The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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The Mitochondrial Free Radical Theory of Aging
line tended to behave the same way, indicating strongly that the nuclear genotype had a
strong influence on the relative selective advantage of the two genomes.
I personally feel, in the light of this and other studies, that the ability of this technique
to tell us about what happens in vivo in aging is less than many people claim; however, a
very recent variation on the technique shows promise of changing that pessimism completely.
The main challenge to the relevance to MiFRA of cybrid mtDNA segregation is that cybrids
are rapidly dividing, and we have long known that rapidly-dividing cells tend to accumulate
far less mutant mtDNA than non-dividing cells. Thus, any major selective advantage of
mutant mtDNA in cybrids is necessarily due either to some curiosity of the particular
mutation—which is in fact quite likely for those mutations that cause inherited diseases
(see Section 6.6.5)—or to some non-physiological aspect of the cultured cells’ environment.
The recent advance 73 is the use of cybrid technology on long-lived, non-dividing
cells—muscle fibers. It has long been known that muscle precursor cells—myoblasts—can
form into muscle fibers in culture but atrophy shortly afterwards. It is also long established
that this atrophy can be prevented, and the fibers kept alive for several months, by coculturing
them with neurons, particularly from the spinal cord. 74 What has now been achieved is the
combination of this technique with the cybrid technique. Muscle precursor cells are first
divested of their endogenous mtDNA, then repopulated with the mtDNA mixture of interest,
and finally induced to fuse and form muscle fibers, which are kept alive by the neurons.
It is thus now possible to study mitochondrial segregation in a population of genetically
well-defined postmitotic cells. I have high hopes that this will lead to rapid progress, not
least in the testing of the details of MiFRA that I will describe in later chapters.
6.6.4. Do mtDNA Point Mutations Accumulate?
Naturally, the discovery of clonal amplification led to a search for a plausible mechanism
whereby mtDNA mutations might derive their selective advantage. It was widely—and
reasonably—felt that this might be easier if there were more information about which
mutations accumulate during aging and which (if any) do not. Efforts to acquire such
information were hampered, however, by the absence of a reliably accurate technology for
measuring the concentration of a particular DNA sequence relative to another: standard
PCR technology can only identify molecules of a different length than normal, so it cannot
pick up point mutations. Also, even among deletions it was very clear that a great many
mutations were possible, and the available techniques (which until recently did not include
long PCR) could only assess the level of a few mutations per experiment. It was established
quite quickly that particular mutations tended to be present at very low concentrations—0.1%
or lower—even in postmitotic tissues of very aged individuals; but it was not possible to
extend this to an estimate of the total mutation “load” in such tissues, because one never
knew how many other, untested mutations were present at similar, or higher, levels or not at
all. 75 It eventually became possible to gauge the overall levels of mtDNA deletions, due to
the development of long PCR; but that still left open the question of the overall levels of
point mutations.
In due course, however, various refinements of PCR were developed which could identify
point mutations, and several were demonstrated to accumulate with age in the same
tissues. 76,77 But then, just as a consensus seemed to be forming on this point, it became
controversial again as a result of experiments in the laboratory of Eric Schon, which
established very convincingly that, in one particular section of the mtDNA, point mutations
seemed never to occur at all—or, if they did, then they were not preferentially amplified in
what others were coming to think of as the usual way. 78 Schon’s experiments used a PCR
variant which was only applicable to a few places in the mitochondrial genome, but which,
in those regions, was likely to give exceptionally accurate measurements of the real