Alternative splice variants of the mitochondrial fission protein DNM1L/Drp1 regulate mitochondrial dynamics and tumor progression in ovarian cancer.

Aberrant mitochondrial fission/fusion dynamics have been reported in cancer cells. While post translational modifications are known regulators of the mitochondrial fission/fusion machinery, we show that alternative splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. Ovarian cancer specimens express an alternative splice transcript variant of Drp1 lacking exon 16 of the variable domain, and high expression of this splice variant relative to other transcripts is associated with poor patient outcome. Unlike the full-length variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration, and TCA cycle metabolites, and is associated with a more metastatic phenotype in vitro and in vivo. These pro-tumorigenic effects can also be inhibited by specific siRNA-mediated inhibition of 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 significance of the pathophysiological consequences of Drp1 alternative splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.

Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators.

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.

Visualizing Cardiolipin In Situ with HKCL-1M, a Highly Selective and Sensitive Fluorescent Probe.

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.

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy.

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1-/D mice). Ckmm-Cre+/- ;Ercc1-/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre+/- ;Ercc1-/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre+/- ;Ercc1-/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre+/- ;Ercc1-/fl and Ercc1-/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.

Fatty acid nitroalkene reversal of established lung fibrosis.

Tissue fibrosis occurs in response to dysregulated metabolism, pro-inflammatory signaling and tissue repair reactions. For example, lungs exposed to environmental toxins, cancer therapies, chronic inflammation and other stimuli manifest a phenotypic shift to activated myofibroblasts and progressive and often irreversible lung tissue scarring. There are no therapies that stop or reverse fibrosis. The 2 FDA-approved anti-fibrotic drugs at best only slow the progression of fibrosis in humans. The present study was designed to test whether a small molecule electrophilic nitroalkene, nitro-oleic acid (NO2-OA), could reverse established pulmonary fibrosis induced by the intratracheal administration of bleomycin in C57BL/6 mice. After 14 d of bleomycin-induced fibrosis development in vivo, lungs were removed, sectioned and precision-cut lung slices (PCLS) from control and bleomycin-treated mice were cultured ex vivo for 4 d with either vehicle or NO2-OA (5 µM). Biochemical and morphological analyses showed that over a 4 d time frame, NO2-OA significantly inhibited pro-inflammatory mediator and growth factor expression and reversed key indices of fibrosis (hydroxyproline, collagen 1A1 and 3A1, fibronectin-1). Quantitative image analysis of PCLS immunohistology reinforced these observations, revealing that NO2-OA suppressed additional hallmarks of the fibrotic response, including alveolar epithelial cell loss, myofibroblast differentiation and proliferation, collagen and α-smooth muscle actin expression. NO2-OA also accelerated collagen degradation by resident macrophages. These effects occurred in the absence of the recognized NO2-OA modulation of circulating and migrating immune cell activation. Thus, small molecule nitroalkenes may be useful agents for reversing pathogenic fibrosis of lung and other organs.

P. aeruginosa augments irradiation injury via 15-lipoxygenase-catalyzed generation of 15-HpETE-PE and induction of theft-ferroptosis.

Total body irradiation (TBI) targets sensitive bone marrow hematopoietic cells and gut epithelial cells, causing their death and inducing a state of immunodeficiency combined with intestinal dysbiosis and nonproductive immune responses. We found enhanced Pseudomonas aeruginosa (PAO1) colonization of the gut leading to host cell death and strikingly decreased survival of irradiated mice. The PAO1-driven pathogenic mechanism includes theft-ferroptosis realized via (a) curbing of the host antiferroptotic system, GSH/GPx4, and (b) employing bacterial 15-lipoxygenase to generate proferroptotic signal - 15-hydroperoxy-arachidonoyl-PE (15-HpETE-PE) - in the intestines of irradiated and PAO1-infected mice. Global redox phospholipidomics of the ileum revealed that lysophospholipids and oxidized phospholipids, particularly oxidized phosphatidylethanolamine (PEox), represented the major factors that contributed to the pathogenic changes induced by total body irradiation and infection by PAO1. A lipoxygenase inhibitor, baicalein, significantly attenuated animal lethality, PAO1 colonization, intestinal epithelial cell death, and generation of ferroptotic PEox signals. Opportunistic PAO1 mechanisms included stimulation of the antiinflammatory lipoxin A4, production and suppression of the proinflammatory hepoxilin A3, and leukotriene B4. Unearthing complex PAO1 pathogenic/virulence mechanisms, including effects on the host anti/proinflammatory responses, lipid metabolism, and ferroptotic cell death, points toward potentially new therapeutic and radiomitigative targets.

