Nuclear Receptor Coactivator 4 Research Articles

Hypoxia inhibits ferritinophagy-mediated ferroptosis in esophageal squamous cell carcinoma via the USP2-NCOA4 axis.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignancy of the digestive system. Hypoxia is a crucial player in tumor ferroptosis resistance. However, the molecular mechanism of hypoxia-mediated ferroptosis resistance in ESCC remains unclear. Here, USP2 expression was decreased in ESCC cell lines subjected to hypoxia treatment and was lowly expressed in clinical ESCC specimens. Ubiquitin-specific protease 2 (USP2) depletion facilitated cell growth, which was blocked in USP2-overexpressing cells. Moreover, USP2 silencing enhanced the iron ion concentration and lipid peroxidation accumulation as well as suppressed ferroptosis, while upregulating USP2 promoted ferroptotic cell death in ESCC cells. Furthermore, knockout of USP2 in ESCC models discloses the essential role of USP2 in promoting ESCC tumorigenesis and inhibiting ferroptosis. In contrast, overexpression of USP2 contributes to antitumor effect and ferroptosis events in vivo. Specifically, USP2 stably bound to and suppressed the degradation of nuclear receptor coactivator 4 (NCOA4) by eliminating the Lys48-linked chain, which in turn triggered ferritinophagy and ferroptosis in ESCC cells. Our findings suggest that USP2 plays a crucial role in iron metabolism and ferroptosis and that the USP2/NCOA4 axis is a promising therapeutic target for the management of ESCC.

Regulating NCOA4-Mediated Ferritinophagy for Therapeutic Intervention in Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke presents a global health challenge, necessitating an in-depth comprehension of its pathophysiology and therapeutic strategies. While reperfusion therapy salvages brain tissue, it also triggers detrimental cerebral ischemia-reperfusion injury (CIRI). In our investigation, we observed the activation of nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy in an oxygen-glucose deprivation/reoxygenation (OGD/R) model using HT22 cells (P < 0.05). This activation contributed to oxidative stress (P < 0.05), enhanced autophagy (P < 0.05) and cell death (P < 0.05) during CIRI. Silencing NCOA4 effectively mitigated OGD/R-induced damage (P < 0.05). These findings suggested that targeting NCOA4-mediated ferritinophagy held promise for preventing and treating CIRI. Subsequently, we substantiated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway effectively regulated the NCOA4-mediated ferritinophagy, by applying the cGAS inhibitor RU.521 and performing NCOA4 overexpression (P < 0.05). Suppressing the cGAS-STING pathway efficiently curtailed ferritinophagy (P < 0.05), oxidative stress (P < 0.05), and cell damage (P < 0.05) of CIRI, while NCOA4 overexpression could alleviate this effect (P < 0.05). Finally, we elucidated the specific molecular mechanism underlying the protective effect of the iron chelator deferoxamine (DFO) on CIRI. Our findings revealed that DFO alleviated hypoxia-reoxygenation injury in HT22 cells through inhibiting NCOA4-mediated ferritinophagy and reducing ferrous ion levels (P < 0.05). However, the protective effects of DFO were counteracted by cGAS overexpression (P < 0.05). In summary, our results indicated that the activation of the cGAS-STING pathway intensified cerebral damage during CIRI by inducing NCOA4-mediated ferritinophagy. Administering the iron chelator DFO effectively attenuated NCOA4-induced ferritinophagy, thereby alleviating CIRI. Nevertheless, the role of the cGAS-STING pathway in CIRI regulation likely involves intricate mechanisms, necessitating further validation in subsequent investigations.

Structural basis for the intracellular regulation of ferritin degradation

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy—a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4–FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.

Therapeutic Effects of Melatonin in the Regulation of Ferroptosis: A Review of Current Evidence.

