Mice live longer and show signs of delayed aging when they express the antioxidant enzyme catalase in their mitochondria, according to a report appearing today in Science online. The results of the study, by Peter Rabinovitch and his colleagues at the University of Washington in Seattle, support the theory that oxidative damage to cells contributes to aging and eventually death in higher animals. Their results also show that mitochondria, now under intense scrutiny for their role in age-related neurodegenerative disorders, are an important source of toxic reactive oxygen species (ROS) in normal cells. ROS are produced as a byproduct of the pathway that generates energy in the mitochondria, the cell’s power plant. During respiration, electrons escape the mitochondria and react with free oxygen to produce highly toxic negative ions. If left alone, the ions inflict irreversible damage to proteins, lipids, and DNA. Cells can defend themselves with detoxifying enzymes like catalase and superoxide dismutase that break down ROS. The balance between production of ROS and their destruction by antioxidant enzymes determines the rate at which oxidative alterations accumulate, and may set an upper limit on how long we can live. Increasing oxidative damage is also hypothesized to trigger aging-related Alzheimer disease and other neurodegenerative disorders (see live discussion of the “mitochondrial cascade hypothesis” of AD).

To determine whether ROS, and in particular hydrogen peroxide (H2O2), do in fact limit lifespan in mammals, first author Samuel Schriner worked with colleagues from the University of California, Irvine, and University of Texas at San Antonio to create transgenic mice overexpressing human catalase in their mitochondria. Catalase, found mainly in peroxisomes, rapidly converts toxic H2O2 into water and oxygen. In two independent lines of mice, the mitochondrial catalase (MCAT) expressers showed about a 20 percent, or a 5-month, increase in median and maximal lifespan compared to wild-type littermates. The ability of catalase to increase longevity was most apparent when the enzyme was targeted to mitochondria: Mice that expressed the enzyme in peroxisomes (PCAT) had a slightly longer median lifespan, but no increase in maximal life. Nuclear catalase expression had no effect on either parameter.

MCAT mice appeared to age more slowly than their control littermates by several measures. While histological comparisons showed little difference between WT and MCAT lines in young mice (9-11 mo), aged transgenic mice (20-25 mo) had significantly less arteriosclerosis and cardiomyopathy than their wild-type siblings. One strain showed a delay in cataract formation in mid-life. Biochemical studies showed that slower aging in the MCAT mice was associated with a lower level of oxidative stress and DNA damage. H2O2 production by cardiac mitochondria from MCAT mice was decreased 25 percent, and mitochondria containing catalase were protected from the toxic effects of H2O2. Age-related increases in oxidative damage to total DNA, and fragmentation of mitochondrial DNA were also slowed in skeletal muscle of MCAT mice.

Despite the indication that MCAT mice were at least partially spared the ravages of time in terms of oxidative damage, catalase is not quite a fountain of youth. Its effects on lifespan, while significant, are much smaller than those observed with caloric restriction or in some genetic models of aging. The question of whether mitochondrial catalase might be additive or synergistic with other life-lengthening treatments was not addressed. The researchers did show that peroxisomal catalase expression together with superoxide dismutase overexpression increased median lifespan more than either alone, but maximum time of survival was not affected. They speculate that because MCAT mice survive longer than PCAT mice, the combination of MCAT and superoxide dismutase may show even more benefit.

