Developing a novel immune infiltration-associated mitophagy prediction model for amyotrophic lateral sclerosis using bioinformatics strategies.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which leads to muscle weakness and eventual paralysis. Numerous studies have indicated that mitophagy and immune inflammation have a significant impact on the onset and advancement of ALS. Nevertheless, the possible diagnostic and prognostic significance of mitophagy-related genes associated with immune infiltration in ALS is uncertain. The purpose of this study is to create a predictive model for ALS using genes linked with mitophagy-associated immune infiltration. Methods: ALS gene expression profiles were downloaded from the Gene Expression Omnibus (GEO) database. Univariate Cox analysis and machine learning methods were applied to analyze mitophagy-associated genes and develop a prognostic risk score model. Subsequently, functional and immune infiltration analyses were conducted to study the biological attributes and immune cell enrichment in individuals with ALS. Additionally, validation of identified feature genes in the prediction model was performed using ALS mouse models and ALS patients. Results: In this study, a comprehensive analysis revealed the identification of 22 mitophagy-related differential expression genes and 40 prognostic genes. Additionally, an 18-gene prognostic signature was identified with machine learning, which was utilized to construct a prognostic risk score model. Functional enrichment analysis demonstrated the enrichment of various pathways, including oxidative phosphorylation, unfolded proteins, KRAS, and mTOR signaling pathways, as well as other immune-related pathways. The analysis of immune infiltration revealed notable distinctions in certain congenital immune cells and adaptive immune cells between the low-risk and high-risk groups, particularly concerning the T lymphocyte subgroup. ALS mouse models and ALS clinical samples demonstrated consistent expression levels of four mitophagy-related immune infiltration genes (BCKDHA, JTB, KYNU, and GTF2H5) with the results of bioinformatics analysis. Conclusion: This study has successfully devised and verified a pioneering prognostic predictive risk score for ALS, utilizing eighteen mitophagy-related genes. Furthermore, the findings indicate that four of these genes exhibit promising roles in the context of ALS prognostic.

OMA1 competitively binds to HSPA9 to promote mitophagy and activate the cGAS-STING pathway to mediate GBM immune escape.

BACKGROUND: Immunotherapy with checkpoint inhibitors, especially those targeting programmed death receptor 1 (PD-1)/PD-1 ligand (PD-L1), is increasingly recognized as a highly promising therapeutic modality for malignancies. Nevertheless, the efficiency of immune checkpoint blockade therapy in treating glioblastoma (GBM) is constrained. Hence, it is imperative to expand our comprehension of the molecular mechanisms behind GBM immune escape (IE). METHODS: Protein chip analysis was performed to screen aberrantly expressed OMA1 protein in PD-1 inhibitor sensitive or resistant GBM. Herein, public databases and bioinformatics analysis were employed to investigate the OMA1 and PD-L1 relation. Then, this predicted relation was verified in primary GBM cell lines through distinct experimental methods. To investigate the molecular mechanism behind OMA1 in immunosuppression, a series of experimental methods were employed, including Western blotting, co-immunoprecipitation (Co-IP), mass spectrometry (MS), immunofluorescence, immunohistochemistry, and qRT-PCR. RESULTS: Our findings revealed that OMA1 competitively binds to HSPA9 to induce mitophagy and mediates the IE of GBM. Data from TCGA indicated a significant correlation between OMA1 and immunosuppression. OMA1 promoted PD-L1 levels in primary cells from patients with GBM. Next, the results of Co-IP and MS conducted on GBM primary cells revealed that OMA1 interacts with HSPA9 and induces mitophagy. OMA1 promoted not only cGAS-STING activity by increasing mitochondrial DNA release but also PD-L1 transcription by activating cGAS-STING. Eventually, OMA1 has been found to induce immune evasion in GBM through its regulation of PD-1 binding and PD-L1 mediated T cell cytotoxicity. CONCLUSIONS: The OMA1/HSPA9/cGAS/PD-L1 axis is elucidated in our study as a newly identified immune therapeutic target in GBM.

Effect of resistance training plus enriched probiotic supplement on sestrin2, oxidative stress, and mitophagy markers in elderly male Wistar rats.