Ferroptosis is implicated in the pathogenesis of multiple diseases, including neurodegenerative diseases, cardiovascular diseases, kidney pathologies, ischemia-reperfusion injury, and cancer. The current review article highlights the involvement of ferroptosis in traumatic brain injury, acute kidney damage, ethanol-induced liver injury, and PM2.5-induced lung injury. Melatonin, a molecule produced by the pineal gland and many other organs, is well known for its anti- aging, anti-inflammatory, and anticancer properties and is used in the treatment of different diseases. Melatonin's ability to activate anti-ferroptosis pathways including sirtuin (SIRT)6/p- nuclear factor erythroid 2-related factor 2 (Nrf2), Nrf2/ antioxidant responsive element (ARE)/ heme oxygenase (HO-1)/SLC7A11/glutathione peroxidase (GPX4)/ prostaglandin-endoperoxide synthase 2 (PTGS2), extracellular signal-regulated kinase (ERK)/Nrf2, ferroportin (FPN), Hippo/ Yes-associated protein (YAP), Phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) and SIRT6/ nuclear receptor coactivator 4 (NCOA4)/ ferritin heavy chain 1 (FTH1) signaling pathways suggests that it could serve as a valuable therapeutic agent for preventing cell death associated with ferroptosis in various diseases. Further research is needed to fully understand the precise mechanisms by which melatonin regulates ferroptosis and its potential as a therapeutic target.

Oxidative medicine and cellular longevity the role and mechanism of NCOA4 in ferroptosis induced by intestinal ischemia reperfusion

BackgroundFerroptosis is an iron-dependent and cystathione-non-dependent non-apoptotic cell death characterized by elevated intracellular free iron levels and reduced antioxidant capacity, leading to the accumulation of lipid peroxides. Nuclear receptor coactivator 4 (NCOA4) mediates ferritinophagy, increasing labile iron levels, which can result in oxidative damage. However, the specific mechanism of NCOA4-mediated ferritinophagy in intestinal ischemia–reperfusion and the underlying mechanisms have not been reported in detail. Object1. To investigate the role of NCOA4 in ferroptosis of intestinal epithelial cells induced by II/R injury in mouse. 2. To investigate the mechanism of action of NCOA4-induced ferroptosis. Methods1. Construct a mouse II/R injury model and detect ferroptosis related markers such as HE staining, immunohistochemistry, ELISA, and WB methods.2. Detect expression of NCOA4 in the intestine of mouse with II/R injury model and analyze its correlation with intestinal ferroptosis in mouse with II/R injury model.3. Construct an ischemia–reperfusion model at the cellular level through hypoxia and reoxygenation, and overexpress/knockdown NCOA4 to detect markers related to ferroptosis. Based on animal experimental results, analyze the correlation and mechanism of action between NCOA4 and intestinal epithelial ferroptosis induced by II/R injury in mouse. Results1. Ferroptosis occurred in the intestinal epithelial cells of II/R-injured mouse, and the expression of critical factors of ferroptosis, ACSL4, MDA and 15-LOX, was significantly increased, while the levels of GPX4 and GSH were significantly decreased. 2. The expression of NCOA4 in the intestinal epithelium of mouse with II/R injure was significantly increased, the expression of ferritin was significantly decreased, and the level of free ferrous ions was significantly increased; the expression of autophagy-related proteins LC3 and Beclin-1 protein was increased, and the expression of P62 was decreased, and these changes were reversed by autophagy inhibitors. 3. Knockdown of NCOA4 at the cellular level resulted in increased ferritin expression and decreased ferroptosis, and CO-IP experiments suggested that NCOA4 can bind to ferritin, which suggests that NCOA4 most likely mediates ferritinophagy to induce ferroptosis. ConclusionThis thesis explored the role of NCOA4 in II/R injury in mice and the mechanism of action. The research results suggest that NCOA4 can mediate ferritinophagy to induce ferroptosis during II/R injury. This experiment reveals the pathological mechanism of II/R injury and provides some scientific basis for the development of drugs for the treatment of II/R injury based on the purpose of alleviating ferroptosis.

