Purpose of reviewIt is increasingly recognized that reactive oxygen and nitrogen species (ROS and RNS) are not only damaging molecules, but also essential signaling molecules regulating functions on organellar, cellular, organ, and organism levels. Nevertheless, damaging capacity of different ROS and RNS are connected to almost all diseases, including (neuro-)inflammation. One of the most frequent neuroinflammatory diseases is multiple sclerosis (MS). In this opinion article, we discuss recent advances in knowledge about oxidative and nitrosative damage, redox regulation, and the related ferroptotic cell death mechanism in initiation and progression of MS.Recent findingsSpecific improvement of supportive or inhibition of harmful redox processes in either brain or immune cells showed great impact on disease severity in MS models. Moreover, (circulating) markers of oxidative damage may hold promise as a supplementary diagnostic tool to differentiate different subtypes of MS.SummaryOxidative damage is well established as driver of MS. Nevertheless, molecular redox mechanisms underlying initiation and progression of MS are not well known. This existing research gaps impedes the establishment of successful antioxidative therapies.

Multiple Sclerosis (MS), an autoimmune disorder, is characterized by severe neuroinflammation, leading to demyelination and neuronal damage in the CNS, resulting in significant clinical impairment. MS progression involves complex pathological processes like immune cell invasion and cytokine-mediated recruitment to the CNS. Experimental autoimmune encephalomyelitis (EAE), widely used as a model for MS, despite its translational limitations, has been crucial for identifying effective treatments. Recent studies have shown that sodium channel (NaV) blockers and monoamine oxidase- (MAO) B inhibitors can alleviate symptoms of EAE and optic neuritis (ON), but their mode of action remains partially unclear. To evaluate the effects and understand the action mechanism of NaV blockers and MAO-B inhibitors (rasagiline, safinamide, flecainide and phenytoin) in neurological conditions, various techniques were used, including optical coherence tomography (OCT), optomotor response measurement (OMR), flow cytometry, histological evaluations, Evans blue assay, blood-brain barrier (BBB) permeability assay, western blot, proliferations assay, and gene expression analyses. The study found that the primary therapeutic effect comes from inhibiting the NaV 1.5 sodium channel, not MAO-B inhibition. Flecainide, a NaV 1.5 channel blocker, significantly reduced EAE disability scores, mitigated neurodegeneration, preserved visual function, and restricted immune cell migration into the CNS. Importantly, blocking the NaV 1.5 channel had an effect on the BBB, limiting lymphocyte entry into the CNS. This research highlights sodium channel blockers' potential in treating EAE. The findings demonstrate induced neuroprotection and reduced disease progression, suggesting a novel therapeutic approach. Crucially, it reveals for the first time that NaV 1.5 channel blockade leads to neuroprotection primarily by affecting the BBB, a key factor in controlling immune cell migration, thus addressing a critical aspect of neuroinflammation.

Redoxins, oxidoreductases of the thioredoxin (Trx) family, are important regulators of signaling processes. The Trx family is characterized by the Trx fold consisting of a four-stranded β-sheet surrounded by three-four α-helices. This article mainly focuses on mammalian Trxs, glutaredoxins, and peroxiredoxins, herein referred to as redoxins. This review article summarizes the current knowledge on redoxin-driven processes related to ferroptosis, a non-apoptotic cell death mechanism based on uncontrolled oxidation of polyunsaturated fatty acids in membranes. To a great extent, the formation of these lipid hydroperoxides depend on the non-enzymatic formation of hydroxyl radicals, the product of the Fenton reaction between hydrogen peroxide and redox active iron. Redoxins are regulators of both redox and iron homeostasis, and some redoxins use lipid hydroperoxides directly as substrates. This review article aims to increase the recognition of redoxins as potential regulators of ferroptosis in both physiological and pathological conditions, while also promoting research to address the numerous gaps in cell specificity and the molecular mechanisms influencing ferroptotic pathways.

