Demethylzeylasteral protects against renal interstitial fibrosis by attenuating mitochondrial complex I-mediated oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Renal interstitial fibrosis (RIF) is a main pathological process in chronic kidney disease (CKD). Demethylzeylasteral (DML), a major component of Tripterygium wilfordii Hook. f., has anti-renal fibrosis effects. However, its mechanism of action remains incompletely understood. AIM OF THE STUDY: The present study was designed to comprehensively examine the effects of DML on RIF and the underlying mechanisms. MATERIALS AND METHODS: Pathological experiments were performed to determine the therapeutic effect of DML on a mouse model of UUO-induced RIF. To determine the novel mechanisms underlying the therapeutic effects of DML against RIF, a comprehensive transcriptomics analysis was performed on renal tissues, which was further verified by a series of experiments. RESULTS: Pathological and immunohistochemical staining showed that DML inhibited UUO-induced renal damage and reduced the expression of fibrosis-related proteins in mice. Transcriptomic analysis revealed that the partial subunits of mitochondrial complex (MC) I and II may be targets by which DML protects against RIF. Furthermore, DML treatment reduced mitochondrial reactive oxygen species (ROS) levels, consequently promoting ATP production and mitigating oxidative stress-induced injury in mice and cells. Notably, this protective effect was attributed to the inhibition of MC I activity, suggesting a crucial role for this specific complex in mediating the therapeutic effects of DML against RIF. CONCLUSIONS: This study provides compelling evidence that DML may be used to treat RIF by effectively suppressing mitochondrial oxidative stress injury mediated by MC I. These findings offer valuable insights into the pharmacological mechanisms of DML and its potential clinical application for patients with CKD.

Respiratory complex I in mitochondrial membrane catalyzes oversized ubiquinones.

NADH-ubiquinone (UQ) oxidoreductase (complex I) couples electron transfer from NADH to UQ with proton translocation in its membrane part. The UQ reduction step is key to triggering proton translocation. Structural studies have identified a long, narrow, tunnel-like cavity within complex I, through which UQ may access a deep reaction site. To elucidate the physiological relevance of this UQ-accessing tunnel, we previously investigated whether a series of oversized UQs (OS-UQs), whose tail moiety is too large to enter and transit the narrow tunnel, can be catalytically reduced by complex I using the native enzyme in bovine heart submitochondrial particles (SMPs) and the isolated enzyme reconstituted into liposomes. Nevertheless, the physiological relevance remained unclear because some amphiphilic OS-UQs were reduced in SMPs but not in proteoliposomes, and investigation of extremely hydrophobic OS-UQs was not possible in SMPs. To uniformly assess the electron transfer activities of all OS-UQs with the native complex I, here we present a new assay system using SMPs, which were fused with liposomes incorporating OS-UQ and supplemented with a parasitic quinol oxidase to recycle reduced OS-UQ. In this system, all OS-UQs tested were reduced by the native enzyme, and the reduction was coupled with proton translocation. This finding does not support the canonical tunnel model. We propose that the UQ reaction cavity is flexibly open in the native enzyme to allow OS-UQs to access the reaction site, but their access is obstructed in the isolated enzyme as the cavity is altered by detergent-solubilizing from the mitochondrial membrane.

Berberine targets the electron transport chain complex I and reveals the landscape of OXPHOS dependency in acute myeloid leukemia with IDH1 mutation.

Metabolic reprogramming, a newly recognized trait of tumor biology, is an intensively studied prospect for oncology medicines. For numerous tumors and cancer cell subpopulations, oxidative phosphorylation (OXPHOS) is essential for their biosynthetic and bioenergetic functions. Cancer cells with mutations in isocitrate dehydrogenase 1 (IDH1) exhibit differentiation arrest, epigenetic and transcriptional reprogramming, and sensitivity to mitochondrial OXPHOS inhibitors. In this study, we report that berberine, which is widely used in China to treat intestinal infections, acted solely at the mitochondrial electron transport chain (ETC) complex I, and that its association with IDH1 mutant inhibitor (IDH1mi) AG-120 decreased mitochondrial activity and enhanced antileukemic effect in vitro andin vivo. Our study gives a scientific rationale for the therapy of IDH1 mutant acute myeloid leukemia (AML) patients using combinatory mitochondrial targeted medicines, particularly those who are resistant to or relapsing from IDH1mi.

