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Autophagy Oct 2021Mitochondrial quality control, which is crucial for maintaining cellular homeostasis, has been considered to be achieved exclusively through mitophagy. Here we report an...
Mitochondrial quality control, which is crucial for maintaining cellular homeostasis, has been considered to be achieved exclusively through mitophagy. Here we report an alternative mitochondrial quality control pathway mediated by extracellular mitochondria release. By performing time-lapse confocal imaging on a stable cell line with fluorescent-labeled mitochondria, we observed release of mitochondria from cells into the extracellular space. Correlative light-electron microscopy revealed that majority of the extracellular mitochondria are in free form and, on rare occasions, some are enclosed in membrane-surrounded vesicles. Rotenone- and carbonyl cyanide m-chlorophenylhydrazone-induced mitochondrial quality impairment promotes the extracellular release of depolarized mitochondria. Overexpression of PRKN (parkin RBR E3 ubiquitin protein ligase), which has a pivotal role in mitophagy regulation, suppresses the extracellular mitochondria release under basal and stress condition, whereas its knockdown exacerbates it. Correspondingly, overexpression of PRKN-independent mitophagy regulators, BNIP3 (BCL2 interacting protein 3) and BNIP3L/NIX (BCL2 interacting protein 3 like), suppress extracellular mitochondria release. Autophagy-deficient cell lines show elevated extracellular mitochondria release. These results imply that perturbation of mitophagy pathway prompts mitochondria expulsion. Presence of mitochondrial protein can also be detected in mouse sera. Sera of PRKN-deficient mice contain higher level of mitochondrial protein compared to that of wild-type mice. More importantly, fibroblasts and cerebrospinal fluid samples from Parkinson disease patients carrying loss-of-function mutations show increased extracellular mitochondria compared to control subjects, providing evidence in a clinical context. Taken together, our findings suggest that extracellular mitochondria release is a comparable yet distinct quality control pathway from conventional mitophagy. ACTB: actin beta; ANXA5: annexin A5; ATP5F1A/ATP5A: ATP synthase F1 subunit alpha; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CM: conditioned media; CSF: cerebrospinal fluid; DMSO: dimethyl sulfoxide; EM: electron microscopy; HSPD1/Hsp60: heat shock protein family D (Hsp60) member 1; KD: knockdown; KO: knockout; MAP1LC3A/LC3: microtubule associated protein 1 light chain 3 alpha; MT-CO1: mitochondrially encoded cytochrome c oxidase I; NDUFB8: NADH:ubiquinone oxidoreductase subunit B8; OE: overexpression; OPA1: OPA1 mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PB: phosphate buffer; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDHB: succinate dehydrogenase complex iron sulfur subunit B; TOMM20: translocase of outer mitochondrial membrane 20; TOMM40: translocase of outer mitochondrial membrane 40; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; WT: wild-type.
Topics: Animals; Autophagy; Humans; Mice; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Mitophagy; Ubiquitin-Protein Ligases
PubMed: 33218272
DOI: 10.1080/15548627.2020.1848130 -
Circulation Research Jun 2023Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7 (autophagy related 7)-dependent...
BACKGROUND
Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7 (autophagy related 7)-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9 (Ras-related protein Rab-9A)-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial. We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are.
METHODS
Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy was evaluated using cardiac-specific Mito-Keima mice. The role of DRP1 was evaluated using tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mice.
RESULTS
Mitophagy was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in MCM mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3 (microtubule-associated protein 1 light chain 3)-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in MCM mice. Activation of alternative mitophagy was also completely abolished in MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 (fission protein 1) only during the chronic, but not acute, phase of HFD consumption.
CONCLUSIONS
DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.
Topics: Animals; Mice; Autophagy; Cardiomyopathies; Dynamins; Heart; Mitochondrial Dynamics; Mitophagy; Obesity
PubMed: 37232152
DOI: 10.1161/CIRCRESAHA.123.322512 -
Cell Death & Disease Sep 2019Acute kidney injury (AKI) is a syndrome of abrupt loss of renal functions. The underlying pathological mechanisms of AKI remain largely unknown. BCL2-interacting protein...
Acute kidney injury (AKI) is a syndrome of abrupt loss of renal functions. The underlying pathological mechanisms of AKI remain largely unknown. BCL2-interacting protein 3 (BNIP3) has dual functions of regulating cell death and mitophagy, but its pathophysiological role in AKI remains unclear. Here, we demonstrated an increase of BNIP3 expression in cultured renal proximal tubular epithelial cells following oxygen-glucose deprivation-reperfusion (OGD-R) and in renal tubules after renal ischemia-reperfusion (IR)-induced injury in mice. Functionally, silencing Bnip3 by specific short hairpin RNAs in cultured renal tubular cells reduced OGD-R-induced mitophagy, and potentiated OGD-R-induced cell death. In vivo, Bnip3 knockout worsened renal IR injury, as manifested by more severe renal dysfunction and tissue injury. We further showed that Bnip3 knockout reduced mitophagy, which resulted in the accumulation of damaged mitochondria, increased production of reactive oxygen species, and enhanced cell death and inflammatory response in kidneys following renal IR. Taken together, these findings suggest that BNIP3-mediated mitophagy has a critical role in mitochondrial quality control and tubular cell survival during AKI.
