Reversible mitochondrial respiratory chain impairment during symptomatic hyperlactatemia associated with antiretroviral therapy

Direct evidence confirming the hypothesis that a dysfunction of the mitochondrial respiratory chain (MRC) underlies the pathogenesis of hyperlactatemia associated with highly active antiretroviral therapy (HAART) is scarce. We studied mitochondrial DNA (mtDNA) content and MRC function in the skeletal muscle of an HIV-infected patient during an episode of symptomatic hyperlactatemia. Skeletal muscle biopsy was performed during the episode when the patient was symptomatic and 3 months later when the patient was clinically recovered. Assessment of mitochondria was performed using histological, polarographic, spectrophotometrical, and Southern blot and real time PCR DNA quantification methods. The histological study disclosed extensive mitochondrial impairment in the form of ragged-red fibers or equivalents on oxidative reactions. These findings were associated with an increase in mitochondrial content and a decrease in both mitochondrial respiratory capacity and MRC enzyme activities. Mitochondrial DNA content declined to 53% of control values. Mitochondrial abnormalities had almost disappeared later when the patient became asymptomatic. Our findings support the hypothesis that MRC dysfunction stands at the basis of HAART-related hyperlactatemia.

Reversible mitochondrial respiratory chain impairment during symptomatic hyperlactatemia associated with antiretroviral therapy

Direct evidence confirming the hypothesis that a dysfunction of the mitochondrial respiratory chain (MRC) underlies the pathogenesis of hyperlactatemia associated with highly active antiretroviral therapy (HAART) is scarce. We studied mitochondrial DNA (mtDNA) content and MRC function in the skeletal muscle of an HIV-infected patient during an episode of symptomatic hyperlactatemia. Skeletal muscle biopsy was performed during the episode when the patient was symptomatic and 3 months later when the patient was clinically recovered. Assessment of mitochondria was performed using histological, polarographic, spectrophotometrical, and Southern blot and real time PCR DNA quantification methods. The histological study disclosed extensive mitochondrial impairment in the form of ragged-red fibers or equivalents on oxidative reactions. These findings were associated with an increase in mitochondrial content and a decrease in both mitochondrial respiratory capacity and MRC enzyme activities. Mitochondrial DNA content declined to 53% of control values. Mitochondrial abnormalities had almost disappeared later when the patient became asymptomatic. Our findings support the hypothesis that MRC dysfunction stands at the basis of HAART-related hyperlactatemia.

Mitochondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial DNA mutator mice

Background: Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings: We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase c, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Dym). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance: These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

EndoG links Bnip3-induced mitochondrial damage and caspase-independent DNA fragmentation in ischemic cardiomyocytes

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-xL. These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells.

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression ofSSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.

Zim17/Tim15 links mitochondrial iron–sulfur cluster biosynthesis to nuclear genome stability

Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron–sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron–sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron–sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants.

Mitochondrial DNA damage and animal longevity: insights from comparative studies

Chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. However, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. Aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. Mitochondria are a main source of reactive oxygen species (ROS) and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own DNA (mtDNA). ROS can modify mtDNA directly at the sugar-phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and DNA mutations. In this scenario, natural selection tends to decrease the mitochondrial ROS generation, the oxidative damage to mtDNA, and the mitochondrial mutation rate in long-lived species, in agreement with the mitochondrial oxidative stress theory of aging.

Hepatocyte nuclear factor 4alpha transactivates the mitochondrial alanine aminotransferase gene in the kidney of Sparus aurata

Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5′-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L-alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.

Analysis of the mitochondrial DNA of Schizophrenia patients by next generation sequencing

Mitochondrial DNA (mtDNA), a maternally inherited 16.6-Kb molecule crucial for energy production, is implicated in numerous human traits and disorders. It has been hypothesized that the presence of mutations in the mtDNA may contribute to the complex genetic basis of schizophreniadisease, due to the evidence of maternal inheritance and the presence of schizophrenia symptoms in patients affected of a mitochondrial disorder related to a mtDNA mutation. The present project aims to study the association of variants of mitochondrial DNA (mtDNA), and an increased risk of schizophrenia in a cohort of patients and controls from the same population. The entire mtDNA of 55 schizophrenia patients with an apparent maternal transmission of the disease and 38 controls was sequenced by Next Generation Sequencing (Ion Torrent PGM, Life Technologies) and compared to the reference sequence. The current method for establishing mtDNA haplotypes is Sanger sequencing, which is laborious, timeconsuming, and expensive. With the emergence of Next Generation Sequencing technologies, this sequencing process can be much more quickly and cost-efficiently. We have identified 14 variants that have not been previously reported. Two of them were missense variants: MTATP6 p.V113M and MTND5 p.F334L ,and also three variants encoding rRNA and one variant encoding tRNA. Not significant differences have been found in the number of variants between the two groups. We found that the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of the bioinformatics analysis and annotation step would be desirable to facilitate the application of NGS in mtDNA analysis.

