Relationship of Cytotoxic and Antimicrobial Effects of Triphenylphosphonium Conjugates with Various Quinone Derivatives.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

Role of Mitochondria in the Regulation of Effector Functions of Granulocytes.

Granulocytes (neutrophils, eosinophils, and basophils) are the most abundant circulating cells in the innate immune system. Circulating granulocytes, primarily neutrophils, can cross the endothelial barrier and activate various effector mechanisms to combat invasive pathogens. Eosinophils and basophils also play an important role in allergic reactions and antiparasitic defense. Granulocytes also regulate the immune response, wound healing, and tissue repair by releasing of various cytokines and lipid mediators. The effector mechanisms of granulocytes include the production of reactive oxygen species (ROS), degranulation, phagocytosis, and the formation of DNA-containing extracellular traps. Although all granulocytes are primarily glycolytic and have only a small number of mitochondria, a growing body of evidence suggests that mitochondria are involved in all effector functions as well as in the production of cytokines and lipid mediators and in apoptosis. It has been shown that the production of mitochondrial ROS controls signaling pathways that mediate the activation of granulocytes by various stimuli. In this review, we will briefly discuss the data on the role of mitochondria in the regulation of effector and other functions of granulocytes.

Current perspectives of mitochondria-targeted antioxidants in cancer prevention and treatment.

Oxidative stress nearly always accompanies all stages of cancer development. At the early stages, antioxidants may help to reduce reactive oxygen species (ROS) production and exhibit anticarcinogenic effects. In the later stages, ROS involvement becomes more complex. On the one hand, ROS are necessary for cancer progression and epithelial-mesenchymal transition. On the other hand, antioxidants may promote cancer cell survival and may increase metastatic frequency. The role of mitochondrial ROS in cancer development remains largely unknown. This paper reviews experimental data on the effects of both endogenous and exogenous antioxidants on cancerogenesis focusing on the development and application of mitochondria-targeted antioxidants. We also discuss the prospects for antioxidant cancer therapy, focusing on the use of mitochondria-targeted antioxidants.

The Distinct Effects of the Mitochondria-Targeted STAT3 Inhibitors Mitocur-1 and Mitocur-3 on Mast Cell and Mitochondrial Functions.

There is accumulating evidence that mitochondria and mitochondrial STAT3 are involved in the activation of mast cells. The mitochondria-targeted curcuminoids Mitocur-1 and Mitocur-3 have been suggested to reduce antigen-dependent mast cell activation by inhibiting mitochondrial STAT3. The aim of the current work was to investigate the mechanisms of action of these mitocurcuminoids on mast cells and mitochondrial functions. The pretreatment of rat basophilic leukemia cells RBL-2H3 with Mitocur-1 and Mitocur-3 decreased antigen-dependent degranulation but did not affect spontaneous degranulation. Both compounds caused mitochondrial fragmentation and increased mitochondrial ROS. Inhibition of Drp1 prevented mitochondrial fragmentation induced by Mitocur-3 but not by Mitocur-1. The antioxidant N-acetylcysteine inhibited mitochondrial fission induced by Mitocur-1 but not Mitocur-3. Mitochondrial fragmentation caused by Mitocur-3 but not Mitocur-1 was accompanied by activation of Drp1 and AMPK. These data suggest a distinct mechanism of action of mitocurcuminoids on the mitochondria of RBL-2H3 cells: Mitocur-3 stimulated AMPK and caused Drp1-dependent mitochondrial fragmentation, while Mitocur-1-induced mitochondrial fission was ROS-dependent. This difference may contribute to the higher toxicity of Mitocur-3 compared to Mitocur-1. The findings contribute to further drug development for inflammatory and allergic diseases.

Altered Mitochondrial Morphology and Bioenergetics in a New Yeast Model Expressing Aß42.

Alzheimer's disease (AD) is an incurable, age-related neurological disorder, the most common form of dementia. Considering that AD is a multifactorial complex disease, simplified experimental models are required for its analysis. For this purpose, genetically modified Yarrowia lipolytica yeast strains expressing Aß42 (the main biomarker of AD), eGFP-Aß42, Aß40, and eGFP-Aß40 were constructed and examined. In contrast to the cells expressing eGFP and eGFP-Aß40, retaining "normal" mitochondrial reticulum, eGFP-Aß42 cells possessed a disturbed mitochondrial reticulum with fragmented mitochondria; this was partially restored by preincubation with a mitochondria-targeted antioxidant SkQThy. Aß42 expression also elevated ROS production and cell death; low concentrations of SkQThy mitigated these effects. Aß42 expression caused mitochondrial dysfunction as inferred from a loose coupling of respiration and phosphorylation, the decreased level of ATP production, and the enhanced rate of hydrogen peroxide formation. Therefore, we have obtained the same results described for other AD models. Based on an analysis of these and earlier data, we suggest that the mitochondrial fragmentation might be a biomarker of the earliest preclinical stage of AD with an effective therapy based on mitochondria- targeted antioxidants. The simple yeast model constructed can be a useful platform for the rapid screening of such compounds.

