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1.
Cell ; 171(3): 628-641.e26, 2017 Oct 19.
Article in English | MEDLINE | ID: mdl-29053969

ABSTRACT

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.


Subject(s)
Acute Kidney Injury/pathology , Asthma/pathology , Brain Injuries, Traumatic/pathology , Cell Death , Phosphatidylethanolamine Binding Protein/metabolism , Acute Kidney Injury/metabolism , Animals , Apoptosis , Asthma/metabolism , Brain Injuries, Traumatic/metabolism , Cell Death/drug effects , Cell Line , Humans , Isoenzymes/metabolism , Lipoxygenase/chemistry , Lipoxygenase/metabolism , Mice , Models, Molecular , Oxazolidinones/pharmacology , Oxidation-Reduction , Phosphatidylethanolamine Binding Protein/chemistry
2.
EMBO Rep ; 25(10): 4281-4310, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39191946

ABSTRACT

Aberrant mitochondrial fission/fusion dynamics are frequently associated with pathologies, including cancer. We show that alternative splice variants of the fission protein Drp1 (DNM1L) contribute to the complexity of mitochondrial fission/fusion regulation in tumor cells. High tumor expression of the Drp1 alternative splice variant lacking exon 16 relative to other transcripts is associated with poor outcome in ovarian cancer patients. Lack of exon 16 results in Drp1 localization to microtubules and decreased association with mitochondrial fission sites, culminating in fused mitochondrial networks, enhanced respiration, changes in metabolism, and enhanced pro-tumorigenic phenotypes in vitro and in vivo. These effects are inhibited by siRNAs designed to specifically target the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the pathophysiological importance of Drp1 alternative splicing, highlight the divergent functions and consequences of changing the relative expression of Drp1 splice variants in tumor cells, and strongly warrant consideration of alternative splicing in future studies focused on Drp1.


Subject(s)
Alternative Splicing , Dynamins , GTP Phosphohydrolases , Microtubule-Associated Proteins , Mitochondria , Mitochondrial Dynamics , Mitochondrial Proteins , Ovarian Neoplasms , Humans , Dynamins/genetics , Dynamins/metabolism , Mitochondrial Dynamics/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Female , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Cell Line, Tumor , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Mitochondria/metabolism , Mitochondria/genetics , Animals , Disease Progression , Exons/genetics , Mice , Gene Expression Regulation, Neoplastic , Protein Isoforms/genetics , Protein Isoforms/metabolism , Microtubules/metabolism , Apoptosis/genetics
3.
Proc Natl Acad Sci U S A ; 120(25): e2218896120, 2023 Jun 20.
Article in English | MEDLINE | ID: mdl-37327313

ABSTRACT

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.


Subject(s)
Ferroptosis , Phosphatidylethanolamine Binding Protein , Glutathione/metabolism , Iron/metabolism , Lipid Peroxidation , Lipids , Oxidation-Reduction , Phosphatidylethanolamine Binding Protein/antagonists & inhibitors
4.
J Cell Sci ; 135(5)2022 03 01.
Article in English | MEDLINE | ID: mdl-33414166

ABSTRACT

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.


Subject(s)
Ferroptosis , Female , Humans , Lipid Peroxidation , Phospholipid Hydroperoxide Glutathione Peroxidase , Placenta , Pregnancy , Trophoblasts
5.
Angew Chem Int Ed Engl ; 63(9): e202314710, 2024 02 26.
Article in English | MEDLINE | ID: mdl-38230815

ABSTRACT

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1-2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.


Subject(s)
Arachidonate 15-Lipoxygenase , Phosphatidylethanolamines , Phosphatidylethanolamines/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Cell Death , Phospholipids/metabolism , Fatty Acids, Unsaturated/metabolism , Lipid Peroxidation
6.
J Am Chem Soc ; 145(20): 11311-11322, 2023 05 24.
Article in English | MEDLINE | ID: mdl-37103240

ABSTRACT

Reliable probing of cardiolipin (CL) content in dynamic cellular milieux presents significant challenges and great opportunities for understanding mitochondria-related diseases, including cancer, neurodegeneration, and diabetes mellitus. In intact respiring cells, selectivity and sensitivity for CL detection are technically demanding due to structural similarities among phospholipids and compartmental secludedness of the inner mitochondrial membrane. Here, we report a novel "turn-on" fluorescent probe HKCL-1M for detecting CL in situ. HKCL-1M displays outstanding sensitivity and selectivity toward CL through specific noncovalent interactions. In live-cell imaging, its hydrolyzed product HKCL-1 efficiently retained itself in intact cells independent of mitochondrial membrane potential (Δψm). The probe robustly co-localizes with mitochondria and outperforms 10-N-nonyl acridine orange (NAO) and Δψm-dependent dyes with superior photostability and negligible phototoxicity. Our work thus opens up new opportunities for studying mitochondrial biology through efficient and reliable visualization of CL in situ.


