Activities Of Respiratory Chain Complexes Research Articles

The Effects of Antofine on the Morphological and Physiological Characteristics of Phytophthora capsici.

Phytophthora capsici is an important plant pathogenic oomycete that causes great losses to vegetable production around the world. Antofine is an important alkaloid isolated from Cynanchum komarovii Al. Iljinski and exhibits significant antifungal activity. In this study, the effect of antofine on the mycelial growth, morphology, and physiological characteristics of P. capsici was investigated using colorimetry. Meanwhile, the activity of mitochondrial respiratory chain complexes of P. capsici was evaluated following treatment with a 30% effective concentration (EC30), as well as EC50 and EC70, of antofine for 0, 12, 24, and 48 h. The results showed that antofine had a significant inhibitory effect against P. capsici, with an EC50 of 5.0795 μg/mL. After treatment with antofine at EC50 and EC70, the mycelia were rough, less full, and had obvious depression; they had an irregular protrusion structure; and they had serious wrinkles. In P. capsici, oxalic acid and exopolysaccharide contents decreased significantly, while cell membrane permeability and glycerol content increased when treated with antofine. Reactive oxygen species (ROS) entered a burst state in P. capsici after incubation with antofine for 3 h, and fluorescence intensity was 2.43 times higher than that of the control. The activities of the mitochondrial respiratory chain complex II, III, I + III, II + III, V, and citrate synthase in P. capsici were significantly inhibited following treatment with antofine (EC50 and EC70) for 48 h compared to the control. This study revealed that antofine is likely to affect the pathways related to the energy metabolism of P. capsici and thus affect the activity of respiratory chain complexes. These results increase our understanding of the action mechanism of antofine against P. capsici.

NDUFA9 and its crotonylation modification promote browning of white adipocytes by activating mitochondrial function in mice

Protein crotonylation plays a role in regulating cellular metabolism, gene expression, and other biological processes. NDUFA9 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9) is closely associated with the activity and function of mitochondrial respiratory chain complex I. Mitochondrial function and respiratory chain are closely related to browning of white adipocytes, it’s speculated that NDUFA9 and its crotonylation are associated with browning of white adipocytes. Firstly, the effect of NDUFA9 on white adipose tissue was verified in white fat browning model mice, and it was found that NDUFA9 promoted mitochondrial respiration, thermogenesis, and browning of white adipose tissue. Secondly, in cellular studies, it was discovered that NDUFA9 facilitated browning of white adipocytes by enhancing mitochondrial function, mitochondrial complex I activity, ATP synthesis, and mitochondrial respiration. Again, the level of NDUFA9 crotonylation was increased by treating cells with vorinostat (SAHA)+sodium crotonate (NaCr) and overexpressing NDUFA9, it was found that NDUFA9 crotonylation promoted browning of white adipocytes. Meanwhile, the acetylation level of NDUFA9 was increased by treating cells with SAHA+sodium acetate (NaAc) and overexpressing NDUFA9, the assay revealed that NDUFA9 acetylation inhibited white adipocytes browning. Finally, combined with the competitive relationship between acetylation and crotonylation, it was also demonstrated that NDUFA9 crotonylation promoted browning of white adipocytes. Above results indicate that NDUFA9 and its crotonylation modification promote mitochondrial function, which in turn promotes browning of white adipocytes. This study establishes a theoretical foundation for the management and intervention of obesity, which is crucial in addressing obesity and related medical conditions in the future.

Matrine attenuating cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity through improved mitochondrial membrane potential and activation of mitochondrial respiratory chain Complex I pathway

The study aimed to demonstrate that matrine can reduce apoptosis in H9c2 cells induced by the cardiotoxic anticancer drug doxorubicin (DOX).The researchers pretreated H9c2 cells with different concentrations of matrine before exposing them to DOX and cultured them for 24 h. They assessed cell survival rates using cell counting kit-8 and MTT assay. Hoechst 33258 dye kits were used to determine apoptosis, while laser confocal JC-1 method was applied to test the mitochondrial membrane potential (MMP). Complex I activities were detected following the manufacturer's protocol. The results indicated that matrine pretreatment significantly increased the survival rate of H9c2 cells injured by DOX. Additionally, matrine reduced apoptosis in H9c2 cells through the improvement of MMP and activity of Complex I, which were damaged by DOX.

