Assaying ATP synthesis in cultured cells: A valuable tool for the diagnosis of patients with mitochondrial disorders

Mitochondrial ATP synthase plays a central role in cell function by synthesising most of the ATP in human tissues. In different cells, active regulation of mitochondrial ATP synthase in response to cellular energy demand has been demonstrated, as well as its alteration under several pathological conditions affecting oxidative phosphorylation (OXPHOS). Traditionally, detection of OXPHOS defects is based on the spectrophotometric measurement of respiratory chain complex activities in muscle biopsies. Considering the broad clinical spectrum of mitochondrial disorders, and the difficulty in arriving at a single diagnostic method, in this study we propose measurement of ATP synthesis in mitochondria from skin fibroblasts as an effective screening tool. In the light of our results this assessment emerges as a useful marker of impaired energy production in primary OXPHOS disorders of childhood and as a tool with the potential to drive further molecular genetic studies.     

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。     

Overexpression of TNF-α in mitochondrial diseases caused by mutations in mtDNA: evidence for signaling through its receptors on mitochondria

Mitochondrial diseases (MDs) are heterogeneous disorders due to impaired respiratory chain function causing defective ATP production. Although the disruption of oxidative phosphorylation is central to the MD pathophysiology, other factors may contribute to these disorders. We investigated the expression and the cellular localization of TNF-α and its receptors, TNFR1 and TNFR2, in muscle biopsies from 15 patients with mitochondrial respiratory chain dysfunction. Our data unambiguously demonstrate that TNF-α is expressed in muscle fibers with abnormal focal accumulations of mitochondria, so-called ragged red fibers, and is delivered to mitochondria where both receptors are localized. Moreover TNF receptors are differentially regulated in patients' muscle in which the expression levels of TNFR1 mRNA are decreased and those of TNFR2 mRNA are increased compared with controls. These findings suggest for the first time that TNF-α could exert a direct effect on mitochondria via its receptors.     

Mitochondrial Involvement in Brain Function and Dysfunction: Relevance to Aging,Neurodegenerative Disorders and Longevity

It is becoming increasingly evident that the mitochondrial genome may play a key role in neurodegenerative diseases. Mitochondrial dysfunction is characteristic of several neurodegenerative disorders, and evidence for mitochondria being a site of damage in neurodegenerative disorders is partially based on decreases in respiratory chain complex activities in Parkinson's disease, Alzheimer's disease, and Huntington's disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant balance perturbation, are thought to underlie defects in energy metabolism and induce cellular degeneration. Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of genes termed vitagenes. A promising approach for the identification of critical gerontogenic processes is represented by the hormesis-like positive effect of stress. In the present review, we discuss the role of energy thresholds in brain mitochondria and their implications in neurodegeneration. We then review the evidence for the role of oxidative stress in modulating the effects of mitochondrial DNA mutations on brain age-related disorders and also discuss new approaches for investigating the mechanisms of lifetime survival and longevity.     

The role of mitochondrial cardiolipin in heart function and its implication in cardiac disease

Mitochondria play an essential role in the energy metabolism of the heart. Many of the essential functions are associated with mitochondrial membranes and oxidative phosphorylation driven by the respiratory chain. Mitochondrial membranes are unique in the cell as they contain the phospholipid cardiolipin. The important role of cardiolipin in cardiovascular health is highlighted by several cardiac diseases, in which cardiolipin plays a fundamental role. Barth syndrome, Sengers syndrome, and Dilated cardiomyopathy with ataxia (DCMA) are genetic disorders, which affect cardiolipin biosynthesis. Other cardiovascular diseases including ischemia/reperfusion injury and heart failure are also associated with changes in the cardiolipin pool. Here, we summarize molecular functions of cardiolipin in mitochondrial biogenesis and morphology. We highlight the role of cardiolipin for the respiratory chain, metabolite carriers, and mitochondrial metabolism and describe links to apoptosis and mitochondria specific autophagy (mitophagy) with possible implications in cardiac disease.     

