Towards a Greater Knowledge of Mitochondrial DNA Damage in Aging

Today I'll point out a very readable scientific commentary on mutations in mitochondrial DNA (mtDNA) and the importance of understanding how these mutations spread within cells. This is a topic of some interest within the field of aging research, as mitochondrial damage and loss of function is very clearly important in the aging process. Mitochondria are, among many other things, the power plants of the cell. They are the evolved descendants of symbiotic bacteria, now fully integrated into our biology, and their primary function is to produce chemical energy store molecules, adenosine triphosphate (ATP), that are used to power cellular operations. Hundreds of mitochondria swarm in every cell, destroyed by quality control processes when damaged, and dividing to make up the numbers. They also tend to promiscuously swap component parts among one another, and sometimes fuse together.

Being the descendants of bacteria, mitochondria have their own DNA, distinct from the nuclear DNA that resides in the cell nucleus. This is a tiny remnant of the original, but a very important remnant, as it encodes a number of proteins that are necessary for the correct operation of the primary method of generating ATP. DNA in cells is constantly damaged by haphazard chemical reactions, and equally it is constantly repaired by a range of very efficient mechanisms. Unfortunately mitochondrial DNA isn't as robustly defended as nuclear DNA. Equally unfortunately, some forms of mutation, such as deletions, seem able to rapidly spread throughout the mitochondrial population of a single cell, even as they make mitochondria malfunction. This means that over time a growing number of cells become overtaken by malfunctioning mitochondria and fall into a state of dysfunction in which they pollute surrounding tissues with reactive molecules. This can, for example, increase the level of oxidized lipids present in the bloodstream, which speeds up the development of atherosclerosis, a leading cause of death at the present time.

The question of how exactly some specific mutations overtake a mitochondrial population so rapidly is still an open one. There is no shortage of sensible theories, for example that it allows mitochondria to replicate more rapidly, or gives them some greater resistance to the processes of quality control that normally cull older, damaged mitochondria. The definitive proof for any one theory has yet to be established, however. In one sense it doesn't actually matter all that much: there are ways to address this problem through medical technology that don't require any understanding of how the damage spreads. The SENS Research Foundation, for example, advocates the path of copying mitochondrial genes into the cell nucleus, a gene therapy known as allotopic expression. For so long as the backup genes are generating proteins, and those proteins make it back to the mitochondria, the state of the DNA inside mitochondria doesn't matter all that much. Everything should still work, and the present contribution of mitochondrial DNA damage to aging and age-related disease would be eliminated. At the present time there are thirteen genes to copy, a couple of which are in commercial development for therapies unrelated to aging, another couple were just this year demonstrated in the lab, and the rest are yet to be done.

Still, the commentary linked below is most interesting if you'd like to know more about the questions surrounding the issue of mitochondrial DNA damage and how it spreads. This is, as noted, a core issue in the aging process. The authors report on recent research on deletion mutations that might sway the debate on how these mutations overtake mitochondrial populations so effectively.

Expanding Our Understanding of mtDNA Deletions

A challenge of mtDNA genetics is the multi-copy nature of the mitochondrial genome in individual cells, such that both normal and mutant mtDNA molecules, including selfish genomes with no advantage for cellular fitness, coexist in a state known as "heteroplasmy." mtDNA deletions are functionally recessive; high levels of heteroplasmy (more than 60%) are required before a biochemical phenotype appears. In human tissues, we also see a mosaic of cells with respiratory chain deficiency related to different levels of mtDNA deletion. Interestingly, cells with high levels of mtDNA deletions in muscle biopsies show evidence of mitochondrial proliferation, a compensatory mechanism likely triggered by mitochondrial dysfunction. In such circumstances, deleted mtDNA molecules in a given cell will have originated clonally from a single mutant genome. This process is therefore termed "clonal expansion."

The accumulation of high levels of mtDNA deletions is challenging to explain, especially given that mitophagy should provide quality control to eliminate dysfunctional mitochondria. Studies in human tissues do not allow experimental manipulation, but large-scale mtDNA deletion models in C. elegans have proved to be helpful, showing some conserved characteristics that match the situation in humans, as well as some divergences. Researchers have used a C. elegans strain with a heteroplasmic mtDNA deletion to demonstrate the importance of the mitochondrial unfolded protein response (UPRmt) in allowing clonal expansion of mutant mtDNAs to high heteroplasmy levels. They demonstrate that wild-type mtDNA copy number is tightly regulated, and that the mutant mtDNA molecules hijack endogenous pathways to drive their own replication.

