Iron and Alzheimer’s Disease: An Update on Emerging Mechanisms

Abstract

Iron is a crucial transition metal for life and is the most abundant transition metal in the brain. However, iron’s biological utility as an effective redox cycling metal also endows it with the potential to catalyze production of noxious free radicals. This “Janus-faced” nature of iron demands a tight regulation of cellular its metabolism. This regulation is crucial in the CNS, where iron plays myriad keystone roles in CNS processes, including mitochondrial energy transduction, enzyme catalysis, mitochondrial function, myelination, neurotransmitter anabolism and catabolism. Aberrations in brain iron homeostasis can elevate levels of this redox-active metal, leading to mislocalization of the metal and catastrophic oxidative damage to sensitive cellular and subcellular structures. Iron dyshomeostasis has been strongly linked to the pathogenesis of Alzheimer’s disease (AD), as well as other major neurodegenerative diseases. Despite the growing societal burden of AD, no disease-modifying therapy exists, necessitating continued investment into both drug-development and the fundamental science investigating the disease-causing mechanisms. Targeting iron dyshomeostasis in the brain represents a rational approach to treat the underlying disease. Here we provide an update on known and emerging iron-associated mechanisms involved in AD. We conclude with an overview of evidence suggesting that, in addition to apoptosis, neuronal loss in AD involves “ferroptosis”, a newly discovered iron- and lipid-peroxidation-dependent form of regulated necrosis. The ferroptosis field is rapidly progressing and may provide key insights for future drug-development with disease-modifying potential in AD.

Keywords

Alzheimer’s disease amyloid-β amyloid-β protein precursor apoptosis astrocytes ferroptosis iron lipid peroxidation oxidative stress neuroinflammation

INTRODUCTION

As the global population ages, enormous resources will be needed to provide adequate care for the growing number of individuals afflicted by Alzheimer’s disease (AD) [1]: the most common cause of dementia, which accounts for up to 80% of all documented cases [2]. AD is an insidious and progressive neurodegenerative disorder, involving substantive cortical and hippocampal neuronal loss [3] that progresses for 20–30 years before clinical onset [4]. Despite the staggering and increasing socioeconomic burden of AD, with >100 million cases predicted by 2050 [1], no disease-modifying therapies are yet available to effectively treat this disease.

The disease is characterized by brain atrophy, extracellular deposition of amyloid-β (Aβ) peptide in senile plaques, the intraneuronal accumulation of hyperphosphorylated tau, neuronal and synaptic loss, chronic inflammation, and oxidative stress [5 –10]. Moreover, despite being the focus of decades of intense research, the cause of AD, especially sporadic AD, is elusive. Although the greatest risk factor for AD is aging [11], the pathophysiological mechanisms underlying the role of aging in the development AD are poorly understood.

Based on the hallmarks of Aβ plaques and neurofibrillary tangles of hyperphosphorylated tau, the “amyloid” and “tau” hypotheses have dominated research into AD etiology. While prevailing drug-development paradigms are predicated on these hypotheses, thus far, effective disease-modifying treatment options remain elusive [3]. This scenario strongly indicates the need to forge new biological models of AD, particularly those that address the advances in our understanding of the underlying etiology of AD-associated neurodegeneration. This approach will be the first step in implementing drug-development strategies that demonstrate disease-modifying activity.

Unlike familial AD, which accounts for <1% cases and is associated with genetic mutations in key proteins and enzymes (e.g., presenilins) associated with amyloid-β protein precursor (AβPP) processing, the initiating event responsible for onset of sporadic AD, particularly early in the prodromal phase of the disease remains masked by uncertain downstream events.

The incidence of AD and oxidative damage to the brain increases with age [11]. Moreover, there is an overwhelming body of evidence that oxidative stress fundamentally contributes to AD pathophysiology and similarly increases with age [6 , 12]. Importantly, the dysregulation of redox-active metals (e.g., iron) within the brain appears to underpin the generation and pathological progression of brain oxidative-stress [3, 13].

Accumulating evidence indicates that AD is closely associated with the cumulative effects of oxidative stress, much of which can be linked to iron, within the brain [8 , 15]. Indeed, levels of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) [16] and lipid hydroperoxides (LOOH) [17], are significantly higher in AD than in healthy control brains. This increase in ROS, as well as their redox-active degradation products, can, at least in part, be attributed to a pathological increase in the levels of redox-active metal ions, particularly iron and copper [13, 18]. Importantly, oxidative stress potentiates the neurotoxic oligomerization of Aβ and tau tangles [3], activation or senescence of astrocytes and microglia, which collectively promote neuroinflammation, and glutamate-induced neurotoxicity (e.g., excitotoxicity and a newly described iron-dependent form of cell death termed “ferroptosis”) [19, 20], all of which ultimately lead to neuronal demise [3, 15]. Therefore, increased knowledge regarding the causes and downstream targets of iron-induced oxidative stress in AD will allow us to forge the way to new and lateral therapeutic targets with disease-modifying activity.

In AD, there is pathological accumulation of iron in the hippocampus and cerebral cortex that co-localizes with classical AD lesions, such as extracellular senile plaques of aggregated Aβ, and intracellular tangles of hyperphosphorylated tau [3, 4]. Iron is considered a central player in oxidative stress in AD because, as a redox-active transition metal, it can cycle between Fe(II) and Fe(III) states in biological systems [21, 22]. While this behavior is responsible for the immense biological utility of iron, when dysregulated, it can drive the formation of highly damaging hydroxyl radicals (OH), and lipid alkoxyl (LO) and peroxyl (LOO) radicals, the catalysis of which involves Fe(II)-induced cleavage of H₂O₂ [15], or LOOH [17 , 23], respectively, via Fenton- and Haber-Weiss-chemistry.

While the downstream iron-dependent effects of H₂O₂ cleavage are apparent, the precise and quantitatively dominant sources of H₂O₂ in AD are unclear. The hallmark AD lesions (e.g., Aβ aggregates and neurofibrillary tangles of tau) are thought to contribute to production H₂O₂, but strong evidence indicates that significant quantities of H₂O₂ also originate from other sources (e.g., dysfunctional mitochondria, and other cellular oxidases such as H₂O₂-producing monoamine and polyamine oxidases, and inflammatory cytokine-activated NADPH oxidases) [15]. Importantly, increasing evidence suggests radical-mediated oxidation of biological substrates (e.g., membrane lipids) is a key feature of AD pathogenesis [23].

The remainder of this review will provide an update on known and emerging roles of iron in the pathogenesis of AD, as well as the aspects of iron biology in the CNS that are relevant to understanding these roles.

IRON AND OXIDATIVE STRESS: KEY REACTIONS AND CONCEPTS

Iron can act as a pro-oxidant by catalyzing the formation of ROS [21]. The classical pro-oxidant reaction of iron, the Fenton reaction [24], results in the formation of highly-reactive OH from H₂O₂, according to the equation: ${Fe}^{2 +} + H_{2} O_{2} \to {Fe}^{3 +} + {HO}^{•} + {HO}^{-} (Reaction 1)$

The one-electron reduction of dioxygen by Fe²⁺ can also generate superoxide anions (O_2•−) according to the following reaction: ${Fe}^{2 +} + O_{2} \to {Fe}^{3 +} + O_{2}^{• -} (Reaction 2)$

These superoxide anions can then be dismutated, either enzymatically by superoxide dismutases (SODs) or non-enzymatically, to yield H₂O₂, according to the following reaction: $2 O_{2}^{• -} + 2 H^{+} \to H_{2} O_{2} + O_{2} (Reaction 3)$

Additionally, or if the activity of SOD activity is rate-limiting, superoxide anions can reduce trace amounts of labile aqueous Fe³⁺ to form dioxygen and regenerate Fe²⁺: ${Fe}^{3 +} + O_{2}^{• -} \to {Fe}^{2 +} + O_{2} (Reaction 4)$

The sum of Reaction 1 (Fenton reaction) and Reaction 4 is commonly known as the Haber-Weiss reaction, with iron as the catalyst [24]: $O_{2}^{• -} + H_{2} O_{2} \to O_{2} + H O^{•} + H O^{-} (Reaction 5)$

The Haber-Weiss reaction illustrates that in the presence of catalytic amounts of redox-active aqueous low-M_r iron, which increase in peripheral tissues under conditions of iron overload, or in the brain in various CNS pathologies (for reviews, see [3 , 25–27]), H₂O₂ may provide a ready source of damaging OH in the presence of a ferrireductants such as superoxide that regenerates the reduced form of the metal. Importantly, other abundant cellular reductants (e.g., ascorbate, α-tocopherol, and GSH) can also reduce ferric ions in an analogous manner to superoxide in Reaction 4 [28 –30]. Thus, cellular reductants such as ascorbate and GSH, which typically function in an anti-oxidative capacity when present at normal physiological levels, can have pro-oxidant activities in the presence of catalytic concentrations of labile iron. Importantly, the iron-catalyzed formation of highly reactive and damaging ROS such as OH, or lipid alkoxyl radicals (in the case of lipid peroxidation), is primarily responsible for the ability of labile iron to cause oxidative stress. In the case of AD, and other CNS diseases associated with increased concentrations of redox-active or labile iron, these iron-driven Haber-Weiss-like reactions underpin much of the oxidative pathology.

IRON TRAFFICKING, STORAGE, AND UTILIZATION: AN OVERVIEW

Iron is essential for the survival of all cells, as demonstrated by cell death following excessive iron depletion [21 , 32]. However, too much iron within cells, tissues, and organs invariably leads to oxidative damage to key macromolecules, including DNA, RNA, proteins, and lipids [33]. Adult humans contain 3-5 g of iron, of which 70-80% is found within erythrocyte hemoglobin, 10-20% is stored within macrophages and hepatocytes, and 3-4% is within heme-bound myoglobin [21, 22]. Within nucleated cells, most iron storage typically occurs within ferritin nanocages [34]. The remainder of the iron is present in other heme-containing proteins (e.g., cytochromes), iron– sulfur cluster (ISC)-containing proteins (e.g., succinate dehydrogenase) [35, 36] and non-heme/non-ISC iron-containing proteins (e.g., 2-oxoglutarate-dependent dioxygenases, BH₄-dependent tyrosine, tryptophan and phenyalanine hydroxylases, as well as the lipoxygenases) [37, 38].

As discussed further below, improperly sequestered iron tends to catalyze the production of toxic ROS through Fenton and Haber-Weiss-type reactions [21]. At the organismal level, iron homeostasis is controlled only through the regulation of iron uptake, as there is no regulated means of the body “ridding” itself of excess iron [21]. In contrast, at the cellular level, iron homeostasis is tightly controlled at several levels, including the import, storage and efflux of iron [21 , 39–41].

