Functional State Of Mitochondria Research Articles

Abstract 7063: Monitoring metabolic plasticity in the tumor microenvironment in vivo by mitophagy generated extracellular vesicles

Abstract Tumor metabolic reprogramming is a hallmark of cancer progression, survival, and therapeutic resistance. A targetable class of cancer metabolic adaptation exploits mitophagy known to be linked to the cancer phenotype. Mitophagy selectively eliminates dysfunctional mitochondria by targeting them, via autophagosome shuttling, to the lysosome for degradation. Cancer cell mitophagy is triggered by elevated oxidative stress and mitochondrial DNA damage caused by hypoxia, radiotherapy, and immunotherapy. A high mitophagy demand can overwhelm the lysosome capacity resulting in the accumulation of damaged mitochondria and can suppress biogenesis of healthy mitochondria. We hypothesize that the newly discovered secretory mitophagy mechanism exports damaged mitochondrial fission-released segments to reduce the overload pressure on the lysosomal system, and thereby sustains cancer cell survival in the face of therapeutic mitochondrial stress. We have discovered a form of secretory mitophagy occurring in vivo in a growing solid tumor. Our molecular analysis of the full repertoire of extracellular vesicles, EVs, shed into the resident tumor interstitial fluid, IF, in vivo yielded a rich set of information about the functional state of mitochondria within the tumor cells, and the host cells. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81 positive IF EVs, including PINK1, DRP1 and FIS1. The export of cellular mitochondria proteins to CD81 positive EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, Mito Tracker export into CD81 positive EVs, ultrastructural characterization and Exoview analyzer. Further, we stimulated mitochondrial oxidative stress and blocked the fusion of the mitophagosome with the lysosome, which markedly stimulated the export of the secretory mitophagy unit. The outcome is new understanding of the importance of secretory mitophagy that can constitute a therapeutic target, and a new clinically relevant means of monitoring the in vivo state of mitophagic flux within the tumor microenvironment by means of mitophagy derived EVs. Citation Format: Purva V. Gade, Marissa Howard, Angela Rojas, Layla Hasanzadah, Lance Liotta, Fatah Kashanchi. Monitoring metabolic plasticity in the tumor microenvironment in vivo by mitophagy generated extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7063.

Laquinimod attenuates oxidative stress-induced mitochondrial injury and alleviates intervertebral disc degeneration by inhibiting the NF-κB signaling pathway

BackgroundLow back pain (LBP) caused by intervertebral disc degeneration (IVDD) is a significant global health concern. It is necessary to investigate the underlying pathological mechanisms leading to IVDD and develop precise treatment strategies for this condition. Considering the well-established anti-inflammatory properties and ability to reduce oxidative stress in various diseases, for the first time we aim to explore the potential of Laquinimod in alleviating IVDD. MethodsWe used hydrogen peroxide (H2O2) to simulate the oxidative stress microenvironment in IVDD, and Laquinimod for intervention purposes. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay were used to measure the expression levels of inflammatory cytokines, catabolic enzymes, and markers of extracellular matrix (ECM) synthesis in nucleus pulposus (NP) cells. In addition, dichlorofluorescin-diacetate (DCFH-DA) and JC-1 fluorescent probes, flow cytometry analysis, and qRT-PCR were used to measure mitochondrial function and apoptosis in NP cells under conditions of oxidative stress. An acupuncture-induced rat model of IVDD was established to further evaluate the efficacy of Laquinimod in alleviating IVDD in vivo. ResultsOur findings showed that Laquinimod significantly reduced the oxidative stress-induced inflammatory response in NP cells, downregulated the expression of catabolic enzymes, and markedly enhanced ECM degradation by inhibiting the NF-κB signaling pathway. The administration of Laquinimod concurrently improved the mitochondrial functional state and reduced apoptosis in NP cells. Additionally, in vivo experiments in rats showed that Laquinimod significantly alleviated acupuncture-induced IVDD. ConclusionsCollectively, the findings of this study provide new insights into the therapeutic potential of Laquinimod as a treatment for oxidative stress-induced IVDD.

