8 replies to this topic

[maestro949]

Nature Genetics Abstract

Mitochondrial point mutations do not limit the natural lifespan of mice

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate. Here, we use a new, highly sensitive assay to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single–base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice.

Letter - Nature Genetics


The Scientist Article

New data contradict aging theory

Point mutations in mitochondrial DNA do not cause signs of aging in mice, according to a report in this week's Nature Genetics. The data, which contradict a prominent theory that mitochondrial mutations drive the aging process, show that mice with mitochondrial mutations 500 times higher than normal levels do not show signs of premature aging.

The finding "supports the idea that the accumulation of these mutations might be correlated with aging but [are] not causative," said Eric Schon of Columbia University in New York, who was not involved in the study.

Previous work has led to the mitochondrial theory of aging, which says that mitochondrial DNA mutations throughout life eventually cause the decline in tissue functioning associated with aging.

During the study, Marc Vermulst of the University of Washington in Seattle and his colleagues measured the accumulation of mitochondrial DNA mutations using a new technique -- an adaptation of the random mutation capture (RMC) assay, a quantitative PCR-based technique that amplifies single molecules to detect mutations but is not limited by polymerase fidelity.

They found that mutation frequency in mouse mitochondria is more than 10 times lower than that reported in previous studies, suggesting earlier work overestimated the rate of these mutations. "The technique we are using is much more sensitive" than previous assays, which had very high background levels, Vermulst said.

The authors compared the mitochondrial mutation frequency in wild-type mice to that in "mutator" mice with abnormally high levels of mitochondrial mutations. Mice homozygous for the mutant gene have 2,500 times the mitochondrial mutations as wild-type mice and also show reduced lifespan and several features of premature aging. However, mice with only one copy of the mutated gene showed no signs of premature aging, even though they have 500 times as many mitochondrial mutations as wild-type mice.

"The homozygous animals have crossed a certain threshold at which these mutations now cause premature aging syndrome," Vermulst said. "The heterzygous animals are below that but are still very high compared to wild-type animals," and still don't age prematurely. This suggests that wild-type animals, with their relatively small rate of mitochondrial mutations, could never accumulate enough to cause aging symptoms, premature or otherwise, he added.

Since their assay examined point mutations only, it remains possible that large deletions in mitochondrial DNA could still underlie aging, said senior author Lawrence Loeb, also of the University of Washington.

The study's result is "a problem for the mitochondrial theory of aging, at least with regards to point mutations," Schon told The Scientist. However, the mutator mice might not be perfect models for aging, he added. These mice begin accumulating mitochondrial mutations early in embryonic development, while normal animals don't accumulate them until after birth.

Given that mutated mice get an early start to these mutations, it's possible that embryonic cells in the mutator mice could "figure out a way to adapt to mutations that adult animals" could not tolerate, agreed Peter Zassenhaus of St. Louis University, who was not involved in the study. "We need to know what the mechanism is" through which mitochondrial mutations might influence tissue function, Zassenhaus told The Scientist. "Without really understanding how these mutations can cause a disease or pathology, it's hard to interpret the mutation frequencies by themselves."

The type of mutation induced in the mutator mice may also affect the results, according to Konstantin Khrapko of Beth Israel Deaconess Medical Center in Boston. These mice have defects in a mitochondrial DNA polymerase, which is not involved in naturally occurring mitochondrial mutations. "These mutations may accumulate in different cell types" than natural mutations, Khrapko told The Scientist in an Email.

These findings present a problem for researchers who believe that mitochondrial DNA mutations are "a central mechanism driving mammalian aging," noted Khrapko, who was not a co-author. But the results don't damage a "softer theory," he added -- that some types of mitochondrial mutations may affect aging only in certain tissues of certain species.

The Scientist

[Karomesis]

so...mutations are different than damage?

or am I misunderstanding?

