Mitochondria-targeted Antioxidant SkQ1 Research Articles

Mitochondrial antioxidant SkQ1 attenuates C26 cancer-induced muscle wasting in males and improves muscle contractility in female tumor-bearing mice.

Mitochondrial dysfunction is a hallmark of cancer cachexia (CC). Mitochondrial reactive oxygen species (ROS) are elevated in muscle shortly after tumor onset. Targeting mitochondrial ROS may be a viable option to prevent CC. The aim of this study was to evaluate the efficacy of a mitochondria-targeted antioxidant, SkQ1, to mitigate CC in both biological sexes. Male and female Balb/c mice were injected bilaterally with colon 26 adenocarcinoma (C26) cells (total 1x106 cells) or PBS (equal volume control). SkQ1 was dissolved in drinking water (~250 nmol/kg body weight/day) and administered to mice beginning seven days following tumor induction, while control groups consumed normal drinking water. In vivo muscle contractility of dorsiflexors, deuterium oxide-based protein synthesis, mitochondrial respiration and mRNA content of mitochondrial, protein turnover and calcium channel-related markers were assessed at endpoint (25 days following tumor induction). Two-way ANOVAs, followed by Tukey's post-hoc test when interactions were significant (p≤0.05), were performed. SkQ1 attenuated cancer-induced atrophy, promoted protein synthesis and abated Redd1 and Atrogin induction in gastrocnemius of C26 male mice. In female mice, SkQ1 decreased muscle mass and increased catabolic signaling in the plantaris of tumor-bearing mice, as well as reduced mitochondrial oxygen consumption, regardless of tumor. However, in females SkQ1 enhanced muscle contractility of the dorsiflexors with concurrent induction of Ryr1, Serca1 and Serca2a in TA. In conclusion, the mitochondria-targeted antioxidant SkQ1 may attenuate CC-induced muscle loss in males, while improving muscle contractile function in tumor-bearing female mice, suggesting sexual dimorphism in the effects of this mitochondrial therapy in CC.

Mitochondrial fragmentation in early differentiation of human MB135 myoblasts: Role of mitochondrial ROS production in the absence of depolarization

AimsStudy of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis. Main methodsMitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria. Key findingsIn immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation. SignificanceMitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.

Exogenous Iron Induces Mitochondrial Lipid Peroxidation, Lipofuscin Accumulation, and Ferroptosis in H9c2 Cardiomyocytes.

Lipid peroxidation plays an important role in various pathologies and aging, at least partially mediated by ferroptosis. The role of mitochondrial lipid peroxidation during ferroptosis remains poorly understood. We show that supplementation of exogenous iron in the form of ferric ammonium citrate at submillimolar doses induces production of reactive oxygen species (ROS) and lipid peroxidation in mitochondria that precede ferroptosis in H9c2 cardiomyocytes. The mitochondria-targeted antioxidant SkQ1 and the redox mediator methylene blue, which inhibits the production of ROS in complex I of the mitochondrial electron transport chain, prevent both mitochondrial lipid peroxidation and ferroptosis. SkQ1 and methylene blue also prevented accumulation of lipofuscin observed after 24 h incubation of cardiomyocytes with ferric ammonium citrate. Using isolated cardiac mitochondria as an in vitro ferroptosis model, it was shown that rotenone (complex I inhibitor) in the presence of ferrous iron stimulates lipid peroxidation and lipofuscin accumulation. Our data indicate that ROS generated in complex I stimulate mitochondrial lipid peroxidation, lipofuscin accumulation, and ferroptosis induced by exogenous iron.

The critical role of mitochondrial lipid peroxidation in ferroptosis: insights from recent studies.