Enhancing radiosensitivity in triple-negative breast cancer through targeting ELOB.

Enhancing radiotherapy sensitivity is crucial for improving treatment outcomes in triple-negative breast cancer (TNBC) patients. In this study, we investigated the potential of targeting Elongin B (ELOB) to enhance radiotherapy efficacy in TNBC. Analysis of TNBC patient cohorts revealed a significant association between high ELOB expression and poor prognosis in patients who received radiation therapy. Mechanistically, we found that ELOB plays a pivotal role in regulating mitochondrial function via modulating mitochondrial DNA expression and activities of respiratory chain complexes. Targeting ELOB effectively modulated mitochondrial function, leading to enhanced radiosensitivity in TNBC cells. Our findings highlight the importance of ELOB as a potential therapeutic target for improving radiotherapy outcomes in TNBC. Further exploration of ELOB's role in enhancing radiotherapy efficacy may provide valuable insights for developing novel treatment strategies for TNBC patients.

Regulation of metastatic potential by drug repurposing and mitochondrial targeting in colorectal cancer cells

BackgroundIncreased mitochondrial activities contributing to cancer cell proliferation, invasion, and metastasis have been reported in different cancers; however, studies on the therapeutic targeting of mitochondria in regulating cell proliferation and invasiveness are limited. Because mitochondria are believed to have evolved through bacterial invasion in mammalian cells, antibiotics could provide an alternative approach to target mitochondria, especially in cancers with increased mitochondrial activities. In this study, we investigated the therapeutic potential of bacteriostatic antibiotics in regulating the growth potential of colorectal cancer (CRC) cells, which differ in their metastatic potential and mitochondrial functions.MethodsA combination of viability, cell migration, and spheroid formation assays was used to measure the effect on metastatic potential. The effect on mitochondrial mechanisms was investigated by measuring mitochondrial DNA copy number by qPCR, biogenesis (by qPCR and immunoblotting), and functions by measuring reactive oxygen species, membrane potential, and ATP using standard methods. In addition, the effect on assembly and activities of respiratory chain (RC) complexes was determined using blue native gel electrophoresis and in-gel assays, respectively). Changes in metastatic and cell death signaling were measured by immunoblotting with specific marker proteins and compared between CRC cells.ResultsBoth tigecycline and tetracycline effectively reduced the viability, migration, and spheroid-forming capacity of highly metastatic CRC cells. This increased sensitivity was attributed to reduced mtDNA content, mitochondrial biogenesis, ATP content, membrane potential, and increased oxidative stress. Specifically, complex I assembly and activity were significantly inhibited by these antibiotics in high-metastatic cells. Significant down-regulation in the expression of mitochondrial-mediated survival pathways, such as phospho-AKT, cMYC, phospho-SRC, and phospho-FAK, and upregulation in cell death (apoptosis and autophagy) were observed, which contributed to the enhanced sensitivity of highly metastatic CRC cells toward these antibiotics. In addition, the combined treatment of the CRC chemotherapeutic agent oxaliplatin with tigecycline/tetracycline at physiological concentrations effectively sensitized these cells at early time points.ConclusionAltogether, our study reports that bacterial antibiotics, such as tigecycline and tetracycline, target mitochondrial functions specifically mitochondrial complex I architecture and activity and would be useful in combination with cancer chemotherapeutics for high metastatic conditions.

Increased O-GlcNAcylation by Upregulation of Mitochondrial O-GlcNAc Transferase (mOGT) Inhibits the Activity of Respiratory Chain Complexes and Controls Cellular Bioenergetics.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. The interplay between O-GlcNAcylation and phosphorylation is critical to control signaling pathways and maintain cellular homeostasis. The addition of O-GlcNAc moieties to target proteins is catalyzed by O-linked N-acetylglucosamine transferase (OGT). Of the three splice variants of OGT described, one is destined for the mitochondria (mOGT). Although the effects of O-GlcNAcylation on the biology of normal and cancer cells are well documented, the role of mOGT remains poorly understood. In this manuscript, the effects of mOGT on mitochondrial protein phosphorylation, electron transport chain (ETC) complex activity, and the expression of VDAC porins were investigated. We performed studies using normal and breast cancer cells with upregulated mOGT or its catalytically inactive mutant. Proteomic approaches included the isolation of O-GlcNAc-modified proteins of the electron transport chain, followed by their analysis using mass spectrometry. We found that mitochondrial OGT regulates the activity of complexes I-V of the respiratory chain and identified a group of 19 ETC components as mOGT substrates in mammary cells. Furthermore, we observed that the upregulation of mOGT inhibited the interaction of VDAC1 with hexokinase II. Our results suggest that the deregulation of mOGT reprograms cellular energy metabolism via interaction with and O-GlcNAcylation of proteins involved in ATP production in mitochondria and its exchange between mitochondria and the cytosol.

Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes

In the last decades enormous advances have been made in characterizing the atomic and molecular structure of respiratory chain supercomplexes [1]. However, it still remains a challenge to stitch this refined spatial atomistic description with functional information provided by biochemical studies of isolated protein material. Development of functional assays that detect respiratory chain complexes in their native membrane environment contribute to address the open questions related to the role played by their association and interactions. We present a characterization assay in which a functionalized gold electrode is modified with mitochondrial membrane fragments that allows monitoring electrochemically the activity of different respiratory chain complexes immersed in the mitochondrial membrane. We measure the intensity of the reducing current of the electron mediator CoQ1 at the electrode surface and its variation upon addition of the corresponding enzymatic substrates. The activities of Complex I, Complex II and Complex III were monitored by the way in which they reduce the current, reflecting the amount of quinone reduced by the complexes in the presence of their substrates. We detect that CoQ1H2 produced by Complex I remains partially trapped within the membrane and is more easily oxidized by Complex III or the electrode than the quinone reduced by Complex II.

L-Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload.

Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinalmicrobiotaimbalance. As a neutral and nonprotein amino acid, L-Citrulline (L-cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L-cit can alleviate iron overload-induced intestinal injury and explores the underlying mechanisms. C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L-cit for 2 weeks. L-cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L-cit-mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2) cells are treated with L-cit or AMP-activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L-cit. Notably, Compound C abolishes L-cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC-J2 cells. L-cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L-cit is a promising therapeutic strategy for iron overload-induced intestinal injury.

Xenobiotics Triggering Acute Intermittent Porphyria and Their Effect on Mouse Brain Respiratory Complexes.

Heme enzyme dysfunction causes a group of diseases called porphyrias. Particularly, a decrease in porphobilinogen deaminase, involved in the third step of heme biosynthesis, leads to acute intermittent porphyria (AIP). Considering our previous works demonstrating the multiplicity of brain metabolisms affected by porphyrinogenic agents, this study aimed to elucidate whether they cause any alteration on the mitochondrial respiratory chain. The activities of respiratory chain complexes (I to IV) were measured in encephalon mitochondria of CF1 male mice receiving volatile anesthetics: isoflurane (2 mL/kg) and sevoflurane (1.5 mL/kg), ethanol (30%), allylisopropylacetamide (AIA) (350 mg/kg), and barbital (167 mg/kg). Moreover, they were compared versus animals with pathological levels of 5-aminolevulinic acid (ALA, 40 mg/kg). Complex I-III activity was induced by isoflurane and decreased by AIA, ethanol, and ALA. Complex II-III activity was increased by sevoflurane and decreased by isoflurane and AIA. Complex II activity was increased by sevoflurane and barbital and decreased by AIA, ethanol, and ALA. Complex IV activity was increased by barbital and ALA and decreased by sevoflurane. The damage to the respiratory chain by ALA could be reflecting the pathophysiological condition of patients with AIP. Better understanding the broad effect of porphyrinogenic drugs and the mechanisms acting on the onset of AIP is vital in translational medicine.

