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Aims: Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age-related histopathological changes were previously reported., Methods: Cervical and lumbar paraspinal muscles were obtained peri-operatively from 13 patients and from six post mortem control cases (age range 18-82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory-deficient muscle fibres (lacking complex IV with intact complex II activity). Real-time polymerase chain reaction, long-range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry., Results: The density of respiratory-deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory-deficient (range 0-10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory-deficient fibres with and without mtDNA deletions or mtDNA depletion., Conclusions: Paraspinal muscles appeared to be particularly susceptible to age-related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age-related postural abnormalities., (© 2012 The Authors. Neuropathology and Applied Neurobiology © 2012 British Neuropathological Society.)

Background: Mutations in the Twinkle (PEO1) gene are a recognized cause of autosomal dominant progressive external ophthalmoplegia (adPEO), resulting in the accumulation of multiple mitochondrial DNA (mtDNA) deletions and cytochrome c oxidase (COX)-deficient fibers in skeletal muscle secondary to a disorder of mtDNA maintenance. Patients typically present with isolated extraocular muscle involvement, with little apparent evidence of the clinical heterogeneity documented in other mtDNA maintenance disorders, in particular POLG-related disease., Methods: We reviewed the clinical, histochemical, and molecular genetics analysis of 33 unreported patients from 26 families together with all previous cases described in the literature to define the clinical phenotype associated with PEO1 mutations., Results: Ptosis and ophthalmoparesis were almost universal clinical features among this cohort, with 52% (17/33) reporting fatigue and 33% (11/33) having mild proximal myopathy. Features consistent with CNS involvement were rarely described; however, in 24% (8/33) of the patients, cardiac abnormalities were reported. Mitochondrial histochemical changes observed in muscle showed remarkable variability, as did the secondary mtDNA deletions, which in some patients were only detected by PCR-based assays and not Southern blotting. Moreover, we report 7 novel PEO1 variants., Conclusions: Our data suggest a shared clinical phenotype with variable mild multiorgan involvement, and that the contribution of PEO1 mutations as a cause of adPEO may well be underestimated. Direct sequencing of the PEO1 gene should be considered in adPEO patients prior to muscle biopsy.

Objectives: Defects of the mitochondrial genome (mtDNA) cause a series of rare, mainly neurological disorders. In addition, they have been implicated in more common forms of movement disorders, dementia and the ageing process. In order to try to model neuronal dysfunction associated with mitochondrial disease, we have attempted to establish a series of transmitochondrial mouse embryonic stem cells harbouring pathogenic mtDNA mutations., Materials and Methods: Transmitochondrial embryonic stem cell cybrids were generated by fusion of cytoplasts carrying a variety of mtDNA mutations, into embryonic stem cells that had been pretreated with rhodamine 6G, to prevent transmission of endogenous mtDNA. Cybrids were differentiated into neurons and assessed for efficiency of differentiation and electrophysiological function., Results: Neuronal differentiation could occur, as indicated by expression of neuronal markers. Differentiation was impaired in embryonic stem cells carrying mtDNA mutations that caused severe biochemical deficiency. Electrophysiological tests showed evidence of synaptic activity in differentiated neurons carrying non-pathogenic mtDNA mutations or in those that caused a mild defect of respiratory activity. Again, however, neurons carrying mtDNA mutations that resulted in severe biochemical deficiency had marked reduction in post-synaptic events., Conclusions: Differentiated neurons carrying severely pathogenic mtDNA defects can provide a useful model for understanding how such mutations can cause neuronal dysfunction.

Aim/hypothesis: Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity., Methods: We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods., Results: There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc., Conclusion/interpretation: Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.

A decline in mitochondrial function has long been shown to exist in neurodegenerative disease. Whether this decline is a secondary consequence of other factors or whether it causes the eventual death of a cell is unknown. In this review, we will discuss some of the major evidence surrounding mitochondrial DNA mutations leading to mitochondrial dysfunction in neurodegenerative disease and discuss their possible role in neurodegeneration.

