Iron homeostasis plays a key role in ferroptosis (Yan and Zhang, 2020). Iron can enter the cell via transferrin 1 receptor (TfR1, also known as TFR1) and be reduced from ferric (Fe³⁺) to ferrous (Fe²⁺) form via metalloreductase STEAP3 in the endosome (Zhang et al., 2012). In this form, iron can be stored in ferritin, or exported from the cell via ferroportin (FPN1) (Chang, 2019). Ferritin degradation via the nuclear receptor coactivator 4 (NCOA4) contributes to ferroptosis by increasing the free intracellular iron levels (Hou et al., 2016). Excessive intracellular iron accumulation can lead to generation of reactive oxygen species (ROS) and oxidative stress via the Fenton reaction (Ward et al., 2014). Iron accumulation-induced ROS, such as superoxide anion (O₂-•) and hydroxyl radical (•OH), possess an unpaired electron at their outer orbit which allows them to react with all cellular components including proteins, lipids and nucleic acid. This results in lipid peroxidation, oxidative damage to membranes and other lipid-containing molecules, and ultimately to cellular damage and ferroptotic cell death (Aprioku, 2013).

On the other hand, inhibition of glutamate/cystine antiporter (system x_c^–, xc-, with xCT as the functional subunit of system x_c^–) and depletion of GSH cause inactivation of GPx4, the critical antioxidant enzyme and regulator of ferroptosis (Seibt et al., 2019). This can lead to ferroptotic cell death through increased lipid peroxidation and accumulation of ROS (Wang et al., 2020). GPx4 reduces hydroperoxides of polyunsaturated-fatty-acid-containing-phospholipids (PL-PUFA(PE)-OOH) to polyunsaturated-fatty-acids (phosphatidylethanol amine)-reduced (PL-PUFA(PE)-OH) (Seibt et al., 2019). GPx4 uses GSH as a reducing substrate and converts it into oxidized form, also referred to as glutathione disulphide (GS-SG) (Cozza et al., 2017). Apart from nuclear factor erythroid 2-related factor 2 (Nrf2, coded by NFE2L2 gene) (Habib et al., 2015), the xCT mRNA can be positively regulated by the activation of transcription factor 4 (ATF4) under oxidative stress (Sato et al., 2004), while its negative regulation by p53 results in cysteine deprivation and increased susceptibility to ferroptosis (Jiang et al., 2015).

Oxidative stress occurs due to the imbalance between generation of free radicals and the ability to neutralize or eliminate them through antioxidants (Birben et al., 2012). One of the main drivers of ferroptosis is ROS-mediated lipid peroxidation, which can result in oxidative stress (Kuang et al., 2020). Inhibition of GPx4 and decrease in GSH levels lead to activation of 12/15-lipoxygenase (12/15-LOX, which is the protein product of the ALOX15 gene). The association of Fe²⁺ with lipoxygenases (LOX, a dioxygenase containing non-heme iron) can lead to oxygenation of polyunsaturated-fatty-acids (PUFA), such as arachidonic acid present in phospholipids, and trigger lipid peroxidation-induced ferroptosis (Kagan et al., 2017). The LOX nomenclature is defined by the specific site of their oxygenation product: in humans there are six LOX isoforms 15-LOX-1, 15-LOX-2, 12-LOX-1, 12-LOX-2, E3-LOX, and 5-LOX, of which 12/15-LOX (15-LOX) are the most abundant. 12/15-LOX are considered as one of the key regulators of ferroptotic cell death (Yang et al., 2016; Kagan et al., 2017). Although, this has been supported by the findings that pharmacological inhibition of 15-LOX-1 exerts a cytoprotective effect (Seiler et al., 2008; Eleftheriadis et al., 2016), some off-target effects of lipoxygenase inhibitors have also been reported (Shah et al., 2018).

