On the other-hand, labile iron taken up by the mitochondrial mitoferrin can be incorporated into Fe–S clusters and heme groups (Hentze et al., 2010). Cellular oxidative stress induced by iron overload is characterized by increased lipid peroxidation and protein and nucleic acid modifications (Ward et al., 2014). These free radicals interact with the surroundings indiscriminately. In the brain, iron plays a role in other enzymatic reactions that produce neurotoxins. The cells which have active iron metabolism are more prone to iron toxicity, such as dopaminergic neurons, which need iron for dopamine metabolism (Hare and Double, 2016). MRI imaging showed that iron deposition was observed in the putamen, globus pallidus, red nucleus and substantia nigra, but yet not all of them have dramatic neuronal loss in PD patients. The major neuronal loss in PD happens in dopaminergic neurons of the substantia nigra pars compacta, which is unique because dopamine metabolism produces several neurotoxins. These toxins are more harmful than widespread oxidative stress caused by iron deposition (Zhou et al., 2010).

Inflammation is commonly observed in the brains of animal models or patients with neurodegenerative diseases like AD and PD (Block and Hong, 2005). Neuroinflammation can produce a variety of proinflammatory factors including TNF-α, IL-6 and nitric oxide (NO). At low levels, NO supports normal neuronal functions via S-nitrosylation of target proteins. However, when NO is produced in a high concentration under pathological conditions such as inflammation, aberrantly S-nitrosylated proteins can initiate neurodegeneration (Nakamura et al., 2013).

We discuss in this review how iron homeostasis is affected in neurodegenerative disease, especially by NO, and how dysregulation of iron can lead to neurodegeneration.

#### IRON HOMEOSTASIS

About half of the total iron is contained in hemoglobin which is needed to transport oxygen along the blood-stream. The other half is, however, stored in complex with ferritin in various cells, mostly in bone marrow, liver and spleen. Only a small portion is circulated in the plasma, bound to transferrin (Conrad and Umbreit, 2000). The absorption of dietary iron takes place in the duodenum. The iron is absorbed by enterocytes of the duodenal lining bound by a protein, such as heme protein, or through divalent metal transporter 1 (DMT1) in ferrous form. The lining cells of the intestine can either bind iron by ferritin for storage, or release it to the body via ferroprotein, the only known mammalian iron exporter (Gropper et al., 2013). Ferroportin (Fpn) can be post-translationally repressed by a peptide hormone of a 25-amino acid length, named hepcidin, Hepcidin binds to Fpn and internalizes it within the cell (Nemeth et al., 2004). Fpn expression can be modulated by the IRP-IRE regulatory mechanism. If the iron concentration is too low, the IRP-IRE binding increases, thus inhibiting Fpn translation. On the other hand, Fpn translation can also be regulated by the microRNA, miR-485-3p (Sangokoya et al., 2013). The ferroxidase, hephaestin, found in the small intestine, oxidizes Fe2+ to Fe3+ and therefore helps Fpn transfer iron across the small intestine basolateral membrane (Ward et al., 2014). Iron metabolism in the body is thus regulated by regulating each of these steps. For instance, under iron-deficient anemic conditions, enterocytes will produce higher levels of Dcytb, DMT1 and transferrin receptor (TfR) proteins (Fleming and Bacon, 2005). Absorption of dietary iron is also enhanced by vitamin C and, in contrast, reduced by excess calcium, zinc, or manganese.

In the brain, iron uptake is through the blood-brain barrier (**Figure 1**). Iron is taken up by the capillary endothelial cell TfR, in the form of transferrin-Fe3+ (Tf-Fe3+). The iron is transported to cerebral compartment from the basolateral membrane of endothelial cells, and is then made available to neurons and glia. Of note, oligodendrocytes stained for most of the detectable iron in the brain. Transferrin is also found predominantly in these cells (Bloch et al., 1985), which is important for myelination formation. Oligodendrocytes support myelin membranes that are many times of the weight of their somata, and need to generate a lot of ATP (Cammer, 1984), hence they need a lot of iron to maintain their metabolism. The TfR is expressed in blood vessels, and in cortical, striatal and hippocampal neurons (Connor and Menzies, 1995). Most types of cells uptake iron primarily through transferrin-mediated endocytosis, via TfR1, TfR2, and GAPDH (West et al., 2000; Kumar et al., 2011; **Figure 1**). Fe3+ is dissociated from transferrin in the endosome, reduced to Fe2+ by a STEAP family reductase, and exported from endosome into cytosol by DMT1 (Hentze et al., 2010). Alternatively, Fe2+ can be absorbed directly via the plasma membrane divalent cation transporters such as DMT1 and ZIP14 (Lane et al., 2015). Fpn is the only protein know for iron efflux so far (Zecca et al., 2004). Neurons and astrocytes express TfR, DMT1, and Fpn (Moos et al., 2007; Zarruk et al., 2015). The astrocyte end feet form intimate contact with brain capillary epithelial cells and play an important role to mediate iron export from blood to brain (Moos et al., 2006). Astrocytes express ceruloplasmins which oxidize Fe2+ to Fe3+ and facilitate iron export from astrocytes (**Figure 1**). During brain inflammation, iron is accumulated in microglia stead of astrocyte. The efflux mechanism of iron in microglia was disrupted in brain

complex. The Hb:Hp complex is bound with the receptor CD163 and undergo endocytosis. The complex are degraded after endocytosis and the iron is absorbed into intracellular iron pool and stored in ferritin. The iron flow direction is indicated by black arrows.

inflammation because Fpn is endocytosed, induced by hepcidin, and microglia cells do not express ferroxidase (Zarruk et al., 2015). In contrast, astrocytes do not accumulate iron, showing robust expression of influx and efflux proteins including Fpn and ferroxidase, with normal iron recycling capability (**Figure 1**). Ferritin, a major iron storage protein in the brain, compromises the H- or L-ferritin monomers. Neurons and oligodendrocyte express H-ferritin, which have a high iron metabolite rate. Microglia express L-ferritin, which is associated with iron storage (Connor et al., 1994). Although oligodendrocytes have the highest iron accumulation in normal aging brain, there is little effect on myelination and oligodendrocyte in neurodegenerative diseases. Oligodendrocytes have high levels of ferritin expression (Connor et al., 1994), which provide more buffering capacity for free iron and therefore affording more protective effect. Fpn needs to couple with ferroxidase to export iron. Astrocytes express ceruloplasmin, while oligodendrocytes express another ferroxidase, hephaestin, to facilitate iron export from oligodendrocyte (Schulz et al., 2011). Oligodendrocytes down regulate TfR1 to reduce iron uptake after maturation (Han et al., 2003). These iron level-regulating mechanisms protect oligodendrocytes in neurodegenerative diseases. The iron absorbed from blood during different age is more than what is accumulated in the brain, indicating that there is a iron efflux mechanism in the brain. The iron export from brain to blood maybe through the cerebrospinal fluid (Moos et al., 2007).

In addition, the highest iron load in the brain is seen in relation to chronic microbleeds. Cerebral microbleeds are likely to be caused by cerebral atherosclerosis in aged people or people with neurodegenerative diseases (Martinez-Ramirez et al., 2014). The iron from hemolysis of erythroid cells may be the major source of iron load in aged or neurogenerative brain (**Figure 1**).

Erythroid cells contain most of the body iron in hemoglobin (Hb). Hb contains 70% of the total body iron in its heme moiety. Hb is not restricted in erythroid cells, but expressed in glia, macrophage and neurons. Hb is an oxygen reservoir inside the cell and regulate mitochondria function. While Hb mainly exists intracellularly, it will be released extracellularly during hemolysis. Haptoglobin (Hp) is mostly produced by hepatic cell and binds free Hb in plasma with very high affinity (Wicher and Fries, 2010). In the brain, Hp is produced locally by oligodendrocyte (Zhao et al., 2009). The Hb–Hp complex has a membrane receptor CD163. The interaction between them leads to internalization of CD163 (**Figure 1**), Hb dissociating from Hp, Hb heme degradation, and iron being absorbed into the intracellular iron pool. Following intravascular hemorrhage, hippocampal and cortical neurons express CD163 in the brain (Garton et al., 2016), which facilitates iron absorption, post-hemolysis.

Although rodent models are widely used for iron study, it is noteworthy that iron accumulation and cellular storage are different between humans and experimental rodent models. Rodent generally have a lower basal level of iron deposition in the brain, especially in young animals (less than one year old), as shown by several groups (Jeong and David, 2006; Liu et al., 2018).

#### IRON DEPOSITION CHANGES IN AD AND PD

In AD, iron hemostasis is disrupted. Transferrin has been shown to be decreased consistently, and in particular in the white matter, White matter is thought to play a major role in neurodegeneration, and increased peroxidative damage to white matter is known to take place in AD (Connor et al., 1992). High concentration of iron is accumulated in Aβ plaques and tau tangles which is characteristic of AD. The Aβ plaques contain a fairly large amount of labile iron, while the neighboring cells express significant levels of ferritin and transferrin receptors (Connor et al., 1995). Moreover, iron influence APP translation via IRP-IRE system. There is an IRE within the 50 -UTR of APP mRNA (Rogers et al., 2002), and in response lowered intracellular iron as a result of chelation by desferrioxamine and clioquinol, translation of APP was selectively down-regulated. APP interacts with Fpn, and it has ferroxidase activity as it possessed the conserved H-ferritin-like active site, and Zn2+ can specifically inhibit this active site (Duce et al., 2010; Bush, 2013). Lei et al. showed that tau deficiency caused iron accumulation in brain and dopaminergic neuron degeneration, which led to parkinsonism in mice with dementia. Tau promotes the export of neuronal iron by facilitating the trafficking of APP to the plasma membrane. The study suggested that Alzheimer disease, Parkinson disease and tauopathies that are associated with the iron toxicity due to the loss of soluble tau could in principle be rescued by a pharmacological agent such as clioquinol, an iron chelator (Lei et al., 2012).

Several studies reported that iron deposition was increased in the substantia nigra according to the severity of the disease in PD patients (SN) (Dexter et al., 1987, 1989; Hirsch et al., 1991). The researchers used plasma spectroscopy to detect iron concentration quantitatively in various brain regions (Dexter et al., 1989). In PD brain, histology studies showed that iron accumulate in neurons and glia in SN (Jellinger et al., 1990). In vitro, the formation of α-synuclein fibrils can be accelerated by the presence of Fe3+ (Uversky et al., 2001) and it was also reported that redox-active iron was sequestrated by Lewis bodies in SN (Castellani et al., 2000). Furthermore, there are reports that a dysfunction in the IRP-IRE system that results in iron accumulation gave rise to α-Syn-induced toxicity (Li et al., 2010; Febbraro et al., 2012), that led to PD pathogenesis (Rocha et al., 2017). Similarly, in almost all PD patient brains the Lewy bodies contained aggregated α-Syn (Wakabayashi et al., 2007).

The reason for iron accumulation in SN is unclear. Several hypotheses proposed include increased brain-blood-barrier (BBB) permeability (Faucheux et al., 1999; Kortekaas et al., 2005), increased neuroinflammation (Block and Hong, 2005; Conde and Streit, 2006; Machado et al., 2011), increased DMT1 isoform expression or function (Block and Hong, 2005; Salazar et al., 2008; Machado et al., 2011; Liu et al., 2018), and altered transferrin or lactoferrin transport (Faucheux et al., 1995; Mastroberardino et al., 2009). Cellular iron accumulation in PD brain may be caused by elevated influx or decreased efflux. Inflammation could contribute to iron accumulation by either increasing DMT1 uptake activity or TfR transport activity. In a mouse model, DMT1 activity was increased to mediate the iron uptake (Salazar et al., 2008), and this increase may be due to direct S-nitrosylation of DMT1 (Liu et al., 2018).

## NO PRODUCTION IN IMMUNE RESPONSE AND NEURODEGENERATION

NO is a gaseous signaling molecule that initially was thought as a dilator in blood vessels, with guanylyl cyclase as the major effector. NO binds to the heme group of guanylyl cyclase and activates it in the presence of iron. High cGMP level are associated with release of neurotransmitters including glutamate, acetylcholine and glycine. NO participates in tumor and bacteria immunity and in the central nervous system, it acts as a retrograde neurotransmitter. In the nervous system, NO has both physiological and pathological functions. For example, NO contributes to long-term potentiation (LTP) and long-term depression (LTD), and thus it plays a role in learning and memory (Schuman and Madison, 1991; Shibuki and Okada, 1991; Lev-Ram et al., 1997; Kakegawa and Yuzaki, 2005; Kakizawa et al., 2012). NO enhances CREB expression to mediate the response to brain-derived neurotrophic factor (Riccio et al., 2006). NO also mediates glutamate-NMDA receptor (NMDAR) signaling as glutamate activates NMDAR in neurons, leading to Ca2+ influx, and activation of nNOS (Xu et al., 2018). The synaptic NMDARs mediate neuroprotection, while the extrasynaptic NMDARs mediate neurodegeneration (Talantova et al., 2013; Molokanova et al., 2014), implicating the diverse signaling pathways triggered by NO. NO also binds to other iron-containing proteins, such as mitochondrial aconitase. Mitochondrial aconitase has a [4Fe-4S]2+ cluster, which is a possible target for NO-induced toxicity

(Drapier and Hibbs, 1986, 1988). The interaction between mitochondrial aconitase and superoxidase are the major cause to mitochondrial damage (Vasquez-Vivar et al., 1999). Most of the cytotoxicity of NO is attributed to the production of peroxynitrite (Pacher et al., 2007), which is more powerful to produce radicals. Peroxynitrite is produced from the diffusion-controlled reaction between NO and superoxide in vivo (Squadrito and Pryor, 1995). Peroxynitrite is a strong oxidant and it interacts with electron-rich groups, including Fe–S cluster. It is reported that peroxynitrite is far more effective to produce hydroxyl radicals than Fenton's reaction (Beckman et al., 1990; Darley-Usmar et al., 1992; Hogg et al., 1992). Peroxynitrite is an important intermediator for protein nitration and oxidation, lipid peroxidation, mitochondria dysfunction, and finally causes apoptosis and necrosis (Radi, 2018).