Ferroptosis is a regulated form of necrotic cell death reliant on iron-catalyzed lipid peroxidation. Although the precise involvement of mitochondria in ferroptosis remains incompletely elucidated, recent research indicates that mitochondrial oxidative events wield a pivotal influence in this mechanism. This article centers on the most recent discoveries, spotlighting the significance of mitochondrial lipid peroxidation in the occurrence of ferroptosis. Modern investigative tools, such as mitochondria-specific dyes responsive to lipid peroxidation and antioxidants targeting mitochondria, have been employed to delve into this phenomenon. The authors' recent empirical evidence demonstrates that mitochondrial lipid peroxidation, quantified using the innovative fluorescent ratiometric probe MitoCLox, takes place prior to the onset of ferroptotic cell death. The mitochondria-targeted antioxidant SkQ1 hinders mitochondrial lipid peroxidation and thwarts ferroptosis, all while leaving unaffected the buildup of reactive oxygen species within the cytoplasm, an antecedent to mitochondrial lipid peroxidation. Similarly, the redox agent methylene blue, impeding the genesis of reactive oxygen species in complex I of the electron transport chain, also imparts a comparable protective effect. These findings collectively imply that reactive oxygen species originating from complex I might hold particular significance in fomenting mitochondrial lipid peroxidation, a pivotal trigger of ferroptosis.

Penetration of Triphenylphosphonium Derivatives through the Cell Envelope of Bacteria of Mycobacteriales Order.

The penetration of substances through the bacterial cell envelope is a complex and underinvestigated process. Mitochondria-targeted antioxidant and antibiotic SkQ1 (10-(plastoquinonyl)decyltriphenylphosphonium) is an excellent model for studying the penetration of substances through the bacterial cell envelope. SkQ1 resistance in Gram-negative bacteria has been found to be dependent on the presence of the AcrAB-TolC pump, while Gram-positive bacteria do not have this pump but, instead, have a mycolic acid-containing cell wall that is a tough barrier against many antibiotics. Here, we report the bactericidal action of SkQ1 and dodecyl triphenylphospho-nium (C12TPP) against Rhodococcus fascians and Mycobacterium tuberculosis, pathogens of plants and humans. The mechanism of the bactericidal action is based on the penetration of SkQ1 and C12TPP through the cell envelope and the disruption of the bioenergetics of bacteria. One, but probably not the only such mechanism is a decrease in membrane potential, which is important for the implementation of many cellular processes. Thus, neither the presence of MDR pumps, nor the presence of porins, prevents the penetration of SkQ1 and C12TPP through the complex cell envelope of R. fascians and M. tuberculosis.

Mitochondria-Targeted Antioxidant SkQ1 Improves Muscle Contractility in Female C26 Tumor-Bearing Mice

Cancer cachexia (CC) affects up to 80% of cancer patients and is responsible for 20-40% of cancer related deaths. Our laboratory recently demonstrated that emission of muscle mitochondrial reactive oxygen species (ROS) is highly elevated shortly after the onset of tumor-bearing in mice. Therefore, targeting of mitochondrial ROS may be a viable option to treat and prevent CC. The aim of this study was to evaluate the effectiveness of a mitochondria-targeted antioxidant, SkQ1, at preventing CC. We hypothesized SkQ1 prevents muscle atrophy and weakness in tumor-bearing mice. To test the effect of SkQ1 on CC, female Balb/c mice were injected bilaterally with colon 26 adenocarcinoma (C26) cells to the hind flanks for a total cell volume of 1x106, while equal volume of PBS was injected in a separate cohort as a sham control. SkQ1 was dissolved in drinking water (250 nmol/kg body weight/day) and administered to mice, while control groups drank normal drinking water. Body weight was assessed every 5 days. In vivo muscle contractility and fatigability were assessed via electrophysiological measurements two days prior to endpoint. At the endpoint (~25 days after C26/PBS injections) body weight and tissue wet weights were measured and normalized by tibia length (mg/mm). Data were analyzed by 2X2 ANOVA with Tukey’s post hoc test where significant interactions were found with p≤0.05. C26 mice had lower tumor-free body weight (p<0.05), as well as the weights of gastrocnemius, tibialis anterior, extensor digitorum longus, quadriceps and heart (p<0.05), accompanied by increased liver and spleen weight (p<0.01) regardless of SkQ1 treatment. An interaction between C26 and SkQ1 was found in plantaris weight (p<0.01) with cancer mice treated by SkQ1 (C26 SkQ1) presenting lower plantaris weight compared to non-treated cancer mice (C26 CON): 0.788 vs. 0.874 mg/mm (p<0.05). The normalized (by tumor-free body weight) maximal isometric torque produced by C26 SkQ1 was higher compared to C26 CON: 0.078 vs 0.066 mN·m/gr (p<0.05). The normalized torques produced at frequencies 80-300 Hz were also higher in C26 SkQ1 compared to C26 CON (p<0.05). Muscle fatiguability was not affected by either cancer or SkQ1. In conclusion, the mitochondria-targeted antioxidant SkQ1 protects cancer-induced muscle weakness in female C26 tumor-bearing mice, although it may accelerate cancer-induced muscle wasting in some muscle groups. Data analysis is ongoing to clarify the effects of SkQ1 on cancer-induced cachexia in both male and female mice. This study was supported by the National Institutes of Health Award Number 5R01AR075794-02. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SKQ1 Improves Trimethylamine-N-Oxide-Induced Endothelial Dysfunction by Attenuating Oxidative Stress and Restoring Junctional Proteins

