Role Of Mitophagy Research Articles

Predicting mitophagy-related genes and unveiling liver endothelial cell heterogeneity in hepatic ischemia-reperfusion injury.

Hepatic Ischemia-Reperfusion Injury (HIRI) is a major complication in liver transplants and surgeries, significantly affecting postoperative outcomes. The role of mitophagy, essential for removing dysfunctional mitochondria and maintaining cellular balance, remains unclear in HIRI. To unravel the role of mitophagy-related genes (MRGs) in HIRI, we assembled a comprehensive dataset comprising 44 HIRI samples alongside 44 normal control samples from the Gene Expression Omnibus (GEO) database for this analysis. Using Random Forests and Support Vector Machines - Recursive Feature Elimination (SVM-RFE), we pinpointed eight pivotal genes and developed a logistic regression model based on these findings. Further, we employed consensus cluster analysis for classifying HIRI patients according to their MRG expression profiles and conducted weighted gene co-expression network analysis (WGCNA) to identify clusters of genes that exhibit high correlation within different modules. Additionally, we conducted single-cell RNA sequencing data analysis to explore insights into the behavior of MRGs within the HIRI. We identified eight key genes (FUNDC1, VDAC1, MFN2, PINK1, CSNK2A2, ULK1, UBC, MAP1LC3B) with distinct expressions between HIRI and controls, confirmed by PCR validation. Our diagnostic model, based on these genes, accurately predicted HIRI outcomes. Analysis revealed a strong positive correlation of these genes with monocytic lineage and a negative correlation with B and T cells. HIRI patients were divided into three subclusters based on MRG profiles, with WGCNA uncovering highly correlated gene modules. Single-cell analysis identified two types of endothelial cells with different MRG scores, indicating their varied roles in HIRI. Our study highlights the critical role of MRGs in HIRI and the heterogeneity of endothelial cells. We identified the macrophage migration inhibitory factor (MIF) and cGAS-STING (GAS) pathways as regulators of mitophagy's impact on HIRI. These findings advance our understanding of mitophagy in HIRI and set the stage for future research and therapeutic developments.

Melatonin protects against myocardial ischemia-reperfusion injury by inhibiting excessive mitophagy through the Apelin/SIRT3 signaling axis

Excessive or uncontrolled mitophagy may result in a drastic shortage of healthy mitochondrial for ATP supply after reperfusion, leading to irreversible myocardial damage. Melatonin, a hormone produced by the pineal gland, has been proven to ameliorate myocardial ischemia-reperfusion (I/R) injury via regulating mitophagy. However, its underlying mechanism has not been fully elucidated. The present study focused on the role of mitophagy in the cardioprotective effects of melatonin by using the myocardial I/R rat model. The rats were pretreated with or without the apelin inhibitor ML221, the sirtuin 3 (SIRT3) inhibitor 3-TYP and then subjected to I/R injury, with melatonin administrated 10 min before reperfusion. The effects of melatonin on myocardial infarct size, biomarkers of myocardial injury, oxidative stress, and mitochondrial function were detected, and the expression of apelin, SIRT3, and mitophagy-related proteins were also measured. Excessive mitophagy was activated after I/R injury and was correlated with oxidative stress and mitochondrial dysfunction. Melatonin pretreatment ameliorated myocardial injury by decreasing oxidative stress, restoring mitochondrial function, and inhibiting excessive mitophagy. However, ML221 or 3-TYP disrupted these beneficial effects of melatonin on I/R injury. Taken together, these results suggest that melatonin pretreatment ameliorates myocardial I/R injury through regulating the apelin/SIRT3 pathway to inhibit excessive mitophagy.

Role of Mitophagy in Cigarette Smoke-induced Lung Epithelial Cell Injury In Vitro.