Qimai Feiluoping decoction inhibits mitochondrial complex I-mediated oxidative stress to ameliorate bleomycin-induced pulmonary fibrosis.

BACKGROUND: Qimai Feiluoping decoction (QM), a Traditional Chinese Medicine formula, has been included in rehabilitation program for functional disorders of discharged COVID-19 patients. QM has been proved to effectively improve the clinical symptoms and imaging signs of PF in COVID-19 convalescent patients. PURPOSE: This study to explore the pharmacological effect of QM against PF from the perspectives of imaging, pathological staining, and molecular mechanisms, and identify possible active components. METHODS: Micro-CT imaging and immunohistochemical staining were investigated to verify the therapeutic effect of QM in the bleomycin (BLM)-induced PF mouse model. The 4D-label-free proteomics analysis of lung tissues was then conducted to explore the novel mechanisms of QM against PF, which were further validated by a series of experiments. The possible components of QM in plasma and lung tissues were identified with UHPLC/IM-QTOF-MS analysis. RESULTS: The results from micro-CT imaging and pathological staining revealed that QM treatment can inhibit BLM-induced lung injury, extracellular matrix accumulation and TGF-ß expression in the mouse model with PF. The 4D-label-free proteomics analysis demonstrated that the partial subunit proteins of mitochondrial complex I and complex II might be potential targets of QM against PF. Furthermore, QM treatment can inhibit BLM-induced mitochondrial ROS content to promote ATP production and decrease oxidative stress injury in the mouse and cell models of PF, which was mediated by the inhibition of mitochondrial complex I. Finally, a total of 13 protype compounds and 15 metabolites from QM in plasma and lung tissues were identified by UHPLC/IM-QTOF-MS, and liquiritin and isoliquiritigenin from Glycyrrhizae radix et rhizoma could be possible active compounds against PF. CONCLUSION: It concludes that QM treatment could treat PF by inhibiting mitochondrial complex I-mediated mitochondrial oxidated stress injury, which could offer new insights into the pharmacological mechanisms of QM in the clinical application of PF patients.

Double administration of self-complementary AAV9NDUFS4 prevents Leigh disease in Ndufs4-/- mice.

Leigh disease, or subacute necrotizing encephalomyelopathy, a genetically heterogeneous condition consistently characterized by defective mitochondrial bioenergetics, is the most common oxidative-phosphorylation related disease in infancy. Both neurological signs and pathological lesions of Leigh disease are mimicked by the ablation of the mouse mitochondrial respiratory chain subunit Ndufs4-/-, which is part of, and crucial for, normal Complex I activity and assembly, particularly in the brains of both children and mice. We previously conveyed the human NDUFS4 gene to the mouse brain using either single-stranded adeno-associated viral 9 recombinant vectors or the PHP.B adeno-associated viral vector. Both these approaches significantly prolonged the lifespan of the Ndufs4-/- mouse model but the extension of the survival was limited to a few weeks by the former approach, whereas the latter was applicable to a limited number of mouse strains, but not to primates. Here, we exploited the recent development of new, self-complementary adeno-associated viral 9 vectors, in which the transcription rate of the recombinant gene is markedly increased compared with the single-stranded adeno-associated viral 9 and can be applied to all mammals, including humans. Either single intra-vascular or double intra-vascular and intra-cerebro-ventricular injections were performed at post-natal Day 1. The first strategy ubiquitously conveyed the human NDUFS4 gene product in Ndufs4-/- mice, doubling the lifespan from 45 to ≈100 days after birth, when the mice developed rapidly progressive neurological failure. However, the double, contemporary intra-vascular and intra-cerebroventricular administration of self-complementary-adeno-associated viral NDUFS4 prolonged healthy lifespan up to 9 months of age. These mice were well and active at euthanization, at 6, 7, 8 and 9 months of age, to investigate the brain and other organs post-mortem. Robust expression of hNDUFS4 was detected in different cerebral areas preserving normal morphology and restoring Complex I activity and assembly. Our results warrant further investigation on the translatability of self-complementary-adeno-associated viral 9 NDUFS4-based therapy in the prodromal phase of the disease in mice and eventually humans.