Iron uptake routes: Transferrin and non-transferrin iron

In terms of the uptake of iron, two major categories of iron-import exist: transferrin (Tf) and non-Tf iron uptake. Under physiological circumstances, particularly in peripheral tissues, virtually all cells favor the import of Tf-bound iron, which is internalized by receptor-mediated endocytosis after binding to Tf receptor 1 (TfR1) [40, 41]. Ferric iron is released from Tf within the endosome after its acidification and is then reduced by an endosomal ferrireductase (e.g., six transmembrane epithelial antigen of the prostate 3 [STEAP3] [42]) [41], or by a novel mechanism involving cellular ascorbate [43 –45]. Following the reduction of ferric iron within the endosomal lumen, the resulting ferrous iron is transported across the endosomal membrane by divalent metal transporter 1 (DMT1) in a proton-coupled manner [46], or in some cases, ZRT/IRT-like protein may also be involved [47]. The protein poly(rC)-binding protein 2 (PCBP2), which is an RNA-binding protein that has been shown to function as an intracellular iron chaperone that delivers iron to ferritin and at least some non-heme-iron enzymes, was recently identified as a DMT1-binding partner that regulates iron influx from Tf across the endosomal membrane to the cytosol [48]. As PCBP2 binds iron and associates with ferritin to deliver iron [37, 49], this may be the first direct evidence of an iron-transport metabolon.

Under physiological conditions, almost all iron in the circulation is bound to Tf, although saturation of Tf with iron is normally ∼30% [21]. In diseases resulting in the excessive loading of tissues with iron, Tf becomes saturated with iron, with excess plasma iron occurring as non-Tf iron [21]. The exact uptake route(s) for non-Tf iron remains unclear but are known to involve one or more cell surface ferrireductases (e.g., duodenal cytochrome b, DCYTB [50]) or the release of cellular reductants, such as ascorbate [43 , 51–53]. These complementary ferrireduction mechanisms reduce ferric non-Tf iron to its ferrous state that can then be imported by transporters such as the transmembrane protein, DMT1 [46], or the ZIPs, ZIP14 or ZIP8 [47].

In Tf- and non-Tf iron uptake, the iron that has entered the cytosol becomes part of a functionally-characterized chelatable or labile iron pool (LIP), which can be utilized for metabolism (e.g., production of iron-containing proteins and enzymes, stored in ferritin or released back to the extracellular space. As such, iron that enters the transitory LIP is either: 1) stored in ferritin; 2) utilized by downstream metabolic pathways (e.g., imported into mitochondria for usage in ISC and heme synthesis, and/or incorporated in cytoplasmic iron-requiring proteins); or 3) released from the cell by the ferrous iron exporter, ferroportin [40, 41].

In non-erythroid cells, including brain cells, the majority (i.e., 70–80%) of this nascently imported iron is thought to be incorporated into ferritin [34]. Ferritin is a multimeric protein composed of 24 subunits that forms a hollow sphere capable of storing ∼4,500 iron atoms as a mineralized ferric, phosphate, and hydroxide (ferrihydrite) core [34 , 55]. In mammals, there are two ferritin subunits: H-ferritin (heavy subunit, encoded by FTH1) and L-ferritin (light subunit, encoded by as FTL), which hetero-polymerize to form different “isoferritins” with tissue-specific distributions [34 , 55]. Ferrous iron that is bound by ferritin is first oxidized to ferric iron by the ferroxidase activity of H-ferritin in an oxygen-dependent manner [34 , 55]. Subsequently, ferric iron core formation commences at carboxyl groups on glutamates of L-ferritin, which is devoid of ferroxidase activity [34 , 55]. This enclosure and sequestration of iron as ferrihydrite is vital, as it maintains iron in a redox-inert state [34 , 55].

Importantly, ferritin will release iron in a tightly-controlled manner under in vivo conditions by targeted autolysosomal proteolysis of the ferritin nanocage, although proteasomal degradation of the protein can occur under specific conditions of therapeutic relevance in which iron chelators are used [34, 56]. The targeting of ferritin for autophagic turnover (i.e., ferritinophagy) has recently been shown to involve nuclear receptor coactivator 4 (NCOA4), which binds to autophagy-related protein 8 (ATG8) proteins on newly formed autophagolysosomes and recruits ferritin as a cargo molecule [57].

Regulation of cellular iron levels: Post-transcriptional control

Due to iron’s ability to promote oxidative stress, cellular iron levels and processing are tightly controlled. One major mechanism by which cellular iron homeostasis is controlled is by a post-transcriptional mechanism that modulates the synthesis of key iron metabolism proteins (e.g., TfR1, ferritin, ferroportin, and AβPP) that are involved in iron uptake, storage and release [31, 39]. Specifically, the iron regulatory protein (IRP)-iron responsive element (IRE) system is responsible for this mode of regulation and allows for rapid changes in the translation of key iron metabolism proteins in response to changing intracellular iron levels [31 , 58]. This system depends on the mRNA-binding proteins, IRPs-1 and -2, which post-transcriptionally control the expression of mRNAs possessing IREs [31 , 58]. IRPs bind to IREs in the 5^′- or 3^′-untranslated regions (UTRs) of key mRNAs involved in iron metabolism with high affinity in iron-depleted cells, either suppressing the translation of the mRNA (i.e., mRNAs in which the IRE is located in the 5^′-UTR; e.g., FTH1, FTL, ferroportin, and AβPP), or by enhancing mRNA stability against nuclease attack (i.e., mRNAs in which the IRE is located in the 3^′-UTR; e.g., TfR1, DMT1-I, etc.) [39, 41].

Under conditions of increased cellular iron, which can be potentiated by endogenous reductants such as ascorbate [43], IRP1 loses its IRE-binding activity by acquiring an ISC (4Fe-4S cluster) [58]. The acquisition of this 4Fe-4S cluster converts IRP1 into a cytosolic aconitase. In the case of IRP2, iron-dependent, proteasomal degradation is the major regulatory mechanism [59].

REGULATION OF BRAIN IRON

Transport and trafficking of iron in the brain

In contrast to cells in the periphery that engage almost exclusively in Tf-bound iron uptake under physiological conditions, different types of brain cells appear to be adapted either for the uptake of Tf-bound iron (e.g., neurons) or non-Tf-bound iron (e.g., astrocytes, oligodendrocytes, and microglia) [60 –62]. The majority of brain iron derives from the Tf-iron in the blood and is thought to transported across the blood-brain barrier (BBB) via brain capillary endothelial cells (BCECs) via a unique mechanism: namely, receptor-mediated endocytosis of Tf-iron from the blood followed by reduction of iron inside BCEC endosomes, followed by retro-endocytosis of apo-transferrin to the luminal surface [60 , 63–65]. Ferrous iron is transferred from the endosome into the cytosol by DMT1 (similar to the classical receptor-mediated endocytosis mechanism of Tf-iron uptake in peripheral tissues), transported to the abluminal side of the BCECs, then exported across the abluminal membrane by ferroportin in a process involving subsequent re-oxidation of the iron to Fe(III) on the extracellular face of the abluminal membrane by the ferroxidases, ceruloplasmin [66] and/or hephaestin [67]. The resulting low-M_r iron is then thought to be complexed by endogenous iron-binding ligands, such as ATP, ascorbate or citrate, which are released by the end-feet of vicinal astrocytes [63]. The iron is then thought to be imported by the end-feet of these astrocytes [60], prior to its redistribution and subsequent trafficking within the brain parenchyma.

While the mechanisms responsible for the uptake of non-Tf iron by astrocytes are not known with certainty [61], at least two major routes of import have been proposed (recently reviewed in Codazzi et al. [68] and Skjørringe et al. [63]). Historically, the first is via DMT1, which has been observed to be highly expressed in astrocytic end-feet in culture [69 –73], as well as in vivo in some studies [74, 75]. DMT1 levels are acutely regulated by cell iron-status in primary astrocyte cultures [70]. Moreover, astrocytes can release ascorbate to promote the uptake of iron by DMT1 under standard culture conditions [53], with the release of ascorbate being enhanced under conditions of hyperglutamatergia [52]. Collectively, these findings support a role for DMT1 in iron uptake in vitro. However, the involvement of DMT1 in astrocytes in vivo, at least under physiological conditions, is less clear (see references in Skjørringe et al. [63]). Notably, under conditions in which intracellular ascorbate is depleted (mimicking chronic oxidative stress), cultured astrocytes demonstrate an apparent preference for the uptake of Fe(III) [53], although the molecular pathway for the putative import of this Fe(III) has yet to be characterized.

Another proposed route for astrocytic ferrous iron uptake in vivo involves transient receptor potential canonical (TRPC) channels, based on studies conducted with quiescent hippocampal astrocytes [76]. Interestingly, astrocytes that have been activated by proinflammatory cytokines (i.e., IL-1β+TNFα) demonstrate a potentiation of non-Tf iron uptake by the de novo expression of the cell-surface DMT1-1A isoform [76], which is the same isoform expressed on the apical membrane of enterocytes, and are required for dietary uptake of low-M_r iron [77]. Accordingly, the de novo expression of this isoform of DMT1 in activated astrocytes was proposed to account for their increased capability to import Fe(II), but not Fe(III) [10 , 79]. Thus, the discrepancies on the importance of DMT1 in astrocytic iron uptake might be ascribed to variation in culture conditions that differentially activate astrocytes [80]. However, and perhaps more importantly, these findings suggest that inflammatory mediators can have profound effects on glia-regulated iron trafficking in the brain, which may be crucial for understanding how iron dysregulation occurs and progresses in AD.

In summary, astrocytes are critical in processing and re-distributing iron upon its entry into the brain across the BBB. Under physiological conditions, astrocytes can import non-Tf iron, which probably occurs via TRCPs, but with an increasing component of DMT1A-mediated iron uptake under conditions of neuro-inflammation, which may be relevant to the role of iron in AD.

IRON IS INCREASED IN THE AD BRAIN: A CONVERGENT PATHOLOGY

Iron is important for maintaining the high energy and metabolic requirements of neuronal tissues in the brain through its involvement in myelin synthesis and neurotransmitter synthesis (e.g., dopamine, serotonin, GABA) and for metabolism [3]. Increased iron content in affected areas of the brain is observed in a growing number of neurodegenerative disorders including Parkinson’s disease, Huntington’s disease, and AD [3 , 82]. In the case of AD, elevated brain iron was first demonstrated in 1953 [83], and remains a widely and consistently reported finding [83 –92]. Importantly, in AD, high Aβ-burden (identified by PET) predicts cognitive decline [93], but the large variability between individuals in the rate of this cognitive decline points to the contribution of other pathologies that synergistically combine with Aβ to accelerate clinical deterioration [94]. The accumulation of brain iron, which is a pathological feature of AD [25], has the potential to promote neurodegeneration through oxidative damage to sensitive subcellular compartments (discussed further below). Indeed, we have shown that elevated CSF ferritin (a biomarker of brain iron burden) predicts poorer cognition and increases the risk of developing AD [95, 96]. This notion of “convergent pathologies” in AD suggests that increased brain iron might combine with increased Aβ, or tau pathology, to increase the rate of disease progression. In support of this model, we recently employed quantitative susceptibility mapping to show that increased iron loading in the hippocampus is a strong predictor of Aβ-related cognitive decline [94].