Due to their role in excretion, renal proximal tubular cells are susceptible to damage by toxic metabolites and xenobiotics. The regenerative capacity of the kidney allows for the replacement of damaged cells, a process involving differentiation programs. However, kidney function tends to decline, suggesting that the replacement cells may not achieve full functionality. To understand possible causes of this decline, we investigated effects of nephrotoxins and oxidants on the differentiation of induced pluripotent stem cells (iPSC) into proximal tubular epithelial-like cells (PTELC). Proliferation, apoptosis, senescence, and expression of oxidative defense genes were analyzed in iPSC, differentiating and differentiated cells treated with cisplatin (CisPt, up to 45 µM), cyclosporin A (CycA, up to 12 µM), and the oxidants menadione (Mena, up to 50 µM) and tert-butylhydroquinone (tBHQ, up to 50 µM). We found that differentiating cells were most sensitive to oxidants and showed increased sensitivity to CisPt, whereas all differentiation stages showed similar sensitivity to CycA. Both oxidative stress and CisPt triggered apoptosis in all differentiation stages, whereas CycA mainly induced senescence. Treatment during differentiation resulted in long-term effects on gene expression in differentiated cells. While oxidants had no effect on transport function of differentiated cells, CisPt and CycA impaired albumin uptake. Our data suggest a substantial sensitivity of differentiating cells to nephrotoxins and oxidants, an aspect that could potentially interfere with regenerative processes.

Ferroptosis is defined as iron dependent non-apoptotic cell death. It is based on peroxidation of polyunsaturated phospholipids and subsequent membrane rupture. Lipid peroxidation is induced mainly by the hydroxyl radical, the product of the Fenton reaction between iron and hydrogen peroxide. Ferroptosis is connected to many organs and diseases including neuroinflammation. The most common inflammatory disease of the central nervous system in Western countries is multiple sclerosis leading to inflammatory demyelination and neurodegeneration. Fumarates, dimethyl fumarate (DMF) and its successor diroximel fumarate (DRF), are approved disease modifying therapies for multiple sclerosis and activate the Nrf2 pathway regulating the expression of many antioxidative enzymes. Since some of these enzymes are anti-ferroptotic, we here investigated whether the therapeutic effect of fumarates is connected to modulated sensitivity towards ferroptosis. Indeed, myelin damage induced by ferroptosis is diminished in presence of DRF or its active metabolite monomethyl fumarate. Moreover, anti-ferroptotic enzymes are upregulated in oligodendrocytes upon DRF treatment as well as in cerebellum of DMF-treated mice and in peripheral blood mononuclear cells of patients receiving DMF. This effect is absent in primary fibroblasts derived from osteo- or rheumatoid arthritis patients. In summary, our data offer a new organ- and disease-specific molecular anti-inflammatory mechanism of DRF.

Aging is a natural process characterized by a progressive physiological decline that undermines health and well-being in the elderly population. Oxidative stress is a widely accepted hallmarks of aging, and its role as one of the main drivers of ferroptosis is quite recent. Ferroptosis is an iron-dependent cell death caused by massive phospholipid peroxidation. The excessive accumulation of intracellular reactive oxygen species and iron, as well as the failure of the main cellular antioxidant systems, cause ferroptotic cell death. While clear roles for ferroptosis in pathological conditions such as cancer or neurodegeneration have been described, its physiological roles and regulators are less understood.Here, using Caenorhabditis elegans as a powerful model organism for aging studies, we uncover a role for ferroptosis in physiological aging mediated by disturbed redox homeostasis. We evaluated healthspan parameters in C. elegans highlighting how several age-related features differentially decline during physiological aging. A progressive loss of the capability to contrast external stressors, with an increase in hydroxyl radicals and a decrease of glutathione demonstrated the disruption of redox homeostasis in older age. Moreover, transcription of selected genes involved in redox metabolism is downregulated with aging. Among them, loss of the fatty acyl-CoA reductase encoded by fard-1 and of the dehydrogenase encoded by dhs-25 display higher sensitivity to ferroptosis, increased lipid peroxidation, lower total glutathione levels and reduced lifespan. Accordingly, the expression of hydroxysteroid 17-beta dehydrogenase 8, one of the closest mammalians dhs-25 homologs, is downregulated in cells which are more sensitive to ferroptosis.Our results clearly prove a causal role for ferroptosis in C. elegans aging driven by mitochondrial redox unbalance, unveiling novel genes involved in this connection that may constitute targets for possible interventions to improve healthy aging.

