Mitochondrial Microheteroplasmy and a Theory of Aging and Age-Related Disease

Interestingness: 6

By Rafal M. Smigrodzki and Shaharyar M. Khan. Rejuvenation Research. Fall 2005, 8(3): 172-198. doi:10.1089/rej.2005.8.172.

Long theoretical paper that tries to make a case for an accumulation of lots of rare mutations in mitchondrial DNA being a major cause of aging. By rare I mean that each individual mutation is rareish (1-2% of mitochondrial genomes), but between all of them it means that most of the mtDNA in a person is mutated in some way or other. The claim is that it is this accumulation of mtDNA mutations across the body plus the inherited component from the person's mother and, importantly, from the mutations accumulated in the mother's egg cell during her life prior to giving birth to the person in question, that causes the deterioration of the body.

The paper focuses a lot on Parkinson's and Alzheimer's disease, understandable since the first author is a neurologist. This made it harder for me to read as a "theory of aging" since I know almost nothing about either and Parkinson's is a relatively rare disease.

A lot of the arguments for why the primary cause should be in mtDNA or in DNA are similar to de Grey's arguments in de Grey's arguments in his version of the mitochondrial theory of aging so I will skip those. There are major differences in the details so I'll concentrate on those.

Low levels of lots of different mutations in the mtDNA (microheteroplasmy) are different from the uniform per-cell mutations of de Grey's. Its existence is much harder to detect but other studies supposedly show such microheteroplasmy exists in all tissue and increases with age, from about 45 mutations per million base pairs at birth to about 200 per million in old age (with a high starting level in the substantia nigra). Each mutation is usually only present in 1-2% of mtDNA across tissue but up to 10-20% in single cells.  By calculations, based on a low estimate of mutation rate, 90% of mitochondrial genomes have a mutation somewhere important. Based on a high estimate of mutation rate, there are on average 3 mutations per mitochondrial genome.  These numbers are very different from the usual numbers quoted and this is due to the trickiness of detecting microheteroplasmy (ie they are not detected at all by the usual methods of sequencing mtDNA) they say. Their referencing of reports of low levels of different types of mutations leading to high levels of mutations overall in sequencing of single neurons and glia are also at odds with the one mutational mtDNA taking over the cell's mitochondria that de Grey tends to reference.

The suggested mechanism of action is only in small part by causing insufficient amounts of ATP to be produced in the cell, but mainly by increasing reactive oxygen species (ROS) production to levels that damage the cell and/or drive it into senescence or apoptosis, with a possible feedback mechanism involved. They also postulate that even post-mitotic cells are regularly killed and replaced by cells derived from stem cells, and that accumulation of microheteroplasmy in the stem cells would lead to quicker reaching of the threshold amount of mutations in the derived cells needed for it to stop functioning properly (due to the above-mentioned mechanism). Overall though, the paper doesn't focus on the mechanism of action and does not rule out a different mechanism of action that would not go through ROS. In private communications with the first author, who gracefully supplied me with a copy of the paper and comments (Thanks Rafal), he mentioned he would downplay the effect of ROS on causing microheteroplasmy nowadays.

The paper has a long list of supporting evidence, but most of it seems quite tenuous. The strongest seems to be the case for Parkinson's disease correlating with mutations in a particular region of the mtDNA, which showed good predictive results in a small study. They mention correlations between Alzheimer's and complex IV dysfunction; cybrid models of both Parkinson's and Alzheimer's producing disease-specific responses (cybrids are hybrids of healthy cells without mitochondria fused with mitochondria from disease patients); faulty mitochondria in relatives and offspring of diabetes patients; oxidative stress and inheritance patterns in hypertension; glycolysis and cancer; metastasis in cancer as being a side-effect of old evolutionary reaction of the cell switching away from mitochondrial respiration as a source of power (due to mutated mtDNA).

The paper describes a particular type of mutation that they call focal microheteroplasmy, where a particular mutation becomes prevalent throughout a person due to it having originated during the mother's life prior to giving birth. While this mutation would be prevalent in the person's tissue, it would be completely different from other people in the family, even identical twins, who would have inherited a different half of the mitochondria. This mechanism is used as a partial explanation of the pattern of inheritance seen in certain diseases (eg Parkinson's and Alzheimer's).