Iron deposition within the brain parenchyma, particularly in vulnerable neuronal populations (e.g., within the hippocampus and cortex), but also in astrocytes, oligodendrocytes, and microglia, potentiates oxidative stress via the Fenton- and Haber-Weiss reactions (see above), as well as by increasing lipid peroxidative stress [19 , 97–99]. The iron-dependent increase in general oxidative stress, particularly of membrane lipids in neurons and glial cells, is increasingly becoming accepted as a keystone contributor to the elevated signs of oxidative stress in the AD brain [100].

Iron enhances Aβ production and oligomerization

As discussed further below, elevated iron in the AD brain contributes to classical features of AD pathology, including Aβ dysfunction and plaque formation [101 –106], tau hyperphosphorylation and neurofibrillary tangles [92 , 107–111], as well as neuronal cell death [112, 113]. An increase in neuronal iron in AD is known to augment Aβ production by several mechanisms, including increasing AβPP expression and its subsequent amyloidogenic processing [106]. First, iron increases the translation of AβPP by virtue of an IRE in the 5′-UTR of its encoding mRNA [114]. This mechanism is essentially the same mechanism by which iron increases the expression of ferritin and ferroportin, both of which possess IREs in the 5‘-UTR of their mRNA (discussed above). Thus, as with ferritin and ferroportin, the translation of AβPP, which is repressed by IRPs under low iron conditions, will be de-repressed under high cellular iron conditions (such as in AD), leading to increased translation of the transcript. Intriguingly, whereas the IREs in ferritin and ferroportin mRNAs can bind either IRP1 or IRP2, which is typical of all classical IREs [33], it has been recently shown by Jack Rogers’ group that only IRP1 binds and regulates the IRE in the AβPP 5^′-UTR [115]. Importantly, the selective regulation of the AβPP mRNA by IRP1 indicates that both AβPP and IRP1, the latter of which is deactivated as an IRE-binding protein by the acquisition of an 4Fe– 4S ISC, may be regulated by the cytosolic (CIA) and mitochondrial ISC biogenesis pathway (for a recent review of the CIA and mitochondrial ISC pathways, see Paul and Lill [116] and Rouault and Maio [117]). The connection between AβPP regulation and ISC biogenesis is worthy of further investigation, particularly as other neurodegenerative diseases, such as Parkinson’s disease and Friedreich’s ataxia exhibit dysfunction in iron homeostasis that is coupled with mitochondrial dysfunction and aberrant ISC metabolism (for reviews, see [27 , 119]).

In contrast to AβPP, although the ferritin IREs can be bound by either IRP1 or IRP2 in vitro [33], in neural cells there is a preference for the binding of IRP2 to the IRE in the 5^′-UTR of the FTH1 mRNA [120]. This may be of relevance to the mechanism of neuronal iron dyshomeostasis and loading in AD, as IRP2, which is selective for ferritin IREs, has been observed to be dysregulated in AD [121]. Indeed, George Perry’s group observed that while IRP1 is present at similar levels in both AD and control brain tissue, IRP2 shows marked differences in expression and localization, being associated with intraneuronal lesions, including neurofibrillary tangles, senile plaque neurites, and neuropil threads [121]. These findings suggest that an increase in IRP2 may contribute to the suppression of ferritin and ferroportin translation within neurons, leading to increased pools of redox-active iron through impaired storage and efflux, respectively. In support of this mechanism, the stabilization of IRP2 in neural stem progenitor cells by the genetic inactivation of the E3 ligase subunit, F-box/LRR-repeat protein 5 (FBXL5), which targets IRP2 for proteasomal degradation, leads to the accumulation of ferrous and ferric iron, as well as the increased production of ROS [122].

In addition to promoting AβPP translation, high iron levels can increase amyloidogenic processing of AβPP, which occurs by the action of ferritin light chain binding to presenilin enhancer 2 (PEN-2), a γ-secretase component, and increasing γ-secretase activity [101]. Chronic iron loading increases amyloidogenic processing of AβPP leading, accelerating Aβ production and neurodegeneration in a mouse model of AD [123]. Importantly, Aβ accumulates in senile plaques, and engages in a positive feedback loop with oxidative stress that increases Aβ generation and oligomerization [124]. Additionally, Aβ is capable of binding transition metals (e.g., copper, zinc and iron), via three His (positions 6, 13, and 14) and 1 Tyr (position 10) residues that are located in the hydrophilic N-terminal region of the peptide [125, 126]. Interestingly, the redox potential of iron is significantly attenuated by Aβ, which may suggest a neuroprotective and chelating role for Aβ in AD pathogenesis that becomes toxic under certain conditions [9]. This feature of Aβ-iron interactions may, at least in part, explain the enrichment of iron in AD plaques that is observed in humans [127] and mouse models [128]. Interestingly, the metal-dependent generation of ROS by Aβ may be a good target for therapeutics. For example, chelation therapy using deferoxamine, a strong, but poorly BBB-permeant Fe(III) chelator, has shown improvement in several key indices in mouse models of AD (provided intranasally) [110 , 130], and has demonstrated clinical improvement in AD patients (provided intramuscularly, five days/week over two years) [131]. Critically, iron-stimulated aggregates of Aβ also demonstrate potentiated cytotoxicity in vitro [112 , 132–135], suggesting that elevated iron and Aβ may synergistically combine to promote AD neuropathology. Moreover, the intranasal delivery of existing and novel iron-binding therapeutics may be a desirable route of administration, given the ability to bypass tight control by the BBB [136].

Iron enhances tau dysfunction and neurofibrillary tangles

Intriguingly, tau also binds iron [107, 108], which causes it to aggregate [109], possibly depositing in vivo as iron-rich tangles in AD brains [92]. In further support of a potentiating role for iron in tau dysfunction, iron-loading of cultured neurons increases tau phosphorylation [137 –140], possibly by virtue of increased glycogen synthase kinase 3 beta (GSK3β) and/or cyclin-dependent kinase 5 (CDK5) activity (which could lead to increased tau phosphorylation), or loss of activity of the major tau phosphatase, protein phosphatase 2 (PP2A), which can occur under conditions of increased oxidative stress [141]. Iron-induced oxidative stress may also have a role in the tau hyperphosphorylation and polymerization. For instance, the oxidation of lipids, which is found to be elevated in AD brains, can facilitate tau polymerization, and may further drive oxidative stress and the formation of the tau fibrillar pathology in AD [142].

Consistent with the importance of iron in tau dysfunction, intranasal deferoxamine decreased the activity of GSK3β (a major tau kinase) in the AβPP/PS1 mouse model of AD, correlating with rescue of reference and working memory, and led to decreases in oxidative stress [130]. Importantly, total tau levels are decreased in AD cortex [143 –146], and we recently demonstrated that loss of tau expression causes iron- and age-dependent cognitive loss and cortical atrophy in mice [147]. Tau is required for the correct trafficking of AβPP to the neuronal membrane [147], where it binds and stabilizes ferroportin in the cell membrane and facilitates iron efflux from neurons [90, 148], which is neuroprotective [149]. Consequently, reduced tau or AβPP levels could lead to iron retention in neurons that is observed in AD. Collectively, such findings suggest that elevated iron promotes pathological alterations in tau behavior in AD. Thus, while AβPP and tau play crucial roles in maintaining iron efflux from neurons [90, 150], chronic iron loading potentiates amyloidogenic processing of AβPP, the toxicity of Aβ aggregates, and tau dysfunction, which further increase iron-mediated lesions and neuropathologies.

Iron enhances neuronal cell death: Apoptosis

Iron-induced oxidative stress has been shown to initiate several apoptotic signaling pathways in neurons [151], and cause oxidative damage to key proteins such as Ca²⁺-ATPase [152 –155], glutamate transporter [19 , 157], ApoE [158, 159], Na⁺/K⁺-ATPase [152 , 161], as well as the NMDA receptor [162 –164], and lipids, such as cholesterol [165 –167], ceramides [168, 169], polyunsaturated fatty acids (PUFAs) [98 , 170–172], and sphingomyelin [173, 174]. There is extensive evidence that oxidative damage to proteins and lipids by iron can cause synaptic dysfunction and neuronal cell death [175], both of which are critical features of AD.

Notably, the type of cell death that occurs in affected areas of the AD brain is still contentious, despite the demonstration that DNA fragmentation and upregulation of pro-apoptotic proteins has been frequently observed (for a review, see [113]). As such, it remains unclear to what extent apoptosis or emerging types of regulated necrosis (e.g., ferroptosis; see below) are responsible for bulk neuronal loss in AD [94]. Human AD brains show a 30- to 50-fold increase of DNA fragmentation in neurons and glial cells, compared to age-matched controls [113], and AD is characterized by dysfunctional DNA repair systems, leading especially to the accumulation of double strand breaks [5]. However, at least on the basis of DNA fragmentation, nuclear alterations suggestive of apoptosis have been reported to be rare in degenerating cells in AD (including neurons, microglia, and oligodendrocytes), except for those that are associated with Aβ deposits and neurofibrillary tangles of tau [176]. These observations suggest that apoptosis may contribute to cell death resulting in AD, even though other studies suggest that degenerating nuclei adjacent to Aβ deposits may not be apoptotic [177]. As an additional consideration, although DNA fragmentation is a classical feature of apoptosis (e.g., as measured by the TUNEL assay), this process can also occur in various models of regulated necrosis, particularly those that are associated with lipid peroxidative damage and glutathione depletion [178]. Such findings suggest that, in addition to apoptosis, other modes of cell death may also be relevant to neurodegeneration in AD.

The emerging role of ferroptosis in neurodegeneration

Although the evidence overwhelmingly suggests that elevations of redox-active iron in vulnerable brain regions in AD (e.g., hippocampus) clearly contribute to neurodegenerative processes and neuronal loss (as discussed above), the precise molecular pathways of cell death, and how iron is involved, remain unclear. Intriguingly, ferroptosis is an emerging pathway of iron-dependent programmed cell death, which is being currently investigated as a possible patho-mechanism in AD [179, 180]. This recently described mode of regulated necrosis was officially discovered and named in 2012 [181], and is distinct from all other known cell death modalities [20 , 181]. Essentially, ferroptosis is an iron- and lipid-peroxidation-dependent pathway of regulated necrotic cell death [182], which exhibits a unique dependence on RAS-RAF signaling, concurring with the original identification of the pathway in RAS-active cancer cells [181]. While an in-depth discussion of ferroptosis is outside the scope of this review, and the field is advancing rapidly, readers are referred to the following recent reviews [179 , 184].

Distinct from other types of cell death, ferroptosis nonetheless exhibits some key dependencies, and/or cross-talk, with other pathways (e.g., autophagy [185, 186] and apoptosis [187, 188]). In a pharmacologic setting, ferroptosis can be initiated by structurally diverse small molecules [179, 184], such as erastin (which inhibits cystine import by system Xc-), sulfasalazine (also inhibits system Xc-), and RSL3 (which inhibits the LOOH-detoxifying selenoenzyme, glutathione peroxidase 4 [GPX4]). Conversely, ferroptosis can be inhibited by: 1) lipophilic antioxidants, such as vitamin E, Trolox and; 2) redox-inactive iron chelators, such as deferoxamine; and 3) the small-molecule aromatic amine inhibitors, ferrostatin-1 and liproxstatin-1 [179, 184], as well as by lipid-soluble diarylamine radical-trapping antioxidants [189] and 1,8-tetrahydronaphthyridinols [190].

