What Is the Role of Ferroptosis in Neurodegeneration?

Ferroptosis is a unique form of programmed cell death pathway that is mechanistically different from necrosis, apoptosis, or autophagy.¹ Ferroptosis is induced by reactive oxygen species (ROS) originating from multiple sources, especially accumulation of free intracellular divalent iron (Fe²⁺), leading to peroxidation of polyunsaturated fatty acids (PUFAs) and resulting in membrane damage (Figure). One major trigger of ferroptosis is decreased availability of intracellular antioxidant enzymes, particularly glutathione peroxidases (GPXs).^1-7 Because of its high lipid concentration and oxygen consumption rate, the nervous tissue is extremely vulnerable to oxidative damage and ferroptosis. With aging, iron accumulates in the brain, which predisposes to and exacerbates these processes.⁸ Iron promotes ferroptosis both directly and as a cofactor required for the activity of enzymes mediating redox reactions and lipid peroxidation.^9,10 Epigenetic regulation can determine cell sensitivity to ferroptosis by affecting intracellular iron levels, oxidative stress, and lipid metabolism.¹¹ Ferroptosis affects all cell types in the nervous system, including neurons, glial cells, and pericytes.¹² Microglia are the primary iron-accumulating cells in disease.^13,14 Recent evidence indicates that iron-laden microglia have a critical role in ferroptosis associated with neurodegeneration.¹⁵ Studies in vitro, in experimental disease models, and in human brain tissue indicate that iron accumulation, oxidative stress, and lipid peroxidation are major disease mechanisms in a wide range of neurologic disorders. These studies also suggest that the different components of the ferroptosis pathway are potential targets for therapeutic intervention. The mechanisms of ferroptosis and its role in neurologic disorders have been the subject of several recent comprehensive reviews.^7,8,16-21 Some of these concepts pertinent to neurodegeneration will be emphasized in this study.

Figure. Iron Metabolisms and Mechanisms of Ferroptosis.

A highly complex network including various transporters, storage, and exporter proteins control iron levels in cells. Ferric iron (Fe³⁺) present in biological fluids binds to transferrin, which is then recognized by the transferrin receptor 1 (TfR1) in cell membranes. This complex transports Fe³⁺ into the endosome where it is converted to ferrous iron (Fe²⁺) by the metalloreductases STEAP2. Ferrous iron is then released through the divalent metal transporter 1 (DMT1) into the labile Fe²⁺ iron pool in the cytoplasm, exported to brain interstitial fluid through ferroportin after oxidation to Fe³⁺ by ceruloplasmin, and then loaded onto transferrin. Ferroportin level is controlled by its interaction with hepcidin. When available iron exceeds the biological needs, it is sequestered by the iron storage protein ferritin. Dynamic changes in the Fe³⁺/Fe²⁺ redox potential determine the sensitivity of cells to ferroptosis. The reaction of Fe²⁺ and hydrogen peroxide (H₂O₂) through the Fenton reaction leads to the formation of hydroxyl radicals (OH⁻), which elicit lipid peroxidation required for the activity of metabolic enzymes used in the redox reaction. The reactive oxygen species triggering ferroptosis may also originate from the mitochondrial respiratory chain through the production of superoxide (O₂^*), which is then converted to H₂O₂ by superoxide dismutases (SODs). The membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) also provides reactive oxygen species. The primary substrates for lipid peroxidation in the process of ferroptosis are polyunsaturated fatty acids (PUFAs) that are incorporated into phospholipids such as phosphatidylethanolamine (PE-PUFA) by sequential action of acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3). Lipid peroxidation occurs not only through the iron-dependent Fenton reaction but also by enzymatic pathways, primarily mediated by lipoxygenases (LOXs), which use iron as a cofactor. Peroxidation of membrane lipids (PE-PUFA-OOH) affects transport along cell membranes, mitochondrial bioenergetics, vesicular traffic, and the machinery for autophagy and mitophagy. This promotes the accumulation of lipofuscin and iron primarily in lysosomes. Ferroptosis is also associated with membrane rupture and release of damage-associated molecular patterns that trigger inflammation. Toxic metabolites resulting from lipid peroxidation, such as 4-hydroxynonenal or malondialdehyde, are also cytotoxic molecules that form adducts with DNA bases, proteins, and other nucleophilic molecules. The primary protective mechanism against ferroptosis is the glutathione (GSH)–glutathione peroxidase 4 (GPx4) system. The availability of GSH in cells depends on the activity of the cystine (Cys)/glutamate (Glu) antiporter (system Xc−), which transports Cys into the cell, where it is reduced to cysteine (CysH) and then combined with glutamate and glycine (Gly) to produce GSH by the action of glutathione synthetase (GS). GPx4 converts GSH to oxidized glutathione (GSSG) and while reducing lipid peroxides to lipid alcohols. The reconstitution of GSH from GSSG is mediated by glutathione reductase (GR). In the plasma membrane, coenzyme Q10 (CoQ10) blocks lipid peroxidation and ferroptosis through an NADPH-mediated action mediated by the oxidoreductase ferroptosis inhibitory protein 1 (FSP1). Nuclear factor erythroid 2–related factor 2 (NRF2) improves cellular tolerance to oxidative stress, lipid peroxidation, and ferroptosis by promoting transcription of proteins related to iron transport and metabolism, NADPH, and GSH metabolism. The calcium-independent phospholipase A2 type G6 (iPLA2G6) degrades hydroxy peroxy-phosphatidylethanolamine also preventing ferroptosis.

