cron-web.org

[A1CR]

[From Summer 2003. Posted on behalf of MR and CRON4healthyfuture]

mtDNA copy number does not change with age; Try again de Grey

CRON4healthyfuture wrote:
>
> These Australians [see below for Abstracts] very carefully analyzed mtDNA copy numbers per cell,
> and found nothing changed with age. In de Grey's original
> conceptualization of aging, the mitochondrial "common deletion" comes
> in and formats the hard drives in all of your mitochondria. But these
> Australians say those hard drives are just as bloated as ever as the
> years roll on by........
>
> I know, I know, the "automatic" stimulus-response retort from a de
> Grey-o-phile would be "Well, a handful of rogue mitochondria is all
> you need.....maybe they wouldn't show up"
>
> Yeah, right.

MR responds:

CRON4healthyfuture, as usual, it'd really be helpful if you actually read de Grey before criticizing his ideas. The athors of this abstract do not assert that mtDNA deletions do not accumulate with aging (a well-established finding on which de Grey's theory is grounded); in fact, they positively assert it -- not as an experimental finding, but as a *given* which they didn't explore. What they have observed is that the total number of mtDNA molecules/cell doesn't change with age -- and nothing in de Grey's theory suggests that it would.

-MR

Nucleic Acids Res. 2003 Jun 1;31(11):e61. Related Articles, Links

Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age.

Miller FJ, Rosenfeldt FL, Zhang C, Linnane AW, Nagley P.

Cardiac Surgical Research Unit, Alfred Hospital and Baker Heart Institute, Commercial Road, Prahran, Victoria 3181, Australia.

Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy beta-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 +/- 920, significantly higher than that in skeletal muscle, 3650 +/- 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.

PMID: 12771225 [PubMed - in process]

[A1CR]

On 28 Jun 2003 MR wrote:

>CRON4healthyfuture, as usual, it'd really be helpful if you actually read de Grey >before criticising his ideas.

CRON4healthyfuture responds:

I'm sorry, I just don't but into the dog turd that is his theory. The fact that the guy doesn't run his own lab tells you everything about his "real" understanding of biology.

He is a dreamer, and there is nothing wrong with dreamers. But most of the greatest contributors to the sciences and the arts were people who toiled in the labs and studios, respectively, and took the next steps for society through tinkering and serendipitous discovery. Not through armchair sophistry and didactic sophistry, but through "roll up your sleeves" hard work. The only thing de Grey works hard at is Othello.

>What they have observed is that the total number of
>mtDNA molecules/cell doesn't change with age -- and nothing in de Grey's
>theory suggests that it would.

Well help me understand de Grey's theory even more than I already do.
Would not the progression towards mitochondrial homoplasmy with deletion-
bearing mtDNA molecules effectively reduce total mtDNA copy number, a
finding that could have been experimentally detected with the technique
established by the investigators in this most recent paper?

[A1CR]

On 29 Jun 2003 another CRONie wrote:


>>Would not the deletions simply reduce the mitochondrial DNA size, but not
>>copy number?

CRON4healthyfuture responds:

Well, that's what I thought at first, but then I noticed that just picked an
arbitrary point on the mitochondrial genome, the cytochrome b gene, and saw
how many times that could be detected using polymerase chain reaction
amplification.

Presumably, if you choose a gene like this as a reference point, that
"reference point" would "disappear" once the "common deletions" start comin'
down the pipeline in association with the "de Grey" conceptualization of
aging.

But, cytochrome b is apparently *not* affected by the common deletion, so
technically, the small number of mitochondria with the common deletion would
not even be seen in this type of assay. Even if there were a lot bearing
this mutation, their choice of cytochrome b would hide that fact from them.

However, that said, I still do not see the importance of mtDNA to the aging
process if ATP production and H2O2 production in the heart are not
modifiable in a significant way through caloric restriction.

[A1CR]

CRON4healthyfuture made the comment that he would not read deGrey's book because he does not read "fairy tales" So somone asked:

>> But... how do you know it's a fairy tale? Who told you?