NO is produced by NOS through the conversion of L-arginine to citrulline. Three distinct NOS isoforms have been identified in the brain (Forstermann et al., 1991). Neuronal NOS (nNOS) is expressed in neurons, while endothelial (eNOS) is expressed in brain endothelial cells. They are Ca2+/calmodulin-dependent and synthesize NO in a short period in response to receptor activation or extracellular stimuli (Moncada et al., 1991).  Inducible NOS produces a large amount of NO upon stimulation by proinflammatory cytokines over a long period of time (Green et al., 1994).

In human immune response, NO is produced by phagocytes such as monocytes, macrophages, and neutrophils. In phagocytes, interferon-gamma (IFN-γ) or tumor necrosis factor (TNF) activates iNOS (Green et al., 1993). On the other hand, transforming growth factor-beta (TGF-β), interleukin-4 (IL-4) or IL-10 weakly inhibits iNOS. As such, phagocytes contribute to inflammatory and immune responses via NO (Green et al., 1994). In an immune response, NO is secreted as free radicals that is toxic to intracellular pathogens. The modes of action are via DNA damage (Wink et al., 1991; Nguyen et al., 1992) and degradation of Fe–S centers into iron ions and ironnitrosyl compounds (Hibbs et al., 1988). The molecular effects of NO depend on two kinds of reactions: S-nitrosylation of thiols and the nitrosylation of some metalloenzymes. Guanylate cyclase, a NO activated heme-containing enzyme, is an essential component of the relaxing function of NO on smooth muscles (Derbyshire and Marletta, 2009). cGMP activates protein kinase G that lead to the re-uptake of Ca2+ and the rise in cytoplasmic Ca2+ activates calcium-activated potassium channels triggering the relaxation of smooth muscle (Rhoades and Tanner, 2003).

In addition to neuro-inflammatory stimuli, induction of iNOS expression in astrocytes, macrophages, and microglia by Aβ oligomers or by toxins such as 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) have been reported to increase NO levels in the degenerating brain (Liberatore et al., 1999; Medeiros et al., 2007; Nakamura et al., 2013). Knockdown of iNOS in APP/PS1 AD mouse model ameliorate AD-related symptom including Aβ plaque formation, premature death, astroglioses and microgliosis (Nathan et al., 2005). However, in the Tg2576 APP AD mouse model, ablation of iNOS exacerbated spatial learning and memory and tau pathology, providing evidence that NO may have a neuroprotective role (Wilcock et al., 2008).

#### NO REGULATION ON IRON HOMEOSTASIS

NO targeted proteins have been partially characterized. NO can interact with Fe–S cluster containing protein and influence their enzyme activity. One of the Fe–S cluster-containing proteins is IRE-binding protein (also termed "iron regulatory protein," IRP). Cytosolic iron concentrations sensed by IRPs could posttranscriptionally adjust the expression of iron metabolizing genes to optimize the availability of labile iron. IRPs bind to iron-responsive elements (IRE), which are specific non-coding mRNA sequences, to control iron metabolism. IREs are of 30 nucleotide in length found along RNA motifs, and they contain the CAGUGN sequence (the classic IRE motif) that form a stemloop structure (Molokanova et al., 2014). IREs are found either within the 30 -UTR (untranslated region) or 50 -UTR regions of a specific mRNA. IRP1 and IRP2 are examples of two RNA-binding proteins that interact with IRE to modulate the translation of either the ferritin or Fpn mRNA, and they also control the stability of TfR and DMT1 mRNAs. The binding of IRPs and IREs is regulated by free iron concentration. Therefore, IRPs can act as either a translation enhancer or inhibitor (Pantopoulos, 2004; Piccinelli and Samuelsson, 2007). As ferritin and Fpn transcripts contain IRE in their 50 -UTRs, their translation will be inhibited by IRPs when there is iron deficiency (Hentze et al., 2010). The decreased expression of ferritin and Fpn reduces free iron binding and export, leading to an increased in availability of labile iron for use by the cell. In contrast, the TfR and DMT1 transcripts contain 30 -UTR IREs that bind IRPs, and when cytoplasmic iron is deficient, the stabilization of transcripts will increase synthesis of TfR and DMT1 proteins to enhance iron uptake (Hentze et al., 2010). Examples of transcripts that contain IREs include those that encode the ferritin subunits L and H, TfR, Fpn, DMT1, mitochondrial aconitase, succinate dehydrogenase, erythroid aminolevulinic acid synthetase, amyloid precursor protein and a-synuclein. These downstream genes suggest that iron has close regulation of iron metabolism, redox and neurodegeneration via the IRP-IRE system. NO could regulate IRP-IRE binding, which in turn regulates many iron metabolism-related proteins (**Figure 2**). NO acts on DMT1 by increasing IRP1 binding to the IRE sequence located at the 30 -UTR, to stabilize and increase DMT1 transcript level, akin to cells exposed to low iron condition (Weiss et al., 1993; Jaffrey et al., 1994; Wallander et al., 2006; Skjorringe et al., 2015). Similarly, NO can regulate ferritin, Fpn and TfR via regulating the interaction of IRP-IRE binding, and hence regulate iron metabolism (**Figure 3**). Another report also showed that NO can enhance iron deposition in the brain via decreasing APP expression (Ayton et al., 2015). The authors reported obvious decrease in expression of APP in substantia nigra of PD brain. APP KO mice have iron-dependent dopaminergic neuron loss, while APP overexpressing mice have protection effect in MPTP mouse model, as APP facilitates iron efflux. NO decreases APP expression via the IRP-IRE system

regulates the transcription of iron-metabolism-related proteins, and elevates intracellular iron level. NO also directly S-nitrosylates DMT1, which enhances DMT1 transporter function. In addition, NO S-nitrosylates Dexras 1 and enhances the binding of Das1-PAP7-DMT1 complex and finally enhances iron uptake.

and this may explain how NO leads to dopaminergic neuron loss in PD.

NO also regulates iron metabolism-related proteins in other ways, such as S-nitrosylation (**Figure 2**). Another pathway to increase iron uptake via DMT1 is by S-nitrosylation of Dexras 1 to indirectly regulate DMT1 function and enhance Fe2+ uptake as reported in PC12 and cortical neurons (Cheah et al., 2006). Recently, we have shown that NO directly modulated DMT1 and enhanced its function via S-nitrosylation. This is unexpected as S-nitrosylation of proteins important in PD such as Parkin and XIAP resulted in compromised functions. Besides, many S-nitrosylated proteins have been identified in the past decade, and of note, those that have been functionally characterized have a loss-of-function (Nakamura et al., 2013). In this regard, enhanced DMT1 activity arising from S-nitrosylation would therefore present a mechanism by which Fe2+ could be accumulated over aging and contribute to age-dependent neurodegeneration (**Figure 3**).

# POTENTIAL THERAPY FOR NEURODEGENERATIVE DISEASES TARGETED TO IRON DEPOSITION

The potential therapeutic use of iron chelators gained much attention in recent years (Ward et al., 2014; Zhou and Tan, 2017). The strategy to target iron deposition is either to chelate iron directly or to regulate iron homeostasis, including NOregulated iron absorption. The candidate compounds should be

BBB-permeant and easily penetrate cell membrane, chelate free iron and minimize the side effect to normal iron metabolism.

Several iron chelators were used to deplete excessive iron and yielded promising clinical outcome. The clinical trial using deferiprone at 30 and 20 mg/kg per day was carried out in PD patient, and the compound was well-tolerated. The syndrome was improved and the iron content in SN significantly decreased as monitored by MRI (Devos et al., 2014). Deferiprone seems quite promising so far, and is waiting to be tested in further clinical trial in larger population. Furthermore, in several in vivo PD models, iron chelators, including deferasirox, deferrioxamine, VAR10303 and D-607, have been used to significantly attenuate DA neuronal loss (Ghosh et al., 2010; Dexter et al., 2011; Bar-Am et al., 2015; Das et al., 2017).

Desferrioxamine has been shown to decelerate AD progression (Rogers and Lahiri, 2004). However, as DFO is unstable with poor BBB permeability (Bandyopadhyay et al., 2010), clioquinol, a BBB-permeant iron chelator, was used instead in clinic to mitigate cognitive loss by reducing plasma Aβ levels in AD patients (Ritchie et al., 2003). As clioquinol has side effects associated with myelopathies (Zhang et al., 2013), PBT2, a second-generation 8-hydroxyquinoline analog metal chaperone which can bind transition metal, was used in clinical trial for AD patients. The patients who received 250 mg/kg PBT2 have substantially reduced Aβ level in cerebral spinal fluid and showed cognitive improvement in a Phase II trial (Lannfelt et al., 2008; Faux et al., 2010). However, in another Phase II trial announced by the Australian company Prana Biotechnoloy in 2014, PTB2 failed to improve brain amyloid deposition, neuronal function, brain atrophy and cognition in a one-year course treatment. Several other iron chelators, such as VK28 (Fe3+ chelator), HLA20 and M30 (Fe3+ chelator with N-propargylamine-like properties), can not only suppress APP expression but also reduce Aβ level in the brain (Bandyopadhyay et al., 2010).

Besides direct chelation of iron, potent nontoxic IRE inhibitors with excellent BBB penetrating capacity were also thought to have high therapeutic significance in neurodegenerative diseases. The IRE inhibitors that downregulate translation of APP and α-Syn and prevent protein aggregation can support survival of neurons. To date, a few promising drug candidates of IRE inhibitors have been characterized and are being tested in various clinical trials for AD and PD patients (Zhou and Tan, 2017). Posiphen is a natural product that has been shown to inhibit translation of both APP and a-synuclein proteins. Furthermore, it is nontoxic and potent (Rogers et al., 2011). The inhibitory effect has been validated by various experiments done in vitro and in vivo (Lahiri

#### REFERENCES

et al., 2007; Yu et al., 2013). A phase I human clinical trial in subjects with mile cognitive impairment have shown that posiphen lowered APP production by 50 % in CSF and was well-tolerated (Maccecchini et al., 2012). The compound is still waiting for further clinical trials to test the efficacy for AD and PD (Bandyopadhyay et al., 2010). Another compound JTR-009, screened from a 110,000-compound library, was identified to have a more potent effect on APP translation than posiphen. JTR-009 is thought to bind to 50 -UTR IRE directly and selectively inhibit APP expression, which was validated in SH-SY5Y cells (Bandyopadhyay et al., 2013). Another case for targeting iron homeostasis is inhibiting DMT1 function. To inhibit NOmediated DMT1 functional increase, we used the NOS inhibitor L-name to reduce NO-mediated iron deposition in LPS-evoked mouse inflammatory model. L-NAME significantly ameliorated SN dopaminergic neuron loss and LPS-induced behavior deficit (Liu et al., 2018).

#### CONCLUSION AND FUTURE PERSPECTIVES

Iron homeostasis is elaborately regulated in the human brain, and iron accumulation is closely associated with neurodegenerative diseases. NO regulates iron deposition at several levels. So far, therapeutic targeting of iron deposition has yielded some promising results, and further clinical trials in larger populations are still needed. There are still some questions that remained unresolved. For example, how iron is transported through brain capillary epithelial cells and more specifically, how S-nitrosylation of DMT1 enhanced its transporter activity. The technique for iron and NO concentration detection is a limitation, especially for accurate detection of Fe2+ and Fe3+ concentrations. A real-time, quantitative and in vivo detection technique will be extremely valuable for the field.

#### AUTHOR CONTRIBUTIONS

CL and MCL drafted the manuscript. TWS critically edited the manuscript. All authors approved the final version of the manuscript.

#### FUNDING

This work was supported by the Singapore National Medical Research Council, NMRC, Grant no: NMRC/CBRG/0042/2013.

of beta-amyloid precursor protein. J. Neurosci. 35, 3591–3597. doi: 10.1523/ JNEUROSCI.3439-14.2015

Bandyopadhyay, S., Cahill, C., Balleidier, A., Huang, C., Lahiri, D. K., Huang, X., et al. (2013). Novel 50 untranslated region directed blockers of iron-regulatory protein-1 dependent amyloid precursor protein translation: implications for down syndrome and Alzheimer's disease. PLoS One 8:e65978. doi: 10.1371/ journal.pone.0065978

Rhoades, R. A., and Tanner, G. A. (2003). Medical Physiology, 2nd Edn. Philadelphia, PA: Lippincott Williams & Wilkins.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Liu, Liang and Soong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fnins-13-00114 February 14, 2019 Time: 19:2 # 10

# The Role of Iron in Friedreich's Ataxia: Insights From Studies in Human Tissues and Cellular and Animal Models

José Vicente Llorens1,2 \*, Sirena Soriano3,4, Pablo Calap-Quintana1,2 , Pilar Gonzalez-Cabo5,6,7 and María Dolores Moltó1,2,8 \*

1 Department of Genetics, Faculty of Biological Sciences, University of Valencia, Valencia, Spain, 2 Unit for Psychiatry and Neurodegenerative Diseases, Biomedical Research Institute INCLIVA, Valencia, Spain, 3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States, 4 Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, United States, 5 Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, Valencia, Spain, 6 Center of Biomedical Network Research on Rare Diseases CIBERER, Valencia, Spain, 7 Associated Unit for Rare Diseases INCLIVA-CIPF, Biomedical Research Institute INCLIVA, Valencia, Spain, 8 Center of Biomedical Network Research on Mental Health CIBERSAM, Valencia, Spain

Edited by: Giorgio Biasiotto, Università degli Studi di Brescia, Italy

#### Reviewed by:

Jordi Tamarit, Universitat de Lleida, Spain Ian Alexander Blair, University of Pennsylvania, United States

#### \*Correspondence:

José Vicente Llorens<EMAIL_ADDRESS>María Dolores Moltó<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 15 November 2018 Accepted: 23 January 2019 Published: 18 February 2019

#### Citation:

Llorens JV, Soriano S, Calap-Quintana P, Gonzalez-Cabo P and Moltó MD (2019) The Role of Iron in Friedreich's Ataxia: Insights From Studies in Human Tissues and Cellular and Animal Models. Front. Neurosci. 13:75. doi: 10.3389/fnins.2019.00075 Friedreich's ataxia (FRDA) is a rare early-onset degenerative disease that affects both the central and peripheral nervous systems, and other extraneural tissues, mainly the heart and endocrine pancreas. This disorder progresses as a mixed sensory and cerebellar ataxia, primarily disturbing the proprioceptive pathways in the spinal cord, peripheral nerves and nuclei of the cerebellum. FRDA is an inherited disease with an autosomal recessive pattern caused by an insufficient amount of the nuclear-encoded mitochondrial protein frataxin, which is an essential and highly evolutionary conserved protein whose deficit results in iron metabolism dysregulation and mitochondrial dysfunction. The first experimental evidence connecting frataxin with iron homeostasis came from Saccharomyces cerevisiae; iron accumulates in the mitochondria of yeast with deletion of the frataxin ortholog gene. This finding was soon linked to previous observations of iron deposits in the hearts of FRDA patients and was later reported in animal models of the disease. Despite advances made in the understanding of FRDA pathophysiology, the role of iron in this disease has not yet been completely clarified. Some of the questions still unresolved include the molecular mechanisms responsible for the iron accumulation and iron-mediated toxicity. Here, we review the contribution of the cellular and animal models of FRDA and relevance of the studies using FRDA patient samples to gain knowledge about these issues. Mechanisms of mitochondrial iron overload are discussed considering the potential roles of frataxin in the major mitochondrial metabolic pathways that use iron. We also analyzed the effect of iron toxicity on neuronal degeneration in FRDA by reactive oxygen species (ROS)-dependent and ROS-independent mechanisms. Finally, therapeutic strategies based on the control of iron toxicity are considered.