Antibiotic therapy does not alter mitochondrial bioenergetics in lymphocytes of patients with septic shock - A prospective cohort study.

Antibiotics may trigger alterations in mitochondrial function, which has been explored in cells culture, and in animal model of sepsis. This study sought to evaluate whether antibiotic therapy affects mitochondrial bioenergetics in a 68-patients clinical study. We studied mitochondrial respiratory rates at two time points: the first day of antibiotic administration and three days after. The Δbasal, ΔCI, ΔCII respiration, and ΔBCE respiratory rates were not different between patients administered with polymyxin, vancomycin, amoxicillin-clavulanate, and azithromycin compared to those who were not administered. Specific beta-lactams are associated with specific modifications in mitochondrial respiratory endpoints - patients who used meropenem had higher delta C2 values compared to those who did not (p = 0.03). Patients who used piperacillin-tazobactam had lower delta C1 (p = 0.03) values than those who did not, but higher delta C2 values (p = 0.02). These mitochondrial metabolic signatures in isolated lymphocytes challenges the proposed effects of antibiotics in mitochondrial bioenergetics of cell cultures, but at current status have an uncertain clinical significance.

Personalized Medicine in Mitochondrial Health and Disease: Molecular Basis of Therapeutic Approaches Based on Nutritional Supplements and Their Analogs.

Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.

Metabolomics analysis highlights Yashtimadhu (Glycyrrhiza glabra L.)-mediated neuroprotection in a rotenone-induced cellular model of Parkinson's disease by restoring the mTORC1-AMPK1 axis in autophagic regulation.

Parkinson's disease (PD) is an age-associated progressive neurodegenerative movement disorder, and its management strategies are known to cause complications with prolonged usage. We aimed to explore the neuroprotective mechanism of the Indian traditional medicine Yashtimadhu, prepared from the dried roots of Glycyrrhiza glabra L. (licorice) in the rotenone-induced cellular model of PD. Retinoic acid-differentiated IMR-32 cells were treated with rotenone (PD model) and Yashtimadhu extract. Mass spectrometry-based untargeted and targeted metabolomic profiling was carried out to discover altered metabolites. The untargeted metabolomics analysis highlighted the rotenone-induced dysregulation and Yashtimadhu-mediated restoration of metabolites involved in the metabolism of nucleic acids, amino acids, lipids, and citric acid cycle. Targeted validation of citric acid cycle metabolites showed decreased α-ketoglutarate and succinate with rotenone treatment and rescued by Yashtimadhu co-treatment. The dysregulation of the citric acid cycle by rotenone-induced energetic stress via dysregulation of the mTORC1-AMPK1 axis was prevented by Yashtimadhu. Yashtimadhu co-treatment restored rotenone-induced ATG7-dependent autophagy and eventually caspases-mediated cell death. Our analysis links the metabolic alterations modulating energy stress and autophagy, which underlies the Yashtimadhu-mediated neuroprotection in the rotenone-induced cellular model of PD.

Regulation of reverse electron transfer at mitochondrial complex I by unconventional Notch action in cancer stem cells.

