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Autophagy Apr 2021Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal...
Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions . Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells. AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.
Topics: Autophagy; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Dopaminergic Neurons; Gene Expression Regulation; Growth Cones; Humans; Induced Pluripotent Stem Cells; Mesencephalon; Mitochondria; Mitophagy; Oxygen Consumption; Pyridines; Pyrimidines; Time Factors
PubMed: 32286126
DOI: 10.1080/15548627.2020.1739441 -
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 -
Molecules (Basel, Switzerland) Aug 2023Polyphenols have attracted attention in the fight against antibiotic-resistant bacteria, as they show antibacterial action. Considering that polyphenols inhibit FF-ATP...
Cirsiliol and Quercetin Inhibit ATP Synthesis and Decrease the Energy Balance in Methicillin-Resistant (MRSA) and Methicillin-Resistant (MRSE) Strains Isolated from Patients.
Polyphenols have attracted attention in the fight against antibiotic-resistant bacteria, as they show antibacterial action. Considering that polyphenols inhibit FF-ATP synthase (ATP synthase) and that bacteria need a constant energy production to maintain their homeostasis, we evaluated the effect of two flavones, cirsiliol (tri-hy-droxy-6,7-dimethoxyflavone) and quercetin (3,3,4,5,7-pentahydroxyflavone), on energy production and intracellular ATP content in a methicillin-resistant (MRSA) strain and a methicillin-resistant (MRSE) strain isolated from patients, comparing the results to those obtained by treating the bacteria with oligomycin, a specific ATP synthase F moiety inhibitor. Real-time quantitative ATP synthesis and total ATP content of permeabilized Gram-positive bacteria were assayed by luminometry. The results showed that cirsiliol and quercetin inhibited ATP synthase and decreased the intracellular ATP levels in both strains, although the effect was higher in MRSE. In addition, while cirsiliol and quercetin acted immediately after the treatment, oligomycin inhibited ATP synthesis only after 30 min of incubation, suggesting that the different responses may depend on the different permeability of the bacterial wall to the three molecules. Thus, cirsiliol and quercetin could be considered potential additions to antibiotics due to their ability to target ATP synthase, against which bacteria cannot develop resistance.
Topics: Humans; Methicillin-Resistant Staphylococcus aureus; Quercetin; Staphylococcus epidermidis; Methicillin Resistance; Flavones; Polyphenols; Adenosine Triphosphate; Anti-Bacterial Agents
PubMed: 37687012
DOI: 10.3390/molecules28176183 -
Toxics Dec 2022Industrial and consumer products, such as pesticides, lubricants, and cosmetics, can contain perfluorinated compounds (PFCs). Although many short-chain PFCs have been...
Industrial and consumer products, such as pesticides, lubricants, and cosmetics, can contain perfluorinated compounds (PFCs). Although many short-chain PFCs have been linked to physiological and behavioral changes in fish, there are limited data on longer-chain PFCs. The objective of this study was to determine the potential impact of perfluorotetradecanoic acid (PFTeDA) exposure on zebrafish () during early developmental stages. We measured several endpoints including gene expression, mitochondrial bioenergetics, and locomotor activity in zebrafish. Survival, timing of hatching, and deformity frequency were unaffected by PFTeDA at the concentrations tested (0.01, 0.1, 1, and 10 µM) over a 7-day exposure period. The expression levels of mitochondrial-related genes ( and ) and oxidative stress-related genes ( and ) were increased in larval fish with exposure to 10 µM PFTeDA; however, there was no change in oxidative respiration of embryos (i.e., basal respiration and oligomycin-induced ATP-linked respiration). Reactive oxygen species were reduced in larvae treated with 10 µM PFTeDA, coinciding with the increased transcription of antioxidant defense genes. Both the visual motor response test and light-dark preference test were conducted on 7 dpf larvae and yielded no significant findings. This study improves current knowledge regarding toxicity mechanisms for longer-chain PFCs such as PFTeDA.