Performance of TMRM and Mitotrackers in mitochondrial morphofunctional analysis of primary human skin fibroblasts

Mitochondrial membrane potential (Δψ) and morphology are considered key readouts of mitochondrial functional state. This morphofunction can be studied using fluorescent dyes (“probes”) like tetramethylrhodamine methyl ester (TMRM) and Mitotrackers (MTs). Although these dyes are broadly used, information comparing their performance in mitochondrial morphology quantification and Δψ-sensitivity in the same cell model is still scarce. Here we applied epifluorescence microscopy of primary human skin fibroblasts to evaluate TMRM, Mitotracker Red CMXros (CMXros), Mitotracker Red CMH2Xros (CMH2Xros), Mitotracker Green FM (MG) and Mitotracker Deep Red FM (MDR). All probes were suited for automated quantification of mitochondrial morphology parameters when Δψ was normal, although they did not deliver quantitatively identical results. The mitochondrial localization of TMRM and MTs was differentially sensitive to carbonyl cyanide-4-phenylhydrazone (FCCP)-induced Δψ depolarization, decreasing in the order: TMRM ≫ CHM2Xros = CMXros = MDR > MG. To study the effect of reversible Δψ changes, the impact of photo-induced Δψ “flickering” was studied in cells co-stained with TMRM and MG. During a flickering event, individual mitochondria displayed subsequent TMRM release and uptake, whereas this phenomenon was not observed for MG. Spatiotemporal and computational analysis of the flickering event provided evidence that TMRM redistributes between adjacent mitochondria by a mechanism dependent on Δψ and TMRM concentration. In summary, this study demonstrates that: (1) TMRM and MTs are suited for automated mitochondrial morphology quantification, (2) numerical data obtained with different probes is not identical, and (3) all probes are sensitive to FCCP-induced Δψ depolarization, with TMRM and MG displaying the highest and lowest sensitivity, respectively. We conclude that TMRM is better suited for integrated analysis of Δψ and mitochondrial morphology than the tested MTs under conditions that Δψ is not substantially depolarized.

Inhibition of mitochondrial fusion via SIRT1/PDK2/PARL axis breaks mitochondrial metabolic plasticity and sensitizes cancer cells to glucose restriction therapy

Mitochondria dynamically change their morphology via fusion and fission, a process called mitochondrial dynamics. Dysregulated mitochondrial dynamics respond rapidly to metabolic cues, and are linked to the initiation and progression of diverse human cancers. Metabolic adaptations significantly contribute to tumor development and escape from tissue homeostatic defenses. In this work, we identified oroxylin A (OA), a dual GLUT1/mitochondrial fusion inhibitor, which restricted glucose catabolism of hepatocellular carcinoma cells and simultaneously inhibited mitochondrial fusion by disturbing SIRT1/PDK2/PARL axis. Based the dual action of OA in metabolic regulation and mitochondrial dynamics, further results revealed that mitochondrial functional status and spare respiratory capacity (SRC) of cancer cells had a close correlation with mitochondrial metabolic plasticity, and played important roles in the susceptibility to cancer therapy aiming at glucose restriction. Cancer cells with healthy mitochondria and high SRC exhibit greater metabolic flexibility and higher resistance to GLUT1 inhibitors. This phenomenon is attributed to the fact that high SRC cells fuse mitochondria in response to glucose restriction, enhancing tolerance to energy deficiency, but undergo less mitochondrial oxidative stress compared to low SRC cells. Thus, inhibiting mitochondrial fusion breaks mitochondrial metabolic plasticity and increases cancer cell susceptibility to glucose restriction therapy. Collectively, these finding indicate that combining a GLUT1 inhibitor with a mitochondrial fusion inhibitor can work synergistically in cancer therapy and, more broadly, suggest that the incorporations of mitochondrial dynamics and metabolic regulation may become the targetable vulnerabilities bypassing the genotypic heterogeneity of multiple malignancies.

