Repeats, longevity and the sources of mtDNA deletions: evidence from 'deletional spectra'.

Perfect direct repeats and, in particular, the prominent 13 bp repeat, are thought to cause mitochondrial DNA (mtDNA) deletions, which have been associated with the aging process. Accordingly, individuals lacking the 13 bp repeat are highly prevalent among centenarians and overall number of perfect repeats in mammalian mitochondrial genomes negatively correlates with species' longevity. However, detailed examination of the distribution of mtDNA deletions challenges a special role of the 13 bp repeat in generating mtDNA deletions. Instead, deletions appear to depend on long and stable, albeit imperfect, duplexes between distant mtDNA segments. Furthermore, significant dissimilarities in breakpoint distributions suggest that multiple mechanisms are involved in creating mtDNA deletions.

Locating the stem cell niche and tracing hepatocyte lineages in human liver.

UNLABELLED: We have used immunohistochemical and histochemical techniques to identify patches of hepatocytes deficient in the enzyme cytochrome c oxidase, a component of the electron transport chain and encoded by mitochondrial DNA (mtDNA). These patches invariably abutted the portal tracts and expanded laterally as they spread toward the hepatic veins. Here we investigate, using mtDNA mutations as a marker of clonal expansion, the clonality of these patches. Negative hepatocytes were laser-capture microdissected and mutations identified by polymerase chain reaction sequencing of the entire mtDNA genome. Patches of cytochrome c oxidase-deficient hepatocytes were clonal, suggesting an origin from a long-lived cell, presumably a stem cell. Immunohistochemical analysis of function and proliferation suggested that these mutations in cytochrome c oxidase-deficient hepatocytes were nonpathogenic. CONCLUSION: These data show, for the first time, that clonal proliferative units exist in the human liver, an origin from a periportal niche is most likely, and that the trajectory of the units is compatible with a migration of cells from the periportal regions to the hepatic veins.

A methodological approach to tracing cell lineage in human epithelial tissues.

Methods for lineage tracing of stem cell progeny in human tissues are currently not available. We describe a technique for detecting the expansion of a single cell's progeny that contain clonal mitochondrial DNA (mtDNA) mutations affecting the expression of mtDNA-encoded cytochrome c oxidase (COX). Because such mutations take up to 40 years to become phenotypically apparent, we believe these clonal patches originate in stem cells. Dual-color enzyme histochemistry was used to identify COX-deficient cells, and mutations were confirmed by microdissection of single cells with polymerase chain reaction sequencing of the entire mtDNA genome. These techniques have been applied to human intestine, liver, pancreas, and skin. Our results suggest that the stem cell niche is located at the base of colonic crypts and above the Paneth cell region in the small intestine, in accord with dynamic cell kinetic studies in animals. In the pancreas, exocrine tissue progenitors appeared to be located in or close to interlobular ducts, and, in the liver, we propose that stem cells are located in the periportal region. In the skin, the origin of a basal cell carcinoma appeared to be from the outer root sheath of the hair follicle. We propose that this is a general method for detecting clonal cell populations from which the location of the niche can be inferred, also affording the generation of cell fate maps, all in human tissues. In addition, the technique allows analysis of the origin of human tumors from specific tissue sites.

Dopaminergic midbrain neurons are the prime target for mitochondrial DNA deletions.

Mitochondrial dysfunction is a consistent finding in neurodegenerative disorders like Alzheimer's (AD) or Parkinson's disease (PD) but also in normal human brain aging. In addition to respiratory chain defects, damage to mitochondrial DNA (mtDNA) has been repeatedly reported in brains from AD and PD patients. Most studies though failed to detect biologically significant point mutation or deletion levels in brain homogenate. By employing quantitative single cell techniques, we were recently able to show significantly high levels of mtDNA deletions in dopaminergic substantia nigra (SN) neurons from PD patients and age-matched controls. In the present study we used the same approach to quantify the levels of mtDNA deletions in single cells from three different brain regions (putamen, frontal cortex, SN) of patients with AD (n = 9) as compared to age-matched controls (n = 8). There were no significant differences between patients and controls in either region but in both groups the deletion load was markedly higher in dopaminergic SN neurons than in putamen or frontal cortex (p < 0.01; ANOVA). This data shows that there is a specific susceptibility of dopaminergic SN neurons to accumulate substantial amounts of mtDNA deletions, regardless of the underlying clinical phenotype.