[niner]

Interesting report. I think the key questions are at the end; perhaps not all mutations are equal with respect to aging, and the mutations due to faulty polymerase may be different than random mutations. Still, nice work, I hope it gets some followup.

[zoolander]

so...mutations are different than damage?

There was a study released in 2006 where a naked mole rat with high oxidative damage in tissue lived just as long as it's distant relative mouse.

Aging Cell. 2006 Dec;5(6):463-71. Epub 2006 Oct 27.

    High oxidative damage levels in the longest-living rodent, the naked mole-rat.

        * Andziak B,
        * O'Connor TP,
        * Qi W,
        * DeWaal EM,
        * Pierce A,
        * Chaudhuri AR,
        * Van Remmen H,
        * Buffenstein R.

    Department of Biology, City College of the City University of New York, New York, NY 10031, USA.

    Oxidative stress is reputed to be a significant contributor to the aging process and a key factor affecting species longevity. The tremendous natural variation in maximum species lifespan may be due to interspecific differences in reactive oxygen species generation, antioxidant defenses and/or levels of accrued oxidative damage to cellular macromolecules (such as DNA, lipids and proteins). The present study tests if the exceptional longevity of the longest living (> 28.3 years) rodent species known, the naked mole-rat (NMR, Heterocephalus glaber), is associated with attenuated levels of oxidative stress. We compare antioxidant defenses (reduced glutathione, GSH), redox status (GSH/GSSG), as well as lipid (malondialdehyde and isoprostanes), DNA (8-OHdG), and protein (carbonyls) oxidation levels in urine and various tissues from both mole-rats and similar-sized mice. Significantly lower GSH and GSH/GSSG in mole-rats indicate poorer antioxidant capacity and a surprisingly more pro-oxidative cellular environment, manifested by 10-fold higher levels of in vivo lipid peroxidation. Furthermore, mole-rats exhibit greater levels of accrued oxidative damage to lipids (twofold), DNA (approximately two to eight times) and proteins (1.5 to 2-fold) than physiologically age-matched mice, and equal to that of same-aged mice. Given that NMRs live an order of magnitude longer than predicted based on their body size, our findings strongly suggest that mechanisms other than attenuated oxidative stress explain the impressive longevity of this species.

    PMID: 17054663 [PubMed - indexed for MEDLINE]

More research....

Physiol Genomics. 2003 Dec 16;16(1):29-37.

    Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging.

        * Van Remmen H,
        * Ikeno Y,
        * Hamilton M,
        * Pahlavani M,
        * Wolf N,
        * Thorpe SR,
        * Alderson NL,
        * Baynes JW,
        * Epstein CJ,
        * Huang TT,
        * Nelson J,
        * Strong R,
        * Richardson A.

    Department of Cellular and Structural Biology at the University of Texas Health Science Center at San Antonio, San Antonio 78229-3900, USA.

    Mice heterozygous for the Sod2 gene (Sod2+/- mice) have been used to study the phenotype of life-long reduced Mn-superoxide dismutase (MnSOD) activity. The Sod2+/- mice have reduced MnSOD activity (50%) in all tissues throughout life. The Sod2+/- mice have increased oxidative damage as demonstrated by significantly elevated levels of 8-oxo-2-deoxyguanosine (8oxodG) in nuclear DNA in all tissues of Sod2+/- mice studied. The levels of 8oxodG in nuclear DNA increased with age in all tissues of Sod2+/- and wild-type (WT) mice, and at 26 mo of age, the levels of 8oxodG in nuclear DNA were significantly higher (from 15% in heart to over 60% in liver) in the Sod2+/- mice compared with WT mice. The level of 8oxodG was also higher in mitochondrial DNA isolated from liver and brain in Sod2+/- mice compared with WT mice. The increased oxidative damage to DNA in the Sod2+/- mice is associated with a 100% increase in tumor incidence (the number of mice with tumors) in old Sod2+/- mice compared with the old WT mice. However, the life spans (mean and maximum survival) of the Sod2+/- and WT mice were identical. In addition, biomarkers of aging, such as cataract formation, immune response, and formation of glycoxidation products carboxymethyl lysine and pentosidine in skin collagen changed with age to the same extent in both WT and Sod2+/- mice. Thus life-long reduction of MnSOD activity leads to increased levels of oxidative damage to DNA and increased cancer incidence but does not appear to affect aging.