Mechanisms of Ferroptosis

Overview of Iron Metabolism

A major trigger of ferroptosis is abnormal intracellular iron accumulation. Iron participates in many fundamental functions in the CNS,^16,22 and its metabolism is a dynamic process involving multiple steps (Figure).^16,23,24 A highly complex network including various transporters, storage, and exporter proteins control iron levels in cells. Ferric iron (Fe³⁺) present in biological fluids binds to transferrin, which is then recognized by the transferrin receptor 1 (TfR1) in cell membranes including those forming the blood-brain barrier (BBB); this complex transports Fe³⁺ into the endosome.²⁵ In the acid milieu of endosomes, Fe³⁺ undergoes reduction to Fe²⁺ by metalloreductases, and Fe²⁺ is then released through the divalent metal transporter 1 (DMT1) into the labile Fe²⁺ iron pool in the cytoplasm.²⁶ Trafficking from the endoplasmic reticulum to the Golgi apparatus may also contribute to the labile iron pool.²⁷ Ferrous iron is oxidized to Fe³⁺ by ceruloplasmin, exported to the brain interstitial fluid through ferroportin, and then loaded onto transferrin.^24,28 Ferric iron bound to transferrin is taken up by neurons and glial cells through TfR1-mediated endocytosis. Ferroportin is a major regulator of intracellular iron levels; in the presence of iron, ferroportin binds to hepcidin, forming a complex that is internalized and degraded in lysosomes.²⁹ When available iron exceeds the biological needs, it is sequestered by the iron-storage protein ferritin. Ferritin is the main iron buffering and storage protein and is found in glial cells but generally not in neurons.³⁰ Catecholaminergic neurons can buffer free iron accumulation through the formation of neuromelanin from oxidized dopamine or norepinephrine metabolites.³¹ In response to change in cellular levels, iron homeostasis is regulated mainly posttranscriptionally by iron regulatory proteins; these proteins bind to specific messenger RNA (mRNA) sequences called iron-responsive elements in untranslated regions of mRNAs encoding TfR1, DMT1, ferritin, and other iron export, storage, and import proteins.³²