In recent years, the role of microbiota and microbial metabolites in the progress of aging and age-related diseases has been attracting increased attention. In this context, trimethylamine N-oxide (TMAO) is a pro-atherogenic and pro-inflammatory metabolite derived from the gut microbial metabolism of trimethylamine and is found to be elevated in patients with age-related cardiovascular disorders. We identified that TMAO treatment in mice has enhanced mitochondrial ROS production, endoplasmic reticulum stress and caused significant endothelial dysfunction. The present study investigated whether mitochondria-targeted antioxidant SKQ1 protected against TMAO-induced endothelial dysfunction. Endothelial cells were treated with or without TMAO (30μM) in the cell culture media. TMAO treated cells showed significant increase in both total ROS as well as mito-ROS production compared to untreated control group (n=8/group; p<0.05). In addition, western blot analysis demonstrated that TMAO treatment significantly decreased tight junction proteins such as occludin, ZO-1 and VE-Cadherin which were restored upon SKQ1 treatment (150nM, 12h). TMAO (30μM) treatment for 24 h induced significant mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential, increased mitochondrial ROS levels, and enhanced endothelial cell permeability compared to the control group (n=8/group; p<0.05). Treatment with a SKQ1 (150nM, 12h) significantly inhibited TMAO-induced ROS production and protected against increased endothelial cell permeability and mitochondrial ROS production (n=8/group; p<0.05). In conclusion, mitochondria-targeted antioxidant SKQ1 protects against TMAO induced endothelial dysfunction by attenuating ROS production and disruption of tight junction proteins. This work is supported by NIH R01 HL148711 award to Saisudha Koka This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells

Iron overload is associated with various pathological changes which contribute to metabolic syndrome, many of which have been proposed to occur via damaging tissue through an excessive amount of reactive oxygen species (ROS) production. In this study, we established a model of iron overload in L6 skeletal muscle cells and observed that iron enhanced cytochrome c release from depolarized mitochondria, assayed by immunofluorescent colocalization of cytochrome c with Tom20 and the use of JC-1, respectively. This subsequently elevated apoptosis, determined via use of a caspase-3/7 activatable fluorescent probe and western blotting for cleaved caspase-3. Using CellROX deep red and mBBr, we observed that iron increased generation of reactive oxygen species (ROS), and that pretreatment with the superoxide dismutase mimetic MnTBAP reduced ROS production and attenuated iron-induced intrinsic apoptosis and cell death. Furthermore, using MitoSox Red we observed that iron enhanced mROS and the mitochondria-targeted anti-oxidant SKQ1 reduced iron-induced ROS generation and cell death. Western blotting for LC3-II and P62 levels as well as immunofluorescent detection of autophagy flux with LC3B and P62 co-localization indicated that iron acutely (2–8 h) activated and later (12–24 h) attenuated autophagic flux. We used autophagy-deficient cell models generated by overexpressing a dominant-negative Atg5 mutant or CRISPR-mediated ATG7 knock out to test the functional significance of autophagy and observed that autophagy-deficiency exacerbated iron-induced ROS production and apoptosis. In conclusion, our study showed that high iron levels promoted ROS production, blunted the self-protective autophagy response and led to cell death in L6 skeletal muscle cells.

