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.

Effect of aerobic exercise on skeletal muscle mitochondrial content and function in older adults

Regular aerobic exercise training, which is touted as a way to ameliorate metabolic diseases, increases aerobic capacity. Aerobic capacity usually declines with advanced age. The decline in aerobic capacity is typically associated by a decrease in the quality of skeletal muscle. At the molecular level, this decreased quality comes in part from perturbations in skeletal muscle mitochondria. Of particular is a decrease in the total amount of mitochondria that occupy the skeletal muscle volume. What is not well established is if this decrease in mitochondrial content is due to inactive lifestyle or the process of aging. Herein, the work of the thesis shows a clear connection between mitochondrial content and aerobic capacity. This indicates that active individuals with higher VChmax levels also contain higher volumes of mitochondria inside their muscle as opposed to sedentary counterparts who have lower levels of mitochondrial content. Upon taking these previously sedentary individuals and entering them into an aerobic exercise intervention, they are able to recover their mitochondrial content as well as function to similar levels of lifelong athletes of the same age. Furthermore, the results of this thesis show that mitochondrial content and function also correlate with exercise efficiency. If one is more efficient, he/she is able to expend less energy for a similar power output. Furthermore, individuals who increase in efficiency also increase in the ability to oxidize and utilize fat during pro-longed exercise. This increased reliance on fat after the intervention is associated with an increased amount of mitochondria, particularly in the intermyofibrillar region of skeletal muscle. Therefore, elderly adults who were once sedentary were able to recover mitochondrial content and function and are able to reap other health benefits from regular aerobic exercise training. Aging per se does not seem to be the culprit that will lead to metabolic diseases but rather it seems to be a lack of physical activity. -- Un entraînement sportif d'endurance, connu pour réduire le risque de développer des maladies métaboliques, augmente notre capacité aérobie. La capacité aérobie diminue généralement avec l'âge. Ce déclin est typiquement associé d'une diminution de la qualité du muscle squelettique. Au niveau moléculaire, cette diminution est due à des perturbations dans les mitochondries du muscle squelettique,, ce qui conduit à une diminution de la quantité totale des mitochondries présentes dans le muscle squelettique. Il n'a pas encore été établi si cette diminution de la teneur mitochondriale est due à un mode de vie sédentaire ou au processus du vieillissement. Ce travail de thèse montre un lien clair entre le contenu mitochondrial et la capacité aérobie. Il indique que des personnes âgées actives, avec des niveaux de V02max plus élevés, possèdent également un volume plus élevé de mitochondries dans leurs muscles en comparaison à leurs homologues sédentaires. En prenant des individus sédentaires et leur faisant pratiquer une activité physique aérobie, il est possible d'accroître leur contenu de même que leur fonction mitochondriale à des niveaux similaires à ceux d'athlètes du même âge ayant pratiqué une activité physique tout au long de leur vie. De plus, les résultats de ce travail démontrent que le contenu et la fonction mitochondriale sont en corrélation avec l'efficiscience lors d'exercice physique. En agumentant l'effiscience, les personnes sont alors capables de dépenser moins d'énergie pour une puissance d'exercice similaire. Donc, un volume mitochondrial accru dans le muscle squelettique, associé à une fonction mitochondriale améliorée, est associté à une augmentation de l'effiscience. En outre, les personnes qui augmentent leur effiscience, augmentent aussi leur capacité à oxyder les graisses durant l'exercice prolongé. Une augmentation du recours au graisses après l'intervention est associée à une quantité accrue de mitochondries, en particulier dans la région inter-myofibrillaire du muscle squelettique. Par conséquent, les personnes âgées autrefois sédentaires sont en mesure de récupérer leur contenu et leur fonction mitochondriale ainsi que d'autres avantages pour la santé grâce à un entraînement aérobie régulier. Le vieillissement en soi ne semble donc pas être le coupable conduisant aux maladies métaboliques qui semblent plutôt être lié à un manque d'activité physique.

