Cytoprotective Genes Research Articles

7447 Rare KSR2 Variants: The Effect of Glycine and N-acetylcysteine (GlyNAC) Supplementation

Abstract Disclosure: J. Youn: None. Y. Carcamo-Bahena: None. E. Lartigue: None. S. Sisley: None. Kinase suppressor of ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Of note, disruption of the kinase domain of KSR2 in humans and mice causes obesity by reducing metabolic rate, suggesting its important role in energy homeostasis. In the present study, we identified two variants (P189L, T290A) with mutations located in the proximal CA2 region from children with severe, early-onset obesity. Since variants in this region have not been confirmed to cause obesity, we generated plasmid constructs containing these KSR2 mutations by site directed mutagenesis. The HEK293 cells transfected with KSR2 plasmid construct containing P189L or T290A had reduced binding affinity with AMPK and phosphorylation of AMPK without affecting KSR2 protein integrity. These variants also significantly reduced cellular metabolic rates measured by Seahorse analysis in C2C12 cells, suggesting that KSR2 variants in the proximal CA2 region can result in impaired KSR2 function, subsequently leading to a decrease in cellular metabolic rate. In addition, these KSR2 variants decreased protein levels of NRF-2, a transcription factor of cytoprotective genes regulating the antioxidant glutathione (GSH) pathway compared to wild type KSR2 which augmented NRF2 levels. We also observed lower levels of total GSH and GSH/Glutathione disulfide (GSSG) ratio and higher levels of thiobarbituric acid reactive substances (TBARS), an indicator of oxidative stress, indicating an imbalance of antioxidant and oxidant with proximal KSR2 variants. However, treatment of cells with Glycine and N-acetylcysteine (GlyNAC) reversed the detrimental effects of KSR2 variants by normalizing protein expression levels of NRF-2, GSH/GSSG ratios and TBARS levels. Taken together, we show strong evidence that two new KSR2 variants, P189L and T290A, cause disruption of KSR2 function. Additionally, we also report a novel role of KSR2 in maintaining redox homeostasis suggesting that increased oxidative stress following GSH deficiency may play an important role in the development of obesity from KSR2 variants. Presentation: 6/1/2024

Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis

ABSTRACT The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner. Abbreviation: ARE: antioxidant response element; Dup: deubiquitinase specific for phosphoribosyl-linked serine ubiquitination; ER: endoplasmic reticulum; h.p.i: hours post infection; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; KEAP1: kelch like ECH associated protein 1; KIR: KEAP1-interacting region; LIR: LC3-interacting region; NES: nuclear export signal; NFKB/NF-κB: nuclear factor kappa B; NLS: nuclear localization signal; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1 domain: Phox1 and Bem1 domain; PR-Ub: phosphoribosyl-linked serine ubiquitination; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; tBHQ: tertiary butylhydroquinone; TUBE2: tandem ubiquitiin binding entity 2; UBA domain: ubiquitin-associated domain.

Discovery and Optimization of a Series of Vinyl Sulfoximine-Based Analogues as Potent Nrf2 Activators for the Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of the central nervous system (CNS), which leads to demyelination, axonal loss, and neurodegeneration. Increased oxidative stress and neurodegeneration have been implicated in all stages of MS, making neuroprotective therapeutics a promising strategy for its treatment. We previously have reported vinyl sulfones with antioxidative and anti-inflammatory properties that activate nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that induces the expression of cytoprotective genes against oxidative stress. In this study, we synthesized vinyl sulfoximine derivatives by modifying the core structure and determined therapeutic potential as Nrf2 activators. Among them, 10v effectively activated Nrf2 (EC50 = 83.5 nM) and exhibited favorable drug-like properties. 10v successfully induced expression of Nrf2-dependent antioxidant enzymes and suppressed lipopolysaccharide (LPS)-induced inflammatory responses in BV-2 microglial cells. We also confirmed that 10v effectively reversed disease progression and attenuated demyelination in an experimental autoimmune encephalitis (EAE) mouse model of MS.

