Homocystinuria (HCU) is an inborn error of metabolism and a conformational disorder chiefly caused by the missense mutations in the cystathionine beta-synthase (CBS) gene. These mutations often cause CBS destabilization, misfolding and dysfunction resulting in CBS deficiency and pathological accumulation of homocysteine. Morphological changes in mitochondria were described in HCU patients and mouse models; however, their functional significance has remained unknown. Here, we characterized the impact of CBS deficiency due to expression of the most common HCU-causing variant CBS I278T on mitochondrial function using three cellular models of HCU: mouse hepatocytes, human fibroblasts and newly developed CRISPR/Cas9-modified HEK293 cells. We found that the expression of the CBS I278T variant resulted in unfolded protein response, oxidative stress and impaired cellular energy metabolism in all three cellular models of HCU. Mitochondrial respiration and ATP production were substantially impaired. Bioenergetic deficit correlated morphologically with mitochondrial swelling and loss of cristae and functionally with the decreased membrane potential and cytosolic mitochondrial DNA release. Impaired clearance of damaged, non-functional mitochondria was caused by the compromised mitophagy activation and dysfunctional lysosomes. Methionine restriction substantially reduced plasma total homocysteine and rescued mitochondrial function of hepatocytes isolated from the treated Tg-I278T HCU mice. Importantly, CBS knockout HEK293 cells showed normal proteostasis and mitochondrial function indicating that CBS I278T misfolding is the main cause and trigger of the described pathological phenotype. These findings provide the first mechanistic insight into the impaired cellular bioenergetics in HCU.
Mitochondrial dysfunction is central to podocyte injury in diabetic kidney disease (DKD). Here, we observed consistent downregulation of dual-specificity phosphatase 8 (DUSP8) in glomeruli from patients with DKD, db/db mice, STZ/HFD-induced DKD mice, and high glucose-treated podocytes. Glomerular DUSP8 levels were positively correlated with the estimated glomerular filtration rate and negatively correlated with the urinary albumin-to-creatinine ratio and serum creatinine level. Functional studies demonstrated that DUSP8 alleviated high glucose-induced mitochondrial fission, reactive oxygen species accumulation, mitochondrial permeability transition pore hyperactivation, loss of mitochondrial membrane potential, and podocyte injury. In vivo, podocyte-specific DUSP8 knockout exacerbated mitochondrial dysfunction, podocyte loss, glomerular pathology, and renal functional decline in STZ/HFD-induced DKD mice. Mechanistically, DUSP8 dephosphorylated PRAS40 at Ser202 via its phosphatase activity, thereby mitigating high glucose-induced mitochondrial injury in podocytes. Consistent with these findings, podocyte-specific DUSP8 knockdown in STZ/HFD-induced DKD mice was positively correlated with PRAS40 hyperphosphorylation at Ser202, mirroring the inverse correlation between DUSP8 expression and PRAS40 phosphorylation observed in patients with DKD. Furthermore, we found that histone deacetylase 1 (HDAC1) epigenetically silenced DUSP8 transcription by deacetylating histone H3 lysine 27 (H3K27) at its promoter. Collectively, we elucidated the HDAC1/DUSP8/PRAS40 axis as a novel regulator of podocyte mitochondrial homeostasis in DKD, providing a mechanistic framework and a potential therapeutic target.
Intercellular mitochondrial transfer has been recognized as an important mechanism for maintaining tissue homeostasis and adapting to stress. Mitochondria can cross cellular boundaries through tunneling nanotubes, extracellular vesicles, and free mitochondrial release. However, the physiological signals coordinating these pathways remain poorly defined. Exercise is a potent inducer of transient redox signaling, generating superoxide and hydrogen peroxide while modulating mitochondrial dynamic remodeling. This review integrates exercise redox biology with redox regulation of transfer machinery characterized in non-exercise models, proposing that exercise-induced redox signaling may function as a candidate regulatory mechanism. The framework emphasizes bidirectional redox coordination, in which oxidant pulses may activate export in donor cells and prepare recipient cells for uptake and antioxidant defense. Exercise-induced mitochondrial transfer has been directly demonstrated in the brain, while observations in skeletal muscle, adipose tissue, and heart remain suggestive but have not been confirmed in exercise models. These findings support a framework in which intercellular mitochondrial transfer contributes to metabolic signaling, antioxidant defense, and distributed quality control across organs. This model represents a working hypothesis requiring direct experimental validation through lineage tracing, tissue-specific mitochondrial reporters, and intravital imaging.
