Metabolic Dysfunction and Altered Mitochondrial Dynamics in the Utrophin-Dystrophin Deficient Mouse Model of Duchenne Muscular Dystrophy

The utrophin-dystrophin deficient (DKO) mouse model has been widely used to understand the progression of Duchenne muscular dystrophy (DMD). However, it is unclear as to what extent muscle pathology affects metabolism. Therefore, the present study was focused on understanding energy expenditure in the whole animal and in isolated extensor digitorum longus (EDL) muscle and to determine changes in metabolic enzymes. Our results show that the 8 week-old DKO mice consume higher oxygen relative to activity levels. Interestingly the EDL muscle from DKO mouse consumes higher oxygen per unit integral force, generates less force and performs better in the presence of pyruvate thus mimicking a slow twitch muscle. We also found that the expression of hexokinase 1 and pyruvate kinase M2 was upregulated several fold suggesting increased glycolytic flux. Additionally, there is a dramatic increase in dynamin-related protein 1 (Drp 1) and mitofusin 2 protein levels suggesting increased mitochondrial fission and fusion, a feature associated with increased energy demand and altered mitochondrial dynamics. Collectively our studies point out that the dystrophic disease has caused significant changes in muscle metabolism. To meet the increased energetic demand, upregulation of metabolic enzymes and regulators of mitochondrial fusion and fission is observed in the dystrophic muscle. A better understanding of the metabolic demands and the accompanied alterations in the dystrophic muscle can help us design improved intervention therapies along with existing drug treatments for the DMD patients.     

Mitochondrial expression and function of GAS-1 in Caenorhabditis elegans

A mutation in the gene gas-1 alters sensitivity to volatile anesthetics, fecundity, and life span in the nematode Caenorhabditis elegans. gas-1 encodes a close homologue of the 49-kDa iron protein subunit of Complex I of the mitochondrial electron transport chain from bovine heart. gas-1 is widely expressed in the nematode neuromuscular system and in a subcellular pattern consistent with that of a mitochondrial protein. Pharmacological studies indicate that gas-1 functions partially via presynaptic effects. In addition, a mutation in the gas-1 gene profoundly decreases Complex I-dependent metabolism in mitochondria as measured by rates of both oxidative phosphorylation and electron transport. An increase in Complex II-dependent metabolism also is seen in mitochondria from gas-1 animals. There is no apparent alteration in physical structure in mitochondria from gas-1 nematodes compared with those from wild type. These data indicate that gas-1 is the major 49-kDa protein of complex I and that the GAS-1 protein is critical to mitochondrial function in C. elegans. They also reveal the importance of mitochondrial function in determining not only aging and life span, but also anesthetic sensitivity, in this model organism.     

线粒体形态学改变与细胞凋亡

近年来,对于线粒体形态学以及其在凋亡过程中的改变和作用的研究打破了传统的观点。正常情况下,线粒体在细胞内相互连接成管网状结构,并发生着频繁的融合与分裂。融合和分裂由一系列蛋白质介导,二者之间的动态平衡维持着线粒体的形态和功能。在细胞凋亡的早期,线粒体融合和分裂失平衡,导致线粒体管网状结构碎裂和嵴的重构,这些改变对线粒体随后的变化以及凋亡的发生具有重要的意义。融合和分裂的蛋白质不仅调控线粒体形态和细胞凋亡过程,也和某些凋亡相关疾病有关。此外,促凋亡的Bcl-2蛋白可能通过改变线粒体的构形来调控凋亡过程。     

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.     