Like the aging brain, AD and Parkinson disease brains show signs of oxidative stress, and mitochondrial dysfunction has been reported in both diseases. In models of AD, ROS appear to enhance Aβ deposition (see ARF related news story), and Aβ itself can be toxic to mitochondria (see ARF related news story), causing an increase in ROS. One of the two MCAT transgenic lines displayed increased catalase expression in the brain, and these animals will no doubt come in handy to further explore the links between the accumulated oxidative insults of normal aging and sporadic AD or other age-related neurodegenerative disease.—Pat McCaffrey

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  1. The findings of Schriner et al. provide an important advance in understanding the contributions of metabolism of reactive oxygen species to aging and lifespan in mammals. Their data strongly suggest that production of hydrogen peroxide in mitochondria is central to the aging process and that, accordingly, manipulations that detoxify hydrogen peroxide can have an "anti-aging" effect, improving healthspan and lifespan. The design of the study, which involved targeting of catalase to mitochondria, peroxisomes, or the nucleus, was excellent and revealed the complexity of reactive oxygen species production and detoxification in regard to subcellular localization. The availability of their mitochondrial catalase transgenic mice to the biomedical research community will be very valuable in determining the role of mitochondrial hydrogen peroxide in various age-related diseases.

    It will be of considerable interest to determine whether the effects of mitochondrial catalase and caloric restriction are complementary or redundant in regard to protection against age-related disease and lifespan extension. While dietary restriction is known to reduce oxidative damage to cells during normal aging and in disease models, and can increase cellular stress resistance (1,2), it is unclear whether there is one action of dietary restriction that is pivotal for lifespan extension. The findings of Schriner et al. suggest that cells are not normally very efficient in removing hydrogen peroxide when it is produced in mitochondria, consistent with previous studies showing that mitochondria-targeted catalase can protect cultured cells against oxidative injury (3). There has been much interest in the role of mitochondria in aging, and further work will be required to establish the specific aspects of mitochondrial function that influence the aging process. Nevertheless, the new findings emphasize the potential for interventions that target mitochondrial reactive oxygen species metabolism in preventing and treating age-related diseases.

    References:

    . The role of oxidative damage and stress in aging. Mech Ageing Dev. 2004 Oct-Nov;125(10-11):811-26. PubMed.

    . Energy Intake, Meal Frequency, and Health: A Neurobiological Perspective. Annu Rev Nutr. 2004 May 21; PubMed.

    . Mitochondrial catalase and oxidative injury. Biol Signals Recept. 2001 May-Aug;10(3-4):189-99. PubMed.

  2. The work by Schriner and colleagues is an important advance in understanding the free radical theory of aging and its implications for healthy aging and longevity. These investigators created transgenic mice that overexpress human catalase localized to peroxisomes, nucleus, and mitochondria, and studied the effects of aging from birth to death in these transgenic mouse lines. Interestingly, but not surprisingly, they found that mice that overexpress human catalase targeted to mitochondria exhibited increased lifespan 5.5 months longer relative to control wild-type mice, suggesting that overexpressed catalase in mitochondria decreases reactive oxygen species (ROS) and boosts the mitochondrial function. These events ultimately lead to an extended lifespan. This finding has tremendous implications for healthy aging, longevity, and age-related illnesses, particularly Alzheimer’s, Parkinson’s, and ALS.

    The free radical theory of aging, one of the prominent aging hypotheses, holds that during aging, an increase in ROS in mitochondria causes mutations in the mitochondrial DNA and damages mitochondrial components, resulting in senescence. Findings from gene expression studies (Lu et al., 2004; Reddy et al., 2004); mitochondrial DNA studies (Lin et al., 2002); and aging animal model studies (Trifunovic et al., 2004; Manczak et al., 2004) also support this hypothesis, suggesting that an age-dependent increase in ROS is a key factor in causing age-related problems and diseases.

    Schriner and colleagues’ exciting work suggests that early treatment with antioxidants may help reduce free radicals and mitochondrial DNA damage, and may increase oxygen consumption in electron transport chain and ultimately their mitochondrial function. The use of antioxidant treatments, alone or in combination with calorie restriction, which has also been found to reduce ROS and to increase the lifespan of rodents (Mattson et al., 2002; Mattson, 2003), may help reduce mitochondrial toxicity, increase lifespan, and improve health during these increased years of aging.