Phylogenetic relationships of Apodemus Kaup, 1829 (Rodentia: Muridae) species in the Eastern Mediterranean inferred from mitochondrial DNA, with emphasis on iranian species

Wood mice of the genus Apodemus are widely distributed in Eurasia, with the Eastern Mediterranean being considered as a hotspot. Indeed, numerous species have been documented in Iran, including A. witherbyi, A. hyrcanicus, A. uralensis, A. avicennicus, A. hermonensis, and A. arianus. In this study, 129 specimens were collected from different Iranian localities and two specimens from Afghanistan. The animals were identified taxonomically and their phylogenetic relationships were investigated using cytochrome b mitochondrial DNA sequences. Five species of the genus Apodemus were identified in Iran, including A. hyrcanicus, A. witherbyi, A. cf. ponticus, A. uralensis, and A. mystacinus, beside, A. pallipes from Afghanistan. This study found no evidence of A. flavicollis or A. sylvaticus in Iran, despite their occurrence in Turkey, shedding doubt on the status of A. flavicollis in Iran, Asia Minor, and the Levant. Phylogenetic analyses imply that A. witherbyi has priority over A. avicennicus, A. hermonensis, and A. iconicus. Estimation of the divergence time for these taxa suggests a separation at around 7.2 Ma for the subgenera Karstomys (including A. mystacinus and A. epimelas) and Sylvaemus (including A. flavicollis, A. sylvaticus, A. uralensis, A. pallipes, A. hyrcanicus, A. witherbyi, and A. cf. ponticus). Within the subgenus Karstomys, the divergence times for A. mystacinus and A. epimelas were between 3.0 and 6.1 Ma, and divergence times within the subgenus Sylvaemus were between 5.2 and 6.9 Ma for A. witherbyi and other species in this subgenus. It is postulated that vicariance events including the uplifting of the Zagros Mountains and Anatolian Plateau in the middle Miocene and climate oscillations during the Messinian Salinity Crisis besides formation of the Hyrcanian tertiary forests during the Neogene probably played substantial roles in the radiation and distribution of the genus Apodemus in the Eastern Mediterranean.

Skeletal Muscle Mitochondrial Function and Fatigability in Older Adults.

BACKGROUND: Fatigability increases while the capacity for mitochondrial energy production tends to decrease significantly with age. Thus, diminished mitochondrial function may contribute to higher levels of fatigability in older adults. METHODS: The relationship between fatigability and skeletal muscle mitochondrial function was examined in 30 participants aged 78.5 ± 5.0 years (47% female, 93% white), with a body mass index of 25.9 ± 2.7 kg/m(2) and usual gait-speed of 1.2 ± 0.2 m/s. Fatigability was defined using rating of perceived exertion (6-20 point Borg scale) after a 5-minute treadmill walk at 0.72 m/s. Phosphocreatine recovery in the quadriceps was measured using (31)P magnetic resonance spectroscopy and images of the quadriceps were captured to calculate quadriceps volume. ATPmax (mM ATP/s) and oxidative capacity of the quadriceps (ATPmax·Quadriceps volume) were calculated. Peak aerobic capacity (VO2peak) was measured using a modified Balke protocol. RESULTS: ATPmax·Quadriceps volume was associated with VO2peak and was 162.61mM ATP·mL/s lower (p = .03) in those with high (rating of perceived exertion ≥10) versus low (rating of perceived exertion ≤9) fatigability. Participants with high fatigability required a significantly higher proportion of VO2peak to walk at 0.72 m/s compared with those with low fatigability (58.7 ± 19.4% vs 44.9 ± 13.2%, p < .05). After adjustment for age and sex, higher ATPmax was associated with lower odds of having high fatigability (odds ratio: 0.34, 95% CI: 0.11-1.01, p = .05). CONCLUSIONS: Lower capacity for oxidative phosphorylation in the quadriceps, perhaps by contributing to lower VO2peak, is associated with higher fatigability in older adults.

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.

Drug-induced mitochondrial dysfunction and cardiotoxicity.