Reno‐protective potential of sudachitin to mitigate paraquat instigated renal toxicity via regulating Nrf2/Keap1 pathway: An inflammatory, apoptotic and histopathological assessment

This investigation was executed to assess the protective effects of SCN to counteract PQ instigated renal damage in albino rats (Rattus norvegicus). Twenty‐four rats were apportioned in 4 different groups i.e., a control group, PQ (5mg/kg) intoxicated, PQ (5mg/kg) + SCN (20mg/kg) exposed & SCN (20mg/kg) only administrated group. Our findings explored that exposure to PQ lessened the expressions of Nrf2 and its cytoprotective genes while escalating the expression of keap1. Furthermore, PQ intoxication reduced the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSR), & glutathione (GSH), while upregulating the levels of malondialdehyde (MDA) & reactive oxygen species (ROS). Moreover, intoxication to PQ significantly increased the levels of neutrophil gelatinous‐associated lipocalin (NGAL), urea, kidney injury molecule‐1(KIM‐1) as well as creatine while reducing creatine clearance. Additionally, exposure to PQ upregulated the levels of inflammatory markers including interleukin‐6 (IL‐6), tumor necrosis‐ α (TNF‐ α), nuclear factor‐ κB (NF‐κB), interleukin 1beta (IL‐1β), & cyclo‐oxygenase‐2 (COX‐2). Moreover, PQ administration upregulated the expression of Bax and Caspase‐3 while downregulating the expressions of Bcl‐2. Besides, PQ exposure prompted various histopathological damages in renal tissues. Nonetheless, SCN substantially restored aforementioned alterations in renal tissues owing to its anti‐oxidative, anti‐inflammatory and anti‐apoptotic potential.

NRF2 Activation by AR-20007 Preserves Renal Tubular Epithelial Cells from Antimycin A-Induced Cell Death via Glutathione Metabolism Regulation.

Oxidative stress plays a crucial role in the development and progression of various kidney diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is the primary transcription factor that protects cells from oxidative stress by regulating cytoprotective genes including those involved in the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and cell death. Antimycin A, an inhibitor of complex III of the electron transport chain, causes oxidative stress and reduces GSH levels. In this study, we induced mitochondrial damage in rat renal proximal tubular cells using antimycin A and investigated cellular viability and levels of NRF2 and GSH. Treatment with antimycin A altered the expression of antioxidant genes, including reduction in the transcription of glutathione-cysteine ligase subunits (Gclc and Gclm) and glutathione reductase (Gsr1), followed by a reduction in total GSH content with a concomitant decrease in NRF2 protein expression. AR-20007, previously described as an NRF2 activator, stabilizes and increases NRF2 protein expression in cells. By stimulating NRF2, AR-20007 increased the expression of antioxidant and detoxifying enzymes, thereby enhancing protection against oxidative stress induced by antimycin A. These data suggest that NRF2 activation effectively inhibits antimycin A-induced oxidative stress and that NRF2 may be a promising therapeutic target for preventing cell death during acute kidney injury.

Lactobacilli Cell-Free Supernatants Modulate Inflammation and Oxidative Stress in Human Microglia via NRF2-SOD1 Signaling

