Background Mutations in CCNF, encoding cyclin F, cause a small proportion of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases. Mutant cyclin F mislocalises from the nucleus to the cytoplasm, impairs the ubiquitin proteasome system, disrupts endoplasmic reticulum (ER) to Golgi transport, and triggers ER stress and apoptosis. Protein disulphide isomerase (PDI), a redox-active ER chaperone of the thioredoxin superfamily, protects against ALS phenotypes induced by mutant SOD1, TDP-43, and FUS, but its activity against mutant cyclin F has not been examined.Objectives To test whether PDI overexpression prevents the cellular and behavioural phenotypes induced by the ALS/FTD-associated cyclin FS621G mutation.Methods Neuro-2a cells were co-transfected with mCherry-tagged wild-type or mutant cyclin FS621G and V5-tagged PDI or empty vector. Subcellular localisation, XBP-1 and CHOP activation, proteasome reporter (GFPu) accumulation, nuclear morphology, and cleaved caspase-3 immunoreactivity were assessed by confocal microscopy. Transient transgenic zebrafish larvae generated by mRNA co-injection of cyclin F and PDI were assessed by photomotor response at 48 h post-fertilisation.Results Mutant cyclin FS621G induced cytoplasmic mislocalisation, ER stress (assessed by XBP-1 and CHOP activation), proteasome dysfunction (indicated by GFPu accumulation), and apoptosis (assessed by nuclear condensation and caspase-3 activation), all of which were significantly attenuated by PDI co-expression. Zebrafish larvae expressing cyclin FS621G swam significantly shorter distances than uninjected or wildtype controls, and co-expression of PDI restored motor function to near-wildtype levels.Conclusion PDI protects against ALS/FTD-associated mutant cyclin F in both cellular and in vivo zebrafish models, identifying PDI as a redox-based therapeutic target for CCNF-linked ALS/FTD.
Introduction To discover novel mechanisms, we compared changes in redox metabolism markers induced by caloric restriction (CR) and sulfur amino acid restriction (SAAR).Methods We fed 16-week-old male F344 rats either a control diet (CD, 0.86% methionine), a sulfur amino acid-restricted diet (SAAR, 0.17% methionine), or a control diet pair-fed to 60% of the CD's ad libitum intake (CR, caloric restriction). We quantified small redox molecule concentrations, mRNA levels, and protein levels of relevant markers.Results Compared with CR, SAAR decreased the hepatic total free glutathione (tGSH), increased GSSG:tGSH, protein-bound GSH:tGSH, and lowered glucose-6-phosphate dehydrogenase levels, indicating a pro-oxidant milieu. SAAR and CR differentially affected the NADPH-generating enzymes. SAAR increased the cytosolic malic enzyme (ME1) without affecting mitochondrial ME3, while CR increased ME3, but not ME1. CR elevated cytosolic methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) without affecting mitochondrial MTHFD2, whereas SAAR increased MTHFD2 without affecting MTHFD1. Folate cycle changes include increases in Aldh1l1 and Aldh1l2 by SAAR and MTHFD1L by CR. SAAR, but not CR, upregulated the gene expression of compartment-specific (de)glutathionylation enzymes; both diets had distinct effects on glutathionylation of the hepatic proteome.Conclusions SAAR and CR engage different pathways to generate compartment-specific NADPH. Protein glutathionylation may contribute to SAAR-induced benefits.
