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FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy.

ABSTRACT: Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from in vitro experiments involving the treatment of mammalian cells with high concentrations of mitochondrial uncouplers, such as CCCP. These chemicals have been shown to mediate off target effects, other than mitochondrial depolarization. A matter of controversy between mitochondrial physiologists and cell biologists is the discrepancy between concentrations of CCCP needed to activate mitophagy (usually >10 ?M), when compared to the much lower concentrations used to depolarize mitochondria (<1 ?M). Thus, there is an urgent need for optimizing the current methods to assess PINK1-PRKN mitophagy in vitro. In this study, we address the utilization of high CCCP concentrations commonly used to activate mitophagy. Combining live fluorescence microscopy and biochemistry, we show that the FBS/BSA in the cell culture medium reduces the ability of CCCP to induce PINK1 accumulation at depolarized mitochondria, subsequent PRKN recruitment and ubiquitin phosphorylation, and ultimately mitochondrial clearance. As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. These data unite mitochondrial physiology and mitophagy studies and are a first step toward a consensus on optimal experimental conditions for PINK1-PRKN mitophagy and mitochondrial physiology investigations to be carried out in parallel. Abbreviations: BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenylhydrazone; DMEM: dulbecco's Modified Eagle's Medium; DNP: 2,4-dinitrophenol; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GSH: glutathione; HBSS: Hanks' balanced salt solution; mtKeima: mitochondria-targeted monomeric keima-red; PBS: phosphate buffered saline; PD: Parkinson disease; PINK1: PTEN induced kinase 1; POE SHSY5Ys: FLAG-PRKN over-expressing SHSY5Y cells; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMRM: tetramethylrhodamine methyl ester; WB: western blot; WT: wild-type; ??m: mitochondrial membrane potential.

SUBMITTER: Soutar MPM

PROVIDER: S-EPMC6844515 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy.

Soutar Marc P M MPM Kempthorne Liam L Annuario Emily E Luft Christin C Wray Selina S Ketteler Robin R Ludtmann Marthe H R MHR Plun-Favreau Hélène H

Autophagy 20190507 11

Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from <i>in vitro</i> experiments involving the treatment of mammalian cells with high concentrations of mitochondrial ...[more]

PMID: 31060423

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Project description:Mitophagy, which is a conserved cellular process for selectively removing damaged or unwanted mitochondria, is critical for mitochondrial quality control and the maintenance of normal cellular physiology. However, the precise mechanisms underlying mitophagy remain largely unknown. Prior studies on mitophagy focused on the events in the mitochondrial outer membrane. PHB2 (prohibitin 2), which is a highly conserved membrane scaffold protein, was recently identified as a novel inner membrane mitophagy receptor that mediates mitophagy. Here, we report a new signaling pathway for PHB2-mediated mitophagy. Upon mitochondrial membrane depolarization or misfolded protein aggregation, PHB2 depletion destabilizes PINK1 in the mitochondria, which blocks the mitochondrial recruitment of PRKN/Parkin, ubiquitin and OPTN (optineurin), leading to an inhibition of mitophagy. In addition, PHB2 overexpression directly induces PRKN recruitment to the mitochondria. Moreover, PHB2-mediated mitophagy is dependent on the mitochondrial inner membrane protease PARL, which interacts with PHB2 and is activated upon PHB2 depletion. Furthermore, PGAM5, which is processed by PARL, participates in PHB2-mediated PINK1 stabilization. Finally, a ligand of PHB proteins that we synthesized, called FL3, was found to strongly inhibit PHB2-mediated mitophagy and to effectively block cancer cell growth and energy production at nanomolar concentrations. Thus, our findings reveal that the PHB2-PARL-PGAM5-PINK1 axis is a novel pathway of PHB2-mediated mitophagy and that targeting PHB2 with the chemical compound FL3 is a promising strategy for cancer therapy.Abbreviations: AIFM1: apoptosis inducing factor mitochondria associated 1; ATP5F1A/ATP5A1: ATP synthase F1 subunit alpha; BAF: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chemical reagent carbonyl cyanide m-chlorophenyl hydrazine; FL3: flavaglines compound 3; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryo fibroblasts; MPP: mitochondrial-processing peptidase; MT-CO2/COX2: mitochondrially encoded cytochrome c oxidase II; MTS: mitochondrial targeting sequence; OA: oligomycin and antimycin A; OPTN: optineurin; OTC: ornithine carbamoyltransferase; PARL: presenilin associated rhomboid like; PBS: phosphate-buffered saline; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PHB: prohibitin; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Roc-A: rocaglamide A; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin beta class I.

