Rebuttal to Jacobs: the mitochondrial theory of aging: alive and well.

We agree with much of what Dr Jacobs argues but would like to comment on some of his statements. He makes many valid comments, comments that were very relevant 4–5 years ago. Studies based on tissue homogenates are, as he mentions, very difficult to interpret and it is exceedingly difficult to correlate mitochondrial changes with physiological outcomes based on those experiments. We would, however, argue that advances in methodologies along with increased attention to the fate of specific cells has greatly enhanced our ability to link changes in the mitochondrial genome with the aging process. The relatively high abundance of cells affected in the mitochondrial myopathies makes it much easier to envision how cellular dysfunction results in tissue dysfunction in these human diseases. The lower frequency of cells affected by mtDNA deletion mutations in aging tissues has resulted in many researchers questioning whether these mtDNA deletion mutations have any physiological relevance. In the past few years, however, there has been a considerable shift in experimental design. Instead of focusing on tissue homogenates, studies have emphasized the analysis of individual cells. These studies indicate that mtDNA deletion mutations and single nucleotide mutations clonally accumulate to high levels within single cells. Our studies indicate that mitochondrial DNA deletion mutations have a physiological impact on skeletal muscle, leading to fibre atrophy and, ultimately, loss of the cells. Rather than rely on in vitro approaches to the analysis of the role of mtDNA mutations in the aging process, we have taken a two-pronged approach in our in vivo studies. The nature of the mutation event and accumulation makes it impossible to identify and then follow a specific mutation event with time (due to the focal and segmental accumulation of the mutations). We use two different animal models, rhesus monkeys and rats, and follow both through aging. For the rhesus monkeys, muscle biopsies are collected at 3-year intervals. Our initial analysis of these samples suggests that the number of ETS abnormal fibres and the number of mtDNA deletion mutations increase with increasing age. For the rat studies, cohorts of animals have been followed longitudinally starting at 18 months of age and analysed at 3-month intervals. In these studies, a variety of different muscle groups (selected based on the degree to which they undergo sarcopenia) have been analysed with respect to mtDNA deletion mutation abundance as well as the number of ETS abnormal fibres. For all samples, a number of tissue sections are characterized such that 1000 μ m of tissue, at 10μ m intervals, are examined. Not only have we demonstrated that the extent of sarcopenia (i.e. degree of muscle fibre loss) is correlated with the number of ETS abnormal fibres and fibre atrophy, we have also clearly demonstrated the link between the presence of ETS abnormalities and the mtDNA deletion mutation (Cao et al ., 2001; Wanagat et al ., 2001; Bua et al ., 2002). The long-term effect of the mtDNA mutations is often subtle, with, in the case of skeletal muscle, a relatively small number of fibres being affected at any one time. Aging is a gradual process. We and others would not argue that the ‘loss of a single neurone might destroy the integrity of a complex neural circuit’ but rather that, with age, the cumulative effect of the loss of neurones would result in physiological declines. A rapid loss of large numbers of neurones, skeletal muscle fibers or other cells would not be aging, but disease. The declines observed with aging are, by definition, much subtler than those observed in disease. The mechanism by which the mtDNA deletion mutations occur is not yet known. It is a very difficult question to address in aging tissues as the event is probably random and occurs at a very low frequency in vivo . Owing to the relatively low frequency of the deletion events in aging tissue, it would be impossible to identify, using currently available methods, the initial deletion event. We have, however, circumstantial evidence to suggest that the initial mutation event is due to oxidative damage. It is generally accepted that caloric restriction decreases oxidative damage and, hence, increasing lifespan. When muscle from rats that have been calorically restricted are compared to age-matched controls, the number of ETS abnormal fibres and the number of mtDNA deletion mutations are reduced. Caloric restriction does not, however, affect the cascade of events that follow a deletion event, i.e. ETS abnormalities, fibre atrophy, etc., all accrue in a pattern similar if not identical to that of ad libitum fed animals. Our studies in rat muscle have also demonstrated a correlation between fibre atrophy and increased levels of oxidative damage, as indicated by the presence of 8-hydroxydeoxyguanosine (Wanagat et al ., 2001). At this time, however, we do not know Correspondence Judd M. Aiken, Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA. Tel.: (608) 262 7362; fax: (608) 262 7420; e-mail: aiken@ahabs.wisc.edu