Cytonuclear coadaptation in Drosophila: disruption of cytochrome c oxidase activity in backcross genotypes

Abstract The cytochrome c oxidase enzyme (COX) is comprised of 10 nuclear-encoded subunits and three mito-chondrial-encoded subunits in close physical association in the inner mitochondrial membrane. COX passes electrons from cytochrome c to molecular oxygen and pumps protons into the inner mitochondrial space for ATP production. Selection on nuclear-mitochondrial interactions within species should lead to coadaptation of the proteins comprising this important enzyme. Under this model, there should be relatively little disruption of COX activity when mitochondrial genomes are crossed among strains within species. A more pronounced disruption of activity is expected when the mitochondrial genome is expressed in the nuclear background of a different species. We test these hypotheses in Drosophila using hybridization and backcrossing among lines of D. simulans and D. mauritiana. Disrupted cytonuclear genotypes were constructed using backcrosses between two lines of D. simulans (siI and si II ) that introduced each divergent mitochondrial DNA (mtDNA) into each nuclear background due to maternal inheritance of mtDNA. Similar crosses were used to introduce eachD. simulans mtDNA into the D. mauritiana maI nuclear background. Reconstituted cytonuclear control genotypes were constructed by backcrossing the initial F1 females to males of the maternal genotype. COX enzyme activities were compared among these disrupted and reconstituted backcross genotypes within and between species. The disruption effect on COX activity was restricted to males of interspecific genotypes. These data support the coadaptation hypothesis and are consistent with predictions that the evolution of modifiers of male mitochondrial dysfunction is hindered by the maternal inheritance of mtDNA. New sequence data for nuclear encoded subunits of COX identified amino acids that may play a role in the disruption effect.     

Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease

Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD.     

Mechanism of ATP synthesis by mitochondrial ATP synthase from beef heart

Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F₁ supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP.The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k ₊₁) and the reverse dissociation step (k _–1).k _–1 was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an additional enhancement ofk _–1 was observed. These energy-dependent dissociations of ATP were sensitive to the uncoupler FCCP.k ₊₁ was affected little by NADH oxidation. The dissociation constant (K _d ATP) increased many orders of magnitude during the transition from nonenergized to energized states.     

The co‐occurrence of mtDNA mutations on different oxidative phosphorylation subunits,not detected by haplogroup analysis,affects human longevity and is population specific

To re‐examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high‐resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.     

线粒体疾病小鼠模型的建立及病理生理学研究

线粒体疾病是机体ATP合成障碍、供能不足引起的多系统疾病。近十年来,随着线粒体疾病小鼠模型的不断建立和完善,发现核DNA（nuclear DNA,nDNA）或（和）线粒体DNA（mitochondrial DNA,mtDNA）突变造成线粒体氧化磷酸化功能缺陷是其发病的主要原因。将着重介绍线粒体氧化磷酸化功能缺陷导致线粒体疾病的小鼠模型的建立及其病理生理学特点。     

Evidence for coevolution of sociality and relative brain size in three orders of mammals

As the brain is responsible for managing an individual's behavioral response to its environment, we should expect that large relative brain size is an evolutionary response to cognitively challenging behaviors. The "social brain hypothesis" argues that maintaining group cohesion is cognitively demanding as individuals living in groups need to be able to resolve conflicts that impact on their ability to meet resource requirements. If sociality does impose cognitive demands, we expect changes in relative brain size and sociality to be coupled over evolutionary time. In this study, we analyze data on sociality and relative brain size for 206 species of ungulates, carnivores, and primates and provide, for the first time, evidence that changes in sociality and relative brain size are closely correlated over evolutionary time for all three mammalian orders. This suggests a process of coevolution and provides support for the social brain theory. However, differences between taxonomic orders in the stability of the transition between small-brained/nonsocial and large-brained/social imply that, although sociality is cognitively demanding, sociality and relative brain size can become decoupled in some cases. Carnivores seem to have been especially prone to this.     