JinChan YiShen TongLuo Formula ameliorate mitochondrial dysfunction and apoptosis in diabetic nephropathy through the HIF-1α-PINK1-Parkin pathway

Ethnopharmacological relevanceThe JinChan YiShen TongLuo (JCYSTL) formula, a traditional Chinese medicine (TCM), has been used clinically for decades to treat diabetic nephropathy (DN). TCM believes that the core pathogenesis of DN is “kidney deficiency and collateral obstruction," and JCYSTL has the effect of “tonifying kidney and clearing collateral," thus alleviating the damage to kidney structure and function caused by diabetes. From the perspective of modern medicine, mitochondrial damage is an important factor in DN pathogenesis. Our study suggests that the regulation of mitophagy and mitochondrial function by JCYSTL may be one of the internal mechanisms underlying its good clinical efficacy. Aim of the studyThis study aimed to investigate the mechanisms underlying the renoprotective effects of JCYSTL. Materials and methodsUnilateral nephrectomy combined with low-dose streptozotocin intraperitoneally injected in a DN rat model and high glucose (HG) plus hypoxia-induced HK-2 cells were used to explore the effects of JCYSTL on the HIF-1α/mitophagy pathway, mitochondrial function and apoptosis. ResultsJCYSTL treatment significantly decreased albuminuria, serum creatinine, blood urea nitrogen, and uric acid levels and increased creatinine clearance levels in DN rats. In vitro, medicated serum containing JCYSTL formula increased mitochondrial membrane potential (MMP); improved activities of mitochondrial respiratory chain complexes I, III, and IV; decreased the apoptotic cell percentage and apoptotic protein Bax expression; and increased anti-apoptotic protein Bcl-2 expression in HG/hypoxia-induced HK-2 cells. The treatment group exhibited increased accumulation of PINK1, Parkin, and LC3-II and reduced P62 levels in HG/hypoxia-induced HK-2 cells, whereas in PINK1 knockdown HK-2 cells, JCYSTL did not improve the HG/hypoxia-induced changes in Parkin, LC3-II, and P62. When mitophagy was impaired by PINK1 knockdown, the inhibitory effect of JCYSTL on Bax and its promoting effect on MMP and Bcl-2 disappeared. The JCYSTL-treated group displayed significantly higher HIF-1α expression than the model group in vivo, which was comparable to the effects of FG-4592 in DN rats. PINK1 knockdown did not affect HIF-1α accumulation in JCYSTL-treated HK-2 cells exposed to HG/hypoxia. Both JCYSTL and FG-4592 ameliorated mitochondrial morphological abnormalities and reduced the mitochondrial respiratory chain complex activity in the renal tubules of DN rats. Mitochondrial apoptosis signals in DN rats, such as increased Bax and Caspase-3 expression and apoptosis ratio, were weakened by JCYSTL or FG-4592 administration. ConclusionThis study demonstrates that the JCYSTL formula activates PINK1/Parkin-mediated mitophagy by stabilizing HIF-1α to protect renal tubules from mitochondrial dysfunction and apoptosis in diabetic conditions, presenting a promising therapy for the treatment of DN.

Yo-Yo Dieting Delays Male Drosophila melanogaster Aging Through Enhanced Mitochondrial Function, Relative to Sustained High-Calorie Diet Feeding.

Weight regain subsequent to weight reduction resulting from dietary interventions represents a prevalent phenomenon recognized as "Yo-yo dieting." However, the impact of prolonged Yo-yo dieting on health, especially in relation to the aging process, remains poorly understood. This study aimed to investigate the influence of Yo-yo dieting on the aging process in male Drosophila melanogaster that have been exposed to a high-calorie (HC) diet. Fruit flies were fed with either a consistent HC diet or an alternating regimen of HC and low-calorie diets every 3 days (referred to as "Yo-yo dieting") for a total of 24 days. Biochemical assays were utilized to quantify levels of oxidative stress and activities of the mitochondrial respiratory chain complexes. The frozen section staining method was employed to assess the presence of lipid droplets, reactive oxygen species, cellular viability, and mitochondrial abundance in tissues. Additionally, we examined the expression of key regulators involved in mitochondrial dynamics and biogenic signaling pathways. Yo-yo dieting resulted in an extension of the fruit flies' lifespan, concomitant with reduced body weight, decreased body protein content, and lower triglyceride levels compared to continuous a HC diet feeding. Furthermore, Yo-yo dieting ameliorated impairments in motility and intestinal barrier function. Importantly, it improved mitochondrial function and upregulated the expression of essential mitochondrial fusion proteins, namely mitofusin 1 and mitofusin 2, optic atrophy 1, and peroxisome proliferator-activated receptor-γ coactivator-1α. Therefore, the practice of Yo-yo dieting extends the lifespan of fruit flies by modulating mitochondrial dynamics and the associated biogenic signaling pathways.