Mitochondria mediate airway inflammatory responses to cigarette smoke (CS). Removal of damaged or defective mitochondrial (mitophagy) may prevent the detrimental impact of CS extract (CSE) on airway and lung epithelial cells. We studied the effect of a mitophagy activator (Urolithin A, UA) and a mitophagy inhibitor (Liensinine diperchlorate, Ld) on CSE-exposed alveolar (A549) and airway (BEAS-2B) epithelial cell proliferation, intracellular and mitochondrial ROS, inflammatory response, mitochondrial membrane potential (Δψm), mitochondrial morphology, mitochondrial complex activities, and protein levels of mitochondrial fission (DRP1, MFF) and mitophagy (SQSTM1/p62, LC3B). In both cell types, CSE exposure led to increased intracellular and mitochondrial oxidative stress, decreased Δψm and resulted in structural disruption of the mitochondrial network. CSE increased the expression of DRP1, MFF and SQSTM1/p62 while decreasing LC3B-II/I protein expression ratio. CSE also increased inflammatory (IL-1β, IL-6, IL-18, CXCL1, CXCL8) and necroptosis factors (RIPK1, RIPK3, MLKL) mRNA expression. Pre-treatment with UA attenuated CSE-induced oxidative stress, inflammatory and necroptosis gene expression and restored mitochondrial structure and function. UA also prevented CSE-evoked increases in DRP1, MFF and SQSTM1/p62 protein expression and increased LC3B-II/I ratio. Conversely, pre-treatment with Ld aggravated CSE-induced cellular and mitochondrial responses. In conclusion, mitophagy mediates CSE-induced damage and inflammation of lung epithelial cells and may represent a therapeutic target in CS-driven diseases.

Mitophagy in human health, ageing and disease.

Maintaining optimal mitochondrial function is a feature of health. Mitophagy removes and recycles damaged mitochondria and regulates the biogenesis of new, fully functional ones preserving healthy mitochondrial functions and activities. Preclinical and clinical studies have shown that impaired mitophagy negatively affects cellular health and contributes to age-related chronic diseases. Strategies to boost mitophagy have been successfully tested in model organisms, and, recently, some have been translated into clinics. In this Review, we describe the basic mechanisms of mitophagy and how mitophagy can be assessed in human blood, the immune system and tissues, including muscle, brain and liver. We outline mitophagy's role in specific diseases and describe mitophagy-activating approaches successfully tested in humans, including exercise and nutritional and pharmacological interventions. We describe how mitophagy is connected to other features of ageing through general mechanisms such as inflammation and oxidative stress and forecast how strengthening research on mitophagy and mitophagy interventions may strongly support human health.

CKLF induces microglial activation via triggering defective mitophagy and mitochondrial dysfunction

ABSTRACT Although microglial activation is induced by an increase in chemokines, the role of mitophagy in this process remains unclear. This study aimed to elucidate the role of microglial mitophagy in CKLF/CKLF1 (chemokine-like factor 1)-induced microglial activation and neuroinflammation, as well as the underlying molecular mechanisms following CKLF treatment. This study determined that CKLF, an inducible chemokine in the brain, leads to an increase in mitophagy markers, such as DNM1L, PINK1 (PTEN induced putative kinase 1), PRKN, and OPTN, along with a simultaneous increase in autophagosome formation, as evidenced by elevated levels of BECN1 and MAP1LC3B (microtubule-associated protein 1 light chain 3 beta)-II. However, SQSTM1, a substrate of autophagy, was also accumulated by CKLF treatment, suggesting that mitophagy flux was reduced and mitophagosomes accumulated. These findings were confirmed by transmission electron microscopy and confocal microscopy. The defective mitophagy observed in our study was caused by impaired lysosomal function, including mitophagosome-lysosome fusion, lysosome generation, and acidification, resulting in the accumulation of damaged mitochondria in microglial cells. Further analysis revealed that pharmacological blocking or gene-silencing of mitophagy inhibited CKLF-mediated microglial activation, as evidenced by the expression of the microglial marker AIF1 (allograft inflammatory factor 1) and the mRNA of proinflammatory cytokines (Tnf and Il6). Ultimately, defective mitophagy induced by CKLF results in microglial activation, as observed in the brains of adult mice. In summary, CKLF induces defective mitophagy, microglial activation, and inflammation, providing a potential approach for treating neuroinflammatory diseases. Abbreviation: 3-MA: 3-methyladenine; AIF1: allograft inflammatory factor 1; ANOVA: analysis of variance; BAF: bafilomycin A1; BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; CKLF/CKLF1: chemokine-like factor 1; CNS: central nervous system; DMEM: Dulbecco’s Modified Eagle Medium; DNM1L: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; IRF3: interferon regulatory factor 3; IgG: immunoglobulin G; LAMP1: lysosomal-associated membrane protein 1; LAPTM4A: lysosomal-associated protein transmembrane 4A; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; mRFP: monomeric red fluorescent protein; mtDNA: mitochondrial DNA; MTORC1: mechanistic target of rapamycin kinase complex 1; OPTN: optineurin; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PINK1: PTEN induced putative kinase 1; PLL: poly-L-lysine; PRKN: parkin RBR E3 ubiquitin protein ligase; qPCR: quantitative polymerase chain reaction; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; VDAC: voltage-dependent anion channel