Microglia are macrophage cells residing in the brain, where they exert a key role in neuronal protection. Through the gut–brain axis, metabolites produced by gut commensal microbes can influence brain functions, including microglial activity. The nuclear factor erythroid 2-related factor 2 (NRF2) is a key regulator of the oxidative stress response in microglia, controlling the expression of cytoprotective genes. Lactobacilli-derived cell-free supernatants (CFSs) are postbiotics that have shown antioxidant and immunomodulatory effects in several in vitro and in vivo studies. This study aimed to explore the effects of lactobacilli CFSs on modulating microglial responses against oxidative stress and inflammation. HMC3 microglia were exposed to lipopolysaccaride (LPS), as an inflammatory trigger, before and after administration of CFSs from three human gut probiotic species. The NRF2 nuclear protein activation and the expression of NRF2-controlled antioxidant genes were investigated by immunoassay and quantitative RT-PCR, respectively. Furthermore, the level of pro- and anti-inflammatory cytokines was evaluated by immunoassay. All CFSs induced a significant increase of NRF2 nuclear activity in basal conditions and upon inflammation. The transcription of antioxidant genes, namely heme oxygenase 1, superoxide dismutase (SOD), glutathione-S transferase, glutathione peroxidase, and catalase also increased, especially after inflammatory stimulus. Besides, higher SOD1 activity was detected relative to inflamed microglia. In addition, CFSs pre-treatment of microglia attenuated pro-inflammatory TNF-α levels while increasing anti-inflammatory IL-10 levels. These findings confirmed that gut microorganisms’ metabolites can play a relevant role in adjuvating the microglia cellular response against neuroinflammation and oxidative stress, which are known to cause neurodegenerative diseases.Graphical Gut-brain crosstalk: molecular point of view. Metabolites contained in the supernatant derived from Lactobacilli can cross the gut barrier and reach the central nervous system, where they are taken up by microglial cells. They induce the activation of the NRF2 pathway and the production of inflammatory mediators. This interaction attenuates two important events: oxidation (with high levels of NRF2) and inflammation (with high levels of IL-10 and low levels of TNF-α).

Molecular basis of the interactions between the disordered Neh4 and Neh5 domains of Nrf2 and CBP/p300 in oxidative stress response.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor that functions in maintaining redox homeostasis in cells. It mediates the transcription of cytoprotective genes in response to environmental and endogenous stresses to prevent oxidative damage. Thus, Nrf2 plays a significant role in chemoprevention. However, aberrant activation of Nrf2 has been shown to protect cancer cells from apoptosis and contribute to their chemoresistance. The interaction between Nrf2 and CBP is critical for the gene transcription activation. CBP and its homologue p300 interact with two transactivation domains in Nrf2, Neh4, and Neh5 domains through their TAZ1 and TAZ2 domains. To date, the molecular basis of this crucial interaction is not known, hindering a more detailed understanding of the regulation of Nrf2. To close this knowledge gap, we have used a set of biophysical experiments to dissect the Nrf2-CBP/p300 interactions. Structural properties of Neh4 and Neh5 and their binding with the TAZ1 and TAZ2 domains of CBP/p300 were characterized. Our results show that the Neh4 and Neh5 domains of Nrf2 are intrinsically disordered, and they both can bind the TAZ1 and TAZ2 domains of CBP/p300 with micromolar affinities. The findings provide molecular insight into the regulation of Nrf2 by CBP/p300 through multi-domain interactions.

The Antinociceptive Role of Nrf2 in Neuropathic Pain: From Mechanisms to Clinical Perspectives.

Neuropathic pain, a chronic condition resulting from nerve injury or dysfunction, presents significant therapeutic challenges and is closely associated with oxidative stress and inflammation, both of which can lead to mitochondrial dysfunction. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, a critical cellular defense mechanism against oxidative stress, has emerged as a promising target for neuropathic pain management. Nrf2 modulators enhance the expression of antioxidant and cytoprotective genes, thereby reducing oxidative damage, inflammation, and mitochondrial impairment. This review explores the antinociceptive effects of Nrf2, highlighting how pharmacological agents and natural compounds may be used as potential therapeutic strategies against neuropathic pain. Although preclinical studies demonstrate significant pain reduction and improved nerve function through Nrf2 activation, several clinical challenges need to be addressed. However, emerging clinical evidence suggests potential benefits of Nrf2 modulators in several conditions, such as diabetic neuropathy and multiple sclerosis. Future research should focus on further elucidating the molecular role of Nrf2 in neuropathic pain to optimize its modulation efficacy and maximize clinical utility.

Abstract We122: Suppression of defender against cell death 1 (Dad1) induces cardiomyocyte death by regulating cell adhesion proteins.