Keywords: Friedreich's ataxia, frataxin, iron, animal models, oxidative stress, lipid deregulation, iron chelators

# INTRODUCTION

fnins-13-00075 February 14, 2019 Time: 19:4 # 2

Nicholaus Friedreich, a German neurologist among a family of professors of medicine, had described a new disease entity in successive papers published between 1863 and 1877 (Koeppen, 2013). The new disease, named later Friedreich's ataxia (FRDA), was characterized by degenerative atrophy of the posterior columns of the spinal cord, leading to the development of progressive ataxia, sensory loss and muscle weakness in patients and was associated with cardiomyopathy in many cases. Clear diagnostic criteria for FRDA were established by Anita Harding in the 1980s (Harding, 1981), who specified the hereditary nature of the disease with an autosomal recessive pattern, an onset during puberty, progressive ataxia of the limbs and gait with the absence of tendon reflexes in the legs, extensor plantar responses, and dysarthria. In 1996, the identification of the causative gene of FRDA and expansion of a trinucleotide repeat as the most frequent mutation (Campuzano et al., 1996) led to the development of a molecular tool for a definitive diagnosis of the disease. It was crucial to address the differential diagnosis with respect to other peripheral neuropathies and ataxias with similar clinical features, as well as the clinical heterogeneity in FRDA, including atypical phenotypes (Bidichandani and Delatycki, 2017). Moreover, the availability of this molecular approach allowed for appropriate genetic counseling.

## Epidemiological, Pathophysiological and Molecular Aspects of FRDA

Friedreich's ataxia (OMIM #229300; ORPHA:95) is a rare neurodegenerative disease affecting 1:20,000–1:50,000 people of Indo-European and North African descendent (Vankan, 2013), and with a carrier frequency of 1:60–1:100, FRDA is the most frequently inherited ataxia in these populations. The neurological signs and symptoms in FRDA are the results of pathological changes that disturb both the central and peripheral nervous systems (NSs) (reviewed in Marmolino, 2011; Koeppen and Mazurkiewicz, 2013). The dorsal root ganglia (DRG) are particularly vulnerable and have been identified as the first site of neuropathology with the degeneration of the large sensory neurons and posterior columns, leading to the loss of position and vibration senses. Next, affectation of the corticospinal and spinocerebellar tracts of the spinal cord occurs, with loss of myelinated fibers. In the cerebellum, dentate nuclei (DN) also show progressive atrophy. Cerebral abnormalities are being progressively reported with the application of neuroimaging techniques (Selvadurai et al., 2018). Non-neurological phenotypes of FRDA mainly include hypertrophic cardiomyopathy, which is the leading cause of mortality (Raman R. et al., 2011; Payne and Wagner, 2012; Weidemann et al., 2013; Koeppen et al., 2015). The heart typically maintains adequate systolic function (Kipps et al., 2009) in FRDA patients who develop a severe hypertrophic cardiomyopathy until shortly before death (Rajagopalan et al., 2010). Additionally, diabetes mellitus and skeletal abnormalities are also present in this disease (Parkinson et al., 2013; Bidichandani and Delatycki, 2017).

Friedreich's ataxia is caused by loss-of-function mutations in the frataxin gene (FXN) (Campuzano et al., 1996). The most common mutation appears in homozygosis in most patients and consists of an abnormally expanded GAA trinucleotide repeat (usually between 600 and 900 repeats) in the first intron of the gene FXN (Campuzano et al., 1996; Monrós et al., 1997). The remaining patients (approximately 4%) are compound heterozygous with the expanded GAA repeat on one allele and another pathogenic variant (point mutation, deletion or insertion) on the other allele (Galea et al., 2016). The pathological GAA expansion affects the expression of FXN by blocking the transcription of mRNA through the formation of sticky DNA triplexes and R-loop structures (reviewed in Kumari and Usdin, 2012) and/or inducing abnormal heterochromatinization (Saveliev et al., 2003). Thus, the level of frataxin is strongly reduced from 5 to 30% of the physiological level (Campuzano et al., 1997). This reduction is related to the disease severity and length of the GAA repeat. In general, longer expansions are present in patients with a more severe phenotype such as earlier onset, faster progression and/or presence of non-neurological features (Parkinson et al., 2013). Although most of the pathogenic point mutations also decrease the level of functional protein, some lead to the production of a less active protein (De Castro et al., 2000; Bridwell-Rabb et al., 2011).

#### Protein Frataxin

Frataxin is a small acidic protein, synthetized in the cytoplasm as a 210-residue polypeptide that is then imported into the mitochondria, where it is proteolytically processed to the mature and most abundant form of 130 residues [frataxin (81–210)] (Condò et al., 2007). Although frataxin has historically been considered a protein exclusively confined to the mitochondrial matrix, several studies have reported the existence of an extramitochondrial pool of frataxin in different human cell types (Acquaviva et al., 2005; Condò et al., 2006; Lu and Cortopassi, 2007). Extramitochondrial frataxin corresponds to the (81–210) mature form of the protein (Condò et al., 2010) and earlier studies suggested that after the initial transport and processing in the mitochondria, mature frataxin was transported back to the cytosol (Acquaviva et al., 2005; Condò et al., 2006). However, a recent study describes an N-terminal acetylated extramitochondrial form of frataxin in erythrocytes. This (76– 210) isoform does not contain the mitochondrial targeting sequence, and remains in the cytosol where it is cleaved to produce an (81–210) protein identical to the mitochondrial mature form (Guo et al., 2018).

The three-dimensional structure of frataxin shows seven antiparallel β-sheets flanked by two α-helices, producing a characteristic globular αβ fold (Dhe-Paganon et al., 2000). The protein is ubiquitously expressed (Campuzano et al., 1996, 1997), but different cells types show distinct susceptibility to its deficiency. This could be explained by frataxin reduction in different tissues being associated with distinct transcriptomic profiles, as described recently (Chandran et al., 2017). Frataxin is a highly conserved protein through evolution, and its cellular function is critical for life in multicellular organisms. A strong reduction of frataxin in Drosophila melanogaster seriously affects

Llorens et al. Iron in Friedreich's Ataxia

viability (Anderson et al., 2005; Llorens et al., 2007). Knockout of the frataxin gene causes embryonic lethality in mice (Cossée et al., 2000) and in the plant Arabidopsis thaliana (Vazzola et al., 2007). In line with this, FRDA patients with non-GAA-repeat mutations in both frataxin alleles, resulting in totally deficient frataxin function, have not been reported.

At the time of FXN identification, there was no evidence about the function of frataxin. Experiments in yeast Saccharomyces cerevisiae promptly suggested a potential role for frataxin in iron homeostasis regulation. Deletion of the ortholog of frataxin in yeast (Yfh1) causes iron accumulation in the mitochondria (Babcock et al., 1997; Foury and Cazzalini, 1997) that was initially suggested at the expense of the cytosolic iron pool (Knight et al., 1998). However, measurements of cytosolic iron in frataxin-deficient yeast cells show that the cytosolic iron levels are not affected by the reduction of the frataxin synthesis (Li et al., 2012). Also associated with frataxin deficiency are reduced activities of mitochondrial iron-sulfur cluster (ISC) enzymes and aconitase, mitochondrial dysfunction and oxidative stress (reviewed in Calabrese et al., 2005; Lupoli et al., 2018). Since then, efforts have been made to answer several questions about iron in FRDA, including whether its accumulation is a primary or secondary event in the disease, what is the mechanism causing the deregulation and buildup of iron, and the role of this metal in the neuropathology of FRDA. Here, we review the contribution of the cellular and animal models of this disease and relevance of the studies using patient samples to gain knowledge about these issues.

#### IRON ACCUMULATION IN FRDA

Iron accumulation is a hallmark in FRDA and initially was suggested to be a primary pathogenic event triggered by frataxin deficiency. Tissues in which iron overload has been reported in FRDA patients are showed in **Supplementary Table S1**. The first observations linking iron and FRDA were made in postmortem heart samples from a few patients, where dense intracytoplasmic deposits of iron particles were detected in the cardiac fibers (Sanchez-Casis et al., 1976; Lamarche et al., 1980). These findings were later corroborated in subsequent studies that use different techniques for iron quantitation, such as Perls Prussian blue staining for histological detection or atomic absorption spectroscopy to measure total iron levels in tissue. The excess iron in the heart of FRDA patients appears as multifocal aggregates highly localized rather than as a diffuse pattern (Ramirez et al., 2012; Koeppen et al., 2015). This regional distribution of the excess of iron could be responsible for the findings of iron levels equivalent to healthy controls in bulk extracts or from random biopsied cardiac samples (Michael et al., 2006; Koeppen et al., 2015; Kruger et al., 2016). In addition to the heart, positive iron staining was observed in the liver and spleen of patients, showing a distribution consistent with a mitochondrial location (Bradley et al., 2000) as has been described in the frataxin-deficient yeast model (Babcock et al., 1997; Foury and Cazzalini, 1997).

In regions of the NS, such as DN and DRG, several scenarios regarding the iron content in FRDA have been reported: normal levels (Bradley et al., 2000; Koeppen et al., 2007, 2013; Solbach et al., 2014), accumulation (Bonilha da Silva et al., 2014; Harding et al., 2016), and limited and regional increases of this metal (Koeppen et al., 2009). These diverse results could be partly due to the technique used for iron measurements and their capability to discriminate between the accumulation and redistribution of iron. In most of these studies, the iron content has been quantified from a small number of patient samples, a situation that could also contribute to the different results reported. A more recent study with a higher number of FRDA subjects analyzed used magnetic resonance imaging, which allows in vivo iron quantitation (Harding et al., 2016). The authors found a significant increase in the iron concentration in the FRDA cohort compared with that in controls, in the DN and Red nucleus in the midbrain. However, the technical approach used in this study did not allow the authors to discriminate between deposition and redistribution or whether the location of the iron was extracellular or intracellular. Changes in the distribution of iron and other metals, such as copper and zinc, in the DN of FRDA patients have also been reported (Koeppen et al., 2012), although the total amount of these metals was similar to that of controls. It is worth highlighting that copper can also generate oxygen radicals and, in the context of FRDA, may exacerbate the oxidative injury caused by the increased amount of iron. Mitochondrial iron accumulation has also been documented in fibroblasts and lymphoblasts of FRDA patients. Although some authors have found an increase in the total iron content (Delatycki et al., 1999) or labile iron fraction (Tan et al., 2001), normal levels of iron have been reported in other studies (Wong et al., 1999; Sturm et al., 2005).

Several mouse models of FRDA have been obtained using different experimental strategies, and most of them show iron overload because of frataxin reduction (**Supplementary Table S2**). However, no iron accumulation was detected in embryonic samples of the frataxin knockout (KO) mice (Cossée et al., 2000), and the conditional model of frataxin deficiency in striated muscle (MCK-KO mice) showed heart intramitochondrial iron buildup only in late stages of the disease (Puccio et al., 2001). This led to the hypothesis that the increase in iron is not the causative pathological mechanism in FRDA and is a secondary effect.

Iron accumulation has been studied, particularly in muscle creatine kinase (MCK) conditional Fxn knockout mice (MCK-KO). This mutant develops a severe cardiac phenotype associated with almost ablation of frataxin in heart and skeletal muscle (Puccio et al., 2001). Different studies on this mouse line have reported distinct ages of onset for the iron overload despite using the same iron detection methodology (**Supplementary Table S2**). Some authors (Puccio et al., 2001) found iron deposits in 10-week-old mice, whereas animals 7 weeks of age did not present iron accumulation but showed deficits in ISC enzyme activities. By contrast, other authors (Huang et al., 2013) observed progressive iron accumulation from 5 to 10 weeks of age, with the presence of the iron deposits being the first observable histological change in the MCK-KO hearts. Therefore, there is no consensus about when iron starts to accumulate and whether the presence of iron deposits is relevant for the development of heart pathology in FRDA.