    PMID: 14679299 [PubMed - indexed for MEDLINE]

plus

J Gerontol A Biol Sci Med Sci. 2000 Jan;55(1):B5-9.

    Ubiquitous overexpression of CuZn superoxide dismutase does not extend life span in mice.

        * Huang TT,
        * Carlson EJ,
        * Gillespie AM,
        * Shi Y,
        * Epstein CJ.

    Department of Pediatrics, University of California, San Francisco 94143-0546, USA. tthuang@itsa.ucsf.edu

    Oxidative damage has been implicated in the aging process and in a number of degenerative diseases. To investigate the role of oxygen radicals in the aging process in mammals, the life spans of transgenic mice on a CD-1 background expressing increased levels of CuZn superoxide dismutase (CuZnSOD), the enzyme that metabolizes superoxide radicals, were determined. Homozygous transgenic mice with a two- to five-fold elevation of CuZnSOD in various tissues showed a slight reduction of life span, whereas hemizygous mice with a 15- to 3-fold increase of CuZnSOD showed no difference in life span from that of the nontransgenic littermate controls. The results suggest that constitutive and ubiquitous overexpression of CuZnSOD alone is not sufficient to extend the life spans of transgenic mice.

    PMID: 10719757 [PubMed - indexed for MEDLINE]

the above mentioned papers, especially the first one, challenge the free-radical theory of aging first suggested by Harman in 1956. These papers force us to reconsider how we look at the situation. That's good!

[JonesGuy]

In science, we love a disproven theory.
In technology ... not so much.

[maestro949]

In technology, we just want the damn thing to work. And when it breaks, we just want it fixed.

zoolander, i've never been too keen on the free-radical theory. Sure, perhaps some of the damage contributes to aging but as the primary cause, pleh. I'm starting to wonder whether there even is a primary cause but rather that there are many interconnected causes where stopping only one or two or three might only delay the progression of aging related changes. With all the expression data that will be pouring out of the next generations of assay technology I suspect we'll be able to build some sharply-defined expert systems for doing the root cause analysis studies that we need to.

[Michael]

All:

As dug up by maestro949:

We measured the in vivo rate of change of the mitochondrial genome at a single–base pair level in mice ... Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice. (1)

Let me first just quote, for emphasis, a few of the comments in the article in The Scientist:

New data contradict aging theory
Since their assay examined point mutations only, it remains possible that large deletions in mitochondrial DNA could still underlie aging, said senior author Lawrence Loeb

This is the biggest one, and I'm pleased that Loeb makes it here (and presumably in the article itself, which I've not yet read, tho' alas I fear that it'll be widely ignored). There is a prevailing misperception amongst life extensionists -- and, unfortunately, most scientists, including even most biogerontologists -- that there is this single entity called the "mitochondrial free radical theory of aging," under which damage to mitochondrial DNA and/or other constituents makes the mitos burn less efficiently, reducing ATP output and/or producing more free radicals, and that this then leads directly and indirectly to yet more mitochondrial (and extramitochondrial) damage in a "vicious circle." In fact, however, this view has long been demonstrably at odds with the experimental evidence (see eg (2)); and while the "vicious circle" theories continued to be trotted out as "the" mitochondrial free radical theory of aging, several distinct mitochondrial free radical theories of aging have subsequently emerged that make a serious effort to grapple with the actual findings as they stand today, as opposed to the more intuitive -- put demonstrably wrong -- version usually presented.

