Free Radical Biology and Medicine

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial RONS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated ROS levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitochondrial ROS, and affects NO{middle dot} bioavailability via mitochondrial peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial RONS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required. HighlightsO_LITranscriptomics data from FSHD muscle indicates enrichment for disturbed mitochondrial pathways C_LIO_LIDisturbed RONS metabolism correlates with mitochondrial membrane polarisation and myotube hypotrophy C_LIO_LIDUX4-induced changes in mitochondrial function precede oxidative stress through mitoROS and affect hypoxia signalling via complex I C_LIO_LIFSHD is sensitive to environmental hypoxia, which increases ROS levels in FSHD myotubes C_LIO_LIHypotrophy in hypoxic FSHD myotubes is efficiently rescued with mitochondria-targeted antioxidants C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/459509v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@16ae1a8org.highwire.dtl.DTLVardef@517caorg.highwire.dtl.DTLVardef@5d0734org.highwire.dtl.DTLVardef@183ef50_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundAmyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease. Accumulating evidence strongly suggests that intrinsic muscle defects exist and contribute to disease progression, including imbalances in whole-body metabolic homeostasis. We have previously reported that tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor inducible 14 (Fn14) are significantly upregulated in skeletal muscle of the SOD1G93A ALS mouse model. While antagonising TWEAK did not impact survival, we did observe positive effects in skeletal muscle. Given that Fn14 has been proposed as the main effector of the TWEAK/Fn14 activity and that Fn14 can act independently from TWEAK in muscle, we suggest that manipulating Fn14 instead of TWEAK in the SOD1G93A ALS mice could lead to differential and potentially improved benefits. MethodsWe thus investigated the contribution of Fn14 to disease phenotypes in the SOD1G93A ALS mice. To do so, Fn14 knockout mice (Fn14-/-) were crossed onto the SOD1G93A background to generate SOD1G93A;Fn14-/- mice. Investigations were performed on both unexercised and exercised (rotarod and/or grid test) animals (wild type (WT), Fn14-/-, SOD1G93A and SOD1G93A;Fn14-/-). ResultsHere, we firstly confirm that the TWEAK/Fn14 pathway is dysregulated in skeletal muscle of SOD1G93A mice. We then show that Fn14-depleted SOD1G93A mice display an increased lifespan and decreased muscle pathology, without an impact on motor function, and that this is dependent on exposure to exercise. Indeed, we observe that endurance (rotarod) and resistance (grid test) exercises influence the positive effects of Fn14 deletion on survival and muscle phenotypes in SOD1G93A mice, which may be further influenced by genotype and disease state. ConclusionsOur study provides further insights on the different roles of the TWEAK/Fn14 pathway in pathological skeletal muscle and how they can be influenced by age, disease and metabolic state. This is particularly relevant in the ALS field, where combinatorial therapies that include exercise regimens are currently being explored. As such, a better understanding and consideration of the interactions between treatments, muscle metabolism and exercise will be of importance in future studies.

Oxidative post-translational modifications on the sulfhydryl group of cysteines can occur spontaneously or enzymatically. The dioxygenation of N-terminal cysteines has emerged as a new oxygen sensing paradigm, catalysed by 2-aminoethanethiol dioxygenase (ADO) in mammals. Conflicting evidence has been reported in recent years on whether this reaction can occur in the absence of ADO. Here we sought to address whether physiological oxidative stress can interfere with ADO-catalysed N-terminal dioxygenation. Using a system to produce titratable intracellular levels of H2O2, we demonstrate that the stability of RGS4 and 5 is not affected by oxidative stress, whether ADO is present or not. However, cytotoxic levels of oxidative stress did induce an increase in RGS4/5 protein levels that occurred independently of the Cys N-degron pathway. This effect of tBHP was reduced by Fe2+ chelation and perturbations of lysosomal function, suggesting the possible involvement of ferroptosis. We conclude that N-terminal cysteine dependent proteolysis of RGS4/5 is not sensitive to physiological oxidative stress, but these proteins can be stabilised during the process of oxidative stress-induced cell death through an N-terminal cysteine independent mechanism.