Mitophagy for cardioprotection

Mitochondrial function is maintained by several strictly coordinated mechanisms, collectively termed mitochondrial quality control mechanisms, including fusion and fission, degradation, and biogenesis. As the primary source of energy in cardiomyocytes, mitochondria are the central organelle for maintaining cardiac function. Since adult cardiomyocytes in humans rarely divide, the number of dysfunctional mitochondria cannot easily be diluted through cell division. Thus, efficient degradation of dysfunctional mitochondria is crucial to maintaining cellular function. Mitophagy, a mitochondria specific form of autophagy, is a major mechanism by which damaged or unnecessary mitochondria are targeted and eliminated. Mitophagy is active in cardiomyocytes at baseline and in response to stress, and plays an essential role in maintaining the quality of mitochondria in cardiomyocytes. Mitophagy is mediated through multiple mechanisms in the heart, and each of these mechanisms can partially compensate for the loss of another mechanism. However, insufficient levels of mitophagy eventually lead to mitochondrial dysfunction and the development of heart failure. In this review, we discuss the molecular mechanisms of mitophagy in the heart and the role of mitophagy in cardiac pathophysiology, with the focus on recent findings in the field.

Parkin-mediated mitophagy protects against aluminum trichloride-induced hippocampal apoptosis in mice via the mtROS-NLRP3 pathway

Aluminum is a neurotoxic food contaminant. Aluminum trichloride (AlCl3) causes hippocampal mitochondrial damage, leading to hippocampal injury. Damaged mitochondria can release mitochondrial reactive oxygen species (mtROS) and activate nucleotide-binding oligomerization domain-like receptor-containing 3 (NLRP3) inflammasomes and apoptosis. E3 ubiquitin ligase PARK2 (Parkin)-mediated mitophagy can attenuate mitochondrial damage. However, the role of mitophagy in AlCl3-induced mice hippocampal damage and its regulatory mechanism remain elusive. First, C57BL/6 N mice were treated with 0, 44.825, 89.65, and 179.3 mg/kg body weight AlCl3 drinking water for 90 d. Apoptosis, NLRP3-inflammasome activation and mitochondrial damage were increased in AlCl3-induced hippocampal damage. In addition, Parkin-mediated mitophagy peaked in the middle-dose group and was slightly attenuated in the high-dose group. Subsequently, we used wild-type and Parkin knockout (Parkin-/-) mice to investigate the AlCl3-induced hippocampal damage. The results showed that Parkin-/- inhibited mitophagy, and aggravated AlCl3-induced mitochondrial damage, NLRP3-inflammasome activation, apoptosis and hippocampal damage. Finally, we administered MitoQ (mtROS inhibitor) and MCC950 (NLRP3 inhibitor) to AlCl3-treated Parkin-/- mice to investigate the mechanism of Parkin-mediated mitophagy. The results showed that inhibition of mtROS and NLRP3 attenuated hippocampal NLRP3-inflammasome activation, apoptosis, and damage in AlCl3-treated Parkin-/- mice. These findings indicate that Parkin-mediated mitophagy protects against AlCl3-induced hippocampal apoptosis in mice via the mtROS-NLRP3 pathway.

Deciphering the implications of mitophagy-related signatures in clinical outcomes and microenvironment heterogeneity of clear cell renal cell carcinoma.

