Search

In human mitochondria, mtDNA encodes for only 13 proteins, all components of the OXPHOS system. The rest of the mitochondrial components, which make up approximately 99% of its proteome, are encoded in the nuclear genome, synthesized in cytosolic ribosomes and imported into mitochondria. Different import machineries translocate mitochondrial precursors, depending on their nature and the final destination inside the organelle. The proper and coordinated function of these molecular pathways is critical for mitochondrial homeostasis. Here, we will review molecular details about these pathways, which components have been linked to human disease and future perspectives on the field to expand the genetic landscape of mitochondrial diseases.

Protein import into mitochondria is facilitated by translocases within the outer and the inner mitochondrial membranes that are dedicated to a highly specific subset of client proteins. The mitochondrial carrier translocase (TIM22 complex) inserts multispanning proteins, such as mitochondrial metabolite carriers and translocase subunits (TIM23, TIM17A/B and TIM22), into the inner mitochondrial membrane. Both types of substrates are essential for mitochondrial metabolic function and biogenesis. Here, we report on a subject, diagnosed at 1.5 years, with a neuromuscular presentation, comprising hypotonia, gastroesophageal reflux disease and persistently elevated serum and Cerebrospinal fluid lactate (CSF). Patient fibroblasts displayed reduced oxidative capacity and altered mitochondrial morphology. Using trans-mitochondrial cybrid cell lines, we excluded a candidate variant in mitochondrial DNA as causative of these effects. Whole-exome sequencing identified compound heterozygous variants in the TIM22 gene (NM_013337), resulting in premature truncation in one allele (p.Tyr25Ter) and a point mutation in a conserved residue (p.Val33Leu), within the intermembrane space region, of the TIM22 protein in the second allele. Although mRNA transcripts of TIM22 were elevated, biochemical analyses revealed lower levels of TIM22 protein and an even greater deficiency of TIM22 complex formation. In agreement with a defect in carrier translocase function, carrier protein amounts in the inner membrane were found to be reduced. This is the first report of pathogenic variants in the TIM22 pore-forming subunit of the carrier translocase affecting the biogenesis of inner mitochondrial membrane proteins critical for metabolite exchange.

Objective: The comparison on mitochondrial function between severe septic patients and healthy control subjects according to mitochondrial deoxyribonucleic acid (mtDNA) haplogroup has not been previously reported; and this was the objective of the current study., Methods: Prospective, multicenter, observational study. We obtained blood samples from 198 severe septic patients at days 1, 4 and 8 of severe sepsis diagnosis and from 96 sex- and age-matched healthy controls to determine mtDNA haplogroup and platelet respiratory complex IV (CIV) specific activity. The endpoint of the study was 30-day mortality., Results: We included 198 severe septic patients (38 with mtDNA haplogroup JT and 160 with mtDNA haplogroup non-JT) and 96 healthy control subjects (16 with mtDNA haplogroup JT and 80 with mtDNA haplogroup non-JT). We have no found statistically significant differences in platelet CIV specific activity between healthy controls and survivor severe septic patients with mtDNA haplogroup JT at days 1, 4 and 8 of severe sepsis diagnosis; and the remaining severe septic patients showed lower platelet CIV specific activity than healthy controls with the same mtDNA haplogroup., Conclusions: The new finding of our study was that survivor severe septic patients and healthy controls with mtDNA haplogroup JT showed no different platelet Civ specific activity., (Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Leber hereditary optic neuropathy (LHON) is a rare, inherited mitochondrial disease. No treatment has shown a clear-cut benefit on a clinically meaningful end-point. Primary open-angle glaucoma (POAG) is a frequent, acquired optic neuropathy. Lowering intraocular pressure (IOP) reduces disease progression. However, current methods to decelerate this progression are recognized as being inadequate. Therefore, there is a clear need to look for new therapeutic approaches. The growing evidence indicates that POAG can also be a mitochondrial optic neuropathy (MON). Several risk elements are common for both diseases and all of them decrease mitochondrial (mt)DNA content. Based on these susceptibility factors and their molecular mechanism, we suggest herein pharmacological therapies targeted to increase mtDNA levels, oxidative phosphorylation (OXPHOS) capability, and mitochondrial energy production as treatments for MONs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The antibiotic linezolid is a ribosomal inhibitor with excellent efficacy. Although the administration period has been reduced to 28 days, side effects, usually of hematologic or neuropathic origin, are still reported due to secondary inhibition of mitochondrial protein synthesis. Susceptibility to linezolid toxicity remains unknown. Therefore, the objective of this study was to gain an understanding of clinical heterogeneity in response to identical linezolid exposures through exhaustive examination of the molecular basis of tissue-dependent mitotoxicity, consequent cell dysfunction, and the association of mitochondrial genetics with adverse effects of linezolid administered for the recommended period. Peripheral blood mononuclear cells (PBMC) and skin nerve fibers from 19 and 6 patients, respectively, were evaluated before and after a 28-day linezolid treatment in order to assess toxic effects on mitochondria and cells. Mitochondrial DNA haplotypes and single nucleotide polymorphisms (SNPs) in ribosomal sequences where linezolid binds to mitochondrial ribosomes were also analyzed to investigate their genetic contributions. We found that linezolid reduced mitochondrial protein levels, complex IV activity, and mitochondrial mass in PBMC and was associated with a trend toward an increase in the rate of apoptosis. In skin tissue, mitochondrial mass increased within nerve fibers, accompanied by subclinical axonal swelling. Mitochondrial haplogroup U, mutations in 12S rRNA, and the m.2706A→G, m.3197T→C, and m.3010G→A polymorphisms in 16S rRNA showed a trend toward an association with increased mitochondrial and clinical adverse effects. We conclude that even when linezolid is administered for a shorter time than formerly, adverse effects are reported by 63% of patients. Linezolid exerts tissue-dependent mitotoxicity that is responsible for downstream cellular consequences (blood cell death and nerve fiber swelling), leading to adverse hematologic and peripheral nervous side effects. Multicentric studies should confirm genetic susceptibility in larger cohorts., (Copyright © 2017 American Society for Microbiology.)

