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Current Neuropharmacology 2023With the advancement in novel drug discovery, biologically active compounds are considered pharmacological tools to understand complex biological mechanisms and the...
With the advancement in novel drug discovery, biologically active compounds are considered pharmacological tools to understand complex biological mechanisms and the identification of potent therapeutic agents. Mitochondria boast a central role in different integral biological processes and mitochondrial dysfunction is associated with multiple pathologies. It is, therefore, prudent to target mitochondrial quality control mechanisms by using pharmacological approaches. However, there is a scarcity of biologically active molecules, which can interact with mitochondria directly. Currently, the chemical compounds used to induce mitophagy include oligomycin and antimycin A for impaired respiration and acute dissipation of mitochondrial membrane potential by using CCCP/FCCP, the mitochondrial uncouplers. These chemical probes alter the homeostasis of the mitochondria and limit our understanding of the energy regulatory mechanisms. Efforts are underway to find molecules that can bring about selective removal of defective mitochondria without compromising normal mitochondrial respiration. In this report, we have tried to summarize and status of the recently reported modulators of mitophagy.
Topics: Humans; Mitophagy; Mitochondria; Membrane Potential, Mitochondrial; Antimycin A
PubMed: 36918785
DOI: 10.2174/1570159X21666230314140528 -
Life Science Alliance Apr 2023Mitochondrial depolarization can initiate reversal activity of ATP synthase, depleting ATP by its hydrolysis. We have recently shown that increased ATP hydrolysis...
Mitochondrial depolarization can initiate reversal activity of ATP synthase, depleting ATP by its hydrolysis. We have recently shown that increased ATP hydrolysis contributes to ATP depletion leading to a maladaptation in mitochondrial disorders, where maximal hydrolytic capacity per CV content is increasing. However, despite its importance, ATP hydrolysis is not a commonly studied parameter because of the limitations of the currently available methods. Methods that measure CV hydrolytic activity indirectly require the isolation of mitochondria and involve the introduction of detergents, preventing their utilization in clinical studies or any high-throughput analyses. Here, we describe a novel approach to assess maximal ATP hydrolytic capacity and maximal respiratory capacity in a single assay in cell lysates, PBMCs, and tissue homogenates that were previously frozen. The methodology described here has the potential to be used in clinical samples to determine adaptive and maladaptive adjustments of CV function in diseases, with the added benefit of being able to use frozen samples in a high-throughput manner and to explore ATP hydrolysis as a drug target for disease treatment.
Topics: Hydrolysis; Adenosine Triphosphate; Mitochondrial Proton-Translocating ATPases; Mitochondria
PubMed: 36918278
DOI: 10.26508/lsa.202201628 -
Cells Feb 2023Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic...
Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.
Topics: Humans; Cytosol; HEK293 Cells; Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Oligomycins; Stress, Physiological
PubMed: 36899830
DOI: 10.3390/cells12050695 -
Cells Feb 2023Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism...
Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin. Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.
Topics: Mice; Animals; Oxidative Phosphorylation; Electron Transport Complex I; Astrocytes; Mitochondria; Oligomycins
PubMed: 36899819
DOI: 10.3390/cells12050683 -
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 -
Journal of Fungi (Basel, Switzerland) Jan 2023infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in are...
infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes and . We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in . Constitutive functional overexpression of wild-type Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in . Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux.
PubMed: 36836283
DOI: 10.3390/jof9020168 -
Journal of Neurophysiology Mar 2023Many studies of Ca effects on mitochondrial respiration in intact cells have used electrical and/or chemical stimulation to elevate intracellular [Ca], and have reported...
Many studies of Ca effects on mitochondrial respiration in intact cells have used electrical and/or chemical stimulation to elevate intracellular [Ca], and have reported increases in [NADH] and increased ADP/ATP ratios as dominant controllers of respiration. This study tested a different form of stimulation: brief temperature increases produced by pulses of infrared light (IR, 1,863 nm, 8-10°C for ∼5 s). Fluorescence imaging techniques applied to single PC-12 cells in low µM extracellular [Ca] revealed IR stimulation-induced increases in both cytosolic (fluo5F) and mitochondrial (rhod2) [Ca]. IR stimulation increased O consumption (porphyrin fluorescence), and produced an alkaline shift in mitochondrial matrix pH (Snarf1), indicating activation of the electron transport chain (ETC). The increase in O consumption persisted in oligomycin, and began during a decrease in NADH, suggesting that the initial increase in ETC activity was not driven by increased ATP synthase activity or an increased fuel supply to ETC complex I. Imaging with two potentiometric dyes [tetramethyl rhodamine methyl ester (TMRM) and R123] indicated a depolarizing shift in ΔΨ that persisted in high [K] medium. High-resolution fluorescence imaging disclosed large, reversible mitochondrial depolarizations that were inhibited by cyclosporin A (CSA), consistent with the opening of transient mitochondrial permeability transition pores. IR stimulation also produced a Ca-dependent increase in superoxide production (MitoSox) that was not inhibited by CSA, indicating that the increase in superoxide did not require transition pore opening. Thus, the intracellular Ca release that follows pulses of infrared light offers new insights into Ca-dependent processes controlling respiration and reactive oxygen species in intact cells. Pulses of infrared light (IR) provide a novel method for rapidly transferring Ca from the endoplasmic reticulum to mitochondria in intact cells. In PC12 cells the resulting ETC activation was not driven by increased ATP synthase activity or NADH. IR stimulation produced a Ca-dependent, reversible depolarization of ΔΨ that was partially blocked by cyclosporin A, and a Ca-dependent increase in superoxide that did not require transition pore opening.