Mitonuclear coevolution as the genesis of speciation and the mitochondrial DNA barcode gap

Mitochondrial genes are widely used in taxonomy and systematics because high mutation rates lead to rapid sequence divergence and because such changes have long been assumed to be neutral with respect to function. In particular, the nucleotide sequence of the mitochondrial gene cytochrome c oxidase subunit 1 has been established as a highly effective DNA barcode for diagnosing the species boundaries of animals. Rarely considered in discussions of mitochondrial evolution in the context of systematics, speciation, or DNA barcodes, however, is the genomic architecture of the eukaryotes: Mitochondrial and nuclear genes must function in tight coordination to produce the complexes of the electron transport chain and enable cellular respiration. Coadaptation of these interacting gene products is essential for organism function. I extend the hypothesis that mitonuclear interactions are integral to the process of speciation. To maintain mitonuclear coadaptation, nuclear genes, which code for proteins in mitochondria that cofunction with the products of mitochondrial genes, must coevolve with rapidly changing mitochondrial genes. Mitonuclear coevolution in isolated populations leads to speciation because population‐specific mitonuclear coadaptations create between‐population mitonuclear incompatibilities and hence barriers to gene flow between populations. In addition, selection for adaptive divergence of products of mitochondrial genes, particularly in response to climate or altitude, can lead to rapid fixation of novel mitochondrial genotypes between populations and consequently to disruption in gene flow between populations as the initiating step in animal speciation. By this model, the defining characteristic of a metazoan species is a coadapted mitonuclear genotype that is incompatible with the coadapted mitochondrial and nuclear genotype of any other population.     

线粒体相关的疾病治疗及干预策略

线粒体疾病是一种累及不同的组织和器官的复杂异质性疾病,由核基因或线粒体基因的遗传缺陷导致,同时也受环境因素的影响。近十年来,有关线粒体疾病的诊断及发生机制的研究进展迅速,而疾病的治疗方法却研究较少。着重介绍线粒体疾病的相关治疗方法和干预策略。     

Role of mitochondrial calcium transport in the control of substrate oxidation

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca²⁺ ions. The mechanism is the activation by Ca²⁺ of four mitochondrial dehydrogenases, viz: glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-IDH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of "downstream" activation by Ca²⁺, likely at the level of the F₁F₀ ATP-ase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca²⁺ ( [Ca²⁺]m ) which act as a signal to these activities. In this article, we review recent work in which ([Ca²⁺]m) is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of ([Ca²⁺]m) relative to changes in cytosol free Ca²⁺ in cells with rapid transients in cytosol Ca²⁺, e.g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of ([Ca²⁺]m) to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, atreptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca²⁺ efflux pathways, thereby raising ([Ca²⁺]m) at a given, time-average value of cytosol free Ca²⁺.     

Increased oxidative damage in nuclear and mitochondrial DNA in mild cognitive impairment

Increasing evidence suggests that oxidative damage is associated with normal aging and several neurodegenerative diseases. Mild cognitive impairment (MCI), the phase between normal aging and early dementia, is a common problem in the elderly with many subjects going on to develop Alzheimer's disease (AD). Although increased DNA oxidation is observed in the AD brain, it is unclear when the oxidative damage begins. To determine if DNA oxidation occurs in the brain of subjects with MCI, we quantified multiple oxidized bases in nuclear and mitochondrial DNA isolated from frontal, parietal and temporal lobes and cerebellum of short post-mortem interval autopsies of eight amnestic patients with MCI and six age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring. We found statistically significant elevations (p < 0.05) of 8-hydroxyguanine, a widely studied biomarker of DNA damage, in MCI nuclear DNA from frontal and temporal lobe and in mitochondrial DNA from the temporal lobe compared with age-matched control subjects. Levels of 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine were significantly elevated in nuclear DNA from all three neocortical regions in MCI. Statistically significant elevations of 4,6-diamino-5-formamidopyrimidine were also observed in mitochondrial DNA of MCI temporal, frontal and parietal lobes. These results suggest that oxidative damage to nuclear and mitochondrial DNA occurs in the earliest detectable phase of AD and may play a meaningful role in the pathogenesis of this disease.     

Effects of amiodarone on cardiac function and mitochondrial oxidative phosphorylation during ischemia and reperfusion

