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Methods in Cell Biology 2023Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through...
Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR) mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.
Topics: Mice; Humans; Animals; Mitophagy; Mitochondria; Cell Line; DNA, Mitochondrial; Reactive Oxygen Species; Autophagy; Neoplasms
PubMed: 36710054
DOI: 10.1016/bs.mcb.2022.09.002 -
Antiviral Research Oct 2021Human cytomegalovirus (HCMV) is a near ubiquitous herpesvirus that relies on host cell metabolism for efficient replication. Although it has been shown that HCMV...
Human cytomegalovirus (HCMV) is a near ubiquitous herpesvirus that relies on host cell metabolism for efficient replication. Although it has been shown that HCMV requires functional host cell mitochondria for efficient replication, it is unknown whether mitochondrial targeted pharmacological agents can be repurposed as antivirals. Here we report that treatment with drugs targeting the electron transport chain (ETC) complexes inhibit HCMV replication. Addition of rotenone, oligomycin, antimycin and metformin resulted in decreased HCMV titers in vitro, independent of HCMV strain. This further illustrates the dependence of HCMV replication on functional mitochondria. Metformin, an FDA approved drug, delays HCMV replication kinetics resulting in a reduction of viral titers. Repurposing metformin as an antiviral is advantageous as its safety profile and epidemiological data are well accepted. Our findings provide new insight into the potential for targeting HCMV infection through host cell metabolism and how these pharmacological interventions function.
Topics: Antiviral Agents; Cells, Cultured; Cytomegalovirus; Electron Transport; Fibroblasts; Foreskin; Humans; Male; Metformin; Oligomycins; Virus Replication
PubMed: 34390771
DOI: 10.1016/j.antiviral.2021.105159 -
CNS Neuroscience & Therapeutics May 2017
Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Brain Ischemia; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Hippocampus; Humans; Inflammation; Lipopolysaccharides; Mice; Microglia; Neuroprotective Agents; Oligomycins; Oxidative Stress; Oxidopamine; Parkinsonian Disorders; Plant Preparations; Rats; Rotenone; Tissue Culture Techniques
PubMed: 28371323
DOI: 10.1111/cns.12693 -
Food Research International (Ottawa,... Jul 2021Flavonoid bioavailability and bioactivity is associated with interindividual variability, which is partially due to differences in health status. Previously, it was...
PURPOSE
Flavonoid bioavailability and bioactivity is associated with interindividual variability, which is partially due to differences in health status. Previously, it was demonstrated that cellular stress, especially mitochondrial stress, increases intracellular quercetin uptake and this is associated with beneficial health effects. Here, the impact of quercetin on mitochondrial dysfunction, induced by stressors targeting different sites of the electron transport chain, is investigated. The influence of the mitochondrial stress on quercetin uptake and subcellular location is studied and the accumulated quercetin metabolites in intestinal Caco-2 cells and mitochondria are characterized.
PRINCIPAL RESULTS
It was observed that quercetin counteracted (i) the carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-induced decrease in maximum oxygen consumption, (ii) the valinomycin-, oligomycin- and FCCP-induced reactive oxygen species production and (iii) the valinomycin-induced disruption of mitochondrial membrane potential. Using confocal microscopy, it was found that upon mitochondrial stress, the intracellular quercetin accumulation increased and was partially located in the mitochondria. Finally, it was demonstrated that quercetin was present as O-methyl, O-methylglucuronide and O-methylsulfate conjugates in the cell lysate and mitochondria-enriched fraction.
MAJOR CONCLUSIONS
This study shows that quercetin can partially restore, especially FCCP-induced, mitochondrial dysfunction and this protective effect was linked with an intracellular quercetin accumulation in the mitochondria of intestinal cells.