    The authors are well-known and respected for their studies of age-related mitochondrial changes in rodents. Schriner and colleagues’ research demonstrates increased mitochondrial function in skeletal muscle, heart, spleen, and other tissues in catalase transgenic mice relative to control wild-type littermates. It would be useful to examine changes in the brain in these transgenic mouse lines in terms of ROS and mitochondrial function/dysfunction. Further, a reasonable next step would be to study exact mechanism(s) of how overexpressed catalase can reduce ROS within mitochondria (matrix or inner membrane), and catalase interactions with mitochondrial proteins, if any, within mitochondria.

    This study is exciting, with implications for longevity, treatments for age-related diseases, and healthy aging.

    References:

    . Gene regulation and DNA damage in the ageing human brain. Nature. 2004 Jun 24;429(6994):883-91. PubMed.

    . Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease. Hum Mol Genet. 2004 Jun 15;13(12):1225-40. PubMed.

    . High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer's disease brain. Hum Mol Genet. 2002 Jan 15;11(2):133-45. PubMed.

    . Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004 May 27;429(6990):417-23. PubMed.

    . Time-course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging. J Neurochem. 2005 Feb;92(3):494-504. PubMed.

    . Modification of brain aging and neurodegenerative disorders by genes, diet, and behavior. Physiol Rev. 2002 Jul;82(3):637-72. PubMed.

    . Gene-diet interactions in brain aging and neurodegenerative disorders. Ann Intern Med. 2003 Sep 2;139(5 Pt 2):441-4. PubMed.

  3. Reactive oxygen species (ROS) are generated in several locations within cells, especially in mitochondria, at the cell membrane and in the endoplasmic reticulum. As a consequence of the electron transport chain, there is a leakage of electrons onto oxygen-forming free radicals, resulting in hydrogen peroxide, hydroxyl radical, and superoxide production. The observation that mitochondrial targeting of catalase prolongs lifespan of mice and limits certain key indices of oxidant and mitochondrial cell damage indicates that free radical generation by mitochondria during "normal" life activities does generate "wear and tear" on mitochondria. In addition, it suggests that individuals or situations (such as diabetes) where there is excess generation of mitochondrial free radicals could be associated with considerably increased morbidity on that basis.

    However, it is important to keep in mind the important role of ROS as mediators of signal transduction processes which contribute to homeostatic and protective mechanisms. At this point, experiments with catalase targeted to mitochondria have not shown protective effects, but the mice generated thus far have not been challenged in many ways (of course, future experiments will address this). On the other hand, it may be that ROS generated at the cell membrane and in the endoplasmic reticulum have a more important role in such signaling events. As recent evidence suggests cross-talk between generation of ROS at the cell membrane and mitochondria, it is possible that these mechanisms do interact and that one should cautiously approach the issue of blocking ROS generation in cells.

    Regardless of these comments, the current paper advances the field by demonstrating a role for ROS during the aging process in a murine model.

  4. Aging, including age-related disease, is currently postulated to be the outcome of a large number of disparate influences. Based on the analysis of familial models of late-onset diseases such as Alzheimer’s, a number of biochemical processes, such as protein folding, posttranslational processing, protein degradation, and accumulation of toxic products (amyloid, synuclein, AGEs, lipofuscin) are believed to be involved. In most of these phenomena, reactive oxygen species (ROS) play an important role prompting many to hypothesize that ROS generation and damage to cellular constituents is the primary agent in aging, if not entirely etiological. ROS are generated in various cellular compartments and exert their immediate effects in close proximity to the site of their production. As such, many have hypothesized that ROS generation by, and damage to, these compartments enables a vicious cycle where free-radicals generated by the cellular constituents cause local damage, further increasing ROS production.

    Unfortunately, tests of the free-radical theory of aging have provided conflicting interpretations. Many animal models of antioxidant enzyme overexpression fail to outlive their nontransgenic littermates and simply increasing ROS burden is insufficient to cause a full-blown aged phenotype. If the free-radical theory of aging is correct, one would expect that reducing free-radical burden would enable organisms to live longer and that increased ROS would be sufficient to cause a progeroid phenotype.

