Biomedical subjects

Nils-Göran Larsson

Publications and source records attributed to Nils-Göran Larsson.

At least 19 recordsLinked to original sources

PubMed2007

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.

Animals↗

PubMed2006

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 gamma. 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.

Animals↗

PubMed2006

Respiratory chain dysfunction in skeletal muscle does not cause insulin resistance.

Insulin resistance in skeletal muscle is a characteristic feature of diabetes mellitus type 2 (DM2). Several lines of circumstantial evidence suggest that reduced mitochondrial oxidative phosphorylation capacity in skeletal muscle is a primary defect causing insulin resistance and subsequent development of DM2. We have now experimentally tested this hypothesis by characterizing glucose homeostasis in tissue-specific knockout mice with progressive respiratory chain dysfunction selectively in skeletal muscle. Surprisingly, these knockout mice are not diabetic and have an increased peripheral glucose disposal when subjected to a glucose tolerance test. Studies of isolated skeletal muscle from knockout animals show an increased basal glucose uptake and a normal increase of glucose uptake in response to insulin. In summary, our findings indicate that mitochondrial dysfunction in skeletal muscle is not a primary etiological event in DM2.

AMP-Activated Protein Kinases↗

PubMed2005

Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production.

The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.

Aging↗

PubMed2005

SOD2 overexpression: enhanced mitochondrial tolerance but absence of effect on UCP activity.

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.

Animals↗

PubMed2005

A family of putative transcription termination factors shared amongst metazoans and plants.

The human mitochondrial transcription termination factor (mTERF) is involved in the regulation of transcription of the mitochondrial genome. Similarity searches and phylogenetic analysis demonstrate that mTERF is a member of large and complex protein family (the MTERF family) shared amongst metazoans and plants. Interestingly, we identify three novel MTERF genes in vertebrates, which all encode proteins with predicted mitochondrial localization. Members of the MTERF family have so far not been detected in fungi, supporting the notion that mitochondrial transcription regulation may have evolved separately in yeast and animal cells.

Amino Acid Sequence↗

PubMed2005

Abnormal Ca(2+) release and catecholamine-induced arrhythmias in mitochondrial cardiomyopathy.

Mitochondrial dysfunction is implicated in numerous cardiac disorders. It has been assumed that the functional defects are directly related to a decreased rate of mitochondrial ATP production, but recent studies have challenged this idea. Here, we used mice with tissue-specific knockout of mitochondrial transcription factor A (Tfam) that leads to progressive cardiomyopathy. The role of changes in the excitation-contraction (E-C) coupling in cardiomyocytes of these mice was studied by measuring the free cytosolic Ca(2+) concentration and by analyzing the expression of genes encoding E-C coupling proteins. Action potential-mediated Ca(2+) transients, measured with the fluorescent indicator fluo-3 in isolated cardiomyocytes, were smaller and faster in Tfam knockout cardiomyocytes when compared with controls. The total sarcoplasmic reticulum (SR) Ca(2+) content was decreased in Tfam knockout cells. The gene for the SR Ca(2+) binding protein calsequestrin-2 (CASQ2), as well as other genes encoding proteins involved in SR Ca(2+) handling, showed decreased expression in Tfam knockout hearts. Decreased CASQ2 levels have been linked to severe arrhythmias triggered by beta-adrenergic stimulation. In line with this, application of the beta-adrenergic agonist isoproterenol resulted in frequent doublet Ca(2+) transients in Tfam knockout cardiomyocytes. In conclusion, our results show that mitochondrial dysfunction in the heart induces specific down-regulation of the expression of genes encoding proteins involved in E-C coupling. These changes predispose to cardiac arrhythmias and terminal heart failure and are thus important in the pathogenesis of mitochondrial cardiomyopathy.

Adrenergic beta-Agonists↗

PubMed2005

Secondary metabolic effects in complex I deficiency.