Introduction: Suppression of cardiomyocyte loss is considered a promising strategy for the prevention of the onset of heart failure (HF). Previously, defender against cell death 1 ( Dad1 ) was identified as a cytoprotective gene. Dad1 forms a complex with staurosporine and temperature sensitive 3A (Stt3A), a catalytic subunit of oligosaccharyltransferase (OST) complex which is responsible for N-glycosylation. However, the function of Dad1 in cardiomyocytes remains unknown. Hypothesis: The disruption of cell-extracellular matrix interactions results in cell death, known as anoikis. Because various cell adhesion proteins are N-glycosylated, we hypothesized that Dad1 suppressed anoikis by regulating N-glycosylation of cell adhesion proteins in cardiomyocytes. Methods: Neonatal rat cardiomyocytes were cultured and the expression of Dad1 or Stt3A was knocked down using siRNA. The protein expression was assessed with immunoblot analysis, and cell viability was evaluated using a CellTiter-Blue assay kit. Results: The suppression of Dad1 using siRNAs reduced cardiomyocyte viability ( p <0.01, n=4). Interestingly, suppression of Dad1 impaired the N-glycosylation of integrin β1 and decreased the expression of mature integrin β1 ( p <0.01, n=3). These changes inactivated integrin-mediated signal transduction through dephosphorylation of focal adhesion kinase ( p <0.01, n=5). In addition, enhanced cell-extracellular matrix interactions by using adhesamine rescued the cardiomyocyte death by Dad1 knockdown ( p <0.01, n=4). Next, we pursued the relationship between Dad1 and Stt3A. Dad1 knockdown decreased the expression of Stt3A ( p <0.01, n=6). Suppression of Stt3A induced cardiomyocyte death accompanied by the changes of cell adhesion proteins ( p <0.01, n=4). Finally, double knockdown of Dad1 and Stt3A did not induce a further reduction in cell viability compared with the knockdown of only Dad1 ( p <0.05, n=4), suggesting that cardiomyocyte death by Dad1 knockdown was dependent on Stt3A. Conclusion: Dad1 is required for the stabilization of Stt3A and suppresses cardiomyocyte anoikis by regulating N-glycosylation of cell adhesion proteins in an OST-dependent manner. Dad1-mediated cardiomyocyte survival could be a therapeutic target against HF.

Use of Microalgae-Derived Astaxanthin to Improve Cytoprotective Capacity in the Ileum of Heat-Induced Oxidative Stressed Broilers.

The gastrointestinal tract has a pivotal role in nutrient absorption, immune function, and overall homeostasis. The ileum segment of the small intestine plays respective roles in nutrient breakdown and absorption. The purpose of this study was to investigate the impact of heat-induced oxidative stress and the potential mitigating effects of an astaxanthin antioxidant treatment on the ileum of broilers. By comparing the growth performance and gene expression profiles among three groups-thermal neutral, heat stress, and heat stress with astaxanthin-thermal neutral temperature conditions of 21-22 °C and heat stress temperature of 32-35 °C, this research aims to elucidate the role of astaxanthin in supporting homeostasis and cellular protection in the ileum. Results showed both treatments under heat stress experienced reduced growth performance, while the group treated with astaxanthin showed a slightly lesser decline. Results further showed the astaxanthin treatment group significantly upregulated in the cytoprotective gene expression for HSF2, SOD2, GPX3, and TXN, as well as the upregulation of epithelial integrity genes LOX, CLDN1, and MUC2. In conclusion, our experimental findings demonstrate upregulation of cytoprotective and epithelial integrity genes, suggesting astaxanthin may effectively enhance the cellular response to heat stress to mitigate oxidative damage and contribute to cytoprotective capacity.

Myeloid Nrf2 Protects against Neonatal Oxidant-Stress-Induced Lung Inflammation and Alveolar Simplification in Mice.

