Mitochondrial DNA Oxidation

Summary: Most of the oxidising damage in mitochondrial DNA (mtDNA) is in bits/fractions of mtDNA, not in the circular form. And iron relaxes mtDNA loop and increases its replication.

Interestingness: 5

Paper by Christoph Richter in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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This paper starts by describing how mtDNA gets oxidised: superoxide radicals (O2-) are formed "when cytochrome oxidase is blocked, when cytochrome c is detached from the inner mitochondrial membrane, " ... and " when mitochondrial oxidative phosphorylation is inhibited". The superoxide radical then gives the electron to a water molecule, which forms hydrogen peroxide (H2O2), which then forms hydroxyl radical (OH.) in the presence of iron or copper (Fenton reaction). The hydroxyl radical is the bastard that then goes and reacts with everything.

It then mentions radical nitrogen species, usual description of mtDNA (16.3 kb pair coding for 13 peptides, 22 tRNAs and 2 rRNAs), how people started thinking of mtDNA damage as important for diseases, measurement of mtDNA damage (usually measuring 8-hydroxyguanine and strand breaks), sidetrack into azidothymidine (AZT, the anti-AIDS drug) causing problems in mitochondria, and Friedreich's ataxia (FA) probably being a problem with oxidation damage in mitochondria.

Now, interesting bit, measurements of amount of oxidative damage in mtDNA differ depending on methodology. Detection of 8-hydroxydeoxyguanosine (8-OHdG) gives big numbers (4 modifications per mtDNA molecule) while numbers from repair enzymes (dunno how it works) give much lower numbers. High number doubted also from seemingly high number of working mitochondria. They do analysis of mtDNA from rat's livers, detecting 8-OHdG. They get 0.051 picomole per microgram of DNA for circular mtDNA, which they say is about one 8-OHdG mutation every two mtDNA molecules, 0.014 picomole per microgram of DNA for nDNA, which is contamination in the sample, but 0.741 picomole per microgram in low molecular mtDNA, ie fractions of floating mtDNA. They don't know what the fractions of mtDNA are doing or why they are so highly oxidised. It could be that they are being actively degraded, or they could be new chunks being made. Having found these fragments, he then hypothesises that these fragments integrate with nDNA, and that this is the main mechanism of aging of mtDNA oxidation damage.

The part that follows is also interesting. Experimenting with iron overload into the mtDNA of rat's livers in vitro they find that it (iron, in the form of Fe3+ gluconate), relaxes mtDNA from the standard supercoiled form to the open circular form. Anti-oxidants prevent some of the change but not all. The iron forms colloids that bind to mtDNA, and there may be a purely physical mechanism of relaxation. They then repeat the experiment in vivo also observing more relaxed circular DNA compared to controls, as well as increased mitochondrial surface and volume density, increased intracellular ferritin and hemosiderin, and higher replication of mtDNA.

It then switches to mtDNA damage prevention, mentions caloric restriction as reducing 8-OHdG counts, AZT leading to higher urinary 8-OHdG but vitamins C and E reducing those levels in AZT-taking people (I thought vitamins C and E didn't enter the mitochondria). Finishes by looking at future studies, evidence that mtDNA inserts in nDNA are more common in tumours, Drosophila overexpressing superoxide dismutase and catalase having increased lifespan, and some wacky suggestion of using bacteria to transfect genes into mitochondria.

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Abstract follows:

Mitochondrial diseases have been known for more than three decades. A refinement of the free radical theory of aging states that oxidative damage to mitochondria, particularly to mitochondrial DNA (mtDNA), is responsible for aging. About 10 years ago, oxidative damage to mtDNA was first reported, and human diseases were related to mutations of mtDNA. Subsequent reports suggested that oxidative mtDNA damage is more pronounced in old individuals and during certain diseases. Studies of animal models indicated that oxidative mtDNA damage can be ameliorated by dietary antioxidants and caloric restriction, an established method to increase life span. More recent data indicate that fragmented mtDNA is the predominant carrier of oxidized mtDNA bases and that fragments constitute a substantial amount of the total mtDNA. This article discusses the emerging relationship among mtDNA oxidation, diseases, and aging, and suggests experiments by which such a relationship can be further substantiated.

A Mechanism Proposed to Explain the Rise in Oxidative Stress During Aging

Summary: Longish speculative chain on how cells dominated by mutant mitochondria, even though they are rare, can cause system wide oxidation damage. The speculation sounds speculative.

Interestingness factor: 6ish

Paper by Aubrey de Grey, published in Journal of Anti-Aging Medicine, Volume 1, Issue 1, Spring 1998. (((can be gotten from http://www.sens.org/files/sens/AdGpubs.htm)))

(((A long theoretical piece. I'm not going to do it justice. Reading about glycolysis and the Krebs cycle helps)))

The previous blog reviewed the article that forms the basis for this paper. Assuming that mutant mitochondria take over individual cells, how can they affect the rest of the body, if, as this paper points out, they seem to only make up about one percent of tissue cells?