Effects of exercise on mitochondrial DNA content in skeletal muscle of patients with COPD

Background Exhausting exercise reduces the mitochondrial DNA (mtDNA) content in the skeletal muscle of healthy subjects due to oxidative damage. Since patients with chronic obstructive pulmonary disease (COPD) suffer enhanced oxidative stress during exercise, it was hypothesised that the mtDNA content will be further reduced. Objective To investigate the effects of exercise above and below the lactate threshold (LT) on the mtDNA content of skeletal muscle of patients with COPD. Methods Eleven patients with COPD (676 8 years; forced expiratory volume in 1s (FEV1)456 8%ref) and 10 healthy controls (666 4 years; FEV1 906 7% ref) cycled 45 min above LT (65% peak oxygen uptake (V9O2 peak)and another 7 patients (656 6 years; FEV1 506 4%ref)and 7 controls (566 9 years;FEV1 926 6%ref) cycled 45 min below their LT (50% V9O2 peak). Biopsies from the vastus lateralis muscle were obtained before exercise, immediately after and 1 h, 1 day and 1 week later to determine by PCR the mtDNA/nuclear DNA (nDNA) ratio (a marker of mtDNA content) and the expression of the peroxisome proliferator-activated receptor- g coactivator-1 a (PGC-1a)mRNA and the amount of reactive oxygen species produced during exercise was estimated from total V9O2. Results Skeletal muscle mtDNA/nDNA fell significantly after exercise above the LT both in controls and in patients with COPD, but the changes were greater in those with COPD. These changes correlated with production of reactive oxygen species, increases in manganese superoxide dismutase and PGC-1 a mRNA and returned to baseline values 1 week later. This pattern of response wa was also observed, albeit minimised, in patients exercising below the LT. Conclusions In patients with COPD, exercise enhances the decrease in mtDNA content of skeletal muscle and the expression of PGC-1 a mRNA seen in healthy subjects probably due to oxidative stress.

The Armc10/SVH gene: Genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1-6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents A beta-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against A beta-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals.

The Armc10/SVH gene: Genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1-6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents A beta-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against A beta-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals.

Reversible mitochondrial respiratory chain impairment during symptomatic hyperlactatemia associated with antiretroviral therapy

Direct evidence confirming the hypothesis that a dysfunction of the mitochondrial respiratory chain (MRC) underlies the pathogenesis of hyperlactatemia associated with highly active antiretroviral therapy (HAART) is scarce. We studied mitochondrial DNA (mtDNA) content and MRC function in the skeletal muscle of an HIV-infected patient during an episode of symptomatic hyperlactatemia. Skeletal muscle biopsy was performed during the episode when the patient was symptomatic and 3 months later when the patient was clinically recovered. Assessment of mitochondria was performed using histological, polarographic, spectrophotometrical, and Southern blot and real time PCR DNA quantification methods. The histological study disclosed extensive mitochondrial impairment in the form of ragged-red fibers or equivalents on oxidative reactions. These findings were associated with an increase in mitochondrial content and a decrease in both mitochondrial respiratory capacity and MRC enzyme activities. Mitochondrial DNA content declined to 53% of control values. Mitochondrial abnormalities had almost disappeared later when the patient became asymptomatic. Our findings support the hypothesis that MRC dysfunction stands at the basis of HAART-related hyperlactatemia.

Reversible mitochondrial respiratory chain impairment during symptomatic hyperlactatemia associated with antiretroviral therapy

Direct evidence confirming the hypothesis that a dysfunction of the mitochondrial respiratory chain (MRC) underlies the pathogenesis of hyperlactatemia associated with highly active antiretroviral therapy (HAART) is scarce. We studied mitochondrial DNA (mtDNA) content and MRC function in the skeletal muscle of an HIV-infected patient during an episode of symptomatic hyperlactatemia. Skeletal muscle biopsy was performed during the episode when the patient was symptomatic and 3 months later when the patient was clinically recovered. Assessment of mitochondria was performed using histological, polarographic, spectrophotometrical, and Southern blot and real time PCR DNA quantification methods. The histological study disclosed extensive mitochondrial impairment in the form of ragged-red fibers or equivalents on oxidative reactions. These findings were associated with an increase in mitochondrial content and a decrease in both mitochondrial respiratory capacity and MRC enzyme activities. Mitochondrial DNA content declined to 53% of control values. Mitochondrial abnormalities had almost disappeared later when the patient became asymptomatic. Our findings support the hypothesis that MRC dysfunction stands at the basis of HAART-related hyperlactatemia.

Geographic patterns of genetic variation in a broadly distributed marine vertebrate: new insights into loggerhead turtle stock structure from expanded mitochondrial DNA sequences

Previous genetic studies have demonstrated that natal homing shapes the stock structure of marine turtle nesting populations. However, widespread sharing of common haplotypes based on short segments of the mitochondrial control region often limits resolution of the demographic connectivity of populations. Recent studies employing longer control region sequences to resolve haplotype sharing have focused on regional assessments of genetic structure and phylogeography. Here we synthesize available control region sequences for loggerhead turtles from the Mediterranean Sea, Atlantic, and western Indian Ocean basins. These data represent six of the nine globally significant regional management units (RMUs) for the species and include novel sequence data from Brazil, Cape Verde, South Africa and Oman. Genetic tests of differentiation among 42 rookeries represented by short sequences (380 bp haplotypes from 3,486 samples) and 40 rookeries represented by long sequences (~800 bp haplotypes from 3,434 samples) supported the distinction of the six RMUs analyzed as well as recognition of at least 18 demographically independent management units (MUs) with respect to female natal homing. A total of 59 haplotypes were resolved. These haplotypes belonged to two highly divergent global lineages, with haplogroup I represented primarily by CC-A1, CC-A4, and CC-A11 variants and haplogroup II represented by CC-A2 and derived variants. Geographic distribution patterns of haplogroup II haplotypes and the nested position of CC-A11.6 from Oman among the Atlantic haplotypes invoke recent colonization of the Indian Ocean from the Atlantic for both global lineages. The haplotypes we confirmed for western Indian Ocean RMUs allow reinterpretation of previous mixed stock analysis and further suggest that contemporary migratory connectivity between the Indian and Atlantic Oceans occurs on a broader scale than previously hypothesized. This study represents a valuable model for conducting comprehensive international cooperative data management and research in marine ecology.