PubMed: 36548609
DOI: 10.3390/toxics10120776 -
Experimental Neurology May 2023Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane that metabolizes amine substrates like serotonin, norepinephrine and dopamine. MAO...
Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane that metabolizes amine substrates like serotonin, norepinephrine and dopamine. MAO inhibitors (MAOIs) are frequently utilized to treat disorders such as major depression or Parkinson's disease (PD), though their effects on brain mitochondrial bioenergetics are unclear. These studies measured bioenergetic activity in mitochondria isolated from the mouse cortex in the presence of inhibitors of either MAO-A, MAO-B, or both isoforms. We found that only 10 μM clorgyline, the selective inhibitor of MAO-A and not MAO-B, increased mitochondrial oxygen consumption rate in State V(CI) respiration compared to vehicle treatment. We then assessed mitochondrial bioenergetics, reactive oxygen species (ROS) production, and Electron Transport Chain (ETC) complex function in the presence of 0, 5, 10, 20, 40, or 80 μM of clorgyline to determine if this change was dose-dependent. The results showed increased oxygen consumption rates across the majority of respiration states in mitochondria treated with 5, 10, or 20 μM with significant bioenergetic inhibition at 80 μM clorgyline. Next, we assessed mitochondrial ROS production in the presence of the same concentrations of clorgyline in two different states: high mitochondrial membrane potential (ΔΨ) induced by oligomycin and low ΔΨ induced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). There were no changes in ROS production in the presence of 5, 10, 20, or 40 μM clorgyline compared to vehicle after the addition of oligomycin or FCCP. There was a significant increase in mitochondrial ROS in the presence of 80 μM clorgyline after FCCP addition, as well as reduced Complex I and Complex II activities, which are consistent with inhibition of bioenergetics seen at this dose. There were no changes in Complex I, II, or IV activities in mitochondria treated with low doses of clorgyline. These studies shed light on the direct effect of MAO-A inhibition on brain mitochondrial bioenergetic function, which may be a beneficial outcome for those taking these medications.
Topics: Mice; Animals; Monoamine Oxidase; Clorgyline; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Reactive Oxygen Species; Monoamine Oxidase Inhibitors; Mitochondria; Respiration
PubMed: 36841465
DOI: 10.1016/j.expneurol.2023.114356 -
British Journal of Pharmacology Nov 2019The permeability transition pore (PTP) is a latent, high-conductance channel of the inner mitochondrial membrane. When activated, it plays a key role in cell death and... (Review)
Review
The permeability transition pore (PTP) is a latent, high-conductance channel of the inner mitochondrial membrane. When activated, it plays a key role in cell death and therefore in several diseases. The investigation of the PTP took an unexpected turn after the discovery that cyclophilin D (the target of the PTP inhibitory effect of cyclosporin A) binds to F F (F)-ATP synthase, thus inhibiting its catalytic activity by about 30%. This observation was followed by the demonstration that binding occurs at a particular subunit of the enzyme, the oligomycin sensitivity conferral protein (OSCP), and that F-ATP synthase can form Ca -activated, high-conductance channels with features matching those of the PTP, suggesting that the latter originates from a conformational change in F-ATP synthase. This review is specifically focused on the OSCP subunit of F-ATP synthase, whose unique features make it a potential pharmacological target both for modulation of F-ATP synthase and its transition to a pore. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.
Topics: Animals; Humans; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proton-Translocating ATPases; Protein Subunits
PubMed: 30291799
DOI: 10.1111/bph.14513 -
Neural Regeneration Research Jan 2024Mitochondrial dysfunction is a significant pathological alteration that occurs in Parkinson's disease (PD), and the Thr61Ile (T61I) mutation in coiled-coil helix...