Subject(s)
Cardiolipins , Fluorescent Dyes , Fluorescent Dyes/chemistry , Cardiolipins/chemistry , Mitochondria/chemistry , Phospholipids/analysis , Mitochondrial Membranes
7.
J Immunol ; 207(6): 1627-1640, 2021 09 15.
Article in English | MEDLINE | ID: mdl-34433619

ABSTRACT

Silicosis is a lethal pneumoconiosis for which no therapy is available. Silicosis is a global threat, and more than 2.2 million people per year are exposed to silica in the United States. The initial response to silica is mediated by innate immunity. Phagocytosis of silica particles by macrophages is followed by recruitment of mitochondria to phagosomes, generation of mitochondrial reactive oxygen species, and cytokine (IL-1ß, TNF-α, IFN-ß) release. In contrast with LPS, the metabolic remodeling of silica-exposed macrophages is unclear. This study contrasts mitochondrial and metabolic alterations induced by LPS and silica on macrophages and correlates them with macrophage viability and cytokine production, which are central to the pathogenesis of silicosis. Using high-resolution respirometer and liquid chromatography-high-resolution mass spectrometry, we determined the effects of silica and LPS on mitochondrial respiration and determined changes in central carbon metabolism of murine macrophage cell lines RAW 264.7 and IC-21. We show that silica induces metabolic reprogramming of macrophages. Silica, as well as LPS, enhances glucose uptake and increases aerobic glycolysis in macrophages. In contrast with LPS, silica affects mitochondria respiration, reducing complex I and enhancing complex II activity, to sustain cell viability. These mitochondrial alterations are associated in silica, but not in LPS-exposed macrophages, with reductions of tricarboxylic acid cycle intermediates, including succinate, itaconate, glutamate, and glutamine. Furthermore, in contrast with LPS, these silica-induced metabolic adaptations do not correlate with IL-1ß or TNF-α production, but with the suppressed release of IFN-ß. Our data highlight the importance of complex II activity and tricarboxylic acid cycle remodeling to macrophage survival and cytokine-mediated inflammation in silicosis.


Subject(s)
Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/immunology , Silicon Dioxide/chemistry , Silicon Dioxide/pharmacology , Silicosis/immunology , Animals , Cell Survival/drug effects , Citric Acid Cycle/drug effects , Crystallization , Cytokines/biosynthesis , Inflammation/immunology , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Phagocytosis/drug effects , Phagosomes/metabolism , RAW 264.7 Cells , Signal Transduction/drug effects , Silicosis/metabolism
8.
Proc Natl Acad Sci U S A ; 117(44): 27319-27328, 2020 11 03.
Article in English | MEDLINE | ID: mdl-33087576

ABSTRACT

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.


Subject(s)
Ferroptosis/physiology , Group VI Phospholipases A2/metabolism , Trophoblasts/metabolism , Animals , Female , Glutathione Peroxidase/metabolism , Group VI Phospholipases A2/genetics , Group VI Phospholipases A2/physiology , Humans , Iron/metabolism , Lipid Peroxides/metabolism , Mice , Mice, Knockout , Phosphatidylethanolamines/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Placenta/metabolism , Pregnancy , Premature Birth/metabolism , Signal Transduction
9.
Proc Natl Acad Sci U S A ; 117(25): 14376-14385, 2020 06 23.
Article in English | MEDLINE | ID: mdl-32513718