The most successful of these is Aubrey de Grey's mitochondrial free radical theory of aging (3-5). Without getting into the details, it predicts precisely several findings that have subsequently received experimental support, including the findings that point mutations in mtDNA will notcontribute to aging, that mitochondria will not], on the whole, produce more free radicals as the organism ages, and that mt ATP production will not decline with age. What matters, in de Grey's theory (for reasons too complex for me to get into here, is the accumulation of mitochondrial DNA deletions -- whose role in aging, as Loeb rightly notes, are not addressed by this study.

Indeed, this is the first study to (apparently) reliably report that point mutations accumulate at all in normal aging -- despite the predictioin of the "vicious circle" theories that they would. They report thi using what is said to be a more sensitive assay method:

Although several studies have quantified either tissue-specific mutation frequencies or mutation accumulation as a function of age, most methods rely heavily on PCR and cloning-based strategies, techniques that are limited in throughput and that may be confounded by polymerase infidelity on damaged templates and by cloning artifacts. Therefore, we explored the relationship between age and mutation accumulation in mtDNA with an adaptation of the random mutation capture (RMC) assay, a quantitative PCR-based approach that relies on PCR amplification of single molecules for mutation detection but is not limited by polymerase fidelity. This methodology allows for exact determination of mutation frequencies in high-throughput screens that interrogate millions of base pairs simultaneously. Approximately 150 million bp were screened for mutation detection in this study.(1)

I don't feel qualified to evaluate the soundness of this technique. Smigrodzki and colleagues have been calling for a better assay than PCR cloning for mtDNA mutations for some time as part of their "microheteroplasmy" theory (one of the OTHER "mitochondrial free radical theorites of aging" that has taken into account the problems in the "vicious circle" theories) (6); while the finding that point mutations really do accumulate with aging is in accordance with their predictions and reports to date, the finding that dramatically increasing them does not also accelerate aging is logically a pretty strong blow against them (7). However, as the article notes:

The type of mutation induced in the mutator mice may also affect the results, according to Konstantin Khrapko of Beth Israel Deaconess Medical Center in Boston. These mice have defects in a mitochondrial DNA polymerase, which is not involved in naturally occurring mitochondrial mutations. "These mutations may accumulate in different cell types" than natural mutations, Khrapko told The Scientist...

Given that mutated mice get an early start to these mutations, it's possible that embryonic cells in the mutator mice could "figure out a way to adapt to mutations that adult animals" could not tolerate, agreed Peter Zassenhaus of St. Louis University,.... "We need to know what the mechanism is" through which mitochondrial mutations might influence tissue function, Zassenhaus told The Scientist. "Without really understanding how these mutations can cause a disease or pathology, it's hard to interpret the mutation frequencies by themselves."

I confess to being so uncreative as to not seeing how this could work on this scale, but I haven't thought too much about it yet.

There was a study released in 2006 where a naked mole rat with high oxidative damage in tissue lived just as long as it's distant relative mouse. [8]

This study finds " poorer antioxidant capacity ... 10-fold higher levels of in vivo lipid peroxidation ... greater levels of accrued oxidative damage to lipids ..., DNA ... and proteins ... than physiologically age-matched mice, and equal to that of same-aged mice." What this tells you is that such damage is not implicated in aging; it doesn't tell you that free radical damage elsewhere isn't. While "oxidative damage" was the culprit in Harman's original theory -- and in Pearson and Shaw, and even now in the literature of supplement hawkers -- the various mitochondrial free radical theories (and especially de Grey's MiFRA) are a lot more specific; indeed, in the latter, it's not even mitochondrial oxidative damage in general that counts, but mitochondrial DNA deletions. If anything, this report tends to support a specifically mitochondrial theory: as de Grey noted years ago (9), endogenous antioxidant levels tend to be either unrelated to, or inversely correlated with, maximum lifespan (and the findings are similar for the CR data). What this may well indicate is precisely that longer-lived organisms get away with lower defenses -- and one explanation for this is that the source of the damage against which these defenses are intended to protect is reduced. On an interspecies basis, and also in CR, there's an obvious factor to finger: the reduced generation of free radicals in the first place at the mitochondrial level.