An interesting result found in the MCK-KO model is that iron levels increase in other tissues besides the heart (**Supplementary Table S2**), such as the liver, kidney and spleen, as well as serum (Huang et al., 2009; Whitnall et al., 2012). Because the amount of frataxin in these non-muscular tissues is within the normal range in the MCK-KO model (Huang et al., 2009; Whitnall et al., 2012), the iron increase was explained as an alteration of systemic iron metabolism orchestrated by the frataxin reduction in heart and skeletal muscle. It could be operated through a signaling mechanism involving hepcidin (Huang et al., 2009; Whitnall et al., 2012), a key regulator of systemic iron metabolism in mammals. These explanations indicate that excess iron plays a significant role in the disease. The mitochondrial iron in the heart of MCK-KO mutants is in the form of biomineral iron, which aggregates with phosphorus and sulfur (Whitnall et al., 2012). This is important because excess of iron that is not bound to ferritin, the main cellular protein that stores iron, can be very toxic by generating reactive oxygen species (ROS).

In the liver of mice with hepatic Fxn deficiency (FxnAlb mice), a fivefold increase in the mitochondrial iron level has been observed at 28 days of age compared with that in control mice. Meanwhile, 14-day-old mutant showed deficits in ISC enzymes in the liver but normal levels of iron (Martelli et al., 2015), suggesting that iron accumulation is a secondary event also in the liver. As in FRDA patients, iron deposition in the NS of FRDA mouse models has not been clarified yet. In conditional knockout models with frataxin reduction in neurons and other tissues (Puccio et al., 2001) or in parvalbumin-positive cells in the DRG, cerebellum and brain (Piguet et al., 2018), no iron accumulation has been observed. Other studies have reported few iron deposits in NS late in disease progression (Simon et al., 2004) or that iron accumulates both intracellularly and extracellularly in the brain following the loss of Fxn (Chen et al., 2016a). To understand these diverse findings, the authors have indicated that the different sensitivities of the techniques used to measure iron could be an explanation, or that, in some mouse models, cells loss may occur before sufficient iron is accumulated to be detected.

Other animal models of FRDA have also shown an increase in iron levels in the mitochondria (**Supplementary Table S2**), as is the case of D. melanogaster (Soriano et al., 2013). In this species, severe loss of frataxin function results in iron accumulation in multiple tissues, including the brain (Chen et al., 2016b). This was the first FRDA model clearly demonstrating that iron can accumulate in the NS. Additionally, frataxin-deficient flies also showed increased levels of other transition metals, such as zinc, copper and manganese, suggesting that frataxin reduction affects the homeostasis of other metals besides iron (Soriano et al., 2016). In the plant A. thaliana, frataxin is localized in both the mitochondria and chloroplasts, where it is suggested to have similar functions; its deficiency alters the iron content, and this metal accumulates in the two organelles (Martin et al., 2009; Turowski et al., 2015). These results indicate that frataxindefective organisms show a common propensity to accumulate Fe into organelles where this protein is located. Although many studies have suggested that iron deposits are not instrumental and represent a late event in the pathogenesis of FRDA, the role of Fe in the disease pathophysiology is not fully understood. Recently, frataxin function has been nicely discussed (Alsina et al., 2018), and it can offer some insight into the mechanisms underlying iron accumulation in FRDA tissues and in model organisms. We review this subject in the following section.

# FRATAXIN FUNCTION AND POTENTIAL MECHANISMS OF MITOCHONDRIAL IRON ACCUMULATION

Iron is vital for the cell because it participates in essential biological processes such as DNA synthesis, gene regulation, cellular respiration, and energy production. The mitochondria play a major role in the cellular metabolism of this element and synthesis of heme and ISCs, which are needed for the function of many proteins involved in such cellular processes (Napier et al., 2005; Richardson et al., 2010). Iron homeostasis must be highly controlled because the depletion of its level can produce cellular death, and an increased level of iron promotes the production of ROS. In its Fe+2 state, iron can interact with oxygen via the Fenton and Haber-Weiss reactions to catalyze the production of ROS, which oxidize and damage proteins, lipids and nucleic acids (Eaton and Qian, 2002). Therefore, precise regulatory systems are needed to ensure the proper amount and oxidative state of iron in the cell and its different compartments.

Although frataxin has been proven to be a mitochondrial ironbinding protein (Foury et al., 2007) involved in the homeostasis of iron, its role remains unclear. Many studies have indicated that this protein could play roles related to iron storage (Park et al., 2003; Huang et al., 2011), heme (Yoon and Cowan, 2004; Napier et al., 2005) and ISC synthesis (Foury et al., 2007; Richardson et al., 2010; Lane et al., 2015) and could function as an iron sensor (Adinolfi et al., 2009) and a metabolic switch (Becker et al., 2002). Some of these possible roles have been more thoroughly tested and proven that others, with the involvement in ISC synthesis being the most accepted one thus far. We discuss these functions next and its possible relationship with mitochondrial iron accumulation in FRDA.

#### Iron Storage

The observation that yeast frataxin (Yfh1), under aerobic conditions and an excess of iron, can form spheroidal oligomers that have ferroxidase activity (Adamec et al., 2000; Gakh et al., 2002; Park et al., 2002) promptly suggested that frataxin could function as an iron storage protein, similar to ferritin (Pandolfo and Pastore, 2009). These Yfh1 multimers have the capacity to bind up to 50 atoms of iron per monomer and maintain it in a redox-inactive state through the ferroxidase activity, which converts redox-active Fe+2 to Fe+3 (Gakh et al., 2006). On this line, frataxin could store iron in a bioavailable form in the mitochondria, thus its deficit would lead to iron loading in this organelle.

Frataxins are proteins capable of binding iron. Nuclear magnetic resonance (NMR) studies predominantly mapped the iron-binding sites in the conserved acidic α1 and β1 regions, which contain a clustering of negatively charged aspartate and glutamate residues (reviewed in Bencze et al., 2006;

Pandolfo and Pastore, 2009). However, the hypothesis of frataxin as an iron storage protein presents several problems. Perhaps, one of most serious questions is that, in the case of human frataxin (FXN), the mature form of the protein is not prone to oligomerization. Only the precursor and intermediate forms of FXN can form oligomers and in an iron-independent manner (Adinolfi et al., 2002; O'Neill et al., 2005). Nevertheless, it has lately reported that the mature form of FXN, under specific experimental conditions can form oligomers, but they are unstable (Ahlgren et al., 2017). Other important caveat to this hypothesis is the redundant role that FXN would play with mitochondrial ferritin. In addition, it has been reported that oligomerization-deficient mutant Yfh1 is able to substitute functionally wild-type Yfh1 (Aloria et al., 2004), indicating that oligomerization is not required for frataxin function.

## Heme Biosynthesis

Frataxin can bind iron directly without forming oligomers (Bencze et al., 2006; Cook et al., 2006). Depending on the organism (bacteria, yeast or human) and state of iron (Fe+2 or Fe+3 ), different possible iron-binding sites in the protein have been described (Yoon and Cowan, 2003; Bou-Abdallah et al., 2004; Cook et al., 2006; Yoon et al., 2007; Huang et al., 2008). The primary site that binds Fe+2 involves the residues 18, 19 and 22 of CyaY (and corresponding ones in Yfh1 and FXN) at the acidic ridge in the α1 helix of frataxin. This raised the possibility of new functions for frataxin in irondependent processes, in which some of the involved machinery components seem to interact with FXN. Frataxin interactions have been reported with mitochondrial aconitase, ferrochelatase (Fech), succinate dehydrogenase and the components of the ISC synthesis machinery (Gerber et al., 2003; Yoon and Cowan, 2003; Bulteau et al., 2004; Bencze et al., 2006). Among these interactions, the binding of frataxin with components of the ISC complex has been reproduced and characterized in independent studies (reviewed in Martelli and Puccio, 2014).

The heme biosynthetic pathway is carried out in mitochondria. This pathway includes several enzymes (Sassa, 2006; Furuyama et al., 2007; Chung et al., 2012; Dailey and Meissner, 2013; Chiabrando et al., 2014), with the 5 aminolevulinic acid synthase (ALAS) being the first one in that pathway and the rate-limiting enzyme. ALAS has two isoenzymes, ALAS1 and ALAS2. Whereas ALAS1 expression is negatively regulated by heme directly bound to a cysteineproline dipeptide motif (Furuyama et al., 2007), the ALAS2 gene contains an iron-responsive element (IRE) that interacts with iron regulatory proteins (IRPs) regulating its expression at the posttranscriptional level. Under iron deficiency conditions, the binding of IRP to ALAS2 mRNA inhibits its translation; by contrast, under iron sufficiency conditions, IRPs detach from the IRE, resulting in increased ALAS2 translation. In this manner, the cytosolic iron availability regulates the rate of heme synthesis (Ponka, 1997).

The final enzyme in the heme biosynthetic pathway is Fech, which inserts ferrous iron into protoporphyrin IX. This enzyme contains a [2Fe-2S] cluster that is necessary for enzymatic activity (Wu et al., 2001). The disruption in ISC synthesis could adversely affect the rate of heme synthesis (Richardson et al., 2010; Huang et al., 2011). It has been shown that mitoferrin (Mfrn1) is responsible for iron transport into mitochondria, allowing the direct transfer of ferrous iron for heme and/or ISC synthesis (Fleming, 2011). The role of frataxin in heme synthesis is not clear, although a reduction of heme synthesis was observed in yeast and mouse frataxin mutants (Lesuisse et al., 2003; Huang et al., 2009). Different studies have suggested a direct role of frataxin in the heme synthesis pathway by binding directly to Fech and acting as an iron donor (Yoon and Cowan, 2004; Napier et al., 2005). Thus, this metal would accumulate because of frataxin deficiency.

#### ISC Biogenesis Chaperone

Iron-sulfur clusters are protein cofactors present in almost all living organisms. They play a critical role in several cellular functions, from electron transport in mitochondrial respiratory complexes to DNA repair or metabolism. The de novo synthesis of ISC occurs in the mitochondria (Lill, 2009; Beilschmidt and Puccio, 2014). The components of the ISC synthesis machinery are the cysteine desulfurase (NFS1), which converts L-cysteine to L-alanine with the generation of sulfur, an accessory protein (ISD11), a reductant (ferredoxin), a scaffold protein (ISCU) and an iron delivery protein that is at the core of the debate (Lill and Mühlenhoff, 2006; Py and Barras, 2010). Besides ISD11, recent findings showed that human NFS1 needs another accessory protein, the mitochondrial acyl carrier protein (ACP), making the cysteine desulfurase complex (NFS1-ISD11-ACP) (Van Vranken et al., 2016). In addition, NFS1 requires FXN for its full function (Tsai and Barondeau, 2010; Fox et al., 2015). Regarding the role of FXN in the ISC synthesis, there are two main hypothesis. It was first suggested that frataxin could be the iron donor (Gerber et al., 2003; Yoon and Cowan, 2003; Layer et al., 2006). Using a combination of different techniques based on isothermal titration calorimetry and multinuclear NMR spectroscopy, Cai et al. (2018) have recently showed that FXN can function as an iron donor in vitro. They found that FXN-Fe2+ interacts weakly to the NFS1-ISD11-ACP complex, but the presence of ISCU largely improves this interaction and that dissociation of Fe2+ from FXN occurs when the L-cysteine and ferredoxin enters into the equation. These results suggest that FXN is the proximal source of iron for ISC assembly, but in vivo characterization is required to confirm this role for frataxin.

Other studies have proposed that frataxin could actually act as a regulator in the synthesis of ISC (Adinolfi et al., 2009; Tsai and Barondeau, 2010; Bridwell-Rabb et al., 2012). The binding of frataxin to the ISCU/NFS1/ISD11 complex can stabilize it, activates the cysteine desulfurase activity and control the entry of iron to the complex (Tsai and Barondeau, 2010; Colin et al., 2013; Pandey et al., 2013).

Mutation of specific aspartate and glutamate residues in the acidic ridge of frataxin, where iron-binding sites were previously mapped, alters the interaction of frataxin with ISCU. Unexpectedly, these mutations does not reduce the total number of iron-binding sites (Schmucker et al., 2011). These results suggested that some of these residues might be critical for frataxin interaction with the core components of the ISC complex, but

not for binding iron. Consequently, some authors (Gentry et al., 2013) proposed that frataxin might have unknown binding sites for this metal. Using different spectroscopic and structural methodologies, a new iron-binding site was identified at His86 in the N-terminus of the mature FXN form, therefore not positioned in the acidic ridge. Interestingly, this residue was essential for iron binding and for in vitro productive synthesis of ISC by ISCU, without showing a direct interaction with the scaffold protein (Gentry et al., 2013). These results support the proposal that frataxin is the iron donor for ISC assembly.

Extramitochondrial frataxin has been shown to physically interact with IscU1, the cytosolic isoform of the human ISC, indicating a role for frataxin in the Fe/S cluster assembly in the cytosol (Acquaviva et al., 2005). The cytosolic form of frataxin has also been reported to interact and possibly regulate the function of the cytosolic aconitase/iron regulatory protein-1 (IRP1) (Condò et al., 2010). Other functions suggested for frataxin in the cytosol include preventing oxidative stress and apoptosis (Condò et al., 2006); and in the nucleus, frataxin could promote DNA repair and telomere stability (Lill et al., 2015).

