN affected people can have movement problems including
spasticity and dystonia. Other neurological issues such as degeneration in the nerves
that carry information to the brain (optic atrophy), dysphagia and incontinence are
linked with MPAN. Mutations in the C19orf12 can cause MPAN. C19orf12 encodes
a mitochondrial membrane protein which can participate in the fatty acid metabolism
and branched-chain amino acids. MPAN is characterized by iron over accumulation
in the globus pallidus and substantia nigra. In MPAN, Lewy bodies containing abnor-
mal alpha-synuclein present throughout in the globus pallidus, deep gray matter, and
neocortex.
Aceruloplasminemia is characterized by the mutation of CP. It was initially
described in 1987. Ceruloplasmin is a multicopper ferroxidase, lost its ferroxidase
activity in aceruloplasminemia condition. As a result, CP may not be able to convert
the ferrous iron form into ferric form to transferrin, which results to fail the export
iron out of brain astrocytes and macrophages to the circulatory system. Patients with
aceruloplasminemia can have higher iron overload in the astrocytes, and these regions
of the brain lose the neurons, which result in the triad of adult-onset neurological
disease and retinal degeneration. The deformity of astrocytes is a prominent charac-
teristic of aceruloplasminemia brains. The proportion of iron deposition is correlated
with the globular structure of the astrocytes [41]. In addition, the iron does not reach
to the neurons due to inactivity of CP, and thus, neurons are susceptible to die.
Neuroferritinopathy is an autosomal dominant neurodegenerative disease caused
by ferritin light chain 1 (FTL1) light chain mutations. Curtis and groups identified
first time neuroferritinopathy in 2001 [7]. Until now, there have been ten mutations
reported to cause this disease. Additional nucleotide inclusion due to a mutation in
the FTL1 can neglect to sequester the iron, and consequently iron leaks out of the
ferritin. This condition makes the cytosol to turn out to be progressively oxidized
environment. Ferritin can also respond to this oxidized cytosolic environment, and
this can further allow more leakage of iron. Furthermore, higher accumulation of iron
can make the IRE-binding proteins to not turn IRE-binding form which further results
in the overproduction of ferritin and ferritin aggregation with iron in the neurons,
substantia nigra, glial cells of the globus pallidus, and cerebellum. Initial failure
of iron sequestration can lead the higher transcription, and reduced translational
repression in the FTL can allow this abnormal protein segregation in the various
regions of the brain. FAHN is caused by the mutation in the gene encoding fatty acid-
2-hydroxylase gene (FA2H) and is involved to produce fatty acid for sphingolipids.
Defect in this gene is associated with abnormal myelination. Studies have shown that
FAHN patients have iron accumulation in the globus pallidus, substantia nigra, and
subcortical and periventricular regions. Also, ferritin-loaded microglial cells enter
into the areas of regenerating tissue for further iron loading. FAHN is characterized
by the spasticity, ataxia/dystonia, and optic atrophy.
14 A. Thirupathi and Y.-Z. Chang

1.5 Current Bottlenecks in the Prevention and Treatment

of Neurological Diseases

1.5.1 The Evaluation of Neurological Therapies

Several serendipitous findings are observed for treating neurological diseases because
these conditions are different from other disorders, and the evaluation therapy has
been designed based on the developing information of biochemistry, physiology,
and anatomy of the brain. For example, Parkinson’s disease is one of the impor-
tant neurological conditions, and treatment for this disease is tightly based on the
above-mentioned merit. Parkinson was believed that the “progress of the disease may
be stopped” and he deliberately inserted a small piece of cork into the blisters and
inflammatory skin for expelling the pressure away from the brain and spinal cord.
Charcot and groups were first established the treatment for PD using belladonna alka-
loids. These alkaloids influence the cholinergic/dopaminergic balance in the striatum
for improving PD. Since then, many active anticholinergic agents have been devel-
oped for treating PD. However, Charcot wanted to use hyoscyamine. He occasionally
used hyoscyamine with rye-based ergot products, a pharmacological dopamine ago-
nist used to invigorate the striatal dopamine receptors. He inferred in 1872 that he
attempted almost everything to treat PD, but all of them were rejected. Then, Char-
cot tried vibratory therapy for managing PD, and he found that patients with PD had
decreased symptoms. However, the tremor was not improved. He used a combina-
tion of hyoscyamine with arsenic, morphia and cannabis, and opium and he observed
that the patients with PD have distinct improvements [19]. The modern therapy is
used cannabis for dopaminergic activation. Although several anticholinergic drugs
were developed in the nineteenth and twentieth centuries, all of them were similar
effects. Holtz discovered an important enzyme called dopa carboxylase that can con-
vert levodopa to dopamine [29]. Loss of dopamine leads the pathogenesis of PD.
Ehringer and Horneykiewicz observed that the depletion of dopamine in the human
brains [13, 28]. Birkmayer and Horneykiewicz injected the levodopa intravenously
first time to the PD patients, and they observed significant improvement in the PD
[3]. For searching levodopa in the naturally available plants, Manyam observed that
cowitch plant (Mucuna pruriens) contains levodopa [37]. It is pointed out that the
current treatment of PD defects, especially the late use of L-DOPA, has no effect,
and the understanding of iron metabolism is helpful for the treatment of PD.

1.5.2 Alzheimer’s Disease

AD is one of the most leading dementia diseases. Although the number of scien-
tific effort has been made to prevent the disease, there are no effective pharma-
cotherapeutic options available for treating AD. To date, several drugs have been
prescribed based on symptomatic approach for counterbalance the neurotransmitter
1 Brain Iron Metabolism and CNS Diseases 15

disturbances in the AD. Cholinergic hypothesis has been linked with AD including
loss of acetylcholine neurons and acetylcholine synthesis and degradation. There
are three approved drugs named cholinesterase inhibitors (CI) used for enhancing
the cholinergic transmission like donepezil, rivastigmine, and galantamine. These
CIs have been shown to improve the cognitive functions of AD. Another therapeutic
option for AD is memantine, and this drug can bind with N-methyl-D-aspartate antag-
onist for protecting neurons. In addition, a combination of donepezil with memantine
significantly improved the cognitive function of moderate to severe AD patients [27,
54]. Another cause for AD is amyloid (Aβ) plaque aggregation. Several researches
have been driven to finding a compound for preventing Aβ aggregation. The only Aβ
aggregation inhibitor is a synthetic glycosaminoglycan 3-amino-1-propanesulfonic
acid (3APS, tramiprosate). Tramiprosate interferes with the Aβ aggregation. How-
ever, data suggest that tramiprosate increases the abnormal aggregation of Aβ [53].
Other compounds such as Colostrinin and scyllo-Inositol can interfere with the Aβ
aggregation. However, the specificity of those compounds is questionable. Drugs
such as PBT2 is a second-generation drug of 8-OH quinoline interferes with the
Cu2+ and Zn2+ mediated toxic oligomerization of Aβ. Also, several compounds that
inhibit BACE (β-site AP-cleaving enzyme inhibition) and γ-secretase are in the pre-
clinical stage. Therefore, the current clinical trials for the treatment of AD drugs have
failed because the mechanism of AD pathogenesis has not been fully understood,
so the understanding of iron metabolism may contribute to the development of AD
drugs.

1.5.3 NBIA Therapies

Therapies for NBIA disorders are symptomatic. NBIA is associated with different
genes mutations that are linked with iron metabolism-regulating proteins. Therefore,
understanding a common feature that shares to induce the pathogenesis in NBIA can
help to develop therapies for NBIA disorders. For example, aceruloplasminemia and
neuroferritinopathy have common defects in handling iron, which results to increase
the oxidative damages, whereas other NBIA disorders such as PLAN and MPAN
are linked with lipid metabolism and mitochondria. Therefore, finding a common
link with membrane homeostasis, mitochondrial function, and iron metabolism may
advantage therapeutic development. Oxidative stress is one of the common condi-
tions of above-mentioned situations. Therefore, understanding of the mechanism
that induces iron metabolism causing oxidative stress and its further consequences
in developing NBIA may help to design a therapy for NBIA-associated disorders.
16 A. Thirupathi and Y.-Z. Chang

1.6 Conclusion

The present drugs for treating iron overload causing neurodegeneration are symp-
tomatic and do not able to prevent the progression of the diseases. Nevertheless, these
drugs are giving consistent improvements for cognition and global status. In addition,
the search for disease-modifying approach for treating these diseases is unsuccess-
ful in demonstrating the efficacy in the clinical stages. Meanwhile, the research is
focusing to target other possible mechanisms that involve progress iron causing neu-
rodegeneration such as iron overloading neuroinflammation and oxidative stress. For
example, clinical trials of antioxidants usage such as Vitamin E and ω3 fatty acids
did not show any significant improvements in patients with neurodegenerative dis-
eases. Before developing a novel compound to treat iron causing neurodegeneration,
it must be needed profound investigations of underlying mechanisms of pathogenesis
of those neurological diseases. For example, disease-modifying approach has been
failed in stage III of the clinical trial for proving its efficacy against AD pathogene-
sis. It needs a better understanding of tau, Aβ, and other direct and indirect factors
to develop successful disease-modifying drugs. Altogether, new strategies need to
be focused to examine the neuroprotective activity of disease-modifying agents in
the presymptomatic stages of neurological diseases. In the development of drugs for
the treatment of these diseases, it is proposed to consider the joint use of drugs and
methods for regulating iron metabolism to improve the prevention and treatment of
diseases.

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Chapter 2
Cellular Iron Metabolism and Regulation

Guofen Gao, Jie Li, Yating Zhang and Yan-Zhong Chang

Abstract Iron is an essential trace element in the human body, but excess iron is toxic
as it contributes to oxidative damage. To keep iron concentration within the optimal
physiologic range, iron metabolism at the cellular level and the whole systemic
level are tightly regulated. Balance of iron homeostasis depends on the expression
levels and activities of iron carriers, iron transporters, and iron regulatory and storage
proteins. Divalent metal transporter 1 (DMT1) at the apical membrane of intestinal
enterocyte brings in non-heme iron from the diet, whereas ferroportin 1 (FPN1) at the
basal membrane exports iron into the circulation. Plasma transferrin (Tf) then carries
iron to various tissues and cells. After binding to transferrin receptor 1 (TfR1), the
complex is endocytosed into the cell, where iron enters the cytoplasm via DMT1
on the endosomal membrane. Free iron is either utilized in metabolic processes,
such as synthesis of hemoglobin and Fe–S cluster, or sequestered in the cytosolic
ferritin, serving as a cellular iron store. Excess iron can be exported from the cell via
FPN1. The liver-derived peptide hepcidin plays a major regulatory role in controlling
FPN1 level in the enterocyte, and thus controls the whole-body iron absorption.
Inside the cells, iron regulatory proteins (IRPs) modulate the expressions of DMT1,
TfR1, ferritin, and FPN1 via binding to the iron-responsive element (IRE) in their
mRNAs. Both the release of hepcidin and the IRP–IRE interaction are coordinated
with the fluctuation of the cellular iron level. Therefore, an adequate and steady iron
supplement is warranted for the utilization of cells around the body. Investigations on
the molecular mechanisms of cellular iron metabolism and regulation could advance
the fields of iron physiology and pathophysiology.

Keywords Iron · DMT1 · TfR1 · FPN1 · Ferritin · IRPs · Hepcidin

G. Gao (B) · J. Li · Y. Zhang · Y.-Z. Chang (B)

Laboratory of Molecular Iron Metabolism, College of Life Sciences, Hebei Normal University,
Shijiazhuang, Hebei Province, China
e-mail: [email protected]
Y.-Z. Chang
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2019 21

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_2
22 G. Gao et al.

2.1 Body Iron and Its Metabolism

Iron (Fe) is one of the essential trace metals in the human body, which is found in
the active centers of many enzymes and oxygen carrier proteins [1]. Iron is a critical
component of cytochromes a, b, and c, cytochrome oxidase, and iron–sulfur (Fe–S)
complexes of the oxidative chain, therefore important for adenosine triphosphate
(ATP) production. Iron is a co-enzyme in ribonucleoside reductase which commits to
DNA synthesis. Iron is also involved in TCA cycle since the succinate dehydrogenase
and aconitase are both Fe-dependent enzymes. In addition, the iron in the brain is
required for myelogenesis and myelin maintenance as well. Therefore, a constant
and readily available supply of iron is important for all the organs of the body [2].
The iron transport, storage, and regulation are tightly regulated at the cellular and
systemic levels [1, 2].
Human bodies contain 4–5 g of iron on average. Dietary iron is absorbed pre-
dominately in the duodenum and enters blood circulation in the small intestine. The
absorption of heme iron is poorly understood. Non-heme iron is transported across
the apical membrane of the intestinal enterocyte by divalent metal transporter 1
(DMT1) and is exported into the circulation via ferroportin 1 (FPN1). Once in blood
circulation, iron binds to apotransferrin and forms Fe-transferrin (Tf) complex [3].
Serum Tf is the major vehicle for iron transport in the body and carries iron to other
cells and tissues through the circulation [3]. At the target cell, Tf binds to transfer-
rin receptor 1 (TfR1) on the cell membrane, and the TfR1-Tf-Fe complex is then
endocytosed into the cell, where the iron is released [4]. Free iron either enters mito-
chondrion for utilization in metabolic processes, such as synthesis of hemoglobin
and Fe–S cluster, or is incorporated into the cytosolic iron storage protein, ferritin
and serves as a cellular store of iron [5]. Excess iron is transported out of the cell by
iron efflux protein ferroportin 1 (FPN1) located on the cell membrane [6].
About 2.5 g iron in the body is contained in the hemoglobin, the red pigment in red
blood cells, which is needed to carry oxygen through the blood. This portion of iron
is circulated in the body by macrophages after they engulf the senile erythrocytes and
release iron. A relatively small amount iron (3–4 mg) circulates through the plasma,
bound to Tf. Most of the rest iron is stored in ferritin complexes that are present in all
cells, but most common in bone marrow, liver, and spleen [1, 5]. Of these iron stores,
the iron in the liver is the primary physiologic source of reserve iron in the body.
There are a lower non-hemoglobin hemoproteins, such as myoglobin, cytochromes,
catalases, heme peroxidase, and endothelial nitric oxide synthase, involved in diverse
biological functions including the transportation of diatomic gases, chemical catal-
ysis, diatomic gas detection, and electron transfer [7]. Because of its toxicity, free
soluble iron in the body is kept in low concentration in the body.
2 Cellular Iron Metabolism and Regulation 23

2.2 Regulation of Iron Metabolism

Iron homeostasis in the body is regulated at two different levels, the systemic level
and the cellular level [8]. The systemic iron level is mainly balanced by the controlled
absorption of dietary iron by intestinal enterocytes, the cells that line at the interior of
the intestines. The cellular iron level is controlled differently by different cell types
due to the expression of particular iron regulatory and transport proteins [9].

2.2.1 Systemic Iron Regulation

At the systemic level, iron homeostasis involves the regulation of a peptide ‘hormone’
hepcidin [10]. Hepcidin is mainly produced by hepatocytes in response to high serum
iron concentration, inflammatory stimuli, or hypoxia [10, 11]. It binds to the extra-
cellular loop of FPN1 and causes its internalization and degradation, and thereby
reduces cellular iron efflux [2, 12]. Hepcidin regulates iron homeostasis mainly via
acting on FPN1 of intestinal enterocytes and FPN1 of macrophages, thereby affect-
ing iron intake from the intestine and iron release from macrophages [13, 14]. The
level of hepcidin is also regulated by the systemic iron level [15]. When body iron is
overloaded, the level of hepcidin increases to reduce circulating iron. When body iron
is deficient, the level of hepcidin decreases to promote small intestinal iron intake.
In addition to the iron level, inflammation, hypoxia, and erythropoiesis regulate the
expression of hepcidin as well [16].

2.2.2 Cellular Iron Regulation

Cellular iron homeostasis is tightly regulated by iron regulatory proteins (IRPs),

including IRP1 and IRP2, which subsequently regulate the levels of iron transporters,
TfR1, divalent metal transporter 1 (DMT1) and FPN1, and ferritin as well [17].
Generally, when the cellular iron concentration is low, the active center of IRPs
binds to the stem-loop structure of the iron-responsive element (IRE) located at the
3 -untranslated region (UTR) of TfR1 and DMT1 mRNAs. This binding stabilizes
TfR1 and DMT1 mRNAs and increases its cellular expression, thereby increasing
iron uptake [18]. When the iron concentration is high, the active center of IRPs is
occupied by four Fe–S, which blocks the binding of IRPs to IRE of TfR1 or DMT1,
resulting in low TfR1 and DMT1 translation levels, and thus reduces iron uptake
[18]. The IRP/IRE system also regulates the stability of FPN1 and ferritin. However,
the binding of IRPs to the IRE of FPN1 or ferritin mRNA located at their 5 -UTR
suppresses their translation and causes lower protein expression [18]. Thus, IRPs
play a key role in the maintenance of cellular iron homeostasis. Studies have shown
24 G. Gao et al.

that the IRPs knockout mice had significant misregulation of iron metabolism and
developed neurodegeneration [18, 19].
In the intestine, the control of ferritin and FPN1 expression by IRPs limits intesti-
nal iron absorption and thus controls the whole-body iron level [20]. In the bone
marrow, IRPs regulate the translation of erythroid 5-aminolevulinate synthase’s
mRNA; therefore, the erythroid heme biosynthesis is coordinated with iron avail-
ability. Besides, the translational control of HIF2α mRNA in the kidney by IRP1
coordinates erythropoietin synthesis with iron and oxygen supply [19, 20].

2.3 Iron Metabolism-Related Proteins

2.3.1 Iron Transport Protein-Tf

Transferrin (Tf) is a serum glycoprotein with a molecular weight of 80 kDa, which

is mainly produced by the liver. It is an important iron chelating protein with serum
iron transport function. Tf transports iron in the blood by picking up free ferric iron
(Fe3+ ) and delivering it into cells [3]. Tf can reversibly bind to Fe3+ . Once binding, a
conformation change in Tf will occur, which facilitates its recognition and binding
to its receptor-TfR1 on the cell membrane [3, 4]. The binding of Tf to TfR1 will
initiate a receptor-mediated endocytotic process, in which the Tf-TfR1 complex is
internalized. Iron is released in the endosome, and the complex is recycled to the cell
surface where the Tf is released [4, 9]. Thus, iron is transported to various organs
and tissues of organisms through blood transportation and utilized for various life
activities.

2.3.2 Iron Uptake Proteins-TfR1 and DMT1

Transferrin receptor (TfR1) is a transmembrane single-chain glycoprotein with two

disulfide subunits, which is composed of ~700 amino acids and has a molecular
weight of about 95 kDa, responsible for iron transport in cells [21, 22]. TfR1 specif-
ically recognizes and binds with Tf at its extracellular domain, with the highest
affinity to Tf carried two Fe3+ ions [4]. The complex formed by TfR1 and Tf initiates
the formation of endocytosis vesicles on the membrane and finishes the endocytosis
process. After iron dissociation, reduction, and translocation, ferrous iron (Fe2+ ) was
released via DMT1, and then TfR1 was recycled to the surface of the cell membrane.
There are IRE sequences located at the 3 -UTR of TfR1 mRNA. Under iron defi-
ciency condition, IRPs bind to the IRE sequence of TfR1 mRNA to prevent TfR1
mRNA degradation, which leads to the increase in TfR1 protein expression. On
the other hand, under iron overload status, IRP is dissociated from IRE of TfR1’s
2 Cellular Iron Metabolism and Regulation 25

mRNA, bound to iron, which leads to the degradation of TfR1 mRNA by nuclease,
and subsequently decreases TfR1 protein synthesis [23].
Divalent metal transporter 1 (DMT1) is a bivalent metal ion transporter with a
molecular weight of approximately 61 kDa [24]. In 1997, both Harvard University
and Yale University reported this transmembrane iron transporter [24, 25]. DMT1
plays a key role in maintaining the homeostasis of bivalent metal ions in cells, espe-
cially iron and manganese [26]. DMT1 can be divided into DMT1 (+IRE) and DMT1
(−IRE) according to whether its 3 -UTR contains IRE sequence. DMT1 (+IRE) is
regulated by IRPs in response to intracellular iron level [18]. In the case of iron
overload, IRPs bind to iron, which reduces the stability of DMT1 (+IRE) mRNA,
promotes its degradation and decreases the level of DMT1 protein. On the contrary,
under iron deficiency status, DMT1 expression increases [18].

2.3.3 Iron Storage Protein-Ferritin and FtMt

Ferritin, a major intracellular iron storage protein, consists of 24 subunits, a spherical

polymer with a huge cavity, which can hold up to 4500 iron atoms [27]. Ferritin can
be divided into two monomer subunits: heavy chain (H-ferritin, 21 kDa) and light
chain (L-ferritin, 19 kDa), respectively [5, 27]. The H-ferritin subunit has the activity
of ferrous oxidase, which oxidizes Fe2+ to Fe3+ . L-ferritin promotes the formation
of iron nuclei and has a larger iron storage capacity than H-ferritin. Ferritin mainly
participates in iron storage and utilization in cells, which limits the unstable labile
iron pool (LIP) in cytoplasm and nucleus, protecting cells from iron-induced damage
[5, 27].
The translation levels of both L-ferritin and H-ferritin are regulated by intracellular
IRPs. There are IRE sequences located at the 5 -UTR of ferritin mRNAs. When the
cell is at iron deficiency status, IRPs bind to IREs to prevent ferritin translation and
downregulate ferritin expression, thereby reducing iron storage. Conversely, when
an iron overload occurs in cells, IRPs cannot bind to IREs, the inhibition of ferritin
translation is relieved, the expression of ferritin increases, and the capacity of iron
storage in cells increases [28].
Mitochondrial ferritin (FtMt) is a kind of iron storage protein located in the mito-
chondrion, which has high homology with H-ferritin and can accumulate specifically
in the mitochondrion [29]. It was found that FtMt could regulate iron distribution
between cytoplasm and mitochondria by incorporating free iron in mitochondria,
which reduces iron content in the cytoplasm and thereby reduces the production of
ROS [30, 31]. FtMt is mainly expressed in mitochondria of some tissues with high
oxygen consumption, such as testis, central nervous system (CNS), etc. [32]. This
tissue-specific distribution may be related to the protection of mitochondria from
iron-dependent oxidative damage caused by high metabolism and high oxygen con-
sumption [33]. FtMt is also expressed in the brain, and the level of FtMt in neurons
was found higher than that in glial cells [34].
26 G. Gao et al.

2.3.4 Iron Efflux Protein-FPN1

Ferroportin (FPN1), also known as MPT1 or IREG1, is the currently only known
multitransmembrane iron export protein in mammalian cells, with a molecular weight
of about 62 kDa [6]. Three groups independently discovered this iron transporter in
2000 [35–37]. FPN1 commonly expresses on the membrane of all the cells, which
allows Fe2+ to pass through. The basal surface of epithelial cells, the cellular surface of
macrophages in liver and spleen, and trophoblast of placenta express relatively higher
levels of FPN1, in order to export iron out of intestinal enterocytes, hepatocytes, and
splenic macrophages to the blood. FPN1 transports ferrous ions out of cells with
the assistance of ferrous oxidase ceruloplasmin (CP) or hephaestin (HP) [38]. The
expression of FPN1 is mainly regulated by two aspects. On the one hand, IRPs
regulate FPN1 expression at the post-transcriptional level via the IRP–IRE system.
The 5 -UTR of FPN1 mRNA has the IRE structure [39]. IRP–IRE binding weakens
the expression of FPN1 protein through translation inhibition. In addition, FPN1
protein level is also regulated by hepcidin. Hepcidin can recognize and bind to FPN1
on the membrane, which induces its internalization [14, 40]. After internalization,
FPN1 and hepcidin are transported to lysosomes and both were degraded [41]. It was
recently reported that hepcidin can also inhibit the activity of FPN1 [42, 43], which
then reduces the release of intracellular iron, thus causing changes in intracellular or
tissue iron levels. In addition, the regulation of hepcidin on FPN1 may also coordinate
with IRPs, together affecting FPN1’s level [44].

2.3.5 Iron Metabolism Regulatory Proteins–IRPs

and Hepcidin

Iron regulatory proteins (IRPs) are cytosolic soluble RNA-binding proteins that bind
to IRE sequences on mRNAs of iron transporters and iron storage proteins, reg-
ulating their expression [17, 45]. IRPs have two forms, iron regulatory protein 1
(IRP1, ~90 kDa) and iron regulatory protein 2 (IRP2, ~105 kDa). IRP1 is rich in
most tissues, while IRP2 is most abundant in the brain and small intestine. When
iron is deficient in cells, IRPs bind to IRE of the target mRNA, and while iron
level is elevated, IRPs dissociate from IRE, regulating protein expression at the
post-transcriptional level [17, 23]. IRPs bound to IREs of TfR1 mRNAs, as well as
DMT1 mRNAs, promote their expression level, thus raising cellular iron absorp-
tion. Meanwhile, IRPs bound to IREs of FPN1 mRNAs reduce cellular iron release
by suppressing FPN1’s translation [39, 45]. Besides, IRPs regulate H-ferritin and
L-ferritin translation, thus adjusting iron storage. These mechanisms have probably
evolved to maintain the cytoplasmic LIP level at a steady level.
Hepcidin, a 25 amino acids peptide, produced mainly by the liver in response
to high serum iron, is the ‘master’ regulator of iron homeostasis at systemic level
[14]. Hepcidin was identified as a urinary antimicrobial peptide synthesized in the
2 Cellular Iron Metabolism and Regulation 27

liver in 2000 [10]. Hepcidin restricts the release of iron by controlling FPN1 on
the membrane, inducing its internalization and degradation [12]. Recently, the study
reported that hepcidin can also downregulate the activity of FPN1 [42]. Hepcidin
level is in turn regulated by body iron demand, through BMP/SMAD and TfR2
pathways. When the body iron is replete, a higher hepcidin concentration reduces
iron absorption at intestine and impairs iron release from iron stores; but when the
body iron is deficient, hepcidin level is low, thereby favoring iron absorption and
delivery to the plasma from storage sites [15]. Inflammation is also an important factor
affecting the expression of hepcidin. Interleukin-6 (IL-6) upregulates the expression
of hepcidin through the STAT3 pathway [46]. In addition, hepcidin is negatively
regulated by erythropoietin, oxygen environment, and sex hormones [16, 47].

2.4 Pathophysiology of Iron

2.4.1 Iron Deficiency

Iron deficiency is a state that the body iron amount is lower than the normal physio-
logic conditions, and the body lacks enough iron to supply its needs. Iron deficiency
may impair the synthesis of essential iron-containing proteins that are required for
normal cellular physiology and result in a range of adverse consequences [48]. When
the loss of iron is not sufficiently compensated by adequate iron intake from the diet,
a state of iron deficiency will develop over time [48].
Untreated iron deficiency can lead to iron deficiency anemia, the most common
type of anemia [49]. In iron deficiency anemia, the body lacks sufficient amounts of
iron to produce the protein hemoglobin, thereby resulting in inadequate red blood
cells (RBCs). Besides, iron deficiency can also be resulted from genetic disturbances
in iron homeostasis. In particular, mutations in the TMPRSS6 gene, which encodes
an upstream regulator of hepcidin, can lead to iron-refractory iron deficiency anemia
[50]. The treatment of mild iron deficiency can be achieved simply by increasing the
amount of bioavailable iron in the diet or by taking iron supplements; while in the
severe case of iron deficiency anemia, an intravenous iron infusion may be used to
efficiently deliver a large amount of iron [51].
The anemia of chronic disease is the second most common form of anemia that is
caused by iron deficiency with inflammation [52]. The anemia of chronic disease is
usually due to the presence of chronic infection, chronic immune activation, malig-
nancy, or various chronic inflammatory states. These conditions all produce massive
elevation of pro-inflammatory cytokine IL-6. IL-6 stimulates hepcidin production
and release from the liver, which in turn reduces the iron efflux protein FPN1, result-
ing in reduced iron uptake at intestine and reduced iron release from iron stores and
macrophages, thereby reducing iron in the circulation. The plasma iron concentra-
tion decreases, and macrophages and other cell types sequester iron. If this condition
persists, the iron supply to the erythroid marrow can be compromised, resulting in
28 G. Gao et al.

reduced erythropoiesis, namely the anemia of chronic disease [52]. Hepcidin antago-
nists or agents that block hepcidin gene synthesis and subsequently increase intestinal
iron absorption have shown promise in the treatment of the anemia of inflammation
[53].

2.4.2 Iron Overload

Iron overload is also associated with a variety of diseases [54]. Excess LIP in the
cell may catalyze reactions that generate ROS and consequently cause oxidative
damage to cells and tissues, which can finally lead to tissue fibrosis and organ dys-
functions [55]. Some iron overload diseases are caused by genetic mutations in
the genome, such as hemochromatosis, atransferrinemia, aceruloplasminemia, and
Friedreich ataxia [54–56]. Some are resulted from the chronic disorders, e.g., chronic
liver disease [57]. Iron overload is also complicated with a range of other diseases,
such as cystic fibrosis, fatty liver disease, and neurodegenerative diseases, including
Alzheimer’s disease (AD), Parkinson’s disease (PD), and so on [54, 58].
Hemochromatosis is a major group of primary iron overload diseases, which is
resulted from mutations in genes that are involved in the transport of iron or the regula-
tion of iron metabolism [59]. Currently, there are five main types of hemochromatosis
are identified due to mutations in the genes encoding hemochromatosis protein HFE,
hemojuvelin (HJV), hepcidin, TfR2, and FPN1 [59, 60]. The common feature of these
different types is a reduced hepcidin expression level [59]. The reduced hepcidin
level leads to a higher FPN1 level on the membrane of intestinal enterocytes, which
increases the absorption of dietary iron, and therefore the body iron load increases.
In these patients, iron accumulation was observed in many organs, particularly in the
liver, heart, and pancreas [54]. Clinical syndromes of the hemochromatosis patients
include hepatic fibrosis and cirrhosis, hepatocellular carcinoma, arthritis, cardiomy-
opathy, and so on [59]. The disease of hemochromatosis is relatively common in the
populations of Northern European origin, but is a rare case in other populations [60].
Hemochromatosis is typically treated via phlebotomy with the removal of ∼0.5 g
Fe/L blood, which is the currently most effective and relatively inexpensive method
[54]. After removal, the serum iron and RBCs in the blood, iron in the storage sites,
notably in the liver, will be subsequently released to replenish the reduced RBCs in
blood for erythropoiesis [54].
Thalassemia is one kind of anemia with iron overload instead of iron deficiency.
In thalassemia, patients have defects in either the α- or β-globin chain, causing the
production of abnormal hemoglobin, thereby characterized by reduced RBCs and
thus anemia [61]. However, blood transfusions for treatment of this inherited disor-
der usually lead to secondary iron overload, with resulting heart or liver diseases,
infections, and osteoporosis. It is most common among people of Italian, Greek, Mid-
dle Eastern, South Asian, and African descent. In the treatment of iron overload in
thalassemias, iron chelators are commonly used [61]. Hepcidin mimetics or agents
2 Cellular Iron Metabolism and Regulation 29

that increase hepcidin expression could also be promising drugs in the treatment of
hemochromatosis and thalassemia [62].
Abnormal iron accumulation has been detected in various neurological diseases in
CNS, including neurodegeneration with brain iron accumulation (NBIA) diseases,
AD, PD, Friedreich’s ataxia, amyotrophic lateral sclerosis, stroke, and so on [63,
64]. Although the causes of most of the neurological disorders have not been fully
understood, common features of these diseases are iron accumulation and increased
oxidative damage [63]. Iron chelators, such as deferoxamine (DFO), have been shown
good efficacy as potential therapeutic agents for iron overload-associated neurolog-
ical disorders [65]. As early as in 1991, McLachlan et al. had used DFO to treat
AD in a clinical trial. They found that continuous administration of DFO could alle-
viate AD-induced cognitive impairment [66]. In the recent years, it was found that
DFO treatment reduced the cognitive decline and the accumulation of amyloid-β in
APP/PS1 mice, which may be related to the induction of M2 activation and inhibition
of M1 activation by DFO in the hippocampus of mice [67]. Researchers have suc-
cessfully developed a brain-targeting peptide-modified nanopolymer encapsulated
DFO, which can cross the blood–brain barrier through receptor-mediated endocy-
tosis [68]. This improvement greatly increased the efficiency of DFO entering the
brain, and significantly decreased the iron content in substantia nigra and striatum,
reduced oxidative stress level, and alleviated dopaminergic neuron damage in PD
mouse model [68].

2.5 Conclusions

Iron is an essential trace element in human body, but it is also toxic in excess. Thus,
iron homeostasis at cellular level and the whole-body iron concentration are tightly
regulated to keep it within the optimal physiologic range. Body iron is acquired from
the diet and exported into the circulation for utilization. Therefore, the amount of iron
uptake at intestinal enterocyte is fairly critical. Both the levels of DMT1 and FPN1
expressed by intestinal enterocytes are must under precise regulations. The liver-
derived peptide hepcidin in serum plays a major regulatory in controlling FPN1
level on the membrane of intestinal enterocytes. While intracellularly, IRPs regulate
the levels of DMT1, TfR1, ferritin and FPN1 in response to iron level changes.
Therefore, an adequate and steady iron supplement is warranted for the utilization of
cells around the body. Similar as the iron regulation in enterocyte, the cellular iron
levels in other cells and tissues are controlled differently by different cell types due to
the expression of particular iron regulatory and transport proteins. Abnormal levels
of body iron, too little or too much, are commonly associated with diseases of iron
deficiency or iron overload. Despite major recent advances have achieved regarding
the mechanisms of cellular iron metabolism, much remains to be learned about iron
physiology and pathophysiology.
30 G. Gao et al.

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Chapter 3
Brain Iron Metabolism and Regulation

Peng Yu and Yan-Zhong Chang

Abstract With the development of research, more and more evidences suggested
that mutations in the genes associated with brain iron metabolism induced diseases in
the brain. Brain iron metabolism disorders might be one cause of neurodegenerative
diseases. This review mainly summarizes the normal process of iron entry into the
brain across the blood–brain barrier, and the distribution and transportation of iron
among neurons and glial cells, as well as the underlying regulation mechanisms. To
understand the mechanisms of iron metabolism in the brain will provide theoretical
basis to prevent and cure brain diseases related to iron metabolism disorders.

Keywords Blood–brain barrier · Transferrin receptor · Ferroportin 1 · Iron

regulatory protein · Hepcidin

3.1 Introduction

Iron is one of the most abundant trace elements and plays great roles in keeping
physiological homeostasis in the body. In the central nervous system (CNS), iron
participates in the synthesis of myelin and neurotransmitters [24, 39]. Iron deficiency
in the brain of infants will lead to abnormal neural development and mental retarda-
tion [21, 70]. While iron accumulation in specific brain regions is also shown to be
related to the patients with neurodegenerative diseases such as Parkinson’s Disease
(PD), Alzheimer’s Disease (AD) and Huntington’s Disease (HD). Excess iron can
react with cellular H2 O2 and generate reactive oxygen species (ROS) through Fenton

P. Yu (B) · Y.-Z. Chang (B)

Laboratory of Molecular Iron Metabolism, Key Laboratory of Animal Physiology, Biochemistry
and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University,
20, Nanerhuan Eastern Road, Shijiazhuang, Hebei Province 050024, China
e-mail: [email protected]
Y.-Z. Chang
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2019 33

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_3
34 P. Yu and Y.-Z. Chang

reaction, and increased ROS could induce a cascade of events to destroy membrane,
protein, and nucleic acid, which caused apoptosis and even cell death [38, 65, 96].
Therefore, it is very important to keep iron homeostasis in the brain.