Evaluation of SYP-34773's resistance risk and its impact on the activity of mitochondrial respiratory electron transport chain complex I in Phytophthora litchii.

SYP-34773 is a low-toxicity pyrimidine amine compound, which was synthesized by modifying the lead compound diflumetorim. Previous literature has shown that it can strongly inhibit the mycelial growth of several important plant pathogens, including Phytophthora litchii. However, the resistance risk of SYP-34773 has not been reported for P. litchii. The mean effective concentration (EC50 ) value of SYP-34773 against the mycelial growth of 111 P. litchii isolates was 0.108 ± 0.008 μg mL-1 , which can be used as the baseline sensitivity for SYP-34773 resistance detection in the future. Six mutants were obtained from two parental strain through fungicide induction, whose resistance factors fell between 194- and 687-fold, with stability. Results regarding mycelial growth, sporangial production, sporangial germination, zoospore release, cystspore germination, and pathogenicity showed that the mutants' compound fitness index values were significantly lower than those of their parental isolate. Furthermore, there was no cross-resistance between SYP-34773 and diflumetorim in P. litchii. Significant inhibition of the mitochondrial complex I enzyme activity in two wild-type P. litchii isolates, but not in mutants, was observed upon treatment with SYP-34773. The resistance risk of SYP-34773 in P. litchii is moderate, and resistance management strategies should be adopted in field use. SYP-34773 is a mitochondrial complex I inhibitor, and SYP-34773-resistant P. litchii isolates did not show cross-resistance against diflumetorim. © 2023 Society of Chemical Industry.

Comparative studies on the chemical composition and pharmacological effects of vinegar-processed antler glue modified from Lei Gong Pao Zhi Lun and traditional water-processed antler glue

Ethnopharmacological relevanceAntler glue is a classic medicinal to enhance sexual function in traditional Chinese medicine (TCM), which was first recorded in Shen Nong Ben Cao Jing (Shennong′s Classic of the Materia Medica). Vinegar-processing is a classic method of processing traditional Chinese medicine. The method of preparing antler glue by boiling antlers in vinegar and then concentrating them is recorded in Lei Gong Pao Zhi Lun (Master Lei′s Discourse on Medicinal Processing). In modern times, the typical processing method of antler glue is water extraction and concentration. However, it is not clear whether there is a difference in the effect of these two processing methods on the chemical composition and pharmacological activity of antler glue. Aim of the studyThe Chinese Pharmacopoeia (2020) records that the processing method of antler glue is water extraction and concentration. But Lei Gong Pao Zhi Lun differs in Chinese Pharmacopoeia (2020), which records the processing method of vinegar extraction and concentration. The effect of the two processing methods on antler glue's chemical composition and pharmacological activity is unknown. So this study aimed to elucidate the difference between different processing methods on the chemical composition and the treatment effect on oligoasthenospermia of antler glue. Materials and methodsSo the automatic amino acid analyzer is used to determine the amino acid content of two different processing methods of antler glue. Proteomics was performed to detect the protein components of two different processing methods of antler glue and analyze them. Cyclophosphamide-induced mice models of oligoasthenospermia were used to study the different pharmacological effects of antler glue in two different processing methods. An automatic sperm analyzer observed the quantity and quality of sperm in mice epididymis. Serum sex hormone testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in mice were tested using the enzyme-linked immunosorbent assay (ELISA) kits. Hematoxylin-eosin (H&E) staining was used to analyze pathological alterations in mouse testicular tissue. The transcriptome has been used to reveal the potential mechanism of antler glue in treating oligoasthenospermia. Mitochondrial complex activity assay kits were used to assay the activity of mitochondrial respiratory chain complex I–V in mouse testicular tissue. Western blot was used to determine the expression of related proteins in mouse testicular tissue. ResultsVinegar-processing can increase the alanine, proline, and glycine content in antler glue, reduce the length of protein peptides in antler glue, and produce a variety of unique proteins. Vinegar-processed antler glue (VAG) increased sperm density, sperm survival, sperm viability, and serum sex hormone levels in oligozoospermic mice. It reversed testicular damage caused by cyclophosphamide, and the effects were differently superior to those of water-processed antler glue (WAG). In addition, transcriptomics and related experiments have shown that VAG can increase the expression of Ndufa2, Uqcr11, Cox6b1, and Atp5i genes and proteins in mouse testis, thus promoting adenosine diphosphate (ATP) synthesis by increasing the activity of mitochondrial respiratory chain complexes I, III, IV and V. By promoting the oxidative phosphorylation process to produce more ATP, VAG can achieve the therapeutic effect of oligoasthenospermia. ConclusionVinegar-processing method can increase the content of active ingredients in antler glue. VAG increases ATP levels in the testes by promoting the process of oxidative phosphorylation to treat oligozoospermia.