This way of reading teh result reconciles this finding (9) with the independent report (10) that the greater lifespan of naked mole rats correlates with the low content of long-chain unsaturated fatty acids (most notably, DHA) relative to their more normally-lived peers: importantly, the mt inner membrane is directly attached to the mtDNA, so that oxidative damage to that membrane is propagated onto it. Similar findings have been reported both for CR, and for other more longevous species (such as similar-sized birds).

More research....
   
Van Remmen H, Ikeno Y, Hamilton M, Pahlavani M, Wolf N, Thorpe SR, Alderson NL, Baynes JW, Epstein CJ, Huang TT, Nelson J, Strong R, Richardson A. 
Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging.
Physiol Genomics. 2003 Dec 16;16(1):29-37.
PMID: 14679299 [PubMed - indexed for MEDLINE]

Crucially, however, Van Remmen's group still hasn't done the key test, which is to see if mtDNA deletions are increased in skeletal muscle and the heart.

plus

Huang TT, Carlson EJ, Gillespie AM, Shi Y, Epstein CJ.  Related Articles, Links
Abstract  Ubiquitous overexpression of CuZn superoxide dismutase does not extend life span in mice.
J Gerontol A Biol Sci Med Sci. 2000 Jan;55(1):B5-9.
PMID: 10719757 [PubMed - indexed for MEDLINE]

... and doesn't reduce mitochondrial DNA deletions, either .

Meanwhile, I see Zoo has also come across (and Shep has posted, for full members -- thanks Shep!) another paper (11) claiming to be "an experimental test of the reductive hotspot hypothesis" (RHH) -- a term for a key part of de Grey's MiFRA. One must wonder if these folks paid any attention to his papers when reading them, or during the construction of their experiment. The measured "The activity of superoxide in muscle microdialysates " in "adult and old mice at rest, [annd] during contractile activity," and found "a significant increase in the superoxide activity in microdialysates from adult muscle but no increase in microdialysates from old muscle ... not due to reduced force generation by these muscles." But how this is supposed to test RHH -- which, building on "survival of the slowest" (SOS -- another important breakthrough in de Grey's MiFRA) is designed precisely to explain how [i]the tiny proportion of cells that actually become taken over by mutant mitochondria can spread oxidative damage throughout the body -- is beyond me: you'd be looking for an increased production of superoxide in >1% of muscle fiber segments, which can't reasonably be expected to show up on "a microdialysis probe ... placed to a depth of 7 mm into the medial head of gastrocnemius muscle of the right hind-limb of ... mice that were killed at 30 min postcontractions."

Meanwhile, the very few interventions that have actually been found to retard aging and extend youthful lifespan in mammals -- CR, IGF1 mutants, and mitochondrially- (but not nuclear- or peroxisome-)targeted catalase (12) -- all reduce the nonenzymatic generation of free radicals, [i]or interdict them in such a way as to reduce the formation of mitochondrial DNA deletions.

the above mentioned papers, especially the first one, challenge the free-radical theory of aging first suggested by Harman in 1956. These papers force us to reconsider how we look at the situation. That's good!

Indeed (2) .

-Michael

1. Vermulst M, Bielas JH, Kujoth GC, Ladiges WC, Rabinovitch PS, Prolla TA, Loeb LA.
Mitochondrial point mutations do not limit the natural lifespan of mice.
Nat Genet. 2007 Mar 4; [Epub ahead of print]
PMID: 17334366 [PubMed - as supplied by publisher]

2. de Grey AD.
Mitochondria in homeotherm aging: will detailed mechanisms consistent with the evidence now receive attention?
Aging Cell. 2004 Apr;3(2):77. No abstract available.
PMID: 15038822 [PubMed - indexed for MEDLINE]

3. de Grey AD. The reductive hotspot hypothesis of mammalian aging: membrane metabolism magnifies mutant mitochondrial mischief. Eur J Biochem. 2002 Apr;269(8):2003-9. Review. PMID: 11985576 [PubMed - indexed for MEDLINE]
http://www.gen.cam.a...sens/mmmmmm.pdf

4. de Grey AD. The mitochondrial free radical theory of aging. 1999; Austin, TX: Landes Bioscience. (ISBN 1-57059-564-X).