The anti-psoriatic drug anthralin accumulates in keratinocyte mitochondria, dissipates mitochondrial membrane potential, and induces apoptosis through a pathway dependent on respiratory competent mitochondria.

Anthralin is a potent topical drug, inducing clearance of psoriatic plaques. Anthralin disrupts mitochondrial function and structure, but its mechanism of action remains undefined. This study aimed to determine whether anthralin induced keratinocyte apoptosis as well as to investigate molecular mechanisms and the role of mitochondria. We studied human keratinocytes and human 143B rho(0) cells, which lack mitochondrial DNA and a functional respiratory chain. We show that anthralin disrupts mitochondrial membrane potential (DeltaPsim) and causes endogenous cytochrome c release, resulting in the activation of caspase-3 and characteristic morphological changes of apoptosis. Disruption of DeltaPsim and cytochrome c release were independent of mitochondrial permeability transition or caspase activation. Human 143B rho(0) cells were resistant to anthralin-induced cell death, disruption of DeltaPsim, and cytochrome c release compared with the isogenic 143B rho+ cell line. Using the intrinsic fluorescence of anthralin, rapid accumulation within mitochondria was observed independent of DeltaPsim. Using assays that measure individual respiratory chain complexes, we show that anthralin specifically interacts with ubiquinone pool. These data indicate that anthralin induces apoptosis through a novel mitochondrial pathway dependent on oxidative respiration and involving electron transfer with the ubiquinone pool. These studies identify keratinocyte apoptosis as a potentially important mechanism involved in the clearance of psoriasis.

The distribution of mitochondrial activity in relation to optic nerve structure.

BACKGROUND: The observation of a buildup of mitochondria at the level of the lamina cribrosa in the optic nerve head has traditionally been attributed to axoplasmic stasis. However, this region is also the transition zone for myelination, resulting in differing energy requirements. OBJECTIVE: To investigate the relationship between myelination and mitochondrial activity in optic nerve tissue. METHODS: Histological, histochemical, and immunocytochemical techniques were used to demonstrate the distribution of myelin, cytochrome-c oxidase activity, and laminar structure in human optic nerve tissue. A study of rabbit optic nerve and retina and unmyelinated human pituitary stalk was also performed. Cytochrome-c oxidase activity in the human optic nerve tissue was measured using microphotometry. RESULTS: There was a striking inverse relationship between myelination and mitochondrial distribution in all tissue studied. Statistical analysis of microphotometric data showed this distribution to be highly significant. CONCLUSION: We caution against the previous inference of a process of axoplasmic stasis and suggest that, instead, the distribution of mitochondria reflects the functional requirement of different regions of the ganglion cell axon. CLINICAL RELEVANCE: Optic neuropathy is associated with several inherited disorders of mitochondria. We suggest that a fine balance exists between energy demand and tissue function in the optic nerve, which may explain why optic nerve pathological features are seen in those with mitochondrial disease.

New treatments for mitochondrial disease-no time to drop our standards.

Mitochondrial dysfunction is a common cause of inherited multisystem disease that often involves the nervous system. Despite major advances in our understanding of the pathophysiology of mitochondrial diseases, clinical management of these conditions remains largely supportive. Using a systematic approach, we identified 1,039 publications on treatments for mitochondrial diseases, only 35 of which included observations on more than five patients. Reports of a positive outcome on the basis of a biomarker of unproven clinical significance were more common in nonrandomized and nonblinded studies, suggesting a publication bias toward positive but poorly executed studies. Although trial design is improving, there is a critical need to develop new biomarkers of mitochondrial disease. In this Perspectives article, we make recommendations for the design of future treatment trials in mitochondrial diseases. Patients and physicians should no longer rely on potentially biased data, with the associated costs and risks.

Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle.

