REVIEW published: 19 February 2019 doi: 10.3389/fnins.2019.00085 Deciphering the Iron Side of Stroke: Neurodegeneration at the Crossroads Between Iron Dyshomeostasis, Excitotoxicity, and Ferroptosis Núria DeGregorio-Rocasolano 1 , Octavi Martí-Sistac 1,2* and Teresa Gasull 1* 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 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 Edited by: to the devastating consequences of excessive iron-induced oxidative stress and, as Isabella Zanella, recently found, to ferroptosis as well. The blood–brain barrier (BBB) protects the brain Università degli Studi di Brescia, Italy from fluctuations in systemic iron. Under pathological conditions, especially in acute Reviewed by: Alessandro Fanzani, brain pathologies such as stroke, the BBB is disrupted, and iron pools from the blood Università degli Studi di Brescia, Italy gain sudden access to the brain parenchyma, which is crucial in mediating stroke- Ashley Ian Bush, The Florey Institute of Neuroscience induced neurodegeneration. Each brain cell type reacts with changes in their expression and Mental Health and The University of proteins involved in iron uptake, efflux, storage, and mobilization to preserve its of Melbourne, Australia internal iron homeostasis, with specific organelles such as mitochondria showing Gladys Oluyemisi Latunde-Dada, King’s College London, specialized responses. However, during ischemia, neurons are challenged with excess United Kingdom extracellular glutamate in the presence of high levels of extracellular iron; this causes *Correspondence: glutamate receptor overactivation that boosts neuronal iron uptake and a subsequent Octavi Martí-Sistac octavi.marti@uab.cat overproduction of membrane peroxides. This glutamate-driven neuronal death can be Teresa Gasull attenuated by iron-chelating compounds or free radical scavenger molecules. Moreover, tgasull@igtp.cat; vascular wall rupture in hemorrhagic stroke results in the accumulation and lysis of teresagasull@yahoo.com iron-rich red blood cells at the brain parenchyma and the subsequent presence of Specialty section: hemoglobin and heme iron at the extracellular milieu, thereby contributing to iron- This article was submitted to induced lipid peroxidation and cell death. This review summarizes recent progresses Neurodegeneration, a section of the journal made in understanding the ferroptosis component underlying both ischemic and Frontiers in Neuroscience hemorrhagic stroke subtypes. Received: 28 October 2018 Keywords: iron, reactive oxygen species, ferroptosis, stroke, excitotoxicity, iron dyshomeostasis, Accepted: 25 January 2019 neurodegeneration, transferrin saturation Published: 19 February 2019 Citation: Abbreviations: AIS, acute ischemic stroke; ALOX5: arachidonic acid lipoxygenase 5; APP, amyloid precursor protein; DeGregorio-Rocasolano N, BBB, brain–blood barrier; CSF, cerebrospinal fluid; Dexras1, dexamethasone-induced Ras protein 1; DFO, deferoxamine; Martí-Sistac O and Gasull T (2019) 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, Deciphering the Iron Side of Stroke: hemoglobin; HO, heme oxygenase; Hp, haptoglobin; Hpx, hemopexin; HTf, holotransferrin; ICH, intracerebral hemorrhage; Neurodegeneration at the Crossroads I/R, ischemia/reperfusion; IREs, iron regulatory elements; IRPs, iron regulatory proteins; NAC, N-acetylcysteine; NMDA, Between Iron Dyshomeostasis, N-methyl-D-aspartate; NTBI, non-transferrin-bound iron; OGD, oxygen and glucose deprivation; PUFA, polyunsaturated Excitotoxicity, and Ferroptosis. fatty acids; ROS, reactive oxygen species; TIM, T-cell immunoglobulin and mucin domain-containing receptor; Tf, Front. Neurosci. 13:85. transferrin; TfR1, transferrin receptor 1; TfR2, transferrin receptor 2; TSAT, saturation of transferrin with iron; UTRs, doi: 10.3389/fnins.2019.00085 untranslated regions; X− c , cystine/glutamate exchanger or antiporter, i.e., SLC7A11/xCT. Frontiers in Neuroscience | www.frontiersin.org 1 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke IRON TRANSPORT TO THE BRAIN: THE Although barrier cells of the brain form tight junctions ROLE OF THE BLOOD–BRAIN BARRIER laterally, epithelial cells of the choroid plexuses are fenestrated and more permeable than the endothelial cells of the BBB Iron is essential for life; numerous proteins require iron as a (Strazielle and Ghersi-Egea, 2016). The abluminal side of cofactor for their activity. Iron acts as an electron donor or brain capillary endothelial cells is surrounded by pericytes and acceptor, and thus is pivotal for oxygen transport (coordinated astrocytes of the neurovascular unit that provide extra structural with hemoglobin), cellular respiration (as a part of heme- and functional support. TfRs are abundantly expressed in the containing cytochromes and proteins containing Fe–S groups in luminal side of membranes of polarized endothelial cells; once the electron transport chain), and DNA synthesis (in ribonuclease they bind Tf, the TfR–Tf complexes undergo endocytosis. The reductase). Moreover, in the nervous system, iron is essential for following steps are still controversial, and different pathways myelinization and neurotransmitter biosynthesis. are thought to be involved in brain iron acquisition (Bradbury, Iron exists both as Fe2+ and Fe3+ ; redox-active and cycling 1997; Khan et al., 2018). Thus far, the main contribution to between these two states lead to the generation of reactive this process is thought to include the following pathways: (1) oxygen species (ROS) through the Fenton reaction. The ROS endocytic vesicles containing TfR–HTf traveling from the luminal promote oxidative stress and cause extensive lipid peroxidation membrane to fuse to the abluminal membrane and exocytose (Gutteridge et al., 1979). The brain is particularly vulnerable to either HTf or free iron into the brain extracellular space; (2) under lipid peroxidation damage because it is rich in polyunsaturated low pH conditions within the endosome of endothelial cells, as fatty acids (PUFAs) and iron, but relatively poor in antioxidant in most cell types, iron is extracted from Tf, converted to Fe2+ defenses (Cobley et al., 2018). The iron content remarkably by endosomal ferrireductases such as the six-transmembrane differs across different brain regions: it is high in the striatum, epithelial antigen of prostate protein 3 (Steap3), and released medium in the hippocampus and cortex, and low in the pons and to the cytosol through divalent metal transporter 1 (DMT1) medulla (Ramos et al., 2014). Although it has recently attracted or the ion channels TRPML, also known as mucolipin 1 or considerable attention, iron metabolism in the brain is yet not MCOLN1 (detailed information can be found in references completely understood as that in other murine tissues (Ashraf Luck and Mason, 2012; Skjorringe et al., 2015). Subsequently, et al., 2018). Brain imports iron from systemic circulation, and the Fe2+ from the cytosol is exported through FPN present in the levels of iron are determined by diet, intestinal iron absorption, abluminal membrane of endothelial cells, which are in contact release of iron initially stored in the hepatocytes, and export with astrocytes expressing ceruloplasmin, that rapidly oxidize of iron recycled from macrophages. In addition, the levels of Fe2+ to Fe3+ (this Tf/TfR endocytosis mechanism is common iron in the plasma are regulated by the hormone hepcidin for endothelial and neuronal cells and is depicted in Figures 1, through its effect on the transmembrane iron export protein 2). A recent study performed using comprehensive mathematical ferroportin (FPN), which is mainly present at the cell membrane models concluded that optimal iron homeostasis is dependent on of macrophages, hepatocytes, and enterocytes. the degree of saturation and the transport of HTf across the BBB In the blood and most other extracellular fluids, iron is from the blood (Khan et al., 2018). essentially carried bound with high affinity to the iron transport In addition, in mice exposed to experimental stroke under protein transferrin (Tf), which shields Fe3+ from redox activity. normal iron conditions, iron deposits in the microvasculature In addition to Tf, iron also associates with other carrier proteins were observed early in the border zone of the damaged areas in the blood such as ferritin (FT), haptoglobin (Hp), Hpx, or (Desestret et al., 2009), suggesting that, when signaled, the BBB albumin. Recently, half a dozen different non-proteinaceous low- might function as an iron reservoir from which iron is released molecular-weight iron species (<10 KDa) have been shown to into the brain. Regarding signaling to release, in bovine retinal exist in the blood (Dziuba and Lindahl, 2018); further studies endothelial cells, the FPN-mediated iron export is known to are required to determine the precise identity and physiological be blocked by the FPN inhibitor hepcidin (Hadziahmetovic role of these species. Once iron is bound to Tf, Fe3+ is rendered et al., 2011). In addition, new insights into the communication redox-inactive and virtually non-exchangeable or displaceable between systemic iron status and brain iron uptake reveal by other physiological metals or molecules. Tf−iron complexes the regulation of brain iron uptake at the level of brain circulate in the blood until they bind transferrin receptors (TfRs) microvascular endothelial cells rather than the BBB acting merely in cellular membranes of target cells. In general, in non-iron- as a retaining wall (Chiou et al., 2018b). In support of this overload conditions, little, if any, non-transferrin-bound iron regulatory hypothesis, endothelial microvascular cell distribution (NTBI) is found in the blood of healthy individuals (Gaasch et al., of TfR is altered in luminal, intracellular, and abluminal 2007). The high affinity of Tf for iron and the large amount of membranes depending on brain iron status (Simpson et al., Tf in the blood exceed the total requirement for iron binding; 2015), and microvascular cell iron availability and efflux have in normal human blood, the saturation of transferrin with iron been found to be tightly modulated by diffusible ceruloplasmin (TSAT) is only of around 30% (De Valk et al., 2000; Van Dijk and hepcidin released from astrocytes surrounding the BBB et al., 2008). Therefore, iron transport to the brain is mainly (McCarthy and Kosman, 2014). Moreover, a recent study showed from iron-loaded transferrin (holotransferrin, HTf) through the that inhibition of DMT1 alters the transport of iron and Tf endothelial cells of the BBB and the epithelium of the choroid across the endothelial cells and proposed that the levels of iron- plexuses and arachnoid membrane, the blood–cerebrospinal fluid devoid Tf (apotransferrin) and iron delivered to the brain are (CSF) barrier. strongly regulated by the ratio apotransferrin:HTf as well as by Frontiers in Neuroscience | www.frontiersin.org 2 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke 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 2004), and that 59 Fe–NTBI seems to be transported faster than (Duck et al., 2017). 59 Fe–TfR into the brain parenchyma of iron-overloaded disease- Further, very recently, a new function for FT heavy chain free mice. Furthermore, the iron storage protein FT has been (FTH) as a transporter of NTBI across the BBB by binding found to be upregulated in epithelial cells of the choroid plexus to the T-cell immunoglobulin and mucin domain-containing 1 and endothelial cells of the brain during systemic iron overload receptor 1 (TIM-1) has been reported in human endothelial cells (Tripathi et al., 2017). (Chiou et al., 2018b). In the context of physiological iron-overload conditions found, for instance, in individuals on iron-rich diets, a significant IRON UPTAKE AND HANDLING BY pool of NTBI is found in the blood and might be readily absorbed PARENCHYMAL BRAIN CELLS by microvascular endothelial cells. This effect might be relevant considering that regional differences in the brain uptake of Once in the brain interstitial fluid, both TBI and NTBI are 59 Fe–NTBI versus 59 Fe–TfR have been reported (Deane et al., potential iron sources for brain cells (Gaasch et al., 2007); unlike Frontiers in Neuroscience | www.frontiersin.org 3 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke FIGURE 2 | Schematic drawing of some of the main players in a neuron under ischemic (left-hand side) and hemorrhagic (right-hand side) stress (see text for details). Abbreviations: APP, amyloid precursor protein; ATf, apotransferrin; CD163, hemoglobin/haptoglobin receptor; CD91, heme/hemopexin receptor; CO, carbon monoxide; CP, ceruloplasmin; DMT1, divalent metal transporter 1; EAAT, excitatory amino acid transporter; Fe2+ , ferrous iron; Fe3+ , ferric iron; Fe–S, iron–sulfur cluster; FPN, ferroportin; FT, ferritin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLS, glutaminase; GPX4, glutathione peroxidase 4; GSH, 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 transporter are only active at normal physiological pH, were iron (Bradbury, 1997). Within the extracellular milieu of the reported to be expressed by hippocampal neurons, and Zip8 was brain parenchyma, iron is carried loaded into Tf or bound to found to play a major role in the accumulation of NTBI by low-molecular-weight molecules such as citrate, ascorbate, or hippocampal neurons (Ji and Kosman, 2015), whereas both Zip8 ATP. Each brain cell type uptakes extracellular TBI or NTBI and Zip14 were considered to play a role in iron homeostasis in depending on the iron-transport receptors and channels present rat retina (Sterling et al., 2017). 