From: Rafal Smigrodzki (rafal@smigrodzki.org)
Date: Wed May 28 2003 - 19:05:17 MDT
Aubrey de Grey wrote:
> Subject: RE: Removing lysosomal aggregates; obviating mitochondrial
> mutations
> Rafal Smigrodzki wrote:
>
>> ### Do you have a hypothesis as to what this difference might be?
>> What are the postulated non-degradable molecular species?
>
> Quite a few are known; luckily, one of my prospective collaborators on
> this work, Keri Carpenter, has been at the forefront of research on
> the chemical composition of atheroma for many years. Mostly they are
> oxidation products of cholesterol, such as 7-ketocholesterol.
### But is 7-ketocholesterol the non-degradable molecular species?
According to: Lyons MA. Brown AJ.Metabolism of an oxysterol,
7-ketocholesterol, by sterol 27-hydroxylase in HepG2 cells.Lipids.
36(7):701-11, 2001 Jul., it is well-metabolized in the liver.
---------------------
>
>>> A key question is, are there any other comparably legitimate
>>> hypotheses?
>>
>> ### The dynamic equilibrium hypothesis.
>
> I don't think you ever explained how that hypothesis can be reconciled
> with the appearance of fatty streaks so young. That's my main problem
> with the hypothesis -- the advance of lipid deposition seems to be a
> continuous, lifelong process.
### If the balance is tilted towards formation of foam cells (e.g. by
ingestion of dietary poisons, like trans-fatty acids, or by genetic factors
in familial hypercholesterolemias), you will have them early on, and you
could have your first heart attack in grade school (or in your forties if
you have only margarine going against you). Dynamic equilibrium integrates a
number of factors and processes, and predicts shifts in the average age of
symptoms onset, as well as at least partial reversibility.
We already discussed reversibility of atherosclerosis, I think. Since you
can observe a bulk reduction in the atheromas in human carotids with some
dietary interventions (vit. B supplementation) as measured by Doppler
sonography, the majority of pathologically relevant material responsible for
artery occlusion is apparently digestible and is removed if the equilibrium
is forced back towards lipid removal. If there are truly indigestible
molecular species present in the atheroma, they would form only a part of
it, and would not have a pathological significance in the age range that
humans currently achieve.
-------------------------------
>
>> ### I found the following: Homma S. Ishii T. Tsugane S. Hirose N.
>> Different effects of hypertension and hypercholesterolemia on the
>> natural history of aortic atherosclerosis by the stage of intimal
>> lesions. [Journal Article] Atherosclerosis. 128(1):85-95, 1997 Jan 3.
>> Apparently even at around age 79 you still find people with no
>> intimal lesions. How did they escape their accumulation?
>
> Thanks for this reference. I would need to consult someone like Keri
> to be sure, but I think the explanation is just random sampling of a
> few sites within the aorta rather than examination of the whole aorta.
> According to this paper's Fig 2, 2/3 of those in their 20s had no foam
> cells; this is ostensibly at odds with the consensus (stated in, for
> example, Jake Lusis's review in Nature 407:233) that they are usually
> present in the first decade.
### Lusis mentions the early fatty streaks without qualifications - but
AFAIK, there are large differences in the age of onset of atherosclerosis
between ethic groups. The Japanese, who were the subject group in Homa's
study, seem to develop lesions later (presumably due to lifestyle factors).
Homa didn't do a random sampling of the aorta - all samples were taken from
predefined locations.
-----------------------------------
>
>> ### I don't understand. The mutants take over mainly as a result of
>> stochastic processes, with no clear mitochondrial proliferative
>> response to partial loss of function. There is a substantial
>> functional reserve of mitochondria, and the nucleus seems to
>> maintain a constant mass of genomes (at least in some experimental
>> paradigms), so there is no mitochondrial hyperproliferation until
>> very late in the process, if at all. Since the stochastic hypothesis
>> makes no reference to the cause of hyperproliferation, merely
>> predicting the emergence of COX-negativity, it is compatible with
>> either hyperproliferation before COX negativity, after, or none at
>> all, if the cell simply decides not to upregulate mitos. These two
>> processes are most likely largely independent, over large range of
>> heteroplasmy levels.
>
> No, Chinnery's model *does* specify the cause of hyperproliferation.
> He says that the cell maintains the same number of *functional* mt
> genomes. Hence, he concludes that the random fluctuation of wild-type
> versus mutant genomes will push the number of mutant genomes up along
> with the wild-type when the wild-type randomly diminish, but won't
> push it down again when the wild-type randomly exceed the necessary
> number. Whereas in real life, just as you say, no clear mitochondrial
> proliferative response to partial loss of function is seen.