Purpose: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease that typically causes bilateral blindness in young men. It is characterized by as yet undisclosed genetic and environmental factors affecting the incomplete penetrance., Methods: We identified 27 LHON subjects who possess heteroplasmic primary LHON mutations. Mitochondrial DNA (mtDNA) copy number was evaluated., Results: The presence of centrocecal scotoma, an edematous, hyperemic optic nerve head, and vascular tortuosity, as well as telangiectasia was recognized in affected subjects. We found higher cellular mtDNA content in peripheral blood cells of unaffected heteroplasmic mutation carriers with respect to the affected., Conclusions: The increase of cellular mtDNA content prevents complete loss of vision despite the presence of a heteroplasmic state of LHON primary mutation, suggesting that it is a key factor responsible for penetrance of LHON.

Background: Several methods have been developed to predict the pathogenicity of missense mutations but none has been specifically designed for classification of variants in mtDNA-encoded polypeptides. Moreover, there is not available curated dataset of neutral and damaging mtDNA missense variants to test the accuracy of predictors. Because mtDNA sequencing of patients suffering mitochondrial diseases is revealing many missense mutations, it is needed to prioritize candidate substitutions for further confirmation. Predictors can be useful as screening tools but their performance must be improved., Results: We have developed a SVM classifier (Mitoclass.1) specific for mtDNA missense variants. Training and validation of the model was executed with 2,835 mtDNA damaging and neutral amino acid substitutions, previously curated by a set of rigorous pathogenicity criteria with high specificity. Each instance is described by a set of three attributes based on evolutionary conservation in Eukaryota of wildtype and mutant amino acids as well as coevolution and a novel evolutionary analysis of specific substitutions belonging to the same domain of mitochondrial polypeptides. Our classifier has performed better than other web-available tested predictors. We checked performance of three broadly used predictors with the total mutations of our curated dataset. PolyPhen-2 showed the best results for a screening proposal with a good sensitivity. Nevertheless, the number of false positive predictions was too high. Our method has an improved sensitivity and better specificity in relation to PolyPhen-2. We also publish predictions for the complete set of 24,201 possible missense variants in the 13 human mtDNA-encoded polypeptides., Conclusions: Mitoclass.1 allows a better selection of candidate damaging missense variants from mtDNA. A careful search of discriminatory attributes and a training step based on a curated dataset of amino acid substitutions belonging exclusively to human mtDNA genes allows an improved performance. Mitoclass.1 accuracy could be improved in the future when more mtDNA missense substitutions will be available for updating the attributes and retraining the model.