    In order to test the free-radical theory of aging, Schriner et al. have created transgenic mice overexpressing catalase targeted to either to the nucleus, peroxisomes, or mitochondria. Catalase is the peroxisomal enzyme responsible for converting hydrogen peroxide into water, thus preventing H2O2 from forming the reactive hydroxyl radical in the presence of reduced metals. Schriner et al. had previously shown that overexpressed catalase transgenes targeted to either the nucleus or the peroxisomes failed to extend lifespan, despite the fact that the nuclear-directed catalase protected nuclear DNA from exogenously applied H202 (Schriner et al., 2000). The failure of both the nuclear and peroxisomal catalase to significantly extend lifespan prompted Schriner et al. to conclude that, contrary to accepted wisdom, H2O2 is not responsible for the accumulation of nuclear DNA damage associated with age and, furthermore, that free-radical damage accounts for a negligible fraction of aged pathology.

    In this most recent ScienceExpress report, Schriner et al. extend their work by targeting human catalase to mitochondria (MCAT). They report an approximately 20% extension of median lifespan in MCAT mice, whereas catalase targeted to peroxisomes and the nucleus yields much reduced benefits of about 12% and 8%, respectively. Their MCAT mice have extended maximum lifespans, reduced cardiac and cataract pathology, slowed accumulation of mitochondrial DNA deletions, and reduced concentrations of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage.

    The immediate implication of this study is that the subcellular location of ROS production and presumably damage is of crucial importance. ROS are rapidly inactivated by frequently destructive interactions with macromolecules, and therefore the damage they cause is likely to be concentrated close to their sources. Catalase expressed in subcellular locations distant from ROS sources would be less effective. The relative magnitude of lifespan prolongation in the three mouse strains corroborates this prediction and may reflect the relative impact of ROS on the three cellular compartments studied here, where mitochondria as the main source of ROS bear the brunt of damage, while the nucleus and peroxisomes appear to be much less affected.

    Furthermore, the results are consistent not only with the free-radical theory of aging, but its important subset, the mitochondrial theory of aging. The mitochondrial theory of aging posits that mitochondrial DNA mutations, whether caused by ROS, failure to repair DNA damage, or by errors in replication, lead to mitochondrial dysfunction—further increasing ROS output, forcing cells to adapt by downregulating oxidative metabolism, relying increasingly on glycolysis and thus fundamentally altering the redox state of the cell. Most interestingly, the transgenics with the most reduced levels of mtDNA deletions had the largest prolongation of lifespan. The fact that mtDNA deletions, even when present at low levels, can sensitize cells to apoptosis may provide a possible explanation (Schoeler et al.). The authors did not analyze levels of mitochondrial DNA mutations, which may have a more profound impact on aging than deletions—this would be a worthwhile measurement (for Review see the upcoming article by Smigrodzki and Khan in Rejuvenation Research).

    The fact that MCAT mice had a significant reduction in cardiac and cataract pathology when compared to their transgenic and nontransgenic littermates emphasizes the roles that free-radical mediated injury to mitochondria and the subsequent mitochondrial dysfunction play in these pathologies (for review see Ballinger and Hirano). The question remains, however, as to what extent human aging is modeled by transgenic animals where mitochondrial damage can be modulated (please see Trifunovic et al., the intriguing paper by Song et al. and for review see Taylor and Turnbull).

    The authors noted a significant degree of mosaic expression of the mitochondrially targeted catalase in the MCAT transgenics, with subsequent loss of catalase expression in following generations; this may be explained by the fact that basal ROS may serve an important role in cellular function, providing selective pressure against transgene expression. The fact that cycling cells utilize oxidative bursts to signal various states and that stem cells produce ROS in concert with the onset of differentiation belies the importance of basal ROS as signal transducers. These findings sound a cautionary note about the use of antioxidants without titration and proper intracellular targeting.