Mitochondrion-targeted antioxidant SkQ1 prevents rapid animal death caused by highly diverse shocks

The response to stress involves the activation of pathways leading either to protection from the stress origin, eventually resulting in development of stress resistance, or activation of the rapid death of the organism. Here we hypothesize that mitochondrial reactive oxygen species (mtROS) play a key role in stress-induced programmed death of the organism, which we called “phenoptosis” in 1997. We demonstrate that the synthetic mitochondria-targeted antioxidant SkQ1 (which specifically abolishes mtROS) prevents rapid death of mice caused by four mechanistically very different shocks: (a) bacterial lipopolysaccharide (LPS) shock, (b) shock in response to intravenous mitochondrial injection, (c) cold shock, and (d) toxic shock caused by the penetrating cation C12TPP. Importantly, under all these stresses mortality was associated with a strong elevation of the levels of pro-inflammatory cytokines and administration of SkQ1 was able to switch off the cytokine storms. Since the main effect of SkQ1 is the neutralization of mtROS, this study provides evidence for the role of mtROS in the activation of innate immune responses mediating stress-induced death of the organism. We propose that SkQ1 may be used clinically to support patients in critical conditions, such as septic shock, extensive trauma, cooling, and severe infection by bacteria or viruses.

Mitochondria-targeted antioxidant SkQ1 inhibits leukotriene synthesis in human neutrophils.

Leukotrienes are among the most potent mediators of inflammation, and inhibition of their biosynthesis, is becoming increasingly important in the treatment of many pathologies. In this work, we demonstrated that preincubation of human neutrophils with the mitochondria targeted antioxidant SkQ1 (100nM) strongly inhibits leukotriene synthesis induced by three different stimuli: the Ca2+ ionophore A23187, the chemotactic formyl-peptide fMLP in combination with cytocholasin B, and opsonized zymosan. The SkQ1 analogue lacking the antioxidant quinone moiety (C12TPP) was ineffective, suggesting that mitochondrial production of reactive oxygen species (ROS) is critical for activating of leukotriene synthesis in human neutrophils. The uncoupler of oxidative phosphorylation FCCP also inhibits leukotriene synthesis, indicating that a high membrane potential is a prerequisite for stimulating leukotriene synthesis in neutrophils. Our data show that activation of mitogen-activated protein kinases p38 and ERK1/2, which is important for leukotriene synthesis in neutrophils is a target for SkQ1: 1) the selective p38 inhibitor SB203580 inhibited fMLP-induced leukotriene synthesis, while the ERK1/2 activation inhibitor U0126 suppressed leukotriene synthesis induced by any of the three stimuli; 2) SkQ1 effectively prevents p38 and ERK1/2 activation (accumulation of phosphorylated forms) induced by all three stimuli. This is the first study pointing to the involvement of mitochondrial reactive oxygen species in the activation of leukotriene synthesis in human neutrophils. The use of mitochondria-targeted antioxidants can be considered as a promising strategy for inhibiting leukotriene synthesis and treating various inflammatory pathologies.

Mitochondrial antioxidant SkQ1 decreases inflammation following hemorrhagic shock by protecting myocardial mitochondria.