18β-Glycyrrhetinic acid protects against deoxynivalenol-induced liver injury via modulating ferritinophagy and mitochondrial quality control

Deoxynivalenol (DON), a widespread mycotoxin, represents a substantial public health hazard due to its propensity to contaminate agricultural produce, leading to both acute and chronic health issues in humans and animals upon consumption. The role of ferroptosis in DON-induced hepatic damage remains largely unexplored. This study investigates the impact of 18β-glycyrrhetinic acid (GA), a prominent constituent of glycyrrhiza, on DON hepatotoxicity and elucidates the underlying mechanisms. Our results indicate that GA effectively attenuates liver injury inflicted by DON. This was achieved by inhibiting nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, as well as by adjusting mitochondrial quality control (MQC). Specifically, GA curtails ferritinophagy by diminishing NCOA4 expression without affecting the autophagic flux. At a molecular level, GA binds to and stabilizes programmed cell death protein 4 (PDCD4), thereby inhibiting its ubiquitination and subsequent degradation. This stabilization of PDCD4 leads to the downregulation of NCOA4 via the JNK-Jun-NCOA4 axis. Knockdown of PDCD4 weakened GA’s protective action against DON exposure. Furthermore, GA improved mitochondrial function and limited excessive mitophagy and mitochondrial division induced by DON. Disrupting GA’s modulation of MQC nullified its anti-ferroptosis effects. Overall, GA offers protection against DON-induced ferroptosis by blocking ferritinophagy and managing MQC. Environmental implicationFood contamination from mycotoxins, is a problem for agricultural and food industries worldwide. Deoxynivalenol (DON), the most common mycotoxins in cereal commodities. A survey in 2023 showed that the positivity rate for DON contamination in food reached more than 70% globally. DON can damage the health of humans whether exposed to high doses for short periods of time or low doses for long periods of time. We have discovered 18β-Glycyrrhetinic acid (GA), a prominent constituent of glycyrrhiza. Liver damage caused by low-dose DON can be successfully treated with GA. This study will support the means of DON control, including antidotes.

Targeted ferritinophagy in gastrointestinal cancer: from molecular mechanisms to implications.

Gastrointestinal cancer is a significant global health burden, necessitating the development of novel therapeutic strategies. Emerging evidence has highlighted the potential of targeting ferritinophagy as a promising approach for the treatment of gastrointestinal cancer. Ferritinophagy is a form of selective autophagy that is mediated by the nuclear receptor coactivator 4 (NCOA4). This process plays a crucial role in regulating cellular iron homeostasis and has been implicated in various pathological conditions, including cancer. This review discusses the molecular mechanisms underlying ferritinophagy and its relevance to gastrointestinal cancer. Furthermore, we highlight the potential therapeutic implications of targeting ferritinophagy in gastrointestinal cancer. Several approaches have been proposed to modulate ferritinophagy, including small molecule inhibitors and immunotherapeutic strategies. We discuss the advantages and challenges associated with these therapeutic interventions and provide insights into their potential clinical applications.

Mechanism of acupuncture in attenuating cerebral ischaemia-reperfusion injury based on nuclear receptor coactivator 4 mediated ferritinophagy.

To explore the effect of acupuncture treatment on cerebral ischaemia-reperfusion injury (CIRI) and reveal the underlying mechanism of the effect based on nuclear receptor coactivator 4 (NCOA4) mediated ferritinophagy. Sprague-Dawley male rats were divided into four groups: the sham group, model group, acupuncture group, and sham acupuncture group. After 2 h of middle cerebral artery occlusion (MCAO), reperfusion was performed for 24 h to induce CIRI. The rats were treated with acupuncture at the Neiguan (PC6) and Shuigou (GV26) acupoints. Their neurological function was evaluated by taking their Bederson scores at 2 h after ischaemia and 24 h after reperfusion. Triphenyltetrazolium chloride staining was applied to assess the cerebral infarct volume at 24 h after reperfusion. The malondialdehyde (MDA) and ferrous iron (Fe2+) levels were observed after 24 h of reperfusion using an assay kit. Western blotting was performed to detect the expression of NCOA4 and ferritin heavy chain 1 (FTH1) at 24 h after reperfusion. Moreover, the colocalization of ferritin with neurons, NCOA4 with microtubule-associated protein 1 light chain 3 (LC3), and NCOA4 with ferritin was visualized using immunofluorescence staining. Acupuncture significantly improved neurological function and decreased cerebral infarct volume in the acupuncture group. Following CIRI, the expression of NCOA4, LC3 and FTH1 was increased, which enhanced ferritinophagy and induced an inappropriate accumulation of Fe2+ and MDA in the ischaemic brain. However, acupuncture dramatically downregulated the expression of NCOA4, LC3 and FTH1, inhibited the overactivation of ferritinophagy, and decreased the levels of MDA and Fe2+. Acupuncture can inhibit NCOA4-mediated ferritinophagy and protect neurons against CIRI in a rat model.

