Daniel Edgar

Random point mutations with major effects on protein-coding genes are the driving force behind premature aging in mtDNA mutator mice.

The mtDNA mutator mice have high levels of point mutations and linear deletions of mtDNA causing a progressive respiratory chain dysfunction and a premature aging phenotype. We have now performed molecular analyses to determine the mechanism whereby these mtDNA mutations impair respiratory chain function. We report that mitochondrial protein synthesis is unimpaired in mtDNA mutator mice consistent with the observed minor alterations of steady-state levels of mitochondrial transcripts. These findings refute recent claims that circular mtDNA molecules with large deletions are driving the premature aging phenotype. We further show that the stability of several respiratory chain complexes is severely impaired despite normal synthesis of the corresponding mtDNA-encoded subunits. Our findings reveal a mechanism for induction of aging phenotypes by demonstrating a causative role for amino acid substitutions in mtDNA-encoded respiratory chain subunits, which, in turn, leads to decreased stability of the respiratory chain complexes and respiratory chain deficiency.

Altered dopamine metabolism and increased vulnerability to MPTP in mice with partial deficiency of mitochondrial complex I in dopamine neurons

A variety of observations support the hypothesis that deficiency of complex I [reduced nicotinamide-adenine dinucleotide (NADH):ubiquinone oxidoreductase] of the mitochondrial respiratory chain plays a role in the pathophysiology of Parkinsons disease (PD). However, recent data from a study using mice with knockout of the complex I subunit NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (Ndufs4) has challenged this concept as these mice show degeneration of non-dopamine neurons. In addition, primary dopamine (DA) neurons derived from such mice, reported to lack complex I activity, remain sensitive to toxins believed to act through inhibition of complex I. We tissue-specifically disrupted the Ndufs4 gene in mouse heart and found an apparent severe deficiency of complex I activity in disrupted mitochondria, whereas oxidation of substrates that result in entry of electrons at the level of complex I was only mildly reduced in intact isolated heart mitochondria. Further analyses of detergent-solubilized mitochondria showed the mutant complex I to be unstable but capable of forming supercomplexes with complex I enzyme activity. The loss of Ndufs4 thus causes only a mild complex I deficiency in vivo. We proceeded to disrupt Ndufs4 in midbrain DA neurons and found no overt neurodegeneration, no loss of striatal innervation and no symptoms of Parkinsonism in tissue-specific knockout animals. However, DA homeostasis was abnormal with impaired DA release and increased levels of DA metabolites. Furthermore, Ndufs4 DA neuron knockouts were more vulnerable to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Taken together, these findings lend in vivo support to the hypothesis that complex I deficiency can contribute to the pathophysiology of PD.

Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.

Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts.

An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O(2)) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

Point mutations are causing progeroid phenotypes in the mtDNA mutator mouse.

