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Aging is closely related to redox regulation. In our previous work, we proposed a new concept, "redox-stress response capacity (RRC)," and found that the decline in RRC was a dynamic characteristic of aging. However, the mechanism of RRC decline during aging remains unknown. In this study, using the senescent human fibroblast cell model and Caenorhabditis elegans model, we identified that peroxiredoxin 2 (PRDX2), as a hydrogen peroxide (H2O2) sensor, was involved in mediating RRC. PRDX2 knockdown led to a decline of RRC and accelerated senescence in fibroblasts and prdx-2 mutant C. elegans also showed decreased RRC. The mechanism study showed that the decreased sensor activity of PRDX2 was related to the increase in hyperoxidation of PRDX2 in senescent cells. Moreover, the level of PRDX2 hyperoxidation also increased in old C. elegans. Simultaneous overexpression of both PRDX2 and sulfiredoxin (SRX) rescued the reduced RRC and delayed senescence. The increase in PRDX2 hyperoxidation in senescent cells led to a decrease in its sensor activity, resulting in the decreased cellular response to H2O2, which is similar to the mechanism of insulin resistance due to the lower insulin receptor sensitivity. Treatment of young cells with a high level of H2O2 to induce a higher level of PRDX2-SO3 resulted in mimicking the RRC decline in senescent cells, which is also similar to a model of insulin resistance induced by high levels of insulin. All these results thrillingly indicate that there is an insulin-resistance-like phenomenon in senescent cells, we named it redox-stress response resistance, RRR. RRR in senescent cells is an important new discovery that explains RRC decline during aging and reveals the internal relationship between redox regulation and aging from a new perspective.
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Resistência à Insulina , Insulinas , Animais , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Senescência Celular , Peróxido de Hidrogênio , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismoRESUMO
Our previous research found that the nuclear factor-E2-related factor 2 (NRF2) protein was sustained activated in malignant transformation of human keratinocyte (HaCaT cells) caused by NaAsO2, but the role of NRF2 in it remains unknown. In this study, malignant transformation of HaCaT cells and labeled HaCaT cells used to detect mitochondrial glutathione levels (Mito-Grx1-roGFP2 HaCaT cells) were induced by 1.0 µM NaAsO2. Redox levels were measured at passages 0, early stage (passages 1, 7, 14), later stage (passages 21, 28 and 35) of arsenite-treated HaCaT cells. Oxidative stress levels increased at early stage. The NRF2 pathway was sustained activated. Cells and mitochondrial reductive stress levels (GSH/GSSG and NADPH/NADP+) increased. The mitochondrial GSH/GSSG levels of Mito-Grx1-roGFP2 HaCaT cells also increased. The indicators of glucose metabolism glucose-6-phosphate, lactate and the glucose-6-phosphate dehydrogenase (G6PD) levels increased, however Acetyl-CoA level decreased. Expression levels of glucose metabolic enzymes increased. After transfection with NRF2 siRNA, the indicators of glucose metabolism were reversed. After transfection with NRF2 or G6PD siRNA, cells and mitochondrial reductive stress levels decreased and the malignant phenotype was reversed. In conclusion, oxidative stress occurred in the early stage and the NRF2 was sustained high expression. In the later stage, increased NRF2/G6PD through glucose metabolic reprogramming induced reductive stress, thereby leading to malignant transformation.
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Arsenitos , Fator 2 Relacionado a NF-E2 , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Dissulfeto de Glutationa , Glucosefosfato Desidrogenase/metabolismo , Arsenitos/toxicidade , Arsenitos/metabolismo , Glucose/metabolismo , Linhagem Celular , Queratinócitos/metabolismo , Estresse Oxidativo , Glutationa/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Fasting is a popular dietary strategy because it grants numerous advantages, and redox regulation is one mechanism involved. However, the precise redox changes with respect to the redox species, organelles and tissues remain unclear, which hinders the understanding of the metabolic mechanism, and exploring the precision redox map under various dietary statuses is of great significance. Twelve redox-sensitive C. elegans strains stably expressing genetically encoded redox fluorescent probes (Hyperion sensing H2O2 and Grx1-roGFP2 sensing GSH/GSSG) in three organelles (cytoplasm, mitochondria and endoplasmic reticulum (ER)) were constructed in two tissues (body wall muscle and neurons) and were confirmed to respond to redox challenge. The H2O2 and GSSG/GSH redox changes in two tissues and three organelles were obtained by confocal microscopy during fasting, refeeding, and satiation. We found that under fasting condition, H2O2 decreased in most compartments, except for an increase in mitochondria, while GSSG/GSH increased in the cytoplasm of body muscle and the ER of neurons. After refeeding, the redox changes in H2O2 and GSSG/GSH caused by fasting were reversed in most organelles of the body wall muscle and neurons. In the satiated state, H2O2 increased markedly in the cytoplasm, mitochondria and ER of muscle and the ER of neurons, while GSSG/GSH exhibited no change in most organelles of the two tissues except for an increase in the ER of muscle. Our study systematically and precisely presents the redox characteristics under different dietary states in living animals and provides a basis for further investigating the redox mechanism in metabolism and optimizing dietary guidance.