Loss of the activity of GPX4, a key glutathione-dependent enzyme specifically involved in protecting cells against ferroptosis, promotes the accumulation of membrane-associated LOOH [179, 184]. These LOOH can form spontaneously in the presence of existing lipid-reactive radicals and dioxygen (often termed “autoxidation”), which is driven by the presence of catalytic concentrations of labile iron, or can be enzymatically produced by the action of the non-heme iron lipoxygenases, ALOX12 or ALOX15, which drive ferroptosis through peroxidation of specific phospholipid-associated PUFAs at the bis-allylic position [191].

Recently, Kagan et al. [192] discovered that ferroptosis involves a highly organized oxygenation center, in which oxidation within ER-associated membrane compartments specifically targets phosphatidylethanolamines (PEs), and moreover, is specific towards two fatty acyls derived from arachidonic acid (AA) and adrenic acid (AdA). Moreover, suppression of AA or AdA esterification into PEs by genetic, or the pharmacological inhibition of acyl-CoA synthase 4 (ACSL4), inhibits ferroptosis [192, 193]. The implicated lipoxygenases (i.e., ALOX12/15) can generate doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species that then act as specific ferroptotic signals, whereas tocopherols and tocotrienols (forms of “vitamin E”) are able to suppress this activity [192]. Indeed, vitamin E is an important endogenous and physiological regulator of ferroptosis, which inhibits the process by directly inhibiting lipoxygenases [192] and/or acting as a membrane-soluble radical-trapping antioxidant [189]. Such considerations are of considerable relevance to AD, as some evidence suggests that vitamin E may be able to delay functional decline in patients with mild to moderate AD [194], as well as showing benefit in animal models of AD [195], although the role of vitamin E in protecting against AD is still under debate [196, 197].

In the context of cancer, ferroptosis may act as an endogenous tumor-suppressive mechanism downstream of p53 that acts, at least in part, by intracellular glutathione depletion (i.e., by decreasing expression of the system Xc- subunit, SLC7A11, that imports cystine that is required for glutathione biosynthesis) [198], and/or by increasing polyamine oxidation [199a] (i.e., by increasing expression of the polyamine N¹-acetyltransferase, SAT1), followed by enhancement of lipid peroxidation. Consistent with these findings, a very recent study has shown that cellular iron depletion markedly suppresses expression of polyamine oxidase (PAOX) [199b], an enzyme that metabolically cooperates with SAT1. Suppression of PAOX would be predicted to decrease polyamine oxidation and suppress ferroptosis-associated lipid peroxidation. From the point of view of the CNS, ferroptosis has recently been implicated in the pathological cell death of brain tissues exposed to pathological levels of glutamate, as well as kidney and heart tissues subjected to ischemia– reperfusion injury [181, 183]. It is, therefore, of great interest to understand how this novel regulated cell death pathway is specifically regulated in the brain, particularly as emerging evidence suggest that the pathophysiology of a range of neurodegenerative diseases may be associated with excessive ferroptosis [99, 200], including AD [179, 180], Parkinson’s disease [201], Huntington’s disease [179], and ischemic stroke [202].

Recent animal studies, in which Gpx4 was conditionally inactivated in neurons, suggest that ferroptosis can be involved in the degeneration of spinal motor neurons and midbrain neurons [203], as well as neurons in forebrain regions, including cerebral cortex and hippocampus that are severely afflicted in AD patients [180]. Indeed, the “Gpx4BIKO” mouse model, in which Gpx4 has been conditionally inactivated in forebrain neurons for 12 weeks (following tamoxifen treatment to trigger gene inactivation), exhibited significant deficits in spatial learning and memory function. Subsequent examinations of the cognitively impaired Gpx4BIKO mice revealed profound hippocampal neurodegeneration [180]. This neurodegeneration was accompanied by markers of ferroptosis, such as elevated lipid peroxidation, ERK activation, and elevated neuroinflammation [180]. Notably, when Gpx4BIKO mice were fed a diet deficient in vitamin E, the rate of hippocampal neurodegeneration and behavioral dysfunction were augmented, providing support for an important role for vitamin E in protecting neurons against ferroptosis. Furthermore, neurodegeneration in these mice could be inhibited by liproxstatin-1 (administered i.p.) [180]. These results strongly suggest that forebrain neurons are susceptible to ferroptosis, particularly in the context of loss of GPX4 activity, further suggesting that ferroptosis may be an important neurodegenerative mechanism in AD.

In AD, the ferroptosis pathway may assist with understanding how iron potentiates the neurotoxicity of other key pathological hallmarks of the disease, such as those associated with Aβ and tau. It is tempting to speculate that the apparent co-dependence of AD pathology on elevated iron and Aβ [94], is that Aβ and tau dysfunction may potentiate the sensitivity of vulnerable neurons to ferroptosis, which could then activate under conditions of elevated iron and/or decreased glutathione and/or GPX4 activity. In support of this hypothesis, a recent study suggests that Aβ (specifically Aβ₄₂) increases RAS-ERK signaling and GSK3β activation, which the authors showed led to phosphorylation of AβPP at Thr668 (potentiating cleavage by γ-secretase) and tau [204]. Furthermore, the authors showed that RAS is hyperactivated in human postmortem AD samples compared to healthy controls [204]. As ferroptosis shows a dependence on RAS activation [181, 205], it may be the case that elevated brain iron “converges” with other key AD pathologies (e.g., those associated with Aβ and tau) that prime neurons for ferroptosis. Consistent with the convergent pathologies hypothesis, the ferroptotic “scales” in AD may be tipped in favor of neurodegeneration and overt neuronal loss in the context of elevated redox-active iron, depletion of cellular antioxidant reserves (e.g., glutathione and vitamin E), loss of GPX4 activity, and/or neuroinflammation that promotes further iron accumulation and oxidative stress.

CONCLUSIONS

Iron is a transition metal that is vital for life and is abundant in the brain, where it plays many vital metabolic roles. Iron is also “Janus-faced”, as its enormous biological utility in being able to readily redox cycle between Fe(II) and Fe(III) states also endows it with the potential to “rust” brain tissue by producing ROS that lead to neurodegeneration and facilitate cell death. Iron contributes to AD pathology at numerous levels, and presently represents a promising and tractable target with untapped disease-modifying potential. Recent evidence points toward a possible role in AD for ferroptosis, a unique mode of programmed cell death that is dependent on redox-active iron and lipid-peroxidative stress and can occur in brain neurons. While this cell death pathway is only beginning to be explored in neurodegenerative diseases, it appears to have important and wide-ranging therapeutic implications for AD, particularly since ferroptosis can be readily prevented by iron chelators and endogenous and synthetic inhibitors of lipid peroxidation [205, 206]. Indeed, drugs specifically targeting components of the ferroptosis pathway (including iron) may show great promise in the treatment of AD and are worthy of further investigation.

DISCLOSURE STATEMENT

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/17-9944).

References

Brookmeyer

, Johnson

, Ziegler-Graham

, Arrighi

. (2007) Forecasting the global burden of Alzheimer’s disease. Alzheimers Dement3, 186–191.

Assoc

. (2017) Alzheimer’s Association Report: 2017 Alzheimer’s disease facts and figures. Alzheimers Dement13, 325–373.

Belaidi

, Bush

. (2016) Iron neumistry in Alzheimer’s disease and Parkinson’s disease: Targets for therapeutics. J Neurochem139(Suppl 1), 179–197roche.

Ayton

, Lei

, Bush

. (2013) Metallostasis in Alzheimer’s disease. Free Radic Biol Med62, 76–89.

Davydov

, Hansen

, Shackelford

. (2003) Is DNA repair compromised in Alzheimer’s disease?. Neurobiol Aging24, 953–968.

Good

, Werner

, Hsu

, Olanow

, Perl

. (1996) Evidence of neuronal oxidative damage in Alzheimer’s disease. Am J Pathol149, 21–28.

Mecocci

, MacGarvey

, Beal

. (1994) Oxidative damage to mitochondrial DNA is increased in Alzheimer’s disease. Ann Neurol36, 747–751.

Pratico

. (2008) Oxidative stress hypothesis in Alzheimer’s disease: A reappraisal. Trends Pharmacol Sci29, 609–615.

Sayre

, Perry

, Harris

, Liu

, Schubert

, Smith

. (2000) In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer’s disease: A central role for bound transition metals. J Neurochem74, 270–279.

10.

Urrutia

, Aguirre

, Esparza

, Tapia

, Mena

, Arredondo

, Gonzalez-Billault

, Nunez

. (2013) Inflammation alters the expression of DMT1, FPN1 and hepcidin, and it causes iron accumulation in central nervous system cells. J Neurochem126, 541–549.

11.

Smith

, Carney

, Starke-Reed

, Oliver

, Stadtman

, Floyd

, Markesbery

. (1991) Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci U S A88, 10540–10543.

12.

Lovell

, Ehmann

, Butler

, Markesbery

. (1995) Elevated thiobarbituric acid-reactive substances and antioxidant enzyme activity in the brain in Alzheimer’s disease. Neurology45, 1594–1601.

13.

Todorich

, Connor JR (2004) Redox metals in Alzheimer’s disease. Ann N Y Acad Sci1012, 171–178.

14.

Bonda

, Wang

, Perry

, Nunomura

, Tabaton

, Zhu

, Smith

. (2010) Oxidative stress in Alzheimer disease: A possibility for prevention. Neuropharmacology59, 290–294.

15.

Zhu

, Su

, Wang

, Smith

, Perry

. (2007) Causes of oxidative stress in Alzheimer disease. Cell Mol Life Sci64, 2202–2210.

16.

Yang

, Yang

, Liang

, Xu

, Moore

, Ran

. (2016) Imaging hydrogen peroxide in Alzheimer’s disease via cascade signal amplification. Sci Rep6, 35613.

17.

Di Paolo

, Kim

. (2011) Linking lipids to Alzheimer’s disease: Cholesterol and beyond. Nat Rev Neurosci12, 284–296.

18.

Gonzalez-Dominguez

, Garcia-Barrera

, Gomez-Ariza

. (2014) Homeostasis of metals in the progression of Alzheimer’s disease. Biometals27, 539–549.

19.

, Guo

, Sun

, Li

, Zhang

, Li

. (2009) Iron is a potential key mediator of glutamate excitotoxicity in spinal cord motor neurons. Brain Res1257, 102–107.

20.

Yang

, Stockwell

. (2016) Ferroptosis: Death by lipid peroxidation. Trends Cell Biol26, 165–176.

21.

Lawen

, Lane

DJR

. (2013) Mammalian iron homeostasis in health and disease: Uptake, storage, transport, and molecular mechanisms of action. Antioxid Redox Signal18, 2473–2507.

22.