BACKGROUND:Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia, with fibroblast-like synoviocytes (FLS) playing a key pathogenic role. Cysteine metabolism is central to redox homeostasis and ferroptosis regulation, yet its mechanisms in RA-FLS remain poorly understood. This study investigates the role of AMD1, a polyamine metabolism enzyme, in regulating cysteine metabolism and ferroptosis sensitivity in RA-FLS. METHODS:Transcriptomic (GSE89408) and single-cell RNA-seq datasets (GSE200815, GSE246416) were analyzed using differential expression, WGCNA, and machine learning (LASSO, Random Forest, SVM). RA-FLS were subjected to AMD1 knockdown or overexpression, with functional assays for proliferation, migration, redox status, and ferroptosis markers. Rescue experiments used exogenous cysteine or Ferrostatin-1 (Fer-1). A collagen-induced arthritis (CIA) mouse model with local AAV-mediated AMD1 knockdown was used for in vivo validation. RESULTS:AMD1 was significantly upregulated in RA synovium and showed prominent expression in FLS. AMD1 knockdown reduced RA-FLS proliferation and migration, decreased polyamine and glutathione levels, increased ROS and lipid peroxidation, and promoted ferroptosis, as evidenced by reduced SLC7A11 and GPX4 expression, increased ACSL4 expression, and Fe2+ accumulation. Exogenous cysteine or Fer-1 partially reversed these effects. In CIA mice, local AMD1 knockdown alleviated joint swelling, cartilage destruction, and synovitis, while modulating ferroptosis-related protein expression. CONCLUSIONS:AMD1 regulates cysteine metabolism and ferroptosis susceptibility in RA-FLS, representing a promising therapeutic target for RA.
Intervertebral disc degeneration (IDD) is associated with the loss of nucleus pulposus derived mesenchymal stem cell (NP-MSC) function, but the contribution of the mitochondrial unfolded protein response (UPRmt) to this process is not well understood. We found that SIRT1 and the UPRmt-related proteins HSP60, ATF5, and CLPP decreased as degeneration progressed in human disc tissues and primary NP-MSCs. In NP-MSCs exposed to tert-butyl hydroperoxide, metformin increased AMPK phosphorylation and SIRT1 expression, enhanced UPRmt signaling, and reduced apoptosis and senescence. Metformin also improved mitochondrial membrane potential and morphology, lowered reactive oxygen species, restored NAD + levels, and favored extracellular matrix synthesis. Blocking SIRT1 with EX527 or SIRT1 siRNA weakened UPRmt activation and largely reversed the mitochondrial, cellular, and matrix effects of metformin. In a rat needle-puncture model, intradiscal metformin preserved disc height and T2 signal, reduced histological degeneration, and increased matrix and UPRmt-related protein expression; these effects were diminished by EX527. Together, these results suggest that reduced AMPK/SIRT1/UPRmt activity contributes to NP-MSC dysfunction during IDD and that metformin may slow disc degeneration by restoring this mitochondrial stress response.
Advanced glycation end products (AGEs) accumulate with aging and have been implicated in neurodegeneration, yet their relationship with the APOE4 genotype and downstream inflammatory signaling remains poorly understood. Here, we show that APOE4 is associated with greater age-dependent AGE accumulation and APOE glycation in the aging brain compared with APOE3 in animal models. These changes are accompanied by mitochondrial dysfunction and increased release of mitochondrial DNA (mtDNA) into the cytosol, providing a potential trigger for innate immune activation. Consistent with enhanced innate immune signaling, APOE4 brains exhibit increased cGAS expression and phosphorylation of STING, TBK1, and IRF3, together with elevated type I interferon and pro-inflammatory responses. This activation is particularly prominent in microglia, as demonstrated by increased cGAS-DNA interactions and greater colocalization of cGAS signaling with Iba1-positive cells. APOE4 mice further display increased levels of cGAMP and IFN-β, as well as enhanced expression of pro-inflammatory cytokines and interferon-stimulated genes. Mechanistically, exposure of primary microglia to AGEs induces cytosolic mtDNA release and activates cGAS-STING signaling, whereas pharmacological inhibition of the receptor for advanced glycation end products (RAGE) attenuates these responses. Together, these findings identify an association between the APOE4-AGE axis, mitochondrial dysfunction, mtDNA release, and enhanced cGAS-STING-related inflammatory signaling in the aging brain, while the in vitro studies support a functional contribution of AGE-RAGE signaling to these responses in primary microglia.
