Literatura académica sobre el tema "Mitochondrial DNA depletion syndrome"

The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Biomolecular and Biomedical Sciences<br>Full Text

Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in several nuclear genes, involved in mitochondrial DNA (mtDNA) maintenance, characterized by a strong reduction of mtDNA copy number in affected tissues and severe defects in mitochondrial functionality. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been specifically associated with hepatocerebral forms of MDS. However, MPV17 protein function is still unclear, although it has been suggested a primary role in mtDNA maintenance. Zebrafish represents a model to clarify this biological question: a mpv17 null mutant (roy orbison) shows a 19 bp deletion resulting in aberrant splicing between the exons 2 and 3 of mpv17 gene and lacks the guanine-based reflective skin cells named iridophores. In our work, we have characterized in details the mitochondrial phenotype of roy larvae and found early severe ultrastructural alterations in liver mitochondria; we could also observe a significant impairment of respiratory chain complexes leading to mitochondrial quality control activation. Our results provide evidences for Mpv17 being really essential in mitochondrial cristae maintenance and OXPHOS functionality, while its effect on mtDNA maintenance seems to be consequential, considering that mtDNA depletion only appeared at later stages of development. Moreover, taking into account that in roy orbison it has been previously postulated a role for Mpv17 in purines availability, and that embryos blocked in their pyrimidine synthesis resemble roy phenotype, we investigated the two alternatives by administrating purine and pyrimidine precursors to homozygous mutant embryos. Interestingly, orotic acid (OA) administration ameliorated roy phenotype, hence linking the loss of Mpv17 to pyrimidine de novo synthesis. In particular, the treatment with OA, currently used as food supplement, significantly increased not only iridophores number but also mtDNA content of mpv17 null mutants, thus opening up a new simple therapeutic approach for MPV17-related MDS.<br>Le sindromi da deplezione del DNA mitocondriale (MDS) sono un gruppo di malattie rare a carattere autosomico recessivo con esordio precoce e prognosi infausta. Le MDS sono causate da mutazioni a carico di diversi geni nucleari, coinvolti nel mantenimento del DNA mitocondriale (mtDNA), e caratterizzate da una forte riduzione del numero di copie del mtDNA nei tessuti interessati, nonchè da gravi difetti nella funzionalità mitocondriale. Le mutazioni a carico di MPV17, un gene nucleare che codifica per una proteina di membrana mitocondriale interna, sono state specificatamente associate a forme epatocerebrali di MDS. Tuttavia, la funzione della proteina MPV17 non è nota, sebbene sia stato ipotizzato un ruolo primario nel mantenimento del mtDNA. Zebrafish rappresenta un modello per rispondere a questa domanda biologica: un mutante nullo per il gene mpv17, detto roy orbison (roy), presenta una delezione di 19 bp che causa uno splicing aberrante tra gli esoni 2 e 3 del gene e, di conseguenza, l’assenza della proteina codificata. Dal punto di vista fenotipico, il mutante roy mostra un difetto di pigmentazione, caratterizzato dall’assenza di cellule, chiamate iridofori, le quali conferiscono alla pelle, grazie al loro contenuto di cristalli di guanina, la sua proprietà riflettente. Il presente lavoro di tesi ha avuto come obiettivo principale la caratterizzazione dettagliata del fenotipo mitocondriale delle larve mutanti per il gene mpv17, nelle quali abbiamo rilevato, già a stadi precoci, importanti alterazioni dell’ultrastruttura mitocondriale nel fegato e una significativa perdita della funzionalità dei complessi della catena respiratoria (OXPHOS). I nostri risultati suggeriscono, pertanto, una funzione essenziale del gene mpv17 nel mantenimento delle creste mitocondriali e della piena attività dell’OXPHOS, mentre il suo effetto sulla stabilità del mtDNA, ipotizzato in letteratura, sembra essere consequenziale, considerando che la deplezione del mtDNA è rilevabile solo in fasi tardive dello sviluppo. Inoltre, considerando che nei mutanti roy orbison era stata precedentemente ipotizzata una funzione di Mpv17 nel metabolismo delle purine e che, inoltre, l’inibizione della sintesi di pirimidine negli embrioni wild-type causa un fenotipo assimilabile a quello dei roy, sono stati somministrati precursori purinici e pirimidinici agli embrioni omozigoti mutanti, al fine di osservare o meno un miglioramento del fenotipo. È interessante notare che la somministrazione di acido orotico (OA), prodotto dell’enzima mitocondriale Diidroorotato deidrogenasi (DHODH) e precursore delle pirimidine, ha migliorato il fenotipo roy, collegando quindi la perdita di Mpv17 alla sintesi de novo dei nucleotidi. In particolare, il trattamento con OA, attualmente usato come integratore alimentare, ha aumentato significativamente non solo il numero di iridofori ma anche il contenuto di mtDNA nei mutanti nulli di mpv17, aprendo così nuove prospettive nel trattamento delle malattie da deplezione del DNA mitocondriale causate da mutazioni a carico di MPV17.