ABSTRACT

Temporally harmonized elimination of damaged or unnecessary organelles and cells is a prerequisite of health. Under Type 2 inflammatory conditions, human airway epithelial cells (HAECs) generate proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) as proximate death signals. Production of 15-HpETE-PE depends on activation of 15-lipoxygenase-1 (15LO1) in complex with PE-binding protein-1 (PEBP1). We hypothesized that cellular membrane damage induced by these proferroptotic phospholipids triggers compensatory prosurvival pathways, and in particular autophagic pathways, to prevent cell elimination through programmed death. We discovered that PEBP1 is pivotal to driving dynamic interactions with both proferroptotic 15LO1 and the autophagic protein microtubule-associated light chain-3 (LC3). Further, the 15LO1-PEBP1-generated ferroptotic phospholipid, 15-HpETE-PE, promoted LC3-I lipidation to stimulate autophagy. This concurrent activation of autophagy protects cells from ferroptotic death and release of mitochondrial DNA. Similar findings are observed in Type 2 Hi asthma, where high levels of both 15LO1-PEBP1 and LC3-II are seen in HAECs, in association with low bronchoalveolar lavage fluid mitochondrial DNA and more severe disease. The concomitant activation of ferroptosis and autophagy by 15LO1-PEBP1 complexes and their hydroperoxy-phospholipids reveals a pathobiologic pathway relevant to asthma and amenable to therapeutic targeting.


Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Asthma/immunology , Autophagy/immunology , Epithelial Cells/pathology , Ferroptosis/immunology , Phosphatidylethanolamine Binding Protein/metabolism , Adult , Animals , Asthma/diagnosis , Asthma/pathology , Bronchoalveolar Lavage Fluid/cytology , Cell Line , Cell Survival/immunology , Epithelial Cells/immunology , Female , Gene Knockout Techniques , Humans , Hydroxyeicosatetraenoic Acids/immunology , Hydroxyeicosatetraenoic Acids/metabolism , Interleukin-13/immunology , Interleukin-13/metabolism , Male , Mice , Microtubule-Associated Proteins/metabolism , Molecular Dynamics Simulation , Phosphatidylethanolamine Binding Protein/genetics , Phosphatidylethanolamines/immunology , Phosphatidylethanolamines/metabolism , Primary Cell Culture , Protein Binding/immunology , Severity of Illness Index
10.
Clin Infect Dis ; 74(9): 1525-1533, 2022 05 03.
Article in English | MEDLINE | ID: mdl-34374761

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.


Subject(s)
COVID-19 , Antibodies, Neutralizing , Biomarkers , COVID-19/diagnosis , Cross-Sectional Studies , Humans , Prospective Studies , RNA, Viral , SARS-CoV-2 , Viremia
11.
Nat Chem Biol ; 16(3): 278-290, 2020 03.
Article in English | MEDLINE | ID: mdl-32080625

ABSTRACT

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.


Subject(s)
Ferroptosis/physiology , Macrophages/metabolism , Nitric Oxide Synthase Type II/metabolism , Animals , Arachidonate 15-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/physiology , Cell Death , Female , Iron/metabolism , Iron/physiology , Leukotrienes/metabolism , Lipid Peroxidation/physiology , Lipid Peroxides/metabolism , Male , Mice , Mice, Inbred C57BL , Microglia/metabolism , Nitric Oxide Synthase Type II/physiology , Oxidation-Reduction , Reactive Oxygen Species/metabolism
12.
FASEB J ; 34(5): 7192-7207, 2020 05.
Article in English | MEDLINE | ID: mdl-32274853

ABSTRACT

Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) alter mitochondrial morphology and result in several subtypes of the inherited peripheral neuropathy Charcot-Marie-Tooth disease; however, the mechanism by which GDAP1 functions has remained elusive. GDAP1 contains primary sequence homology to the GST superfamily; however, the question of whether GDAP1 is an active GST has not been clearly resolved. Here, we present biochemical evidence, suggesting that GDAP1 has lost the ability to bind glutathione without a loss of substrate binding activity. We have revealed that the α-loop, located within the H-site motif is the primary determinant for substrate binding. Using structural data of GDAP1, we have found that critical residues and configurations in the G-site which canonically interact with glutathione are altered in GDAP1, rendering it incapable of binding glutathione. Last, we have found that the overexpression of GDAP1 in HeLa cells results in a mitochondrial phenotype which is distinct from oxidative stress-induced mitochondrial fragmentation. This phenotype is dependent on the presence of the transmembrane domain, as well as a unique hydrophobic domain that is not found in canonical GSTs. Together, we data point toward a non-enzymatic role for GDAP1, such as a sensor or receptor.