Ginsenoside Rg5 inhibits glioblastoma by activating ferroptosis via NR3C1/HSPB1/NCOA4

BackgroundThe utilization of Chinese medicine as an adjunctive therapy for cancer has recently gained significant attention. Ferroptosis, a newly regulated cell death process depending on the ferrous ions, has been proved to be participated in glioma stem cells inactivation. PurposeWe aim to study whether ginsenoside Rg5 exerted inhibitory effects on crucial aspects of glioma stem cells, including cell viability, tumor initiation, invasion, self-renewal ability, neurosphere formation, and stemness. MethodsThrough comprehensive sequencing analysis, we identified a compelling association between ginsenoside Rg5 and the ferroptosis pathway, which was further validated through subsequent experiments demonstrating its ability to activate this pathway. ResultsTo elucidate the precise molecular targets affected by ginsenoside Rg5 in gliomas, we conducted an intersection analysis between differentially expressed genes obtained from sequencing and a database-predicted list of transcription factors and potential targets of ginsenoside Rg5. This rigorous approach led us to unequivocally confirm NR3C1 (Nuclear Receptor Subfamily 3 Group C Member 1) as a direct target of ginsenoside Rg5, a finding consistently supported by subsequent experimental investigations. Moreover, we uncovered NR3C1′s capacity to transcriptionally regulate ferroptosis -related genes HSPB1 and NCOA4. Strikingly, ginsenoside Rg5 induced notable alterations in the expression levels of both HSPB1 (Heat Shock Protein Family B Member 1) and NCOA4 (Nuclear Receptor Coactivator 4). Finally, our intracranial xenograft assays served to reaffirm the inhibitory effect of ginsenoside Rg5 on the malignant progression of glioblastoma. ConclusionThese collective findings strongly suggest that ginsenoside Rg5 hampers glioblastoma progression by activating ferroptosis through NR3C1, which subsequently modulates HSPB1 and NCOA4. Importantly, this novel therapeutic direction holds promise for advancing the treatment of glioblastoma.

ISRIB improves white matter injury following TBI by inhibiting NCOA4-mediated ferritinophagy

Traumatic brain injury (TBI) often results in persistent neurological dysfunction, which is closely associated with white matter injury. The mechanisms underlying white matter injury after TBI remain unclear. Ferritinophagy is a selective autophagic process that degrades ferritin and releases free iron, which may cause ferroptosis. Although ferroptosis has been demonstrated to be involved in TBI, it is unclear whether ferritinophagy triggers ferroptosis in TBI. Integrated stress response inhibitor (ISRIB) has neuroprotective properties. However, the effect of ISRIB on white matter after TBI remains uncertain. We aimed to investigate whether ferritinophagy was involved in white matter injury following TBI and whether ISRIB can mitigate white matter injury after TBI by inhibiting ferritinophagy. In this study, controlled cortical impact (CCI) was performed on rats to establish the TBI model. Ferritinophagy was measured by assessing the levels of nuclear receptor coactivator 4 (NCOA4), which regulates ferritinophagy, ferritin heavy chain 1(FTH1), LC3, ATG5, and FTH1 colocalization with LC3 in the white matter. Increased NCOA4 and decreased FTH1 were detected in our study. FTH1 colocalization with LC3 enhanced in the white matter after TBI, indicating that ferritinophagy was activated. Immunofluorescence co-localization results also suggested that ferritinophagy occurred in neurons and oligodendrocytes after TBI. Furthermore, ferroptosis was assessed by determining free iron content, MDA content, GSH content, and Perl’s staining. The results showed that ferroptosis was suppressed by NCOA4 knockdown via shNCOA4 lentivirus infection, indicating that ferroptosis in TBI is triggered by ferritinophagy. Besides, NCOA4 deletion notably improved white matter injury following TBI, implying that ferritinophagy contributed to white matter injury. ISRIB treatment reduced the occurrence of ferritinophagy in neurons and oligodendrocytes, attenuated ferritinophagy-induced ferroptosis, and alleviated white matter injury. These findings suggest that NCOA4-mediated ferritinophagy is a critical mechanism underlying white matter injury after TBI. ISRIB holds promise as a therapeutic agent for this condition.