The paper has an interesting list of corollaries/predictions of its hyptothesis:

• Focal microheteroplasmy in Parkinson's, Alzheimer's, diabetes and hypertension.
• Locations of the mutations in focal microheteroplasmy to be more detrimental than other locations of microheteroplasmy or of homoplasmic mutations.
• Accumulation rate of microheteroplasmy to be modulated by level of ROS.
• Menopause caused by microheteroplasmy, and trisomy 21 to protect the cell from microheteroplasmic-induced apoptosis
• Therapeutic interventions hammering secondary responses (eg amyloid in Alzheimer's) to be harmful
• Replacement of mitochondrial genomes to reverse many symptoms of aging

Overall, I'm not particularly fond of the mechanism of action being mainly down to ROS action. The predictions are quite good though, and if microheteroplasmy is as prevalent as stated then it should become quite clear shortly with the amount and variety of sequencing going on nowadays.

Premature ageing in mice expressing defective mitochondrial DNA polymerase

Interestingness: 8

By Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT and Larsson NG, in Nature, on the 27th of May, 2004. 429(6990):417-23. http://www.ncbi.nlm.nih.gov/pubmed/15164064


This isn't a paper from Rejuvenation Research, but since it was referenced in the last post, I read it and found it very interesting, notwithstanding de Grey's comments on it not being as interesting as it seems.

They created mice with a mutant version of the mtDNA polymerase instead of the regular version, that resulted in 3-5 times the usual number of mutations in their mtDNA and many more mtDNA deletions (30% less full-length mtDNA than wild type).  Mutations were uniform throughout the whole mtDNA.  The method of creating these mutant mice is interesting enough, but I won't describe that here.

These mice live for about a year. They have much smaller testes by the 3 month mark. They then get some fucked-up looking back deformations (kyphosis), start losing their hair, losing weight, become anaemic (with larger than usual and paler red blood cells), and get enlarged spleens by around the six-month mark.  They develop osteoporosis and enlarged left ventricles at around the 9 month mark.

If de Grey's explanation is right then it's not significant, but if he is wrong, then mtDNA mutations become more important.

Possible Influence of Metabolic Activity on Aging

Summary: Details of ATP production control mechanism in mitochondria

Interestingness: 4

Paper by Bernhard Kadenbach, Elisabeth Bender, Annette Reith, Andreas Becker, Shahla Hammerschmidt, Icksoo Lee, Susanne Arnold and Maik Hüttemann in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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This is more of a mitochondria biochem details piece, not directly related to aging. Most of it is too detailed for me to summarise or keep in memory or even follow.

Some interesting bits at the front that are not usually spelt out: out of the 13 proteins that mtDNA codes for, seven code for parts (out of 45) of NADH (nicotinamide adenine dinucleotide, protonated form) dehydrogenase (aka complex I), one for ubiquinol-cytochrome c oxidoreductase (aka complex III) (out of 11), three for cytochrome c oxidase (aka complex IV) (out of 13), and two for ATP synthase (out of some number I couldn't find). There's 5-10 mtDNA copies per mitochondrion, and 100-1000 mitochondria per cell.

It then describes two separate mechanisms of respiratory control. The first being due to the stimulation of ATP synthase by ADP triggering a lower proton motive force (deltaP) which trigger the proton pumps of the respiratory chain (NADH dehydrogenase, cytochrome c oxidoreductase and cytochrome c oxidase), kind of like an inverted system I think, with the final step pressuring the steps that come before it, but I imagine talking about the order here is completely wrong, they all happen at the same time. The second being due to the ATP/ADP ratio, with high ATP/ADP intramitochondrial ratio triggering a shut down of cytochrome c oxidase. This second method of control is bypassed by the presence of certain molecules, including 3,5-diiodo-L-thyronine, suggested as the mechanism of the short-term effects of thyoroid hormones, and palmitate (but not stearate, oleate or arachidonate).