A New Mouse Strain with a Mutation in the NFE2L2 (NRF2) Gene.

Transcription factor NRF2 is involved in inflammatory reactions, maintenance of redox balance, metabolism of xenobiotics, and is of particular interest for studying aging. In the present work, the CRISPR/Cas9 genome editing technology was used to generate the NRF2ΔNeh2 mice containing a substitution of eight amino acid residues at the N-terminus of the NRF2 protein, upstream of the functional Neh2 domain, which ensures binding of NRF2 to its inhibitor KEAP1. Heterozygote NRF2wt/ΔNeh2 mice gave birth to homozygous mice with lower than expected frequency, accompanied by their increased embryonic lethality and visual signs of anemia. Mouse embryonic fibroblasts (MEFs) from the NRF2ΔNeh2/ΔNeh2 homozygotes showed impaired resistance to oxidative stress compared to the wild-type MEFs. The tissues of homozygous NRF2ΔNeh2/ΔNeh2 animals had a decreased expression of the NRF2 target genes: NAD(P)H:Quinone oxidoreductase-1 (Nqo1); aldehyde oxidase-1 (Aox1); glutathione-S-transferase A4 (Gsta4); while relative mRNA levels of the monocyte chemoattractant protein 1 (Ccl2), vascular cell adhesion molecule 1 (Vcam1), and chemokine Cxcl8 was increased. Thus, the resulting mutation in the Nfe2l2 gene coding for NRF2, partially impaired function of this transcription factor, expanding our insights into the functional role of the unstructured N-terminus of NRF2. The obtained NRF2ΔNeh2 mouse line can be used as a model object for studying various pathologies associated with oxidative stress and inflammation.

Mitochondria-Targeted Antioxidant SkQ1 Prevents the Development of Experimental Colitis in Mice and Impairment of the Barrier Function of the Intestinal Epithelium.

Mitochondria-targeted antioxidants have become promising candidates for the therapy of various pathologies. The mitochondria-targeted antioxidant SkQ1, which is a derivative of plastoquinone, has been successfully used in preclinical studies for the treatment of cardiovascular and renal diseases, and has demonstrated anti-inflammatory activity in a number of inflammatory disease models. The present work aimed to investigate the therapeutic potential of SkQ1 and C12TPP, the analog of SkQ1 lacking the antioxidant quinone moiety, in the prevention of sodium dextran sulfate (DSS) experimental colitis and impairment of the barrier function of the intestinal epithelium in mice. DSS-treated animals exhibited weight loss, bloody stool, dysfunction of the intestinal epithelium barrier (which was observed using FITC-dextran permeability), reduced colon length, and histopathological changes in the colon mucosa. SkQ1 prevented the development of clinical and histological changes in DSS-treated mice. SkQ1 also reduced mRNA expression of pro-inflammatory molecules TNF, IL-6, IL-1ß, and ICAM-1 in the proximal colon compared with DSS-treated animals. SkQ1 prevented DSS-induced tight junction disassembly in Caco-2 cells. Pretreatment of mice by C12TPP did not protect against DSS-induced colitis. Furthermore, C12TPP did not prevent DSS-induced tight junction disassembly in Caco-2 cells. Our results suggest that SkQ1 may be a promising therapeutic agent for the treatment of inflammatory bowel diseases, in particular ulcerative colitis.

The First Yarrowia lipolytica Yeast Models Expressing Hepatitis B Virus X Protein: Changes in Mitochondrial Morphology and Functions.

Chronic hepatitis B virus infection is the dominant cause of hepatocellular carcinoma, the main cause of cancer death. HBx protein, a multifunctional protein, is essential for pathogenesis development; however, the underlying mechanisms are not fully understood. The complexity of the system itself, and the intricate interplay of many factors make it difficult to advance in understanding the mechanisms underlying these processes. The most obvious solution is to use simpler systems by reducing the number of interacting factors. Yeast cells are particularly suitable for studying the relationships between oxidative stress, mitochondrial dynamics (mitochondrial fusion and fragmentation), and mitochondrial dysfunction involved in HBx-mediated pathogenesis. For the first time, genetically modified yeast, Y. lipolytica, was created, expressing the hepatitis B virus core protein HBx, as well as a variant fused with eGFP at the C-end. It was found that cells expressing HBx experienced stronger oxidative stress than the control cells. Oxidative stress was alleviated by preincubation with the mitochondria-targeted antioxidant SkQThy. Consistent with these data, in contrast to the control cells (pZ-0) containing numerous mitochondrial forming a mitochondrial reticulum, in cells expressing HBx protein, mitochondria were fragmented, and preincubation with SkQThy partially restored the mitochondrial reticulum. Expression of HBx had a significant influence on the bioenergetic function of mitochondria, making them loosely coupled with decreased respiratory rate and reduced ATP formation. In sum, the first highly promising yeast model for studying the impact of HBx on bioenergy, redox-state, and dynamics of mitochondria in the cell and cross-talk between these parameters was offered. This fairly simple model can be used as a platform for rapid screening of potential therapeutic agents, mitigating the harmful effects of HBx.