This study aimed to determine the effects of resistance training combined with a probiotic supplement enriched with vitamin D and leucine on sestrin2, oxidative stress, antioxidant defense, and mitophagy markers in aged Wistar rats. Thirty-five male rats were randomly assigned to two age groups (old with 18-24 months of age and young with 8-12 weeks of age) and then divided into five groups, including (1) old control (OC: n = 5 + 2 for reserve in all groups), (2) young control (YC: n = 5), (3) old resistance training (OR: n = 5), (4) old resistance training plus supplement (ORS: n = 5), and old supplement group (OS: n = 5). Training groups performed ladder climbing resistance training 3 times per week for 8 weeks. Training intensity was inserted progressively, with values equal to 65, 75, and 85, determining rats' maximal carrying load capacity. Each animal made 5 to 8 climbs in each training session, and the time of each climb was between 12 and 15 s, although the time was not the subject of the evaluation, and the climbing pattern was different in the animals. Old resistance plus supplement and old supplement groups received 1 ml of supplement 5 times per week by oral gavage in addition to standard feeding, 1 to 2 h post training sessions. Forty-eight hours after the end of the training program, 3 ml of blood samples were taken, and all rats were then sacrificed to achieve muscle samples. After 8 weeks of training, total antioxidant capacity and superoxide dismutase activity levels increased in both interventions. A synergistic effect of supplement with resistance training was observed for total antioxidant capacity, superoxide dismutase, and PTEN-induced kinase 1. Sestrin 2 decreased in intervention groups. These results suggest that resistance training plus supplement can boost antioxidant defense and mitophagy while potentially decreasing muscle strength loss.

Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

Mitophagy mediated by HIF-1α/FUNDC1 signaling in tubular cells protects against renal ischemia/reperfusion injury.

Acute kidney injury (AKI) is associated with a high mortality rate. Pathologically, renal ischemia/reperfusion injury (RIRI) is one of the primary causes of AKI, and hypoxia-inducible factor (HIF)-1α may play a defensive role in RIRI. This study assessed the role of hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy in protection against RIRI in vitro and in vivo. The human tubular cell line HK-2 was used to assess hypoxia/reoxygenation (H/R)-induced mitophagy through different in vitro assays, including western blotting, immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reactive oxygen species (ROS) measurement. Additionally, a rat RIRI model was established for evaluation by renal histopathology, renal Doppler ultrasound, and transmission electron microscopy to confirm the in vitro data. The selective HIF-1α inhibitor LW6 reduced H/R-induced mitophagy but increased H/R-induced apoptosis and ROS production. Moreover, H/R treatment enhanced expression of the FUN14 domain-containing 1 (FUNDC1) protein. Additionally, FUNDC1 overexpression reversed the effects of LW6 on the altered expression of light chain 3 (LC3) BII and voltage-dependent anion channels as well as blocked the effects of HIF-1α inhibition in cells. Pretreatment of the rat RIRI model with roxadustat, a novel oral HIF-1α inhibitor, led to decreased renal injury and apoptosis in vivo. In conclusion, the HIF-1α/FUNDC1 signaling pathway mediates H/R-promoted renal tubular cell mitophagy, whereas inhibition of this signaling pathway protects cells from mitophagy, thus aggravating apoptosis, and ROS production. Accordingly, roxadustat may protect against RIRI-related AKI.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

Chlorantraniliprole induces mitophagy, ferroptosis, and cytokine homeostasis imbalance in grass carp (Ctenopharyngodon idella) hepatocytes via the mtROS-mitochondrial fission/fusion axis.