    Implications for Alzheimer’s Disease
    Oxidative damage in Alzheimer disease brain is a common finding. The source of that damage remains controversial, with many investigators indicting β amyloid. Yet, some investigators consistently find that regions of amyloid deposition fail to colocalize with markers of oxidative stress (see the work of George Perry and Mark Smith). As the emphasis of the AD community has shifted from deposited amyloid in fibrillar and plaque form to oligomeric forms, the failure of deposited amyloid to colocalize with ROS damage may be partially explained. This raises the question as to what is the locus of action of oligomeric amyloid in eliciting ROS. One strong possibility is mitochondria where investigators not only find amyloid but the entire γ-secretase complex (Hansson et al.) and APP (Anandatheerthavarada et al.). Furthermore, AD brains possess a tenfold higher oxidative damage to DNA, with mtDNA providing the bulk of the difference (Wang et al.), and control region mutations in mtDNA are more numerous in AD (Coskun et al.). (See Reddy and Beal for an excellent review of the mitochondrial involvement in AD, as well as Swerdlow and Khan for the mitochondrial cascade hypothesis of AD).

    Here is where the transgenic models developed by Schriner et al. may be of particular use to the AD community. Crossing the various subcellular compartment-targeted catalase mice with Tg2576 or other models of AD may allow investigators to determine the relative role oxidative damage plays in AD, and whether and how limiting oxidative damage within differing subcellular compartments affects amyloid and tau.

    To summarize, targeted reduction of ROS in mitochondria, but not nuclei or peroxisomes, reduces the accumulation of at least some forms of mtDNA mutations and prolongs median and maximal life spans. This is a result of importance to the AD research community, given the increasing number of reports implicating ROS in the pathogenesis of this disease and the looming question regarding the source and recipient of that oxidative stress.

    References:

    . Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells. J Cell Biol. 2003 Apr 14;161(1):41-54. PubMed.

    . Mitochondrial dysfunction in cardiovascular disease. Free Radic Biol Med. 2005 May 15;38(10):1278-95. PubMed.

    . Alzheimer's brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication. Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10726-31. PubMed.

    . Nicastrin, presenilin, APH-1, and PEN-2 form active gamma-secretase complexes in mitochondria. J Biol Chem. 2004 Dec 3;279(49):51654-60. PubMed.

    . Transmitochondrial mice: proof of principle and promises. Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):401-3. PubMed.

    . Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease. Hum Mol Genet. 2004 Jun 15;13(12):1225-40. PubMed.

    . Are mitochondria critical in the pathogenesis of Alzheimer's disease?. Brain Res Brain Res Rev. 2005 Nov;49(3):618-32. PubMed.

    . Levels of DNA damage are unaltered in mice overexpressing human catalase in nuclei. Free Radic Biol Med. 2000 Oct 1;29(7):664-73. PubMed.

    . DNA precursor asymmetries in mammalian tissue mitochondria and possible contribution to mutagenesis through reduced replication fidelity. Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):4990-5. PubMed.

    . A "mitochondrial cascade hypothesis" for sporadic Alzheimer's disease. Med Hypotheses. 2004;63(1):8-20. PubMed.

    . Mitochondrial DNA mutations in human disease. Nat Rev Genet. 2005 May;6(5):389-402. PubMed.

    . Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004 May 27;429(6990):417-23. PubMed.

    . Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease. J Neurochem. 2005 May;93(4):953-62. PubMed.

References

News Citations

  1. Aβ Production Linked to Oxidative Stress
  2. ABAD, aka ERAB: Mitochondrial Miscreant Returns

Other Citations

  1. “mitochondrial cascade hypothesis”

Further Reading

Primary Papers

  1. . Extension of murine life span by overexpression of catalase targeted to mitochondria. Science. 2005 Jun 24;308(5730):1909-11. PubMed.