Mice Deficient in the Respiratory Chain Gene Cox6a2 Are Protected against High-Fat Diet-Induced Obesity and Insulin Resistance

Oxidative phosphorylation in mitochondria is responsible for 90% of ATP synthesis in most cells. This essential housekeeping function is mediated by nuclear and mitochondrial genes encoding subunits of complex I to V of the respiratory chain. Although complex IV is the best studied of these complexes, the exact function of the striated muscle-specific subunit COX6A2 is still poorly understood. In this study, we show that Cox6a2-deficient mice are protected against high-fat diet-induced obesity, insulin resistance and glucose intolerance. This phenotype results from elevated energy expenditure and a skeletal muscle fiber type switch towards more oxidative fibers. At the molecular level we observe increased formation of reactive oxygen species, constitutive activation of AMP-activated protein kinase, and enhanced expression of uncoupling proteins. Our data indicate that COX6A2 is a regulator of respiratory uncoupling in muscle and we demonstrate that a novel and direct link exists between muscle respiratory chain activity and diet-induced obesity/insulin resistance.     

Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends Lifespan in Caenorhabditis elegans

The oxidative stress theory of aging postulates that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS) generated during normal metabolism. Superoxide dismutases (SODs) counteract this process by detoxifying superoxide. It has previously been shown that elimination of either cytoplasmic or mitochondrial SOD in yeast, flies, and mice results in decreased lifespan. In this experiment, we examine the effect of eliminating each of the five individual sod genes present in Caenorhabditis elegans. In contrast to what is observed in other model organisms, none of the sod deletion mutants shows decreased lifespan compared to wild-type worms, despite a clear increase in sensitivity to paraquat- and juglone-induced oxidative stress. In fact, even mutants lacking combinations of two or three sod genes survive at least as long as wild-type worms. Examination of gene expression in these mutants reveals mild compensatory up-regulation of other sod genes. Interestingly, we find that sod-2 mutants are long-lived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on known pathways of lifespan extension reveals a clear interaction with genes that affect mitochondrial function: sod-2 deletion markedly increases lifespan in clk-1 worms while clearly decreasing the lifespan of isp-1 worms. Combined with the mitochondrial localization of SOD-2 and the fact that sod-2 mutant worms exhibit phenotypes that are characteristic of long-lived mitochondrial mutants—including slow development, low brood size, and slow defecation—this suggests that deletion of sod-2 extends lifespan through a similar mechanism. This conclusion is supported by our demonstration of decreased oxygen consumption in sod-2 mutant worms. Overall, we show that increased oxidative stress caused by deletion of sod genes does not result in decreased lifespan in C. elegans and that deletion of sod-2 extends worm lifespan by altering mitochondrial function.     

Relationship Between Catalase and Life Span in Recombinant Inbred Strains of Caenorhabditis elegans

Johnson and Wood constructed recombinant inbred strains of Caenorhabditis elegans with life spans ranging from 10 to 31 days. Using these strains, we have demonstrated previously that hyperoxia and methyl viologen inhibited development at rates inversely correlated with life span. The growth rates of the short-lived recombinant inbred strains were more profoundly inhibited by oxidative stress than were those of the long-lived strains. Here we report a positive correlation between life span and catalase levels in these same strains. Specifically, when compared to short-lived strains at 10 days after fertilization, the long-lived strains possessed higher levels of total enzymatic catalase. Northern blots indicated a similar relationship between life span and clt-1mRNA (the cytosolic catalase). This suggests that at least some of the polygenes that influence life span are also responsible for regulating gene expression of catalase, an important defense component against oxidative stress.     

Altered Spatiotemporal Dynamics of the Mitochondrial Membrane Potential in the Hypertrophied Heart

Chronically elevated levels of oxidative stress resulting from increased production and/or impaired scavenging of reactive oxygen species are a hallmark of mitochondrial dysfunction in left ventricular hypertrophy. Recently, oscillations of the mitochondrial membrane potential (ΔΨ_m) were mechanistically linked to changes in cellular excitability under conditions of acute oxidative stress produced by laser-induced photooxidation of cardiac myocytes in vitro. Here, we investigate the spatiotemporal dynamics of ΔΨ_m within the intact heart during ischemia-reperfusion injury. We hypothesize that altered metabolic properties in left ventricular hypertrophy modulate ΔΨ_m spatiotemporal properties and arrhythmia propensity.     