The data suggests that the expansion of mtDNA deletions involves nuclear signaling to upregulate the UPRmt and increase total mtDNA copy number. The nature of the mito-nuclear signal in this C. elegans model may have been the transcription factor ATFS-1 (activating transcription factor associated with stress-1), which fails to be imported by depolarized mitochondria, mediates UPRmt activation by mtDNA deletions. A long-standing hypothesis proposes that deleted mtDNA molecules clonally expand because they replicate more rapidly due to their smaller size. To address this question, researchers examined the behavior of a second, much smaller mtDNA deletion molecule. They found no evidence for a replicative advantage of the smaller genome, and clonal expansion to similar levels as the larger deletion. In human skeletal muscle, mtDNA deletions of different sizes also undergo clonal expansion to the same degree. Furthermore, point mutations that do not change the size of the total mtDNA molecule also successfully expand to deleterious levels, indicating that clonal expansion is not driven by genome size. Thus, similar mechanisms may be operating across organisms. In the worm, this involves mito-nuclear signaling and activation of the UPRmt.

There is some debate over interpretation of results. One paper indicates that UPRmt allows the mutant mtDNA molecules to accumulate by reducing mitophagy. Another demonstrates that the UPRmt induces mitochondrial biogenesis and promotes organelle dynamics (fission and fusion). Both papers show that by downregulating the UPRmt response, mtDNA deletion levels fall, which may allow a therapeutic approach in humans. Could there be a similar mechanism in humans, especially since some features detected in C. elegans are also present in human tissues, including the increase in mitochondrial biogenesis and the lack of relationship between mitochondrial genome size and expansion? It is likely that there will be a similar mechanism to preserve deletions since, as in the worm, deletions persist and accumulate in human tissues, despite an active autophagic quality-control process. Although the UPRmt has not been characterized in humans as it has in the worm, and no equivalent protein to ATFS-1 has been identified in mammals, proteins such as CHOP, HSP-60, ClpP, and mtHSP70 appear to serve similar functions in mammals as those in C. elegans and suggest that a similar mechanism may be present.


I asked Aubrey on one of his Reddit AMAs why you don't just use CRISPR on a model animal embryo to allotopically express all 13 protein coding mtDNA genes with targeting sequences to see what happens.

His reply was "That's not how you build a rocket ship". And I get that this approach won't tell you where you are going wrong in the likely event that it doesn't work and extend lifespan/healthspan on the first attempt. How could you tell if individual mts (mitochondrial targeting sequences) are actually working for example.

But with 4 genes 'proven' to work (ND4, ND1, ATP6, ATP8), I've got to wonder what would happen if you used gene editing to put these into Killifish embryos?

Posted by: Jim at July 26th, 2016 7:03 AM


Hi Jim ! I wager not much for Killfish, the embryos would not die of MELAS lethality disease like in humans but would go on to have mitochondrial assmebly and function (their life would be 'normal') but it would not extend the very-short lifespan of this animal very much beyond 20-40% avg and MLSP (if taking fly example), or less since Killifish lives much longer than a fly.

In the first study (below) Ndi1 increases fly lifespan by allowing Complex I assembly (also seen in humans with a study in the eye where Nd1 reassembles
Complex I in mitochondria and vision is restored) and so allowing more mitochondrial respiratory ATP energy efficiency/per O2 molecule, increasing lifespan.
If you were to ask me would this translate into humans living longer, possibly, since maximu lifespan was increased, though not much.
Still, any study in short-lived animal, translates as very weak results in long-lived animals (like us humans) as such it may not have any
effect whatsoever. In this study, there was a 30-40% increase in mean and MLSP for males only..females living longer..and the effect was smaller...
This Demonstrates..that if the specie is Already Optimized (the female fly, the individual of this gender) than the effect is weak or null. So for us humans, perhaps, only male
humans would really have a significant effect on lifespan by ND1 reassembly of Complex I, and, mostly only, men that are short-lived (biologically older) or that have MELAS type
mitochondrial mutations disease. Not for long-lived healthy men (biologically younger) or even less for women (who already outlive men by better longevity gene and protection
by their estrogen activation of hTERT estrogenic receptors, and XX female chromosomes vs Xy more unstable male chromosomes with a weak smaller y chromsome).

''NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction,
and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity
and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation
of markers of oxidative damage in aged flies.''