Cadmium exposure induced neuronal ferroptosis and cognitive deficits via the mtROS-ferritinophagy pathway

Exposure to environmental cadmium (Cd) is known to cause neuronal death and cognitive decline in humans. Ferroptosis, a novel iron-dependent type of regulated cell death, is involved in various neurological disorders. In the present study, Cd exposure triggered ferroptosis in the mouse hippocampus and in the HT22 murine hippocampal neuronal cell line, as indicated by significant increases in ferroptotic marker expression, intracellular iron levels, and lipid peroxidation. Interestingly, ferroptosis of hippocampal neurons in response to Cd exposure relied on the induction of autophagy since the suppression of autophagy by 3-methyladenine (3-MA) and chloroquine (CQ) substantially ameliorated Cd-induced ferroptosis. Furthermore, nuclear receptor coactivator 4 (NCOA4)-mediated degradation of ferritin was required for the Cd-induced ferroptosis of hippocampal neurons, demonstrating that NCOA4 knockdown decreased intracellular iron levels and lipid peroxidation and increased cell survival, following Cd exposure. Moreover, Cd-induced mitochondrial reactive oxygen species (mtROS) generation was essential for the ferritinophagy-mediated ferroptosis of hippocampal neurons. Importantly, pretreatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively attenuated Cd-induced hippocampal neuronal death and cognitive impairment in mice. Taken together, these findings indicate that ferroptosis is a novel mechanism underlying Cd-induced neurotoxicity and cognitive impairment and that the mtROS-ferritinophagy axis modulates Cd-induced neuronal ferroptosis.

BRCA1-mediated dual regulation of ferroptosis exposes a vulnerability to GPX4 and PARP co-inhibition in BRCA1-deficient cancers.

Resistance to poly (ADP-ribose) polymerase inhibitors (PARPi) limits the therapeutic efficacy of PARP inhibition in treating breast cancer susceptibility gene 1 (BRCA1)-deficient cancers. Here we reveal that BRCA1 has a dual role in regulating ferroptosis. BRCA1 promotes the transcription of voltage-dependent anion channel 3 (VDAC3) and glutathione peroxidase 4 (GPX4); consequently, BRCA1 deficiency promotes cellular resistance to erastin-induced ferroptosis but sensitizes cancer cells to ferroptosis induced by GPX4 inhibitors (GPX4i). In addition, nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and defective GPX4 induction unleash potent ferroptosis in BRCA1-deficient cancer cells upon PARPi and GPX4i co-treatment. Finally, we show that xenograft tumors derived from BRCA1-mutant breast cancer patients with PARPi resistance exhibit decreased GPX4 expression and high sensitivity to PARP and GPX4 co-inhibition. Our results show that BRCA1 deficiency induces a ferroptosis vulnerability to PARP and GPX4 co-inhibition and inform a therapeutic strategy for overcoming PARPi resistance in BRCA1-deficient cancers.

