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The mitochondrial theory of aging: Insight from transgenic and knockout mouse models
Experimental Gerontology 44 (2009) 256–260
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Mini review
The mitochondrial theory of aging: Insight from transgenic and knockout mouse models Youngmok C. Jang a,b, Holly Van Remmen a,b,c,* a
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MSC 7762, San Antonio, TX 78229-3900, USA Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA c South Texas Veterans Health Care System, San Antonio, TX 78229-4404, USA b
a r t i c l e
i n f o
Article history: Received 13 October 2008 Received in revised form 17 December 2008 Accepted 31 December 2008 Available online 12 January 2009 Keywords: Mitochondria Mitochondrial theory of aging Oxidative stress Aging Transgenic and knockout mouse models
a b s t r a c t A substantial body of evidence has accumulated over the past 35 years in support of a role for oxidative damage to the mitochondrial respiratory chain and mitochondrial DNA in the determination of mammalian lifespan. The goal of this review is to provide a concise summary of recent studies using transgenic and knockout mouse models with altered expression of mitochondrial antioxidant enzymes (MnSOD (Sod2Tg and Sod2+/ ), thioredoxin 2 (Trx2+/ ), mitochondrial targeted catalase (mCAT) and mutant mice models that have been genetically manipulated to increase mitochondrial deletions or mutations (PolcD257A/D257A mutant mice) to examine the role of mitochondrial oxidative stress in aging. The majority of studies using these strategies do not support a clear role for mitochondrial oxidative stress or a vicious cycle of oxidative damage in the determination of lifespan in mice and furthermore do not support the free radical theory of aging. However, several key questions remain to be addressed and clearly more studies are required to fully understand the role of mitochondria in age-related disease and aging. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The free radical or oxidative stress theory of aging proposed by Denham Harman in 1956 states that the age-related loss of physiological function is due to the progressive accumulation of oxidative damage and that this ultimately determines the lifespan of an organism ( Harman, 1956). Shortly after the discovery of the mitochondrial genome (mtDNA), Harman modified his original theory to incorporate the contribution/role of mitochondria in oxidative stress and proposed the mitochondrial theory of aging (Harman, 1972). In subsequent years, the mitochondrial theory of aging was further refined and developed by Miquel and colleagues (Miquel et al., 1980), who suggested that the accumulation of somatic mutations in the mtDNA induced by oxidative stress is the major contributor of aging and age-related degenerative diseases. Currently, the mitochondrial theory of aging can be summarized as follows: reactive oxygen species (ROS) emanating from the mitochondrial respiratory chain damages macromolecules, especially mtDNA. As a result, an accumulation of mtDNA mutations leads to production of defective mitochondrial respiration, further increasing ROS generation and oxidative damage. This so-called
* Corresponding author. Address: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MSC 7762, San Antonio, TX 78229-3900, USA. Tel.: +1 210 562 6141; fax: +1 210 562 6110. E-mail address: [email protected] (H.V. Remmen). 0531-5565/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2008.12.006
‘vicious cycle’ of ROS generation and concomitant oxidative damage is proposed as the ultimate determinant of mammalian lifespan. Over the past 35 years, a significant amount of correlative evidence has been accumulated in support of this theory, including studies that demonstrate an increase in ROS generation with age, reduced mitochondrial function, and increased oxidative damage to mtDNA (Van Remmen and Richardson, 2001; Boveris and Navarro, 2008; Figueiredo et al., 2008). However, in recent years, studies that have used transgenic/knockout animal models to more directly test the theory have produced inconsistent results, and some have strongly challenged the core principle of the mitochondrial theory of aging. In this review, we will provide a concise summary of the recent literature, narrowing the focus to studies utilizing transgenic/knockout mouse models, with particular emphasis on models with altered expression of mitochondrial antioxidant enzymes and mutant mice models with increased rates of mtDNA mutagenesis. 2. Mitochondrial oxidative damage and mitochondrial dysfunction with age The core principle of the mitochondrial theory of aging is based on the fact that mitochondrial respiratory chain, mainly through complex I and complex III, is the major source of superoxide anion (O2 ) (Muller et al., 2004; Andreyev et al., 2005; Jezek and Hlavata, 2005). Although mitochondria contain an intricate antioxidant
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defense system that rapidly scavenges ROS to a non-toxic form, the balance between ROS generation and antioxidant defense is believe to be disrupted in aging and age-related diseases, resulting in cumulative oxidative damage of pivotal biological macromolecules. An age-related increase in oxidative damage to lipids, proteins, and DNA has been demonstrated by several investigators in a variety of tissues in humans and several experimental animal models (Muller et al., 2007). Among the various oxidative insults to mitochondrial components, oxidative damage to mtDNA has been the major focus of the mitochondrial theory of aging. Unlike nuclear DNA, mtDNA lacks extensive protection by histones and only possesses a limited capacity for a DNA repair mechanism (Yakes and Van Houten, 1997; Croteau et al., 1999). Also, it is plausible to assume that mtDNA is highly susceptible to oxidative stress because of its close proximity to the main source of ROS (Ames et al., 1993). Oxidative damage to DNA is to known to cause modification to purine and pyrimidine bases, single and double-stranded breaks, and cross-linking to other molecules (Croteau et al., 1999). Indeed, it has been well documented that mtDNA accumulates alterations (deletions, rearrangements, or point mutations) with age in various animal model systems as well as in humans (Ames et al., 1993). Oxidative damage to mtDNA has been suggested as the major cause of instability and mutation of the mitochondrial genome (Beckman and Ames, 1998). Moreover, damage to mtDNA can be particularly critical to overall cellular function because instability of mtDNA can be propagated as mitochondria and cells divide, amplifying the damage (Beckman and Ames, 1998). Results from studies using long-lived mice and experimental manipulations that extend lifespan, such as