Streptococcus pneumoniae (Spn) causes pneumonia that kills millions through acute toxicity and invasion of the lung parenchyma. During aerobic respiration, Spn releases hydrogen peroxide (Spn-H2O2), as a by-product of enzymes SpxB and LctO, and causes cell death with signs of both apoptosis and pyroptosis by oxidizing unknown cell targets. Hemoproteins are molecules essential for life and prone to oxidation by H2O2. We recently demonstrated that during infection-mimicking conditions, Spn-H2O2 oxidizes the hemoprotein hemoglobin (Hb), releasing toxic heme. In this study, we investigated details of the molecular mechanism(s) by which the oxidation of hemoproteins by Spn-H2O2 causes human lung cell death. Spn strains, but not H2O2-deficient Spn{Delta}spxB{Delta}lctO strains caused time-dependent cell cytotoxicity characterized by the rearrangement of the actin, the loss of the microtubule cytoskeleton and nuclear contraction. Disruption of the cell cytoskeleton correlated with the presence of invasive pneumococci and an increase of intracellular reactive oxygen species. In cell culture, the oxidation of Hb or cytochrome c (Cytc) caused DNA degradation and mitochondrial dysfunction from inhibition of complex I-driven respiration, which was cytotoxic to human alveolar cells. Oxidation of hemoproteins resulted in the creation of a radical, which was identified as a protein derived side chain tyrosyl radical by using electron paramagnetic resonance (EPR). Thus, we demonstrate that Spn invades lung cells, releasing H2O2 that oxidizes hemoproteins, including Cytc, catalyzing the formation of a tyrosyl side chain radical on Hb and causing mitochondrial disruption, that ultimately leads to the collapse of the cell cytoskeleton.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease that affects motor neurons. This disease is associated with oxidative stress especially in mutant superoxide dismutase 1 (mutSOD1) patients. However, less is known for the most prevalent sporadic ALS due to a lack of disease models. Here, we studied oxidative the stress profiles in lymphoblasts from ALS patients with mutSOD1 or unknown (undSOD1) mutations. mutSOD1 and undSOD1 lymphoblasts, as well as sex/age-matched controls (3/group) were obtained from Coriell and divided in 46 years-old-men (C1), 46 years-old-women (C2) or 26/27 years-old-men (C3) cohorts. Growth curves were performed, and several parameters associated with redox homeostasis were evaluated, including SOD activity and expression, general oxidative stress levels, lipid peroxidation, response to oxidative stimulus, glutathione redox cycle, catalase expression, and activity, and Nrf2 transcripts. Pooled (all cohorts) and paired (intra-cohort) statistical analyses were performed, followed by clustering and principal component analyses (PCA). Although a high heterogeneity among lymphoblast redox profiles was found between cohorts, clustering analysis based on 7 parameters with high chi-square ranking (total SOD activity, oxidative stress levels, catalase transcripts, SOD1 protein levels, metabolic response to mM concentrations of tert-butyl hydroperoxide, glutathione reductase activity, and Nrf2 transcript levels) provided a perfect separation between samples from healthy controls and ALS (undSOD1 and mutSOD1), also visualized in the PCA analysis. Our results show distinct redox signatures in lymphoblasts from mutSOD1, undSOD1, and healthy controls that can be used as therapeutic targets for ALS drug development. Highlights Lymphoblasts from ALS patients present altered redox properties Redox profiling evidenced total SOD and glutathione reductase activities, SOD1 protein levels, DCF fluorescence, catalase and Nrf2 transcripts, and tert-butyl hydroperoxide cytotoxicity, as discriminant features for experimental groups. Lymphoblast redox profiles may be helpful for patient stratification and precision medicine