Sporadic inclusion body myositis (sIBM) is one of the most common myopathies in elderly people. Mitochondrial abnormalities at the histological level are present in these patients. We hypothesize that mitochondrial dysfunction may play a role in disease aetiology. We took the following measurements of muscle and peripheral blood mononuclear cells (PBMCs) from 30 sIBM patients and 38 age- and gender-paired controls: mitochondrial DNA (mtDNA) deletions, amount of mtDNA and mtRNA, mitochondrial protein synthesis, mitochondrial respiratory chain (MRC) complex I and IV enzymatic activity, mitochondrial mass, oxidative stress and mitochondrial dynamics (mitofusin 2 and optic atrophy 1 levels). Depletion of mtDNA was present in muscle from sIBM patients and PBMCs showed deregulated expression of mitochondrial proteins in oxidative phosphorylation. MRC complex IV/citrate synthase activity was significantly decreased in both tissues and mitochondrial dynamics were affected in muscle. Depletion of mtDNA was significantly more severe in patients with mtDNA deletions, which also presented deregulation of mitochondrial fusion proteins. Imbalance in mitochondrial dynamics in muscle was associated with increased mitochondrial genetic disturbances (both depletion and deletions), demonstrating that proper mitochondrial turnover is essential for mitochondrial homoeostasis and muscle function in these patients., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Oxidative phosphorylation dysfunction has been found in many different disorders. This biochemical pathway depends on mitochondrial protein synthesis. Thus, mutations in components of the mitochondrial translation system can be responsible for some of these pathologies. We identified a new homozygous missense mutation in the mitochondrial translation elongation factor Ts gene in a patient suffering from slowly progressive childhood ataxia and hypertrophic cardiomyopathy. Using cell, biochemical and molecular-genetic protocols, we confirm it as the etiologic factor of this phenotype. Moreover, as an important functional confirmation, we rescued the normal molecular phenotype by expression of the wild-type TSFM cDNA in patient's fibroblasts. Different TSFM mutations can produce the same or very different clinical phenotypes, going from abortions to moderately severe presentations. On the other hand, the same TSFM mutation can also produce same or different phenotypes within the same range of presentations, therefore suggesting the involvement of unknown factors.

Background: Mutations causing Leber hereditary optic neuropathy are usually homoplasmic, show incomplete penetrance, and many of the affected positions are not well conserved through evolution. A large percentage of patients harbouring these mutations have no family history of disease. Moreover, the transfer of the mutation in the cybrid model is frequently not accompanied by the transfer of the cellular, biochemical and molecular phenotype. All these features make difficult their classification as the etiologic factors for this disease. We report a patient who exhibits typical clinical features of Leber hereditary optic neuropathy but lacks all three of the most common mitochondrial DNA mutations., Methods: The diagnosis was made based on clinical studies. The mitochondrial DNA was completely sequenced, and the candidate mutation was analysed in more than 18 000 individuals around the world, its conservation index was estimated in more than 3100 species from protists to mammals, its position was modelled in the crystal structure of a bacteria ortholog subunit, and its functional consequences were studied in a cybrid model., Results: Genetic analysis revealed an m.3472T>C transition in the MT-ND1 gene that changes a phenylalanine to leucine at position 56. Bioinformatics, molecular-genetic analysis and functional studies suggest that this transition is the etiological factor for the disorder., Conclusions: This mutation expands the spectrum of deleterious changes in mitochondrial DNA-encoded complex I polypeptides associated with this pathology and highlights the difficulties in assigning pathogenicity to new homoplasmic mutations that show incomplete penetrance in sporadic Leber hereditary optic neuropathy patients., (© 2014 Royal Australian and New Zealand College of Ophthalmologists.)

FK506 is an important immunosuppressive medication. However, it can provoke neurotoxicity, nephrotoxicity, and diabetes as adverse side effects. The decrease in oxygen consumption of rat cells treated with pharmacologically relevant concentrations of FK506, along with other evidences, has insinuated that some of the toxic effects are probably caused by drug-induced mitochondrial dysfunction at the level of gene expression. To confirm this suggestion, we have analyzed cell respiration and mitochondrial protein synthesis in human cell lines treated with FK506. This drug provokes an important decrease in oxygen consumption, accompanied by a slight reduction in the synthesis of mitochondria DNA-encoded proteins. These results are similar to those triggered by rapamycin, another macrolide with immunosuppressive properties, therefore insinuating a common toxic pathway.

Multiple sclerosis is likely caused by a complex interaction of multiple genes and environmental factors. The contribution of mitochondrial DNA genetic backgrounds has been frequently reported. To evaluate the effect of mitochondrial DNA haplogroups in the same genetic and environmental circumstances, we have built human transmitochondrial cell lines and simulated the effect of axon demyelination, one of the hallmarks of multiple sclerosis pathology, by altering the ionic gradients through the plasmalemma and increasing ATP consumption. In this model, mitochondrial biogenesis is observed. This process is larger in Uk cybrids, which mirrors their lower oxidative phosphorylation capacity in basal conditions. This model replicates a process occurring in both patients suffering from multiple sclerosis and several animal models of axon demyelination. Therefore, it can be used to analyze the contribution of various mitochondrial DNA genotypes to multiple sclerosis. In this sense, a longer or stronger energy stress, such as that associated with demyelinated axons in multiple sclerosis, could make Uk individuals more susceptible to this pathology. Finally, pharmacologic compounds targeted to mitochondrial biogenesis could be a potential therapy for multiple sclerosis.

