The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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The Mitochondrial Free Radical Theory of Aging
The amplification phenomenon has since been studied in some detail in a variety of
systems. 61-62c Several subtleties have emerged which have a large impact on the search for a
plausible mechanism; the rest of this chapter deals with these details in turn.
6.6.1. Segmental OXPHOS Deficiency in Muscle
Müller-Höcker’s studies on muscle all involved staining of a sample of muscle that was
cut perpendicular to the direction of the fibers, so that each fiber appeared as a disc. (This is
termed a transverse section of the muscle.) A different approach is to dissect a sample
longitudinally: to extract an entire fiber (or a substantial length of it) from a sample and
stain it all the way along. This technique 59-61 reveals that the loss of, for example, cytochrome
c oxidase activity does not extend throughout the fiber, but is localised to short segments,
almost always under 1 mm in length. (The same result can be obtained by studying transverse
sections if a series of consecutive sections are lined up so that the slices through a particular
fiber can be identified.) 62a The obvious question arises: if the mutation is clonally amplified
within part of the fiber, why not within all of it?
This distribution is not so curious, however, when one considers the structure of muscle
fibers. They are syncytial, comprising a single cytosolic compartment, but they have nuclei
dotted along their entire length. Thus, it is theoretically possible for each nucleus to maintain
its own mitochondrial population independent of the other nuclei to either side. One might
imagine that mitochondria would randomly move along the fiber, into neighbouring nuclei’s
fiefdoms, so that a mutant with a selective advantage would march along taking over
successive sections of fiber. This may indeed happen, since the regions very near to an affected
segment are unusually prone to morphological changes, 61 but if so then it is clearly very
slow, or else much longer mutant segments would sometimes be observed. A simple
explanation can be seen, however, from the way that muscle fibers arrange their mitochondria.
They do not float free in the cytosol, but are attached to the contractile filaments to which
they supply energy. 63 They must necessarily be free for short periods after mitochondrial
division, but once fixed in place they may stay put effectively forever. The tight packing of
mitochondria may also impede the diffusion of cytosolic ATP along the fiber.
6.6.2. Rate of Acquisition of Selective Advantage
A shortcoming of in situ assays of tissue is their low sensitivity. One can detect a mtDNA
deletion by in situ hybridisation to a mtDNA segment within that deletion only if the deletion
is present in large quantity in a cell, so that the wild-type mtDNA (which is the molecule to
which the probe hybridises) is perceptibly depleted. It is theoretically possible to design
probes that bind selectively to the deleted mtDNA, so allowing detection down to lower
levels: such probes identify the new junction between the ends of the deletion, which is not
present in the wild-type molecule. But in practice this technique is rather inexact, largely
because the most common deletions are formed by illegitimate recombination between
direct repeats (see Section 2.4.5), so that the new junction is in fact a sequence that is
present—twice—in the wild-type molecule.
Much lower levels of mtDNA mutations can be detected, however, if the mtDNA is
extracted and subjected to the polymerase chain reaction (PCR). 64 Moreover, by using a
relatively new variation called long PCR, 65 which is capable of amplifying the whole mtDNA
in one go, one can identify most of the different mtDNA deletions present in a tissue sample,
in contrast to the original technique which could only detect a small proportion of sequence
variants. 66,67 When this technique is applied to one or a small number of muscle fibers, 56,62
it is found that, though the proportion of fibers with high levels of any deletion is comparable
to that seen by in situ hybridisation, a much higher proportion—perhaps as high as
4% 56 —carry some deletion at very low levels.