calorie restriction, have provided a strong correlation between oxidative damage to the mitochondria and lifespan (Barja and Herrero, 2000; Trinei et al., 2002; Barja, 2004). Although many of these studies mentioned above support the mitochondrial theory of aging, the data are correlative and do not prove the theory directly. A simple approach to directly test the mitochondrial theory of aging is to manipulate the amount of oxidative damage to the mitochondria by increasing the expression or by deleting a key antioxidant enzyme in order to either reduce or increase the oxidative insults to mitochondria and determine whether this manipulation alters lifespan. This approach has been put forward by several investigators using invertebrate animal models. For example, Longo et al. demonstrated that in Saccharomyces cerevisiae, deletion of the key antioxidant enzyme, manganese superoxide dismutase (MnSOD, Sod2), which is located in mitochondrial matrix, dramatically accelerates chronological aging and overexpression unambiguously increases the organism’s lifespan (Longo et al., 1996). Likewise, Tower’s group reported a similar extension of lifespan by the overexpression of MnSOD in adult Drosophila melanogaster (Sun et al., 2002). However, as outlined below (Table 1), mammalian transgenic/knockout models with alterations in key mitochondrial antioxidant enzymes have produced mixed results and do not fully support the mitochondrial theory of aging. 2.1. MnSOD knockout and transgenic mice MnSOD (Sod2) is the main antioxidant enzyme that scavenges superoxide in the mitochondrial matrix. MnSOD is considered the first line of defense against superoxide generated towards the mitochondrial matrix by mitochondrial electron transport chain. Complete ablation of MnSOD causes dilated cardiomyopathy and neurodegeneration leading to early postnatal death. These mice exhibit severe oxidative damage to mitochondria and also are extremely sensitive to hyperoxia (Li et al., 1995; Lebovitz et al., 1996). Although the studies using Sod2 / mice provide critical evidence
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that mitochondrial superoxide toxicity can have a profound effect on cellular function and lifespan, this evidence does not necessarily prove that mitochondrial superoxide per se accelerates aging. However, if as the mitochondrial theory of aging predicts, elevated superoxide levels and increased oxidative stress to mitochondria are in fact a primary cause of aging, a modest reduction in MnSOD activity also should have a ‘dose’ response in the aging process. In order to address this idea, our laboratory has extensively studied the heterozygous knockout of Sod2 (Sod2+/ mice), which have approximately 50% reduction in MnSOD activity in all of the tissues we analyzed. Sod2+/ mice do display some of the phenotypes that are indicative of decreased mitochondrial function (i.e., decreased aconitase activity, decreased complex I activity, decreased ATP generation) and increased oxidative damage such as 30–80% increase in both nuclear and mitochondrial DNA oxidation (8-oxodG). However, the lifespan of Sod2+/ mice in a pure C57B6/J background, did not show a statistical difference from that of wild-type mice (n = 70 per group) (Van Remmen et al., 1999; 2003). Although, some oxidative stress markers were elevated (e.g., 8-oxo-dG), it is important to keep in mind that not all oxidative stress markers were elevated in Sod2+/ mice. For example, F2-isoprostanes (measured in plasma as an indicator of overall oxidative damage), the level of protein carbonyls (protein oxidation marker), and the rate of H2O2 production from isolated mitochondria were all unchanged in Sod2+/ mice compared at different ages to age-matched, wildtype mice (Mansouri et al., 2006). In addition, biomarkers of aging, such as cataract formation, defective immune responses, and formation of glycoxidation products, were unaltered, whereas the incidence of cancer was higher in Sod2+/ mice (Van Remmen et al., 2003). Despite the fact that a 50% reduction in MnSOD is not sufficient to limit lifespan, the possibility remains that a further reduction in MnSOD activity initiated after development might in fact increase mitochondrial oxidative damage and reduce lifespan. To address this possibility, similar studies could be conducted using a conditional knockout approach in which MnSOD activity is ablated post-development. Although reducing MnSOD activity in Sod2 knockout mice tests whether superoxide toxicity in mitochondria is limiting to lifespan, the alternative approach is to test the whether an increase in mitochondrial MnSOD activity can reduce in vivo oxidative damage by superoxide and to determine whether increased protection against oxidative stress can extend lifespan. As mentioned earlier, overexpression of MnSOD in Drosophila was shown to have a beneficial effect, extending lifespan, and supporting the mitochondrial theory of aging (Sun et al., 2002). Moreover, in some genetically manipulated mouse models with extended lifespan, investigators have reported an increase in MnSOD activity and enhanced response against oxidative stress (Yamamoto et al., 2005; Taguchi et al., 2007). Thus, we and others have tested whether the overexpression of MnSOD activity in all tissues throughout the lifespan has any effect on oxidative damage and longevity in a mouse model. Recently, Hu et al. reported that approximately 3-fold overexpression of human SOD2 gene, driven by b-actin promoter, increased mean lifespan by 4% and that 18% of the SOD2 transgenic mice lived longer than 40 months while the longest surviving wild-type mouse was 36 months of age (n = 24–30) (Hu et al., 2007). In contrast, in our laboratory, an Sod2 transgenic mouse line that overexpresses MnSOD in all tissues under control of the endogenous mouse Sod2 promoter to a similar level (2- to 2.5-fold increase in MnSOD activity) failed to show increased lifespan even when the transgenic mice generated less superoxide (Jang et al., submitted for publication). When the lifespan of our colony is compared to the study by Hu et al., the maximum lifespan of MnSOD transgenic mice is similar (1245 days for our colony vs. 1290 days for Hu et al.); however, the lifespan of the wild-type control is notably different (1260 days for our colony vs. 1095 days in Hu et al.). In
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Table 1 Phenotype of mitochondrial transgenic/knockout mice. Gene name
Main function
Mn superoxide dismutase Sod2 /
Scavenger of superoxide
Sod2+/
Scavenger of superoxide
Sod2 transgenic
Scavenger of superoxide
Catalase Mitochondrial targeted CAT transgenic
Scavenger of H2O2
Thioredoxin 2 Trx2+/
Mitochondrial polymerase gamma PolcD257A/D257A
Reduce substrates to methionine sulfoxide and peroxiredoxins
Phenotype
Lifespan
Support mitochondria theory of aging?