Chlorantraniliprole (CAP) is a bis-amide pesticide used for pest control mainly in agricultural production activities and rice-fish co-culture systems. CAP residues cause liver damage in non-target organism freshwater fish. However, it is unclear whether CAP-exposure-induced liver injury in fish is associated with mitochondrial dysfunction-mediated mitophagy, ferroptosis, and cytokines. Therefore, we established grass carp hepatocyte models exposed to different concentrations of CAP (20, 40, and 80 µM) in vitro. MitoSOX probe, JC-1 staining, immunofluorescence double staining, Fe2+ staining, lipid peroxidation staining, qRT-PCR, and Western blot were used to verify the physiological regulatory mechanism of CAP induced liver injury. In the present study, the CAP-treated groups exhibited down-regulation of antioxidant-related enzyme activities and accumulation of peroxides. CAP treatment induced an increase in mitochondrial reactive oxygen species (mtROS) levels and altered expression of mitochondrial fission/fusion (Drp1, Fis1, Mfn1, Mfn2, and Opa1) genes in grass carp hepatocytes. In addition, mitophagy (Parkin, Pink1, p62, LC3II/I, and Beclin-1), ferroptosis (GPX4, COX2, ACSL4, FTH, and NCOA4), and cytokine (IFN-γ, IL-18, IL-17, IL-6, IL-10, IL-1ß, IL-2, and TNF-α)-related gene expression was significantly altered. Collectively, these findings suggest that CAP exposure drives mitophagy activation, ferroptosis occurrence, and cytokine homeostasis imbalance in grass carp hepatocytes by triggering mitochondrial dysfunction mediated by the mtROS-mitochondrial fission/fusion axis. This study partly explained the physiological regulation mechanism of grass carp hepatocyte injury induced by insecticide CAP from the physiological and biochemical point of view and provided a basis for evaluating the safety of CAP environmental residues to non-target organisms.

Chronic replication stress invokes mitochondria dysfunction via impaired parkin activity.

Replication stress is a major contributor to tumorigenesis because it provides a source of chromosomal rearrangements via recombination events. PARK2, which encodes parkin, a regulator of mitochondrial homeostasis, is located on one of the common fragile sites that are prone to rearrangement by replication stress, indicating that replication stress may potentially impact mitochondrial homeostasis. Here, we show that chronic low-dose replication stress causes a fixed reduction in parkin expression, which is associated with mitochondrial dysfunction, indicated by an increase in mtROS. Consistent with the major role of parkin in mitophagy, reduction in parkin protein expression was associated with a slight decrease in mitophagy and changes in mitochondrial morphology. In contrast, cells expressing ectopic PARK2 gene does not show mtROS increases and changes in mitochondrial morphology even after exposure to chronic replication stress, suggesting that intrinsic fragility at PARK2 loci associated with parkin reduction is responsible for mitochondrial dysfunction caused by chronic replication stress. As endogenous replication stress and mitochondrial dysfunction are both involved in multiple pathophysiology, our data support the therapeutic development of recovery of parkin expression in human healthcare.

SIRT3 alleviates painful diabetic neuropathy by mediating the FoxO3a-PINK1-Parkin signaling pathway to activate mitophagy.

INTRODUCTION: Painful diabetic neuropathy (PDN) is a common complication of diabetes. Previous studies have implicated that mitochondrial dysfunction plays a role in the development of PDN, but its pathogenesis and mechanism have not been fully investigated. METHODS: In this study, we used high-fat diet/low-dose streptozotocin-induced rats as a model of type 2 diabetes mellitus. Behavioral testing, whole-cell patch-clamp recordings of dorsal root ganglion (DRG) neurons, and complex sensory nerve conduction velocity studies were used to assess peripheral neuropathy. Mitochondrial membrane potential (MMP), ATP, tissue reactive oxygen species, and transmission electron microscopy were used to evaluate the function and morphology of mitochondria in DRG. Real-time PCR, western blot, and immunofluorescence were performed to investigate the mechanism. RESULTS: We found that damaged mitochondria were accumulated and mitophagy was inhibited in PDN rats. The expression of sirtuin 3 (SIRT3), which is an NAD+-dependent deacetylase in mitochondria, was inhibited. Overexpression of SIRT3 in DRG neurons by intrathecally administered LV-SIRT3 lentivirus ameliorated neurological and mitochondrial dysfunctions. This was evidenced by the reversal of allodynia and nociceptor hyperexcitability, as well as the restoration of MMP and ATP levels. Overexpression of SIRT3 restored the inhibited mitophagy by activating the FoxO3a-PINK1-Parkin signaling pathway. The effects of SIRT3 overexpression, including the reversal of allodynia and nociceptor hyperexcitability, the improvement of impaired mitochondria and mitophagy, and the restoration of PINK1 and Parkin expression, were counteracted when FoxO3a siRNA was intrathecally injected. CONCLUSION: These results showed that SIRT3 overexpression ameliorates PDN via activation of FoxO3a-PINK1-Parkin-mediated mitophagy, suggesting that SIRT3 may become an encouraging therapeutic strategy for PDN.