Mito-TEMPO mitigates 5-fluorouracil-induced intestinal injury via attenuating mitochondrial oxidative stress, inflammation, and apoptosis: an in vivo study

BackgroundRecent evidences highlight role of mitochondria in the development of 5-fluorouracil (5-FU)-induced intestinal toxicity. Mitochondria-targeted antioxidants are well-known for their protective effects in mitochondrial oxidative stress- mediated diseases. In the present study, we investigated protective effect of Mito-TEMPO in 5-FU-induced intestinal toxicity.MethodsMito-TEMPO (0.1 mg/kg b.w.) was administered intraperitoneally to male BALB/c mice for 7 days, followed by co-administration of 5-FU for next 4 days (intraperitoneal 12 mg/kg b.w.). Protective effect of Mito-TEMPO on intestinal toxicity was assessed in terms of histopathological alterations, modulation in inflammatory markers, apoptotic cell death, expression of 8-OhDG, mitochondrial functional status and oxidative stress.Results5-FU administered animals showed altered intestinal histoarchitecture wherein a shortening and atrophy of the villi was observed. The crypts were disorganized and inflammatory cell infiltration was noted. Mito-TEMPO pre-protected animals demonstrated improved histoarchitecture with normalization of villus height, better organized crypts and reduced inflammatory cell infiltration. The inflammatory markers and myeloperoxidase activity were normalized in mito-TEMPO protected group. A significant reduction in intestinal apoptotic cell death and expression of 8-OhDG was also observed in mito-TEMPO group as compared to 5-FU group. Further, mtROS, mtLPO and mitochondrial antioxidant defense status were improved by mito-TEMPO.ConclusionMito-TEMPO exerted significant protective effect against 5-FU-induced intestinal toxicity. Therefore, it may be used as an adjuvant in 5-FU chemotherapy.

A mitochondria-targeted dual-response sensor for monitoring viscosity and peroxynitrite in living cells with distinct fluorescence signals

Viscosity and peroxynitrite (ONOO−) are two significant indicators to affect and evaluate the mitochondrial functional status, which are nearly relational with pathophysiological process in many diseases. Developing suitable analytical methods for monitoring mitochondrial viscosity changes and ONOO− is thus of great importance. In this research, a new mitochondria-targeted sensor DCVP-NO2 for the dual determination of viscosity and ONOO− was exploited based on the coumarin skeleton. DCVP-NO2 displayed a red fluorescence “turn-on” response toward viscosity along with about 30-fold intensity increase. Meanwhile, it could be used as ratiometric probe for detection of ONOO− with excellent sensitivity and extraordinary selectivity for ONOO− over other chemical and biological species. Moreover, thanks to its good photostability, low cytotoxicity and ideal mitochondrion-targeting capability, DCVP-NO2 was successfully utilized for fluorescence imaging of viscosity variations and ONOO− in mitochondria of living cells through different channels. In addition, the results of cell imaging revealed that ONOO− would lead to the increase of viscosity. Taken together, this work provides a potential molecular tool for researching biological functions and interactions of viscosity and ONOO− in mitochondria.

Abstract 4829: Monitoring metabolic plasticity in the tumor microenvironment in vivo

Abstract Tumor metabolic reprogramming is a hallmark of cancer progression, survival, and therapeutic resistance. A targetable class of cancer metabolic adaptation exploits mitophagy, a specialized autophagy pathway known to be linked to the cancer phenotype. Mitophagy selectively eliminates dysfunctional mitochondria by targeting them, via autophagosome shuttling, to the lysosome for degradation. Cancer cell mitophagy is triggered by elevated oxidative stress and mitochondria DNA damage caused by hypoxia, radiotherapy, molecular therapy, and immunotherapy. A high mitophagy demand can overwhelm the lysosome capacity resulting in the accumulation of damaged mitochondria that is harmful to the cell, and can suppress biogenesis of healthy mitochondria. We hypothesize that the newly discovered process of secretory mitophagy exports damaged mitochondrial fission-released segments to reduce the overload pressure on the lysosomal system, and thereby sustains cancer cell survival in the face of therapeutic mitochondrial stress. We have discovered a form of secretory mitophagy occurring in vivo in a growing solid tumor. Our molecular analysis of the full repertoire of extracellular vesicles (EV) shed into the resident tumor interstitial fluid (IF) in vivo yielded a rich set of information about the functional state of mitochondria within the tumor cells, and the host cells. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; including PINK1 and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial pinching), VDAC-1 (ubiquitination state triggers mitophagy away from apoptosis), and VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, MitoTracker export into CD81+ EVs, and ultrastructural characterization. Further, we stimulated mitochondrial oxidative stress and blocked the fusion of the mitophagosome with the lysosome, which markedly stimulated the export of the secretory mitophagy unit. We also found that mitophagy inducer PINK1 cleavage status (full length versus cleaved), is prominently reflected in the set of mitochondrial proteins exported within IF EVs, and may constitute a new quantitative measurement tool to monitor the real-time state of tumor intracellular mitophagy. The outcome is new understanding of the importance of secretory mitophagy that can constitute an important therapeutic target, and a new clinically relevant means of monitoring the in vivo state of mitophagic flux within the tumor microenvironment. Citation Format: Marissa Ashton Howard. Monitoring metabolic plasticity in the tumor microenvironment in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4829.