Background: Hemorrhagic shock (HS) is a type of hypovolemic shock characterized by hemodynamic instability, tissue hypoperfusion and cellular hypoxia. In pathophysiology, the gradual accumulation of reactive oxygen species (ROS) damages the mitochondria, leading to irreversible cell damage and the release of endogenous damage-associated molecular patterns (DAMPs) including mitochondrial DAMPs (MTDs), eventually triggering the inflammatory response. The novel mitochondria-targeted antioxidant SkQ1 (Visomitin) effectively eliminate excessive intracellular ROS and exhibits anti-inflammatory effects; however, the specific role of SkQ1 in HS has not yet been explicated. Methods and results: A 40% fixed-blood-loss HS rat model was established in this study. Transmission electron microscopy showed that after HS, the myocardial mitochondrial ultrastructure was damaged and the mtDNA release in circulation was increased and the differentially expressed genes were significantly enriched in mitochondrial and ROS-related pathways. Mitochondria-targeted antioxidant SkQ1 attenuated the increased ROS induced by HS in myocardial tissues and by oxygen-glucose deprivation (OGD) in cardiomyocytes. Ultrastructurally, SkQ1 protected the myocardial mitochondrial structure and reduced the release of the peripheral blood mtDNA after HS. RNA-seq transcriptome analysis showed that 56.5% of the inflammation-related genes, which altered after HS, could be significantly reversed after SkQ1 treatment. Moreover, ELISA indicated that SkQ1 significantly reversed the HS-induced increases in the TNF-α, IL-6, and MCP-1 protein levels in rat peripheral blood. Conclusion: HS causes damage to the rat myocardial mitochondrial structure, increases mtDNA release and ROS contents, activates the mitochondrial and ROS-related pathways, and induces systemic inflammatory response. The mitochondrial antioxidant SkQ1 can improve rat myocardial mitochondria ultrastructure, reduce mtDNA and ROS contents, and decrease inflammation by protecting myocardial mitochondria, thereby playing a novel protective role in HS.

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-1β, 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.

Effect of Antioxidants on the Production of MCP-1 Chemokine by EA.hy926 Cells in Response to IL-6.

An elevated level of circulatory interleukin 6 (IL-6) is a biomarker for cytokine storm of various etiologies, including COVID-19, and contributes to poor prognosis. Vascular endothelial cells are one of the main targets of pathological action of IL-6. IL-6 activates the trans-signaling pathway via the formation of the IL-6/sIL-6Ra/gp130 receptor complex and subsequent activation of the JAK/STAT3 signaling pathway and PI3K/AKT and MEK/ERK kinases in some cases. Previously, it was shown by the authors' group and other researchers that reactive oxygen species (ROS), including mitochondrial ROS (mito-ROS), contribute to the induction of IL-6 expression in the endothelium, mainly due to increased activation of the transcription factor NF-kB. We have also shown that the mitochondria-targeted antioxidant SkQ1 (Plastoquinolyl-10(6'-decyltriphenyl)phosphonium) prevented tumor necrosis factor (TNF)-induced cytokine storm and death in mice. In the aortas of these animals, SkQ1 also prevented the increase in the expression of NF-kB-dependent genes, including the cytokine IL-6 and the chemokine MCP-1. In the current work, the hypothesis of mito-ROS involvement in the IL-6-signaling-mediated proinflammatory gene expression in endothelial cells is tested. SkQ1 suppressed the expression and secretion of the MCP-1 chemokine, induced by IL-6 in combination with sIL-6-Ra, but not the expression of ICAM-1 adhesion molecules in EA.hy926 human endothelial cells. Using specific inhibitors, the authors have shown that, in EA.hy926 cells, IL-6-induced expression of MCP-1 and ICAM-1 depends on the signaling protein and transcription activator STAT3 and, in some cases, on JNK, PI3K, and MEK1/2 kinases and is independent of p38 kinase. In this model, IL-6 induced rapid STAT3 activation, while ERK1/2 activation was less pronounced, and there was no IL-6 effect on Akt and JNK activation. SkQ1 partially suppressed STAT3 and ERK1/2 activation. Thus, we have shown that SkQ1 suppresses not only NF-kB-dependent expression of IL-6 and other proinflammatory genes but also IL-6-induced activation of JAK/STAT3 and STAT3-dependent expression of MCP-1, which probably contributes to the overall therapeutic effect of SkQ1.