In addition to iron accumulation-induced generation of ROS, mitochondria also contribute to ROS production. Electrons leak from complex I and III of the electron transport chain (ETC) located on the inner membrane of mitochondria (Zhao et al., 2019). This can result in the formation of ROS such as O₂-• and hydrogen peroxides (H₂O₂), and potentially can lead to loss of mitochondrial membrane potential (ΔΨm) (Gao et al., 2019). Reduced ΔΨm was associated with ferroptosis and involves different regulatory mechanisms than apoptosis (Kuang et al., 2020). GSH depletion-induced activation of 12/15-LOX can increase cytosolic Ca²⁺ via both (1) the import from the extracellular compartment and (2) release from mitochondria and endoplasmic reticulum (Maher et al., 2018). Decrease in GSH levels can also lead to dysregulation of Ca²⁺ transport in and out of mitochondria by voltage dependant anion channels (VDAC) and mitochondrial Ca²⁺ uniporter (MCU) (Zorov et al., 2014; DeHart et al., 2018). This results in mitochondrial Ca²⁺ overload and collapse of the mitochondrial function which activates Ca²⁺-dependant proteases (Zorov et al., 2014; DeHart et al., 2018; Marmolejo-Garza and Dolga, 2021). Consequently, ROS-induced transactivation of BH3 interacting-domain death agonist (BID) to mitochondria and Ca²⁺overload-induced translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus causes the cell to die (Neitemeier et al., 2017). This caspase-independent process is accompanied by mitochondrial fragmentation and enlarged cristae (Dixon et al., 2012). The rescue of mitochondria (Jelinek et al., 2018), decrease of mitochondria-associated endoplasmic reticulum membranes (MAMs) interaction (Guo et al., 2019) and small conductance calcium-activated potassium (K_Ca2/SK) channel activation have the potential to protect from ferroptotic cell death (Krabbendam et al., 2020).

Advanced age is associated with iron dysregulation affecting most of our organs (Xu et al., 2012; Picca et al., 2019). Many studies show that iron dysregulation can also contribute to AD pathology (Bush, 2013; Nuñez and Chana-Cuevas, 2018). With aging, iron deposits in the brain (Acosta-Cabronero et al., 2016), which can increase the formation of Aβ plaques (Becerril-Ortega et al., 2014) and tau hyperphosphorylation in AD transgenic mouse brain (Guo et al., 2013). Imaging and histological experiments support this by showing increased iron deposition in AD-specific brain regions (Altamura and Muckenthaler, 2009; Bush, 2013; Apostolakis and Kypraiou, 2017; Lee and Lee, 2019). Magnetic resonance imaging (MRI) studies revealed increased iron levels in the putamen, pulvinar thalamus, red nucleus, hippocampus, and temporal cortex of AD patients (Langkammer et al., 2014). Later, quantitative susceptibility mapping showed higher iron levels in caudate and putamen nucleus of AD patients than in controls. Interestingly, the increased iron level in the left caudate nucleus correlated with the degree of cognitive impairment (Du et al., 2018). Finally, higher iron levels in the frontal cortex were associated with AD severity (Bulk et al., 2018b). This evidence suggested that iron contributes to AD pathology and presented an important avenue for therapy development (Masaldan et al., 2019).

Ferroptosis can be induced by compounds interfering with system x_c^–, such as erastin, which induces cysteine deprivation, GSH depletion, endoplasmic reticulum stress, and cell death (Dixon et al., 2012, 2014; Sato et al., 2018). System x_c^– can also be inhibited by adding small concentrations of sorafenib (Lachaier et al., 2014), glutamate (Jiang et al., 2020) and sulfasalazine (Yu et al., 2019) to the extracellular compartment. Inhibition of gamma-glutamylcysteine synthetase (γ-GCS) by buthionine sulphoximine (BSO) results in GSH depletion and can lead to ferroptosis (Griffith, 1982). Irreversible and direct inhibition of GPx4 by the (1S,3R)-RSL3 (RSL3), causes the production of polyunsaturated-fatty-acid-containing-phospholipid hydroperoxides, which leads to lipid peroxidation and ferroptotic cell death (Liang et al., 2019). In addition to pharmacological compounds, genetic modifications targeting regulators of the system x_c^– can induce ferroptosis. The Gpx4BI-KO mouse was generated by a conditional deletion of Gpx4 in forebrain neurons by administration of tamoxifen. In this mouse model, 75–85% decrease of Gpx4 was shown to induce hippocampal neuronal loss, lipid peroxidation, neuroinflammation and spatial learning deficits (Hambright et al., 2017). Similarly, the knockout of Gpx1, facilitated memory impairment induced by β-Amyloid in mice (Joo et al., 2020). The Western blot analysis of AD post mortem brain tissue revealed enhanced expression of the light-chain subunit of the xCT (Ashraf et al., 2020). These results suggest that impaired GSH metabolism might play a role in ferroptosis during AD pathology (Ashraf et al., 2020).