Membrane-associated NADPH oxidase of malignant cells drives intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling. Cells from late stages of oncogenesis, i. e. bona fide tumor cells, interfere with these signaling pathways through expression of membrane-associated catalase. Transformed cells and tumor cells with inhibited catalase show mutual exclusivity of apoptosis-inducing NO/peroxynitrite and HOCl signaling. H2O2 is the substrate for HOCl synthesis by DUOX-coded peroxidase. However, at high concentrations of H2O2, HOCl signaling is abrogated through the reaction between excess H2O2 and HOCl. Experimental increase in the NO concentration through addition of NO donors, induction of NO synthase (NOS), addition or the NOS substrate arginine, or inhibition of NO dioxygenase(NOD) leads to the inhibition of HOCl signaling, but also to abrogation of H2O2-mediated interference with HOCl signaling. Vice versa, an increase in extracellular H2O2 through increasing the cell density, gamma irradiation or addition of glucose oxidase inhibits NO/peroxynitrite signaling. These mutual reactions between NO and H2O2/HOCl are catalyzed by an iron-mediated reaction cycle, which allows consumption of NO by H2O2/HOCl and vice versa. These reactions explain the preferences of tumor cell lines for NO/peroxynitrite or HOCl signaling after inhibition of their protective catalase, as well as the differential expression of these pathways dependent on the degree of catalase inhibition. The understanding of these reactions allows to rationally modulate quality and strength of ROS/RNS-dependent apoptosis-inducing signaling pathways of malignant cells. The knowledge about these reactions should allow to optimize strategies for tumor therapy that are based on reactivation of intercellular ROS/RNS-dependent apoptosis-inducing signaling of tumor cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/498603v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@4fbdb6org.highwire.dtl.DTLVardef@130063forg.highwire.dtl.DTLVardef@1ea2e67org.highwire.dtl.DTLVardef@18c85cb_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights* NADPH oxidase and peroxidase drive apoptosis-inducing HOCl signaling * NADPH oxidase and NO synthase drive apoptosis-inducing NO/peroxynitrite signaling * H2O2 establishes and interferes with HOCl signaling, dependent on its concentration * NO inhibits HOCl signaling and abrogates H2O2-dependent inhibition of HOCl signaling * H2O2 interferes with NO/peroxnitrite signaling * Mutual interactions of NO and H2O2 are catalysed by an iron-mediated reaction cycle

Decreased ryanodine receptor type 1 (RyR1) protein is a hallmark of recessive RYR1-related myopathies (RyR1-RM), which are caused by recessive mutations in the RYR1 gene. It is not clear how the decrease in the RyR1 protein triggers muscular disorders. Furthermore, it is a hot topic whether a decrease in RyR1 protein levels can also occur during non-RYR1-related myopathies. In this study, we first show that reduced RYR1 transcripts are associated with various human myopathies, and that RyR1 protein levels are significantly decreased in muscle samples analysed in inflammatory myopathies (IM) and mitochondrial myopathies (MM), both of which are non-RYR1-RM. Secondly, proteomic data show that exclusive depletion of RyR1 protein in vitro recapitulates the common altered molecular pathways observed during myopathies. RyR1 protein depletion impairs ER-mitochondria tethering and Ca2+ transfer to mitochondria, decreases mitophagy genes and induces an accumulation of dysfunctional mitochondria. This phenomenon is also associated with altered lipid homeostasis with an increase in deleterious sphingolipid species. Finally, decreased RyR1 protein levels lead to an increase in the ER stress markers GRP78-Bip and CHOP in muscle cell in vitro, and in mouse and human muscles. Overall, our results indicate an important role of RyR1 protein depletion and ER stress in the pathogenesis of myopathies.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum sensing (QS) transcription factor MvfR by interrogating five days post-infection its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs ATP generation, oxidative phosphorylation (OXPHOS), and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR- mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients.

Different muscles exhibit varied susceptibility to degeneration in Amyotrophic Lateral Sclerosis (ALS), a fatal neuromuscular disorder. Extraocular muscles (EOMs) are particularly resistant to ALS progression, and exploring the underlying molecular nature may offer significant therapeutic value. Reactive aldehyde 4-hydroxynonenal (HNE) is implicated in ALS pathogenesis, and Aldh3a1 is an inactivation-resistant intracellular aldehyde dehydrogenase that detoxifies 4-HNE to protect eyes against UV-induced oxidative stress. We detected prominently higher levels of Aldh3a1 in mouse EOMs compared to other muscles under normal physiological conditions. In an ALS mouse model (hSOD1G93A) reaching end-stage, Aldh3a1 expression was maintained high in EOMs, substantially elevated in soleus and diaphragm, but only moderately increased in extensor digitorum longus (EDL) muscle, which endured the most severe pathological remodeling, as demonstrated by unparalleled upregulation of a denervation marker Ankrd1. Importantly, sciatic nerve transection in wildtype mice further confirmed induced Aldh3a1 and Ankrd1 expression in an inverse manner across muscle types in response to denervation. Mechanistically, whole-muscle RNA-Seq and pharmacological tests indicate that higher basal levels of lipid oxidation in soleus and diaphragm muscles may predispose them to stronger Nrf2 antioxidant responses under pathological stress compared to EDL, leading to more prominent Aldh3a1 upregulation. Additionally, the identification of the myoblast fusion marker Mymk as an EOM signature gene suggests that the spontaneous activation of satellite cells contributes to high levels of Aldh3a1 in EOMs. Functionally, adeno-associated virus-mediated overexpression of Aldh3a1 protected myotubes from 4-HNE-induced DNA fragmentation and plasma membrane leakage. It also restored MG53-mediated membrane repair, highlighting its potential for clinical applications.

