Ferroptosis Mechanisms Involved in Neurodegenerative Diseases

Abstract

Ferroptosis is a type of cell death that was described less than a decade ago. It is caused by the excess of free intracellular iron that leads to lipid (hydro) peroxidation. Iron is essential as a redox metal in several physiological functions. The brain is one of the organs known to be affected by iron homeostatic balance disruption. Since the 1960s, increased concentration of iron in the central nervous system has been associated with oxidative stress, oxidation of proteins and lipids, and cell death. Here, we review the main mechanisms involved in the process of ferroptosis such as lipid peroxidation, glutathione peroxidase 4 enzyme activity, and iron metabolism. Moreover, the association of ferroptosis with the pathophysiology of some neurodegenerative diseases, namely Alzheimer's, Parkinson's, and Huntington's diseases, has also been addressed.

Keywords: Alzheimer’s disease; GSH; Huntington’s disease; Parkinson’s disease; cell death; ferroptosis; glutathione peroxidase 4; iron metabolism; neurodegenerative diseases; system xc−.

Figure 1

Ferroptosis pathway. Ferroptosis can be initiated through transferrin endocytosis linked to transferrin receptor 1 (TFR1). After endocytosis, ferric iron is released from the Transferrin–TRF1 complex and is reduced to ferrous iron (Fe²⁺). Fe²⁺ can be stored in ferritin or remain in the cytoplasm as a labile iron Pool (LIP). The LIP is composed mainly of Fe²⁺, which through Fenton reaction generates species such as: the hydroxyl radical that reacts with membrane lipids, providing the lipid peroxidation of arachidonic acid (AA) or adrenic acid (AdA). Lipid peroxidation can also occur via enzyme. However, it is necessary for the free polyunsaturated fatty acids (PUFAs) to be esterified as membrane PUFA by the enzymes ACSL4 and LPCAT3, forming arachidonic or adrenic acids esterified in phosphatidyl ethanolamine (PE-AA/PE-AdA). Dioxigenation by 15-LOX generates PE-AA/AdA-OOH, which reacts with other membrane lipids, forming pores in the lipid bilayer, destabilizing it and then rupturing the membrane. Ferroptosis is inhibited by GPx4, which converts PE-AA/AdA-OOH to alcohol and water. This reaction occurs through the use of glutathione (GSH) as a substrate. GSH synthesis occurs via the entry of cystine into the cell by system xc⁻.

Figure 2

Glutathione (GSH) biosynthesis pathway. GSH is known as one of the small-molecule water-soluble antioxidants, the most important of somatic cells. GSH is a linear tripeptide formed by three amino acids: glutamic acid, cysteine and glycine. The thiol group present in the amino acid cysteine is considered the active site responsible for the antioxidant biochemical properties of glutathione. In biological systems, glutathione can be found in reduced form (GSH) or in oxidized form (GSSG). The oxidized form is a heterodimerization of the reduced form. The GSH/GSSG ratio is used to estimate the redox state of biological systems [51]. The rate-limiting compound of GSH synthesis is the non-essential amino acid cysteine. Cysteine can be imported into cells directly or in its oxidized form, cystine, through the cystine/glutamate antiporter system xc⁻. In humans, on chromosome 4, the SLC7A11 gene (solute carrier family 7A11) encodes the SLCA11 antiporter, which is part of a system called system xc⁻. The structure of this protein is heterodimeric and includes two chains: a specific light chain, xCT (SLCA11), and a heavy chain, 4F2hc (SLC3A2), which are linked by a disulfide bridge. The xCT chain has 12 transmembrane domains consisting of 501 amino acids, with the N and C terminal regions located intracellularly; it is not glycosylated and has a molecular mass of approximately 55 kDa. The heavy chain, 4F2hc, is a type II glycoprotein with a single transmembrane domain, an intracellular NH 2 terminal and a molecular weight of approximately 85 kDa. The 4F2hc chain is a subunit common to amino acid transport systems, while the xCT chain is unique to cystine/glutamate exchange. System xc⁻ transports amino acids, independently of sodium and dependent on chloride, which are specific to import cystine and export glutamate at the same time through the plasma membrane. Both amino acids are transported in anionic form. The ratio of counter transport between cystine and glutamate is 1:1. Currently, it is known that system xc⁻ is involved in (a) cystine uptake to maintain the extracellular balance of cysteine/redox cystine, (b) cysteine/cystine uptake for GSH synthesis and (c) non-vesicular glutamate export [68]. Within the cell, cystine is reduced to cysteine. This reduction reaction can be performed by intracellular GSH or by the enzyme thioredoxin reductase 1 (TRR1) [69]. The beginning of GSH synthesis is the formation of the γ-glutamylcysteine molecule, which is catalyzed by the enzyme glutamate cysteine ligase (GCL). GCL catalyzes the binding of glutamate and cysteine in the presence of adenosine triphosphate (ATP). Then, the enzyme GSH synthase (GS) catalyzes the formation of GSH through the link between γ-glutamylcysteine and glycine [69]. GSH reduces radicals (R•) non-enzymatically and organic hydroperoxides catalyzed by GSH peroxidase (GPx) and is thus converted to GSH disulfide (GSSG). GSSG is recycled to GSH by GSH reductase (GR), a reaction that uses reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor [69]. GSH S-transferase (GST) forms GSH (GS-R) adducts from organic molecules (R) and GSH, which together with GSH and GSSG are exported from the cell by ABC transporters, mainly ABC-1 and ABC-G2 [70,71]. Extracellular GSH is metabolized by the γ-glutamyl transferase (GGT) ectoenzyme, which transfers the γ-glutamyl residue to different acceptor amino acids, leading to the formation of a dipeptide containing γ-glutamyl and the cysteine glycine dipeptide, which is cleaved by extracellular dipeptides to generate cysteine and glycine that can be taken up by cells, starting the glutathione biosynthesis cycle [69].

