Marc Vermulst

Mitochondrial point mutations do not limit the natural lifespan of mice

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate. Here, we use a new, highly sensitive assay to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single–base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice.

DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice.

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in many age-related pathologies. Here we identify mtDNA deletions as a driving force behind the premature aging phenotype of mitochondrial mutator mice, and provide evidence for a homology-directed DNA repair mechanism in mitochondria that is directly linked to the formation of mtDNA deletions. In addition, our results demonstrate that the rate at which mtDNA mutations reach phenotypic expression differs markedly among tissues, which may be an important factor in determining the tolerance of a tissue to random mitochondrial mutagenesis.

Overexpression of Catalase Targeted to Mitochondria Attenuates Murine Cardiac Aging

Background— Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%. Methods and Results— We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin–nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%. Conclusions— This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species–related cardiovascular diseases.

Decreased Mitochondrial DNA Mutagenesis in Human Colorectal Cancer

Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C∶G to T∶A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage.

Transcription errors induce proteotoxic stress and shorten cellular lifespan

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimers disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging.

On Mitochondria, Mutations, and Methodology

Whether mutations in mitochondrial DNA (mtDNA) are causal to, or merely correlated with, aging is an area of intense debate. Recently, this debate has intensified with the development of mitochondrial mutator mice. These mice carry an error-prone copy of DNA polymerase gamma (PolgA), the enzyme that replicates the mitochondrial genome. We found that heterozygous carriers of this error-prone allele display a >100-fold elevation in point mutation frequency without manifesting features of premature aging (Vermulst et al., 2007xVermulst, M., Bielas, J.H., Kujoth, G.C., Ladigies, W.C., Rabinovitch, P.S., Prolla, T.A., and Loeb, L.A. Nat. Genet. 2007; 39: 540–543Crossref | PubMed | Scopus (201)See all ReferencesVermulst et al., 2007). These results imply that mtDNA point mutations do not limit the life span of normal mice.Interestingly, homozygous carriers of this allele do display extensive mitochondrial dysfunction and suffer from a premature aging-like syndrome (Trifunovic et al., 2004xTrifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J.N., Rovio, A.T., Bruder, C.E., Bohlooly-Y, M., Gidlof, S., Oldfors, A., Wibom, R. et al. Nature. 2004; 429: 417–423Crossref | PubMed | Scopus (1249)See all ReferencesTrifunovic et al., 2004). We found that this progeroid syndrome correlated best with a large increase in clonally expanded mtDNA mutations (which could be either point mutations or deletions) and a 10- to 90-fold increase in mtDNA deletions (Vermulst et al., 2008xVermulst, M., Wanagat, J., Kujoth, G.C., Bielas, J.H., Rabinovitch, P.S., Prolla, T.A., and Loeb, L.A. Nat. Genet. 2008; 40: 392–394Crossref | PubMed | Scopus (189)See all ReferencesVermulst et al., 2008). MtDNA deletions are associated with a number of age-related pathologies, including muscle wasting and neuronal dysfunction. We reasoned that, if the frequency of deletions in WT animals is sufficient to perturb the function of aging neurons and muscle fibers, a substantial increase in their frequency should have a profound physiological impact. For instance, a much milder increase in mtDNA deletions results in late onset mitochondrial disease in Twinkle-deficient mice. Moreover, mtDNA deletions are suppressed in certain long-lived mice. A recent paper in Cell Metabolism by Edgar et al. (2009)xEdgar, 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 (85)See all ReferencesEdgar et al. (2009) now challenges the role of mtDNA deletions in the progeroid syndrome of mitochondrial mutator mice.In their study, Edgar et al. find that protein supercomplexes in the electron transport chain are unstable in the progeroid mice, and they argue that this type of dysfunction could not have been generated by mtDNA deletions, which they find to be too infrequent. Thus, mtDNA deletions are not responsible for the progeroid syndrome of homozygous mutator mice.We would like to point out that the instability of the supercomplexes is an interesting feature of the mutator mice, but Edgar et al. provide no experiments to show that this instability is indeed causal to their progeroid syndrome. Thus, it seems incorrect to argue that mtDNA deletions are not important for the phenotype of the mice because they have no role in supercomplex instability.Second, the assays used to quantify mtDNA deletions and, thus, discard them as irrelevant are inappropriate for their detection. Previously, we demonstrated that mtDNA deletions in the mutator mice are generated randomly. As a result, the majority of deleted molecules are unique and of a different size. Although these molecules may clonally expand within single cells, this variability precludes their detection on a Southern blot, since no single-sized molecule predominates. Furthermore, random deletions are unlikely to be detected with the PCR assay used. To illustrate, if a thousand molecules are present in a PCR reaction and one hundred of these carry an identical deletion, this deletion is easily detected. However, if the size of each deletion is different, the signal of any deleted molecule will be too faint to detect. Importantly, the control experiments that demonstrate a 0.1% sensitivity of detection are done on clonally expanded mtDNA deletions and are thus inappropriate for the detection of random deletions. Similarly, due to their uneven distribution and location, it is also not expected that these deletions will cause a drop in protein levels, especially when a large group of cells is assayed simultaneously, and the signal of each individual cell is averaged out.Finally, in human pathology, low numbers of deletions are thought to contribute significantly to muscle wasting and neuronal degradation. Thus, a low level of mtDNA deletions in mitochondrial mutator mice does not preclude an impact on animal physiology. If the hypothesis by Edgar et al. holds true though and point mutations are the sole cause of their progeroid syndrome, the mutator mice can no longer be regarded as an accurate model for normal aging, as point mutations do not cause aging in wild-type mice (Vermulst et al., 2007xVermulst, M., Bielas, J.H., Kujoth, G.C., Ladigies, W.C., Rabinovitch, P.S., Prolla, T.A., and Loeb, L.A. Nat. Genet. 2007; 39: 540–543Crossref | PubMed | Scopus (201)See all ReferencesVermulst et al., 2007). Moreover, if a 10- to 90-fold increase in deletions is negligible in terms of pathology, this partially conflicts with the mitochondrial theory of aging. However, we conclude that the methodology used by Edgar et al. is insufficient to resolve this controversy (Kraytsberg et al., 2009xKraytsberg, Y., Simon, D.K., Turnbull, D.M., and Khrapko, K. Aging Cell. 2009; 8: 502–506Crossref | PubMed | Scopus (25)See all ReferencesKraytsberg et al., 2009).