Panax notoginseng saponins prevent dementia and oxidative stress in brains of SAMP8 mice by enhancing mitophagy.

BACKGROUND: Mitochondrial dysfunction is one of the distinctive features of neurons in patients with Alzheimer's disease (AD). Intraneuronal autophagosomes selectively phagocytose and degrade the damaged mitochondria, mitigating neuronal damage in AD. Panax notoginseng saponins (PNS) can effectively reduce oxidative stress and mitochondrial damage in the brain of animals with AD, but their exact mechanism of action is unknown. METHODS: Senescence-accelerated mouse prone 8 (SAMP8) mice with age-related AD were treated with PNS for 8 weeks. The effects of PNS on learning and memory abilities, cerebral oxidative stress status, and hippocampus ultrastructure of mice were observed. Moreover, changes of the PTEN-induced putative kinase 1 (PINK1)-Parkin, which regulates ubiquitin-dependent mitophagy, and the recruit of downstream autophagy receptors were investigated. RESULTS: PNS attenuated cognitive dysfunction in SAMP8 mice in the Morris water maze test. PNS also enhanced glutathione peroxidase and superoxide dismutase activities, and increased glutathione levels by 25.92% and 45.55% while inhibiting 8-hydroxydeoxyguanosine by 27.74% and the malondialdehyde production by 34.02% in the brains of SAMP8 mice. Our observation revealed the promotion of mitophagy, which was accompanied by an increase in microtubule-associated protein 1 light chain 3 (LC3) mRNA and 70.00% increase of LC3-II/I protein ratio in the brain tissues of PNS-treated mice. PNS treatment increased Parkin mRNA and protein expression by 62.80% and 43.80%, while increasing the mRNA transcription and protein expression of mitophagic receptors such as optineurin, and nuclear dot protein 52. CONCLUSION: PNS enhanced the PINK1/Parkin pathway and facilitated mitophagy in the hippocampus, thereby preventing cerebral oxidative stress in SAMP8 mice. This may be a mechanism contributing to the cognition-improvement effect of PNS.

Exquisite visualization of mitophagy and monitoring the increase of lysosomal micro-viscosity in mitophagy with an unusual pH-independent lysosomal rotor.

BACKGROUND: Mitophagy plays indispensable roles in maintaining intracellular homeostasis in most eukaryotic cells by selectively eliminating superfluous components or damaged organelles. Thus, the co-operation of mitochondrial probes and lysosomal probes was presented to directly monitor mitophagy in dual colors. Nowadays, most of the lysosomal probes are composed of groups sensitive to pH, such as morpholine, amine and other weak bases. However, the pH in lysosomes would fluctuate in the process of mitophagy, leading to the optical interference. Thus, it is crucial to develop a pH-insensitive probe to overcome this tough problem to achieve exquisite visualization of mitophagy. RESULTS: In this study, we rationally prepared a pH-independent lysosome probe to reduce the optical interference in mitophagy, and thus the process of mitophagy could be directly monitored in dual color through cooperation between IVDI and MTR, depending on Förster resonance energy transfer mechanism. IVDI shows remarkable fluorescence enhancement toward the increase of viscosity, and the fluorescence barely changes when pH varies. Due to the sensitivity to viscosity, the probe can visualize micro-viscosity alterations in lysosomes without washing procedures, and it showed better imaging properties than LTR. Thanks to the inertia of IVDI to pH, IVDI can exquisitely monitor mitophagy with MTR by FRET mechanism despite the changes of lysosomal pH in mitophagy, and the reduced fluorescence intensity ratio of green and red channels can indicate the occurrence of mitophagy. Based on the properties mentioned above, the real-time increase of micro-viscosity in lysosomes during mitophagy was exquisitely monitored through employing IVDI. SIGNIFICANCE AND NOVELTY: Compared with the lysosomal fluorescent probes sensitive to pH, the pH-inert probe could reduce the influence of pH variation during mitophagy to achieve exquisite visualization of mitophagy in real-time. Besides, the probe could monitor the increase of lysosomal micro-viscosity in mitophagy. So, the probe possesses tremendous potential in the visualization of dynamic changes related to lysosomes in various physiological processes.

[Research progress on the role of TANK-binding kinase 1 in PINK1/Parkin-dependent and -independent mitophagy].

