Respiratory Chain Function Research Articles

Differential expression of long no-coding RNA in human intracranial arteriovenous malformation tissue

Objective To investigate the differential expression of long no-coding RNA(LncRNA)in human brain arteriovenous malformation(AVM)tissue using an Arraystar-LncRNA chip. Methods The vascular nidi of 14 patients with AVM used as the specimens of the test group and operated in Beijing Tiantan Hospital, Capital Medical University were selected. The scalp blood vessel or superficial temporal artery segment of the same patient was used as a control specimen. Arraystar-LncRNA chip was used to conduct the expression profile analysis of LncRNA and mRNA. The maximum expression difference of 8 LncRNAs was selected for PCR validation. Results After screening and validation, in the 2 8012 detectable LncRNAs, 1 931 were up-regulated for ≥2 times and 450 were up-regulated for ≥4 times compared with the control group; 1 852 were down-regulated for ≥2 times and 719 were down-regulated for ≥4 times. In the 21 780 detectable mRNAs, 1 577 were up-regulated for ≥2 times, 450 were up-regulated for ≥4 times; and 1 699 were down-regulated for ≥2 times and 681 were down-regulated for ≥4 times. The bioinformatics and pathway analyses for mRNAs showed that the differential expression of 8-code mRNAs(FDXR, SYNE2, FCGR3A, NDUFS4, COX5A, OPA1, ETFA, and LPL)were most obvious in comparison of the test group and the control group. These mRNAs were associated with mitochondrial electron respiratory chain function, interaction between cytokines, stress response, lipoprotein lipase, and cytoskeleton associated protein. PCR validation for mRNAs associated with these LncRNAs showed that there were significant differences in the differential expression of 4 LncRNAs(codes ENS0423394(P=0.03), ENS0444114(P=0.01), TCONS_00013855(P=0.01), and ENS0452148(P=0.01). Conclusion The occurrence and development mechanism of intracranial AVM may be associated with the mitochondrial NADPH oxidoreductase, lipoprotein lipase, and optic atrophy associated protein-related down-regulation of LncRNA expression. Key words: Intracranial arteriovenous malformations; Gene expression profile; Biological processes; Long no-coding RNA

The study of the mechanism of arsenite toxicity in respiration-deficient cells reveals that NADPH oxidase-derived superoxide promotes the same downstream events mediated by mitochondrial superoxide in respiration-proficient cells

We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O2.-) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5μM, arsenite elicited selective formation of O2.- in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O2.- triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O2.- because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O2.- mediated by NADPH oxidase. Interestingly, extramitochondrial O2.- triggered the same downstream events and an identical mode of death previously observed in RP cells.Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O2.- availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis.

Abstract 329: Reprogramming glucose metabolism and energy production with a small molecule HJC0152 suppresses breast cancer development and progression to metastasis