Linking mitochondrial dynamics to mitochondrial protein quality control

Over the last decade, countless discoveries have been made that have expanded our knowledge of mitochondrial biology, and more often than not, these discoveries provided fascinating new insights into the etiology of human disease. For example, advances in mitochondrial genetics exposed the role of mitochondrial mutations in cancer progression, and the discovery of mitophagy highlighted the role of mitochondrial quality control in Parkinsons disease. Additional discoveries underscored the importance of the mTor pathway in aging and disease, and more recently, the mitochondrial unfolded protein response was implicated in the regulation of mammalian lifespan. Some of the most fundamental discoveries though, were made in the context of mitochondrial fusion and fission. The balance between these two opposing forces shapes the mitochondrial population in our cells, and influences mitochondrial function at every level. Here, we highlight the basic biology that underlies mitochondrial fusion and fission, explain how these processes promote human health by solving a problem that is innate to multifarious organelles, and make a novel prediction: that fusion and fission are intimately linked to mitochondrial protein quality control.

The landscape of transcription errors in eukaryotic cells

This paper provides the first comprehensive analysis of the fidelity of transcription in eukaryotic cells. Accurate transcription is required for the faithful expression of genetic information. To understand the molecular mechanisms that control the fidelity of transcription, we used novel sequencing technology to provide the first comprehensive analysis of the fidelity of transcription in eukaryotic cells. Our results demonstrate that transcription errors can occur in any gene, at any location, and affect every aspect of protein structure and function. In addition, we show that multiple proteins safeguard the fidelity of transcription and provide evidence suggesting that errors that evade these layers of RNA quality control profoundly affect the physiology of living cells. Together, these observations demonstrate that there is an inherent limit to the faithful expression of the genome and suggest that the impact of mutagenesis on cellular health and fitness is substantially greater than currently appreciated.

Effects of calorie restriction on the lifespan and healthspan of POLG mitochondrial mutator mice.

Mitochondrial DNA (mtDNA) mutations are thought to have a causative role in age-related pathologies. We have shown previously that mitochondrial mutator mice (PolgD257A/D257A), harboring a proofreading-deficient version of the mtDNA polymerase gamma (POLG), accumulate mtDNA mutations in multiple tissues and display several features of accelerated aging. Calorie restriction (CR) is known to delay the onset of age-related diseases and to extend the lifespan of a variety of species, including rodents. In the current study we investigated the effects of CR on the lifespan and healthspan of mitochondrial mutator mice. Long-term CR did not increase the median or maximum lifespan of PolgD257A/D257A mice. Furthermore, CR did not reduce mtDNA deletions in the heart and muscle, accelerated sarcopenia, testicular atrophy, nor improve the alterations in cardiac parameters that are present in aged mitochondrial mutator mice. Therefore, our findings suggest that accumulation of mtDNA mutations may interfere with the beneficial action of CR in aging retardation.

Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans

SUMMARY Genetic instability of the mitochondrial genome (mtDNA) plays an important role in human aging and disease. Thus far, it has proven difficult to develop successful treatment strategies for diseases that are caused by mtDNA instability. To address this issue, we developed a model of mtDNA disease in the nematode C. elegans, an animal model that can rapidly be screened for genes and biological pathways that reduce mitochondrial pathology. These worms recapitulate all the major hallmarks of mtDNA disease in humans, including increased mtDNA instability, loss of respiration, reduced neuromuscular function, and a shortened lifespan. We found that these phenotypes could be rescued by intervening in numerous biological pathways, including IGF-1/insulin signaling, mitophagy, and the mitochondrial unfolded protein response, suggesting that it may be possible to ameliorate mtDNA disease through multiple molecular mechanisms.