Cardiomyopathy Hemolytic anemia Increased incidence of cancer Genomic instability Spongiform encephalopathy Optic neuropathy Movement disorders Neurodegeneration High oxidative stress
0–4 weeks
Yes
Partial increase in oxidative stress increased incidence of cancer vascular dysfunction Increased apoptosis
Normal
No change in mitochondrial oxidative damage Reduced lipid peroxidation with age
Normal
Reduced mitochondrial oxidative stress Reduced H2O2 Reduced mtDNA mutation Delayed cardiac pathology Delayed cataract formation Reduced tumor burden Reduced cardiac lesions
20% Increase
Yes
Decreased ATP, ETC activity Increase in H2O2 Increased mitochondrial oxidative damage
Preliminary results suggest no change
No
Weight loss, hair graying, hearing loss, infertility, kyphosis, decreased bone density, anemia, muscle loss Increased in mtDNA deletion/point mutation Decreased ETC activity No change in ROS/oxidative damage Increase in mtDNA point mutation No change in mtDNA deletion
70% Decrease
Yes
Normal
No
No
No
Proofreading newly synthesized mtDNA
PolcD257A/+
addition, the mean lifespan extension of the Sod2 transgenic mice is not statistically different (4% vs. 2%). To reliably conclude that overexpression of MnSOD can prolong survival in mice; both the mean and the maximum lifespan should increase. However, when using a large sample size (n = 47–50), we did not see the any changes in either the mean or the maximum lifespan. It is also possible that the disparity between the survival curves of two studies is due to the differences in the genetic background used and the regulatory element used to drive the transgene expression. In all, ectopically increasing the MnSOD activity throughout the lifespan in mice to augment mitochondrial antioxidant protection does not unequivocally result in increased lifespan. 2.2. Mitochondrial targeted catalase transgenic mice Transgenic mice overexpressing catalase targeted to peroxisomes, nuclei, or mitochondria were generated by Schriner et al. (2005). Although peroxisome and nuclear overexpression showed a trend toward increased lifespan, only the mitochondrial targeted construct provided the maximal benefit, increasing both median and maximal lifespan by 20%. Mitochondrial hydrogen peroxide (H2O2) production and oxidative inactivation of aconitase were reduced in isolated cardiac mitochondria of the mitochondrial targeted catalase transgenic mice (mCAT mice) (Schriner et al., 2005). In addition, DNA oxidation and levels of mitochondrial deletions were also reduced in the skeletal muscle of these mice.
Furthermore, cardiac pathology and cataract development were also delayed in the mitochondria targeted catalase transgenic mice (Schriner et al., 2005). More recently, the same group of authors further analyzed the end-of-life pathology and concluded that mCAT mice had reduced age-associated pathologies such as malignant nonhematopoietic tumors and cardiac lesions (Treuting et al., 2008) and thus not only do mice expressing mitochondrial catalase have an extension in lifespan , but these mice also have an increase in ‘healthspan’. Data from the mCAT mice undoubtedly support the mitochondrial theory of aging in that a reduction in mitochondrial oxidative stress and damage in these mice is associated with an increase in lifespan; however, the authors pointed out in the original study that when the transgenic mice were backcrossed to a nearly pure C57BL6 background, the lifespan extension phenotype appears to be diminished. Before we make conclusions about this model, genetic background issues must be resolved. In comparison, mice that overexpress a modest level of catalase in the peroxisome (the compartment in which catalase is normally localized) did not show an extension of either mean or the maximum lifespan. 2.3. Thioredoxin 2 knockout mice Thioredoxin (Trx) is a 12 kDa protein with two redox-sensitive cysteine-thiols in its active site. Thioredoxin is kept in the reduced state by the flavoenzyme thioredoxin reductase, in a NADPHdependent reaction. The thioredoxins play an essential role in the
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antioxidant defense mechanism (Yoshida et al., 2003), can scavenge peroxides via peroxiredoxins, and reduce disulfide bonds and methionine sulfoxide either directly or through the action of other oxidoreductases. The mitochondrial form of thioredoxin, thioredoxin 2 (Trx2) has been implicated as a key player in maintaining the mitochondrial redox environment and protecting against mitochondrial oxidative damage (Yoshida et al., 2003). The homozygous knockout of the Trx2 in mice is embryonic lethal. Pérez et al. studied the effect of partial deficiency of Trx2 in mitochondrial function and oxidative stress (Pérez et al., 2008). They reported that mice with 50% reduction in Trx2 activity (Trx2+/ mice) exhibit a decrease in mitochondrial function as measured by the decrease in ATP production, decline in ETC activity, and increased generation of mitochondrial H2O2. In addition, compared to their age-matched counterparts, Trx2+/ mice have higher oxidative damage to protein (protein carbonyl), lipid (F2-isoprostane), and DNA (8-oxo-dG). Furthermore, Trx2+/ mice are more sensitive to diquat-induced oxidative damage and apoptosis (Pérez et al., 2008). Because of the increase in mitochondrial oxidative stress and reduced mitochondrial function, the Trx2+/ mice would be predicted by the mitochondrial theory of aging to have a reduced lifespan. However, preliminary results from a pilot study using Trx2+/ mice on a mixed genetic background suggest that the lifespan is not reduced in these mice (Pérez et al., 2009). Interestingly, overexpression of thioredoxin 1 (the cytosolic form) has been shown to increase lifespan in mice (Mitsui et al., 2002). 3. Mitochondrial DNA instability: accumulation of mtDNA mutation with age Because mitochondrial ROS is undoubtedly capable of inducing mtDNA instability as observed in aging tissue, a majority of studies that have attempted to test the mitochondrial theory of aging have focused on mtDNA mutations. Recently, mouse models of mtDNA instability have provided direct evidence of its role in the aging process. Using a gene ‘knock-in’ strategy, two independent investigators have generated a mouse model that has a point mutation in a catalytic subunit of mtDNA polymerase (POLG) that is responsible for proofreading newly synthesized mtDNA (Trifunovic et al., 2004; Kujoth et al., 2005). Mice homozygous for this mutant mtDNA polymerase (PolcD257A/D257A) exhibit a progressive accumulation of mtDNA point mutations and show a 70% reduction in lifespan compared to wild-type littermates. In addition, these mice show phenotype and pathology that are similar to aging such as weight loss, hearing loss, hair graying, infertility, kyphosis, decreased bone density, anemia, and muscle loss (Kujoth et al., 2005). Yet, as Richard Miller has argued, ‘accelerated aging phenotypes’ seen in these ‘mutator mice’ are similar to phenotypes of aging humans but perhaps not the phenotypes necessarily observed in normal aging, inbred mice (Miller et al., 2005). Nevertheless, the most surprising findings of the report by Kujoth et al. is that, in spite of the large mutation load and decrease in ETC activity, mitochondrial ROS production and the level of oxidative damage (lipid peroxidation and DNA oxidation) did not show any difference compared to their wild-type counterparts (Kujoth et al., 2005). A simple explanation of these observations is that the dramatic aging phenotype and pathology seen in these mice are not directly mediated by mitochondrial ROS production. An alternative interpretation is that massive mutations throughout the mitochondrial genome may prevent the normal assembly of the respiratory chain thus eliminating the site of ROS generation. For example, if the loss of proofreading activity causes a large scale deletion of the mitochondrial genome, this mutation could eliminate the genes for the subunit of ETC (possibly complex I and complex III). More importantly, if the tRNA gene is deleted, the