Topics: Caco-2 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Quercetin; Reactive Oxygen Species
PubMed: 34112387
DOI: 10.1016/j.foodres.2021.110430 -
Physiological Research Dec 2022Five-sixths nephrectomy is a widely used experimental model of chronic kidney disease (CKD) that is associated with severe mitochondrial dysfunction of the remnant...
Five-sixths nephrectomy is a widely used experimental model of chronic kidney disease (CKD) that is associated with severe mitochondrial dysfunction of the remnant tissue. In this study, we assessed the effect of CKD on mitochondrial respiration separately in the rat kidney cortex and medulla 10 weeks after induction of CKD by subtotal 5/6 nephrectomy (SNX). Mitochondrial oxygen consumption was evaluated on mechanically permeabilized samples of kidney cortex and medulla using high-resolution respirometry and expressed per mg of tissue wet weight or IU citrate synthase (CS) activity. Mitochondrial respiration in the renal cortex of SNX rats was significantly reduced in all measured respiratory states if expressed per unit wet weight and remained lower if recalculated per IU citrate synthase activity, i.e. per mitochondrial mass. In contrast, the profound decrease in the activity of CS in SNX medulla resulted in significantly elevated respiratory states expressing the OXPHOS capacity when Complexes I and II or II only are provided with electrons, LEAK respiration after oligomycin injection, and Complex IV-linked oxygen consumption per unit CS activity suggesting compensatory hypermetabolic state in remaining functional mitochondria that is not sufficient to fully compensate for respiratory deficit expressed per tissue mass. The results document that CKD induced by 5/6 nephrectomy in the rat is likely to cause not only mitochondrial respiratory dysfunction (in the kidney cortex), but also adaptive changes in the medulla that tend to at least partially compensate for mitochondria loss.
Topics: Rats; Animals; Citrate (si)-Synthase; Kidney; Kidney Cortex; Renal Insufficiency, Chronic; Mitochondria
PubMed: 36647910
DOI: 10.33549/physiolres.935000 -
Molecules (Basel, Switzerland) Feb 2021Herein, we report the neuroprotective and antioxidant activity of 1,1'-biphenyl nitrones () - as α-phenyl---butylnitrone analogues prepared from commercially available...
Herein, we report the neuroprotective and antioxidant activity of 1,1'-biphenyl nitrones () - as α-phenyl---butylnitrone analogues prepared from commercially available [1,1'-biphenyl]-4-carbaldehyde and [1,1'-biphenyl]-4,4'-dicarbaldehyde. The neuroprotection of - has been measured against oligomycin A/rotenone and in an oxygen-glucose deprivation in vitro ischemia model in human neuroblastoma SH-SY5Y cells. Our results indicate that - have better neuroprotective and antioxidant properties than α-phenyl---butylnitrone (), and they are quite similar to -acetyl-L-cysteine (), which is a well-known antioxidant agent. Among the nitrones studied, homo-bis-nitrone , bearing two --Bu radicals at the nitrone motif, has the best neuroprotective capacity (EC = 13.16 ± 1.65 and 25.5 ± 3.93 μM, against the reduction in metabolic activity induced by respiratory chain blockers and oxygen-glucose deprivation in an in vitro ischemia model, respectively) as well as anti-necrotic, anti-apoptotic, and antioxidant activities (EC = 11.2 ± 3.94 μM), which were measured by its capacity to reduce superoxide production in human neuroblastoma SH-SY5Y cell cultures, followed by mononitrone , with one -Bn radical, and , with only one --Bu substituent. The antioxidant activity of - has also been analyzed for their capacity to scavenge hydroxyl free radicals (82% at 100 μM), lipoxygenase inhibition, and the inhibition of lipid peroxidation (68% at 100 μM). Results showed that although the number of nitrone groups improves the neuroprotection profile of these , the final effect is also dependent on the substitutent that is being incorporated. Thus, bearing --Bu and -Bn groups show better neuroprotective and antioxidant properties than those substituted with Me. All these results led us to propose homo--nitrone as the most balanced and interesting nitrone based on its neuroprotective capacity in different neuronal models of oxidative stress and in vitro ischemia as well as its antioxidant activity.