5. de Grey ADNJ. A mechanism proposed to explain the rise in oxidative stress during aging. J Anti-Aging Med 1998; 1(1):53-66.
http://www.gen.cam.ac.uk/sens/pmor.pdf

6. Smigrodzki RM, Khan SM. Related Articles, Links
Abstract Mitochondrial microheteroplasmy and a theory of aging and age-related disease.
Rejuvenation Res. 2005 Fall;8(3):172-98. Review.
PMID: 16144471 [PubMed - indexed for MEDLINE]

7. de Grey AD. Related Articles, Links
Abstract Falsifying falsifications: the most critical task of theoreticians in biology.
Med Hypotheses. 2004;62(6):1012-20.
PMID: 15142666 [PubMed - indexed for MEDLINE]
http://www.sens.org/m17prep.pdf

8. Andziak B, O'Connor TP, Qi W, DeWaal EM, Pierce A, Chaudhuri AR, Van Remmen H, Buffenstein R. Related Articles, Links
Abstract High oxidative damage levels in the longest-living rodent, the naked mole-rat.
Aging Cell. 2006 Dec;5(6):463-71. Epub 2006 Oct 27.
PMID: 17054663 [PubMed - indexed for MEDLINE]

9. de Grey ADNJ. The non-correlation between maximum longevity and enzymatic antioxidant levels among homeotherms; implications for retarding human aging. J Anti-Aging Med 2000; 3(1):25-36.
http://www.sens.org/mims.pdf

10. Hulbert AJ, Faulks SC, Buffenstein R. Related Articles, Links
Abstract Oxidation-resistant membrane phospholipids can explain longevity differences among the longest-living rodents and similarly-sized mice.
J Gerontol A Biol Sci Med Sci. 2006 Oct;61(10):1009-18.
PMID: 17077193 [PubMed - indexed for MEDLINE]

11. Close GL, Kayani AC, Ashton T, McArdle A, Jackson MJ. Related
Release of superoxide from skeletal muscle of adult and old mice: an experimental test of the reductive hotspot hypothesis.
Aging Cell. 2007 Feb 27; [Epub ahead of print]
PMID: 17328687 [PubMed - as supplied by publisher]

12. Schriner SE, Linford NJ, Martin GM, Treuting P, Ogburn CE, Emond M, Coskun PE, Ladiges W, Wolf N, Van Remmen H, Wallace DC, Rabinovitch PS. Related Articles, Links
Abstract Extension of murine life span by overexpression of catalase targeted to mitochondria.
Science. 2005 Jun 24;308(5730):1909-11. Epub 2005 May 5.
PMID: 15879174 [PubMed - indexed for MEDLINE]

[John Schloendorn]

the finding that dramatically increasing them does not also accelerate aging is logically a pretty strong blow against (Smigrodzki)

Against their theory of aging, but hopefully not against their therapeutic approach. In theory, their mitochondrial gene therapy should deal with large deletions or any other sort of mutations equally well, if perfected.

[maestro949]

Jan Vijg chimes in via Science

This claim may be a bit premature, however, cautions Jan Vijg, a molecular geneticist at the Buck Institute, a nonprofit research center in Novato, California. Point mutations are tissue-specific, he says, meaning that some tissues may be more prone to mtDNA mutations than others. Vermulst's study looked only at tissues from the heart, brain, and embryonic fibroblasts. In addition, says Vijg, the mutator mouse Loeb used may not be the best model for these studies. Mutations in these animals began to amass during embryonic development, while normal animals don't begin to accumulate mutations until after birth.