Abstract Mitochondrial DNA depletion and deletions are related to mutations in the nuclear genes responsible for replication and maintenance of mitochondrial DNA (mtDNA). The POLG1 gene encodes the enzyme responsible for replication of mtDNA. A particular feature of the POLG1 mutations is an increased risk of acute liver failure (ALF) upon exposure to sodium valproate (VPA), but the pathomechanism is not resolved. The present work studies the molecular genetic aetiology and clinical phenotypes associated with mtDNA depletion and deletion. Another objective was an investigation of clinical phenotypes in POLG1 mutations and disentangling the pathomechanism of VPA-induced ALF in POLG1 mutations. Mitochondrial toxicity of VPA was examined using HepG2 cells as an experimental in vitro model. In this work, mtDNA depletion was associated with severe neonatal-onset encephalopathy. Furthermore, mtDNA depletion was found in muscle dystrophy as a secondary finding to muscle degradation. Multiple mitochondrial DNA deletions were found in two patients with Kearns-Sayre syndrome suggesting a genetic origin of the disease. POLG1 p.R722H mutation has been previously reported as a neutral polymorphism, but we found evidence suggesting that POLG1 p.R722H could be a pathogenic mutation in a homozygous or compound heterozygous state. We identified retrospectively five patients, who required liver transplant after VPA-induced ALF. All five patients harboured POLG1 mutations supporting the evidence of POLG1 mutations as a risk factor for VPA-induced ALF. Previously, patients with POLG1 mutations have been considered unsuitable for liver transplantation, but we found that homozygous POLG1 mutations and adolescent or adult-onset disease predicted a good outcome following liver transplantation. In vitro studies on HepG2 cells showed that VPA disturbs mitochondrial respiration. Our results expand the phenotypes and molecular genetic features in mitochondrial DNA depletion and deletion syndromes. We found evidence that POLG1 mutations are not a contraindication for liver transplantation; rather, mutation status and age at onset affect survival. This finding should be taken in consideration in the treatment of VPA-induced ALF. Furthermore, our findings indicate that sodium valproate is toxic to mitochondria and should be avoided in patients with mitochondrial disease<br>Tiivistelmä Mitokondrion DNA:n (mtDNA) kahdentumisesta ja ylläpidosta vastaavien tuman geenien mutaatiot voivat johtaa mtDNA:n määrän vähenemiseen (depleetioon) ja katkoksiin (deleetioihin). MtDNA:n kahdentumisesta vastaavaa entsyymiä koodaa tuman POLG1-geeni. POLG1-mutaatioihin liittyy kohonnut riski sairastua natriumvalproaatin (VPA) aiheuttamaan akuuttiin maksavaurioon. Tutkimuksen tavoitteena oli tutkia mtDNA:n depleetion ja deleetioiden molekyyligeneettistä etiologiaa ja kliinisiä taudinkuvia. Tutkimuksessa selvitettiin myös POLG1-mutaatioihin liittyviä taudinkuvia ja POLG1-mutaatioihin liittyvän akuutin maksavaurion patomekanismia. VPA:n vaikutusta mitokondrioiden toimintaan tutkittiin in vitro HepG2-solumallissa. Tutkimuksessa todettiin mtDNA:n depleetion liittyvän vaikeaan varhain alkavaan aivosairauteen. Depleetio todettiin myös sekundaarisena merosiini-negatiivisessa lihasdystrofiassa. Kahdella Kearns-Sayren syndroomaa sairastavalla potilaalla todettiin multippelit mtDNA:n deleetiot, mikä viittaa syndrooman geneettisen alkuperään. POLG1 p.R722H-mutaatiota on aiemmin pidetty neutraalina polymorfiana, mutta tutkimuksen tulokset viittasivat siihen, että homotsygoottisena tai yhdistelmäheterotsygoottisena mutaatio on tautia aiheuttava. Helsingin yliopistollisen sairaalan elinsiirtorekisteristä tunnistettiin retrospektiivisesti viisi potilasta, jotka olivat saaneet maksansiirteen VPA:n aiheuttaman maksavaurion vuoksi. Kaikilla viidellä potilaalla todettiin POLG1-geenin mutaatio, mikä vahvistaa käsitystä geenin yhteydestä VPA:n aiheuttamaan maksavaurioon. POLG1-mutaatioita on pidetty vasta-aiheena maksansiirrolle, mutta tutkimuksessa todettiin homotsygoottisena esiintyvän POLG1-mutaation ja nuoruusiällä tai varhaisella aikuisiällä alkaneen taudin liittyvän parempaan maksansiirron jälkeiseen ennusteeseen. HepG2-solumallilla tehdyt tutkimukset osoittivat VPA:n haittaavan mitokondrioiden solyhengitystä. Tutkimuksen tulokset tuovat lisätietoa mtDNA:n depleetioon ja deleetioihin liittyvistä taudinkuvista ja molekyyligeneettisestä taustasta. POLG1-mutaatiot eivät ole ehdoton vasta-aihe maksansiirrolle; potilaan geneettinen status ja ikä taudin alkamishetkellä vaikuttavat ennusteeseen, mikä tulisi huomioida potilaiden hoidossa. Tulokset myös osoittivat VPA:n olevan mitokondriotoksinen lääke, jonka käyttöä tulisi välttää mitokondriotautipotilaiden hoidossa