Cardioprotective potential of mitochondria-targeted antioxidant, mito-TEMPO, in 5-fluorouracil-induced cardiotoxicity

PurposeThe mitochondria-targeted antioxidants (MTAs) are known to offer protection against mitochondrial oxidative stress. The recent evidences support their role in mitigating oxidative stress-induced diseases, including cancer. Therefore, this study investigated cardioprotective potential of mito-TEMPO against 5-FU-induced cardiotoxicity.MethodsMito-TEMPO was administered to male BALB/C mice (intraperitoneally, 0.1 mg/kg b.w. for 7 days) followed by intraperitoneal administration of 5- FU (12 mg/kg b.w. for 4 days). During this period, mito-TEMPO treatment was also continued. The cardioprotective potential of mito-TEMPO was assessed by evaluating cardiac injury markers, extent of non-viable myocardium and histopathological alterations. Mitochondrial functional status and mitochondrial oxidative stress were assessed in cardiac tissue. 8-OHdG expression and apoptotic cell death were assessed using immunohistochemical techniques.ResultsThe level of cardiac injury markers CK-MB and AST were significantly (P ≤ 0.05) decreased in mito-TEMPO pre-protected group which was further reflected in histopathology as decrease in the percentage of non-viable myocardial tissue, disorganization, and loss of myofibrils. Mito-TEMPO ameliorated mtROS, mtLPO and conserved mitochondrial membrane potential. Further, it had significantly (P ≤ 0.05) improved the activity of mitochondrial complexes and mitochondrial enzymes. A significant (P ≤ 0.05) increase in the level of mtGSH, activity of mitochondrial glutathione reductase, glutathione peroxidase, and mitochondrial superoxide dismutase was observed. A decreased expression of 8-OHdG and reduced apoptotic cell death were observed in mito-TEMPO pre-protected group.ConclusionMito-TEMPO effectively mitigated 5-FU-induced cardiotoxicity by modulating mitochondrial oxidative stress, hence may serve as a protective agent/adjuvant in 5-FU-based combinatorial chemotherapy.

Adaptogenic Properties of 1-(Germatran-1-il)-Oxyethylamine

Background: Germanium is a biologically active trace element, and it is present in almost all organs and tissues. Its biological activity was revealed in the 20th century. However, the study on the possibility of using germanium for medical purposes was first undertaken by the Japanese scientist Dr. Kazuhiko Asai in 1940. In 1965, academician M.G.Voronkov and colleagues synthesized tricyclic esters of triethanolamine germanium with the general formula XGe(OCH2CH2)3N and studied their biological activity. However, the adaptogenic properties of these compounds have not been sufficiently studied. In this regard, there is an urgent need to study the adaptogenic properties of these drugs. Objective: As the resistance of the organism to stress factors primarily depends on energy metabolism, the aim of our work was to study the influence of stress and 1- (germatran-1-il) –oxyethylamine (GM) on the functional state of mitochondria. Methods: The functional state of mitochondria was studied as per the rate of mitochondria respiration by the level of lipid peroxidation and fatty acid composition of mitochondrial membranes by chromatography technique. Results: It was shown that the drug in concentrations of 10-5, 10-6, and 10-11M reduced the intensity of LPO in the membranes of "aged" mitochondria. This may serve as evidence regarding the presence of anti-stress properties in the drug. Injection of GM at a dose of 10-5 mol/kg to rats prevented the activation of LPO in the membranes of the liver mitochondria in conditions of acute hypobaric hypoxia. Restricting lipid peroxidation, GM prevented changes in the content of C18 and C22 fatty acids in mitochondrial membranes, which probably contributed to maintaining the bioenergetic characteristics of mitochondria at the control level. Conclusion: It is assumed that the anti-stress activity of the drug is associated with its antioxidant properties and its effect on the complex I of the mitochondrial respiratory chain.