Project description:Mitochondrial dynamics is highly implicated in a plethora of cellular processes including apoptosis and mitophagy. However, little is known about the scope and precise functions of mitochondrial dynamics proteins for mitochondrial quality control and cellular homeostasis. Whether mitochondrial dynamics proteins serve in cellular processes reliant on mitochondrial fission-fusion is still not fully explored. MIEF1/MiD51 (mitochondrial elongation factor 1) is known to promote mitochondrial fission via the recruitment of GTPase protein DNM1L/DRP1 (dynamin 1 like), but the fundamental understandings of MIEF1 for mitochondrial-dependent cellular processes are largely elusive. Here, we report novel roles of MIEF1 in responding to apoptotic stimuli and mitochondrial damage. Given our result that staurosporine (STS) treatment induced the degradation of MIEF1 via the ubiquitin-proteasome system (UPS), we are motivated to explore the role of MIEF1 in apoptosis. MIEF1 loss triggered the imbalance of BCL2 family members on the mitochondria, consequently initiating the translocation of BAX onto the mitochondria, catalyzing the decrease of mitochondrial membrane potential and promoting the release of DIABLO/SMAC (diablo IAP-binding mitochondrial protein) and CYCS (cytochrome c, somatic). We further demonstrate that MIEF1 deficiency impaired mitochondrial respiration and induced mitochondrial oxidative stress, sensitizing cells to PINK1-PRKN-mediated mitophagy. The recruitment of PRKN to depolarized mitochondria modulated the UPS-dependent degradation of MFN2 (mitofusin 2) and FIS1 (fission, mitochondrial 1) specifically, to further promote mitophagy. Our findings uncover a bridging role of MIEF1 integrating cell death and mitophagy, unlikely dependent on mitochondrial dynamics, implying new insights to mechanisms determining cellular fate.Abbreviations: ActD: actinomycin D; BAX: BCL2 associated X, apoptosis regulator; BAK1: BCL2 antagonist/killer 1; BCL2L1: BCL2 like 1; BMH: 1,6-bismaleimidohexane; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHX: cycloheximide; CQ: chloroquine; CYCS: cytochrome c, somatic; DIABLO: diablo IAP-binding mitochondrial protein; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; FIS1: fission, mitochondrial 1; GFP: green fluorescent protein; IP: immunoprecipitation; MFN1: mitofusin 1; MFN2: mitofusin 2; MG132: carbobenzoxy-Leu-Leu-leucinal; MIEF1/MiD51: mitochondrial elongation factor 1; MIEF2/MiD49: mitochondrial elongation factor 2; MOMP: mitochondrial outer membrane permeabilization; MTR: MitoTracker Red; OA: oligomycin plus antimycin A; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; SD: standard deviation; STS: staurosporine; TNF: tumor necrosis factor; UPS: ubiquitin-proteasome system; VDAC1: voltage dependent anion channel 1.

Project description:Lacking a self-contained metabolism network, viruses have evolved multiple mechanisms for rewiring the metabolic system of their host to hijack the host's metabolic resources for replication. Newcastle disease virus (NDV) is a paramyxovirus, as an oncolytic virus currently being developed for cancer treatment. However, how NDV alters cellular metabolism is still far from fully understood. In this study, we show that NDV infection reprograms cell metabolism by increasing glucose utilization in the glycolytic pathway. Mechanistically, NDV induces mitochondrial damage, elevated mitochondrial reactive oxygen species (mROS) and ETC dysfunction. Infection of cells depletes nucleotide triphosphate levels, resulting in elevated AMP:ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and MTOR crosstalk mediated autophagy. In a time-dependent manner, NDV shifts the balance of mitochondrial dynamics from fusion to fission. Subsequently, PINK1-PRKN-dependent mitophagy was activated, forming a ubiquitin chain with MFN2 (mitofusin 2), and molecular receptor SQSTM1/p62 recognized damaged mitochondria. We also found that NDV infection induces NAD+-dependent deacetylase SIRT3 loss via mitophagy to engender HIF1A stabilization, leading to the switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis. Overall, these studies support a model that NDV modulates host cell metabolism through PINK1-PRKN-dependent mitophagy for degrading SIRT3.Abbreviations: AMPK: AMP-activated protein kinase; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECAR: extracellular acidification rate; hpi: hours post infection LC-MS: liquid chromatography-mass spectrometry; mito-QC: mCherry-GFP-FIS1[mt101-152]; MFN2: mitofusin 2; MMP: mitochondrial membrane potential; mROS: mitochondrial reactive oxygen species; MOI: multiplicity of infection; 2-NBDG: 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose; NDV: newcastle disease virus; OCR: oxygen consumption rate; siRNA: small interfering RNA; SIRT3: sirtuin 3; TCA: tricarboxylic acid; TCID50: tissue culture infective doses.

Dataset Information

FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy.

Publications

FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy.

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