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synthesis of all 13 mtDNA encoded proteins can be compromised. In this scenario, a significant decrease in ETC activity will occur and no ROS can be generated. Recently, Vermulst et al., (2007) used a new and more sensitive methodology (Random Mutation Capture Assay) to assess mtDNA point mutations, demonstrating that heterozygous mice, which have only one mutant allele (Polc+/D257A), do not exhibit any premature aging phenotype despite having an approximately 500-fold increase in mtDNA point mutations. Moreover, Polc+/D257A mice have a normal lifespan similar to wild-type littermates, disproving the role of mtDNA point mutations as a lifespan-limiting factor. These authors have followed up their study by modifying the random mutation capture assay to detect mtDNA deletions. Intriguingly, they reported that mtDNA deletion was significantly elevated in heart and brain tissue with age. However, unlike the point mutation, only the homogenous mutant PolcD257A/D257A mice had an accelerated rate of deletion mutations. In addition, they demonstrated that the rate at which mtDNA mutations reached the phenotypical expression varies greatly among different tissues. They concluded that the mtDNA deletion is the driving force behind the premature aging phenotype of the Polc mutant mice (Vermulst et al., 2008). Conversely, a previous report by Tyynismaa et al. showed that the transgenic mice with a mutated allele of mitochondrial helicase TWINKLE did not exhibit any aging phenotype despite having elevated levels of mtDNA deletions (Tyynismaa et al., 2005). However, it is noteworthy that this discrepancy in phenotype may be due to differences in the gene overexpression strategy. In Polc mutant mice, a knock-in technique was used to target the transgene in a selected locus, whereas in the TWINKLE study, overexpression was achieved by random integration of mutant transgene. These observations, along with those showing the lack of increased oxidative stress in the PolcD257A/D257A mutant mice, raise doubts regarding the direct connection between mtDNA mutation and ROS production and the vicious cycles proposed as part of the mitochondrial theory of aging. However, before we make any conclusions, there are still many unanswered questions regarding the role of mtDNA instability in the aging process. For example, in normal aging mice, is the age-related increase in mtDNA mutations due to infidelity of Polc or due to oxidative damage to mtDNA? All in all, the mutator mice provide a model with which to examine mechanistic features of the aging process that relate to mitochondrial genome integrity and the response to mtDNA mutations. 4. Conclusions and future directions Recent studies using the transgenic/knockout strategies have challenged the core principles of the mitochondria theory of aging as well as its parent theory, the free radical or oxidative stress theory of aging. Based on the studies to date, most studies do not support or remain inconclusive on whether mitochondrial dysfunction and oxidative stress determine lifespan. However, several questions still need to be addressed. First, most of the studies using the knockout and transgenic approach do not take into account the effect of oxidative stress on development. It is possible that the genetic manipulations are affecting development and not necessarily the aging process. For example, in the study by Trifunovic et al., the mutations are uniform among tissues, suggesting that much of the mutation accumulation occurs during embryonic or fetal development (Trifunovic et al., 2004). This issue can be resolved in future studies using the inducible knockout/knockin approach. Secondly, our understanding of the mitochondrial biology is far from complete. Inter-organelle interaction, mitochondrial biogenesis, fission and fusion, and ‘mitophagy’-autophagic removal of damaged mitochondria during the aging process are only some
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of the examples that have not been thoroughly investigated. Interestingly, in S. cerevisiae and P. anserina, the deletion of a protein that promotes mitochondrial fission (dynamin related protein 1dnm1p) extended lifespan without lowering fitness or reproduction (Scheckhuber et al., 2007). In addition, the increase in ROS generation in normal cells was delayed in the cells that lack dnm1p (Scheckhuber et al., 2007). It remains to be seen if any of the proteins involved in turnover and maintenance of mitochondria affect the lifespan of higher model organisms. Finally, in genetic mouse studies, different strains of inbred mice might have different phenotypes in response to certain genetic manipulations. In fact, using the quantitative trait locus mapping (QTL), Rikke et al. showed different strains of mice show a significant genetic variation in weight loss in response to a 40% calorie-restricted (CR) diet (Rikke et al., 2006). Therefore, it could be misleading to state that a life extension or reduction effect seen in one strain proves or disproves the mitochondrial theory of aging. In summary, the majority of the initial pioneering studies in mice to test the mitochondrial theory of aging have yielded results that either do not support the theory or remain inconclusive. An exception is a single study involving the overexpression of catalase in mitochondria, but the findings may not be applicable to all genetic backgrounds. Clearly, more work is required to clarify whether mitochondrial dysfunction determines aging and the lifespan of an organism. In addition, future studies should take into account phenotypic changes as an indication of healthspan as well as changes in lifespan. References Ames, B.N., Shigenaga, M.K., et al., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. 90 (17), 7915–7922. Andreyev, A.Y., Kushnareva, Y.E., et al., 2005. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Moscow) 70 (2), 200–214. Barja, G., 2004. Aging in vertebrates, and the effect of caloric restriction: a mitochondrial free radical production-DNA damage mechanism?. Biol. Rev. Camb. Philos. Soc. 79 (2), 235–251. Barja, G., Herrero, A., 2000. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals. FASEB J. 14 (2), 312–318. Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 78 (2), 547–581. Boveris, A., Navarro, A., 2008. Brain mitochondrial dysfunction in aging. IUBMB Life 60 (5), 308–314. Croteau, D.L., Stierum, R.H., et al., 1999. Mitochondrial DNA repair pathways. Mutat. Res. 434 (3), 137–148. Figueiredo, P.A., Mota, M.P., et al., 2008. The role of mitochondria in aging of skeletal muscle. Biogerontology 9 (2), 67–84. Harman, D., 1956. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300. Harman, D., 1972. The biologic clock: the mitochondria?. J. Am. Geriatr. Soc. 20 (4), 145–147. Hu, D., Cao, P., et al., 2007. Hippocampal long-term potentiation, memory, and longevity in mice that overexpress mitochondrial superoxide dismutase. Neurobiol. Learn. Mem. 87 (3), 372–384. Jang, Y.C., Pérez, V.I., Song, W., Lustgarten, M.S., Salmon, A.B., Mele, J., Qi, W., Liu, Y., Liang, H., Chaudhuri, A., Ikeno, Y., Epstein, C.J., Van Remmen, H., Richardson, A., submitted for publication. Overexpression of Mn Superoxide Dismutase (MnSOD) protects against oxidative stress but does not increase lifespan in mice. J. Gerontol. Jezek, P.L., Hlavata, L., 2005. Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int. J. Biochem. Cell. Biol. 37 (12), 2478–2503.