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm⁻¹) and SEN (469±13 MNs×mm⁻¹). Nav1.5 positive fibers (1548±70 µm²) were 35% smaller than Nav1.5 negative fibers (2367±78 µm²; P<0.05) in SEN muscle, whereas Nav1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 µm²; P<0.05) where no Nav1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.

Developmental and pathological changes in the human cardiac muscle mitochondrial DNA organization, replication and copy number.

Adult human heart mitochondrial DNA (mtDNA) has recently been shown to have a complex organization with abundant dimeric molecules, branched structures and four-way junctions. In order to understand the physiological significance of the heart-specific mtDNA maintenance mode and to find conditions that modify human heart mtDNA structure and replication, we analyzed healthy human heart of various ages as well as several different heart diseases, including ischemic heart disease, dilated as well as hypertrophic cardiomyopathies, and several mitochondrial disorders. By using one- and two-dimensional agarose gel electrophoresis, various enzymatic treatments and quantitative PCR we found that in human newborns heart mtDNA has a simple organization, lacking junctional forms and dimers. The adult-type branched forms are acquired in the early childhood, correlating with an increase in mtDNA copy number. Mitochondrial disorders involving either mutations in the mtDNA polymerase gamma (PolGalpha) or mtDNA helicase Twinkle, while having no obvious cardiac manifestation, show distinct mtDNA maintenance phenotypes, which are not seen in various types of diseased heart or in mitochondrial disorders caused by point mutations or large-scale deletions of mtDNA. The findings suggest a link between cardiac muscle development, mtDNA copy number, replication mode and topological organization. Additionally, we show that Twinkle might have a direct role in the maintenance of four-way junctions in human heart mtDNA.

Do mtDNA deletions drive premature aging in mtDNA mutator mice?

Deletions in mitochondrial DNA (mtDNA) have long been suspected to be involved in mammalian aging, but their role remains controversial. Recent research has demonstrated that relatively higher levels of mtDNA deletions correlate with premature aging in mtDNA mutator mice, which led to the conclusion that premature aging in these mice is driven by mtDNA deletions. However, it is reported here that the absolute level of deletions in mutator mice is quite low, especially when compared with the level of point mutations in these mice. It is thus argued that the available data are insufficient to conclude that mtDNA mutations drive premature aging in mtDNA mutator mice. It remains possible that clonal expansion of mtDNA deletions may result in sufficiently high levels to play a role in age-related dysfunction in some cells, but assessing this possibility will require studies of the distribution of these deletions among different cell types and in individual cells.

OPA1 deficiency associated with increased autophagy in retinal ganglion cells in a murine model of dominant optic atrophy.

PURPOSE: To examine retinal ganglion cell (RGC) and axonal abnormalities in an ENU-induced mutant mouse carrying a protein-truncating nonsense mutation in OPA1. Mutations in the OPA1 gene cause autosomal dominant optic atrophy (ADOA) in which loss of RGCs followed by myelin degeneration in the optic nerve leads to progressive decrease in visual acuity. METHODS: Ultrastructure of the optic nerve was examined in heterozygous mutants and wild-type littermate controls at 6, 9, and 24 months using electron microscopy. The RGC layer was examined at 6 and 24 months. RESULTS: There was an increase in the number of autophagosomes in the RGC layer in heterozygous mutants compared with wild type at 24 months. Signs of optic nerve degeneration were seen as early as 9 months in Opa1(+/-) mice, with more severe degeneration by 24 months. By 24 months, degeneration of axons was also seen in control mice. Numbers of opaque mitochondria in the Opa1(+/-) mice increased at 6 and 24 months, possibly representing an increase in the density of cristae to fulfill the energy requirements of the axon. In addition, mitochondria with vesiculation of the inner membranes, similar to the mutant mitochondria described in a mouse model of Charcot-Marie-Tooth type 2A, were observed. CONCLUSIONS: Mutations in OPA1 cause pathologic changes to optic nerve axons that are similar to, but occur earlier than, age-related degeneration. Increased autophagy is likely to result from an increase in abnormal mitochondria and could be one mechanism contributing to RGC loss and subsequent optic atrophy seen in ADOA.