#### Iron Sensor and Metabolic Switch

As indicate above, frataxin could act as an iron sensor to tightly regulate ISC synthesis (Adinolfi et al., 2009). In E. coli, CyaY, far from facilitating iron delivery, can inhibit the ISC synthesis process. With normal iron levels, CyaY has a low affinity for the IscS-IscU system (the bacterial ISC synthesis machinery), and ISC is loaded to the final acceptor. However, under conditions of iron excess, the affinity of CyaY for IscS would increase, slowing the rate of ISC synthesis and avoiding an overproduction of ISC. This would avoid the degradation of ISC not inserted into an acceptor protein and the release of free iron that could catalyze the production of ROS. However, the consequence of frataxin deficiency in FRDA patients is a decrease in ISC protein function, suggesting that FXN might operate as an activator of the ISC synthesis. In vitro studies also support the role of FXN as an allosteric activator of the ISC assembly (Tsai and Barondeau, 2010). In contrast, both FXN and CyaY can complement a yeast strain with deletion of Yfh1, indicating that eukaryotic and bacterial frataxins can at least partially conserve their functions (Cavadini et al., 2000; Bedekovics et al., 2007). To clarify this controversial results, Bridwell-Rabb et al. (2012) conducted enzyme kinetic assays in which analogous components of the human and E. coli ISC complex were interchanged. Interestingly, they found that CyaY could substitute FXN as an activator of the cysteine desulfurase in the human ISC complex. Conversely, FXN could also replace CyaY as an inhibitor in the assembly of the bacterial ISC. Therefore, both frataxin proteins have the property to modulate the ISC formation, but the mechanism of action of any of them, as an activator or inhibitor of ISC biogenesis, depends on another component of the ISC assembly machinery. This component was found to be the cysteine desulfurase, which controls how the frataxin protein operates in the ISC complex (Bridwell-Rabb et al., 2012). Differences between the human and bacteria cysteine desulfurase might explain the different role of frataxin in humans with respect to E. coli and by extension in eukaryotes vs prokaryotes. Further studies are needed to define the nature of these differences that dictate how frataxin acts in the process of the ISC synthesis.

Examination of the role of frataxin in ISC and heme metabolism in differentiating erythroid cells has suggested that frataxin acts as a metabolic switch in the diversion of iron from one metabolic pathway to another. This was supported by the observation that an increased level of protoporphyrin IX (PPIX) leads to the downregulation of frataxin expression and potential diversion of iron from ISC synthesis toward heme synthesis (Becker et al., 2002). The molar ratio between frataxin and Fech affects the rate of heme synthesis (Yoon and Cowan, 2004), and these findings have suggested that frataxin could function as a metabolic switch in regulating mitochondrial iron metabolism, independently of the expression levels of PPIX or Fech. This metabolic switch allows the mitochondria to favor heme or ISC syntheses, depending on the frataxin levels. The possible mechanism behind this switching may be the relative affinities of frataxin for Fech vs ISCU. This may result in the preferential binding of frataxin to Fech over ISCU at relatively lower frataxin levels (Richardson et al., 2010).

When the frataxin level is reduced, there would be no control in the rate of ISC formation, causing iron accumulation (Adinolfi et al., 2009). Regarding the metabolic switch role of frataxin, its depletion would provoke the enhancement in the reduction of ISC formation at the ISCU machinery level and at the end, subjecting the cell to increased iron accumulation and ROS production (Yoon and Cowan, 2004).

# Dysregulation of Iron Homeostasis in FRDA

Although the mechanisms responsible for mitochondrial iron loading in FRDA are not fully understood, there are several reasonable hypotheses that could explain this alteration. In the MCK-KO mouse, Huang et al. reported altered heme and ISC synthesis and mitochondrial iron storage. Significantly diminished activity and expression of ISC enzymes, such as NFS1 and ISCU1/2, were observed that agreed with the reduction in the expression of succinate dehydrogenase complex, subunit A (Sdha) (Huang et al., 2009). At the same time, other studies have shown a downregulation in the number of key heme synthesis enzymes and decreased heme levels in the heart of the MCK KO model (Schoenfeld et al., 2005; Huang et al., 2009). A decrease in the synthesis of different products that require iron in the mitochondria could explain the accumulation of this metal because it cannot be exported from the organelle being part of those products. However, frataxin deficiency can also affect the expression of several other proteins, some of them critical for the proper control of iron metabolism (**Figure 1**). In DN, Koeppen et al. (2007) observed modification of the expression of proteins such as transferrin receptor 1 (TFR1), ferritins (FRTs) and ferroportin (FPN), alterations shared among animal models of FRDA. In the MCK-KO mouse (Martelli and Puccio, 2014), has been described marked TFR1 upregulation, leading to increased iron uptake, and the downregulation of FPN, decreasing cellular iron release from the cytosol. There are also decreased levels of ferritin light chain 1 (FRTL1)

and heavy chain 1 (FRTH), implying reduced cytosolic iron incorporation and storage. Finally, an increased expression of Sec15l1 (an exocyst complex protein) and the mitochondrial iron importer mitoferrin-2 (MFRN2) in the mutant could facilitate mitochondrial iron influx, which was seen to be enhanced in this model (Whitnall et al., 2008).

The link between frataxin deficiency and alteration in the expression of some of these proteins could be IRP1 and IRP2. Both proteins bind to IRE elements in the 50 and 30 UTR of the mRNAs of iron metabolism-related proteins (Chiang et al., 2016). When bound to a 50 IRE, IRP1 and IRP2 repress the translation of the mRNAs while binding of the IRPs to a 30 IRE increases the stability and translation of the mRNAs. The activity of both IRPs is increased by the low levels of cellular iron by different mechanisms. Interestingly, IRP1 activity is controlled by the insertion of an ISC, which converts the protein into a cytosolic aconitase and makes it unable to bind to IRE elements. Since frataxin depletion reduces ISC synthesis, it is reasonable to suggest a role for IRP1 in the deregulation of iron metabolism. Nevertheless, the activity of IRP2, which is controlled by irondependent controlled ubiquitination and degradation, is also increased in MCK-KO mouse, possibly due to cytosolic iron depletion (Whitnall et al., 2008). More recently, Martelli et al. (2015) studied a double KO mouse with complete ablation of Irp1 and liver-specific deletion of Fxn. In these mice, the authors observed that alterations in the expression of proteins such as TFR1 and divalent metal transporter 1 (DMT1) were indeed due, a least partially, to IRP1, while the regulatory protein seemed to exert little effect on the expression of other proteins such as FRTL and FPN. Other models of the disease also manifest similar alterations in the expression of proteins involved in iron metabolism. Frataxin depletion in D. melanogaster produces alterations in the expression of mitoferrin, IRP-1A and ferritin, while genetic modifiers for Malvolio, Tsf1 and Tsf3 (Drosophila homologs of DMT1 and transferrins) and IRP-1A and IRP-1B could correct the motor deficiency phenotype of frataxindeficient flies (Soriano et al., 2016; Calap-Quintana et al., 2018; Monnier et al., 2018). Although some hints point to the possible causes of iron dysregulation in FRDA, more research is needed to propose a more precise and final hypothesis.

# MECHANISMS OF IRON NEUROTOXICITY IN FRDA

Initially, it was postulated that iron overload in FRDA activates the formation of ROS via the Fenton reaction, which would arbitrate the pathogenesis of the disease. The highly redox active environment in the mitochondria could promote ROS generation when the free iron content increases. Nevertheless, an ROS independent mechanism mediated by iron has recently been proposed for FRDA neurodegeneration. These two mechanisms are described in the following paragraphs (**Figure 2**).

# ROS-Dependent Mechanism of Iron Neurotoxicity

Iron accumulation in the NS is a common feature in neurodegenerative diseases, including Alzheimer's disease (Connor et al., 1992), Parkinson's disease (Gorell et al., 1995), Huntington's disease (Dumas et al., 2012) or amyotrophic lateral sclerosis (Kwan et al., 2012). In these disorders, iron deposits are observed in specific regions of the NS. As indicated previously, in Friedreich ataxia patients, deposits of iron have not been clearly reported in NS, and the increment of iron levels in the DN remains controversial (Waldvogel et al., 1999; Koeppen et al., 2007; Harding et al., 2016). This region is interesting because the patients develop neuronal atrophy and abnormal proliferation of the synaptic terminal, called grumose degeneration. The Koeppen's group confirmed the presence of iron metabolism alteration in the NS of FRDA patients by changes in the expression of proteins related to iron, such as FPN, FRT and TFR1, in the DN (Koeppen et al., 2012) and DRG (Koeppen et al., 2009). They observed a relocation of iron from the affected neurons to the microglia, in the case of DN, or from neurons to the satellite cells in the DRGs. The increment of iron levels correlates with the production of ROS in these areas, suggesting that alterations of iron homeostasis could contribute to the pathogenesis of the disease through ROS production. Additionally, frataxin deficiency is related to low antioxidant defenses (Chantrel-Groussard et al., 2001; Paupe et al., 2009), making the nervous cells have an oxidative stress-prone phenotype.

The toxicity of iron may be due to its localization, biochemical form or cell ability to prevent the generation and propagation of iron. In living organisms, iron can be found in its reduced form, ferrous iron (Fe2+), and its oxidized form, ferric iron (Fe3+). Most iron binds, as a prosthetic group, to proteins such as hemoglobin, myoglobin, cytochromes or storage proteins; only less than 5% of iron in the cell is in the labile iron pool

(LIP) (Li and Reichmann, 2016). The cellular LIP is defined as a pool of chelatable and redox-active iron complexes (Kakhlon and Cabantchik, 2002). LIP has the capacity to participate in the formation of harmful free radicals including the hydroxyl radical. Fe2+ is catalyzed to Fe3+ in a chemical reaction mediated by hydrogen peroxide (H2O2), which leads to the formation of hydroxide radical (OH•). Cytotoxic events are started in the cell via the Fenton reaction. To avoid the negative effects of excess free iron, it is coupled to proteins such as FRT, which is responsible for iron storage inside the cell. In the satellite cells of DRG from FRDA patients, an increment of cytosolic FRT (Koeppen et al., 2009) was observed; in DN, ferritin is expressed mostly in oligodendrocytes, satellite cells around the neurons. As the disease progresses, the neurons begin to undergo atrophy, and these cells disappear, producing microglia FRT-positive cells (Koeppen et al., 2007). Noteworthy, mitochondrial FRT has not been detected in DRG neurons, a protein responsible to store free mitochondrial iron and with a very important role in antioxidant activity (Koeppen et al., 2007; Campanella et al., 2009). FPN is a transmembrane protein that exports iron and is expressed in the central NS and DRG. Interestingly, in the DRG from FRDA patients, the expression of FPN is exclusive to satellite cells (Koeppen et al., 2009), probably as a response to the iron release from DRG neurons to these cells (Koeppen et al., 2016). In the synaptic terminals of neurons from the DN of FRDA patients, is observed an increment of the ferroportin levels (Koeppen et al., 2007). The nerve endings present an elevated number of mitochondria, suggesting a response in the biosynthesis of this protein due to the dyshomeostasis of iron. Thus, the toxicity of this metal could be an important factor in grumose degeneration.

Because mitochondria are an important generator of ROS, the continuous presence of iron in the mitochondrial matrix makes it susceptible to new production of ROS. Proteins with ISC clusters, such as complex I, II and III of the electron transport chain, can be damaged by ROS. These complexes show reduced activity in FRDA patients (Rötig et al., 1997), probably because of iron toxicity. Moreover, these proteins will generate new free iron that will participate in the Fenton reaction and carry out new oxidative processes. In plasma and urine from FRDA patients (Emond et al., 2000; Schulz et al., 2000), similar to in all the disease models generated (González-Cabo and Palau, 2013), oxidative stress markers have been detected.

In the last years, neuronal models of the disease have been developed to better understand the neurodegenerative process. Oxidative stress, mitochondrial dysfunction, and iron dyshomeostasis are features shared among the neuronal models. For example, the granulate cells of the cerebellum from YG8R mice show an increment of ROS, lipid peroxidation and reduced glutathione levels (Bradshaw et al., 2016); the neurons of DRG from YG8R mice present an increment of ROS, dysfunctional mitochondria, and calcium dyshomeostasis (Mollá et al., 2017); the motor neuron cell line (NSC34) with reduced expression of the frataxin gene shows an increment in the GSSG/GSH ratio and more production of ROS (Piermarini et al., 2016) and alteration in complex I activity of the electron transport chain (Carletti et al., 2014); reduction of Drosophila frataxin (fh) expression in glial cells produces massive accumulation of fatty acids and increased sensitivity to oxidative stress (Navarro et al., 2010).

Under oxidative stress, the transcription factor Nrf2 binds to antioxidant response element (ARE) and modulates the expression of many genes involved in the antioxidant defense. It has been reported that Nrf2 signaling is defective in fibroblasts from FRDA patients (Paupe et al., 2009), the DRG and cerebellum of YG8R mice (Shan et al., 2013) and motor neuron and

Llorens et al. Iron in Friedreich's Ataxia

Schwann cell models (D'Oria et al., 2013; Shan et al., 2013), making these cells and tissues more vulnerable to oxidative stress damage and the neurodegeneration process. Concretely, frataxin deficiency in neurons promotes axonal dystrophy with multiple axonal spheroid formation mainly due to Ca2+ imbalance and oxidative stress (Mollá et al., 2017). The axonal swellings are associated with specific cytoskeletal changes (Mincheva-Tasheva et al., 2014; Piermarini et al., 2016; Mollá et al., 2017), improper axonal transport and autophagic flux (Shidara and Hollenbeck, 2010; Mollá et al., 2017). Moreover, the oxidative stress damage effects to the neurites growth leads to shorter dendrites due to alterations in the dynamics of the microtubules (Shan et al., 2013; Carletti et al., 2014; Piermarini et al., 2016). When oxidative stress is reduced in these neurons, either by direct addition of antioxidants (Piermarini et al., 2016) or through drugs that increase the expression of Nrf2 (Petrillo et al., 2017), axonal growth is improved. In any case, these alterations affect the cellular viability of both neurons and glia, triggering cell death processes either by apoptosis (Mincheva-Tasheva et al., 2014; Igoillo-Esteve et al., 2015; Katsu-Jiménez et al., 2016) or autophagy (Bolinches-Amorós et al., 2014; Edenharter et al., 2018). It has been shown that iron is a key element in the induction of cell death (Dixon et al., 2012). This new process, called ferroptosis, is dependent on iron and is accompanied by an increase in ROS and lipid peroxidation and a decrease in GSH. As we have reviewed previously, these characteristics are observed in frataxin-deficient cells; therefore, new studies are needed to determine the possible cell death due to a ferroptosis process.