Editors Note: The December 2009 issue of Cell Metabolism included a Letter to the Editor from Vermulst and colleagues (“On Mitochondria, Mutations, and Methodology”). This Response to that Letter, from Edgar et al., was inadvertently omitted. We present here the Response, with our sincere apologies to all involved for the omission.The mtDNA mutator mice provide genetic evidence linking mtDNA mutations to aging phenotypes. These mice develop high levels of random point mutations within mtDNA (20–30 per genome) and contain ∼25% linear deleted mtDNA molecules. We have demonstrated that the point mutations cause progressive respiratory chain deficiency, which, we propose, leads to premature aging (Edgar et al., 2009xEdgar, D., Shabalina, I., Camara, Y., Wredenberg, A., Calvaruso, M.A., Nijtmans, L., Nedergaard, J., Cannon, B., Larsson, N.G., and Trifunovic, A. Cell Metab. 2009; 10: 131–138Abstract | Full Text | Full Text PDF | PubMed | Scopus (92)See all ReferencesEdgar et al., 2009). This interpretation has been challenged by Loeb and coworkers, who argue that the two types of mtDNA mutations we found at very high levels in mtDNA mutator mice do not cause the phenotype. Instead, they argue that third type of mutation, circular deleted mtDNA molecules, are the culprit. We feel the published data from this group do not justify their conclusions, and we therefore performed the study recently published in Cell Metabolism (Edgar et al., 2009xEdgar, D., Shabalina, I., Camara, Y., Wredenberg, A., Calvaruso, M.A., Nijtmans, L., Nedergaard, J., Cannon, B., Larsson, N.G., and Trifunovic, A. Cell Metab. 2009; 10: 131–138Abstract | Full Text | Full Text PDF | PubMed | Scopus (92)See all ReferencesEdgar et al., 2009).Loeb and coworkers reported a substantial increase of circular mtDNA molecules with deletions by using the random mutation capture (RMC) method. A recent study has given concerns about the performance of the RMC method (Greaves et al., 2009xGreaves, L.C., Beadle, N.E., Taylor, G.A., Commane, D., Mathers, J.C., Khrapko, K., and Turnbull, D.M. Aging Cell. 2009; 8: 566–572Crossref | PubMed | Scopus (20)See all ReferencesGreaves et al., 2009), and the results should therefore be interpreted with caution. We analyzed serial dilutions of DNA from mice that accumulate random multiple mtDNA deletions (Tyynismaa et al., 2005xTyynismaa, H., Mjosund, K.P., Wanrooij, S., Lappalainen, I., Ylikallio, E., Jalanko, A., Spelbrink, J.N., Paetau, A., and Suomalainen, A. Proc. Natl. Acad. Sci. USA. 2005; 102: 17687–17692Crossref | PubMed | Scopus (155)See all ReferencesTyynismaa et al., 2005) and robustly detected deletions if present above 0.1% of total mtDNA in these control samples. In contrast, no such deletions were detected with the same assay in mtDNA mutator samples. Our results are supported by a recent study using an independent method; the single molecule PCR technique (Kraytsberg et al., 2009xKraytsberg, Y., Simon, D.K., Turnbull, D.M., and Khrapko, K. Aging Cell. 2009; 8: 502–506Crossref | PubMed | Scopus (28)See all ReferencesKraytsberg et al., 2009). We find it impossible to conclude that a very rare type of mtDNA mutation should override the importance of the two very abundant forms. Further support for our conclusion comes from the fact that mouse strains with substantially higher levels of single or multiple deleted circular mtDNAs show no features of premature aging (Tyynismaa et al., 2005xTyynismaa, H., Mjosund, K.P., Wanrooij, S., Lappalainen, I., Ylikallio, E., Jalanko, A., Spelbrink, J.N., Paetau, A., and Suomalainen, A. Proc. Natl. Acad. Sci. USA. 2005; 102: 17687–17692Crossref | PubMed | Scopus (155)See all ReferencesTyynismaa et al., 2005).Loeb and coworkers also mistakenly state that we report that “supercomplexes in the electron transport chain are unstable.” We report no data on supercomplexes in our paper; however, we do report that the respiratory chain complexes are unstable, consistent with the conclusion that point mutations of mtDNA lead to the synthesis of respiratory chain subunits with amino acid substitutions that impair complex stability (Edgar et al., 2009xEdgar, D., Shabalina, I., Camara, Y., Wredenberg, A., Calvaruso, M.A., Nijtmans, L., Nedergaard, J., Cannon, B., Larsson, N.G., and Trifunovic, A. Cell Metab. 2009; 10: 131–138Abstract | Full Text | Full Text PDF | PubMed | Scopus (92)See all ReferencesEdgar et al., 2009). In summary, we feel the conclusions of our paper are well justified and that there is no convincing experimental evidence for a causative role for circular deleted mtDNA molecules in creating the progeroid syndrome of mtDNA mutator mice.