Abstract Currently there are no targeted therapeutic strategies for estrogen receptor (ER)-negative breast cancer (ENBC), which constitutes 30-40% of breast cancer cases and is prone to metastasize and recur. Distant metastasis accounts for 90% of cancer-associated deaths. The majority of deaths from breast cancer are caused by distant metastasis developed in lung, liver, bone, or brain. However, to date it remains a challenge and unmet need to treat existing metastasis and block new metastasis in cancer patients. Dysregulated glucose and energy metabolism is critically involved in the development and progression of various cancers via promoting aberrant cell growth, malignant transformation and metastasis, but the potential role of glucose/energy metabolism in ENBC progression and metastasis has scarcely been explored heretofore, thus representing a key knowledge gap and a potential avenue for anticancer targeting. A number of anticancer metabolic and biogenetic therapies have been developed, yet none of them has progressed to clinical use, due to their limited potency, specificity or drug properties such as toxicity and poor bioavailability. We recently identified a novel small molecule HJC0152 that significantly suppresses ENBC xenograft tumor growth and blocks ER-negative mammary tumor development in mouse models. HJC0152 treatment for 24-72 hours differentially modulates protein expression of HK1, PFK-L, PFKFB2, ENO2, PDH, PDK1, PGAM1 and ALDOA in a time-dependent manner. HJC0152 also regulates the transcription of genes involved in glucose and mitochondrial energy metabolism, including the subunits of mitochondrial respiratory chain complexes. Functional assessments of mitochondrial complexes demonstrate that HJC0152 significantly inhibits Complexes IV but increases Complex V (ATP synthase) function, while Complexes I and II function is minimally affected. Migration and invasion of MDA-MB-231 cells are significantly inhibited by HJC0152 treatment. In vivo, HJC0152 administrated either before, concurrent with or after tail vein injection of MDA-MB-231 cells dramatically blocks the development of lung macro- and micro-metastasis in all groups. These results suggest that HJC0152 can specifically reprogram/restore the dysregulated glucose metabolism by inducing specific glycolytic enzyme expression and mitochondrial respiratory chain function, likely via targeting one or more upstream signal molecule(s) that regulates glucose and energy metabolism, thereby suppressing breast cancer progression to metastasis. This work was supported by Grants P50 CA097007, P30DA028821, and R21MH093844 (J.Z.) from the NIH, CPRIT (J.Z.), John Sealy Memorial Endowment Fund (J.Z.), DFI Seed Grants from MD Anderson Cancer Center (Q.S.), and Holden Family Research Grant in Breast Cancer Prevention from the Prevent Cancer Foundation (Q.S). Citation Format: Hao Zou, Na Ye, Hui Pang, Dan Zhang, Ruping Yan, Haijun Chen, Guoshuai Cai, Lili Wang, Zhengduo Yang, Haiying Chen, Grace Xu, Yingchao Zhang, Ritu Arora, Ming Tan, Yongchang Wei, Jia Zhou, Qiang Shen. Reprogramming glucose metabolism and energy production with a small molecule HJC0152 suppresses breast cancer development and progression to metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 329.

Abstract 1248: Ibrutinib inhibits platelet aggregation through inhibition of mitochondrial respiratory chain function and suppression of Syk activation

Abstract Ibrutinib represents a therapeutic improvement in hematologic cancers, but it is associated with bleeding-related adverse events. To minimize such adverse events and improve the therapeutic utility of this drug, it is important to understand the underlying mechanisms responsible for the adverse events. In this study, we observed that platelets isolate from the blood samples of chronic lymphocytic leukemia (CLL) treated with Ibrutinib exhibited deficiencies in collagen-mediated aggregation. Mechanistic studies identified the mitochondrial respiratory chain as a potential mediator of ibrutinib-induced impairment of platelet function. A short-term treatment of platelets isolated from human blood with ibrutinib for less than one hour led to reactive oxygen species (ROS) generation associated with inhibition of mitochondrial respiration, as evident by a reduction in oxygen consumption rate measured by a Seahorse metabolic analyser. Long-term incubation of megakaryocytes with ibrutinib markedly suppressed the expression of mitochondrial electron transport chain components in platelets and a reduction in mitochondrial respiration. Interference with the respiratory chain by rotenone also resulted in similar reduction in collagen-induced platelet aggregation. Interestingly, ROS generation upon collagen stimulation induced tyrosine phosphorylation of Syk (spleen tyrosine kinase) to activate platelet, while impairment of respiratory chain induced by ibrutinib diminished collagen-evoked activation of the Syk pathway. These data suggest that ibrutinib suppresses mitochondrial respiration, leading to defects in collagen-induced platelet aggregation, potentially through inhibiting the activation of the Syk pathway. Citation Format: Jie Huang, Helen Pelicano, Feng Li, Michael J. Keating, Peng Huang. Ibrutinib inhibits platelet aggregation through inhibition of mitochondrial respiratory chain function and suppression of Syk activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1248.