## ROS-Independent Mechanisms of Iron Neurotoxicity

The extent to which oxidative stress is necessary for the pathogenesis of FRDA is, however, controversial. The deficit in ISC enzymes and increase in iron levels were not paired with an increase in ROS in the MCK-KO mouse (Seznec et al., 2005), in FxnAlb mice with specific depletion of Fxn in liver (Martelli et al., 2015), and in the inducible and reversible FRDAkd mouse model (Chandran et al., 2017). Similarly, increased oxidative stress was not detected in several Drosophila knockdown models, and overexpression of ROS scavengers did not rescue the viability, heart dysfunction or neurodegeneration phenotypes (Anderson et al., 2005; Shidara and Hollenbeck, 2010; Tricoire et al., 2014; Chen et al., 2016b). Moreover, FRDA neural tissue did not show differences in the susceptibility to oxidative stress in a study using patient samples (Macevilly and Muller, 1997). The absence of ROS markers in these studies might be attributed to differences in methodology or the distinctive features of each of the animal models or tissues employed. Alternatively, it was suggested that the discrepancies in the presence of ROS would be explained by differences in the timepoints studied, in a scenario where oxidative stress would be a secondary effect rather than a primary cause, occurring later in the progression of FRDA (discussed in Lupoli et al., 2018). Recent findings in the fly and mouse models of FRDA actually support a mechanism that is independent of ROS that links neurodegeneration and iron accumulation via the sphingolipid pathway (Chen et al., 2016a,b).

Alterations in the homeostasis of lipids are not exclusive of FRDA (Huang et al., 1978; Filla et al., 1980; Thurman and Draper, 1989; Raman S.V. et al., 2011; Worth et al., 2014) and have been reported in neurodegenerative disorders, including Parkinson's, Alzheimer's, Huntington's, multiple sclerosis and Niemann–Pick diseases (reviewed in Shamim et al., 2018). In the case of FRDA, lipid droplet accumulation was first described in the cardiac muscle of the NSE-KO mouse model (Puccio et al., 2001) and was further characterized as pathological in a glial frataxin knockdown model in Drosophila (Navarro et al., 2010). The accumulation and dyshomeostasis of lipids have been reported as well in several other cellular and animal models of the disease (Martelli et al., 2012; Schiavi et al., 2013; Obis et al., 2014). More recently, the expression of a mutant allele of the Drosophila fh in mosaic clones produced an age-dependent degeneration of photoreceptor neurons with impaired mitochondrial function, increased levels of Fe2+ and Fe3+ in multiple tissues and dramatic lipid droplet accumulation in glial cells (Chen et al., 2016b), consistent with the work from Navarro et al. (2010). Because the Fenton reaction can induce lipid peroxidation, oxidative stress has been classically proposed to be the link between abnormal lipid homeostasis and neurodegeneration in FRDA. However, Chen et al. did not observe an increase in free radical production and, neither providing the antioxidant AD4 nor overexpressing the ROS scavengers catalase, SOD1 and SOD2, suppressed neurodegeneration (Chen et al., 2016b). Moreover, reducing the number of lipid droplets by overexpressing brummer lipase or lipase 4 was insufficient to improve the neurodegeneration defect in the knockout photoreceptors. Because high iron conditions increase sphingolipid synthesis in wild-type yeast (Lee et al., 2012), Chen et al. (2016b) pursued the characterization of this pathway in their fly model. The synthesis of sphingolipids, including dihydrosphingosine, dihydroceramide and sphingosine, was actually increased in the fh mutants. Both the genetic knockdown and chemical inhibition of the fly serine palmitoyltransferase, an enzyme essential for the de novo synthesis of sphingolipids, rescued neurodegeneration. Additionally, they found that downstream targets of the transcription factor Mef2 were upregulated in response to the increase in sphingolipids in fh mutants and knocking down the Pdk1/Mef2 pathway led to the suppression of photoreceptor degeneration. To ask whether this novel pathway described in Drosophila was conserved in vertebrates, Chen et al. (2016a) developed a mouse model with reduced Fxn levels in the NS. Both ferrous and ferric iron were found to be increased in the cerebral cortex and, consistent with their observation in the fly model, they could not find increased ROS production measured as levels of lipid peroxidation. Loss of Fxn in the NS led to the activation of the PDK1/Mef2 pathway in the mouse before the appearance of the neurological manifestations. Sphingolipids and PDK1 activation were also found to be increased in cardiac tissue from FRDA patients (Chen et al., 2016a). Nevertheless, alterations in the PDK1/Mef2 pathway were not replicated in the FRDAkd-inducible mouse model of FRDA (Chandran et al., 2017). In this study, the authors did not find changes in PDK phosphorylation in the brain, muscle, heart or liver in the chronic

adult knockdown mice. The levels in the cerebellum and heart of five target genes of Mef2 were analyzed as well and displayed changes that were inconsistent across tissues. Alternatively, other biochemical pathways could be altered in relation to iron under frataxin-deficient conditions. In line, several genes involved in hemochromatosis, an iron-overload disorder, are upregulated in the FRDAkd-inducible mouse model, in which no increase in ROS and no strong evidence of Pdk1/Mef2 activation were observed (Chandran et al., 2017).

## POTENTIAL TREATMENT OF FRDA BY REDUCING IRON TOXICITY

An optimal therapy in FRDA could consist of providing patients with sufficient frataxin amount to restore the functions of this protein. Several strategies are being developed to obtain this objective, such as gene therapy (Perdomini et al., 2014; Piguet et al., 2018), transplanting hematopoietic stem and progenitor cells (Rocca et al., 2017) or improving FXN transcription (Soragni and Gottesfeld, 2016). While advancing the development of such strategies for its application to patients, rational treatment could be considered to remove excess iron.

The utilization of iron chelators is one of the first strategies proposed to treat FRDA patients to eliminate excess iron accumulation in the mitochondria. To that end, an iron chelator should be able to cross the cell membranes, access mitochondria and redistribute the excess labile iron. Deferiprone (DFP, 1, 2 dimethyl-3-hydroxy-pyrid-4-one), an orally administered lipid soluble iron chelator can easily cross the blood–brain barrier and cellular membranes. DFP shows relatively low affinity for iron and can transfer this metal to circulating transferrin, as well as to cellular acceptors—e.g., for heme synthesis (Sohn et al., 2008). Therefore, its use is less probable to produce iron depletion than other known chelators. For all these reasons, DFP has been considered as the prototypic compound for iron redeployment (Cabantchik et al., 2013). It is also used to treat iron-overloaded patients with β-thalassemia who need continuous blood transfusions (Totadri et al., 2015), and their properties make it an interesting therapeutic compound for FRDA.

The use of DFP in cellular and animal models of FRDA has beneficial results, e.g., frataxin-deficient HEK-293 cells could restore mitochondrial dysfunction after DFP treatment. It was attributed to the ability of this compound to make available the iron accumulated into mitochondria (Kakhlon et al., 2008). These conclusions were also supported by our group using a Drosophila FRDA model, in which DFP improved the survival and climbing ability phenotypes because of the removal of excess free mitochondrial iron, thereby preventing its toxic effects (Soriano et al., 2013). Recently, it has been reported that DFP is effective to reduce ROS production and improve calcium handling kinetics in an FRDA humaninduced pluripotent stem cell-derived cardiomyocyte model (Lee et al., 2016).

The efficacy of DFP has already been assessed in clinical trials in FRDA patients, but the results are inconclusive. However, considering these studies, DFP has shown beneficial effects on cardiac parameters achieved at low doses of the compound, while treatment with higher doses of DFP could aggravate the disorder by affecting the ataxia phenotype (Pandolfo et al., 2014). A high dose of DFP could remove too much iron, provoking a deficiency of this metal, which could affect the cellular functions that require iron. On the other hand, the positive action of this drug on the heart compared with NS has also been related to the initiation of treatment before cardiac dysfunction is present (Elincx-Benizri et al., 2016). Overall, DFP seems to have therapeutic value in FRDA and is quite safe at lower doses, but systematic, large scale, placebo controlled longer studies are needed to evaluate its usefulness (Strawser et al., 2017). In line with these issues, our results using a Drosophila model of the disease indicate that DFP treatments at the larval stage recover FRDA-like phenotypes in flies, but no improvement was observed when the drug is administered to adults (Soriano et al., 2013). Additionally, we found that reducing the excess of iron accumulating into mitochondria through a genetic strategy has benefit in this FRDA model (Navarro et al., 2015; Soriano et al., 2016). These results show a causal relationship between iron and frataxin deficiency. Thus, iron is a critical factor in the pathophysiology of FRDA and appears not be a simple epiphenomenal event in the disease. Consequently, research on how to deal with the iron excess and eradicate the iron toxicity is still an area of great interest.

In the last decades, some small molecules with pharmacophore characteristics for iron chelation are being explored for treatment in neurodegenerative diseases with regional increase of iron levels (reviewed in Singh et al., 2018). These compounds are derivatives of the 8-hydroxyquinoline, hydroxypyridone, and arylhydrazones, and some of them are able to reduce selectively the iron excess in the mitochondria (Mena et al., 2015). The use of pro-chelating compounds, which are activated in the target region, have the benefit to remove the excess of iron in the affected regions. Thus, this strategy would protect cells in non-affected areas from the risk of iron subtraction. Novel pro-chelators triggered by H2O2 were obtained as promising candidates for management of Parkinson's disease (Perez et al., 2008). Similarly, this approach might be useful in FRDA since H2O2 is a critical pathogenic agent associated to frataxin deficiency (Anderson et al., 2008). Finally, new compounds with multifunctional actions, as previously indicated for DFP, are also being examined for effective treatment of neurodegenerative diseases (Singh et al., 2018). Several of these diseases, including FRDA, share some pathological features such as regional iron increase, oxidative stress and dysregulation of calcium signaling. Recently, a novel compound, named CT51 (N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2 yl)-2-(7-hydroxy-2-oxo-2H-chromen-4 yl) acetamide), has been synthesized that seems to suppress these cellular impairments (García-Beltrán et al., 2017). CT51 can easily permeate the cell membrane and distribute to both mitochondria and the cytoplasm. It works as a highly selective iron chelator and possesses significant capability for free radical quenching, protecting cells against oxidative damage. The use of CT51 in primary hippocampal neurons decreases the continuous release

of calcium provoked by an agonist of ryanodine receptorcalcium channels, supporting a protective role of this compound in neuronal function. Although developments of this kind of drugs are not a curative strategy, because the frataxin deficiency will persist, they might improve the current pharmacological treatments and might produce increases in quality of life and slowing of disease progression.

#### CONCLUSION

The relationship between neurodegeneration and iron accumulation is well established. However, in Friedreich's ataxia, it remains unclear whether iron loading is the cause or consequence of the pathophysiology. The studies in model organisms suggested both hypotheses in relation to the role of iron in this disease: a key role in the pathophysiologic mechanisms or a secondary event that appeared late in the disease progression. It was also debated the extent to which oxidative stress is crucial to explain the pathogenesis of FRDA. Indeed, additional pathways might be involved in the toxicity of iron independent of ROS production. Differences in the distinctive features of each of the FRDA models studied, vulnerability of the different tissues and cells to frataxin deficit and timepoints analyzed could partially explain the contradictory results. Nevertheless, from the therapeutic point of view, it is interesting to explore whether new iron chelators showing

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#### AUTHOR CONTRIBUTIONS

JL and MM planned the manuscript. JL, SS, PC-Q, PG-C, and MM wrote the manuscript and agreed on the finally submitted version of the manuscript.

## FUNDING

Financial support of this work came from Generalitat Valenciana, Spain (PROMETEOII/2014/067) to MM, the Spanish Ministry of Economy and Competitiveness [SAF2015-66625-R] within the framework of the National R+D+I Plan to PG-C, Fundación Ramón Areces, Spain (CIVP18A3899) to PG-C and MM. PC-Q was a recipient of a fellowship from Generalitat Valenciana, Spain, and JL is supported by a research contract from FARA and FARA Ireland.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.00075/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Llorens, Soriano, Calap-Quintana, Gonzalez-Cabo and Moltó. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Deciphering the Iron Side of Stroke: Neurodegeneration at the Crossroads Between Iron Dyshomeostasis, Excitotoxicity, and Ferroptosis

Núria DeGregorio-Rocasolano1 , Octavi Martí-Sistac1,2 \* and Teresa Gasull1 \*

1 Cellular and Molecular Neurobiology Research Group, Department of Neurosciences, Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain, 2 Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain

#### Edited by:

Isabella Zanella, Università degli Studi di Brescia, Italy

#### Reviewed by:

Alessandro Fanzani, Università degli Studi di Brescia, Italy Ashley Ian Bush, The Florey Institute of Neuroscience and Mental Health and The University of Melbourne, Australia Gladys Oluyemisi Latunde-Dada, King's College London, United Kingdom

#### \*Correspondence:

Octavi Martí-Sistac<EMAIL_ADDRESS>Teresa Gasull<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 28 October 2018 Accepted: 25 January 2019 Published: 19 February 2019

#### Citation:

DeGregorio-Rocasolano N, Martí-Sistac O and Gasull T (2019) Deciphering the Iron Side of Stroke: Neurodegeneration at the Crossroads Between Iron Dyshomeostasis, Excitotoxicity, and Ferroptosis. Front. Neurosci. 13:85. doi: 10.3389/fnins.2019.00085 In general, iron represents a double-edged sword in metabolism in most tissues, especially in the brain. Although the high metabolic demands of brain cells require iron as a redox-active metal for ATP-producing enzymes, the brain is highly vulnerable to the devastating consequences of excessive iron-induced oxidative stress and, as recently found, to ferroptosis as well. The blood–brain barrier (BBB) protects the brain from fluctuations in systemic iron. Under pathological conditions, especially in acute brain pathologies such as stroke, the BBB is disrupted, and iron pools from the blood gain sudden access to the brain parenchyma, which is crucial in mediating strokeinduced neurodegeneration. Each brain cell type reacts with changes in their expression of proteins involved in iron uptake, efflux, storage, and mobilization to preserve its internal iron homeostasis, with specific organelles such as mitochondria showing specialized responses. However, during ischemia, neurons are challenged with excess extracellular glutamate in the presence of high levels of extracellular iron; this causes glutamate receptor overactivation that boosts neuronal iron uptake and a subsequent overproduction of membrane peroxides. This glutamate-driven neuronal death can be attenuated by iron-chelating compounds or free radical scavenger molecules. Moreover, vascular wall rupture in hemorrhagic stroke results in the accumulation and lysis of iron-rich red blood cells at the brain parenchyma and the subsequent presence of hemoglobin and heme iron at the extracellular milieu, thereby contributing to ironinduced lipid peroxidation and cell death. This review summarizes recent progresses made in understanding the ferroptosis component underlying both ischemic and hemorrhagic stroke subtypes.