Topics: Acute Kidney Injury; Animals; Apoptosis; Kidney; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitophagy; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction
PubMed: 31515472
DOI: 10.1038/s41419-019-1899-0 -
Autophagy Mar 2022There is increasing evidence that mitophagy, a specialized form of autophagy to degrade and clear long-lived or damaged mitochondria, is impaired in aging and...
There is increasing evidence that mitophagy, a specialized form of autophagy to degrade and clear long-lived or damaged mitochondria, is impaired in aging and age-related disease. Previous study has demonstrated the obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. However, it remains unknown whether mitophagy functions in oocyte and what's the regulatory mechanism in oocyte aging. In the study, when fully grown oocytes were treated with CCCP, an uncoupling agent to induce mitophagy, we found the activation of the PRKN-mediated mitophagy pathway accompanied the blockage of meiosis at metaphase I stage. Our result then demonstrated its association with the decreased activity of RAB7 and all the observed defects in CCCP treated oocytes could be effectively rescued by microinjection of mRNA encoding active RAB7 or treatment with the RAB7 activator ML098. Further study indicated PRKN protein level as a rate-limiting factor to facilitate degradation of RAB7 and its GEF (guanine nucleotide exchange factor) complex CCZ1-MON1 through the ubiquitin-proteasome system. In GV oocytes collected during ovarian aging, we found the age-related increase of PINK1 and PRKN proteins and a significant decrease of RAB7 which resulted in defects of mitophagosome formation and the accumulation of damaged mitochondria. The age-related retardation of female fertility was improved after in vivo treatment of ML098. Thus, RAB7 activity is required to maintain the balance between mitophagy and chromosome stability and RAB7 activator is a good candidate to ameliorate age-related deterioration of oocyte quality. ATG9: autophagy related 9A; ATP: adenosine triphosphate; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CCZ1: CCZ1 vacuolar protein trafficking and biogenesis associated; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAPs: GTPase-activating proteins; GEF: guanine nucleotide exchange factor; GV: germinal vesicle; GVBD: germinal vesicle breakdown; LAMP1: lysosomal-associated membrane protein 1; MI: metaphase I stage of meiosis; MII: metaphase II stage of meiosis; Mito: MitoTracker; mtDNA: mitochondrial DNA; MON1: MON1 homolog, secretory trafficking associated; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB7: RAB7, member RAS oncogene family; ROS: reactive oxygen species; TEM: transmission electron microscopy; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; UB: ubiquitin.
Topics: Animals; Autophagy; Carbonyl Cyanide m-Chlorophenyl Hydrazone; DNA, Mitochondrial; Female; Guanine Nucleotide Exchange Factors; Meiosis; Mitophagy; Oocytes; Protein Kinases; Quality Control; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 34229552
DOI: 10.1080/15548627.2021.1946739 -
Circulation Sep 2020Increased fatty acid oxidation (FAO) has long been considered a culprit in the development of obesity/diabetes mellitus-induced cardiomyopathy. However, enhancing...
BACKGROUND
Increased fatty acid oxidation (FAO) has long been considered a culprit in the development of obesity/diabetes mellitus-induced cardiomyopathy. However, enhancing cardiac FAO by removing the inhibitory mechanism of long-chain fatty acid transport into mitochondria via deletion of acetyl coenzyme A carboxylase 2 (ACC2) does not cause cardiomyopathy in nonobese mice, suggesting that high FAO is distinct from cardiac lipotoxicity. We hypothesize that cardiac pathology-associated obesity is attributable to the imbalance of fatty acid supply and oxidation. Thus, we here seek to determine whether further increasing FAO by inducing ACC2 deletion prevents obesity-induced cardiomyopathy, and if so, to elucidate the underlying mechanisms.
METHODS
We induced high FAO in adult mouse hearts by cardiac-specific deletion of ACC2 using a tamoxifen-inducible model (ACC2 iKO). Control and ACC2 iKO mice were subjected to high-fat diet (HFD) feeding for 24 weeks to induce obesity. Cardiac function, mitochondria function, and mitophagy activity were examined.