Splicing Defect in Mitochondrial Seryl-tRNA Synthetase Gene Causes Progressive Spastic Paresis Instead of HUPRA Syndrome

Mitochondrial aminoacyl-tRNA synthetases are an important group of disease genes typically underlying either a disorder affecting an isolated tissue or a distinct syndrome. Missense mutations in the mitochondrial seryl-tRNA synthetase gene, SARS2, have been identified in HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis). We report here a homozygous splicing mutation in SARS2 in a patient with progressive spastic paresis. We show that the mutation leads to diminished levels of the synthetase in patient's fibroblasts. This has a destabilizing effect on the tRNASer(AGY) isoacceptor, but to a lesser degree than in HUPRA syndrome patients. tRNASer(UCN) is largely unaffected in both phenotypes. In conclusion, the level of tRNASer(AGY) instability may be a factor in determining tissue manifestation in patients with SARS2 mutations. This finding exemplifies the sensitivity of the nervous system to partially reduced aminoacylation, which is sufficient in other tissues to maintain respiratory chain function.

Specific requirements of nonbilayer phospholipids in mitochondrial respiratory chain function and formation.

Mitochondrial membrane phospholipid composition affects mitochondrial function by influencing the assembly of the mitochondrial respiratory chain (MRC) complexes into supercomplexes. For example, the loss of cardiolipin (CL), a signature non-bilayer-forming phospholipid of mitochondria, results in disruption of MRC supercomplexes. However, the functions of the most abundant mitochondrial phospholipids, bilayer-forming phosphatidylcholine (PC) and non-bilayer-forming phosphatidylethanolamine (PE), are not clearly defined. Using yeast mutants of PE and PC biosynthetic pathways, we show a specific requirement for mitochondrial PE in MRC complex III and IV activities but not for their formation, whereas loss of PC does not affect MRC function or formation. Unlike CL, mitochondrial PE or PC is not required for MRC supercomplex formation, emphasizing the specific requirement of CL in supercomplex assembly. Of interest, PE biosynthesized in the endoplasmic reticulum (ER) can functionally substitute for the lack of mitochondrial PE biosynthesis, suggesting the existence of PE transport pathway from ER to mitochondria. To understand the mechanism of PE transport, we disrupted ER-mitochondrial contact sites formed by the ERMES complex and found that, although not essential for PE transport, ERMES facilitates the efficient rescue of mitochondrial PE deficiency. Our work highlights specific roles of non-bilayer-forming phospholipids in MRC function and formation.

Novel roles for uncoupling proteins in brown adipose tissue mitochondrial respiratory chain function and supermolecular organization

Brown adipose tissue (BAT) has been a hot topic since its discovery. BAT is responsible for the generation of heat during non‐shivering thermogenesis, the mechanism by which mammals maintain their temperature during prolonged cold exposure. This process is dependent upon the presence and activation of the inner membrane mitochondrial protein, uncoupling protein 1 (UCP1). UCP1 directly dissipates the mitochondrial proton gradient, such that the oxidation of metabolites produces heat instead of ATP. This property, along with recent evidence that adult humans may have greater amounts of BAT than previously appreciated has garnered significant clinical attention in invoking BAT thermogenesis as an anti‐obesity strategy for intervention in metabolic disease. While BAT thermogenesis has been known to be UCP1‐ dependent for three decades, how UCP1 regulates BAT mitochondrial function apart from its uncoupling function remain largely uncharacterized. Using UCP1‐null, and UCP3‐null and double‐null mouse mutants, this study aims to characterize how the presence of uncoupling proteins in BAT affect mitochondrial supercomplex organization in addition to their effects on bioenergetics. Studies in mitochondrial dynamics and function have shown that defects in the mitochondrial architecture can adversely affect cold sensitivity in animals. Specifically, it has been shown that the ability of the mitochondrial respiratory chain to form supercomplexes (MRSC) is necessary to maintain temperature during acute exposure to cold. MRSC are physical associations of the mitochondrial respiratory chain (I–IV) that increase the efficiency of mitochondrial substrate oxidation in addition to lowering reactive oxygen species generation. We discovered that the loss of UCP1 leads to a dramatic increase in intrascapular BAT size that is accompanied by profound changes in MRSC complex composition, as determined by blue native polyacrylamide gel electrophoresis and western blotting. In addition, we have found that UCP1 natively co‐migrates with mitochondrial respiratory chain components, which provides evidence that UCP1 could be directly influencing MRSC formation. Finally, we found that isolated BAT mitochondria from UCP1 and double null animals show decreased maximal (FCCP stimulated) substrate oxidation rates. These data invoke a mechanism by which UCP1 may act to stabilize mitochondrial respiratory chain components, while also necessitating increased mitochondrial respiratory chain component expression to maintain mitochondrial membrane potential both in stimulated and basal conditions. Future studies will further elucidate the role of uncoupling proteins in this functionally important and novel phenomenon.