COVID-19, caused by SARS-CoV-2 involves multiple organs including cardiovascular, pulmonary and central nervous system. Understanding how SARS-CoV-2 infection afflicts diverse organ systems remains challenging1,2. Particularly vexing has been the problem posed by persistent organ dysfunction known as "long COVID," which includes cognitive impairment3. Here we provide evidence linking SARS-CoV-2 infection to activation of TGF-{beta} signaling and oxidative overload. One consequence is oxidation of the ryanodine receptor/calcium (Ca2+) release channels (RyR) on the endo/sarcoplasmic (ER/SR) reticuli in heart, lung and brains of patients who succumbed to COVID-19. This depletes the channels of the stabilizing subunit calstabin2 causing them to leak Ca2+ which can promote heart failure4,5, pulmonary insufficiency 6 and cognitive and behavioral defects7-9. Ex-vivo treatment of heart, lung, and brain tissues from COVID-19 patients using a Rycal drug (ARM210)10 prevented calstabin2 loss and fixed the channel leak. Of particular interest is that neuropathological pathways activated downstream of leaky RyR2 channels in Alzheimers Disease (AD) patients were activated in COVID-19 patients. Thus, leaky RyR2 Ca2+ channels may play a role in COVID-19 pathophysiology and could be a therapeutic target for amelioration of some comorbidities associated with SARS-CoV-2 infection.

Cellular energy metabolism varies depending on tissue and cell type, as well as the availability of energy substrates and energy demands. We recently investigated the variations in cellular metabolism and antioxidant responses in primary bovine vascular endothelial cells (BAECs) under different energetic substrate conditions in vitro, specifically glucose or galactose. In this context, pharmacological agents may affect cells differently depending on their energy metabolism status. In this study, we aimed to characterize the effects of diphenyl diselenide ((PhSe)2), a redox-active molecule known for its prominent cardiovascular effects, on redox-bioenergetic cellular pathways under glycolytic or oxidative conditions in BAECs. Under glucose conditions, (PhSe)2 positively impacted mitochondrial oxidative capacity, as assessed by respirometry, and was associated with changes in mitochondrial cellular dynamics. However, these changes were not observed in cells cultured with galactose. Although (PhSe)2 induced the nuclear translocation of the redox sensitive nuclear factor erythroid 2-related factor 2 (Nrf2) in both glucose and galactose media, Nrf2 remained in the nuclei of cells cultured in galactose for a longer duration. Additionally, activation of another redox sensitive transcription factor, forkhead O3 (FOXO3a) was only detected in galactose media. Notably, (PhSe)2 induced the expression of genes controlling mitochondrial antioxidant capacity and glutathione synthesis and recycling in glucose media, whereas its effects in galactose media were primarily focused on glutathione homeostasis. In conclusion, our findings underscore the critical influence of cellular metabolic status on the antioxidant capacity of redox-active molecules such as (PhSe)2.

AbstractLysine Specific Demethylase 1 (KDM1A / LSD1) regulates mitochondrial respiration and stabilizes HIF-1A (hypoxia-inducible factor 1A). HIF-1A modulates reactive oxygen species (ROS) levels by increasing cellular glucose uptake, glycolysis, and endogenous antioxidants. The role of KDM1A in cellular ROS response has not previously been described. We determined the role of KDM1A in regulating the ROS response and the utility of KDM1A inhibitors in combination with ROS-inducing cancer therapies. Our results show that KDM1A inhibition sensitized cells to oxidative stress and increased total cellular ROS, which was mitigated by treatment with the antioxidant N-acetyl cysteine. KDM1A inhibition decreased basal mitochondrial respiration and impaired induction of HIF-1A after ROS exposure. Overexpression of HIF-1A salvaged cells from KDM1A inhibition enhanced sensitivity to ROS. Thus we found that increased sensitivity of ROS after KDM1A inhibition was mediated by HIF-1A and depletion of endogenous glutathione. We also show that KDM1A-specific inhibitor bizine synergized with antioxidant-depleting therapies, buthionine sulfoximine, and auranofin in rhabdomyosarcoma cell lines (Rh28 and Rh30). In this study, we describe a novel role for KDM1A in regulating HIF- 1A functions under oxidative stress and found that dual targeting of KDM1A and antioxidant systems may serve as an effective combination anticancer strategy.