Bronchopulmonary dysplasia (BPD) is a chronic condition affecting preterm infants, characterized by lung alveolar simplification/hypoalveolarization and vascular remodeling. The nuclear factor erythroid 2 like 2 (Nfe2l2, or Nrf2) plays a critical role in the cytoprotective response to neonatal hyperoxia, and its global deficiency exacerbates hypoalveolarization in mice. The abnormal recruitment and activation of myeloid cells are associated with the pathogenesis of BPD. Therefore, we employed a genetic approach to investigate the role of myeloid Nrf2 in regulating hyperoxia-induced hypoalveolarization. Pups, both wild-type (Nrf2f/f) and those with a myeloid Nrf2 deletion (abbreviated as Nrf2∆/∆mye), were exposed to hyperoxia for 72 h at postnatal day 1 (Pnd1), and then sacrificed at either Pnd4 or Pnd18 following a two-week recovery period. We analyzed the hypoalveolarization, inflammation, and gene expression related to cytoprotective and inflammatory responses in the lungs of these pups. The hypoalveolarization induced by hyperoxia was significantly greater in Nrf2∆/∆mye pups compared to their Nrf2f/f counterparts (35.88% vs. 21.01%, respectively) and was accompanied by increased levels of inflammatory cells and IL-1β activation in the lungs. Antioxidant gene expression in response to neonatal hyperoxia was lower in Nrf2∆/∆mye pups compared to their Nrf2f/f counterparts. Furthermore, Nrf2-deficient macrophages exposed to hyperoxia exhibited markedly decreased cytoprotective gene expression and increased IL-1β levels compared to Nrf2-sufficient cells. Our findings demonstrate the crucial role of myeloid Nrf2 in mitigating hyperoxia-induced lung hypoalveolarization and inflammatory responses in neonatal mice.

Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study.

Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear. We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd. Male wild-type (WT), global Nrf2-knockout () and myeloid-specific Nrf2 knockout [Nrf2(M)-KO] mice were administered Cd (50 or ) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or ) with a combination of genetic and chemical modulations targeting NRF2 and NRF1. Compared with relevant control mice, global or Nrf2(M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2-deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to ) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L-Nrf1 silenced (via lentiviral means) Nrf2-knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2-KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants -acetyl-l-cysteine () and mitoquinone mesylate (MitoQ; ) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2-KD cells. This invivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

Keap1 Negatively Regulates Transcription of Three Counter-Defense Genes and Susceptibility to Plant Toxin Gossypol in Helicoverpa armigera.

Expressions of a wide range of cytoprotective counter-defense genes are mainly regulated by the Keap1-Nrf2-ARE signaling pathway in response to oxidative stress from xenobiotics. Gossypol is the major antiherbivore secondary metabolite of cotton, but how the polyphagous pest Helicoverpa armigera copes with this phytochemical to utilize its favorite host plant cotton remains largely elusive. In this study, we first suppressed the Keap1 gene in newly hatched larvae of cotton bollworm by feeding them the siRNA diet for 4 days. All of the larvae were subsequently fed the artificial diet supplied with gossypol or the control diet for 5 days. We identified that the knockdown of the Keap1 gene significantly decreased larval mortality and significantly increased the percentages of larval survival, reaching the fourth instar, compared with ncsiRNA when exposed to a diet containing gossypol. Three counter-defense genes CYP9A17, CYP4L11 and UGT41B3, which were related to the induction or metabolism of gossypol according to the report before, were all significantly up-regulated after the knockdown of the Keap1 gene. The Antioxidant Response Elements (AREs) were also detected in the promoter regions of the three counter-defense genes above. These data indicate that the suppression of the Keap1 gene activates the Keap1-Nrf2-ARE signaling pathway, up-regulates the expressions of counter-defense genes involved in the resistance of oxidative stress and finally contributes to reducing the susceptibility of gossypol. Our results provide more knowledge about the transcriptional regulation mechanisms of counter-defense genes that enable the cotton bollworm to adapt to the diversity of host plants including cotton.