Real-time imaging of asthmatic epithelial cells identifies migratory deficiencies under type-2 conditions.

BACKGROUND: The epithelium is increasingly recognized as a pathologic contributor to asthma and its phenotypes. Although delayed wound closure by asthmatic epithelial cells is consistently observed, underlying mechanisms remain poorly understood, partly due to difficulties in studying dynamic physiologic processes involving polarized multilayered cell systems. Although type-2 immunity has been suggested to play a role, the mechanisms by which repair is diminished are unclear. OBJECTIVES: This study sought to develop and utilize primary multilayered polarized epithelial cell systems, derived from patients with asthma, to evaluate cell migration in response to wounding under type-2 and untreated conditions. METHODS: A novel wounding device for multilayered polarized cells, along with time-lapse live cell/real-time confocal imaging were evaluated under IL-13 and untreated conditions. The influence of inhibition of 15 lipoxygenase (15LO1), a type-2 enzyme, on the process was also addressed. Cell migration patterns were analyzed by high-dimensional frequency modulated Möbius for statistical comparisons. RESULTS: IL-13 stimulation negatively impacts wound healing by altering the total speed, directionality, and acceleration of individual cells. Inhibition 15LO1 partially improved the wound repair through improving total speed. CONCLUSIONS: Migration abnormalities contributed to markedly slower wound closure of IL-13 treated cells, which was modestly reversed by 15LO1 inhibition, suggesting its potential as an asthma therapeutic target. These novel methodologies offer new ways to dynamically study cell movements and identify contributing pathologic processes.

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

Metabolic Adaptation of Macrophages as Mechanism of Defense against Crystalline Silica.

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.

Yes-Associated Protein (Yap) Is Up-Regulated in Heart Failure and Promotes Cardiac Fibroblast Proliferation.

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFß and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFß/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFß/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFß/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.

RIP3-dependent necroptosis contributes to the pathogenesis of chronic obstructive pulmonary disease.

Necroptosis has emerged as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we found that markers of necroptosis, including high mobility group box 1 release and phosphorylation of mixed lineage kinase domain-like protein (p-MLKL), were markedly induced in the late stage of cigarette smoking-induced (CS-induced) emphysema in mouse lung tissue as well as in lung epithelial cells and organoids with higher dosage of or more prolonged exposure to cigarette smoking extract (CSE). Apoptotic signals were also detected and maximally induced in the early stage of CS-exposed mice and CSE-treated epithelial cells. Inhibition of apoptosis by Z-VAD, a pan-caspase inhibitor, switched the cellular stress to enhanced necroptosis in lung epithelial cells and organoids treated with CSE. Depletion or inhibition of receptor-interacting protein kinase 3 (RIP3) or MLKL attenuated the CSE-induced cell death, suggesting that necroptosis contributes to CSE-induced cell death. Silencing or inhibition of RIP1 had no protective effect, indicating a RIP1-independent RIP3 activation pathway. CSE-induced necroptosis released more damage-associated molecular patterns and evoked greater engulfment but slower clearance by bone marrow-derived macrophages, leading to enhanced expression of proinflammatory cytokines Tnfα and Il6. Finally, our in vivo data verified that inhibition of necroptosis by RIP3 inhibitor GSK'872 protected mice from CS-induced emphysema and suppressed the lung inflammation. In conclusion, we provide evidence that necroptosis contributes to the pathogenesis of COPD. Targeting RIP3 and its downstream pathway may be an effective therapy for COPD.

Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.

Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.

Apoptosis of hematopoietic progenitor-derived adipose tissue-resident macrophages contributes to insulin resistance after myocardial infarction.

Patients with insulin resistance have high risk of cardiovascular disease such as myocardial infarction (MI). However, it is not known whether MI can initiate or aggravate insulin resistance. We observed that patients with ST-elevation MI and mice with MI had de novo hyperglycemia and features of insulin resistance, respectively. In mouse models of both myocardial and skeletal muscle injury, we observed that the number of visceral adipose tissue (VAT)-resident macrophages decreased because of apoptosis after these distant organ injuries. Patients displayed a similar decrease in VAT-resident macrophage numbers and developed systemic insulin resistance after ST-elevation MI. Loss of VAT-resident macrophages after MI injury led to systemic insulin resistance in non-diabetic mice. Danger signaling-associated protein high mobility group box 1 was released by the dead myocardium after MI in rodents and triggered macrophage apoptosis via Toll-like receptor 4. The VAT-resident macrophage population in the steady state in mice was transcriptomically distinct from macrophages in the brain, skin, kidney, bone marrow, lungs, and liver and was derived from hematopoietic progenitor cells just after birth. Mechanistically, VAT-resident macrophage apoptosis and de novo insulin resistance in mouse models of MI were linked to diminished concentrations of macrophage colony-stimulating factor and adiponectin. Collectively, these findings demonstrate a previously unappreciated role of adipose tissue-resident macrophages in sensing remote organ injury and promoting MI pathogenesis.