Cold atmospheric plasma and plasma-treated liquids (PTLs) are emerging promising tools for tumor-targeted cancer treatment, as they preferentially injure tumor cells more than non-malignant cells. Oxidative stress is critical to the antitumor effect, but the oxidant mediating the effect is debatable. Previously, we reported that air plasma-activated medium (APAM) has tumor-selective cytotoxicity in vitro and in vivo. Moreover, an unusual mitochondrial positioning named monopolar perinuclear mitochondrial clustering (MPMC) and nuclear damage proceeds to cell death. We noticed that air plasma generation was accompanied by ozone (O3) formation, leading to suppose the possible role of O3 in the effect of APAM. In this study, we produced an O3-dissolved medium (ODM) and comparatively analyzed its biological effect with APAM. Both agents had comparable amounts of dissolved O3 (dO3), while APAM, but not ODM, contained nitrite and nitrate. Like APAM, ODM could induce apoptosis, nonapoptotic cell death, tubulin remodeling, MPMC, and nuclear shrinkage. Catalase mitigated all these events. The increases in various intracellular and mitochondrial reactive oxygen species (ROS) and lipid peroxides proceeded to cell death, and catalase also prevented them. Conversely, suppressing cellular H2O2 removal systems augmented mitochondrial ROS production and cell death. In contrast, like APAM, ODM minimally increased ROS production and MPMC in non-malignant cells. These results indicate that dO3 is a critical mediator of the actions of APAM, including tumor-selective induction of MPMC and cell death. Our findings suggest ODM could be a more chemically-defined alternative to PTLs in cancer treatment.

Amyotrophic lateral sclerosis (ALS)-associated mutations in Cu/Zn superoxide dismutase (SOD1) reduce folding stability, resulting in misfolding, aggregation, and ultimately cellular toxicity. A great deal of effort has focused on preventing the misfolding and aggregation of SOD1 as a potential therapy for ALS, however, the results have been mixed. Here, we utilise a small-molecule polytherapy of CuATSM and ebselen to mimic the metal delivery and disulfide bond promoting activity of SOD1s cellular chaperone, the copper chaperone for SOD1 (CCS). We find that polytherapy using CuATSM and ebselen is highly effective at reducing inclusion formation in a cell model of SOD1 aggregation, reduces mutant SOD1-associated cell death, and promotes effective maturation of SOD1 beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may be an effective method of treating SOD1-associated ALS.

Aging and degenerative diseases are characterized by the progressive decline in cellular, tissue, and organ function, resulting in a significant reduction in quality of life and posing major medical challenges. This highlights the urgent need to elucidate the underlying mechanisms and to develop innovative therapeutic approaches. In this study, we identify Erdr1 and Mid1 as shared risk factors for aging and multiple degenerative diseases. We propose that they contribute to disease progression by modulating oxidative stress, a well-established driver of aging and degenerative processes. We demonstrate that Erdr1 and Mid1 are both involved in oxidative stress regulation. Notably, Erdr1 undergoes alternative splicing in response to oxidative stress, resulting in reduced production of its antioxidant isoforms (Erdr1-177 and Erdr1-209), while promoting the secretion of its pro-oxidant isoform (Erdr1-145). Moreover, Erdr1-145 exacerbates oxidative damage by activating Mid1, a key inducer of oxidative stress. The Erdr1-Mid1-oxidative stress axis provides a molecular mechanistic basis for their shared role as risk factors for aging and degenerative diseases. Furthermore, we propose therapeutic strategies to mitigate cellular damage by regulating Erdr1 levels, implying a straightforward and effective approach for in situ repair of damage associated with aging and degenerative diseases. HighlightsO_LIErdr1 and Mid1 are shared risk factors in aging and multiple degenerative diseases. C_LIO_LIErdr1 and Mid1 contribute to cellular damage and degeneration by modulating oxidative stress. C_LIO_LIErdr1 undergoes alternative splicing in response to oxidative stress, downregulating antioxidant isoforms (Erdr1-177 and 209) while promoting the secretion of prooxidant isoform (Erdr1-145). C_LIO_LIErdr1-145 robustly primes oxidative stress by promoting Mid1 activation, thereby highlighting the role of the Erdr1-Mid1-oxidative stress axis in cellular damage. C_LIO_LIModulating Erdr1 indicates promising strategies for in situ repair of damage associated with aging and degenerative diseases. C_LI