Mitochondria has an essential role in myocardial tissue homeostasis; thus deterioration in mitochondrial function eventually leads to cardiomyocyte and endothelial cell death and consequent cardiovascular dysfunction. Several chemical compounds and drugs have been known to directly or indirectly modulate cardiac mitochondrial function, which can account both for the toxicological and pharmacological properties of these substances. In many cases, toxicity problems appear only in the presence of additional cardiovascular disease conditions or develop months/years following the exposure, making the diagnosis difficult. Cardiotoxic agents affecting mitochondria include several widely used anticancer drugs [anthracyclines (Doxorubicin/Adriamycin), cisplatin, trastuzumab (Herceptin), arsenic trioxide (Trisenox), mitoxantrone (Novantrone), imatinib (Gleevec), bevacizumab (Avastin), sunitinib (Sutent), and sorafenib (Nevaxar)], antiviral compound azidothymidine (AZT, Zidovudine) and several oral antidiabetics [e.g., rosiglitazone (Avandia)]. Illicit drugs such as alcohol, cocaine, methamphetamine, ecstasy, and synthetic cannabinoids (spice, K2) may also induce mitochondria-related cardiotoxicity. Mitochondrial toxicity develops due to various mechanisms involving interference with the mitochondrial respiratory chain (e.g., uncoupling) or inhibition of the important mitochondrial enzymes (oxidative phosphorylation, Szent-Györgyi-Krebs cycle, mitochondrial DNA replication, ADP/ATP translocator). The final phase of mitochondrial dysfunction induces loss of mitochondrial membrane potential and an increase in mitochondrial oxidative/nitrative stress, eventually culminating into cell death. This review aims to discuss the mechanisms of mitochondrion-mediated cardiotoxicity of commonly used drugs and some potential cardioprotective strategies to prevent these toxicities.

Synaptic dysfunction, memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons.

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.

The Vitamin A Derivative All-Trans Retinoic Acid Repairs Amyloid-β-Induced Double-Strand Breaks in Neural Cells and in the Murine Neocortex.

The amyloid-β peptide or Aβ is the key player in the amyloid-cascade hypothesis of Alzheimer's disease. Aβ appears to trigger cell death but also production of double-strand breaks (DSBs) in aging and Alzheimer's disease. All-trans retinoic acid (RA), a derivative of vitamin A, was already known for its neuroprotective effects against the amyloid cascade. It diminishes, for instance, the production of Aβ peptides and their oligomerisation. In the present work we investigated the possible implication of RA receptor (RAR) in repair of Aβ-induced DSBs. We demonstrated that RA, as well as RAR agonist Am80, but not AGN 193109 antagonist, repair Aβ-induced DSBs in SH-SY5Y cells and an astrocytic cell line as well as in the murine cortical tissue of young and aged mice. The nonhomologous end joining pathway and the Ataxia Telangiectasia Mutated kinase were shown to be involved in RA-mediated DSBs repair in the SH-SY5Y cells. Our data suggest that RA, besides increasing cell viability in the cortex of young and even of aged mice, might also result in targeted DNA repair of genes important for cell or synaptic maintenance. This phenomenon would remain functional up to a point when Aβ increase and RA decrease probably lead to a pathological state.

Synthesis of a tetrahydroimidazo-[2',1':2,3]thiazolo[5,4-c]pyridine derivative with Met inhibitory activity

A straightforward synthesis of the Met antagonist JLK1360 involving an alkylationcyclocondensation process using aminothiazole 1 and nitrophenacyl bromide 2, reduction of the nitro group, and coupling of the resulting tetracyclic aniline 5 with an appropriate N-acyl alanine derivative, is reported.

Synthesis of a tetrahydroimidazo-[2',1':2,3]thiazolo[5,4-c]pyridine derivative with Met inhibitory activity

A straightforward synthesis of the Met antagonist JLK1360 involving an alkylationcyclocondensation process using aminothiazole 1 and nitrophenacyl bromide 2, reduction of the nitro group, and coupling of the resulting tetracyclic aniline 5 with an appropriate N-acyl alanine derivative, is reported.

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.