Some ribosomal antibiotics used in clinical practice to fight pathogenic bacteria can provoke serious adverse drug reactions in patients. Sensitivity to the antibiotics is a multifactorial trait but the genetic variation of sensitive individuals to off-target effects of the drugs might be one of the factors contributing to this condition. Thus, the protein synthesis apparatus of mitochondria is similar to that of bacteria because of its endosymbiotic origin and, therefore, mitochondrial ribosomes are frequently unintended off-targets of these antibiotics. Because of the limitations of epidemiologic studies of pharmacogenomics, we constructed 25 transmitochondrial cell lines using platelets from individuals belonging to high-frequency European mitochondrial DNA (mtDNA) haplogroups and grew them in the absence or presence of commonly used ribosomal antibiotics. Next, we analyzed the mitochondrial synthesis of proteins and the mitochondrial oxygen consumption to ascertain whether some side effects of ribosomal drugs are due to their interaction with particular mtDNA haplogroup-defining polymorphisms. The amount of mitochondrial translation products, the p.MT-CO1/succinate dehydrogenase subunit A ratio and the ratio of respiratory complex IV quantity to citrate synthase (CS)-specific activity were significantly lower, after the treatment with linezolid, in cybrids harboring the highly frequent m.3010A allele. These results suggest that mitochondrial antibiograms should be implemented for at least the most frequent mitochondrial ribosomal RNA (rRNA) polymorphisms and combinations of polymorphisms and the most frequently used ribosomal antibiotics. In this way, we would obtain individualized barcodes for antibiotic therapy, avoid the side effects of the antibiotics and enable appropriate personalized medicine.

Introduction: We recently found that platelet cytochrome c oxidase (COX) activities and quantities in 6-month-survival septic patients are significantly higher than those of patients who died before 6 months. Other studies suggested that the mitochondrial DNA (mtDNA) genotype could play a major role in sepsis survival. Given that COX catalytic subunits are encoded by mtDNA, the objective of the present study was to explore whether mtDNA population genetic variation could affect COX activity and quantity and favors sepsis survival., Methods: A prospective, multicenter, observational study was carried out in six Spanish ICUs. We included 96 patients with severe sepsis. We determined the mtDNA haplogroup, the COX specific activity/citrate synthase specific activity (COXa/CSa) ratio and the COX quantity/citrate synthase specific activity (COXq/CSa) ratio in circulating platelets at the time of diagnosis, day 4 and day 8. We used survival at 1 and 6 months as endpoints., Results: Patients with the JT mtDNA haplogroup (n=15) showed higher COXq/CSa ratio at day 4 (P=0.04) and day 8 (P=0.02) than those with other haplogroups (n=81). Logistic regression analysis showed that the JT mtDNA haplogroup (odds ratio=0.18; 95% confidence interval=0.04 to 0.94; P=0.04) and COXq/CSa ratio (odds ratio=0.53; 95% confidence interval=0.30 to 0.93; P=0.03) were associated with 1-month survival after controlling for age and lactic acid levels., Conclusions: The novel findings of our study are that 1-month surviving septic patients showed higher COXq/CSa ratio than nonsurviving individuals, that patients from the JT mtDNA haplogroup showed a higher COXq/CSa ratio and that JT patients had a higher 1-month survival than patients from other mtDNA haplogroups.

Background: Mitochondrial DNA is an ideal source of information to conduct evolutionary and phylogenetic studies due to its extraordinary properties and abundance. Many insights can be gained from these, including but not limited to screening genetic variation to identify potentially deleterious mutations. However, such advances require efficient solutions to very difficult computational problems, a need that is hampered by the very plenty of data that confers strength to the analysis., Results: We develop a systematic, automated methodology to overcome these difficulties, building from readily available, public sequence databases to high-quality alignments and phylogenetic trees. Within each stage in an autonomous workflow, outputs are carefully evaluated and outlier detection rules defined to integrate expert knowledge and automated curation, hence avoiding the manual bottleneck found in past approaches to the problem. Using these techniques, we have performed exhaustive updates to the human mitochondrial phylogeny, illustrating the power and computational scalability of our approach, and we have conducted some initial analyses on the resulting phylogenies., Conclusions: The problem at hand demands careful definition of inputs and adequate algorithmic treatment for its solutions to be realistic and useful. It is possible to define formal rules to address the former requirement by refining inputs directly and through their combination as outputs, and the latter are also of help to ascertain the performance of chosen algorithms. Rules can exploit known or inferred properties of datasets to simplify inputs through partitioning, therefore cutting computational costs and affording work on rapidly growing, otherwise intractable datasets. Although expert guidance may be necessary to assist the learning process, low-risk results can be fully automated and have proved themselves convenient and valuable.

