Nils-Göran Larsson

Premature ageing in mice expressing defective mitochondrial DNA polymerase

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.

Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA.

Characterization of the basic transcription machinery of mammalian mitochondrial DNA (mtDNA) is of fundamental biological interest and may also lead to therapeutic interventions for human diseases associated with mitochondrial dysfunction. Here we report that mitochondrial transcription factors B1 (TFB1M) and B2 (TFB2M) are necessary for basal transcription of mammalian mitochondrial DNA (mtDNA). Human TFB1M and TFB2M are expressed ubiquitously and can each support promoter-specific mtDNA transcription in a pure recombinant in vitro system containing mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor A. Both TFB1M and TFB2M interact directly with POLRMT, but TFB2M is at least one order of magnitude more active in promoting transcription than TFB1M. Both factors are highly homologous to bacterial rRNA dimethyltransferases, which suggests that an RNA-modifying enzyme has been recruited during evolution to function as a mitochondrial transcription factor. The presence of two proteins that interact with mammalian POLRMT may allow flexible regulation of mtDNA gene expression in response to the complex physiological demands of mammalian metabolism.

The role of mitochondria in aging

Over the last decade, accumulating evidence has suggested a causative link between mitochondrial dysfunction and major phenotypes associated with aging. Somatic mitochondrial DNA (mtDNA) mutations and respiratory chain dysfunction accompany normal aging, but the first direct experimental evidence that increased mtDNA mutation levels contribute to progeroid phenotypes came from the mtDNA mutator mouse. Recent evidence suggests that increases in aging-associated mtDNA mutations are not caused by damage accumulation, but rather are due to clonal expansion of mtDNA replication errors that occur during development. Here we discuss the caveats of the traditional mitochondrial free radical theory of aging and highlight other possible mechanisms, including insulin/IGF-1 signaling (IIS) and the target of rapamycin pathways, that underlie the central role of mitochondria in the aging process.

Unraveling the Biological Roles of Reactive Oxygen Species

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.

Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression

Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation and may also have a role in ageing and acquired diseases of old age. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA (Refs 6,7). Using a loxP-flanked Tfam allele (TfamloxP; ref. 8) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter9,10, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.

Impaired insulin secretion and β-cell loss in tissue-specific knockout mice with mitochondrial diabetes

Mitochondrial dysfunction is an important contributor to human pathology and it is estimated that mutations of mitochondrial DNA (mtDNA) cause approximately 0.5–1% of all types of diabetes mellitus. We have generated a mouse model for mitochondrial diabetes by tissue-specific disruption of the nuclear gene encoding mitochondrial transcription factor A (Tfam, previously mtTFA; ref. 7) in pancreatic β-cells. This transcriptional activator is imported to mitochondria, where it is essential for mtDNA expression and maintenance. The Tfam-mutant mice developed diabetes from the age of approximately 5 weeks and displayed severe mtDNA depletion, deficient oxidative phosphorylation and abnormal appearing mitochondria in islets at the ages of 7–9 weeks. We performed physiological studies of β-cell stimulus–secretion coupling in islets isolated from 7–9-week-old mutant mice and found reduced hyperpolarization of the mitochondrial membrane potential, impaired Ca2+-signalling and lowered insulin release in response to glucose stimulation. We observed reduced β-cell mass in older mutants. Our findings identify two phases in the pathogenesis of mitochondrial diabetes; mutant β-cells initially display reduced stimulus–secretion coupling, later followed by β-cell loss. This animal model reproduces the β-cell pathology of human mitochondrial diabetes and provides genetic evidence for a critical role of the respiratory chain in insulin secretion.

Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons

Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson′s disease (PD), a common age-associated neurodegenerative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. We have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD.

Proteolytic Processing of OPA1 Links Mitochondrial Dysfunction to Alterations in Mitochondrial Morphology

Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase γ. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.

Strong Purifying Selection in Transmission of Mammalian Mitochondrial DNA

There is an intense debate concerning whether selection or demographics has been most important in shaping the sequence variation observed in modern human mitochondrial DNA (mtDNA). Purifying selection is thought to be important in shaping mtDNA sequence evolution, but the strength of this selection has been debated, mainly due to the threshold effect of pathogenic mtDNA mutations and an observed excess of new mtDNA mutations in human population data. We experimentally addressed this issue by studying the maternal transmission of random mtDNA mutations in mtDNA mutator mice expressing a proofreading-deficient mitochondrial DNA polymerase. We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection. There are striking similarities between the mutational patterns in our experimental mouse system and human mtDNA polymorphisms. These data show strong purifying selection against mutations within mtDNA protein-coding genes. To our knowledge, our study presents the first direct experimental observations of the fate of random mtDNA mutations in the mammalian germ line and demonstrates the importance of purifying selection in shaping mitochondrial sequence diversity.

Somatic Mitochondrial DNA Mutations in Mammalian Aging

Mitochondrial dysfunction is heavily implicated in the multifactorial aging process. Aging humans have increased levels of somatic mtDNA mutations that tend to undergo clonal expansion to cause mosaic respiratory chain deficiency in various tissues, such as heart, brain, skeletal muscle, and gut. Genetic mouse models have shown that somatic mtDNA mutations and cell type-specific respiratory chain dysfunction can cause a variety of phenotypes associated with aging and age-related disease. There is thus strong observational and experimental evidence to implicate somatic mtDNA mutations and mosaic respiratory chain dysfunction in the mammalian aging process. The hypothesis that somatic mtDNA mutations are generated by oxidative damage has not been conclusively proven. Emerging data instead suggest that the inherent error rate of mitochondrial DNA (mtDNA) polymerase gamma (Pol gamma) may be responsible for the majority of somatic mtDNA mutations. The roles for mtDNA damage and replication errors in aging need to be further experimentally addressed.