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Mini review
The mitochondrial theory of aging: Insight from transgenic and knockout mouse models Youngmok C. Jang a,b, Holly Van Remmen a,b,c,* a
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MSC 7762, San Antonio, TX 78229-3900, USA Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA c South Texas Veterans Health Care System, San Antonio, TX 78229-4404, USA b
a r t i c l e
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Article history: Received 13 October 2008 Received in revised form 17 December 2008 Accepted 31 December 2008 Available online 12 January 2009 Keywords: Mitochondria Mitochondrial theory of aging Oxidative stress Aging Transgenic and knockout mouse models
a b s t r a c t A substantial body of evidence has accumulated over the past 35 years in support of a role for oxidative damage to the mitochondrial respiratory chain and mitochondrial DNA in the determination of mammalian lifespan. The goal of this review is to provide a concise summary of recent studies using transgenic and knockout mouse models with altered expression of mitochondrial antioxidant enzymes (MnSOD (Sod2Tg and Sod2+/ ), thioredoxin 2 (Trx2+/ ), mitochondrial targeted catalase (mCAT) and mutant mice models that have been genetically manipulated to increase mitochondrial deletions or mutations (PolcD257A/D257A mutant mice) to examine the role of mitochondrial oxidative stress in aging. The majority of studies using these strategies do not support a clear role for mitochondrial oxidative stress or a vicious cycle of oxidative damage in the determination of lifespan in mice and furthermore do not support the free radical theory of aging. However, several key questions remain to be addressed and clearly more studies are required to fully understand the role of mitochondria in age-related disease and aging. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The free radical or oxidative stress theory of aging proposed by Denham Harman in 1956 states that the age-related loss of physiological function is due to the progressive accumulation of oxidative damage and that this ultimately determines the lifespan of an organism ( Harman, 1956). Shortly after the discovery of the mitochondrial genome (mtDNA), Harman modified his original theory to incorporate the contribution/role of mitochondria in oxidative stress and proposed the mitochondrial theory of aging (Harman, 1972). In subsequent years, the mitochondrial theory of aging was further refined and developed by Miquel and colleagues (Miquel et al., 1980), who suggested that the accumulation of somatic mutations in the mtDNA induced by oxidative stress is the major contributor of aging and age-related degenerative diseases. Currently, the mitochondrial theory of aging can be summarized as follows: reactive oxygen species (ROS) emanating from the mitochondrial respiratory chain damages macromolecules, especially mtDNA. As a result, an accumulation of mtDNA mutations leads to production of defective mitochondrial respiration, further increasing ROS generation and oxidative damage. This so-called
* Corresponding author. Address: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MSC 7762, San Antonio, TX 78229-3900, USA. Tel.: +1 210 562 6141; fax: +1 210 562 6110. E-mail address: [email protected] (H.V. Remmen). 0531-5565/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2008.12.006
‘vicious cycle’ of ROS generation and concomitant oxidative damage is proposed as the ultimate determinant of mammalian lifespan. Over the past 35 years, a significant amount of correlative evidence has been accumulated in support of this theory, including studies that demonstrate an increase in ROS generation with age, reduced mitochondrial function, and increased oxidative damage to mtDNA (Van Remmen and Richardson, 2001; Boveris and Navarro, 2008; Figueiredo et al., 2008). However, in recent years, studies that have used transgenic/knockout animal models to more directly test the theory have produced inconsistent results, and some have strongly challenged the core principle of the mitochondrial theory of aging. In this review, we will provide a concise summary of the recent literature, narrowing the focus to studies utilizing transgenic/knockout mouse models, with particular emphasis on models with altered expression of mitochondrial antioxidant enzymes and mutant mice models with increased rates of mtDNA mutagenesis. 2. Mitochondrial oxidative damage and mitochondrial dysfunction with age The core principle of the mitochondrial theory of aging is based on the fact that mitochondrial respiratory chain, mainly through complex I and complex III, is the major source of superoxide anion (O2 ) (Muller et al., 2004; Andreyev et al., 2005; Jezek and Hlavata, 2005). Although mitochondria contain an intricate antioxidant
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defense system that rapidly scavenges ROS to a non-toxic form, the balance between ROS generation and antioxidant defense is believe to be disrupted in aging and age-related diseases, resulting in cumulative oxidative damage of pivotal biological macromolecules. An age-related increase in oxidative damage to lipids, proteins, and DNA has been demonstrated by several investigators in a variety of tissues in humans and several experimental animal models (Muller et al., 2007). Among the various oxidative insults to mitochondrial components, oxidative damage to mtDNA has been the major focus of the mitochondrial theory of aging. Unlike nuclear DNA, mtDNA lacks extensive protection by histones and only possesses a limited capacity for a DNA repair mechanism (Yakes and Van Houten, 1997; Croteau et al., 1999). Also, it is plausible to assume that mtDNA is highly susceptible to oxidative stress because of its close proximity to the main source of ROS (Ames et al., 1993). Oxidative damage to DNA is to known to cause modification to purine and pyrimidine bases, single and double-stranded breaks, and cross-linking to other molecules (Croteau et al., 1999). Indeed, it has been well documented that mtDNA accumulates alterations (deletions, rearrangements, or point mutations) with age in various animal model systems as well as in humans (Ames et al., 1993). Oxidative damage to mtDNA has been suggested as the major cause of instability and mutation of the mitochondrial genome (Beckman and Ames, 1998). Moreover, damage to mtDNA can be particularly critical to overall cellular function because instability of mtDNA can be propagated as mitochondria and cells divide, amplifying the damage (Beckman and Ames, 1998). Results from studies using long-lived mice and experimental manipulations that extend lifespan, such as calorie restriction, have provided a strong correlation between