XBP1 modulates endoplasmic reticulum and mitochondria crosstalk via regulating NLRP3 in renal ischemia/reperfusion injury

The functional status of mitochondria and the endoplasmic reticulum are central to renal ischemia/reperfusion injury (IRI). X-box binding protein 1 (XBP1) is an important transcription factor in endoplasmic reticulum stress. NLR family pyrin domain containing-3 (NLRP3) inflammatory bodies are closely related to renal IRI. In vivo and in vitro, we examined the molecular mechanisms and functions of XBP1-NLRP3 signaling in renal IRI, which influences ER-mitochondrial crosstalk. In this study, mice were subjected to 45 min of unilateral renal warm ischemia, the other kidney resected, and reperfusion was performed for 24 h in vivo. In vitro, murine renal tubular epithelial cells (TCMK-1) were exposed to hypoxia for 24 h and reoxygenation for 2 h. Tissue or cell damage was evaluated by measuring blood urea nitrogen and creatinine levels, histological staining, flow cytometry, terminal deoxynucleotidyl transferase-mediated nick-end labeling, diethylene glycol staining, and transmission electron microscopy (TEM). Western blotting, immunofluorescence staining, and ELISA were used to analyze protein expression. Whether XBP1 regulates the NLRP3 promoter was evaluated using a luciferase reporter assay. Kidney damage was reduced with decreasing blood urea nitrogen, creatinine, interleukin-1β, and interleukin-18 levels. XBP1 deficiency reduced tissue damage and cell apoptosis, protecting the mitochondria. Disruption of XBP1 was associated with reduced NLRP3 and cleaved caspase-1 levels and markedly improved survival. In vitro in TCMK-1 cells, XBP1 interference inhibited caspase-1-dependent mitochondrial damage and reduced the production of mitochondrial reactive oxygen species. The luciferase assay showed that spliced XBP1 isoforms enhanced the activity of the NLRP3 promoter. These findings reveal that XBP1 downregulation suppresses the expression of NLRP3, a potential regulator of endoplasmic reticulum mitochondrial crosstalk in nephritic injury and a potential therapeutic target in XBP1-mediated aseptic nephritis.

Red light photobiomodulation rescues murine brain mitochondrial respiration after acute hypobaric hypoxia

Low-level laser therapy, or photobiomodulation, utilizes red or near-infrared light for the treatment of pathological conditions due to the presence of intracellular photoacceptors, such as mitochondrial cytochrome c oxidase, that serve as intermediates for the therapeutic effects. We present an in-detail analysis of the effect of low-intensity LED red light irradiation on the respiratory chain of brain mitochondria. We tested whether low-level laser therapy at 650nm could alleviate the brain mitochondrial dysfunction in the model of acute hypobaric hypoxia in mice. The irradiation of the mitochondrial fraction of the left cerebral cortex with low-intensity LED red light rescued Complex I-supported respiration during oxidative phosphorylation, normalized the initial polarization of the inner mitochondrial membrane, but has not shown any significant effect on the activity of Complex IV. In comparison, the postponed effect (in 24h) of the similar transcranial irradiation following hypoxic exposure led to a less pronounced improvement of the mitochondrial functional state, but normalized respiration related to ATP production and membrane polarization. In contrast, the similar irradiation of the mitochondria isolated from control healthy animals exerted an inhibitory effect on CI-supported respiration. The obtained results provide significant insight that can be beneficial for the development of non-invasive phototherapy.