Sex-Dependent Increases of Hepatic Acylcarnitines by Myocardial Infarction

Many different diseases and environmental stressors can lead to an increase in oxidative stress. Urban air pollution leads to inhalation of airborne particulate matter in high traffc environments. This has been linked to chronic oxidative stress and increased risk or severity of myocardial infarction (MI). MI is one cause of acute oxidative stress. A key player for cellular defense against oxidative stress is activation of Nrf2, a transcription factor that controls the expression of key antioxidant and cytoprotective genes. Although Nrf2 has been reported to regulate genes for anabolic or lipid metabolism, little is known about the impact of Nrf2 or MI on the biochemical metabolism in the liver. Because the liver is a center for metabolism, metabolomic landscapes were determined in a model of chronic oxidative stress by Nrf2 knockout or acute oxidative stress by induction of MI via left anterior descending coronary artery ligation. 1353 compounds were detected with a quality control coeffcient variation value <20% using LC-MS/MS based non-targeted metabolomics. Differences in the baseline metabolic profiles of male versus female lead to comparison of Nrf2 knockout or MI in a sex specific manner. In male wild type mice, the liver responded to MI by elevating several carnitine derivatives, including linoleoyl carnitine, palmitoyl carnitine, and (11Z, 14Z)-eicosadienoyl carnitine. However, none of these changes were observed in female wild type mice. Either male or female Nrf2 knockout mice failed to respond to MI by elevating carnitines. These acylcarnitine increases were confirmed with a non-targeted lipidomics analysis. The elevated acylcarnitine levels correlate with an increase in carnitine palmitoyl transferase 1, CPT1, the enzyme responsible for the conjugation of carnitine and long-chain fatty acids. After this conjugation, acylcarnitines are transported into the mitochondria via carnitine-acylcarnitine translocase. Once inside the mitochondria, the carnitine moiety is removed via CPT2 and the long-chain fatty acid can be used for beta-oxidation to produce ATP. This data suggest that male mice are responding to MI by altering energy metabolism and increasing carnitines to compensate for energy production. Nrf2 appears to play a role in such metabolic reprogramming. The research programs under the direction of Dr. Qin M. Chen have been supported by NIH R01 GM125212, R01 GM126165, Holsclaw Endowment, the University of Arizona College of Pharmacy start-up fund, and NIEHS T32 ES007091. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of the mitochondrial integrated stress response and ATF4 by acute contractile activity in mouse muscle

In the context of energetically demanding conditions, such as exercise, skeletal muscle relies on mitochondria to support its metabolic requirements. Maintenance of mitochondria and their bioenergetic availability is thus critical to sustain the metabolic flexibility of muscle. The quality of the organelle network is highly regulated by the coordination of mitochondrial quality control (MQC) processes. One MQC pathway that has garnered recent interest is the integrated stress response (ISR), which is activated in response to a variety of stimuli. The transcription factor ATF4, denoted as the main effector of this response pathway, serves to ameliorate cellular stress by upregulating a plethora of cytoprotective genes, such as CHOP and ATF5. Current literature has shown that the ISR is activated upon mitochondrial stress/perturbations to restore optimal organelle function and health. However, the precise mechanism that constitutes the activation of ISR and thus the response of ATF4 following acute exercise-induced stress is unknown. Therefore, our objective is to determine the extent of ISR/ATF4 induction in vivo and to test whether this pathway is required for the maintenance of muscle function. To investigate this, a mouse in situ hindlimb protocol was utilized to acutely stimulate muscles at 0.25-1 tetani/per second for 9 mins, followed by a 1-hour recovery period. The kinases, CAMKIIα and JNK2, were robustly phosphorylated/activated 6-fold immediately following the protocol. The initial stage of ISR activation, reflected in the ratio of phosphorylated to total-eIF2α protein levels, was also increased in response to acute contractile activity, evident in the recovery phase. Downstream of ISR activation, the total protein expression of ATF4 remained unchanged, however, contractile activity induced an increase in the localization of ATF4 to the nucleus. Robust 2-fold increases in the mRNA expression of ATF4 and CHOP were also observed and enhanced following the recovery period. Changes in ATF4 mRNA appeared to be independent of transcriptional activation, as assessed using an ATF4 promoter-reporter plasmid. An in vitro cell free mRNA decay assay revealed an increase in ATF4 mRNA stability post-stimulation, possibly as a result of cellular compartmental changes in the RNA binding proteins, HuR and CUGBP1. RNA sequencing analyses also confirmed the observed increase in related ISR genes following an exhaustive bout of exercise. These data suggest that acute contractile activity is indeed suffcient to induce mitochondrial stress and activate the ISR, corresponding to the induction of ATF4. Future work will reveal the underlying mitochondrial signal responsible for activating this pathway, as well as the downstream consequences for the regulation of MQC processes in maintaining mitochondrial homeostasis. This work was supported by funds from the Canadian Institutes for Health Research (CIHR). Victoria C. Sanfrancesco is the recipient of the CIHR Frederick Banting and Charles Best Canada Graduate Scholarship-Master's (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of Nrf2 signaling pathways in nerve damage repair.