PEBP1 acts as a rheostat between prosurvival autophagy and ferroptotic death in asthmatic epithelial cells.

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.

Toll interacting protein protects bronchial epithelial cells from bleomycin-induced apoptosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by altered epithelial cell phenotypes, which are associated with myofibroblast accumulation in the lung. Atypical alveolar epithelial cells in IPF express molecular markers of airway epithelium. Polymorphisms within and around Toll interacting protein (TOLLIP) are associated with the susceptibility to IPF and mortality. However, the functional role of TOLLIP in IPF is unknown. Using lung tissues from IPF and control subjects, we showed that expression of TOLLIP gene in the lung parenchyma is globally lower in IPF compared to controls. Lung cells expressing significant levels of TOLLIP include macrophages, alveolar type II, and basal cells. TOLLIP protein expression is lower in the parenchyma of IPF lungs but is expressed in the atypical epithelial cells of the distal fibrotic regions. Using overexpression and silencing approaches, we demonstrate that TOLLIP protects cells from bleomycin-induced apoptosis using primary bronchial epithelial cells and BEAS-2B cells. The protective effects are mediated by reducing mitochondrial reactive oxygen species (ROS) levels and upregulating autophagy. Therefore, global downregulation of the TOLLIP gene in IPF lungs may predispose injured lung epithelial cells to apoptosis and to the development of IPF.

Structural and functional divergence of GDAP1 from the glutathione S-transferase superfamily.

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.

Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity.

Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.

Most life processes require the energy produced by small cellular compartments called mitochondria. Many internal and external factors can harm these miniature powerhouses, potentially leading to cell death. For instance, in patients with Parkinson's or Alzheimer's disease, dying neurons often show mitochondrial damage. However, it is unclear exactly how injured mitochondria trigger the demise of these cells. Gaining a better understanding of this process requires studying the impact of mitochondrial damage in live neurons, something that is still difficult to do. As a response to this challenge, Xie, Jiao, Bai, Ilin et al. designed a new tool that can specifically injure mitochondria in the neurons of live zebrafish larvae at will, and fine-tune the amount of damage inflicted. The zebrafish are genetically engineered so that the mitochondria in their neurons carry a protein which can bind to a chemical compound called MG2I. When attached to each other, MG2I and the protein respond to far-red light by locally creating highly damaging chemicals. This means that whenever far-red light is shone onto the larvae, mitochondria in their neurons are harmed ­ the brighter the light, the stronger the damage. Zebrafish larvae exposed to these conditions immediately stopped swimming: mitochondria in their neurons could not produce enough energy and these cells could therefore no longer communicate properly. The neurons then started to die about 24 hours after exposure to the light, suggesting that the mitochondrial damage triggered other downstream processes that culminated in cell death. This new light-controlled tool could help to understand the consequences of mitochondrial damage, potentially revealing new ways to rescue impaired neurons in patients with Parkinson's or Alzheimer's disease. In the future, the method could be adapted to work in any type of cell and deactivate other cell compartments, so that it can be used to study many types of diseases.

Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.

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.

Nrf2 activation protects against lithium-induced nephrogenic diabetes insipidus.

Lithium (Li) is the mainstay pharmacotherapeutic mood stabilizer in bipolar disorder. Its efficacious use is complicated by acute and chronic renal side effects, including nephrogenic diabetes insipidus (NDI) and progression to chronic kidney disease (CKD). The nuclear factor erythroid-derived 2-related factor 2 (Nrf2) pathway senses and coordinates cellular responses to oxidative and electrophilic stress. Here, we identify that graded genetic activation of Nrf2 protects against Li-induced NDI (Li-NDI) and volume wasting via an aquaporin 2-independent mechanism. Renal Nrf2 activity is differentially expressed on functional segments of the nephron, and its activation along the distal tubule and collecting duct directly modulates ion transporter expression, mimicking paradoxical effects of diuretics in mitigating Li-NDI. In addition, Nrf2 reduces cyclooxygenase expression and vasoactive prostaglandin biosynthesis. Pharmacologic activation of Nrf2 confers protective effects, confirming this pathway as a potentially novel druggable target for the prevention of acute and chronic renal sequelae of Li therapy.