Impact of Airborne Exposure to PM10 Increases Susceptibility to P. aeruginosa Infection.

The effects of exposure to airborne particulate matter with a size of 10 µm or less (PM10) on C57BL/6 mouse corneas, their response to Pseudomonas aeruginosa (PA) infection, and the protective effects of SKQ1 were determined. C57BL/6 mouse corneas receiving PBS or SKQ1 were exposed to control (air) or PM10 for 2 weeks, infected, and the disease was documented by clinical score, PMN quantitation, bacterial plate count, RT-PCR and Western blot. PBS-treated, PM10-exposed corneas did not differ at 1 day postinfection (dpi), but exhibited earlier (3 dpi) corneal thinning compared to controls. By 3 dpi, PM10 significantly increased corneal mRNA levels of several pro-inflammatory cytokines, but decreased IL-10, NQO1, GR1, GPX4, and Nrf2 over control. SKQ1 reversed these effects and Western blot selectively confirmed the RT-PCR results. PM10 resulted in higher viable bacterial plate counts at 1 and 3 dpi, but SKQ1 reduced them at 3 dpi. PM10 significantly increased MPO in the cornea at 3 dpi and was reduced by SKQ1. SKQ1, used as an adjunctive treatment to moxifloxacin, was not significantly different from moxifloxacin alone. Exposure to PM10 increased the susceptibility of C57BL/6 to PA infection; SKQ1 significantly reversed these effects, but was not effective as an adjunctive treatment.

10-(6-Plastoquinonyl) decyltriphenylphosphonium imparts anti-colitogenic protection through recovery of mitochondrial dysfunction in ulcerated murine colon: Implications in ulcerative colitis.

AIMS: To elucidate the impact of 10-(6-plastoquinonyl) decyltriphenylphosphonium (SkQ1) as an anti-colitogenic agent for maintenance of colon epithelial tract in ulcerated mice through recovery of mitochondrial dysfunction and mitochondrial stress by virtue of its free radical scavenging properties. MAIN METHODS: DSS induced ulcerated BALB/c mice were treated with SkQ1 for 14 days @ 30 nmol/kg/body wt./day/mice. Post-treatment, isolated colonic mitochondria were utilized for spectrophotometric and spectrofluorometric biochemical analysis of various mitochondrial functional variables including individual mitochondrial respiratory enzyme complexes. Confocal microscopy was utilized for measuring mitochondrial membrane potential in vivo. ELISA technique was adapted for measuring colonic nitrite and 3-nitrotyrosine (3-NT) content. Finally in vitro cell line study was carried out to substantiate in vivo findings and elucidate the involvement of free radicals in UC using antioxidant/free radical scavenging regimen. KEY FINDINGS: Treatment with SkQ1 in vivo reduced histopathological severity of colitis, induced recovery of mitochondrial respiratory complex activities and associated functional variables, improved oxidative stress indices and normalized mitochondrial cardiolipin content. Importantly, SkQ1 lowered nitrite concentration and 3-nitrotyrosine formation in vivo. In vitro SkQ1 restored mitochondrial functions wherein the efficacy of SkQ1 proved equal or better compared to SOD and DMSO indicating predominant involvement of O2- and OH in UC. However, NO and ONOO- also seemed to play a secondary role as MEG and L-NAME provided lesser protection as compared to SOD and DMSO. SIGNIFICANCE: SkQ1 can be considered as a potent anti-colitogenic agent by virtue of its free radical scavenging properties in treating UC.

Supplementation of SkQ1 Increases Mouse In Vitro Oocyte Maturation and Subsequent Embryonic Development by Reducing Oxidative Stress.

In vitro oocyte maturation (IVM) technology is important for assisted animal and human reproduction. However, the maturation rates and developmental potential of in vitro-matured oocytes are usually lower than those of in vivo-matured oocytes. Oxidative stress is a main factor that causes the lower maturation rates and quality of in vitro-matured oocytes. The purpose of this study was to investigate the effects of treatment with SkQ1, a mitochondria-targeted antioxidant, on mouse IVM and subsequent embryonic development. The results demonstrated that the supplementation of SkQ1 during IVM improves the maturation rates of mouse oocytes and the subsequent developmental competence of in vitro-fertilized embryos. The addition of SkQ1 to the IVM medium also decreased oxidative stress and apoptosis, and increased mitochondrial membrane potential in matured mouse oocytes. This study provides a new method through which to enhance the maturation rates and the quality of in vitro-matured mouse oocytes, thus promoting the application and development of assisted animal and human reproductive technology.