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 Receptors Involved in Iron Uptake by are cell type-specific, and the list of proteins involved in iron Brain Cells homeostasis is still growing. As an example of iron-related new In addition to the well-known TfR1 as a main player of the proteins, only 3 years ago, the members of the ZIP family of TfR/Tf endocytic pathway in most brain cells, other proteins, e.g., metal ion transporters Zip8 and Zip14, which, unlike the DMT1 transferrin receptor 2 (TfR2) (Kawabata, 2018) or glyceraldehyde- Frontiers in Neuroscience | www.frontiersin.org 4 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke 3-phosphate dehydrogenase (GAPDH) (Raje et al., 2007), have with endosomes/lysosomes. Further, neurons express the iron been described as receptors for Tf and are currently being export molecule FPN (Burdo et al., 2001; Zhou et al., 2017). investigated. TfR1 binds HTf with a Kd of 1 nM; it is expressed Astrocytes express TfR1, DMT1, and FPN (Yang et al., 2012; in response to changes in iron levels through modulation by Huang et al., 2014). Hence, although TfR1 is considered to be iron regulatory elements (IREs) present in its mRNA and the main gate for cellular iron import, control astrocytes show binds hereditary hemochromatosis HFE protein, a molecule that low TfR1 levels, and most mature oligodendrocytes or microglial competes with HTf for binding to TfR1. In contrast, TfR2 has cells do not express TfR1 (Kaur and Ling, 1995; Rathnasamy lower affinity for HTf; its levels are not modulated by IREs (West et al., 2016). However, under hypoxia, both astrocytes and et al., 2000) and, although still controversial, seems to bind HFE oligodendrocytes are found to upregulate TfR (Yang et al., 2012; protein in a sequence non-overlapping to that of the Tf-binding Rathnasamy et al., 2016). Moreover, oligodendrocytes and cells of site (for a review see Worthen and Enns, 2014). choroid plexuses, but not other brain cell types, express Tf under The TfR2 is present in two main isoforms, TfR2α and TfR2β. normal condition (Leitner and Connor, 2012). All the above The TfR2α form has been reported to be more abundant in the are examples of the important heterogeneity in the expression brain, especially in the hippocampus; brain TfR2α levels were and induction of iron homeostatic or IRPs observed in different reported to significantly differ across animals fed iron-deficient brain cell types and situations. Most of the proteins involved or iron-enriched diets (Pellegrino et al., 2016). A specific role in iron metabolism, including cytosolic FT levels, are mainly for TfR2 in the control of the iron regulatory network in the regulated by iron, oxidative stress, and inflammation. The iron- brain tissue has been recently suggested, as altered brain IRPs, dependent regulation occurs mainly at the post-transcriptional iron accumulation, and reactive microglia signaling were found level via the IREs and iron regulatory protein (IRP) machinery. in TfR2-KO mice (Pellegrino et al., 2016). In contrast, a previous When iron levels are high, iron binds to the IRP, which then study on TfR2 mutant mice showed changes in hepatic, but not loses affinity for the IRE sequence in FT mRNA, resulting in brain iron levels, although that study reported a 30% increase in increased FT expression (the cytosolic iron handling is depicted brain FT levels (Acikyol et al., 2013). in Figure 2) (Arosio et al., 2017). Several studies have shown In addition to TfR1 and TfR2, the multifunctional molecule that the mobilization of iron from FT requires FT degradation GAPDH has been reported to work as a TfR in different cell either by lysosome–autophagy or ubiquitin proteasome pathways types (Raje et al., 2007; Kumar et al., 2012) with a role in iron (Asano et al., 2011; Mancias et al., 2015). In addition to its role uptake and/or iron export (Sheokand et al., 2016) and as a in storing iron within the cell, FT can be secreted (Truman- chaperone for intracellular heme traffic (Sweeny et al., 2018). Rosentsvit et al., 2018). Relevant to brain and excitotoxicity, GAPDH interacts with the In addition to post-transcriptional regulation, transcriptional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype regulation has been reported for some of the proteins involved of glutamate receptors in the neurons without glutamate in iron homeostasis through the hypoxia-inducible factor family stimulation and has also been reported to be pivotal for axon of transcription factors or the heme-dependent transcription development (Lee et al., 2016). factors (e.g., for TfR1 or FPN) (Marro et al., 2010; Man-man et al., Another receptor-mediated means to import iron into brain 2017). Moreover, the master regulator of bodily iron homeostasis cells, at least in oligodendrocytes, is the uptake of FTH through hepcidin is subjected to a sophisticated transcriptional regulation TIM-1/2 receptors (Todorich et al., 2008, 2011; Chiou et al., through the bone morphogenetic protein/SMAD pathway, 2018a). In other non-neuronal cells, FTH internalize through interleukin 6 pathway, or erythropoietin/erythroferrone TfR1 (Li et al., 2010), whereas scavenger receptor class A member pathways when regulated according to the iron, inflammatory, 5 (SCARA5) has been found to play also a role as a receptor or erythropoietic demands (Sangkhae and Nemeth, 2017). The for FT light chain uptake (Li et al., 2009). To the best of our regulation of cellular iron balance has been recently reviewed knowledge, the role of SCARA5 has not yet been investigated in (Recalcati et al., 2017). any brain cell type. 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 Mechanisms and Proteins Involved in apparatus, and contain the respiratory complexes that need iron- Cytosolic Iron Handling in Different Brain containing cofactors. In addition, mitochondria are a major Cell Types source of endogenous ROS. Sustained iron exposure increased Inside the cell cytosolic and nuclear compartments, iron mitochondrial ROS levels in dopaminergic neuroblastoma SH- chaperone poly(rC)-binding proteins serve the binding and SY5Y cells (Huang et al., 2017), and scavenging of mitochondrial delivery of iron to iron-requiring enzymes, ubiquitous protein ROS was protective against iron overload damage in hippocampal of iron storage FT, or the iron export membrane protein FPN neurons, preserving mitochondrial morphological integrity and (Philpott et al., 2017). In the cytosol, unused iron is stored membrane potential (Pelizzoni et al., 2011). In addition, a recent into FT, a molecule composed of 24 subunits of a variable study showed that sequestration of iron by mitochondrial FT was proportion of heavy and light chains, that shields up to 4500 neuroprotective against oxidative stress and played an important iron atoms in a redox inert state. Neurons express both TfR1 role in preventing neuronal damage in some other conditions and DMT1 (Pelizzoni et al., 2012; DeGregorio-Rocasolano et al., (You et al., 2016; Guan et al., 2017). Moreover, mitochondrial 2018); DMT1 is mainly found in the cytoplasm colocalizing FT overexpression significantly reduced cellular labile iron pool, Frontiers in Neuroscience | www.frontiersin.org 5 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke prevented H2 O2 -induced elevation of labile iron, and rescued observed in amyloid precursor protein (APP)-knockout mice cells from H2 O2 -induced damage (Gao et al., 2017). (Belaidi et al., 2018). 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 IRON OVERLOAD WORSENS mitochondria (Das et al., 2016; Hamdi et al., 2016) through the NEUROLOGICAL DAMAGE BOTH IN “kiss-and-run” hypothesis. ISCHEMIC STROKE AND INTRACEREBRAL HEMORRHAGE IRON DYSHOMEOSTASIS IN THE BRAIN Stroke is a life-threatening disease that causes high rates of Brain iron abnormalities are associated to rare, but severe, permanent disability subsequent to neuronal loss. There are two neurodegenerative conditions, and there is growing evidence major types of strokes: ischemic stroke or AIS, which is caused by that the more common systemic iron overload disorders such a blood clot blocking an artery and accounts for 85% of all strokes, as hereditary hemochromatosis exert important effects on the and hemorrhagic stroke or ICH, caused by leakage or rupture of brain iron content and pathological brain iron deposition an artery and accounts for 15% of stroke cases. (Nandar and Connor, 2011; Kumar et al., 2016). Moreover, the hemochromatosis H63D polymorphism of the HFE gene could be Iron Overload Condition in Stroke a disease-modifying gene in frontotemporal lobar degeneration, Damage and Outcome fostering iron deposition in the basal ganglia (Gazzina et al., In ischemic stroke, neurons die because of interrelated processes 2016), or even macro- and microanatomically altering some that include glutamate excitotoxicity, excess of free radical brain structures associated with changes in Tf levels in the production, and inflammation (Nagy and Nardai, 2017). Each blood (Jahanshad et al., 2012). Recently, abnormal recycling of these mechanisms has already been targeted in clinical trials, of TfR1 due to reduced post-translational palmitoylation has although unsuccessfully (for a review see Chamorro et al., 2016). been reported to be crucial to affect the iron import in the so- Moreover, free radical-induced oxidative stress has been for long called neurodegeneration with brain iron accumulation disease recognized as a pathogenic factor in the ischemia/reperfusion (Drecourt et al., 2018). Further, neurodegeneration with brain (I/R) injury that follows reoxygenation. The hydroxyl radical, iron accumulation disease-linked genes that showed altered the most harmful of the free radicals, is generated through expression in response to iron loading has been recently reactions catalyzed by iron (Kell, 2009; Chen H. et al., 2011). Of reported to be directly or indirectly related to myelin metabolism relevance to stroke damage and final outcome, several clinical (Heidari et al., 2016). studies indicate that a previous systemic iron overload condition, A list of mild conditions, including aging (Ward et al., measured as high levels of serum FT at admission, is associated 2014), continuous uptake of some bioavailable iron sources with increased brain damage and worse outcome induced by (Peters et al., 2018), or obesity (Han et al., 2017), have been ischemic stroke (Davalos et al., 1994; Millan et al., 2007). reported to alter brain iron content and/or distribution. In A similar detrimental effect (increased ischemic damage) was addition, a recent report showed that iron administration observed in iron-overloaded animals exposed to experimental increases iron levels in the brains of healthy rats, which induces ischemic stroke (Castellanos et al., 2002; Gamez et al., 2003; brain changes and triggers a hormetic response that reduces Mehta et al., 2004; García-Yébenes et al., 2012; DeGregorio- oxidative damage (Piloni et al., 2018). Moreover, other studies Rocasolano et al., 2018). Iron overload is a major source of reveal a complex interplay between inflammation and brain oxidative stress in ischemic brains (Carbonell and Rama, 2007). iron homeostasis (Righy et al., 2018; Sankar et al., 2018), In addition, iron overload exacerbated the risk of hemorrhagic with acute inflammation increasing non-Tf iron uptake by transformation in animal models of transient ischemia plus early brain microglia (McCarthy et al., 2018). This dyshomeostasis reperfusion (García-Yébenes et al., 2012; García-Yébenes et al., is especially important in acute pathologies such as stroke, in 2018); therefore, in iron-overloaded animals, an initial ischemic which the impairment of the BBB regulatory role is rapidly event has higher probability to become an ICH. The contribution and massively affected following either rupture of an artery of iron overload to widespread the damage in the ICH is (ICH) or aberrant increase of brain microvascular endothelial supported by the fact that systemic iron overload (estimated using permeability following ischemic stroke (AIS). Dysregulation of high serum FT levels) was associated with higher perihematoma iron resulting in the accumulation of free iron and brain iron edema and poor outcomes in patients with ICH (Mehdiratta et al., overload, which can increase the production of ROS, is evident 2008; Perez de la Ossa et al., 2010; Bakhshayesh et al., 2014; in aging and stroke-related pathologies and is also a hallmark Garton et al., 2017). Further, a combination of high serum FT of chronic and long-term neurodegenerative pathologies. In and low serum iron and Tf was associated with poor outcome this regard, iron overload in the brain has been reported in in ICH patients (Yang et al., 2016). Therefore, systemic iron Huntington, Parkinson, or Alzheimer diseases [readers interested overload impacts the outcome of both ischemic and hemorrhagic in the topic are referred to recent detailed reviews by Morris et al. stroke patients. (2018) and Uranga and Salvador (2018)]. Interestingly, marked The brain is physiologically sheltered from fluctuations in age-related changes in brain iron homeostasis have also been systemic iron because of the BBB, even under experimentally Frontiers in Neuroscience | www.frontiersin.org 6 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke induced diet iron overload conditions or intraperitoneal iron inflammatory response, direct toxic effects of iron and heme, and administration (Castellanos et al., 2002; Millerot et al., 2005). glutamate-induced excitotoxicity (Righy et al., 2016). 