3.2 Iron Uptake into the Brain

3.2.1 Iron Enters the Brain Across Blood–Brain Barrier

(BBB)

Iron homeostasis in the brain was regulated strictly in mammalian animals, and it
depends on BBB and many molecules involved in the transportation of iron from the
blood circulation. In CNS, the microvascular endothelial cell (BMVEC), together
with astrocyte end-feet and pericytes, forms BBB which is the primary gatekeeper
for iron entry into the brain. Because there are tight junctions between BMVEC,
iron was first uptake into BMVEC from blood and then across BBB with the help of
many iron metabolism-related proteins [1, 44].
Long time ago, it is thought that iron can cross BBB into the brain mainly during
infancy before the formation of BBB. Now, new data demonstrated that iron can also
enter the brain across BBB in the adult brain, which depends on transferrin receptor
1 (TfR1) [56] and ferroportin 1 (FPN1) [50, 71, 92]. Iron can be bound in transferrin
(Tf) and transport in the blood, and Tf-TfR1 has been known as the main major route
of iron transport across the luminal membrane of the capillary endothelium from the
blood into endothelial cells [8, 47, 56]. First, Tf-bound iron in serum reached BBB,
the Tf-Fe binds to TfR1 in BMVEC, which forms endosomes after endocytosis of Tf-
TfR1. When pH value decreased to 5.5~6.5 in the endosomes, iron dissociated from
Tf and reduced to ferrous iron which released from the endosomes into endothelial
cytoplasm with the help of divalent metal transporter 1 (DMT1) [80, 81]. After
dissociation of iron from Tf-TfR1, apo-Tf-TfR1 returned to the apical surface of
BMVEC for the next round of iron uptake [9, 74], and intracellular iron is then either
incorporated into functional proteins, stored in ferritin, or continued to transport
across the abluminal membrane of the endothelial cell into the brain interstitium and
parenchyma [8, 56]. During the process of iron transport across the basal membrane
of intestinal epithelial cells, ferroportin 1 (FPN1) is an iron exporter and responsible
for iron efflux from epithelia into the blood in the presence of hephaestin (HP) and
ceruloplasmin (CP). Our previous results showed that FPN1 was distributed in murine
brain such as cerebral cortex, hippocampus, and striatum [88]. Others also reported
that FPN1 was also expressed in BBB, ependymocytes and choroid plexus, and that
FPN1 was especially expressed in the basal surface of BMVEC [90]. Moreover, CP
was also mainly expressed in the end-feet of astrocytes surrounding microvascular
vessels in the brain [41, 42, 68]. These evidences suggested that FPN1/CP might
play critical roles in iron efflux into brain parenchyma from BMVEC just similar to
that of enterocytes in duodenum. It is also reported that astrocytes can also directly
3 Brain Iron Metabolism and Regulation 35

uptake ferrous iron from BMVEC through the end-feet [49, 64]. However, there are
no direct evidences in vivo.
Besides Tf-TfR1 dependent iron uptake pathway, there are Tf-independent iron
transport ways. Ferritin (H-ferritin) can also deliver iron to the brain across BBB,
which is an iron transport system independent of Tf-TfR system. Ferritin might bind
to ferritin receptor or be transcytosed across the BBB [24]. Lactoferrin (Lf) and
its receptor (LfR), glycosylphosphatidylinositol anchored form of melanotransferrin
(p97, MTf), and soluble form of MTf also play roles in iron uptake across BBB from
the blood [22, 59]. Lf belongs to the Tf family and it is an iron-binding glycoprotein
which is distributed in milk or other secretion in peripheral tissues. In CNS, Lf is
expressed and secreted from activated microglia [2, 23], and LfR is expressed in
neurons, BMVEC, and some glial cells. Lf-LfR might play similar roles as Tf-TfR1
in iron transport into the brain parenchyma [21]. MTf is a transferrin homolog, and
GPI-MTf also plays similar roles as Tf-TfR1 in iron transport across BBB, and
secreted MTf seems to play more important roles in iron metabolism of patients
[59].
However, it is still not fully understood the detailed regulatory mechanisms of the
crosstalk between brain iron uptake and release at the BBB in vivo.

3.2.2 Iron Enters the Brain Across Blood Cerebrospinal

Fluid Barrier (BCB)

It is speculated that iron cross the blood–cerebrospinal fluid barrier (BCB) from
the blood is similar to iron transport at BBB. In situ hybridization found that the
choroid plexus expressed high levels of TfR1, DMT1, FPN1, Dcytb, and soluble
form of CP and HP, which suggested that choroid plexus might mediate significant
iron transport in CNS [52, 67, 78]. The choroid plexus can synthesize and secret
Tf, which might be involved in iron transport from BCB to the brain. But choroid
plexus-derived Tf does not seem too important to bind iron and transport iron in the
brain interstitium because [59 Fe125 I]Tf injected intracerebroventricularly was never
observed in regions distant from the CSF. Tf in the CSF probably plays a significant
role in export to the blood of iron [55].
Stromal cell-derived receptor 2 (SDR2) is expressed in the choroid plexus and
ependymal cells lining the four ventricles with in situ hybridization analysis [85].
SDR2 is the homolog of duodenal cytochrome b that is a ferric reductase, and DMT1
is also located in the choroid plexus [29], so SDR2 might also have ferric reductase
activity and participate in iron uptake with DMT1 at BCB.
36 P. Yu and Y.-Z. Chang

3.3 The Distribution and Transportation of Iron

in the Brain

3.3.1 Iron Is Distributed Unevenly in Different Brain Regions

After iron is released into brain interstitial fluid, it is distributed and transported
among different cell types in different brain regions. It is reported that 10% of non-
heme iron entered the brain across BBB from the vertebrate blood and that iron levels
are different in different brain regions [11, 77]. Iron contents are most abundant in
the extrapyramidal system, especially in the basal ganglia region [58, 60], and iron
concentrations are high in subtantia nigra (SN), red nucleus, and cerebellar dentate
gyrus, while iron is relatively low in cerebral cortex, and iron levels are the lowest
in white matter and medulla oblongata [17, 58, 60].

3.3.2 Distribution and Transportation of Iron in Different

Cell Types of the Brain

Iron is released into the brain interstitial fluid across BBB, and it is absorbed by
neurons and different types of glial cells, but iron is distributed unevenly in these
cells [57, 77]. Neuroglia expressed almost all the iron transporters and distinct profiles
of iron metabolism related proteins [6, 32, 78, 89]. Data from primary cultures of
neurons, astrocytes, and microglia showed that iron retention in microglia was three
times higher than that of neurons when they were incubated with iron [7]. Studies in
a hippocampal slice culture mode also supported that ferritin mRNA was induced by
iron predominantly in microglia and oligodendrocytes, followed by astrocytes, but
never in neurons [31]. So neuroglia are indispensable to maintain iron homeostasis
and normal physiological function in the brain.
As for neurons, they express TfR1 and acquire iron through classical Tf-TfR1-
dependent iron uptake pathway from the brain interstitial fluid [30]. Neurons can also
uptake ferrous and ferric iron through DMT1 and trivalent cation-specific transporter
(TCT1), respectively [4, 39, 73].
As for astrocytes, the major glial cell type in CNS, they are important components
of BBB, so they play crucial roles in iron transport and are responsible for iron distri-
bution in the brain [10, 57]. They exclusively express glycosylphosphatidylinositol-
anchored CP (GPI-CP) in the end-feet surrounding BBB [35, 67]. Astrocytes also
secrete a soluble form of CP into interstitium which also facilitates iron trafficking
across BBB into the brain [52, 83]. Both GPI-CP and soluble form of CP are required
for the stability of FPN1 in the membrane and iron release from BMVEC. Moreover,
hephaestin present in astrocytes [10, 86] can also oxidize BMVEC released ferrous
iron to ferric iron so as to facilitate incorporation of iron into Tf and the following
transportation [13, 79, 94]. Astrocytes acquire iron across the interstitial space using
3 Brain Iron Metabolism and Regulation 37

NTBI mechanisms such as citrate, ATP, ascorbic acid, DMT1, and Zip [45, 69, 91].
They release iron through FPN1, and the released iron can be acquired by neurons
and other types of glial cells.
Oligodendrocytes are unique to the brain, because they are the only cells with
synthesized Tf and releasing Tf, and they express the Tim2 (T cell immunoglobulin
and mucin domain protein-2) ferritin transporter and contain a large amount of the
brain’s lipids and cholesterol [70], and they have high levels of iron and require large
amounts of iron in the myelination of neuronal axons to form the white matter in the
brain [5].
Microglia compromise approximately 12% of cells in the brain, and the resident
microglia are very stable. Microglia express the transporters or molecules involved in
iron metabolism, and they are most efficient in accumulating iron as mentioned above
[7, 83]. Under inflammation and environmental and endogenous stimuli, microglia
can sense and recognize the changes, and then, they become activated, iron retention
and affect the iron metabolism and function of neurons [83, 84]. Immunofluorescence
and hybridization in situ results revealed that activated microglia could synthesize
and secrete lactoferrin [2, 23], which can also affect the iron metabolism of lactoferrin
receptor expressing cells through the receptor-mediated pathway [91].
After iron is used for neuronal and glial metabolism and stored in ferritin, excess
iron is released via FPN1. The excess iron can be exported back to the interstitial
fluid or released into the cerebrospinal fluid in the brain ventricles [8, 27, 97], and the
choroidal epithelia capture iron by TfR1-dependent or TfR1-independent pathway
and then transport it back to the blood circulation [27].

3.4 The Regulation of Iron Metabolism in the Brain

Iron homeostasis is very important for normal brain functions. Iron homeostasis
depends on iron uptake, storage, and release [34]. Iron regulatory proteins (IRPs)
and hepcidin are critical regulators and coordinate with each other to maintain iron
homeostasis [28, 87].

3.4.1 IRPs Regulate Brain Iron Metabolism from Cellular

Level

IRP1 share high sequence homology with IRP2, and IRPs will regulate iron uptake,
iron storage, and iron release proteins with iron responsive element (IRE) in the gene
transcripts [61]. When cellular iron levels change, IRPs will regulate iron metabolism
through IRE-IRP regulatory systems at post-transcription level [40, 66].
In 2006, Rouault lab reported that IRP1−/− IRP2−/− double knockout mice were
embryonically lethal [82], which demonstrated the critical roles of IRPs in life.
38 P. Yu and Y.-Z. Chang

However, IRP1 and IRP2 have different distribution patterns, only IRP2 is highly
expressed in the brain [54]. Rouault lab and others also generated IRP2−/− mice,
and it was reported that excessive iron is accumulated in the brain of IRP2−/− mice
and neurodegenerative diseases like impairments were observed in aging IRP2−/−
mice [12, 25, 46, 66, 98], which further confirmed the crucial role of IRP2 in the
regulation of brain iron metabolism.
When cellular iron concentrations change, the expression of IRP2 will be altered
accordingly. Decreased cellular iron contents can increase the expression of IRP2,
which can bind to IRE in 5 -untranslated region (UTR) of the mRNA such as ferritin
mRNA, IRE in 3 UTR of transferrin receptor 1 (TfR1) mRNA, and DMT1 with IRE
[DMT1 (+IRE)] mRNA, thus induce the inhibition of ferritin expression, elevation
of TfR1 and DMT1 (+IRE) expression, which increase iron uptake and decrease iron
storage to relieve the iron deficiency in the cell. The vice is versa [20, 54, 61, 66,
93]. Therefore, IRE-IRP system can maintain iron homeostasis responding to altered
cellular iron levels in the brain.

3.4.2 Hepcidin Regulates Brain Iron Metabolism

from Systemic Level

Hepcidin, an iron-regulatory hormone, plays an essential role in maintaining body

iron homeostasis [33, 62, 63]. Hepcidin regulates organismal iron concentration and
tissue iron distribution by controlling intestinal iron absorption, iron recycling by
macrophages, and iron mobilization from hepatic stores [33, 43, 62]. In peripheral
tissues, hepcidin is upregulated responding to inflammation [48] and iron overload
in the blood; then, hepcidin binds to the extracellular loop of FPN1 and causes its
internalization and degradation, thereby reducing cellular iron efflux from intestine
enterocytes and macrophages, which relieves the iron overload status and decreases
the iron levels to the normal range in the blood [3, 14, 16, 63, 75].
Recent studies have also revealed that hepcidin mRNA levels are different in
different brain regions, and that hepcidin immunoreactivity can be detected in both
neurons and GFAP-positive glial cells [95], which suggests that hepcidin can be also
involved in the regulation of brain iron metabolism [19, 72, 88, 95]. FPN1-dependent
iron export systems might also play a key role in iron transport across BBB similar
to that in the abluminal membrane of enterocytes in the gut [15, 53].
Several studies have provided evidences of an important regulatory role for hep-
cidin in brain iron metabolism. It is reported that hepcidin mRNA levels increase with
aging and injection of hepcidin peptide into the lateral cerebral ventricle decreases
FPN1 levels, resulting in brain iron overload [88]. So hepcidin can respond to changes
of systemic iron levels and inflammation and then regulates FPN1 to keep iron home-
ostasis [26]. In brain microvascular endothelial cells, it is demonstrated that hepcidin
downregulates iron efflux from hBMVECs by internalization of FPN1 in vitro [51]. In
primary cultures of astrocytes, extracellular hepcidin triggers the decreased expres-
3 Brain Iron Metabolism and Regulation 39

sion of TfR1, DMT1 and FPN1, and hepcidin inhibits TfR1 expression via a cyclic
AMP-protein kinase A pathway, which implies the existence of a novel hepcidin-
receptor on the membrane of astroglia [18, 83]. However, there are no direct evi-
dences to show that brain hepcidin regulates the content of brain iron through effects
on FPN1 of BMVEC in vivo.
In fact, hepcidin mRNA levels in the brain have been shown to be much lower
than those in non-neural tissues or blood by qPCR experiments [88], but most
researches draw the conclusions of hepcidin-mediated regulation of FPN1 expres-
sion in BMVECs or neurons based on the usage of a considerably high concentration
of supraphysiological hepcidin (approximately 20-fold higher than that in blood).
Based on these data and facts, it is still to be elucidated how the low concentration
of hepcidin in the brain can regulate FPN1 in BMVECs and other types of cells.
It is worth noting that hepcidin and IRE-IRP systems must have communications
and coordination in their regulation of brain iron metabolisms [87]. In addition,
erythroferrone (ERFE) is a newly identified protein by Ganz group, and ERFE could
inhibit hepcidin and thus regulate iron metabolism in response to erythropoietin
stimulation under the stress condition [36, 37]. ERFE is also detected in the brain
with real-time PCR [37], but it is still unclear about its regulation of brain iron
metabolism. It is also unknown if there is any other functions of ERFE in the brain,
which needs to be further investigated.

3.5 Conclusion

Although brain iron metabolism and its regulation are complicated and there are still
some puzzles to be unraveled, the regulation profile is becoming clearer for us. Iron
is transported in the blood and entered the brain across BBB. Then iron is acquired by
neurons and different types of glial cells through iron uptake proteins, and released
from these cells through FPN1. Brain iron homeostasis is coordinately regulated by
IRP2 and hepcidin at post-transcriptional and protein levels, respectively. However,
there are still some problems to be answered and understood, such as how the iron
was released from the brain? Is the recently concerned glymphatic pathway involved
in it [76]? Which kind of signal initiates the uptake of iron and release from the
brain? The answers to these problems will not only enrich the theories of brain
iron metabolism and regulation, but also provide strategies to prevent and treat iron
metabolism disorders and related neurodegenerative diseases.

Acknowledgements The work was supported by the National Natural Science Foundation of China
(31520103908, 31970905), Hebei Provincial Natural Science Foundation (C2017205140), Hebei
Provincial Education Department Foundation of China (ZD2015105), Hebei Provincial Fund for
studying abroad and returning to China (C201862), and Hebei normal University Outstanding Youth
Fund (L2018J05).
40 P. Yu and Y.-Z. Chang

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Chapter 4
Iron Pathophysiology in Parkinson
Diseases

Hong Jiang, Ning Song, Qian Jiao, Limin Shi and Xixun Du

Abstract The key molecular events that provoke Parkinson’s disease (PD) are not
fully understood. Iron deposit was found in the substantia nigra pars compacta
(SNpc) of PD patients and animal models, where dopaminergic neurons degeneration
occurred selectively. The mechanisms involved in disturbed iron metabolism remain
unknown, however, considerable evidence indicates that iron transporters dysregula-
tion, activation of L-type voltage-gated calcium channel (LTCC) and ATP-sensitive
potassium (KATP) channels, as well as N-methyl-D-aspartate (NMDA) receptors
(NMDARs) contribute to this process. There is emerging evidence on the structural
links and functional modulations between iron and α-synuclein, and the key player
in PD which aggregates in Lewy bodies. Iron is believed to modulate α-synuclein
synthesis, post-translational modification, and aggregation. Furthermore, glia, espe-
cially activated astroglia and microglia, are involved in iron deposit in PD. Glial
contributions were largely dependent on the factors they released, e.g., neurotrophic
factors, pro-inflammatory factors, lactoferrin, and those undetermined. Therefore,
iron chelation using iron chelators, the extracts from many natural foods with iron
chelating properties, may be an effective therapy for prevention and treatment of the
disease.

Keywords Iron · Parkinson’s disease · α-Synuclein · Glia · Iron chelation

4.1 Iron Metabolism Dysfunction in Parkinson’s Diseases

Parkinson’s disease (PD), the second common neurodegenerative disorder of the

elderly, is characterized by the death of dopaminergic neurons in the substantia nigra
(SN) pars compacta (SNpc), leading to striatal dopamine (DA) deficiency [1–3].
PD affects 1–2% of those over 60 years of age by causing motor dysfunctions such
as bradykinesia, resting tremor, rigidity, postural instability, as well as non-motor
symptoms, such as cognition, hyposmia, sleep disturbances, depression, constipa-

H. Jiang (B) · N. Song · Q. Jiao · L. Shi · X. Du

Department of Physiology, Medical College of Qingdao University, Qingdao 266071, China
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2019 45

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_4
46 H. Jiang et al.

tion, and other dysautonomic symptoms [4–6]. Up to now, the key molecular events
that provoke neurodegeneration are not fully understood. In addition to misfolding
of proteins, impairment of the ubiquitin proteasome, dysfunction of mitochondria,
oxidative stress, there is mounting evidence implicated that iron accumulation is
pivotal to PD pathogenesis [7, 8].
In the late 1980s, several groups reported elevated iron levels in the SN of parkin-
sonian patients compared to age-matched control [9–12]. In other parts of brain,
including cortex (Brodmann area 21), hippocampus, putamen, and globus pallidus,
the contents of iron were unchanged [9]. Especially, it was reported that SNpc is a
selective area of iron accumulation and the ratio of ferrous to ferric iron decreased
in parkinsonian patients [13]. Furthermore, a variety of methods, such as inductively
coupled plasma spectroscopy, magnetic resonance imaging, laser microprobe mass
analysis, susceptibility-weighted imaging, and enhanced T2-star-weighted angiog-
raphy, have proved the increased iron levels in the SNpc of PD brains [7]. The
disruption of iron metabolism in parkinsonian patients is also demonstrated by a
previous work that the iron levels are significantly decreased in the temporal cor-
tex in brain when compared with age-matched healthy controls, suggesting a likely
re-distribution pattern in PD [14].
The iron metabolism disturbance was also present in PD animal and cell models.
The transgenic PD mice model overexpressing the mutant human A53T α-synuclein
(A53T mice) show an accumulation of aggregates comprising α-synuclein and
develop age-dependent motor deficits. A53T mice are more vulnerable to accumulate
iron in the SN than the wild type [15]. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP), an inhibitor of complex I of the mitochondrial electron transport chain, is
commonly used to induce PD animal models that exhibit the damage of dopaminergic
neurons [1, 16]. Injection of MPTP into the caudate or putamen of monkeys resulted
in the increase of ferric iron-reaction products in the ipsilateral SNpc [17, 18]. In
hemiparkinsonian monkeys, iron contents were significantly increased from 4.5 to
18 months in the ipsilateral SNpc after MPTP injection by unilateral internal carotid
artery [19]. Moreover, the increasing extent was significantly correlated to the extent
of dopaminergic cell death [17, 19, 20]. Additionally, the phenomena that nigral
iron levels elevation is also observed in MPTP-induced PD mice model [21–23].
6-hydroxydopamine (6-OHDA) is a neurotoxin, which induces dopaminergic neu-
rons death and therefore has been implicated in PD rat models. It was reported that
iron levels increased in the ipsilateral SN from 1 to 14 days in 6-OHDA unilaterally
lesioned rats [24]. Actually, both increased iron levels and dopaminergic neurons
loss are apparent in the SN as promptly as one day following 6-OHDA injection
[24]. Iron levels increased in SN with 6-OHDA injection into rats have been proved
by peer laboratories [21, 25–27]. In vitro studies, iron uptake significantly increased
both in 1-methyl-4-phenylpyridinium (MPP+ )- and 6-OHDA-induced PD cell mod-
els [28, 29]. In other models of PD, including lipopolysaccharide-induced inflam-
mation models, the loss of dopaminergic neurons was accompanied by increased
iron and ferritin levels in glial cells of the SN pars reticulate [30, 31]. Moreover, in
iron-overloaded animal models, such as direct injection of ferric iron into the SN,
high dietary iron supplements and peripheral iron overload, they develop degener-
4 Iron Pathophysiology in Parkinson Diseases 47

ation of dopaminergic neurons and further illustrated the relationship between iron
and PD pathogenesis [15, 32]. With the common focusing on neurotoxins MPTP
and 6-OHDA, the environment toxins exiting in food and drinking water, including
paraquat, rotenone, and mancozeb, may be directly or indirectly involved and affect
iron metabolism [33, 34].
The unbalanced distribution of iron in the brain of parkinsonian patients indicates
that iron plays a key role in the degeneration of dopaminergic neurons. Potential
mechanisms have been offered to explain why iron metabolism disturbance occurs
(see below), as well as how elevated iron behaviors leading to dopaminergic neurons
degeneration [7]. Among these, iron and DA toxic couple has to be highlighted. DA
is the central neurotransmitter involved in PD. DA easily forms toxic metabolites
and these processes occur predominantly in the cytoplasm [35]. Physiologically,
H2 O2 is produced by a DA enzymatic process via monoamine oxidase. The revealed
aspects of iron-DA coupling are largely derived from their pro-oxidant properties
[36]. The high-level iron in dopaminergic neurons aggravates the oxidative stress
by Fenton reaction that iron reacts with H2 O2 produced in the enzymatic process
in DA metabolism and then can generate OH that can damage proteins, nucleic
acids, and membrane phospholipids [37]. An alternative mechanism has been raised
that the oxidation of DA by iron forms 6-OHDA. As a commonly used neurotoxin
in PD models, 6-OHDA even liberates iron from ferritin and produced H2 O2 in
its metabolism. Recently, it was reported that high-level iron inside cells caused
ferroptosis which is a form of regulated cell death characterized by the iron-dependent
accumulation of lipid hydroperoxides to lethal levels [38, 39]. Elevated iron likely
contributes in dopaminergic neurons degeneration in PD, which could be further
validated by the facts that using pharmacological or genetic chelation of iron in
animal models of PD has neuroprotective effects on dopaminergic neurons (see
below) [40, 41].

4.2 Possible Mechanisms Underlying Iron Metabolism

Disturbance

4.2.1 Roles of Iron Transporters

The mechanisms involved in this iron accumulation remain unknown. Consider-

able evidence indicates that dysregulation of several iron transporters (responsible
for either iron influx/uptake or iron efflux/release) contributes to the abnormal iron
deposit. Thus, understanding iron transporters dysregulation, particularly in the con-
text of PD, might be beneficial for exploring therapeutic strategies via restoring iron
homeostasis.
Generally, cellular iron uptake from transferrin (Tf) is accomplished by
glycoprotein-receptor-mediated endocytosis via transferrin receptor (TfR), which
is considered as a major pathway for iron import in brain. There are two types of
48 H. Jiang et al.

TfR, namely TfR1 and TfR2. TfR1, expressed abundantly in the central nervous
system, especially in neurons, constitutes the main receptor for Tf and internalizes
it through receptor-mediated endocytosis. Furthermore, TfR1 mRNA contains iron-
responsive elements (IRE) in the 5 -untranslated region, thus it can be regulated
by intracellular iron levels via iron response proteins (IRP) [42]. In contrast, TfR2,
mainly expressed in the mitochondria of nigral dopaminergic neurons [43], does not
contain the IRE, thus it cannot be regulated by iron levels. Although TfR2 has a lower
affinity for Tf than TfR1, it can serve as an iron sensor in the regulation of hepcidin
[44]. Recent studies found that 1-methyl-4-phenylpyridinium (MPP+ ) treatment led
to the increased expression of TfR, suggesting a role for TfR-dependent iron influx
in the selective iron accumulation in PD [45]. The localization of TfR2 in the mito-
chondria of dopaminergic neurons and a dramatic increase of oxidized Tf in the SN
raised the possibility that the Tf-TfR2 system might also contribute to iron deposit
in PD [46].
The non-transferrin-bound iron (NTBI) is mainly transported through divalent
metal transporter 1 (DMT1), which is responsible for ferrous iron uptake. There
are at least four distinct DMT1 mRNA isoforms: 1A, 1B, +IRE and −IRE [47, 48].
DMT1, also known as natural resistance-associated macrophage protein 2 (Nramp 2)
or divalent cation transporter 1 (DCT1), was first discovered to transport iron in
the duodenal lumen. In the SN, DMT1 is widely expressed in neurons, astrocytes
and microglia [49], which mediates iron transmembrane transport as well as from
the endosomes to the cytoplasm [50]. The expression of DMT1 can be regulated
post-transcriptionally through IRE/IRP, or post-translationally through the ubiquitin-
proteasome system (UPS). Previous studies have shown that the E3 ligase parkin is
responsible for the proteasomal degradation of DMT1 and regulates iron transport
[51]. Moreover, DMT1 can be ubiquitinated by the neural precursor cell expressed
developmentally down-regulated protein 4 (Nedd4) family member WW domain-
containing protein 2 (WWP2), and this requires Nedd4 family interacting protein
1 (Ndfip1) and/or Ndfip2 [52]. In the postmortem brains of PD patients and ani-
mal models, the expression of nigral DMT1 was significantly up-regulated, thus
contributed to the intracellular iron accumulation and dopaminergic neurons degen-
eration [28, 53, 54]. Modulation of DMT1 proteasomal degradation via UPS might
also be associated with DMT1 dysregulation and participate in iron deposit in PD
[51, 55, 56].
Another iron uptake mechanism involves an iron transporter called lactoferrin
(Lf), which acts as an iron-binding protein, belonging to the Tf family. In a similar
manner to Tf, Lf-bound ferric iron can bind to Lf receptor (LfR), resulting in iron
transport across the plasma membrane. The affinity of Lf for iron is 300 times higher
than that of Tf [57]. In the brain, Lf is only synthesized and released by activated
microglia [58]. LfR is present in blood vessels and nigral dopaminergic neurons
[59], suggesting that Lf-LfR pathway may be involved in iron transport into brain
parenchyma and dopaminergic neurons. Moreover, an increased expression of Lf
and LfR on dopaminergic neurons was found in PD patients [58, 59], which may
account for the excessive accumulation of iron. And this increase was occurred in
4 Iron Pathophysiology in Parkinson Diseases 49

the most severely affected dopaminergic neurons, indicating a relationship between

Lf-LfR increase and dopaminergic neurons degeneration.
Up to now, ferroportin 1 (FPN1) is the only known iron transporter responsible
for intracellular iron export. Iron is exported in the form of ferrous iron, therefore
the ferroxidase activity of hephaestin (HP) or ceruloplasmin (CP) is needed, which
oxidizes ferrous iron (Fe2+ ) into the ferric form (Fe3+ ). FPN1 and HP co-localized
in neurons, astrocytes, oligodendrocytes, and microglia, indicating that HP could
facilitate FPN1-mediated iron efflux [60]. CP acts as a copper-dependent ferroxi-
dase, which can exist as a soluble form in plasma or as a membrane-bound form
highly expressed in astrocytes. The absence of CP in aceruloplasminemia results
in iron accumulation in the basal ganglia accompanied by neuronal degeneration
[61]. It has been also reported that iron exporter FPN1 was reduced in the brain of
6-OHDA/MPTP/lipopolysaccharide-treated animal models [31, 60, 62]. In addition,
several evidence implicates the role of CP in the pathogenesis of PD. Reduced fer-
roxidase activity of CP was found in the CSF of PD patients [63]. The decreased
expression of CP in the SN is involved in the nigral iron accumulation in 6-OHDA-
lesioned rats [64].

4.2.2 Roles of L-Type Voltage-Gated Calcium Channel

(LTCC)

The LTCC of SN dopaminergic neurons contains pore-forming Cav1.2 or Cav1.3

subunits in complex with an accessory β and α2-δ subunit [65]. Compared with ven-
tral tegmental area (VTA) dopaminergic neurons, LTCC are selectively activated in
the nigral dopaminergic neurons during pacemaking and cause an oscillating cal-
cium burden inducing mitochondrial stress. Otherwise, the δ subunits of LTCC were
obviously up-regulated in the proteome analysis of human SN in PD. These studies
suggest that LTCC might be related to the pathogenesis of PD [66]. In cardiomy-
ocytes, secretory cells and neurons, LTCC have the similar functional properties,
which could mediate iron import into cells which were iron overload [67, 68]. Some
evidence has demonstrated that iron could compete with calcium via LTCC for entry
into NGF-treated rat PC12 cells and murine N2a cells, which suggest that LTCC
could provide an alternative route for iron entering into neuronal cells under the
iron-overloaded conditions [69]. In the SN of rats, the protective effect of LTCC
blocker nifedipine against iron overload-induced dopaminergic neurons degenera-
tion and iron accumulation has been demonstrated [70]. Based on these studies,
LTCC might mediate the iron overload in the dopaminergic neurons and be involved
in the selective iron accumulation in the SN in PD.
50 H. Jiang et al.

4.2.3 Roles of N-Methyl-D-Aspartate (NMDA) Receptors

(NMDARs)

NMDARs are cation channels which mediate Na+ and Ca2+ ions entering the cells and
could be activated by glutamate, glycine, or D-serineare. NMDARs activation appro-
priately plays a critical role in several physiological processes, e.g., synaptic plas-
ticity and excitatory neurotransmission. However, excessive activation of NMDARs
contributes to pathological changes in the brain. NMDARs mediated excessive Ca2+
influx might induce neuronal death in several neurodegenerative diseases, including
PD [71, 72]. NR1, a very important subunit of NMDARs, which expression was
increased in surviving dopaminergic neurons from PD brains compared with the
controls [73]. Some studies also revealed that NMDARs activation could signifi-
cantly promote Fe2+ entry into cells. The underlying mechanisms might be involved.
First, NMDARs activation increases NTBI influx. Fe2+ could compete with Ca2+ for
NMDARs to enter the cultured neurons [74, 75]. The increased iron levels may induce
a corresponding ROS overproduction and higher susceptibility of neurons. Another
investigation has identified a novel signaling cascade for NMDARs regulated iron
uptake in brain [76]. It was reported that glutamate via NMDARs trigger Ca2+ influx,
then activated neuronal nitric oxide (NO) synthase (NOS) produce NO, which induce
excitability toxicity [76–78]. And neuronal NOS (nNOS) binding to CAPON could
cause NO delivery to Dexrase1 and S-nitrosylation of Dexrase1, which promote iron
uptake by regulating the function of DMT1 [79]. Rhes (Dexras2), a homolog of
Dexras1, is selectively localized to the corpus striatum. Rhes was also reported to
be involved in NMDARs-induced iron influx into the striatal neurons and regulating
striatal iron homeostasis by PKA-Rhes-DMT1 pathway [80].
Second, NMDARs activation enhanced iron releasing from lysosome. The iron
in lysosome serves as a main source for intracellular iron, in which DMT1 plays a
critical role in iron recycling from lysosome to cytoplasm. Recently, several evidence
showed that Dexras1/PAP7/DMT1 complex located on the lysosome. Either impaired
DMT1 function or the collapsed proton gradient could reduce iron pool in cytoplasm,
which indicated that Dexras1/PAP7/DMT1 complex plays a role in iron release from
lysosome to cytoplasm [81].
Finally, NMDARs activation increased DMT1 expression. It has been reported
that the mRNA levels of DMT1 were increased after 5 min treated with 50 μM
NMDA to primary hippocampal cultures. With exposure to the transcription inhibitor
actinomycin D and NMDARs antagonist MK-801, DMT1 up-regulation induced by
NMDAs or 6-OHDA could be restored [82]. Interestingly, both excessive activation
of NMDAs and up-regulation of DMT1 were observed in the process of degeneration
of dopaminergic neurons in PD, indicating the close links between NMDARs and
DMT1 in PD [83].
4 Iron Pathophysiology in Parkinson Diseases 51

4.2.4 Roles of ATP-Sensitive Potassium (KATP ) Channels

Functional KATP channels are composed of four pore-forming Kir6 (Kir6.1 and
Kir6.2) subunits and four regulatory SUR (SUR1, SUR2A, and SUR2B) subunits.
Different subunit compositions decided the different biophysical, pharmacological,
and metabolic properties of KATP channels [84]. In dopaminergic neurons in the SN
and VTA, KATP channels are both expressed with Kir6.2 and SUR1 subunits. But
only the KATP channels of dopaminergic neurons in the SN were selectively activated
when treated with MPP+ . And dopaminergic neurons in the SN but not those in the
VTA in the MPTP model were selectively rescued after genetic inactivation of Kir6.2
subunits [85]. It was also reported that mRNA levels of the regulatory subunit SUR1
were increased in survived dopaminergic neurons in the SN from PD patients. Other-
wise, mRNA levels of SUR2 and Kir6.2 were not changed [73]. These results suggest
that the activation of KATP channels might be involved in the selective degeneration
of dopaminergic neurons in the SN in PD. The activation of KATP channels would
induce the membrane potential hyperpolarized, which is important for the membrane
oscillations that underlie bursting firing generated [86]. KATP channels’ gated burst
firing could aggravate excitotoxicity and increase calcium loading with NMDARs
and LTCCs synergistically in the dopaminergic neurons of SN, where metabolic
state have already challenged [87]. This led to the insufficient mitochondrial calcium
buffering capacities and acceleration of calcium-induced ROS production [88], which
in turn activated KATP channels in highly vulnerable neurons [73].
Selective activation of KATP channels in the SN was suggested to contribute to
iron accumulation. The transport function of DMT1 is proton-coupled and dependent
on the cell membrane potential. It was reported that hyperpolarized potentials could
promote the iron uptake via DMT1 [89]. There is evidence that diazoxide, a KATP
channel opener, could increase intracellular iron levels after ferrous iron treatment
[90]. The subsequent ATP depletion and ROS production would induce additional
KATP channels activated in a feed-forward cycle [73]. Accordingly, inhibition of
KATP channels dramatically decreases the iron uptake and inhibits cell damage [90].
These provide evidence that the selective iron accumulation in the SN might be
related to KATP channels activation; further investigation should be done to reveal
the underlying mechanisms.

4.2.5 Iron and Gene Mutation

Parkin, α-synuclein, DJ-1, leucine-rich repeat kinase 2 (LRRK2), PTEN-induced

kinase 1 (PINK1), and several other genes were believed to be involved in the patho-
genesis of PD. As a matter of fact, these genes were demonstrated to be linked to iron
accumulation in PD [51, 91]. PD patients with Parkin, α-synuclein, DJ-1, LRRK2,
or PINK1 mutations showed significantly larger echogenicity in the SN compared
with the healthy control group evaluated by transcranial sonography [43], which
52 H. Jiang et al.

indicated an increased nigral iron level. Thus, hyperechogenicity has been partially
shown to be increased iron levels in the SN [92], which has also been deemed to be
a typical sign in idiopathic PD. A subset of cellular proteins not degraded attributed
to Parkin mutations are currently supposed to be the most common reasons of famil-
ial Parkinsonism [93–95]. Iron could induce altered Parkin solubility and result in
its intracellular aggregation. And with the depletion of soluble, functional forms of
Parkin, proteasomal activities were impaired with cell damage [96]. In 1998, Parkin
mutation was first identified in autosomal recessive juvenile parkinsonism (ARJP),
and iron staining in the SN was reported to be more intense than that of controls, as
well as sporadic PD patients [91]. It was hypothesized that iron accumulation might
be related to the loss of the Parkin gene.
More recently, Parkin was reported to be responsible for ubiquitination of
DMT1 + IRE. The expression of 1B-DMT1 isoforms was decreased if SH-SY5Y
cells overexpressed with Parkin [51]. When fed with iron-supplemented diet, trans-
genic mice with DMT1 overexpression showed selectively accumulated iron in the
SN, meanwhile, the expression of Parkin is also up-regulated, likely a neuroprotec-
tive response [97]. In human lymphocytes containing a homozygous deletion of exon
4 of Parkin or in the brains of Parkin knockout animals, expression of DMT1 + IRE
was also shown to be elevated.