Subject(s)
Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Catalytic Domain/genetics , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/metabolism , Crystallography, X-Ray , Glutathione/metabolism , Glutathione Transferase/genetics , HeLa Cells , Humans , Mitochondria/metabolism , Mitochondria/ultrastructure , Models, Molecular , Mutation , Nerve Tissue Proteins/genetics , Oxidative Stress , Phenotype , Protein Domains , Protein Structure, Quaternary , Substrate Specificity
13.
Angew Chem Int Ed Engl ; 59(40): 17435-17441, 2020 09 28.
Article in English | MEDLINE | ID: mdl-32585075

ABSTRACT

Hydrogen peroxide (H2 O2 ) mediates the biology of wound healing, apoptosis, inflammation, etc. H2 O2 has been fluorometrically imaged with protein- or small-molecule-based probes. However, only protein-based probes have afforded temporal insights within seconds. Small-molecule-based electrophilic probes for H2 O2 require many minutes for a sufficient response in biological systems. Here, we report a fluorogenic probe that selectively undergoes a [2,3]-sigmatropic rearrangement (seleno-Mislow-Evans rearrangement) with H2 O2 , followed by acetal hydrolysis, to produce a green fluorescent molecule in seconds. Unlike other electrophilic probes, the current probe acts as a nucleophile. The fast kinetics enabled real-time imaging of H2 O2 produced in endothelial cells in 8 seconds (much earlier than previously shown) and H2 O2 in a zebrafish wound healing model. This work may provide a platform for endogenous H2 O2 detection in real time with chemical probes.


Subject(s)
Fluorescent Dyes/chemistry , Hydrogen Peroxide/chemistry , Acetals/chemistry , Animals , Disease Models, Animal , Endothelial Cells/cytology , Endothelial Cells/metabolism , HeLa Cells , Humans , Hydrogen Peroxide/metabolism , Hydrolysis , Mice , Microscopy, Fluorescence , Molecular Conformation , Optical Imaging , Oxidation-Reduction , RAW 264.7 Cells , Selenium/chemistry , Wounds and Injuries/diagnostic imaging , Zebrafish/metabolism
14.
J Neurosci ; 38(34): 7505-7515, 2018 08 22.
Article in English | MEDLINE | ID: mdl-30030401

ABSTRACT

Dysregulation of mitochondrial biogenesis is implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). However, it is not clear how mitochondrial biogenesis is regulated in neurons, with their unique compartmentalized anatomy and energetic demands. This is particularly relevant in PD because selectively vulnerable neurons feature long, highly arborized axons where degeneration initiates. We previously found that exposure of neurons to chronic, sublethal doses of rotenone, a complex I inhibitor linked to PD, causes early increases in mitochondrial density specifically in distal axons, suggesting possible upregulation of mitochondrial biogenesis within axons. Here, we directly evaluated for evidence of mitochondrial biogenesis in distal axons and examined whether PD-relevant stress causes compartmentalized alterations. Using BrdU labeling and imaging to quantify replicating mitochondrial DNA (mtDNA) in primary rat neurons (pooled from both sexes), we provide evidence of mtDNA replication in axons along with cell bodies and proximal dendrites. We found that exposure to chronic, sublethal rotenone increases mtDNA replication first in neurites and later extending to cell bodies, complementing our mitochondrial density data. Further, isolating axons from cell bodies and dendrites, we discovered that rotenone exposure upregulates mtDNA replication in distal axons. Utilizing superresolution stimulated emission depletion (STED) imaging, we identified mtDNA replication at sites of mitochondrial-endoplasmic reticulum contacts in axons. Our evidence suggests that mitochondrial biogenesis occurs not only in cell bodies, but also in distal axons, and is altered under PD-relevant stress conditions in an anatomically compartmentalized manner. We hypothesize that this contributes to vulnerability in neurodegenerative diseases.SIGNIFICANCE STATEMENT Mitochondrial biogenesis is crucial for maintaining mitochondrial and cellular health and has been linked to neurodegenerative disease pathogenesis. However, regulation of this process is poorly understood in CNS neurons, which rely on mitochondrial function for survival. Our findings offer fundamental insight into these regulatory mechanisms by demonstrating that replication of mitochondrial DNA, an essential precursor for biogenesis, can occur in distal regions of CNS neuron axons independent of the soma. Further, this process is upregulated specifically in axons as an early response to neurodegeneration-relevant stress. This is the first demonstration of the compartmentalized regulation of CNS neuronal mitochondrial biogenesis in response to stress and may prove a useful target in development of therapeutic strategies for neurodegenerative disease.