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 BRAIN REGULATION OF IRON increase in transport through a still somehow intact BBB is METABOLISM DURING STROKE 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 Endogenous Mechanisms Involved in the exchange of brain and blood molecules occurs relatively early Protection of Brain Cells From Excess following ischemic stroke (within the first hours of post-symptom Iron Following AIS onset) showing a biphasic behavior in some studies (Klohs Increased brain Tf or TfR1 levels (Ding et al., 2011; DeGregorio- et al., 2009). Moreover, leakage through the BBB varies in Rocasolano et al., 2018) were observed in lethal ischemia. different experimental stroke models (Klohs et al., 2009; Abulrob Consensus exists about the protective role of increased et al., 2011). Of note, post-stroke BBB leakage also varies in FT expression during experimental stroke, and the same different areas of the brain, whereas the administration of the applies to hepcidin in AIS; however, the role of blunting broadly used thrombolytic agent tissue plasminogen activator the ischemia-induced upregulation of Tf and TfR remains increases leakage (Jaffer et al., 2013), resulting in hemorrhagic controversial. Reductions of Tf and TfR were considered to be transformation. Moreover, a recent evidence of the pivotal associated with neuroprotection in one specific stroke model (Lo contribution of BBB leakage to ischemic stroke damage has been et al., 2007). However, other neuroprotective conditions were not revealed by a study in which a vascular leakage blocker reduced associated with Tf and/or TfR reductions (He et al., 2012). stroke damage (Zhang et al., 2017). Hepcidin seems to protect from neurodegeneration in AIS. It is an iron-inducible peptide hormone that is considered to be Effect of Stroke on Brain Iron the main regulator of iron homeostasis. Hepcidin is produced Regarding iron content of the brain following ischemic stroke, as a propeptide mainly in the liver and then secreted to the an increase in total iron was observed in the ischemic areas after blood where it interacts with the only known iron exporter permanent or transient ischemic stroke (Helal, 2008; Millerot- FPN located at the cell membrane. Hepcidin acts as a negative Serrurot et al., 2008; Tuo et al., 2017). In addition, increased TfR regulator of cellular iron release by binding FPN and causing and iron levels were observed in susceptible hippocampal areas its internalization and degradation; this inhibits FPN-mediated following transient global cerebral ischemia (Park et al., 2011), iron egression from enterocytes, macrophages, hepatocytes and, or increased extravasation of iron-loaded HTf was observed as recently found, some other cell types (e.g., endothelial cells following acute focal ischemic stroke (DeGregorio-Rocasolano or neurons). The inhibition of iron egression, mainly from et al., 2018). In fact, HTf from blood accumulates in the brain macrophages and hepatocytes, prevents the presence of NTBI parenchyma of the rat ischemic brain hemisphere at 1 h after in the blood as well as reduces the TSAT in the blood. In line reperfusion (DeGregorio-Rocasolano et al., 2018). Subsequently, with its main function, hepcidin deficiency leads to iron overload iron accumulates into the endothelial cells of brain capillaries (Nicolas et al., 2001), and hepcidin overexpression causes severe near ischemic areas (Desestret et al., 2009), whereas increased iron deficiency (Nicolas et al., 2002a). Hepcidin has a rapid and levels of Tf were observed 24 h following a transient experimental direct impact on circulating iron pools. In fact, even a single stroke at the ischemic hemisphere (Lo et al., 2007). In fact, this Tf intraperitoneal administration of hepcidin reduces the TSAT in seems to accumulate in neurons since Tf immunoreactivity was blood to less than half the normal levels for 6−12 h (Sillerova found located at the neuronal cytoplasm 24 h after stroke onset et al., 2012). Both blood hepcidin and HTf/apotransferrin ratio (DeGregorio-Rocasolano et al., 2018). might be considered as the master regulatory factors of systemic In the ICH stroke type, the systemic iron pools play a direct iron metabolism, since hepcidin senses iron by sensing HTf role in brain damage since the rupture of the microvascular wall levels in the circulation and iron-FT levels inside hepatocytes. and concomitant blood release results in neurons, astrocytes, and Relevant to this review on iron and stroke, hepcidin expression other cells around the hemorrhagic area to become immediately is also responsive to hypoxia (Nicolas et al., 2002b; Sonnweber exposed to blood-derived iron, either free or bound to Tf et al., 2014) mainly by the oxygen-regulated hypoxia-inducible (Figures 1, 2). Moreover, the brain is exposed to a bulk factor. In general, hypoxia inhibits hepcidin expression to allow amount of erythrocytes that undergo lysis and subsequent spill the mobilization of iron to sustain erythropoietic expansion. of hemoglobin (Hb). Erythrocyte lysis occurs within minutes However, a recent study showed that the exposure of mice to an and continues for days after the formation of brain hematoma, hypoxic environment induces biphasic modulation of hepcidin leading to the release of red blood cell-derived products. Cells expression with a transient increase in liver expression within the of the brain are then sequentially exposed to aberrantly high first 6 h and a reduction after 15 h of hypoxia (Ravasi et al., 2018). extracellular levels of Hb, the oxidized Hb form methemoglobin, Among ischemic stroke patients, who have areas of the and free heme or the iron ferric heme form hemin. The main brain exposed to oxygen and glucose deprivation (OGD), blood mechanisms of neurodegeneration in hemorrhagic stroke are hepcidin levels increased within the first 6 h from symptom iron- and heme-induced ROS production, amplification of an onset compared to those in controls; serum hepcidin levels in Frontiers in Neuroscience | www.frontiersin.org 7 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke ICH patients were similar to those of controls (unpublished endothelial cells and further draining of iron into circulation, results from our laboratory). This early increase in blood hepcidin leading to the aggravation of oxidative brain injury (Xiong during ischemic stroke is in agreement with the early increase et al., 2016). Conversely, addition of hepcidin or adenovirus- reported in the hypoxic mice model (Ravasi et al., 2018) driven overexpression of hepcidin has been shown to protect and with the two other reports on stroke patients (Petrova neurons from hemin-induced injury (Zhou et al., 2017); this et al., 2015; Slomka et al., 2015) and might be a reflection protection was thought to be related to the effect of hepcidin of an increased expression and secretion of hepcidin from reducing hemin-induced iron uptake and reduced expression of the ischemic brain cells to the extracellular medium, and a TfR1 and DMT1. subsequent spillover out of the brain during stroke. Although Moreover, importantly, stroke has an inflammatory hepcidin expression in the brain is thought to be low in basal component, and some inflammatory inducers or mediators conditions, hepcidin is now emerging as an important player (e.g., lipopolysaccharide or interleukin 6) have been found to in brain iron homeostasis (Vela, 2018). Hepcidin mRNA levels increase the expression of hepcidin in astrocytes, microglia, or increased early in the brain in response to ischemia in a model neurons (for a recent review see Vela, 2018), although the effect of endothelin-induced vasoconstriction (Bickford et al., 2014), on neurons is usually weaker (Ma et al., 2018). and hepcidin levels were significantly elevated in the ischemic The brain has protective mechanisms that can be activated side of the brain 24 h after occlusion in a stroke model due to after ICH and erythrocyte lysis, the most important one being middle cerebral artery occlusion; this ischemic side also showed the Hb-scavenging molecule haptoglobin (Hp) and the heme- increased TfR1 and FT levels and reduced FPN levels (Ding et al., scavenging system hemopexin (Hpx) (Figure 2). Hb itself is 2011). In addition, knock-down of hepcidin slows the ischemic- avidly bound by Hp to form a complex that prevents the release mediated changes in brain FT and FPN, revealing an important of the heme group from Hb or methemoglobin. However, Hp contribution of this molecule to parenchyma iron regulation cannot prevent the release of the heme group from the highest in cerebral ischemia. Further, normoxic iron-overloaded rats, oxidative Hb form ferrylHb. In the brain, Hp is almost exclusively hepcidin, either overexpressed in the brain or administrated to synthesized by oligodendrocytes, and mice overexpressing Hp the brain parenchyma, reduced brain uptake of iron in vivo as were more resistant, whereas Hp-knockout mice were more well as reduced iron uptake in cultures of neurons or brain susceptible, to ICH injury (Zhao et al., 2011). The complex microvascular endothelial cells, owing to the dowregulation of Hp:Hb binds to the Hp-specific receptor CD163 that is present in iron-transport proteins (Du et al., 2015). In microglial cells, macrophages, allowing them to remove Hb from the extracellular the bone morphogenetic protein antagonist noggin abrogated space. In addition, CD163 is upregulated in neurons after ICH the OGD-induced changes in hepcidin, FPN, and FT levels, (Liu et al., 2017). The role of Hp in neuroprotection was not and this inhibition of bone morphogenetic protein/hepcidin devoid of controversy since Hp was found to exacerbate the axis in vitro shifted reactive microglia from an iron-storing to vulnerability of CD163-expressing neurons to Hb, an effect that an iron-releasing phenotype after OGD plus reperfusion (Shin was reduced by iron chelators (Chen-Roetling and Regan, 2016). et al., 2018). Factors released from noggin- and OGD-exposed However, most Hb was not bound to Hp, indicating that the microglial culture medium were found to increase myelin CD163–Hb–Hp system is saturated, and that the primary route production in oligodendrocyte cell cultures; since this effect is for Hb clearance from the CNS occurs through the BBB (Galea blunted by the iron chelator deferoxamine (DFO), iron released et al., 2012; Righy et al., 2018). A good clearance is associated from the microglia is proposed to serve in the remyelination in with neuroprotection since lower CSF Hp levels were found in the ischemic brain (Shin et al., 2018). ICH patients without clinical and/or radiological evidence of delayed cerebral ischemia (Galea et al., 2012). Hb accumulated in the brain parenchyma is converted to ferrylHb, which Endogenous Mechanisms Involved in the causes the upregulation of proinflammatory adhesion molecules Protection of Brain Cells From Excess and increases the recruitment of inflammatory neutrophils or Iron Following ICH macrophages (Jeney et al., 2013). Similar to that observed in AIS, subarachnoid hemorrhage has Iron–hemin uptake has been reported in neurons and been shown to increase hepcidin expression in neurons (Zhao astrocytes and is especially high in microglial cell cultures; et al., 2018) associated with a reduction of its downstream neurons benefit from a high export of iron following the target FPN (Tan et al., 2016). However, in this hemorrhage initial iron–hemin loading, and this export was exacerbated model, hepcidin seems to be harmful since its administration by treatment with DFO, a compound that attenuates hemin potentiated and its siRNA decreased apoptosis and early brain neurotoxicity (Chen-Roetling et al., 2014). As already mentioned, damage (Tan et al., 2016). In agreement with a harmful the brain produces the heme scavenger protein Hpx that effect of hepcidin on brain hemorrhage, higher blood hepcidin protects from deleterious effects of heme. In this regard, levels were observed 1−7 days following ICH in patients deletion/knockout of the Hpx aggravates brain injury by ICH with poorer outcomes (Xiong et al., 2015). The rationale (Chen L. et al., 2011; Ma et al., 2016) and, although endogenous for this harmful effect of hepcidin following ICH might be levels of Hpx are insufficient to counteract the massive heme an impaired clearance of iron from brain parenchyma, since overload following ICH, increasing brain Hpx levels has been increased hepcidin expression caused by inflammation was found found to improve the outcome after ICH (Leclerc et al., 2018). to inhibit intracellular iron efflux from brain microvascular Conversely, Hpx has recently been reported to increase the Frontiers in Neuroscience | www.frontiersin.org 8 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke neurotoxicity of Hb in the absence of Hp (Chen-Roetling et al., in both ischemic and hemorrhagic stroke subtypes. Although 2018). Therefore, although both Hp and Hpx might provide a the concept is still evolving, ferroptosis is now considered a robust line of defense during ICH, additional knowledge on the unique form of regulated cell death characterized by cytosolic fine tuning of this system is still required to design new possible accumulation of iron and executed by the massive peroxidation neuroprotective treatments for ICH. The low-density lipoprotein of PUFA in the plasma membrane and associated with receptor-related protein CD91 is expressed in several cell reduced glutathione (GSH). Compounds that induce only either types, including macrophages and neurons, and was identified cytosolic or mitochondrial ROS production are not considered as the Hpx–heme receptor, mediating Hpx–heme uptake by as ferroptosis inducers. A recent study proposed that both endocytosis (Hvidberg et al., 2005). Once inside the cell, the Fenton reactions, in which Fe2+ converts to Fe3+ producing heme oxygenase (HO) system is responsible for cellular heme ROS, and enzymatic lipoxygenase-mediated reactions in cellular degradation. In neurons, HO generates biliverdin, which has been membranes contribute to ferroptosis (Feng and Stockwell, 2018). reported to scavenge free radicals. Astrocytes rapidly upregulate Some authors considered that the ferroptosis concept and the HO1 and FT and are resistant to heme-mediated injury, and previously described oxidative oxytosis concept have many astrocyte HO1 is known to play a robust neuroprotective role common features to be regarded as independent processes after hemorrhage (Chen-Roetling et al., 2017). A recent study (Lewerenz et al., 2018). In fact, the widely used oxidative indicated that Hpx activates endothelial progenitor cells and stress stimulus tert-butylhydroperoxide was found to induce reduces synaptic damage in a rodent ischemic stroke model; these neuronal cell death that was blocked by ferroptosis inhibitors, effects require HO1 activity (Yang et al., 2018). thereby implying a crosstalk between the initial oxidative damage Moreover, the presence of heme groups in the extracellular and the occurrence of ferroptosis (Wu et al., 2018). However, milieu activates the endothelial expression of adhesion molecules ferroptosis is considered a major driver of cell death in many and promotes the activation of neutrophil extracellular traps neurodegenerative and neurological diseases (Lewerenz et al., through a mechanism dependent on ROS generation (Chen et al., 2018; Morris et al., 2018). 