4.3 Iron Deposit and α-Synuclein Pathology in PD

Among the contributors in PD pathogenesis, α-synuclein was considered to be a key

player which aggregates in Lewy bodies (LBs), the neuropathological hallmarks in
both familial and sporadic PD. As a coexisting factor with α-synuclein in LB, iron was
most pronouncedly stained in the LBs core of the remaining dopaminergic neurons
of SNpc [98]. Fe3+ has the direct, high affinity for α-synuclein at D121, N122, and
E123 [99]. The potential bridging by polyvalent iron is proposed to be a key factor
in the metal-induced conformational changes of α-synuclein, leading to significant
accelerations in the rate of α-synuclein fibril formation [100]. Fe3+ also was able to
alter the morphology of α-synuclein fibrils and accelerates aggregation in both wild
and mutant α-synuclein, as demonstrated by transmission electron microscopy mon-
itoring α-synuclein aggregation [101]. With the emerging evidence on the structural
links and functional modulations between iron and α-synuclein, iron is believed to
modulate on α-synuclein synthesis, post-translational modification and aggregation.
It was first predicted that 46 nucleotide in the 5 -UTR of α-synuclein tran-
script form a single RNA stem-loop, showing similarity to the IREs in the 5 -UTRs
of mRNAs encoding H-ferritin, L-ferritin, FPN1, and erythroid 5-aminolevulinate
(eALAS) and mitochondrial aconitase, including 100% homology at the five
nucleotide apex (CAGUG) [102]. This provides the evidence that iron might be
able to regulate α-synuclein synthesis by IRPs-IRE post-transcriptional machinery.
In human SK-N-SH cells, α-synuclein mRNA level was up-regulated with IRP1
knockdown, mimicking the condition that IRPs failed to bind IRE with iron [103].
4 Iron Pathophysiology in Parkinson Diseases 53

Iron accumulation and oxidative stress induced by ferrous ammonium was able to
up-regulate α-synuclein protein levels in these cells. The data showed the 5 -UTR
in α-synuclein is a positive mediator of promoting translation, but not of mRNA
stability/expression, consistent with the fact that IRPs serve to block transcription
activation [104]. On the other hand, in HEK293 cells iron chelator deferoxamine
(DFO, also known as desferal) decreased human α-synuclein mRNA levels [105].
Together with the evidence of an IRE presence in the 5 -UTR of the Alzheimer’s
amyloid precursor protein (APP) transcript [106], these evidence suggests that iron
might contribute to neurodegenerative disease course by up-regulating α-synuclein
or APP levels.
Alpha-synuclein undergoes a variety of post-translational modifications, which
have been linked to the aggregation and pathology of α-synuclein [107, 108]. Nitra-
tion and phosphorylation were believed to be associated with iron and iron-induced
oxidative stress. Nitrated α-synuclein is present in LBs, as well as in the insoluble
fractions of affected brain regions in PD [109]. It is suggested that nitration of only
a single tyrosine residue is sufficient to induce profound changes in catalytic activ-
ities and structural conformations of proteins. The attachment of a nitro molecule
to α-synuclein at tyrosine residues (Y39, Y125, Y133, and Y136), is sufficient to
induce profound changes in α-synuclein [110]. Nitrated of α-synuclein monomers
and dimers could be mixed into the fibrils and accelerate the rate of fibril formation
of unmodified α-synuclein [111, 112]. It was demonstrated that nitrated monomeric
α-synuclein was degraded at a slower rate; together with diminished binding to
lipid vesicles, nitrated α-synuclein were responsible for increasing fibrils formation
and intracytoplasmic inclusions [113]. Excessive iron levels are associated with the
increase of oxidative/nitrative stress, leading to elevated levels of tyrosine nitration.
Iron supplements in rats markedly increased the extent of lipid peroxidation, protein
carbonyls tyrosine nitration and oxidative metabolism of NO in liver [114]. There are
currently no reports on the effects of iron on α-synuclein nitration. However, oxida-
tive stress, the most common deleterious condition induced by iron, was believed to
be involved in the nitrated process of α-synuclein. In vitro evidence demonstrated that
microglial activation could induce NO-dependent oxidative stress in dopaminergic
neurons and then cause α-synuclein nitration. These nitrated, aggregated α-synuclein
during oxidative stress responses, in turn, incurs inflammatory microglial functions
[115, 116]. In vivo, nitrated-α-synuclein (Tyr125, Tyr133) levels was elevated in the
thymus of MPTP induced PD mice; nitrated recombinant α-synuclein was able to
activate microglia and astrocytes lead to the degeneration of dopaminergic neurons
[117, 118].
Phosphorylation of α-synuclein represents more than 90% of the total α-synuclein
found in LB, whereas only 4% of the soluble monomeric α-synuclein is phosphory-
lated physiologically [119]. Phosphorylation at S129 is the major post-translational
modification of α-synuclein [120]. Genetic approaches mimicking phosphorylated
α-synuclein at S129 was associated with pathology in a transgenic Drosophila model,
as well as in MPTP-treated monkeys. The mutation of serine 129 to aspartate signif-
icantly promotes α-synuclein phosphorylation and aggregation and thus dopaminer-
gic neurons death. However, altering serine 129 to alanine prevents phosphorylation
54 H. Jiang et al.

and suppresses dopaminergic neuronal loss [121]. Phosphorylated S129 α-synuclein

aggregation was even correlated with the extent of dopaminergic neuron loss in the
SN, further supporting that phosphorylation of α-synuclein might be harmful in PD
[122]. There is also contradictory evidence that phosphorylation has no accelerating
effects on α-synuclein toxicity or even neuroprotective. Phosphorylation mimicking
S129 phosphorylation did not cause motor dysfunction and growth retardation; or
even reduce α-synuclein pathology, suppress dopaminergic neurodegeneration and
restore motor impairments by promoting α-synuclein clearance [123–125]. Together,
there is still no consensus on whether α-synuclein phosphorylation is promoting
aggregation or degradation, or neurotoxic or neuroprotective [126]. However, there
is clear evidence that iron-derived oxidative stress promotes phosphorylation. Iron
overload in 3D5 cells caused inclusion formation and phosphorylated α-synuclein
aggregation [127]. Similarly, iron caused α-synuclein phosphorylation at S129 in
SH-SY5Y cells, which was linked to ROS formation and mitochondrial dysfunc-
tion [128]. Furthermore, the binding affinity between α-synuclein and iron might
be altered by phosphorylation at either S129 or Y125. It was suggested that phos-
phorylation at S129 or Y125 increases the binding affinity for Fe2+ but not Fe3+ at
C-terminal region of α-synuclein [129].
It was hypothesized that blocking the metal modulated α-synuclein translation
might provide a potential pathway to ameliorate the pathologies of both metal and
α-synuclein [130]. As well, targeting iron and α-synuclein interactions might be a
plausible therapeutic strategy in PD.

4.4 Glial Contributions in Iron Metabolism Disturbance

Neurodegenerative processes trigger universal and conserved glial reactions repre-

sented by astroglial dysfunction and microglial activation. As the most abundant
glial cell types in the brain, astroglia were physiologically thought of as passive sup-
port cells mopping up transmitters and maintaining extracellular ion levels, and thus
necessary to ensure optimal neuronal functioning [131]. Astroglia in the SNpc were
relatively low distributed compared to other brain region. In autopsies of the SN from
PD patient, reactive astrocytosis is absent although the activation of microglia was
consistently observed [132]. Less astroglial neuroprotective factors are likely to con-
tribute to increased vulnerability to any PD insults. However, reactive astroglia may
cause or contribute to neurodegenerative processes. Stressed/dysfunctional astroglia
might cease to support the dopaminergic neurons, which in turn contributes to the
degeneration. This is referred as a dual role of astroglia since they are capable of
releasing ROS and pro-inflammatory factors, further exacerbating neuroinflamma-
tion and brain damage [133, 134]. As for microglia, as the resident innate immune
cells in the brain, they were proposed to constantly move and monitor the area in
which they reside for immune insults and invading pathogens. These were regarded
as beneficial effects due to the clearance of damaged cells and pathogens, as well as
detrimental effects by the release of neurotoxic factors. Among many factors related
4 Iron Pathophysiology in Parkinson Diseases 55

to PD pathology, microglia-mediated neuroinflammation is one of the most striking

hallmarks [135–137].
Both astroglia and microglia contribute to well-balanced iron metabolism in the
brain. They expressed almost all the iron transporters and distinct profiles of iron
metabolism proteins [44, 138–140]. Astroglia do not have very much ferritin and
iron, indicating that these cells provide little iron storage [132, 141, 142]. However,
they are more resistant than neurons and other glial cells to iron-induced toxicity,
which might be due to the large antioxidants system aiding in the protection, e.g.,
metallothioneins [143, 144]. In primary cultures enriched in neurons, astroglia, or
microglia, microglia were found to be the most efficient in accumulating iron, fol-
lowed by astroglia, and then neurons. Iron retention in microglia was three times
higher than that in neurons when they were exposed to iron, suggesting microglia
have the capacity to accumulate and store large quantities of iron [145]. In autop-
sies of the SN from PD patient, ferritin immune reactivity was observed in activated
microglia, indicating the abundant ferritin levels might reflect the large iron storage
capacity in microglia [132]. The relationship between the accumulation of iron and
ferritin in microglia during neuroinflammation was not fully elucidated [146, 147].
However, iron-containing microglia/macrophages are consistently present in the spe-
cific regions where the neurodegeneration occurred, providing a possible explanation
of the increasing concentration of iron [148].
Iron elevation is evident in astroglia and microglia in the SN of PD [149], which
might be capable of buffering iron accumulation. Meanwhile, iron accumulation in
the specific region inevitably aggravates astroglia and microglia activation, which
are in turn relevant to iron deposit in the diseased state of PD. That means iron affects
these neuroglia responses in PD, at the same time, the latter affects neuronal iron
metabolism. Brain-derived neurotrophic factor (BDNF), which is mainly derived
from astroglia to protect neurons against oxidative stress and apoptosis, act on intra-
cellular IRPs and thus post-transcriptionally target iron importer DMT1 to ameliorate
iron accumulation in neurons [150]. These could be the beneficial effects exerted by
astroglia, both on normal conditions and in the diseased state of PD. Also, these
results provide novel perspectives to view the advantages of neurotrophic factors in
PD. Meanwhile, neurotoxin 6-OHDA, inducing iron accumulation in neurons, might
also be capable of promoting iron transport rate in primary cultured ventral mesen-
cephalic astroglia, indicating a different response between neurons and astroglia in
PD [151]. It was supposed when applied to in vivo circumstances, astroglia are capa-
ble of transporting excess iron outside thus preventing themselves, their neighboring
cells and even the regions from iron overload. More recently, lipocalin-2 (LCN2),
a member of the highly heterogeneous secretory protein family of lipocalins, was
reported to increase mainly in the reactive astroglia in both the SN and striatum in
PD. Astroglial LCN2 up-regulation induced the nigrostriatal dopaminergic neuro-
toxicity was aggravated by iron repletion [152], reinforcing the associations among
astroglia, iron, and neurodegeneration.
Only activated microglia contained both Lf and its messenger, indicating microglia
are the Lf producing cells [58, 153]. As we discussed above, by binding to LfR,
iron transport crossing the plasma membrane is achieved through iron-saturated Lf
56 H. Jiang et al.

internalization by a receptor-mediated pathway. LfR immunoreactivity was increased

and more pronounced in the region where the loss of dopaminergic neurons is severe,
suggesting that increased LfR on vulnerable neurons may increase intraneuronal
iron levels. However, Lf, either iron-saturated or not, may be protective because it
might function as a mitochondrial calcium modulator or a specialized iron scavenger
[154–156]. The mechanisms that how microglia released Lf influence other cells
like neurons, especially in terms of iron metabolism in PD need to be established
further. However, it is clear now that the pro-inflammatory cytokines, e.g., IL-1β and
TNF-α release were significantly increased when microglia were activated. More
striking, the release was enhanced in activated microglia with iron overload [157].
On the other hand, the activated microglia perturb neuronal iron homeostasis via these
releasing cytokines. IL-1β/6 and TNF-α were proved to up-regulate iron importer-
DMT1, whereas down-regulated iron exporter-FPN1, thus causing iron accumulation
in neurons. Both intracellular IRPs and hepcidin levels contributed to this modulation
[157, 158], thus provide powerful evidence that microglia might deteriorate iron-
mediated neuropathologies in PD. This is meaningful to understand the detrimental
situation with the presence of iron deposit and microglia activation at the foci of
lesion.
Based on the fact that astroglia and microglia contribute to neuronal iron
metabolism, as well as that their functions might be altered in the diseased status,
we suppose that there is a feedback loop between glial contributions and neuronal
iron metabolism disturbance. Therefore, modulating the factors, e.g., neurotrophic
factors or deleterious pro-inflammatory cytokines, might be valuable targets in
drug discovery, especially in the aspects of ameliorating iron metabolism distur-
bance in PD.

4.5 Iron Chelation as a Therapy for PD

The role of iron in the pathology of PD has logically led to the theory that iron
chelation may be an effective therapy for prevention and treatment of the dis-
ease [4, 159–163]. Mice with low iron diets for six weeks may provide protec-
tion against MPTP-inducing insults [164]. Similarly, rats received an iron-deficient
diet attenuated kainate-induced neurotoxicity [165]. The clinically iron chelators,
DFO, deferiprone, and deferasirox, have been shown to attenuate 6-OHDA, MPTP
or iron-induced oxidative stress, mitochondrial dysfunction and prevent α-synuclein
aggregation both in vivo and in vitro [166–170]. Recent phase II clinical trial data
also demonstrated that deferiprone improved motor symptoms of PD patients [41].
8-hydroxyquinoline (8-HQ), a highly lipophilic complex, has been utilized as the
base for VK-28, M30, clioquinol, and Q1. These iron chelators readily penetrate cell
membranes and the blood-brain barrier and have been shown to be effective in both
cell culture and the PD animal models [40, 171–175].
Recently, several novel iron chelators have been explored and synthesized.
Lipophilic analogs of desferrioxamine B (conjugating ancillary compounds onto
4 Iron Pathophysiology in Parkinson Diseases 57

the amine terminus) attenuated neuronal loss in SK-N-BE2-M17 cell lines and
MPTP-treated mice [176, 177]. A novel hybrid iron chelator, (-)-12 (D-607), res-
cued PC12 cells from toxicity induced by iron [178]. Iron chelation therapeutic
nanoparticles protected by a zwitterionic poly(2-methacryloyloxyethyl phosphoryl-
choline) (PMPC), which delay the saturation of iron chelators in blood circula-
tion and prolong the in vivo lifetime, provided a PD phenotype reversion therapy
and significantly improved the living quality of the PD mice [179]. 7,8-dihydroxy-
4-((methylamino)methyl)-2H-chromen-2-one (DHC12) chelated mitochondrial and
cytoplasmic labile iron, protected mitochondria against lipid peroxidation, and antag-
onized MPTP-induced neuronal death [180]. Polysubstituted piperazine derivatives
(a selection of 1-hydroxypyridin-2-one (1,2-HOPO) metal chelators) and PBT434
(an orally bioavailable 8-hydroxyquinazolin-4(3H)-one) have also been designed as
new iron chelators and proved to be neuroprotective in dopaminergic cell lines and
multiple models of PD [181–183].
It has been found that the extracts from many natural foods have iron-chelating
properties. For example, epigallocatechin gallate (EGCG), a major polyphenol in
green tea, regulated the iron-export protein FPN1 and exerted a neurorescuing effect
in MPTP-induced PD mice and α-synuclein transfected-PC12 cells [184, 185].
Indeed, epidemiological evidence showed that drinking two cups of tea a day could
reduce the risk of PD [186]. Several natural flavonoids, such as ginkgetin (a nat-
ural bioflavonoid isolated from leaves of Ginkgo biloba L), curcumin (1,7-bis[4-
hydroxy 3-methoxy phenyl]-1,6-heptadiene-3,5-dione, the polyphenolic flavonoid
from Curcuma longa), myricetin and quercetin (bioflavonoids found in abundance
in fruits, vegetables, and herbs) exerted neuroprotective effects against neuroinjury
in PD model induced by MPTP or 6-OHDA via chelating iron [187–190]. Baicalin,
a major active ingredient of the plant Scutellaria baicalensis Georgi, has been shown
to reduce iron accumulation and have protective effects on dopaminergic neurons
in the SN of rotenone-induced PD rats, as well as in C6 cells [191, 192]. Ginseno-
sides, the principal active components of ginseng, also reduced nigral iron levels in
MPTP/MPP+ -treated models via regulating the expression of DMT1 and FPN1 [21,
193, 194]. A major advantage of these natural chelators is the mixture of neuropro-
tective properties they possess. In addition to iron chelation, they have been shown
to exert a variety of beneficial effects, including antioxidant, anti-inflammatory, anti-
apoptosis, and anticancer effects. Therefore, this allows for a more comprehensive
usage in the prevention and treatment of PD.
It is important to realize that whether and how iron chelators could remove excess
iron from the specified brain areas, without affecting systemic iron homeostasis.
Indeed, iron is an essential element in various physiological processes, e.g., DNA
synthesis, mitochondrial respiration, and oxygen transport. Future studies should
seek to identify more cellular specificity therapies to prevent and attenuate brain iron
metabolism dysfunction in PD.
58 H. Jiang et al.

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Chapter 5
Iron Pathophysiology in Alzheimer’s
Diseases

Tao Wang, Shuang-Feng Xu, Yong-Gang Fan, Lin-Bo Li and Chuang Guo

Abstract Alzheimer’s disease (AD) is a multifactorial neurodegenerative condition

associated with pathological accumulation of amyloid plaques and with the appear-
ance of deposit of neurofibrillary tangles. Increasing evidence suggests that disorders
of metal ion metabolism in the brain are one of the risk factors for the pathogenesis
of AD. Iron, one of the endogenous metal ions, involves in many important physi-
ological activities in the brain. Iron metabolism mainly depends on iron regulatory
proteins including ferritin, transferrin and transferrin receptor, hepcidin, ferroportin,
lactoferrin. Abnormal iron metabolism generates hydroxyl radicals through the Fen-
ton reaction, triggers oxidative stress reactions, damages cell lipids, protein and DNA
structure and function, leads to cell death, and ultimately influences the process of
β-amyloid (Aβ) misfolding and plaque aggregation. Although the results are dif-
ferent, in general, iron has deposition in different brain regions of AD patients,
which may impair normal cognitive function and behavior. Therefore, neuroimaging
changes have so far been largely attributed to focal iron deposition accompanying
the plaques at preclinical stages of AD, and iron-targeted therapeutic strategies have
become a new direction. Iron chelators have received a great deal of attention and
have obtained good results in scientific experiments and some clinical trials. Future
research will also focus on iron as an opportunity to study the mechanism of the
occurrence and development of AD from the iron steady state to more fully clarify
the etiology and prevention strategies.

Keywords Alzheimer’s disease · Iron · Oxidative stress · Mitochondria · Chelators

Tao Wang, Shuang-Feng Xu, Yong-Gang Fan, Lin-Bo Li—These authors contributed equally to
this work

T. Wang · S.-F. Xu · Y.-G. Fan · L.-B. Li · C. Guo (B)

College of Life and Health Sciences, Northeastern University, No. 195, Chuangxin Road, Hunnan
District, Shenyang 110169, China
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2019 67

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_5
68 T. Wang et al.

5.1 Introduction

Alzheimer’s disease (AD) is a chronic neurodegenerative disease that occurs mostly

in older people over the age of 65 [167]. In 1906, the German psychotic and neu-
ropathologist Alois Alzheimer first described the disease that was later to carry
his name as a syndrome that involves progressive cognitive decline and behavioral
changes underpinned by senile plaques (SPs) and neurofibrillary tangles (NFTs) in
the autopsied brains of people with AD [90]. As the society ages, AD is the main
cause of dementia and one of the great healthcare challenges of the twenty-first cen-
tury [246]. According to the World Alzheimer’s Disease Report of 2018 issued by
the International Alzheimer’s Association (ADI), there are about 50 million demen-
tia patients in the world in 2018, mainly dementia caused by AD. According to the
prevalence of AD and the aging process of society, by 2050, the number of dementia
patients worldwide is estimated to reach 152 million, equivalent to the size of Russia
or Bangladesh. At the same time, the current global cost of preventing and treating
dementia is about $1 trillion, and it is expected to double by 2030 [297]. However, to
date, no cure or therapeutic intervention is available for AD that can treat the under-
lying cause of AD or slow disease progression [139]. It can be seen that AD not
only seriously affects the quality of life of the elderly, but also increases the burden
on families and society, making it a huge challenge for the medical community and
society [9, 167].

5.2 Clinical and Pathological Hallmarks of Alzheimer

Disease

Clinically, AD presents as familial AD (fAD) or sporadic AD (sAD). Although

the vast majority of AD is sporadic, the presence of mutations in one of three
genes—amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2
(PSEN2)—can lead to rare (<0.5%) familial form of AD [22]. Symptoms of fAD
develop earlier than sporadic AD, usually between 30 and 50 years of age [163]. The
clinical symptoms of AD are mainly cognitive decline, decreased ability to live and
abnormal mental behavior. According to recommendations from the working groups
convened by National Institute of Aging and the Alzheimer’s Association in 2011,
the dementia stages of AD were divided into the preclinical/presymptomatic stage
of AD, the symptomatic predementia stage called mild cognitive impairment (MCI)
due to AD, and AD dementia [9, 261]. At present, the MCI can be found through
neuropsychological testing, which can be used as a preliminary clinical diagnostic
criterion for AD [38, 52]. The neuropsychological characteristics of MCI are mainly
memory impairment. In fact, memory impairment is an important change in brain
function during normal aging, and the core symptoms and pre-early characteristic
symptoms of AD are also memory disorders [261]. Thus, it can be said that memory
impairment (memory loss) runs through normal aging to pre-early AD. Similar to the
pre-symptoms, early symptoms of AD are also memory disorders, especially near-
5 Iron Pathophysiology in Alzheimer’s Diseases 69

memory disorders; that is, short-term events cannot be recalled. As the pathology of
AD progresses, the patient’s personality and behavior will change, severe cognitive
function declines, mental disorders appear, speaking and acting very difficult, and
losing self-care ability in the late stage, and following extremely high mortality from
multiple complications [9].
Pathologically, the typical pathological features of AD, the deposition of NFTs
formed by hyperphosphorylation of tau protein in neurons and extracellular amyloid-
β (Aβ) plaques, can be detected in early AD [167]. NFTs and Aβ are also detected
on PET scanning in healthy human brains, but normal organisms metabolize them or
allow them to dissolve in cell fluids to perform normal functions [167]. In the brain
of AD patients, these proteins are excessively present, and the clearance is blocked,
which affects cell survival along with synaptic and neuronal losses particularly in
cortex and hippocampus [9, 90] and hence atrophy of brain tissue. Hippocampus is
associated with memory function; hippocampal atrophy is regarded as an important
indicator of memory impairment. When performing neurological examination and
cranial CT or magnetic resonance imaging (MRI) in early AD, most of them have
positive findings [167]. As the pathology of AD progresses, the plaque and tangles
formed by the middle of the brain in the memory and thinking area of the AD patients
are more, resulting in impaired memory and thinking ability of the patient. At this
stage, plaque and tangles spread to the sensory area responsible for speaking and
understanding of the language and for the body to be in contact with the surrounding
objects. Its pathological manifestations are atrophy of the cerebral cortex, flatten-
ing of the brain, widening of the sulcus, enlargement of the ventricles, and weight
reduction of brain. The atrophy of the temporal lobe, parietal lobe, prefrontal cortex,
and hippocampus was the most obvious, and it was more significant than the early
patients. In the advanced stage of AD, most of the patient’s cerebral cortex has been
severely damaged, neurons have died, and the brain has severely shrunk [221, 282].

5.3 Iron Dyshomeostasis and AD

The etiology of AD is complex and diverse, and it is the result of a combination

of genetics, environment, and life, but the specific mechanism of its onset is still
unclear, and there is no corresponding treatment. Several hypotheses of AD pathology
have been discovered including extracellular deposition of Aβ, intracellular accumu-
lation of hyperphosphorylated tau, mitochondrial structure and function disorders,
oxidative stress, disorders of metal ion metabolism, damage to N-methyl-D-aspartate
receptor (NMDAR)-associated signaling pathways, abnormal lipid metabolism, and
abnormal cell cycle in some neurons [39, 226]. Note that metals are essential for
life and play a central role in many biochemical pathways. Environmental exposure,
aging, genetic dysfunction, inadequate dietary intake, and drug interaction can all
induce an alteration in their homeostasis leading to deleterious effects and neurotox-
icity [307]. In recent years, there is growing evidence that impaired iron homeostasis
is an early event in the progression of AD [150, 176]. Abnormal iron content results
70 T. Wang et al.

in the loss of function of several enzymes that require iron as a cofactor, forming
toxic oxidizing species, and increasing β-amyloid production [226]. Several com-
mon genetic polymorphisms that cause elevated levels of iron and disorders in the
body are associated with AD [63, 198], but the pathology is unclear.
It is widely believed that aging is a major risk factor for neurodegenerative dis-
eases. Iron will accumulate in several brain regions of healthy elderly people and will
exhibit a large degree of accumulation in the brains of patients with neurodegenera-
tive diseases [53, 55, 56, 119, 295]. In vitro, increasing iron concentration accelerates
Aβ plaque and pTau tangles and increases its toxicity [240]. Autopsy results showed
that total iron deposition was positively correlated with age, and altered iron levels
were observed in specific brain areas of AD patients (the hippocampus) compared
with the age-matched control group [232, 308]. Iron elevation in the AD brain was
first reported in 1953 [108], and its association with Aβ and NFTs or with ferritin in
peripheral glial cells was confirmed in some studies [55, 56, 150, 176, 295]. Clin-
ical studies have also shown that the disorder of iron metabolism is the key to the
pathology of AD. It is based on the observation that the patient’s content in the
cortex, subcortex, and white matter is significantly increased [233]. MRI analysis
showed that iron levels in the hippocampus increased in the early stage of AD and
were negatively correlated with memory test performance [68]. Moreover, the data
of MRI and histological analysis suggest that increased iron load in the brain is also
associated with Aβ plaque formation, which locally binds to the Aβ core and halo
regions and is entangled with hyperphosphorylated tau (pTau) in humans and animals
[107, 182, 260]. At the cellular level, oligodendrocytes require iron as a cofactor for
enzymes involved in myelin production and maintenance and have the highest iron
content of all cell types in CNS, which increases with age and even further in AD
[21]. Compared to other cells, oligodendrocytes contain low levels of anti-oxidants
[273], which renders them vulnerable to oxidative stress that can accompany a rapid
increase in intracellular iron [199]. Thus, the observation that neuroimaging-defined
white matter abnormalities are characteristic of AD is relatively new, which may be
radiological manifestations in preclinical AD [85, 199]. In addition, the successful
application of iron chelators in patients with early AD and several AD animal models
[62, 78, 115, 114, 116, 142, 237, 309] provided strong evidence that iron-overloading
in specific brain areas with age is associated with the pathophysiological progres-
sion of AD. Of note, an important consideration in studying iron levels in AD is
to emphasize that the increase in total iron content in the brain is not necessary for
brain oxidative stress alone, but that the body may favor oxidation of Aβ and NFTs
by inducing an iron-rich environment, causing agitation and cell death, while other
brain regions may suffer from impaired nerve function due to iron deficiency [24].

5.4 The Role of Iron in the Pathogenesis of AD

Iron is a necessary element demanded for virtually all living organisms and compli-
cates in numerous biological reactions such as electron transfer oxygen transport,
oxidative metabolism, and gene regulation. In recent years, numerous studies have
5 Iron Pathophysiology in Alzheimer’s Diseases 71

revealed that abnormally elevated brain iron is the cause of certain neurodegen-
erative diseases [160]. Nonetheless, why iron accumulates in these disorders and
other important questions regarding their pathogenesis remain unanswered [188].
The abnormally increased iron ions in the brain of AD patients can interact with
H2 O2 to produce highly active hydroxyl radicals in the body. Hydroxyl radicals act
on the proteins, nucleic acids, and cell membrane containing a large amount of unsat-
urated fatty acids and then cause cell damage, called oxidative stress damage [161].
Mitochondria are an energy processing plant in cells. It is also an important site
for generating free radicals. Excessive iron destroys the mitochondrial respiratory
chain, the destruction of any part of the respiratory chain causes the formation of
free radicals, and then the biological macromolecules are damaged, eventually the
cells death [8, 131]. Accumulated iron can also damage mitochondrial membrane
potential, activate caspase-3-dependent apoptotic pathway leading to cells apoptosis,
and ultimately lead to the occurrence of AD [164].

5.4.1 Iron and APP/Tau in AD

In AD, Fe accumulation has been observed in and around the Aβ, SPs and NFTs
[260]. Cerebral iron deposition may be related to the metal-binding sites of Aβ
and tau proteins and may be related to abnormal expression of various brain iron
metabolism-related proteins in the brain of AD patients.
Interactions between iron and Aβ or APP
APP is expressed in most cell types, both brain-derived and other tissues, including
blood cells [238]. In the amyloidogenic pathway, APP is a transmembrane met-
alloprotein [194] that is cleaved by β-secretase enzyme and γ-secretase enzyme
to generate the neurotoxic 40–42-amino-acid prefibrillar amyloid [286]. During the
non-amyloidogenic path of APP metabolism, α-secretase enzyme is the zinc metallo-
protease that cleaves APP in the Aβ-peptide domain to release the 90 kDa ectodomain
of APP. According to reports, we know that α-secretase activation produces a 90 kDa
APP(s) and APP mRNA translation upregulation after interleukin (IL)-1-mediated
stress response, ultimately resulting in secretion of the APP, is increased [19]. Some
study findings showed that APP provides protection against catalyzed oxidative
stress, for example, the lesions during the pathogenesis of AD or lesion and reper-
fusion after stroke [91].
Aggregation of Aβ in brain is the main pathological characteristic of AD, which
is known to be triggered by metals such as iron. Aβ can directly bind iron through
its His6, His13, His14, due to the universality of the binding of ferrous iron to
Aβ, and the interaction between Aβ and iron is believed to be beneficial to smaller
microenvironments such as the brain [32, 298]. Recently, a study uncovering the
relationships between iron biochemistry and AD pathology in APP/PS1 mouse cortex
revealed that iron is an integral part of the SP and also provided the evidence that
Aβ-induced reduction of iron can be occurred in vivo [269]. The iron-aggregated Aβ
72 T. Wang et al.

produces substantial ROS which would partially be conducive to the neurotoxicity

present within the iron-enriched regions around SPs [96, 166].
Iron metabolism can also be modulated by Aβ. The proteins required to regulate
iron metabolism in brain are quite similar to that in periphery. The iron response
proteins (IRPs) 1 and 2 bind to the iron regulatory elements (IREs) in the 3 or 5 -
untranslated region of an mRNA to regulate the iron regulatory proteins’ expression.
Iron can not only accelerate the Aβ deposition, but also regulate the Aβ production.
The intracellular iron was reported to modulate the APP translation via targeting
the IRE in the 5 -untranslated region of APP transcript; therefore, the increased
concentration of intracellular iron translationally upregulates the APP expression [50,
238]. Interestingly, the APP was recently proved to be as an anchor for ferroportin
(FPN) which stables the cell-surface expression of FPN, thereby promoting the efflux
of iron [75]. Depletion of APP in neuronal cultures or in mouse models resulted in the
retention of iron in cells [75], while exogenously add the APP or overexpression of
APP rescues this phenomenon [287]. Based on these observations, the iron overload
modulates the Aβ generation via regulating the expression of APP; in return, the
APP regulates the intracellular iron homeostasis by stabling the cell-surface FPN to
increase iron efflux.

Iron and Tau

Tauopathies are mainly characterized by NFTs, which compose of spiral fibers by

consisting of hyperphosphorylated tau. Tau promotes microtubule stabilization and
regulation, regulates axonal transport [71, 174], and may play an important role in
neurotransmission and iron homeostasis through the trafficking of APP to the cell
surface [158]. These behaviors may be interrupted following hyperphosphorylation
of tau proteins, and iron has been clarified to mediate the modification in keeping with
its redox state. Hyperphosphorylation of tau, one of the key steps toward aggregation,
may be induced by iron via CDK5/P25 complex and GSK-3β [115]. In addition,
Fe3+ may mediate nitration of tau, which prevents microtubule stabilization and
is commonly found in NFTs and SPs [303]. The accumulation of tau in tangles
also results in induction of heme oxygenase (HO) 1, an antioxidant that facilitates
the release of the redox-energetic Fe2+ , which releases loose radicals to produce
oxidative stress [294]. This in flip promotes hyperphosphorylation and aggregation
of tau [152]. In addition, iron accumulation leads to loss of tau function in the cells,
which prevents iron export through impaired transport of APP to the cell membrane,
where it stabilizes ferroportin resulting in a vicious cycle of iron accumulation and
tau pathology [299]. Other studies however suggest that iron chelator was reported to
dissociate the binding of iron associated with hyperphosphorylated tau [258], which
might indicate better therapeutic strategies to suppress tau phosphorylation in AD
[115].
5 Iron Pathophysiology in Alzheimer’s Diseases 73

5.4.2 Iron and Oxidative Stress in AD

Iron plays an important role in the brain. On the one hand, it maintains the brain’s
extremely high respiratory activity for the benefit of myelination and the produc-
tion of various neurotransmitters [37]. On the other hand, due to the high oxygen
metabolism rate of brain tissue and metal ions such as iron, and the relative lack
of antioxidant protection mechanism, it is easy to cause a loss of balance between
oxidative damage and antioxidant defense of brain tissue [49]. Oxidative stress is
a common pathological feature inside the development and pathogenesis of sev-
eral neurodegenerative diseases, which includes AD [161, 291]. Empirical evidence
shows that oxidative stress occurs prior to the advent of SPs and NFTs, thus leading to
the signs and symptoms of dementia [291]. Clinical data further prove an association
between oxidative stress and AD-like cognitive dysfunction [140]. Oxidative stress
contributes to the production of Aβ and hence aggravates its neurotoxicity [193].
In recent years, lots of studies have confirmed that substantially all intracellular
macromolecules, such as lipid, protein and nucleic acid, existing in oxidized form
in the brain tissue of AD patients, and the level of oxidized form are significantly
increased.

Lipid peroxidation

It has been excellently revealed that lipid peroxidation comprises three distinct mech-
anisms, free radical-mediated oxidation, free radical-independent, nonenzymatic oxi-
dation, and enzymatic oxidation [205, 250, 256]. Lipid peroxidation has been shown
to induce disturbance to membrane organization and functional disorder of proteins
and DNA. However, lipid peroxidation products also act as redox signaling mediators
[311].
The aggregation of Aβ is recommended to play a central role in the pathogenesis
of AD which is the most common cause of dementia, Aβ, and small oligomers can
insert into the lipid bilayer and generate hydrogen peroxide [135]. In the presence
of redox-active transition metal ions, consisting of iron and copper, hydrogen per-
oxide undergoes a Fenton reaction resulting in the formation of hydroxyl radicals.
The reaction then develops lipid peroxidation and causes oxidative impairment to
proteins and biomolecules [40]. On the other hand, using an animal model of AD,
it has been shown that oxidative stress and lipid peroxidation can induce Aβ accu-
mulation during the progression of AD [223]. The growing body of evidence for the
involvement of lipid peroxidation and general oxidative stress as a driving force of
AD has generated much interest in using antioxidants as therapeutics [94]. Since the
antioxidant capacity of a cell is dependent on a cocktail of multiple antioxidants, it
is possible that supplementation with one or two antioxidants is not sufficient for
changing the course of disease in humans. Additionally, since lipid peroxidation can
be initiated at very early stages of the disease, antioxidant therapy may be more
effective in patients with early stages of AD, rather than late-stage patients [218].
74 T. Wang et al.

DNA/RNA oxidation and accumulation

In the foregoing text of the present article, we have already mentioned that the brain
is susceptible to oxidative stress and ROS caused by the presence of iron. Homeosta-
sis of iron is tightly regulated under normal physiological conditions as excessive
amounts of iron are known to cause cellular susceptibility to oxidative stress excess,
and then injury DNA and/or RNA via ROS mechanism [128]. The accumulation of
DNA and RNA oxidative damage is observed in cortical and hippocampal neurons
from AD brains at early stages of pathology [283]. An increase in oxidative DNA and
RNA damage occurs in a subset of cortical and hippocampal neurons at the onset of
AD [33]. Non-repaired nucleic acid damage is highly deleterious in neurons, as it can
trigger transcriptional or translational dysregulation and chromosomal instability, as
observed in AD [58]. However, it is poorly comprehended about the mechanisms
underlying the alteration of nucleic acid integrity in neurons all through the early
tiers of AD pathogenesis [128]. In unique, the capability role of tau pathology in
nucleic acid integrity has not been explored.
Several groups have documented the accumulation of oxidative DNA damage
during aging in the brain [121, 127]. Oxidative damage to DNA may be one of the
maximum crucial elements in neuron degeneration of AD due to the critical role of
DNA in cell function. Lu et al. [171] located that oxidative DNA damage preferen-
tially accumulates in the promoter regions of several genes concerned in synaptic
plasticity, vesicular transport, and mitochondrial function. It has been mentioned that
differential accumulation of oxidative DNA damage in the brain regions indicates
that certain regions of the brain are more susceptible to oxidative DNA modifications
than other regions [42, 101]. Without efficient repair ability in the brain in AD, muta-
tions in nDNA and mtDNA may result in neuron death through defects in oxidative
phosphorylation [93, 170]. Furthermore, higher concentrations of oxidized pyridines
and purines were detected in lymphocytes and leukocytes of AD patients compared
with controls [185, 190], and increased levels of 8-oxoG were observed in DNA from
ventricular CSF of AD patients [170]. Recent findings indicate increased oxidative
damage in leukocytes [190] and brain tissue [288] of subjects with MCI, indicating
that DNA oxidation may constitute an early occasion in the AD progression, prior to
cytopathological changes, and might make contributions to the pathogenesis of the
disease [184].
Abundant evidence considerably supports the early involvement of RNA oxidation
in the pathological of neurodegeneration, particularly in AD. RNA oxidation was
observed in postmortem brains of early AD cases [208], a presymptomatic case
with familial AD mutation [207]. Oxidized RNAs may demote faster than other
types of oxidatively modified macromolecules, while protein carbonyl compounds,
lipid peroxidation products, and glycoxidation products are stable at the point of
production [210]. Researches proved the observations contrast remarkably with the
pattern of the RNA oxidation, a steady-state marker, which is prominent in neurons
without pathology [208, 209].
As for RNA oxidization, a study has shown that the Fenton reaction oxidized
and cleaved rRNA in cultured primary neurons and astrocytes [69]. It is known that
5 Iron Pathophysiology in Alzheimer’s Diseases 75

redox-active iron has been shown to also bind with RNA at the metal-binding site
[25] and that redox-active iron bound to ribosomes oxidizes rRNA in AD [130]. Of
course, mRNA oxidation is not a negligible event and may play an important role in
the pathogenesis of AD [255].
Mitochondrial dysfunction
Mitochondrial dysfunction has long been associated with several neurodegenerative
diseases including AD [278]. Mitochondria are essential for regulating the equilib-
rium of iron throughout the cell [219]. Mitochondrial function is critical for normal
cellular processes. It is particularly notable that many neurodegenerative diseases are
directly related to iron metabolism disorders in mitochondria [278, 293]. For exam-
ple, an abnormal increase in iron in brain tissue of patients with AD and PD is likely
to be one of the important causes of neuronal death [20]. Mitochondrial damage and
oxidative stress have been greatly implicated in the progression of aging, along with
the pathogenesis of age-related neurodegenerative diseases, such as AD, PD, and
ALS [79]. Several mitochondria-targeted strategies, such as promoting antioxidant
bioavailability through novel delivery systems, figuring out unique mitochondrial
proteins as precise drug targets, studying signaling pathways for mitochondrial bio-
genesis and kinetics, and identifying effective natural products may be effective
resistant to neurodegenerative diseases associated with mitochondrial dysfunction
[268].
In fact, the early stage of AD is identified via morphologically and functionally
impaired mitochondria, which are prone to over-form ROS, resulting in reduced
brain energy due to ATP reduction [102]. Once formed, Aβ corresponds with the
mitochondria and causes further mitochondrial defect. Aβ inhibits complex IV and
binds Aβ-bound alcohol dehydrogenase (ABAD). ABAD produces ROS. It has been
reported that mitochondria contain an active γ-secretase complex that is involved in
processing of APP to form Aβ, and the non-soluble APP can affect outer mitochon-
drial membrane and interfere with protein shuttle import [102]. Impaired crosstalk
between endoplasmic reticulum and mitochondria also seems to play an important
role in AD pathogenesis. In addition, mitochondrial dysfunction and apoptosis are
associated with Aβ-mediated toxicity [251]. In brief, mitochondria function as both
the source and target of toxic ROS because mitochondrial dysfunction and oxida-
tive stress are important in age-related neurodegenerative diseases, particularly AD
[281]. However, whether mitochondrial iron accumulation is a founding event or a
consequence of upstream events in a particular disease is a matter of debate [187].