Each cell type develops and maintains a specific oxidative phosphorylation (OXPHOS) capacity to satisfy its metabolic and energetic demands. This implies that there are differences between tissues in mitochondrial number, function, protein composition and morphology. The OXPHOS system biogenesis requires the coordinated expression of both mitochondrial and nuclear genomes. Mitochondrial DNA (mtDNA) expression can be regulated at different levels (replication, transcription, translation and post-translational levels) to contribute to the final observed OXPHOS activities. By analyzing five mammalian tissues, we evaluated the differences in the cellular amount of mtDNA and its correlation with the final observed mitochondrial activity., (Copyright © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Many epidemiologic studies have associated human mitochondrial haplogroups to rare mitochondrial diseases like Leber's hereditary optic neuropathy or to more common age-linked disorders such as Parkinson's disease. However, cellular, biochemical and molecular-genetic evidence that is able to explain these associations is very scarce. The etiology of multifactorial diseases is very difficult to sort out because such diseases are due to a combination of genetic and environmental factors that individually only contribute in small part to the development of the illness. Thus, the haplogroup-defining mutations might behave as susceptibility factors, but they could have only a small effect on oxidative phosphorylation (OXPHOS) function. Moreover, these effects would be highly dependent on the 'context' in which the genetic variant is acting. To homogenize this 'context' for mitochondrial DNA (mtDNA) mutations, a cellular approach is available that involves the use of what is known as 'cybrids'. By using this model, we demonstrate that mtDNA and mtRNA levels, mitochondrial protein synthesis, cytochrome oxidase activity and amount, normalized oxygen consumption, mitochondrial inner membrane potential and growth capacity are different in cybrids from the haplogroup H when compared with those of the haplogroup Uk. Thus, these inherited basal differences in OXPHOS capacity can help to explain why some individuals more quickly reach the bioenergetic threshold below which tissue symptoms appear and progress toward multifactorial disorders. Hence, some population genetic variants in mtDNA contribute to the genetic component of complex disorders. The existence of mtDNA-based OXPHOS differences opens possibilities for the existence of a new field, mitochondrial pharmacogenomics. New sequence accession nos: HM103354-HM103363.

Mitochondrial background has been demonstrated to influence maximal oxygen uptake (VO(2max), in mLkg(-1)min(-1)), but this genetic influence can be compensated for by regular exercise. A positive correlation among electron transport chain (ETC) coupling, ATP and reactive oxygen species (ROS) production has been established, and mitochondrial variants have been reported to show differences in their ETC performance. In this study, we examined in detail the VO(2max) differences found among mitochondrial haplogroups. We recruited 81 healthy male Spanish Caucasian individuals and determined their mitochondrial haplogroup. Their VO(2max) was determined using incremental cycling exercise (ICE). VO(2max) was lower in J than in non-J haplogroup individuals (P=0.04). The H haplogroup was responsible for this difference (VO(2max); J vs. H; P=0.008) and this group also had significantly higher mitochondrial oxidative damage (mtOD) than the J haplogroup (P=0.04). In agreement with these results, VO(2max) and mtOD were positively correlated (P=0.01). Given that ROS production is the major contributor to mtOD and consumes four times more oxygen per electron than the ETC, our results strongly suggest that ROS production is responsible for the higher VO(2max) found in the H variant. These findings not only contribute to a better understanding of the mechanisms underneath VO(2max), but also help to explain some reported associations between mitochondrial haplogroups and mtOD with longevity, sperm motility, premature aging and susceptibility to different pathologies.

Migrations into Africa from the Levant have greatly determined the mitochondrial genetic landscape of North Africa. After analyzing samples from North Morocco to Spain, we show that three fourths of the Moroccan individuals belong to Western Eurasian haplogroups and the frequencies of these are much more similar to those of the Iberian Peninsula than to those of the Middle East. This is particularly true for the mitochondrial haplogroups H1, H3 and V, which experienced a late-glacial expansion from this region, that repopulated much of Central and Northern Europe. Iberian Peninsula was also a source for prehistoric migrations to North Africa.