The paper then does some studies showing that cAMP-dependent phosphorilation of complex IV enhances this ATP/ADP ratio control mechanism, and mitochondrial protein phosphatases reverse this enhancement. This second effect is shown mainly by adding a potassium fluoride which acts as a phosphatase inhibitor, and seeing the cAMP effect be stronger.

They also confirmed that it is mostly one mutant species of mtDNA that dominates a muscle fiber. They mapped a common deletion of mtDNA, probably that mtDNA4977 that was seen a couple of posts ago, and its occurrence varied between 0 and 0.06%, but corresponded with the bits of tissue that had malfunctioning complex IV.

They then speculate on how this phosphorilation/dephosphorilation mechanism is usually in balance, and is controlled by stressors and how when the ATP/ADP control mechanism is working, the proton gradient voltage is lower, and so less leakage of protons across the membrane occur, and less reactive oxide species are produced, and how this would be normally bypassed in a high caloric diet by the presence of palmitic acid, but the chain of reasoning is long and requires more concentration than I was willing to give it.

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Abstract follows:

The mitochondrial hypothesis on aging suggests stochastic stomatic mutations of mitochondrial DNA (mtDNA) as an important cause of respiratory-defective cells and the decline of energetic capabilities with increasing age. Reactive oxygen species (ROS), which are produced in the respiratory chain under stress conditions, are assumed to cause deletions and/or mutations of mtDNA. Using quantitative PCR, the stochastic distribution of the "common deletion" of mtDNA in human skeletal muscle tissue is shown. Recent data suggest that in vivo, under normal conditions, respiration is controlled by the intramitochondrial ATP/ADP ratio, via interaction of the nucleotides with subunit IV of cytochrome c oxidase, representing the rate-limiting step of the respiratory chain. Kinetic data are presented indicating that this "second mechanism of respiratory control" is turned on by cAMP-dependent phosphorylation of the enzyme and turned off by mitochondrial protein phosphatases. It is proposed that dephosphorylation of cytochrome c oxidase via "deleterious stress signals" results in increased mitochondrial membrane potentials and stimulated production of ROS in the mitochondrial respiratory chain. As a consequence, mutations of mtDNA would increase and aging would be accelerated. The inhibition of cytochrome c oxidase at high ATP/ADP ratios can also be abolished by low concentrations of free palmitate and high substrate pressure in the respiratory chain, supporting the notion that low caloric diet supports longevity.

Modeling the Role of Mitochondrial Mutations in Cellular Aging

Summary: Model of what happens if mitochondria with damaged DNA both reproduces and degrades slower than intact mitochondria, and how it fits observed data

Interestingness: 7

Paper by Axel Kowald and Thomas BL Kirkwood in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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They start by claiming there is a problem with the then-present theory of how damaged mitochondria are preferentially disseminated/take over cells by noting that it, the theory, is inconsistent with experimental results that show that damaged mitochondria is more prevalent in senescent cells than in dividing cells, and that the cells, or at least the muscle fibres, are taken over by one mutant type of mitochondria (by one I mean one type per case, not one common type for all cases) like we just saw in the last post http://readingrejuvenationresearch.blogspot.com/2010/12/segmental-nature-of-age-associated.html. Those are the main problems they want to see if they can patch with their model.

Their model starts at de Grey's model http://readingrejuvenationresearch.blogspot.com/2010/01/proposed-refinement-of-mitochondrial.html that basically hypothesises that mutant mitochondria produce less holes in their membranes and so are degraded less often. They justify the apparent contradiction in mutant mitochondria producing less holes with the "well known" fact that they produce more radicals by saying that most radicals are O2.- radicals but only the perhydroxy radical (HO2.-) can rip protons from lipids. Mutant mitochondria have a lower proton gradient so they produce lower amounts of HO2.- even if they produce more O2.-. Would seem good to get actual measurements, but they say that these aren't available and that they would be hard to get.