Ferroptosis, an iron-dependent form of oxidative cell death, is predominantly regulated by glutathione peroxidase 4 (GPX4), making it a promising target for cancer therapy. However, the majority of GPX4 inhibitors, most of which contain a chloroacetamide moiety such as RSL3, are limited by poor pharmacokinetic properties and off-target effects, hindering their preclinical translation. Utilizing a range of interdisciplinary methodologies, we show that sodium aurothiomalate (ATM), a drug approved by many agencies, induces ferroptosis by covalently targeting GPX4 via formation of a selenenylsulfide bond. In preclinical models of neuroblastoma and acute myeloid leukemia (AML), ATM combined with ferric ammonium citrate (FAC) yields a synergistic effect, resulting in a significant reduction in tumor growth. Mechanistically, ATM disrupts GPX4 activity by covalently binding thiomalate to the active site selenocysteine, while modification of specific cysteine residues leads to destabilization of the protein and impaired binding to phospholipids. We propose that these covalent modifications are achieved through a unique reaction mechanism, in which the gold component of ATM acts as a thiol-masking carrier and is only transiently present, being subsequently displaced and allowing the reaction of the thiomalate moiety with the target selenocysteine or cysteine. Our data lay the foundation for development of novel, drug-like thiol-based GPX4 inhibitors. ### Competing Interest Statement The authors have declared no competing interest.

DNA damage is a major risk factor for the decline of neuronal functions with age and neurodegenerative diseases. The connection between DNA damage and neurodegeneration is extensively investigated, however, the mechanisms limiting the propagation of damaged DNA from highly replicative neural stem/progenitor cells (NSPCs) to post-mitotic neurons remains largely unknown. Here, we describe that enzymatic activity of the histone deacetylase sirtuin 1 (SIRT1) is important for the homologues recombination-dependent repair of double- stranded DNA breaks induced by etoposide. Furthemore, SIRT1 abolishes neuronal fate of murine NSPCs following induction of DNA damage. Pharmacological inhibition or genetic inactivation of SIRT1 rescues etoposide-mediated inhibition of neuronal differentiation in NSPCs and hippocampal slice cultures and promotes transcription of pro-neuronal genes. Inhibition of Ataxia-telangiectasia mutated (ATM), the central regulator of the DNA damage response, mimics the SIRT-dependent effect of DNA damage on neuronal differentiation, indicating that the ATM/SIRT1 axis inhibits formation of neuronal cells harbouring damaged DNA. These data are consistent with the role of SIRT1 in genome stability, healthy ageing and protection from the development of ageing-associated neurodegenerative diseases. ### Competing Interest Statement The authors have declared no competing interest. German Research Foundation (DFG), 417677437GRK2578

Selenium-dependent glutathione peroxidase 4 (GPX4) is the guardian of ferroptosis, preventing unrestrained (phospho)lipid peroxidation by reducing phospholipid hydroperoxides (PLOOH). However, the contribution of other phospholipid peroxidases in ferroptosis protection remains unclear. We show that cells lacking GPX4 still exhibit substantial PLOOH-reducing capacity, suggesting a contribution of alternative PLOOH peroxidases. By scrutinizing potential candidates, we found that although overexpression of peroxiredoxin 6 (PRDX6), a thiol-specific antioxidant enzyme with reported PLOOH-reducing activity, failed to prevent ferroptosis, its genetic loss sensitizes cancer cells to ferroptosis. Mechanistically, we uncover that PRDX6, beyond its known peroxidase activity, acts as a selenium-acceptor protein, facilitating intracellular selenium utilization and efficient selenium incorporation into selenoproteins, including GPX4. Its physiological significance was demonstrated by reduced GPX4 expression in Prdx6-deficient mouse brains and increased sensitivity to ferroptosis in PRDX6-deficient tumor xenografts in mice. Our study highlights PRDX6 as a critical player in directing cellular selenium utilization and dictating ferroptosis sensitivity.

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (M pro ), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that M pro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit M pro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.