HIGHLIGHTSO_LID-PUFA diet prevents muscle atrophy in STZ-induced diabetic mice. C_LIO_LID-PUFA diet prevents muscle weakness depending on increased calcium release in STZ-induced diabetic mice. C_LIO_LID-PUFA diet may show a trend to decrease blood glucose in STZ-induced diabetic mice. C_LIO_LID-PUFA diet does not alter ferroptosis-related protein profiles including ACSL4, LPCAT3, ALOX12, and Gpx4. C_LI Oxidative stress and reactive oxygen species (ROS) have been linked to muscle atrophy and weakness. Diabetes increases the oxidative status of lipoproteins in nearly all tissues, including muscle tissues, but the role of lipid ROS on diabetes-induced muscle atrophy is not fully understood. Deuterium reinforced polyunsaturated fatty acids (D-PUFA) are more resistant to ROS-initiated chain reaction of lipid peroxidation than regular hydrogenated PUFA (H-PUFA). In this study, we tested the hypothesis that D-PUFA would protect muscle atrophy induced by diabetes driven by an accumulation of lipid hydroperoxides (LOOH). C57BL/6J mice were dosed with H-PUFA or D-PUFA for four weeks through dietary supplementation and then injected with streptozotocin (STZ) to induce insulin-deficient diabetes. After two weeks, muscles tissues were analyzed for individual muscle mass, force generating capacity and cross-sectional area. Skeletal muscle fibers from diabetic mice exhibited increased total ROS and LOOH. This was abolished by the D-PUFA supplementation regardless of accumulated iron. D-PUFA were found to be protective against muscle atrophy and weakness from STZ-induced diabetes. Prevention of muscle atrophy and weakness by D-PUFA might be independent of ACSL4/LPCAT3/15-LOX pathway. These findings provide novel insights into the role of LOOH in the mechanistic link between oxidative stress and diabetic myopathy and suggest a novel therapeutic approach to diabetes-associated muscle weakness.

Irritation causes the recruitment and activation of neutrophils in the stressed airways. This process is known as neutrophilic inflammation. This process results in myeloperoxidase (MPO), an enzyme contained inside neutrophil azurophilic granules, being released as neutrophil extracellular traps (NETs), which also contain genomic DNA, modified histones, and other proteins. In the airways, released MPO can be taken up by bystander tissue epithelial cells. MPO is the only mammalian peroxidase enzyme that under physiological conditions produces hypochlorite (HOCl). Intracellularly produced HOCl may damage the cell genome, with the intermediacy of DNA-centered free radicals, which upon reaction with molecular oxygen decay to mutagenic end-oxidation products, such as 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxo-dGuo). Herein, we aimed to test whether HOCl-induced DNA-centered radicals precede the oxidation of DNA and mutagenesis in A549 human lung epithelial cells as an in vitro model that resembles neutrophilic inflammation in irritated airways. Interestingly, by trapping HOCl-induced DNA-centered radicals, the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) blocks the formation of 8-oxo-dGuo and possibly other end-oxidation products, forming DNA-DMPO nitrone adducts, thus reducing mutagenesis in the hypoxanthine phosphoribosyl transferase (hrpt) gene, one of the most sensitive genes to oxidative damage. P53 is a transcription factor known as the master regulator of the cell response to genomic damage. By trapping DNA-centered radicals, DMPO also blocks the translocation of p53 to the cell nucleus, suggesting that by trapping DNA-centered radicals with DMPO, end-oxidation products are prevented, and the cell response to genomic damage is not sensed. DMPO traps DNA-centered radicals, reduces 8-oxo-dGuo accumulation, and blocks hrpt gene mutation. Trapping DNA-centered radicals to reduce the accumulation of HOCl-induced mutagenic end-oxidation products in the genome of bystander cells, which have taken MPO from the inflammatory milieu, will provide new therapeutic avenues to reduce genotoxic damage at sites of neutrophilic inflammation, such as in the irritated airways.