They, instead, produce a model with two assumptions: the first is that damaged mitochondria are destroyed slower than ones with intact mtDNA, and secondly, one introduced by them, that damaged mitochondria grow slower, which they justify by the energy shortage produced by the lower proton gradient. They split mitochondria into six groups, for little membrane damage, medium membrane damage and high membrane damage, each with intact mtDNA or mutant mtDNA. Radicals can increase the level of membrane damage or switch the mitochondria from an intact to a damaged mtDNA state. They give different turnover rates for mitochondria in each of the membrane damage classes, independent of their mtDNA state. The corresponding half lives for each damage class are 10, 2 and 1 week for low, medium and high damage. They used a factor of 2 as the increase in rate of free radicals that a mutant mitochondria produces compared to intact mitochondria, that mutants produced membrane damage at a rate 10 times lower than intact, and that intact grew 5 times quicker. I guess these numbers were half-guesses, and probably important in the results they got.

The model replicates the features from experiments they were looking to replicate, with one mutant taking over cells, and senescent cells having larger proportion of mutants than dividing cells, due to cell replication being a purifier of mitochondria. This purification happens because of the growth advantage of the intact mitochondria. This effect dominates when large amounts of mitochondria are to be produced, as in dividing cells, but the rate of destruction dominates when few mitochondria are being synthesised. They have some graphs showing what happens when the parameters are very different: if the mitochondria destruction rate are a bit lower, the population eventually collapses, if they are much higher, they collapse very quickly, along with other graphs showing the effects of different rates of cell reproduction and how that affects mitochondria population and stability (quick enough cell reproduction can fix higher rates of mutation).

From the model they also predict differences in importance between telomere shortening and mitochondrial damage in vivo vs in vitro. They claim that because in vitro conditions cells are replicated quickly, their collection of mitochondria will be pure through the process talked about above, so they will reach their Hayflick limit with nary an issue in their mitochondria, while in vivo, where cells replicate more slowly, mitochondrial damage will accumulate earlier and keeping telomeres long will not have an effect on cell lifespan.

(Interesting little factoid in the paper that I didn't fit in anywhere else: oxygen radicals are estimated to amount to 1-4% of consumed oxygen which sounded like a lot)

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Abstract follows:

The mitochondrial theory of aging suggests that an accumulation of defective mitochondria leads to loss of cell viability. The challenge is to explain how mitochondrial defects accumulate within cells, and why this process is more evident in postmitotic than in dividing cells. We describe a new mathematical model incorporating two critical features: (a) defective mitochondria are turned over more slowly than intact ones, and (b) defective mitochondria suffer a growth disadvantage. We also model the effect of cell division on the accumulation of defective mitochondria. The results support the mitochondrial theory and explain many of the observed data. The relationship of the mitochondrial theory to the suggested role of telomere loss in cell replicative senescence is discussed. We suggest that because of differences in the kinetics of their impact on cells, these two mechanisms have different relative importance for in vivo and in vitro cell aging.

Segmental Nature of Age-Associated, Skeletal Muscle Mitochondrial Abnormalities Necessitates Three-Dimensional Analyses

Summary: Mitochondria with abnormal electron transport chain activity are grouped along the fibre in muscle tissue

Interestingness: 4

Paper by Nathan L Van Zeeland, Jonathan Wanagat, Marisol E Lopez and Judd M Aiken in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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They looked at low cytochrome c oxidase (COX, complex IV) activity and high succinate dehydrogenase (SDH, complex II) activity in muscle tissue, which are supposedly common markers for age-related mitochondrial abnormalities. They are colocated with mitochondrial DNA (mtDNA) deletion (mtDNA4977). Also, muscle fibres with these abnormal mitochondrial activity are more commonly atrophied/have much lower cross-sections in rhesus monkeys. Part of the COX enzyme is encoded in the mtDNA, while all of the SDH enzyme is encoded in the nuclear DNA. (That explains why COX activity goes down, but why does the SDH activity go up?)

They measured COX and SDH activity in muscles of old (3-year old) rat and old (33 year old) rhesus monkey, making 200 slices across the muscle fibre so that they got a cross-section of the muscle at each slice. Each slice was about 10 microns thick, and they followed the muscle for about 1.6 millimetres in the monkey and 2 in the rat.