Aims/hypothesis: The aims of this study were (1) to determine the prevalence and rate of progression in diabetes secondary to mitochondrial DNA (mtDNA) mutations; and (2) to determine whether percentage heteroplasmy predicts clinical outcome in patients carrying the m.3243A>G mutation., Methods: We prospectively assessed 242 patients attending a specialist neuromuscular clinic using a validated mitochondrial disease rating scale. Retrospective clinical data on these patients from up to 25 years of follow-up were also included. Percentage heteroplasmy in blood, urine and muscle was determined for the m.3243A>G group and correlated against clinical features., Results: Patients carrying the m.3243A>G mutation formed the largest group of patients with diabetes (31/81 patients). The highest prevalence of diabetes was in the m.12258C>A group (2/2 patients), the lowest in the multiple mtDNA deletions group (3/43 patients). The earliest age of onset was in the m.3243A>G group (37.9 years) with the highest age of presentation in the multiple deletion group (56.3 years). Of patients presenting with m.3243A>G, 12.9% required insulin; an additional 32.3% progressed to insulin requirement over a mean of 4.2 years after presentation. Percentage heteroplasmy in blood, urine or muscle did not predict progression of diabetes or risk of developing complications. Early age of presentation with diabetes did predict poor clinical outcome., Conclusions/interpretation: Although patients carrying the m.3243A>G mutation account for the majority of cases of diabetes secondary to mtDNA mutations, several other genotypes are also associated with the development of diabetes, some with high penetrance. All show a gradual progression to insulin requirement. Percentage heteroplasmy is a poor predictor of severity of diabetes in the m.3243A>G group.

Background: In clinical practice, mitochondrial disease is seldom considered until a variable combination of seizures, alteration in tone, muscle weakness, and developmental problems is evident. However, it is not uncommon for one symptom to occur in isolation and dominate the clinical phenotype. We report six patients from two families where dystonia was the principal clinical manifestation. A mitochondrial etiology was considered in each case because of the association of dystonia with other less prominent clinical features such as epilepsy., Methods: Histochemical and biochemical analyses were undertaken in skeletal muscle biopsies from individuals in both families. Sequencing of skeletal muscle mtDNA was also performed and suspected mutations were quantified by hot last cycle PCR-RFLP or primer extension assay. Functional consequences of one of the mutations were investigated by measurement of steady state levels of mitochondrial tRNA., Results: Two distinct mitochondrial pathologies were identified: a novel, homoplasmic mitochondrial tRNA(Cys) (MTTC) mutation and the primary, m.11778G>A Leber hereditary optic neuropathy (LHON) mutation. The mild nature of both mutations has permitted very high levels of mutated mtDNA to accumulate. Patients with the mutation in the MTTC gene have no wild type mtDNA detectable and although the LHON mutation is heteroplasmic in the patients we report, it is commonly observed to be homoplasmic., Conclusions: The mitochondrial etiology identified in these patients emphasizes the pathologic potential of homoplasmic mutations and has important implications for the investigation and genetic counseling of families where dystonia is the principal clinical feature. We advocate that mitochondrial disease should be given serious consideration in patients with familial, progressive dystonia, particularly when additional neurologic features such as epilepsy are present.

The authors sequenced POLG1, C10ORF2, and ANT1 in 38 sporadic progressive external ophthalmoplegia patients with multiple mitochondrial DNA (mtDNA) deletions. Causative mutations were identified in approximately 10% of cases, with two unrelated individuals harboring a novel premature stop codon mutation (1356T>G). None had a mutation in C10ORF2 or ANT1. In the majority of patients, the primary nuclear genetic defect is likely to affect other unknown genes important for mtDNA maintenance.

Aim: To investigate a possible association between mitochondrial haplogroups and primary open angle glaucoma (POAG)., Methods: Genomic DNA was extracted from 140 POAG patients and 75 healthy individuals. Restriction enzyme digest analysis of polymerase chain reaction (PCR) amplified fragments was used to determine the mitochondrial haplogroup of each patient and control., Results: The median age was 73 years for the POAG patients (range 51-87, SD 8.01) and 78 years for the controls (range 68-90, SD 4.4). Mean IOP was 20.8 mm Hg for the patients (SD 2.6) and 16.2 mm Hg for the controls (SD 3.4). Median cup/disc ratio was 0.8 and 0.3 for patients and controls respectively. No statistically significant difference was found in the haplogroup distribution between the POAG patients and the healthy individuals (Fisher's exact test)., Conclusion: In this cohort, mitochondrial haplogroups do not appear to contribute to the pathogenesis of POAG.