This study was carried out in order to determine if the efficiency of amiodarone, a class III antiarrhythmic agent, is associated with changes in mitochondrial oxidative phosphorylation. A population of 30 rats were treated with amiodarone (100 mg/kg/day) for 5 days. A second population receiving only vehicle was used as control. The hearts were perfused according to the working mode. After 15 min of normoxic perfusion, the left main coronary artery was ligated and the ligation was maintained for 20 min. The ligation was removed and reperfusion continued for a further 30 min. The electrocardiogram was monitored continuously. At the end of perfusion, the ischemic and non ischemic areas were visually separated and mitochondria were harvested from each area. Their oxidative and energy metabolism were assessed with palmitoylcarnitine as substrate in 2 respiration media differing in their free calcium concentration (0 or 0.34 m). In normoxic conditions, amiodarone treatment increased the cardiac metabolic efficiency (mechanical work to oxygen consumption ratio). The local ischemia decreased the aortic and coronary flows without modifying the cardiac metabolic efficiency. Amiodarone treatment maintained the aortic flow at a significantly higher value; the duration of severe arrhythmias was significantly decreased by the drug. The reperfusion of the ischemic area allowed the partial recovery of fluid dynamics. The coronary flow was restored to 89% of the pre ischemic value. Conversely, the aortic flow never exceeded that measured at the end of ischemia, partly due to the important development of severe arrhythmias. The recovery of aortic flow and metabolic efficiency during reperfusion was improved by amiodarone treatment; ventricular tachycardia and fibrillation duration were reduced. In the mitochondria issued from the normoxic area, the energy metabolism was not altered by the amiodarone treatment, but the presence of calcium in the respiration medium modified the oxidative phosphorylation. The divalent cation slightly decreased the state III respiration rate and increased noticeably the state IV respiration rate. This was associated with an important mitochondrial AMP production and maintenance of ADP in the respiration medium. This energy wasting was reported to decrease the mitochondrial metabolic efficiency. After an ischemia-reperfusion sequence, mitochondrial oxidation phosphorylation was reduced and amiodarone treatment amplified this decrease. This was presumably due to an increased mitochondrial calcium accumulation. Thus, the beneficial properties of amiodarone during reperfusion are supposed to be due to a protection against the deleterious effect of excess matrix calcium on mitochondrial energy metabolism.     

Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for β-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures.     

The biogeography of mitochondrial and nuclear discordance in animals

Combining nuclear (nuDNA) and mitochondrial DNA (mtDNA) markers has improved the power of molecular data to test phylogenetic and phylogeographic hypotheses and has highlighted the limitations of studies using only mtDNA markers. In fact, in the past decade, many conflicting geographic patterns between mitochondrial and nuclear genetic markers have been identified (i.e. mito-nuclear discordance). Our goals in this synthesis are to: (i) review known cases of mito-nuclear discordance in animal systems, (ii) to summarize the biogeographic patterns in each instance and (iii) to identify common drivers of discordance in various groups. In total, we identified 126 cases in animal systems with strong evidence of discordance between the biogeographic patterns obtained from mitochondrial DNA and those observed in the nuclear genome. In most cases, these patterns are attributed to adaptive introgression of mtDNA, demographic disparities and sex-biased asymmetries, with some studies also implicating hybrid zone movement, human introductions and Wolbachia infection in insects. We also discuss situations where divergent mtDNA clades seem to have arisen in the absence of geographic isolation. For those cases where foreign mtDNA haplotypes are found deep within the range of a second taxon, data suggest that those mtDNA haplotypes are more likely to be at a high frequency and are commonly driven by sex-biased asymmetries and/or adaptive introgression. In addition, we discuss the problems with inferring the processes causing discordance from biogeographic patterns that are common in many studies. In many cases, authors presented more than one explanation for discordant patterns in a given system, which indicates that likely more data are required. Ideally, to resolve this issue, we see important future work shifting focus from documenting the prevalence of mito-nuclear discordance towards testing hypotheses regarding the drivers of discordance. Indeed, there is great potential for certain cases of mitochondrial introgression to become important natural systems within which to test the effect of different mitochondrial genotypes on whole-animal phenotypes.     

Inheritance of organelle genomes in citrus somatic cybrids

Restriction fragment length polymorphisms (RFLPs) were used for the characterization of citrus organelle inheritance in somatic cybrids produced during six different citrus protoplast fusions. All the cybrids in this work inherited their mitochondrial genome from the embryogenic fusion partner (callus or cell suspension). In some of the combinations, non-parental bands were observed among the mitochondrial configurations. In contrast, the cybrids inherited plastid DNA from either the embryogenic or the nonembryogenic (leaf) fusion partner. The relative abundance of organelle DNAs in the embryogenic and leaf cells was in accordance with these inheritance patterns. Stochastic processes may therefore influence the outcome of somatic cell fusions with respect to organelle genomes.     