The coupling of mitoproteolysis and oxidative phosphorylation enables tracking of an active mitochondrial state through MitoTimer fluorescence

The regulation of mitochondria function and health is a central node in tissue maintenance, ageing as well as the pathogenesis of various diseases. However, the maintenance of an active mitochondrial functional state and its quality control mechanisms remain incompletely understood. By studying mice with a mitochondria-targeted reporter that shifts its fluorescence from “green” to “red” with time (MitoTimer), we found MitoTimer fluorescence spectrum was heavily dependent on the oxidative metabolic state in the skeletal muscle fibers. The mitoproteolytic activity was enhanced in an energy dependent manner, and accelerated the turnover of MitoTimer protein and respiratory chain substrate, responsible for a green predominant MitoTimer fluorescence spectrum under the oxidative conditions. PGC1α, as well as anti-ageing regents promoted enhanced mitoproteolysis. In addition, cells with the green predominant mitochondria exhibited lower levels of MitoSox and protein carbonylation, indicating a favorable redox state. Thus, we identified MitoTimer as a probe for mitoproteolytic activity in vivo and found a heightened control of mitoproteolysis in the oxidative metabolic state, providing a framework for understanding the maintenance of active oxidative metabolism while limiting oxidative damages.

Functional state of mitochondria in chronic respiratory diseases

Introduction. Chronic respiratory diseases are one of the most common types of non-communicable diseases and are an important problem of our time. The induction of oxidative stress, chronic inflammation and hypoxia, which underlie the pathogenesis of chronic diseases of the bronchopulmonary system, can be determined at the cellular and molecular level by impaired mitochondrial functioning.Aim. This review is devoted to the prospects for assessing the functional state of mitochondria as a fine indicator of the course of chronic respiratory diseases.Results. The data of domestic and foreign sources on the most important parameters of mitochondrial functioning in normal and chronic bronchopulmonary pathology were analyzed. It has been shown that mitochondria are highly sensitive to changes in both exogenous and endogenous homeostasis. Functional parameters of mitochondria, the level of mitochondrial reactive oxygen species, mitochondrial membrane potential, and fatty acid composition of mitochondrial membranes can be used as diagnostic and prognostic criteria for chronic respiratory diseases. The data presented in the review indicate the need for further studies of the functional state of mitochondria in chronic bronchopulmonary pathology.

The activator/repressor Hap1 binds to the yeast eIF5A-encoding gene TIF51A to adapt its expression to the mitochondrial functional status.

Mitochondrial activity adapts to cellular energetic and metabolic demands; its dysfunction is a hallmark of ageing and many human diseases. The evolutionarily conserved translation elongation factor eIF5A is involved in maintaining mitochondrial function. In humans, eIF5A is encoded by two highly homologous but differentially expressed genes; in yeast, these are TIF51A and TIF51B. We show that yeast transcription factor Hap1 constitutively binds to the TIF51A promoter to activate its expression under respiration, but represses its expression under nonrespiration conditions by recruiting the corepressor Tup1. Hap1 indirectly regulates TIF51B expression by binding to and activating the TIF51B repressor genes ROX1 and MOT3 under respiration and repressing them under nonrespiration. Thus, the levels of eIF5A isoforms are adapted to the mitochondrial functional status.

EFFECT OF GABA AND GLUTAMIC ACID DERIVATIVES ON THE FUNCTIONAL ACTIVITY OF LIVER CELL MITOCHONDRIA IN VITRO 147 UNDER CONDITIONS OF OXIDATIVE STRESS

The aim of the study was to study the effect of GABA derivatives on the functional activity of intact and oxidative stress-damaged mitochondria. Isolated mitochondria were obtained by differential centrifugation. Oxidative damage was simulated by incubation with tert-Butylhydroperoxide (200 nmol/ml, 10 min). The studied substances (baclofen, mefebut, neuroglutam, RGPU-238 compound, salifen, tolibut, phenibut, phenotropil) were added to intact and damaged mitochondria (1*10-5 M per 100 μl, 10 min of incubation), the functional state of mitochondria was studied using a polarographic method. It was revealed that salifen and phenotropil had a pronounced protective effect on the respiratory complexes of the electron transport chain.