Do Extracellular Vesicles Derived from Mesenchymal Stem Cells Contain Functional Mitochondria?

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV.

Extrusion of mitochondria: Garbage clearance or cell-cell communication signals?

Mitochondria are dynamic organelles that regulate various intracellular signaling pathways, including the mechanisms of programmed cell death, differentiation, inflammation, and so on. Mitochondria may be extruded as membrane enveloped or as free organelles during developmental processes, inflammatory activation, and in the process of "garbage clearance" of damaged mitochondria in postmitotic cells. Extracellular mitochondria can be engulfed by immune and nonimmune cells and trigger intracellular signaling leading to an inflammatory response. At the same time, it was reported that the release of extracellular vesicles containing mitochondria from mesenchymal stem cells contributes to their therapeutic anti-inflammatory effects. Numerous studies claim that engulfed mitochondria improve cellular bioenergetics, but this assumption requires further investigation. This review aims at a critical discussion of the mechanisms of mitochondrial extrusion in mammals, the reception of mitochondrial components, and the responses of recipient cells to extracellular mitochondria.

Mitochondria-targeted triphenylphosphonium-based compounds inhibit FcεRI-dependent degranulation of mast cells by preventing mitochondrial dysfunction through Erk1/2.

AIMS: FcεRI-dependent activation and degranulation of mast cells (MC) play an important role in allergic diseases. We have previously demonstrated that triphenylphosphonium (TPP)-based antioxidant SkQ1 inhibits mast cell degranulation, but the exact mechanism of this inhibition is still unknown. This study focused on investigating the influence of TPP-based compounds SkQ1 and C12TPP on FcεRI-dependent mitochondrial dysfunction and signaling during MC degranulation. MAIN METHODS: MC were sensitized by anti-dinitrophenyl IgE and stimulated by BSA-conjugated dinitrophenyl. The degranulation of MC was estimated by ß-hexosaminidase release. The effect of TPP-based compounds on FcεRI-dependent signaling was determined by Western blot analysis for adapter molecule LAT, kinases Syk, PI3K, Erk1/2, and p38. Fluorescent microscopy was used to evaluate mitochondrial parameters such as morphology, membrane potential, reactive oxygen species and ATP level. KEY FINDINGS: Pretreatment with TPP-based compounds significantly decreased FcεRI-dependent degranulation of MC. TPP-based compounds also prevented mitochondrial dysfunction (drop in mitochondrial ATP level and mitochondrial fission), and decreased Erk1/2 kinase phosphorylation. Selective Erk1/2 inhibition by U0126 also reduced ß-hexosaminidase release and prevented mitochondrial fragmentation during FcεRI-dependent degranulation of MC. SIGNIFICANCE: These findings expand the fundamental understanding of the role of mitochondria in the activation of MC. It also contributes to the rationale for the development of mitochondrial-targeted drugs for the treatment of allergic diseases.

Does Nrf2 Play a Role of a Master Regulator of Mammalian Aging?

For a long time Nrf2 transcription factor has been attracting attention of researchers investigating phenomenon of aging. Numerous studies have investigated effects of Nrf2 on aging and cell senescence. Nrf2 is often considered as a key player in aging processes, however this needs to be proven. It should be noted that most studies were carried out on invertebrate model organisms, such as nematodes and fruit flies, but not on mammals. This paper briefly presents main mechanisms of mammalian aging and role of inflammation and oxidative stress in this process. The mechanisms of Nrf2 activity regulation, its involvement in aging and development of the senescence-associated secretory phenotype (SASP) are also discussed. Main part of this review is devoted to critical analysis of available experimental data on the role of Nrf2 in mammalian aging.

Gene Expression Pattern of Peyer's Patch Lymphocytes Exposed to Kagocel Suggests Pattern-Recognition Receptors Mediate Its Action.

