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1.
Nat Chem Biol ; 13(1): 81-90, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27842066

ABSTRACT

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.


Subject(s)
Arachidonic Acid/metabolism , Fatty Acids, Unsaturated/metabolism , Phospholipids/metabolism , Animals , Arachidonic Acid/antagonists & inhibitors , Cell Death/drug effects , Cell Line , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/deficiency , Coenzyme A Ligases/metabolism , Fatty Acids, Unsaturated/antagonists & inhibitors , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
3.
Res Sq ; 2023 Jan 16.
Article in English | MEDLINE | ID: mdl-36711642

ABSTRACT

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies that includes steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with insulin resistance and type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD, particularly in the transition from steatosis to NASH. Mitophagy is a mitochondrial quality control mechanism that allows for the selective removal of damaged mitochondria from the cell via the autophagy pathway. While past work demonstrated a negative association between liver fat content and rates of mitophagy, when changes in mitophagy occur during the pathogenesis of NAFLD and whether such changes contribute to the primary endpoints associated with the disease are currently poorly defined. We therefore undertook the studies described here to establish when alterations in mitophagy occur during the pathogenesis of NAFLD, as well as to determine the effects of genetic inhibition of mitophagy via conditional deletion of a key mitophagy regulator, PARKIN, on the development of steatosis, insulin resistance, inflammation and fibrosis. We find that loss of mitophagy occurs early in the pathogenesis of NAFLD and that loss of PARKIN hastens the onset but not severity of key NAFLD disease features. These observations suggest that loss of mitochondrial quality control in response to nutritional stress may contribute to mitochondrial dysfunction and the pathogenesis of NAFLD.

4.
Mech Ageing Dev ; 180: 97-106, 2019 06.
Article in English | MEDLINE | ID: mdl-31002926

ABSTRACT

Cellular senescence is a phenotype characterized by irreversible growth arrest, chronic elevated secretion of proinflammatory cytokines and matrix proteases, a phenomenon known as senescence-associated secretory phenotype (SASP). Biomarkers of cellular senescence have been shown to increase with age and degeneration of human disc tissue. Senescent disc cells in culture recapitulate features associated with age-related disc degeneration, including increased secretion of proinflammatory cytokines, matrix proteases, and fragmentation of matrix proteins. However, little is known of the metabolic changes that underlie the senescent phenotype of disc cells. To assess the metabolic changes, we performed a bioenergetic analysis of in vitro oxidative stress-induced senescent (SIS) human disc cells. SIS disc cells acquire SASP and exhibit significantly elevated mitochondrial content and mitochondrial ATP-linked respiration. The metabolic changes appear to be driven by the upregulated protein secretion in SIS cells as abrogation of protein synthesis using cycloheximide decreased mitochondrial ATP-linked respiration. Taken together, the results of the study suggest that the increased energy generation state supports the secretion of senescent associated proteins in SIS disc cells.


Subject(s)
Cellular Senescence , Energy Metabolism , Intervertebral Disc/metabolism , Mitochondria/metabolism , Oxidative Stress , Oxygen Consumption , Adult , Female , Humans , Intervertebral Disc/pathology , Male , Middle Aged , Mitochondria/pathology
5.
Biotechniques ; 43(1 Suppl): 14-9, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17933097

ABSTRACT

The use of confocal and multiphoton microscopy for in vivo studies in animals continues to be an area of exciting technical and commercial development. However the application of these technologies at high resolution, such that molecular and subcellular information is collected, remains an elusive goal. This review discusses the practical and performance limitations and the potential uses of currently available systems. We also highlight the ongoing developments in both miniaturized and bench-mounted systems for single and multiphoton optical sectioning studies in animals and in human clinical trials.


Subject(s)
Fiber Optic Technology/instrumentation , Fiber Optic Technology/trends , Image Enhancement/instrumentation , Microscopy, Confocal/instrumentation , Microscopy, Confocal/veterinary , Equipment Design , Feasibility Studies , Fiber Optic Technology/methods , Forecasting , Image Enhancement/methods , Microscopy, Confocal/methods , Microscopy, Confocal/trends , Miniaturization , Technology Assessment, Biomedical
6.
Mitochondrion ; 30: 67-77, 2016 09.
Article in English | MEDLINE | ID: mdl-27374852

ABSTRACT

Benzyl isothiocyanate (BITC) is a highly promising phytochemical abundant in cruciferous vegetables with preclinical evidence of in vivo efficacy against breast cancer in xenograft and transgenic mouse models. Mammary cancer chemoprevention by BITC is associated with apoptotic cell death but the underlying mechanism is not fully understood. Herein, we demonstrate for the first time that altered mitochondrial dynamics is an early and critical event in BITC-induced apoptosis in breast cancer cells. Exposure of MCF-7 and MDA-MB-231 cells to plasma achievable doses of BITC resulted in rapid collapse of mitochondrial filamentous network. BITC treatment also inhibited polyethyleneglycol-induced mitochondrial fusion. In contrast, a normal human mammary epithelial cell line (MCF-10A) that was derived from fibrocystic breast disease, was resistant to BITC-mediated alterations in mitochondrial dynamics as well as apoptosis. Transient or sustained decrease in levels of proteins engaged in regulation of mitochondrial fission and fusion was clearly evident after BITC treatment in both cancer cell lines. A trend for a decrease in the levels of mitochondrial fission- and fusion-related proteins was also observed in vivo in tumors of BITC-treated mice compared with control. Immortalized mouse embryonic fibroblasts from Drp1 knockout mice were resistant to BITC-induced apoptosis when compared with those from wild-type mice. Upon treatment with BITC, Bak dissociated from mitofusin 2 in both MCF-7 and MDA-MB-231 cells suggesting a crucial role for interaction of Bak and mitofusins in BITC-mediated inhibition of fusion and morphological dynamics. In conclusion, the present study provides novel insights into the molecular complexity of BITC-induced cell death.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Isothiocyanates/pharmacology , Mitochondrial Dynamics/drug effects , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Mice, Knockout
7.
EBioMedicine ; 11: 239-248, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27523807

ABSTRACT

BACKGROUND: Transfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. METHODS: We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). FINDINGS: Human and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. INTERPRETATION: The common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage.


Subject(s)
Blood Preservation , Erythrocyte Transfusion , Erythrocytes/metabolism , Hemolysis , Sickle Cell Trait/blood , Animals , Blood Preservation/adverse effects , Clodronic Acid/pharmacology , Clodronic Acid/therapeutic use , Disease Models, Animal , Erythrocytes/pathology , Erythrocytes/ultrastructure , Erythrocytes, Abnormal/ultrastructure , Female , Hemoglobin A/genetics , Hemoglobin A/metabolism , Humans , Male , Mice , Mice, Transgenic , Osmotic Fragility/genetics , Sickle Cell Trait/mortality , Sickle Cell Trait/therapy , Splenectomy
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