Regular exercise attenuates alcoholic myopathy in zebrafish by modulating mitochondrial homeostasis.

Alcoholic myopathy is caused by chronic consumption of alcohol (ethanol) and is characterized by weakness and atrophy of skeletal muscle. Regular exercise is one of the important ways to prevent or alleviate skeletal muscle myopathy. However, the beneficial effects and the exact mechanisms underlying regular exercise on alcohol myopathy remain unclear. In this study, a model of alcoholic myopathy was established using zebrafish soaked in 0.5% ethanol. Additionally, these zebrafish were intervened to swim for 8 weeks at an exercise intensity of 30% of the absolute critical swimming speed (Ucrit), aiming to explore the beneficial effects and underlying mechanisms of regular exercise on alcoholic myopathy. This study found that regular exercise inhibited protein degradation, improved locomotion ability, and increased muscle fiber cross-sectional area (CSA) in ethanol-treated zebrafish. In addition, regular exercise increases the functional activity of mitochondrial respiratory chain (MRC) complexes and upregulates the expression levels of MRC complexes. Regular exercise can also improve oxidative stress and mitochondrial dynamics in zebrafish skeletal muscle induced by ethanol. Additionally, regular exercise can activate mitochondrial biogenesis and inhibit mitochondrial unfolded protein response (UPRmt). Together, our results suggest regular exercise is an effective intervention strategy to improve mitochondrial homeostasis to attenuate alcoholic myopathy.

Carnation Italian Ringspot Virus p36 Expression Induces Mitochondrial Fission and Respiratory Chain Complex Impairment in Yeast.

Positive-strand RNA virus replication invariably occurs in association with host cell membranes, which are induced to proliferate and rearrange to form vesicular structures where the virus replication complex is assembled. In particular, carnation Italian ringspot virus (CIRV) replication takes place on the mitochondrial outer membrane in plant and yeast cells. In this work, the model host Saccharomyces cerevisiae was used to investigate the effects of CIRV p36 expression on the mitochondrial structure and function through the determination of mitochondrial morphology, mitochondrial respiratory parameters, and respiratory chain complex activities in p36-expressing cells. CIRV p36 ectopic expression was shown to induce alterations in the mitochondrial network associated with a decrease in mitochondrial respiration and the activities of NADH-cyt c, succinate-cyt c (C II-III), and cytochrome c oxidase (C IV) complexes. Our results suggest that the decrease in respiratory complex activity could be due, at least in part, to alterations in mitochondrial dynamics. This yeast-based model will be a valuable tool for identifying molecular targets to develop new anti-viral strategies.

Specific role of NAD+ biosynthesis reduction mediated mitochondrial dysfunction in vascular endothelial injury induced by chronic intermittent hypoxia.