oxidative damage to the mitochondria and lifespan (Barja and Herrero, 2000; Trinei et al., 2002; Barja, 2004). Although many of these studies mentioned above support the mitochondrial theory of aging, the data are correlative and do not prove the theory directly. A simple approach to directly test the mitochondrial theory of aging is to manipulate the amount of oxidative damage to the mitochondria by increasing the expression or by deleting a key antioxidant enzyme in order to either reduce or increase the oxidative insults to mitochondria and determine whether this manipulation alters lifespan. This approach has been put forward by several investigators using invertebrate animal models. For example, Longo et al. demonstrated that in Saccharomyces cerevisiae, deletion of the key antioxidant enzyme, manganese superoxide dismutase (MnSOD, Sod2), which is located in mitochondrial matrix, dramatically accelerates chronological aging and overexpression unambiguously increases the organism’s lifespan (Longo et al., 1996). Likewise, Tower’s group reported a similar extension of lifespan by the overexpression of MnSOD in adult Drosophila melanogaster (Sun et al., 2002). However, as outlined below (Table 1), mammalian transgenic/knockout models with alterations in key mitochondrial antioxidant enzymes have produced mixed results and do not fully support the mitochondrial theory of aging. 2.1. MnSOD knockout and transgenic mice MnSOD (Sod2) is the main antioxidant enzyme that scavenges superoxide in the mitochondrial matrix. MnSOD is considered the first line of defense against superoxide generated towards the mitochondrial matrix by mitochondrial electron transport chain. Complete ablation of MnSOD causes dilated cardiomyopathy and neurodegeneration leading to early postnatal death. These mice exhibit severe oxidative damage to mitochondria and also are extremely sensitive to hyperoxia (Li et al., 1995; Lebovitz et al., 1996). Although the studies using Sod2 / mice provide critical evidence
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that mitochondrial superoxide toxicity can have a profound effect on cellular function and lifespan, this evidence does not necessarily prove that mitochondrial superoxide per se accelerates aging. However, if as the mitochondrial theory of aging predicts, elevated superoxide levels and increased oxidative stress to mitochondria are in fact a primary cause of aging, a modest reduction in MnSOD activity also should have a ‘dose’ response in the aging process. In order to address this idea, our laboratory has extensively studied the heterozygous knockout of Sod2 (Sod2+/ mice), which have approximately 50% reduction in MnSOD activity in all of the tissues we analyzed. Sod2+/ mice do display some of the phenotypes that are indicative of decreased mitochondrial function (i.e., decreased aconitase activity, decreased complex I activity, decreased ATP generation) and increased oxidative damage such as 30–80% increase in both nuclear and mitochondrial DNA oxidation (8-oxodG). However, the lifespan of Sod2+/ mice in a pure C57B6/J background, did not show a statistical difference from that of wild-type mice (n = 70 per group) (Van Remmen et al., 1999; 2003). Although, some oxidative stress markers were elevated (e.g., 8-oxo-dG), it is important to keep in mind that not all oxidative stress markers were elevated in Sod2+/ mice. For example, F2-isoprostanes (measured in plasma as an indicator of overall oxidative damage), the level of protein carbonyls (protein oxidation marker), and the rate of H2O2 production from isolated mitochondria were all unchanged in Sod2+/ mice compared at different ages to age-matched, wildtype mice (Mansouri et al., 2006). In addition, biomarkers of aging, such as cataract formation, defective immune responses, and formation of glycoxidation products, were unaltered, whereas the incidence of cancer was higher in Sod2+/ mice (Van Remmen et al., 2003). Despite the fact that a 50% reduction in MnSOD is not sufficient to limit lifespan, the possibility remains that a further reduction in MnSOD activity initiated after development might in fact increase mitochondrial oxidative damage and reduce lifespan. To address this possibility, similar studies could be conducted using a conditional knockout approach in which MnSOD activity is ablated post-development. Although reducing MnSOD activity in Sod2 knockout mice tests whether superoxide toxicity in mitochondria is limiting to lifespan, the alternative approach is to test the whether an increase in mitochondrial MnSOD activity can reduce in vivo oxidative damage by superoxide and to determine whether increased protection against oxidative stress can extend lifespan. As mentioned earlier, overexpression of MnSOD in Drosophila was shown to have a beneficial effect, extending lifespan, and supporting the mitochondrial theory of aging (Sun et al., 2002). Moreover, in some genetically manipulated mouse models with extended lifespan, investigators have reported an increase in MnSOD activity and enhanced response against oxidative stress (Yamamoto et al., 2005; Taguchi et al., 2007). Thus, we and others have tested whether the overexpression of MnSOD activity in all tissues throughout the lifespan has any effect on oxidative damage and longevity in a mouse model. Recently, Hu et al. reported that approximately 3-fold overexpression of human SOD2 gene, driven by b-actin promoter, increased mean lifespan by 4% and that 18% of the SOD2 transgenic mice lived longer than 40 months while the longest surviving wild-type mouse was 36 months of age (n = 24–30) (Hu et al., 2007). In contrast, in our laboratory, an Sod2 transgenic mouse line that overexpresses MnSOD in all tissues under control of the endogenous mouse Sod2 promoter to a similar level (2- to 2.5-fold increase in MnSOD activity) failed to show increased lifespan even when the transgenic mice generated less superoxide (Jang et al., submitted for publication). When the lifespan of our colony is compared to the study by Hu et al., the maximum lifespan of MnSOD transgenic mice is similar (1245 days for our colony vs. 1290 days for Hu et al.); however, the lifespan of the wild-type control is notably different (1260 days for our colony vs. 1095 days in Hu et al.). In
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Table 1 Phenotype of mitochondrial transgenic/knockout mice. Gene name
Main function
Mn superoxide dismutase Sod2 /
Scavenger of superoxide
Sod2+/
Scavenger of superoxide
Sod2 transgenic
Scavenger of superoxide
Catalase Mitochondrial targeted CAT transgenic
Scavenger of H2O2
Thioredoxin 2 Trx2+/
Mitochondrial polymerase gamma PolcD257A/D257A
Reduce substrates to methionine sulfoxide and peroxiredoxins
Phenotype
Lifespan
Support mitochondria theory of aging?