It has been clearly established that mitochondrial variants, among other potential factors, influence on VO(2max). With this study we sought to determine whether this genetic predisposition could be modified by steady exercise. Mitochondrial genetic variants were determined in 70 healthy controls (CON) and in 77 athletes who trained regularly (50 cyclists, aerobic training (AER), and 27 runners of 400m, anaerobic training (NoAER)). All of them were male Spanish Caucasian individuals. A maximum graded exercise test (GXT) in cycle-ergometer was performed to determine VO(2max) (mL kg(-1)min(-1)). Our results confirmed that, in CON, VO(2max) (P=0.007) was higher in Non-J than J individuals. Furthermore, we found that AER and NoAER showed, as it could be expected, higher VO(2max) than CON, but not differences between mitochondrial variants have been found. According with these findings, the influence of mitochondrial DNA (mtDNA) variants on VO(2max) has been confirmed, and a new conclusion has arisen: the steady exercise is able to remove this influence. The interest of these promising findings in muscular performance should be further explored, in particular, the understanding of potential applications in sport training and in muscle pathological syndromes.

Despite the strong purifying selection that occurs during embryonic development, the particular location and features of mitochondrial DNA make it especially susceptible to accumulating point mutations, giving rise to a large number of mitochondrial DNA variants. Many of these will have moderate or no phenotypic effects but others will be the cause of very dramatic diseases, usually known as mitochondriopathies. Because of the abundance of different mitochondrial DNA variants, it is not easy to determine whether a new mutation is pathogenic. To facilitate this task, different criteria have been proposed, but they are often either too severely or too loosely applied. Citing examples from the literature, in this paper we discuss some critical aspects of these criteria.

This work investigates if human mitochondrial variants influence on maximal oxygen consumption (VO(2max)). With this purpose we recruited, as a uniform population in term of nutritional habits and life style, 114 healthy male Spanish subjects that practiced fitness exercises 3-4 times a week. Once mtDNA haplogroups were determined, we found that J presents with lower VO(2max) (P=0.02) than nonJ variants. J has been related with a lower efficiency of electron transport chain (ETC), diminished ATP and ROS production. Thus, the difficult to compensate the mitochondrial energetic deficiency could explain the accumulation of J haplogroup in LHON and multiple sclerosis. Furthermore, the lower ROS production associated to J could also account for the accrual of this variant in elderly people consequent to a decreased oxidative damage.

Aim: To describe the scientific contributions and biography of Giuseppe Attardi (1923-2008), in particular his work on the human mitochondrial genetic system of crucial importance for the discovery and understanding of the mechanisms that produce the mitochondrial diseases., Development: Giuseppe Attardi was an Italian-born medical doctor that worked with outstanding researchers as James Watson, Francois Jacob, and Renato Dulbecco, all Nobel Prizes of Medicine until 1963 that was promoted to Professor of the California Institute of Technology in Pasadena (USA). In 1967 discovered the human mitochondrial RNA, a little later the mitochondrial ribosomes and proteins encoded in the genome, in 1981 showed the genetic and transcription map of the mitochondrial DNA, and in 1983 described the proteins codified in this genome, all of them components of the oxidative phosphorylation system, metabolic pathway that leads to the synthesis of ATP. Later, he developed a technique, widely used nowadays, to study the mechanism by which the mutations in the mitochondrial DNA altered the cellular function and originated the mitochondrial diseases. In 1999, demonstrated the role of the mitocondrial DNA, and the mutations produced along the life, in aging.

We have studied the production of reactive oxygen species (ROS) in transmitochondrial cells, harboring homoplasmic levels of the T14487C mtDNA mutation in the ND6 gene of mitochondrial DNA (mtDNA). Previous work has shown that this mutation causes complex I deficiency. Here, we show that this mutation causes an overproduction of ROS leading to an increase in the oxidation of lipids and mtDNA without modification of antioxidant enzyme activities. We suggest that mutations in mtDNA affecting complex I activity may result in oxidative cellular damage, and reinforce the possible role of ROS-mediated mechanisms participating in some mtDNA-related disorders.

The molecular mechanisms involved in the regulation of the balance between rRNA and mRNA in mitochondria are poorly understood. The mitochondrial transcription termination factor (mTERF) was highlighted as a potential transcription-control point. In this study, rat mTERF has been expressed in vitro and in Escherichia coli. The mature protein, in addition to the expected specific DNA-binding capacity for the sequence required for termination, has a new DNA-binding activity, and is able to bind to rat mitochondrial promoter region. This finding suggests communication between transcription initiation and termination regions. However, the results of a competition experiment argue against the formation of a complex between rat mTERF and the termination probe and promoter probe simultaneously, although it remains to be investigated whether another factor(s) might be involved in this interaction. In addition, recombinant human mTERF is also able to bind to human mitochondrial promoter region.