2014). Further, inflammatory response is a well-documented Ferroptosis can be triggered by the inhibitors of the lipid mechanism of brain damage after ICH (Wang and Doré, 2007), repair enzyme glutathione peroxidase 4 (GPX4), such as the and the mechanisms underlying heme-induced inflammation RAS-selective lethal 3 compound, or depletion of the antioxidant have been reviewed elsewhere (Dutra and Bozza, 2014). Iron GSH by starving the cells of GSH precursors [inhibiting and heme associated with pro-inflammatory mediators in the the cystine/glutamate antiporter, i.e., system X− − c (Xc ) with CNS following hemorrhagic stroke and inflammatory profiles erastin, sulfasalazine, sorafenib, or, alternatively, challenging the are associated with poorer prognosis. Similarly, iron-induced exchange capacity of X− c with high extracellular glutamate]. damage following ischemic stroke in iron-overloaded animals has Extracellular Tf in its iron-loaded HTf form is considered as also been related to increased inflammatory responses associated an inducer of ferroptosis as well, and inhibiting TfR expression with increased serum levels of interleukin 6 and TNF-alpha prevents ferroptosis (Gao et al., 2015). (Castellanos et al., 2002). At present, the existence of X−c in neurons is still controversial, with one report showing expression lack of X− c in neurons (Ottestad-Hansen et al., 2018), whereas another showing neurons THE FERROPTOTIC COMPONENT OF expressing significant X− c levels (Soria et al., 2016). However, STROKE-INDUCED neurons in culture overexpress X− c shortly after being exposed to OGD (Soria et al., 2014). The physiological role of X− c as a NEURODEGENERATION cystine importer is linked to its antiporter glutamate export and requires that extracellular levels of glutamate and other excitatory The Concept of Ferroptosis: Molecular amino acids are maintained low by the action of excitatory Players Involved in Ferroptosis in the amino acid transporters (EAATs). Thus, one of these glutamate Neurons transporters, the sodium-dependent EAAT3, might be selectively The concept of ferroptosis was introduced as a form of cell expressed in neurons and could also be an unidirectional death, which is morphologically, biochemically, and genetically cysteine import channel, with extracellular glutamate inhibiting distinct from apoptosis, necrosis, and autophagy; it is associated its cysteine import capacity (Watts et al., 2014). EAAT3 might with excessive iron-mediated accumulation of lipid ROS in be as important as X− c in providing GSH precursors to neurons, cancer cells as well as in hippocampal slice cultures exposed to considering that mice lacking EAAT3 have lower GSH levels glutamate excitotoxicity (Dixon et al., 2012). As excitotoxicity and age prematurely (Aoyama et al., 2006). Notably, low GSH is the main mechanism involved in neuronal death in ischemic levels might also inhibit GPX4 activity since GPX4 uses GSH as stroke, the involvement of ferroptosis in ischemic stroke could be a cofactor to catalyze the reduction of lipid peroxides and protect suspected. In addition, the role of high systemic iron exacerbating cells membranes against peroxidation (see Latunde-Dada, 2017). stroke-induced damage, concomitant with increased brain iron Conversely, ferroptosis can be prevented by (1) iron- after ischemic stroke onset (reviewed in the previous section), binding compounds such as DFO and ciclopirox; (2) small further suggested a possible ferroptosis component in stroke; molecules, free radical scavengers, or lipophilic antioxidants in the recent past, mounting evidence indicated that ferroptosis such as ferrostatin-1, α-tocopherol, butylated hydroxytoluene, or plays such a pivotal role in stroke-induced neurodegeneration liproxstatin-1; (3) treatments with molecules such as deuterated Frontiers in Neuroscience | www.frontiersin.org 9 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke PUFA, inhibitors of acyl-CoA synthetase long-chain family in addition to the well-known NMDAR-mediated calcium member 4, lipoxygenase inhibitors, glutaminolysis inhibitors, or influx, an NMDAR-mediated iron uptake. They also identified cycloheximide; or (4) boosting cystine and/or cysteine import by a signaling cascade whereby NMDAR regulated neuronal reducing extracellular glutamate. iron homeostasis and excitotoxicity through an NMDAR/nitric Glutamate is the most abundant excitatory neurotransmitters oxide/dexamethasone-induced Ras protein 1 (Dexras1) pathway in the vertebrate nervous system, with 90% of the synaptic that increased both non-Tf and Tf-mediated iron uptake from the neurotransmission considered glutamatergic (Hofmeijer and Van extracellular medium through the neuronal plasma membrane Putten, 2012; Mayor and Tymianski, 2018). Under physiological (Cheah et al., 2006). A similar increase of iron uptake in conditions, extracellular glutamate levels are low, and glutamate hippocampal neurons was observed in a subsequent study in exerts its physiological role mainly at the neuronal synapse. which either synaptic activity or NMDAR activity was boosted The ability of astrocytes near glutamatergic neurons to rapidly (Pelizzoni et al., 2011). Moreover, either ischemia or NMDA clear synaptic glutamate after its physiological action is critical overactivation increased the uptake of HTf, resulting in an in the functioning of synapses and neuronal circuits, and the increase of cytosolic redox-active iron in neurons, whereas initial increase of extracellular glutamate is inhibited mainly blockade of TfR1 reduced iron dyshomeostasis and NMDA- by glial EAAT1 and 2. Neuronal glutamate transporters, induced excitotoxicity (DeGregorio-Rocasolano et al., 2018). mainly EAAT3, also play a role in clearing extracellular The original work from Snyder’s laboratory hypothesized that glutamate excess. However, ischemia leads to energy failure iron in its free form gains access to the neuronal cytosol and subsequent electrochemical gradient impairment, causing through DMT1 present in the neuronal membrane. However, EAATs to act in a reversed manner, thereby extruding non- this would not explain the uptake of Tf-bound iron since the vesicular intracellular glutamate and resulting in large excess DMT1 isoform endogenously overexpressed in primary cultures of extracellular glutamate and impairment of EAAT3 as a of hippocampal neurons exposed to NMDA is DMT1-1B/IRE(+) unidirectional cysteine import channel. (Haeger et al., 2010) and is found in endosomes/lysosomes in The impact of excess extracellular glutamate on neuronal the cytoplasm (Pelizzoni et al., 2012), thereby not supporting survival is more than that on the cystine/glutamate exchanger X− c . a direct role of DMT1 in iron entry through the neuronal In fact, most of the effect is produced by glutamate binding to membrane. Following the above rationale of crossroads between specific glutamate receptors present in cell membranes of several excitotoxicity and ferroptosis, several in vitro and in vivo cell types and, especially in neurons, N-methyl-D-aspartate studies showed that neurons survive otherwise lethal NMDA (NMDA) subtype of glutamate receptors (NMDARs) (Guirao or glutamate concentrations when treated with iron chelators et al., 2017) that are coupled to an intracellular downstream such as DFO or deferasirox (Yu et al., 2009; Tian et al., complex of signaling effectors, which are known to initiate 2017; Sakamoto et al., 2018). Moreover, a study showed that excitotoxic neuronal death following ischemia. the deletion of Dexras1 reduced NMDA-induced iron uptake Although both glutamate and iron are essential for neuronal and NMDA toxicity in cortical neurons and retinal ganglion survival, excess glutamate can be toxic, and excess free iron cells (Chen et al., 2013). Moreover, Dexras1-KO mice showed through the Fenton reaction is a potentially toxic substance increased expression of the NR2A subunit of the NMDAR that can catalyze the production of extremely damaging (Carlson et al., 2016), a subunit that is preferentially located ROS. During stroke, the impairment of BBB function allows at synapses and related to NMDAR pro-survival signaling; increased access to brain of iron-carrying substances and, this strongly suggests the existence of a functional crosstalk preceding neurodegeneration, iron accumulates in the ischemic between the NMDA-induced iron intracellular signaling and the areas (Helal, 2008); therefore, not surprisingly, iron overload physiological NMDAR function. Interestingly, intracellular iron conditions are associated with larger areas of brain damage in released from lysosomes has been shown to modulate NMDAR animals exposed to experimental ischemic stroke (Castellanos synaptic excitability via Dexras1 (White et al., 2016), and iron is et al., 2002; Gamez et al., 2003; Mehta et al., 2004; García- needed post-synaptically to activate Ca2+ -dependent pathways Yébenes et al., 2012; DeGregorio-Rocasolano et al., 2018). downstream NMDAR (Muñoz et al., 2011). Further, glutamate- Following experimental stroke or NMDAR overactivation, induced neuronal excitotoxicity was described as ferroptosis prevention of NMDA-induced neuronal iron uptake was found (Dixon et al., 2012). to be neuroprotective (DeGregorio-Rocasolano et al., 2018). In the widely reported scenario of elevated extracellular Therefore, the crosstalk between neuronal homeostasis of iron glutamate during ischemic stroke, NMDAR-induced iron import and glutamatergic signaling is increasingly being supported was upregulated in neurons associated with increased ROS by evidence and should not be disregarded as a therapeutic production (Cheah et al., 2006), and system X− c and EAAT3 intervention target. cystine/cysteine import are impaired by high extracellular glutamate (Conrad and Sato, 2012), thus compromising neuronal iron homeostasis and GSH-mediated free radical detoxification, Glutamate-Induced Iron Uptake in eventually leading to an excess lipid oxidative stress that Neurons and Ferroptosis causes ferroptotic damage. High GSH stores are pivotal for Cheah et al. (2006) from Dr. Snyder’s laboratory published a cell survival since GSH is required for GPX4 activity and study before the concept of ferroptosis was introduced and is also thought to be the only molecular species having a showed that the overactivation of NMDAR in neurons induces, significant role as a cytosolic ligand for Fe2+ (Philpott and Frontiers in Neuroscience | www.frontiersin.org 10 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke Ryu, 2014). Even when ischemia is resolved in the brain owing and susceptible to be chelated, precedes the increase in total to a successful reperfusion, other mechanisms involved in iron. In experimental models of transient ischemic stroke, ferroptotic damage might become dangerous. In this regard, a DFO administered intramuscularly (Xing et al., 2009) or recent study on Sertoli cells revealed that GPX4 is inactivated intranasally (Hanson et al., 2009) after the induction of via GSH depletion following I/R injury (Li et al., 2018), and ischemia significantly decreased infarct volume. Another iron several studies showed that GPX4 gene inactivation causes chelator, the aforementioned 2,2’-dipyridyl was found to be hippocampal neurodegeneration (Seiler et al., 2008; Hambright cytoprotective after permanent arterial occlusion in rats; this was et al., 2017) and a remarkable degeneration of motor neurons associated with reduced infarct size and preservation of GSH (Chen et al., 2015). levels (Demougeot et al., 2004) or reduction of the apoptotic component and prevention of the enlargement of the lesion (Van Hoecke et al., 2005). Ferroptosis in AIS and ICH Stroke The role of iron in ICH neurodegeneration has been known Subtypes: Effect of Ferroptosis Inhibitors for long. Recently, neuronal death following ICH was found and Iron Chelators to have features similar to those of ferroptosis, since chemical In addition to a pivotal role of ferroptosis in I/R-induced damage inhibitors of ferroptosis protected against Hb- and hemin- in several peripheral organs (Friedmann Angeli et al., 2014; induced neurotoxicity (Zille et al., 2017). The nonheme iron Linkermann et al., 2014; Gao et al., 2015), a role of ferroptosis present in brain parenchyma after ICH is a source of redox- has been specifically identified in I/R-induced brain damage in active iron. The marked increase in brain nonheme iron was transient ischemia, as brain damage was