Subject(s)
Axons/ultrastructure , DNA Replication , DNA, Mitochondrial/biosynthesis , Mitochondria/metabolism , Organelle Biogenesis , Parkinson Disease/metabolism , Animals , Axons/drug effects , Axons/metabolism , Cerebral Cortex/cytology , DNA Replication/drug effects , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex I/metabolism , Electron Transport Complex IV/analysis , Endoplasmic Reticulum/ultrastructure , Female , Humans , Male , Mitochondria/drug effects , Mitochondria/ultrastructure , Mitochondrial Dynamics/drug effects , Mitochondrial Proton-Translocating ATPases/analysis , Neurites/drug effects , Neurites/ultrastructure , Neurons/drug effects , Neurons/ultrastructure , Oxidative Stress , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/analysis , Rats , Rats, Sprague-Dawley , Rotenone/toxicity , Uncoupling Agents/toxicity
15.
Am J Physiol Lung Cell Mol Physiol ; 316(6): L1150-L1164, 2019 06 01.
Article in English | MEDLINE | ID: mdl-30892078

ABSTRACT

Pulmonary hypertension (PH) is a leading cause of death in sickle cell disease (SCD) patients. Hemolysis and oxidative stress contribute to SCD-associated PH. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and, by interacting with its receptor CD47, limits vasodilation of distal pulmonary arteries ex vivo. We hypothesized that the TSP1-CD47 interaction may promote PH in SCD. We found that TSP1 and CD47 are upregulated in the lungs of Berkeley (BERK) sickling (Sickle) mice and patients with SCD-associated PH. We then generated chimeric animals by transplanting BERK bone marrow into C57BL/6J (n = 24) and CD47 knockout (CD47KO, n = 27) mice. Right ventricular (RV) pressure was lower in fully engrafted Sickle-to-CD47KO than Sickle-to-C57BL/6J chimeras, as shown by the reduced maximum RV pressure (P = 0.013) and mean pulmonary artery pressure (P = 0.020). The afterload of the sickle-to-CD47KO chimeras was also lower, as shown by the diminished pulmonary vascular resistance (P = 0.024) and RV effective arterial elastance (P = 0.052). On myography, aortic segments from Sickle-to-CD47KO chimeras showed improved relaxation to acetylcholine. We hypothesized that, in SCD, TSP1-CD47 signaling promotes PH, in part, by increasing reactive oxygen species (ROS) generation. In human pulmonary artery endothelial cells, treatment with TSP1 stimulated ROS generation, which was abrogated by CD47 blockade. Explanted lungs of CD47KO chimeras had less vascular congestion and a smaller oxidative footprint. Our results show that genetic absence of CD47 ameliorates SCD-associated PH, which may be due to decreased ROS levels. Modulation of TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH.


Subject(s)
Anemia, Sickle Cell/pathology , CD47 Antigen/metabolism , Hypertension, Pulmonary/pathology , Pulmonary Artery/pathology , Thrombospondin 1/metabolism , Anemia, Sickle Cell/genetics , Animals , CD47 Antigen/antagonists & inhibitors , CD47 Antigen/genetics , Cells, Cultured , Endothelial Cells/pathology , Humans , Hypertension, Pulmonary/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pulmonary Artery/cytology , Reactive Oxygen Species/metabolism , Ventricular Function, Right/physiology
16.
Nat Methods ; 13(3): 263-8, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26808669

ABSTRACT

Upon illumination, photosensitizer molecules produce reactive oxygen species that can be used for functional manipulation of living cells, including protein inactivation, targeted-damage introduction and cellular ablation. Photosensitizers used to date have been either exogenous, resulting in delivery and removal challenges, or genetically encoded proteins that form or bind a native photosensitizing molecule, resulting in a constitutively active photosensitizer inside the cell. We describe a genetically encoded fluorogen-activating protein (FAP) that binds a heavy atom-substituted fluorogenic dye, forming an 'on-demand' activated photosensitizer that produces singlet oxygen and fluorescence when activated with near-infrared light. This targeted and activated photosensitizer (TAPs) approach enables protein inactivation, targeted cell killing and rapid targeted lineage ablation in living larval and adult zebrafish. The near-infrared excitation and emission of this FAP-TAPs provides a new spectral range for photosensitizer proteins that could be useful for imaging, manipulation and cellular ablation deep within living organisms.


