The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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CHAPTER 12 Some Testable Predictions of MiFRA It has often been observed that gerontology is heavy on data but light on theory. A consequence of this is that, when a theory is propounded which fits the data already available, it may be unusually difficult to identify experiments to test it: the problem is that so many of the experiments have already been done in the attempt to inspire a theory in the first place. Nonetheless, several tests of MiFRA are available. Before introducing them, it may be worth stressing that each really only tests a component of MiFRA. In a sense this is inevitable for a theory with so much structure and detail; furthermore, it inspires confidence that, if MiFRA is broadly correct but some details are wrong, the falsification of those details will lead more rapidly to a corrected hypothesis than is typical when an entire theory has to be abandoned. 12.1. Some Predictions of SOS The hypothesis that SOS is the mechanism of mtDNA decline with aging makes a specific prediction with regard to which mutations will and will not accumulate in affected cells. As noted earlier, SOS is proposed to occur when the mutation has the effect of reducing the proton gradient at the inner membrane of its host mitochondrion. Any mutation that inhibits the respiratory chain will have this effect, but a mutation that inhibits the ATP synthase, without at the same time inhibiting the respiratory chain, will not. Therefore, point mutations in the two ATPase subunits should not accumulate in postmitotic cells. I noted in Section 8.5.4 that there is already preliminary evidence in support of this. Müller-Höcker has reported seeing cells which lack ATPase activity, but that all such cells had also lost cytochrome c oxidase activity. 1 However, this result must be considered preliminary since the antibody used stains the wholly nuclear-coded F1-ATPase as well as the Fº. Similarly, Schon has reported a failure of accumulation of point mutations in the region 8991-8995 within ATPase 6, determined by an assay which did show slight accumulation at a tRNA site 2 (see Section 6.6.4). Again, though, the fact that this study only assayed for mutations at five of the 844 base pairs in the ATPase genes weakens the confidence with which one can cite it in support of SOS. What is needed is a systematic survey of point mutations throughout the mitochondrial genome, to establish which do and do not accumulate. It must be stressed that such a survey should, if at all possible, look at mutations that are known (or seem very likely) seriously or totally to disrupt the relevant gene’s function. The preference of most studies to date has been to pursue point mutations which have been shown to cause inherited diseases, but the fact that such mutations are inherited strongly suggests that they have only a rather slight deleterious effect on gene function: if they were more serious, they would cause embryonic lethality. This is clear from an examination of the range of mutations so far identified as causing inherited diseases: all are missense mutations, which change one amino acid into another, as opposed to nonsense mutations which turn an amino acid codon into a stop codon and thereby truncate the protein 3 (see Section 6.6.5). The Mitochondrial Free Radical Theory of Aging, by Aubrey D.N.J. de Grey. ©1999 R.G. Landes Company.