Retinoprotective Effect of SkQ1, Visomitin Eye Drops, Is Associated with Suppression of P38 MAPK and ERK1/2 Signaling Pathways Activity.

Visomitin eye drops are the first and, so far, the only drug based on SkQ1 - the mitochondria-targeted antioxidant 10-(6'-plastoquinonyl) decyltriphenylphosphonium, developed in the laboratories of Moscow State University under the leadership of Academician V. P. Skulachev. SkQ1 is considered as a potential tool to combat the aging program. We have previously shown that it is able to prevent and/or suppress development of all manifestations of accelerated senescence in OXYS rats, including retinopathy, similar to the age-related macular degeneration (AMD). Here, we assessed the effect of Visomitin instillations on progression of the AMD-like pathology and p38 MAPK and ERK1/2 activity in the OXYS rat retina (from the age of 9 to 12 months). Wistar and OXYS rats treated with placebo (composition identical to Visomitin with the exception of SkQ1) were used as controls. Ophthalmological examination showed that in the OXYS rats receiving placebo, retinopathy progressed and severity of clinical manifestations did not differ from the intact OXYS rats. Visomitin suppressed progression of the AMD-like pathology in the OXYS rats and significantly improved structural and functional parameters of the retinal pigment epithelium cells and state of microcirculation in the choroid, which, presumably, contributed to preservation of photoreceptors, associative and ganglion neurons. It was found that the activity of p38 MAPK and ERK1/2 in the retina of 12-month-old OXYS rats is higher than that of the Wistar rats of the same age, as indicated by the increased content of phosphorylated forms of p38 MAPK and ERK1/2 and their target protein tau (at position T181 and S396). Visomitin decreased phosphorylation of p38 MAPK, ERK1/2, and tau indicating suppression of activity of these MAPK signaling cascades. Thus, Visomitin eye drops are able to suppress progression of the AMD-like pathology in the OXYS rats and their effect is associated with the decrease in activity of the MAPK signaling cascades.

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.

Mechanisms of PM10 Disruption of the Nrf2 Pathway in Cornea.

We have previously shown that PM10 exposure causes oxidative stress and reduces Nrf2 protein levels, and SKQ1 pre-treatment protects against this damage in human corneal epithelial cells (HCE-2). The current study focuses on uncovering the mechanisms underlying acute PM10 toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 µg/mL PM10. Cell viability, oxidative stress markers, programmed cell death, DNA damage, senescence markers, and pro-inflammatory cytokines were analyzed. Nrf2 cellular location and its transcriptional activity were determined. Effects of the Nrf2 inhibitor ML385 were similarly evaluated. Data showed that PM10 decreased cell viability, Nrf2 transcriptional activity, and mRNA levels of antioxidant enzymes, but increased p-PI3K, p-NFκB, COX-2, and iNOS proteins levels. Additionally, PM10 exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and ß-galactosidase (ß-gal) staining, which confirmed the senescence. SKQ1 pre-treatment reversed these effects. ML385 lowered the Nrf2 protein levels and mRNA levels of its downstream targets. ML385 also abrogated the protective effects of SKQ1 against PM10 toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM10 induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.

SkQ3 Exhibits the Most Pronounced Antioxidant Effect on Isolated Rat Liver Mitochondria and Yeast Cells.

Oxidative stress is involved in a wide range of age-related diseases. A critical role has been proposed for mitochondrial oxidative stress in initiating or promoting these pathologies and the potential for mitochondria-targeted antioxidants to fight them, making their search and testing a very urgent task. In this study, the mitochondria-targeted antioxidants SkQ1, SkQ3 and MitoQ were examined as they affected isolated rat liver mitochondria and yeast cells, comparing SkQ3 with clinically tested SkQ1 and MitoQ. At low concentrations, all three substances stimulated the oxidation of respiratory substrates in state 4 respiration (no ADP addition); at higher concentrations, they inhibited the ADP-triggered state 3 respiration and the uncoupled state, depolarized the inner mitochondrial membrane, contributed to the opening of the mPTP (mitochondrial permeability transition pore), did not specifically affect ATP synthase, and had a pronounced antioxidant effect. SkQ3 was the most active antioxidant, not possessing, unlike SkQ1 or MitoQ, prooxidant activity with increasing concentrations. In yeast cells, all three substances reduced prooxidant-induced intracellular oxidative stress and cell death and prevented and reversed mitochondrial fragmentation, with SkQ3 being the most efficient. These data allow us to consider SkQ3 as a promising potential therapeutic agent to mitigate pathologies associated with oxidative stress.