Mitophagy is a process that selectively removes excess or damaged mitochondria and plays an important role in regulating intracellular mitochondrial mass and maintaining mitochondrial energy metabolism. TANK-binding kinase 1 (TBK1) is a multifunctional serine/threonine protein kinase, which is involved in the regulation of PTEN-induced putative kinase 1 (PINK1)/Parkin-dependent and -independent mitophagy. Recent studies have shown that TBK1 phosphorylates the autophagy related proteins, such as optineurin (OPTN), p62/sequestosome-1, Ras-related GTP binding protein 7 (Rab7), and mediates the binding of nuclear dot protein 52 (NDP52) to UNC-51 like autophagy activating kinase 1 (ULK1) complex, as well as the binding of TAX1-binding protein 1 (TAX1BP1) to microtubule-associated protein 1 light chain 3 (LC3), thereby enhancing PINK1/Parkin-dependent mitophagy. In addition, TBK1 is a direct substrate of AMP-activated protein kinase (AMPK)/ULK1 pathway, and its activation phosphorylates dynamin-related protein 1 (Drp1) and Rab7 to promote PINK1/Parkin-independent mitophagy. This article reviews the role and mechanism of TBK1 in regulating PINK1/Parkin-dependent and -independent mitophagy.

XinJiaCongRongTuSiZiWan protects triptolide-induced rats from oxidative stress injury via mitophagy mediated PINK1/Parkin signaling pathway.

PURPOSE: XinJiaCongRongTuSiZiWan (XJCRTSZW) is a traditional Chinese medicine compound for invigorating the kidney, nourishing blood, and promoting blood circulation. This study aimed to explore the effect of XJCRTSZW on triptolide (TP)-induced oxidative stress injury. METHODS: Adult female Sprague-Dawley rats and human ovarian granulosa cell lines were treated with TP and XJCRTSZW. Hematoxylin and eosin staining, enzyme-linked immunosorbent assay, flow cytometry, CCK-8, JC-1 staining, transmission electron microscopy, reverse transcription-quantitative polymerase chain reaction, and Western blotting were performed in this study. RESULTS: XJCRTSZW treatment observably ameliorated the TP-induced pathological symptoms. Furthermore, XJCRTSZW treatment observably enhanced the TP-induced reduction of estradiol, anti-Mullerian hormone, progesterone, superoxide dismutase, ATP content, mitochondrial membrane potential, p62, and Hsp60 mRNA, and protein levels in vivo and in vitro (p < 0.05). However, TP-induced elevation of follicle stimulating hormone and luteinizing hormone concentrations, malondialdehyde levels, reactive oxygen species levels, apoptosis rate, mitophagy, and the mRNA and protein expressions of LC3-II/LC3-I, PTEN-induced kinase 1 (PINK1), and Parkin were decreased (p < 0.05). In addition, XJCRTSZW treatment markedly increased cell viability in vitro (p < 0.05). CONCLUSIONS: XJCRTSZW protects TP-induced rats from oxidative stress injury via the mitophagy-mediated PINK1/Parkin pathway.

BAG3 localizes to mitochondria in cardiac fibroblasts and regulates mitophagy.

The co-chaperone Bcl2-associated athanogene 3 (BAG3) is a central node in protein quality control in the heart. In humans and animal models, decreased BAG3 expression is associated with cardiac dysfunction and dilated cardiomyopathy. Although previous studies focused on BAG3 in cardiomyocytes, cardiac fibroblasts are also critical drivers of pathologic remodeling. Yet, the role of BAG3 in cardiac fibroblasts is almost completely unexplored. Here, we show that BAG3 is expressed in primary rat neonatal cardiac fibroblasts and preferentially localizes to mitochondria. Knockdown of BAG3 reduces mitophagy and enhances fibroblast activation, which is associated with fibrotic remodeling. Heat shock protein 70 (Hsp70) is a critical binding partner for BAG3 and inhibiting this interaction in fibroblasts using the drug JG-98 decreased autophagy, decreased mitofusin-2 expression, and disrupted mitochondrial morphology. Together, these data indicate that BAG3 is expressed in cardiac fibroblasts, where it facilitates mitophagy and promotes fibroblast quiescence. This suggests that depressed BAG3 levels in heart failure may exacerbate fibrotic pathology, thus contributing to myocardial dysfunction through sarcomere-independent pathways.NEW & NOTEWORTHY We report BAG3's localization to mitochondria and its role in mitophagy for the first time in primary ventricular cardiac fibroblasts. We have also collected the first evidence showing that loss of BAG3 increases cardiac fibroblast activation into myofibroblasts, which are major drivers of cardiac fibrosis and pathological remodeling during heart disease.