Figure 3

Human Iron metabolism. Iron concentration in the body is maintained through diet and the recycling of senescent erythrocytes. The daily diet provides approximately 1–2 mg of iron. Enterocytes, present in the duodenum and in the proximal portion of the jejunum, can absorb both ferrous iron (heme iron) and ferric iron (non-heme). However, it is necessary to reduce ferric iron to ferrous iron, by apical ferric reductase enzymes, such as enzyme duodenal cytochrome b (Dcytb), for absorption to occur. Then, iron is transported by the divalent metal type transporter-1 (DMT-1) and stored inside the cell [86]. Ferrous iron from the diet is internalized by the heme-1 carrier protein (HCP) in cells, where it is stored as hemosiderin and/or ferritin, to prevent the Fenton reaction. Physiologically, iron stores are mobilized from intracellular to the extracellular by ferroportin (FPN) when the serum iron is low. Iron released in its ferrous state is oxidized to ferric iron and binds to serum apotransferrin to be transported through the body, giving rise to holotransferrin. This oxidation reaction occurs through the action of oxidase enzymes: hephestine present in enterocytes, ceruloplasmin present in hepatocytes macrophages. The distribution of iron to the tissues occurs through the endocytosis of holotransferrin, mediated by the binding to transferrin receptors type 1 (TFR1) and type 2 (TFR2). Ferroportin mediates the efflux of iron within cells, maintaining systemic iron homeostasis. This process is negatively regulated by hepcidin, which promotes ferroportin endocytosis and then proteolysis in lysosomes by induced ubiquitination [83]. The recycling of iron by macrophages occurs through the phagocytosis of senescent erythrocytes and hemoglobin and the heme group of intravascular hemolysis. Once internalized in the macrophage, the heme group releases ferrous iron through the activity of the enzyme heme oxygenase, which can be exported to the extracellular medium by ferroportin or stored as ferritin [83,84].

Figure 4

Alzheimer’s disease. The development and progression of Alzheimer’s disease (AD) lead to atrophy, loss and dysfunction of both neurons and glial cells. AD begins in the dorsal raphe nucleus with subsequent progression to the cortex, which is the center of information processing and memory storage. The factors that promote the development of AD are still unknown. However, it seems that the intracellular accumulation in neurons of the phosphorylated Tau protein (neurofibrillary tangle) and the formation of amyloid-B plaque (senile plaque) in the extracellular environment and brain tissue both lead to neuron loss and dysfunction. In addition, the formation of neurofibrillary tangle and senile plaque alters the functions of glial cells, such as oligodendrocytes (responsible for the myelination of neurons), microglia cells (phagocytic cells) and astrocytes (responsible for the absorption and exchange of nutrients between neurons and blood vessels). Dysregulation of cholesterol transport and iron metabolism in the central nervous system contributes to poor prognosis of Alzheimer’s disease. All these associated factors lead to an increase in neuroinflammation and oxidative stress associated with mitochondrial dysfunction, compromising the production of ATP, altering the concentration of neurotransmitters in the synaptic cleft, finally promoting cell death.

Figure 5

Parkinson’s disease. Parkinson’s disease (PD) occurs due to the decrease in and/or oxidation of dopamine in the substantia nigra, involving the motor system. The incorrect folding of α-synuclein leads to the accumulation of protein (Lewy body) in nervous tissue. The formation of Lewy bodies may be due to a highly pro-oxidative environment, due to dysfunction in the transport of lipids, iron, inflammation and mitochondrial changes. The increase in Lewy bodies is the trigger for the development of dementia, neurotoxicity and neuronal death.

Figure 6

Huntington’s disease. Huntington’s disease is caused by the repetition of autosomal dominant CAG trinucleotide in the Huntingtin gene (HTT gene) on chromosome 4, giving rise to the mutant huntingtin protein. The mutated protein translocates to the nucleus and remains in the cytoplasm. In the nucleus, association, oligomerization and aggregation with other proteins occurs, leading to the formation of inclusions. Protein inclusions disrupt the transcriptional process in nerve tissue cells. In the cytoplasm, the oligomerization, aggregation and precipitation of the huntingtin protein occurs. This process alters the metabolism and both intra- and extra-cellular signaling pathways. The increase in oxidative stress, lipid peroxidation and iron dyshomeostasis contribute to the aggregation and oligomerization of huntingtin protein with other cytoplasmic proteins. Aberrant protein aggregation increases the excitotoxicity of glutamate. Mitochondrial dysfunction changes autophagy mechanisms, and transport in the neuronal axon, leading to nerve cell degeneration.