Sperm motility is dependent on mitochondrial ATP production that relies on the coordinated expression of the mitochondrial and nuclear genomes. It is generally accepted that mammalian ejaculated spermatozoa retain the ability to synthesize mtDNA-encoded proteins but not most of the nuclear ones. This implies an asynchronous regulation of the oxidative phosphorylation-related genes encoded by each genome. Trying to investigate this issue, we unexpectedly found that ejaculated human spermatozoa do not synthesize mtDNA-encoded proteins. Moreover, we estimated that the discrepancy between our observations and those published elsewhere was due to a chloramphenicol-sensitive protein synthesis attributed to mitochondria that instead corresponds to contaminating bacteria.

Herpes simplex virus thymidine kinase (HSV-tk) converts ganciclovir (GCV) into an active compound, which can be incorporated into DNA molecules and terminate DNA synthesis. Gene transfer of HSV-tk followed by GCV administration has been used with success to treat experimental cancer and this strategy has entered into clinical trials. Although it is thought that the cytotoxic effect occurs mainly in tumoral dividing cells, where mitotic activity favors integration of the genotoxic compound into nuclear DNA, there are concerns of potential damage to normal nondividing cells. In the present work we have explored the mechanisms of HSV-tk/GCV toxicity and in particular whether this therapy may cause lesions of mitochondrial DNA (mtDNA) and mitochondrial dysfunction. We found that the administration of GCV to rats injected with adenovirus encoding HSV-tk induced hepatocellular damage characterized by the presence of apoptotic bodies, ballooning of hepatocytes, and severe hepatic steatosis with mitochondria enlargement and cristae dissolution at the ultrastructural level. Remarkably, Southern blot analysis showed substantial reduction in the amount of mtDNA in the liver. Using radiolabeled GCV we could demonstrate incorporation of this compound into both nuclear and mtDNA in HSV-tk-transduced rat hepatocytic cell line MCA-RH7777 and subsequent alteration of mitochondrial function. Our observations confirm that GCV can damage both nuclear and mtDNA in cells transduced with HSV-tk and that this effect could be responsible for severe mitochondrial dysfunction and toxicity in normal nondividing cells. These data are relevant for the design of clinical trials using adenoviral vectors encoding HSV-tk.

In the present work, the RNA polymerase activity of the human mitochondrial RNA polymerase mature protein (h-mtRPOLm) is shown, and its molecular activity calculated (2.1+/-0.9 min(-1)). An activity analysis of h-mtRPOLm and deleted versions of it has demonstrated that the entire recombinant protein is required for this activity. In addition, h-mtRPOLm alone or in presence of the known mitochondrial transcription factors (human mitochondrial transcription factor A and/or human mitochondrial transcription termination factor) is not able to initiate transcription from the specific human mitochondrial promoters pointing to the existence of a human mitochondrial transcription factor B-like protein.

A variety of mtDNA mutations responsible for human diseases have been associated with molecular defects in the OXPHOS system. It has been proposed that mtDNA genetic alterations can also be responsible for sperm dysfunction. In addition, it was suggested that if sperm dysfunction is the main phenotypic consequence, these mutations could be fixed as stable mtDNA variants, because mtDNA is maternally inherited. To test this possibility, we have performed an extensive analysis of the distribution of mtDNA haplogroups in white men having fertility problems. We have found that asthenozoospermia, but not oligozoospermia, is associated with mtDNA haplogroups in whites. Thus, haplogroups H and T are significantly more abundant in nonasthenozoospermic and asthenozoospermic populations, respectively, and show significant differences in their OXPHOS performance.

Until now, little attention has been paid to the contribution of mitochondrial dysfunction to germinal tissue disorders. The target of this study was to investigate the relationship between sperm motility and mitochondrial respiratory chain enzyme activities. The results obtained showed that semen samples of control individuals (n = 33) have substantially higher activities of complexes I, II, and IV compared with those of asthenozoospermic subjects (n = 86). Moreover, a direct and positive correlation was found in the whole population studied between spermatozoa motility and all the mitochondrial respiratory complex activities assayed (I, II, I+III, II+III, and IV). The ratio of these enzymes to citrate synthase (a reliable enzymatic marker of mitochondrial volume) activities did not correlate with sperm motility. This suggests that motility depends largely on the mitochondrial volume within the sperm midpiece. These observations could be of physiopathological relevance because they suggest that factors affecting the mitochondrial energy production could be then responsible for particular cases of idiopathic asthenozoospermia.