Kagocel is a synthetic carboxymethylcellulose derivative copolymerized with gossypol. Clinical data evidence its safety and efficiency for the treatment of flu and other viral infections via enhancement of interferon production. The gut-associated lymphoid tissue seems a likely site of kagocel action. The study was aimed to investigate the molecular mechanisms of its action using murine Peyer's patches lymphocytes as a test system and the cytokines production and gene expression patterns as the primary outcomes. The Peyer's patches lymphocytes isolated from BALB/c mice were stimulated with concanavalin A, or, to mimic viral infection, with a combination of concanavalin A and TLR3 ligand poly I:C. After 24 h of stimulation the cells were treated with saline, 30, 100, or 300 µg/ml of kagocel, or, as positive controls, 300 µg/ml oats b-D-glucan or 300 µg/ml lentinan. After 24 and 72 h of incubation with these drugs cytokines production was analyzed with ELISA and gene expression pattern was investigated using nCounter Inflammation panel chips followed by bioinformatics analysis. Expression of genes involved in the inflammatory response, antiviral defense, lymphocytes survival and proliferation (C1qa, C2, C3, Ccl21a, Il11, Il1b, Il23a, Il5, Ltb4r2, Alox15, Pla2g4a, Ptger1, Mapkapk5, Hras, Ifna1, Tlr2, Mrc1, Mx2) was upregulated in kagocel-treated Peyer's patches lymphocytes. A list of plausible transcription factors (CEBPs, IRF, NFκB, RXR, Stat, Tead4, and ZSCAN) and master-regulators has been identified (cIAP, CIKS, dock9, MEKK1, FXR, IKK, IRAK, TRAF, dsRNA:TLR3:TRIF). The changes in gene expression pattern and the outcomes of bioinformatics analysis suggest that pattern recognition receptors, TLRs and dectin-1, are the key mediators of kagocel immunomodulatory action, with the possible involvement of interferon autocrine loop. The genes upregulated with kagocel include diverse components of the innate immune defense system.

Corrigendum: The Role Played by Mitochondria in FcϵRI-Dependent Mast Cell Activation.

[This corrects the article .].

The Role Played by Mitochondria in FcεRI-Dependent Mast Cell Activation.

Mast cells play a key role in the regulation of innate and adaptive immunity and are involved in pathogenesis of many inflammatory and allergic diseases. The most studied mechanism of mast cell activation is mediated by the interaction of antigens with immunoglobulin E (IgE) and a subsequent binding with the high-affinity receptor Fc epsilon RI (FcεRI). Increasing evidences indicated that mitochondria are actively involved in the FcεRI-dependent activation of this type of cells. Here, we discuss changes in energy metabolism and mitochondrial dynamics during IgE-antigen stimulation of mast cells. We reviewed the recent data with regards to the role played by mitochondrial membrane potential, mitochondrial calcium ions (Ca2+) influx and reactive oxygen species (ROS) in mast cell FcεRI-dependent activation. Additionally, in the present review we have discussed the crucial role played by the pyruvate dehydrogenase (PDH) complex, transcription factors signal transducer and activator of transcription 3 (STAT3) and microphthalmia-associated transcription factor (MITF) in the development and function of mast cells. These two transcription factors besides their nuclear localization were also found to translocate in to the mitochondria and functions as direct modulators of mitochondrial activity. Studying the role played by mast cell mitochondria following their activation is essential for expanding our basic knowledge about mast cell physiological functions and would help to design mitochondria-targeted anti-allergic and anti-inflammatory drugs.

Mitochondria-targeted triphenylphosphonium-based compounds do not affect estrogen receptor α.

BACKGROUND: Targeting negatively charged mitochondria is often achieved using triphenylphosphonium (TPP) cations. These cationic vehicles may possess biological activity, and a docking study indicates that TPP-moieties may act as modulators of signaling through the estrogen receptor α (ERα). Moreover, in vivo and in vitro experiments revealed the estrogen-like effects of TPP-based compounds. Here, we tested the hypothesis that TPP-based compounds regulate the activity of ERα. METHODS: We used ERa-positive and ERα-negative human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231, respectively). Cell proliferation was measured using a resazurin cell growth assay and a real-time cell analyzer assay. Cell cycle progression was analyzed using flow cytometry. Real-time PCR was used to assess mRNA expression of endogenous estrogen-responsive genes. Luciferase activity was measured to evaluate transcription driven by estrogen-responsive promoters in cells transfected with an estrogen response element (ERE)3-luciferase expression vector. RESULTS: The TPP-based molecules SkQ1 and C12TPP, as well as the rhodamine-based SkQR1, did not increase the proliferation or alter the cell cycle progression of MCF-7 cells. In contrast, 17ß estradiol increased the proliferation of MCF-7 cells and the proportion of cells in the S/G2/M-phases of the cell cycle. TPP-based compounds did not affect the induction of transcription of an ERE-luciferase expression vector in vitro, and SkQ1 did not alter the levels of expression of estrogen-dependent genes encoding GREB1, TFF1, COX6, and IGFBP4. CONCLUSION: TPP-based compounds do not possess properties typical of ERα agonists.

Mild depolarization of the inner mitochondrial membrane is a crucial component of an anti-aging program.

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.