Metabolic flexibility is a hallmark of many cancers where mitochondrial respiration is critically involved, but the molecular underpinning of mitochondrial control of cancer metabolic reprogramming is poorly understood. Here, we show that reverse electron transfer (RET) through respiratory chain complex I (RC-I) is particularly active in brain cancer stem cells (CSCs). Although RET generates ROS, NAD+/NADH ratio turns out to be key in mediating RET effect on CSC proliferation, in part through the NAD+-dependent Sirtuin. Mechanistically, Notch acts in an unconventional manner to regulate RET by interacting with specific RC-I proteins containing electron-transporting Fe-S clusters and NAD(H)-binding sites. Genetic and pharmacological interference of Notch-mediated RET inhibited CSC growth in Drosophila brain tumor and mouse glioblastoma multiforme (GBM) models. Our results identify Notch as a regulator of RET and RET-induced NAD+/NADH balance, a critical mechanism of metabolic reprogramming and a metabolic vulnerability of cancer that may be exploited for therapeutic purposes.

Plant hvu-MIR168-3p enhances expression of glucose transporter 1 (SLC2A1) in human cells by silencing genes related to mitochondrial electron transport chain complex I.

Diet is a crucial factor for preventing most diseases. Edible plant extracts are known to contain exosome-like nanoparticles, in which food-derived plant microRNAs are included and may serve as a novel functional component in human health. Here, we demonstrated that hvu-MIR168-3p included in the nanoparticles of rice aleurone cells down-regulated the expression of the genes related to mitochondrial electron transport chain complex I in human cells. Subsequently, hvu-MIR168-3p enhanced protein and RNA expression levels of glucose transporter I and caused a decrease in the blood glucose level, which findings were obtained by in vitro and in vivo experiments, respectively. These findings suggest that a cross-kingdom relationship between plants and humans with respect to hvu-MIR168-3p exists and may contribute to preventive medicine for GLUT1-related dysfunctions including glucose metabolism, aging, and tumor immunology.

A value-added application of eugenol as acaricidal agent: The mechanism of action and the safety evaluation.

Introduction: Eugenol is a major component of essential oils of several plants, it exhibits significant antiparasitic and acaricidal activities, yet its molecular targets remain unknown. Objectives: We aimed to systematically investigate the mechanism of action and the potential targets of eugenol against P. cuniculi, and evaluate the safety for laying the theoretical foundation for clinical application as an acaricide. Methods: Using RNA-Seq analysis, surface plasmon resonance analysis and RNA interference assay, the mode of action of eugenol against Psoroptes cuniculi was investigated. The effect on the mitochondrial membrane potential and complex I of PC12 cells and C6/36 cells was assayed to investigate the species specificity of eugenol in insects and mammals. Finally, a safety evaluation of eugenol in vivo was performed. Results: Eugenol inhibited complex I activity of the mitochondrial respiratory chain in the oxidative phosphorylation pathway by binding to NADH dehydrogenase chain 2 and resulted in the death of mites. The inhibition rates were 37.89% for 50 µg/mL and 60.26% for 100 µg/mL, respectively. Further experiments indicated that the difference in the complex I sequence between insects and mammals led to the different affinity of eugenol to specific peptide, resulting in species specificity. Eugenol exhibited significant inhibitory effects against the mitochondrial membrane potential and complex I in Aedes albopictus C6/36 cells but was not active in rat PC12 cells. Insect cells were particularly sensitive to eugenol. In contrast to the known inhibitor rotenone, eugenol had better safety and did not result in Parkinson's disease or other diseases in rats. Conclusion: This is the first report on acaricidal eugenol targeting complex I of the mitochondrial respiratory chain. This work lays the foundation for the development of eugenol as an environmentally alternative acaricidal agent.

Downregulation of the tyrosine degradation pathway extends Drosophila lifespan.

Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived Drosophila melanogaster. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity.

Functional interactions between complex I and complex II with nNOS in regulating cardiac mitochondrial activity in sham and hypertensive rat hearts.