Role of Iron and Oxidative Stress in Ferroptosis

Dynamic changes in the Fe³⁺/Fe²⁺ redox potential mediate several fundamental cell processes such as mitochondrial respiration and have a major role in determining the sensitivity of cells to ferroptosis.¹⁶ The reaction of Fe²⁺ and hydrogen peroxide through the Fenton reaction leads to the formation of hydroxides and hydroxyl radicals, which elicits lipid peroxidation.^23,33 Iron is also a cofactor required for the activity of metabolic enzymes used in redox reactions and lipid peroxidation, such as lipoxygenase (LOX).^9,10 In addition to Fenton reaction, the ROS triggering ferroptosis may originate from the mitochondria, which are regulatory hubs for cell death.³⁴ Mitochondrial ROS production by the electron transport chain, fatty acid synthesis, and DNA stress all promote lipid peroxidation.^35,36 Mitochondrial DNA stress also triggers autophagic-dependent ferroptotic cell death.^35,37 Selective forms of autophagy, such as ferritinophagy and lipophagy, drive cells toward ferroptosis.³⁸ For example, ROS-induced autophagy promotes the degradation of ferritin and induction of TfR1, resulting in intracellular iron accumulation.³⁹ Mitochondrial energy sensors such as adenosine monophosphate–activated protein kinase may bidirectionally regulate ferroptosis by affecting lipid synthesis⁴⁰ and autophagy.⁴¹ Another source of ROS for ferroptosis is the activity of membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX),⁵ which is primarily expressed in microglia.^42,43 NOX family members, like other enzymes involved in ferroptosis, require iron for their normal function.⁴⁴

Mechanisms and Consequences of Lipid Peroxidation

The primary substrates for lipid peroxidation are PUFAs, such as arachidonic acid and adrenic acid.^10,45 These PUFAs are incorporated into cellular phospholipids, especially phosphatidylethanolamine, by sequential action of acyl-CoA synthetase long-chain family member 4 and lysophosphatidylcholine acyltransferase 3.^10,45-47 Other sources of lipid substrates for peroxidation include polyunsaturated ether phospholipids derived from peroxisomes⁴⁸ and fatty acids produced by lipophagy.⁴⁹ Lipid peroxidation occurs not only through hydroxyl radicals generated during the iron-dependent Fenton reaction³³ but also through enzymatic pathways primarily mediated by LOXs, which use iron as a cofactor.⁵⁰ Cytochrome P450 oxidoreductase transfers electrons from NADPH to the mitochondria, which also promotes lipid peroxidation.⁵¹

Peroxidation of membrane lipids results in increased membrane rigidity and affects membrane transport, mitochondrial bioenergetics, vesicular traffic, and the machinery for autophagy and mitophagy.^52-54 These processes interact with each other to promote ferroptosis. Impaired mitophagy promotes the accumulation of ROS and damaged proteins and lipids in mitochondria; release of iron by mitochondria into autolysosomes triggers further Fenton reactions, lipid peroxidation, and impaired autophagy.^53,54 This leads to the accumulation of lipofuscin and iron which, in turn, increase oxidative stress and lipid peroxidation.⁵⁵ Ferroptosis is a type of lytic cell death associated with membrane rupture and release of damage-associated molecular patterns that trigger inflammation.^38,56 Cathepsin B, a lysosomal cysteine protease, is one of the mediators of ferroptosis, causing DNA damage after translocation from lysosomes to the nucleus.^57,58 Toxic metabolites resulting from lipid peroxidation, such as 4-hydroxynonenal or malondialdehyde, are also cytotoxic molecules that form adducts with DNA bases, proteins, and other nucleophilic molecules, including proteins involved in autophagosome and autolysosome formation, thereby further decreasing autophagic flux.^45,59