The role of mitophagy in various cancer-associated biological processes is well recognized. Nonetheless, the comprehensive implications of mitophagy in clear cell renal cell carcinoma (ccRCC) necessitate further exploration. Based on the transcriptomic data encompassing 25 mitophagy-related genes (MRGs), we identified the distinct mitophage patterns in 763 ccRCC samples. Subsequently, a mitophage-related predictive signature with machine learning algorithms was constructed, designated as RiskScore, to quantify the individual mitophagy status in ccRCC patients. Employing multispectral immunofluorescence (mIF) and immunohistochemistry (IHC) staining, we detected the effect of PTEN-induced putative kinase 1 (PINK1) in the prognosis and immune microenvironment of ccRCC. Our analysis initially encompassed a comprehensive assessment of the expression profiling, genomic variations, and interactions among the 25 MRGs in ccRCC. Subsequently, the consensus clustering algorithm was applied to stratify ccRCC patients into three clusters with distinct prognostic outcomes, tumor microenvironment (TME) characteristics, and underlying biological pathways. We screened eight pivotal genes (CLIC4, PTPRB, SLC16A12, ENPP5, FLRT3, HRH2, PDK4, and SCD5) to construct a mitophagy-related predictive signature, which showed excellent prognostic value for ccRCC patients. Moreover, patient subgroups divided by the RiskScore showed contrasting expression levels of immune checkpoints (ICPs), abundance of immune cells, and immunotherapy response. Additionally, a nomogram was established with robust predictive power integrating the RiskScore and clinical features. Notably, we observed that PINK1 expression markedly correlated with favorable treatment response and advanced maturation stages of tertiary lymphoid structures, which potentially shed light on enhancing anti-tumor immunity of ccRCC. Collectively, this study initially developed a signature associated with mitophagy, which demonstrated an excellent ability to predict the clinical prognosis, TME characterization, and responsiveness to targeted therapy and immunotherapy for ccRCC patients. Of particular note is the pivotal role of PINK1 in mediating the treatment response and immune microenvironment for ccRCC patients.

Comprehensive analysis of mitophagy-related genes in diagnosis and heterogeneous endothelial cells in chronic rhinosinusitis: based on bulk and single-cell RNA sequencing data.

Background: Chronic rhinosinusitis (CRS) is a complex inflammatory disorder affecting the nasal and paranasal sinuses. Mitophagy, the process of selective mitochondrial degradation via autophagy, is crucial for maintaining cellular balance. However, the role of mitophagy in CRS is not well-studied. This research aims to examine the role of mitophagy-related genes (MRGs) in CRS, with a particular focus on the heterogeneity of endothelial cells (ECs). Methods: We employed both bulk and single-cell RNA sequencing data to investigate the role of MRGs in CRS. We compiled a combined database of 92 CRS samples and 35 healthy control samples from the Gene Expression Omnibus (GEO) database and we explored the differential expression of MRGs between them. A logistic regression model was built based on seven key genes identified through Random Forests and Support Vector Machines - Recursive Feature Elimination (SVM-RFE). Consensus cluster analysis was used to categorize CRS patients based on MRG expression patterns and weighted gene co-expression network analysis (WGCNA) was performed to find modules of highly correlated genes of the different clusters. Single-cell RNA sequencing data was utilized to analyze MRGs and EC heterogeneity in CRS. Results: Seven hub genes-SQSTM1, SRC, UBA52, MFN2, UBC, RPS27A, and ATG12-showed differential expression between two groups. A diagnostic model based on hub genes showed excellent prognostic accuracy. A strong positive correlation was found between the seven hub MRGs and resting dendritic cells, while a significant negative correlation was observed with mast cells and CD8+ T cells. CRS could be divided into two subclusters based on MRG expression patterns. WGCNA analysis identified modules of highly correlated genes of these two different subclusters. At the single-cell level, two types of venous ECs with different MRG scores were identified, suggesting their varying roles in CRS pathogenesis, especially in the non-eosinophilic CRS subtype. Conclusion: Our comprehensive study of CRS reveals the significant role of MRGs and underscores the heterogeneity of ECs. We highlighted the importance of Migration Inhibitory Factor (MIF) and TGFb pathways in mediating the effects of mitophagy, particularly the MIF. Overall, our findings enhance the understanding of mitophagy in CRS, providing a foundation for future research and potential therapeutic developments.

The Role of Parkin in Rat Pancreatic Beta Cells Fate.

Parkin is a member of the mitochondrial quality control system that plays a major role in mitophagy. Although the loss of function mutations in the Parkin gene has been associated with the Familial Parkinson's phenotype, research in recent years points out that Parkin's function is not limited to neurodegenerative diseases. Parkin's function impressing key cellular quality control mechanisms, including the ubiquitin-proteasome and autophagy-lysosome systems, makes it an important player in the maintenance of cellular homeostasis. In this study, we investigated whether Parkin affects cell viability and ER stress responses under lipotoxic conditions in INS-1E cells. Our results may suggest that silencing Parkin may affect autophagy in addition to apoptosis. We also showed that Parkin may have a protective effect against lipo-toxic effects in INS-1E cells. Consistent with previous studies, we observed that stress responses were different for high and low palmitic acid doses. The Parkin being inhibited under high-dose PA treatment and active under low-dose PA treatment indicate that regulation of stress responses is controlled by environmental conditions. Our preliminary findings may suggest that in low lipotoxic conditions, Parkin affects the ER stress response by modulating Chop activity and Ca2+ release from the ER to the cytoplasm.