RESULTS
Despite both control and ACC2 iKO mice exhibiting a similar obese phenotype, increasing FAO oxidation by deletion of ACC2 prevented HFD-induced cardiac dysfunction, pathological remodeling, and mitochondria dysfunction, as well. Similarly, increasing FAO by knockdown of ACC2 prevented palmitate-induced mitochondria dysfunction and cardiomyocyte death in vitro. Furthermore, HFD suppressed mitophagy activity and caused damaged mitochondria to accumulate in the heart, which was attenuated, in part, in the ACC2 iKO heart. Mechanistically, ACC2 iKO prevented HFD-induced downregulation of parkin. During stimulation for mitophagy, mitochondria-localized parkin was severely reduced in control HFD-fed mouse heart, which was restored, in part, in ACC2 iKO HFD-fed mice.
CONCLUSIONS
These data show that increasing cardiac FAO alone does not cause cardiac dysfunction, but protects against cardiomyopathy in chronically obese mice. The beneficial effect of enhancing cardiac FAO in HFD-induced obesity is mediated, in part, by the maintenance of mitochondria function through regulating parkin-mediated mitophagy. Our findings also suggest that targeting the parkin-dependent mitophagy pathway could be an effective strategy against the development of obesity-induced cardiomyopathy.
Topics: Acetyl-CoA Carboxylase; Animals; Cardiomyopathies; Diet, High-Fat; Fatty Acids; Mice; Mice, Knockout; Mitochondria, Heart; Mitophagy; Oxidation-Reduction; Ubiquitin-Protein Ligases
PubMed: 32597196
DOI: 10.1161/CIRCULATIONAHA.119.043319 -
Autophagy Apr 2020PRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged...
PRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged mitochondria. Deubiquitinating enzymes (DUBs) functionally reversing PRKN ubiquitination are critical in controlling the magnitude of PRKN-mediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its pro-mitophagy effect remains to be identified and characterized. Here, we demonstrated that USP33/VDU1 is localized at the outer membrane of mitochondria and serves as a PRKN DUB through their interaction. Cellular and assays illustrated that USP33 deubiquitinates PRKN in a DUB activity-dependent manner. USP33 prefers to remove K6, K11, K48 and K63-linked ubiquitin conjugates from PRKN, and deubiquitinates PRKN mainly at Lys435. Mutation of this site leads to a significantly decreased level of K63-, but not K48-linked PRKN ubiquitination. deficiency enhanced both K48- and K63-linked PRKN ubiquitination, but only K63-linked PRKN ubiquitination was significantly increased under mitochondrial depolarization. Further, knockdown increased both PRKN protein stabilization and its translocation to depolarized mitochondria leading to the enhancement of mitophagy. Moreover, silencing protects SH-SY5Y human neuroblastoma cells from the neurotoxin MPTP-induced apoptotic cell death. Our findings convincingly demonstrate that USP33 is a novel PRKN deubiquitinase antagonizing its regulatory roles in mitophagy and SH-SY5Y neuron-like cell survival. Thus, inhibition may represents an attractive new therapeutic strategy for PD patients. CCCP: carbonyl cyanide 3-chlorophenylhydrazone; DUB: deubiquitinating enzymes; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OMM: outer mitochondrial membrane; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; TM: transmembrane; Ub: ubiquitin; UBA1: ubiquitin like modifier activating enzyme 1; UBE2L3/UbcH7: ubiquitin conjugating enzyme E2 L3; USP33: ubiquitin specific peptidase 33; WT: wild type.
Topics: Autophagy; Humans; Mitochondria; Mitophagy; Protein Kinases; Reactive Oxygen Species; Ubiquitin Thiolesterase; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 31432739
DOI: 10.1080/15548627.2019.1656957 -
The EMBO Journal Jul 2023To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of...
To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCF , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.
Topics: Autophagy; DNA, Mitochondrial; Mitochondria; Mitochondrial Proteins; Mitophagy; Phagocytosis; Humans; Animals; Mice
PubMed: 37161784
DOI: 10.15252/embj.2022112767 -
Autophagy Jan 2023Aminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It...
Aminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It is well known that damaged mitochondria are degraded by mitophagy, an important mitochondrial quality control system that maintains mitochondrial homeostasis and ensures cell survival. However, it is unclear whether dysregulation of mitophagy contributes to aminoglycoside-induced hair cell injury. In the current study, we found that PINK1-PRKN-mediated mitophagy was impaired in neomycin-treated hair cells. Our data suggested that mitochondrial recruitment of PRKN and phagophore recognition of damaged mitochondria during mitophagy were blocked following neomycin treatment. In addition, the degradation of damaged mitochondria by lysosomes was significantly decreased as indicated by the mitophagic flux reporter mt-mKeima. Moreover, we demonstrated that neomycin disrupted mitophagy through transcriptional inhibition of expression, the key initiator of mitophagy. Moreover, we found that neomycin impaired mitophagy by inducing ATF3 expression. Importantly, treatment with a mitophagy activator could rescue neomycin-treated hair cells by increasing mitophagy, indicating that genetic modulation or drug intervention in mitophagy may have therapeutic potential for aminoglycoside-induced hearing loss. AAV: adeno-associated virus; ABR: auditory brainstem response; ATF3: activating transcription factor 3; ATOH1/MATH1: atonal bHLH transcription factor 1; BafA1: bafilomycin A; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; COX4I1/COXIV: cytochrome c oxidase subunit 4I1; CTBP2/RIBEYE: C-terminal binding protein 2; DFP: deferiprone; EGFP: enhanced green fluorescent protein; FOXO3: forkhead box O3; GRIA2/GLUR2: glutamate receptor, ionotropic, AMPA2 (alpha 2); HC: hair cell; HSPD1/HSP60: heat shock protein 1 (chaperonin); IHC: inner hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MYO7A: myosin VIIA; OPTN: optineurin; OMM: outer mitochondrial membrane; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RT-qPCR: real-time quantitative polymerase chain reaction; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; USP30: ubiquitin specific peptidase 30; XBP1: X-box binding protein 1.
Topics: Mitophagy; Autophagy; Aminoglycosides; Protein Kinases; Ubiquitin-Protein Ligases; Anti-Bacterial Agents; Neomycin; Hair Cells, Auditory
PubMed: 35471096
DOI: 10.1080/15548627.2022.2062872 -
Computers in Biology and Medicine Sep 2023Gastric carcinoma (GC) is the fourth leading cause of cancer-related mortality worldwide. Patients with advanced GC tend to have poor prognoses and shortened survival....
Identifying mitophagy-related genes as prognostic biomarkers and therapeutic targets of gastric carcinoma by integrated analysis of single-cell and bulk-RNA sequencing data.
Gastric carcinoma (GC) is the fourth leading cause of cancer-related mortality worldwide. Patients with advanced GC tend to have poor prognoses and shortened survival. Finding novel predictive biomarkers for GC prognosis is an urgent need. Mitophagy is the selection degradation of damaged mitochondria to maintain cellular homeostasis, which has been shown to play both pro- and anti-tumor effects. This study combined single-cell sequencing data and transcriptomics to screen mitophagy-related genes (MRGs) associated with GC progression and analyze their clinical values. Reverse transcription-quantitative PCR (RT-qPCR) and immunochemistry (IHC) further verified gene expression profiles. A total of 18 DE-MRGs were identified after taking an intersection of single-cell sequencing data and MRGs. Cells with a higher MRG score were mainly distributed in the epithelial cell cluster. Cell-to-cell communications among epithelial cells with other cell types were significantly upregulated. We established and validated a reliable nomogram model based on DE-MRGs (GABARAPL2 and CDC37) and traditional clinicopathological parameters. GABARAPL2 and CDC37 displayed different immune infiltration states. Given the significant correlation between hub genes and immune checkpoints, targeting MRGs in GC may supplement more benefits to patients who received immunotherapy. In conclusion, GABARAPL2 and CDC37 may be prognostic biomarkers and candidate therapeutic targets of GC.
Topics: Humans; RNA; Mitophagy; Prognosis; Stomach Neoplasms; Sequence Analysis, RNA; Carcinoma
PubMed: 37413850
DOI: 10.1016/j.compbiomed.2023.107227 -
Expansion of T memory stem cells with superior anti-tumor immunity by Urolithin A-induced mitophagy.Immunity Nov 2022T memory stem cells (T) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell...
T memory stem cells (T) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell responses. T cells can be expanded by Urolithin A (UA), which is produced by the commensal gut microbiome from foods rich in ellagitannins and is known to improve mitochondrial health. Oral UA administration to tumor-bearing mice conferred strong anti-tumor CD8 T cell immunity, whereas ex vivo UA pre-treated T cells displayed improved anti-tumor function upon adoptive cell transfer. UA-induced T formation depended on Pink1-mediated mitophagy triggering cytosolic release of the mitochondrial phosphatase Pgam5. Cytosolic Pgam5 dephosphorylated β-catenin, which drove Wnt signaling and compensatory mitochondrial biogenesis. Collectively, we unravel a critical signaling pathway linking mitophagy to T formation and suggest that the well-tolerated metabolic compound UA represents an attractive option to improve immune therapy.
Topics: Mice; Animals; Mitophagy; Coumarins; Wnt Signaling Pathway; Stem Cells; Immunologic Memory
PubMed: 36351375
DOI: 10.1016/j.immuni.2022.09.014