Nitric Oxide-Mediated Inhibition of the Mitochondrial Respiratory Chain in Cultured Astrocytes

Abstract: The Ca²⁺-independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 µg/ml) plus interferon-γ (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH-ubiquinone-1 reductase (complex I) and succinate-cytochrome c reductase (complex II–III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate-cytochrome c reductase activity, but NADH-ubiquinone-1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor N ^G-monomethyl- l -arginine. The lipopolysaccharide/interferon-γ treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide-mediated neurotoxicity.     

Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte

CLH-3a and CLH-3b are swelling-activated, alternatively spliced Caenorhabditis elegans ClC anion channels that have identical membrane domains but exhibit marked differences in their cytoplasmic NH₂ and COOH termini. The major differences include a 71-amino acid CLH-3a NH₂-terminal extension and a 270-amino acid extension of the CLH-3b COOH terminus. Splice variation gives rise to channels with striking differences in voltage, pH, and Cl^– sensitivity. On the basis of structural and functional insights gained from crystal structures of bacterial ClCs, we suggested previously that these functional differences are due to alternative splicing of the COOH terminus that may change the accessibility and/or function of pore-associated ion-binding sites. We recently identified a mutant worm strain harboring a COOH-terminal deletion mutation in the clh-3 gene. This mutation removes 101 COOH-terminal amino acids unique to CLH-3b and an additional 64 upstream amino acids shared by both channels. CLH-3b is expressed in the worm oocyte, which allowed us to characterize the mutant channel, CLH-3bC, in its native cellular environment. CLH-3bC exhibits altered voltage-dependent gating as well as pH and Cl^– sensitivity that resemble those of CLH-3a. This mutation also alters channel inhibition by Zn²⁺, prevents ATP depletion-induced activation, and dramatically reduces volume sensitivity. These results suggest that the deleted COOH-terminal region of CLH-3bC functions to modulate channel sensitivity to voltage and extracellular ions. This region also likely plays a role in channel regulation and cell volume sensitivity. Our findings contribute to a growing body of evidence indicating that cytoplasmic domains play key roles in the gating and regulation of eukaryotic ClCs. chloride; cell volume; voltage-gated anion channel     

Biogenesis of the bc1 Complex of the Mitochondrial Respiratory Chain

The oxidative phosphorylation system contains four respiratory chain complexes that connect the transport of electrons to oxygen with the establishment of an electrochemical gradient over the inner membrane for ATP synthesis. Due to the dual genetic source of the respiratory chain subunits, its assembly requires a tight coordination between nuclear and mitochondrial gene expression machineries. In addition, dedicated assembly factors support the step-by-step addition of catalytic and accessory subunits as well as the acquisition of redox cofactors. Studies in yeast have revealed the basic principles underlying the assembly pathways. In this review, we summarize work on the biogenesis of the bc₁ complex or complex III, a central component of the mitochondrial energy conversion system.     

Mitochondrial dynamics and autophagy aid in removal of persistent mitochondrial DNA damage in Caenorhabditis elegans

Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mitochondrial DNA (mtDNA) by important environmental genotoxins and endogenous metabolites. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. We report that removal of such mtDNA damage is detectable by 48 h in Caenorhabditis elegans, and requires mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the in vivo relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.     

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPR^mt) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPR^mt, suggesting that the UPR^mt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPR^mt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPR^mt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPR^mt and longevity.     

Inhibition of the Mitochondrial Respiratory Chain by Phenylalanine in Rat Cerebral Cortex

Phenylketonuria (PKU) is biochemically characterized by the accumulation of phenylalanine (Phe) and its metabolites in tissues of affected children. Neurological damage is the clinical hallmark of PKU, and Phe is considered the main neurotoxic metabolite in this disorder. However, the mechanisms of neurotoxicity are poorly known. The main objective of the present work was to measure the activities of the mitochondrial respiratory chain complexes (RCC) and succinate dehydrogenase (SDH) in brain cortex of Wistar rats subjected to chemically induced hyperphenylalaninemia (HPA). We also investigated the in vitro effect of Phe on SDH and RCC activities in the cerebral cortex of 22-day-old rats. HPA was induced by subcutaneous administration of 2.4 mol/g body weight -methylphenylalanine, a phenylalanine hydroxylase inhibitor, once a day, plus 5.2 M/g body weight phenylalanine, twice a day, from the 6th-21st postnatal day. The results showed a reduction of SDH and complex I + III activity in brain cortex of rats subjected to HPA. We also verified that Phe inhibited the in vitro activity of complexes I + III, possibly by competition with NADH. Considering the importance of SDH and RCC for the maintenance of energy supply to brain, our results suggest that energy deficit may contribute to the Phe neurotoxicity in PKU.     