Subject(s)
Apoptosis/radiation effects , Infrared Rays , Photochemotherapy/methods , Photosensitizing Agents/chemistry , Photosensitizing Agents/radiation effects , Recombinant Proteins/genetics , Apoptosis/physiology , Dose-Response Relationship, Radiation , HEK293 Cells , Humans , Radiation Dosage , Recombinant Proteins/therapeutic use
17.
Circulation ; 133(8): 717-31, 2016 Feb 23.
Article in English | MEDLINE | ID: mdl-26813102

ABSTRACT

BACKGROUND: Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available, and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in endothelial nitric oxide synthase null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in preclinical models of pulmonary hypertension. METHODS AND RESULTS: To evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a 2-hit PH-HFpEF model in rats with multiple features of metabolic syndrome attributable to double-leptin receptor defect (obese ZSF1) with the combined treatment of vascular endothelial growth factor receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose-lowering effect of nitrite was abolished in SIRT3-deficient human skeletal muscle cells, and in SIRT3 knockout mice fed a high-fat diet, as well. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMP-activated protein kinase. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMP-activated protein kinase. CONCLUSIONS: These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Heart Failure/metabolism , Hyperglycemia/metabolism , Hypertension, Pulmonary/metabolism , Sirtuin 3/metabolism , Stroke Volume/physiology , Animals , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Activation/physiology , Heart Failure/drug therapy , Humans , Hyperglycemia/drug therapy , Hypertension, Pulmonary/drug therapy , Male , Metformin/pharmacology , Metformin/therapeutic use , Mice , Mice, 129 Strain , Mice, Knockout , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Rats , Rats, Zucker , Sodium Nitrite/pharmacology , Sodium Nitrite/therapeutic use , Stroke Volume/drug effects
18.
Biochem J ; 473(12): 1821-30, 2016 06 15.
Article in English | MEDLINE | ID: mdl-27099338

ABSTRACT

Lysine acetylation is tightly coupled to the nutritional status of the cell, as the availability of its cofactor, acetyl-CoA, fluctuates with changing metabolic conditions. Recent studies have demonstrated that acetyl-CoA levels act as an indicator of cellular nourishment, and increased abundance of this metabolite can block the induction of cellular recycling programmes. In the present study we investigated the cross-talk between mitochondrial metabolic pathways, acetylation and autophagy, using chemical inducers of mitochondrial acetyl-CoA production. Treatment of cells with α-lipoic acid (αLA), a cofactor of the pyruvate dehydrogenase complex, led to the unexpected hyperacetylation of α-tubulin in the cytosol. This acetylation was blocked by pharmacological inhibition of mitochondrial citrate export (a source for mitochondria-derived acetyl-CoA in the cytosol), was dependent on the α-tubulin acetyltransferase (αTAT) and was coupled to a loss in function of the cytosolic histone deacetylase, HDAC6. We further demonstrate that αLA slows the flux of substrates through autophagy-related pathways, and severely limits the ability of cells to remove depolarized mitochondria through PTEN-associated kinase 1 (PINK1)-mediated mitophagy.


Subject(s)
Mitochondria/metabolism , Thioctic Acid/pharmacology , Tubulin/metabolism , Acetyl Coenzyme A/metabolism , Acetylation/drug effects , Acetyltransferases/metabolism , Animals , Autophagy/drug effects , COS Cells , Chlorocebus aethiops , Hep G2 Cells , Histone Deacetylase Inhibitors/pharmacology , Humans , Microscopy, Confocal , Mitochondria/drug effects , Signal Transduction/drug effects
19.
J Immunol ; 192(8): 3837-46, 2014 Apr 15.
Article in English | MEDLINE | ID: mdl-24623132