''NDI1 expression had a clear effect on lifespan, both in the heterozygous
(w1118∕Dahomey w-) background (Fig. S4A) and after
backcrossing over 11 generations to the Dahomey w- reference
strain (Fig. S4B).
Median, mean, and maximum lifespan were
substantially increased. The increase was more pronounced in
males (30-40%) than females (10-20%), and was highly significant
when NDI1-expressors were compared with all nonexpressor
control groups (Fig. S4B).
Importantly, it was independent, in
both sexes, of the lifespan enhancement caused by dietary restriction
(Fig. 4A and Fig. S4C). Consistent with this, and despite the
altered steady-state ratio of NADþ∕NADH, NDI1 expression
caused no alteration in sirtuin activity (Fig. 4B).
To gain insight into the mechanism promoting lifespan extension
by NDI1, we analyzed respiratory functions in aging flies,
at approximately 2∕3 of the median lifespan of nonexpressors.
In contrast to young flies (Fig. S5), isolated mitochondria from
aging NDI1-expressing flies showed clearly increased substrate
oxidation and decreased mitochondrial ROS production compared
with nonexpressors, when supplied with a complex I-linked
substrate mix (Fig. 5A).''

I've said before, ROS, are major culprits, they are important signals for stress response compensation mechanism (Nrf2, mitoHormesis) but
they are a double-edged sword, they still damage when they are out of balance between reduction and oxidation (redox properties); meaning
as the fly ages ROS accumulate more and more; and thus the oxidation/reduction balance is lost - giving weight to ROS destruction of mitochondrial
DNA rather than just 'more' signaling. I'm very surprised that NDI1 was capable of increasing - maximum lifespan - that is incredible, and goes
to show that mitochondrias are important heart to our specie maximum lifespan; every study on length lifespan seem to emanate from one place,
the mitochondria - because they are the 'energy-producing' engine centers; without energy the cell dies. It, also, shows that Complex I is the major
culprit, as NDI1 reassembles Complex I and OXPHOS, restores respiratory ATP energy production, solving serious mitochondrial mutations (like MELAS in humans).
In fact, one study studied 3 MELAS patients (see link 3. below) average 15 years old, and all three had NDI1 defects and Complex I defects in their fibroblasts.
Thus showing, energy deficit (by mitochondrial impossibility to have a Complex I fuctionning/assembled) created the lethal disease.

Ironically, in the second study, ATP synthase subunit is KO and it increases lifespan depending on diet composition (I expressed in another post
that it's not CR, it's diet nutrient composition/density per calorie that alters life much strongly than calorie count restriction).
That means, that ATP synthase is important for development and 'healthy' functionning (avoid deadly disease like MELAS and mitochondrial mutations
pathologies), but in this example; it seems ATP synthase can be dispensable. I am waging for flies it works, but it would never work for humans
who suffer from many mitochondrial mutations (MELAS being the worse). This also demonstrate the mootness of ATP synthase in having any effect
on 'normal' healthy lifespan with no pathologies - it won't increase it. In this example, they Remove/KO it..and lifespan increases...I understand it's not ATP6 or ATP8 genes but its related it's a ATP subunit
that is just as critical at ATP6 or 8. Thus, we could infer that ATP6 or ATP8 would have mild or no effect on lifespan in humans, but definitely
improve healthspan; especially for people affected with mitochondrial mutations at Complex I (it always happens there, the engine and ROS producer)
since Complex I is needed for respiratory ATP production in human mitochondria.

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction


A Mitochondrial ATP synthase Subunit Interacts with TOR Signaling to Modulate Protein Homeostasis and Lifespan in Drosophila


Mutations of the mitochondrial ND1 gene as a cause of MELAS


Posted by: CANanonymity at July 26th, 2016 6:08 PM

PS: One study after another regarding mitochondrias give the feeling that SENS or any other therapy trying to alter Complex I and other mito allotopic expression will be the equivalent of MELAS resolution, but no more. As such remain a health improvement and possibly, a light effect on MLSP, but Nothing Ever to Reach LEV. It would take way more than that, by combining all 7 thérapies, perhaps. But this one alone doesn't make much diff for regular 'intrisinc' maximum lifespan of healthy 'normal' aging humans reaching 122 years limit. I still say Redox is the only way if all 7 SENS ones can't do it and remain therapeutic health-improvement (it was said we could get 30-40 years lifespan increase, meaning a 80 years old would reach 120) but not true lifespan extension LE (going Above 122 years old limit) and then LEV (infinite lifespan) in the future next 50 years.

Posted by: CANanonymity at July 26th, 2016 7:07 PM

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