The hypothesis starts with an explanation of how these cells survive at all. It uses data gathered from experiments with cells that lack mitochondrial DNA altogether (p0 cells), that can survive if supplemented with pyruvate or a whole bunch of other molecules.

The problem to solve for them, aside from the lower amounts of energy available, is how to restore levels of NAD+ that get converted to NADH during glycolysis. The proposed solution is that they do this by reduction of extracellular molecules through an enzyme that sits on the membrane called plasma membrane oxidoreductase (PMOR) that exist in every cell. That is, they do this by exporting electrons. Evidence presented is that succinate dehydrogenase is upregulated in these p0 cells, and that since it is part of the Krebs cycle that consumes pyruvate, it shows that the main alternative method of exporting those electrons (by reducing pyruvate to lactate with NADH and then exporting the lactate) is probably not being used. (((Which doesn't show me how some other third method is not what is really going on)))

Those exported electrons primarily go to vitamin C in extracellular fluid, but once you run out of that, they'd go to oxygen, creating superoxide radicals. Most of this would be cleaned up by superoxide dismutase, but some would escape and react with the iron in haemin (((non-protein bit of haemoglobin))) (other iron options are well protected) which would then oxidase LDL particles. This last part, the oxidation of LDL particles by haemin, seems to have some evidence behind it.

The oxidation of some LDL would raise the intake of somewhat oxidised LDL by all cells in the body, which would raise the amount of oxidation damage that all cells have to deal with. Since this mechanism would be going on constantly, ie those mutant mtDNA cells would be constantly spewing electrons, quite a lot of oxidised LDL would be created.

The paper then moves onto methods of testing the suggested chain of events:

• Seeing if cells that do not have a functioning electron transport chain (by assessing cytochrome c oxidase activity) have high PMOR activity.
• Seeing if there's high levels of superoxide near cells with busted mitochondria
• Checking if LDL is highly oxidised near mutant cells
• Checking if oxidised LDL particles stress normal cells anti-oxidant system.

And then onto methods for checking that it affects aging:

• Restoring the function of the mutant mitochondria by importing the proteins encoded by the mitochondrial DNA and seeing what happens (((hard project)))
• Targeting those zombie cells controlled by mutant mitochondria and destroying them, then seeing what happens. (((I like it)))

(((Conclusion: The chain of events suggested here sounds much more dubious to me than the one suggested in the previous article. The electrons from NADH might be used some other way inside the cells, or the electrons might be quenched in some benign way outside the cell, or oxidation of LDL might not have any major effect on aging (outside of the effects on cardiovascular disease). The tests seem simple enough though, and they'll pop up regardless, if they haven't already. It would be good if this was correct since killing the mutant cells doesn't sound insanely hard to me. Easier than curing cancer since these cells don't reproduce, as determined by the method that they come into existence)))

(((This is the last paper that I'll write about in the first issue. The rest consists of some futurist speculation, a meeting report and literary review. While interesting, they are already in summary form)))

Abstract follows:

Most phenotypes of aging in vertebrates may be caused by a progressive decline in the ability of antioxidant defences to maintain cellular and systemic homeostasis. This is due both to a diminished efficacy of those defences and to an enhanced level of pro-oxidant toxicity; the imbalance between the two has been termed oxidative stress. However, the cause of this increasing imbalance remains obscure. This article proposes a mechanism by which spontaneously mutant mitochondrial DNA (mtDNA), despite being present only in very small quantities in the body, may be the main generator of oxidative stress. Mutant mtDNA is distributed very unevenly within a tissue: some cells apparently contain no wild-type mtDNA whatever. Those cells must rely on glycolysis for ATP production; furthermore, they require a system to stabilize their NAD+/NADH ratio. This can only be achieved by an efflux of electrons from the cell, most probably mediated by the plasma membrane oxidoreductase (PMOR). It is proposed that the required rate of electron efflux from these anaerobic cells exceeds the local electron-accepting capacity of "safe" acceptors in plasma such as dehydroascorbate, with the result that reactive species, such as Superoxide, are formed. This leads to increased oxidation of lipids in the plasma, notably of low-density lipoprotein (LDL) particles, which are subsequently imported into mitochondrially healthy cells. This oxidized lipoprotein must be destroyed by the recipient cells' antioxidant defences. That task diverts the cell from the degradation of pro-oxidants that it is itself generating; thus, it imposes oxidative stress on the cell. As the number of anaerobic cells in the body rises, so does oxidative stress in all cells. The consistency of this hypothesis with known facts is discussed, and technically feasible tests are suggested both of the proposed mechanism and of its overall contribution to mammalian aging, including plausible interventions to retard the process.