Cardiomyopathy Hemolytic anemia Increased incidence of cancer Genomic instability Spongiform encephalopathy Optic neuropathy Movement disorders Neurodegeneration High oxidative stress
0–4 weeks
Yes
Partial increase in oxidative stress increased incidence of cancer vascular dysfunction Increased apoptosis
Normal
No change in mitochondrial oxidative damage Reduced lipid peroxidation with age
Normal
Reduced mitochondrial oxidative stress Reduced H2O2 Reduced mtDNA mutation Delayed cardiac pathology Delayed cataract formation Reduced tumor burden Reduced cardiac lesions
20% Increase
Yes
Decreased ATP, ETC activity Increase in H2O2 Increased mitochondrial oxidative damage
Preliminary results suggest no change
No
Weight loss, hair graying, hearing loss, infertility, kyphosis, decreased bone density, anemia, muscle loss Increased in mtDNA deletion/point mutation Decreased ETC activity No change in ROS/oxidative damage Increase in mtDNA point mutation No change in mtDNA deletion
70% Decrease
Yes
Normal
No
No
No
Proofreading newly synthesized mtDNA
PolcD257A/+
addition, the mean lifespan extension of the Sod2 transgenic mice is not statistically different (4% vs. 2%). To reliably conclude that overexpression of MnSOD can prolong survival in mice; both the mean and the maximum lifespan should increase. However, when using a large sample size (n = 47–50), we did not see the any changes in either the mean or the maximum lifespan. It is also possible that the disparity between the survival curves of two studies is due to the differences in the genetic background used and the regulatory element used to drive the transgene expression. In all, ectopically increasing the MnSOD activity throughout the lifespan in mice to augment mitochondrial antioxidant protection does not unequivocally result in increased lifespan. 2.2. Mitochondrial targeted catalase transgenic mice Transgenic mice overexpressing catalase targeted to peroxisomes, nuclei, or mitochondria were generated by Schriner et al. (2005). Although peroxisome and nuclear overexpression showed a trend toward increased lifespan, only the mitochondrial targeted construct provided the maximal benefit, increasing both median and maximal lifespan by 20%. Mitochondrial hydrogen peroxide (H2O2) production and oxidative inactivation of aconitase were reduced in isolated cardiac mitochondria of the mitochondrial targeted catalase transgenic mice (mCAT mice) (Schriner et al., 2005). In addition, DNA oxidation and levels of mitochondrial deletions were also reduced in the skeletal muscle of these mice.
Furthermore, cardiac pathology and cataract development were also delayed in the mitochondria targeted catalase transgenic mice (Schriner et al., 2005). More recently, the same group of authors further analyzed the end-of-life pathology and concluded that mCAT mice had reduced age-associated pathologies such as malignant nonhematopoietic tumors and cardiac lesions (Treuting et al., 2008) and thus not only do mice expressing mitochondrial catalase have an extension in lifespan , but these mice also have an increase in ‘healthspan’. Data from the mCAT mice undoubtedly support the mitochondrial theory of aging in that a reduction in mitochondrial oxidative stress and damage in these mice is associated with an increase in lifespan; however, the authors pointed out in the original study that when the transgenic mice were backcrossed to a nearly pure C57BL6 background, the lifespan extension phenotype appears to be diminished. Before we make conclusions about this model, genetic background issues must be resolved. In comparison, mice that overexpress a modest level of catalase in the peroxisome (the compartment in which catalase is normally localized) did not show an extension of either mean or the maximum lifespan. 2.3. Thioredoxin 2 knockout mice Thioredoxin (Trx) is a 12 kDa protein with two redox-sensitive cysteine-thiols in its active site. Thioredoxin is kept in the reduced state by the flavoenzyme thioredoxin reductase, in a NADPHdependent reaction. The thioredoxins play an essential role in the
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antioxidant defense mechanism (Yoshida et al., 2003), can scavenge peroxides via peroxiredoxins, and reduce disulfide bonds and methionine sulfoxide either directly or through the action of other oxidoreductases. The mitochondrial form of thioredoxin, thioredoxin 2 (Trx2) has been implicated as a key player in maintaining the mitochondrial redox environment and protecting against mitochondrial oxidative damage (Yoshida et al., 2003). The homozygous knockout of the Trx2 in mice is embryonic lethal. Pérez et al. studied the effect of partial deficiency of Trx2 in mitochondrial function and oxidative stress (Pérez et al., 2008). They reported that mice with 50% reduction in Trx2 activity (Trx2+/ mice) exhibit a decrease in mitochondrial function as measured by the decrease in ATP production, decline in ETC activity, and increased generation of mitochondrial H2O2. In addition, compared to their age-matched counterparts, Trx2+/ mice have higher oxidative damage to protein (protein carbonyl), lipid (F2-isoprostane), and DNA (8-oxo-dG). Furthermore, Trx2+/ mice are more sensitive to diquat-induced oxidative damage and apoptosis (Pérez et al., 2008). Because of the increase in mitochondrial oxidative stress and reduced mitochondrial function, the Trx2+/ mice would be predicted by the mitochondrial theory of aging to have a reduced lifespan. However, preliminary results from a pilot study using Trx2+/ mice on a mixed genetic background suggest that the lifespan is not reduced in these mice (Pérez et al., 2009). Interestingly, overexpression of thioredoxin 1 (the cytosolic form) has been shown to increase lifespan in mice (Mitsui et al., 2002). 3. Mitochondrial DNA instability: accumulation of mtDNA mutation with age Because mitochondrial ROS is undoubtedly capable of inducing mtDNA instability as observed in aging tissue, a majority of studies that have attempted to test the mitochondrial theory of aging have focused on mtDNA mutations. Recently, mouse models of mtDNA instability have provided direct evidence of its role in the aging process. Using a gene ‘knock-in’ strategy, two independent investigators have generated a mouse model that has a point mutation in a catalytic subunit of mtDNA polymerase (POLG) that is responsible for proofreading newly synthesized mtDNA (Trifunovic et al., 2004; Kujoth et al., 2005). Mice homozygous for this mutant mtDNA polymerase (PolcD257A/D257A) exhibit a progressive accumulation of mtDNA point mutations and show a 70% reduction in lifespan compared to wild-type littermates. In addition, these mice show phenotype and pathology that are similar to aging such as weight loss, hearing loss, hair graying, infertility, kyphosis, decreased bone density, anemia, and muscle loss (Kujoth et al., 2005). Yet, as Richard Miller has argued, ‘accelerated aging phenotypes’ seen in these ‘mutator mice’ are similar to phenotypes of aging humans but perhaps not the phenotypes necessarily observed in normal aging, inbred mice (Miller et al., 2005). Nevertheless, the most surprising findings of the report by Kujoth et al. is that, in spite of the large mutation load and decrease in ETC activity, mitochondrial ROS production and the level of oxidative damage (lipid peroxidation and DNA oxidation) did not show any difference compared to their wild-type counterparts (Kujoth et al., 2005). A simple explanation of these observations is that the dramatic aging phenotype and pathology seen in these mice are not directly mediated by mitochondrial ROS production. An alternative interpretation is that massive mutations throughout the mitochondrial genome may prevent the normal assembly of the respiratory chain thus eliminating the site of ROS generation. For example, if the loss of proofreading activity causes a large scale deletion of the mitochondrial genome, this mutation could eliminate the genes for the subunit of ETC (possibly complex I and complex III). More importantly, if the tRNA gene is deleted, the