substantially reduced not cleared for weeks following the ICH event, and brain by the ferroptosis inhibitor liproxstatin-1 in an AIS model (Tuo Tf, TfR, and FT levels were also increased 72 h after ICH, et al., 2017). These authors also showed that a reduction of thereby making FT upregulation long-lasting (Wu et al., 2003). tau levels, a protein that facilitates iron export by trafficking Iron chelators such as DFO reduce hemin- and iron-mediated APP to membranes to stabilize FPN, was found to precede a neurotoxicity, perihematoma edema, and neuronal damage, pro-ferroptotic iron accumulation in the brain following I/R leading to good neurologic outcomes after ICH (Nakamura (Tuo et al., 2017). APP mRNA possesses a functional IRE et al., 2004; Hatakeyama et al., 2013). Other recent studies with sequence homology to that of FT, and its translation is indicated that the specific inhibitor of ferroptosis ferrostatin-1 responsive to free iron levels in the cytosol (Duce et al., 2010). reduces neuron degeneration and improves neurological deficit In addition, APP has been reported to interact with FPN to in experimental ICH models (Li Q. et al., 2017; Zhang et al., 2018), promote iron release in several brain cell types (Duce et al., and GPX4 levels were found to be reduced after ICH, whereas 2010; McCarthy et al., 2014). APP overexpression reduces free pharmacological inhibition of GPX or genetic knockdown iron content in neuroblastoma cells (Wan et al., 2012), and exacerbated brain injury after ICH (Zhang et al., 2018). Moreover, either preventing APP transport to the cell surface or knocking inhibitors of ALOX5 or inhibition of the ALOX5 metabolic down of APP results in iron accumulation in primary cortical products protected against hemin- or ICH-induced ferroptosis neurons in culture (Lei et al., 2012). The complete role of APP (Karuppagounder et al., 2018). in ferroptosis and neurodegeneration in AIS requires additional Moreover, DFO significantly reduced hemorrhagic studies. However, further evidence supporting the involvement transformation after focal I/R (Xing et al., 2009; Li Y. et al., of ferroptosis mechanisms in AIS brain damage is provided by 2017). Some studies have shown that the effect of DFO in the overexpression of lipoxygenases following brain ischemia and preventing ischemic neuronal death is related to the DFO- the protection afforded by treatment with lipoxygenase inhibitors induced expression of hypoxia-inducible factor 1 and its (Cui et al., 2010; Karatas et al., 2018). Further, a recent study downstream targets genes rather than with its iron-chelating showed that N-acetylcysteine (NAC), an FDA-approved cysteine properties or, recently, with microglial/macrophage HO1 prodrug, prevents hemin-induced ferroptosis by neutralizing expression in ICH (LeBlanc et al., 2016). A systematic review toxic lipids generated by arachidonate-dependent activity of 5- and meta-analysis in experimental models of ICH showed that lipoxygenases (ALOX5) (Karuppagounder et al., 2018). They DFO was neuroprotective, e.g., reduction of brain edema and reported that ALOX5 inhibitors such as zileuton and molecules improvement of neurobehavioral outcomes, especially when such as NAC that inhibit toxic metabolic products of ALOX5 it was administered 2−4 h after ICH induction (Cui et al., or prevent ALOX5 recruitment to its site of action, protect 2015). In clinical studies, conclusive evidence of the benefit of against hemin or ICH-induced ferroptosis (Karuppagounder DFO treatment in ICH (Zeng et al., 2018) or AIS patients is et al., 2018). Notably, NAC has been previously reported to still lacking; some clinical trials of DFO in ICH and AIS are prevent neuronal damage induced by AIS (Khan et al., 2004; currently ongoing. Turkmen et al., 2016). The rationale for iron chelation as a neuroprotectant relies Further, the role of free iron in ferroptosis and primarily on the idea that chelation removes free iron present neurodegeneration is further supported by the protective in the blood and enhances iron elimination in the urine. After effect of treatments with exogenous iron chelators, e.g., DFO gaining access to other body compartments, DFO could also or 2,20 -dipyridyl, in animal models of stroke. After permanent act by chelating free extracellular and cytosolic iron, whereas ischemic stroke (Millerot-Serrurot et al., 2008), the increase its effect on blood TSAT or blood total iron levels is modest in free iron, the iron form prone to induce lipid peroxidation (Tauchenová et al., 2016). In fact, DFO was shown to have Frontiers in Neuroscience | www.frontiersin.org 11 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke an extremely slow removal rate of iron from Tf (Abergel has been known for long, since blood spillover results and Raymond, 2008). This, together with the fact that DFO in iron-Hb and iron–heme complexes at the extracellular half-life in the plasma is extremely short (20 min), poses milieu of brain parenchymal cells, thereby contributing to serious doubts about the effectiveness of the treatment with iron-induced lipid peroxidation and neuronal death. The intravenous DFO unless administered with continuous infusion. review documents the beneficial effects of iron chelators Recently, another iron-regulator, apotransferrin, administered such as DFO, physiological molecules with neuroprotective after stroke AIS onset, was found to be neuroprotective because potential, and specific inhibitors of ferroptosis liproxstatin-1 or of the systemic reduction of blood TSAT and a reduction of ferrostatin-1 in reducing neuron degeneration and improving NMDA ischemia-induced neuronal iron import (DeGregorio- neurologic deficits induced by ischemic and hemorrhagic Rocasolano et al., 2018). stroke subtypes. CONCLUSION AUTHOR CONTRIBUTIONS Increasing number of studies show that ferroptosis is involved in several neurodegenerative diseases. This review summarizes The three authors have contributed in a similar way to the writing the knowledge available thus far regarding the role of iron and of the article. ferroptosis in the execution of neuronal cell death during AIS and ICH. In AIS-induced cell death, the role of systemic iron, Tf, TSAT, and hepcidin on excitotoxic damage, infarct size, and FUNDING neurological outcome in rodent models of stroke might have important clinical implications. Following the AIS event, high This study was supported by the following grants: Instituto levels of extracellular glutamate and impairment of the BBB de Salud Carlos III (ISCIII) INVICTUS PLUS RD16/0019/0020 allow the NMDAR-mediated influx of iron, either bound or not that was susceptible to be co-financed by FEDER funds and to Tf, into neurons. In addition, high extracellular glutamate Agència de Gestió d’Ajuts Universitaris i de Recerca 2017 SGR prevents the neuronal production of the antioxidant GSH or the 1520. We acknowledge the funding received from “la Caixa” function of GPX4, thereby promoting a form of cell death in Foundation CI15-00009 and from the European Institute of which glutamate receptor overactivation boosts neuronal iron Innovation and Technology (EIT) PoC-2016-SPAIN-04. EIT uptake, which in turn promotes an overwhelming production of receives support from the European Union’s Horizon 2020 membrane peroxides. The role of iron in ICH neurodegeneration research and innovation program. REFERENCES Belaidi, A. A., Gunn, A. P., Wong, B. X., Ayton, S., Appukuttan, A. T., Roberts, B. R., et al. (2018). Marked age-related changes in brain iron homeostasis in Abergel, R. J., and Raymond, K. N. (2008). Terephthalamide-containing ligands: amyloid protein precursor knockout mice. Neurotherapeutics 15, 1055–1062. fast removal of iron from transferrin. J. Biol. Inorg. Chem. 13, 229–240. doi: doi: 10.1007/s13311-018-0656-x 10.1007/s00775-007-0314-y Bickford, J. S., Ali, N. F., Nick, J. A., Al-Yahia, M., Beachy, D. E., Doré, S., et al. Abulrob, A., Brunette, E., Slinn, J., Baumann, E., and Stanimirovic, D. (2011). (2014). Endothelin-1-mediated vasoconstriction alters cerebral gene expression In vivo optical imaging of ischemic blood-brain barrier disruption. Methods in iron homeostasis and eicosanoid metabolism. Brain Res. 1588, 25–36. doi: Mol. Biol. 763, 423–429. doi: 10.1007/978-1-61779-191-8_29 10.1016/j.brainres.2014.09.022 Acikyol, B., Graham, R. M., Trinder, D., House, M. J., Olynyk, J. K., Scott, R. J., Bradbury, M. W. (1997). Transport of iron in the blood-brain-cerebrospinal fluid et al. (2013). Brain transcriptome perturbations in the transferrin receptor 2 system. J. Neurochem. 69, 443–454. doi: 10.1046/j.1471-4159.1997.69020443.x mutant mouse support the case for brain changes in iron loading disorders, Burdo, J. R., Menzies, S. L., Simpson, I. A., Garrick, L. M., Garrick, M. D., including effects relating to long-term depression and long-term potentiation. Dolan, K. G., et al. (2001). Distribution of divalent metal transporter 1 and Neuroscience 235, 119–128. doi: 10.1016/j.neuroscience.2013.01.014 metal transport protein 1 in the normal and Belgrade rat. J. Neurosci. Res. 66, Aoyama, K., Sang, W. S., Hamby, A. M., Liu, J., Wai, Y. C., Chen, Y., et al. (2006). 1198–1207. doi: 10.1002/jnr.1256 Neuronal glutathione deficiency and age-dependent neurodegeneration in the Carbonell, T., and Rama, M. (2007). Iron, oxidative stress and early neurological EAAC1 deficient mouse. Nat. Neurosci. 9, 119–126. doi: 10.1038/nn1609 deterioration in ischemic stroke. Curr. Med. Chem. 14, 857–874. doi: 10.2174/ Arosio, P., Elia, L., and Poli, M. (2017). Ferritin, cellular iron storage and 092986707780363014 regulation. IUBMB Life 69, 414–422. doi: 10.1002/iub.1621 Carlson, G. C., Lin, R. E., Chen, Y., Brookshire, B. R., White, R. S., Lucki, I., Asano, T., Komatsu, M., Yamaguchi-Iwai, Y., Ishikawa, F., Mizushima, N., and et al. (2016). Dexras1 a unique ras-GTPase interacts with NMDA receptor Iwai, K. (2011). Distinct mechanisms of ferritin delivery to lysosomes in iron- activity and provides a novel dissociation between anxiety, working memory depleted and iron-replete cells. Mol. Cell. Biol. 31, 2040–2052. doi: 10.1128/ and sensory gating. Neuroscience 322, 408–415. doi: 10.1016/j.neuroscience. MCB.01437-10 2016.02.063 Ashraf, A., Clark, M., and So, P. W. (2018). The aging of iron man. Front. Aging Castellanos, M., Puig, N., Carbonell, T., Castillo, J., Martinez, J. M., Rama, R., et al. Neurosci. 10:65. doi: 10.3389/fnagi.2018.00065 (2002). Iron intake increases infarct volume after permanent middle cerebral Bakhshayesh, B., Hosseininezhad, M., Saadat, S., Ansar, M., Ramezani, H., artery occlusion in rats. Brain Res. 952, 1–6. doi: 10.1016/S0006-8993(02) and Saadat, S. (2014). Iron overload is associated with perihematoma 03179-7 edema growth following intracerebral hemorrhage that may contribute Chamorro, Á, Dirnagl, U., Urra, X., and Planas, A. M. (2016). Neuroprotection to in-hospital mortality and long-term functional outcome. Curr. in acute stroke: targeting excitotoxicity, oxidative and nitrosative stress, Neurovasc. Res. 11, 248–253. doi: 10.2174/15672026116661405301 and inflammation. Lancet Neurol. 15, 869–881. doi: 10.1016/S1474-4422(16) 24855 00114-9 Frontiers in Neuroscience | www.frontiersin.org 12 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke Cheah, J. H., Kim, S. F., Hester, L. D., Clancy, K. W., Patterson, S. E., Deane, R., Zheng, W., and Zlokovic, B. V. (2004). Brain capillary endothelium and Papadopoulos, V., et al. (2006). NMDA receptor-nitric oxide transmission choroid plexus epithelium regulate transport of transferrin-bound and free iron mediates neuronal iron homeostasis via the GTPase Dexras1. Neuron 51, into the rat brain. J. Neurochem. 88, 813–820. doi: 10.1046/j.1471-4159.2003. 431–440. doi: 10.1016/j.neuron.2006.07.011 02221.x Chen, G., Zhang, D., Fuchs, T. A., Manwani, D., Wagner, D. D., and Frenette, DeGregorio-Rocasolano, N., Martí-Sistac, O., Ponce, J., Castelló-Ruiz, M., P. S. (2014). Heme-induced neutrophil extracellular traps contribute to the Millán, M., Guirao, V., et al. (2018). Iron-loaded transferrin (Tf) is detrimental pathogenesis of sickle cell disease. Blood 123, 3818–3827. doi: 10.1182/blood- whereas iron-free Tf confers protection against brain ischemia by modifying 2013-10-529982 blood Tf saturation and subsequent neuronal damage. Redox Biol. 15, 143–158. Chen, H., Yoshioka, H., Kim, G. S., Jung, J. E., Okami, N., Sakata, H., et al. doi: 10.1016/j.redox.2017.11.026 (2011). Oxidative stress in ischemic brain damage: mechanisms of cell death Demougeot, C., Van Hoecke, M., Bertrand, N., Prigent-Tessier, A., Mossiat, C., and potential molecular targets for neuroprotection. Antioxid. Redox Signal. 14, Beley, A., et al. (2004). Cytoprotective efficacy and mechanisms of the 1505–1517. doi: 10.1089/ars.2010.3576 liposoluble iron chelator 2,2’-dipyridyl in the rat photothrombotic ischemic Chen, L., Zhang, X., Chen-Roetling, J., and Regan, R. F. (2011). Increased striatal stroke model. J. Pharmacol. Exp. Ther. 311, 1080–1087. doi: 10.1124/jpet.104. injury and behavioral deficits after intracerebral hemorrhage in hemopexin 072744 knockout mice. J. Neurosurg. 114, 1159–1167. doi: 10.3171/2010.10.JNS10861 Desestret, V., Brisset, J. C., Moucharrafie, S., Devillard, E., Nataf, S., Honnorat, J., Chen, L., Hambright, W. S., Na, R., and Ran, Q. (2015). Ablation of the ferroptosis et al. (2009). Early-stage investigations of ultrasmall superparamagnetic iron inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron oxide-induced signal change after permanent middle cerebral artery occlusion degeneration and paralysis. J. Biol. Chem. 290, 28097–28106. doi: 10.1074/jbc. in mice. Stroke 40, 1834–1841. doi: 10.1161/STROKEAHA.108.531269 M115.680090 Ding, H., Yan, C. Z., Shi, H., Zhao, Y. S., Chang, S. Y., Yu, P., et al. (2011). Hepcidin Chen, Y., Khan, R. S., Cwanger, A., Song, Y., Steenstra, C., Bang, S., is involved in iron regulation in the ischemic brain. PLoS One 6:e25324. doi: et al. (2013). Dexras1, a small GTPase, is required for glutamate-NMDA 10.1371/journal.pone.0025324 neurotoxicity. J. Neurosci. 