Defense Mechanisms Against Ferroptosis

The primary mechanism that protects cells from ferroptosis is the glutathione (GSH)-GPX4 system (Figure).^1,2,7,17 In the brain, GPX4 is expressed in neurons and glial cells and has a free radical–scavenging and antioxidant function.⁶⁰ The availability of GSH in cells depends on the activity of the cystine/glutamate antiporter (system Xc-), which transports cystine into the cell; cystine is reduced to cysteine and then combined with glutamate and glycine to produce GSH by the action of glutathione synthetase.⁶¹ Because neurons lack this cystine transport system, they primarily depend on GSH synthesis and release from surrounding astrocytes.⁶² Glutathione peroxidase 4 converts GSH to oxidized glutathione (GSSG) while reducing lipid peroxides to lipid alcohols.⁶³ The reconstitution of GSH from GSSG is mediated by glutathione reductase using NADPH. In addition to the GSH-GPX4 system, there are other mechanisms that protect against oxidative stress and ferroptosis. In the plasma membrane, coenzyme Q10 (CoQ10) functions as a lipophilic free radical–trapping antioxidant that blocks lipid peroxidation through an NADPH-mediated action mediated by the oxidoreductase ferroptosis inhibitory protein 1.^5,64 The mevalonate pathway provides substrates for the synthesis of both GPX4 and CoQ10.⁶⁵ Other ferroptosis protection mechanisms include the mitochondrial dihydroorotate dehydrogenase–dihydroubiquinone system⁶⁶ and the guanosine triphosphate cyclohydrolase–1–tetrahydrobiopterin pathway.^67,68 The relative contribution of each antioxidant mechanism may vary in different cells and tissues.⁶⁹

Nuclear factor erythroid 2–related factor 2 (NRF2) is a transcription factor that has a major role in promoting cellular tolerance to oxidative stress, lipid peroxidation, and ferroptosis.^70-73 NRF2 is expressed mainly in astrocytes and promotes the transcription of proteins related to iron transport and metabolism, such as ferroportin 1 and ferritin light and heavy chains; enzymes related to NADPH regeneration such as glucose-6-phosphate dehydrogenase; and proteins and enzymes related to GSH metabolism such as subunits of the cystine/glutamate transporter protein and glutathione synthetase.^62,70,71 Other mechanisms can prevent ferroptosis by affecting lipid composition and repair of cell membranes. For example, the calcium-independent phospholipase A2 type G6 (PLA2G6) degrades membrane hydroxyperoxy-phosphatidylethanolamine.⁷⁴ The membrane scission machinery that depends on endosomal sorting complex required for transport-III can repair the cell membrane.⁷⁵

Role of Ferroptosis in Neurologic Disease

Biochemical and transcriptomic analyses of mediators of iron metabolism, antioxidant defenses, oxidative stress, and lipid peroxidation in experimental disease models, autopsy material, and induced pluripotent stem cell (iPSCs) derived from patients indicate that ferroptosis is a major mechanism of cell death in a wide range of neurologic disorders. These include Alzheimer disease (AD),^64,72,76-82 Parkinson disease (PD),^55,83-87 amyotrophic lateral sclerosis (ALS),^88-92 Huntington disease,^93-95 Friedreich ataxia,^96-98 multiple sclerosis,^99-101 ischemic stroke,^47,102-105 intracerebral hemorrhage,^106,107 traumatic brain injury,¹⁰⁸ spinal cord injury,^109,110 epilepsy,^111-113 and glioma,¹¹⁴ among others. This topic has been extensively reviewed,^{16-21,115,116} and only few concepts will be emphasized in this study.

Cerebral iron deposition is a typical finding associated with aging and neurodegeneration.^8,117 Studies in experimental models and human autopsy material indicate that several interacting mechanisms may result in iron accumulation and oxidative stress in both neurons and glial cells in vulnerable areas. Mitochondrial dysfunction with excessive ROS production, autophagy disruption, lipofuscin accumulation, and lipid peroxidation are common pathomechanisms in these disorders.^{34,55,115,116,118,119} Indirect evidence of ferroptosis include abnormal levels of iron import, storage, and export proteins indicating iron dyshomeostasis, reduced expression of GSH and other antioxidants, upregulation of the NRF2, and accumulation of products from lipid peroxidation, such as malonyldialdehyde and 4-hydroxynonenal.^16-21