Mitochondrial enzymes discriminate between mitochondrial disorders and chronic fatigue syndrome

We studied the extent of mitochondrial involvement in chronic fatigue syndrome (CFS) and investigated whether measurement of mitochondrial respiratory chain complex (RCC) activities discriminates between CFS and mitochondrial disorders. Mitochondrial content was decreased in CFS compared to healthy controls, whereas RCC activities corrected for mitochondrial content were not. Conversely, mitochondrial content did not discriminate between CFS and two groups of mitochondrial disorders, whereas ATP production rate and complex I, III and IV activity did, all with higher activities in CFS. We conclude that the ATP production rate and RCC activities can reliably discriminate between mitochondrial disorders and CFS.     

Diseases caused by mutations in mitochondrial DNA

Mitochondrial diseases associated with mutations within mitochondrial genome are a subgroup of metabolic disorders since their common consequence is reduced metabolic efficiency caused by impaired oxidative phophorylation and shortage of ATP. Although the vast majority of mitochondrial proteins (approximately 1500) is encoded by nuclear genome, mtDNA encodes 11 subunits of respiratory chain complexes, 2 subunits of ATP synthase, 22 tRNAs and 2 rRNAs. Up to now, more than 250 pathogenic mutations have been described within mtDNA. The most common are point mutations in genes encoding mitochondrial tRNAs such as 3243A-->G and 8344T-->G that cause, respectively, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) or MIDD (maternally-inherited diabetes and deafness) and MERRF (myoclonic epilepsy with ragged red fibres) syndromes. There have been also found mutations in genes encoding subunits of ATP synthase such as 8993T-->G substitution associated with NARP (neuropathy, ataxia and retinitis pigmentosa) syndrome. It is worth to note that mitochondrial dysfunction can also be caused by mutations within nuclear genes coding for mitochondrial proteins.     

Pluripotent Stem Cells for Uncovering the Role of Mitochondria in Human Brain Function and Dysfunction

Mitochondrial dysfunctions are a known pathogenetic mechanism of a number of neurological and psychiatric disorders. At the same time, mutations in genes encoding for components of the mitochondrial respiratory chain cause mitochondrial diseases, which commonly exhibit neurological symptoms. Mitochondria are therefore critical for the functionality of the human nervous system. The importance of mitochondria stems from their key roles in cellular metabolism, calcium handling, redox and protein homeostasis, and overall cellular homeostasis through their dynamic network. Here, we describe how the use of pluripotent stem cells (PSCs) may help in addressing the physiological and pathological relevance of mitochondria for the human nervous system. PSCs allow the generation of patient-derived neurons and glia and the identification of gene-specific and mutation-specific cellular phenotypes via genome engineering approaches. We discuss the recent advances in PSC-based modeling of brain diseases and the current challenges of the field. We anticipate that the careful use of PSCs will improve our understanding of the impact of mitochondria in neurological and psychiatric disorders and the search for effective therapeutic avenues.     

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

A peep into mitochondrial disorder:multifaceted from mitochondrial DNAmutations to nuclear gene modulation

Mitochondrial genome is responsible for multiple human diseases in a maternal inherited pattern, yet phenotypes of patients in a same pedigree frequently vary largely. Genes involving in epigenetic modification, RNA processing, and other biological pathways, rather than “threshold effect” and environmental factors, provide more specific explanation to the aberrant phenotype. Thus, the double hit theory, mutations both in mitochondrial DNA and modifying genes aggravating the symptom, throws new light on mitochondrial dysfunction processes. In addition, mitochondrial retrograde signaling pathway that leads to reconfiguration of cell metabolism to adapt defects in mitochondria may as well play an active role. Here we review selected examples of modifier genes and mitochondrial retrograde signaling in mitochondrial disorders, which refine our understanding and will guide the rational design of clinical therapies.     

Mitochondrial Dysfunction and Chronic Liver Disease

Mitochondria are generally considered the powerhouse of the cell, a small subcellular organelle that produces most of the cellular energy in the form of adenosine triphosphate (ATP). In addition, mitochondria are involved in various biological functions, such as biosynthesis, lipid metabolism, oxidative phosphorylation, cell signal transduction, and apoptosis. Mitochondrial dysfunction is manifested in different aspects, like increased mitochondrial reactive oxygen species (ROS), mitochondrial DNA (mtDNA) damage, adenosine triphosphate (ATP) synthesis disorder, abnormal mitophagy, as well as changes in mitochondrial morphology and structure. Mitochondrial dysfunction is related to the occurrence and development of various chronic liver diseases, including hepatocellular carcinoma (HCC), viral hepatitis, drug-induced liver injury (DILI), alcoholic fatty liver (AFL), and non-alcoholic fatty liver (NAFL). In this review, we summarize and discuss the role and mechanisms of mitochondrial dysfunction in chronic liver disease, focusing on and discussing some of the latest studies on mitochondria and chronic liver disease.     