Steinbicker

, Muckenthaler

. (2013) Out of balance—systemic iron homeostasis in iron-related disorders. Nutrients5, 3034–3061.

23.

Bradley-Whitman

, Lovell

. (2015) Biomarkers of lipid peroxidation in Alzheimer disease (AD): An update}. Arch Toxicol89, 1035–1044.

24.

Winterbourn

. (1995) Toxicity of iron and hydrogen peroxide: The Fenton reaction. Toxicol Lett82-83, 969–974.

25.

Ayton

, Lei

, Bush

. (2015) Biometals and their therapeutic implications in Alzheimer’s disease. Neurotherapeutics12, 109–120.

26.

Bandyopadhyay

, Rogers

. (2014) Alzheimer’s disease therapeutics targeted to the control of amyloid precursor protein translation: Maintenance of brain iron homeostasis. Biochem Pharmacol88, 486–494.

27.

Horowitz

, Greenamyre

. (2010) Mitochondrial iron metabolism and its role in neurodegeneration. J Alzheimers Dis20(Suppl 2), 551–568.

28.

Buettner

, Jurkiewicz

. (1996) Catalytic metals, ascorbate and free radicals: Combinations to avoid. Radiat Res145, 532–541.

29.

Lane

DJR

, Lawen

. (2009) Ascorbate and plasma membrane electron transport–enzymes vs efflux. Free Radic Biol Med47, 485–495.

30.

Sagrista

, Garcia

, Africa De Madariaga

, Mora

. (2002) Antioxidant and pro-oxidant effect of the thiolic compounds N-acetyl-L-cysteine and glutathione against free radical-induced lipid peroxidation. Free Radic Res36, 329–340.

31.

Muckenthaler

, Galy

, Hentze

. (2008) Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network. Annu Rev Nutr28, 197–213.

32.

Richardson

, Lane

DJR

, Becker

, Huang

, Whitnall

, Rahmanto

, Sheftel

, Ponka

. (2010) Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol. Proc Natl Acad Sci U S A107, 10775–10782.

33.

Lane

, Merlot

, Huang

, Bae

, Jansson

, Sahni

, Kalinowski

, Richardson

. (2015) Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease. Biochim Biophys Acta1853, 1130–1144.

34.

Arosio

, Levi

. (2010) Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage. Biochim Biophys Acta1800, 783–792.

35.

Lill

. (2009) Function and biogenesis of iron-sulphur proteins. Nature460, 831–838.

36.

Rouault

. (2012) Biogenesis of iron-sulfur clusters in mammalian cells: New insights and relevance to human disease. Dis Model Mech5, 155–164.

37.

Philpott

, Ryu

. (2014) Special delivery: Distributing iron in the cytosol of mammalian cells. Front Pharmacol5, 173.

38.

Philpott

. (2012) Coming into view: Eukaryotic iron chaperones and intracellular iron delivery. J Biol Chem287, 13518–13523.

39.

Hentze

, Kuhn

. (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A93, 8175–8182.

40.

Richardson

, Ponka

. (1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta1331, 1–40.

41.

Hentze

, Muckenthaler

, Galy

, Camaschella

. (2010) Two to tango: Regulation of mammalian iron metabolism. Cell142, 24–38.

42.

Ohgami

, Campagna

, Greer

, Antiochos

, McDonald

, Chen

, Sharp

, Fujiwara

, Barker

, Fleming

. (2005) Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells. Nat Genet37, 1264–1269.

43.

Lane

, Richardson

. (2014) The active role of vitamin C in mammalian iron metabolism: Much more than just enhanced iron absorption!. Free Radic Biol Med75C, 69–83.

44.

Lane

DJR

, Chikhani

, Richardson

. (2013) Transferrin iron uptake is stimulated by ascorbate an intracellular reductive mechanism. Biochim Biophys Acta1833, 1527–1541.

45.

Escobar

, Gaete

, Nunez

. (1992) Effect of ascorbate in the reduction of transferrin-associated iron in endocytic vesicles. J Bioenerg Biomembr24, 227–233.

46.

Gunshin

, Mackenzie

, Berger

, Gunshin

, Romero

, Boron

, Nussberger

, Gollan

, Hediger

. (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature388, 482–488.

47.

Jenkitkasemwong

, Wang

, Mackenzie

, Knutson

. (2012) Physiologic implications of metal-ion transport by ZIP14 and ZIP8. Biometals25, 643–655.

48.

Yanatori

, Yasui

, Tabuchi

, Kishi

. (2014) Chaperone protein involved in transmembrane transport of iron. Biochem J462, 25–37.

49.

Leidgens

, Bullough

, Shi

, Li

, Shakoury-Elizeh

, Yabe

, Subramanian

, Hsu

, Natarajan

, Nandal

, Stemmler

, Philpott

. (2013) Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin. J Biol Chem288, 17791–17802.

50.

McKie

, Barrow

, Latunde-Dada

, Rolfs

, Sager

, Mudaly

, Richardson

, Barlow

, Bomford

, Peters

, Raja

, Shirali

, Hediger

, Farzaneh

, Simpson

. (2001) An iron-regulated ferric reductase associated with the absorption of dietary iron. Science291, 1755–1759.

51.

Lane

DJR

, Lawen

. (2008) Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells. J Biol Chem283, 12701–12708.

52.

Lane

DJR

, Lawen

. (2013) The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes. Cell Biochem Biophys65, 107–119.

53.

Lane

, Robinson

, Czerwinska

, Bishop

, Lawen

. (2010) Two routes of iron accumulation in astrocytes: Ascorbate-dependent ferrous iron uptake via the divalent metal transporter (DMT1) plus an independent route for ferric iron. Biochem J432, 123–132.

54.

Harrison

, Arosio

. (1996) The ferritins: Molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta1275, 161–203.

55.

Chasteen

, Harrison

. (1999) Mineralization in ferritin: An efficient means of iron storage. J Struct Biol126, 182–194.

56.

Domenico De

, Ward

, Kaplan

. (2009) Specific iron chelators determine the route of ferritin degradation. Blood114, 4546–4551.

57.

Mancias

, Wang

, Gygi

, Harper

, Kimmelman

. (2014) Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature509, 105–109.

58.

Rouault

. (2006) The role of iron regulatory proteins in mammalian iron homeostasis and disease. Nat Chem Biol2, 406–414.

59.

Guo

, Phillips

, Yu

, Leibold

. (1995) Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome. J Biol Chem270, 21645–21651.

60.

Moos

, Nielsen

, Skjørringe

, Morgan

. (2007) Iron trafficking inside the brain. J Neurochem103, 1730–1740.

61.

Dringen

, Bishop

, Koeppe

, Dang

, Robinson

. (2007) The pivotal role of astrocytes in the metabolism of iron in the brain. Neurochem Res32, 1884–1890.

62.

Bishop

, Dang

, Dringen

, Robinson

. (2011) Accumulation of non-transferrin-bound iron by neurons, astrocytes, and microglia. Neurotox Res19, 443–451.

63.

Skjorringe

, Burkhart

, Johnsen

, Moos

. (2015) Divalent metal transporter 1 (DMT1) in the bra, Imlications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology. Front Mol Neurosci8, 19.

64.

Moos

, Skjoerringe

, Gosk

, Morgan

. (2006) Brain capillary endothelial cells mediate iron transport into the brain by segregating iron from transferrin without the involvement of divalent metal transporter 1. J Neurochem98, 1946–1958.

65.

Benarroch

. (2009) Brain iron homeostasis and neurodegenerative disease. Neurology72, 1436–1440.

66.

Burkhart

, Skjorringe

, Johnsen

, Siupka

, Thomsen

, Nielsen

, Thomsen

, Moos

. (2016) Expression of iron-related proteins at the neurovascular unit supports reduction and reoxidation of iron for transport through the blood-brain barrier. Mol Neurobiol53, 7237–7253.

67.

McCarthy

, Kosman

. (2013) Ferroportin and exocytoplasmic ferroxidase activity are required for brain microvascular endothelial cell iron efflux. J Biol Chem288, 17932–17940.

68.

Codazzi

, Pelizzoni

, Zacchetti

, Grohovaz

. (2015) Iron entry in neurons and astrocytes: A link with synaptic activity. Front Mol Neurosci8, 18.

69.

Jeong

, David

. (2003) Glycosylphosphatidylinositol-anchored ceruloplasmin is required for iron efflux from cells in the central nervous system. J Biol Chem278, 27144–27148.

70.

Erikson

, Aschner

. (2006) Increased manganese uptake by primary astrocyte cultures with altered iron status is mediated primarily by divalent metal transporter. Neurotoxicology27, 125–130.

71.

Song

, Jiang

, Wang

, Xie

J-X

. (2007) Divalent metal transporter 1 up-regulation is involved in the 6-hydroxydopamine-induced ferrous iron influx. J Neurosci Res85, 3118–3126.

72.

Tulpule

, Robinson

, Bishop

, Dringen

. (2010) Uptake of ferrous iron by cultured rat astrocytes. J Neurosci Res88, 563–571.

73.

Lis

, Barone

, Paradkar

, Plunkett

, Roth

. (2004) Expression and localization of different forms of DMT1 in normal and tumor astroglial cells. Mol Brain Res122, 62–70.

74.

Burdo

, Menzies

, Simpson

, Garrick

, Dolan

, Haile

, Beard

, Connor

. (2001) Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat. J Neurosci Res66, 1198–1207.

75.

Wang

, Ong

, Connor

. (2001) A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus. J Neurocytol30, 353–360.

76.

Pelizzoni

, Zacchetti

, Campanella

, Grohovaz

, Codazzi

. (2013) Iron uptake in quiescent and inflammation-activated astrocytes: A potentially neuroprotective control of iron burden. Biochim Biophys Acta1832, 1326–1333.

77.

Hubert

, Hentze

. (2002) Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: Implications for regulation and cellular function. Proc Natl Acad Sci U S A99, 12345–12350.

78.

Rathore

, Redensek

, David

. (2012) Iron homeostasis in astrocytes and microglia is differentially regulated by TNF-a and TGF-b1. Glia60, 738–750.

79.

Pelizzoni

, Zacchetti

, Smith

, Grohovaz

, Codazzi

. (2012) Expression of divalent metal transporter 1 in primary hippocampal neurons: Reconsidering its role in non-transferrin-bound iron influx. J Neurochem120, 269–278.

80.

Saura

. (2007) Microglial cells in astroglial cultures: A cautionary note. J Neuroinflammation4, 26.

81.

Ayton

, Lei

. (2014) Nigral iron elevation is an invariable feature of Parkinson’s disease and is a sufficient cause of neurodegeneration. Biomed Res Int2014, 581256.

82.

Ayton

, Lei

, Adlard

, Volitakis

, Cherny

, Bush

, Finkelstein

. (2014) Iron accumulation confers neurotoxicity to a vulnerable population of nigral neurons: Implications for Parkinson’s disease. Mol Neurodegener9, 27.

83.

Goodman

. (1953) Alzheimer’s disease; a clinico-pathologic analysis of twenty-three cases with a theory on pathogenesis. J Nerv Ment Dis118, 97–130.