Lipopolysaccharide From E. coli Increases Glutamate-Induced Disturbances of Calcium Homeostasis, the Functional State of Mitochondria, and the Death of Cultured Cortical Neurons

Lipopolysaccharide (LPS), a fragment of the bacterial cell wall, specifically interacting with protein complexes on the cell surface, can induce the production of pro-inflammatory and apoptotic signaling molecules, leading to the damage and death of brain cells. Similar effects have been noted in stroke and traumatic brain injury, when the leading factor of death is glutamate (Glu) excitotoxicity too. But being an amphiphilic molecule with a significant hydrophobic moiety and a large hydrophilic region, LPS can also non-specifically bind to the plasma membrane, altering its properties. In the present work, we studied the effect of LPS from Escherichia coli alone and in combination with the hyperstimulation of Glu-receptors on the functional state of mitochondria and Ca2+ homeostasis, oxygen consumption and the cell survival in primary cultures from the rats brain cerebellum and cortex. In both types of cultures, LPS (0.1–10 μg/ml) did not change the intracellular free Ca2+ concentration ([Ca2+]i) in resting neurons but slowed down the median of the decrease in [Ca2+]i on 14% and recovery of the mitochondrial potential (ΔΨm) after Glu removal. LPS did not affect the basal oxygen consumption rate (OCR) of cortical neurons; however, it did decrease the acute OCR during Glu and LPS coapplication. Evaluation of the cell culture survival using vital dyes and the MTT assay showed that LPS (10 μg/ml) and Glu (33 μM) reduced jointly and separately the proportion of live cortical neurons, but there was no synergism or additive action. LPS-effects was dependent on the type of culture, that may be related to both the properties of neurons and the different ratio between neurons and glial cells in cultures. The rapid manifestation of these effects may be the consequence of the direct effect of LPS on the rheological properties of the cell membrane.

Fluorescence and Photoacoustic Spectroscopy-Based Assessment of Mitochondrial Dysfunction in Oral Cancer Together with Machine Learning: A Pilot Study.

The current study reports an integrated approach of machine learning and tryptophan fluorescence and photoacoustic spectral properties to assess the mitochondrial status under oral pathological conditions. The mitochondria in the study were isolated from oral cancer tissues and adjacent normal counterparts, and the corresponding fluorescence and photoacoustic spectra of tryptophan were recorded at 281 nm pulsed laser excitations. A set of features were selected from the pre-processed spectra and were used to classify the data using support vector machine (SVM) learning in the MATLAB platform. SVM analysis demonstrated clear differentiation between mitochondria isolated from normal and cancer tissues for fluorescence (sensitivity, 86.6%; specificity, 90%) and photoacoustic (sensitivity, 86.6%; specificity, 96.6%) measurements. Further investigation into the influence of change in protein conformation on the nature of tryptophan spectral properties was evaluated by 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence assay. The impact of protein structural changes on the mitochondrial functions was also estimated by mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and cytochrome c oxidase (COX) assays, suggesting an altered mitochondrial function. The findings indicate that tryptophan fluorescence and photoacoustic spectral properties together with machine learning algorithms may delineate the mitochondrial functional status in vitro, indicating its translational potential.

Selective striatal cell loss is ameliorated by regulated autophagy of the cortex

AimsThe harmful cellular environment leads to brain damage, and each brain subregion exhibits a differential vulnerability to its effects. This study investigated the causes of selectively striatal cell loss in systemic 3-nitropropionic acid (3-NP) infused mice. Main methodsThis study was performed in the neuronal cell line, primary neuron, cultured mouse brain, and mice brain tissues. The 3-NP solution was delivered using an osmotic mini-pump system for 7 days. ROS in brain tissue were detected and evaluated with the signals of CM-H2DCFDA for total cellular ROS and MitoSOX Red for mitochondrial ROS. Cellular ROS and the functional status of mitochondria were assessed with a detection kit and analyzed using flow cytometry. To quantify oxidative damaged DNA, apurinic/apyrimidinic (AP) site numbers in DNA were measured. The protein expression level was assessed using Western blotting, and immunohistochemistry was performed. Cleaved caspase-3 activities were measured by using an enzyme-linked immunosorbent assay (ELISA) kit. Key findingsBy 3-NP, mitochondrial dysfunction was higher in the striatum than in the cortex, and mitochondria-derived ROS levels were higher in the striatum than in the cortex. However, autophagy that may restore the energy depletion resulting from mitochondrial dysfunction occurred comparably less in the striatum than in the cortex. Inhibition of ASK1 by NQDI1 regulates MAPK signaling, apoptosis, and autophagy. Regulated autophagy of the cortex improved non-cell autonomously striatal damaged condition. SignificanceThis study illustrated that the different vulnerabilities of the brain subregions, striatum or cortex, against 3-NP are rooted in different mitochondria-derived ROS amounts and autophagic capacity.