Impaired mitophagy induces antimicrobial responses in macrophages infected with Mycobacterium tuberculosis

BackgroundMitophagy, mitochondrial selective autophagy, plays a pivotal role in the maintenance of cellular homeostasis in response to cellular stress. However, the role of mitophagy in macrophages during infection has not been elucidated. To determine whether mitophagy regulates intracellular pathogen survival, macrophages were infected with Mycobacterium tuberculosis (Mtb), an intracellular bacterium.ResultsWe showed that Mtb-infected macrophages induced mitophagy through BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) activation. In contrast, BNIP3-deficient macrophages failed to induce mitophagy, resulting in reduced mitochondrial membrane potential in response to Mtb infection. Moreover, the accumulation of damaged mitochondria due to BNIP3 deficiency generated higher levels of mitochondrial reactive oxygen species (mROS) compared to the control, suppressing the intracellular survival of Mtb. We observed that siBNIP3 suppressed intracellular Mtb in mice lungs.ConclusionWe found that BNIP3 plays a critical role in the regulation of mitophagy during Mtb infection. The inhibition of mitophagy suppresses Mtb growth in macrophages through increased mROS production. Therefore, BNIP3 might be a novel therapeutic target for tuberculosis treatment.

FBXO7/ntc and USP30 antagonistically set the ubiquitination threshold for basal mitophagy and provide a target for Pink1 phosphorylation in vivo.

Functional analyses of genes linked to heritable forms of Parkinson's disease (PD) have revealed fundamental insights into the biological processes underpinning pathogenic mechanisms. Mutations in PARK15/FBXO7 cause autosomal recessive PD and FBXO7 has been shown to regulate mitochondrial homeostasis. We investigated the extent to which FBXO7 and its Drosophila orthologue, ntc, share functional homology and explored its role in mitophagy in vivo. We show that ntc mutants partially phenocopy Pink1 and parkin mutants and ntc overexpression supresses parkin phenotypes. Furthermore, ntc can modulate basal mitophagy in a Pink1- and parkin-independent manner by promoting the ubiquitination of mitochondrial proteins, a mechanism that is opposed by the deubiquitinase USP30. This basal ubiquitination serves as the substrate for Pink1-mediated phosphorylation that triggers stress-induced mitophagy. We propose that FBXO7/ntc works in equilibrium with USP30 to provide a checkpoint for mitochondrial quality control in basal conditions in vivo and presents a new avenue for therapeutic approaches.

Characterization of SH3GLB1 in the auditory system and its potential role in mitophagy

Characterization of SH3GLB1 in the auditory system and its potential role in mitophagy

Pooled analysis of frontal lobe transcriptomic data identifies key mitophagy gene changes in Alzheimer's disease brain.

The growing prevalence of Alzheimer's disease (AD) is becoming a global health challenge without effective treatments. Defective mitochondrial function and mitophagy have recently been suggested as etiological factors in AD, in association with abnormalities in components of the autophagic machinery like lysosomes and phagosomes. Several large transcriptomic studies have been performed on different brain regions from AD and healthy patients, and their data represent a vast source of important information that can be utilized to understand this condition. However, large integration analyses of these publicly available data, such as AD RNA-Seq data, are still missing. In addition, large-scale focused analysis on mitophagy, which seems to be relevant for the aetiology of the disease, has not yet been performed. In this study, publicly available raw RNA-Seq data generated from healthy control and sporadic AD post-mortem human samples of the brain frontal lobe were collected and integrated. Sex-specific differential expression analysis was performed on the combined data set after batch effect correction. From the resulting set of differentially expressed genes, candidate mitophagy-related genes were identified based on their known functional roles in mitophagy, the lysosome, or the phagosome, followed by Protein-Protein Interaction (PPI) and microRNA-mRNA network analysis. The expression changes of candidate genes were further validated in human skin fibroblast and induced pluripotent stem cells (iPSCs)-derived cortical neurons from AD patients and matching healthy controls. From a large dataset (AD: 589; control: 246) based on three different datasets (i.e., ROSMAP, MSBB, & GSE110731), we identified 299 candidate mitophagy-related differentially expressed genes (DEG) in sporadic AD patients (male: 195, female: 188). Among these, the AAA ATPase VCP, the GTPase ARF1, the autophagic vesicle forming protein GABARAPL1 and the cytoskeleton protein actin beta ACTB were selected based on network degrees and existing literature. Changes in their expression were further validated in AD-relevant human in vitro models, which confirmed their down-regulation in AD conditions. Through the joint analysis of multiple publicly available data sets, we identify four differentially expressed key mitophagy-related genes potentially relevant for the pathogenesis of sporadic AD. Changes in expression of these four genes were validated using two AD-relevant human in vitro models, primary human fibroblasts and iPSC-derived neurons. Our results provide foundation for further investigation of these genes as potential biomarkers or disease-modifying pharmacological targets.