Type 2 diabetes is an age-related condition and is characterised by a progressive decline in insulin secretion. Mitochondria play a key role in energy generation for insulin secretion. We previously reported an age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. TFAM, mtDNA Transcription Factor A, regulates mtDNA transcription and mtDNA copy number. Aims: We aimed to replicate the percentage decrease in mtDNA copy number that we observed with ageing in human islets, and to explore whether this affected mitochondrial function and insulin secretion. Methods: Two independent models of mtDNA depletion were created. The first model knocked down TFAM gene expression using siRNA technology. The second model subjected cells to didanosine, a nucleoside analogue of adenosine with a high affinity to POLG, a mtDNA polymerase. Results: Both models produced comparable levels of mtDNA depletion. Upon investigating the effects of partial mtDNA depletion on mitochondrial function, we found that both mtDNA depletion models displayed reduced mtDNA gene transcription and translation. However, neither model of mtDNA depletion affected ATP content or mitochondrial membrane potential. Glucose-stimulated insulin secretion was decreased following mtDNA depletion in the TFAM knock down cells which was rescued following treatment with the insulin secretagogue glibenclamide. Conversely, didanosine-induced mtDNA depleted cells showed increased insulin secretion. Conclusions: Both models generated a similar degree of mtDNA depletion, which was comparable to the percentage decrease seen in human islets with ageing. Both models were seen to impair mitochondrial function, but with opposing effects on insulin secretion. The TFAM model findings are in line with previous studies of severe mtDNA depletion, suggesting that the increase in insulin secretion seen with didanosine is due to drug off target effects. Strategies to slow islet mtDNA depletion in man could help to preserve insulin secretion and delay the development of Type 2 diabetes.