84.

Connor

, Menzies

, St Martin

, Mufson

. (1992) A histochemical study of iron, transferrin, and ferritin in Alzheimer’s diseased brains. J Neurosci Res31, 75–83.

85.

Connor

, Snyder

, Beard

, Fine

, Mufson

. (1992) Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer’s disease. J Neurosci Res31, 327–335.

86.

Collingwood

, Mikhaylova

, Davidson

, Batich

, Streit

, Terry

, Dobson

. (2005) In situ characterization and mapping of iron compounds in Alzheimer’s disease tissue. J Alzheimers Dis7, 267–272.

87.

Baltes

, Princz-Kranz

, Rudin

, Mueggler

. (2011) Detecting amyloid-beta plaques in Alzheimer’s disease. Methods Mol Biol711, 511–533.

88.

Meadowcroft

, Connor

, Smith

, Yang

. (2009) MRI and histological analysis of beta-amyloid plaques in both human Alzheimer’s disease and APP/PS1 transgenic mice. J Magn Reson Imaging29, 997–1007.

89.

Collingwood

, Chong

, Kasama

, Cervera-Gontard

, Dunin-Borkowski

, Perry

, Posfai

, Siedlak

, Simpson

, Smith

, Dobson

. (2008) Three-dimensional tomographic imaging and characterization of iron compounds within Alzheimer’s plaque core material. J Alzheimers Dis14, 235–245.

90.

Duce

, Tsatsanis

, Cater

, James

, Robb

, Wikhe

, Leong

, Perez

, Johanssen

, Greenough

, Cho

H-H

, Galatis

, Moir

, Masters

, Mclean

, Tanzi

, Cappai

, Barnham

, Ciccotosto

, Rogers

, Bush

. (2010) Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer’s disease. Cell142, 857–867.

91.

Andrasi

, Farkas

, Scheibler

, Reffy

, Bezur

. (1995) Al, Zn, Cu, Mn and Fe levels in brain in Alzheimer’s disease. Arch Gerontol Geriatr21, 89–97.

92.

Smith

, Harris

, Sayre

, Perry

. (1997) Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A94, 9866–9868.

93.

Pietrzak

, Lim

, Ames

, Harrington

, Restrepo

, Martins

, Rembach

, Laws

, Masters

, Villemagne

, Rowe

, Maruff

, Australian Imaging, Biomarkers and Lifestyle (AIBL) Research Group (2015) Trajectories of memory decline in preclinical Alzheimer’s disease: Results from the Australian Imaging, Biomarkers and Lifestyle Flagship Study of ageing. Neurobiol Aging36, 1231–1238.

94.

Ayton

, Fazlollahi

, Bourgeat

, Raniga

, Ng

, Lim

, Diouf

, Farquharson

, Fripp

, Ames

, Doecke

, Desmond

, Ordidge

, Masters

, Rowe

, Maruff

, Villemagne

, Australian Imaging B, Lifestyle Research G, Salvado

, Bush

. (2017) Cerebral quantitative susceptibility mapping predicts amyloid-β-related cognitive decline. Brain140, 2112–2119.

95.

Ayton

, Faux

, Bush

. (2017) Association of cerebrospinal fluid ferritin level with preclinical cognitive decline in APOE-ɛ4 carriers. JAMA Neurol74, 122–125.

96.

Ayton

, Faux

, Bush

, Alzheimer’s Disease Neuroimaging Initiative (2015) Ferritin levels in the cerebrospinal fluid predict Alzheimer’s disease outcomes and are regulated by APOE. Nat Commun6, 6760.

97.

Braughler

, Duncan

, Chase

. (1986) The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation. J Biol Chem261, 10282–10289.

98.

Dougherty

, Croft

, Hoekstra

. (1981) Effects of ferrous chloride and iron dextran on lipid peroxidation in vivo in vitamin E and selenium adequate and deficient rats. J Nutr111, 1784–1796.

99.

Morris

, Berk

, Carvalho

, Maes

, Walker

, Puri

. (2018) Why should neuroscientists worry about iron? The emerging role of ferroptosis in the pathophysiology of neuroprogressive diseases. Behav Brain Res341, 154–175.

100.

Hare

, Ayton

, Bush

, Lei

. (2013) A delicate balance: Iron metabolism and diseases of the brain. Front Aging Neurosci5, 34.

101.

, Liu

, Zheng

, Yao

, Cheng

, Bu

, Xu

, Zhang

. (2013) Ferritin light chain interacts with PEN-2 and affects gamma-secretase activity. Neurosci Lett548, 90–94.

102.

Peters

, Pollack

, Cheng

, Sun

, Saido

, Haaf

, Yang

, Connor

, Meadowcroft

. (2018) Dietary lipophilic iron alters amyloidogenesis and microglial morphology in Alzheimer’s disease knock-in APP mice. Metallomics10, 426–443.

103.

Gurel

, Cansev

, Sevinc

, Kelestemur

, Ocalan

, Cakir

, Aydin

, Kahveci

, Ozansoy

, Taskapilioglu

, Ulus

, Basar

, Sahin

, Tuzuner

, Baykal

. (2018) Early stage alterations in CA1 extracellular region proteins indicate dysregulation of IL6 and iron homeostasis in the 5XFAD Alzheimer’s disease mouse model. J Alzheimers Dis61, 1399–1410.

104.

Ayton

, Diouf

, Bush

. (2018) Evidence that iron accelerates Alzheimer’s pathology: A CSF biomarker study. J Neurol Neurosurg Psychiatry89, 456–460.

105.

Telling

, Everett

, Collingwood

, Dobson

, van der Laan

, Gallagher

, Wang

, Hitchcock

. (2017) Iron biochemistry is correlated with amyloid plaque morphology in an established mouse model of Alzheimer’s disease. Cell Chem Biol24, 1205–1215.e1203

106.

Greenough

. (2016) The role of presenilin in protein trafficking and degradation-implications for metal homeostasis. J Mol Neurosci60, 289–297.

107.

de Ancos García

, Correas

, Avila

. (1993) Differences in microtubule binding and self-association abilities of bovine brain tau isoforms. J Biol Chem268, 7976–7982.

108.

Ledesma

, Avila

, Correas

. (1995) Isolation of a phosphorylated soluble tau fraction from Alzheimer’s disease brain. Neurobiol Aging16, 515–522.

109.

Yamamoto

, Shin

R-W

, Hasegawa

, Naiki

, Sato

, Yoshimasu

, Kitamoto

. (2002) Iron (III) induces aggregation of hyperphosphorylated tau and its reduction to iron (II) reverses the aggregation: Implications in the formation of neurofibrillary tangles of Alzheimer’s disease. J Neurochem82, 1137–1147.

110.

Guo

, Wang

, Zhong

, Wang

, Huang

, Li

, Wang

. (2013) Deferoxamine inhibits iron induced hippocampal tau phosphorylation in the Alzheimer transgenic mouse brain. Neurochem Int62, 165–172.

111.

Wang

, Wang

. (2017) Metal ions influx is a double edged sword for the pathogenesis of Alzheimer’s disease. Ageing Res Rev35, 265–290.

112.

Kuperstein

, Yavin

. (2003) Pro-apoptotic signaling in neuronal cells following iron and amyloid beta peptide neurotoxicity. J Neurochem86, 114–125.

113.

Radi

, Formichi

, Battisti

, Federico

. (2014) Apoptosis and oxidative stress in neurodegenerative diseases. J Alzheimers Dis42(Suppl 3), 125–152.

114.

Rogers

, Randall

, Cahill

, Eder

, Huang

, Gunshin

, Leiter

, McPhee

, Sarang

, Utsuki

, Greig

, Lahiri

, Tanzi

, Bush

, Giordano

, Gullans

. (2002) An iron-responsive element type II in the 5’-untranslated region of the Alzheimer’s amyloid precursor protein transcript. J Biol Chem277, 45518–45528.

115.

Cho

, Cahill

, Vanderburg

, Scherzer

, Wang

, Huang

, Rogers

. (2010) Selective translational control of the Alzheimer amyloid precursor protein transcript by iron regulatory protein-1. J Biol Chem285, 31217–31232.

116.

Paul

, Lill

. (2015) Biogenesis of cytosolic and nuclear iron-sulfur proteins and their role in genome stability. Biochim Biophys Acta1853, 1528–1539.

117.

Rouault

, Maio

. (2017) Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways. J Biol Chem292, 12744–12753.

118.

Isaya

. (2014) Mitochondrial iron-sulfur cluster dysfunction in neurodegenerative disease. Front Pharmacol5, 29.

119.

Mena

, Urrutia

, Lourido

, Carrasco

, Nunez

. (2015) Mitochondrial iron homeostasis and its dysfunctions in neurodegenerative disorders. Mitochondrion21, 92–105.

120.

Rogers

, Bush

, Cho

, Smith

, Thomson

, Friedlich

, Lahiri

, Leedman

, Huang

, Cahill

. (2008) Iron and the translation of the amyloid precursor protein (APP) and ferritin mRNAs: Riboregulation against neural oxidative damage in Alzheimer’s disease. Biochem Soc Trans36, 1282–1287.

121.

Smith

, Wehr

, Harris

, Siedlak

, Connor

, Perry

. (1998) Abnormal localization of iron regulatory protein in Alzheimer’s disease. Brain Res788, 232–236.

122.

Yamauchi

, Nishiyama

, Moroishi

, Kawamura

, Nakayama

. (2017) FBXL5 inactivation in mouse brain induces aberrant proliferation of neural stem progenitor cells. Mol Cell Biol37, 00470–16.

123.

Becerril-Ortega

, Bordji

, Freret

, Rush

, Buisson

. (2014) Iron overload accelerates neuronal amyloid-b production and cognitive impairment in transgenic mice model of Alzheimer’s disease. Neurobiol Aging35, 2288–2301.

124.

Zhang

, Zhao

, Yew

, Kusiak

, Roth

. (1997) Processing of Alzheimer’s amyloid precursor protein during H₂O₂-induced apoptosis in human neuronal cells. Biochem Biophys Res Commun235, 845–848.

125.

Atwood

, Scarpa

, Huang

, Moir

, Jones

, Fairlie

, Tanzi

, Bush

. (2000) Characterization of copper interactions with Alzheimer amyloid beta peptides: Identification of an attomolar-affinity copper binding site on amyloid beta1-42. J Neurochem75, 1219–1233.

126.

Atwood

, Moir

, Huang

, Scarpa

, Bacarra

, Romano

, Hartshorn

, Tanzi

, Bush

. (1998) Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem273, 12817–12826.

127.

Lovell

, Robertson

, Teesdale

, Campbell

, Markesbery

. (1998) Copper, iron and zinc in Alzheimer’s disease senile plaques. J Neurol Sci158, 47–52.

128.

James

, Churches

, de Jonge

, Birchall

, Streltsov

, McColl

, Adlard

, Hare

. (2017) Iron, copper, and zinc concentration in Aβ plaques in the APP/PS1 mouse model of Alzheimer’s disease correlates with metal levels in the surrounding neuropil. ACS Chem Neurosci8, 629–637.