### Chinnery specified the "maintenance of wild-type mechanism", yes, but
AFAIK he didn't say that random fluctuations are irreversible and cannot
push the total number of genomes back down (except when driven to
homoplasmy). His model applies to functionally neutral mutations (Am J Hum
Genet 2001; 68: 80206.), with the maintenance of wild-type being an add-on
to the hypothesis, meant to help explain MERRF-like phenomena. There are
threshold effects for derangements of mito function by various mutations,
ranging from 30 to 95% mutated genomes needed to produce a detectable
pathology. The cell cannot read the quality of its mtDNA genomes directly
(at least, so we believe), so it must use a biochemical sensor to decide
when to push mtDNA replication higher. Since the biochemical derangements
appear late because of the thresholds, even if the cell tries to maintain a
certain constant level of mito function, it will only react with
proliferation to advanced heteroplasmy, and in an inconsistent, tissue- and
mutation-specific manner, just as seen in real life.
-----------------------------
>
> He has a new paper out about this in J Theor Biol which explains and
> extends the model somewhat.
### Is it in review? I can't find it on Medline.
----------------------------------
>
>> ### In the CNS there are neurons with enormous differences in
>> cellular mass, from tiny interneurons to the giant alpha motor
>> neurons. The giant ones do have a very active metabolism. If indeed
>> single deleterious mutations had a significant replicative
>> advantage, the large neurons would die much faster than the
>> interneurons, which is not the case.
>
> Um, hang on -- there are at least three things wrong with this logic.
> First, neurons die of lots of things, not just OXPHOSlessness.
> Second, they don't necessarily die even of OXPHOSlessness, because if
> they did then we wouldn't see COX-negative ones accumulating with
> age. And third, most brain regions (of whatever size) have far fewer
> COX-negative neurons than the substantia nigra. It could be that
> this is because they resist apoptosis in the substantia nigra more
> than elsewhere, but chances are that it's mutation rate, because of
> the H2O2 production associated with dopamine synthesis.
### Well, one way or another, large neurons would be generally much more
likely to be either COX-negative or dying, by more than an order of
magnitude.
----------------------------------
>
>> Also, we know that the mutation level in single neurons in elderly
>> adults is in the range of 50 to 800 diverse aminoacid-changing
>> mutations per million bases (according to Simon and Beal), meaning
>> that more than ninety percent of genomes are mutated, and without
>> doubt many of them are deleterious. This leaves little place for a
>> preferential amplification of low psi or otherwise impaired mitos.
>
> Ah, not so fast. Are you referring to Hum Mol Genet 11(2):133? I'm
> keeping an open mind about that study until the appearance of a ms in
> preparation that that article cites, but it's not clear to me that
> they made a compelling case for the reliability of their quantitation.
> For example, they made no allowance for the isolation of mtDNA that
> was in the process of being degraded because of damage (see Suter and
> Richter, Biochemistry 38(1):459). The acid test is the single-cell
> analysis being done so effectively by Khrapko's group, who have now
> started serious work on neurons (see Mech Ageing Dev 123(8):891 as a
> starting point). It's possible that only deletions clonally expand
> and point mutations don't, but I doubt it.
>
### Yes, Hum Mol Genet 11(2):133, and Simon & Beal did a poster at the
recent AAN meeting, showing more data on single neurons and homogenates. I
also refer to our unpublished data which agree with their findings.
According to the poster, each single cell has an accumulations of a small
number of types of mutations but each present at higher frequency (10-15%),
which in the homogenate changes to thousands of types of mutations present
each at a very low level (less than 1%).
Thanks for the Suter ref - very useful (actually I should say - extremely
useful, I just added it to the discussion in my article). Khrapko seems to
lean towards the random drift model in the article you quoted.
The possibility that the mutation rates calculated for mtDNA could be
influenced by oxidative damage to the DNA has to be taken seriously, of
course, but our data show that there is a significant difference between the
frequency of mutations in the D-loop and in the protein-coding regions, by
almost an order of magnitude. If all the mutations we observe were due to
degradation of the DNA, then we would expect that the frequency would be the
same for both areas, unless there is a significant influence from e.g. SSB
vs. MTFA interactions. Also, the size of the fragmented, highly-oxidized
mtDNA in Suter's article is about .2 to .5 Mb. We amplified fragments of 1
kb to 4 kb, and gel-purified the DNA to exclude smaller fragments, in both
cases finding the same overall mutation frequencies, indicating that the
mutations are not from the oxidized mtDNA which would be excluded by our
approach, especially with the longer PCR products. Finally, most types of
DNA damage seem to block PCR rather than introduce errors, which is the
basis of some methods for adduct detection.
BTW, I just read the abstract of your paper in Protoplasma - sounds
intriguing, but the full text is not available online. Could you send me a
copy?
Rafal
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