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synthesis of all 13 mtDNA encoded proteins can be compromised. In this scenario, a significant decrease in ETC activity will occur and no ROS can be generated. Recently, Vermulst et al., (2007) used a new and more sensitive methodology (Random Mutation Capture Assay) to assess mtDNA point mutations, demonstrating that heterozygous mice, which have only one mutant allele (Polc+/D257A), do not exhibit any premature aging phenotype despite having an approximately 500-fold increase in mtDNA point mutations. Moreover, Polc+/D257A mice have a normal lifespan similar to wild-type littermates, disproving the role of mtDNA point mutations as a lifespan-limiting factor. These authors have followed up their study by modifying the random mutation capture assay to detect mtDNA deletions. Intriguingly, they reported that mtDNA deletion was significantly elevated in heart and brain tissue with age. However, unlike the point mutation, only the homogenous mutant PolcD257A/D257A mice had an accelerated rate of deletion mutations. In addition, they demonstrated that the rate at which mtDNA mutations reached the phenotypical expression varies greatly among different tissues. They concluded that the mtDNA deletion is the driving force behind the premature aging phenotype of the Polc mutant mice (Vermulst et al., 2008). Conversely, a previous report by Tyynismaa et al. showed that the transgenic mice with a mutated allele of mitochondrial helicase TWINKLE did not exhibit any aging phenotype despite having elevated levels of mtDNA deletions (Tyynismaa et al., 2005). However, it is noteworthy that this discrepancy in phenotype may be due to differences in the gene overexpression strategy. In Polc mutant mice, a knock-in technique was used to target the transgene in a selected locus, whereas in the TWINKLE study, overexpression was achieved by random integration of mutant transgene. These observations, along with those showing the lack of increased oxidative stress in the PolcD257A/D257A mutant mice, raise doubts regarding the direct connection between mtDNA mutation and ROS production and the vicious cycles proposed as part of the mitochondrial theory of aging. However, before we make any conclusions, there are still many unanswered questions regarding the role of mtDNA instability in the aging process. For example, in normal aging mice, is the age-related increase in mtDNA mutations due to infidelity of Polc or due to oxidative damage to mtDNA? All in all, the mutator mice provide a model with which to examine mechanistic features of the aging process that relate to mitochondrial genome integrity and the response to mtDNA mutations. 4. Conclusions and future directions Recent studies using the transgenic/knockout strategies have challenged the core principles of the mitochondria theory of aging as well as its parent theory, the free radical or oxidative stress theory of aging. Based on the studies to date, most studies do not support or remain inconclusive on whether mitochondrial dysfunction and oxidative stress determine lifespan. However, several questions still need to be addressed. First, most of the studies using the knockout and transgenic approach do not take into account the effect of oxidative stress on development. It is possible that the genetic manipulations are affecting development and not necessarily the aging process. For example, in the study by Trifunovic et al., the mutations are uniform among tissues, suggesting that much of the mutation accumulation occurs during embryonic or fetal development (Trifunovic et al., 2004). This issue can be resolved in future studies using the inducible knockout/knockin approach. Secondly, our understanding of the mitochondrial biology is far from complete. Inter-organelle interaction, mitochondrial biogenesis, fission and fusion, and ‘mitophagy’-autophagic removal of damaged mitochondria during the aging process are only some
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of the examples that have not been thoroughly investigated. Interestingly, in S. cerevisiae and P. anserina, the deletion of a protein that promotes mitochondrial fission (dynamin related protein 1dnm1p) extended lifespan without lowering fitness or reproduction (Scheckhuber et al., 2007). In addition, the increase in ROS generation in normal cells was delayed in the cells that lack dnm1p (Scheckhuber et al., 2007). It remains to be seen if any of the proteins involved in turnover and maintenance of mitochondria affect the lifespan of higher model organisms. Finally, in genetic mouse studies, different strains of inbred mice might have different phenotypes in response to certain genetic manipulations. In fact, using the quantitative trait locus mapping (QTL), Rikke et al. showed different strains of mice show a significant genetic variation in weight loss in response to a 40% calorie-restricted (CR) diet (Rikke et al., 2006). Therefore, it could be misleading to state that a life extension or reduction effect seen in one strain proves or disproves the mitochondrial theory of aging. In summary, the majority of the initial pioneering studies in mice to test the mitochondrial theory of aging have yielded results that either do not support the theory or remain inconclusive. An exception is a single study involving the overexpression of catalase in mitochondria, but the findings may not be applicable to all genetic backgrounds. Clearly, more work is required to clarify whether mitochondrial dysfunction determines aging and the lifespan of an organism. In addition, future studies should take into account phenotypic changes as an indication of healthspan as well as changes in lifespan. References Ames, B.N., Shigenaga, M.K., et al., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. 90 (17), 7915–7922. Andreyev, A.Y., Kushnareva, Y.E., et al., 2005. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Moscow) 70 (2), 200–214. Barja, G., 2004. Aging in vertebrates, and the effect of caloric restriction: a mitochondrial free radical production-DNA damage mechanism?. Biol. Rev. Camb. Philos. Soc. 79 (2), 235–251. Barja, G., Herrero, A., 2000. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals. FASEB J. 14 (2), 312–318. Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 78 (2), 547–581. Boveris, A., Navarro, A., 2008. Brain mitochondrial dysfunction in aging. IUBMB Life 60 (5), 308–314. Croteau, D.L., Stierum, R.H., et al., 1999. Mitochondrial DNA repair pathways. Mutat. Res. 434 (3), 137–148. Figueiredo, P.A., Mota, M.P., et al., 2008. The role of mitochondria in aging of skeletal muscle. Biogerontology 9 (2), 67–84. Harman, D., 1956. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300. Harman, D., 1972. The biologic clock: the mitochondria?. J. Am. Geriatr. Soc. 20 (4), 145–147. Hu, D., Cao, P., et al., 2007. Hippocampal long-term potentiation, memory, and longevity in mice that overexpress mitochondrial superoxide dismutase. Neurobiol. Learn. Mem. 87 (3), 372–384. Jang, Y.C., Pérez, V.I., Song, W., Lustgarten, M.S., Salmon, A.B., Mele, J., Qi, W., Liu, Y., Liang, H., Chaudhuri, A., Ikeno, Y., Epstein, C.J., Van Remmen, H., Richardson, A., submitted for publication. Overexpression of Mn Superoxide Dismutase (MnSOD) protects against oxidative stress but does not increase lifespan in mice. J. Gerontol. Jezek, P.L., Hlavata, L., 2005. Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int. J. Biochem. Cell. Biol. 37 (12), 2478–2503.
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