Keywords: iron, reactive oxygen species, ferroptosis, stroke, excitotoxicity, iron dyshomeostasis, neurodegeneration, transferrin saturation

**Abbreviations:** AIS, acute ischemic stroke; ALOX5: arachidonic acid lipoxygenase 5; APP, amyloid precursor protein; BBB, brain–blood barrier; CSF, cerebrospinal fluid; Dexras1, dexamethasone-induced Ras protein 1; DFO, deferoxamine; DMT1, divalent metal transporter 1; EAATs, excitatory amino acid transporters; FPN, ferroportin; FT, ferritin; FTH, ferritin heavy chain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPX4, glutathione peroxidase 4; GSH, glutathione; Hb, hemoglobin; HO, heme oxygenase; Hp, haptoglobin; Hpx, hemopexin; HTf, holotransferrin; ICH, intracerebral hemorrhage; I/R, ischemia/reperfusion; IREs, iron regulatory elements; IRPs, iron regulatory proteins; NAC, N-acetylcysteine; NMDA, N-methyl-D-aspartate; NTBI, non-transferrin-bound iron; OGD, oxygen and glucose deprivation; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; TIM, T-cell immunoglobulin and mucin domain-containing receptor; Tf, transferrin; TfR1, transferrin receptor 1; TfR2, transferrin receptor 2; TSAT, saturation of transferrin with iron; UTRs, untranslated regions; X− c , cystine/glutamate exchanger or antiporter, i.e., SLC7A11/xCT.

#### IRON TRANSPORT TO THE BRAIN: THE ROLE OF THE BLOOD–BRAIN BARRIER

Iron is essential for life; numerous proteins require iron as a cofactor for their activity. Iron acts as an electron donor or acceptor, and thus is pivotal for oxygen transport (coordinated with hemoglobin), cellular respiration (as a part of hemecontaining cytochromes and proteins containing Fe–S groups in the electron transport chain), and DNA synthesis (in ribonuclease reductase). Moreover, in the nervous system, iron is essential for myelinization and neurotransmitter biosynthesis.

Iron exists both as Fe2+ and Fe3+; redox-active and cycling between these two states lead to the generation of reactive oxygen species (ROS) through the Fenton reaction. The ROS promote oxidative stress and cause extensive lipid peroxidation (Gutteridge et al., 1979). The brain is particularly vulnerable to lipid peroxidation damage because it is rich in polyunsaturated fatty acids (PUFAs) and iron, but relatively poor in antioxidant defenses (Cobley et al., 2018). The iron content remarkably differs across different brain regions: it is high in the striatum, medium in the hippocampus and cortex, and low in the pons and medulla (Ramos et al., 2014). Although it has recently attracted considerable attention, iron metabolism in the brain is yet not completely understood as that in other murine tissues (Ashraf et al., 2018). Brain imports iron from systemic circulation, and the levels of iron are determined by diet, intestinal iron absorption, release of iron initially stored in the hepatocytes, and export of iron recycled from macrophages. In addition, the levels of iron in the plasma are regulated by the hormone hepcidin through its effect on the transmembrane iron export protein ferroportin (FPN), which is mainly present at the cell membrane of macrophages, hepatocytes, and enterocytes.

In the blood and most other extracellular fluids, iron is essentially carried bound with high affinity to the iron transport protein transferrin (Tf), which shields Fe3+ from redox activity. In addition to Tf, iron also associates with other carrier proteins in the blood such as ferritin (FT), haptoglobin (Hp), Hpx, or albumin. Recently, half a dozen different non-proteinaceous lowmolecular-weight iron species (<10 KDa) have been shown to exist in the blood (Dziuba and Lindahl, 2018); further studies are required to determine the precise identity and physiological role of these species. Once iron is bound to Tf, Fe3+ is rendered redox-inactive and virtually non-exchangeable or displaceable by other physiological metals or molecules. Tf−iron complexes circulate in the blood until they bind transferrin receptors (TfRs) in cellular membranes of target cells. In general, in non-ironoverload conditions, little, if any, non-transferrin-bound iron (NTBI) is found in the blood of healthy individuals (Gaasch et al., 2007). The high affinity of Tf for iron and the large amount of Tf in the blood exceed the total requirement for iron binding; in normal human blood, the saturation of transferrin with iron (TSAT) is only of around 30% (De Valk et al., 2000; Van Dijk et al., 2008). Therefore, iron transport to the brain is mainly from iron-loaded transferrin (holotransferrin, HTf) through the endothelial cells of the BBB and the epithelium of the choroid plexuses and arachnoid membrane, the blood–cerebrospinal fluid (CSF) barrier.

Although barrier cells of the brain form tight junctions laterally, epithelial cells of the choroid plexuses are fenestrated and more permeable than the endothelial cells of the BBB (Strazielle and Ghersi-Egea, 2016). The abluminal side of brain capillary endothelial cells is surrounded by pericytes and astrocytes of the neurovascular unit that provide extra structural and functional support. TfRs are abundantly expressed in the luminal side of membranes of polarized endothelial cells; once they bind Tf, the TfR–Tf complexes undergo endocytosis. The following steps are still controversial, and different pathways are thought to be involved in brain iron acquisition (Bradbury, 1997; Khan et al., 2018). Thus far, the main contribution to this process is thought to include the following pathways: (1) endocytic vesicles containing TfR–HTf traveling from the luminal membrane to fuse to the abluminal membrane and exocytose either HTf or free iron into the brain extracellular space; (2) under low pH conditions within the endosome of endothelial cells, as in most cell types, iron is extracted from Tf, converted to Fe2+ by endosomal ferrireductases such as the six-transmembrane epithelial antigen of prostate protein 3 (Steap3), and released to the cytosol through divalent metal transporter 1 (DMT1) or the ion channels TRPML, also known as mucolipin 1 or MCOLN1 (detailed information can be found in references Luck and Mason, 2012; Skjorringe et al., 2015). Subsequently, Fe2+ from the cytosol is exported through FPN present in the abluminal membrane of endothelial cells, which are in contact with astrocytes expressing ceruloplasmin, that rapidly oxidize Fe2+ to Fe3+ (this Tf/TfR endocytosis mechanism is common for endothelial and neuronal cells and is depicted in **Figures 1**, **2**). A recent study performed using comprehensive mathematical models concluded that optimal iron homeostasis is dependent on the degree of saturation and the transport of HTf across the BBB from the blood (Khan et al., 2018).

In addition, in mice exposed to experimental stroke under normal iron conditions, iron deposits in the microvasculature were observed early in the border zone of the damaged areas (Desestret et al., 2009), suggesting that, when signaled, the BBB might function as an iron reservoir from which iron is released into the brain. Regarding signaling to release, in bovine retinal endothelial cells, the FPN-mediated iron export is known to be blocked by the FPN inhibitor hepcidin (Hadziahmetovic et al., 2011). In addition, new insights into the communication between systemic iron status and brain iron uptake reveal the regulation of brain iron uptake at the level of brain microvascular endothelial cells rather than the BBB acting merely as a retaining wall (Chiou et al., 2018b). In support of this regulatory hypothesis, endothelial microvascular cell distribution of TfR is altered in luminal, intracellular, and abluminal membranes depending on brain iron status (Simpson et al., 2015), and microvascular cell iron availability and efflux have been found to be tightly modulated by diffusible ceruloplasmin and hepcidin released from astrocytes surrounding the BBB (McCarthy and Kosman, 2014). Moreover, a recent study showed that inhibition of DMT1 alters the transport of iron and Tf across the endothelial cells and proposed that the levels of irondevoid Tf (apotransferrin) and iron delivered to the brain are strongly regulated by the ratio apotransferrin:HTf as well as by

FIGURE 1 | Schematic drawing of some of the main players in iron transport to the brain in control (left hand upper side), iron overload (right-hand upper side), ischemic stroke (left-hand lower side), or intracerebral hemorrhage (right-hand lower side). The thicker the arrows the more increased the transport. Abbreviations: ATf, apotransferrin; FPN, ferroportin; HTf, holotransferrin; FT, ferritin, GPX4, glutathione peroxidase 4; GSH, glutathione; Hb, hemoglobin; NTBI, non-transferrin-bound iron; ROS, reactive oxygen species; TJ, tight junction; ZA, zonula occludens.

hepcidin levels in the extracellular fluid of each brain region (Duck et al., 2017).

Further, very recently, a new function for FT heavy chain (FTH) as a transporter of NTBI across the BBB by binding to the T-cell immunoglobulin and mucin domain-containing 1 receptor 1 (TIM-1) has been reported in human endothelial cells (Chiou et al., 2018b).

In the context of physiological iron-overload conditions found, for instance, in individuals on iron-rich diets, a significant pool of NTBI is found in the blood and might be readily absorbed by microvascular endothelial cells. This effect might be relevant considering that regional differences in the brain uptake of 59Fe–NTBI versus 59Fe–TfR have been reported (Deane et al., 2004), and that 59Fe–NTBI seems to be transported faster than 59Fe–TfR into the brain parenchyma of iron-overloaded diseasefree mice. Furthermore, the iron storage protein FT has been found to be upregulated in epithelial cells of the choroid plexus and endothelial cells of the brain during systemic iron overload (Tripathi et al., 2017).

## IRON UPTAKE AND HANDLING BY PARENCHYMAL BRAIN CELLS

Once in the brain interstitial fluid, both TBI and NTBI are potential iron sources for brain cells (Gaasch et al., 2007); unlike

glutathione; GSSG, glutathione disulfide; Hb, hemoglobin; HFE, hereditary hemochromatosis protein; HIF, hypoxia-inducible factor; HO1, heme oxygenase 1; Hp, haptoglobin; Hpx, hemopexin; HTf, holotransferrin; IRE, iron-responsive element; IRPs, iron regulatory proteins; LIP, labile iron pool; MCOLN1, mucolipin 1; MtFT, mitochondrial ferritin; NCOA, nuclear receptor coactivator; NMDAR, NMDA receptor; NTBI, non-transferrin-bound iron; OH, hydroxyl radical; PCBP, poly(rC)-binding protein; PUFA, poly-unsaturated fatty acid; PUFA-OOH, PUFA peroxide; ROS, reactive oxygen species; Se, selenium; Steap, six-transmembrane epithelial antigen of prostate; system X− c , cystine–glutamate antiporter; TfR1, transferrin receptor 1; TfR2, transferrin receptor 2; ZIP8/14, zinc transporters 8/14. The picture depicts the effect of one of the major players in the pathophysiology of brain I, the disruption of glutamatergic neurotransmission homeostasis that produces elevated extracellular glutamate levels, overactivation of the NMDA subtype of glutamate receptors (NMDAR), and excitotoxic cell death. In the recent past, different authors have shown that this NMDAR overactivation boosts neuronal iron uptake and produces an iron-dependent form of neuronal death associated with massive lipid peroxidation that fits with the definition of ferroptosis. Further, in the hemorrhage type of stroke, iron derived from hemoglobin and/or heme enters neurons and produces massive lipid peroxidation. Damage by ischemic and hemorrhagic stroke is alleviated by the inhibitors of ferroptosis.

blood Tf, CSF Tf is considered to be completely saturated with iron (Bradbury, 1997). Within the extracellular milieu of the brain parenchyma, iron is carried loaded into Tf or bound to low-molecular-weight molecules such as citrate, ascorbate, or ATP. Each brain cell type uptakes extracellular TBI or NTBI depending on the iron-transport receptors and channels present in their membranes and, physiologically, safely handles iron by using the network of proteins they specifically express to import, store, traffic, and export iron. Players of cellular iron homeostasis are cell type-specific, and the list of proteins involved in iron homeostasis is still growing. As an example of iron-related new proteins, only 3 years ago, the members of the ZIP family of metal ion transporters Zip8 and Zip14, which, unlike the DMT1 transporter are only active at normal physiological pH, were reported to be expressed by hippocampal neurons, and Zip8 was found to play a major role in the accumulation of NTBI by hippocampal neurons (Ji and Kosman, 2015), whereas both Zip8 and Zip14 were considered to play a role in iron homeostasis in rat retina (Sterling et al., 2017).

# Receptors Involved in Iron Uptake by Brain Cells

In addition to the well-known TfR1 as a main player of the TfR/Tf endocytic pathway in most brain cells, other proteins, e.g., transferrin receptor 2 (TfR2) (Kawabata, 2018) or glyceraldehyde-

3-phosphate dehydrogenase (GAPDH) (Raje et al., 2007), have been described as receptors for Tf and are currently being investigated. TfR1 binds HTf with a Kd of 1 nM; it is expressed in response to changes in iron levels through modulation by iron regulatory elements (IREs) present in its mRNA and binds hereditary hemochromatosis HFE protein, a molecule that competes with HTf for binding to TfR1. In contrast, TfR2 has lower affinity for HTf; its levels are not modulated by IREs (West et al., 2000) and, although still controversial, seems to bind HFE protein in a sequence non-overlapping to that of the Tf-binding site (for a review see Worthen and Enns, 2014).