Vascular Endothelial Cell-Derived Exosomal Sphingosylphosphorylcholine Attenuates Myocardial Ischemia-Reperfusion Injury through NR4A2-Mediated Mitophagy.

Cardiomyocyte survival is a critical contributing process of host adaptive responses to cardiovascular diseases (CVD). Cells of the cardiovascular endothelium have recently been reported to promote cardiomyocyte survival through exosome-loading cargos. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, mediates protection against myocardial infarction (MI). Nevertheless, the mechanism of SPC delivery by vascular endothelial cell (VEC)-derived exosomes (VEC-Exos) remains uncharacterized at the time of this writing. The present study utilized a mice model of ischemia/reperfusion (I/R) to demonstrate that the administration of exosomes via tail vein injection significantly diminished the severity of I/R-induced cardiac damage and prevented apoptosis of cardiomyocytes. Moreover, SPC was here identified as the primary mediator of the observed protective effects of VEC-Exos. In addition, within this investigation, in vitro experiments using cardiomyocytes showed that SPC counteracted myocardial I/R injury by activating the Parkin and nuclear receptor subfamily group A member 2/optineurin (NR4A2/OPTN) pathways, in turn resulting in increased levels of mitophagy within I/R-affected myocardium. The present study highlights the potential therapeutic effects of SPC-rich exosomes secreted by VECs on alleviating I/R-induced apoptosis in cardiomyocytes, thereby providing strong experimental evidence to support the application of SPC as a potential therapeutic target in the prevention and treatment of myocardial infarction.

Cr(VI) induces ferroptosis in DF-1 cells by simultaneously perturbing iron homeostasis of ferritinophagy and mitophagy.

Hexavalent chromium [Cr(VI)] is an environmental pollutant known for its strong oxidizing and carcinogenic effects. However, its potential to induce ferroptosis in poultry remains poorly understood. This study aims to investigate the induction of ferroptosis by Cr(VI) in DF-1 cells and elucidate the underlying mechanisms. DF-1 cells exposed to Cr(VI) showed increased lipid reactive oxygen species and changes in ferroptosis marker genes (decreased expression of GPX4 and increased expression of COX2). Notably, the addition of the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) can reverse this effect. During the cell death process, Cr(VI) induced ferritinophagy, disrupting iron homeostasis and releasing labile iron ions. We predicted by docking that these iron ions would bind to mitochondrial membrane proteins through virtual docking. This binding was validated through colocalization analysis. In addition, Cr(VI) caused mitophagy, which releases additional ferrous ions. Therefore, Cr(VI) can induce the simultaneous release of ferrous ions through these pathways, thereby exacerbating lipid peroxidation and ultimately triggering ferroptosis in DF-1 cells. This study demonstrates that Cr(VI) can induce ferroptosis in DF-1 cells by disrupting intracellular iron homeostasis and providing valuable insights into the toxic effects of Cr(VI) in poultry and potentially other organisms.

Sirt3 Protects Retinal Pigment Epithelial Cells From High Glucose-Induced Injury by Promoting Mitophagy Through the AMPK/mTOR/ULK1 Pathway.