Cryptochrome 1 regulates ovarian granulosa cell senescence through NCOA4-mediated ferritinophagy

Age-associated decreases in follicle number and oocyte quality result in a decline in female fertility, which is associated with increased infertility. Granulosa cells play a major role in oocyte development and maturation both in vivo and in vitro. However, it is unclear whether a reduction in cryptochrome 1 (Cry1) expression contributes to granulosa cell senescence, and further exploration is needed to understand the underlying mechanisms. In this study, we investigated the role of Cry1, a core component of the molecular circadian clock, in the regulation of senescence in ovarian granulosa cells. Western blotting and qRT-PCR showed that Cry1 expression was downregulated in aged human ovarian granulosa cells and was correlated with age and anti-Müllerian hormone (AMH) levels. RNA-seq analysis suggested that ferritinophagy was increased after Cry1 knockdown in KGN cells. MDA, iron, and reactive oxygen species (ROS) assays were used to detect cellular ferritinophagy levels. Ferroptosis inhibitors, iron chelators, autophagy inhibitors, and nuclear receptor coactivator 4 (NCOA4) knockdown alleviated KGN cell senescence induced by Cry1 knockdown. Immunofluorescence, immunoprecipitation, and ubiquitination assays indicated that Cry1 affected NCOA4 ubiquitination and degradation through HERC2, thereby affecting NCOA4-mediated ferritinophagy and causing granulosa cell senescence. KL201, a Cry1 stabilizer, enhanced ovarian function in naturally aged mice by reducing ferritinophagy. Our study reveals the potential mechanisms of action of Cry1 during ovarian aging and provides new insights for the clinical treatment of age-related fertility decline.

Ferritinophagy-Mediated Hippocampus Ferroptosis is Involved in Cognitive Impairment in Immature Rats Induced by Hypoxia Combined with Propofol.

Propofol is a clinically common intravenous general anesthetic and is widely used for anesthesia induction, maintenance and intensive care unit (ICU) sedation in children. Hypoxemia is a common perioperative complication. In clinical work, we found that children with hypoxemia who received propofol anesthesia experienced significant postoperative cognitive changes. To explore the causes of this phenomenon, we conducted the study. In this study, our in vivo experiments found that immature rats exposed to hypoxia combined with propofol (HCWP) could develop cognitive impairment. We performed the RNA-seq analysis of its hippocampal tissues and found that autophagy and ferroptosis may play a role in our model. Next, we verified the participation of the two modes of death by detecting the expression of autophagy-related indexes Sequestosome 1 (SQSTM1) and Beclin1, and ferroptosis-related indicators Fe2+, reactive oxygen species (ROS) and glutathione peroxidase 4 (GPX4). Meanwhile, we found that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, could improve cognitive impairment in immature rats caused by HCWP. In addition, we found that nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, which acted as a key junction between autophagy and ferroptosis, was also involved. Finally, our in vitro experiments concluded that autophagy activation was an upstream factor in HCWP-induced hippocampus ferroptosis through the intervention of autophagy inhibitor 3-methyladenine (3-MA). Our study was expected to provide an attractive therapeutic target for cognitive impairment that occurred after HCWP exposures.

Ferritinophagy Is Critical for Deoxynivalenol-Induced Liver Injury in Mice.

Background: Deoxynivalenol (DON) contamination, pervasive throughout all stages of food production and processing, presents a significant threat to human health. The degradation of ferritin mediated by nuclear receptor coactivator 4 (NCOA4), termed ferritinophagy, plays a crucial role in maintaining iron homeostasis and regulating ferroptosis. Aim: This study aims to elucidate the role of ferritinophagy and ferroptosis in DON-induced liver injury. Methods: Male mice and AML12 cells were subjected to varying doses of DON, serving as in vivo and in vitro models, respectively. Protein expression was assessed by using immunofluorescence and western blot techniques. Co-immunoprecipitation was employed to investigate the protein-protein interactions. Results: Our findings demonstrate that DON triggers hepatocyte ferroptosis in a ferritinophagy-dependent manner. Specifically, DON impedes the activation of the mammalian target of rapamycin complex 1 (mTORC1) by inhibiting RAC1's binding to mTOR, thereby ultimately inducing autophagy. Concurrently, DON amplifies NCOA4's affinity for ferritin by facilitating NCOA4 phosphorylation through the ataxia-telangiectasia mutated kinase (ATM), thus promoting the autophagy-dependent degradation of ferritin. Both autophagy inhibition and NCOA4 expression suppression ameliorate DON-induced ferroptosis. Conclusion: Our study concludes that DON facilitates NCOA4-mediated ferritinophagy via the ATM-NCOA4 pathway, subsequently inducing ferroptosis in the liver.