Cardiolipin externalization mediates prion protein (PrP) peptide 106-126-associated mitophagy and mitochondrial dysfunction.

Proper mitochondrial performance is imperative for the maintenance of normal neuronal function to prevent the development of neurodegenerative diseases. Persistent accumulation of damaged mitochondria plays a role in prion disease pathogenesis, which involves a chain of events that culminate in the generation of reactive oxygen species and neuronal death. Our previous studies have demonstrated that PINK1/Parkin-mediated mitophagy induced by PrP106-126 is defective and leads to an accumulation of damaged mitochondria after PrP106-126 treatment. Externalized cardiolipin (CL), a mitochondria-specific phospholipid, has been reported to play a role in mitophagy by directly interacting with LC3II at the outer mitochondrial membrane. The involvement of CL externalization in PrP106-126-induced mitophagy and its significance in other physiological processes of N2a cells treated with PrP106-126 remain unknown. We demonstrate that the PrP106-126 peptide caused a temporal course of mitophagy in N2a cells, which gradually increased and subsequently decreased. A similar trend in CL externalization to the mitochondrial surface was seen, resulting in a gradual decrease in CL content at the cellular level. Inhibition of CL externalization by knockdown of CL synthase, responsible for de novo synthesis of CL, or phospholipid scramblase-3 and NDPK-D, responsible for CL translocation to the mitochondrial surface, significantly decreased PrP106-126-induced mitophagy in N2a cells. Meanwhile, the inhibition of CL redistribution significantly decreased PINK1 and DRP1 recruitment in PrP106-126 treatment but had no significant decrease in Parkin recruitment. Furthermore, the inhibition of CL externalization resulted in impaired oxidative phosphorylation and severe oxidative stress, which led to mitochondrial dysfunction. Our results indicate that CL externalization induced by PrP106-126 on N2a cells plays a positive role in the initiation of mitophagy, leading to the stabilization of mitochondrial function.

Haploinsufficiency of progranulin causes cell type specific impairments in PINK1/Parkin mitophagy

AbstractBackgroundGRN mutations that result in progranulin haploinsufficiency cause frontotemporal dementia (FTD). Mitophagy, the selective autophagy of damaged mitochondria, is impaired in several neurodegenerative diseases. A number of genes linked to FTD (e.g. OPTN, SQSTM, VCP and TBK1) are also known to play a role in mitophagy. Xenophagy, the selective autophagy of non‐host pathogens, relies on some of the same proteins as mitophagy (Parkin and TBK1) and is reduced in GRN knockout mice. Progranulin has also been found to play a role in mitophagy in mouse kidney cells. We therefore hypothesised that loss of progranulin could lead to defective mitophagy in neurons, astrocytes and microglia.MethodsWe investigated PINK1/Parkin mitophagy in astrocytic‐like H4 cells and neuron‐like Parkin overexpressing SHSY5Y cells (PoE‐5Ys) +/‐ siRNA against GRN. We also examined induced pluripotent stem cells (iPSCs) from four controls, three patients with FTD‐associated GRN mutations (R493X and C31fs) and a CRISPR series from the human iPSC Neurodegenerative Disease Initiative1,2,3 (iNDI) with an isogenic control, heterozygous and homozygous R493X mutation lines. The iPSCs were differentiated to cortical neurons, astrocytes and microglia. PINK1/Parkin mitophagy was induced using Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor). PINK1 accumulation, levels of S65 phosphorylated ubiquitin (pUb), other mitophagy markers as well as proteins hypothesised to play a role in the mechanism were examined using western blotting and immunofluorescence (ICC).ResultsLower levels of pUb were detected in PoE‐5Ys treated with oligomycin/antimycin following GRN knockdown. There was a significant reduction in mitophagy in GRN siRNA treated H4 cells, detected by ICC and western blotting of mitophagy markers. Initial results from iPSC neurons found no significant difference in mitophagy between control and patient lines. Work is ongoing in a larger set of iPSC neurons, astrocytes and microglia. Preliminary results suggest that reducing progranulin affects mitophagy in astrocytes but has a limited effect on mitophagy in neurons.ConclusionsThe results suggest that progranulin has a cell specific role in mitophagy with progranulin regulating stability and/or activity of PINK1. Current work aims to understand the mechanisms of this process and to dissect cell‐type specific contributions of progranulin to mitophagy.