5.4.3 Neuroinflammation

Neuroinflammation plays an important role in the pathogenesis of many of the neu-

rodegenerative diseases. Increasing evidence suggests that the pathogenesis of AD
may be caused by inappropriate activation of the inflammatory response in the brain,
and a strong immune response may cause cell damage or death [124, 126]. In AD
76 T. Wang et al.

neuroinflammation, Aβ deposition alone may be sufficient to trigger an inflammatory

reaction by binding to pattern recognition receptors on micro- and astroglia, charac-
terized by the release of inflammatory mediators, which subsequently contributes to
cognitive decline and disease progression. Given the possibility that Aβ deposition
precedes cognitive deficits, one may speculate that exogenous or endogenous factors,
including systemic inflammation, obesity, iron accumulation, and traumatic brain
injury, can modify the innate immune response mounted by Aβ-exposed microglia
[124, 183].
Indeed, both animal studies and clinical AD patients demonstrate SPs and NFTs
formation in vicinity to the activated glial cells in the brain, and thus, immune
response is aggravated [24, 129, 76]. The microglia and astrocyte activation stim-
ulated by Aβ deposition induce reactive gliosis and pro-inflammatory signaling
cascade [145]. Activation of astrocytes patients is often secondary to activation of
microglia in the brain of AD, and activation of astrocytes enhances β-amyloid depo-
sition by overexpression of APP, further activation microglia to form a vicious circle
[245]. A renowned mark of neuroinflammation is the activation and increased intake
of extracellular iron and subsequent downregulation of iron-interacting proteins,
causing the intracellular iron sequestration [272]. Alter of morphology and quantity
of microglia and astrocyte cells are observed in the AD brain, and both are involved in
iron homeostasis [183]. Microglia isolates excess iron in ferritin to protect neurons.
The incidence of L-ferritin positive active microglia is notably increased in the AD
brain, and the characterization of L-ferritin positive cells showed that they are merely
microglia in accordance with morphological and immunohistochemical staining [54,
169]. Microglial cells interact with both non-Tf-bound iron and Tf-bound iron [92,
206], and pathways for each transport substrate have been characterized [180].
Elevated brain concentrations of inflammatory cytokines have been associated
with AD [245, 310]. Interestingly, accumulation of intracellular iron is associated
with neuronal degeneration that underlies most neurological disorders [302], and
microglial secretion of the inflammatory cytokines TNF-α and IL-1β enhances neu-
ronal iron uptake [290]. In turn, these pro-inflammatory mediators have been shown
to strongly influence microglia iron transport and metabolism [129, 144, 272, 277].
In addition, research findings pointed lipopolysaccharide (LPS)-induced expression
of hepcidin in astrocytes is regulated by IL-6-activated microglia. Astrocyte-derived
hepcidin is tightly related to neurons’ apoptosis by iron accumulation in the neu-
rons. These data indicate that a few iron-related proteins, including hepcidin, are not
only a factor in the regulation of iron metabolism but also a critical linker between
iron homeostasis and the modulation of acute inflammatory responses therefore fur-
ther elaborate the pathogenesis of neurodegenerative diseases [306]. Despite the
connection between inflammation and iron metabolism has been established, the
relationship and mechanisms between neuroinflammation and iron metabolism in
neurodegenerative diseases are not fully understood.
5 Iron Pathophysiology in Alzheimer’s Diseases 77

5.4.4 Iron Affected NMDAR-Mediated Synaptic

Transmission in AD

NMDARs, a cation channel, adjust the entry of Na+ and Ca2+ ions when are activated
by the co-agonists glutamate (or NMDA) and glycine or D-serine. Appropriate acti-
vation of NMDARs plays a critical role in physiological functions such as excitatory
neurotransmission, synaptic plasticity [45]. Activated NMDARs cause speedy open-
ing of Ca2+ channels on the cell membrane, followed by an increase in free Ca2+ ions
in the cytoplasm with the aid of a CNS signaling cascade [215]. However, excitotoxic-
ity induced by excessive activation of NMDARs contributes to pathological changes
in the CNS [106]. Hippocampal neurons require iron to generate calcium signals
after NMDARs’ stimulation [196]. Studies of NMDARs involvement in the NTBI
influx pathways implied that activation of NMDARs drastically promotes Fe2+ entry
into cells in primary neurons [48, 216]. Moreover, iron has been shown to block
the influx of Ca2+ in the NMDARs channel, suggesting that Fe2+ competes with
Ca2+ for NMDARs in cultured neurons [197]. In addition, NMDAR’s activation has
been mentioned to promote iron accumulation by accelerating DMT1-mediated iron
influx [48], amplifying iron releasing from lysosome and regulating DMT1 expres-
sion [296]. Interestingly, it is the increased expression of DMT1+IRE mRNA rather
than DMT1-IRE mRNA after treatment of NMDA in primary hippocampal neurons
[118]. In fact, iron deficiency can inhibit cognitive ability [43, 81, 196]; thus, it is of
potential value to ensure an adequate iron supply through the pathway of iron entry
from DMT1. This means that NMDAR’s activation may contribute to the formation
of hippocampal spatial memory by stimulating the expression of DMT1+IRE.
Of note, a role for iron in NMDAR-mediated synaptic plasticity has begun to
remind. A previous study suggest that ROS generated by iron are required for
NMDA-induced and RyR-mediated ERK1/2 phosphorylation/nuclear translocation
(a requisite step of sustained synaptic plasticity) in primary hippocampal neurons,
and electrophysiological studies also found that iron addition resulted in a signifi-
cant increase of the long-term potentiation (LTP), but the increase can be prevented
by iron chelation [196]. Recent studies reported that NMDARs were substantially
improved within the prefrontal cortex and hippocampus of iron-loaded rats, sug-
gesting that altered iron status by iron supplementation in the brain regulates gluta-
matergic neurotransmission pathway [122]. In the hippocampus, studies found that
intracellular iron signaling could modulate neuronal excitability [296], suggesting
that iron can make in a feedback method to modulate NMDA function to maintain
neuronal excitability under normal condition. Further study showed that iron-induced
ROS generation is required for Ca2+ release for long-term potentiation in primary hip-
pocampal neurons [196]. However, iron overload might promote NMDARs mediated
excitotoxicity possibly by enhancing Ca2+ release and calcium-induced cell damage.
Both iron dyshomeostasis and NMDAR-mediated neurotoxicity have been shown to
have an important role in neurological diseases such as AD [301].
78 T. Wang et al.

5.4.5 Iron and Neuronal Damage

Synaptic loss and neuronal death are one of the major pathological hallmarks of
AD, and the endurance of neuronal loss is correlated with the progression of the
disease. Synapses are formed by the junction between two neurons, which allows
neuronal cells to transmit signals to another cell. This channel is usually damaged
or lost in many neurodegenerative diseases, such as AD [143, 257]. Accumulat-
ing evidence suggests that dysfunction and loss of synaptic connections may be
an important early event underlying AD progression, more than other pathological
changes [12, 57, 262]. Damage to synapses by Aβ oligomers includes changes in
synaptic numbers, synaptic transmission, and changes in synaptic plasticity [80, 224,
262]. Comparably, evidence also shows that pathological changes in tau are toxic for
synapses [222]. The molecular mechanisms that lead to synapse degeneration and
neuronal loss downstream of Aβ and tau are less well established, but different path-
ways are implicated such as metal dyshomeostasis, oxidative stress, mitochondrial
dysfunction, and neuroinflammation [64]. Some results suggested that iron impacts
mitochondrial dynamics, possibly trigging synaptic loss and apoptotic, and suggests
that iron chelators have to take into consideration as a capability molecule with
memory restoring and neuroprotective properties, observed in the remedy of cogni-
tive deficits in neurodegenerative disorders [259, 117]. Reduced synaptophysin may
be associated with iron-induced cognitive deficits observed in AD model, as these
researchers have previously reported that iron-induced hippocampal synaptophysin
levels continue to decline in rats [259, 155]. Recently, some data on iron deficit that
may play a role in synaptic processes, as well as preceding studies on the effects of
iron deficiency on cognitive processes and synaptic function, can generate valuable
information to prevent against the effects of iron deficiency and iron overload [195].
Thus, insightful synapse degeneration in AD is characterized by the worsening of
cognitive function, synapse loss, and neuronal cell death.
Morphologically, advanced AD manifests postmortem as a significant shrinkage
in cortical volume, especially throughout the cortex and hippocampus. This is caused
by massive neuronal cell death, whose etiology is not clear [88]. Notably, the type of
cell death that occurs in affected areas of the AD brain is still contentious, despite the
demonstration that DNA fragmentation and upregulation of pro-apoptotic proteins
has been frequently observed [227]. As such, it remains unclear to what extent apop-
tosis or emerging types of regulated necrosis are responsible for bulk neuronal loss
in AD [16, 227]. We now know that abnormal elevation of iron may be the initiat-
ing factor of neuronal death in the brain of patients with neurodegenerative diseases
[225]. Iron-induced oxidative stress has been shown to initiate several apoptotic sig-
naling pathways in neurons [243] and cause oxidative damage to key proteins. There
is extensive evidence that oxidative damage to proteins and lipids by iron can cause
synaptic dysfunction and neuronal cell death [178], both of which are critical features
of AD.
Ferroptosis is the fourth cell death pattern proposed by scholars based on the
unique pathological state caused by intracellular iron accumulation, which is differ-
5 Iron Pathophysiology in Alzheimer’s Diseases 79

ent from necrosis, apoptosis, and autophagy [73]. Ferroptosis signs a newly located
form of cell death that is not associated with the caspase pathway and is involved in
iron disorders, lipid peroxidation, and inflammation as predominant markers [72].
The disorder in cellular redox homeostasis leads to accumulation of lipid ROS and
hence lipid peroxidation [1, 97]. Morphological changes affecting the outcomes
involve mitochondrial volume reduction, reduced density of mitochondrial mem-
branes, chromatin condensation, eventually rupture of mitochondrial outer mem-
brane, and plasma membrane [73, 89]. Several powerful ferroptosis inducers have
been identified [267] in which the small-molecule erastin effectively inhibits xCTcys-
tine back transporter to further induce ferroptosis, as cystine is vital for glutathione
synthesis. Glutathione peroxidase 4 (GPX4) [305] utilizes reduced glutathione to
detoxify phospholipid hydroperoxides and hydrogen peroxides [279]. It should be
noted that GPX4 knockout indicates iron imbalance, lipid peroxidation, and inflam-
mation in mice, at the same time shows early signs of AD phenotype, including
behavior defect and hippocampal neurodegenerative disease [120, 253]. Intriguingly,
small-molecule inhibitors of ferroptosis can mitigate neurodegeneration and inflam-
mation observed in GPX4 knockout mice, suggesting that ferroptosis is a potential
neurodegenerative mechanism affecting neurons important for spatial function [120].

5.4.6 Iron and White Matter

Over the past several years, neuroimaging studies have implicated micro- and
macrostructural abnormalities in white matter in the risk and progression of AD
[34, 35, 157]. Analysis of postmortem brain tissue of AD patients found out a chem-
ical change in white matter compared to non-demented patients: The amounts of
general protein, myelin basic protein, myelin proteolipid protein, cyclic nucleotide
phosphohydrolase, and cholesterol are notably reduced, indicating myelin loss [239].
The abnormal white matter in AD is mainly manifested in changes in myelin and
oligodendrocytes [41, 199]. Myelin loss and the inability of the oligodendrocytes,
the cells responsible for the production and maintenance of myelin, to repair myelin
damage may be additional central features of AD [41]. Due to the essential role of
oligodendrocyte lineage in the process of myelination and remyelination, changes in
the variety of oligodendrocytes or their precursor cells and/or their dysfunction can
affect myelin integrity and thus may be related to AD pathogenesis [179].
In the brain, iron is a key substance involved in the process of myelin protein and
fatty acid synthesis in oligodendrocytes. Iron deposition in the brain of AD patients,
which in turn causes an increase in oxidative stress, may result in increased cell
damage. It is reported that myelinating oligodendrocytes contains a lower amount of
antioxidant than other glial cells [141]. Therefore, high iron content and low antiox-
idant content make oligodendrocytes one of the most susceptible to oxidative stress
in the CNS [276]. Rather, iron deficiency during infancy can lead to neurocognitive
developmental delay, impaired learning and memory and in some cases can lead to
mental illness, in part due to impaired myelination [228]. Due to normal aging, loss
80 T. Wang et al.

of white matter increases free iron, which is toxic to the brain. MRI analysis showed
that white matter damage in AD patients preceded gray matter damage compared
with age-matched controls [3].

5.5 Iron-Binding Proteins Implicated in AD

Ferritin

Ferritin is a highly conserved supramolecular protein, which composes of two differ-

ent subunit types, H (heavy) and L (light). These two subunits co-assemble into a 24-
subunit heteropolymer, with tissue-specific distributions, to form shell-like protein.
A single ferritin molecule can sequester up to thousands of iron atoms, in the form
of insoluble inorganic ferrihydrite core [14]. As a ubiquitous and well-characterized
iron storage and detoxification protein, ferritin is the most efficient way to protect
cells from the toxic effect of elevated levels of labile iron cations. In bacteria, plants,
fish, as well as amphibians, the ferritins are generally homopolymers which consist
of H-type subunits, while the ferritin is a heteropolymer typically composing of 24
subunits of two types, H and L in animals [14, 186]. The inner cavity of ferritin
is separated from the outside environment by the resultant protein nanocage which
is remarkably stable in the pH range of 3–9 and temperatures up to 80 °C. Small
molecules and various cations can be able to enter the ferritin interior through eight
narrow hydrophilic threefold channels also maybe through six hydrophobic fourfold
channels as well [14]. The natural Fe(II) cations rapidly diffuse into the ferritin shell,
where the Fe(II) cations are oxidized to insoluble iron(III) cations to form the inor-
ganic ferrihydrite core. The different subunits of ferritin own different bio-functions.
In mammals, the H-subunit is charging of the rapid oxidation of Fe(II) to Fe(III) by
oxidative molecules at a dinuclear center, whereas the L-subunit helps iron export
from the ferritin interior in support of iron nucleation and mineralization [31, 270].
It should also be noted that ferritin consisting exclusively of L-subunits is still able
to oxidative Fe(II) cations, although this bio-activity is very low. It was reported that
ferritin in plasma and cerebrospinal fluid is significantly increased in neocortical
amyloid-β loaded participants, and ferritin is also observed to correlate positively
with amyloid-β load[15, 109]. Subsequent studies implicate that CSF ferritin levels
were able to act as a predictive biomarker of disease progression in the prodromal
stages of AD [70].

Mitochondrial ferritin

Mitochondrial ferritin (FtMt), specifically located in mitochondria, is a new ferritin

type identified in 2001. The human FtMt gene locates at chromosome 5q23.1 which
encodes a ∼1 kb mRNA that translates to a 242 amino acids FtMt precursor protein.
The FtMt precursor protein is translocated to mitochondria after synthesis and is
further processed to the mature type as the subunit to form typical ferritin shells [61].
The mature human FtMt has a 79% identity to the H-chain ferritin in the sequence
5 Iron Pathophysiology in Alzheimer’s Diseases 81

of amino acids. The inner cavity of FtMt and H-ferritin shares a highly conserved
sequence and a fully overlapped crystallographic structure [153], suggesting that
these two proteins have similar functions. Unlike ferritin, the expression of FtMt
is tissue-specific, and it is predominantly expressed in the testis and brain [244].
The major bio-function of FtMt is also sequestering excess free iron. Although the
ferroxidase activity of FtMt is not as strong as H-ferritin, the iron sequestering ability
is high. Overexpression of FtMt in Hela cells resulted in reduced expression of
cytosolic ferritin and cytosolic iron deficiency [61]. Elevated expression of FtMt
was observed in the brains of AD patients and was associated with anti-oxidative
activity [304]. Furthermore, FtMt deletion exacerbated Aβ-induced neurotoxicity
[292], whereas FtMt overexpression attenuated Aβ-induced neurotoxicity [300].

Lactoferrin

In 1939, the lactoferrin (Lf) was firstly identified in bovine milk and subsequently
isolated in 1960 from both human and bovine milk. Human Lf is a glycoprotein
which consists of 691 amino acids, which is constitutively expressed and secreted
by glandular epithelial cells and by neutrophils following induction. The Lf pos-
sesses multi-functional bioactivities, such as anti-microbe, anti-inflammation, and
immunomodulation; meanwhile, it is an iron-binding glycoprotein, which is impor-
tant for the modulation of iron homeostasis. Like Tf, Lf has been shown to reversibly
bind iron as Fe3+ with high affinity (K D ∼ 10−22 M), as well as retain Fe3+ until pH
values less than 3.0. The Fe3+ -binding activity of Lf is dependent on the concomitant
and synergistic binding of carbonate, CO3 2− [5]. Spectroscopic studies and the 3D
structure suggest that Lf binds the CO3 2− firstly, thus neutralizing the positive charge
of the arginine residue (Arg-121 in the N-lobe and Arg-465 in the C-lobe) [18]. The
participation of the CO3 2− in the iron binding to Lf is important for iron reversible
binding since the protonation of the CO3 2− is likely a first step to break up the iron
site at low pH values [2]. Lf has two main conformational states, the open metal-free
(apo-lactoferrin, apo-Lf) state and the closed metal-bound (holo-lactoferrin, holo-Lf)
state. Metal binding and release are determined by the large-scale conformational
changes of Lf, by which the domains open to release the irons or close over the
bound irons [99]. The metal-free apo-Lf form is flexible and more prone to thermal
denaturation and digestion by proteases. However, the metal-bound holo-Lf is con-
formationally rigid and very stable, and the bound irons can only be removed by
the very low pH or some iron-chelating reagents. Of note, the iron-binding activ-
ity of Lf is not specific, and other trivalent metal ions such as Ga3+ and Al3+ can
also bind to Lf, although the affinity is lesser than Fe3+ [17]. The functions of Lf
in AD pathology remain largely unknown. A recent study reported that intranasal
human Lf effectively inhibits the cognitive decline of APP/PS1 mice via activation
of ADAM10 expression [116]. Meanwhile, salivary Lf was identified as a diagnostic
biomarker for mild cognitive impairment and AD due to it is more accurate than the
cerebrospinal fluid total tau and Aβ42 [44].
82 T. Wang et al.

Hepcidin

In 2000, a defensin-like disulfide-bonded 25 amino acid peptide with antimicrobial

activity was respectively identified by two independent research groups in human
ultrafiltrate and urine [151, 214]. This peptide was named hepcidin, and structural
study further uncovered that this peptide contains eight disulfide-bonded cysteines
forming a structure of β-turns, loops, and distorted β-sheets [214]. Hepcidin is pre-
dominantly synthesized and secreted by hepatocytes. In iron-overloaded mice, a
dose-dependent induction of hepcidin mRNA transcript was observed [220]. How-
ever, substantial irons were accumulated in liver, pancreas, and serum, while the
splenic iron store was depleted, in the mice with lack of hepcidin gene expression
[202]. These studies revealed that hepcidin plays a vital role in regulating home-
ostasis of body iron. Upon binding to FPN, hepcidin induces covalent modification
of FPN and subsequently causes FPN internalization and degradation in lysosomes,
which reduces the export of iron from reticuloendothelial macrophages, hepatocytes,
as well as duodenal enterocytes [201]. Hepcidin decreases the cell face stability of
FPN, thereby inhibiting the entry of iron into plasma. Decreasing iron efflux from
cells coupled with the ongoing iron uptake of erythropoiesis through the bone marrow
leads to decreased serum iron and reduced transferrin (TF) saturation. Agelong high
levels of hepcidin lead to insufficient serum iron for erythropoiesis which increases
the risk of iron-restricted anemias [203]. In AD brains, hepcidin expression was
decreased and restricted to the blood vessels, neuropil and damaged neurons; more-
over, hepcidin was accompanied with heme-positive granular deposits in the region
of damaged blood vessels [229]. The reasons for the reduction in hepcidin levels are
less clear which need to be further investigated.

Ferroportin

FPN, encoded by the SLC40A1 gene, a 571 amino acid transmembrane protein,
plays a critical role in iron metabolism. Although cells possess several mechanisms
to uptake iron, FPN is the only known cellular iron exporter [125]. Iron efflux is in the
form of ferrous iron; therefore, the activity of ferroxidase is necessary for FPN. The
ferroxidase converts the divalent iron into ferric iron, which is mainly accomplished
by the positive membrane iron transport accessory proteins or ceruloplasmin. Fpn
controls iron entry into the plasma through duodenal enterocytes (1–2 mg/day) or
macrophages that recycle near 20–30 mg/day of iron from senescent erythrocytes
[74]. Recently, FPN has been recognized as the receptor for hepcidin, a master reg-
ulator of iron homeostasis [235]. Upon binding to hepcidin, FPN is internalized and
subsequently submitted to lysosomal degradation [201], which results in a reduc-
tion of plasma iron concentration and an increase of intracellular ferritin. The FPN
expression in AD brain is reduced with uncertain reasons [229]. Considering that APP
stabilizes the cell-surface FPN expression [75] and substantial APP variants were
observed in AD patients, the decreased FPN expression may be highly associated
with APP mutations.
5 Iron Pathophysiology in Alzheimer’s Diseases 83

Transferrin and transferrin receptor

TF–transferrin receptor (TFR) axon is an important pathway for iron homeostasis. All
vertebrates have functional TF, which is a 76 KD glycoprotein that mainly expressed
in and secreted by the liver with a half-life of approximately 8 days in serum. TF
contains two highly homological lobes at the N- and C-termini, and these lobes are
connected by a short linking region [104]. Each lobe can bind one metal ion such as
zinc, gallium, and iron with different affinities; therefore, one TF can maximum bind
two ferric Fe. Most iron in the serum is bound to the TF; with the blood circulations,
the iron can be transported to every tissue and organ. Serum TF can exist in non-
bound (apo-TF), monoferric, or differric (holo-TF) forms. TF-bound iron enters cells
which needs TFR, and TFR is a 97 KD type 2 membrane protein expressed in as
a homodimer in cell membrane. On the cell surface, holo-TF binds to TFR and
subsequently initiates the endocytic process to intake iron [156]. The interaction of
TF and TFR is determined by the pH value: at pH 7.4, and TFR binds to holo-TF
but not apo-TF; inversely, at a lower pH value in endosomes, TFR binds to apo-
TF [285]. In the acidic endosomes, the ferric Fe is dissociated from TF, and it is
further converted to ferrous Fe by metalloreductases [211]. The TF-TFR complex
in the endosomes is then transferred to the cell surface by recycling endosomes and
subsequently release of apo-TF [74]. In AD plasma, the iron is decreased due to TF
desaturation [123]. Furthermore, the phosphorylated TF in brain and serum from an
AD animal model are significantly reduced, and the cerebrospinal fluid and serum
from AD patients also exert an aberrant phosphorylated TF [241].
DMT1
DMT1 is the firstly identified iron transporter in mammalian, and it transports various
ferrous metal ions including Mn2+ , Co2+ , and Fe2+ [138]. DMT1 has 12 transmem-
brane helices with aqueous cavity and a metal ion binding site formed between
residues in α-helices 1 and 6. DMT1 is well known to play a vital role in iron
metabolism. In the proximal small intestine, DMT1 couples with the transport of
H and Fe2+ into enterocytes. Dietary iron in the small intestine is in the form of
Fe3+ ; therefore, a reduction step mediated by the apical reductase Dcytb is required
prior to the uptake of iron by DMT1 [181]. DMT1 is abundantly expressed in brain,
in aged rat brain; the expression of DMT1 is increased in an age-dependent man-
ner which associated with elevated brain iron content [172]. This study indicates that
age-induced DMT1 upregulation is associated with increased brain iron in AD brain.
Melanotransferrin
Melanotransferrin (MTf) is a TF homolog that is found mainly bound to the cell mem-
brane via a glycosylphosphatidylinositol anchor. It shares an identical N-terminal
Fe-binding site with serum TF, by which, MTf can bind one Fe-atom [36]. MTf can
be secreted by cells at a very low level; however, its ability to donate Fe to cells is
far less effective than TF [87]. Furthermore, the secreted MTf cannot be bound by
the transferrin receptors, despite its high structural homology to TF [87]. Substantial
in vitro studies have been performed to uncover the relationship between MTf and
84 T. Wang et al.

iron metabolism. However, the in vitro data all suggested that MTf does not play a
vital role in Fe transport and metabolism [149, 234]. In addition, the MTf knockout
mice showed normal fertility and development, with no morphological or histolog-
ical abnormalities [77, 254]. Taken together, although the high-affinity Fe-binding
site of MTf is structurally and functionally similar to that in Tf, MTf is not necessary
in Fe transport and metabolism. It has been reported that MTf is expressed in reactive
microglia and the secreted MTf is increased in sera in AD patients [148]. To date,
the function of MTf in AD is largely unknown, which need to be further elucidated.

HFE
HFE is a protein that comprised of 343 amino acids, which interacts with β2 M to
form a heterodimer expressed at the cell surface [83]. HFE contains a signal peptide,
an extracellular transferrin receptor-binding region (α1 and α2), an immunoglobulin-
like domain (α3), a transmembrane region, and a short cytoplasmic tail [83]. Gly-
cosylation of HFE is important for its normal intracellular trafficking and functions.
HFE can be glycosylated at residues N110, N130, and N234 during transport to the
cell membrane [26]. HFE plays an important part in the regulation of iron homeosta-
sis. In iron-loaded HFE knockout mice, liver hepcidin was significantly decreased
[4]. Furthermore, iron overload was rescued in HFE knockout mice with constitutive
overexpression of hepcidin [204]. These data suggested that HFE acts as an upstream
regulator of hepcidin expression. HFE can interact with TFR1 and TFR2 in the cell
membrane, and any changes in the expression of either HFE or TFR2 can cause
the downregulation of hepcidin [111, 146]. The binding affinity of HFE for TFR
is similar to the binding affinity of iron-loaded TF, and the HFE can competitively
bind to the iron-loaded TF-binding site of TFR, thereby inhibiting the iron uptake
[84]. The associations between HFE polymorphisms and AD were investigated in
some studies. The HFE H63D mutation is associated with prolonged endoplasmic
reticulum stress and increased the risk of AD [165, 175]. The clear functions of HFE
in AD pathology remain unexplored, possibly involved in cholesterol modulation
and iron regulation [6].

HO-1

HO-1 is one of the three isoforms of heme oxygenase (HO) family which act as
key rate-limiting enzyme in the degradation of iron-containing heme [173]. It is
widely distributed in many mammalian tissue cells and is also recognized as heat
shock protein 32 (Hsp32). The level of HO-1 is low at basic conditions and can
be highly increased in the response of numerous pro-oxidant agents and stimuli
including heat shock, heavy metals, ultraviolet rays and cytokines. After induction,
HO-1 plays a pleiotropic, protective role and shows antioxidant, anti-apoptotic, and
anti-inflammatory effects in various conditions [168]. The cytoprotective effects of
HO-1 depend on multiple cellular processes including the generation of bilirubin,
an antioxidant molecule, from the degradation of heme; the induction of ferritin,
a strong chelator of free molecular iron; and the liberation of CO, responsible for
HO-1 major anti-inflammatory and anti-apoptotic effects [274]. Ninety percent of
5 Iron Pathophysiology in Alzheimer’s Diseases 85

the body’s iron is derived from the iron of aging red blood cells, which is the iron
produced by the decomposition of hemoglobin. Therefore, the role of HO-1 in iron
metabolism is crucial. Studies showed that sustained HO-1 activity decreased the
expression of transferrin receptor-1 and increased the proteasomal degradation of
ferritin [162]. Because of the distinct transcriptional regulation hemoglobin have
from iron, the change of HO-1 also affect FPN1 and hepcidin [177]. Besides, HO-1
is also related to neurodegeneration diseases [247]. The expression levels of HO-1 in
reactive astrocytes are markedly increased in the hippocampus and cerebral cortex of
patients with AD relative to age-matched, non-demented controls [249]. In AD mice,
long-term overexpression of HO-1 causes phosphorylation of tau protein, which
accumulates in the brain and forms neuronal fiber tangles, aggravating the condition
[137]. To review most of the literature, the glial HO-1 response may serve as a robust
transducer of noxious stimuli, an important driver of relevant neuropathology and a
potentially disease-modifying therapeutic target [248].

Lipocalin-2

Lipocalins are small secreted proteins that play roles in many processes including
retinol transport and prostaglandin synthesis. The lipocalins are capable of binding
low-molecular-weight ligands due to a common secondary and tertiary structure
corresponds to a single, eight-stranded antiparallel β-barrel around a central pocket
[46]. Lipocalin-2 (LCN2), also known as oncogene 24p3 or neutrophil gelatinase-
associated lipocalin (NGAL), is a 25 kDa soluble and secretory protein and reportedly
functions as a mediator of inflammation and iron ion transport [191]. Studies showed
that LCN2 mediates iron ion transport by association with a siderophore. LCN2
saturated with iron (holo-form) can increase intracellular iron levels by moving and
releasing iron into the cytoplasm. Iron-free form of LCN2 (apo-form) can deplete
intracellular iron and transport it to the extracellular space via its receptor NGAL-R
[66]. It can increase or decrease intracellular iron content based on the body iron store,
making it a malleable factor in the maintenance of iron homoeostasis [47]. Of note,
LCN2 protein levels in blood increase with age and mild cognitive impairment [51]
and are increased in human postmortem brain tissues of AD [65, 200]. In the context
of AD pathology, in vitro studies demonstrated that astrocytes generate high levels
of LCN2 in response to Aβ and that LCN2 sensitizes brain cells more vulnerable to
Aβ-induced cell death [189, 200]. However, more insight into such effects of LCN2
using LCN2 KO mice is required to understand its importance in AD and its potential
as a therapeutic target [65].

Furin

Furin is a member of the mammalian proprotein convertases’ family. It is a ubiq-

uitously expressed calcium-dependent serine endoproteases that is expressed in all
vertebrates and many invertebrates. It is expressed as a 794 amino acid zymogen that
undergoes two autocatalytic cleavage steps before it becomes fully active [10]. As a
proprotein convertase, furin is known to control the activation of diverse functional
proteins by cleaving inactive protein precursors in the secretory pathway. Studies
86 T. Wang et al.

showed that hepcidin is produced in the liver and processed into the mature active
peptide by furin. The hepcidin gene encodes a precursor prepropeptide of 84 amino
acids. Furin recognizes the consensus sequence and participates the two sequential
cleavages to produce the mature peptide of 25 amino acids [280]. Few studies point
out that furin plays an important role in the activation of both α- and β-secretase. Both
ADAM10 and ADAM17 (the α-secretase) contain a consensus furin motif which is
essential for their activation. The β-secretase, known as BACE, also requires prote-
olytic removal of its proregion by furin at a minimal furin site [271].

5.6 Alteration of Iron Content for Clinical Diagnosis of AD

Progression

The role of iron in the pathology of AD has only received attention in the past few
decades, in part because of inconsistencies in the literature regarding whether iron
increase is a major or minor event in AD brain [252]. Moreover, iron deficiency
during infancy can lead to neurocognitive developmental delay, impaired learning,
and memory and in some cases can lead to mental illness, in part due to impaired
myelination [228]. However, an early inductively coupled plasma mass spectrometry
(ICPMS) study showed that even in the normal brain region, there is a regionally
specific change in metal content [230]. Thus, it is important to distinguish between
large organizations and specific areas of research for the iron status in tissues of AD
patients relative to age-matched controls, including the blood, cerebrospinal fluid
(CSF), and brain tissue.

5.6.1 CSF Iron Level in AD

Using ICPMS technology, Gerhardsson et al. [98] detected CSF iron levels in 174
AD patients (122 females, 52 males) and 54 control subjects (36 females, 18 males).
There was no significant difference in CSF iron levels between the two groups, and
the same results were obtained by using the same method in different laboratories [30,
134]. Lavados et al. [154] used atomic absorption spectrometry (AAS) to detect iron
levels in the CSF of 7 AD patients and 12 control groups. There was no significant
difference in CSF iron levels between the two groups, consistent with the previous
study [192]. Interestingly, in the above studies, there was no significant difference,
but the CSF iron in the control group was slightly higher than the experimental
group. Taken together, the iron content in the CSF of AD patients did not change
significantly in its pathological process.
5 Iron Pathophysiology in Alzheimer’s Diseases 87

5.6.2 Blood Iron Level in AD

For iron levels in AD blood, the researchers used different experimental methods to
verify the iron content in serum, plasma and whole blood of healthy controls and
AD patients. Alimonti et al. [7] used inductively coupled plasma atomic emission
spectrometry (ICPAES) to detect serum iron levels in 53 AD patients (36 females, 17
males) and 124 control subjects (43 females, 81 males). They found that the serum
iron levels in the control group were higher than those in the AD group. Using ICPMS,
several research teams also detected that healthy controls have higher serum iron
levels than patients with AD [23, 27, 30]. However, Giambattistelli et al. [100] used a
turbidity immunoassay kit to determined serum iron levels in 107 AD patients and 52
healthy controls, and there was no significant difference in serum iron levels between
the two groups. Similar findings were detected by both Vitros 5.1 [275] and FerroZine
colorimetry [264, 265], respectively. Using AAS, the observation of several research
teams also confirmed that there was no difference in serum iron content between
normal people and AD patients [136, 192, 263], while Ozcankaya et al. (Ozcankaya
and Delibas [213]; Vural et al. [284]) detected that serum iron levels were significantly
increased in AD patients than in the control group. Different conclusions from various
studies may be due to different experimental methods, different data collection times,
individual sample differences, and different geographical distribution of samples.
Therefore, changes in iron levels in the blood may not be sufficient to diagnose AD.
Compared with blood and CSF, changes in iron content in the brain have reference
significance for the diagnosis of AD.

5.6.3 Brain Iron Level in AD

Iron has been extensively studied in different brain regions of AD patients, and the
results are different. Corrigan et al. [60] used ICPMS to detect hippocampal iron
levels in 12 AD patients (10 females, 2 males) and 12 control subjects (4 females,
8 males). The hippocampal iron levels in AD patients were significantly increased
compared to the control group. Gu et al. [113] used the same method to detect cortical
iron levels in 12 AD patients and 11 control groups and found that cortical iron levels
were significantly increased compared to the control group. House et al. [133] used
ICPAES to detect 5 AD patients (2 females, 3 males) and 5 control groups (2 females,
3 males). The iron levels of amygdala, corpus callosum, cingulate cortex, caudate
nucleus, entorhinal cortex, frontal cortex, globuspallidus, hippocampus, parietal cor-
tex, lateral temporal cortex, thalamus, subcortical frontal white matter, and temporal
white matter found that except for lateral temporal cortex, iron levels were signifi-
cantly increased compared to the control group, and the iron content in the other brain
regions did not change significantly. Srivastava et al. [266] used ICPMS to detect iron
content in the cerebellum and parietal cortex of 4 AD patients and 4 control groups
and found no significant difference between the two groups. Galazka-Friedman et al.
88 T. Wang et al.