Mitochondria-targeted SkQ1 nanoparticles for dry eye disease: Inhibiting NLRP3 inflammasome activation by preventing mitochondrial DNA oxidation.

Dry eye disease (DED) is a multifactorial ocular surface disorder mutually promoted by reactive oxygen species (ROS) and ocular surface inflammation. NLRP3 is the key regulator for inducing ocular surface inflammation in DED. However, the mechanism by which ROS influences the bio-effects of NLRP3, and the consequent development of DED, largely remains elusive. In the present study, we uncovered that robust ROS can oxidate mitochondrial DNA (ox-mtDNA) along with loss of mitochondria compaction causing the cytosolic release of ox-mtDNA and subsequent co-localization with cytosolic NLRP3, which can promote the activation of NLRP3 inflammasome and stimulate NLRP3-mediated inflammation. Visomitin (also known as SkQ1), a mitochondria-targeted anti-oxidant, could reverse such a process by in situ scavenging of mitochondrial ROS. To effectively deliver SkQ1, we further developed a novel mitochondria-targeted SkQ1 nanoparticle (SkQ1 NP) using a charge-driven self-assembly strategy. Compared with free SkQ1, SkQ1 NPs exhibited significantly higher cytosolic- and mitochondrial-ROS scavenging activity (1.7 and 1.9 times compared to levels of the free SkQ1 group), thus exerting a better in vitro protective effect against H2O2-induced cell death in human corneal epithelial cells (HCECs). After topical administration, SkQ1 NPs significantly reduced in vivo mtDNA oxidation, while suppressing the expressions of NLRP3, Caspase-1, and IL-1ß, which consequently resulted in better therapeutic effects against DED. Results suggested that by efficiently scavenging mitochondrial ROS, SkQ1 NPs could in situ inhibit DED-induced mtDNA oxidation, thus blocking the interaction of ox-mtDNA and NLRP3; this, in turn, suppressed NLRP3 inflammasome activation and NLRP3-mediated inflammatory signaling. Results suggested that SkQ1 NPs have great potential as a new treatment for DED.

Repurposing an Antioxidant to Kill Mycobacterium tuberculosis by Targeting the 50S Subunit of the Ribosome.

Tuberculosis and drug-resistant TB remain serious threats to global public health. It is urgent to develop novel anti-TB drugs in order to control it. In addition to redesigning and developing new anti-TB drugs, drug repurposing is also an innovative way to develop antibacterial drugs. Based on this method, we discovered SKQ-1 in the FDA-approved drug library and evaluated its anti-TB activity. In vitro, we demonstrated that SKQ-1 engaged in bactericidal activity against drug-sensitive and -resistant Mtb and confirmed the synergistic effects of SKQ1 with RIF and INH. Moreover, SKQ-1 showed a significant Mtb-killing effect in macrophages. In vivo, both the SKQ-1 treatment alone and the treatment in combination with RIF were able to significantly reduce the bacterial load and improve the survival rate of G. mellonella infected with Mtb. We performed whole-genome sequencing on screened SKQ-1-resistant strains and found that the SNP sites were concentrated in the 50S ribosomal subunit of Mtb. Furthermore, we proved that SKQ-1 can inhibit protein translation. In summary, from the perspective of drug repurposing, we discovered and determined the anti-tuberculosis effect of SKQ-1, revealed its synergistic effects with RIF and INH, and demonstrated its mechanism of action through targeting ribosomes and disrupting protein synthesis, thus making it a potential treatment option for DR-TB.

Cardiolipin-mediated temporal response to hydroquinone toxicity in human retinal pigmented epithelial cell line.