THE EMERGING ROLE OF IDH2-MEDIATED MITOPHAGY AND MTUPR IN ATHEROSCLEROSIS

Objective: Mitochondrial dysfunction is a risk factor for vascular disease by overexpressing ROS caused by some stimuli in mitochondria which causes oxidative stress and enhances vascular inflammatory response. Isocitrate dehydrogenase 2 (IDH2) is NADP+ dependent mitochondrial enzyme and an antioxidant enzyme that produces NADPH in the antioxidant system. Mitophagy and mitochondrial Unfolding Protein Response (mtUPR) are internal defense mechanism in mitochondria. In this study, we investigated whether IDH2 knockdown causes mitochondrial dysfunction then Mitophagy and mtUPR in vitro in HUVECs and in vivo in IDH2 knock out mice. Design and method: We examined expression of mitophagy and mtUPR-related gene in IDH2-deficient human umbilical vein endothelial cells (HUVECs) and its role in mitophagy and mtUPR in the endothelial cell and IDH2 KO animal tissues. Mitophagy and mtUPR were measured using the western blotting and qPCR. Results: We showed that knockdown of IDH2 expression induced depolarization of mitochondrial membrane potential (MMP). Mitochondrial dynamics is mitochondrial fusion and fission. Knockdown of IDH2 increased Drp1 (fission protein) and mfn1 (fusion protein) compared with Tom20 (control). IDH2 deficiency increase Mitophagy related protein PINK-1 and Parkin expression and mRNA level (PINK-1, Parkin, BNIP3, NIX, FUNDC-1). Moreover, knockdown of IDH2 induced mtUPR mRNA level (USP30, Clpp) in vitro. In addition, IDH2 deficiency increases mtUPR mRNA level and decreases PINK-1 and Parkin protein expression in vivo. Conclusions: Our data show that IDH2 deficiency induces mitochondrial dysfunction and then Mitophagy and mtUPR expression in endothelial cells. These findings provide novel strategy for the development of therapeutic agents for restoring mitochondrial and endothelial function.

A TNFα/Miz1-positive feedback loop inhibits mitophagy in hepatocytes and propagates non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a chronic inflammatory disease that can further progress to cirrhosis and hepatocellular carcinoma. However, the key molecular mechanisms behind this process have not been clarified. We analyzed human NASH and normal liver tissue samples by RNA-sequencing and liquid chromatography-mass spectrometry, identifying hepatocyte cytosolic protein Myc-interacting zinc-finger protein 1 (Miz1) as a potential target in NASH progression. We established a Western diet+fructose-induced NASH model in hepatocyte-specific Miz1 knockout and adeno-associated virus type 8-overexpressing mice. Human NASH liver organoids were used to confirm the mechanism, and immunoprecipitation and mass spectrometry were used to detect proteins that could interact with Miz1. We demonstrate that Miz1 is reduced in hepatocytes in human NASH. Miz1 is shown to bind to peroxiredoxin 6 (PRDX6), retaining it in the cytosol, blocking its interaction with mitochondrial Parkin at Cys431, and inhibiting Parkin-mediated mitophagy. In NASH livers, loss of hepatocyte Miz1 results in PRDX6-mediated inhibition of mitophagy, increased dysfunctional mitochondria in hepatocytes, and production of proinflammatory cytokines, including TNFα, by hepatic macrophages. Crucially, the increased production of TNFα results in a further reduction in hepatocyte Miz1 by E3-ubiquitination. This produces a positive feedback loop of TNFα-mediated hepatocyte Miz1 degradation, resulting in PRDX6-mediated inhibition of hepatocyte mitophagy, with the accumulation of dysfunctional mitochondria in hepatocytes and increased macrophage TNFα production. Our study identified hepatocyte Miz1 as a suppressor of NASH progression via its role in mitophagy; we also identified a positive feedback loop by which TNFα production induces degradation of cytosolic Miz1, which inhibits mitophagy and thus leads to increased macrophage TNFα production. Interruption of this positive feedback loop could be a strategy to inhibit the progression of NASH. Non-alcoholic steatohepatitis (NASH) is a chronic inflammatory disease that can further develop into cirrhosis and hepatocellular carcinoma. However, the key molecular mechanism of this process has not been fully clarified. Herein, we identified a positive feedback loop of macrophage TNFα-mediated hepatocyte Miz1 degradation, resulting in PRDX6-mediated inhibition of hepatocyte mitophagy, aggravation of mitochondrial damage and increased macrophage TNFα production. Our findings not only provide mechanistic insight into NASH progression but also provide potential therapeutic targets for patients with NASH. Our human NASH liver organoid culture is therefore a useful platform for exploring treatment strategies for NASH development.