M6A reader YTHDC2 mediates NCOA4 mRNA stability affecting ferritinophagy to alleviate secondary injury after intracerebral haemorrhage

ABSTRACT Oxidative stress and neuronal dysfunction caused by intracerebral haemorrhage (ICH) can lead to secondary injury. The m6A modification has been implicated in the progression of ICH. This study aimed to investigate the role of the m6A reader YTHDC2 in ICH-induced secondary injury. ICH models were established in rats using autologous blood injection, and neuronal cell models were induced with Hemin. Experiments were conducted to overexpress YTH domain containing 2 (YTHDC2) and examine its effects on neuronal dysfunction, brain injury, and neuronal ferritinophagy. RIP-qPCR and METTL3 silencing were performed to investigate the regulation of YTHDC2 on nuclear receptor coactivator 4 (NCOA4). Finally, NCOA4 overexpression was used to validate the regulatory mechanism of YTHDC2 in ICH. The study found that YTHDC2 expression was significantly downregulated in the brain tissues of ICH rats. However, YTHDC2 overexpression improved neuronal dysfunction and reduced brain water content and neuronal death after ICH. Additionally, it reduced levels of ROS, NCOA4, PTGS2, and ATG5 in the brain tissues of ICH rats, while increasing levels of FTH and FTL. YTHDC2 overexpression also decreased levels of MDA and Fe2+ in the serum, while promoting GSH synthesis. In neuronal cells, YTHDC2 overexpression alleviated Hemin-induced injury, which was reversed by Erastin. Mechanistically, YTHDC2-mediated m6A modification destabilized NCOA4 mRNA, thereby reducing ferritinophagy and alleviating secondary injury after ICH. However, the effects of YTHDC2 were counteracted by NCOA4 overexpression. Overall, YTHDC2 plays a protective role in ICH-induced secondary injury by regulating NCOA4-mediated ferritinophagy.

C5b-9 promotes ferritinophagy leading to ferroptosis in renal tubular epithelial cells of trichloroethylene-sensitized mice

Trichloroethylene (TCE) is a common environmental contaminant that can cause a severe allergic reaction called TCE hypersensitivity syndrome, which often implicates the patient's kidneys. Our previous study revealed that C5b-9-induced tubular ferroptosis is involved in TCE-caused kidney damage. However, the study did not explain how tubule-specific C5b-9 causes free iron overload, a key event in ferroptosis. Here, we aimed to explore the role of NCOA4-mediated ferritinophagy in C5b-9-induced iron overload and ferroptosis in TCE-sensitized mice. Our results showed that TCE sensitization does not affect iron import or export, but does affect iron storage, causing ferritin degradation and free iron overload. In addition, mitochondrial ROS was upregulated, and these changes were blocked by C5b-9 inhibition. Interestingly, TCE-induced ferritin degradation and ferroptosis were significantly antagonized by the application of the mitochondrial ROS inhibitor, Mito-TEMPO. Moreover, all of these modes of action were further verified in C5b-9-attack signalling HK-2 cells. Further investigation demonstrated that C5b-9-upregulated mitochondrial ROS induced a marked increase in nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy. In addition, the application of NCOA4 small interfering RNA not only significantly reversed ferritinophagy caused by C5b-9 but also reduced C5b-9-induced ferroptosis in HK-2 cells. Taken together, these results suggest that tubule-specific C5b-9 deposition activates NCOA4 through the upregulation of mitochondrial ROS, causing ferritin degradation and elevated free iron, which ultimately leads to tubular epithelial cell ferroptosis and kidney injury in TCE-sensitized mice.

L-Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload.

Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinalmicrobiotaimbalance. As a neutral and nonprotein amino acid, L-Citrulline (L-cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L-cit can alleviate iron overload-induced intestinal injury and explores the underlying mechanisms. C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L-cit for 2 weeks. L-cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L-cit-mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2) cells are treated with L-cit or AMP-activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L-cit. Notably, Compound C abolishes L-cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC-J2 cells. L-cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L-cit is a promising therapeutic strategy for iron overload-induced intestinal injury.

Role of circPSEN1 in carbon black and cadmium co-exposure induced autophagy-dependent ferroptosis in respiratory epithelial cells

Carbon black and cadmium (Cd) are important components of atmospheric particulate matter and cigarette smoke that are closely associated with the occurrence and development of lung diseases. Carbon black, particularly carbon black nanoparticles (CBNPs), can easily adsorbs metals and cause severe lung damage and even cell death. Therefore, this study aimed to explore the mechanisms underlying the combined toxicity of CBNPs and Cd. We found that the combined exposure to CBNPs and Cd promoted significantly greater autophagosome formation and ferroptosis (increased malonaldehyde (MDA), reactive oxygen species (ROS), and divalent iron ions (Fe2+) levels and altered ferroptosis-related proteins) compared with single exposure in both 16HBE cells (human bronchial epithelioid cells) and mouse lung tissues. The levels of ferroptosis proteins, transferrin receptor protein 1 (TFRC) and glutathione peroxidase 4 (GPX4), were restored by CBNPs-Cd exposure following treatment with a 3-MA inhibitor. Additionally, under CBNPs-Cd exposure, circPSEN1 overexpression inhibited increases in the autophagy proteins microtubule-associated protein 1 light chain 3 (LC3II/I) and sequestosome-1 (P62). Moreover, increases in TFRC and Fe2+, and decreases in GPX4were inhibited. Knockdown of circPSEN1 reversed these effects. circPSEN1 interacts with autophagy-related gene 5 (ATG5) protein and upregulates nuclear receptor coactivator 4 (NCOA4), the co-interacting protein of ATG5, thereby degrading ferritin heavy chain 1 (FTH1) and increasing Fe2+ in 16HBE cells. These results indicated that the combined exposure to CBNPs and Cd promoted the binding of circPSEN1 to ATG5, thereby increasing autophagosome synthesis and ATG5-NCOA4-FTH1 axis activation, ultimately inducing autophagy-dependent ferroptosis in 16HBE cells and mouse lung tissues. This study provides novel insights into the toxic effects of CBNPs and Cd in mixed pollutants.

Glyphosate drives autophagy-dependent ferroptosis to inhibit testosterone synthesis in mouse Leydig cells

Glyphosate (GLY), a widely used herbicide, can adversely affect the male reproductive health by inhibiting testosterone synthesis. Ferroptosis is a form of iron-dependent oxidative cell death that contributes to inhibition of testosterone secretion. However, it still remains unclear whether ferroptosis is involved in GLY-inhibited testosterone synthesis. Hereby, an in vitro model of 1 mM GLY-exposed testicular Leydig (TM3) cells was established to elucidate this issue. Data firstly showed that GLY causes cytotoxicity and testosterone synthesis inhibition via ferroptosis, while accumulation of lipid peroxides due to intracellular ferrous ion (Fe2+) overload and glutathione depletion is confirmed as a determinant of ferroptosis. Blockage of ferroptosis via chelation of Fe2+ or inhibition of lipid peroxidation can markedly mitigate GLY-induced testosterone synthesis inhibition. Also, autophagy activation is revealed in GLY-treated TM3 cells and nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy is involved in ferroptosis through the release of excess Fe2+. GLY-induced cytotoxicity and testosterone synthesis inhibition are significantly alleviated by NCOA4 knockdown, demonstrating the crucial role of NCOA4-mediated ferritinophagy in GLY-inhibited testosterone synthesis. In summary, this study provides solid evidence that NCOA4-mediated ferritinophagy promotes ferroptosis to inhibit testosterone synthesis, highlighting that targeting NCOA4 may be a potential therapeutic approach in GLY-induced male reproductive toxicity.