Background: Redox control seems to be indispensable for proper embryonic development. The ratio between glutathione (GSH) and its oxidized disulfide (GSSG) is the most abundant cellular redox circuit. Methods: We used zebrafish harboring the glutaredoxin 1-redox sensitive green fluorescent protein (Grx1-roGFP) probe either in mitochondria or cytosol to test the hypothesis that the GSH:GSSG ratio is strictly regulated through zebrafish embryogenesis to sustain the different developmental processes of the embryo. Results: Following the GSSG:GSH ratio as a proxy for the GSH-dependent reduction potential (EhGSH) revealed increasing mitochondrial and cytosolic EhGSH during cleavage and gastrulation. During organogenesis, cytosolic EhGSH decreased, while that of mitochondria remained high. The similarity between EhGSH in brain and muscle suggests a central regulation. Modulation of GSH metabolism had only modest effects on the GSSG:GSH ratios of newly hatched larvae. However, inhibition of GSH reductase directly after fertilization led to dead embryos already 10 h later. Exposure to the emerging environmental pollutant Perfluorooctane Sulfonate (PFOS) disturbed the apparent regulated EhGSH as well. Conclusions: Mitochondrial and cytosolic GSSG:GSH ratios are almost identical in different organs during zebrafish development indicating that the EhGSH might follow H2O2 levels and rather indirectly affect specific enzymatic activities needed for proper embryogenesis. General significance: Our data confirm that vertebrate embryogenesis depends on strictly regulated redox homeostasis. Disturbance of the GSSG:GSH circuit, e.g. induced by environmental pollution, leads to malformation and death.

Iron-regulatory protein 1 (IRP1), a central regulator of iron metabolism in vertebrates, also affects cellular response to hypoxia. IRP1 binds to the iron-responsive element (IRE) in the mRNA encoding hypoxia-inducible factor (HIF) 2α, thereby blocking the translation of the HIF2α-mRNA, and allowing the transcriptional regulation of, e.g., erythropoiesis. Here, we characterize the oxidoreductase thioredoxin 1 (Trx1) as a new regulator of hypoxia signaling. Human and murine Trx1 complex iron-sulfur clusters using one of the active site cysteinyl residues and a vertebrate-specific additional cysteinyl residue outside the active site. FeS-Trx1 is inactive, activated apo-Trx1 reduces cysteinyl residues in the binding pocket of IRP1/apo-Aconitase 1, which allows IRP1 to bind IREs in regulated mRNAs. Therefore, translation of the HIF2α mRNA requires either sufficient iron supply or the lack of reducing power of the Trx system under iron-limiting conditions. FeS-Trx1 thus links both redox and iron homeostasis to hypoxia responses. ### Competing Interest Statement The authors have declared no competing interest.

Ferroptosis is an iron-dependent form of regulated cell death arising from excessive lipid peroxidation. While seminal work described that oncogenic RAS transformation drives synthetic lethal vulnerability to archetypal ferroptosis inducers including erastin (eradicator of RAS and ST-expressing cells) and RSL3 (Ras selective lethal 3), more recent work suggest that oncogenic RAS signaling may confer ferroptosis resistance. Thus, the impact of oncogenic RAS on the cellular response to ferroptosis is still unclear. Here, we provide unifying evidence across multiple cellular models that oncogenic RAS signaling suppresses ferroptosis. Using integrated proteo- and transcriptomic analyses, we uncovered that oncogenic RAS signaling upregulates the ferroptosis suppressor GTP cyclohydrolase I (GCH1) via transcriptional induction by the transcription factor ETS1 downstream of the RAS-MAPK signaling cascade. Targeted repression of Gch1 or of the tetrahydrobiopterin (BH4) synthesis pathway, which is mediated by GCH1, was sufficient to sensitize oncogenic RAS transformed cells to ferroptosis in 2D and 3D cell models, highlighting a mechanism through which RAS promotes resistance to ferroptosis induction. Furthermore, we found that GCH1 expression is clinically relevant and correlates with RAS signaling activation in human cancers. Overall, this study redefines oncogenic RAS signaling to be a ferroptosis suppressor, and identifies GCH1 as a mediator of this effect and a potential vulnerability for targeting RAS driven cancers.### Competing Interest StatementThe authors have declared no competing interest.