In order to test the hypothesis that mitochondrial transcription factor A (mtTFA) regulates mitochondrial transcription in vivo, mtTFA was overexpressed in HeLa cells and imported into isolated rat liver mitochondria. Five hours after transfection with an eukaryotic expression vector, mitochondrial transcripts for cytochrome-c-oxidase subunit I and 12 S rRNA were increased over controls. In the presence of rat liver mitochondria, the 29 kDa mtTFA, generated by in vitro translation, was processed to a 24 kDa protein which was protected from protease digestion. This demonstrates that mtTFA was imported into the matrix. Incorporation of 32P-UTP into mitochondrial transcripts was stimulated following import of mTFA. We conclude that the intracellular and intramitochondrial concentration of mtTFA, respectively, indeed regulates mitochondrial transcription.

1. A system for studying RNA synthesis in isolated sheep brain mitochondria was set up to investigate the transcriptional activity of different brain regions (cortex, cerebellum and brain stem). In this system, mitochondrial DNA is transcribed and RNA processed in a way that faithfully reproduces the in vivo process. 2. The comparison of the electrophoretic patterns of the mitochondrial DNA transcription products showed that although they were qualitatively similar, there were large differences in the rate of mitochondrial DNA transcription of the three regions studied, cerebellum and brain stem showing transcriptional rates which were 34 and 18% respectively of that of cerebral cortex.

Isolated rat brain mitochondria, when incubated in the presence of [alpha-32P]UTP in an appropriate incubation buffer, in which the energy requirements are provided by exogenous ADP in the presence of an oxidizable substrate, are able to support mitochondrial DNA transcription and RNA processing in a way faithfully resembling the in vivo process. Furthermore, we have strikingly found a saturation of the synthesis of mature 16 S and 12 S rRNA under conditions in which their RNA precursor as well as the mature mRNAs continue being synthesized. This suggests that synthesis of mature rRNAs could be regulated at the level of processing of their precursor rather than at the level of transcription.

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.

Two mitochondrial populations with cytochrome c oxidase activity, and densities of 1.07 and 1.08 g/ml have been separated by means of a dextran-sucrose gradient. The highest enzymatic specific activity appeared in the 1.07 g/ml band. The mitochondria assayed by their in vivo cycloheximide resistant leucine incorporation appeared at the 1.08 g/ml density band of the gradient. Some amount of activity of cytochrome c oxidase, and leucine incorporation appeared at the bottom of the gradient. The cytochrome spectra in liquid nitrogen of each band showed different proportion of cytochrome(s) a, b and c. The highest ratio of cytochrome a and cytochrome b in respect to cytochrome c appeared in the 1.08 g/ml band.

The distribution of mitochondria from Aspergillus nidulans on a dextran-sucrose gradient resulted in the fractionation of three bands. The lightest band presented the highest respiratory and cytochrome c oxidase activities and was also the most active in the incorporation of tritiated glycerol. Pulse-chase experiments with 3H-glycerol suggested that mitochondria from the light band was transformed to denser organelles as the organism grew.

A 3'-end proximal segment of most of the putative mRNAs encoded in the heavy strand of HeLa cell mtDNA has been partially sequences and aligned with the DNA sequence. In all cases, the 3'-end nucleotide of the individual mRNA coding sequences has been found to be immediately contiguous to a tRNA gene or another mRNA coding sequence. These and previous results indicate that the heavy (H) strand sequences coding for the rRNA, poly(A)-containing RNA and tRNA species form a continuum extending over almost the entire length of this strand. We propose that the H strand is transcribed into a single polycistronic RNA molecule, which is processed later into mature species by precise endonucleolytic cleavages which occur, in most cases, immediately before and after a tRNA sequence.

Background: Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome have been associated to m.8993T>G/C mutations in the subunit 6 of the ATP synthase (p.MT-ATP6). Methods: We have performed a mutational screening of the mitochondrial DNA gene encoding for this protein in 62 patients with the disease, that do not carry any of the common mutations described to date. Results: We report clinical and molecular data in one patient who harbours a de novo insertion in the MT-ATP6 gene that results in a truncated subunit. The mutation was heteroplasmic (85%) in muscle DNA and the BN-PAGE analysis showed a clear decrease in the amount of ATP synthase. Conclusion: Molecular analysis of NARP patients cannot be limited to the search of the m.8993T>G/C and either the ATP6 or the whole mtDNA should be sequenced. [ABSTRACT FROM AUTHOR]