Pathogenic Role of Endoplasmic Reticulum Stress in Diabetic Corneal Endothelial Dysfunction.

PurposeProgressive corneal edema and endothelial cell loss represent the major corneal complications observed in diabetic patients after intraocular surgery. However, the underlying pathogenesis and potential treatment remain incompletely understood.MethodsWe used streptozotocin-induced type 1 diabetic mice and db/db type 2 diabetic mice as diabetic animal models. These mice were treated with the endoplasmic reticulum (ER) stress agonist thapsigargin; 60-mmHg intraocular pressure (IOP) with the ER stress antagonist 4-phenylbutyric acid (4-PBA); mitochondria-targeted antioxidant SkQ1; or reactive oxygen species scavenger N-acetyl-l-cysteine (NAC). Corneal thickness and endothelial cell density were measured before and after treatment. Human corneal endothelial cells were treated with high glucose with or without 4-PBA. The expression of corneal endothelial- and ER stress–related genes was detected by western blot and immunofluorescence staining. Mitochondrial bioenergetics were measured with an Agilent Seahorse XFp Analyzer.ResultsIn diabetic mice, the appearance of ER stress preceded morphological changes in the corneal endothelium. The persistent ER stress directly caused corneal edema and endothelial cell loss in normal mice. Pharmacological inhibition of ER stress was sufficient to mitigate corneal edema and endothelial cell loss in both diabetic mice after high IOP treatment. Mechanistically, inhibiting ER stress ameliorated the hyperglycemia-induced mitochondrial bioenergetic deficits and improved the barrier and pump functional recovery of the corneal endothelium. When compared with NAC, 4-PBA and SkQ1 exhibited better improvement of corneal edema and endothelial cell loss in diabetic mice.ConclusionsHyperglycemia-induced ER stress contributes to the dysfunction of diabetic corneal endothelium, and inhibiting ER stress may offer therapeutic potential by improving mitochondrial bioenergetics.

Changes in Glial Support of the Hippocampus during the Development of an Alzheimer's Disease-like Pathology and Their Correction by Mitochondria-Targeted Antioxidant SkQ1.

Astrocytes and microglia are the first cells to react to neurodegeneration, e.g., in Alzheimer’s disease (AD); however, the data on changes in glial support during the most common (sporadic) type of the disease are sparse. Using senescence-accelerated OXYS rats, which simulate key characteristics of sporadic AD, and Wistar rats (parental normal strain, control), we investigated hippocampal neurogenesis and glial changes during AD-like pathology. Using immunohistochemistry, we showed that the early stage of the pathology is accompanied by a lower intensity of neurogenesis and decreased astrocyte density in the dentate gyrus. The progressive stage is concurrent with reactive astrogliosis and microglia activation, as confirmed by increased cell densities and by the acquisition of cell-specific gene expression profiles, according to transcriptome sequencing data. Besides, here, we continued to analyze the anti-AD effects of prolonged supplementation with mitochondria-targeted antioxidant SkQ1. The antioxidant did not affect neurogenesis, partly normalized the gene expression profile of astrocytes and microglia, and shifted the resting/activated microglia ratio toward a decrease in the activated-cell density. In summary, both astrocytes and microglia are more vulnerable to AD-associated neurodegeneration in the CA3 area than in other hippocampal areas; SkQ1 had an anti-inflammatory effect and is a promising modality for AD prevention and treatment.