Mitochondrial defects associated with β-alanine toxicity: relevance to hyper-beta-alaninemia.

Hyper-beta-alaninemia is a rare metabolic condition that results in elevated plasma and urinary β-alanine levels and is characterized by neurotoxicity, hypotonia, and respiratory distress. It has been proposed that at least some of the symptoms are caused by oxidative stress; however, only limited information is available on the mechanism of reactive oxygen species generation. The present study examines the hypothesis that β-alanine reduces cellular levels of taurine, which are required for normal respiratory chain function; cellular taurine depletion is known to reduce respiratory function and elevate mitochondrial superoxide generation. To test the taurine hypothesis, isolated neonatal rat cardiomyocytes and mouse embryonic fibroblasts were incubated with medium lacking or containing β-alanine. β-alanine treatment led to mitochondrial superoxide accumulation in conjunction with a decrease in oxygen consumption. The defect in β-alanine-mediated respiratory function was detected in permeabilized cells exposed to glutamate/malate but not in cells utilizing succinate, suggesting that β-alanine leads to impaired complex I activity. Taurine treatment limited mitochondrial superoxide generation, supporting a role for taurine in maintaining complex I activity. Also affected by taurine is mitochondrial morphology, as β-alanine-treated fibroblasts undergo fragmentation, a sign of unhealthy mitochondria that is reversed by taurine treatment. If left unaltered, β-alanine-treated fibroblasts also undergo mitochondrial apoptosis, as evidenced by activation of caspases 3 and 9 and the initiation of the mitochondrial permeability transition. Together, these data show that β-alanine mediates changes that reduce ATP generation and enhance oxidative stress, factors that contribute to heart failure.

The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early-onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin-/PINK1-mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.

Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6.

Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.

Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients.

Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.

Genotoxic risk of quinocetone and its possible mechanism in in vitro studies.

Quinoxalines possessing the quinoxaline-1,4-dioxide (QdNOs) basic structure are used for their antibacterial action, although their mechanism of genotoxicity is not clear. After comparing the sensitivity of V79 cells and HepG2 cells to quinocetone (QCT) and other QdNOs, it was found that HepG2 cells are more sensitive. The results show that QCT induces the generation of O2˙- and OH˙ during metabolism. Free radicals could then attack guanine and induce 8-hydroxy-deoxyguanine (8-OHdG) generation, causing DNA strand breakage, the inhibition of topoisomerase II (topo II) activity, and alter PCNA, Gadd45 and topo II gene expression. QCT also caused mutations in the mtDNA genes COX1, COX3 and ATP6, which might affect the function of the mitochondrial respiratory chain and increase the production of reactive oxygen species (ROS). Nuclear extracts from HepG2 cells treated with QCT had markedly reduced topo II activity, as judged by the inability to convert pBR322 DNA from the catenated to the decatenated form by producing stable DNA-topo II complexes. This study suggests that QCT electrostatically bound to DNA in a groove, affecting the dissociation of topo II from DNA and impacting DNA replication. Taken together, these data reveal that DNA damage induced by QCT resulted from O2˙- and OH˙ generated in the metabolism process. This data throws new light onto the genotoxicity of quinoxalines.

Early Infantile Epileptic Encephalopathy in an STXBP1 Patient with Lactic Acidemia and Normal Mitochondrial Respiratory Chain Function.