Nitric oxide (NO) affects mitochondrial activity through its interactions with complexes. Here, we investigated regulations of complex I (C-I) and complex II (C-II) by neuronal NO synthase (nNOS) in the presence of fatty acid supplementation and the impact on left ventricular (LV) mitochondrial activity from sham and angiotensin II (Ang-II)-induced hypertensive (HTN) rats. Our results showed that nNOS protein was expressed in sham and HTN LV mitochondrial enriched fraction. In sham, oxygen consumption rate (OCR) and intracellular ATP were increased by palmitic acid (PA) or palmitoyl-carnitine (PC). nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC), did not affect OCR or cellular ATP increment by PA or PC. However, SMTC increased OCR with PA + malonate (a C-II inhibitor), but not with PA + rotenone (a C-I inhibitor), indicating that nNOS attenuates C-I with fatty acid supplementation. Indeed, SMTC increased C-I activity but not that of C-II. Conversely, nNOS-derived NO was increased by rotenone + PA in LV myocytes. In HTN, PC increased the activity of C-I but reduced that of C-II, consequently OCR was reduced. SMTC increased both C-I and C-II activities with PC, resulted in OCR enhancement in the mitochondria. Notably, SMTC increased OCR only with rotenone, suggesting that nNOS modulates C-II-mediated OCR in HTN. nNOS-derived NO was partially reduced by malonate + PA. Taken together, nNOS attenuates C-I-mediated mitochondrial OCR in the presence of fatty acid in sham and C-I modulates nNOS activity. In HTN, nNOS attenuates C-I and C-II activities whereas interactions between nNOS and C-II maintain mitochondrial activity.

Magnolia extract is effective for the chemoprevention of oral cancer through its ability to inhibit mitochondrial respiration at complex I.

BACKGROUND: Magnolia extract (ME) is known to inhibit cancer growth and metastasis in several cell types in vitro and in animal models. However, there is no detailed study on the preventive efficacy of ME for oral cancer, and the key components in ME and their exact mechanisms of action are not clear. The overall goal of this study is to characterize ME preclinically as a potent oral cancer chemopreventive agent and to determine the key components and their molecular mechanism(s) that underlie its chemopreventive efficacy. METHODS: The antitumor efficacy of ME in oral cancer was investigated in a 4-nitroquinoline-1-oxide (4NQO)-induced mouse model and in two oral cancer orthotopic models. The effects of ME on mitochondrial electron transport chain activity and ROS production in mouse oral tumors was also investigated. RESULTS: ME did not cause detectable side effects indicating that it is a promising and safe chemopreventive agent for oral cancer. Three major key active compounds in ME (honokiol, magnolol and 4-O-methylhonokiol) contribute to its chemopreventive effects. ME inhibits mitochondrial respiration at complex I of the electron transport chain, oxidizes peroxiredoxins, activates AMPK, and inhibits STAT3 phosphorylation, resulting in inhibition of the growth and proliferation of oral cancer cells. CONCLUSION: Our data using highly relevant preclinical oral cancer models, which share histopathological features seen in human oral carcinogenesis, suggest a novel signaling and regulatory role for mitochondria-generated superoxide and hydrogen peroxide in suppressing oral cancer cell proliferation, progression, and metastasis. Video abstract.

Pathogenic variants in NUBPL result in failure to assemble the matrix arm of complex I and cause a complex leukoencephalopathy with thalamic involvement.

Disorders of the white matter are genetically very heterogeneous including several genes involved in mitochondrial bioenergetics. Diagnosis of the underlying cause is aided by pattern recognition on neuroimaging and by next-generation sequencing. Recently, genetic changes in the complex I assembly factor NUBPL have been characterized by a consistent recognizable pattern of leukoencephalopathy affecting deep white matter including the corpus callosum and cerebellum. Here, we report twin boys with biallelic variants in NUBPL, an unreported c.351 G > A; p.(Met117Ile) and a previously reported pathological variant c. 693 + 1 G > A. Brain magnetic resonance imaging showed abnormal T2 hyperintense signal involving the periventricular white matter, external capsule, corpus callosum, and, prominently, the bilateral thalami. The neuroimaging pattern evolved over 18 months with marked diffuse white matter signal abnormality, volume loss, and new areas of signal abnormality in the cerebellar folia and vermis. Magnetic resonance spectroscopy showed elevated lactate. Functional studies in cultured fibroblasts confirmed pathogenicity of the genetic variants. Complex I activity of the respiratory chain was deficient spectrophotometrically and on blue native gel with in-gel activity staining. There was absent assembly and loss of proteins of the matrix arm of complex I when traced with an antibody to NDUFS2, and incomplete assembly of the membrane arm when traced with an NDUFB6 antibody. There was decreased NUBPL protein on Western blot in patient fibroblasts compared to controls. Compromised NUBPL activity impairs assembly of the matrix arm of complex I and produces a severe, rapidly-progressive leukoencephalopathy with thalamic involvement on MRI, further expanding the neuroimaging phenotype.