Electrophysiological and psychophysical studies in assessment of visual functions in patients with hereditary optic neuropathy

To study the capabilities of electrophysiological and psychophysical examination methods for assessment of the functional state of ganglion cells, retina and optic nerve in patients with hereditary optic neuropathy (HON). The study included 60 patients (118 eyes) with a genetically confirmed diagnosis of HON. All study patients underwent visual field test (VFT), spectral optical coherence tomography (OCT), flash and pattern visual evoked potentials (VEP) (Flash-VEP, FVEP; Pattern-VEP, PVEP), photopic electroretinography with photonegative response (PhNR) registration and the color vision test. In 24 patients (46 eyes), these parameters were assessed before the start of treatment and one year later. The treatment involved the mitochondria-targeted antioxidant SkQ1 - plastoquinonyl-decyl-triphenylphosphonium bromide (PDTP) in the form of eye drops. The main PVEP components for 1.0° and 0.3° were registered in 20% and in 14% of patient eyes with HON and high visual functions, respectively. After one year of PDTP use, a significant decrease in P100 peak latency was found only in the group with disease duration of ≤1.5 years as of the time of treatment start (p<0.05). Significant differences were observed in the PhNR amplitude (p<0.004) between patients of the main and the control groups, as well as in the PhNR amplitude between patients with visual acuity of ≤0.1 and ≥0.13 (p<0.01). Patients with high visual functions were found to have a correlation between the PhNR amplitude, GCC thickness and the global loss index (GLV). Along with VFT, OCT and color vision tests, electrophysiological studies are one of the main methods of examining patients with HON. After one year of PDTP use, there was a significant decrease in the FVEP P2 peak latency in the group with a disease duration of ≤1.5 years as of the time of treatment start. The PhNR amplitude in patients with high visual functions was found to correlate with structural changes in the ganglion cell layer and the retinal nerve fiber layer.

Mitochondrial Antioxidant SkQ1 Has a Beneficial Effect in Experimental Diabetes as Based on the Analysis of Expression of microRNAs and mRNAs for the Oxidative Metabolism Regulators.

Diabetes mellitus and related complications are among the most important problems of the world-leading healthcare systems. Despite their priority, molecular and genetic aspects of diabetes pathogenesis are poorly understood; however, the involvement of oxidative stress in this process is undoubted. Rats with experimental diabetes induced by the intraperitoneal injection of alloxan were subjected to the antioxidant pre-therapy with a series of mitochondria-targeted 10-(6’-plastoquinonyl)decyltriphenylphosphonium (SkQ1) injections and analyzed for the expression of mRNAs and microRNAs by real-time quantitative polymerase chain reaction to identify potential predictors of diabetes. Animals that received SkQ1 before diabetes induction demonstrated lower blood glucose levels compared to the diabetic animals not subjected to the therapy. SkQ1 caused changes in the mRNA levels of genes involved in the cellular defense against free radicals, which indicates a beneficial effect of the pre-therapy. Moreover, similar changes were observed on the epigenetic level, as the microRNA expression patterns not only proved the SkQ1 efficacy but also correlated with the expression levels of their mRNA targets. Oxidative stress and macromolecule damage by free radicals are determining factors in diabetes, which suggests that strategies aimed at restoring the antioxidant status of the cell can be beneficial. Mitochondria-targeted antioxidant SkQ1 demonstrates positive effects on several levels, from the normalization of the blood glucose content to genetic and epigenetic changes. Our results can serve as a basis for the development of novel therapeutic and diagnostic strategies.

440-P: Dual Long-Term Effects of Free Fatty Acid on Human Endothelial Barrier In Vitro Are Mediated by NO, Mitochondrial ROS, and Dicarbonyl Stress