Оксидантный стресс и тканевая гипоксия как факторы, способствующие развитию дисфункции предстательной железы и мочевого пузыря при метаболическом синдроме

Introduction. The development of oxidative stress and nonspecific inflammation is one of the leading factors in the development of benign prostatic hyperplasia (BPH) and associated urination disorders in metabolic syndrome (MS). However, the specific mechanisms of these processes are not entirely clear. The purpose of the study. To study the activity of reactive oxygen species production and the functional state of mitochondria in the prostate and bladder and their role in the dysfunction of these organs using an experimental model of MS induction in rats. Material and methods. In 10 adult mongrel male rats MS was induced by keeping them on a high-calorie diet with an increased content of carbohydrates and fats for 3 months. 10 rats kept on a standard vivarium diet served as controls. The development of MS was confirmed by characteristic changes in the biochemical analysis of blood (hyperglycemia, hyperuricemia, dyslipidemia, hyperinsulinemia). In both series of rats, sections of the native prostate and bladder were examined by laser confocal microscopy and stained with fluorescent probes that characterize the activity of the production of reactive oxygen species (dichlorofluorescein-DCF) and the functional state of the mitochondria (tetramethylrodamine ether – TMRE). The activity of a number of intracellular enzymes (AST, ALT. Alkaline phosphatase, LDH) was investigated in the tissues and urine. Results. In rats with MS, the development of BPH and hypertrophy of the bladder were revealed, confirmed histologically. The study of sections of both organs by confocal microscopy revealed a significant increase in the production of reactive oxygen species by their cells and a decrease in the functional activity of mitochondria, which indicated the development of oxidant stress and tissue hypoxia. In the prostate, this was accompanied by a decrease in the secretory activity of the prostate glands, and in the bladder – the release of cytoplasmic enzymes from damaged cells into the urine, indicating cell damage. Conclusion. The causes of the development of a non-specific inflammatory process in the prostate and bladder, leading to dysfunction of these organs in MS, are increased production of reactive oxygen species and the development of tissue hypoxia.

SGLT2 inhibitors break the vicious circle between heart failure and insulin resistance: targeting energy metabolism.

Heart failure (HF) often coexists with insulin resistance (IR), and the incidence of HF in type 2 diabetes mellitus (T2DM) patients is significantly higher. The reciprocal relationship between HF and IR has long been recognized, and the integration complicates the therapy of both. A number of mechanisms ascribe to the progression of cardiac IR, in which the main factors are the shift of myocardial substrate metabolism. Studies have found that SGLT2 inhibitors, an anti-diabetic drug, can improve the cardiac prognosis of patients with T2DM, which may be at least partially due to the relief of cardiac IR. Basic and clinical studies have revealed the important role of cardiac IR in the pathogenesis and progression of HF, and studies suggest that energy metabolism plays an important role in the pathogenesis of cardiac IR and HF. SGLT2 inhibitors mediated cardiovascular benefits through various mechanisms such as improving substrate utilization and improving myocardial energy. The regulation of SGLT2 inhibitors on cardiac energy status including carbohydrates, fatty acids (FA), amino acids and ketones, ATP transfer to the cytoplasm, and mitochondrial functional status have received extensive attention in HF, but its specific mechanism of action is still unclear. Therefore, this article reviews the relationship between IR and HF from the perspective of energy metabolism; subsequently, targeting energy metabolism discusses the pivotal role of SGLT2 inhibitors in improving cardiac IR and HF based on basic and clinical research evidences, and sought to clarify the molecular mechanism involved. (Fig.1).