Purpose: The regulation of mitophagy by Sirt3 has rarely been studied in ocular diseases. In the present study, we determined the effects of Sirt3 on AMPK/mTOR/ULK1 signaling pathway-mediated mitophagy in retinal pigment epithelial (RPE) cells in a high glucose environment. Methods: The mRNA expression levels of Sirt3, AMPK, mTOR, ULK1, and LC3B in RPE cells under varying glucose conditions were measured by real-time polymerase chain reaction (RT-PCR). The expressions of Sirt3, mitophagy protein, and AMPK/mTOR/ULK1 signaling pathway-related proteins were detected by Western blotting. Lentivirus (LV) transfection mediated the stable overexpression of Sirt3 in cell lines. The experimental groups were NG (5.5 mM glucose), hypertonic, HG (30 mM glucose), HG + LV-GFP, and HG + LV-Sirt3. Western blotting was performed to detect the expressions of mitophagy proteins and AMPK/mTOR/ULK1-related proteins in a high glucose environment during the overexpression of Sirt3. Reactive oxygen species (ROS) production in a high glucose environment was measured by DCFH-DA staining. Mitophagy was detected by labeling mitochondria and lysosomes with MitoTracker and LysoTracker probes, respectively. Apoptosis was detected by flow cytometry. Results: Sirt3 expression was reduced in the high glucose group, inhibiting the AMPK/mTOR/ULK1 pathway, with diminished mitophagy and increased intracellular ROS production. The overexpression of Sirt3, increased expression of p-AMPK/AMPK and p-ULK1/ULK1, and decreased expression of p-mTOR/mTOR inhibited cell apoptosis and enhanced mitophagy. Conclusions: Sirt3 protected RPE cells from high glucose-induced injury by activating the AMPK/mTOR/ULK1 signaling pathway. Translational Relevance: By identifying new targets of action, we aimed to establish effective therapeutic targets for diabetic retinopathy treatment.

Involvement of DJ-1 in the pathogenesis of intervertebral disc degeneration via hexokinase 2-mediated mitophagy.

Intervertebral disc degeneration (IDD) is an important pathological basis for degenerative spinal diseases and is involved in mitophagy dysfunction. However, the molecular mechanisms underlying mitophagy regulation in IDD remain unclear. This study aimed to clarify the role of DJ-1 in regulating mitophagy during IDD pathogenesis. Here, we showed that the mitochondrial localization of DJ-1 in nucleus pulposus cells (NPCs) first increased and then decreased in response to oxidative stress. Subsequently, loss- and gain-of-function experiments revealed that overexpression of DJ-1 in NPCs inhibited oxidative stress-induced mitochondrial dysfunction and mitochondria-dependent apoptosis, whereas knockdown of DJ-1 had the opposite effect. Mechanistically, mitochondrial translocation of DJ-1 promoted the recruitment of hexokinase 2 (HK2) to damaged mitochondria by activating Akt and subsequently Parkin-dependent mitophagy to inhibit oxidative stress-induced apoptosis in NPCs. However, silencing Parkin, reducing mitochondrial recruitment of HK2, or inhibiting Akt activation suppressed DJ-1-mediated mitophagy. Furthermore, overexpression of DJ-1 ameliorated IDD in rats through HK2-mediated mitophagy. Taken together, these findings indicate that DJ-1 promotes HK2-mediated mitophagy under oxidative stress conditions to inhibit mitochondria-dependent apoptosis in NPCs and could be a therapeutic target for IDD.

DDIT3/CHOP promotes LPS/ATP-induced pyroptosis in osteoblasts via mitophagy inhibition.

Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As a key factor of ER stress, DNA damage-inducible transcript 3 (DDIT3)/CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is upregulated in osteoblasts during inflammatory stimulation. DDIT3/CHOP may therefore regulate osteoblast pyroptosis in inflammatory conditions. During this investigation, we found that lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) stimulation in vitro induced osteoblasts to undergo pyroptosis, and the expression of DDIT3/CHOP was increased during this process. The overexpression of DDIT3/CHOP further promoted osteoblast pyroptosis as evidenced by the increased expression of the inflammasome NLR family pyrin domain containing 3 (NLRP3) and ratios of caspase-1 p20/caspase-1 and cleaved gasdermin D (GSDMD)/GSDMD. To explore the specific mechanism of this effect, we found through fluorescence imaging and Western blot analysis that LPS/ATP stimulation promoted PTEN-induced kinase 1 (PINK1)/E3 ubiquitin-protein ligase parkin (Parkin)-mediated mitophagy in osteoblasts, and this alteration was suppressed by the DDIT3/CHOP overexpression, resulting in increased ratio of pyroptosis compared with the control groups. The impact of DDIT3/CHOP on pyroptosis in osteoblasts was reversed by the application of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a specific mitophagy agonist. Therefore, our data demonstrated that DDIT3/CHOP promotes osteoblast pyroptosis by inhibiting PINK1/Parkin-mediated mitophagy in an inflammatory environment.