Topics: Rats; Animals; Mitochondrial Membrane Transport Proteins; Cyclosporine; Superoxides; NAD; Mitochondria; Adenosine Triphosphate; Calcium
PubMed: 36752512
DOI: 10.1152/jn.00293.2022 -
Cellular and Molecular Gastroenterology... 2023Many studies have revealed crucial roles of the gut microbiota and its metabolites in liver disease progression. However, the mechanism underlying their effects on liver...
BACKGROUND & AIMS
Many studies have revealed crucial roles of the gut microbiota and its metabolites in liver disease progression. However, the mechanism underlying their effects on liver ischemia/reperfusion (I/R) injury remain largely unknown. Here, we investigate the function of gut microbiota and its metabolites in liver I/R injury.
METHODS
C57BL/6 mice was pretreated with an antibiotic cocktail. Then, we used multi-omics detection methods including 16s rRNA sequencing, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) to explore the changes of gut microbiota and metabolites in both feces and portal blood to reveal the mechanism of their protective effect in liver I/R injury.
RESULTS
We found that antibiotic pretreatment (ABX) could significantly reduce the severity of I/R-induced hepatic injury, and this effect could be transferred to germ-free mice by fecal microbiota transplantation (FMT), suggesting a protective role of the gut microbiota depletion. During I/R, the rates of serum α-ketoglutarate (αKG) production and glutamate reduction, downstream products of gut microbiota-derived glutamine, were more significant in the ABX mice. Then, we showed that αKG could promote alternative (M2) macrophage activation through oxidative phosphorylation, and oligomycin A could inhibit M2 macrophage polarization and reversed this protective effect.
CONCLUSIONS
These findings show that the gut microbiota and its metabolites play critical roles in hepatic I/R injury by modulating macrophage metabolic reprogramming. Potential therapies that target macrophage metabolism, including antibiotic therapies and novel immunometabolism modulators, can be exploited for the treatment of liver I/R injury.
Topics: Mice; Animals; Glutamine; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Chromatography, Liquid; Tandem Mass Spectrometry; Mice, Inbred C57BL; Liver; Macrophages; Reperfusion Injury; Anti-Bacterial Agents; Ischemia
PubMed: 36706918
DOI: 10.1016/j.jcmgh.2023.01.004 -
Cell Death & Disease Jan 2023The mitochondrial protein IF1 binds to the catalytic domain of the ATP synthase and inhibits ATP hydrolysis in ischemic tissues. Moreover, IF1 is overexpressed in many...
The mitochondrial protein IF1 binds to the catalytic domain of the ATP synthase and inhibits ATP hydrolysis in ischemic tissues. Moreover, IF1 is overexpressed in many tumors and has been shown to act as a pro-oncogenic protein, although its mechanism of action is still debated. Here, we show that ATP5IF1 gene disruption in HeLa cells decreases colony formation in soft agar and tumor mass development in xenografts, underlining the role of IF1 in cancer. Notably, the lack of IF1 does not affect proliferation or oligomycin-sensitive mitochondrial respiration, but it sensitizes the cells to the opening of the permeability transition pore (PTP). Immunoprecipitation and proximity ligation analysis show that IF1 binds to the ATP synthase OSCP subunit in HeLa cells under oxidative phosphorylation conditions. The IF1-OSCP interaction is confirmed by NMR spectroscopy analysis of the recombinant soluble proteins. Overall, our results suggest that the IF1-OSCP interaction protects cancer cells from PTP-dependent apoptosis under normoxic conditions.
Topics: Humans; HeLa Cells; Mitochondrial Proton-Translocating ATPases; Mitochondria; Nitric Oxide Synthase; Apoptosis; Adenosine Triphosphate; Neoplasms
PubMed: 36690622
DOI: 10.1038/s41419-023-05572-y -
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