Background Reactive oxygen species (ROS) regulate stem-cell function, but sustained exposure causes oxidative damage, mitochondrial dysfunction and exhaustion. Multilineage-differentiating Stress-Enduring (MUSE) cells, a rare SSEA-3⁺ MSC subset, tolerate ROS-rich environments, although the mechanisms remain unclear.Methods We compared MUSE, Non-MUSE (SSEA-3⁻) and MSCs after H₂O₂ exposure, assessing ROS clearance, mtDNA integrity, 8-oxo-dG, antioxidant and BER responses, mitochondrial morphology/function, mitophagy and biogenesis, with focus on the PGC-1α/NRF2 axis. Three independent biological replicates were analyzed by imaging, qPCR, flow cytometry and biochemical assays.Results MUSE cells showed greater resistance to oxidative stress than MSCs and Non-MUSE cells. After H₂O₂, they displayed lower ROS accumulation (~15–25%), preserved mtDNA integrity, reduced 8-oxo-dG, rapid induction of antioxidant and BER-related genes, and maintenance of mitochondrial morphology, membrane potential and respiratory activity. MUSE cells also showed enhanced mitophagy, with >2-fold increased LAMP1–MitoTracker colocalization and induction of PINK1/phospho-Ub-S65, together with increased biogenesis, including ~45% higher COX-I/SDHA ratio, TFAM induction and PGC-1α activation at 48 h.Conclusion MUSE cells coordinate antioxidant defense, DNA repair, mitophagy and biogenesis through a remove-and-replace program that preserves mitochondrial function during oxidative stress.
Objective Ferroptosis has been shown to participate in luteolysis, yet the upstream signals and metabolic remodeling that trigger it remain unknown. This study aims to clarify these mechanisms.Methods Physiological postpartum and PGF2α-induced luteolysis models were established in mice. Targeted energy metabolomics, pharmacological inhibition and adenoviral shRNA-mediated knockdown were employed in vivo and in primary luteal cells to measure ferroptosis markers, G6PD activity, NADP⁺/NADPH ratio and NOX2/NOX4 expression.Results In both models, luteal regression was accompanied by increased ACSL4, decreased GPx4 and GSH, and elevated Fe2⁺ and lipid peroxidation and NOX2 and NOX4 expression. Metabolic analysis revealed 6-phosphogluconate accumulation, reduced pyruvate and pyruvate kinase M activity, and increased G6PD expression and activity, but NADPH depletion and redox imbalance. In vivo, combined NOX2/NOX4 knockdown reduced ROS accumulation, suppressed G6PD hyperactivation, alleviated ferroptotic injuryand delayed regression Moreover, G6PD inhibition with 6-AN similarly reduced oxidative stress and ferroptosis and partially restored progesterone secretion. In primary luteal cells, G6PD knockdown or 6-AN treatment attenuated PGF2α-induced ferroptosis and improved cell viability. Simultaneously, NOX2/NOX4 silencing, N-acetylcysteine, or Ferrostatin-1 suppressed PGF2α-induced G6PD activation, and attenuated oxidative stress and ferroptosis.Conclusion NOX2/NOX4-associated oxidative stress contributes to G6PD-associated metabolic remodeling and ferroptosis during mice luteolysis.
Objectives: Reactive oxygen and nitrogen species (RONS) act as signalling molecules under physiological conditions; however, how oxidative eustress regulates glucose uptake in skeletal muscle remains poorly defined. We investigated the role of moderate oxidation in skeletal muscle glucose uptake, focusing on AKT/AMPK phosphorylation, GLUT4 translocation and functional glucose uptake. Methods: AKT and AMPK phosphorylation were analysed in C2C12 myoblasts/myotubes exposed to insulin and redox-modulating stimuli associated with oxidative eustress, including hydrogen peroxide, nitric oxide donors and angiotensin II. GLUT4 translocation was assessed by quantitative immunocytochemistry and confocal fluorescence microscopy in cells expressing a GLUT4 reporter. Glucose uptake was evaluated in isolated skeletal muscle fibres using 6-NBDG. Results: Oxidative eustress was associated with increased AKT and AMPK phosphorylation and enhanced GLUT4 translocation to the plasma membrane. Hydrogen peroxide, nitric oxide donors and angiotensin II increased GLUT4 presence at the plasma membrane and enhanced glucose uptake, with hydrogen peroxide showing a dose-dependent effect. Increased glucose uptake was consistent with GLUT4 translocation. Discussion: These findings support oxidative eustress as a physiological redox mechanism linking AKT/AMPK activation, GLUT4 translocation and glucose uptake in skeletal muscle. Redox signalling may therefore contribute to skeletal muscle glucose metabolism.