The TfR2 is present in two main isoforms, TfR2α and TfR2β. The TfR2α form has been reported to be more abundant in the brain, especially in the hippocampus; brain TfR2α levels were reported to significantly differ across animals fed iron-deficient or iron-enriched diets (Pellegrino et al., 2016). A specific role for TfR2 in the control of the iron regulatory network in the brain tissue has been recently suggested, as altered brain IRPs, iron accumulation, and reactive microglia signaling were found in TfR2-KO mice (Pellegrino et al., 2016). In contrast, a previous study on TfR2 mutant mice showed changes in hepatic, but not brain iron levels, although that study reported a 30% increase in brain FT levels (Acikyol et al., 2013).

In addition to TfR1 and TfR2, the multifunctional molecule GAPDH has been reported to work as a TfR in different cell types (Raje et al., 2007; Kumar et al., 2012) with a role in iron uptake and/or iron export (Sheokand et al., 2016) and as a chaperone for intracellular heme traffic (Sweeny et al., 2018). Relevant to brain and excitotoxicity, GAPDH interacts with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors in the neurons without glutamate stimulation and has also been reported to be pivotal for axon development (Lee et al., 2016).

Another receptor-mediated means to import iron into brain cells, at least in oligodendrocytes, is the uptake of FTH through TIM-1/2 receptors (Todorich et al., 2008, 2011; Chiou et al., 2018a). In other non-neuronal cells, FTH internalize through TfR1 (Li et al., 2010), whereas scavenger receptor class A member 5 (SCARA5) has been found to play also a role as a receptor for FT light chain uptake (Li et al., 2009). To the best of our knowledge, the role of SCARA5 has not yet been investigated in any brain cell type.

# Mechanisms and Proteins Involved in Cytosolic Iron Handling in Different Brain Cell Types

Inside the cell cytosolic and nuclear compartments, iron chaperone poly(rC)-binding proteins serve the binding and delivery of iron to iron-requiring enzymes, ubiquitous protein of iron storage FT, or the iron export membrane protein FPN (Philpott et al., 2017). In the cytosol, unused iron is stored into FT, a molecule composed of 24 subunits of a variable proportion of heavy and light chains, that shields up to 4500 iron atoms in a redox inert state. Neurons express both TfR1 and DMT1 (Pelizzoni et al., 2012; DeGregorio-Rocasolano et al., 2018); DMT1 is mainly found in the cytoplasm colocalizing with endosomes/lysosomes. Further, neurons express the iron export molecule FPN (Burdo et al., 2001; Zhou et al., 2017). Astrocytes express TfR1, DMT1, and FPN (Yang et al., 2012; Huang et al., 2014). Hence, although TfR1 is considered to be the main gate for cellular iron import, control astrocytes show low TfR1 levels, and most mature oligodendrocytes or microglial cells do not express TfR1 (Kaur and Ling, 1995; Rathnasamy et al., 2016). However, under hypoxia, both astrocytes and oligodendrocytes are found to upregulate TfR (Yang et al., 2012; Rathnasamy et al., 2016). Moreover, oligodendrocytes and cells of choroid plexuses, but not other brain cell types, express Tf under normal condition (Leitner and Connor, 2012). All the above are examples of the important heterogeneity in the expression and induction of iron homeostatic or IRPs observed in different brain cell types and situations. Most of the proteins involved in iron metabolism, including cytosolic FT levels, are mainly regulated by iron, oxidative stress, and inflammation. The irondependent regulation occurs mainly at the post-transcriptional level via the IREs and iron regulatory protein (IRP) machinery. When iron levels are high, iron binds to the IRP, which then loses affinity for the IRE sequence in FT mRNA, resulting in increased FT expression (the cytosolic iron handling is depicted in **Figure 2**) (Arosio et al., 2017). Several studies have shown that the mobilization of iron from FT requires FT degradation either by lysosome–autophagy or ubiquitin proteasome pathways (Asano et al., 2011; Mancias et al., 2015). In addition to its role in storing iron within the cell, FT can be secreted (Truman-Rosentsvit et al., 2018).

In addition to post-transcriptional regulation, transcriptional regulation has been reported for some of the proteins involved in iron homeostasis through the hypoxia-inducible factor family of transcription factors or the heme-dependent transcription factors (e.g., for TfR1 or FPN) (Marro et al., 2010; Man-man et al., 2017). Moreover, the master regulator of bodily iron homeostasis hepcidin is subjected to a sophisticated transcriptional regulation through the bone morphogenetic protein/SMAD pathway, interleukin 6 pathway, or erythropoietin/erythroferrone pathways when regulated according to the iron, inflammatory, or erythropoietic demands (Sangkhae and Nemeth, 2017). The regulation of cellular iron balance has been recently reviewed (Recalcati et al., 2017).

Iron also plays an important role in specific organelles; this is especially true for mitochondria. Mitochondria house the assembly of heme, the majority of the Fe–S cluster biosynthesis apparatus, and contain the respiratory complexes that need ironcontaining cofactors. In addition, mitochondria are a major source of endogenous ROS. Sustained iron exposure increased mitochondrial ROS levels in dopaminergic neuroblastoma SH-SY5Y cells (Huang et al., 2017), and scavenging of mitochondrial ROS was protective against iron overload damage in hippocampal neurons, preserving mitochondrial morphological integrity and membrane potential (Pelizzoni et al., 2011). In addition, a recent study showed that sequestration of iron by mitochondrial FT was neuroprotective against oxidative stress and played an important role in preventing neuronal damage in some other conditions (You et al., 2016; Guan et al., 2017). Moreover, mitochondrial FT overexpression significantly reduced cellular labile iron pool,

prevented H2O2-induced elevation of labile iron, and rescued cells from H2O2-induced damage (Gao et al., 2017).

In several cell types, although not yet studied in neurons, mitochondria−endosome interactions have been observed, suggesting a possible direct transfer of iron from endosomes to mitochondria (Das et al., 2016; Hamdi et al., 2016) through the "kiss-and-run" hypothesis.

#### IRON DYSHOMEOSTASIS IN THE BRAIN

Brain iron abnormalities are associated to rare, but severe, neurodegenerative conditions, and there is growing evidence that the more common systemic iron overload disorders such as hereditary hemochromatosis exert important effects on the brain iron content and pathological brain iron deposition (Nandar and Connor, 2011; Kumar et al., 2016). Moreover, the hemochromatosis H63D polymorphism of the HFE gene could be a disease-modifying gene in frontotemporal lobar degeneration, fostering iron deposition in the basal ganglia (Gazzina et al., 2016), or even macro- and microanatomically altering some brain structures associated with changes in Tf levels in the blood (Jahanshad et al., 2012). Recently, abnormal recycling of TfR1 due to reduced post-translational palmitoylation has been reported to be crucial to affect the iron import in the socalled neurodegeneration with brain iron accumulation disease (Drecourt et al., 2018). Further, neurodegeneration with brain iron accumulation disease-linked genes that showed altered expression in response to iron loading has been recently reported to be directly or indirectly related to myelin metabolism (Heidari et al., 2016).

A list of mild conditions, including aging (Ward et al., 2014), continuous uptake of some bioavailable iron sources (Peters et al., 2018), or obesity (Han et al., 2017), have been reported to alter brain iron content and/or distribution. In addition, a recent report showed that iron administration increases iron levels in the brains of healthy rats, which induces brain changes and triggers a hormetic response that reduces oxidative damage (Piloni et al., 2018).  This dyshomeostasis is especially important in acute pathologies such as stroke, in which the impairment of the BBB regulatory role is rapidly and massively affected following either rupture of an artery (ICH) or aberrant increase of brain microvascular endothelial permeability following ischemic stroke (AIS). Dysregulation of iron resulting in the accumulation of free iron and brain iron overload, which can increase the production of ROS, is evident in aging and stroke-related pathologies and is also a hallmark of chronic and long-term neurodegenerative pathologies. In this regard, iron overload in the brain has been reported in Huntington, Parkinson, or Alzheimer diseases [readers interested in the topic are referred to recent detailed reviews by Morris et al. (2018) and Uranga and Salvador (2018)]. Interestingly, marked age-related changes in brain iron homeostasis have also been observed in amyloid precursor protein (APP)-knockout mice (Belaidi et al., 2018).

#### IRON OVERLOAD WORSENS NEUROLOGICAL DAMAGE BOTH IN ISCHEMIC STROKE AND INTRACEREBRAL HEMORRHAGE

Stroke is a life-threatening disease that causes high rates of permanent disability subsequent to neuronal loss. There are two major types of strokes: ischemic stroke or AIS, which is caused by a blood clot blocking an artery and accounts for 85% of all strokes, and hemorrhagic stroke or ICH, caused by leakage or rupture of an artery and accounts for 15% of stroke cases.

#### Iron Overload Condition in Stroke Damage and Outcome

In ischemic stroke, neurons die because of interrelated processes that include glutamate excitotoxicity, excess of free radical production, and inflammation (Nagy and Nardai, 2017). Each of these mechanisms has already been targeted in clinical trials, although unsuccessfully (for a review see Chamorro et al., 2016). Moreover, free radical-induced oxidative stress has been for long recognized as a pathogenic factor in the ischemia/reperfusion (I/R) injury that follows reoxygenation. The hydroxyl radical, the most harmful of the free radicals, is generated through reactions catalyzed by iron (Kell, 2009; Chen H. et al., 2011). Of relevance to stroke damage and final outcome, several clinical studies indicate that a previous systemic iron overload condition, measured as high levels of serum FT at admission, is associated with increased brain damage and worse outcome induced by ischemic stroke (Davalos et al., 1994; Millan et al., 2007).  Iron overload is a major source of oxidative stress in ischemic brains (Carbonell and Rama, 2007). In addition, iron overload exacerbated the risk of hemorrhagic transformation in animal models of transient ischemia plus early reperfusion (García-Yébenes et al., 2012; García-Yébenes et al., 2018); therefore, in iron-overloaded animals, an initial ischemic event has higher probability to become an ICH. The contribution of iron overload to widespread the damage in the ICH is supported by the fact that systemic iron overload (estimated using high serum FT levels) was associated with higher perihematoma edema and poor outcomes in patients with ICH (Mehdiratta et al., 2008; Perez de la Ossa et al., 2010; Bakhshayesh et al., 2014; Garton et al., 2017). Further, a combination of high serum FT and low serum iron and Tf was associated with poor outcome in ICH patients (Yang et al., 2016). Therefore, systemic iron overload impacts the outcome of both ischemic and hemorrhagic stroke patients.

The brain is physiologically sheltered from fluctuations in systemic iron because of the BBB, even under experimentally

induced diet iron overload conditions or intraperitoneal iron administration (Castellanos et al., 2002; Millerot et al., 2005). However, in the ischemic brain, the detrimental effect of iron overload might result from systemic iron pools reaching the brain parenchyma. Following artery occlusion, a physiological increase in transport through a still somehow intact BBB is noted, and thereafter pathological halt of the BBB function that results in leaky or broken capillaries that allow blood spill out (**Figure 1**). Some BBB impairment with aberrant exchange of brain and blood molecules occurs relatively early following ischemic stroke (within the first hours of post-symptom onset) showing a biphasic behavior in some studies (Klohs et al., 2009). Moreover, leakage through the BBB varies in different experimental stroke models (Klohs et al., 2009; Abulrob et al., 2011). Of note, post-stroke BBB leakage also varies in different areas of the brain, whereas the administration of the broadly used thrombolytic agent tissue plasminogen activator increases leakage (Jaffer et al., 2013), resulting in hemorrhagic transformation. Moreover, a recent evidence of the pivotal contribution of BBB leakage to ischemic stroke damage has been revealed by a study in which a vascular leakage blocker reduced stroke damage (Zhang et al., 2017).

#### Effect of Stroke on Brain Iron

Regarding iron content of the brain following ischemic stroke, an increase in total iron was observed in the ischemic areas after permanent or transient ischemic stroke (Helal, 2008; Millerot-Serrurot et al., 2008; Tuo et al., 2017). In addition, increased TfR and iron levels were observed in susceptible hippocampal areas following transient global cerebral ischemia (Park et al., 2011), or increased extravasation of iron-loaded HTf was observed following acute focal ischemic stroke (DeGregorio-Rocasolano et al., 2018).  Subsequently, iron accumulates into the endothelial cells of brain capillaries near ischemic areas (Desestret et al., 2009), whereas increased levels of Tf were observed 24 h following a transient experimental stroke at the ischemic hemisphere (Lo et al., 2007). In fact, this Tf seems to accumulate in neurons since Tf immunoreactivity was found located at the neuronal cytoplasm 24 h after stroke onset (DeGregorio-Rocasolano et al., 2018).

In the ICH stroke type, the systemic iron pools play a direct role in brain damage since the rupture of the microvascular wall and concomitant blood release results in neurons, astrocytes, and other cells around the hemorrhagic area to become immediately exposed to blood-derived iron, either free or bound to Tf (**Figures 1**, **2**). Moreover, the brain is exposed to a bulk amount of erythrocytes that undergo lysis and subsequent spill of hemoglobin (Hb). Erythrocyte lysis occurs within minutes and continues for days after the formation of brain hematoma, leading to the release of red blood cell-derived products. Cells of the brain are then sequentially exposed to aberrantly high extracellular levels of Hb, the oxidized Hb form methemoglobin, and free heme or the iron ferric heme form hemin. The main mechanisms of neurodegeneration in hemorrhagic stroke are iron- and heme-induced ROS production, amplification of an inflammatory response, direct toxic effects of iron and heme, and glutamate-induced excitotoxicity (Righy et al., 2016).

# BRAIN REGULATION OF IRON METABOLISM DURING STROKE

# Endogenous Mechanisms Involved in the Protection of Brain Cells From Excess Iron Following AIS

Increased brain Tf or TfR1 levels (Ding et al., 2011; DeGregorio-Rocasolano et al., 2018) were observed in lethal ischemia. Consensus exists about the protective role of increased FT expression during experimental stroke, and the same applies to hepcidin in AIS; however, the role of blunting the ischemia-induced upregulation of Tf and TfR remains controversial. Reductions of Tf and TfR were considered to be associated with neuroprotection in one specific stroke model (Lo et al., 2007). However, other neuroprotective conditions were not associated with Tf and/or TfR reductions (He et al., 2012).