[95] and Griffiths et al. [112] used AAS to detect iron content in hippocampus of
AD patients and healthy controls and found that the iron content in hippocampus of
AD patients increased slightly, but there was no significant difference. Andrási et al.
[11] used instrumental neutron activation analysis (INAA) to detect iron content in
the whole brain of 5 AD patients and 5 control groups. No significant difference
was found between the two groups, but Cornett et al. [59] using the same method
found that the iron content in multiple brain regions of AD patients was significantly
increased. From the results of autopsy, iron is unevenly distributed in the brain of
AD patients, and there are certain metabolic disorders, which may be involved in the
pathological process of AD. Although the results are different, in general, iron has
deposition in different brain regions of AD patients. This phenomenon may be one
of the causes of memory loss in AD patients. Therefore, the increase of iron content
in the brain may be one of the methods for the diagnosis of AD.
In fact, the diagnosis of AD is difficult because pathologic AD findings are not
sufficient for symptoms. To investigate AD pathological development and biomarkers
for early-stage AD identification, we learned that neuroimaging data do not directly
measure AD-related pathology but suggest neurodegeneration and achieved many
meaningful results; particularly, the noninvasive detectability of Aβ plaques in MRI
has so far been largely attributed to focal iron deposition accompanying the plaques
at preclinical stages of AD [105].

5.7 Targeting Iron Dysregulation

The issue of iron dysregulation has been the target of therapeutic attempts for the
treatment of AD. In this regard, iron chelation is a potential therapeutic strategy
in both oxidative stress and protein aggregation processes but remains a matter for
debate. For a metal chelator to effectively exert chelation, it must have the following
characteristics: (1) target iron-enriched areas without depleting transferrin-bound
iron from plasma; (2) remove chelated iron from iron accumulation sites or transfer
it to other biological proteins, such as circulating transferrin; (3) easy to penetrate
the cell membrane and blood-brain barrier (BBB); and (4) no side effects or minor
side effects on the body [28].
Several iron chelators including desferrioxamine (DFO) and clioquinol (CQ) have
been recently designed for their potential use in the treatment of AD [242]. DFO, a
hexadentate ligand with a higher binding affinity for Fe3+ than Fe2+ , is able to inhibit
the formation of β-pleated sheets of Aβ(1–42) and dissolve preformed β-pleated
sheets of plaque-like amyloid in vitro [132]. Despite a variety of promising results
in some clinical trials [62], experimental mouse models, the molecular size, and
hydrophilicity of DFO prevent it from freely crossing the BBB, when administered
orally [231]. Again, long-term administrated DFO leads many side effects, including
allergic reactions, anemia, liver and renal dysfunction, and neuronal hearing loss
[29]. Our recent studies demonstrated that intranasal DFO treatment inhibits iron-
induced amyloidogenic APP processing, tau hyperphosphorylation, and impaired
5 Iron Pathophysiology in Alzheimer’s Diseases 89

spatial learning and memory [115, 114], suggesting that intranasal administration of
DFO may be a safe and noninvasive therapeutic strategy for AD therapy. CQ, a small
lipophilic iron chelator that can cross the BBB, has been used in phase II clinical trials
for moderate AD cases [236]. However, CQ is not a iron selective and produces very
toxic effects. Surprisingly, recent research implicating the role of CQ in the inhibition
of cAMP efflux might represent promise for the treatment of neurodegenerative
diseases [217]. In addition, plant polyphenols were proposed for the treatment of
AD because of their antioxidant activities and strong iron-binding affinity [176],
but their capacity to be absorbed at the gastrointestinal tract and be transported
subsequently through the BBB is limited. Clearly, the development of an effective
non-toxic therapeutic agent for AD-like brain disorders remains a challenging task.
α-lipoic acid (LA), a small-molecule compound that can be naturally synthesized
in mammals, is mainly used for the treatment of diabetic polyneuropathy, and it
was recently found that 129 patients with AD who had been treated with LA for a
period of time had an effective remission of cognitive decline [82]. In fact, in vitro
studies have demonstrated the antioxidant properties of LA and a similar role as
metal chelating agents [212]. Studies showed that LA not only reduced iron uptake
rates but also decreases dynamic iron pools by increasing iron stores within the lens
epithelial cells [110]. In vivo, LA can enhance Aβ solubility the frontal cortex of APP
transgenic mice and human AD brain tissues, suggesting that LA, like other transition
metal chelators, reduces deposition of amyloid protein through Aβ re-dissolution
[86]. Most recently, our study showed that chronic treatment with LA effectively
inhibited tau hyperphosphorylation by alleviation of oxidative stress, inflammation,
and ferroptosis in the brains of an AD mouse model [309].
Surprisingly, the recent study found that Lf as an iron chelator and a therapeutic
agent has been investigated to identify its roles in AD. The affinity of Lf for iron is
300 times higher than that of TF. In weakly acidic environments, the affinity is further
enhanced, which may be related to the transfer of iron from transferrin to Lf during
inflammation [67]. Several experiments showed that Lf is expressed in many neurons
and in some astrocytes, microglia, and oligodendrocytes of the human brain [13, 147]
and that it could involve in aging processes [159, 226]. Further research findings sug-
gested that Lf is highly expressed in both extracellular SPs composed of aggregated
Aβ peptide and in intracellular NFTs composed of aggregated hyperphosphorylated
tau protein, which are suggesting the link of Lf and AD-like pathologies [147, 289].
Therefore, the use of exogenous Lf as a therapeutic agent has been investigated to
identify its roles in AD. Administrated Lf could stimulate the non-amyloidogenic
processing of APP and α-secretase catalytic activity and expression in AD model
mice and Lf enhanced the α-secretase-dependent APP process through the ERK1/2-
CREB and HIF-1α pathways in vitro and in vivo [116]. In combination with other
biological functions of Lf, such as its participation in metal ion metabolism and the
regulation of cell proliferation and apoptosis, we infer that Lf may be a potential
therapeutic agent to delay the pathological progress of AD.
90 T. Wang et al.

5.8 Conclusion

Why is it so hard to find a cure to AD? The main explanation is that the complex
causes of AD. It presents itself typically late in life and under the influence of many
combined genetic and lifestyle risk modifiers such as metabolic disorder of metal
ions, smoking, alcohol use, diabetes, body weight, hypertension, and physical and
intellectual activity levels, and perhaps even certain diets. Researchers have done
a lot of research on these pathogenic factors [103, 246, 282]. Recently, the study
of the pathogenesis of AD using iron as a target has become a research hotspot [8,
32, 56, 160, 161, 164, 176, 242]. The use of iron chelators to chelate excess iron
in the brains of AD patients has also become a new strategy for the treatment of
AD [78]. However, many therapeutic drugs for AD are still difficult to translate into
clinical application, largely due to the existence of BBB, which prevents drugs from
entering the brain to play a role. Therefore, effective drugs for AD must be easy to
pass through BBB. In our bodies, many proteins or molecules bind to iron to form
active complexes, such as LA and Lf. Some researchers have confirmed that treating
AD transgenic mice with LA and LF can alleviate their pathological symptoms [116,
309], but the specific mechanism of its action is still unclear, and further studies are
needed. Hopefully, in the meantime, Lf-based nano-drug molecules are also under
study. This drug molecule exhibits excellent biocompatibility and can easily cross
BBB to reach the lesion site, showing effective therapeutic effect. Future research
will also use iron as an opportunity to study the mechanism of the occurrence and
development of AD from the iron homeostasis to more fully explain the causes of
AD.

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Chapter 6
Iron Pathophysiology in Stroke

Mohammed M. A. Almutairi, Grace Xu and Honglian Shi

Abstract Ischemic and hemorrhagic stroke are the common types of stroke that lead
to brain injury neurological deficits and mortality. All forms of stroke remain a seri-
ous health issue, and there is little successful development of drugs for treating stroke.
Incomplete understanding of stroke pathophysiology is considered the main barrier
that limits this research progress. Besides mitochondria and free radical-producing
enzymes, labile iron is an important contributor to oxidative stress. Although iron
regulation and metabolism in cerebral stroke are not fully understood, much progress
has been achieved in recent years. For example, hepcidin has recently been recog-
nized as the principal regulator of systemic iron homeostasis and a bridge between
inflammation and iron regulation. This review discusses recent research progress
in iron pathophysiology following cerebral stroke, focusing molecular regulation of
iron metabolism and potential treatment targets.

Keywords Stroke · Iron · Hepcidin · FPN1 · HIF-1α · Anesthesia

6.1 Introduction

Stroke is one of the leading causes of death and long-term severe disability world-
wide. Stroke can be classified into ischemic or hemorrhagic forms according to
its pathophysiological nature. The majority of all stroke cases (around 90%) are
ischemic, which occur due to a sudden decrease or loss of blood supply to the brain

M. M. A. Almutairi · H. Shi (B)

Department of Pharmacology and Toxicology, School of Pharmacy, University of Kansas,
Lawrence, KS 66045, USA
e-mail: [email protected]
M. M. A. Almutairi
Department of Pharmacology and Toxicology, School of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
G. Xu
Department of Anesthesiology, School of Medicine, University of Kansas, Kansas City,
KS 66160, USA

© Springer Nature Singapore Pte Ltd. 2019 105

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_6
106 M. M. A. Almutairi et al.

by thrombi and/or emboli. Intracerebral and/or subarachnoid rapturing blood vessels

cause hemorrhagic stroke, comprising about 10% of all strokes [44, 82]. Even though
the incidence percentage of hemorrhagic stroke is lower than ischemic stroke, it can-
not be ignored because this type is more dangerous compared to the ischemic form
[21]. In the last ten years, research has focused on identifying effective therapeu-
tic approaches for reducing death and disability related to stroke. Numerous agents
have been tested in animal models and clinical trials, but there has been limited
progress in developing effective therapeutic strategies for stroke [14, 26, 104, 111].
An incomplete understanding of the mechanism responsible for stroke-induced neu-
ronal injuries is a major issue underlying this limited progress. Disturbance of brain
iron homeostasis contributes to neuronal injury following cerebral stroke [43, 49,
94]. Recently, there has been exciting progress in understanding iron metabolism in
stroke. For example, hepcidin has recently been recognized as the central regulator
of iron homeostasis and a bridge between inflammation and iron regulation [39, 83].
Many studies have shown that hepcidin enhances iron-dependent neuronal damage
and subsequently has become a target for the development of novel therapeutics for
iron toxicity in stroke [24, 101]. This review summarizes the pathophysiological role
of iron in stroke as well as manipulation of iron-dependent pathways as a potential
therapeutic approach to improve the outcome of stroke patients.

6.2 Brain Iron Regulation

Iron is essential for life as it is needed for many biological processes. In the brain, iron
plays an important role in neurotransmission, myelination, cell division, and oxygen
transport processes [110]. Brain cells maintain iron homeostasis by regulating iron
import, storage, metabolism, and export [50, 51]. Many studies have demonstrated the
role of transferrin, divalent metal transporter 1 (DMT1), ceruloplasmin (CP), ferritin,
and ferroportin1 (FPN1) in the regulation of brain iron [70, 87]. Under physiological
conditions, iron binds to transport proteins such as transferrin. Transferrin-bound iron
is transported into brain cells via receptor-mediated endocytosis. Emerging evidence
suggests that uptake of transferrin-bound iron into the brain cells by transferrin
receptor-mediated endocytosis is the major known pathway of brain iron transport
across the blood–brain barrier (BBB) [87]. This route of iron transport consists of
several steps, including transferrin-iron binding, endocytosis of transferrin-bound
iron, dissociation of iron from transferrin, and translocation of the iron inside brain
cells. Unbound iron can gain access into brain cells through DMT1, but the majority
of iron in brain cells is bound to storage proteins, such as ferritin. CP promotes iron
release from intracellular stores. FPN1 is the first and solely known vertebrate iron
exporter expressed in the brain. It has been shown that FPN1 mediates iron efflux
from brain cells. Since iron import, storage, and export are tightly regulated, any
alterations in these steps may disturb iron homeostasis in the brain resulting in iron
neurotoxicity [2, 87]. Deficiency in FPN1 activity results in iron retention and further
6 Iron Pathophysiology in Stroke 107

iron toxicity [28]. Increased free iron levels have been observed in ischemic and
hemorrhagic brains; however, the exact mechanism behind it is not fully understood
[16, 94].

6.3 Alterations of Iron Homeostasis in Ischemic

and Hemorrhagic Stroke

Iron plays a critical role in oxygen transport [110]. Under ischemic conditions,
required levels of oxygen in the brain increase, resulting in high demand on iron
transport and metabolism inside preserved brain regions [94]. Ferritin-bound iron is
released in response to changes from ischemic insults, including acidosis and inflam-
matory mediators. Many studies have shown that the binding affinity of transferrin
for iron decreases at low pH levels, resulting in dissociation of iron from transferrin
[64]. Upon dissociation, unbound iron can easily gain access to the extracellular
space where it is transported into neurons, resulting in increased intracellular iron
levels [79, 94]. Recent observations have demonstrated that decreased iron efflux
from neuronal cells contributes to putative alterations in iron homeostasis following
ischemic stroke [62]. Furthermore, growing evidence indicates that iron accumu-
lation in neurons following ischemia increases brain susceptibility to iron-induced
damage [41, 64, 94]. In hemorrhagic stroke condition, many studies showed iron
overload in the brain after intracerebral hemorrhage (ICH) [16, 88]. Red blood cell
release and lysis have been considered the major factors mediating iron-induced
brain damage after ICH [53]. Collagenase, which is commonly used to induce ICH,
results in iron overload [113]. Moreover, iron overload may contribute to brain edema
following ICH [43]. However, the potential mechanism, underlying iron-mediated
neurotoxicity, is unsatisfactorily understood.

6.4 Iron Toxicity

Many studies have suggested that the severity of neuronal damage after ischemic
stroke is proportional to the magnitude of brain iron accumulation [94]. For example,
Patt et al. demonstrated that gerbils with low brain iron exhibited fewer neurological
deficits compared to those with high brain iron levels [81]. Consistently, rats with
iron overload exhibited about 60% greater infarct volume than rats with lower iron
load [12]. Iron accumulation in rat brains following ischemia [58, 94] induces lipid
peroxidation and neuronal injury by catalyzing conversion of superoxide and hydro-
gen peroxide into highly reactive toxic hydroxyl radicals [54]. Increased formation
of these free radicals results in oxidative stress and subsequent ischemic neuronal
death [11]. In addition, it has been shown that experimental ICH causes brain injury
via iron-induced oxidation and DNA damage [71, 114]. Results from animal studies
108 M. M. A. Almutairi et al.

indicated that Tirilazad mesylate, an inhibitor of iron-dependent lipid peroxidation,

reduces neuronal injury in global and focal cerebral ischemia [31, 47]. Moreover,
deferoxamine (DFO), a potent iron-chelating agent, reduces infract volume and neu-
rological deficits in ischemic rats [48, 97] and human patients [93]. Rosenthal et al.
have reported that DFO improves neurological status and reduces mortality rate in
rats subjected to a 6.5 min-cardiac arrest [90]. DFO also improves neurologic func-
tion in collagenase-induced ICH model [113]. Together, these results support the
notion that iron induces neurotoxicity following cerebral stroke.

6.5 Molecular Mechanisms Involved in Iron Toxicity

6.5.1 Ferroportin in Ischemic and ICH Stroke

Ferroportin 1 (FPN1, encoded by SLC40A1) is a transmembrane protein, which

contains 12 domains and is expressed in the duodenal enterocytes, placenta,
macrophages, and brain [1, 7, 67]. In the brain, FPN1 is predominately expressed in
neurons of the cerebral cortex, hippocampus, and cerebellum [7, 9] where it functions
as the sole protein capable of exporting iron from cells [37]. In 2000, Donovan et al.
found that a complete loss of FPN1 resulted in severe iron deficiency anemia and
caused embryonic death in zebrafish and mammals [27]. Additionally, FPN1-mutated
mice exhibited severe cellular iron accumulation [28, 125]. Emerging evidence shows
that ischemic stroke disrupted the mechanisms that maintaining brain iron homeosta-
sis and further resulted in iron neurotoxicity [56]. Li et al. demonstrated that FPN1
expression was down-regulated in the cerebral cortex and hippocampus after cerebral
ischemia. A decrease in FPN1 expression in an ischemic brain may result in iron
overload and hepcidin synthesis [62]. Since brain endothelial cells express FPN1,
this transporter plays an important role in ICH-induced iron overload. Many studies
have showed that FPN1 significantly increased after ICH [107]. Despite the largely
unknown neurophysiological role of FPN1 in the brain, FPN1 has been shown to act
as a receptor for hepcidin in cultured neuronal cells [74].

6.5.2 Hepcidin

Hepcidin was first discovered in human blood and urine as a bactericidal peptide
synthesized in the liver [46, 55]. Since its discovery, many reports have demon-
strated that hepcidin plays a critical role in systemic regulation of iron metabolism
in mammals including human, rat, mouse, and pig [33, 50, 72]. The gene encoding
hepcidin (HAMP) is expressed in a growing list of tissues, including liver, brain,
lungs, intestines, and heart [15, 35, 38, 68, 109]. Hepcidin is a central regulator of
iron homeostasis, but the exact mechanism of how hepcidin regulates iron acquisi-
6 Iron Pathophysiology in Stroke 109

tion has not been fully characterized. Nicolas et al. unambiguously recognized that
inactivation of HAMP results in severe iron accumulation in the liver and pancreas
[76]. Additionally, transgenic mice overexpressing HAMP exhibit a severe anemia
[77]. Human patients with hepcidin mutations suffer from juvenile hemochromato-
sis [89]. Treating cultured cells expressing an FPN1-GFP with hepcidin results in
translocation of FPN1-GFP from the cell surface into lysosomes where it is degraded
[19]. Moreover, the injection of hepcidin into the cerebral ventricle suppresses FPN1
protein levels in different regions of the brain, such as the cerebral cortex, hippocam-
pus, and striatum [24]. Xiong et al. reported that both brain and serum hepcidin was
up-regulated after ICH. This increased expression of hepcidin reduces intracellu-
lar iron efflux from brain microvessel endothelial cells via regulating FPN1 [117].
Studies have shown that hepcidin binds to FPN1 and induces intracellular iron accu-
mulation via interaction with FPN1 and causes its internalization and degradation
in endolysosomes (Fig. 6.1) [19, 20]. It has been identified that residue Cys326 of
FPN1 is required for hepcidin interaction and mutation of this residue induces FPN1
resistance to hepcidin binding [20]. Fernandes et al. proposed that a thiol subunit
of the Cys326 residue initiates a disulfide bond with a cysteine residue in hepcidin
[17, 32]. These findings indicate that increased iron accumulation and decreased
iron efflux might be caused by the action of hepcidin. In physiological conditions,
intercellular iron is mainly stored in ferritin and FPN1 mediates efflux of un-needed
or excess iron. During many pathological conditions, including stroke, hepcidin is

Fig. 6.1 Mechanism of hepcidin action. Hepcidin increases intracellular iron levels by causing the
degradation of FPN1, a cellular membrane protein that export iron to extracellular space
110 M. M. A. Almutairi et al.

up-regulated and binds to FPN1 and causes its internalization and degradation in
endolysosomes. FPN1 degradation leads to iron accumulation in neurons and fur-
ther results in neuronal death.

6.5.3 Regulation of Hepcidin–FPN1 Pathway

Hepcidin expression is regulated and modulated at the transcriptional level by many

factors, including iron status and inflammation [61]. For example, the inflamma-
tory cytokine interleukin (IL)-6, a major inflammatory mediator, up-regulates hep-
cidin expression and reduces serum iron levels in humans and experimental animal
models [73, 112]. Additional studies have suggested that hepcidin expression is up-
regulated by inflammatory cytokines following infection and post-ischemic inflam-
matory conditions [1, 105]. The mechanisms underlying inflammation-induced hep-
cidin expression are complex and only partially understood. There is increasing
evidence that inflammation positively regulates hepcidin expression, mainly through
induction of signal transducer and activator of transcription3 (STAT3) pathway [106].
Upon inflammatory stimuli, IL-6 activates Janus Kinase (JAK), a kinase responsi-
ble for the STAT3 phosphorylation. Phosphorylated STAT3 (p-STAT3) translocates
into the nucleus and binds its response element in the hepcidin promoter, resulting
in the stimulation of hepcidin transcription (Fig. 6.2) [112]. At transcriptional lev-
els, knockdown of STAT3 significantly down-regulates endogenous hepcidin mRNA
expression, indicating that STAT3 is involved in the transcriptional control of hep-
cidin [106]. Recent studies show that administration of lipopolysaccharide (LPS),
a key inflammatory mediator, increases hepcidin expression [108]. Furthermore, an
in-vitro study demonstrated that iron-overloaded microglial cells exhibit increased
production of proinflammatory cytokines, suggesting that hepcidin may provide a
link between iron accumulation and inflammatory responses in the brain [92, 105].
Furthermore, iron disposition and IL-6 secretion are enhanced in mouse and rat
brains following experimental ischemic stroke and ICH [24, 115]. This intracellu-
lar iron disposition results from reduced iron export in ischemic brain due to the
down-regulated expression of FPN1 by hepcidin [119]. It has also been reported that
ICH models exhibited increased hepcidin and intercellular iron accumulation due
to decreased iron efflux [117]. These findings indicate that hepcidin may promote a
crucial association between inflammation and iron overload in brain tissues.

6.5.4 Iron-Mediated Hypoxia-Inducible Factor 1α (HIF-1α)

Degradation

HIF-1 has been suggested to be a critical regulator of neurological outcomes follow-

ing ischemic stroke due to its function as a central role in regulating neuronal adap-
6 Iron Pathophysiology in Stroke 111

Fig. 6.2 Inflammation-

induced hepcidin
transcription. Inflammation
plays a key role in
upregulating hepcidin
expression. Stroke initiates
the production of
inflammatory stimuli. Upon
inflammatory stimuli,
interleukin (IL)-6 activates
Janus Kinase (JAK), a kinase
responsible for the STAT3
phosphorylation.
Phosphorylated STAT3
(p-STAT3) translocates into
the nucleus and binds its
response element in the
hepcidin promoter, resulting
in the stimulation of hepcidin
transcription action

tive responses to hypoxia. HIF-1 regulates genes that promote energy metabolism,
angiogenesis, and erythropoiesis, all of which are critical in promoting cell survival
in hypoxia. HIF-1 is a heterodimer, consisting of a constitutively expressed β sub-
unit and a regulated α subunit. HIF-1α is preferentially degraded under normoxic
conditions while under hypoxic conditions, it is stabilized, dimerizes with the β sub-
unit, and subsequently induces adaptive gene expression and promotes cell survival.
Under normoxic conditions, HIF-1α is degraded through the ubiquitin–proteasome
system after recognized by the von Hippel Lindau tumor suppressor (pVHL), a com-
ponent of the E3 ubiquitin ligase. The recognition by pVHL requires the action of a
family of HIF prolyl hydroxylase domain (PHD) that hydroxylates proline residues
on HIF-1α in the presence of oxygen. Ferrous ion is a cofactor of the enzyme PHD.
Increase in free iron promotes HIF-1α degradation through the 26S pathway by
elevating its hydroxylation and thus compromises cellular resistance to ischemic
injury (Fig. 6.3). Conversely, reducing iron levels would be lower PHD activity and
increase HIF-1α stability/accumulation. In fact, Hishikawa et al. have reported that
DFO significantly attenuated basilar artery vasospasm and reduction of brainstem
blood flow in a rat model of hemorrhagic stroke by increase in HIF-1α protein level
and HIF-1 activity [52]. Similarly, in ischemic stroke DFO induced HIF-1 DNA bind-
ing and transcription of erythropoietin in vivo and ischemic tolerance [5, 86]. More
recently, Sorond et al. reported that DFO infusion improved cerebrovascular function
in older individuals, possibly by increased the activity of HIF-1-regulated pathways
[99]. Although it is arguable if the protective effect of DFO can be all attributed to
112 M. M. A. Almutairi et al.

Fig. 6.3 New targets for increasing HIF-1 activity. Iron reduction could be an effective pharma-
cological approach to activate HIF-1 and to enhance cerebrovascular function acute and chronic
ischemic conditions. Hepcidin and FPN1 may provide additional targets for increasing HIF-1 levels

HIF-1 activity [122], iron reduction could be an effective pharmacological approach

to activate HIF-1 and to enhance cerebrovascular function acute and chronic ischemic
conditions. Hepcidin and FPN1 may provide additional targets for increasing HIF-1
levels (Fig. 6.3).

6.5.5 Post-ischemic and ICH Inflammation Regulates

Neuronal Damage

Neuro-inflammation following ischemia plays a crucial role in neuronal injury [25].

The inflammatory response may promote cerebral swelling, which is often lethal
[96]. Microglial cells, which are the resident macrophages of the brain, modulate
neuronal damage by releasing several proinflammatory mediators such as cytokines
and chemokines in response to post-ischemic inflammation [29, 59]. It has been
reported that the number of activated microglia significantly increases in rat brains
subjected to 2-h ischemia [65]. Many cytokines, including ILs and tumor necrosis
factor-α (TNF-α), are up-regulated in ischemic brains [59, 118, 123]. Additionally,
elevated cytokine levels following ischemia exaggerate the infarct severity and induce
further neuronal death [59]. Intraventricular injection of recombinant IL-1β worsens
the infract severity in ischemic mouse brains when compared with controls [118].
However, IL-1β deficient mice exhibit less neuronal damage in comparison with wild-
type [8]. In addition to cytokines, chemokine levels are elevated in ischemic animal
models while their deficiency rescues neurons from ischemic injury [57, 98]. Con-
sistently, mice lacking chemokine receptors exhibit less brain injury after ischemia
[23]. Similar to ischemic stroke, microglial activation, cytokines, and chemokines
release have been reported in ICH [124]. These findings suggest that inflammation
may contribute to neuronal injury and expand the brain lesion upon ischemia and
ICH. Recent studies have reported that inflammation mediates iron accumulation in
CNS cells [105]. As mentioned previously, increased iron deposition has been linked
to neuronal damage following ischemia and ICH [94, 117]. These findings indicate
6 Iron Pathophysiology in Stroke 113

that inflammation may drive iron accumulation in cerebral stroke. However, the
hypothesis that hepcidin acts as a link between inflammation and iron neurotoxicity
requires further investigation.

6.5.6 Effect of Anesthesia on Iron Regulation in Stroke

Postoperative cognitive dysfunction (POCD) refers to a decline in cognition after

major surgery and anesthesia. Age is one major factor for cognitive complications
following surgery [116]. Based on a prospective longitudinal study, Monk et al.
found that stroke is another major independent factor that increases the incidence
of prolonged POCD [69]. The authors argued that the increased incidence of pro-
longed POCD may be related to the concept of cognitive reserve because the patients
may have lost critical neural mass with their stroke, leading to a decrease in their
cognitive reserve and an increased susceptibility to POCD. Alternatively, the latest
clinical evidence establishes a positive relationship between general anesthesia and
development of POCD in the elderly [6, 22, 66, 95, 100]. It has been suggested
that anesthetics may promote inflammation, increase oxidative stress, reduce neu-
rotrophin expression, and cholinergic dysfunction, which contribute to POCD. We
speculate that iron accumulation might be one contributing factor to anesthesia-
mediated POCD due to the following factors. (1) Iron overload has direct links to
key components of POCD symptoms such as in Alzheimer’s diseases [3, 45]. (2) It
seems that oxidative stress contributes to the progression of POCD, and iron overload
can cause oxidative stress. (3) It has been reported that anesthetics could increase the
production of inflammation mediators, which can upregulate hepcidin expression,
causing iron overload. (4) Preconditioning is to stimulate the endogenous protection
mechanism with a mild stress. Sevoflurane and isoflurane preconditioning amelio-
rates inflammation, cerebral lipid peroxidation, and histologic injury in a rat model
of focal cerebral ischemia [4], implying that the prolonged anesthetic exposure could
cause adverse effects such as inflammation and lipid peroxidation. Future studies are
needed to explore the potential role of iron metabolism in anesthetics-mediated brain
dysfunction such as POCD.

6.6 Iron as a Treatment Target

In the last ten years, our understanding of the pathogenesis of stroke-induced brain
injury has advanced. However, therapeutic options for cerebral stroke are still very
limited. Current approved therapies rely mainly on the use of thrombolytic treatment
in acute ischemic cases [104]. Recombinant tissue plasminogen activator (r-tPA) is
the only thrombolytic drug that has been approved for clinical use to reduce neuro-
logical injury and improve the functional recovery of patients with cerebral ischemia
[91]. The time window for effective treatment with r-tPA is limited to three hours
114 M. M. A. Almutairi et al.

after symptom onset. Because the large majority of patients with ischemic stroke
are not aware of initial symptoms, many do not seek care within the three-hour time
frame. Consequently, due to its short therapeutic window and potential hemorrhagic
risk, the number of patients that benefit from this treatment is limited [104]. In
term of ICH, several reports have shown that majority of ICH-patients who received
anticoagulant and antiplatelet treatments had experienced the incidence of severe
anticoagulant-associated ICH [34]. In addition, emerging evidence demonstrated
that antithrombotic drugs such as warfarin increased the risk of ICH by seven folds
and is associated disability and death [30, 40]. This major challenge increases the
demand for safe and effective therapeutic approaches to improve the outcome of this
pathological condition. This section discusses some prospective therapeutic strate-
gies that can protect the brain from the negative consequences of cerebral stroke.
The negative consequences of stroke that could be beneficial therapeutic targets,
including post-stroke inflammation and brain iron accumulation [94, 123, 124].

6.6.1 Anti-inflammatory Treatment Approach

As mentioned previously, post-ischemic inflammation is a large effector in the pathol-

ogy of cerebral stroke [96, 123, 124]. Therefore, targeting inflammatory pathways
can be a protective therapeutic strategy to reduce or limit brain injury after the stroke
[104]. Many anti-inflammatory molecules have been tested as neuroprotective agents
in acute ischemic stroke. Although most of these agents have failed to restore neuro-
logical functions in patients with ischemic stroke, blocking post-ischemic inflamma-
tion may improve pro-regenerative effects following ischemia. IL-10 and transform-
ing growth factor beta (TGF-β) are major anti-inflammatory mediators in ischemic
and ICH brain injury [42, 96, 103]. Many studies have been conducted to determine
the beneficial effect of these anti-inflammatory mediators in ischemic stroke treat-
ment. Ooboshi et al. reported that overexpression of IL-10 protects neurons against
focal and global brain ischemia. Their results also found that IL-10-treated ischemic
brains exhibit less brain infarction and immune cell infiltrations in comparison with
controls [78]. Recently, Cekanaviciute et al. demonstrated that TGF-β inhibits suba-
cute neuroinflammation and preserves brain function in the peri-infarct cortex after
ischemic stroke [13]. Clinically, minocycline is one of the most anti-inflammatory
drugs that have been tested in the treatment of human acute ischemic stroke and ICH
[10, 104]. It has been found that minocycline improves the prognosis of patients
or animals with stroke compared with placebo treatment [18, 60]. Although the
exact mechanism as well as the onset effect of these anti-inflammatory molecules
in ischemia treatment remain to be clarified, controlling post-ischemic inflamma-
tory pathway shows a promising potential target to treat ischemic stroke [96]. The
next section discusses whether iron-chelating agents could be a neuroprotective tool
against inflammation-induced iron neurotoxicity in ischemic stroke.
6 Iron Pathophysiology in Stroke 115

6.6.2 Iron-Chelating Therapy

Considerable evidence suggests that iron-mediated neurotoxicity plays a critical role

in the pathogenesis of ischemic and ICH stroke [43, 94]. Additionally, it has been
found that iron accumulation in the brain is induced by post-ischemic and ICH
inflammation [18, 24]. Based on these notions, many studies have examined whether
decreasing iron overload in the brain during ischemia decreases neurological defects
and improves the outcome of ischemic stroke. Iron chelators bind excess iron in a sta-
ble complex to prevent iron accumulation and maintain a safe iron status [84]. In fact,
DFO reduces ischemic and ICH stroke damage in rodent models [75, 80]. Addition-
ally, DFO significantly decreases cortical infarct volume and improves neurologic
deficits following ischemia in rat brains [48]. Furthermore, these neuroprotective
effects of DFO are mediated by suppression of iron-induced free radical formation
in animal models after ischemic stroke [93]. As discussed previously in this review,
iron accumulation, FPN1 reduction and hepcidin up-regulation are all induced during
ischemia-induced neuroinflammation [24, 105]. DFO dramatically blocks increased
iron levels and reduces FPN1 down-regulation in LPS-treated neurons[121]. These
findings suggest that DFO may also attenuate neuroinflammation-induced iron accu-
mulation in neurons upon stroke via regulation of hepcidin pathway.

6.6.3 Targeting Hepcidin Pathway in Ischemic and ICH

Stroke

Hepcidin, a negative regulator of the sole cellular iron exporter FPN1, plays a crucial
role in intracellular iron accumulation [19, 20]. Therefore, therapeutic approaches
that decrease hepcidin synthesis and/or increase FPN1 activity seem to be potential
candidates to treat patient with iron overload disorders [85]. Recent studies have
showed that hepcidin siRNA treatment induces the FPN1 expression and rescues
iron accumulation in cerebral cortex, hippocampus, and corpus striatum of ischemic
mouse brains [24]. In addition, hepcidin knockout mice ICH models exhibit reduced
brain iron accumulation compared with wild-type mice [117]. These findings sug-
gest that iron overload following ischemia and ICH is negatively correlated with
increased FPN1 levels in hepcidin knockout models. Additionally, alteration of
hepcidin-FPN1 interaction with a thiol-reactive compound fursultiamine prevents
FPN1 internalization and degradation via sequestering the Cys326 residue of FPN1,
a necessary residue for hepcidin binding to FPN1 [85]. Consequently, fursultiamine
reduces intracellular iron accumulation via enhanced cellular iron export despite the
presence of hepcidin [36]. As discussed previously, activation of IL6/STAT3 path-
way induces hepcidin transcription [24]. Therefore, targeting IL6/STAT3 pathway
with anti-IL6 antibodies can down-regulate hepcidin expression. Siltuximab and
tocilizumab, anti-IL6 antibody drugs, are in clinical use to reduce hepcidin effects
and improve cellular iron export in patients with anemia [85]. Recent studies have
116 M. M. A. Almutairi et al.

shown that anti-IL6 antibody significantly decreases the expression of IL-6 protein in
the brain of ischemic animal models [102]. Furthermore, anti-IL6 antibody rescues
the blood–brain barrier from ischemic injury in the fetal sheep model [120]. In addi-
tion, urocortin, anti-inflammatory neuropeptide, suppresses inflammatory cytokine
production (e.g., IL-6 and TNF-α) and reduces neurological deficits following ICH
[63]. Taken together, targeting hepcidin and/or corresponding pathways could be an
effective therapeutic approach to protect the brain from stroke injury.

6.7 Conclusion and Future Perspectives

Inflammation and iron accumulation play a crucial role in neuronal injuries caused by
ischemic stroke. Hepcidin binds FPN1 and reduces its activity in excreting excess iron
from the brain cells. The major factors that positively regulate hepcidin include iron
status and inflammation. Modifying iron metabolism and post-stroke inflammation
can reduce brain injury in stroke and the hepcidin pathway seems to be a promising
molecular target for stroke therapy.
Increased hepcidin expression has emerged as a causative factor in several com-
mon iron disorders such as cerebral stroke. Targeting hepcidin pathways and/or
interfering hepcidin–FPN1 interaction have become promising approaches for the
development of novel therapeutics for iron disorders. However, multiple potential
opportunities for cross communication among these pathways can cause off-target
effects such as an increased risk of infection. For example, IL-6 receptor-targeted ther-
apeutics impair host defense. Moreover, interfering hepcidin–FPN1 interaction by
fursultiamine antagonizes hepcidin effects rather than reducing its expression. There-
fore, lowering hepcidin expression directly in cerebral stroke where it is elevated may
have less possible off-target effects in comparison with other therapeutic approaches
mentioned previously. In addition, the direct targeting of hepcidin in stroke models
would help us to understand the specific role of hepcidin in stroke-induced brain
injury. The major challenge of systemic administration of anti-hepcidin is that some
negative effects need to be taken into account. Deficiency of systemic hepcidin results
in increased serum iron, dietary iron hyperabsorption, and iron-dependent oxidative
stress. To overcome this challenge, anti-hepcidin could be delivered into the stroke-
injured brain by using microinjection or nanoparticles as drug delivery systems. In
the following, some experimental approaches are proposed in order to refine our
understanding of the mechanistic role of hepcidin in cerebral stroke.
In summary, these future directions may improve our understating of the roles
of hepcidin in iron-mediated neurotoxicity following cerebral stroke. Based on the
results, novel therapeutic interventions might be developed to protect the brain from
stroke injury.

Acknowledgements This investigation was supported by the University of Kansas General

Research Fund. Mohammed M. A. Almutairi was supported by a scholarship from King Saud
University (Riyadh, Saudi Arabia).
6 Iron Pathophysiology in Stroke 117

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Chapter 7
Iron Pathophysiology in Friedreich’s
Ataxia

Kuanyu Li

Abstract Friedreich’s ataxia (FRDA) is a degenerative disease that affects both the
central and the peripheral nervous systems and non-neural tissues including, mainly,
heart, and endocrine pancreas. It is an autosomal recessive disease caused by a
GAA triplet-repeat localized within an Alu sequence element in intron 1 of frataxin
(FXN) gene, which encodes a mitochondrial protein FXN. This protein is essential
for mitochondrial function by the involvement of iron–sulfur cluster biogenesis. The
effects of its deficiency also include disruption of cellular, particularly mitochondrial,
iron homeostasis, i.e., relatively more iron accumulated in mitochondria and less
iron presented in cytosol. Though iron toxicity is commonly thought to be mediated
via Fenton reaction, oxidative stress seems not to be the main problem to result
in detrimental effects on cell survival, particularly neuron survival. Therefore, the
basic research on FXN function is urgently demanded to understand the disease.
This chapter focuses on the outcome of FXN expression, regulation, and function
in cellular or animal models of FRDA and on iron pathophysiology in the affected
tissues. Finally, therapeutic strategies based on the control of iron toxicity and iron
cellular redistribution are considered. The combination of multiple therapeutic targets
including iron, oxidative stress, mitochondrial function, and FXN regulation is also
proposed.