Age related mitochondrial degenerative disorders in humans

Disorders caused by mitochondrial respiratory chain deficiency due to mutations in mitochondrial DNA have varied phenotypes but many involve neurological features often associated with cell loss within specific brain regions. These disorders, along with the increasing evidence of decline in mitochondrial function with ageing, have raised speculation that primary changes in mitochondria could have an important role in age-related neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Evidence supporting a role for mitochondria in common neurodegenerative diseases comes from studies with the toxin MPP+ and familial PD, which has been shown to involve proteins such as DJ-1 and Pink1 (both of which are predicted to have a role in mitochondrial function and oxidative stress). Mutations within the mitochondrial genome have been shown to accumulate with age and in common neurodegenerative diseases. Mitochondrial DNA haplogroups have also been shown to be associated with certain neurodegenerative conditions. This review covers the primary mitochondrial diseases but also discuss the potential role of mitochondria and mitochondrial DNA mutations in mitochondrial and neurodegenerative diseases, in particular in PD and in AD.     

Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation

Dimerization or oligomerization of ATP synthase has been proposed to play an important role for mitochondrial cristae formation and to be involved in regulating ATP synthase activity. We found comparable oligomycin-sensitive ATPase activity for monomeric and oligomeric ATP synthase suggesting that oligomerization/monomerization dynamics are not directly involved in regulating ATP synthase activity. Binding of the natural IF1 inhibitor protein has been shown to induce dimerization of F1-subcomplexes. This suggested that binding of IF1 might also dimerize holo ATP synthase, and possibly link dimerization and inhibition. Analyzing mitochondria of human rho zero cells that contain mitochondria but lack mitochondrial DNA, we identified three subcomplexes of ATP synthase: (i) F1 catalytic domain, (ii) F1-domain with bound IF1, and (iii) F1-c subcomplex with bound IF1 and a ring of subunits c. Since both IF1 containing subcomplexes were present in monomeric state and exhibited considerably reduced ATPase activity as compared to the third subcomplex lacking IF1, we postulate that inhibition and induction of dimerization of F1-subcomplexes by IF1 are independent events. F1-subcomplexes were also found in mitochondria of patients with specific mitochondrial disorders, and turned out to be useful for the clinical differentiation between various types of mitochondrial biosynthesis disorders. Supramolecular associations of respiratory complexes, the "respirasomes", seem not to be the largest assemblies in the structural organization of the respiratory chain, as suggested by differential solubilization of mitochondria and electron microscopic analyses of whole mitochondria. We present a model for a higher supramolecular association of respirasomes into a "respiratory string".     

Enhanced Mitochondrial Radical Production in Patients with Rheumatoid Arthritis Correlates with Elevated Levels of Tumor Necrosis Factor alpha in Plasma

Mitochondrial dysfunction contributes to cell damage in a number of human diseases. One significant mechanism by which mitochondria damage cells is by producing reactive oxygen species from the respiratory chain. In this study we measured the production of reactive oxygen species by leukocyte mitochondria in blood from rheumatoid arthritis patients. To do this we used the chemiluminescence of lucigenin, which is accumulated by mitochondria within cells and reacts with superoxide to form a chemiluminescent product. By using specific inhibitors we could distinguish between the production of reactive oxygen species by mitochondria and by NADPH oxidase. There was a five-fold increase in mitochondrial reactive oxygen species production in whole blood and monocytes from patients with rheumatoid arthritis, when compared to healthy subjects or patients with non-rheumatic diseases. There was no increase in mitochondrial reactive oxygen species production by neutrophils from rheumatoid arthritis patients. The enhanced mitochondrial radical production in rheumatoid arthritis patients correlated significantly with increased levels of tumor necrosis factor alpha in plasma (p<0.0001). As tumor necrosis factor alpha is known to increase mitochondrial reactive oxygen species production the elevated mitochondrial radical formation seen in rheumatoid arthritis patients may be due to activation of the mitochondrial radical production. These data suggest that elevated mitochondrial oxidative stress contributes to the pathology of rheumatoid arthritis.     