A set of 96 complete mtDNA sequences that belong to the three major African haplogroups (L1, L2, and L3) was analyzed to determine if mtDNA has evolved as a molecular clock. Likelihood ratio tests (LRTs) were carried out with each of the haplogroups and with combined haplogroup sequence sets. Evolution has not been clock-like, neither for the coding region nor for the control region, in combined sets of African haplogroup L mtDNA sequences. In tests of individual haplogroups, L2 mtDNAs showed violations of a molecular clock under all conditions and in both the control and coding regions. In contrast, haplogroup L1 and L3 sequences, both for the coding and control regions, show clock-like evolution. In clock tests of individual L2 subclades, the L2a sequences showed a marked violation of clock-like evolution within the coding region. In addition, the L2a and L2c branch lengths of both the coding and control regions were shorter relative to those of the L2b and L2d sequences, a result that indicates lower levels of sequence divergence. Reduced median network analyses of the L2a sequences indicated the occurrence of marked homoplasy at multiple sites in the control region. After exclusion of the L2a and L2c sequences, African mtDNA coding region evolution has not significantly departed from a molecular clock, despite the results of neutrality tests that indicate the mitochondrial coding region has evolved under nonneutral conditions. In contrast, control region evolution is clock-like only at the haplogroup level, and it thus appears to have evolved essentially independently from the coding region. The results of the clock tests, the network analyses, and the branch length comparisons all caution against the use of simple mtDNA clocks.

The authors describe a patient who presented with headache, seizures, and severe cerebral edema in whom they identified a novel mutation in the mitochondrial (mt-) tRNA(His) gene. This G12147A transition is heteroplasmic, predicted to disrupt a highly conserved base pair, and segregates with the cytochrome c oxidase deficiency in single muscle fibers.

This article provides evidence that selection has been a significant force during the evolution of the human mitochondrial genome. Both gene-by-gene and whole-genome approaches were used here to assess selection in the 560 mitochondrial DNA (mtDNA) coding-region sequences that were used previously for reduced-median-network analysis. The results of the present analyses were complex, in that the action of selection was not indicated by all tests, but this is not surprising, in view of the characteristics and limitations of the different analytical methods. Despite these limitations, there is evidence for both gene-specific and lineage-specific variation in selection. Whole-genome sliding-window approaches indicated a lack of selection in large-scale segments of the coding region. In other tests, we analyzed the ratio of nonsynonymous-to-synonymous substitutions in the 13 protein-encoding mtDNA genes. The most straightforward interpretation of those results is that negative selection has acted on the mtDNA during evolution. Single-gene analyses indicated significant departures from neutrality in the CO1, ND4, and ND6 genes, although the data also suggested the possible operation of positive selection on the AT6 gene. Finally, our results and those of other investigators do not support a simple model in which climatic adaptation has been a major force during human mtDNA evolution.

Mitochondrial genome instability has recently been demonstrated in a wide variety of human tumours and is implicated in the development of the myelodysplastic syndromes, a heterogeneous group of haematological disorders with an increased risk of malignant transformation. We therefore investigated the incidence of somatic mitochondrial DNA (mtDNA) mutations in patients with adult-onset leukaemia. We sequenced the entire mitochondrial genome from both normal tissue (buccal epithelial cells) and the leukaemia from 24 patients with adult-onset leukaemia. Somatic mtDNA mutation was present in nine individuals ( approximately 40%) and in each case the tumour genome differed from the normal genome sequence by a single sequence change. Using PCR-RFLP analysis and real-time PCR, we have studied in detail the mutation present in one patient with acute lymphatic leukaemia, demonstrating that the mutation is associated specifically with the leukaemia.

A 67-year-old man presented with cognitive deficits, status epilepticus, left hemiparesis, and severe lactic acidosis. Respiratory chain enzyme analysis of skeletal muscle revealed a defect in complex I activity, associated with a heteroplasmic C11777A mutation in the mitochondrial ND4 gene. This case is remarkable not only because of the late onset of symptoms, but because this mutation affects the identical ND4 codon as the G11778A mutation that causes Leber hereditary optic neuropathy.