129.

Guo

, Wang

, Zheng

, Shan

, Teng

, Wang

. (2013) Intral deferoxamine reverses iron-induced memory deficits and inhibits amyloidogenic APP processing in a transgenic mouse model of Alzheimer’s disease. Neurobiol Aging34, 562–575.

130.

Fine

, Renner

, Forsberg

, Cameron

, Galick

, Le

, Conway

, Stroebel

, Frey

2nd , Hanson

. (2015) Intral deferoxamine engages multiple pathways to decrease memory loss in the APP/PS1 model of amyloid accumulation. Neurosci Lett584, 362–367.

131.

Crapper McLachlan

, Dalton

, Kruck

, Bell

, Smith

, Kalow

, Andrews

. (1991) Intramuscular desferrioxamine in patients with Alzheimer’s disease. Lancet337, 1304–1308.

132.

Schubert

, Chevion

. (1995) The role of iron in beta amyloid toxicity. Biochem Biophys Res Commun216, 702–707.

133.

Liu

, Moloney

, Meehan

, Morris

, Thomas

, Serpell

, Hider

, Marciniak

, Lomas

, Crowther

. (2011) Iron promotes the toxicity of amyloid beta peptide by impeding its ordered aggregation. J Biol Chem286, 4248–4256.

134.

Rottkamp

, Raina

, Zhu

, Gaier

, Bush

, Atwood

, Chevion

, Perry

, Smith

. (2001) Redox-active iron mediates amyloid-beta toxicity. Free Radic Biol Med30, 447–450.

135.

Rival

, Page

, Chandraratna

, Sendall

, Ryder

, Liu

, Lewis

, Rosahl

, Hider

, Camargo

, Shearman

, Crowther

, Lomas

. (2009) Fenton chemistry and oxidative stress mediate the toxicity of the beta-amyloid peptide in a Drosophila model of Alzheimer’s disease. Eur J Neurosci29, 1335–1347.

136.

Hanson

, Frey

2nd . (2008) Intral delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci9(Suppl 3), 5.

137.

Lovell

, Xiong

, Xie

, Davies

, Markesbery

. (2004) Induction of hyperphosphorylated tau in primary rat cortical neuron cultures mediated by oxidative stress and glycogen synthase kinase-3.659-671; discussion. J Alzheimers Dis6, 673–681.

138.

Chan

, Shea

. (2006) Dietary and genetically-induced oxidative stress alter tau phosphorylation: Influence of folate and apolipoprotein E deficiency. J Alzheimers Dis9, 399–405.

139.

Huang

, Dai

, Huang

, Zhang

, Bhanot

, Pelle

. (2007) Deferoxamine synergistically enhances iron-mediated AP-1 activation: A showcase of the interplay between extracellular-signal-regulated kinase and tyrosine phosphatase. Free Radic Res41, 1135–1142.

140.

Muñoz

, Zavala

, Castillo

, Aguirre

, Hidalgo

, Nuñez

. (2006) Effect of iron on the activation of the MAPK/ERK pathway in PC12 neuroblastoma cells. Biol Res39, 189–190.

141.

Jin Jung

, Hyun Kim

, Kyeong Lee

, Woo Song

, Pal Yu

, Young Chung

. (2013) Oxidative stress induces inactivation of protein phosphatase 2A, promoting proinflammatory NF-kB in aged rat kidney. Free Radic Biol Med61, 206–217.

142.

Gamblin

, King

, Kuret

, Berry

, Binder

. (2000) Oxidative regulation of fatty acid-induced tau polymerization. Biochemistry39, 14203–14210.

143.

Ksiezak-Reding

, Binder

, Yen

S-HC

. (1988) Immunochemical and biochemical characterization of tau proteins in normal and Alzheimer’s disease brains with Alz 50 and Tau-1. J Biol Chem263, 7948–7953.

144.

Khatoon

, Grundke-Iqbal

, Iqbal

. (1994) Levels of normal and abnormally phosphorylated tau in different cellular and regional compartments of Alzheimer disease and control brains. FEBS Lett351, 80–84.

145.

Zhukareva

, Sundarraj

, Mann

, Sjogren

, Blenow

, Clark

, McKeel

, Goate

, Lippa

, Vonsattel

, Growdon

, Trojanowski

, Lee

. (2003) Selective reduction of soluble tau proteins in sporadic and familial frontotemporal dementias: An international follow-up study. Acta Neuropathol105, 469–476.

146.

van Eersel

, Bi

, Ke

, Hodges

, Xuereb

, Gregory

, Halliday

, Gotz

, Kril

, Ittner

. (2009) Phosphorylation of soluble tau differs in Pick’s disease and Alzheimer’s disease brains. J Neural Transm116, 1243–1251.

147.

Lei

, Ayton

, Finkelstein

, Spoerri

, Ciccotosto

, Wright

, Wong

BXW

, Adlard

, Cherny

, Lam

, Roberts

, Volitakis

, Egan

, Mclean

, Cappai

, Duce

, Bush

. (2012) Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export. Nat Med18, 291–295.

148.

McCarthy

, Park

, Kosman

. (2014) sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin. EMBO Rep15, 809–815.

149.

Ayton

, Zhang

, Roberts

, Lam

, Lind

, McLean

, Bush

, Frugier

, Crack

, Duce

. (2014) Ceruloplasmin and beta-amyloid precursor protein confer neuroprotection in traumatic brain injury and lower neuronal iron. Free Radic Biol Med69, 331–337.

150.

Wong

, Tsatsanis

, Lim

, Adlard

, Bush

, Duce

. (2014) b-Amyloid precursor protein does not possess ferroxidase activity but does stabilize the cell surface ferrous iron exporter ferroportin. PLoS One9, 114174.

151.

Salvador

, Uranga

, Giusto

. (2010) Iron and mechanisms of neurotoxicity. Int J Alzheimers Dis2011, 720658.

152.

Mark

, Hensley

, Butterfield

, Mattson

. (1995) Amyloid b-peptide impairs ion-motive ATPase activities: Evidence for a role in loss of neuronal Ca²⁺ homeostasis and cell death. J Neurosci15, 6239–6249.

153.

Ahuja

, Borchman

, Dean

, Paterson

, Zeng

, Zhang

, Ferguson-Yankey

, Yappert

. (1999) Effect of oxidation on Ca²⁺ -ATPase activity and membrane lipids in lens epithelial microsomes. Free Radic Biol Med27, 177–185.

154.

Moreau

, Castilho

, Ferreira

, Carvalho-Alves

. (1998) Oxidative damage to splasmic reticulum Ca²⁺-ATPase at submicromolar iron concentrations: Evidence for metal-catalyzed oxidation. Free Radic Biol Med25, 554–560.

155.

Kaplan

, Matejovicova

, Mezesova

. (1997) Iron-induced inhibition of Na⁺, K+-ATPase and Na⁺/Ca²⁺ exchanger in synaptosomes: Protection by the pyridoin dole stobadine. Neurochem Res22, 1523–1529.

156.

Gnana-Prakasam

, Thangaraju

, Liu

, Ha

, Martin

, Smith

, Ganapathy

. (2009) Absence of iron-regulatory protein Hfe results in hyperproliferation of retinal pigment epithelium: Role of cystine/glutamate exchanger. Biochem J424, 243–252.

157.

Mitchell

, Lee

, Simmons

, Connor

. (2011) HFE polymorphisms affect cellular glutamate regulation. Neurobiol Aging32, 1114–1123.

158.

Pedersen

, Fu

, Keller

, Markesbery

, Appel

, Smith

, Kasarskis

, Mattson

. (1998) Protein modification by the lipid peroxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients. Ann Neurol44, 819–824.

159.

Pedersen

, Chan

, Mattson

. (2000) A mechanism for the neuroprotective effect of apolipoprotein E: Isoform-specific modification by the lipid peroxidation product 4-hydroxynonenal. J Neurochem74, 1426–1433.

160.

Keller

, Mark

, Bruce

, Blanc

, Rothstein

, Uchida

, Waeg

, Mattson

. (1997) 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience80, 685–696.

161.

Strugatsky

, Gottschalk

, Goldshleger

, Bibi

, Karlish

. (2003) Expression of Na⁺,K⁺-ATPase, Analysis of wild tye and D369N mutant proteins by Fe²⁺-catalyzed oxidative cleavage and molecular modeling. J Biol Chem278, 46064–46073.

162.

Munoz

, Humeres

, Elgueta

, Kirkwood

, Hidalgo

, Nunez

. (2011) Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity. J Biol Chem286, 13382–13392.

163.

Jiang

, Mu

, Biran

, Faustino

, Chang

, Rincon

, Sheldon

, Ferriero

. (2008) Activated Src kinases interact with the N-methyl-D-aspartate receptor after neonatal brain ischemia. Ann Neurol63, 632–641.

164.

Nakamichi

, Ohno

, Nakamura

, Hirai

, Kuramoto

, Yoneda

. (2002) Blockade by ferrous iron of Ca²⁺ influx through N-methyl-D-aspartate receptor channels in immature cultured rat cortical neurons. J Neurochem83, 1–11.

165.

Shinkyo

, Guengerich

. (2011) Cytochrome P450 7A1 cholesterol 7alpha-hydroxylation: Individual reaction steps in the catalytic cycle and rate-limiting ferric iron reduction. J Biol Chem286, 4632–4643.

166.

Graham

, Chua

, Carter

, Delima

, Johnstone

, Herbison

, Firth

, O’Leary

, Milward

, Olynyk

, Trinder

. (2010) Hepatic iron loading in mice increases cholesterol biosynthesis. Hepatology52, 462–471.

167.

Kraml

, Klein

, Huang

, Nareika

, Lopes-Virella

. (2005) Iron loading increases cholesterol accumulation and macrophage scavenger receptor I expression in THP-1 mononuclear phagocytes. Metabolism54, 453–459.

168.

Francis

, Wong

, Hampson

, Wang

, Young

, Deigner

, Salmon

, Scheel-Toellner

, Lord

. (2011) Lactoferrin inhibits neutrophil apoptosis via blockade of proximal apoptotic signaling events. Biochim Biophys Acta1813, 1822–1826.

169.

Yurkova

, Kisel

, Arnhold

, Shadyro

. (2005) Iron-mediated free-radical formation of signaling lipids in a model system. Chem Phys Lipids137, 29–37.

170.

Yehuda

, Rabinovitz-Shenkar

, Carasso

. (2011) Effects of essential fatty acids in iron deficient and sleep-disturbed attention deficit hyperactivity disorder (ADHD) children. Eur J Clin Nutr65, 1167–1169.

171.

LeBlanc

, Fiset

, Surette

, Turgeon O’Brien

, Rioux

. (2009) Maternal iron deficiency alters essential fatty acid and eicosanoid metabolism and increases locomotion in adult guinea pig offspring. J Nutr139, 1653–1659.

172.