Araucaria angustifolia (Bertol.) Kuntze has neuroprotective action through mitochondrial modulation in dopaminergic SH-SY5Y cells.

Brain disorders (BD) including neuropsychiatric and neurodegenerative diseases, are often associated with impairments in mitochondrial function and oxidative damage that can lead to neuronal injury. The mitochondrial complex I enzyme is one of the main sites of ROS generation and is implicated in many BD pathophysiologies. Despite advances in therapeutics for BD management, conventional pharmacotherapy still cannot efficiently control neuronal redox imbalance and mitochondrial dysfunction. Araucaria angustifolia is one of the main pine species in South America and presents a notable therapeutic history in folk medicine. A. angustifolia extract (AAE), obtained from the natural waste named bracts, is rich in flavonoids; molecules able to regulate cell redox metabolism. We examined the effects of AAE on rotenone-induced mitochondrial complex I dysfunction in human dopaminergic SH-SY5Y cells. AAE restored complex I assembly and activity mainly through overexpression of NDUFS7 protein and NDUFV2 gene levels. These findings were accompanied by a reduction in the generation of neuronal reactive oxygen species and lipid peroxidation. Our data demonstrates, for the first time, that AAE exerts in vitro neuroprotective effects, thus making it an interesting source for future drug development in BD-associated mitochondrial dysfunctions.

Targeting of reactive isolevuglandins in mitochondrial dysfunction and inflammation.

Inflammation is a major cause of morbidity and mortality in Western societies. Despite use of multiple drugs, both chronic and acute inflammation still represent major health burdens. Inflammation produces highly reactive dicarbonyl lipid peroxidation products such as isolevuglandins which covalently modify and cross-link proteins via lysine residues. Mitochondrial dysfunction has been associated with inflammation; however, its molecular mechanisms and pathophysiological role are still obscure. We hypothesized that inflammation-induced isolevuglandins contribute to mitochondrial dysfunction and mortality. To test this hypothesis, we have (a) investigated the mitochondrial dysfunction in response to synthetic 15-E2-isolevuglandin (IsoLG) and its adducts; (b) developed a new mitochondria-targeted scavenger of isolevuglandins by conjugating 2-hydroxybenzylamine to the lipophilic cation triphenylphosphonium, (4-(4-aminomethyl)-3-hydroxyphenoxy)butyl)-triphenylphosphonium (mito2HOBA); (c) tested if mito2HOBA protects from mitochondrial dysfunction and mortality using a lipopolysaccharide model of inflammation. Acute exposure to either IsoLG or IsoLG adducts with lysine, ethanolamine or phosphatidylethanolamine inhibits mitochondrial respiration and attenuates Complex I activity. Complex II function was much more resistant to IsoLG. We confirmed that mito2HOBA markedly accumulates in isolated mitochondria and it is highly reactive with IsoLGs. To test the role of mitochondrial IsoLGs, we studied the therapeutic potential of mito2HOBA in lipopolysaccharide mouse model of sepsis. Mito2HOBA supplementation in drinking water (0.1 g/L) to lipopolysaccharide treated mice increased survival by 3-fold, improved complex I-mediated respiration, and histopathological analyses supported mito2HOBA-mediated protection of renal cortex from cell injury. These data support the role of mitochondrial IsoLG in mitochondrial dysfunction and inflammation. We conclude that reducing mitochondrial IsoLGs may be a promising therapeutic target in inflammation and conditions associated with mitochondrial oxidative stress and dysfunction.