They found that the mutations were grouped along each muscle fibre. They found that in their sample, 3% of the rat's fibers had abnormal activity at some point along its length, and 0.31% of the monkey's (a 25-year old monkey though, not sure what happened to the other monkey), and contrasted these with the much lower values they would have gotten if they would have just sliced at one point (about six times lower). Through some dodgy extrapolation, they claim that 50% of the muscles fibers in the rat's case would be abnormal at some point if they had followed it through the whole length of the muscle, although they say that further studies by them point the number to be closer to 25%
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Abstract follows:

Age-associated electron transport system (ETS) abnormalities in skeletal muscle are distributed in a mosaic and segmental fashion; thus, histological techniques examining a single cross-section of tissue underestimate the number of fibers harboring such mitochondrial abnormalities. Analyses of consecutive cross-sections along the length of a muscle are necessary to determine the absolute number of ETS abnormal fibers within a given skeletal muscle. Two hundred serial cross-sections of old rat and rhesus monkey skeletal muscle were obtained by cryostat sectioning. Sections were stained and examined for cytochrome c oxidase and succinate dehydrogenase activity at regular intervals spanning a 1,600-micrometre region of muscle. All fibers staining negative for cytochrome c oxidase activity or hyperreactive for succinate dehydrogenase activity were then followed along their lengths to determine the extent of the ETS abnormal regions. ETS abnormalities in both animal models were found to be distributed in localized regions of individual muscle fibers (i.e., segmental). Examination of fibers along their length lead to a fourfold increase in detection of rat muscle fibers bearing mitochondrial abnormalities. In situ histological techniques that examine numerous sections at multiple positions along the length of skeletal muscles are particularly well suited for determining numbers and assessing the cellular impact of skeletal muscle fibers harboring age-related mitochondrial abnormalities.

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.

Area-Specific Differences in OH8dG and mtDNA4977 Levels in Alzheimer Disease Patients and Aged Controls

Summary: Mitochondrial DNA in the brain gets damaged at different rates across brain regions depending on type of damage, age, and Alzheimer's diseasedness.

Interestingness: 1

Paper by AMS Lezza, P Mecocci, A Cormio, M Flint Beal, A Cherubini, P Cantatore, U Senin and MN Gadaleta in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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They track two different common mutations to mitochondrial DNA (mtDNA) in post-mortem brains of 14 people, 8 with Alzheimer's, 6 control. One type of mutation is a deletion of 4977 bases in the mtDNA, which, going by the large amount of google results, seems to be quite a common thing to check for. The other is a product of oxidation, 8-hydroxy-2'-deoxyguanosine (OH8dG).

It seems like very little data to be taking the conclusions seriously, but the abstract is a good summary of the results. If nothing else, it seems that Alzheimer's disease patients have more oxidised mtDNA than non-Alzheimer's disease patients.
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Abstract follows:

The levels of mitochondrial DNA (mtDNA) 4977-bp deletion (mtDNA4977) and 8-hydroxy-2'-deoxyguanosine (OH8dG) have been measured in different brain areas of aged controls and Alzheimer disease patients. An area-specific distribution of the OH8dG level among three cortices and the cerebellum in aged controls as well as in Alzheimer disease patients has been found. It seems that in control subjects the age-related oxidative damage to mtDNA, represented by OH8dG content, shows a faster increase in the temporal and parietal cortices than in the frontal and in the cerebellum. In Alzheimer disease patients, where the OH8dG values are always higher than those of the control counterparts, such an area-specific distribution is maintained, but with a less significant difference among the cortices. The mtDNA4977 levels, on the other hand, are very different between frontal and parietal cortices on one side and temporal cortex and cerebellum on the other, both in control subjects and in Alzheimer disease patients. In general, it seems that the lowest mtDNA4977 levels coexist with the highest OH8dG contents in controls and, even more, in Alzheimer disease patients. This suggests that oxidative stress takes place both in aging and in Alzheimer disease, where it is amplified; however, mtDNA4977 level correlates with OH8dG content only in the frontal cortex of controls.