Mitochondrial dysfunction is a significant pathological alteration that occurs in Parkinson's disease (PD), and the Thr61Ile (T61I) mutation in coiled-coil helix coiled-coil helix domain containing 2 (CHCHD2), a crucial mitochondrial protein, has been reported to cause Parkinson's disease. F1F0-ATPase participates in the synthesis of cellular adenosine triphosphate (ATP) and plays a central role in mitochondrial energy metabolism. However, the specific roles of wild-type (WT) CHCHD2 and T61I-mutant CHCHD2 in regulating F1F0-ATPase activity in Parkinson's disease, as well as whether CHCHD2 or CHCHD2 T61I affects mitochondrial function through regulating F1F0-ATPase activity, remain unclear. Therefore, in this study, we expressed WT CHCHD2 and T61I-mutant CHCHD2 in an MPP-induced SH-SY5Y cell model of PD. We found that CHCHD2 protected mitochondria from developing MPP-induced dysfunction. Under normal conditions, overexpression of WT CHCHD2 promoted F1F0-ATPase assembly, while T61I-mutant CHCHD2 appeared to have lost the ability to regulate F1F0-ATPase assembly. In addition, mass spectrometry and immunoprecipitation showed that there was an interaction between CHCHD2 and F1F0-ATPase. Three weeks after transfection with AAV-CHCHD2 T61I, we intraperitoneally injected 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into mice to establish an animal model of chronic Parkinson's disease and found that exogenous expression of the mutant protein worsened the behavioral deficits and dopaminergic neurodegeneration seen in this model. These findings suggest that WT CHCHD2 can alleviate mitochondrial dysfunction in PD by maintaining F1F0-ATPase structure and function.
PubMed: 37488867
DOI: 10.4103/1673-5374.378010 -
Journal of Animal Science Aug 2022Beta-adrenergic agonists (β-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known...
Beta-adrenergic agonists (β-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which β-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCRs) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no β-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by β-AA treatment in ovine or porcine cells. These findings indicate that β-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to β-AA incubation also demonstrates differing physiological responses to β-AA across species, which helps to explain the variation in its effectiveness as a growth supplement.
Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Animals; Cattle; Female; Myoblasts; Oxidative Phosphorylation; Phenethylamines; Protons; Sheep; Sheep, Domestic; Swine
PubMed: 35908785
DOI: 10.1093/jas/skac208 -
Endocrinology Dec 2021Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of...
CONTEXT
Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs).
OBJECTIVE
This work sought a role for mitochondria in OAT adipogenesis in GO.
METHODS
Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white adipose tissue in culture medium compared with culture medium containing a mixed hormonal cocktail as adipogenic medium (ADM), or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial biosubstrate adenosine 5'-diphosphate/guanosine 5'-diphosphate (ADP/GDP), AICAR (adenosine analogue), or inhibitor oligomycin-A for 17 days. Main outcome measures included oil-red-O staining and foci count of differentiated adipocytes for in vitro adipogenesis, flow cytometry, relative quantitative polymerase chain reaction, MTS-assay/106 cells, total cellular-ATP detection kit, and Seahorse-XFe96-Analyzer for mitochondria and oxidative-phosphorylation (OXPHOS)/glycolysis-ATP production analysis.
RESULTS
During early adipogenesis before adipocyte formation (days 0, 4, and7), we observed OAT-specific cellular ATP production via mitochondrial OXPHOS in PFs both from OAT-H and OAT-GO, and substantially disrupted OXPHOS-ATP/glycolysis-ATP production in PFs from OAT-GO, for example, a 40% reduction in OXPHOS-ATP and trend-increased glycolysis-ATP production on days 4 and 7 compared with day 0, which contrasted with the stable levels in OAT-H. FA supplementation in culture-medium triggered adipogenesis in PFs both from OAT-H and OAT-GO, which was substantially enhanced by 1-mM GDP reaching 7% to 18% of ADM adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed a significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/glycolysis-ATP through adipogenesis of PFs from OAT-GO.
CONCLUSION
Our study confirmed that FA uptake can drive OAT adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.
Topics: Adipocytes; Adipogenesis; Adipose Tissue; Cell Differentiation; Cells, Cultured; Fatty Acids; Fibroblasts; Graves Ophthalmopathy; Humans; Lipid Metabolism; Mitochondria; Orbit; Oxidative Phosphorylation
PubMed: 34473251
DOI: 10.1210/endocr/bqab188