The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

History of the Mitochondrial Free Radical Theory of Aging, 1954-1995
This is something of a paradox, since we know (from the rarity of fibers with reduced
but non-zero OXPHOS function—see Section 6.6) that the selective advantage of mutant
molecules is very great. One might therefore predict that these 4% of fibers with low levels
of mutant mtDNA would become completely OXPHOS-deficient within months, whereas
in fact even very elderly individuals never exhibit such levels.
The answer explains the title of this section. When a mtDNA mutation first occurs, it
probably has no impact whatever on its host mitochondrion’s function, because that
mitochondrion also possesses several wild-type genomes. Accordingly it is a matter of pure
chance, unbiased by any selective pressure, whether that mitochondrion divides (and its
mtDNA is replicated) or is destroyed by lysosomal digestion (see Section 2.4.6). If it does
divide, then it is again a matter of chance whether the two copies of the mutant molecule
segregate into the same daughter mitochondrion or one into each. Cumulatively, therefore,
the chance that a given spontaneous mutation will end up homozygous in some
mitochondrion is rather low, since it involves several mitochondrial divisions going the
right way. It is probably not until then that the mutation will have any significant effect on
its host mitochondrion’s OXPHOS functionality. In fact, that functionality is likely to be
largely retained for a couple of mitochondrial generations even after homozygosity is
achieved, since some of the host mitochondrion’s membrane proteins are inherited from
previous generations when it still had some wild-type genomes.
This means that, on average, a given mutation must have survived for a considerable
while by pure luck in order to exert a phenotypic effect, and thus in order to begin enjoying
a selective advantage. Most mutations will be snuffed out (by chance, not by any selective
pressure) before this. But many of those that are snuffed out will progress part of this way,
being randomly replicated up to five or ten copies. And that sort of level, among a total of
perhaps 10 4 or 10 5 molecules total in a sample, is enough to be detected by PCR. This means
that the high proportion of fibers which harbour detectable levels of mutations are not a
paradox after all: most of those mutations were not destined to take over their cellular
environment anyway.
6.6.3. Cybrids
An in vitro technology was developed in the 1980s 68,69 which has had a dramatic impact
on, among other things, the study of selective advantage of mtDNA species. King and Attardi
developed a method for removing all the mtDNA in a human cell culture and yet keeping
the culture alive. A summary of the technique appears in Section 9.2; its relevance to this
chapter is in how these cells (termed ρ 0 cells) have been used to study mtDNA selection. The
value of ρ 0 cells is that one can repopulate them with mitochondria of one’s choice. Thus,
for example, if one has a patient suffering from a mtDNA-linked disease, whose cells contain
some mutant mtDNA and some wild-type, then one can take platelets 70 (which have
mitochondria but no nucleus), or other cells whose nucleus has been artificially removed,
and fuse them with ρ 0 cells. The resulting fusion cells are called cytoplasmic hybrids, usually
contracted to cybrids. Then one can follow the relative abundance of two species of mtDNA
in a culture of these fused cells; and, crucially, one can repeat the experiment with a different
source of mtDNA, from a different patient, but with the same ρ 0 line, thereby eliminating
complications of interpretation due to differing nuclear genotypes. It was quickly
demonstrated 71 that the mutant mtDNA genotype of a well-studied mutation often took
over such cultures.
Unfortunately, since then the picture has become altogether muddier. A study 72 which
essentially repeated this experiment found that some such cultures behaved as reported, 71
but others exhibited the opposite behaviour—mitochondria of the mutant genotype
dwindled in number. Moreover, multiple cultures created from the same recipient (ρ 0 ) cell
77