A set of iron-responsive elements in 5′untranslated regions of mRNA can bind excessive iron and fold mRNA into loops that affect transcription of proteins such as amyloid precursor protein (APP), α-synuclein, and prion protein; many of these proteins interact with iron, which may initiate a potential feedback to accelerate ferroptosis.¹²⁰ Excessive iron accumulation correlates with accelerated cognitive decline in AD.¹²¹ In this disorder, iron accumulates in β-amyloid (Aβ) aggregates and neurofibrillary tangles^77,122,123 and promotes the aggregation of both toxic Aβ oligomers and hyperphosphorylated tau.¹²⁴ Consistent with the role of ferroptosis in AD, studies in human autopsy material and experimental models show decreased GSH levels, GPX4 downregulation, and accumulation of malonyldialdehyde in brain tissue.¹²² Ferroptosis may be a prominent cell death mechanism in PD. Both oxidative stress due to impaired mitochondrial complex I and decreased CoQ, GSH, and GPX4 levels and iron dyshomeostasis, reflected by increased TfR1 and DMT1 and decreased ceruloplasmin and ferritin expression, promote abnormal iron accumulation and ferroptosis in both dopaminergic neurons and glial cells.^28,83,125 α-Synuclein can form complexes with iron, act as a ferrireductase, and increase DMT1-mediated iron uptake in the cytosol.¹²⁶ In human dopaminergic neuron models, endogenous levels of α-synuclein can affect the availability of ether-linked phospholipids required for ferroptosis.^127,128 Aggregated α-synuclein also induces the production of ROS and formation of lipid peroxidation products, which in turn, promote α-synuclein aggregation, thus perpetuating the pathogenic cycle.^129,130 Misfolded α-synuclein also activates microglia, which release interleukin-6 that induces changes in the neuronal transcriptome promoting iron uptake and decreases iron export.¹³¹

Glial cells also have a major role in the mechanisms of ferroptosis. Astrocytes express TfR1, store iron through DMT1, and release iron through ferroportin, thereby regulating iron distribution in neural tissues in both normal and pathologic conditions.^132,133 Production of GSH by astrocytes may also have neuroprotective effects both by attenuating neuroinflammation¹³⁴ and maintaining BBB stability.¹³⁵ Recent evidence indicates that microglia are especially vulnerable to ferroptosis and iron accumulation in microglia has deleterious effects of neuronal viability.^14,15 Single-cell RNA sequencing studies in both transgenic models and microglia derived from human iPSCs revealed that iron-laded microglia express markers of ferroptosis activation, upregulation of oxidative stress pathways, dampening of transcriptional activation of proinflammatory and anti-inflammatory mediators, and impaired phagocytosis.^14,15 This iron-induced transcriptomic signature is similar to that found in microglia from postmortem tissue from patients with PD, AD, ALS, and MS.^15,136,137 In transgenic mice that overexpress APP and presenilin 1 (PS1; APP/PS1 mice), stimulation of cultured microglia with interferon gamma and Aβ induced these cells to retain iron and have reduced phagocytic and chemotactic functions.¹³⁸ A study using human iPSC–derived triculture containing microglia, astrocytes, and neurons showed that exposure to iron and a GPX4 inhibitor resulted in marked microglial cell death followed by neuronal cell death.¹⁵ Omission of microglia from the culture markedly reduced neuronal death.¹⁵ These studies thus show that iron accumulation in microglia induces oxidative stress, dampens inflammatory activation, and triggers neurodegeneration.^14,15 Iron also accumulates in the choroid plexus and may contribute to the damage of the BBB and blood-CSF barrier.¹³⁹