The objective of this study was to investigate clinical, biochemical, and genetic features in 7 probands (a total of 11 patients) with nicotine-amide adenine dinucleotide (NADH) dehydrogenase (complex I) deficiency. We screened the mitochondrial DNA for mutations and found pathogenic mutations in complex I genes (mitochondrial NADH dehydrogenase subunit (MTND) genes) in three probands. The 10191T>C mutation in MTND3 and the 14487T>C mutation in MTND6 were present in two probands with Leigh's-like and Leigh's syndrome, respectively. Four siblings with a syndrome consisting of encephalomyopathy with hearing impairment, optic nerve atrophy, and cardiac involvement had the 11778G>A mutation in MTND4, previously associated with Leber hereditary optic neuropathy. These findings demonstrate that mutations in MTND genes are relatively frequent in patients with complex I deficiency. Biochemical measurements of respiratory chain function in muscle mitochondria showed that all patients had a moderate decrease of the mitochondrial adenosine triphosphate production rate. Interestingly, the complex I deficiency caused secondary metabolic alterations with decreased oxaloacetate-induced inhibition of succinate dehydrogenase (complex II) and excretion of Krebs cycle intermediates in the urine. Our results thus suggest that altered regulation of metabolism may play an important role in the pathogenesis of complex I deficiency.

Adenosine Triphosphate↗

PubMed2004

The transcription machinery in mammalian mitochondria.

Initiation of transcription at mitochondrial promoters in mammalian cells requires the simultaneous presence of a monomeric mitochondrial RNA polymerase, mitochondrial transcription factor A, and either transcription factor B1 or B2. We here review recent progress in our understanding of how these basal factors cooperate in the initiation and regulation of mitochondrial transcription. We describe the evolutionary origin of individual transcription factors and discuss how these phylogenetic relationships may facilitate a molecular understanding of the mitochondrial transcription machinery.

Animals↗

PubMed2004

The mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells.

Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position -1 to -2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.

Animals↗

PubMed2004

Understanding aging: revealing order out of chaos.

Aging is often described as an extremely complex process affecting all of the vital parameters of an individual. In this article, we review how understanding of aging evolved from the first analyses of population survival to the identification of the molecular mechanisms regulating life span. Abundant evidence implicates mitochondria in aging and we focus on the three main components of the mitochondrial theory of aging: (1) increased reactive oxygen species (ROS) production, (2) mitochondrial DNA (mtDNA) damage accumulation, and (3) progressive respiratory chain dysfunction. Experimental evidence shows a relationship between respiratory chain dysfunction, ROS damage, and aging in most of the model organisms. However, involvement of the mtDNA mutations in the aging process is still debated. We recently created a mutant mouse strain with increased levels of somatic mtDNA mutations causing a progressive respiratory chain deficiency and premature aging. These mice demonstrate the fundamental importance of the accumulation of mtDNA alterations in aging. We present here an integrative model where aging is provoked by a single primary event leading to a variety of effects and secondary causes.

Aging↗

PubMed2004

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.

Adipose Tissue↗

PubMed2004

Mitochondrial transcription factor A regulates mtDNA copy number in mammals.

Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse Tfam gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.

Animals↗

PubMed2004

A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient mouse hearts.

We performed global gene expression analyses in mouse hearts with progressive respiratory chain deficiency and found a metabolic switch at an early disease stage. The tissue-specific mitochondrial transcription factor A (Tfam) knockout mice of this study displayed a progressive heart phenotype with depletion of mtDNA and an accompanying severe decline of respiratory chain enzyme activities along with a decreased mitochondrial ATP production rate. These characteristics were observed after 2 weeks of age and became gradually more severe until the terminal stage occurred at 10-12 weeks of age. Global gene expression analyses with microarrays showed that a metabolic switch occurred early in the progression of cardiac mitochondrial dysfunction. A large number of genes encoding critical enzymes in fatty acid oxidation showed decreased expression whereas several genes encoding glycolytic enzymes showed increased expression. These alterations are consistent with activation of a fetal gene expression program, a well-documented phenomenon in cardiac disease. An increase in mitochondrial mass was not observed until the disease had reached an advanced stage. In contrast to what we have earlier observed in respiratory chain-deficient skeletal muscle, the increased mitochondrial biogenesis in respiratory chain-deficient heart muscle did not increase the overall mitochondrial ATP production rate. The observed switch in metabolism is unlikely to benefit energy homeostasis in the respiratory chain-deficient hearts and therefore likely aggravates the disease. It can thus be concluded that at least some of the secondary gene expression alterations in mitochondrial cardiomyopathy do not compensate but rather directly contribute to heart failure progression.