In diabetes-associated hyperlipidemia, free fatty acids (FFAs) are thought to induce insulin resistance and endothelial dysfunction leading to vascular injury and cardiovascular T2DM complications. However, evidence is scarce regarding the effects of FFA on vascular endothelial barrier. We used human umbilical vein endothelial cells (HUVEC) to model, in vitro, the endothelial barrier and reveal its responses to experimental hyperlipidemia induced by long-term treatment with palmitic acid in complex with albumin. Palmitate was continuously present in the growth medium at 0.8 mM to reproduce FFA levels typical for T2DM. We developed conditions to maintain the HUVEC monolayers in the presence of the high palmitate concentration for up to 2 weeks by adding exogenous carnitine to facilitate mitochondrial β-oxidation of FFA. In this model system, palmitate readily augmented the transendothelial electric resistance (TER) of HUVEC via increased NO production by endothelial NO-synthase (eNOS). Western blots showed that palmitate neither disturbed insulin signaling cascade towards eNOS nor upregulated eNOS expression. After the initial 3-4-day rise in TER, further treatment with palmitate led to barrier stabilization and attenuation as compared to FFA-free conditions. This later phase was associated with increased ROS and malondialdehyde (MDA) accumulation, both prevented by the mitochondria-targeted antioxidant SkQ1. Treatment of HUVEC with exogenous MDA impaired the TER and barrier suggesting that accumulation of MDA-modified proteins may contribute to the barrier dysfunction upon long-term experimental hyperlipidemia. Altogether, obtained evidence suggests that prolonged exposure of vascular endothelium to high levels of palmitate compromises endothelium barrier capacity and insulin-mediated eNOS regulation turning the enzyme permanently active. Disclosure M. Samsonov: None. V. P. Shirinsky: None. N. Podkuychenko: None. A. Y. Khapchaev: None. T. Vlasik: None. V. Lankin: None. M. Skulachev: Other Relationship; Self; Mitotech LLC. I. Stafeev: None. A. V. Vorotnikov: None. M. V. Shestakova: None. Funding Russian Science Foundation (19-15-00361)

Anthelmintic Activity of Antioxidants: In Vitro Effects on the Liver Fluke Opisthorchis felineus.

Currently, molecular parasitologists are searching for new agents against trematodiases. Redox metabolism is important for parasites as far as long-lived adult parasites inside a mammalian host are exposed to redox challenges. Antioxidants have been poorly studied as anthelmintic agents, in particular against the foodborne trematodes. Study of in vitro anthelmintic activity of nonenzymatic natural and synthetic antioxidants of various chemical structures was performed using standard motility and mortality assays against juvenile and adult Opisthorchis felineus worms. Promising agents have been found among both natural and synthetic compounds. The mitochondria-targeted antioxidant SkQ1 [10-(6′-plastoquinonyl)decyltriphenylphosphonium] in motility assays was as effective (half-maximal inhibitory concentration [IC50] 0.6–1.4 μM) as praziquantel (IC50 0.47–1.4 μM), and SkQ1 was significantly more effective than praziquantel in mortality assays. Moreover, extensive tegument damage of the adult fluke was revealed after SkQ1 treatment. Flavonoids manifested potency too, with IC50 values in a micromolar range (5.1–17.4 μM). Other natural and synthetic compounds tested against helminths were significantly less effective than praziquantel. Results of our study indicate that SkQ1 and flavonoids have high anthelmintic activities against the liver flukes. We propose that structure–activity relationship research might be worthwhile based on the structures of the most effective substances.

Autophagy as a Target for the Retinoprotective Effects of the Mitochondria-Targeted Antioxidant SkQ1.

Age-related macular degeneration (AMD) is a complex neurodegenerative disease, a main cause of vision loss in elderly people. The pathogenesis of dry AMD, the most common form of AMD (~ 80% cases), involves degenerative changes in the retinal pigment epithelium (RPE), which are closely associated with the age-associated impairments in autophagy. Reversion of these degenerative changes is considered as a promising approach for the treatment of this incurable disease. The purpose of our study was to assess the relationship between previously identified retinoprotective effects of the mitochondrial antioxidant plastoquinonyl-decyl-triphenylphosphonium (SkQ1) and its influence on the autophagy process in senescence-accelerated OXYS rats characterized by the development of AMD-like retinopathy (Wistar rats were used as a control). The treatment with SkQ1 (250 nmol/kg body weight) during the period of active disease progression (from 12 to 18 months of age) completely prevented progression of clinical manifestations of retinopathy in the OXYS rats, suppressed atrophic changes in the RPE cells and activated autophagy in the retina, which was evidenced by a significant decrease in the content of the multifunctional adapter protein p62/Sqstm1 and increase in the level of the Beclin1 gene mRNA. In general, the results obtained earlier and in the present study have shown that SkQ1 is a promising agent for prevention and suppression of AMD.