The brain is the most vulnerable organ to oxidative stress. It represents only 2% of the body but uses 20% of the total oxygen supply (Sokoloff, 1999). Oxidative stress plays a key role in AD pathology by initiating the generation and enhancing of both Aβ plaques and hyperphosphorylation of Tau (p-Tau) (Huang et al., 2016; Nassireslami et al., 2016). Oxidative stress can be enhanced in AD via metal accumulation. In addition to iron, the Aβ precursor protein (APP) has a high affinity to binding zinc and copper at the N terminal metal-binding sites (Barnham et al., 2003). Additionally, high concentrations of these metals were also found in Aβ plaques in mouse and human brain (Plascencia-Villa et al., 2016; James et al., 2017). As copper is the potent mediator of •OH, and the binding of zinc leads to production of toxic Aβ and further uncontrolled zinc release, these metals can contribute to the increase of oxidative stress in AD (Strozyk et al., 2009). Post mortem tissue from AD patients shows higher levels of oxidized bases in the frontal, parietal and temporal lobes compared to control subjects (Wang et al., 2005), which correlates with imbalanced levels of copper, zinc and iron (Deibel et al., 1996). Other studies have shown higher level of lipid peroxidation, in diseased regions of AD brain compared to controls (Montine et al., 1998; Lovell et al., 2001; Bradley-Whitman and Lovell, 2015). These results support that oxidative stress might be an important factor contributing to the development and progression of AD (Zhao and Zhao, 2013).

Our transcriptomic analysis revealed that FTH1, component responsible for iron storage, is differentially expressed in early and late stages of AD. Furthermore, excessive iron deposition in specific brain areas contributes to AD pathology (Antharam et al., 2012; Moon et al., 2016). Therefore, an increased interest in the development of therapeutic strategies targeting iron has emerged in the past years. In animal models, DFO treatment decreased AD hallmarks, iron overload, iron-induced kinase activity [cyclin-dependent kinase 5 (CDK5), glycogen synthase kinase 3β (GSK3β)], mitochondrial dysfunction, synaptic loss, and neuronal damage (Fine et al., 2012; Guo et al., 2013, 2015; Sripetchwandee et al., 2016). DFO increased expression of transferrin receptor (TfR1) and brain-derived neurotrophic factor (BDNF), leading to reduced iron-induced memory deficits in rodents (Fine et al., 2012; C. Guo et al., 2013, 2015; Sripetchwandee et al., 2016). In a clinical trial, DFO slowed down the progression of AD in patients (McLachlan et al., 1991, 1993). However, the dosing regimens need to be standardized before DFO could be implemented in the clinical setting (Farr and Xiong, 2021). In addition, to reduce DFO-related cytotoxicity and prolong its presence into circulation, new DFO component-containing nanogels were proposed as promising alternatives for iron-chelation in AD (Wang et al., 2018). Besides AD, DFO alone or co/treatment with ferrostatin (Fer-1, inhibitor of lipid peroxidation) also improved α-synuclein-induced pathology in a PD animal model (Febbraro et al., 2013). PBT1, a drug inhibiting zinc and copper ions from binding to Aβ, reduced Aβ deposition, attenuated astrogliosis and prevented memory impairment in AD animal models AD (Cherny et al., 2001; Grossi et al., 2009). In pilot-phase 2 clinical trial, PBT1 reduced Aβ plasma levels and, when looked specifically on severely affected AD patients, PBT1 was able to slow down the clinical decline (Ritchie et al., 2003). PBT2, a second-generation 8-hydroxyquinoline analog produced as a successor to clioquinol, induced GSK3β phosphorylation and prevented formation of Aβ in neuroblastoma SH-SY5Y cells (Crouch et al., 2011). In animal models of AD, PBT2 induced Aβ plaque degradation, decreased p-tau, rescued decreased spine density, increased brain-levels of BDNF and improved cognitive performance (Adlard et al., 2008, 2011). PBT2 was also assessed in a phase 2 clinical trial, where it lead to reduced levels of Aβ in cerebrospinal fluid and improved executive function compared to placebo (Lannfelt et al., 2008). However, PBT2 did not show any significant effect on cognition. Currently, deferiprone (DFP), a compound that alleviates symptoms related to PD pathology (Devos et al., 2014; Grolez et al., 2015; Gutbier et al., 2020), is evaluated a in phase 2 randomized placebo-controlled clinical trial with AD patients (NCT03234686). As previously reported, iron chelators can attenuate symptoms and slow down the progression of AD, which shows the potential for novel therapeutic approaches (Nuñez and Chana-Cuevas, 2018).