To verify the impact of mutations in ANT1, Twinkle, and POLG1 genes in sporadic progressive external ophthalmoplegia associated with multiple mitochondrial DNA (mtDNA) deletions, DNA samples from 15 Italian and 12 British patients were screened. Mutations in ANT1 were found in one patient, in Twinkle in two patients, and in POLG1 in seven patients. Irrespective of the inheritance mode, screening of these genes should be performed in all patients with progressive external ophthalmoplegia with multiple mtDNA deletions.

We have extended our previous analysis of the pedigree rate of control-region divergence in the human mitochondrial genome. One new germline mutation in the mitochondrial DNA (mtDNA) control region was detected among 185 transmission events (generations) from five Leber hereditary optic neuropathy (LHON) pedigrees. Pooling the LHON pedigree analyses yields a control-region divergence rate of 1.0 mutation/bp/10(6) years (Myr). When the results from eight published studies that used a similar approach were pooled with the LHON pedigree studies, totaling >2,600 transmission events, a pedigree divergence rate of 0.95 mutations/bp/Myr for the control region was obtained with a 99.5% confidence interval of 0.53-1.57. Taken together, the cumulative results support the original conclusion that the pedigree divergence rate for the control region is approximately 10-fold higher than that obtained with phylogenetic analyses. There is no evidence that any one factor explains this discrepancy, and the possible roles of mutational hotspots (rate heterogeneity), selection, and random genetic drift and the limitations of phylogenetic approaches to deal with high levels of homoplasy are discussed. In addition, we have extended our pedigree analysis of divergence in the mtDNA coding region. Finally, divergence of complete mtDNA sequences was analyzed in two tissues, white blood cells and skeletal muscle, from each of 17 individuals. In three of these individuals, there were four instances in which an mtDNA mutation was found in one tissue but not in the other. These results are discussed in terms of the occurrence of somatic mtDNA mutations.

Aims/hypothesis: To examine whether there is a high content of mutated mitochondrial DNA in individual pancreatic beta cells from a patient with the A3243G mitochondrial DNA mutation., Methods: Tissues were available from a patient with diabetes and the A3243G mutation including pancreatic tissue. We quantified the amount of mutated mitochondrial DNA in tissue homogenates and single pancreatic beta cells using hot last cycle PCR., Results: The percentage ratio of mutated to wild-type mtDNA was high in tissues such as muscle and brain (>60%), but surprisingly low in both pancreatic islets and in individual beta cells from these islets. The islets were smaller in the patient than in control subjects in keeping with a decreased beta-cell mass., Conclusions/interpretation: These observations suggest that either the beta cells show increased sensitivity to the effects mtDNA mutations on respiratory chain function, and/or cells with a high mutant load are preferentially removed leading to a progressive decrease in the islet beta-cell mass.

A heteroplasmic T to C transition at nucleotide position 14709 in the mitochondrial tRNA glutamic acid (tRNA(Glu)) gene has previously been associated with maternally inherited diabetes and deafness (MIDD). To investigate the pathogenic mechanism of the T14709C mutation, we have constructed transmitochondrial cell lines by transferring fibroblasts mitochondria from a patient with the mutation into human cells lacking mitochondrial DNA (mtDNA) (rho degrees cells). Clonal cybrid cell lines were obtained containing various levels of the heteroplasmic mutation, or exclusively mutated or wild-type mtDNA. Measurement of respiratory chain enzymatic activities failed to detect a difference between the homoplasmic mutant and homoplasmic wild-type cybrid cell lines. However, a subtle decrease in the steady-state levels of tRNA(Glu) transcripts in some mutant clones. Our studies suggest that the T14709C mutation is insufficient to lead impairment of mitochondrial function in homoplasmic osteosarcoma cybrid clones, and that we cannot exclude that the T14709C mutation affects mitochondrial function by a yet unidentified mechanism.

