The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

History of the Mitochondrial Free Radical Theory of Aging, 1954-1995
most often muscle, which he analyzed in various ways—with DNA probes, with antibodies,
or with enzymatic assays—in order to identify the distribution of certain mitochondrial
defects. He made two pivotal discoveries. The first of these was that the loss of mitochondrial
function which others had detected was not distributed evenly across the tissue, but was
localised in just a few cells or muscle fibres, and that these fibres (actually, as it turned out,
short segments of them) were totally devoid of aerobic respiration. 54 Even stranger was
that he only occasionally detected fibres in which the level of respiration was reduced but
non-zero. 54,55 This showed that, if the vicious cycle theory was correct, then (a) it went on
in each cell independently of its neighbours, and (b) it was very, very vicious. Cells independently,
for whatever reason, reached a point where the cycle took off; after that, they must plummet to
aerobic oblivion in (at most) a matter of months, or else we would catch more of them in the
act of plummeting.*
The second, and even more important, observation came when he assayed the same
sample of muscle with two different mtDNA probes. 57 He found that the cells which were
completely lacking in respiration were lacking for genetically different reasons. In one such
cell there would be almost complete loss of hybridisation to one probe but absolutely normal
reaction with the other, while in another cell it would be the opposite way around.
Some cells had lost affinity for both probes; some had lost neither (indicating a mutation
elsewhere). This meant that the vicious cycle theory had to be radically revised. It was no
longer possible to say that stress caused more mtDNA mutations causing more stress,
because a cycle like that would necessarily give a virtually identical spectrum of mutations
in each and every affected cell, irrespective of which ones were present first in a given cell.
We see the exact opposite—each cell taken over by, ostensibly, just one mutation. (A single
mutation was able to remove both probed regions in Müller-Höcker’s experiments, by
being a large deletion or a complete loss of the whole mtDNA (mtDNA depletion, of which
more in Section 10.11); in histochemical assays the same effect would also result from
mutation of a mitochondrial tRNA gene.) This meant that there was not an intracellular
vicious cycle—at least, not at the level of mtDNA mutation. Rather, the cell was somehow
allowing its mitochondrial population to be taken over by copies of a single initial mutation:
the mutant molecule was somehow experiencing a selective advantage.
It is appropriate—and salutory—at this point to take a brief leap back in time. In
1974, just two years after Harman had suggested the involvement of mitochondria in the
free radical theory, Comfort 58 identified the existence of mitochondrial turnover as a serious
challenge to that possibility. He reasoned that damaged mitochondria would obviously be
got rid of and replaced by functional ones, so damage could never accumulate. He was
wrong, but his point that mutant and wild-type mitochondria have independent
propensities to be maintained in the cell—they compete, effectively—was key. In hindsight,
it is highly unlikely that the two genotypes will be exactly equally matched in this context,
so one or other will inevitably win out; since we see an accumulation of mutant mtDNA,
we should have immediately expected that it would be caused mainly by clonal amplification.
Yet this idea was not picked up on for twenty years.
*Another way, not involving intact tissue samples, in which it was established that mutations are not
distributed evenly across all cells is to analyse the DNA of only a few cells. If a homogenate of many thousands
of muscle fibres is analysed, it is found always to contain the “common deletion” (see Section 2.4.5) at levels
around 0.1%. If the same sample is divided into small bundles of only ten fibres, however, nearly all the
bundles show no evidence of the deletion whereas one or two show high concentrations of it. 56
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