The revealed differential gene expression of GPX4 and GSS suggests that modifying the expression or/and the activity of these gene-encoded proteins might be beneficial to treat AD. The expression of GPx4 can be directly upregulated by α-Lipoic acid (LA) (Zhang et al., 2018). LA treatment on P301S Tau transgenic mice enhanced the activity of system x_c^–, GPx4, superoxide dismutase 1 (Sod1), CDK5, GSK3β, TfR1 and FPN1 (Zhang et al., 2018). LA reduced the hippocampal levels of glial fibrillary acidic protein (GFAP), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), as well as the calcium (Ca²⁺) content, p-tau, calpain1 levels, and synaptic loss. As a result, these processes led to enhanced memory function (Zhang et al., 2018). Apart from LA, GPx4 can be activated in an indirect manner through Nrf2. Nrf2 plays an important role in neurodegeneration and ferroptosis by regulating a wide range of genes (Song and Long, 2020). In addition to the activation of GPx4 and GSH synthesis (Dodson et al., 2019), it can also affect the activity of glucose-6-phosphate dehydrogenase, GSH reductase, glutamate-cysteine ligase modifier subunit (GLCM), solute carrier family 7 member 11 (SLC7A11) and others as previously summarized by Song and Long (2020). Nrf2 can be upregulated using a human lentiviral vector or compounds such as sodium arsenite, triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-methylamide (CDDO-MA), dl-3-n-butylphthalide (DI-NBP), kavalactone methysticin, carnosic acid (CA) and sulforaphane (SFN). Nrf2 upregulation increased heme oxygenase-1 (HMOX1) levels and decreased AD hallmarks, hippocampal inflammation, oxidative stress, and Aβ-induced memory deficits in AD mouse models (Dumont et al., 2009; Kanninen et al., 2009; Kim et al., 2013; Lipton et al., 2016; Nassireslami et al., 2016; Wang et al., 2016; Fragoulis et al., 2017). Finally, genetic downregulation of Kelch-like ECH-associated protein 1 (Keap1), the negative regulator of Nrf2, in ArcAβ42 flies, activated Nrf2, induced Aβ42 degradation, prevented neuronal toxicity in response to Aβ42 peptide, rescued neuronal-specific motor defects and increased life span (Kerr et al., 2017).

Altogether, these results suggest that inhibition of ferroptosis by targeting GSH metabolism is an important avenue for the development of new therapies for AD (Ashraf et al., 2020).

Lipid peroxidation represents an important hallmark of AD (Sultana et al., 2013), which was also supported by the observed differential expression of ACSL3 and 4 in the course of the pathology (Tables 1, 2). In many studies, oxidative stress was targeted to reduce neuronal damage and alleviate symptoms related to AD pathology. Anti-ferroptotic compounds that reduce oxidative stress include liproxstatin 1 (Lip-1) (inhibitor of ROS and lipid peroxidation), chalcones 14a-c (inhibitor of Aβ and lipid peroxidation), Selenomethionine (Se-Met) (inhibitor of lipid peroxidation), CMC121 (fatty acid synthase inhibitor), N-acetylcysteine (NAC) (free radical scavenger), Vitamin E (Vit E) and PD146176 (15-LOX-1 inhibitor). Studies using in vitro and in vivo models of AD have shown that targeting oxidative stress has a positive effect on neural degeneration, inflammation, Aβ1-42 aggregation, p-tau formation, GSH levels, iron overload, mitochondrial function, motor dysfunction and learning and memory (Fu et al., 2006; Sripetchwandee et al., 2016; Hambright et al., 2017; Zhang et al., 2017; Cong et al., 2019; Ates et al., 2020). In concordance with these results, clinical trials have shown that NAC and co-treatment of NAC, Vit E and Se-Met improved behavioral symptoms, general well-being, and neuropsychiatric and cognitive scores of AD patients (Adair et al., 2001; McCaddon and Davies, 2005; Remington et al., 2009). Although Vitamin E treatment had no beneficial effect on patients with mild cognitive impairment (Marder, 2005), it was able to improve symptoms related to other neurodegenerative diseases such as PD (Taghizadeh et al., 2017) and cerebellar ataxia (Gabsi et al., 2001). Considering the lack of adverse events of these antioxidants, ferroptosis inhibition by targeting oxidative stress is a new promising therapeutic strategy for AD.