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

Background: Nucleoside reverse transcriptase inhibitors (NRTIs) interfere with mitochondrial DNA polymerase gamma causing significant toxic effects, including fatal lactic acidosis. Little is known about mitochondrial DNA (mtDNA) in human immunodeficiency virus (HIV) infected children who face a lifetime exposure to these agents. We performed a cross sectional observation of mtDNA levels in whole blood in a pediatric population to ascertain the relationship between mtDNA, NRTI regimens and parameters of HIV-infection severity. Methods: Whole blood mt:nDNA ratios were determined by real-time PCR in three groups: 27 presumed HIV-negative, 89 HIV-infected, NRTI-treated and 62 HIV-infected treatment-naive children. Multivariate analysis was used to identify variables independently associated with mtDNA depletion. Results: Mean mt:nDNA ratios were lower (P < 0.001) at 77% of control in the HIVinfected antiretroviral treatment (ART) Naïve group and 73% of control in the ART group, but not different between the two HIV-infected groups. Mt:nDNA ratios were negatively associated with age (P = 0.029), HIV status (P < 0.0001) and Log10 of the HIV-1 viral load (P = 0.035) and positively associated with CD4 % (p = 0.032). A 6 stavudine vs zidovudine based regimen was associated with lower but not significant levels of mtDNA (P = 0.1). Conclusions: Depletion of whole blood mtDNA in children is associated independently with HIV-infection and markers of HIV infection severity, and does not improve with either stavudine or zidovudine based ART despite virological control, suggesting that these agents also deplete mtDNA.

Abstract This study describes clinical and biochemical consequences of the 3243A→G mutation in the tRNALeu(UUR) gene of the mitochondrial DNA. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) is usually caused by this mutation. Demyelinating polyneuropathy was observed as a novel feature in a patient with the mutation. Based on electrodiagnostic examination the polyneuropathy was defined as being of the demyelinating, mixed (motor more than sensory) type. In a 1 year follow-up an approximately 7% reduction in both the motor and sensory nerve conduction velocities were observed. The effect and mechanism of action of nicotinamide treatment in a MELAS patient with the 3243A→G was studied. The blood NAD concentration increased linearly, being 24-fold elevated at 6 weeks of treatment. Blood lactate and pyruvate concentration decreased by 50% within three days and 24 h urine lactate content within 2 weeks. A clinical improvement together with a decrease in the lesion volume in magnetic resonance imaging within the first month were observed. Alleviation of the lactate accumulation during the nicotinamide treatment suggested that an increase in the cellular NAD+NADH concentration led to enhancement of the oxidation of reducing equivalents, suggesting that complex I of respiratory chain operates at non-saturating substrate concentration. Myoblasts cultured from patients carrying the 3243A→G mutation and from controls were used to measure ATP, ADP, catalase and superoxide dismutase activity, population growth, apoptotic cell death and the morphology of cytoskeletal components. ATP and ADP concentrations were decreased, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, suggesting an increased production of reactive oxygen species due to respiratory chain dysfunction. No increase in apoptotic cell death was observed in proliferating myoblasts, but randomization of vimentin filament direction and length was observed and decreased population growth was associated with the mutation. The results show that the 3243A→G mutation leads to numerous secondary pathophysiological events. Based on the literature and the results of this study, similarities were found between the pathophysiology of 3243A→G mutation and other neurodegenerative diseases and aging.

Plant mitochondrial genomes are large and complex, and the mechanisms for maintaining mitochondrial DNA (mtDNA) remain unclear. Arabidopsis thaliana has two DNA polymerase genes, polIA and polIB, that have been shown to be dual localized to mitochondria and chloroplasts but are unequally expressed within primary plant tissues involved in cell division or cell expansion. PolIB expression is observed at higher levels in both shoot and root apexes, suggesting a possible role in organelle DNA replication in rapidly dividing or expanding cells. It is proposed that both polIA and polIB are required for mtDNA replication under wild type conditions. An Arabidopsis T-DNA polIB mutant has a 30% reduction in mtDNA levels but also a 70% induction in polIA gene expression. The polIB mutant shows an increase relative to wild type plants in the number of mitochondria that are significantly smaller in relative size, observed within hypocotyl epidermis cells that have a reduced rate of cell expansion. These mutants exhibit a significant increase in gene expression for components of mitorespiration and photosynthesis, and there is evidence for an increase in both light to dark (transitional) and light respiration levels. There is not a significant difference in dark adjusted total respiration between mutant and wild type plants. Chloroplast numbers are not significantly different in isolated mesophyll protoplasts, but mesophyll cells from the mutant are significantly smaller than wild type. PolIB mutants exhibit a three-day delay in chloroplast development but after 7dpi (days post-imbibition) there is no difference in relative plastid DNA levels between the mutant and wild type. Overall, the polIB mutant exhibits an adjustment in cell homeostasis, which enables the maintenance of functional mitochondria but at the cost of normal cell expansion rates.