The protein, Nuclear factor-E2-related factor 2 (Nrf2), is a transitory protein that acts as a transcription factor and is involved in the regulation of many cytoprotective genes linked to xenobiotic metabolism and antioxidant responses. Based on the existing clinical and experimental data, it can be inferred that neurodegenerative diseases are characterized by an excessive presence of markers of oxidative stress (OS) and a reduced presence of antioxidant defense systems in both the brain and peripheral tissues. The presence of imbalances in the homeostasis between oxidants and antioxidants has been recognized as a substantial factor in the pathogenesis of neurodegenerative disorders. The dysregulations include several cellular processes such as mitochondrial failure, protein misfolding, and neuroinflammation. These dysregulations all contribute to the disruption of proteostasis in neuronal cells, leading to their eventual mortality. A noteworthy component of Nrf2, as shown by recent research undertaken over the last decade, is to its role in the development of resistance to OS. Nrf2 plays a pivotal role in regulating systems that defend against OS. Extant research offers substantiation for the protective and defensive roles of Nrf2 in the context of neurodegenerative diseases. The purpose of this study is to provide a comprehensive analysis of the influence of Nrf2 on OS and its function in regulating antioxidant defense systems within the realm of neurodegenerative diseases. Furthermore, we evaluate the most recent academic inquiries and empirical evidence about the beneficial and potential role of certain Nrf2 activator compounds within the realm of therapeutic interventions.

Mechano-induced cell metabolism disrupts the oxidative stress homeostasis of SAOS-2 osteosarcoma cells.

There has been an increasing focus on cancer mechanobiology, determining the underlying-induced changes to unlock new avenues in the modulation of cell malignancy. Our study used LC-MS untargeted metabolomic approaches and real-time polymerase chain reaction (PCR) to characterize the molecular changes induced by a specific moderate uniaxial stretch regimen (i.e., 24h-1Hz, cyclic stretch 0,5% elongation) on SAOS-2 osteosarcoma cells. Differential metabolic pathway analysis revealed that the mechanical stimulation induces a downregulation of both glycolysis and the tricarboxylic acid (TCA) cycle. At the same time, the amino acid metabolism was found to be dysregulated, with the mechanical stimulation enhancing glutaminolysis and reducing the methionine cycle. Our findings showed that cell metabolism and oxidative defense are tightly intertwined in mechanically stimulated cells. On the one hand, the mechano-induced disruption of the energy cell metabolism was found correlated with an antioxidant glutathione (GSH) depletion and an accumulation of reactive oxygen species (ROS). On the other hand, we showed that a moderate stretch regimen could disrupt the cytoprotective gene transcription by altering the expression levels of manganese superoxide dismutase (SOD1), Sirtuin 1 (SIRT1), and NF-E2-related factor 2 (Nrf2) genes. Interestingly, the cyclic applied strain could induce a cytotoxic sensitization (to the doxorubicin-induced cell death), suggesting that mechanical signals are integral regulators of cell cytoprotection. Hence, focusing on the mechanosensitive system as a therapeutic approach could potentially result in more effective treatments for osteosarcoma in the future.