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc₁ complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.     

苯并噻唑醌类化合物对呼吸链酶系的抑制作用

合成了2-氯-5-正十二硫烷基-6-甲基-4,7-苯并噻唑醌(2-Cl-DMMDBT)和2-氯-5-正丁烷氨基-6-甲基-4,7-苯并噻唑醌(2-Cl-BAMDBT)两种化合物,研究了它们对线粒体呼吸链酶系的抑制作用.结果表明:2-Cl-DMMDBT和2-C1-BAMDBT对琥珀酸氧化酶及泛醌氧化酶的电子传递活性均表现一定的抑制作用,而对细胞色素氧化酶无作用,说明二者的抑制作用发生在泛醌反应区.二者对NADH氧化酶的抑制行为略有不同,2-Cl-DMMDBT是一个逐渐加强的过程,最终可致酶活性完全抑制,而2-Cl-BAMDBT则表现为瞬间抑制.比较了2-Cl-DMMDBT和2-Cl-BAMDBT对琥珀酸氧化酶的抑制能力,长侧链的2-Cl-DMMDBT比短侧链的2-Cl-BAMDBT抑制能力强很多.     

Mitochondrial electron transport is a key determinant of life span in Caenorhabditis elegans.

Increased protection from reactive oxygen species (ROS) is believed to increase life span. However, it has not been clearly demonstrated that endogenous ROS production actually limits normal life span. We have identified a mutation in the Caenorhabditis elegans iron sulfur protein (isp-1) of mitochondrial complex III, which results in low oxygen consumption, decreased sensitivity to ROS, and increased life span. Furthermore, combining isp-1(qm150) with a mutation (daf-2) that increases resistance to ROS does not result in any significant further increase in adult life span. These findings indicate that both isp-1 and daf-2 mutations increase life span by lowering oxidative stress and result in the maximum life span increase that can be produced in this way.     

Effect of Electroconvulsive Shock on Mitochondrial Respiratory Chain in Rat Brain

It is well described that impairment of energy production has been implicated in the pathogenesis of a number of diseases. Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy (ECT) to minimize its side effects, little progress has been made in understanding its mechanism of action. In this work, our aim was to measure the activities of mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase from rat brain after acute and chronic electroconvulsive shock (ECS). Our results showed that mitochondrial respiratory chain enzymes activities were increased after acute ECS in hippocampus, striatum and cortex of rats. Besides, we also demonstrated that complex II activity was increased after chronic ECS in cortex, while hippocampus and striatum were not affected. Succinate dehydrogenase, however, was inhibited after chronic ECS in striatum, activated in cortex and not affected in hippocampus. Finally, complex IV was not affected by chronic ECS in hippocampus, striatum and cortex. Our findings demonstrated that brain metabolism is altered by ECS.     

An Altered Method of Feeding RNAi That Knocks Down Multiple Genes Simultaneously in the Nematode Caenorhabditis elegans

In reverse genetics, RNA interference (RNAi) which is substitutable for gene-disruption, is an outstanding method for knockdown of a gene’s function. In Caenorhabditis elegans, feeding RNAi is most convenient, but this RNAi is not suitable for knockdown of multiple genes. Hence, we attempted to establish an efficient method of feeding RNAi for multiple knockdown. We produced bacteria yielding three distinct double-stranded RNAs bound to one another, and fed those bacteria to C. elegans. Quantitative RT-PCR and observation of phenotypes indicated that our method is much more efficient than the traditional one. Our method is useful for investigating genes’ functions in C. elegans.