Improved health-span and lifespan in mtDNA mutator mice treated with the mitochondrially targeted antioxidant SkQ1

MtDNA mutator mice exhibit marked features of premature aging. We find that these mice treated from age of ≈100 days with the mitochondria-targeted antioxidant SkQ1 showed a delayed appearance of traits of aging such as kyphosis, alopecia, lowering of body temperature, body weight loss, as well as ameliorated heart, kidney and liver pathologies. These effects of SkQ1 are suggested to be related to an alleviation of the effects of an enhanced reactive oxygen species (ROS) level in mtDNA mutator mice: the increased mitochondrial ROS released due to mitochondrial mutations probably interact with polyunsaturated fatty acids in cardiolipin, releasing malondialdehyde and 4-hydroxynonenal that form protein adducts and thus diminishes mitochondrial functions. SkQ1 counteracts this as it scavenges mitochondrial ROS. As the results, the normal mitochondrial ultrastructure is preserved in liver and heart; the phosphorylation capacity of skeletal muscle mitochondria as well as the thermogenic capacity of brown adipose tissue is also improved. The SkQ1-treated mice live significantly longer (335 versus 290 days). These data may be relevant in relation to treatment of mitochondrial diseases particularly and the process of aging in general.

Leydig cell steroidogenesis unexpectedly escapes mitochondrial dysfunction in prematurely aging mice

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading‐deficient form of mtDNA polymerase γ, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels. Although mtDNA mutator mice showed a dramatic reduction in testicle weight (only 15% remaining) and similar decreases in number of spermatozoa, testosterone levels in mtDNA mutator mice were unexpectedly fully unchanged. Leydig cell did not escape mitochondrial damage (only 20% of complex I and complex IV remaining) and did show high levels of reactive oxygen species (ROS) production (>5‐fold increased), and permeabilized cells demonstrated absence of normal mitochondrial function. Nevertheless, within intact cells, mitochondrial membrane potential remained high, and testosterone production was maintained. This implies development of a compensatory mechanism. A rescuing mechanism involving electrons from the pentose phosphate pathway transferred via a 3‐fold up‐regulated cytochrome b5 to cytochrome c, allowing for mitochondrial energization, is suggested. Thus, the Leydig cells escape mitochondrial dysfunction via a unique rescue pathway. Such a pathway, bypassing respiratory chain dysfunction, may be of relevance with regard to mitochondrial disease therapy and to managing ageing in general.—Shabalina, I. G., Landreh, L., Edgar, D., Hou, M., Gibanova, N., Atanassova, N., Petrovic, N., Hultenby, K., Söder, O., Nedergaard, J. Svechnikov, K. Leydig cell steroidogenesis unexpectedly escapes mitochondrial dysfunction in prematurely aging mice. FASEB J. 29, 3274‐3286 (2015). www.fasebj.org

DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity

The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat.The transcription factor DAF-16/FOXO is a downstream effector of insulin/insulin-like growth factor signaling and plays an important role in stress resistance and longevity. Here, the authors show that DAF-16/FOXO can form a complex with HLH-30/TFEB to synergistically regulate transcription of target genes in response to certain stress stimuli.

Mitochondria: The Dark Side

Mitochondria are small organelle found in almost every cell of an organism (Fig. 1). They are the size of bacteria and form a dynamic network that is constantly changing. A typical eukaryotic cell contains about 2,000 mitochondria, which occupy roughly one fifth of its total volume [1]. Mitochondria {fg115-01} are considered to be the power generators of the cell, converting oxygen and nutrients into adenosine triphosphate (ATP), through a process of oxidative phosphorylation. Although mitochondria are involved in various other important cellular processes such as the beta-oxidation of fatty acids and the biosynthesis of pyrimidines, amino acids, nucleotides, phospholipids, and heme, ATP synthesis is likely to be the most important function of these organelles. Without mitochondria, higher animals would likely not exist because their cells would not be able to obtain enough energy. In fact, mitochondria enable cells to produce 15 times more ATP than they could otherwise. Mitochondrial energy production is a foundation for health and well being. It is necessary for physical strength, stamina, and consciousness [1]. Even subtle insufficiency in mitochondrial function can cause weakness, fatigue, and cognitive difficulties [2]. Furthermore, chemicals which strongly interfere with mitochondrial function are known to be potent poisons.