Mitochondrial biogenesis in cultured animal cells. I. Effect of chloramphenicol on morphology and mitochondrial respiratory enzymes.

The effects of chloramphenicol on the morphology and respiratory enzymes of BHK-21 cells in spinner culture have been examined with time. Cells treated with chloramphenicol double twice before growth ceases; these cells have increased size as measured by several techniques. Mitochondria are enlarged and appear to degenerate with prolonged treatment. Cytochrome c oxidase and succinate cytochrome c reductase activities are reduced while there is no decrease in the activities of monoamine oxidase, glutamate dehydrogenase or NADPH-cytochrome c reductase. Cytochromes aa3 and b disappear on treatment while cytochromes c + c1 appears to be unaffected. All these effects are reversible if chloramphenicol is removed within a limited period of time.     

Mitochondrial respiratory chain dysfunction: implications in neurodegeneration

For decades mitochondria have been considered static round-shaped organelles in charge of energy production. In contrast, they are highly dynamic cellular components that undergo continuous cycles of fusion and fission influenced, for instance, by oxidative stress, cellular energy requirements, or the cell cycle state. New important functions beyond energy production have been attributed to mitochondria, such as the regulation of cell survival, because of their role in the modulation of apoptosis, autophagy, and aging. Primary mitochondrial diseases due to mutations in genes involved in these new mitochondrial functions and the implication of mitochondrial dysfunction in multifactorial human pathologies such as cancer, Alzheimer and Parkinson diseases, or diabetes has been demonstrated. Therefore, mitochondria are set at a central point of the equilibrium between health and disease, and a better understanding of mitochondrial functions will open new fields for exploring the roles of these mitochondrial pathways in human pathologies. This review dissects the relationships between activity and assembly defects of the mitochondrial respiratory chain, oxidative damage, and alterations in mitochondrial dynamics, with special focus on their implications for neurodegeneration.     

线粒体呼吸链与活性氧

已知有氧真核生物细胞吸收的氧分子绝大部分都是在线粒体呼吸链末端细胞色素氧化酶上通过四步单电子还原生成水。但同时也有1％-2％的氧可在呼吸链中途接受单电子或双电子被部分还原生成超氧（O2·^-和过氧化氢（H2O2）作为呼吸作用的正常代谢产物。此种来源于线粒体呼吸链的O2·^-和H2O2不但在多种病理的氧化损伤中起关键作用,同样它们也是正常生理条件下对多种细胞过程具有基本调控意义的氧还信号。基于Chance实验室约自20世纪70到90年代的早期研究贡献以及20世纪90年代后其他各实验室的研究新进展,我们聚焦于下述四个相关问题的评述和讨论：（1）由于线粒体内膜面积及其含有的呼吸链复合体酶活力远远高出细胞中所有膜系数量和相关酶活力之总和,因而线粒体呼吸链产生的O2·^-和H2O2构成生物体内最大数量ROS的恒定来源;（2）线粒体呼吸链复合体III的Q循环中Qo位点中半醌自由基（UQH·）已明确是O2·^-的单电子来源;还原细胞色素C-P66^SHC是生成H2O2的双电子供体。虽然复合体I也是产生线粒体基质内O2·^-的主要来源,但由于其确切生成位点尚未明确,在invivo条件下能否产生大量O2·^-也尚有争议;（3）线粒体呼吸链产生O2·^-后的分配和跨膜转移涉及其生理病理作用机制和作用靶点等复杂而重要的问题,直到目前尚未意见一致。“质子和O2·^-循环双回路解偶联模型”整合了目前提出的几种假说的联系点,指出H^＋和O2·^-相互作用生成HO2·及其跨膜很可能是这一复杂问题的中心环节,并与O2·^-对“脂肪酸shuttling model”或O2·^-对“UCPS激活”模型形成了内在的联系;（4）线粒体呼吸形成的△P（△ψ和△pH）能直接控制呼吸链的ROS生成,并以非线性（非欧姆）相关方式通过影响Q循环中的Qo半醌的氧还态和寿命来调节O2·^-生成的急速?     