NRF-2α and mitophagy underlie enhanced mitochondrial functions and biogenesis induced by T-2 toxin in GH3 cells.

T-2 toxin is a natural contaminant in grain cereals produced by species of Fusarium. Studies indicate that T-2 toxin can positively affect mitochondrial function, but the underlying mechanism is unclear. In this study, we examined the role of nuclear respiratory factor 2α (NRF-2α) in T-2 toxin-activated mitochondrial biogenesis and the direct target genes of NRF-2α. Furthermore, we investigated T-2 toxin-induced autophagy and mitophagy, and the role of mitophagy in changes in mitochondrial function and apoptosis. It was found that T-2 toxin significantly increased NRF-2α levels and nuclear localization of NRF-2α was induced. NRF-2α deletion significantly increased the production of reactive oxygen species (ROS), abrogated T-2 toxin-induced increases in ATP and mitochondrial complex I activity, and inhibited the mitochondrial DNA copy number. Meanwhile, With chromatin immunoprecipitation sequencing (ChIP-Seq), various novel NRF-2α target genes were identified, such as mitochondrial iron-sulphur subunits (Ndufs 3,7) and mitochondrial transcription factors (Tfam, Tfb1m, and Tfb2m). Some target genes were also involved in mitochondrial fusion and fission (Drp1), mitochondrial translation (Yars2) and splicing (Ddx55), and mitophagy. Further studies showed that T-2 toxin induced Atg5 dependent autophagy and Atg5/PINK1-dependent mitophagy. In addition, mitophagy defects increase ROS production, inhibit ATP levels and the expression of genes related to mitochondrial dynamics, and promote apoptosis in the presence of T-2 toxins. Altogether, these results suggest that NRF-2α plays a critical role in promoting mitochondrial function and biogenesis through regulation of mitochondrial genes, and, interestingly, mitophagy caused by T-2 toxin positively affected mitochondrial function and protected cell survival against T-2 toxin.

HIF1α-BNIP3-mediated mitophagy protects against renal fibrosis by decreasing ROS and inhibiting activation of the NLRP3 inflammasome

Chronic kidney disease affects approximately 14.3% of people worldwide. Tubulointerstitial fibrosis is the final stage of almost all progressive CKD. To date, the pathogenesis of renal fibrosis remains unclear, and there is a lack of effective treatments, leading to renal replacement therapy. Mitophagy is a type of selective autophagy that has been recognized as an important way to remove dysfunctional mitochondria and abrogate the excessive accumulation of mitochondrial-derived reactive oxygen species (ROS) to balance the function of cells. However, the role of mitophagy and its regulation in renal fibrosis need further examination. In this study, we showed that mitophagy was induced in renal tubular epithelial cells in renal fibrosis. After silencing BNIP3, mitophagy was abolished in vivo and in vitro, indicating the important effect of the BNIP3-dependent pathway on mitophagy. Furthermore, in unilateral ureteral obstruction (UUO) models and hypoxic conditions, the production of mitochondrial ROS, mitochondrial damage, activation of the NLRP3 inflammasome, and the levels of αSMA and TGFβ1 increased significantly following BNIP3 gene deletion or silencing. Following silencing BNIP3 and pretreatment with mitoTEMPO or MCC950, the protein levels of αSMA and TGFβ1 decreased significantly in HK-2 cells under hypoxic conditions. These findings demonstrated that HIF1α-BNIP3-mediated mitophagy played a protective role against hypoxia-induced renal epithelial cell injury and renal fibrosis by reducing mitochondrial ROS and inhibiting activation of the NLRP3 inflammasome.