Oxylipins, produced from polyunsaturated fatty acids (PUFA) by lipoxygenases (LOX), play pivotal roles in inflammation. While 5-LOX-derived leukotrienes promote inflammation, the specialized pro-resolving mediators (SPMs) contribute to inflammation resolution. The biosynthesis of the SPM members lipoxins and resolvins (RVs) supposedly relies on two sequential PUFA oxygenation steps, involving metabolism by 5-LOX and 12-/15-lipoxygenating paralogues (e.g., 12-LOX, 15-LOX-1/-2). These oxygenations may occur within one cell where both LOX isoforms are expressed or via transcellular metabolism between different cell types with complementary LOX expression. Here, we investigated monocellular incubations and (patho-)physiologically relevant co-incubations of human 5-LOX-positive polymorphonuclear leukocytes (PMNL) or monocytes with 12-LOX-positive platelets or with 15-LOX-1-rich M2a or dexamethasone-treated 15-LOX-2-rich MDex macrophages, reflecting possible transcellular SPM-biosynthetic routes. Comprehensive metabololipidomics using UPLC-MS/MS was employed for oxylipin profiling, and LOX-transfected HEK293 cells served as supportive model. While we confirmed the interplay of 5-LOX and 12-LOX in lipoxin formation in PMNL-platelet and monocyte-platelet co-incubations, we uncovered cooperation between 5-LOX and 15-LOX-2 yielding strong RvD5 and RvE4 formation in PMNL/MDex co-incubations. In contrast, 15-LOX-1-rich M2a produced substantial RvD5, while co-incubations with PMNL surprisingly impaired formation of RvD5 and other 15-LOX products. Moreover, we show that 15-LOX-1 prefers arachidonic acid and eicosapentaenoic acid as substrates, while 15-LOX-2 favors docosahexaenoic acid and 5- or 7-monohydroxylated precursors for SPM formation; in this context, 5- or 7-lipoxygenation is seemingly the first and rate-limiting oxygenation step. Conclusively, 15-LOX-1 and -2 isoforms exert distinct roles in conjunction with 5-LOX in the sequential biosynthesis of SPMs, which differs in monocellular versus co-incubations.
Oxaliplatin (OXA) is widely used to treat advanced gastric cancer, but acquired resistance limits its benefit. How resistant cells avoid ferroptosis remains unclear. Quantitative proteomics identified UGT8, a glycosphingolipid biosynthetic enzyme, as an upregulated protein in OXA-resistant gastric cancer cells. UGT8 protein was also increased in tumors from clinically OXA-resistant patients treated with oxaliplatin-based chemotherapy, and high UGT8 expression was associated with shorter survival. UGT8 overexpression increased OXA tolerance, while UGT8 depletion sensitized resistant cells and patient-derived organoids (PDOs) from clinically OXA-resistant patients to OXA. UGT8 depletion increased lipid reactive oxygen species and malondialdehyde, reduced glutathione, and aggravated ferroptosis-associated mitochondrial damage. Immunoprecipitation followed by mass spectrometry identified USP10 as a UGT8-associated protein. USP10 increased UGT8 stability and limited its K48-linked polyubiquitination. K307 was identified by mutational analysis as a functionally important residue involved in USP10-regulated UGT8 ubiquitination. UGT8 increased NRF2 nuclear accumulation and maintained HO-1, SLC7A11, and GPX4 expression. Restoring UGT8 or pharmacologically enhancing NRF2-associated antioxidant signaling reduced the effects of USP10 or UGT8 depletion on OXA sensitivity and ferroptosis-associated changes. Disrupting the USP10-UGT8 pathway also improved the OXA response in xenografts. These results support a role for USP10-dependent UGT8 stabilization in sustaining NRF2-associated antioxidant signaling, suppressing ferroptosis, and promoting OXA resistance.