CRON4healthyfuture responds:

I used to try and find things out for myself, but that was before I found
out a little secret that nobody told me: forming your own opinions is a
complete waste of time!

I wish somebody had told me many years ago that forming your own opinions
was a complete waste of time. Why have your own opinions when there are
plenty of folks out there waiting to give you theirs! I can save so much
time and energy by simply taking their opinions and using them off the
shelf! Boy, this sure makes life so much easier! I'm glad I have found
this new way of settling life's "controeversees", because it sure makes my
head hurt less!

>>Was there a research paper claiming that?

"Screening for the presence of the common deletion yielded no significant
differences in relative distribution, neither between astrocytes and
neurons, nor between healthy young and old humans." - PMID: 12559408

"Using a highly sensitive PCR methodology, we identified the presence of
the CD in leukocytes from all 71 individuals (age range-8 months-99 years)
including all infants and children (n=15) which in addition were free of
any known mitochondrial diseases. This is important because the few reports
of the CD in infants have been linked to mitochondrial disease. These
results question the significance of the CD as a universal DNA marker of
ageing and subsequent mitochondrial dysfunction and provide support for the
possibility for maternal transmission of deletions." - PMID: 12742534

"The occurrence of large deletions in mtDNA from brain, skeletal, and heart
muscles and other tissues of old subjects at relatively low levels has been
well documented. We discuss their POSSIBLE functional relevance for the
aging processes." - PMID: 12729587

"However, the amount of intact mtDNA, the mRNA of all mitochondrially
encoded subunits of the entire respiratory chain, the amount of mtTFA, and
the enzymatic activity of complex III and complex IV, which also contain
mitochondrially encoded subunits, were normal compared with donor hearts,
excluding generalized disturbance of mitochondrial gene expression.
Retrospective analysis of drug therapy before transplantation identified
beta-blockers as one putative protection against this disturbance.
CONCLUSIONS: In terminally failing human myocardium of patients receiving
drug therapy, complex I depression is not caused by mtDNA damage and
disturbed mitochondrial gene expression. The ABSENCE of mtDNA damage should
facilitate recovery of the overloaded myocardium, if effective unloading
could be achieved." - PMID: 12505231

"In situ hybridization using wild and chimera (5 kb) mtDNA probes revealed
positive signals in damaged mitochondria from the vascular endothelium and
in perivascular cells of lesioned microvessels close to regions of large
amyloid deposition. These features WERE ABSENT in undamaged regions of
human AD tissues, YAC and C57B6/SJL Tg (+) mouse tissues, and in *AGE-
MATCHED* control subjects." - PMID: 12480733

"There was no significant difference in the number or distribution of
neurons with the common deletion or the average of the mean densities (AMD)
of staining with the common deletion in nigral neurons among patients with
PD, MSA-P, PSP, DLB, or age-matched controls. In addition, there was no
difference in the number or distribution of neurons with the common
deletion in nigral neurons between any age group, although there was a
tendency for the common deletion to increase in the non-nigral neurons in
older patients. These data indicate that accumulation of the 4977-bp common
deletion in mitochondrial DNA in midbrain occurred primarily in neurons,
and by this cytological approach, it was NOT ASSOCIATED with nigral
neurodegeneration in the common movement disorders or aging." -
PMID: 12039426

"Lifelong caloric restriction, which prolongs maximum life span in animals,
did not attenuate the age-related decline in ATP content or rate of
production in skeletal muscle and had no effect on the heart. 8-Oxo-7,8-
dihydro-2'-deoxyguanosine in skeletal muscle mtDNA was unaffected by aging
but decreased 30% with caloric restriction, suggesting that the mechanisms
that decrease oxidative stress in these tissues with caloric restriction
are independent from ATP availability. The generation of reactive oxygen
species, as indicated by H2O2 production in isolated mitochondria, did not
change significantly with age in skeletal muscle or in the heart." -
PMID: 12388443