Hydroquinone, a potent toxic agent of cigarette smoke, damages retinal pigmented epithelial cells by triggering oxidative stress and mitochondrial dysfunction, two events causally related to the development and progression of retinal diseases. The inner mitochondrial membrane is enriched in cardiolipin, a phospholipid susceptible of oxidative modifications which determine cell-fate decision. Using ARPE-19 cell line as a model of retinal pigmented epithelium, we analyzed the potential involvement of cardiolipin in hydroquinone toxicity. Hydroquinone exposure caused an early concentration-dependent increase in mitochondrial reactive oxygen species, decrease in mitochondrial membrane potential, and rise in the rate of oxygen consumption not accompanied by changes in ATP levels. Despite mitochondrial impairment, cell viability was preserved. Hydroquinone induced cardiolipin translocation to the outer mitochondrial membrane, and an increase in the colocalization of the autophagosome adapter protein LC3 with mitochondria, indicating the induction of protective mitophagy. A prolonged hydroquinone treatment induced pyroptotic cell death by cardiolipin-mediated caspase-1 and gasdermin-D activation. Cardiolipin-specific antioxidants counteracted hydroquinone effects pointing out that cardiolipin can act as a mitochondrial "eat-me signal" or as a pyroptotic cell death trigger. Our results indicate that cardiolipin may act as a timer for the mitophagy to pyroptosis switch and propose cardiolipin-targeting compounds as promising approaches for the treatment of oxidative stress-related retinal diseases.

Mitochondrial Oxidative Stress and Mitophagy Activation Contribute to TNF-Dependent Impairment of Myogenesis.

Many muscular pathologies are associated with oxidative stress and elevated levels of the tumor necrosis factor (TNF) that cause muscle protein catabolism and impair myogenesis. Myogenesis defects caused by TNF are mediated in part by reactive oxygen species (ROS), including those produced by mitochondria (mitoROS), but the mechanism of their pathological action is not fully understood. We hypothesized that mitoROS act by triggering and enhancing mitophagy, an important tool for remodelling the mitochondrial reticulum during myogenesis. We used three recently developed probes-MitoTracker Orange CM-H2TMRos, mito-QC, and MitoCLox-to study myogenesis in human myoblasts. Induction of myogenesis resulted in a significant increase in mitoROS generation and phospholipid peroxidation in the inner mitochondrial membrane, as well as mitophagy enhancement. Treatment of myoblasts with TNF 24 h before induction of myogenesis resulted in a significant decrease in the myoblast fusion index and myosin heavy chain (MYH2) synthesis. TNF increased the levels of mitoROS, phospholipid peroxidation in the inner mitochondrial membrane and mitophagy at an early stage of differentiation. Trolox and SkQ1 antioxidants partially restored TNF-impaired myogenesis. The general autophagy inducers rapamycin and AICAR, which also stimulate mitophagy, completely blocked myogenesis. The autophagy suppression by the ULK1 inhibitor SBI-0206965 partially restored myogenesis impaired by TNF. Thus, suppression of myogenesis by TNF is associated with a mitoROS-dependent increase in general autophagy and mitophagy.

Airborne Exposure of the Cornea to PM10 Induces Oxidative Stress and Disrupts Nrf2 Mediated Anti-Oxidant Defenses.

The purpose of this study is to test the effects of whole-body animal exposure to airborne particulate matter (PM) with an aerodynamic diameter of <10 µm (PM10) in the mouse cornea and in vitro. C57BL/6 mice were exposed to control or 500 µg/m3 PM10 for 2 weeks. In vivo, reduced glutathione (GSH) and malondialdehyde (MDA) were analyzed. RT-PCR and ELISA evaluated levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and inflammatory markers. SKQ1, a novel mitochondrial antioxidant, was applied topically and GSH, MDA and Nrf2 levels were tested. In vitro, cells were treated with PM10 ± SKQ1 and cell viability, MDA, mitochondrial ROS, ATP and Nrf2 protein were tested. In vivo, PM10 vs. control exposure significantly reduced GSH, corneal thickness and increased MDA levels. PM10-exposed corneas showed significantly higher mRNA levels for downstream targets, pro-inflammatory molecules and reduced Nrf2 protein. In PM10-exposed corneas, SKQ1 restored GSH and Nrf2 levels and lowered MDA. In vitro, PM10 reduced cell viability, Nrf2 protein, and ATP, and increased MDA, and mitochondrial ROS; while SKQ1 reversed these effects. Whole-body PM10 exposure triggers oxidative stress, disrupting the Nrf2 pathway. SKQ1 reverses these deleterious effects in vivo and in vitro, suggesting applicability to humans.