Topics: Antioxidants; Cyclic N-Oxides; Humans; Hydroxyl Radical; Lipid Peroxidation; Lipoxygenase; Lipoxygenase Inhibitors; Molecular Structure; Neuroprotective Agents; Tumor Cells, Cultured
PubMed: 33672652
DOI: 10.3390/molecules26041127 -
Biochimica Et Biophysica Acta.... Feb 2024Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear....
Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear. We have used N2a mouse neuroblastoma cells and primary cultures of mouse neurons and 1-methyl-4-phenylpyridinium (MPP), a known mitochondrial complex I inhibitor and the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), known to trigger a cascade of events associated with PD neuropathological features. Simultaneously, we utilized other mitochondrial toxins, including antimycin A, oligomycin, and carbonyl cyanide chlorophenylhydrazone. MPP treatment resulted in elevated levels of total cholesterol and in a Niemann Pick type C1 (NPC1)-like phenotype characterized by accumulation of cholesterol in lysosomes. Interestingly, NPC1 mRNA levels were specifically reduced by MPP. The decrease in NPC1 levels was also seen in midbrain and striatum from MPTP-treated mice and in primary cultures of neurons treated with MPP. Together with the MPP-dependent increase in intracellular cholesterol levels in N2a cells, we observed an increase in 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and a concomitant increase in the phosphorylated levels of mammalian target of rapamycin (mTOR). NPC1 knockout delayed cell death induced by acute mitochondrial damage, suggesting that transient cholesterol accumulation in lysosomes could be a protective mechanism against MPTP/MPP insult. Interestingly, we observed a negative correlation between NPC1 protein levels and disease stage, in human PD brain samples. In summary, MPP decreases NPC1 levels, elevates lysosomal cholesterol accumulation and alters mTOR signaling, adding to the existing notion that PD may rise from alterations in mitochondrial-lysosomal communication.
Topics: Animals; Humans; Mice; Cholesterol; Mammals; Mechanistic Target of Rapamycin Complex 1; Niemann-Pick C1 Protein; Parkinson Disease; Phenotype; TOR Serine-Threonine Kinases
PubMed: 38061599
DOI: 10.1016/j.bbadis.2023.166980 -
PLoS Pathogens Mar 2020Biofilm-associated prosthetic joint infections (PJIs) cause significant morbidity due to their recalcitrance to immune-mediated clearance and antibiotics, with...
Biofilm-associated prosthetic joint infections (PJIs) cause significant morbidity due to their recalcitrance to immune-mediated clearance and antibiotics, with Staphylococcus aureus (S. aureus) among the most prevalent pathogens. We previously demonstrated that S. aureus biofilm-associated monocytes are polarized to an anti-inflammatory phenotype and the adoptive transfer of pro-inflammatory macrophages attenuated biofilm burden, highlighting the critical role of monocyte/macrophage inflammatory status in dictating biofilm persistence. The inflammatory properties of leukocytes are linked to their metabolic state, and here we demonstrate that biofilm-associated monocytes exhibit a metabolic bias favoring oxidative phosphorylation (OxPhos) and less aerobic glycolysis to facilitate their anti-inflammatory activity and biofilm persistence. To shift monocyte metabolism in vivo and reprogram cells to a pro-inflammatory state, a nanoparticle approach was utilized to deliver the OxPhos inhibitor oligomycin to monocytes. Using a mouse model of S. aureus PJI, oligomycin nanoparticles were preferentially internalized by monocytes, which significantly reduced S. aureus biofilm burden by altering metabolism and promoting the pro-inflammatory properties of infiltrating monocytes as revealed by metabolomics and RT-qPCR, respectively. Injection of oligomycin alone had no effect on monocyte metabolism or biofilm burden, establishing that intracellular delivery of oligomycin is required to reprogram monocyte metabolic activity and that oligomycin lacks antibacterial activity against S. aureus biofilms. Remarkably, monocyte metabolic reprogramming with oligomycin nanoparticles was effective at clearing established biofilms in combination with systemic antibiotics. These findings suggest that metabolic reprogramming of biofilm-associated monocytes may represent a novel therapeutic approach for PJI.