Mitochondrial respiratory chain and free radical generation in stroke

Being the second most common cause of death in the industrial countries and one of the major causes of death and disability, stroke has a great effect on public health and is the neurological disease which accounts for the largest number of hospitalizations. In order to develop new treatments, biochemical mechanisms involved in brain damage have been investigated. Among them, oxidant species generated during stroke have been implicated as critical mediators of neuronal injury in this condition, although neuroprotective roles have also been demonstrated. This review is focused on the role of the mitochondrial respiratory chain as both source and target of reactive oxygen and nitrogen species such as nitric oxide, superoxide and peroxynitrite produced in cerebral ischemia. The neuroprotective role of antioxidants or other molecules acting on the mitochondrial respiratory chain and ATP synthesis in the setting of cerebral ischemia is discussed.     

Alternative respiratory chain enzymes: Therapeutic potential and possible pitfalls

The alternative respiratory chain (aRC), comprising the alternative NADH dehydrogenases (NDX) and quinone oxidases (AOX), is found in microbes, fungi and plants, where it buffers stresses arising from restrictions on electron flow in the oxidative phosphorylation system. The aRC enzymes are also found in species belonging to most metazoan phyla, including some chordates and arthropods species, although not in vertebrates or in Drosophila. We postulated that the aRC enzymes might be deployed to alleviate pathological stresses arising from mitochondrial dysfunction in a wide variety of disease states. However, before such therapies can be contemplated, it is essential to understand the effects of aRC enzymes on cell metabolism and organismal physiology. Here we report and discuss new findings that shed light on the functions of the aRC enzymes in animals, and the unexpected benefits and detriments that they confer on model organisms. In Ciona intestinalis, the aRC is induced by hypoxia and by sulfide, but is unresponsive to other environmental stressors. When expressed in Drosophila, AOX results in impaired survival under restricted nutrition, in addition to the previously reported male reproductive anomalies. In contrast, it confers cold resistance to developing and adult flies, and counteracts cell signaling defects that underlie developmental dysmorphologies. The aRC enzymes may also influence lifespan and stress resistance more generally, by eliciting or interfering with hormetic mechanisms. In sum, their judicious use may lead to major benefits in medicine, but this will require a thorough characterization of their properties and physiological effects.     

Mitochondrial biogenesis: recent developments and insights

Biosynthesis of a functional mitochondrion requires the coordinate expression of genes in both mitochondrial and nuclear DNAs. In yeast, three mitochondrial genes are split and RNA splicing plays a pivotal role in their expression. The recent finding that some introns are capable of self-splicing activity in vitro has permitted analysis of the mechanisms involved in RNA catalysis and may eventually shed light on the evolution of splicing mechanisms in general. Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and imported by the organelle. The availability of cloned genes coding for several constituent subunits of the ubiquinol-cytochrome c reductase, which are imported by mitochondria, has allowed study of selected steps in the addressing of proteins to mitochondria and their intercompartmental sorting within the organelle. Recent developments are discussed.     

Bcs1, a AAA protein of the mitochondria with a role in the biogenesis of the respiratory chain

The family of AAA+ proteins in eukaryotes has many members in various cellular compartments with a broad spectrum of functions in protein unfolding and degradation. The mitochondrial AAA protein Bcs1 plays an unusual role in protein translocation. It is involved in the topogenesis of the Rieske protein, Rip1, and thereby in the biogenesis of the cytochrome bc(1) complex of the mitochondrial respiratory chain. Bcs1 mediates the export of the folded FeS domain of Rip1 across the mitochondrial inner membrane and the insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc(1) complex. We discuss structural elements of the Bcs1 protein compared to other AAA proteins in an attempt to understand the mechanism of its function. In this context, we discuss human diseases caused by mutations in Bcs1 that lead to different properties of the protein and subsequently to different symptoms.     

Mitochondrial localization unveils a novel role for GRK2 in organelle biogenesis

Metabolic stimuli such as insulin and insulin like growth factor cause cellular accumulation of G protein coupled receptor kinase 2 (GRK2), which in turn is able to induce insulin resistance. Here we show that in fibroblasts, GRK2 is able to increase ATP cellular content by enhancing mitochondrial biogenesis; also, it antagonizes ATP loss after hypoxia/reperfusion. Interestingly, GRK2 is able to localize in the mitochondrial outer membrane, possibly through one region within the RGS homology domain and one region within the catalytic domain. In vivo, GRK2 removal from the skeletal muscle results in reduced ATP production and impaired tolerance to ischemia. Our data show a novel sub-cellular localization of GRK2 in the mitochondria and an unexpected role in regulating mitochondrial biogenesis and ATP generation.     