Impaired Mitochondrial Function and Dynamics in the Pathogenesis of FXTAS

Mitochondrial involvement plays an important role in neurodegenerative diseases. At least one-third of adult carriers of a FMR1 premutation (55-200 CGG repeats) are at risk of presenting an adult-onset neurodegenerative disorder known as fragile X-associated tremor/ataxia syndrome (FXTAS). In an attempt to provide new insights into the mechanisms involved in the pathogenesis of FXTAS, we characterized mitochondrial function and dynamics by the assessment of oxidative respiratory chain function, mitochondrial content, oxidative stress levels, and mitochondrial network complexity. Regarding mitochondrial function, we found that mitochondrial respiratory capacity is compromised in skin fibroblasts whereas in blood, no differences were observed between the FXTAS and control groups. Furthermore, fibroblasts from FXTAS patients presented altered mitochondrial architecture, with more circular and less interconnected mitochondria being observed. Mitochondrial function and dynamics deregulation and characteristic of neurological disorders are present in FXTAS patients. These features might be limiting temporal and spatial bioenergetics cells supply and thus contributing to disease pathogenesis.     

A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies

Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.     

Mitochondrial pathobiology in ALS

Amyotrophic lateral sclerosis (ALS) is the third most common human adult-onset neurodegenerative disease. Some forms of ALS are inherited, and disease-causing genes have been identified. Nevertheless, the mechanisms of neurodegeneration in ALS are unresolved. Genetic, biochemical, and morphological analyses of human ALS as well as cell and animal models of ALS reveal that mitochondria could have roles in this neurodegeneration. The varied functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations. Changes occur in mitochondrial respiratory chain enzymes and mitochondrial programmed cell death proteins in ALS. Transgenic mouse models of ALS reveal possible principles governing the biology of neurodegeneration that implicate mitochondria and the mitochondrial permeability transition pore. This paper reviews how mitochondrial pathobiology might contribute to the mechanisms of neurodegeneration in ALS.     

Energy,ageing, fidelity and sex: oocyte mitochondrial DNA as a protected genetic template

Oxidative phosphorylation couples ATP synthesis to respiratory electron transport. In eukaryotes, this coupling occurs in mitochondria, which carry DNA. Respiratory electron transport in the presence of molecular oxygen generates free radicals, reactive oxygen species (ROS), which are mutagenic. In animals, mutational damage to mitochondrial DNA therefore accumulates within the lifespan of the individual. Fertilization generally requires motility of one gamete, and motility requires ATP. It has been proposed that oxidative phosphorylation is nevertheless absent in the special case of quiescent, template mitochondria, that these remain sequestered in oocytes and female germ lines and that oocyte mitochondrial DNA is thus protected from damage, but evidence to support that view has hitherto been lacking. Here we show that female gametes of Aurelia aurita, the common jellyfish, do not transcribe mitochondrial DNA, lack electron transport, and produce no free radicals. In contrast, male gametes actively transcribe mitochondrial genes for respiratory chain components and produce ROS. Electron microscopy shows that this functional division of labour between sperm and egg is accompanied by contrasting mitochondrial morphology. We suggest that mitochondrial anisogamy underlies division of any animal species into two sexes with complementary roles in sexual reproduction. We predict that quiescent oocyte mitochondria contain DNA as an unexpressed template that avoids mutational accumulation by being transmitted through the female germ line. The active descendants of oocyte mitochondria perform oxidative phosphorylation in somatic cells and in male gametes of each new generation, and the mutations that they accumulated are not inherited. We propose that the avoidance of ROS-dependent mutation is the evolutionary pressure underlying maternal mitochondrial inheritance and the developmental origin of the female germ line.     

M19 modulates skeletal muscle differentiation and insulin secretion in pancreatic β-cells through modulation of respiratory chain activity

Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.