33, 3582–3587. doi: 10.1523/JNEUROSCI.1497-12. Dixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev, E. M., Gleason, 2013 C. E., et al. (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell Chen-Roetling, J., Cai, Y., Lu, X., and Regan, R. F. (2014). Hemin uptake and release death. Cell 149, 1060–1072. doi: 10.1016/j.cell.2012.03.042 by neurons and glia. Free Radic. Biol. Med. 48, 200–205. doi: 10.3109/10715762. Drecourt, A., Babdor, J., Dussiot, M., Petit, F., Goudin, N., Garfa-Traoré, M., 2013.859386 et al. (2018). Impaired transferrin receptor palmitoylation and recycling in Chen-Roetling, J., Kamalapathy, P., Cao, Y., Song, W., Schipper, H. M., and Regan, neurodegeneration with brain iron accumulation. Am. J. Hum. Genet. 102, R. F. (2017). Astrocyte heme oxygenase-1 reduces mortality and improves 266–277. doi: 10.1016/j.ajhg.2018.01.003 outcome after collagenase-induced intracerebral hemorrhage. Neurobiol. Dis. Du, F., Qian, Z. M., Luo, Q., Yung, W. H., and Ke, Y. (2015). Hepcidin suppresses 102, 140–146. doi: 10.1016/j.nbd.2017.03.008 brain iron accumulation by downregulating iron transport proteins in iron- Chen-Roetling, J., Ma, S.-K., Cao, Y., Shah, A., and Regan, R. F. (2018). overloaded rats. Mol. Neurobiol. 52, 101–114. doi: 10.1007/s12035-014-8847-x Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is Duce, J. A., Tsatsanis, A., Cater, M. A., James, S. A., Robb, E., Wikhe, K., et al. (2010). absent. J. Neurochem. 145, 464–473. doi: 10.1111/jnc.14328 An iron-export ferroxidase activity of APP is inhibited by zinc in Alzheimer’s Chen-Roetling, J., and Regan, R. (2016). Haptoglobin increases the vulnerability disease. Cell 142, 857–867. doi: 10.1016/j.cell.2010.08.014 of CD163-expressing neurons to hemoglobin. J. Neurochem. 139, 586–589. Duck, K. A., Simpson, I. A., and Connor, J. R. (2017). Regulatory mechanisms for doi: 10.1111/jnc.13720 iron transport across the blood-brain barrier. Biochem. Biophys. Res. Commun. Chiou, B., Lucassen, E., Sather, M., Kallianpur, A., and Connor, J. (2018a). 494, 70–75. doi: 10.1016/j.bbrc.2017.10.083 Semaphorin4A and H-ferritin utilize Tim-1 on human oligodendrocytes: a Dutra, F. F., and Bozza, M. T. (2014). Heme on innate immunity and inflammation. novel neuro-immune axis. Glia 66, 1317–1330. doi: 10.1002/glia.23313 Front. Pharmacol. 5:115. doi: 10.3389/fphar.2014.00115 Chiou, B., Neal, E. H., Bowman, A. B., Lippmann, E. S., Simpson, I. A., and Dziuba, N., and Lindahl, P. A. (2018). Low-molecular-mass iron in healthy blood Connor, J. R. (2018b). Endothelial cells are critical regulators of iron transport plasma is not predominately ferric citrate. Metallomics 10, 802–817. doi: 10. in a model of the human blood–brain barrier. J. Cereb. Blood Flow Metab. 1039/c8mt00055g doi: 10.1177/0271678X18783372 [Epub ahead of print]. Feng, H., and Stockwell, B. R. (2018). Unsolved mysteries: how does lipid Cobley, J. N., Fiorello, M. L., and Bailey, D. M. (2018). 13 Reasons why the brain peroxidation cause ferroptosis? PLoS Biol. 16:e2006203. doi: 10.1371/journal. is susceptible to oxidative stress. Redox Biol. 15, 490–503. doi: 10.1016/j.redox. pbio.2006203 2018.01.008 Friedmann Angeli, J. P., Schneider, M., Proneth, B., Tyurina, Y. Y., Tyurin, V. A., Conrad, M., and Sato, H. (2012). The oxidative stress-inducible cystine/glutamate Hammond, V. J., et al. (2014). Inactivation of the ferroptosis regulator Gpx4 antiporter, system Xc-: cystine supplier and beyond. Amino Acids 42, 231–246. triggers acute renal failure in mice. Nat. Cell Biol. 16, 1180–1191. doi: 10.1038/ doi: 10.1007/s00726-011-0867-5 ncb3064 Cui, H. J., He, H. Y., Yang, A. L., Zhou, H. J., Wang, C., Luo, J. K., et al. (2015). Gaasch, J. A., Lockman, P. R., Geldenhuys, W. J., Allen, D. D., and Van Der Schyf, Efficacy of deferoxamine in animal models of intracerebral hemorrhage: a C. J. (2007). Brain iron toxicity: differential responses of astrocytes, neurons, systematic review and stratified meta-analysis. PLoS One 10:e0127256. doi: and endothelial cells. Neurochem. Res. 32, 1196–1208. doi: 10.1007/s11064-007- 10.1371/journal.pone.0127256 9290-4 Cui, L., Zhang, X., Yang, R., Liu, L., Wang, L., Li, M., et al. (2010). Baicalein Galea, J., Cruickshank, G., Teeling, J. L., Boche, D., Garland, P., Perry, V. H., et al. is neuroprotective in rat MCAO model: role of 12/15-lipoxygenase, mitogen- (2012). The intrathecal CD163-haptoglobin-hemoglobin scavenging system in activated protein kinase and cytosolic phospholipase A2. Pharmacol. Biochem. subarachnoid hemorrhage. J. Neurochem. 121, 785–792. doi: 10.1111/j.1471- Behav. 96, 469–475. doi: 10.1016/j.pbb.2010.07.007 4159.2012.07716.x Das, A., Nag, S., Mason, A. B., and Barroso, M. M. (2016). Gamez, A., Carbonell, T., and Rama, R. (2003). Does nitric oxide contribute to Endosome–mitochondria interactions are modulated by iron release iron-dependent brain injury after experimental cerebral ischaemia? J. Physiol. from transferrin. J. Cell Biol. 214, 831–845. doi: 10.1083/jcb.201 Biochem. 59, 249–254. doi: 10.1007/BF03179881 602069 Gao, G., Zhang, N., Wang, Y., Wu, Q., Yu, P., Shi, Z., et al. (2017). Mitochondrial Davalos, A., Fernandez-Real, J. M., Ricart, W., Soler, S., Molins, A., Planas, E., et al. ferritin protects hydrogen peroxide-induced neuronal cell damage. Aging Dis. (1994). Iron-related damage in acute ischemic stroke. Stroke 25, 1543–1546. 8, 458–470. doi: 10.14336/AD.2016.1108 doi: 10.1161/01.STR.25.8.1543 Gao, M., Monian, P., Quadri, N., Ramasamy, R., and Jiang, X. (2015). De Valk, B., Addicks, M. A., Gosriwatana, I., Lu, S., Hider, R. C., and Marx, Glutaminolysis and transferrin regulate ferroptosis. Mol. Cell 59, 298–308. doi: J. J. M. (2000). Non-transferrin-bound iron is present in serum of hereditary 10.1016/j.molcel.2015.06.011 haemochromatosis heterozygotes. Eur. J. Clin. Invest. 30, 248–251. doi: 10.1046/ García-Yébenes, I., García-Culebras, A., Peña-Martínez, C., Fernández-López, D., j.1365-2362.2000.00628.x Díaz-Guzmán, J., Negredo, P., et al. (2018). Iron overload exacerbates the risk Frontiers in Neuroscience | www.frontiersin.org 13 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke of hemorrhagic transformation after tPA (tissue-type plasminogen activator) Huang, H., Chen, J., Lu, H., Zhou, M., Chai, Z., and Hu, Y. (2017). Iron-induced administration in thromboembolic stroke mice. Stroke 49, 2163–2172. doi: generation of mitochondrial ROS depends on AMPK activity. BioMetals 30, 10.1161/STROKEAHA.118.021540 623–628. doi: 10.1007/s10534-017-0023-0 García-Yébenes, I., Sobrado, M., Moraga, A., Zarruk, J. G., Romera, V. G., Pradillo, Huang, S., Du, F., Li, L., Liu, Y., Liu, Y., Zhang, C., et al. (2014). J. M., et al. (2012). Iron overload, measured as serum ferritin, increases brain Angiotensin II inhibits uptake of transferrin-bound iron but not non- damage induced by focal ischemia and early reperfusion. Neurochem. Int. 61, transferrin-bound iron by cultured astrocytes. Neuropeptides 48, 161–166. doi: 1364–1369. doi: 10.1016/j.neuint.2012.09.014 10.1016/j.npep.2014.04.001 Garton, A. L. A., Gupta, V. P., Christophe, B. R., and Connolly, E. S. (2017). Hvidberg, V., Maniecki, M. B., Jacobsen, C., Højrup, P., Møller, H. J., and Biomarkers of functional outcome in intracerebral hemorrhage: interplay Moestrup, S. K. (2005). Identification of the receptor scavenging hemopexin- between clinical metrics, CD163, and ferritin. J. Stroke Cerebrovasc. Dis. 26, heme complexes. Blood 106, 2572–2579. doi: 10.1182/blood-2005-03-1185 1712–1720. doi: 10.1016/j.jstrokecerebrovasdis.2017.03.035 Jaffer, H., Adjei, I. M., and Labhasetwar, V. (2013). Optical imaging to map Gazzina, S., Premi, E., Zanella, I., Biasiotto, G., Archetti, S., Cosseddu, M., blood-brain barrier leakage. Sci. Rep. 3:3117. doi: 10.1038/srep03117 et al. (2016). Iron in frontotemporal lobar degeneration: a new subcortical Jahanshad, N., Kohannim, O., Hibar, D. P., Stein, J. L., McMahon, K. L., de pathological pathway? Neurodegener. Dis. 16, 172–178. doi: 10.1159/000440843 Zubicaray, G. I., et al. (2012). Brain structure in healthy adults is related to Guan, H., Yang, H., Yang, M., Yanagisawa, D., Bellier, J. P., Mori, M., et al. (2017). serum transferrin and the H63D polymorphism in the HFE gene. Proc. Natl. Mitochondrial ferritin protects SH-SY5Y cells against H2O2-induced oxidative Acad. Sci. 109, E851–E859. doi: 10.1073/pnas.1105543109 stress and modulates α-synuclein expression. Exp. Neurol. 291, 51–61. doi: Jeney, V., Eaton, J. W., Balla, G., and Balla, J. (2013). Natural history of the bruise: 10.1016/j.expneurol.2017.02.001 formation, elimination, and biological effects of oxidized hemoglobin. Oxid. Guirao, V., Martí-Sistac, O., DeGregorio-Rocasolano, N., Ponce, J., Dávalos, A., Med. Cell. Longev. 2013:703571. doi: 10.1155/2013/703571 and Gasull, T. (2017). Specific rescue by ortho-hydroxy atorvastatin of cortical Ji, C., and Kosman, D. J. (2015). Molecular mechanisms of non-transferrin- GABAergic neurons from previous oxygen/glucose deprivation: role of pCREB. bound and transferring-bound iron uptake in primary hippocampal neurons. J. Neurochem. 143, 359–374. doi: 10.1111/jnc.14210 J. Neurochem. 133, 668–683. doi: 10.1111/jnc.13040 Gutteridge, J. M., Richmond, R., and Halliwell, B. (1979). Inhibition of the Karatas, H., Eun Jung, J., Lo, E. H., and van Leyen, K. (2018). Inhibiting 12/15- iron-catalysed formation of hydroxyl radicals from superoxide and of lipid lipoxygenase to treat acute stroke in permanent and tPA induced thrombolysis peroxidation by desferrioxamine. Biochem. J. 184, 469–472. doi: 10.1042/ models. Brain Res. 1678, 123–128. doi: 10.1016/j.brainres.2017.10.024 bj1840469 Karuppagounder, S. S., Alin, L., Chen, Y., Brand, D., Bourassa, M. W., Dietrich, K., Hadziahmetovic, M., Song, Y., Ponnuru, P., Iacovelli, J., Hunter, A., Haddad, N., et al. (2018). N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and et al. (2011). Age-dependent retinal iron accumulation and degeneration in can synergize with PGE 2 to inhibit ferroptosis and improve outcomes following hepcidin knockout mice. Investig. Ophthalmol. Vis. Sci. 52, 109–118. doi: 10. hemorrhagic stroke in mice. Ann. Neurol. 84, 854–872. doi: 10.1002/ana.25356 1167/iovs.10-6113 Kaur, C., and Ling, E. A. (1995). Transient expression of transferrin receptors Haeger, P., Alvarez, A., Leal, N., Adasme, T., Núñez, M. T., and Hidalgo, C. and localisation of iron in amoeboid microglia in postnatal rats. J. Anat. 186, (2010). Increased hippocampal expression of the divalent metal transporter 165–173. 1 (DMT1) mRNA variants 1B and +IRE and DMT1 protein after NMDA- Kawabata, H. (2018). Transferrin and transferrin receptors update. Free Radic. Biol. receptor stimulation or spatial memory training. Neurotox. Res. 17, 238–247. Med. doi: 10.1016/j.freeradbiomed.2018.06.037 [Epub ahead of print]. doi: 10.1007/s12640-009-9096-z Kell, D. B. (2009). Iron behaving badly: inappropriate iron chelation as a major Hambright, W. S., Fonseca, R. S., Chen, L., Na, R., and Ran, Q. (2017). Ablation of contributor to the aetiology of vascular and other progressive inflammatory and ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes degenerative diseases. BMC Med. Genomics 2:2. doi: 10.1186/1755-8794-2-2 cognitive impairment and neurodegeneration. Redox Biol. 12, 8–17. doi: 10. Khan, A. I., Liu, J., and Dutta, P. (2018). Iron transport kinetics through blood- 1016/j.redox.2017.01.021 brain barrier endothelial cells. Biochim. Biophys. Acta Gen. Subj. 1862, 1168– Hamdi, A., Roshan, T. M., Kahawita, T. M., Mason, A. B., Sheftel, A. D., and 1179. doi: 10.1016/j.bbagen.2018.02.010 Ponka, P. (2016). Erythroid cell mitochondria receive endosomal iron by a Khan, M., Sekhon, B., Jatana, M., Giri, S., Gilg, A. G., Sekhon, C., et al. (2004). “kiss-and-run” mechanism. Biochim. Biophys. Acta 1863, 2859–2867. doi: 10. Administration of N-Acetylcysteine after focal cerebral ischemia protects brain 1016/j.bbamcr.2016.09.008 and reduces inflammation in a rat model of experimental stroke. J. Neurosci. Han, J., Plummer, J., Liu, L., Byrd, A., Aschner, M., and Erikson, K. M. (2017). The Res. 76, 519–527. doi: 10.1002/jnr.20087 impact of obesity on brain iron levels and α-synuclein expression is regionally Klohs, J., Steinbrink, J., Bourayou, R., Mueller, S., Cordell, R., Licha, K., et al. (2009). dependent. Nutr. Neurosci. doi: 10.1080/1028415X.2017.1387720 [Epub ahead Near-infrared fluorescence imaging with fluorescently labeled albumin: a novel of print]. method for non-invasive optical imaging of blood-brain barrier impairment Hanson, L. R., Roeytenberg, A., Martinez, P. M., Coppes, V. G., Sweet, D. C., Rao, after focal cerebral ischemia in mice. J. Neurosci. Methods 180, 126–132. doi: R. J., et al. (2009). Intranasal deferoxamine provides increased brain exposure 10.1016/j.jneumeth.2009.03.002 and significant protection in rat ischemic stroke. J. Pharmacol. Exp. Ther. 330, Kumar, N., Rizek, P., Sadikovic, B., Adams, P. C., and Jog, M. (2016). Movement 679–686. doi: 10.1124/jpet.108.149807 disorders associated with hemochromatosis. Can. J. Neurol. Sci. 43, 801–808. Hatakeyama, T., Okauchi, M., Hua, Y., Keep, R. F., and Xi, G. (2013). Deferoxamine doi: 10.1017/cjn.2016.286 reduces neuronal death and hematoma lysis after intracerebral hemorrhage in Kumar, S., Sheokand, N., Mhadeshwar, M. A., Raje, C. I., and Raje, M. (2012). aged rats. Transl. Stroke Res. 4, 546–553. doi: 10.1007/s12975-013-0270-5 Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel He, Y., Karabiyikoglu, M., Hua, Y., Keep, R. F., and Xi, G. (2012). Ischemic transferrin receptor. Int. J. Biochem. Cell Biol. 44, 189–199. doi: 10.1016/j.biocel. preconditioning attenuates brain edema after experimental intracerebral 2011.10.016 hemorrhage. Transl. Stroke Res. 3, 180–187. doi: 10.1007/s12975-012-0171-z Latunde-Dada, G. O. (2017). Ferroptosis: role of lipid peroxidation, iron and Heidari, M., Johnstone, D. M., Bassett, B., Graham, R. M., Chua, A. C. G., House, ferritinophagy. Biochim. Biophys. Acta Gen. Subj. 1861, 1893–1900. doi: 10. M. J., et al. (2016). Brain iron accumulation affects myelin-related molecular 1016/j.bbagen.2017.05.019 systems implicated in a rare neurogenetic disease family with neuropsychiatric LeBlanc, R. H., Chen, R., Selim, M. H., and Hanafy, K. A. (2016). Heme features. Mol. Psychiatry 21, 1599–1607. doi: 10.1038/mp.2015.192 oxygenase-1-mediated neuroprotection in subarachnoid hemorrhage via Helal, G. K. (2008). Systemic administration of Zn2+during the reperfusion phase intracerebroventricular deferoxamine. J. Neuroinflammation 13:244. doi: 10. of transient cerebral ischaemia protects rat hippocampus against iron-catalysed 1186/s12974-016-0709-1 postischaemic injury. Clin. Exp. Pharmacol. Physiol. 