Keywords Friedreich ataxia · Frataxin · Iron · Iron–sulfur cluster biogenesis

7.1 Friedreich’s Ataxia (FRDA)

FRDA, described by Nikolaus Friedreich in 1863, is a single-gene, recessive, and pro-

gressive neurodegenerative disorder that classically appears around puberty, rarely
in early childhood, and in some cases later in life. On average, in ten or fifteen years
after the onset of the disease, patients lose the ability to walk, to stand or to sit up

K. Li (B)
Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing
210093, People’s Republic of China
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2019 125

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_7
126 K. Li

unaided. The disease occurs mainly in Caucasians. It is believed that the restricted
distribution in Caucasians is due to a single founder mutation that occurred between
500 and 800 generations ago [1].
The essential clinical and pathological features of the disease include the degen-
eration in the spinal cord of the posterior columns, which contain axons of dorsal-
root-ganglia sensory neurons. Most patients succumb to cardiomyopathy, and many
become diabetic during the course of the disease. Typically, cognitive functions are
preserved well. The presence of comorbid depression in FRDA is recently recog-
nized [2]. In 1996, came the breakthrough identification of the disease gene, frataxin
(FXN), for FRDA [3].
The last 20 years have seen remarkable progress in elucidating the function of the
encoded protein, FXN, which localizes to the mitochondria [4] and is involved in
iron-sulfur cluster biogenesis [5]. The focus of this chapter is on iron dysregulation
in FRDA, with a particular emphasis on the most recent findings.

7.2 FXN Gene and Its Function

FXN is a highly conserved gene present from gram-negative bacteria to eukary-

otes, including yeast and mammals. In human, FXN maps on chromosome 9, at
9q13–q21.1. The prevalent mutation is an abnormal expansion of a GAA triplet-
repeat localized within an Alu sequence element in intron 1, which accounts for
greater than 98% cases of mutation in patients. Homozygotes (bi-allelic expansion)
are found in the majority (96–98%) of FRDA patients, and the remaining ones are
compound of heterozygotes of the (GAA)n expansion and a point mutation (mis-
sense, nonsense or splicing) [4, 5] or an intragenic deletion [6] or intronic deletion
[7].

7.2.1 FXN Gene Mutation and Its Expression of Isoforms

Ages of onset and death as a function of GAA trinucleotide repeat expansions are
closely correlated with expression levels of FXN. This GAA expansion ranges from
fewer than 200 to as many as 1500 repeats, in which size of the expansion correlates
negatively with expression of the encoded protein and positively with the ages of onset
and severity of the disease. This expansion leads to transcriptional silencing of FXN
through a commonly accepted mechanism involving modifications of the chromatin
structure of the locus, resulting in expression of a structurally and functionally normal
FXN but at levels that are estimated at 5–30% of normal [8, 9]. The decreased histone
acetylation and extensive methylation of CpG regions upstream of the GAA repeats
are observed in patient cell lines and tissues [10, 11], suggesting that the enhanced
heterochromatin formation might impede the transcription of FXN, leading to the
lower levels of FXN protein [12–14]. Studies employing an experimental histone
7 Iron Pathophysiology in Friedreich’s Ataxia 127

deacetylase inhibitor (HDACi) in a mouse model (KIKI with GAA insertion) of

FRDA revealed that this drug can substantially increase FXN mRNA and protein
levels [13]. Unexpectedly, the phase III clinical trial did not prove the effects of
HDACi on clinical symptoms.
Reduction of FXN transcription may result from reduced accessibility of tran-
scriptional regulatory factors to the promoter region and/or trinucleotide repeat region
[12]. However, the identity and number of the regulatory factors influencing FXN
expression are largely unknown. The known human transcription factors have been
proposed such as SRF and AP2 [15], HIF1 [16], and p53 [17] to regulate FXN
expression. The entity of the transcriptional regulatory machinery involved in FXN
expression needs to be further investigated to aid the identification of drugs or ther-
apies directed at restoring FXN protein levels in affected tissues of FRDA.
FXN is synthesized as a precursor protein (210 amino acids, FXN1–210) that
is then matured in two steps to give an intermediate form (FXN42–210) and the
major mature form (FXN81–210) in the mitochondrial matrix [15, 18]. A few stud-
ies support the presence of the extra-mitochondrial isoforms [19–22], and the ori-
gin of the extra-mitochondrial FXN is proposed [21, 22] although the functional
rational is controversial [22, 23]. More isoforms, e.g., in erythrocytes, cerebellum,
and heart, are revealed [21, 22, 24], and increasing the abundance of the heart-
specific isoform III significantly increased the mitochondrial aconitase activity, while
over-expression of the cerebellum-specific isoform II protected against oxidative
damage of Fe–S cluster-containing aconitase [22]. More specifically, monomeric
FXN81–210 donates Fe(2+) for Fe–S cluster assembly on ISCU, whereas oligomeric
FXN42–210 donates either Fe(2+) or Fe(3+) based on important functional differ-
ences observed between FXN81–210 and oligomeric FXN42–210 [25]. In the same
study, authors even proposed that isoform FXN81-210 takes a role in steady stage,
while FXN42-210 does under conditions of increased Fe-S cluster demand. More
work needs to be done to understand the mechanism of tissue-specific pathology in
FRDA.

7.2.2 FXN Function

FXN is mainly localized within the eukaryotic mitochondria and is ubiquitously

expressed in mammals. The structure of FXN is unique and conserved between
species: a small globular acidic protein composed of a long N-terminal alpha helix
and a short C-terminal alpha helix that both interact with a central beta-sheet structure
[26, 27].
To understand the role and pathophysiological implication of iron in the disease,
it is therefore essential to understand the function of FXN and how its deficit can lead
to cellular iron dysregulation. Initially, iron got extensive attention because of the
impaired activities of iron–sulfur cluster-containing enzymes including mitochon-
drial aconitase, respiratory complex I, II, and III in affected cells derived from FRDA
patients [28]. The consequence is the dramatic reduction in ATP production. Hence,
128 K. Li

mitochondrial dysfunction was suspected of contributing to the pathophysiology of

FRDA.
Though multiple functions of FXN have been proposed, a commonly accepted
hypothesis is that FXN is primarily involved in Fe–S cluster biogenesis. Because
Fe–S cluster is a very important prosthetic group fulfilling functions in electron
transport, enzyme catalysis, homeostatic regulation, and sulfur activation, FXN
deficiency-induced Fe–S cluster deficits reduce the enzymatic activities of Fe–S
proteins in all eukaryotes [29]. In order to investigate the exact function of FXN,
interactants were revealed with different methods. However, data from mammals,
yeast, and bacteria were quite controversial as to the direct FXN protein partner in
Fe–S biogenesis [30–33]. The only interaction partners of FXN have been exten-
sively and convincingly identified to be NFS1(+ISC11) and ISCU, which complex
is considered to be the core components of Fe–S cluster biosynthesis machinery
(reviewed in [29]).

7.2.2.1 Iron Chaperone and Iron Storage

Studies suggested that FXN might be the iron donor or iron chaperone for the assem-
bly of the Fe–S cluster [25, 34–36]. It was proposed in early studies that FXN could
act as a metal chaperone because the structure indicated the iron-binding sites of
FXN [37] and the purified protein could coordinate iron [38]. There is also uncer-
tainty on the number of metal ions that are bound per FXN monomer, whereas 2
ferrous iron or 6–7 ferric iron ions per FXN monomer were suggested using a group
of exposed acidic residues [37, 38]. These amino acids are different from the canon-
ical iron-binding motifs, which normally are cysteine and/or histidine. The rational
for the iron-binding activity of FXN was proposed by the interaction between FXN
and ISCU [30] and between FXN and ferrochelatase (FECH) [39]. The former com-
plex facilitates the transfer of iron from holo-FXN to nucleation sites for [2Fe–2S]
cluster formation on ISU [38], while the latter complex mediates iron insertion into
protoporphyrin IX to make heme [40]. The destination of iron delivery by FXN
was determined by the cellular physiological activities, i.e., mainly to FECH for
heme biosynthesis in erythrocytes by down-regulation of FXN and to both ISCU and
FECH in normal cells to fulfill the two major iron-dependent pathways [40]. How-
ever, a recent study demonstrated that no changes in heme synthesis was observed in
human FRDA erythroid progenitor cells compared with healthy control cells, which
was supported by that FRDA patients do not have abnormal hematological symp-
toms. As for the distinct high binding affinities of holo-FXN to the target protein
FECH, the binding between might be a negative mechanism for removal of FXN
during erythropoiesis. In non-erythrocytes, it is relevant that holo-FXN delivers iron
to ISCU, particularly in cells with highly expressed FXN, for instance in FRDA-
affected tissues.
More recently, Isaya and colleagues found that iron binding to in vitro purified
FXN promoted its oligomerization to form a complex of high molecular weight
(850–1100 kDa) [42, 43]. These oligomeric forms resemble ferritin structure con-
7 Iron Pathophysiology in Friedreich’s Ataxia 129

taining thousands of iron ions for iron storage [42, 44]. Therefore, FXN is proposed
to act as ferritin for mitochondrial iron storage. Indeed, the ferroxidase activity of
FXN was revealed in yeast [45, 46], further supporting the iron-binding activity.
However, another study revealed that human mitochondrial ferritin improves respi-
ratory function in yeast mutants deficient in iron–sulfur cluster biogenesis, but is
not a functional homologue of yeast FXN [47]. Concerning the iron-binding activity
and iron-induced oligomerization, more detailed investigation has elucidated that a
[FXN42–210]24 :[NFS1]24 :[ISD11]24 :[ISCU]24 complex is consistent with the mea-
sured 1:1:1:1 stoichiometry of its four components [35]. This structure of the complex
does not only recapitulate the known mechanism for sulfur donation from NFS1 to
ISCU but also provides a path for iron donation from FXN42–210 to ISCU. How-
ever, isoforms FXN42–210 and FXN81–210 have strikingly different biochemical
properties (reviewed in [48]). The former one has not yet accepted abundant in heart,
cerebellum, and dividing lymphoblasts, and the later one only forms monomeric
complex in vivo. Overall, it was suggested that the iron binding of the mature form
is served for delivery of iron to biochemical pathways, while FXN42–210 could be
able to oligomerize and store iron as ferritin.

7.2.2.2 Regulator of Cysteine Desulfurase in Fe–S Cluster Biogenesis

The role of FXN in orchestrating Fe–S cluster formation is widely accepted. Iron is
considered first to be imported into the mitochondria by mitoferrins (MFRN1 and
MFRN2) [49]. MFRN1 is inducibly expressed during erythropoiesis, and MFRN2 is
ubiquitously expressed in all tissues, probably, for basal mitochondrial iron uptake.
The iron is then majorly used for Fe–S cluster and heme biogenesis within mito-
chondria though how the iron was delivered into the process is in debate. De novo
Fe–S cluster biogenesis involves assembly of inorganic sulfur and iron on a scaffold
protein ISCU, with which a complex consists of NFS1-ISD11, the cysteine desul-
furase providing the sulfur, and FXN. A few studies support that FXN regulates the
NFS1 activity and the entry of iron within the complex via stabilizing the complex
[50–52], which is in consistence with the observation that the binding of FXN with
NFS1 is not iron dependent [23]. These results would not support FXN functions
as an iron donor for Fe–S cluster biosynthesis, rather than as an allosteric regulator
for enhancing NFS1 function to form persulfide on NFS1 and to transfer sulfur from
NFS1 to ISCU as an initial step in Fe–S cluster biosynthesis [52]. Still, how FXN
stabilizes the quaternary complex of core components and controls iron entry to the
complex and how iron gets to the entry and is transferred to scaffold protein ISCU
are mysterious.

7.2.2.3 Other Putative Functions

Though the functions of FXN have been proposed many, for instance, involvements
against oxidative stress and heme biogenesis, they may be the secondary effects of
130 K. Li

Fe–S cluster deficiency. The lack of FXN prevents the assembly of Fe–S clusters with
consecutive mitochondrial iron overload [53]. The toxic radicals through the Fenton
reaction are generated at an excessively high level in FXN-deficient mitochondria.
Meanwhile, due to the lack of Fe–S clusters as important cofactors in complex I, II,
and III of electron transport chain, the respiratory chain is defective, which makes
reactive oxygen species (ROS) produced more severely. These ROS can oxidize
ferrous iron and disrupt the cellular redox homeostasis. The consequence will be the
damage of macromolecules including DNA, proteins, and lipids, which may be the
pathological mechanism of FRDA. Interestingly, not all mitochondria-rich tissues
are affected in FRDA patients, but clearly cerebellum, spinal cord, and heart.
Because ferrochelatase, the last enzyme for heme biogenesis, needs Fe–S cluster to
gain the enzymatic activity for insertion of iron into protoporphyrin IX to form heme,
the lack of Fe–S cluster induced by FXN deficiency would affect heme biogenesis.
Therefore, FRDA patients would develop hematological disorder. FXN was found to
interact with ferrochelatase, which hints that FXN delivers iron to ferrochelatase to
produce heme in cells, particularly in erythroblasts. However, FRDA patients do not
present typical hematological disorder [41], which suggests that FXN may also play
a role in determining the recipient of this prosthetic group after assembly, favorably
being transferred to mitochondrial respiratory complexes.

7.3 Working Models for FRDA Disease

The functions of FXN have been proposed involving biogenesis of iron–sulfur clus-
ters, iron chaperoning, iron storage, and control of iron-mediated oxidative tissue
damage (reviewed in [54]). As demonstrated in knockout animals, complete absence
of FXN leads to early embryonic lethality [55], indicating that at least some FXN
function is essential for survival. Despite the difficulty in generating perfect FRDA
models, a multitude of complementary models have been generated enabling signif-
icant advances in understanding the function of FXN in model organisms as yeast,
fruit fly, nematode, and mouse.
In yeast, the Yfh1 protein (encoded by yeast FXN homolog Yfh1p) is confirmed
to be involved in iron homeostasis and mitochondrial function [56]. The deletion of
the Yfh1 gene makes mitochondrial iron accumulation (up to 10 times more than the
wild strain) makes the cells highly sensitive to oxidative stress, and also makes the
strain unable to grow on a non-fermentable source of carbon [53, 56], suggesting that
it could not carry out oxidative phosphorylation. In drosophila, FXN homolog (dfh)
was identified, a much smaller genomic region than the human gene [57]. Its genomic
composition just includes a single intron dividing the coding region into two exons.
Moderate systemic reduction of dfh expression with RNA interference (RNAi) to
levels of 30% of normal dfh-mRNA represents the situation in patients of FRDA, in
which flies develop with normal embryonic stage, but shortened mean and maximum
life span of 60 and 32%, respectively [58]. Reduction of FXN drastically compro-
mises aconitase activity and increases the levels of reactive oxygen species (ROS)
7 Iron Pathophysiology in Friedreich’s Ataxia 131

through inactivation of Fe–S enzymes of the respiratory chain [58]. Interestingly,

the ectopic expression of enzymes that scavenge H2 O2 suppresses the deleterious
phenotypes associated with FXN deficiency [59]. In contrast, genetic augmentation
with enzymes that scavenge superoxide is ineffective, suggesting that H2 O2 is an
important pathogenic substrate in FRDA with promising therapeutic implications
(reviewed in [60]).
The genome of the nematode Caenorhabditis elegans is unusual among eukary-
otes due to the operon organization, in which the C. elegans FXN gene, frh-1, is
encoded in the operon CEOP2232 with complex structure and regulation [61]. Some
work was carried out with C. elegans as a model for FRDA study. A transient knock-
down by RNAi showed a consistent pleiotropic phenotype that includes poor growth,
short lifespan, and increased sensitivity to oxidative stress [62]. Flavin adenine din-
ucleotide may rescue the phenotype of Frh-1 deficiency, very likely by enhancing
the respiratory capacity [63]. Interestingly, the extended lifespan has been observed
when frh-1 was knocking down by RNAi [64–66]. The difference may result from
the efficiency of knockdown, i.e., the varied protein levels of frh-1 or the complexity
of frh-1-containing operon.
Generation of a representative mouse model of FRDA is considered very impor-
tant for the understanding of disease pathology and the testing of potential thera-
peutic strategies. Initially, a homozygous knockout of the murine FXN gene leads
to early embryonic lethality without evidence of iron accumulation, indicating that
FXN plays an important role during early embryonic development [55]. Later, a
striated muscle Fxn-deficient line and a neuron/cardiac muscle FXN-deficient line
were generated [67] to confirm the function of FXN involving in Fe–S cluster bio-
genesis. Though the ablation of Fxn in adult mice led to progressive neurological
symptoms, the clinical symptoms could not well resemble FRDA. Since GAA repeat
expansion within the first intron of the human FXN gene is the genetic basis of the
disease, a mouse model harboring GAA repeats was created. The semizygous mice
(knockin-knockout, KIKO) expressing 25–36% of wild-type FXN levels carry a
(GAA)230 repeat expansion in the first intron of the mouse Fxn gene [68]. Unex-
pectedly, the mice did not present phenotypes of FRDA notwithstanding meiotically
and mitotically stable GAA repeats [68]. Probably, the protein levels of Fxn are still
above the threshold of the needs. In an attempt to generate a more relevant mouse
model, human FXN YAC transgenic mice containing GAA repeat expansion derived
from FRDA patient were created [69, 70]. These mice closely represent the first
GAA repeat expansion-based FRDA models, which exhibit progressive FRDA-like
pathology due to deficit of the important roles of FXN in mitochondrial function
and iron metabolism. More recently, an inducible and reversible mouse model of
Fxn deficiency was developed [71], which provides a broad use in therapeutic devel-
opment and in refining our understanding of the relative contribution of reversible
cellular dysfunction at different stages in disease.
132 K. Li

7.4 FRDA and Iron

Iron relation was first evidenced for myocardial iron deposits in FRDA hearts [72].
Much later, iron involvement was recognized by the finding of deficiencies of aconi-
tase and succinate dehydrogenase [28], both of which are mitochondrial iron–sulfur
cluster-containing enzymes.
FXN deficiency leads to mitochondrial iron overload, defective energy supply,
and generation of reactive oxygen species [67] (also see reviews [34, 73]). To date,
no data provide a clear answer on the nature of the iron that is accumulated in affected
mitochondria, but few interesting hints are available. The deposited mitochondrial
iron was identified to be a biomineral aggregate distinctly different from mammalian
ferritin [74] due to low expression of mitochondrial ferritin and high expression of
mitochondrial importer mitoferrin 2 (MFRN2) [75]. What drives high expression of
mitoferrin and how the excess toxic iron is trapped in the mitochondrial matrix are
still unknown. It seems that mitochondrial iron overload is commonly taken place
when Fe–S cluster biogenesis or transfer is disturbed. A similar increase of MFRN2
mRNA has been revealed in skeletal muscle biopsies from ISCU myopathy patients
[76]. These results suggest the existence of a Fe–S cluster-dependent regulation of
mitochondrial iron homeostasis. Does it mean that a particular transcription factor,
likely, regulated by a Fe–S-dependent peptide or protein, modulates mitochondrial
iron acquisition and retention [77]?

7.4.1 Iron in Nervous System

The neurological phenotypes of FRDA reflect lesions in the central nervous sys-
tem and peripheral nervous system. The major lesions are located in the dorsal root
ganglia of spinal cord and the dentate nucleus of the cerebellum [78]. The sen-
sory peripheral nerves are often affected. Optic atrophy or hearing loss is relatively
uncommon though it may be severe enough to cause deafness in approximately 1%
of patients [79, 80]. Despite normal pure tone audiometry, patients with FRDA may
have problems with speech perception [81]. Because dentate nucleus (DN) and dorsal
root ganglions (DRGs) of individuals with FRDA are severely affected tissues, they
have been extensively studied to investigate iron dysregulation by Dr. Koeppen AH
[74]. Dentate nucleus is an iron-rich cerebellar structure that shows signs of neurode-
generation in patients with FRDA [78, 82, 83]. The much smaller size of the dentate
nucleus in FRDA than in normal specimen is particularly apparent on a macro-stain
for iron [84]. It lacks the distinct gray matter ribbon of the normal nucleus, and the
intensity of the iron staining is lower than normal [82]. Further studies revealed that
ferritin detected by immunohistochemistry is most abundant in oligodendroglia of the
DN hilus and hypertrophic microglia in the atrophic DN gray matter of FRDA [85].
So far, the presence of mitochondrial iron accumulation in FRDA-affected neurons
is unclear although it is common in heart of model organisms and FRDA patients.
7 Iron Pathophysiology in Friedreich’s Ataxia 133

Despite a report of difference in the MRI signals that suggests an overall increase
of iron in the dentate nucleus of individuals with FRDA [86, 87], no difference in
iron concentrations was demonstrated using autopsies [82]. However, modification
of the expression of iron-related proteins such as transferrin receptor1, ferritins,
and ferroportin was observed, thereby suggesting a change in iron metabolism [82],
supporting that iron was relocating, likely, from dying neurons to microglia of dentate
nucleus.
Similarly, DRGs from individuals with FRDA do not show overall iron concentra-
tions above normal [88]. However, the expression of ferritin, the iron-storage protein,
increases in satellite cells surrounding affected DRG neurons and decreases in DRG
neurons [78, 88]. Meanwhile, not only the overall size reduction of subcapsular nerve
cells in the DRG, but also the more proliferated glial cells surrounding the degener-
ated neurons are observed in DRG, in consistence with the much thicker satellite layer
around neurons in patients than in normal subjects, whereas sural nerves in FRDA
showed no convincing change in ferritin and ferroportin, militating against local iron
dysmetabolism. Surprisingly, mitochondrial iron deposits have never been reported
in neurons from FRDA individuals [89] and were not observed in an inducible con-
ditional mouse model reproducing the neuronal phenotypes (PrPmice) [90]. The
results stand out in contrast to the previously reported changes in dorsal spinal roots
of patients with FRDA. Therefore, we are not sure about any indication on the pri-
mary involvement of iron dysregulation in the development of dorsal root ganglion
in FRDA patients.

7.4.2 Iron in Heart

FRDA is an autosomal recessive disease with a complex neurological phenotype, but

the most common cause of death is heart failure. Most FRDA patients succumb to
cardiomyopathy, which progressively induces heart failure during the course of their
disease. Heart is the first place to be revealed that iron accumulation occurs in FRDA
patient tissues [72, 91]. It is relatively easy to find that the sections of the autopsy
specimens display iron-positive granules in cardiac muscle fibers and all examined
subjects presented Fe-positive inclusions though the degree of fiber hypertrophy
and fibrosis varied [91]. More evidences showed that iron metabolism dysregulation
occurred in heart autopsies of individuals with FRDA and the iron deposit is within
mitochondria of cardiomyocytes [92]. However, the total iron obtained by assay of
bulk digests of normal and FRDA left ventricular wall, right ventricular wall, and
ventricular septum did not reveal a net increase [93] though a trend toward higher Fe
concentrations was evident [91]. The mitochondrial iron accumulation and deposits
were observed in the conditional mouse model reproducing the cardiac phenotype
(MCK mouse) [67], which was thought to reflect progression.
Inflammation is an important factor in the pathogenesis of FRDA cardiomyopathy
but may be more evident in advanced stages of the disease [91]. In comparison
with the normal section of heart, the inflammatory nodules with macrophages in
134 K. Li

pathological areas contained granular iron and iron-laden phagocytes invade and
replace sarcoplasm and myofibrils of single fibers. The further molecular evidence
was that CD68-positive inflammatory cells express cytosolic ferritin and hepcidin,
an iron-regulatory hormone [91]. The retention of iron in macrophages is a likely
contributor to heart failure in FRDA due to the highly presented hepcidin to keep
the iron from export by ferroportin. The unevenly distributed iron in heart suggested
that iron accumulation progressed from a few small granules to coarse aggregates in
phagocytized cardiomyocytes or infiltrated macrophages to result in elevated levels
of iron in highly localized, randomly distributed regions of the heart.
Altogether, these observations led to the early assumption of a pathophysiological
implication of iron-dependent pathways in FRDA. However, it is not clear why the
restricted accumulation of cardiac iron in FRDA should be much more damaging
than global iron overload and what drives the heterogeneous distribution of iron in
heart with variance from systemic iron overload.

7.4.3 Iron in Pancreas

Cardiomyopathy or neurological signs are the first clinical manifestation, whereas

the late onset of diabetes mellitus is often observed in the course of the illness, but
not present in all patients. Early occurrence of diabetes and rapid progression of
ataxia in FRDA patient were also observed [94]. The incidence rates vary between
8 and 32% indicating that FXN deficiency is a risk factor of diabetes. In a recent
study of Friedreich ataxia, a much higher percentage (49%) of the participants had
impaired fasting glucose and/or impaired glucose tolerance without a preexisting
clinical diagnosis of diabetes [95]. Interestingly, in non-FRDA subjects, the level
of FXN expression is also much lower in T2DM islets [96]. In the same study,
a positive correlation was apparent between response to glucose stimulation and
response to FXN gene expression, suggesting that FXN levels of human pancreatic
islets contribute to the prevention from T2DM. And a shared mechanism of apoptosis
between β-cells and neurons in FRDA is revealed [97] that FXN deficiency in β-
cells induces mitochondrial oxidative stress and activates the intrinsic pathway of
apoptosis. Puzzlingly, no mitochondrial iron accumulation has been reported in β-
cells of pancreatic islets in FRDA patients.
Why FXN deficiency mainly compromises neurons, cardiomyocytes, and pancre-
atic β-cells is elusive. Although these cells have a distinct embryonic origin, they need
mitochondria to fulfill many functional activities. In addition, three types of cells in
those tissues are very long lived. What very fundamental features are common in the
long-lived cells may tell us more the pathogenesis of FRDA.
7 Iron Pathophysiology in Friedreich’s Ataxia 135

7.5 FRDA Treatment by Manipulation of Iron Status

At present, there is no any approved cure treatment for this progressive condition.
However, the efforts for a potential pharmacological treatment have been made con-
siderably in the past two decades with many potential therapeutic agents proposed
to delay disease progression (reviewed in Ref. [98]). An optimal therapy in FRDA
would consist of providing sufficient intracellular FXN to restore the physiological
functions of cells, including the gene therapy [99, 100] and drugs increasing FXN
expression [101]. Interestingly, two very recent studies have reported that bone mar-
row transplantation stimulates neural repair and ameliorates deficits in FRDA mice
[102, 103]. Here, we only take attention on iron-related therapies in mouse model
and human.
It is often observed that iron accumulation in mitochondria occurs in case of
impairment of Fe–S cluster biogenesis (reviewed in [29]). Iron accumulation was
first observed in the heart of FRDA patients [72] as a secondary effect of FXN
deficiency [55, 67]. The excess iron may play destructive effects in several cellular
functions due to its unique physiochemical properties. Interestingly, it seems still
controversial in the central neuronal system [82, 87]. One possibility could be the
ratio of Fe2+ /Fe3+ shifted as observed in Parkinson disease (PD) patients [104]. In this
mentioned study, iron ratio was reported to be shifted from 1:2 in healthy subjects to
2:1 in PD patients in substantia nigra pars compacta regions of the brain. The total
iron content measured with some common methods may not distinguish the Fe2+ and
Fe3+ . The increase in the concentration of highly reactive iron species, Fe2+ , results in
excess production of ROS via Fenton chemistry. Nevertheless, iron chelation therapy
was proposed and assessed by Cabantchik group [86]. In that study, they found that
a membrane-permeant and orally administered chelator, deferiprone (DFP), reduced
neuropathy and ataxic gait only in the youngest FRDA patients by shuttling chelated
iron from cells to transferrin to reduce brain iron accumulation without systemic
anemia. The results indicate that DFP is able to cross the blood–brain barrier. It
was commonly observed in a few clinical trials that DFP administration at low dose
was associated with a significant reduction in cardiac hypertrophy, or an abnormal
enlargement, or thickening, of the heart muscle (reviewed in [105]). The effects of
long-term treatment at low doses are suggested to be worthy to be evaluated later
on since most of the patients die of cardiac failure. However, the results were some-
how puzzling as different doses showed opposite effects, if any (reviewed in [106]).
Moreover, our previous results demonstrated that iron deficiency downregulated FXN
expression [107] and reduced aconitase activity even with low concentration of DFP
(Li, unpublished data) in cells derived from healthy controls and FRDA patients.
Therefore, the rationale is further questioned behind the use of chelation therapy in
FRDA. Larger studies with hard endpoints are still missing.
Things might get a turn for a hope. A combined therapy of a low dose of DFP with
idebenone is relatively safe and might improve neurological function and heart hyper-
trophy in a 6-month randomized, double-blind placebo-controlled study [108]. Some
novel bifunctional or multifunctional agents have been synthesized with the ability
136 K. Li

of iron chelation and monoamine oxidase inhibitory (MAOI) activities for ironing
out iron from the brain and preventing iron-induced Fenton reaction from hydroxyl
radical toxicity [109]. Though these pharmacophores as iron chelators are being
examined for treatment in other neurodegenerative diseases with regional increase
of iron levels (reviewed in [110, 111]), such as Parkinson and Alzheimer diseases,
the similar strategies are worth to be tried in the treatment of FRDA. Besides iron
chelation, the radical scavenging ability, the mitochondrial activity enhancing, and
upregulation of FXN expression may also be under consideration to be combined.
Recently, we used a mitochondria-targeted agent SS-31, a tetrapeptide with the abil-
ity to enrich in mitochondria more than 1000-folds than in other organelles within
cells by binding cardiolipin [112]. This binding is thought to maintain the mitochon-
drial structure and integrity to improve the mitochondrial function. Interestingly, not
only as observed to reduce the oxidative stress, but also increase of FXN expression
at translational levels was revealed in a model of cells derived from FRDA patients
[101]. When we used the GAA-containing mouse model [69], the FRDA pheno-
types were reversed and the similar mechanism was revealed to that observed in cell
model of FRDA (in preparation for publication). Therefore, drugs designed based
on iron chelation, even on more accurate characteristics for iron shuttling between
organelles, are not enough. Due to the important role of FXN in mitochondrial func-
tion, development of therapeutic strategies should end at targeting more pathways in
combination with a few drugs or simply end at elevating FXN protein levels. Gene
therapy and transplantation of stem cells are also recommended.

7.6 Challenges Associated with Iron Chelators

Iron involvement in the pathophysiology of FRDA does not seem to be a simple

event of iron toxicity. The consequence of neurodegeneration is solidly connected
with mitochondrial dysfunction, rather than with iron accumulation in the disease.
Particularly, the iron accumulation only occurs in some tissues, more specifically
in mitochondria of the affected cells in these tissues. Though DFP may shuttle iron
between subcellular compartments or transfer iron from iron-overloaded cells to
extracellular apotransferrin and pre-erythroid cells for heme synthesis [106], DFP
still is a systemic iron chelator, with which iron is complexed and excreted in the
urine within 5–6 h of administration (https://pubchem.ncbi.nlm.nih.gov). It is spec-
ulated that the iron stores in the body would decrease when DFP was administrated
though the anemia was not clearly observed in a 6-months trial [86]. We observed
that DFP treatment reduced the mitochondrial aconitase activity even in a low dose in
both wild-type and mutant cells derived from FRDA patients (unpublished). On the
other hand, excess mitochondrial iron uptake and sequestration cause cytosolic iron
depletion, which can further diminish FXN expression by decreasing FXN transcrip-
tion [107]. Thus, the decrease in FXN levels caused by the trinucleotide repeat may
be worsened by DFP administration. As cells accumulate mitochondrial iron and
deplete cytosolic iron stores, we postulate that it may form a negative feedback loop
7 Iron Pathophysiology in Friedreich’s Ataxia 137

to contribute to progression of disease in long-lived cells, particularly neurons and

cardiomyocytes [107]. On the contrary, a seemingly paradox method was suggested
that addition of an iron supplementation regime that would replenish cytosolic iron
might be of value in conjunction with iron chelators, such as DFP that would reduce
mitochondrial iron stores [107], or with antioxidants that would mitigate the effects
of mitochondrial iron overload [113]. These therapeutic approaches could potentially
increase FXN expression or at least maintain FXN levels, in affected cells in FRDA
patients, which management might improve the quality of life and slow the disease
progression.

7.7 Summary

The Friedreich’s Ataxia Research Alliance (FARA) provides a comprehensive out-

line of current therapeutic agents with a treatment pipeline (http://www.curefa.
org/pipeline). No study has successfully achieved well-accepted endpoint thus far
because the exact function of FXN is still undermining. Though it is well accepted
that the primary consequence of FXN deficiency is mitochondrial dysfunction, any
drugs that can promote mitochondrial function do not widely target on all affected
steps. The combination of a few mitochondrial function-improving drugs might be
an option to be considered. Meanwhile, the defect of Fe–S biogenesis compromises
a large range of enzymes within and extra-mitochondria; therefore, drugs targeting
extra-mitochondrial process may be also a wise choice.

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Chapter 8
The Role of Iron in Amyotrophic Lateral
Sclerosis

Xian-Le Bu, Yang Xiang and Yansu Guo

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative

disorder characterized by the degeneration of motor neurons in the motor cortex,
brainstem, and spinal cord. The etiology and pathogenesis of this devastating disease
remain largely unknown. Increasing evidence suggests that iron accumulation is
involved in the onset and progression of ALS. In this review, we discuss the regulation
of iron homoeostasis in the brain, the misregulation of iron homeostasis in ALS, and
its possible roles in the mechanism of the disease. Finally, we summarize the recent
progress and problems with respect to iron chelator therapies on ALS, aiming to
propose a new therapeutic strategy to ameliorate the progression of the disease.

Keywords Amyotrophic lateral sclerosis · Pathogenesis · Iron · Iron chelator

8.1 Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive and devastating neurodegener-

ative disorder characterized with the degeneration of upper and lower motor neurons
in the spinal anterior horn and motor cortex and loss of axons in the lateral columns
of the spinal cord [1]. ALS is a clinically heterogeneous disease as patients present a
large variability in age, onset site, initial symptoms, and speed of disease progression.
Almost 90% of ALS patients are sporadic form and the rest are familial form caused
by genetic mutations. However, the clinical manifestations and the pathological hall-
marks are similar, and no effective treatment is available now. The pathogenesis of
this fatal neurodegenerative disease remains largely unknown. Numerous evidences

X.-L. Bu
Department of Neurology and Centre for Clinical Neuroscience, Daping Hospital, Third Military
Medical University, Chongqing, China
Y. Xiang
Department of Neurology, Chengdu Military General Hospital, Chengdu, China
Y. Guo (B)
Beijing Geriatric Healthcare Center, Xuanwu Hospital, Capital Medical University, Beijing, China
e-mail: [email protected]
© Springer Nature Singapore Pte Ltd. 2019 145
Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_8
146 X.-L. Bu et al.

suggest that redox metals, mitochondrial dysfunction, oxidative stress, and neuroin-
flammation are involved in the loss of motor neurons [2–4].
Iron has been proved to play a crucial role in oxygen transportation, cell prolifera-
tion and differentiation, DNA synthesis, myelin production, synthesis of neurotrans-
mitters, and the generation of oxidative stress [5]. Many studies indicate that iron
overload may be involved in ALS pathogenesis. In this review, we summarize the
current knowledge of iron metabolism disturbances and its pathogenic role in ALS,
and discuss the recent progress and problems with respect to iron chelator therapy
on ALS, aiming to propose a new therapeutic strategy to ameliorate the progression
of the disease.

8.2 Iron Homoeostasis in the Brain

Transferrin receptor 1 (TFR1) is responsible for the endocytosis of the iron uptake,
and it is highly expressed on the luminal side of endothelial cells [6]. Blood iron enters
the endothelial cells of the blood–brain barrier via TFR1-mediated endocytosis [7–9].
Iron could also be released into the extracellular environment and then be taken up
by neurons, microglia, and astrocytes [5, 10].
Neurons are thought to uptake iron via the transferrin receptors and divalent metal
transporter 1 (DMT 1). Meanwhile, neurons express ferroportin, which is involved
in the efflux of iron [11]. It has been shown that inflammatory stimuli could increase
DMT1 expression while decrease the expression of ferroportin [12], causing the iron
accumulation in neurons. Astrocyte, the important component of the blood-brain
barrier, plays a key role in regulating brain iron absorption [13]. Although astrocytes
do not express TFR, it is able to take up iron via the DMT1 that expressed in the
perivascular end-foot processes of astrocytes [14]. The glycosylphosphatidylinositol-
anchored form of ceruloplasmin expressed by astrocytes plays a role in oxidizing
ferrous iron, and could help iron efflux from cells [15]. It shows that lack of cerulo-
plasmin can lead to iron accumulation in the brain and neurodegeneration [16]. Iron
is required for axon myelination in oligodendrocytes. It has been proved that oligo-
dendrocytes can take up interstitial ferritin via the ferritin receptor. On the other hand,
oligodendrocyte is able to uptake iron from blood vessels [5]. Inflammatory stim-
uli increases iron accumulation in microglia by increasing the expression of DMT1
protein [17, 18]. It showed that iron uptake was increased and iron exportation was
down-regulated when the microglia is activated [19]. Moreover, inflammatory stim-
uli could increase hepcidin concentrations in astrocytes and microglia, and decrease
ferroportin level in astrocytes, microglia, and neurons [12].
Neuroinflammation is a chronic activation of the immune response in the central
nervous system and related to neuronal dysfunction in neurodegenerative diseases.
Microglia could be switched from their normal quiescent state to an activated state by
the systemic inflammation with aging [20]. Age-dependent enhanced neuroinflam-
matory processes in the brain refer to increased microglia and astrocyte numbers
and proinflammatory cytokines secreted by activated glial cells with aging [20]. Iron
8 The Role of Iron in Amyotrophic Lateral Sclerosis 147

levels in the substantia nigra, putamen, globus pallidus, caudate nucleus, and cortices
increase with age [5]. Additionally, the blood–brain barrier permeability increases
in the process of aging. Therefore, brain iron load may increase during aging.