Animals↗

PubMed2003

Characterization of the mouse genes for mitochondrial transcription factors B1 and B2.

We have recently fully reconstituted the basal human mitochondrial transcription machinery in a pure in vitro system. Surprisingly, we found two different transcription factors (TFB1M and TFB2M) that each interact with mitochondrial RNA polymerase in human mitochondria, whereas there is only one such factor in budding yeast mitochondria. This unexpected finding raised important questions concerning the regulation of mitochondrial transcription in mammals in general and in other metazoans. We have now further analyzed putative homologs to TFB1M and TFB2M in different species. We mapped the mouse homologs, Tfb1m and Tfb2m, by linkage analysis to mouse Chr 17 and Chr 1, respectively. These regions display conserved linkage synteny with human Chr 6 and Chr 1, where TFB1M and TFB2M map. The intron-exon arrangements of Tfb1m and TFB1M and of Tfb2m and TFB2M were identical, and the promoter regions had similar predicted recognition elements for transcriptional factors NRF2 and Sp1. Northern blot analyses showed that Tfb1m and Tfb2m were ubiquitously expressed and had expression patterns that were very similar to the previously reported expression patterns for TFB1M and TFB2M. These findings show that Tfb1m and Tfb2m indeed are orthologs to TFB1M and TFB2M. Bioinformatic analyses indicated that most metazoans have two TFBM genes, since putative homologs to both TFB1M and TFB2M were found in D. melanogaster. Our data thus suggest that a duplication event of the TFBM gene in early metazoan evolution has permitted a more flexible regulation of mtDNA transcription, possibly in response to the complex physiological demands of multicellular organisms.

Animals↗

PubMed2002

Increased mitochondrial mass in mitochondrial myopathy mice.

We have generated an animal model for mitochondrial myopathy by disrupting the gene for mitochondrial transcription factor A (Tfam) in skeletal muscle of the mouse. The knockout animals developed a myopathy with ragged-red muscle fibers, accumulation of abnormally appearing mitochondria, and progressively deteriorating respiratory chain function in skeletal muscle. Enzyme histochemistry, electron micrographs, and citrate synthase activity revealed a substantial increase in mitochondrial mass in skeletal muscle of the myopathy mice. Biochemical assays demonstrated that the increased mitochondrial mass partly compensated for the reduced function of the respiratory chain by maintaining overall ATP production in skeletal muscle. The increased mitochondrial mass thus was induced by the respiratory chain deficiency and may be beneficial by improving the energy homeostasis in the affected tissue. Surprisingly, in vitro experiments to assess muscle function demonstrated that fatigue development did not occur more rapidly in myopathy mice, suggesting that overall ATP production is sufficient. However, there were lower absolute muscle forces in the myopathy mice, especially at low stimulation frequencies. This reduction in muscle force is likely caused by deficient formation of force-generating actin-myosin cross bridges and/or disregulation of Ca(2+) homeostasis. Thus, both biochemical measurements of ATP-production rate and in vitro physiological studies suggest that reduced mitochondrial ATP production might not be as critical for the pathophysiology of mitochondrial myopathy as thought previously.

Animals↗

PubMed2002

Manipulation of mitochondrial DNA gene expression in the mouse.

Mitochondrial dysfunction due to impaired respiratory chain function is increasingly recognized as an important cause of human disease. Mitochondrial disorders are relatively common and have an estimated incidence of 1:10,000 live births. There are more than 100 different point mutations and numerous large rearrangements of mitochondrial DNA (mtDNA; mainly single deletions) that cause human disease. We aimed at obtaining an animal model to study physiological aspects of mtDNA mutation disorders. There are as yet unsolved technical problems associated with transfection of mammalian mitochondria. We therefore choose to manipulate mtDNA expression by targeting of the nuclear gene encoding Tfam. We utilised the cre-loxP recombination system to disrupt Tfam since this system allows manipulation of respiratory chain function in selected mouse tissues. We have found increased cell death or apoptosis induction in both germ line and tissue-specific Tfam knockouts. Our results further suggest that increased production of reactive oxygen species (ROS) is not a prominent feature in cells with impaired mtDNA expression.

Animals↗

PubMed2002

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.

Amino Acid Sequence↗