Purpose: To evaluate somatic mitochondrial (mt)DNA mutations in the macula during ageing., Methods: Ten 30-microm cryostat sections from the macula (foveal and perifoveal regions) and peripheral retina of 14 donors (aged 14-94 years) were cut for cytochrome c oxidase cytochemistry. The photoreceptor layer was microdissected and DNA extracted for 4977-bp mtDNA (mtDNA(4977)) quantification using PCR. Dual cytochemistry for cytochrome c oxidase and succinate dehydrogenase allowed the detection of cytochrome c oxidase-deficient cones., Results: Findings showed a progressive accumulation of mtDNA(4977) from ages 14 to 94 years. From ages 14 to 60 years there was an increase from 0.006% to 0.25%, and from ages 60 to 94 years there was a steeper increase from 0.25% to 5.39%. Counts of cones in the dual-reacted preparations showed more cytochrome c oxidase-deficient cones in the foveal region than elsewhere., Conclusions: The results show that mitochondrial DNA deletions and cytochrome c oxidase-deficient cones accumulate in the ageing retina, particularly in the foveal region. These defects may contribute to the changes in macular function observed in ageing and age-related maculopathy.

Mitochondrial DNA (mtDNA) defects are responsible for symptom complexes that are characteristically heterogeneous but are typically represented by muscle weakness and neurological deficits. One common feature of mitochondrial disease is deafness. This report details the assessment and outcome of a patient with a previously undescribed mtDNA rearrangement who underwent cochlear implantation. The patient shows a marked improvement in sentence recognition tests and recognition of environmental sounds. Patients with profound sensorineural hearing loss due to mtDNA defects should be considered as candidates for cochlear implantation when they no longer benefit from conventional hearing aids.

Mutations in mitochondrial DNA (mtDNA) cause a spectrum of human pathologies, which predominantly affect skeletal muscle and the central nervous system. In patients, mutated and wild-type mtDNAs often co-exist in the same cell (mtDNA heteroplasmy). In the absence of pharmacological therapy, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative to the single-stranded replication intermediate, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Previous studies have shown the efficacy of this anti-genomic approach in vitro, targeting pathogenic mtDNA templates with only a single point mutation. Pathogenic molecules harbouring deletions, however, present a more difficult problem. Deletions often occur at the site of two short repeat sequences (4-13 residues), only one of which is retained in the deleted molecule. With the more common larger repeats it is therefore difficult to design an anti-genomic molecule that will bind selectively across the breakpoint of the deleted mtDNA. To address this problem, we have used linker-substituted oligodeoxynucleotides to bridge the repeated residues. We show that molecules can be designed to bind more tightly to the deleted as compared to the wild-type mtDNA template, consistent with the nucleotide sequence on either side of the linker co-operating to increase binding affinity. Furthermore, these bridging molecules are capable of sequence-dependent partial inhibition of replication in vitro.

Studies of single cells have previously shown intracellular clonal expansion of mitochondrial DNA (mtDNA) mutations to levels that can cause a focal cytochrome c oxidase (COX) defect. Whilst techniques are available to study mtDNA rearrangements at the level of the single cell, recent interest has focused on the possible role of somatic mtDNA point mutations in ageing, neurodegenerative disease and cancer. We have therefore developed a method that permits the reliable determination of the entire mtDNA sequence from single cells without amplifying contaminating, nuclear-embedded pseudogenes. Sequencing and PCR-RFLP analyses of individual COX-negative muscle fibres from a patient with a previously described heteroplasmic COX II (T7587C) mutation indicate that mutant loads as low as 30% can be reliably detected by sequencing. This technique will be particularly useful in identifying the mtDNA mutational spectra in age-related COX-negative cells and will increase our understanding of the pathogenetic mechanisms by which they occur.

In mammals, mitochondrial DNA (mtDNA) is the only autonomously replicating source of DNA outside the nucleus. Housed in the mitochondrial matrix, this molecule encodes thirteen polypeptides, all of which are believed to be essential components of the mitochondrial respiratory chain. Defects of the mitochondrial genome can cause severe neurological and multi-systemic disorders. As the genetic defect causes a dysfunction in the terminal stage of oxidative metabolism, there is little potential for pharmacological intervention. Thus, there is currently no effective therapy for these chronic progressive disorders. In the disease state, pathogenic mtDNA molecules often cohabit the same cell and tissue with wild type mtDNA, a situation termed heteroplasmy. Manifestation of biochemical and clinical defects occur only when a threshold level of heteroplasmy has been passed. The mitochondrial genome must be continually turned over. Consequently, if a pathogenic mtDNA molecule were to be targeted to prevent it from replicating, the wild type copy would be given a propagative advantage. Over time, therefore, the biochemical and, potentially, the clinical deficiency could be reversed. This manuscript summarises our attempts to identify such an antigenomic molecule, to localise this molecule to mitochondria and to assess its function in whole cells. Finally, we discuss the importance of identifying and designing new antigenomic molecules which may prove effective in treating patients with disorders of the mitochondrial genome.