A wide range of clinical findings have been associated with mutations in Syntaxin Binding Protein 1 (STXBP1), including multiple forms of epilepsy, nonsyndromic intellectual disability, and movement disorders. STXBP1 mutations have recently been associated with mitochondrial pathology, although it remains unclear if this phenotype is a part of the core feature for this gene disorder. We report a 7-year-old boy who presented for diagnostic evaluation of intractable epilepsy, episodic ataxia, resting tremor, and speech regression following a period of apparently normal early development. Mild lactic acidemia was detected on one occasion at the time of an intercurrent illness. Due to the concern for mitochondrial disease, ophthalmologic evaluation was performed that revealed bilateral midperiphery pigmentary mottling. Optical coherence tomography (OCT) testing demonstrated a bilaterally thickened ganglion cell layer in the perifovea. Skeletal muscle biopsy analysis showed no mitochondrial abnormalities or respiratory chain dysfunction. Exome sequencing identified a de novo c.1651C>T (p.R551C) mutation in STXBP1. Although mitochondrial dysfunction has been reported in some individuals, our proband had only mild lactic acidemia and no skeletal muscle tissue evidence of mitochondrial disease pathology. Thus, mitochondrial dysfunction is not an obligate feature of STXBP1 disease.

Novel Homozygous Missense Mutation in SPG20 Gene Results in Troyer Syndrome Associated with Mitochondrial Cytochrome c Oxidase Deficiency.

Troyer syndrome is an autosomal recessive form of hereditary spastic paraplegia (HSP) caused by deleterious mutations in the SPG20 gene. Although the disease is associated with a loss of function mechanism of spartin, the protein encoded by SPG20, the precise pathogenesis is yet to be elucidated. Recent data indicated an important role for spartin in both mitochondrial maintenance and function. Here we report a child presenting with progressive spastic paraparesis, generalized muscle weakness, dysarthria, impaired growth, and severe isolated decrease in muscle cytochrome c oxidase (COX) activity. Whole exome sequencing identified the homozygous c.988A>G variant in SPG20 gene (p.Met330Val) resulting in almost complete loss of spartin in skeletal muscle. Further analyses demonstrated significant tissue specific reduction of COX 4, a nuclear encoded subunit of COX, in muscle suggesting a role for spartin in proper mitochondrial respiratory chain function mediated by COX activity. Our findings need to be verified in other Troyer syndrome patients in order to classify it as a form of HSP caused by mitochondrial dysfunction.

Whole Exome Sequencing Identifies the Genetic Basis of Late-Onset Leigh Syndrome in a Patient with MRI but Little Biochemical Evidence of a Mitochondrial Disorder.

Leigh syndrome is a subacute necrotising encephalomyopathy proven by post-mortem analysis of brain tissue showing spongiform lesions with vacuolation of the neuropil followed by demyelination, gliosis and capillary proliferation caused by mutations in one of over 75 different genes, including nuclear- and mitochondrial-encoded genes, most of which are associated with mitochondrial respiratory chain function. In this study, we report a patient with suspected Leigh syndrome presenting with seizures, ptosis, scoliosis, dystonia, symmetrical putaminal abnormalities and a lactate peak on brain MRS, but showing normal MRC enzymology in muscle and liver, thereby complicating the diagnosis. Whole exome sequencing uncovered compound heterozygous mutations in NADH dehydrogenase (ubiquinone) flavoprotein 1 gene (NDUFV1), c.1162+4A>C (NM_007103.3), resulting in skipping of exon 8, and c.640G>A, causing the amino acid substitution p.Glu214Lys, both of which have previously been reported in a patient with complex I deficiency. Patient fibroblasts showed a significant reduction in NDUFV1 protein expression, decreased complex CI and complex IV assembly and consequential reductions in the enzymatic activities of both complexes by 38% and 67%, respectively. The pathogenic effect of these variations was further confirmed by immunoblot analysis of subunits for MRC enzyme complexes in patient muscle, liver and fibroblast where we observed 90%, 60% and 95% reduction in complex CI, respectively. Together these studies highlight the importance of a comprehensive, multipronged approach to the laboratory evaluation of patients with suspected Leigh syndrome.