35, 775–781. doi: 10.1111/ Leclerc, J. L., Santiago-Moreno, J., Dang, A., Lampert, A. S., Cruz, P. E., Rosario, j.1440-1681.2007.04858.x A. M., et al. (2018). Increased brain hemopexin levels improve outcomes after Hofmeijer, J., and Van Putten, M. J. (2012). Ischemic cerebral damage: an appraisal intracerebral hemorrhage. J. Cereb. Blood Flow Metab. 38, 1032–1046. doi: of synaptic failure. Stroke 43, 607–615. doi: 10.1161/STROKEAHA.111.632943 10.1177/0271678X16679170 Frontiers in Neuroscience | www.frontiersin.org 14 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke Lee, F. H. F., Su, P., Xie, Y. F., Wang, K. E., Wan, Q., and Liu, F. (2016). Disrupting McCarthy, R. C., Sosa, J. C., Gardeck, A. M., Baez, A. S., Lee, C.-H., and Wessling- GluA2-GAPDH interaction affects axon and dendrite development. Sci. Rep. Resnick, M. (2018). Inflammation-induced iron transport and metabolism by 6:30458. doi: 10.1038/srep30458 brain microglia. J. Biol. Chem. 293, 7853–7863. doi: 10.1074/jbc.RA118.001949 Lei, P., Ayton, S., Finkelstein, D. I., Spoerri, L., Ciccotosto, G. D., Wright, D. K., Mehdiratta, M., Kumar, S., Hackney, D., Schlaug, G., and Selim, M. (2008). et al. (2012). Tau deficiency induces parkinsonism with dementia by impairing Association between serum ferritin level and perihematoma edema volume APP-mediated iron export. Nat. Med. 18, 291–295. doi: 10.1038/nm.2613 in patients with spontaneous intracerebral hemorrhage. Stroke 39, 1165–1170. Leitner, D. F., and Connor, J. R. (2012). Functional roles of transferrin in the brain. doi: 10.1161/STROKEAHA.107.501213 Biochim. Biophys. Acta Gen. Subj. 1820, 393–402. doi: 10.1016/j.bbagen.2011. Mehta, S. H., Webb, R. C., Ergul, A., Tawfik, A., and Dorrance, A. M. (2004). 10.016 Neuroprotection by tempol in a model of iron-induced oxidative stress in acute Lewerenz, J., Ates, G., Methner, A., Conrad, M., and Maher, P. (2018). Oxytosis / ischemic stroke. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R283–R288. ferroptosis —( re- ) emerging roles for oxidative cell death in diseases of the doi: 10.1152/ajpregu.00446.2002 central nervous system. Front. Neurosci. 12:214. doi: 10.3389/fnins.2018.00214 Millan, M., Sobrino, T., Castellanos, M., Nombela, F., Arenillas, J. F., Riva, E., Li, J. Y., Paragas, N., Ned, R. M., Qiu, A., Viltard, M., Leete, T., et al. (2009). et al. (2007). Increased body iron stores are associated with poor outcome after Scara5 Is a ferritin receptor mediating non-transferrin iron delivery. Dev. Cell thrombolytic treatment in acute stroke. Stroke 38, 90–95. doi: 10.1161/01.STR. 16, 35–46. doi: 10.1016/j.devcel.2008.12.002 0000251798.25803.e0 Li, L., Fang, C. J., Ryan, J. C., Niemi, E. C., Lebron, J. A., Bjorkman, P. J., et al. Millerot, E., Prigent-Tessier, A. S., Bertrand, N. M., Faure, P. J. C., Mossiat, C. M., (2010). Binding and uptake of H-ferritin are mediated by human transferrin Giroud, M. E., et al. (2005). Serum ferritin in stroke: a marker of increased receptor-1. Proc. Natl. Acad. Sci. 107, 3505–3510. doi: 10.1073/pnas.0913 body iron stores or stroke severity? J. Cereb. Blood Flow Metab. 25, 1386–1393. 192107 doi: 10.1038/sj.jcbfm.9600140 Li, L., Hao, Y., Zhao, Y., Wang, H., Zhao, X., Jiang, Y., et al. (2018). Ferroptosis Millerot-Serrurot, E., Bertrand, N., Mossiat, C., Faure, P., Prigent-Tessier, A., is associated with oxygen- glucose deprivation / reoxygenation- induced Garnier, P., et al. (2008). Temporal changes in free iron levels after Sertoli cell death. Int. J. Mol. Med. 41, 3051–3062. doi: 10.3892/ijmm.2018. brain ischemia. Relevance to the timing of iron chelation therapy 3469 in stroke. Neurochem. Int. 52, 1442–1448. doi: 10.1016/j.neuint.2008. Li, Q., Han, X., Lan, X., Gao, Y., Wan, J., Durham, F., et al. (2017). Inhibition 04.002 of neuronal ferroptosis protects hemorrhagic brain. JCI Insight 2:e90777. doi: Morris, G., Berk, M., Carvalho, A. F., Maes, M., Walker, A. J., and Puri, B. K. 10.1172/jci.insight.90777 (2018). Why should neuroscientists worry about iron? The emerging role of Li, Y., Yang, H., Ni, W., and Gu, Y. (2017). Effects of deferoxamine on blood-brain ferroptosis in the pathophysiology of neuroprogressive diseases. Behav. Brain barrier disruption after subarachnoid hemorrhage. PLoS One 12:e0172784. doi: Res. 341, 154–175. doi: 10.1016/j.bbr.2017.12.036 10.1371/journal.pone.0172784 Muñoz, P., Humeres, A., Elgueta, C., Kirkwood, A., Hidalgo, C., and Núñez, M. T. Linkermann, A., Skouta, R., Himmerkus, N., Mulay, S. R., Dewitz, C., De Zen, F., (2011). Iron mediates N-Methyl-D-aspartate receptor-dependent stimulation et al. (2014). Synchronized renal tubular cell death involves ferroptosis. Proc. of calcium-induced pathways and hippocampal synaptic plasticity. J. Biol. Natl. Acad. Sci. 111, 16836–16841. doi: 10.1073/pnas.1415518111 Chem. 286, 13382–13392. doi: 10.1074/jbc.M110.213785 Liu, R., Cao, S., Hua, Y., Keep, R. F., Huang, Y., and Xi, G. (2017). CD163 Nagy, Z., and Nardai, S. (2017). Cerebral ischemia/repefusion injury: from bench expression in neurons after experimental intracerebral hemorrhage. Stroke 48, space to bedside. Brain Res. Bull. 134, 30–37. doi: 10.1016/j.brainresbull.2017. 1369–1375. doi: 10.1161/STROKEAHA.117.016850 06.011 Lo, A. C. Y., Cheung, A. K. H., Hung, V. K. L., Yeung, C. M., He, Q. Y., Chiu, J. F., Nakamura, T., Keep, R. F., Hua, Y., Schallert, T., Hoff, J. T., and Xi, G. (2004). et al. (2007). Deletion of aldose reductase leads to protection against cerebral Deferoxamine-induced attenuation of brain edema and neurological deficits ischemic injury. J. Cereb. Blood Flow Metab. 27, 1496–1509. doi: 10.1038/sj. in a rat model of intracerebral hemorrhage. J. Neurosurg. 100, 672–678. doi: jcbfm.9600452 10.3171/jns.2004.100.4.0672 Luck, A. N., and Mason, A. B. (2012). Transferrin-mediated cellular iron delivery. Nandar, W., and Connor, J. R. (2011). HFE gene variants affect iron in the brain. Curr. Top. Membr. 69, 3–35. doi: 10.1016/B978-0-12-394390-3.00001-X J. Nutr. 141, 729S–739S. doi: 10.3945/jn.110.130351 Ma, B., Day, J. P., Phillips, H., Slootsky, B., Tolosano, E., and Doré, S. (2016). Nicolas, G., Bennoun, M., Devaux, I., Beaumont, C., Grandchamp, B., Kahn, A., Deletion of the hemopexin or heme oxygenase-2 gene aggravates brain et al. (2001). Lack of hepcidin gene expression and severe tissue iron overload injury following stroma-free hemoglobin-induced intracerebral hemorrhage. in upstream stimulatory factor 2 (USF2) knockout mice. Proc. Natl. Acad. Sci. J. Neuroinflammation 13, 1–12. doi: 10.1186/s12974-016-0490-1 98, 8780–8785. doi: 10.1073/pnas.151179498 Ma, J., Li, F., Gan, Z., Yu, Z., Bao, X., Shen, Y., et al. (2018). Different characteristics Nicolas, G., Bennoun, M., Porteu, A., Mativet, S., Beaumont, C., Grandchamp, B., of hepcidin expression in IL-6 + / + and IL- 6 - / - neurons and astrocytes et al. (2002a). Severe iron deficiency anemia in transgenic mice expressing liver treated with lipopolysaccharides. Neurochem. Res. 43, 1624–1630. doi: 10.1007/ hepcidin. Proc. Natl. Acad. Sci. 99, 4596–4601. s11064-018-2577-9 Nicolas, G., Chauvet, C., Viatte, L., Danan, J. L., Bigard, X., Devaux, I., et al. (2002b). Mancias, J. D., Vaites, L. P., Nissim, S., Biancur, D. E., Kim, A. J., Wang, X., The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, et al. (2015). Ferritinophagy via NCOA4 is required for erythropoiesis and hypoxia, and inflammation. J. Clin. Invest. 110, 1037–1044. is regulated by iron dependent HERC2-mediated proteolysis. eLife 4, 1–19. Ottestad-Hansen, S., Hu, Q. X., Follin-Arbelet, V. V., Bentea, E., Sato, H., doi: 10.7554/eLife.10308 Massie, A., et al. (2018). The cystine-glutamate exchanger (xCT, Slc7a11) is Man-man, X. U., Jun, W., and Jun-xia, X. I. E. (2017). Regulation of iron expressed in significant concentrations in a subpopulation of astrocytes in the metabolism by hypoxia-inducible factors. Sheng Li Xue Bao 69, 598–610. mouse brain. Glia 66, 951–970. doi: 10.1002/glia.23294 Marro, S., Chiabrando, D., Messana, E., Stolte, J., Turco, E., Tolosano, E., et al. Park, U. J., Lee, Y. A., Won, S. M., Lee, J. H., Kang, S. H., Springer, J. E., (2010). Heme controls ferroportin1 (FPN1) transcription involving Bach1, Nrf2 et al. (2011). Blood-derived iron mediates free radical production and and a MARE/ARE sequence motif at position -7007 of the FPN1 promoter. neuronal death in the hippocampal CA1 area following transient forebrain Haematologica 95, 1261–1268. doi: 10.3324/haematol.2009.020123 ischemia in rat. Acta Neuropathol. 121, 459–473. doi: 10.1007/s00401-010- Mayor, D., and Tymianski, M. (2018). Neurotransmitters in the mediation of 0785-8 cerebral ischemic injury. Neuropharmacology 134, 178–188. doi: 10.1016/j. Pelizzoni, I., Macco, R., Morini, M. F., Zacchetti, D., Grohovaz, F., and Codazzi, F. neuropharm.2017.11.050 (2011). Iron handling in hippocampal neurons: activity-dependent iron entry McCarthy, R. C., and Kosman, D. J. (2014). Glial cell ceruloplasmin and hepcidin and mitochondria-mediated neurotoxicity. Aging Cell 10, 172–183. doi: 10. differentially regulate iron efflux from brain microvascular endothelial cells. 1111/j.1474-9726.2010.00652.x PLoS One 9:e89003. doi: 10.1371/journal.pone.0089003 Pelizzoni, I., Zacchetti, D., Smith, C. P., Grohovaz, F., and Codazzi, F. (2012). McCarthy, R. C., Park, Y.-H., and Kosman, D. J. (2014). sAPP modulates iron Expression of divalent metal transporter 1 in primary hippocampal neurons: efflux from brain microvascular endothelial cells by stabilizing the ferrous iron reconsidering its role in non-transferrin-bound iron influx. J. Neurochem. 120, exporter ferroportin. EMBO Rep. 15, 809–815. doi: 10.15252/embr.201338064 269–278. doi: 10.1111/j.1471-4159.2011.07578.x Frontiers in Neuroscience | www.frontiersin.org 15 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke Pellegrino, R. M., Boda, E., Montarolo, F., Boero, M., Mezzanotte, M., Saglio, G., Sillerova, T., Zivny, J., Vyoral, D., and Petrak, J. (2012). Nutritional hepatic iron et al. (2016). Transferrin receptor 2 dependent alterations of brain iron overload is not prevented by parenteral hepcidin substitution therapy in mice. metabolism affect anxiety circuits in the mouse. Sci. Rep. 6:30725. doi: 10.1038/ Br. J. Nutr. 108, 1723–1725. doi: 10.1017/S0007114512000116 srep30725 Simpson, I. A., Ponnuru, P., Klinger, M. E., Myers, R. L., Devraj, K., Coe, C. L., Perez de la Ossa, N., Sobrino, T., Silva, Y., Blanco, M., Millan, M., Gomis, M., et al. et al. (2015). A novel model for brain iron uptake: introducing the concept of (2010). Iron-related brain damage in patients with intracerebral hemorrhage. regulation. J. Cereb. Blood Flow Metab. 35, 48–57. doi: 10.1038/jcbfm.2014.168 Stroke 41, 810–813. doi: 10.1161/STROKEAHA.109.570168 Skjorringe, T., Burkhart, A., Johnsen, K. B., and Moos, T. (2015). Divalent metal Peters, D. G., Purnell, C. J., Haaf, M. P., Yang, Q. X., Connor, J. R., and transporter 1 (DMT1) in the brain: implications for a role in iron transport at Meadowcroft, M. D. (2018). Dietary lipophilic iron accelerates regional brain the blood-brain barrier, and neuronal and glial pathology. Front. Mol. Neurosci. iron-load in C57BL6 mice. Brain Struct. Funct. 223, 1519–1536. doi: 10.1007/ 8:19. doi: 10.3389/fnmol.2015.00019 s00429-017-1565-1 Slomka, A., Switoñska, M., and Zekanowska, E. (2015). Hepcidin Petrova, J., Manolov, V., Hadjidekova, S., Hadjiev, E., Bogov, B., Marinov, B., et al. levels are increased in patients with acute ischemic stroke: (2015). Is there a link between changes in levels of hepcidin and stroke? Clin. preliminary report. J. Stroke Cerebrovasc. Dis. 24, 1570–1576. doi: Lab. 61, 1935–1939. 10.1016/j.jstrokecerebrovasdis.2015.03.031 Philpott, C. C., and Ryu, M. S. (2014). Special delivery: distributing iron in the Sonnweber, T., Nachbaur, D., and Schroll, A. (2014). Hypoxia induced cytosol of mammalian cells. Front. Pharmacol. 5:173. doi: 10.3389/fphar.2014. downregulation of hepcidin is mediated by platelet derived growth factor BB. 00173 Gut 63, 1951–1959. doi: 10.1136/gutjnl-2013-305317 Philpott, C. C., Ryu, M. S., Frey, A., and Patel, S. (2017). Cytosolic iron chaperones: Soria, F. N., Pérez-samartín, A., Martin, A., Gona, K. B., Llop, J., Szczupak, B., et al. proteins delivering iron cofactors in the cytosol of mammalian cells. J. Biol. (2014). Extrasynaptic glutamate release through cystine / glutamate antiporter Chem. 292, 12764–12771. doi: 10.1074/jbc.R117.791962 contributes to ischemic damage. J. Clin. Inverstig. 