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Introduction: Loss of neurons in the neural retina is a leading cause of vision loss. While humans do not possess the capacity for retinal regeneration, zebrafish can achieve this through activation of resident Müller glia. Remarkably, despite the presence of Müller glia in humans and other mammalian vertebrates, these cells lack an intrinsic ability to contribute to regeneration. Upon activation, zebrafish Müller glia can adopt a stem cell-like state, undergo proliferation and generate new neurons. However, the underlying molecular mechanisms of this activation subsequent retinal regeneration remains unclear. Methods/Results: To address this, we performed single-cell RNA sequencing (scRNA-seq) and report remarkable heterogeneity in gene expression within quiescent Müller glia across distinct dorsal, central and ventral retina pools of such cells. Next, we utilized a genetically driven, chemically inducible nitroreductase approach to study Müller glia activation following selective ablation of three distinct photoreceptor subtypes: long wavelength sensitive cones, short wavelength sensitive cones, and rods. There, our data revealed that a region-specific bias in activation of Müller glia exists in the zebrafish retina, and this is independent of the distribution of the ablated cell type across retinal regions. Notably, gene ontology analysis revealed that injury-responsive dorsal and central Müller glia express genes related to dorsal/ventral pattern formation, growth factor activity, and regulation of developmental process. Through scRNA-seq analysis, we identify a shared genetic program underlying initial Müller glia activation and cell cycle entry, followed by differences that drive the fate of regenerating neurons. We observed an initial expression of AP-1 and injury-responsive transcription factors, followed by genes involved in Notch signaling, ribosome biogenesis and gliogenesis, and finally expression of cell cycle, chromatin remodeling and microtubule-associated genes. Discussion: Taken together, our findings document the regional specificity of gene expression within quiescent Müller glia and demonstrate unique Müller glia activation and regeneration features following neural ablation. These findings will improve our understanding of the molecular pathways relevant to neural regeneration in the retina.
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Oxidative stress in aging has attracted much attention; however, the role of reductive stress in aging remains largely unknown. Here, we report that the endoplasmic reticulum (ER) undergoes reductive stress during replicative senescence, as shown by specific glutathione and H2O2 fluorescent probes. We constructed an ER-specific reductive stress cell model by ER-specific catalase overexpression and observed accelerated senescent phenotypes accompanied by disrupted proteostasis and a compromised ER unfolded protein response (UPR). Mechanistically, S-nitrosation of the pivotal ER sulfhydryl oxidase Ero1α led to decreased activity, therefore resulting in reductive stress in the ER. Inhibition of inducible nitric oxide synthase decreased the level of Ero1α S-nitrosation and decreased cellular senescence. Moreover, the expression of constitutively active Ero1α restored an oxidizing state in the ER and successfully rescued the senescent phenotypes. Our results uncover a new mechanism of senescence promoted by ER reductive stress and provide proof-of-concept that maintaining the oxidizing power of the ER and organelle-specific precision redox regulation could be valuable future geroprotective strategies.
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Estresse do Retículo Endoplasmático , Peróxido de Hidrogênio , Senescência Celular , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Peróxido de Hidrogênio/metabolismo , Nitrosação , Resposta a Proteínas não DobradasRESUMO
The redox homeostasis system regulates many biological processes, intracellular antioxidant production and redox signaling. However, long noncoding RNAs (lncRNAs) involved in redox regulation have rarely been reported. Herein, we reported that downregulation of MAGI2-AS3 decreased the superoxide level in Human fibroblasts (Fbs), a replicative aging model, as detected by the fluorescent probes dihydroethidium (DHE) and MitoSOX™ Red. RNA pulldown combined with mass spectrometry showed that HSPA8 is a novel interacting protein of MAGI2-AS3, which was further confirmed by photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Downregulation of MAGI2-AS3 decreased the hydrogen peroxide (H2O2) content by stabilizing the HSPA8 protein level via inhibiting the protesome degradation of HSPA8. Further evidence showed that MAGI2-AS3 interacted with the C-terminal domain (CTD) of HSPA8. Downregulation of MAGI2-AS3 delayed cell senescence, while this antiaging effect was abolished by HSPA8 knockdown. The underlying molecular mechanism by which MAGI2-AS3 knockdown inhibited cell senescence was mediated via suppression of the ROS/MAP2K6/p38 signaling pathway. Taken together, these findings revealed that downregulation of lncRNA MAGI2-AS3 decreased the H2O2 content and delayed cell senescence by stabilizing the HSPA8 protein level, identifying a potential antiaging application.
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Proteínas de Choque Térmico HSC70 , MicroRNAs , RNA Longo não Codificante , Linhagem Celular Tumoral , Proliferação de Células/genética , Senescência Celular , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genéticaRESUMO
Significance: The redox balance of cells provides a stable microenvironment for biological macromolecules to perform their physiological functions. As redox imbalance is closely related to the occurrence and development of a variety of diseases, antioxidant therapies are an attractive option. However, redox-based therapeutic strategies have not yet shown satisfactory results. To find the key reason is of great significance. Recent Advances: We emphasize the precise nature of redox regulation and elucidate the importance and necessity of precision redox strategies from three aspects: differences in redox status, differences in redox function, and differences in the effects of redox therapy. We then propose the "5R" principle of precision redox in antioxidant pharmacology: "Right species, Right place, Right time, Right level, and Right target." Critical Issues: Redox status must be considered in the context of species, time, place, level, and target. The function of a biomacromolecule and its cellular signaling role are closely dependent on redox status. Accurate evaluation of redox status and specific interventions are critical for the success of redox treatments. Precision redox is the key for antioxidant pharmacology. The precise application of antioxidants as nutritional supplements is also key to the general health of the population. Future Directions: Future studies to develop more accurate methods for detecting redox status and accurately evaluating the redox state of different physiological and pathological processes are needed. Antioxidant pharmacology should consider the "5R" principle rather than continuing to apply global nonspecific antioxidant treatments. Antioxid. Redox Signal. 34, 1069-1082.