Brown

, Dennery

, Ridnour

, Fimmel

, Kladney

, Brunt

, Spitz

. (2003) Effect of iron overload and dietary fat on indices of oxidative stress and hepatic fibrogenesis in rats. Liver Int23, 232–242.

173.

Isaac

, Fredriksson

, Danielsson

, Eriksson

, Bergquist

. (2006) Brain lipid composition in postnatal iron-induced motor behor alterations following chronic neuroleptic administration in mice. FEBS J273, 2232–2243.

174.

Jenkins

, Kramer

. (1988) Effect of excess dietary iron on lipid composition of calf liver, heart, and skeletal muscle. J Dairy Sci71, 435–441.

175.

Mattson

. (2004) Metal-catalyzed disruption of membrane protein and lipid signaling in the pathogenesis of neurodegenerative disorders. Ann N Y Acad Sci1012, 37–50.

176.

Lassmann

, Bancher

, Breitschopf

, Wegiel

, Bobinski

, Jellinger

, Wisniewski

. (1995) Cell death in Alzheimer’s disease evaluated by DNA fragmentation in situ. Acta Neuropathol89, 35–41.

177.

Woodhouse

, Dickson

, West

, McLean

, Vickers

. (2006) No difference in expression of apoptosis-related proteins and apoptotic morphology in control, pathologically aged and Alzheimer’s disease cases. Neurobiol Dis22, 323–333.

178.

Higuchi

. (2003) Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochem Pharmacol66, 1527–1535.

179.

Stockwell

, Friedmann Angeli

, Bayir

, Bush

, Conrad

, Dixon

, Fulda

, Gascon

, Hatzios

, Kagan

, Noel

, Jiang

, Linkermann

, Murphy

, Overholtzer

, Oyagi

, Pagnussat

, Park

, Ran

, Rosenfeld

, Salnikow

, Tang

, Torti

, Toyokuni

, Woerpel

, Zhang

. (2017) Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease. Cell171, 273–285.

180.

Hambright

, Fonseca

, Chen

, Na

, Ran

. (2017) Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration. Redox Biol12, 8–17.

181.

Dixon

, Lemberg

, Lamprecht

, Skouta

, Zaitsev

, Gleason

, Patel

, Bauer

, Cantley

, Yang

, Morrison

, Stockwell

. (2012) Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell149, 1060–1072.

182.

Galluzzi

, Vitale

, Aaronson

, Abrams

, Adam

, Agostinis

, Alnemri

, Altucci

, Amelio

, Andrews

, Annicchiarico-Petruzzelli

, Antonov

, Arama

, Baehrecke

, Barlev

, Bazan

, Bernassola

, Bertrand

MJM

, Bianchi

, Blagosklonny

, Blomgren

, Borner

, Boya

, Brenner

, Campanella

, Candi

, Carmona-Gutierrez

, Cecconi

, Chan

, Chandel

, Cheng

, Chipuk

, Cidlowski

, Ciechanover

, Cohen

, Conrad

, Cubillos-Ruiz

, Czabotar

, D’Angiolella

, Dawson

, De Laurenzi

, De Maria

, Debatin

, DeBerardinis

, Deshmukh

, Di Daniele

, Di Virgilio

, Dixit

, Dixon

, Duckett

, Dynlacht

, El-Deiry

, Elrod

, Fimia

, Fulda

, Garcia-Saez

, Garg

, Garrido

, Gavathiotis

, Golstein

, Gottlieb

, Green

, Greene

, Gronemeyer

, Gross

, Hajnoczky

, Hardwick

, Harris

, Hengartner

, Hetz

, Ichijo

, Jaattela

, Joseph

, Jost

, Juin

, Kaiser

, Karin

, Kaufmann

, Kepp

, Kimchi

, Kitsis

, Klionsky

, Knight

, Kumar

, Lee

, Lemasters

, Levine

, Linkermann

, Lipton

, Lockshin

, Lopez-Otin

, Lowe

, Luedde

, Lugli

, MacFarlane

, Madeo

, Malewicz

, Malorni

, Manic

, Marine

, Martin

, Martinou

, Medema

, Mehlen

, Meier

, Melino

, Miao

, Molkentin

, Moll

, Munoz-Pinedo

, Nagata

, Nunez

, Oberst

, Oren

, Overholtzer

, Pagano

, Panaretakis

, Pasparakis

, Penninger

, Pereira

, Pervaiz

, Peter

, Piacentini

, Pinton

, Prehn

JHM

, Puthalakath

, Rabinovich

, Rehm

, Rizzuto

, Rodrigues

CMP

, Rubinsztein

, Rudel

, Ryan

, Sayan

, Scorrano

, Shao

, Shi

, Silke

, Simon

, Sistigu

, Stockwell

, Strasser

, Szabadkai

, Tait

SWG

, Tang

, Tavernarakis

, Thorburn

, Tsujimoto

, Turk

, Vanden Berghe

, Vandenabeele

, Vander Heiden

, Villunger

, Virgin

, Vousden

, Vucic

, Wagner

, Walczak

, Wallach

, Wang

, Wells

, Wood

, Yuan

, Zakeri

, Zhivotovsky

, Zitvogel

, Melino

, Kroemer

. (2018) Molecular mechanisms of cell death: Recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ25, 486–541.

183.

Cao

, Dixon

. (2016) Mechanisms of ferroptosis. Cell Mol Life Sci73, 2195–2209.

184.

Xie

, Hou

, Song

, Yu

, Huang

, Sun

, Kang

, Tang

. (2016) Ferroptosis: Process and function. Cell Death Differ23, 369–379.

185.

Kang

, Tang

. (2017) Autophagy and ferroptosis - what’s the connection?. Curr Pathobiol Rep5, 153–159.

186.

Gao

, Monian

, Pan

, Zhang

, Xiang

, Jiang

. (2016) Ferroptosis is an autophagic cell death process. Cell Res26, 1021–1032.

187.

Neitemeier

, Jelinek

, Laino

, Hoffmann

, Eisenbach

, Eying

, Ganjam

, Dolga

, Oppermann

, Culmsee

. (2017) BID links ferroptosis to mitochondrial cell death pathways. Redox Biol12, 558–570.

188.

Jelinek

, Heyder

, Daude

, Plessner

, Krippner

, Grosse

, Diederich

, Culmsee

. (2018) Mitochondrial rescue prevents glutathione peroxidase-dependent ferroptosis. Free Radic Biol Med117, 45–57.

189.

Shah

, Margison

, Pratt

. (2017) The potency of diarylamine radical-trapping antioxidants as inhibitors of ferroptosis underscores the role of autoxidation in the mechanism of cell death. ACS Chem Biol12, 2538–2545.

190.

Zilka

, Shah

, Li

, Friedmann Angeli

, Griesser

, Conrad

, Pratt

. (2017) On the mechanism of cytoprotection by ferrostatin-1 and liproxstatin-1 and the role of lipid peroxidation in ferroptotic cell death. ACS Cent Sci3, 232–243.

191.

Yang

, Kim

, Gaschler

, Patel

, Shchepinov

, Stockwell

. (2016) Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A113, 4966–4975.

192.

Kagan

, Mao

, Qu

, Angeli

, Doll

, Croix

, Dar

, Liu

, Tyurin

, Ritov

, Kapralov

, Amoscato

, Jiang

, Anthonymuthu

, Mohammadyani

, Yang

, Proneth

, Klein-Seetharaman

, Watkins

, Bahar

, Greenberger

, Mallampalli

, Stockwell

, Tyurina

, Conrad

, Bayir

. (2017) Oxidized arachidonic and adrenic PEs ngate cells to ferroptosis. Nat Chem Biol13, 81–90.

193.

Doll

, Proneth

, Tyurina

, Panzilius

, Kobayashi

, Ingold

, Irmler

, Beckers

, Aichler

, Walch

, Prokisch

, Trumbach

, Mao

, Qu

, Bayir

, Fullekrug

, Scheel

, Wurst

, Schick

, Kagan

, Angeli

, Conrad

. (2017) ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat Chem Biol13, 91–98.

194.

Epperly

, Dunay

, Boice

. (2017) Alzheimer disease: Pharmacologic and nonpharmacologic therapies for cognitive and functional symptoms. Am Fam Physician95, 771–778.

195.

Gugliandolo

, Bramanti

, Mazzon

. (2017) Role of vitamin E in the treatment of Alzheimer’s disease: Evidence from animal models. Int J Mol Sci18, 2504.

196.

Kryscio

, Abner

, Caban-Holt

, Lovell

, Goodman

, Darke

, Yee

, Crowley

, Schmitt

. (2017) Association of antioxidant supplement use and dementia in the Prevention of Alzheimer’s Disease by Vitamin E and Selenium Trial (PREADViSE). JAMA Neurol74, 567–573.

197.

Cervantes

, Ulatowski

. (2017) Vitamin E and Alzheimer’s disease-is it time for personalized medicine?Antioxidants (Basel)6, 45.

198.

Jiang

, Kon

, Li

, Wang

, Su

, Hibshoosh

, Baer

, Gu

. (2015) Ferroptosis as a p53-mediated activity during tumour suppression. Nature520, 57–62.

199.

, Wang

, Li

, Chu

, Gu

. (2016) Activation of SAT1 engages polyamine metabolism with p53-mediated ferroptotic responses. Proc Natl Acad Sci U S A113, E6806–e6812.

200.

Lane

DJR

, Bae

D-H

, Siafakas

, Rahmanto

, Al-Akra

, Jansson

, Casero

Jr , Richardson

(2018) Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes. BBA - Mol Basis Dis [Epub ahead of print] doi: 10.1016/j.bbadis.2018.05.007

201.

Fricker

, Tolkovsky

, Borutaite

, Coleman

, Brown

. (2018) Neuronal cell death. Physiol Rev98, 813–880.

202.

Guiney

, Adlard

, Bush

, Finkelstein

, Ayton

. (2017) Ferroptosis and cell death mechanisms in Parkinson’s disease. Neurochem Int104, 34–48.

203.

Tuo

, Lei

, Jackman

, Li

, Xiong

, Li

, Liuyang

, Roisman

, Zhang

, Ayton

, Wang

, Crouch

, Ganio

, Wang

, Pei

, Adlard

, Lu

, Cappai

, Wang

, Liu

, Bush

. (2017) Tau-mediated iron export prevents ferroptotic damage after ischemic stroke. Mol Psychiatry22, 1520–1530.

204.

Chen

, Hambright

, Na

, Ran

. (2015) Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis. J Biol Chem290, 28097–28106.

205.

Kirouac

, Rajic

, Cribbs

, Padmanabhan

. (2017) Activation of Ras-ERK signaling and GSK-3 by amyloid precursor protein and amyloid beta facilitates neurodegeneration in Alzheimer’s disease. eNeuro4, ENEURO.0149162017.

206.

Yagoda

, von Rechenberg

, Zaganjor

, Bauer

, Yang

, Fridman

, Wolpaw

, Smukste

, Peltier

, Boniface

, Smith

, Lessnick

, Sahasrabudhe

, Stockwell

. (2007) RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature447, 864–868.

207.

Yang

, Stockwell

. (2008) Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol15, 234–245.