A proposed refinement of the mitochondrial free radical theory of aging

(((This isn't really part of the series. I was reading "A Mechanism Proposed to Explain the Rise in Oxidative Stress During Aging" in the series but it assumes that the reader is familiar with this paper. Since I found it interesting, I'm writing a summary of this one as well)))

Summary: Hypothesis is that cells get filled up with mutant mitochondria. This is because their membrane gets destroyed at a slower rate due to them being slower in producing free radicals. This leads to them not being destroyed by the cell recycling mechanism.

Interestingness factor: 7ish

Paper by Aubrey de Grey, published in BioEssays, 1997, volume 19, issue 2. (((can be gotten from http://www.sens.org/files/sens/AdGpubs.htm)))

(((Harder to summarise theoretical papers, since most of it tends to be important for the theory to hold)))

The paper makes the case that the replication of mutant mitochondria fills up cells with mostly useless mitochondria that do not feed the cell enough ATP, and that this is important for mammalian aging. I'll focus on the description of the mechanism of how mutant mitochondria supposedly get selectively replicated and come to represent most/all of the mitochondria in a cell, since that's the bit that's relevant to the paper in the Anti-Aging journal. I'll mostly ignore the importance of this to aging.

(((Reading up on mitochondria, and http://en.wikipedia.org/wiki/Electron_transport_chain#Electron_transport_chains_in_mitochondria helps)))

Mitochondria reproduce more frequently that the cells that contain them, especially if those cells don't replicate at all
(senescent). Their DNA (mtDNA) is not as well protected as nuclear DNA and is close to the reactive molecules that they produce. There are 13 genes in mtDNA that are not duplicated in the nuclear DNA and so are essential to the functioning of the mitochondria. They include proteins that form part of the respiration chain and the ATP generation. Therefore point mutations in those parts of their DNA would interfere with those functions. If there was a selective process which would preferentially replicate these mitochondria over the non-mutant ones, then mutants would dominate the cell and all/most mitochondria in the cell would have non-working or slow ATP production.

(((The most speculative part is the following))) Mitochondria damage their cell membrane in the production of the proton gradient. The process creates radical molecules that attack the lipids in their inner membrane. If the membrane is damaged enough small molecules from the interior of the mitochondrion will leak into the cytoplasm. The cell uses these as markers of damaged mitochondria and destroys it (by lysosomal degradation). Because the mutant mitochondria have faulty electron transport chains, their membrane degrades slower. This means they are left alive and reproduced when the cell thinks it needs more ATP (cell has to pick from the mitochondria that are alive). (((Tada!)))

Evidence offered for this theory: 1) Mitochondria in cells tend to share the same mutations, and these are different from the mutations in the cell next door. 2) Mutations that affect the ATP synthetising enzymes do not become popular among cells, because their transport chain is intact and therefore their membranes are just as damaged as non-mutant mitochondria (((this is offered as a prediction in the paper but he claims the result is provisionally known to be true)))

Refutations of potential counter-arguments. (((I'm restricting to only the ones that refer to the spread of the mitochondria again)))

1) Objection: Mitochondria have many copies of mtDNA, single mutation in one copy won't make much of an impact. Counter-counter: Some mutations will hang around and become homozyguous in all copies of a mitochondrion by genetic drift. Also, even arecessive mutation would have some small effect on electron transport and the mitochondrion only has to survive a little bit longer than the rest to be replicated.

2) Objections: Membranes get repaired. Counter-counter: Not all damage can be fixed.

3) Objection/question: Does a mitochondria with a damaged electron transport chain mechanism actually cause less damage on their inner membrane? Counter-counter: Yes. Three paths of lipid peroxidation: perhydroxy levels lower on the outside of an inner membrane of a slowly respiring mitochondrion, so will levels of ubisemiquinone. Metal-catalysed pathway still just as active probably (((I'm just parroting this bit. No idea if it's complete nonsense)))


Abstract follows:

Over recent years, evidence has been accumulating in favour of the free radical theory of aging, first proposed by Harman. Despite this, an understanding of the mechanism by which cells might succumb to the effects of free radicals has proved elusive. This paper proposes such a mechanism, based on a previously unexplored hypothesis for the proliferation of mutant mitochondrial DNA: that mitochondria with reduced respiratory function, due to a mutation or deletion affecting the respiratory chain, suffer less frequent lysosomal degradation, because they inflict free radical damage more slowly on their own membranes. Once such a mutation occurs in a mitochondrion of a non-dividing cell, therefore, mitochondria carrying it will rapidly populate that cell, thereby destroying the cell's respiratory capability. The accumulation of cells that have undergone this transition results in aging at the organismal level. The consistency of the hypothesis with known facts is discussed, and technically feasible tests are suggested, of both the proposed mechanism and its overall contribution to mammalian aging.