Glioblastoma (GBM) is the most commonly occurring and most aggressive primary brain tumor. Transcriptomics-based tumor subtype classification has established the mesenchymal lineage of GBM (MES-GBM) as cancers with particular aggressive behavior and high levels of therapy resistance. Previously it was show that Trihexyphenidyl (THP), a market approved M1 muscarinic receptor-targeting oral drug can suppress proliferation and survival of GBM stem cells from the classical transcriptomic subtype. In a series of in vitro experiments, this study confirms the therapeutic potential of THP, by effectively suppressing the growth, proliferation and survival of MES-GBM cells with limited effects on non-tumor cells. Transcriptomic profiling of treated cancer cells identified genes and associated metabolic signaling pathways as possible underlying molecular mechanisms responsible for THP-induced effects. In vivo trials of THP in immunocompromised mice carry orthotopic MES-GBMs showed moderate response to the drug. This study further highlights the potential of THP repurposing as an anti-cancer treatment regimen but mode of action and d optimal treatment procedures for in vivo regimens need to be investigated further.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

With a global increase in chronic kidney disease patients, alternatives to dialysis and organ transplantation are needed. Stem cell-based therapies could be one possibility to treat chronic kidney disease. Here, we used multipotent urine-derived renal progenitor cells (UdRPCs) to study nephrogenesis. UdRPCs treated with the JNK inhibitor—AEG3482 displayed decreased proliferation and downregulated transcription of cell cycle-associated genes as well as the kidney progenitor markers—SIX2, SALL1 and VCAM1. In addition, levels of activated SMAD2/3, which is associated with the maintenance of self-renewal in UdRPCs, were decreased. JNK inhibition resulted in less efficient oxidative phosphorylation and more lipid peroxidation via ferroptosis, an iron-dependent non-apoptotic cell death pathway linked to various forms of kidney disease. Our study is the first to describe the importance of JNK signalling as a link between maintenance of self-renewal and protection against ferroptosis in SIX2-positive renal progenitor cells.

Table S1: The redox status of the cell correlates with sensitivity to MTH1 inhibition. Table S2: Data collection and refinement statistics. Table S3: Kinetic parameters of zfMTH1. Table S4: Primer sequences. Figure S1: BSO treatment sensitizes U2OS and U343 cells against MTH1 inhibition. Figure S2: pre-treatment with PEG-catalase protects cancer cells against MTH1 inhibition. Figure S3: Alignment of human and zebrafish MTH1. Figure S4: Purification of zebrafish MTH1. Figure S5: Electron density for TH588 in the structure of Zebrafish MTH1. Figure S6: knock-down of VHL in HAEB cells sensitizes to the MTH1 inhibitor TH588. Figure S7: TH588 is well tolerated of zebrafish embryos. Figure S8: Expression of MTH1 in VHL knock-out background. Figure S9: Experimental setup for DMOG exposure of zebrafish embryos. Figure S10: ZebROS exposed to reducing and oxidizing agent.

BACKGROUND & AIMS: The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells. METHODS: Kcnk9(-/- )mice were challenged with 3% dextran sulfate sodium (DSS) to induce experimental acute colitis. Primary cultures of intestinal epithelial cells were generated, and expression of potassium channels as well as cytosolic calcium levels and susceptibility to apoptosis were evaluated. Furthermore, we evaluated whether KCNK9 deficiency was compensated by the closely related 2-pore potassium channel KCNK3 in vivo or in vitro. RESULTS: Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9-dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk(-9-) mice but did not prevent apoptosis after DSS exposure. Conversely, increased levels of KCNK9 in Kcnk3(-/- )mice were associated with an ameliorated course of DSS-induced colitis. CONCLUSIONS: KCNK9 enhances mitochondria' stability, reduces apoptosis, and thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3(-/-) resulted in less mitochondria' damage and apoptosis and was associated with beneficial outcomes in DSS-induced colitis.

Thioredoxins and glutaredoxins are key proteins in the control of the protein thiol state and thereby, for redox signaling. These proteins share structural features such as the thioredoxin fold, but differ in the ability to coordinate iron sulfur (FeS) clusters. Moreover, the coordination of the FeS cluster itself is different in subfamilies of the glutaredoxins and determines the functional activities of these subfamilies. In contrast to CxxC-type glutaredoxins and thioredoxins, CGFS-type glutaredoxins are not oxidoreductases but are important for iron metabolisms, especially iron transport and biosynthesis of FeS clusters. Here, we describe the cellular functions of FeS coordinating thioredoxins and glutaredoxins in the mammalian system, their impact on diseases, and how small structural differences in wildtype proteins and engineered mutants define these distinct functions. This chapter is written in memoriam to Arne Holmgren, the godfather of thio- and glutaredoxins, who sadly passed away in January 2020.