ABSTRACT

Macrophages play a fundamental role in innate immunity and the pathogenesis of silicosis. Phagocytosis of silica particles is associated with the generation of reactive oxygen species (ROS), secretion of cytokines, such as TNF, and cell death that contribute to silica-induced lung disease. In macrophages, ROS production is executed primarily by activation of the NADPH oxidase (Phox) and by generation of mitochondrial ROS (mtROS); however, the relative contribution is unclear, and the effects on macrophage function and fate are unknown. In this study, we used primary human and mouse macrophages (C57BL/6, BALB/c, and p47(phox-/-)) and macrophage cell lines (RAW 264.7 and IC21) to investigate the contribution of Phox and mtROS to silica-induced lung injury. We demonstrate that reduced p47(phox) expression in IC21 macrophages is linked to enhanced mtROS generation, cardiolipin oxidation, and accumulation of cardiolipin hydrolysis products, culminating in cell death. mtROS production is also observed in p47(phox-/-) macrophages, and p47(phox-/-) mice exhibit increased inflammation and fibrosis in the lung following silica exposure. Silica induces interaction between TNFR1 and Phox in RAW 264.7 macrophages. Moreover, TNFR1 expression in mitochondria decreased mtROS production and increased RAW 264.7 macrophage survival to silica. These results identify TNFR1/Phox interaction as a key event in the pathogenesis of silicosis that prevents mtROS formation and reduces macrophage apoptosis.


Subject(s)
Mitochondria/metabolism , NADPH Oxidases/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Silicosis/metabolism , Animals , Cell Death , Cell Line , Disease Models, Animal , Female , Gene Expression Regulation , Lung Injury/etiology , Lung Injury/metabolism , Lung Injury/pathology , Macrophages/metabolism , Mice , Mice, Knockout , NADPH Oxidases/genetics , Protein Binding , Protein Transport , Reactive Oxygen Species/metabolism , Silicon Dioxide/adverse effects , Silicon Dioxide/metabolism , Silicosis/genetics
20.
J Cell Biochem ; 116(4): 524-32, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25358453

ABSTRACT

Artificial trans fatty acids promote atherosclerosis by blocking macrophage clearance of cell debris. Classical fatty-acid response mechanisms include TLR4-NF-κB activation, and Erk1/2 phosphorylation, but these may not indicate long-term mechanisms. Indeed, nuclear NF-κB was increased by 60 min treatment by 30 µM of the 18 carbon trans unsaturated fatty acid elaidic acid (elaidate), the physiological cis-unsaturated fatty acid oleic acid (oleate), and the 18 or 16 carbon saturated fatty acids stearic and palmitic acid (stearate or palmitate). However, except for stearate, effects on related pathways were minimal at 44 h. To determine longer term effects of trans fatty acids, we compared mRNA expression profiles of (trans) elaidate to (cis) oleate, 30 µM, at 44 h in human macrophages. We found that elaidate changed Zn(2+) -homeostasis gene mRNAs markedly. This might be important because Zn(2+) is a major regulator of macrophage activity. Messenger RNAs of seven Zn(2+) -binding metallothioneins decreased 2-4-fold; the zinc importer SLC39A10 increased twofold, in elaidate relative to oleate-treated cells. Results were followed by quantitative PCR comparing cis, trans, and saturated fatty acid effects on Zn(2+) -homeostasis gene mRNAs. This confirmed that elaidate uniquely decreased metallothionein expression and increased SLC39A10 at 44 h. Further, intracellular Zn(2+) was measured using N-(carboxymethyl)-N-[2-[2-[2(carboxymethyl) amino]-5-(2,7,-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-phenoxy]-ethoxy]-4-methoxyphenyl]glycine, acetoxymethyl ester (FluoZin-3-AM). This showed that, at 44 h, only cells treated with elaidate had increased Zn(2+) . The durable effect of elaidate on Zn(2+) activation is a novel and specific effect of trans fatty acids on peripheral macrophage metabolism.


Subject(s)
Cation Transport Proteins/genetics , Metallothionein/genetics , Oleic Acid/pharmacology , Zinc/metabolism , Cells, Cultured , Fatty Acids/physiology , Gene Expression Profiling , Gene Expression Regulation/drug effects , Homeostasis/drug effects , Humans , Macrophages/chemistry , Macrophages/drug effects , Macrophages/metabolism , Oleic Acids
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