Role of microRNA in Oxidative Stress

An imbalance between the formation of reactive oxygen species (ROS) and the reaction of antioxidant proteins is referred to as oxidative stress. NFE2L2/Nrf2, also known as nuclear factor erythroid-derived 2-related factor 2, is a critical enabler of cytoprotective responses to oxidative and electrophilic insults. When Nrf2 is activated, it triggers the transcription of numerous cytoprotective genes, whose promoter regions contain antioxidant response elements (AREs). In recent times, the regulation of Nrf2 by miRNAs has garnered significant attention, among the various mechanisms that govern Nrf2 signaling. It has been reported that a number of miRNAs directly suppress the expression of Nrf2s, which in turn negatively regulates the Nrf2-dependent cellular cytoprotective response. Furthermore, it has been shown that Nrf2 itself regulates miRs, which carry out some of Nrf2’s unique metabolic regulation functions. Here, we provide an overview of the functions and mechanisms of action of miRs as downstream effectors of Nrf2, as well as in their regulation of its activity.

The Antioxidant Drug Edaravone Binds to the Aryl Hydrocarbon Receptor (AHR) and Promotes the Downstream Signaling Pathway Activation.

A considerable effort has been spent in the past decades to develop targeted therapies for the treatment of demyelinating diseases, such as multiple sclerosis (MS). Among drugs with free radical scavenging activity and oligodendrocyte protecting effects, Edaravone (Radicava) has recently received increasing attention because of being able to enhance remyelination in experimental in vitro and in vivo disease models. While its beneficial effects are greatly supported by experimental evidence, there is a current paucity of information regarding its mechanism of action and main molecular targets. By using high-throughput RNA-seq and biochemical experiments in murine oligodendrocyte progenitors and SH-SY5Y neuroblastoma cells combined with molecular docking and molecular dynamics simulation, we here provide evidence that Edaravone triggers the activation of aryl hydrocarbon receptor (AHR) signaling by eliciting AHR nuclear translocation and the transcriptional-mediated induction of key cytoprotective gene expression. We also show that an Edaravone-dependent AHR signaling transduction occurs in the zebrafish experimental model, associated with a downstream upregulation of the NRF2 signaling pathway. We finally demonstrate that its rapid cytoprotective and antioxidant actions boost increased expression of the promyelinating Olig2 protein as well as of an Olig2:GFP transgene in vivo. We therefore shed light on a still undescribed potential mechanism of action for this drug, providing further support to its therapeutic potential in the context of debilitating demyelinating conditions.

Ceramide kinase-mediated C1P metabolism attenuates acute liver injury by inhibiting the interaction between KEAP1 and NRF2

Acute liver injury is the basis of the pathogenesis of diverse liver diseases. However, the mechanism underlying liver injury is complex and not completely understood. In our study, we revealed that CERK, which phosphorylates ceramide to produce ceramide-1-phosphate (C1P), was the sphingolipid pathway-related protein that had the most significantly upregulated expression during acute liver injury. A functional study confirmed that CERK and C1P attenuate hepatic injury both in vitro and in vivo through antioxidant effects. Mechanistic studies have shown that CERK and C1P positively regulate the protein expression of NRF2, which is a crucial protein that helps maintain redox homeostasis. Furthermore, our results indicated that C1P disrupted the interaction between NRF2 and KEAP1 by competitively binding to KEAP1, which allowed for the nuclear translocation of NRF2. In addition, pull-down assays and molecular docking analyses revealed that C1P binds to the DGR domain of KEAP1, which allows it to maintain its interaction with NRF2. Importantly, these findings were verified in human primary hepatocytes and a mouse model of hepatic ischemia‒reperfusion injury. Taken together, our findings demonstrated that CERK-mediated C1P metabolism attenuates acute liver injury via the binding of C1P to the DGR domain of KEAP1 and subsequently the release and nuclear translocation of NRF2, which activates the transcription of cytoprotective and antioxidant genes. Our study suggested that the upregulation of CERK and C1P expression may serve as a potential antioxidant strategy to alleviate acute liver injury.