We describe a 42-year-old man who presented with a progressive history of epilepsy, stroke-like episodes, bilateral optic atrophy, and cognitive decline. Investigation of his muscle biopsy revealed a specific defect in complex I activity. Subsequent analysis of the mitochondrial genome identified a novel heteroplasmic T10191C mutation in the ND3 gene. The mutation was present at lower levels in blood from the patient and unaffected maternal relatives and is the first pathogenic mitochondrial DNA mutation in the ND3 gene to be described.

Mitochondrial abnormalities, in particular the accumulation of mitochondrial DNA mutations, have been proposed as a potential cause of normal ageing. One group of patients with mtDNA disorders have a nuclear DNA defect which accelerates the chronological accumulation of mitochondrial DNA mutations. These patients provide an ideal means of investigating whether accelerated mitochondrial DNA defects can cause accelerated ageing pathology. The choroid plexus demonstrates a robust accumulation of pathological changes, in the form of Biondi bodies, with normal ageing. We have therefore examined the choroid plexus of a case with multiple mitochondrial DNA deletions for evidence of accelerated ageing and compared it with two cases with point mutation mitochondrial DNA disorders and several age-matched and elderly controls with and without clinical and neuropathological evidence of neurodegenerative disease. We also demonstrate that the choroid plexus of the mitochondrial DNA cases contain cells with levels of mitochondrial DNA mutation sufficient to cause a biochemical deficiency in the oxidative phosphorylation pathway. As previously reported, both cases with point mutation mitochondrial DNA disorders exhibit a characteristic oncocytic type transformation of the choroidal epithelial cells. However, in the case with multiple mitochondrial DNA deletions we demonstrate pathological changes in choroid plexus that are strongly suggestive of accelerated ageing. We believe that this finding supports the theory that the accumulation of mitochondrial DNA mutations can lead to pathological changes typical of ageing cells.

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium-PNA (ph-PNA) conjugates of 3.4-4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph-PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph-PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph-PNA uptake. The ph-PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease 'myoclonic epilepsy and ragged red fibres' (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph-PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph-PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Human tissues acquire somatic mitochondrial DNA (mtDNA) mutations with age. Very high levels of specific mtDNA mutations accumulate within individual cells, causing a defect of mitochondrial oxidative metabolism. This is a fundamental property of nondividing tissues, but it is not known how it comes about. To explore this problem, we developed a model of mtDNA replication within single human cells. Using this model, we show that relaxed replication of mtDNA alone can lead, through random genetic drift, to the clonal expansion of single mutant events during human life. Significant expansions primarily develop from mutations acquired during a critical period in childhood or early adult life.

The last ten years have seen a huge increase in the number of different genetic defects found in patients with mitochondrial disorders, but the true impact of mitochondrial disease is only just becoming apparent. Mitochondrial diseases are far more common than was anticipated. Although there have also been major advances in our understanding of mitochondrial pathology, the clinical management of patients with mitochondrial disease is largely supportive. In this article, we focus on primary disorders of the mitochondrial respiratory chain and mtDNA defects. We review the available epidemiological data, outline current strategies for the management of mitochondrial disease, and highlight new therapeutic approaches that may prove useful in the future., (Copyright 2001 Wiley-Liss, Inc.)

Recent observations in cultured human fibroblasts suggest that the accumulation of point mutations in the noncoding control region of mtDNA may be important in human aging. We studied the mtDNA control region in brain tissue from 31 normal elderly individuals, from 35 individuals who had Alzheimer disease, and from 47 individuals who had dementia with Lewy bodies. We found no evidence that these somatic mtDNA point mutations accumulate either in the brains of normal elderly individuals or in the brains of individuals with neurodegenerative disease.

We measured the proportion of mutant mtDNA (mutation load) in 82 primary oocytes from a woman who harbored the A3243G mtDNA mutation. The frequency distribution of mutation load indicates that random drift is the principal mechanism that determines the level of mutant mtDNA within individual oocytes.