124, 3645–3655. doi: 10.1172/ Piloni, N. E., Caro, A. A., and Puntarulo, S. (2018). Iron overload prevents oxidative JCI71886 damage to rat brain after chlorpromazine administration. BioMetals doi: 10. Soria, F. N., Zabala, A., Pampliega, O., Palomino, A., Miguelez, C., Ugedo, L., et al. 1007/s10534-018-0104-8 [Epub ahead of print]. (2016). Cystine/glutamate antiporter blockage induces myelin degeneration. Raje, C. I., Kumar, S., Harle, A., Nanda, J. S., and Raje, M. (2007). The macrophage Glia 64, 1381–1395. doi: 10.1002/glia.23011 cell surface glyceraldehyde-3-phosphate dehydrogenase is a novel transferrin Sterling, J., Gutthaa, S., Song, Y., Song, D., Hadziahmetovic, M., and Dunaief, J. L. receptor. J. Biol. Chem. 282, 3252–3261. doi: 10.1074/jbc.M608328200 (2017). Iron importers Zip8 and Zip14 are expressed in retina and regulated Ramos, P., Santos, A., Pinto, N. R., Mendes, R., Magalhães, T., and Almeida, A. by retinal iron levels JacobHHS public access. Exp. Eye Res. 155, 15–23. doi: (2014). Iron levels in the human brain: a post-mortem study of anatomical 10.1016/j.exer.2016.12.008 region differences and age-related changes. J. Trace Elem. Med. Biol. 28, 13–17. Strazielle, N., and Ghersi-Egea, J.-F. (2016). Potential pathways for CNS drug doi: 10.1016/j.jtemb.2013.08.001 delivery across the blood-cerebrospinal fluid barrier. Curr. Pharm. Des. 22, Rathnasamy, G., Murugan, M., Ling, E. A., and Kaur, C. (2016). Hypoxia- 4942–4958. doi: 10.2174/1381612822666160726112115 induced iron accumulation in oligodendrocytes mediates apoptosis by eliciting Sweeny, E. A., Singh, A. B., Chakravarti, R., Martinez-Guzman, O., Saini, A., endoplasmic reticulum stress. Mol. Neurobiol. 53, 4713–4727. doi: 10.1007/ Haque, M. M., et al. (2018). Glyceraldehyde 3-phosphate dehydrogenase is a s12035-015-9389-6 chaperone that allocates labile heme in cells. J. Biol. Chem. 293, 14557–14568. Ravasi, G., Pelucchi, S., Buoli Comani, G., Greni, F., Mariani, R., Pelloni, I., doi: 10.1074/jbc.RA118.004169 et al. (2018). Hepcidin regulation in a mouse model of acute hypoxia. Eur. J. Tan, G., Liu, L., He, Z., Sun, J., Xing, W., and Sun, X. (2016). Role of hepcidin and Haematol. 100, 636–643. doi: 10.1111/ejh.13062 its downstream proteins in early brain injury after experimental subarachnoid Recalcati, S., Gammella, E., Buratti, P., and Cairo, G. (2017). Molecular regulation hemorrhage in rats. Mol. Cell. Biochem. 418, 31–38. doi: 10.1007/s11010-016- of cellular iron balance. IUBMB Life 69, 389–398. doi: 10.1002/iub.1628 2730-1 Righy, C., Bozza, M. T., Oliveira, M. F., and Bozza, F. A. (2016). Molecular, cellular Tauchenová, L., Køížová, B., Kubánek, M., Fraòková, S., Melenovskı, V., and clinical aspects of intracerebral hemorrhage: are the enemies within? Curr. Tintìra, J., et al. (2016). Successful treatment of iron-overload cardiomyopathy Neuropharmacol. 14, 392–402. doi: 10.2174/1570159X14666151230110058 in hereditary hemochromatosis with deferoxamine and deferiprone. Can. J. Righy, C., Turon, R., de Freitas, G., Japiassu, A. M., Faria Neto, H. C. C., Bozza, M., Cardiol. 32, 1574.e1–1574.e3. doi: 10.1016/j.cjca.2016.07.589 et al. (2018). Hemoglobin metabolism by-products are associated with an Tian, Y., He, Y., Song, W., Zhang, E., and Xia, X. (2017). Neuroprotective effect of inflammatory response in patients with hemorrhagic stroke. Rev. Bras. Ter. deferoxamine on N-methyl-d-aspartate-induced excitotoxicity in RGC-5 cells. Intens. 30, 21–27. doi: 10.5935/0103-507x.20180003 Acta Biochim. Biophys. Sin. 49, 827–834. doi: 10.1093/abbs/gmx082 Sakamoto, K., Suzuki, T., Takahashi, K., Koguchi, T., Hirayama, T., Mori, A., Todorich, B., Zhang, X., and Connor, J. R. (2011). H-ferritin is the major source of et al. (2018). Iron-chelating agents attenuate NMDA-Induced neuronal injury iron for oligodendrocytes. Glia 59, 927–935. doi: 10.1002/glia.21164 via reduction of oxidative stress in the rat retina. Exp. Eye Res. 171, 30–36. Todorich, B., Zhang, X., Slagle-Webb, B., Seaman, W. E., and Connor, J. R. (2008). doi: 10.1016/j.exer.2018.03.008 Tim-2 is the receptor for H-ferritin on oligodendrocytes. J. Neurochem. 107, Sangkhae, V., and Nemeth, E. (2017). Regulation of the iron homeostatic hormone 1495–1505. doi: 10.1111/j.1471-4159.2008.05678.x hepcidin. Adv. Nutr. Int. Rev. J. 8, 126–136. doi: 10.3945/an.116.013961 Tripathi, A. K., Karmakar, S., Asthana, A., Ashok, A., Desai, V., Baksi, S., et al. Sankar, S. B., Donegan, R. K., Shah, K. J., Reddi, A. R., and Wood, L. B. (2018). (2017). Transport of non-transferrin bound iron to the brain: implications for Heme and hemoglobin suppress amyloid β–mediated inflammatory activation Alzheimer’s disease. J. Alzheimer’s Dis. 58, 1109–1119. doi: 10.3233/JAD-170097 of mouse astrocytes. J. Biol. Chem. 293, 11358–11373. doi: 10.1074/jbc.RA117. Truman-Rosentsvit, M., Berenbaum, D., Spektor, L., Cohen, L. A., Belizowsky- 001050 Moshe, S., Lifshitz, L., et al. (2018). Ferritin is secreted via 2 distinct nonclassical Seiler, A., Schneider, M., Förster, H., Roth, S., Wirth, E. K., Culmsee, C., et al. vesicular pathways. Blood 131, 342–352. doi: 10.1182/blood-2017-02-768580 (2008). Glutathione peroxidase 4 senses and translates oxidative stress into Tuo, Q., Lei, P., Jackman, K. A., Li, X., Xiong, H., Li, X., et al. (2017). Tau-mediated 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab. 8, iron export prevents ferroptotic damage after ischemic stroke. Mol. Psychiatry 237–248. doi: 10.1016/j.cmet.2008.07.005 22, 1520–1530. doi: 10.1038/mp.2017.171 Sheokand, N., Malhotra, H., Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Turkmen, S., Gonenc, O. C., Karaca, Y., Mentese, A., Demir, S., Beyhun, E., et al. (2016). Reverse overshot water-wheel retroendocytosis of apotransferrin et al. (2016). The effect of ethyl pyruvate and N -acetylcysteine on ischemia- extrudes cellular iron. J. Cell Sci. 129, 843–853. doi: 10.1242/jcs.180356 reperfusion injury in an experimental model of ischemic stroke. Am. J. Emerg. Shin, J. A., Kim, Y. A., Kim, H. W., Kim, H. S., Lee, K. E., Kang, J. L., et al. (2018). Med. 34, 1804–1807. doi: 10.1016/j.ajem.2016.06.003 Iron released from reactive microglia by noggin improves myelin repair in the Uranga, R. M., and Salvador, G. A. (2018). Unraveling the burden of iron in ischemic brain. Neuropharmacology 133, 202–215. doi: 10.1016/j.neuropharm. neurodegeneration: intersections with amyloid beta peptide pathology. Oxid. 2018.01.038 Med. Cell. Longev. 2018:2850341. doi: 10.1155/2018/2850341 Frontiers in Neuroscience | www.frontiersin.org 16 February 2019 | Volume 13 | Article 85 DeGregorio-Rocasolano et al. The Iron Side of Stroke Van Dijk, B. A. C., Laarakkers, C. M. M., Klaver, S. M., Jacobs, E. M. G., Van Tits, Yang, L., Fan, M., Du, F., Gong, Q., Bi, Z. G., Zhu, Z. J., et al. (2012). L. J. H., Janssen, M. C. H., et al. (2008). Serum hepcidin levels are innately Hypoxic preconditioning increases iron transport rate in astrocytes. Biochim. low in HFE-related haemochromatosis but differ between C282Y-homozygotes Biophys. Acta Mol. Basis Dis. 1822, 500–508. doi: 10.1016/j.bbadis.2011. with elevated and normal ferritin levels. Br. J. Haematol. 142, 979–985. doi: 12.004 10.1111/j.1365-2141.2008.07273.x Yang, Y., Dong, B., Lu, J., Wang, G., and Yu, Y. (2018). Hemopexin reduces blood- Van Hoecke, M., Prigent-Tessier, A., Bertrand, N., Prevotat, L., Marie, C., and brain barrier injury and protects synaptic plasticity in cerebral ischemic rats Beley, A. (2005). Apoptotic cell death progression after photothrombotic focal by promoting EPCs through the HO-1 pathway. Brain Res. 1699, 177–185. cerebral ischaemia: effects of the lipophilic iron chelator 2,2’-dipyridyl. Eur. J. doi: 10.1016/j.brainres.2018.08.008 Neurosci. 22, 1045–1056. doi: 10.1111/j.1460-9568.2005.04297.x You, L. H., Li, Z., Duan, X. L., Zhao, B. L., Chang, Y. Z., and Shi, Z. H. (2016). Vela, D. (2018). Hepcidin, an emerging and important player in brain iron Mitochondrial ferritin suppresses MPTP-induced cell damage by regulating homeostasis. J. Transl. Med. 16:25. doi: 10.1186/s12967-018-1399-5 iron metabolism and attenuating oxidative stress. Brain Res. 1642, 33–42. doi: Wan, L., Nie, G., Zhang, J., and Zhao, B. (2012). Overexpression of human wild- 10.1016/j.brainres.2016.03.023 type amyloid-β protein precursor decreases the iron content and increases Yu, J., Guo, Y., Sun, M., Li, B., Zhang, Y., and Li, C. (2009). Iron the oxidative stress of neuroblastoma SH-SY5Y cells. J. Alzheimer’s Dis. 30, is a potential key mediator of glutamate excitotoxicity in spinal cord 523–530. doi: 10.3233/JAD-2012-111169 motor neurons. Brain Res. 1257, 102–107. doi: 10.1016/j.brainres.2008. Wang, J., and Doré, S. (2007). Inflammation after intracerebral hemorrhage. 12.030 J. Cereb. Blood Flow Metab. 27, 894–908. doi: 10.1038/sj.jcbfm.9600403 Zeng, L., Tan, L., Li, H., Zhang, Q., Li, Y., and Guo, J. (2018). Deferoxamine therapy Ward, R. J., Zucca, F. A., Duyn, J. H., Crichton, R., and Zecca, L. (2014). The for intracerebral hemorrhage: a systematic review. PLoS One 13:e0193615. doi: role of iron in brain ageing and neurodegenerative disorders. Lancet Neurol. 10.1371/journal.pone.0193615 13, 1045–1060. doi: 10.1016/S1474-4422(14)70117-6 Zhang, H., Park, J. H., Maharjan, S., Park, J. A., Choi, K. S., Park, H., et al. (2017). Watts, S. D., Torres-Salazar, D., Divito, C. B., and Amara, S. G. (2014). Cysteine Sac-1004, a vascular leakage blocker, reduces cerebral ischemia-reperfusion transport through excitatory amino acid transporter 3 (EAAT3). PLoS One injury by suppressing blood-brain barrier disruption and inflammation. 9:e109245. doi: 10.1371/journal.pone.0109245 J. Neuroinflammation 14:122. doi: 10.1186/s12974-017-0897-3 West, A. P., Bennett, M. J., Sellers, V. M., Andrews, N. C., Enns, C. A., and Zhang, Z., Wu, Y., Yuan, S., Zhang, P., Zhang, J., Li, H., et al. (2018). Bjorkman, P. J. (2000). Comparison of the interactions of transferrin receptor Glutathione peroxidase 4 participates in secondary brain injury through and transferrin receptor 2 with transferrin and the hereditary hemochromatosis mediating ferroptosis in a rat model of intracerebral hemorrhage. Brain Res. protein HFE. J. Biol. Chem. 275, 38135–38138. doi: 10.1074/jbc.C000664200 1701, 112–125. doi: 10.1016/j.brainres.2018.09.012 White, R. S., Bhattacharya, A. K., Chen, Y., Byrd, M., McMullen, M. F., Siegel, S. J., Zhao, J., Xiang, X., Zhang, H., Jiang, D., Liang, Y., Qing, W., et al. (2018). CHOP et al. (2016). Lysosomal iron modulates NMDA receptor-mediated excitation induces apoptosis by affecting brain iron metabolism in rats with subarachnoid via small GTPase, Dexras1. Mol. Brain 9:38. doi: 10.1186/s13041-016-0220-8 hemorrhage. Exp. Neurol. 302, 22–33. doi: 10.1016/j.expneurol.2017. Worthen, C. A., and Enns, C. A. (2014). The role of hepatic transferrin receptor 12.015 2 in the regulation of iron homeostasis in the body. Front. Pharmacol. 5:34. Zhao, X., Song, S., Sun, G., Zhang, J., Strong, R., Zhang, L., et al. (2011). doi: 10.3389/fphar.2014.00034 Cytoprotective role of haptoglobin in brain after experimental intracerebral Wu, C., Zhao, W., Yu, J., Li, S., Lin, L., and Chen, X. (2018). Induction of ferroptosis hemorrhage. Acta Neurochir. Suppl. 111, 107–112. doi: 10.1007/978-3-7091- and mitochondrial dysfunction by oxidative stress in PC12 cells. Sci. Rep. 8:574. 0693-8_17 doi: 10.1038/s41598-017-18935-1 Zhou, Y. F., Zhang, C., Yang, G., Qian, Z. M., Zhang, M. W., Ma, J., et al. (2017). Wu, J., Hua, Y., Keep, R. F., Nakamura, T., Hoff, J. T., and Xi, G. (2003). Iron and Hepcidin protects neuron from hemin-mediated injury by reducing iron. Front. iron-handling proteins in the brain after intracerebral hemorrhage. Stroke 34, Physiol. 8:332. doi: 10.3389/fphys.2017.00332 2964–2969. doi: 10.1161/01.STR.0000103140.52838.45 Zille, M., Karuppagounder, S. S., Chen, Y., Gough, P. J., Bertin, J., Finger, J., et al. Xing, Y., Hua, Y., Keep, R. F., and Xi, G. (2009). Effects of deferoxamine on brain (2017). Neuronal death after hemorrhagic stroke in vitro and in vivo shares injury after transient focal cerebral ischemia in rats with hyperglycemia. Brain features of ferroptosis and necroptosis. Stroke 48, 1033–1043. doi: 10.1161/ Res. 1291, 113–121. doi: 10.1016/j.brainres.2009.07.032 STROKEAHA.116.015609 Xiong, X. Y., Chen, J., Zhu, W. Y., Zhao, T., Zhong, Q., Zhou, K., et al. (2015). Serum hepcidin concentrations correlate with serum iron level and outcome Conflict of Interest Statement: The authors declare that the research was in patients with intracerebral hemorrhage. Neurol. Sci. 36, 1843–1849. doi: conducted in the absence of any commercial or financial relationships that could 10.1007/s10072-015-2266-2 be construed as a potential conflict of interest. Xiong, X. Y., Liu, L., Wang, F. X., Yang, Y. R., Hao, J. W., Wang, P. F., et al. (2016). Toll-like receptor 4/MyD88-mediated signaling of hepcidin expression Copyright © 2019 DeGregorio-Rocasolano, Martí-Sistac and Gasull. This is an open- causing brain iron accumulation, oxidative injury, and cognitive impairment access article distributed under the terms of the Creative Commons Attribution after intracerebral hemorrhage. Circulation 134, 1025–1038. doi: 10.1161/ License (CC BY). The use, distribution or reproduction in other forums is permitted, CIRCULATIONAHA.116.021881 provided the original author(s) and the copyright owner(s) are credited and that the Yang, G., Hu, R., Zhang, C., Qian, C., and Luo, Q. (2016). A combination of serum original publication in this journal is cited, in accordance with accepted academic iron, ferritin and transferrin predicts outcome in patients with intracerebral practice. No use, distribution or reproduction is permitted which does not comply hemorrhage. Sci. Rep. 6:21970. doi: 10.1038/srep21970 with these terms. Frontiers in Neuroscience | www.frontiersin.org 17 February 2019 | Volume 13 | Article 85