160 The Mitochondrial Free Radical Theory of Aging It is also conceivable that certain phenotypically silent mtDNA mutations may affect the rate of SOS, by varying the tendency of the mtDNA to undergo spontaneous mutation (see Section 10.12). We do not as yet know what proportion of amplified mutations are deletions, but we do know that the most commonly seen deletions are flanked by direct repeats, and in particular that the longest relevant (see Section 2.4.5) direct repeat in the mtDNA, 13 base pairs, 4 flanks a deletion which is seen much more commonly than any other. 5 The 26 base pairs which form this repeat are not all “fixed”: some of them are third bases in protein-coding genes, which could be changed without affecting the encoded amino acid sequence. What they would be expected to affect, though, is the frequency with which the common deletion occurs, since the direct repeat would no longer be perfect. Thus, if it turns out that deletions are much commoner than point mutations in anaerobic cells, there would be value in assessing whether silent polymorphisms in these 26 base pairs correlate with longevity. Since such polymorphisms would be maternally heritable, such a study can make use of databases of historical records, such as that maintained by the Mormons in Salt Lake City. (That database has been used for such studies in the past). 6 One characteristic of mitochondria that is superficially promising for testing SOS, but less so on closer analysis, is the rate of mitochondrial turnover. Turnover rate was first measured over thirty years ago, 7-9 so the experimental aspect is not particularly problematic; what is difficult is to identify predictions of SOS that such measurements might test. An illustrative example comes from a recent comparison of turnover rates (or of their presumed effects) in the central nervous system versus the peripheral nervous system. 10 The CNS has higher energy utilisation and shows higher levels of mtDNA damage with aging than the PNS, but CNS mitochondria appear to be recycled more slowly than PNS ones. This might be thought incompatible with SOS, since SOS predicts that the rate of turnover is increased by more rapid membrane damage (which should correlate with mtDNA damage). But a simple resolution derives from the observation that the CNS must generate a lot of heat purely to maintain brain temperature, whereas PNS neurons do not need to do so since their surrounding muscle does it for them. This suggests that CNS mitochondria are probably maintained in a leakier state than PNS ones, which is most easily done by recycling them more slowly. The description of SOS given in Section 8.5.2 made no attempt to suggest a detailed biochemical pathway for the targeting of lysosomes to unacceptably leaky mitochondria. This is another area in which specialists in the relevant cellular components (lysosomes, in this case) may be able to lend weight for or against SOS by identification of such a pathway. Finally, it should be possible to test SOS in vitro using human cells. The experiments of Chambers and Gingold 11 (see Section 8.3) cannot be copied in every detail, because human cells are not so obliging as yeast in their cell cycle; but culture conditions should be obtainable in which the rate of cell division is substantially less than that of mitochondria, and so in which lysosomal degradation of mitochondria is occurring. The recent work of King’s laboratory 12 on cybrid muscle fibres (see Section 6.6.3) is a highly promising step in this direction. 12.2. Some Predictions of the Reductive Hotspot Hypothesis This component of MiFRA is rather less comprehensively supported by existing data, so is correspondingly more amenable to testing. One prediction is that the PMOR system itself should be much more active in cells which have lost OXPHOS function. Antibodies to some of the respiratory chain enzymes, notably cytochrome c oxidase (Complex IV) are widely available. Histochemical assays for the PMOR are likewise already established. 13 Studies of muscle tissue may be able, therefore, to establish whether PMOR hyperactivity colocalises with inactivity of Complex IV, as has
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COMPANY de Grey The Mitochondrial F
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ISBN: 1-57059-564-X MOLECULAR BIOLO
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7. The Status of Gerontological The
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17. Conclusion: The Role of the Ger
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I cannot overstate my profound grat
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CHAPTER 1 Introduction 1.1. What Is
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Introduction Some may feel that all
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6 The Mitochondrial Free Radical Th
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8 The Mitochondrial Free Radical Th
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10 The Mitochondrial Free Radical T
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18 Fig. 2.7. The respiratory chain.
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24 Fig. 2.9. Types of protein impor
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26 Fig. 2.10. Why the DNA bases pai
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CHAPTER 3 An Introduction to Free R
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An Introduction to Free Radicals Ta
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An Introduction to Free Radicals Fi
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CHAPTER 6 History of the Mitochondr
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History of the Mitochondrial Free R
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CHAPTER 7 The Status of Gerontologi
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The Status of Gerontological Theory
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The Status of Gerontological Theory
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CHAPTER 8 The Search for How Mutant
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The Search for How Mutant mtDNA is
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CHAPTER 9 The Search for How So Few
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The Search for How So Few Anaerobic
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The Search for How So Few Anaerobic
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Page 181 and 182: CHAPTER 15 Transgenic Copies of mtD
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Page 195 and 196: Prospective Impact on the Healthy H
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Page 203 and 204: Index Symbols “~”, 19 A Acetald
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MOLECULAR BIOLOGY INTELLIGENCE UNIT
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