8.3 Misregulation of Iron Homeostasis in Amyotrophic

Lateral Sclerosis

Numerous studies have examined the iron levels in ALS patients. A prior study
reported in 1985 showed that the iron concentrations of erythrocytes were the same
between ALS and controls [21]. Goodall et al. declared that the serum ferritin levels
were significantly increased in ALS patients, while no significant differences in the
concentrations of iron, transferrin, iron saturation, or total iron binding capacity were
found [22]. It also reported that ALS patients had significantly higher plasma levels
of ferritin but lower concentrations of transferrin [23, 24], and the patients with a
higher ferritin level had a shorter survival time [25]. A recent study firstly showed
that the serum iron, ferritin, and transferrin saturation coefficient were significantly
elevated in ALS patients, and the iron status was associated with body weight loss
[26]. And serum ferritin may be a candidate biomarker for ALS aggravation [27]. Free
iron level was also increased in the cerebrospinal fluid of ALS patients compared
to controls [28, 29]. And twofold increased level of inappropriate iron ligands in
cerebrospinal fluid (CSF) are found in patients with ALS, which may increase iron
redox activity and reactive oxygen species (ROS) production [30].
Autopsy study demonstrated that iron load in gray matter from the frontal cortex
of ALS was increased significantly than that of controls [31]. The concentration of
iron was also increased in the spinal cord of ALS patients [32–34]. Disruption in
the expression of brain iron transporters such as lactotransferrin receptor, melan-
otransferrin, and ceruloplasmin is related to iron accumulation in ALS [35]. Iron
accumulation could also be observed in the motor cortex using magnetic resonance
imaging (MRI), which appears to be a very promising biomarker for ALS [36, 37].
MRI also found showed increased iron deposits in the globus pallidus of frontotem-
poral dementia with ALS [38]. 7T MRI showed that ALS patients had motor cortex
hypointensity, and the postmortem studies showed that the increased iron accumu-
lation in microglial cells may be responsible for the location of the signal changes
on MRI [39]. Recent researches confirmed that iron level was significantly higher in
the motor cortex of ALS patients by susceptibility-weighted imaging (SWI), which
may be useful biomarkers for the diagnosis of clinically suspected ALS [40, 41].
148 X.-L. Bu et al.

8.4 The Link of Iron with Amyotrophic Lateral Sclerosis

Iron is a redox ion and can aggravate oxidative stress by generating ROS, which
may lead to neuronal cell damage, aging, and apoptosis. It demonstrated that the
increased iron load can form redox active complexes which participates in the Fen-
ton reaction and produces the toxic hydroxyl radical [42–44]. Meanwhile, ROS can
induce the release of iron from the iron storage proteins such as mitochondrial iron—
sulfur cluster proteins [45]. Increased oxidative stress occurred in ALS could also
be promoted by the genetic abnormalities (e.g., superoxide dismutase mutations)
[46]. Iron accumulation also increases the enzymatic activity of TNF-α converting
enzyme, leading to secretion of TNF-α and promoting neuroinflammation [47]. A
number of studies have demonstrated that mutations of genes encoding the proteins
involved in iron regulation are related to neuronal cell death also in ALS [48, 49]. The
mutation of HFE gene, which encoding protein interacts with the transferrin receptor
and regulates transferrin-mediated iron uptake, altered brain iron profiles, increased
oxidative stress, and accelerated disease process in mice [50, 51]. The mutation of
SLC11A2 gene that encoding DMT1 is associated with shorter disease duration in
ALS cases [52]. On the other hand, activation of genes involved in iron homeostasis
can induce ferritin overexpression and thus limit toxic hydroxyl radical and neuronal
loss [53]. Thus, iron misregulation promotes oxidative stress, which disrupts cellular
iron balance and causes progressive loss of motor neurons and consequent oxidative
stress, thus giving rise to a vicious circle.

8.5 Iron Chelator Therapy on Amyotrophic Lateral

Sclerosis

In view of the key role of iron in the pathogenesis of ALS, researchers explore the
efficacy and therapeutic potential of brain-permeable iron-chelating drugs for ALS in
animal models. An earlier study found that intraperitoneal injection of iron porphyrin
significantly decreased neuronal death and improved motor function in G93A mutant
SOD1 transgenic mouse model of ALS [54]. VAR10303, the brain-permeable iron-
chelating radical scavenging drug, exerted several beneficial effects in SOD1G93A
transgenic ALS mice, including alleviating the iron accumulation and motoneuron
loss in the spinal cord, improving motor performance and prolonging survival time
[55]. Iron-chelating drugs M30 and HLA20 showed neuroprotective effects against
oxidative stress and can extend the survival time of ALS transgenic mice [56]. Iron
chelators VK-28 also significantly alleviated the increased levels of iron and oxygen
free radicals and suppressed glial activation in the spinal cords of the ALS mice
[57]. What’s more, it was able to decrease TDP-43 protein aggregation and the
proapoptotic molecule Bax and to increase the expression of antiapoptotic protein
Bcl-2 [57]. A recent pilot clinical trial showed that deferiprone could remarkably
decrease the iron levels in the cervical spinal cord, medulla oblongata, and motor
8 The Role of Iron in Amyotrophic Lateral Sclerosis 149

cortex, the levels of oxidative stress markers and neurofilament light chains in the
CSF, and attenuate the disability progression and weight loss in patients with ALS
[58]. Therefore, brain-permeable iron-chelating drugs hold a promise as therapeutic
agents for ALS by targeting multiple key pathways of the disease pathogenesis. And
the effectiveness of iron chelators in ALS therapy supports the role of iron in the
ALS pathogenesis.

8.6 Conclusion

In summary, abundant evidence shows that iron accumulation in motor cortex and
spinal cord is a hallmark of ALS, and increased iron load, in particular, in regions of
central nervous system causes neuronal cell damage. However, it is uncertain whether
iron accumulation is a primary cause of ALS or consequential pathological change.
And why iron accumulation in ALS is selective for some brain areas is also unclear.
To further clarify the pathophysiological role of iron in ALS pathogenesis, future
studies should focus on the chemical state of iron in healthy control and ALS patients,
and its specifically pathogenic role in ALS different from other neurodegenerative
disorders.
More randomized, double-blind, placebo-controlled, multicenter trials are needed
to evaluate the effect of iron chelators on patients with ALS.

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Chapter 9
Iron Pathophysiology
in Neurodegeneration with Brain Iron
Accumulation

Sonia Levi, Anna Cozzi and Paolo Santambrogio

Abstract Neurodegeneration with brain iron accumulation (NBIA) is a group of

seriously devastating and life-threatening rare monogenic diseases characterized by
focal iron accumulation in the brain. The main symptoms of NBIA comprise pro-
gressive movement disorder, often including painful dystonia, parkinsonism, mental
disability, and early death. Currently, a single established therapy is not available
to reverse the progression of these debilitating disorders. The complexity of NBIA
emerged from the identification of various causative genes, and up to 15 genes have
been identified to date. Although the NBIA genes are involved in different cellular
biochemical pathways, they show the common characteristic of generating severe
iron accumulation in the basal ganglia of the patients’ brains. Thus, the molecular
events that lead to brain iron overload and their important roles in the pathophysiol-
ogy of the diseases are not easy to identify and are poorly understood. This review
summarizes the current knowledge on NBIA disorders, with a particular focus on
the data describing the role of iron in the pathogenic mechanisms.

Keywords Iron · Neurodegenerative disorders · NBIA

9.1 Introduction

Neurodegeneration with brain iron accumulation (NBIA) comprises a wide spectrum

of clinically and genetically heterogeneous diseases (Table 9.1). The heterogeneity is
due to the variability in the onset of clinical symptoms that are recapitulated in dysto-
nia, pyramidal and extrapyramidal signs, cognitive decline, behavioral abnormalities,
and retinal and axonal neuropathy [47]. Nevertheless, these diseases are classified
as a unique group of disorders because they are characterized by iron overload in

S. Levi (B)
Division of Neuroscience, San Raffaele Scientific Institute, Vita-Salute San Raffaele University,
20132 Milan, Italy
e-mail: [email protected]
A. Cozzi · P. Santambrogio
Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy

© Springer Nature Singapore Pte Ltd. 2019 153

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_9
Table 9.1 Neurodegeneration with brain iron accumulation disorders
154

Gene Function Inheritance Human disorder Brain iron % in NBIA

FLT1; L-Ferritin (ferritin Iron storage AD Neuroferritinopathy, Dense ferritin-Fe spheroid Rare
light chain) hereditary ferritinopathy, inclusions throughout
NBIA type III neurons, oligodendrocytes,
and microglia in the
forebrain and cerebellum
Cp; Ceruloplasmin Cu-dependent-ferroxidase AR Aceruloplasminemia Iron accumulation in the Rare
basal ganglia
PANK2; Pantothenate Coenzyme A synthesis and AR PKAN, NBIA type I, Iron accumulation in the 35–50
kinase-2 fatty acid metabolism Hallervorden–Spatz basal ganglia (eye of the
syndrome tiger)
COASY CoA synthase AR CoPAN, COASY Iron deposition in the globus Rare
protein-associated pallidus and substantia nigra
neurodegeneration
PLA2G6; iPLA2; Arachidonic acid release AR PLAN, NBIA type II, Iron deposition in the globus 20
INAD1, Karak syndrome pallidus and substantia nigra
(in <50% of patients)
C19orf12 Unknown, lipid metabolism? AR MPAN, mitochondrial Iron deposition in the globus 6–10
membrane-associated pallidus and substantia nigra
neurodegeneration
FA2H Fatty acid hydroxylase AR FAHN, fatty acid Iron deposition in the globus Rare
hydroxylase-associated pallidus and substantia nigra
neurodegeneration in some cases
WDR45 Autophagy XLD BPAN, β-propeller Predominant iron deposition 1–2
protein-associated in the globus pallidus
neurodegeneration compared with the substantia
nigra
(continued)
S. Levi et al.
Table 9.1 (continued)
Gene Function Inheritance Human disorder Brain iron % in NBIA
SCP2 Peroxisomal enzyme AR Leukoencephalopathy with Iron deposition in the Rare
dystonia and motor thalamus
neuropathy
CRAT Carnitine acetyl transferase, AR CRAT-associated Iron accumulation in the Rare
β-oxidation neurodegeneration globus pallidus and
substantia nigra
AP4M1 Vesicle formation AR AP4M1-associated Iron in globus pallidus in a Rare
neurodegeneration single family
REPS1 Endocytosis and vesicle AR RESP1-associated Iron accumulation in the Rare
transport neurodegeneration globus pallidus and
peduncles.
ATP13A2 Lysosomal cation pump, AR Kufor–Rakeb syndrome Few patients show iron Rare
autophagosome formation deposition in the striatum
DCAF17 (C2orf37) Nucleolar protein AR Woodhouse–Sakati Iron deposition in the globus Rare
syndrome pallidus and substantia nigra
is only detected in some
patients
9 Iron Pathophysiology in Neurodegeneration with Brain Iron …

GTPBP2 Unknown, mRNA/ribosome AR GTPBP2-associated Iron overload in the globus Rare

stability? neurodegeneration pallidus and substantia nigra
155
156 S. Levi et al.

specific brain areas, mainly in the basal ganglia, even if this feature is not present in
all patients [71]. The brain iron burden has been visualized in vivo using MRI [30],
and in several cases, it was also confirmed by a histopathological examinations in
postmortem brain samples [64]. Indeed, MRI is the main method used to analyze
areas of iron accumulation in the brain, and this feature, combined with the variations
in the structures of certain areas, is the basis of distinguishing between types of NBIA
[79]. Thus, the radiological evidence of iron accumulation together with the clinical
symptoms is the preliminary diagnostic tool that suggests to clinicians to undertake
a screening to identify the specific causative gene [99]. Currently, 15 genes have
been associated with different subtypes of NBIA, the main characteristics of which
are described in Table 9.1. Among these diseases, only two, neuroferritinopathy and
aceruloplasminemia, are linked to mutations in genes that are directly involved in iron
metabolism, FTL1 and CP, respectively. All the other forms (PKAN, CoPAN, PLAN,
MPAN, FAHN, BPAN, leukoencephalopathy with dystonia and motor neuropa-
thy, Kufor–Rakeb syndrome, Woodhouse–Sakati syndrome, and GTPBP2-, CRAT-,
REPS1-, and AP4M1-associated neurodegeneration) are characterized by altered
iron metabolism and its accumulation in the brain via an as-yet-unidentified mech-
anism, possibly implicating other pathways. These disorders are indeed caused by
mutations in genes linked to lipid metabolism (PLAN, FAHN and leukoencephalopa-
thy with dystonia and motor neuropathy), mitochondrial activities (PKAN, CoPAN,
MPAN and CRAT-associated neurodegeneration), autophagy (BPAN, Kufor–Rakeb
syndrome and REPS1- and AP4M1-associated neurodegeneration) and some still
unknown functions (Woodhouse–Sakati syndrome, GTPBP2-associated neurode-
generation), all of which are apparently unrelated to iron metabolism (Table 9.1).
Interestingly, numerous genes associated with inherited NBIA encode proteins
localized in the mitochondria (PANK2, COPAN, PLA2G6, C19orf12, ATP13A2,
and CRAT). Thus, these pathologies represent an important opportunity to study and
understand the molecular mechanisms involved in brain iron management and the
connections between mitochondria function and neurodegeneration. In this review,
we focus on the reported data regarding iron alterations, with the aim of highlighting
the knowledge of the involvement of this metal in the pathogenesis of NBIA disorders.

9.2 Fe-Related NBIA

Aceruloplasminemia, which is associated with recessive mutations in CP, and Neu-

roferritinopathy, which is linked to mutations in FTL1, are the two NBIA subtypes
caused by proteins that are important in maintaining iron homeostasis and therefore
have been extensively investigated in this context.
9 Iron Pathophysiology in Neurodegeneration with Brain Iron … 157

9.2.1 Aceruloplasminemia

Aceruloplasminemia is the first disease in which the alteration of an iron protein

has been associated with neurodegeneration (OMIM #604290). CP is located on
chromosome 3q 21–24 and encodes ceruloplasmin (Cp), a protein with ferroxidase
function. Aceruloplasminemia is an autosomal recessive inherited disease that was
originally described in a Japanese female in 1987 [82]. Indeed, it particularly affects
the Japanese population, with a prevalence of approximately one per 2,000,000. A
genetic analysis of patients affected by aceruloplasminemia revealed approximately
50 distinct causative mutations identified in more than 60 families worldwide [60,
81].
The symptoms include neurological signs such as blepharospasm, grimacing,
facial and neck dystonia, tremors, chorea, and ataxia, with the first appearance in
adulthood (fourth or fifth decade of life). Cognitive dysfunction is also observed in
a large portion of affected subjects. Generally, the neurological symptoms are pre-
ceded by diabetes mellitus, retinal degeneration, and microcytic anemia [59]. The
other hematological parameters displaying alterations include undetectable serum
ceruloplasmin levels, high serum ferritin levels, and low levels of sideremia [85].
The striatum, thalamus, and dentate nucleus are the regions of the brain affected by
iron deposition, as revealed by abnormally low intensities on T1- and T2-weighted
MRIs [80]. Neuropathology indicates a severe destruction of the basal ganglia and
dentate nucleus, with considerable iron deposition in neuronal and glial cells [38, 56,
114]. Iron was localized to terminal astrocytic processes, and, accordingly, enlarged
or deformed astrocytes and spheroid-like globular structures are characteristic neu-
ropathological findings in patients with aceruloplasminemia [38]. Iron overload was
also detected in the cerebral cortex [56], retina and cerebellum, as well as in the
pancreas and myocardium [56].
Cp is a glycoprotein present in the β2-globulin fraction of serum. It is a multicopper
ferroxidase, containing 95% of the copper in the plasma. In the CNS, Cp is expressed
as a glycosylphosphatidylinositol (GPI)-linked form in astrocytes [89]. Its function
is to facilitate the export of iron mediated by ferroportin, oxidizing Fe2+ to Fe3+ and
facilitating Fe3+ binding to the transferrin present in the extracellular environment.
This action is essential in astrocytes, for which ceruloplasmin is the only existing
ferroxidase [55]. CP mutations cause structural modifications in the protein that
induces the retention of Cp in the ER, miss-incorporation of copper into Cp or
impaired ferroxidase activity [27, 48, 62, 63]. In all these cases, iron export from the
cell is impaired, leading to cellular iron overload.
The molecular pathogenesis of aceruloplasminemia was examined by analyzing
mammalian cell cultures expressing CP mutants [27, 48, 62, 63] and by characterizing
several different murine models [45, 89, 106, 112]. The overall data collected from
these models indicated that when the function of Cp is altered, the iron entering
in the CNS as ferrous iron is not oxidized. Cells exposed to excess ferrous iron
internalize it in large quantities through non-regulated internalization pathways of
non-transferrin-bound iron [14]. The excess iron import combined with the inability
158 S. Levi et al.

of ferroportin to export iron caused by the absence of Cp leads to the accumulation

of the metal in astrocytes, as observed in patients. Iron may remain in astrocytes
and does not reach the neurons, which then die due to the iron deficiency and from
exposure to toxins released by dying astrocytes. Nevertheless, some mutated forms
of CP also accumulate in aggregates and lead to the death of astrocytes through an
iron-independent pathway [61].
Other cells in the CNS, including oligodendrocytes, do not depend on the ferro-
oxidative function of Cp, but require the function of hephaestin; this finding explains
the specificity of astrocytes and neuronal death. Only the contemporary ablation
of the CP and hephaestin in mice effectively produces symptoms consistent with
patients with aceruloplasminemia [44, 103]. These animals exhibit iron accumula-
tion in oligodendrocytes present in both the gray and white matter [103], macular
degeneration, iron overload, and increased oxidative stress in the retina [42]. In
this animal model, the administration of the oral iron chelator deferiprone protected
against the increase in oxidative stress, retinal degeneration, and lipofuscin accumu-
lation and promoted a reduction in ataxia and an increase in lifespan [43]. Despite the
good results obtained from mice models, the administer iron chelation therapies to
patients partially controls systemic iron deposition but was ineffective against neu-
rodegeneration [56, 96]. However, iron-chelating therapy appeared to be effective in
reducing the hepatic and pancreatic iron overload [35]. Another therapeutic approach
is ceruloplasmin replacement therapy. The parental administration of human ceru-
loplasmin to Cp-/- mice restored the protein levels and ferroxidase activity in the
brain in a recent study [115]. Interestingly, in ceruloplasmin-treated mice, reduced
brain iron deposition was associated with the amelioration of impairments in motor
coordination, indicating that enzyme replacement therapy may be a promising strat-
egy [115]. This finding further supports the overall data collected, indicating that
oxidative stress driven by heavy metal accumulation represents the primary cellular
cytotoxic process that determines the neuronal damage in affected brain regions.

9.2.2 Neuroferritinopathy

Neuroferritinopathy (NF, OMIM#606159) is an autosomal dominant inherited dis-

ease linked to mutations in FTL1. The gene product is L-ferritin subunit that com-
plexes with the H-ferritin subunit to form the major iron storage protein. NF was first
described in 2001 in a large English family carrying an adenine insertion at position
460–461 (c.460dupA) of the FTL1 gene [22]. After this first identification, 10 differ-
ent mutations (Fig. 9.1) were described in families from different parts of the world
[22, 26, 66, 76, 83, 84, 86, 105, 108]. The peculiarity of these DNA alterations is the
restricted area of the gene in which they arise. In fact, 9 of these causative mutations
are duplications of one or more nucleotides in exon 4 of the gene. All these variants
cause a frameshift of different lengths in the sequence that modifies the C-terminal
sequence of the L-peptide (Fig. 9.1) [72]. Moreover, the missense Ala96Thr mutation
in intron 3 that modifies the middle of the FTL1 sequence is associated with NF [74].
9 Iron Pathophysiology in Neurodegeneration with Brain Iron … 159

Fig. 9.1 Schematic representation of L-ferritin sequence and alignment of NF variants. Human
L-ferritin sequence is matched with a schematic diagram of protein secondary structure elements
that include five α-helices segments (blue cylinders), conventionally named with letters from A to
E, connected by four loop elements and of two additional short amino acid sequences at the N and C
termini. The blue rectangle indicates the sequence interval involved in the so far detected frameshift
variants. Frameshift mutations are responsible for amino acidic changes at the C-terminus starting
from different sequence position depending on the duplication starting point. In the lower panel, the
alignment of the frameshift altered regions compared with the wild-type FtL sequence is shown.
Dashes correspond to unchanged position, while modified residues are defined

Other cases may not yet have been identified; however, NF is a very rare disorder
and thus the prevalence is not able to be calculated.
The c.460dupA mutation has been detected in the largest number of cases, 41 of
which have been carefully described [18]. The mean age of onset was approximately
40 years, and the majority of patients presented with focal onset chorea or focal dys-
tonia and reported a family history of movement disorders. The serum ferritin level
was generally low. MR imaging was abnormal in all cases. The disease progressed
relentlessly and became generalized in 5–10 years, leading to aphonia, dysphagia,
and motor disability, with cognitive dysfunction as a late feature. Characteristic facial
dystonia was common. Signs of parkinsonism, dementia, postural tremor, and cere-
bellar ataxia were also described in patients with other types of mutations [26, 86].
In general, the disorders appear to exhibit full penetrance at the age of 60 years,
with heterogeneous clinical features associated with early movement disorders fol-
lowed by minor cognitive impairments. The major and common feature is MRI signs,
with similar abnormalities observed in all affected patients. In a comparison of the
different NBIA disorders, NF was characterized by lesions in the globus pallidus,
putamen, and dentate nuclei that differed from the other NBIA subtypes. Interest-
ingly, two patients with NF showed the eye-of-the-tiger sign, which is considered
pathognomonic almost exclusively for PKAN [78].
Brain histopathology has been performed on two patients with a c.460dupA muta-
tion [22], one with a c.442dupC mutation [76, 91] and one with a c.497_498dup
160 S. Levi et al.

mutation [109]. Patients with c.460dupA mutations showed prominent abundant

inclusions in the globus pallidus that contained ferritin and iron. Cysts formed in the
basal ganglia centered on the site of the globus pallidus. Signs of neurodegeneration,
such as axonal swelling and immunoreactivity for neurofilament, ubiquitin, and tau
proteins, were observed throughout the white matter of the brain. Biochemical and
hematological parameters were in the normal range, with the exception of low serum
ferritin levels [22]. The patient with the c.442dupC mutation showed neurons and
glia with highly distinctive swollen to vacuolated nuclei containing iron and fer-
ritin [76]. Hyaline deposits were observed, which were positive for ferritin and iron.
Moreover, the iron concentration in the putamen was >40-fold higher than in the con-
trol. The brain showed signs of oxidative damage and mitochondrial abnormalities.
Further immunohistochemical staining of the specimen showed that the ferritin/iron
deposits also contained neuroglobin [91], p53, and activated caspase-3, suggesting
the activation of an apoptotic pathway. The patient with the c.497_498dup mutation
presented with mild cerebral and cerebellar atrophy and cavitation of the putamen
[109]. A major pathological alteration was the presence of intranuclear and intra-
cytoplasmic bodies containing ferritin and iron in astrocytes and oligodendroglia in
the gray and white matter. The ferritin bodies in the glia were more abundant in the
caudate nucleus, putamen, and globus pallidus, where nerve loss was more severe.
Similar bodies were observed in the fibroblasts of the papillary dermis, in the tubular
epithelium of the kidney and in the endothelial cells of muscle capillaries.
According to the results from biochemical studies, the ferritin shells carrying
the mutated subunits expressed from the c.460dupA and c.497_498dup alleles were
less stable than wild-type ferritin and displayed largely different solubility and a
lower capacity to incorporate iron [5, 6, 21]. Cellular models showed that the expres-
sion of these ferritin mutants not only increased the cellular free iron pool but also
induced oxidative damage and impaired proteasome activity [20, 21]. Transgenic
mice overexpressing the human c.497_498dup mutant showed movement disorders,
the presence of cytosolic and nuclear inclusions typical of the human disease, and
abnormalities in brain iron homeostasis [7, 110]. These data were further extended
by a study of another c.497_498dup transgenic model that showed oxidative alter-
ations in the brain [73]. Indeed, the examination of the postnatal hippocampal neurons
obtained from this latter animal model revealed a major susceptibility to chronic iron
overload and/or acute oxidative stress compared to wild-type neurons. Furthermore,
the brain ultrastructural analyses highlighted an accumulation of lipofuscin granules
associated with iron deposits that were particularly enriched in the cerebellum and
striatum during aging [73]. Thus, oxidative stress and lipofuscin formation might be
involved in the etiopathogenesis of human NF. The only data on human cells reported
to date concerned fibroblasts obtained from a patient carrying the c.497_498dupTC
mutation. These cells showed a consistent increase in the intracellular iron content,
altered iron management, the accumulation of ferritin, and markers of oxidative
stress, as detected both in patients and in the transgenic mice. ROS levels were
significantly increased in these cells compared to controls [8].
Similar to aceruloplasminemia, the use of iron chelator (desferrioxamine, DFO, or
deferiprone, DFP)-based therapies was tested both in animal models and in patients,
9 Iron Pathophysiology in Neurodegeneration with Brain Iron … 161

obtaining similar results. A DFP treatment exerted remarkable effects on systemic

iron homeostasis in mice with iron overload [36], while only marginal effects on iron
deposition in the brain were observed [17], without signs of amelioration of the CNS
pathology. In patients, iron chelator-based therapies cause substantial iron depletion
without clinical benefits [18, 66].
The pathogenic process of NF hypothesized from the overall data predicts that
ferritin/iron deposition triggers cellular oxidative damage and the subsequent cascade
of oxidative events that lead to proteasomal and lysosomal dysfunction. Long-term
impairments in the cellular recycling system permit the accumulation of lipofuscin
in the brain tissue. The lysosome may undergo rupture that results in two effects: (i)
direct cell damage by the hydrolytic enzymes and (ii) the release of lipofuscin in the
cytosol, where it may function as a seed for further iron precipitation [73].

9.3 NBIA Related to CoA Biosynthesis

Two forms of NBIA are due to mutations in genes involved in the first and last steps of
the same biosynthetic pathway that produces Coenzyme A (CoA). Mutations in these
two genes, PANK2 and COASY, cause the NBIA subtypes known as pantothenate
kinase-associated neurodegeneration (PKAN, OMIM#234200) and CoA synthase
protein-associated neurodegeneration (CoPAN, OMIM*609855), respectively.
PKAN approximately accounts for 50% of all NBIA cases and, depending on
the age of onset and the rate of progression, is classified into two forms: the clas-
sical and the atypical forms. CoPAN appears to be more rare, as it has only been
identified in five subjects to date [3, 31, 33]. These two disorders display strikingly
similar phenotypes, presenting with early-onset spastic–dystonic paraparesis with a
later appearance of parkinsonian features, cognitive impairment, obsessive–compul-
sive disorder, and brain iron accumulation [31]. PKAN usually manifests in early
childhood as gait disturbances and rapidly progresses to a severe movement deficit
with dystonia, dysarthria, and dysphagia [68]. The hallmark of this disease is the
eye-of-the-tiger signal in the globus pallidus (GP) on T2-weighted magnetic res-
onance imaging, which reflects the focal accumulation of iron in this area [119].
The substantia nigra (SN) is also affected in patients with CoPAN and to a lesser
extent in patients with PKAN. Iron accumulation correlates with neural damage and
mitochondrial lesions and is often associated with axonal expansions (spheroids) but
also appears as a granular form in perivascular sites [64].
Although iron accumulation is a hallmark of PKAN and CoPAN, its relation-
ship with CoA dysfunctional biosynthesis remains unclear. CoA is a key molecule
involved in more than 100 metabolic processes, among which CoA derivatives are
crucial substrates for ATP generation mediated by the tricarboxylic acid cycle, fatty
acid metabolism, cholesterol and ketone body biosynthesis, and histone and non-
histone protein acetylation [1]. The CoA biosynthetic pathway involves five universal
enzymatic steps that use pantothenate (vitamin B5), ATP, and cysteine. This pathway
is initiated by PANK2, which converts pantothenic acid into 4 -phosphopantothenic
162 S. Levi et al.

acid. The last two steps of CoA biosynthesis are mediated by COASY. Although
these processes are vital in any cell type, researchers have not yet explained why the
disease primarily affects the central nervous system.
Several animal models have been developed in an attempt to clarify the molecular
events that generate these disorders. Pank2 null mice showed reduced growth, male
infertility due to azoospermia [67], and impaired mitochondrial function [16], but did
not suffer from movement disorders or exhibit signs of neurodegeneration, implying
that the other Pank genes may compensate for the Pank2 deficiency in mice. Only by
modulating the fat content in the diet of these mice, it was possible to elicit neurode-
generation and cause a movement disorder associated with a general bioenergetic
failure [15]. The Drosophila Pank2 KO model (fumble) displays brain lesions and
defective neurological functions, resulting in severe motor impairment [113]. The
downregulation of pank2 in zebra fish induces defects in neuronal development and
in the formation of the vascular system [118].
A yeast model was used to study a human COASY pathogenic mutation. It showed
mitochondrial dysfunction, and both the iron and lipid contents were altered [12].
Downregulation of zebra fish coasy expression led to a generalized reduction in size,
poor definition of brain structures, and alteration of the vasculature [57]. Although
all of these animal models were informative tools for studying pathological mecha-
nisms, they share only a few neuropathological signs with milder severity than the
human disorder, limiting their impact for predicting novel therapies [71]. In fact,
while presenting some levels of neurodegeneration, these models lack any evidence
of brain iron mishandling, preventing researchers from obtaining any insights into
the causative link between CoA deficiency and brain iron deposition. Fibroblasts
from patients with PKAN have been instrumental in revealing some defects in mito-
chondrial activity and iron metabolism associated with the PANK2 deficiency [100];
however, their specific contributions to the pathological neurodegenerative processes
have not been identified in these cells.
Insights into the neurodegenerative process began to emerge in an investigation
of human neurons differentiated from induced pluripotent stem cells (iPSC) [87].
Compared with neurons from 3 healthy subjects, iPSC-derived glutamatergic neurons
from 3 patients with PKAN showed alterations in mitochondrial function and impair-
ments in mitochondrial iron-dependent biosynthesis that caused iron dyshomeostasis
and subsequently increased ROS production, leading to major membrane excitability
defects. In particular, the inability of the cells to maintain Fe/S biosynthesis simulates
a cellular iron-deficient phenotype that promotes cellular iron uptake and increases
the translation of TfR1 [87]. In addition, CoA supplementation prevented neuronal
death and ROS formation by restoring mitochondrial and neuronal functions, sug-
gesting that a CoA treatment is a possible therapeutic intervention [87]. A second
paper analyzed iPSC-derived cortical neuronal cells obtained from fibroblasts from
a patient with atypical PKAN [4]. Consistent with the milder phenotype, the Pank2
peptide was not abolished as described in the previous study but was present at lower
levels than in the control cells. In this case, CoA homeostasis and cellular iron han-
dling were normal; however, mitochondrial function was affected, with increased
generation of reactive oxygen species and lipid peroxidation in patient-derived neu-
9 Iron Pathophysiology in Neurodegeneration with Brain Iron … 163

rons [4]. Notably, an iron chelator treatment exacerbated the mitochondrial phenotype
in neuronal cells from both control subjects and patients. Thus, both of these inves-
tigations using human neurons concluded that mitochondrial impairment is an early
feature of the disease process and the subsequent ROS formation plays a main role in
the pathogenesis; however, the iron chelator treatment did not appear to be a viable
option. Indeed, the data on the efficacy of the DFP treatment collected from different
clinical trials are still under investigation. Two pilot studies confirmed the safety and
efficacy of the DFP treatment in reducing brain iron levels in patients with PKAN
[19, 119], but the results from the still ongoing clinical trial involving approximately
100 patients with PKAN are needed to define the clinical outcomes of iron chelator
treatments.

9.4 NBIA Caused by Defects in Proteins Related to Lipid

Metabolism

Since sequencing has become a method for diagnosing NBIA, many similar cases
in terms of symptomatology have been distinguished in different groups based on
the genes in which the mutations were identified. The main group is composed of
disorders associated with mutations in genes encoding proteins involved in lipid
homeostasis. These diseases typically begin in childhood with symptoms such as
developmental delay, regression or attention deficits associated with hyperactivity,
spastic paraparesis, dystonia, and visual pathway disorders. Atypical variants begin
in adolescence or adulthood, with a variable combination of dystonia, dysarthria,
parkinsonism, and psychiatric symptoms. Iron accumulation is not a universal feature
at the time when symptoms begin and is often documented only in the later stages,
suggesting that iron overload, which is typically present in the GP and sometimes in
the SN, is not the primary cause of the disease but contributes to neurodegeneration
[29].

9.4.1 Neurodegeneration Associated

with Phospholipase-A2G6 Defects (PLAN)

The first pathology to be distinguished from PKAN by sequencing was PLAN (or
INAD, infantile neuroaxonal dystrophy, OMIM#256600), which is caused by defects
in the PLA2G6 gene located on chromosome 22q13.1 that encodes a calcium-
independent phospholipase involved in cell membrane homeostasis [116]. Defects in
the protein may lead to altered lipid composition of the plasma, vesicular, and endo-
somal membranes, with subsequent alterations in membrane maintenance, causing
neuronal damage [75]. This feature typically occurs in the first year of life as neu-
roaxonal childhood dystrophy with psychomotor regression and axial hypotonia that
164 S. Levi et al.

may be accompanied by neuropathy with spheroidal bodies in the peripheral nerves,

which is detectable by biopsy. Brain MRI shows cerebellar atrophy in most patients,
while only 40–50% of patients develop iron deposits. The atypical form manifests
in the second or third decade of life as a form of dystonia-parkinsonism that is often
accompanied by psychosis and dementia.
Studies on the Pla2g6 -/- mouse, which were also confirmed in a Drosophila
model [58], suggested that one of the first pathological signs in the cells of the ner-
vous system was the disruption of the crests of the inner mitochondrial membrane,
followed by the destruction of the mitochondria, the release of cytochrome c, and
the formation of swollen and degenerated axons as the disease progresses [75]. Even
the presynaptic membranes were altered in this animal model, indicating that the
mitochondria require the remodeling activity of this phospholipase [10]. A more
recent study of the Pla2g6 -/- mouse model revealed that these mice showed age-
dependent iron accumulation in the SN, ST, and GP regions of the brain, similar to
findings in affected patients. This iron deposition is associated with increased lev-
els of iron-related proteins such as DMT1, TfR1, and IRPs, decreased FPN1 levels,
enhanced lipid peroxidation, and mitochondrial dysfunction [9]. These features are
observed before signs of neuronal loss, suggesting that iron-dependent ROS forma-
tion might be a primary cause of neurodegeneration [9]. Evidence of an impairment
in the recycling of transferrin and transferrin receptor from endosomes due to the
loss of PLA2G6 was also reported in vitro in HeLa cells [23], further highlight-
ing the connection between the iron imbalance and impaired dynamics of cellular
membranes.

9.4.2 Mitochondrial Membrane Protein-Associated

Neurodegeneration (MPAM)

MPAM accounts for approximately 30% of the NBIA cases. It is caused by a mutation
in C19orf12 (OMIM*614297) that encodes a mitochondrial protein with an unknown
function but is probably involved in fatty acid metabolism and in the degradation of
the branched amino acid chains [46, 88]. The main symptoms of MPAN are pro-
gressive spastic para/tetraparesis, generalized dystonia, motor axonal neuropathy,
parkinsonism, psychiatric symptoms, retinal abnormalities, and optic atrophy [46,
50, 70]. Patients with MPAN develop Lewy bodies containing α-synuclein in the
GP and midbrain, as well as perivascular iron deposits similar to those observed in
patients with PKAN [49]. The C19orf12-encoded peptide is a transmembrane glycine
zipper-containing protein with two isoforms originating from different start codons.
In the literature, few reports have characterized the protein. An analysis of HeLa sub-
cellular fractions indicated that the longer isoform was located in the mitochondria,
endoplasmic reticulum, and a small fraction in the MAM [107], suggesting a role
in the transport of phospholipids. Fibroblasts from one patient showed an alteration
of mitochondrial calcium homeostasis and increased H2 O2 -induced apoptosis com-
9 Iron Pathophysiology in Neurodegeneration with Brain Iron … 165

pared to controls [107]. The only available animal disease model is the knockdown
of the C19orf12 orthologues in Drosophila that showed signs of neurological defects
and the presence of vacuoles in the brain and optical lobe, but not iron accumulation
[53].

