Experimental Evidence that Is not Readily Explained by the Mitochondrial Theory of Aging

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Despite the wealth of experimental evidence in support of the mitochondrial theory of aging, there is a very significant and growing body of data that appear to be at odds with it. Thus, the rate of ROS generation by mitochondria under physiological conditions, which is a cornerstone of the mitochondrial theory of aging, has been recently critically reexamined by several groups. Hansford et al. have found that active H2O2 production (an indirect measure of O2*_ generation) requires both a high fractional reduction of complex I (indexed by NADH/NAD+ + NADH ratio) and a high membrane potential, AC. These conditions are achieved only with supraphysiologi-cal concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of

H2O2 formation are much lower (less than 0.1% of respiratory chain electron flux). This H2O2 production may be stimulated by the complex III inhibitor antimycin A, but not by myxothiazol (Hansford et al., 1997). Staniek and Nohl further reported that mitochondria respiring on complex I and complex II substrates generate detectable H2O2 only in the presence of the antimycin A. They also suggested that the rates of mitochondrial H2O2 production reported by others are artificially high due to flaws in experimental design (Staniek and Nohl, 2000). Martin Brand's group capitalized on these findings and used an improved experimental design to show that mitochondria do not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates, but release significant amounts of superoxide from complex I when respiring on palmitoyl carnitine (St-Pierre et al., 2002). However, even at saturating concentrations of palmitoyl carnitine, in their estimation only 0.15% of the electron flow gives rise to H2O2 under resting conditions with a respiration rate of 200 nmol of electrons/min/mg mitochondrial protein. Under physiological conditions, this rate should be even lower due to a) lower partial oxygen pressure, b) lower concentration of palmitoyl carnitine, and c) lower mitochondrial membrane potential. Therefore, under physiological conditions in cells with uncompromised antioxidant defenses, ROS are produced by ETC in quantities that should be efficiently scavenged by mitochondrial antioxidant systems. As a consequence, no significant oxidative damage can be expected in mtDNA due to an electron leak from ETC, provided that cells have normal levels of antioxidants. This conclusion is in agreement with the observations of Orr et al. (2003), who recently have reexamined their earlier findings and those of others on the effect of overexpression of antioxidant enzymes on extension of Drosophila lifespan. They have found that significant increases in the activities of both CuZn-SOD and CAT had no beneficial effect on survivorship in relatively long-lived yw mutant flies, and were associated with slightly decreased life spans in WT flies of the Oregon-R strain. The introduction of additional transgenes encoding Mn-SOD or thioredoxin reductase in the same genetic background also failed to cause life span extension. These authors conclude that increasing the activities of major antioxidative enzymes above WT levels does not decrease the rate of aging in long-lived strains of Drosophila, although there may be some effect in relatively short-lived strains (Orr et al., 2003). In line with this conclusion, Van Remmen et al., in their study of mice heterozygous for the MnSOD gene knockout, have found that although life-long reduction of MnSOD activity leads to increased levels of oxidative damage to mitochondrial and nuclear DNA and increased cancer incidence, it does not appear to affect aging (Van Remmen et al., 2003).

As mentioned earlier, accumulation of 8-oxodG in mtDNA correlates with mitochondrial dysfunction in aging. In addition, mtDNA 8-oxodG levels inversely correlate with mammalian life span. 8-OxodG-induced mutagenesis is prevented by removal of the lesion from mtDNA via base excision repair (BER), which involves recognition and incision of the modified base by OGG1. MtDNA from mice deficient for OGG1 accumulates 8-oxodG to 20-fold higher levels than WT controls by six months of age (de Souza-Pinto et al., 2001). This elevation of 8-oxodG levels in the mtDNA of Ogg1—/— mice is almost an order of magnitude greater than is observed in old mice. One can predict, in line with mito-chondrial theory of aging, that these mice would accumulate mutations in their mtDNA, which would result in the production of defective subunits for and decline in activity of mitochondrial respiratory complexes (especially complexes I and IV, to which most of mitochondrially encoded polypeptides cater), which in turn would result in accelerated aging. However, these mice are not reported to age prematurely, nor do they show any decline in mitochondrial respiratory function in the heart and liver, or signs of oxidative stress as judged by protein carbonyl content (Stuart et al., 2005).

Rasmussen et al. assayed 13 different enzyme activities using optimized preparation techniques and found that the central bioenergetic systems, including pyruvate dehydrogenase, tricarboxylic acid cycle, respiratory chain, and ATP synthesis, appeared unaltered with age (Rasmussen et al., 2003). Maklashina and Ackrell have recently critically examined the literature regarding the role of ETC dysfunction in aging (Maklashina and Ackrell, 2004). They conclude that the evidence for age-related inactivation of the respiratory chain can be challenged on the grounds of preparation purity and the use of inadequate assay procedures, and that recent experimental evidence does not support the mitochondrial theory of aging (Maklashina and Ackrell, 2004).