Single molecule mass photometry was used to study the dynamic equilibria of the ubiquitous and highly abundant 2-Cysteine peroxiredoxins (2-CysPRX). 2-CysPRXs adopt distinct functions in all cells dependent on their oligomeric conformation ranging from dimers to decamers and high molecular weight aggregates (HMW). The oligomeric state depends on the redox state of their catalytic cysteinyl residues. To which degree they interconvert, how the interconversion is regulated, and how the oligomerisation propensity is organism specific remains, however, poorly understood. The dynamics differs between wild-type and single point mutants affecting the oligomerization interfaces, with concomitant changes to function. Titrating concentration and redox state of Arabidopsis thaliana and human 2-CysPRXs revealed features conserved among all 2-CysPRX and clear differences concerning oligomer transitions, the occurrence of transition states and the formation of HMW which are associated with chaperone activity or storage. The results indicate functional differentiation of human 2-CysPRXs. Our results point to a diversified functionality of oligomerization for 2-CysPRXs and illustrate the power of mass photometry to non-invasively quantify oligomer distributions in a redox environment. This knowledge is important to fully address and model PRX function in cell redox signaling e.g., in photosynthesis, cardiovascular and neurological diseases or carcinogenesis.

Mitochondria are essential for metabolic homeostasis and neuronal function, extending beyond ATP production to roles in cell signaling, inflammation, and stress responses. Mitochondrial dysfunction, marked by abnormal morphology, ATP deficiency, and oxidative stress, is a key feature of aging-related diseases and neurodegenerative disorders like Parkinsons. Given the importance of mitochondrial homeostasis to brain function, this study aimed to determine the possible vitamin D (VD3) effects on mitochondrial susceptibility to Ca2+-induced mitochondrial permeability transition pore (mPTP), bioenergetics in brain mitochondria, and redox balance. We demonstrated that VD3 protects isolated brain mitochondria. Male rats were divided into control and VD3-treated groups. Brain mitochondria were isolated for assessments of Ca2+-induced mitochondrial swelling secondary to MPTP opening, oxygen consumption (states 3 - ADP-stimulated and state 4 - in the presence of oligomycin), and the respiratory control ratio (RCR). Oxidative stress parameters (nitrite and lipid peroxidation), superoxide dismutase (SOD) activity, and reduced glutathione (GSH) levels were also evaluated. The results revealed that VD3 treatment blocked Ca2+-induced mitochondrial swelling secondary to MPTP opening. Additionally, VD3 improved mitochondrial RCR compared to controls, in the presence of complex I (malate/glutamate) and complex II (succinate) substrates, reduced mitochondrial succinate-driven H2O2 release, and enhanced SOD activity and GSH levels. These changes occurred in parallel with decreased nitrite and TBARS formation. These results suggest that vitamin D{square} confers mitochondrial neuroprotection, emphasizing its prospective role in maintaining neuronal homeostasis and mitigating neurodegenerative processes.

Cu/Zn superoxide dismutase 1 (SOD1) is essential for maintaining neural health. Its functions include modulating metabolism, maintaining redox balance, regulating transcription, besides eliminating superoxide radicals, which are achieved through various post-translational modifications (PTMs). Consequently, unusual PTMs in SOD1 can impair its functionality and stability, leading to the accumulation of misfolded SOD1 and the increase of oxidative stress markers, hallmarks of Amyotrophic Lateral Sclerosis (ALS). Although SOD1 has been extensively studied, especially regarding its role in ALS, relatively little is known about how aging and mutations affect SOD1 PTMs. This study aimed to evaluate the effect of oxidative stress induced by chronological aging on PTMs of human SOD1: wild-type (WT) and A5V SOD1, a severe ALS-related mutant. To do this, both hSOD1 forms were expressed in Saccharomyces cerevisiae lacking the SOD1 gene, and then purified from extracts of stressed and non-stressed cells. PTMs were analyzed using mass spectrometry, observing the modification of WT and mutant human SOD1 in both conditions. We observed changes in the levels of damage, including oxidation, formylation, and carboxylation, such as oxidized tryptophan 33, associated with prion-like propagation of SOD1 misfolding. Increased levels of this PTM appeared in WT SOD1 after aging and in A5V SOD1. Acetylation and succinylation were also found on lysines. Some of these modifications already have described functions in the literature, while others still lack a defined role. Interestingly, the levels of these physiological PTMs differed between WT and mutant SOD1, providing important information for elucidating the molecular mechanisms of ALS involving SOD1.