The severity of rat liver injury by fructose and high fat depends on the degree of respiratory dysfunction and oxidative stress induced in mitochondria.

BACKGROUND: High fat or fructose induces non-alcoholic fatty liver disease (NAFLD) accompanied of mitochondrial dysfunction and oxidative stress. Controversy remains about whether fructose or fat is more deleterious for NAFLD development. To get more insights about this issue and to determine if the severity of liver disease induced by fructose or fat is related to degree of mitochondrial dysfunction, we compared the effects of diets containing high fat (HF), fructose (Fr) or high fat plus fructose (HF + Fr) on NAFLD development, mitochondrial function, ROS production and lipid peroxidation. METHODS: Wistar rats were assigned to four groups: Control, fed with standard rodent chow; High fat (HF), supplemented with lard and hydrogenated vegetable oil; Fructose (Fr), supplemented with 25% fructose in the drinking water; High fat plus fructose group (HF + Fr), fed with both HF and Fr diets. Rats were sacrificed after 6 weeks of diets consumption and the liver was excised for histopathological analysis by hematoxylin and eosin staining and for mitochondria isolation. Mitochondrial function was evaluated by measuring both mitochondrial respiration and complex I activity. Lipid peroxidation and ROS production were evaluated in mitochondria by the thiobarbituric acid method and with the fluorescent ROS probe 2,4-H2DCFDA, respectively. RESULTS: Fr group underwent the lower degree of both liver damage and mitochondrial dysfunction that manifested like less than 20% of hepatocytes with microvesicular steatosis and partial decrease in state 3 respiration, respectively. HF group displayed an intermediate degree of damage as it showed 40% of hepatocytes with microvesicular steatosis and diminution of both state 3 respiration and complex I activity. HF + Fr group displayed more severe damage as showed microvesicular steatosis in 60% of hepatocytes and inflammation, while mitochondria exhibited fully inhibited state 3 respiration, impaired complex I activity and increased ROS generation. Exacerbation of mitochondrial lipid peroxidation was observed in both the Fr and HF + Fr groups. CONCLUSION: Severity of liver injury induced by fructose or fat was related to the degree of dysfunction and oxidative damage in mitochondria. Attention should be paid on the serious effects observed in the HF + Fr group as the typical Western diet is rich in both fat and carbohydrates.

Complex I syndrome in striatum and frontal cortex in a rat model of Parkinson disease.

Mitochondrial dysfunction named complex I syndrome was observed in striatum mitochondria of rotenone treated rats (2 mg rotenone/kg, i. p., for 30 or 60 days) in an animal model of Parkinson disease. After 60 days of rotenone treatment, the animals showed: (a) 6-fold increased bradykinesia and 60% decreased locomotor activity; (b) 35-34% decreases in striatum O2 uptake and in state 3 mitochondrial respiration with malate-glutamate as substrate; (c) 43-57% diminished striatum complex I activity with 60-71% decreased striatum mitochondrial NOS activity, determined both as biochemical activity and as functional activity (by the NO inhibition of active respiration); (d) 34-40% increased rates of mitochondrial O2•- and H2O2 productions and 36-46% increased contents of the products of phospholipid peroxidation and of protein oxidation; and (e) 24% decreased striatum mitochondrial content, likely associated to decreased NO-dependent mitochondrial biogenesis. Intermediate values were observed after 30 days of rotenone treatment. Frontal cortex tissue and mitochondria showed similar but less marked changes. Rotenone-treated rats showed mitochondrial complex I syndrome associated with cellular oxidative stress in the dopaminergic brain areas of striatum and frontal cortex, a fact that describes the high sensitivity of mitochondrial complex I to inactivation by oxidative reactions.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.