In contrast, Jacobs does not challenge experimental evidence supporting the mitochondrial theory of aging but rather points out that all the evidence available to date is indirect in its nature (Jacobs, 2003). He argues that studies performed so far do not address the critical issue of cause and effect; that is, does the somatic mutation of mtDNA result in OXPHOS dysfunction and increased oxidative stress? Does increased oxidative stress promote mtDNA mutagenesis? Finally, he points out that the results from his own lab suggest that, at least in a tissue culture model, progressive accumulation of mtDNA mutations due to expression of mutant DNA polymerase gamma (Poly) does not lead to significant phenotypic changes despite the accumulation of mtDNA mutations at a level three times greater than that found in aged tissues.

Very recently, however, Nils-Goran Larsson and colleagues reported the generation of a homozygous knock-in mice that express a proofreading-deficient catalytic subunit of Poly, the only DNA polymerase found in mammalian mitochondria (Trifunovic et al., 2004).

These mice develop a mtDNA mutator phenotype with a three- to five-fold increase in the levels of mtDNA point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and the premature onset of age-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anemia, reduced fertility, and heart enlargement. Thus results of this study provide the best evidence so far for a causative link between mtDNA mutations and aging phenotypes in mammals.

However, as these authors concede, the detailed kinetics of the accumulation of somatic mtDNA mutations remains to be elucidated. The mutation load in the brain of mutator mice at two months of age is already two- to three-fold greater than in six-month old WT littermates. This, and the rather uniform mutation loads between tissues, suggests that much of the accumulation of mutations may occur during embryonic and/or fetal development. Also, the onset of premature aging in this model is not accompanied, temporally, by a large de novo accumulation of mtDNA mutations around six months. Therefore, it appears plausible that the premature onset of aging in this model may be the result of the cumulative physiological damage caused by the high mutation load present during adult life and/or to segregation or clonal expansion of specific mutations. This is supported by the observed mosaicism for the respiratory chain deficiency found in the heart. However, since the effects of high mutational burden in mtDNA during embryonic and fetal development are poorly understood, and since very substantial mutation loads were observed in the mtDNA at the earliest time point tested in this study, we have to consider the possibility that premature aging in this model could be predetermined at the prenatal, rather than postnatal, stage (developmental programming; Cameron and Demerath 2002).

As Aubrey de Grey (2004) has pointed out recently, the only tissues directly affected by the elevated mtDNA mutation rate in these mice are those that are mitotically active. This suggests that embryonic development can be compromised by high mtDNA mutational loads, as embryonic tissues undergo active mitosis. If this holds true, then the issue of a causative relationship between mtDNA mutations and normal aging is likely to remain open, since, in normal aging, accumulation of mtDNA mutations is not observed during embryonic development. Thus, a substantial mutational load in mtDNA during embryonic development might be an important limitation of this model, and a model with an inducible mtDNA mutator phenotype should help to resolve this and other outstanding issues. Finally, mtDNA mutations in this study are generated by a mutator Poly rather than by oxidative DNA damage. Therefore, this study does not address one of the central premises of the mitochondrial theory of aging, namely, that oxidative mtDNA damage is the driving force behind the accumulation of mtDNA mutations.

Earlier we discussed the support provided for the mitochondrial theory of aging by the discovery of mitochondrial disease. We might then reasonably expect, based on the mitochondrial theory of aging, that mito-chondrial ROS would be causative in a significant fraction of pathogenic mtDNA mutations. We have analyzed 188 pathogenic mtDNA point mutations (Brandon et al., 2005) and found that the mutagenic effect of 8-oxodG, widely regarded as a prime lesion resulting from an oxidative insult to DNA, can be implicated in the etiology of only a few mutations. Indeed, unrepaired 8-oxodG in mtDNA can pair with both C and A with almost equal efficiency resulting in G to T (and C to A on complementary strand) transversions, which account for only 5.9% of pathogenic mtDNA mutations. Even when the potentially mutagenic pool of 8-oxo deoxyguanosine triphosphate (8-oxo-dGTP, the product of cytoplasmic/ matrix dGTP pool oxidation) is taken in consideration (T to G and A to C transversions), the cumulative impact of both types of mutation is still only 8.5%. For comparison, 82% (almost 10 times as many) of the pathogenic point mutations in mtDNA can be attributed to deami-nation of adenine and cytosine. Similarly, it was found that 8-oxodG-mediated transversions can account for only about 25% of mutations detected in both frontal cortex and substantia nigra of old subjects (Simon et al., 2004). The efficient repair of 8-oxodG by BER pathways in mitochondria (Thorslund et al., 2002) explains these phenomena and argues against it being the prime mutagenic lesion. Transversions, the type of mutations caused by 8-oxodG, are rare events in both nuclear and mitochondrial DNA, which also argues against 8-oxodG being the prime mutagenic lesion. Thus, the exact factors required for the accumulation of point mutations in mtDNA are yet to be fully defined. Oxidative DNA damage can produce a variety of base lesions whose mutagenic potential has not been fully elucidated (Evans et al., 2004). Therefore, it is possible that other, at present unidentified, lesions are responsible for the bulk of ROS-mediated mutagenesis. Alternatively, it can be postulated that ROS do not play a major role in mtDNA mutagenesis.

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