Tissue-specific expression atlas of murine mitochondrial tRNAs

Mammalian mitochondrial tRNA (mt-tRNA) plays a central role in the synthesis of the 13 subunits of the oxidative phosphorylation complex system (OXPHOS). However, many aspects of the context-dependent expression of mt-tRNAs in mammals remain unknown. To investigate the tissue-specific effects of mt-tRNAs, we performed a comprehensive analysis of mitochondrial tRNA expression across five mice tissues (brain, heart, liver, skeletal muscle, and kidney) using Northern blot analysis. Striking differences in the tissue-specific expression of 22 mt-tRNAs were observed, in some cases differing by as much as tenfold from lowest to highest expression levels among these five tissues. Overall, the heart exhibited the highest levels of mt-tRNAs, while the liver displayed markedly lower levels. Variations in the levels of mt-tRNAs showed significant correlations with total mitochondrial DNA (mtDNA) contents in these tissues. However, there were no significant differences observed in the 2-thiouridylation levels of tRNA^Lys, tRNA^Glu, and tRNA^Gln among these tissues. A wide range of aminoacylation levels for 15 mt-tRNAs occurred among these five tissues, with skeletal muscle and kidneys most notably displaying the highest and lowest tRNA aminoacylation levels, respectively. Among these tissues, there was a negative correlation between variations in mt-tRNA aminoacylation levels and corresponding variations in mitochondrial tRNA synthetases (mt-aaRS) expression levels. Furthermore, the variable levels of OXPHOS subunits, as encoded by mtDNA or nuclear genes, may reflect differences in relative functional emphasis for mitochondria in each tissue. Our findings provide new insight into the mechanism of mt-tRNA tissue-specific effects on oxidative phosphorylation.     

Mitochondrial DNA and respiratory chain function in spinal cords of ALS patients

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. In order to address the question of a putative role of mitochondrial dysfunction in the pathogenesis of ALS, we studied the mitochondrial DNA (mtDNA) and mitochondrial respiratory chain enzyme activities in spinal cords of ALS patients and in control subjects without neuropathologic abnormalities. Using a "double PCR and digestion" technique to estimate the levels of randomly distributed point mutations in two small regions of the mtDNA, we found significantly higher levels of mutant mtDNA in the spinal cord of ALS patients compared to controls. No large-scale rearrangements were found, but the amount of mtDNA, measured by Southern blot, was significantly lower in the ALS samples. This reduction correlated well with a decrease of citrate synthase (CS) activity, a mitochondrial marker, as were the activities of respiratory chain complexes I + III, II + III, and IV, suggesting a loss of mitochondria in ALS spinal cords.     

Chlamydia trachomatis targets mitochondrial dynamics to promote intracellular survival and proliferation

Chlamydia trachomatis is an obligate intracellular bacterium that scavenges host metabolic products for its replication. Mitochondria are the power plants of eukaryotic cells and provide most of the cellular ATP via oxidative phosphorylation. Several intracellular pathogens target mitochondria as part of their obligatory cellular reprogramming. This study was designed to analyse the mitochondrial morphological changes in response to C. trachomatis infection in HeLa cells. Mitochondrial elongation and fragmentation were found at the early stages and late stages of C. trachomatis infection, respectively. C. trachomatis infection‐induced mitochondrial elongation was associated with the increase of mitochondrial respiratory activity, ATP production, and intracellular growth of C. trachomatis. Silencing mitochondrial fusion mediator proteins abrogated the C. trachomatis infection‐induced elevation in the oxygen consumption rate and attenuated chlamydial proliferation. Mechanistically, C. trachomatis induced the elevation of intracellular cAMP at the early phase of infection, followed by the phosphorylation of fission‐inactive serine residue 637 (S637) of Drp1, resulting in mitochondrial elongation. Accordingly, treatment with adenylate cyclase inhibitor diminished mitochondrial elongation and bacterial growth in infected cells. Collectively, these results strongly indicate that C. trachomatis promotes its intracellular growth by targeting mitochondrial dynamics to regulate ATP synthesis via inhibition of the fission mediator Drp1.     

线粒体功能缺陷和神经系统疾病

线粒体呼吸链缺陷一直被认为是诱发线粒体疾病的重要因素,这有助于研究人员阐释其遗传和临床多样性。然而,线粒体的其他功能也具有重要意义,包括蛋白质运输、细胞器动力学和细胞凋亡。调控这些功能的基因缺陷不仅导致神经和精神疾病,而且还导致年龄相关的神经变性疾病。因此,引起越来越多的关注。在讨论呼吸链缺陷引起相关神经系统疾病的一些致病难题后,就线粒体动力学改变引起的相关神经系统疾病病因和常见神经变性疾病的病理生理机制作一综述。