9.4.3 Fatty Acid Hydroxylase-Associated Neurodegeneration

(FAHN)

FAHN (OMIM#612319) is caused by mutations in the gene encoding FA2H (fatty

acid-2-hydroxylase). The disorder was initially identified in 2 consanguineous fam-
ilies, one Italian and one Albanian [65]. To date, 51 patients have been reported
to carry missense mutations [77], some of whom were previously diagnosed with
leukodystrophy with spastic paraparesis and dystonia and a form of paraplegia spas-
ticity [101]. The pathology is initially characterized by spasticity, ataxia, dystonia,
optic atrophy, and oculomotor abnormalities, followed by cognitive disorders and
epilepsy. Magnetic resonance studies in patients have shown iron overload at the
level of the GP, SN, subcortical, and periventricular regions [40], as well as pro-
found changes in the white matter distribution, cerebellar atrophy, and a thinning
of the corpus callosum. In the mouse model, an accumulation of microglial cells
was observed in areas exhibiting greater degeneration of white matter [90]. FA2H
is a membrane-bound protein involved in the synthesis of ceramide, an essential
component of myelin, and its deficit caused defects in myelination and a subsequent
impairment in axonal function [90].
To date, no specific analysis of iron metabolism has been reported using models
of this disorder. The evidence that the protein includes a cytochrome b5-like domain,
a heme group, and a catalytic di-iron cluster involved in the enzymatic activity of the
protein suggests a possible direct interaction with other iron-containing proteins. A
recent computational analysis of the effect of the causative missense mutations on the
3D protein structure indicated that these mutations interfere with the heme binding
affinity, affecting protein stability, or alter the structure of the catalytic hydroxylase
domain, disturbing enzymatic activity [77]. However, researchers have not deter-
mined how the impaired enzymatic activity leads to brain iron accumulation; we can
only state that the impairment in myelin synthesis is associated with alterations in
brain iron homeostasis.

9.4.4 Leukoencephalopathy with Dystonia and Motor

Neuropathy

Leukoencephalopathy with dystonia and motor neuropathy (LKDMN, OMIM#

613724) is an autosomal recessive disorder caused by SCP2 mutations that was
166 S. Levi et al.

only recently classified as an NBIA disorder. It is characterized by azoospermia,

tremor, slight cerebellar ataxia, and balance and gait impairments. MRI shows bilat-
eral hyperintense signals in the thalamus, butterfly-like lesions in the pons, and lesions
in the occipital region [34, 51]. The sterol carrier protein-2 (SCP2) gene encodes two
different proteins located in peroxisomes: sterol carrier protein X (SCPx) and sterol
carrier protein 2 (SCP2). These proteins possess the thiolase activity required to
degrade branched-chain fatty acids [111]. Patients carrying SCP2 mutations show
abnormal fatty acid acyl-CoA metabolism, suggesting a common pathogenic mech-
anism between this disorder and the NBIA related to CoA biosynthesis.

9.4.5 CRAT- and REPS1-Associated Neurodegeneration

Two other genes involved in lipid homeostasis have recently been identified as
causative for NBIA [28]. The genes are CRAT (OMIM*600184), which is involved
in the transfer of acyl groups from carnitine to CoA and in the transport of fatty acids
for beta-oxidation, and REPS1 (OMIM*614825), which is engaged in endocytosis
and vesicle transport. Interestingly, the authors also described a new perspective of
the NBIA pathogenic mechanism. They observed a common phenotype in fibrob-
lasts derived from patients with CRAT and REPS1 mutations, as well as fibroblasts
derived from patients with PANK2, PLA2G6, C19orf12, and FA2H mutations. All
these cells exhibited reduced TfR1 palmitoylation that affected the recycling of the
receptor [28]. This anomaly in cellular iron incorporation led to iron overload that
was decreased by treatment with the antimalarial drug artesunate. This drug promotes
receptor palmitoylation, restoring the normal TfR1 recycling [28]. This report was
the first time to experimentally confirm a common pathogenic mechanism for NBIA
disorders.

9.5 NBIA Related to Lysosome Regulation/Autophagosome

Formation

Although the role of lysosomes in iron metabolism is known [69], some lysosomal
disorders have only recently been included in the NBIA categories. These disorders
are caused by mutations in genes encoding lysosomal proteins and show a variable age
of onset and severity of the phenotype, depending on the degree of dysfunctionality
of the mutated proteins. An explanation for why only a portion of the affected patients
exhibit iron overload in the brain is unknown, and researchers have proposed that
iron is not the primary cause of the disease in this case [29].
9 Iron Pathophysiology in Neurodegeneration with Brain Iron … 167

9.5.1 Kufor–Rakeb Disease (KRD)

KRD (OMIM#606693) was originally described in a Jordanian family, but the

causative gene was identified only later in a large group of Chilean brothers [11].
Then, other cases and new mutations have been identified in several countries, includ-
ing Brazil, Pakistan, Afghanistan, Japan, and Italy [101]. The gene associated with
this pathology is the ATP13A2 gene [94] located on chromosome 1p36.13. It contains
29 exons and encodes a P-type ATPase. In the human brain, this protein is expressed
in the pyramidal neurons of the cerebral cortex and the dopaminergic neurons of the
SN [95]. KRD usually manifests as a juvenile form of parkinsonism (also classified
as PARK9) that is responsive to levodopa. Patients show pyramidal symptoms, cog-
nitive decline, and visual hallucinations; patients with the late-onset form may also
exhibit ataxia and axonal neuropathy [32]. The majority of patients do not present
with iron overload; yet, when it is present, it is mainly found in the striatum region.
The peptide encoded by ATP13A2 is located in lysosomes and late endosomes and
was proposed to remodel the endocytic pathway toward export [25]. The ablation of
this gene in mice led to lysosomal dysfunction, lipofuscin accumulation, α-synuclein
aggregation, and age-dependent sensorimotor deficits [102]. Recently, the ATP13A2
gene was identified as a novel HIF1α target and therefore regulated by the PHD2-
HIF1α signaling pathway. Indeed, ATP13A2 knockdown abrogated the maintenance
of cellular iron homeostasis elicited by PHD2 inhibition, which occurred under con-
ditions of mitochondrial stress both in vivo and in cultured dopaminergic cells [93].
The authors concluded that ATP13A2 is involved in maintaining lysosomal iron
stores.

9.5.2 β-Propeller Protein-Associated Neurodegeneration

(BPAM)

β-Propeller-associated neurodegeneration (BPAN) (OMIM#300894) is caused by

mutations in the WD repeat domain 45 (WDR45) gene located on the X chromosome.
To date, all affected individuals are the only ones in their family with the disease
and most are females. The causative mutations are de novo mutations and are lethal
to most male individuals [39]. Indeed, patients of both genders displayed similar
symptoms due to the presence of somatic mosaicism in male patients and germline
or somatic mutations in females, as well as skewing of X chromosome inactivation
[41]. Affected individuals exhibited a developmental delay during infancy while
developing progressive parkinsonism associated with dementia during adolescence
and adulthood [41]. Magnetic resonance studies have revealed iron accumulation
in the GP and SN. In particular, T1-weighted images revealed a hypo-intense zone
surrounded by a hyperintense area within the SN.
The WDR45 gene encodes a protein belonging to the WD40 scaffold-protein fam-
ily that is involved in various cellular processes, such as cell cycle progression, signal
168 S. Levi et al.

transduction, apoptosis, and regulation of gene expression. In particular, Atg18, the

yeast orthologue of the human WDR45 gene, is a crucial component of the complex
required for autophagosome formation [92]. Recent reports have highlighted the role
of iron in the pathogenesis of the BPAN disorder. Studies on fibroblasts from two
individuals with BPAN revealed that an upregulation of the ferrous iron transport
(-)IRE/DMT1 and downregulation of TfR1 compared with control subjects. This
alteration in the levels of iron transporter proteins occurs concomitantly with the
increase in intracellular ferrous iron levels under starvation conditions [52], suggest-
ing that the compromised autophagy in BPAN cells may determine iron overload [52].
A further study of WDR45 mutant fibroblasts and iPSC-derived midbrain neurons
confirmed and extended these observations. The authors confirmed that the loss of
WDR45 increased cellular iron levels and promoted oxidative stress, accompanied by
mitochondrial abnormalities, autophagy defects, and diminished lysosomal function
[104]. Therefore, the close relationship between the diminished lysosomal function
and the onset of iron accumulation has also been highlighted in these disorders.

9.5.3 AP4M1-Associated Neurodegeneration

A new candidate NBIA disorder was reported in a recent study that identified iron
deposition in the brains of 3 patients afflicted with an autosomal recessive form
of hereditary spastic paraplegia (HSP) (OMIM#602296) [98] that is characterized
by early-onset developmental delay, tetraparesis, motor function deterioration, and
intellectual disability. This very rare neurodevelopmental disorder is due to mutations
in AP4M1. The AP4M1 gene encodes the subunits of adaptor protein 4 (AP-4), a
heterotetrameric complex involved in intracellular protein trafficking [24]. No data
on iron involvement in the pathophysiology of the disorder have been reported;
however, the finding that fibroblasts from AP-4-deficient patients and neurons from
AP-4-deficient mice show impaired autophagy further emphasizes the stringent link
between alterations in autophagy and iron deposition.

9.6 NBIA Related to Genes with Unknown Functions

Two other disorders are classified as NBIA, although not all patients show signs of
brain iron deposition. These disorders are briefly described here because published
data concerning the role of iron in the pathological process do not exist.
9 Iron Pathophysiology in Neurodegeneration with Brain Iron … 169

9.6.1 Woodhouse–Sakati Syndrome

Woodhouse–Sakati syndrome (OMIM#241080) is a rare autosomal condition caused

by mutations in the DCAF17 gene (also known as C2orf37), which encodes a
nucleolar protein with an as-yet-unknown function. The disorder manifests as
hypogonadism, deafness, alopecia, and diabetes mellitus; subsequently, with the
progression of the disease, dysarthria, dystonia, chorea, and cognitive decline are
observed. Magnetic resonance studies revealed lesions in the white matter and iron
deposits in SN and GP in a portion of affected patients [2].

9.6.2 GTPBP2-Associated Neurodegeneration

A splice site mutation in GTPBP2 (OMIM*607434) was identified in 3 affected

siblings in a consanguineous family. The patients exhibited mental retardation, ataxia,
and dystonic features [54]. Iron accumulation in the brains of the patients was a
notable phenotypic trait. However, recently, biallelic inactivating variants of GTPBP2
were reported in three unrelated families with a neurological phenotype that differed
from the previously published family and did not display signs of iron accumulation
[13]. The GTPBP2 gene encodes a GTP-binding protein that is a member of the
GTPase superfamily that is capable of binding GTP or GDP. The precise function
of GTPBP2 is not yet defined, but it has been proposed to be a regulator of BMP
and Wnt signaling [37] and to be involved in the dysregulation of mRNA translation
[117].

9.7 Conclusions

Advances in genetic screens based on whole-exome sequencing and the recent

insights into the biochemical basis of NBIA have begun to delineate the role of
iron in the pathophysiology of these disorders. In fact, even if a genetic cause has
not been defined in all cases of NBIA, the recent identification of new genes has
assisted researchers in outlining some crucial interrelated cellular pathways that,
when impaired, induce iron accumulation in the CNS. The pathways encompass
mitochondrial functions, lipid metabolism, membrane remodeling, and autophagy.
Alterations in all these pathways result in the dysregulation of iron management.
Mitochondrial function is essential for the transformation of iron in its biological form
(i.e., iron–sulfur cluster and heme cofactors) and for the preservation of energy pro-
duction processes that depend on the availability of the same iron cofactors. Indeed,
the levels of these cofactors are also decreased in patients with a CoA deficiency,
further highlighting the relationship between iron availability, energy resources, and
lipid homeostasis. Lipid metabolism also plays a crucial role in myelin perturba-
170 S. Levi et al.

tion, where iron is required for its biosynthesis [97]. Membrane remodeling and
autophagy appear to be essential to avoid iron deposition in the brain. In particular,
the impaired ability of cells to disrupt the iron-containing macromolecules (i.e., fer-
ritin) and organelles (mitochondria) through the lysosomal pathway appears to be a
common phenotype in patients with various NBIAs.
Although additional in vitro and in vivo studies are needed to define whether
iron dysregulation has a primary or an auxiliary role in the pathophysiology of these
diseases, the clarification of the pathogenic mechanisms of NBIAs will add important
knowledge required for a better understanding of the relationship between iron and
the neurodegenerative process that is necessary to define the therapeutic options.

Acknowledgements The financial support from Telethon-Italia (Grant nos. GGP10099,

GGP11088 and GGP16234 to SL) and AISNAF (to SL) is gratefully acknowledged. The authors
are grateful to Dr. Ermanna Rovida for assisting with the figure preparation.

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Chapter 10
Diagnostics and Treatments
of Iron-Related CNS Diseases

Huan Xiong, Qing-zhang Tuo, Yu-jie Guo and Peng Lei

Abstract Iron has been proposed to be responsible for neuronal loss in several
diseases of the central nervous system, including Alzheimer’s disease (AD), Parkin-
son’s disease (PD), stroke, Friedreich’s ataxia (FRDA), multiple sclerosis (MS),
amyotrophic lateral sclerosis (ALS). In many diseases, abnormal accumulation of
brain iron in disease-affected area has been observed, without clear knowledge of the
contribution of iron overload to pathogenesis. Recent evidences implicate that key
proteins involved in the disease pathogenesis may also participate in cellular iron
metabolism, suggesting that the imbalance of brain iron homeostasis is associated
with the diseases. Considering the complicated regulation of iron homeostasis within
the brain, a thorough understanding of the molecular events leading to this phenotype
is still to be investigated. However, current understanding has already provided the
basis for the diagnosis and treatment of iron-related CNS diseases, which will be
reviewed here.

Keywords Iron · Alzheimer’s disease · Iron homeostasis · Parkinson’s disease ·

Stroke

10.1 Introduction

In the central nervous system (CNS), iron is involved in multiple crucial biological
processes including oxygen transportation, DNA synthesis, mitochondrial respira-
tion, myelin synthesis, and the synthesis and metabolism of neurotransmitters [1].
It plays an important role in electron transfer and is cofactors for varies enzymes
[2]. Iron homoeostasis is needed to maintain normal physiological brain function,

H. Xiong · Q. Tuo · P. Lei (B)

Department of Neurology and State Key Laboratory of Biotherapy, West China Hospital,
Sichuan University, Sichuan 610041, China
e-mail: [email protected]
Y. Guo · P. Lei
West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University,
Sichuan 610041, China

© Springer Nature Singapore Pte Ltd. 2019 179

Y.-Z. Chang (ed.), Brain Iron Metabolism and CNS Diseases,
Advances in Experimental Medicine and Biology 1173,
https://doi.org/10.1007/978-981-13-9589-5_10
180 H. Xiong et al.

whereas abnormal iron homoeostasis can induce cellular damage through reactive
oxygen species (ROS), notably the hydroxyl radical [3]. ROS can cause the oxidation
and modification of lipids, proteins, carbohydrates, and DNA [4, 5]. Peroxidation of
polyunsaturated fatty acids in membrane lipids by ROS can generate highly reac-
tive aldehydes, such as 4-hydroxynonenal, which irreversibly modify proteins by
carbonylation [6–8]. ROS can induce the release of iron from mitochondrial iron-
sulfur cluster proteins of the respiratory chain and other iron storage proteins, which
will lead to further ROS production via Fenton’s reaction [9]. Iron-related CNS dis-
eases that result from iron toxicity can lead to apoptosis [10] and ferroptosis [11],
an iron-specific form of non-apoptotic cell death [12].
In various iron-related CNS diseases, changes in iron homoeostasis result in
altered cellular iron distribution and accumulation [13–21]. Magnetic resonance
imaging (MRI) can often identify these changes, thus providing a potential diag-
nostic biomarker of iron-related CNS diseases. An important avenue to reduce iron
accumulation is the use of iron chelators that are able to cross the blood–brain barrier,
penetrate cells, and reduce excessive iron accumulation, thereby affording neuropro-
tection. These will provide new ideas for the diagnostics and treatment of iron-related
CNS diseases.

10.2 Potential Diagnostic Value of Iron for CNS Diseases

10.2.1 Alzheimer’s Disease (AD)

Making a diagnosis of AD on purely clinical grounds is challenging [22], not only

in the prodromal stage in which patients only have subtle cognitive symptoms but
also in the dementia phase. With the recognition that the pathological changes occur
years prior to symptoms, and the advent of biomarkers of β-amyloid (Aβ) and tau
pathology and MRI measures of atrophy, diagnostic criteria have evolved to allow for
the diagnosis to be made both earlier and with increased molecular specificity. The
most recent diagnostic criteria from both the National Institute of Aging (NIA) and
the International Working Group (IWG-2) now incorporate one or more preclinical
AD phases, where biomarker evidence of AD pathology exists in the absence of
symptoms [23–25].
The mainstay of the diagnosis of AD remains the clinical assessment, and in
particular the clinical interview with the patient and an informant, and a cognitive
and focused physical examination. Neuropsychology allows for the quantification of
both the pattern and severity of cognitive deficits against age-related norms.
Latest MRI showed that iron levels in the bilateral hippocampus (HP), parietal
cortex (PC), frontal white matter, putamen (PU), caudate nucleus (CN), and dentate
nucleus (DN) subregions of patients with AD were significantly higher than the
healthy controls. Moreover, these brain iron levels, especially those in the PC at the
early stages of AD, were positively correlated with the severity of patients’ cognitive
10 Diagnostics and Treatments of Iron-Related CNS Diseases 181

impairment [26]. These data indicated that iron in specific brain regions may be used
as a biomarker to evaluate the progression of AD. Australian researchers have applied
quantitative susceptibility mapping (QSM), a relatively new MRI method sensitive
to tissue iron, to assess the relationship between iron, Aβ load, and cognitive decline.
They found that QSM level is associated with Aβ deposition, which is consistent
with brain iron increasing during the natural history of AD [27]. This study showed
that higher levels of brain iron can also predict clinical deterioration.
Ferritin is the major iron storage protein of the body; by using cerebrospinal fluid
(CSF) levels of ferritin as an index, researchers explored whether brain iron status
impacts the longitudinal outcomes in the AD Neuroimaging Initiative (ADNI) cohort.
Results showed that CSF ferritin levels were negatively associated with cognitive
performance over 7 year’s period and predicted the conversion from mild cognitive
impairment (MCI) to AD [28]. These findings reveal that ferritin levels in the CSF
may predict AD outcomes.

10.2.2 Parkinson’s Disease (PD)

Parkinson’s disease is a common neurodegenerative disease that mainly affects the

motor system appears as tremor, rigidity, and bradykinesia, and especially involved
people over 60 years old [29]. The cause of PD is unknown, and there is no effective
means to cure PD. The primary pathology of PD is dopaminergic neuron loss in
substantia nigra (SN). The standard of diagnosis PD mainly included assessment
using Unified PD Rating Scale (UPDRS) motor score based on the motor symptoms,
but a single-photon emission computerized tomography (SPECT) scan for dopamine
transporter may also be helpful. In addition, iron may play an important role in PD.
An epidemic research proceeded by Powers et al. showed that dietary iron intake
exhibited a positive dose–response gradient with PD risk in men, through investigated
newly diagnosed Parkinson’s disease case (n = 420) and normal control (n = 560) in
Washington State with Willett Food Frequency Questionnaire (FFQ) [30]. Whereas
a Mendelian randomization research about 20000 PD patients and 80000 normal
controls had shown that increased of serum iron level would reduce the risk of PD
[31], while the CSF level of iron was not changed in PD patients in a research about
37 PD patients [32], the mechanism underlying remains unclear.
Transcranial sonography (TCS) and later T*2 -weighted MRI serve as general tech-
nologies clinically to detect iron in SN of PD, for the laden iron will increase
echogenicity in these technologies [33, 34], the positive predictive value is 85.7%
[35]. In a 3-year clinical study on PD patients and normal controls, the iron content
measured by T*2 -weighted MRI is increased in SN and caudal putamen in PD group
while not changed in control [35]. In addition, Zhang et al. found that iron concen-
tration in SN of PD patients was a positive correlation with the UPDRS of these
PD patients by using high-resolution susceptibility-weighted magnetic resolution
imaging [36].
182 H. Xiong et al.

Some studies in postmortem PD brains also get the similar conclusion that iron
is overloaded in SN in PD. For instance, detection the concentration of metal ion in
SN with Electrothermal Atomic Absorption Spectrometry (ETAAS), discovered that
the labile iron concentration but not copper was significantly higher in PD patients
than in healthy control [37]. Lei et al. also found that iron elevation in PD patients
and demonstrated that it may be associated with tau deficiency [15].
Diseases that affect iron status also have influences on PD. Mutation of gene
encoding ferritin light polypeptide would cause iron deposition and lead to adult-
onset basal ganglia disease such as parkinsonism symptom [38, 39]. Ceruloplasmin
(Cp) is a copper oxidase and plays a role in iron export from cell [40]. About 176
PD patients from UK Brain Bank with increased SN iron level were examined for
mutations of Cp gene and found that five missense variations about Cp were related to
PD [41]. Mutation of pantothenate kinase 2 (PANK2) will lead to iron accumulation
in basal ganglia [42], while two siblings with this PANK2 mutation come from
consanguineous parents have developed parkinsonism [43].

10.2.3 Stroke

Stroke is a leading cause of mortality and permanent disability [44]. In general,

the stroke is classicized as ischemic and hemorrhagic stroke. Ischemic stroke is the
most common type, accounting for about 80% of the total stroke events [45]. Clinical
studies indicated that there was iron-mediated neurotoxicity in patients with ischemic
stroke. The increased iron deposition, measured by MRI, was found in the basal
ganglia, thalami, and white matter in children with severe ischemic-anoxic insult
and subsequent resuscitation [17]. In addition, the serum transferrin, a main protein
regulating iron homeostasis, and ceruloplasmin were correlated with the ischemic
lesion volume and hydroperoxides, respectively [46]. High level of serum ferritin has
been found to be associated with poor outcome in patients with ischemic stroke [47].
In further research, plasma ferritin concentrations >275 ng/ml with 24 h from the
onset of ischemic stroke increased 33-fold the risk of subsequent stroke progression
[48]. These results indicated that the systemic iron level may be associated with the
clinical states of ischemic stroke.
In a study of hemorrhagic stroke, there was a significant correlation between
the serum ferritin level and the relative perihematoma edema volume at 3–4 days
after hemorrhagic stroke onset [49]. The serum ferritin level was upregulated after
hemorrhagic stroke, and the higher levels of serum ferritin, the poorer outcome in
patients [50, 51]. Another clinical study showed that the hematoma iron content,
measured by MRI, correlated with the relative perihematoma edema volume [52].
Therefore, iron-related signals and tests may help the diagnosis of stroke.
10 Diagnostics and Treatments of Iron-Related CNS Diseases 183

10.2.4 Friedreich’s Ataxia (FRDA)

Most of FRDA patients caused by trinucleotide amplification of GAA in frataxin

(FXN) gene [53]. The FXN protein, an iron chaperone in iron–sulfur cluster, is
related to iron accumulation in mitochondria [54]. Although a conclusive diagnosis
of FRDA can only be made via genetic testing, other helpful tests, such as the iron
concentrations and the expression of iron-related proteins, may be performed to be
a potential biomaker of FRDA. Clinical study showed the iron accumulation in the
dentate nucleus and the dorsal root ganglia of FRDA patients [18, 55, 56]. And
the expression of iron-related proteins changed in individuals with FRDA [57]. The
mitochondrial iron–sulfur-cluster-containing enzymes were found to be deficient by
biochemical studies of heart biopsies [58]. Compared with control neurons, FRDA
neurons exhibited higher labile iron pool, reactive oxygen species, and lower reduced
GSH levels, as well as enhanced sensitivity to oxidants [59]. In a yeast model of
FRDA, GSH peroxidase activity increased with the reduction of total GSH levels
[60].

10.2.5 Restless Legs Syndrome (RLS)

RLS is a chronic neurological disorder with unique clinical symptoms. The diag-
nosis of RLS used to be based on the subjective description of symptoms, lacking
of available biomarkers or objective tests [61]. Since RLS can be exacerbated by
systemic iron deficiency, all newly diagnosed patients as well as those with wors-
ening symptoms should have iron-level tests [61]. Clinical study showed that the
SN iron index, determined by MRI, significantly decreased in the early-onset RLS
patients. And there was a significant negative correlation between SN iron index and
the Johns Hopkins RLS severity scale [62]. In addition, iron deficiency was found
in red nucleus, pallidum, thalamus, and putamen of RLS patients [63, 64]. Further-
more, compared to healthy control, the CSF ferritin and transferrin levels from RLS
patients significantly decreased and increased, respectively [65].

10.2.6 Multiple Sclerosis (MS)

The diagnosis of MS needs the integration of clinical, imaging, and laboratory evi-
dences. Besides, the magnetic field correlations value, indicating iron deposition, was
increased in the putamen, thalamus, and globus pallidus in MS patients compared
with the healthy control [66]. And MS lesions had 47 μg of iron per gram of tissue
more than the surrounding tissue on average, quantified by susceptibility-weighted
imaging [67]. In chronic MS, iron levels reduced in normal-appearing white matter
and correlated with disease duration. The ferritin and frataxin levels were upregulated
184 H. Xiong et al.

and downregulated in initial lesions, respectively [68]. Previous studies indicated that
the level of iron deposition might be related to disease duration and severity [69, 70].

10.2.7 Amyotrophic Lateral Sclerosis (ALS)

ALS, also known as ‘Lou Gehrig’s disease,’ is a neurodegenerative disorder [71].

The elevation of iron levels has been found in the motor cortex, the spinal neurons,
and the CSF of patients with ALS, as well as in the spinal cord in mouse models of
ALS [19, 72–74]. The expression of several iron regulators changed in ALS, such
as mitochondrial ferritin, TfR1, DMT1, and FPN1 [75]. Moreover, the CSF ferric
reducing ability decreased [76] and serum ferritin levels increased in ALS patients
[77]. Interestingly, transferrin has been observed in the Bunina bodies which are the
typical pathological symptoms of ALS. Besides cystatin C, transferrin is the only
protein localized in the Bunina bodies [78]. Furthermore, mutation of HFE gene,
associated with the iron overload disease and hemochromatosis, increased in ALS
patients [79].

10.3 Treatment of Iron-Related CNS Diseases

10.3.1 Alzheimer’s Disease

Aging is the single highest risk factor for AD with an incidence of 50% in people over
the age of 85 [80]. Over more than thirty years of research, no disease-modifying
treatment has been approved by either the US Food and Drug Administration (FDA)
or the European Medicines Agency (EMA) for AD treatment.
Given the pathological iron accumulation in AD, iron removal from specific brain
region without causing a systematic iron deficiency should be taken into considera-
tion [81, 82]. McLachlan and colleagues [83] conducted a two-year, single-blinded
study to investigate whether the progression of dementia could be slowed by an
iron chelator, desferrioxamine (DFO), and showed a substantial reduction in the
rate of deterioration of daily living skills in 48 patients with Alzheimer’s disease
who were given DFO (125 mg intramuscularly twice daily, 5 days per week, for
24 months) when compared with Alzheimer’s disease given placebo. Despite the
promising results, no other clinical trials using DFO for AD have been investigated
so far. And yet, iron chelation using intranasal deferoxamine has been investigated
in mouse models of AD, was shown to reverse iron-induced memory deficits, and
inhibits amyloidogenic APP processing and Aβ aggregation [84–86].
5-Chloro-7-iodoquinolin-8-ol (clioquinol) is a moderate chelator of iron, copper,
and zinc, and for this reason, it was explored as a drug candidate for AD [87]. Indeed,
clioquinol has shown efficacy in patients with AD and models of diseases. Clioquinol
10 Diagnostics and Treatments of Iron-Related CNS Diseases 185

inhibits Aβ oligomer formation and protects against cell loss in an Aβ-injection

model [88–90]. Nine weeks of oral clioquinol treatment lowered plaque burden and
improved cognitive performance in AD mice [91, 92]. In a phase 2 clinical trial
of 32 patients, clioquinol prevented cognitive deterioration (Alzheimer’s Disease
Assessment Scale-Cognitive) and lowered plasma Aβ42 levels over 36 weeks [93].
While these data were promising, complications with the large-scale manufacturing
of the compound made further development of this drug unviable.
PBT2 (5,7-dichloro-2-[(dimethylamino)-methyl]-8-hydroxyquinoline), the sec-
ond generation of clioquinol derivative, has been shown to promote amyloid plaque
degradation and relocate excess released metals to other depleted neuronal com-
partments [94–96]. Given orally to two types of amyloid-bearing transgenic mouse
models of AD (APP/PS1 transgenic mice and Tg2576 transgenic mice), PBT2 out-
performed clioquinol by markedly decreasing soluble interstitial brain Aβ within
hours and improving cognitive performance to exceed that of normal littermate con-
trols within days [94]. In a phase 2a clinical trial of 78 patients with Alzheimer’s
disease, PBT2 was given at a dose of 250 mg/day or 50 mg/day, and patients receiving
highest dose showed a substantial reduction in CSF of Aβ concentration, and some
cognitive improvement (executive function) was noted [97, 98].
A randomized, multi-center, double-blinded, placebo-controlled Phase 2a trial
using deferiprone (DFP) for AD is currently ongoing in Melbourne (Deferiprone to
Delay Dementia, the 3D study), and the results are due in 2021.

10.3.2 Parkinson’s Disease

The primary treatment strategies for Parkinson’s disease include medications such as
carbidopa-levodopa, dopamine agonists, MAO-B inhibitors, as well as anticholiner-
gics, and deep brain stimulation surgery if necessary. Given the substantial evidences
linking iron accumulation with PD, a few treatments targeting iron were trialed.
The iron chelator deferiprone had been trialed for PD. A patient with symptoms
of dysarthria, orofacial dystonia, moderate parkinsonism, iron accumulation in the
substantia nigra, red nuclei, internal globi pallidi, and dentate nuclei detected by
T2*-weighted brain MRI had developed worsen during 5 years even was given med-
ications of levodopa, baclofen, oxitriptan, and buspirone. She was given 30 mg/kg
deferiprone per day for 32 months and found that her symptoms were improved 30%
after 6 months treatment, and the UPDRS decreased 30% after 1 year, while iron
accumulation in bilateral nuclei, substantia nigra were decreased, but not changed in
the red nuclei after 32 months [99]. PD is associated with iron deposition in SN and
iron deposition will increase oxidative damage. Grolez et al. carried out a pilot clini-
cal trial about treating PD patients with deferiprone or placebo for 6–12 months and
found that the iron level in SN and the UPDSR was significantly decreased; in addi-
tion, if the patients have gene mutation about iron metabolism, the effect of reducing
iron was stronger [100]. Devos et al. detected cytoprotection of deferiprone treated
cell models of PD or MPTP-inject mouse model of PD, and found a high level
186 H. Xiong et al.

cytoprotection of deferiprone. Whereafter they did a randomized clinical trial on

early-stage PD patients whom were treated with deferiprone for 12 months, revealed
that deferiprone definite lower the iron deposition and UPDRS, and the effect would
come earlier when the treat begin sooner [101].
Except deferiprone, the efficacy of other iron chelators or iron modulation ther-
apy for PD has been investigated in animal models of PD. Tau knockout mice which
develop iron deposition in SN and parkinsonism motor disorder would have reduced
iron content in the brain and reversed motor deficit after 5-month clioquinol treat-
ment [102]. In PD, Cp activity decrease may contribute to the iron accumulation, and
peripheral infusion of Cp weakens iron deposition in SN of PD mouse model [103].
VAR10303 (VAR) was a brain-permeable iron chelator attached with lipoperoxida-
tion inhibitory potency, and animal models of PD include 6-hydroxydopamine stereo-
tactically injected rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperi-
toneally injected mice were treated with VAR and revealed that VAR can increase
tyrosine-hydroxylase level and improve synaptic plasticity and some other neuro-
protective and neurorestorative effects [104]. Multifunctional iron chelators similar
to VAR, such as VK-28 and M30, have also been designed and tested in PD models
in vitro or in vivo [105–107].
Interestingly, Kaur et al. treated MPTP-inject mouse model of PD with transgenic
expression of ferritin, detected reduced oxidative stress of dopaminergic neurons in
SN, and found genetic and pharmacological means both efficient to protect dopamin-
ergic neurons [108].

10.3.3 Stroke

Injection of tissue plasminogen activator (tPA, also called alteplase) is the only
evidence-based treatment option for ischemic stroke [109]. However, limited by
the narrow therapeutic window (less than 4.5 h after the onset of ischemic stroke)
and other safety concerns such as potential bleeding in the brain, less than 5% of
patients have received this treatment [110]. Therefore, other effective therapies are
needed. Deferoxamine mesylate has exhibited neuroprotective effects by suppression
of iron-induced hydroxyl radical formation. In a clinical trial, the serum levels of per-
oxides had decreased and the levels of total radical-trapping antioxidant capacity had
increased after 3-day treatment with deferoxamine mesylate in stroke patients [111].
Tirilazad mesylate, a nonglucocorticoid, 21-aminosteroid, and iron-dependent per-
oxidation inhibitor, has been investigated. In a randomized, double-blinded, placebo-
controlled trial, patients with acute stroke were treated with the tirilazad mesylate
beginning at a median of 4.3 h after stroke. While the overall functional outcome
had not been improved measured by the Glasgow Outcome Scale and the Barthel
Index 3 months later [112], a higher dose showed 14% absolute reduction in mortal-
ity [113]. In a rat model of ischemic stroke, intranasally administered deferoxamine
improved the outcome [114], and inhibitors of ferroptosis protected from ischemic
injury in MCAO mouse model [115].
10 Diagnostics and Treatments of Iron-Related CNS Diseases 187

As to treat hemorrhagic stroke, five randomized, placebo-controlled trials were

performed, and the tirilazad had not shown effective on clinical outcome, but
decreased symptomatic vasospasm in patients with subarachnoid hemorrhage [116].

10.3.4 Friedreich’s Ataxia (FRDA)

Since excess mitochondrial iron may worse the symptom of FRDA, iron chelators
were considered to treat FRDA. Deferoxamine cannot cross the blood–brain barrier
[117] and therefore might induce extracellular iron deficiency before the reduc-
tion of mitochondrial iron [118]. Another orally iron chelator deferiprone can cross
the blood–brain barrier with low affinity to iron [117]. Clinical trials indicated that
deferiprone at low concentrations had beneficial effects, but worse the symptoms of
FRDA at high doses [55, 119]. In another clinical trial, deferiprone and idebenone,
latter of which was an analog of coenzyme Q10 , were combined to treat FRDA. After
11 months of therapy, iron deposits in dentate nucleus, determined by MRI, as well as
heart hypertrophy parameters, were improved significantly. However, posture scores
and gait got worse [120].

10.3.5 Restless Legs Syndrome (RLS)

Clinical studies suggested that in certain cases correcting the iron deficiency by
iron supplementation alone can relieve RLS without any further intervention [61].
In an open-label study, a single 1000 mg intravenous (IV) infusion of iron dextran
to treat RLS was evaluated. The results indicated that IV iron dextran significantly
improved the RLS symptom severity, duration, and total sleep time two weeks later.
In addition, brain iron concentration as determined by MRI was increased in the
SN and the prefrontal cortex [121]. In a randomized, parallel-group double-blinded
study, IV iron sucrose treatment significantly increased the CSF ferritin level and
reduced the RLS symptom severity, but did not change MRI iron index or periodic
leg movements of sleep [122].

10.3.6 Multiple Sclerosis (MS)

Although iron chelation therapy works in animal model of MS [123], clinical studies
have failed to show its efficacy. In one clinical study, desferrioxamine was given to
9 patients up to 8 courses over 2 years. During the trial, 1 patient was improved, 3
patients unchanged, and 5 patients got worse, although the drug was well tolerated
[124, 125]. A preliminary data indicated that the symptoms of 2–4 patients with
progressive MS could be improved by the combination of iron depletion and ery-
188 H. Xiong et al.

thropoietin [126]. Therefore, the effectiveness of iron chelators for MS needs further
investigation.

10.3.7 Amyotrophic Lateral Sclerosis (ALS)

Recently, iron chelation for ALS was assessed in a pilot clinical trial. The results
showed that the body mass index and the ALS Functional Rating Scale significantly
decreased for the first 3 months of deferiprone therapy. The iron levels in the cervical
spinal cord, motor cortex, and medulla oblongata reduced after deferiprone treatment.
These data encourage further attempt to test iron chelation for ALS.

10.4 Conclusion

Evidences have shown that iron deposition in brain was closely related with the
pathogenesis of multiple CNS diseases, and treatments to reduce iron in brain have
been investigated in clinical and animal studies. With positive outcomes, there are
still challenges to use iron chelation as therapy for brain disorders. Therefore, future
investigations are required to develop iron-trapping agents that are highly selective,
low affinity, BBB permeable, low in toxicity. With the right agent, the right time
window, and the right patients to intervene, iron will be a tractable target for CNS
diseases.

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