Silver nanoparticles (AgNPs) are widely used for their antimicrobial properties in consumer and biomedical products, prompting concern about their potential neurotoxicity. However, the mechanisms by which AgNPs drive neuronal death across different exposure durations remain poorly defined. In this study we investigated the temporal dynamics and mechanisms by which AgNPs induce neurotoxicity in hippocampal neurons, focusing on the cystine-glutamate antiporter solute carrier family 7 member 11 (SLC7A11). Using combined in vivo (1-day acute and 28-day subacute intranasal exposure in mice) and in vitro (HT22 hippocampal neurons) models, together with inhibitors (deferoxamine, ferrostatin-1, TCEP), SLC7A11 siRNA, and surface-enhanced Raman spectroscopy (SERS) spectroscopy, we delineated a time-dependent switch between disulfidptosis and ferroptosis. Early (24 h) AgNPs exposure produced SLC7A11 up-regulation, cystine accumulation, NADPH depletion, GLUT1 down-regulation and F-actin collapse-features of disulfidptosis that were rescued by TCEP or SLC7A11 knockdown. With prolonged/subacute exposure, SLC7A11 and glutathione peroxidase 4 (GPX4) expression fell, iron homeostasis was disrupted, mitochondria showed ferroptotic morphology, hippocampal silver accumulated, and mice exhibited spatial memory deficits. SERS of AgNPs-cystine mixtures revealed cleavage of cystine disulfide bonds and formation of Ag-S complexes, a chemical transition that likely attenuates disulfide stress and facilitates the shift toward iron-dependent ferroptosis. Collectively, these data position SLC7A11 as a context-dependent metabolic switch governing the balance between disulfidptosis and ferroptosis, and provide a mechanistic basis to refine their safe and targeted application, particularly in biomedical contexts.
Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an emerging non-invasive marker across cancers. Yet, in gastric cancer (GC), its relationship to tissue mtDNA content, oxidative remodeling and somatic mtDNA variants, and thus its basis in mitochondrial homeostasis, remains poorly defined. We analyzed 169 individuals: 70 GC patients, 29 with precancerous gastric lesions and 70 healthy controls. MtDNA copy number was measured by quantitative PCR, and plasma biomarkers of oxidative damage (8-hydroxy-2'-deoxyguanosine, 8OH-dG; 4-hydroxynonenal, 4HNE) and of antioxidant capacity (glutathione peroxidase-1, GPX-1) were measured by ELISA. MtDNA variants were identified by next-generation sequencing. In MKN-28 GC cells, mitochondrial transcription factor A (TFAM) was manipulated with lentiviral vectors to alter mtDNA content. The results showed that GC progression was accompanied by higher ccf-mtDNA, shifts in plasma oxidative damage and antioxidant markers, and accumulation of mtDNA variants, alongside lower mtDNA content in cancers than in adjacent tissues. In GC cells, lowering mtDNA content by TFAM silencing promoted the malignant phenotype and increased intracellular superoxide-related fluorescence. By multiplex immunohistochemistry, tissue markers of mitochondrial maintenance and mtDNA release declined without a rise in caspase-3. D-loop variants already present at the precancerous stage suggest early mitochondrial changes, whereas predicted deleterious coding variants affecting respiratory chain components were observed in GC tissues. These findings support a redox-associated model of mitochondrial homeostasis that links impaired mitochondrial maintenance, mtDNA instability, and ccf-mtDNA accumulation, and provide a tissue-anchored mechanistic basis for the circulating mtDNA changes detected by plasma-based approaches in GC.
Lipid homeostasis is essential for oocyte quality and developmental competence, and uncoupling protein 1 (UCP1) has been identified as a critical regulator of lipid metabolism in oocytes. However, the downstream molecular mechanism by which UCP1 regulates lipid homeostasis remains unexplored. In this study, we demonstrate that UCP1 inhibition impairs oocyte developmental competence, accompanied by increased lipid accumulation, elevated oxidative stress, and disrupted mitochondrial function. Further investigation revealed that UCP1 inhibition markedly elevated mitochondrial calcium levels, which were associated with upregulation of the mitochondrial calcium uniporter (MCU). Functional inhibition of MCU with Ru360 reduced mitochondrial calcium accumulation and alleviated mitochondrial function, redox homeostasis, and lipid metabolic balance, ultimately improving oocyte developmental competence. These findings suggest that UCP1 regulates oocyte lipid homeostasis and developmental competence through MCU-dependent mitochondrial calcium uptake, establishing a mechanistic link between mitochondrial uncoupling, calcium homeostasis, and oocyte quality.