Mitochondrial electron transfer in the wheat pathogenic fungus Septoria tritici: on the role of alternative respiratory enzymes in fungicide resistance

Certain phytopathogenic fungi are able to express alternative NADH- and quinol-oxidising enzymes that are insensitive to inhibitors of the mitochondrial respiratory Complexes I and III. To assess the extent to which such enzymes confer tolerance to respiration-targeted fungicides, an understanding of mitochondrial electron transfer in these species is required. An isolation procedure has been developed which results in intact, active and coupled mitochondria from the wheat pathogen Septoria tritici, as evidenced by morphological and kinetic data. Exogenous NADH, succinate and malate/glutamate are readily oxidised, the latter activity being only partly (approx. 70%) sensitive to rotenone. Of particular importance was the finding that azoxystrobin (a strobilurin fungicide) potently inhibits fungal respiration at the level of Complex III. In some S. tritici strains investigated, a small but significant part of the respiratory activity (approx. 10%) is insensitive to antimycin A and azoxystrobin. Such resistant activity is sensitive to octyl gallate, a specific inhibitor of the plant alternative oxidase. This enzyme, however, could not be detected immunologically. On the basis of the above findings, a conceptual mitochondrial electron transfer chain is presented. Data are discussed in terms of developmental and environmental regulation of the composition of this chain.     

Mitochondrial biogenesis during fungal spore germination: respiratory cytochromes of dormant and germinating spores of Botryodiplodia.

The mitochondrial respiratory cytochrome contents of dormant and germinating conidia of Botryodiplodia theobromae were examined. Oxidized versus reduced difference spectra at 77 degrees K of whole mitochondria from physiologically mature germinated spores showed a typical a-band pattern for cytochromes c, b, and a, with absorption maxima at 549, 554 + 559, and 604 nm, respectively, whereas the difference spectrum of the counterpart mitochondrial fraction from dormant spores showed no cytochrome a bands. However, a fraction prepared from dormant spore mitochondria by detergent extraction and (NH4)2SO4 fractionation contained readily detectable quantities of cytochromes c and b (as shown by the a and Soret absorption bands), but it did not contain the a or Soret bands of cytochrome a observed in a counterpart preparation from germinated spores. The pyridine hemochromogen preparation from the dormant spore mitochondria contained no material that is spectroscopically characteristic of a-type heme and protoheme. These results suggest that cytochrome a is not present as a functional molecule in dormant spores. The first spectroscopically detectable cytochromes were observed in whole mitochondria at 210 min of spore germination, and the amount of each of the cytochromes increased with cell growth. A precursor of the heme porphyrin, delta-[4-14C]aminolevulinic acid, was first incorporated (at accelerating rates) into acid-insoluble spore material at 180 min of germination, which appears to be the approximate time of organization of new mitochondria in these spores.     

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster–specific decrease in mitochondrial DNA–encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.     

Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients

Mutations in the human TAZ gene are associated with Barth Syndrome, an often fatal X-linked disorder that presents with cardiomyopathy and neutropenia. The TAZ gene encodes Tafazzin, a putative phospholipid acyltranferase that is involved in the remodeling of cardiolipin, a phospholipid unique to the inner mitochondrial membrane. It has been shown that the disruption of the Tafazzin gene in yeast (Taz1) affects the assembly and stability of respiratory chain Complex IV and its supercomplex forms. However, the implications of these results for Barth Syndrome are restricted due to the additional presence of Complex I in humans that forms a supercomplex with Complexes III and IV. Here, we investigated the effects of Tafazzin, and hence cardiolipin deficiency in lymphoblasts from patients with Barth Syndrome, using blue-native polyacrylamide gel electrophoresis. Digitonin extraction revealed a more labile Complex I/III(2)/IV supercomplex in mitochondria from Barth Syndrome cells, with Complex IV dissociating more readily from the supercomplex. The interaction between Complexes I and III was also less stable, with decreased levels of the Complex I/III(2) supercomplex. Reduction of Complex I holoenzyme levels was observed also in the Barth Syndrome patients, with a corresponding decrease in steady-state subunit levels. We propose that the loss of mature cardiolipin species in Barth Syndrome results in unstable respiratory chain supercomplexes, thereby affecting Complex I biogenesis, respiratory activities and subsequent pathology.     

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.