Cardiovascular diseases (CVD) are prevalent among those with obstructive sleep apnea (OSA) and are the leading cause of death in these individuals. However, due to clinical confounders, the mechanism by which OSA induces CVD is still unclear. Previous studies have shown that chronic intermittent hypoxia (CIH) and high cholesterol diet (HCD) induce distinct characteristics of atherosclerotic plaques, highlighting the specific mechanisms involved in CIH-induced vascular endothelial injury. This study aims to investigate whether nicotinamide adenine dinucleotide (NAD+) biosynthesis reduction-mediated mitochondrial dysfunction is responsible for vascular endothelial injury induced by CIH and to elucidate its specific role in this process. Models were established to stimulate human umbilical vein endothelial cells (HUVECs) with CIH and oxidized low-density lipoprotein (ox-LDL), and the NAD+ biosynthesis-related indicators, such as NAD+ levels and nicotinamide phosphoribosyltransferase (NAMPT) enzyme activity, were measured in this model. Additionally, interventions were performed by supplementing NAD+ levels with nicotinamide mononucleotide (NMN), inhibiting NAD+ synthesis with FK866, and evaluating mitochondrial function, oxidative stress status, vascular constriction and dilation function, and endothelial adhesion function in these models. A comparative study was conducted to assess the effects of these interventions. We found that under CIH conditions, NAMPT enzyme activity was inhibited, leading to a reduction in NAD+ biosynthesis and a decrease in NAD+/NADH ratio. At the same time, CIH caused mitochondrial dysfunction in HUVECs, including a decrease in adenosine triphosphate (ATP) content and mitochondrial membrane potential, as well as the activity of respiratory chain complex I and III, induced an increase in oxidative stress levels in endothelial cells, impaired vascular constriction and dilation function, and significantly increased expression of adhesion factors. The impact of CIH on endothelial cell-related mitochondrial function and endothelial function was restored by supplementing NMN. Although ox-LDL also causes multi-level endothelial injury, it does not involve the NAD+ pathway, as there were no significant changes in the related indicators, and the impaired endothelial function under ox-LDL conditions was not restored by supplementing NMN. CIH-induced vascular endothelial injury may be associated with NAD+ biosynthesis reduction-mediated mitochondrial dysfunction. Supplementing NAD+ precursors to increase its levels may be a potential intervention to ameliorate CIH-induced vascular endothelial injury, while it does not have a significant effect on endothelial injury caused by ox-LDL.

Weakened Contractile Performance and Mitochondrial Respiratory Complex Activity in Skeletal Muscle Improve during Interbout Arousal in Hibernating Daurian Ground Squirrel, Spermophilus dauricus

Mammalian hibernation is composed of multiple episodes of torpor bout, separated by phases of interbout arousal. During torpor, the skeletal muscles of mammals are undoubtedly inactive, but it has been proven to mitigate disuse atrophy. While interbout arousal has been implicated in the prevention of muscle atrophy, the underlying mechanisms sustaining muscle contraction remain to be explored. In the present study, Daurian ground squirrels (Spermophilus dauricus) were divided into four groups: pre-hibernation (PRE), torpor (TOR), interbout arousal (IBA), and post-hibernation (POST). The contractile performance of slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL) was detected both in situ and in vitro. Concurrently, mitochondrial respiratory chain complex activity in these muscles was quantified. Our findings revealed that in situ contractile properties of both muscles, including force, power output, time duration, and force development/relaxation rates of twitch contraction, and force and power output of tetanic contraction declined in the TOR group compared to the PRE group, but improved in the IBA and POST groups. Fatigue resistance of muscles, determined by the power output of repetitive tetanic contractions in situ, decreased in the TOR group but recovered in the IBA and POST groups. In vitro studies demonstrated that tetanic contraction power output in isolated muscles increased with muscle temperature in both TOR and IBA groups. However, at the same temperature, power output was consistently lower in the TOR group compared to the IBA group. Moreover, the activity of the mitochondrial respiratory chain complex, especially Complexes I and II, decreased in the TOR group but showed recovery in the IBA and POST groups. These findings suggest that both the contractile performance and fatigue resistance of mammalian skeletal muscle are compromised during torpor but can be improved during interbout arousal and post-hibernation. The rebound in body temperature and rise in mitochondrial respiratory chain complex activity in skeletal muscle are involved in enhancing contractile performance and fatigue resistance. This study suggests that interbout arousal functions as a vital temporal interval during which skeletal muscles can transition from the inactivity induced by torpor to a state of restored contractile functionality. Thus, interbout arousal serves as a behavioral safeguard against disuse-induced damage to skeletal muscles during hibernation.

METTL14 Regulates the m6A Modification of TRAF6 to Suppress Mitochondrial Dysfunction and Ferroptosis in Dopaminergic Neurons via the cGAS-STING Pathway.