Resveratrol Directly Binds to Mitochondrial Complex I and Increases Oxidative Stress in Brain Mitochondria of Aged Mice.

Resveratrol is often described as a promising therapeutic molecule for numerous diseases, especially in metabolic and neurodegenerative disorders. While the mechanism of action is still debated, an increasing literature reports that resveratrol regulates the mitochondrial respiratory chain function. In a recent study we have identified mitochondrial complex I as a direct target of this molecule. Nevertheless, the mechanisms and consequences of such an interaction still require further investigation. In this study, we identified in silico by docking study a binding site for resveratrol at the nucleotide pocket of complex I. In vitro, using solubilized complex I, we demonstrated a competition between NAD+ and resveratrol. At low doses (<5μM), resveratrol stimulated complex I activity, whereas at high dose (50 μM) it rather decreased it. In vivo, in brain mitochondria from resveratrol treated young mice, we showed that complex I activity was increased, whereas the respiration rate was not improved. Moreover, in old mice with low antioxidant defenses, we demonstrated that complex I activation by resveratrol led to oxidative stress. These results bring new insights into the mechanism of action of resveratrol on mitochondria and highlight the importance of the balance between pro- and antioxidant effects of resveratrol depending on its dose and age. These parameters should be taken into account when clinical trials using resveratrol or analogues have to be designed.

Analysis of Bj fibroblasts mitochondrial respiratory chain function under glucose starvation and exposure to different doses of rotenone: Implications for neurogenerative diseases

Analysis of Bj fibroblasts mitochondrial respiratory chain function under glucose starvation and exposure to different doses of rotenone: Implications for neurogenerative diseases

Titration of mitochondrial fusion rescues Mff-deficient cardiomyopathy.

Defects in mitochondrial fusion or fission are associated with many pathologies, raising the hope that pharmacological manipulation of mitochondrial dynamics may have therapeutic benefit. This approach assumes that organ physiology can be restored by rebalancing mitochondrial dynamics, but this concept remains to be validated. We addressed this issue by analyzing mice deficient in Mff, a protein important for mitochondrial fission. Mff mutant mice die at 13 wk as a result of severe dilated cardiomyopathy leading to heart failure. Mutant tissue showed reduced mitochondrial density and respiratory chain activity along with increased mitophagy. Remarkably, concomitant deletion of the mitochondrial fusion gene Mfn1 completely rescued heart dysfunction, life span, and respiratory chain function. Our results show for the first time that retuning the balance of mitochondrial fusion and fission can restore tissue integrity and mitochondrial physiology at the whole-organ level. Examination of liver, testis, and cerebellum suggest, however, that the precise balance point of fusion and fission is cell type specific.

Impact of levosimendan and ischaemia-reperfusion injury on myocardial subsarcolemmal mitochondrial respiratory chain, mitochondrial membrane potential, Ca2+ cycling and ATP synthesis.