Some forms of neurodegeneration with brain iron accumulation (NBIA), such as hereditary ferritinopathy and aceruloplasminemia, are caused by pathogenic variants in genes coding for proteins directly involved in the iron regulatory pathway.¹¹⁷ However, the relationship between iron accumulation and neurodegeneration in these disorders is incompletely understood. For example, in neuroferritinopathy, abnormal ferritin light chain aggregates lead to impaired proteostasis while derangement of iron metabolism may occur as a secondary event. Other disorders in the NBIA group are caused by variants in genes not directly related to iron metabolism. Many of these disorders share features such as mitochondrial dysfunction, oxidative stress, altered phospholipid metabolism, and neuroinflammation.^140-142 Of potential relevance is NBIA caused by pathogenic variants of the PLA2G6 gene, causing PLA2G6-associated neurodegeneration.^{55,74,140,141,143,144} As mentioned earlier in this review, PLA2G6 degrades membrane hydroxyperoxy-phosphatidylethanolamine, thus protecting against ferroptosis.⁷⁴ In cellular models, reduced PLA2G6 function alters vesicular transport (including retromer formation), impairs mitophagy and autophagy, and leads to lysosomal dysfunction and accumulation of lipofuscin.^145,146 Lipofuscin recruits iron, promoting the formation of free radicals and thus lipid peroxidation in lysosomes.^147,148

Perspective

The evidence that intracellular free iron accumulation, oxidative stress, and lipid peroxidation resulting in ferroptosis have a major role in neurodegeneration has led to multiple approaches to interrupt these processes. Studies in experimental models suggest that iron chelators such as deferoxamine and deferiprone; antioxidants and inhibitors of lipid peroxidation such as α-tocopherol, CoQ10, tetrahydrobiopterin, or edaravone; GPX4 inducers such as ferrostatin-1 and lipostatin-1; GSH precursors such as N-acetylcysteine; or activators of NRF2 signaling such as dimethyl fumarate could potentially have a neuroprotective effect.^19,149 However, the role of iron chelation as a neuroprotective approach in human neurologic disorders is yet to be established. For example, a recent multicenter phase 2 randomized double-blind trial in patients with newly diagnosed PD showed that patients treated with oral deferiprone over the course of 36 weeks had worse scores in measures of parkinsonism despite a higher reduction of iron in the substantia nigra compared with those receiving placebo.¹⁵⁰ Whereas iron chelation has also been suggested as a potential neuroprotective approach for AD,¹⁵¹ it has proven unsuccessful in patients with ALS.¹⁵² In a placebo-controlled double-blind multicenter trial in patients with pantothenate kinase–associated neurodegeneration, deferiprone reduced iron load in the basal ganglia and showed a trend to slowing of disease progression but did not produce any significant change in symptoms.¹⁵³ Similarly, despite evidence from preclinical models, strong clinical evidence is still lacking for the benefit of antioxidant therapy as a neuroprotective approach, although edaravone may provide potential benefits in patients with ALS.¹⁵⁴ Given the complex interactions among iron metabolism, oxidative stress, organelle dysfunction, and neuroinflammation at different disease stages, it is unlikely that targeting an individual component of the ferroptosis pathway will provide substantial clinical benefit. Novel therapies, such as epigenetic approaches¹¹ or multitargeting drugs with iron-chelating, antioxidant, antiferroptotic, and anti-inflammatory properties may provide potential alternatives.

Glossary

Aβ

β-amyloid

AD

Alzheimer disease

ALS

amyotrophic lateral sclerosis

APP

amyloid precursor protein

BBB

blood-brain barrier

CoQ10

coenzyme Q10

DMT1

divalent metal transporter 1

Fe²⁺

iron

Fe³⁺

ferric iron

GPX

glutathione peroxidase

GSH

glutathione

GSSG

oxidized glutathione

iPSC

induced pluripotent stem cell

LOX

lipoxygenase

mRNA

messenger RNA

NADPH

nicotinamide adenine dinucleotide phosphate

NBIA

neurodegeneration with brain iron accumulation

NOX

nicotinamide oxidase

NRF2

nuclear factor erythroid 2–related factor 2

PD

Parkinson disease

PLA2G6

phospholipase A2 type G6

PS1

presenilin 1

PUFA

polyunsaturated fatty acid

ROS

reactive oxygen species

TfR1

transferrin receptor 1

Study Funding

No targeted funding reported.

Disclosure

The authors report no relevant disclosures. Go to Neurology.org/N for full disclosures.