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.

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.

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 glutamate/GABA system in the retina of male rats: effects of aging, neurodegeneration, and supplementation with melatonin and antioxidant SkQ1.

Glutamate and -aminobutyric acid (GABA) are the most abundant amino acids in the retina. An imbalance of the glutamate/GABA system is involved in the pathogenesis of various neurodegenerative disorders. Here we for the first time analyzed alterations of expression of glutamate- and GABA-synthesizing enzymes, transporters, and relevant receptors in the retina with age in Wistar rats and in senescence-accelerated OXYS rats who develop AMD-like retinopathy. We noted consistent age-dependent expression changes of GABAergic-system proteins (GAD67, GABA-T, and GAT1) in OXYS and Wistar rats: upregulation by age 3 months and downregulation at age 18 months. At a late stage of AMD-like retinopathy in OXYS rats (18 months), there was significant upregulation of glutaminase and downregulation of glutamine synthetase, possibly indicating an increasing level of glutamate in the retina. AMD-like-retinopathy development in the OXYS strain was accompanied by underexpression of glutamate transporter GLAST. Prolonged supplementation with both melatonin and SkQ1 (separately) suppressed the progression of the AMD-like pathology in OXYS rats without affecting the glutamate/GABA system but worsened the condition of the Wistar rat's retina during normal aging. We observed decreasing protein levels of glutamine synthetase, GLAST, and GABAAR1 and an increasing level of glutaminase in Wistar rats. In summary, both melatonin and mitochondrial antioxidant SkQ1 had different effect on the retinal glutamate / GABA in healthy Wistar and senescence-accelerated OXYS rats.

[Electrophysiological and psychophysical studies in assessment of visual functions in patients with hereditary optic neuropathy]. / Elektrofiziologicheskie i psikhofizicheskie issledovaniya v otsenke zritel'nykh funktsii pri nasledstvennoi opticheskoi neiropatii.

PURPOSE: 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). MATERIAL AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Targeting whole body metabolism and mitochondrial bioenergetics in the drug development for Alzheimer's disease.

Aging is by far the most prominent risk factor for Alzheimer's disease (AD), and both aging and AD are associated with apparent metabolic alterations. As developing effective therapeutic interventions to treat AD is clearly in urgent need, the impact of modulating whole-body and intracellular metabolism in preclinical models and in human patients, on disease pathogenesis, have been explored. There is also an increasing awareness of differential risk and potential targeting strategies related to biological sex, microbiome, and circadian regulation. As a major part of intracellular metabolism, mitochondrial bioenergetics, mitochondrial quality-control mechanisms, and mitochondria-linked inflammatory responses have been considered for AD therapeutic interventions. This review summarizes and highlights these efforts.

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.

SkQ1 as a Tool for Controlling Accelerated Senescence Program: Experiments with OXYS Rats.

According to the concept suggested by V. P. Skulachev and co-authors, aging of living organisms can be considered as a special case of programmed death of an organism - phenoptosis, and mitochondrial antioxidant SkQ1 is capable of inhibiting both acute and chronic phenoptosis (aging). The authors of the concept associate effects of SkQ1 with suppression of the enhanced generation of ROS in mitochondria. Numerous studies have confirmed the ability of SkQ1 to inhibit manifestations of the "healthy", or physiological, aging. According to the results of our studies, SkQ1 is especially effective in suppressing the program of genetically determined accelerated senescence in OXYS rats, which appears as an early development of a complex of age-related diseases: cataracts, retinopathy (similar to the age-related macular degeneration in humans), osteoporosis, and signs of Alzheimer's disease. Accelerated senescence in OXYS rats is associated with mitochondrial dysfunction, but no direct associations with oxidative stress have been identified. Nevertheless, SkQ1 is able to prevent and/or suppress development of all manifestations of accelerated senescence in OXYS rats. Its effects are due to impact on the activity of many signaling pathways and processes, but first of all they are associated with restoration of the structural and functional parameters of mitochondria. It could be suggested that the use of SkQ1 could represent a promising strategy in prevention of accelerated phenoptosis - early development of a complex of age-related diseases (multimorbidity) in people predisposed to it.