Levosimendan (LS) is increasingly used in case of myocardial failure after cardiac surgery. The impact of LS on myocardial mitochondrial functions, such as respiratory chain function (RCF), mitochondrial membrane potential (ΔΨm), Ca(2+) handling, mitochondrial permeability transition pore (mPTP) opening and ATP during ongoing ischaemia/reperfusion (IR) injury, is not well understood. Depending on LS, I/R injury or the combination of both, we analysed myocardial functions in a retrograde Langendorff-model followed by the analysis of subsarcolemmal mitochondrial (SSM) functions. Rat hearts were divided into four study groups; two were subjected to 30 min of perfusion without (control) or with the application of 1.4 µmol/20 min LS (Levo). Experiments were repeated with hearts being subjected to 40 min of normothermic stop-flow ischaemia and 30 min of reperfusion without (IR) or with LS application (Levo-IR). Systolic left ventricular pressure (LVPsys), left ventricular contractility (LVdp/dtmax) and coronary flow were determined. SSM were analysed regarding RCF, ΔΨm, ATP, and Ca(2+) retention capacity (CRC), Ca(2+)-induced swelling and Ca(2+) fluxes after (re)perfusion. I/R injury suppressed LVdp/dtmax (1381 ± 927 vs 2464 ± 913 mmHg/s; P = 0.01 at 30 min (re-)perfusion time). IR revealed complex I-V state3 (19.1 ± 7.4 vs 27.6 ± 11.0 nmolO2/min; P < 0.044) and II-V state3 (20.6 ± 6.8 vs 37.3 ± 9.10 molO2/min; P < 0.0001) suppression and Levo limited I-V (14.8 ± 11.1 vs 27.6 ± 11.0 nmolO2/min; P < 0.001) and II-V (24.1 ± 6.4 vs 37.3 ± 9.10 molO2/min; P < 0.0001) function. After energizing, ΔΨm hypopolarization was observed in Levo (0.76 ± 0.04 vs 0.84 ± 0.04; P = 0.02), IR (0.75 ± 0.06 vs 0.84 ± 0.04; P = 0.007) and Levo-IR (0.75 ± 0.06 vs 0.06 ± 0.04; P = 0.01). IR (AUC: 626 vs 292; P = 0.023) and Levo-IR (AUC: 683 vs 292, P = 0.003) increased Ca(2+)-induced mPTP-opening susceptibility. CRC declined in IR (6.4 ± 2.1 vs 10.5 ± 2.6; P = 0.04) or Levo (6.5 ± 2.0 vs 10.5 ± 2.6; P = 0.023). Ca(2+) uptake was delayed in IR and Levo-IR without LS impact (P < 0.0001). Ca(2+) liberation was increased in Levo-IR. ATP synthesis was reduced in Levo (0.49 ± 0.14 vs 0.74 ± 0.14; P = 0.002) and Levo-I/R (0.34 ± 0.18 vs 0.74 ± 0.14; P < 0.002). LS limited RCF at complex IV and V with ΔΨm hypopolarization suggesting a specific [Formula: see text]-dependent pathway. Ca(2+) redistribution from SSM by LS during I/R injury possibly prevents from Ca(2+) overload due to mPTP flickering. LS-induced mPTP flickering did not promote permanent Ca(2+)-induced mPTP opening. LS-dependent inhibition of ATP generation presumably resulted from complex IV and V limitations and lowered ΔΨm. However, a resulting impact of limited ATP synthesis on myocardial recovery remains arguable.

Mitochondrial functions modulate neuroendocrine, metabolic, inflammatory, and transcriptional responses to acute psychological stress

The experience of psychological stress triggers neuroendocrine, inflammatory, metabolic, and transcriptional perturbations that ultimately predispose to disease. However, the subcellular determinants of this integrated, multisystemic stress response have not been defined. Central to stress adaptation is cellular energetics, involving mitochondrial energy production and oxidative stress. We therefore hypothesized that abnormal mitochondrial functions would differentially modulate the organism's multisystemic response to psychological stress. By mutating or deleting mitochondrial genes encoded in the mtDNA [NADH dehydrogenase 6 (ND6) and cytochrome c oxidase subunit I (COI)] or nuclear DNA [adenine nucleotide translocator 1 (ANT1) and nicotinamide nucleotide transhydrogenase (NNT)], we selectively impaired mitochondrial respiratory chain function, energy exchange, and mitochondrial redox balance in mice. The resulting impact on physiological reactivity and recovery from restraint stress were then characterized. We show that mitochondrial dysfunctions altered the hypothalamic-pituitary-adrenal axis, sympathetic adrenal-medullary activation and catecholamine levels, the inflammatory cytokine IL-6, circulating metabolites, and hippocampal gene expression responses to stress. Each mitochondrial defect generated a distinct whole-body stress-response signature. These results demonstrate the role of mitochondrial energetics and redox balance as modulators of key pathophysiological perturbations previously linked to disease. This work establishes mitochondria as stress-response modulators, with implications for understanding the mechanisms of stress pathophysiology and mitochondrial diseases.