The degeneration of dopaminergic (DA) neurons has emerged as a crucial pathological characteristic in Parkinson's disease (PD). To enrich the related knowledge, we aimed to explore the impact of the METTL14-TRAF6-cGASSTING axis in mitochondrial dysfunction and ferroptosis underlying DA neuron degeneration. 1-methyl-4-phenylpyridinium ion (MPP+) was used to treat DA neuron MN9D to develop the PD cell models. Afterward, a cell counting kit, flow cytometer, DCFH-DA fluorescent probe, and Dipyrromethene Boron Difluoride staining were utilized to measure the cell viability, iron concentration, ROS level, and lipid peroxidation, respectively. Meanwhile, the mitochondrial ultrastructure, the activity of mitochondrial respiratory chain complexes, and levels of malondialdehyde and glutathione were monitored. In addition, reverse transcription-quantitative polymerase chain reaction and western blot assays were adopted to measure the expression of related genes. cGAS ubiquitylation and TRAF6 messenger RNA (mRNA) N6-methyladenosine (m6A) levels, the linkages among METTL14, TRAF6, and the cGAS-STING pathway were also evaluated. METTL14 expression was low, and TRAF6 expression was high after MPP+ treatment. In MPP+-treated MN9D cells, METTL14 overexpression reduced ferroptosis, ROS generation, mitochondrial injury, and oxidative stress (OS) and enhanced mitochondrial membrane potentials. TRAF6 overexpression had promoting impacts on mitochondrial dysfunction and ferroptosis in MPP+-treated MN9D cells, which was reversed by further overexpression of METTL14. Mechanistically, METTL14 facilitated the m6A methylation of TRAF6 mRNA to down-regulate TRAF6 expression, thus inactivating the cGAS-STING pathway. METTL14 down-regulated TRAF6 expression through TRAF6 m6A methylation to inactivate the cGAS-STING pathway, thereby relieving mitochondrial dysfunction and ferroptosis in DA neurons.

Diversities in Leigh Syndrome Associated with MT-ATP6 Gene Variants.

Leigh syndrome (LS) is clinically and genetically heterogeneous and presents defective mitochondrial bioenergetics. Patients present neurological symptoms and imagiological features that may result in early death [1]. The LS has been associated with mitochondrial DNA (mtDNA) variants, e.g., m.8993T>G (L156R) and m.8993T>C (L156P), in the MT-ATP6 gene. They lead to the substitution of a highly conserved amino acid in subunit 6 of ATP synthase, affecting the F0 domain and ATP synthesis [1-3]. We present five cases with m.8993T>G and a family harbouring m.8993T>C+m.1555A>G (proband and four relatives). Our laboratory received 48 samples from LS-suspected patients. The samples (various tissues) were assessed for bioenergetics (activity of mitochondrial respiratory chain (MRC) complexes, ubiquinone content) and genetic analyses (mtDNA copy number, Sequencing and PCR-RFLP) by established protocols. Bioenergetics were assessed in 5 patients (various tissues) with varying levels of MRC/ATP synthase impairment. Six cases had a mtDNA pathogenic variant in the 8993 nucleotide associated with LS. Five cases presented the m.8993T>G variant, one of which (P5) possibly de novo. This variant was homoplasmy (P1-3) or very high heteroplasmy (P4/5, 90-95%). Of the four patients with bioenergetics assessment, three (P1/3/4) had deficiencies of MRC complexes, and P5 had small deficits. The other case (familial, proband and 4 relatives) presented a combination of m.1555A>G (homoplasmy) and m.8993T>C (heteroplasmy) variants. The proband presents m.8993T>C in 95% heteroplasmy and 85-35% in three relatives. All have m.1555A>G in homoplasmy, including the fourth relative without m.8993T>C. A deficiency (31%) was found in complex V activity in muscle for proband. We present a case series of patients harbouring pathogenic variants in the 8993 nucleotide of mtDNA, which have been associated with LS and impairment of MRC's complex V. These cases highlight the variability in clinical symptoms and their severity, as well as genetic heterogeneity within LS. Many patients will not present a classic pathogenic variant and there are many cases of asymptomatic relatives (carriers). It is important to get a broader view of the cases - classical methods and multiple tissue analysis are still valuable tools for the comprehensive characterization of patients.

Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism

Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.