Topics: Animals; Biofilms; Cellular Reprogramming; Implants, Experimental; Inflammation; Mice; Monocytes; Oligomycins; Oxidative Phosphorylation; Staphylococcal Infections; Staphylococcus aureus
PubMed: 32142554
DOI: 10.1371/journal.ppat.1008354 -
Journal of Fungi (Basel, Switzerland) Jun 2023Concern about the global emergence of multidrug-resistant fungal pathogens led us to explore the use of combination therapy to combat azole resistance in . Clorgyline...
Concern about the global emergence of multidrug-resistant fungal pathogens led us to explore the use of combination therapy to combat azole resistance in . Clorgyline had previously been shown to be a multi-target inhibitor of Cdr1 and Mdr1 efflux pumps of and . A screen for antifungal sensitizers among synthetic analogs of Clorgyline detected interactions with the efflux pump azole substrates Posaconazole and Voriconazole. Of six Clorgyline analogs, M19 and M25 were identified as potential sensitizers of azole resistance. M19 and M25 were found to act synergistically with azoles against resistant clade I isolates and recombinant strains overexpressing efflux pumps. Nile Red assays with the recombinant strains showed M19 and M25 inhibited the activity of Cdr1 and Mdr1 efflux pumps that are known to play key roles in azole resistance in clades I, III, and IV. While Clorgyline, M19 and M25 uncoupled the Oligomycin-sensitive ATPase activity of Cdr1 from and , their mode of action is yet to be fully elucidated. The experimental combinations described herein provides a starting point to combat azole resistance dominated by overexpression of CauCdr1 in clades I and IV and CauMdr1 in clade III.
PubMed: 37367600
DOI: 10.3390/jof9060663 -
Free Radical Biology & Medicine Nov 2021Whether from known or unknown causes, loss of epithelial repair plays a central role in the pathogenesis of pulmonary fibrosis. Recently, diminished mitochondrial...
Whether from known or unknown causes, loss of epithelial repair plays a central role in the pathogenesis of pulmonary fibrosis. Recently, diminished mitochondrial function has been implicated as a factor contributing to the loss of epithelial repair but the mechanisms mediating these changes have not been defined. Here, we investigated the factors contributing to mitochondrial respiratory dysfunction after bleomycin, a widely accepted agent for modeling pulmonary fibrosis in mice and in vitro systems. In agreement with previous reports, we found that mitochondrial respiration was decreased in lung epithelial cells exposed to bleomycin, but also observed that responses differed depending on the type of metabolic fuel available to cells. For example, we found that mitochondrial respiration was dramatically reduced when glucose served as the primary fuel. Moreover, this associated with a marked decrease in glucose uptake, expression of glucose uptake transport 1 and capacity to augment glycolysis to either glucose or oligomycin. Conversely, mitochondrial respiration was largely preserved if glutamine was present in culture medium. The addition of glutamine also led to increased intracellular metabolite levels, including multiple TCA cycle intermediates and the glycolytic intermediate lactate, as well as reduced DNA damage and cell death to bleomycin. Taken together, these findings indicate that glutamine, rather than glucose, supports mitochondrial respiration and metabolite production in injured lung epithelial cells, and suggest that this shift away from glucose utilization serves to protect the lung epithelium from injury.
Topics: Animals; Bleomycin; Epithelial Cells; Glucose; Glutamine; Glycolysis; Mice; Mitochondria; Respiration
PubMed: 34634441
DOI: 10.1016/j.freeradbiomed.2021.10.006