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.

Reactive oxygen species production in the mitochondrial matrix: implications for the mechanism of mitochondrial mutation accumulation

Interestingness: 3

By Aubrey D.N.J. De Grey. Rejuvenation Research. Spring 2005, 8(1): 13-17. doi:10.1089/rej.2005.8.13.

Comparison between three different theories of mitochondrial DNA mutation, and the how to interpret new findings that alpha-ketoglutarate dehydrogenase (AKDH), an enzyme in the mitochondrial matrix, makes hydrogen peroxide and possibly superoxide when exposed to high concentrations of NADH.

One of the theories is the vicious cycle hypothesis, in which mutations in the mtDNA trigger creation of superoxides which then trigger more mtDNA mutations, etc. Another is de Grey's own survival of the slowest (SOS) hypothesis described earlier in the blog. The third is the crippled mitochondrion (CM) (underspecified) hypothesis in which mutant mitochondria are stimulated to replicate by some mechanism internal to the mitochondria.

de Grey claims that the vicious cycle theory is refuted by the commonality of mutations that would get rid of the possibility to make superoxide (mtDNA deletions that get rid of the genes encoding for Complex I and III, and also get rid of at least one tRNA, for which there are no redundancies in the mtDNA), and also by the observation that the mutations in all the mitochondria's DNA tend to be the same within any one individual cell.

Differences in predictions by the other two theories:

SOS	CM
Autophagy of mitochondria selects mitochondria with damaged membranes	No prediction
Loss of ATP-synthase does not preferentially replicate	All functional losses replicate
No prediction	Cell overpopulated with mitochondria comes before or at the same time as loss of respiratory function
ROS production by mutant mitochondria eliminated	No prediction

That last prediction by the SOS hypothesis, lack of ROS production, seems to run counter with findings that oxidation-damaged DNA and RNA are found in respiration-deficient muscle fiber segments. This is the reason for bringing up the new findings of generation of ROS by AKDH when under high concentration of NADH, a state de Grey claims would be more common in mitochondria with broken respiratory chains, giving the oxidised DNA an alternate cause.

Selective Mitochondrial Autophagy, or Mitophagy, as a Targeted Defense Against Oxidative Stress, Mitochondrial Dysfunction, and Aging

Interestingness: 6

By Dr. John J. Lemasters. Rejuvenation Research. Spring 2005, 8(1): 3-5. doi:10.1089/rej.2005.8.3.

Short speculative piece about the importance of a mitochondrial outer membrane protein, Uth1p, in aging. Yeast cells with mutant versions of the gene have lower levels of mitochondrial autophagy when induced by rapamycin and starvation. If this means that the particular mitochondria that don't express Uth1p are not recycled, then it would give an "evolutionary" advantage for mitochondria to have mutant versions of this gene, as long as they are picked as often as others for mitochondrial replication. If this is the case, then other mutations in the same mitochondrion as ones with Uth1p mutations would proliferate throughout the cell.

As a downer, it seems there isn't (wasn't?) much information about the human analogue/s of the gene.

Mitochondrial Mutations in Mammalian Aging: An Over-Hasty About-Turn?

Interestingness: 7

By Aubrey DNJ De Grey, in Rejuvenation Research, Northern Autumn 2004, 7(3): 171-174. doi:10.1089/rej.2004.7.171.

A short piece of commentary trying to raise the profile of, I think, this paper:  "Construction of transgenic mice with tissue-specific acceleration of mitochondrial DNA mutagenesis", by Zhang D et al (http://www.ncbi.nlm.nih.gov/pubmed/11031098), but mainly about lowering the importance of "Premature ageing in mice expressing defective mitochondrial DNA polymerase" by Trifunovic A et al (http://www.ncbi.nlm.nih.gov/pubmed/15164064 ) . I haven't read the former, and I've only read the latter just now (separate post).

Zhang's paper is  about mice with a mutant mtDNA polymerase which produced more errors, but is only activated in the heart, and only after birth.  Trifunovic's paper is a similar concept but they made it active in all tissue. Also, while in Trifunovic's experiment they modified the built-in version of the mtDNA polymerase, I am not sure if that is what was done in Zhang's version, or if they just added a second copy.

The results of the modifications are that in Zhang's, the mice get cardiomyopathy, while Trifunovic's mice get fucked up all over and die at around the year mark.

de Grey's objection to the importance of Trifunovic's paper is that the effects seem to hit mainly tissue undergoing rapid replication (the spleen, the skin, testes and blood), and that the heart issues were a compensation mechanism for the anaemia.  His claim is that this failure of rapidly replicating tissue cannot cause a shortening of lifespan since the failing cells can just be replaced by the replication of the still-good cells, unless the cells are failing faster than they can be replaced, which is what he thinks is happening during the experiment.

It would be good if he'd have addressed specifically the coincidence that in both cases the mice suffered from enlarged hearts, but according to him due to separate causes (ie is it a coincidence?).  I don't know the timing of the cardiomyopathy in the Zhang experiment though, so it might have come around much later and there might not be anything to address.

Antioxidant Genes, Hormesis, and Demographic Longevity

Interestingness:5

By Robert Arking and Craig Giroux in the Journal of Anti-Aging Medicine, June 2001, pp125-136. doi:10.1089/10945450152466170.

It didn't end up being as interesting as expected, since they skipped/assumed the question of the effect of late-life mortality deceleration or even decrease being real in humans.  They mostly work on fruit flies, and that's where most of their data comes from.

I still find overarching theories interesting though, so it was a decent read, even though there isn't much more there to summarise than what was on the abstract. 

Their hypothesis can be summarised as follows: there are some stressors that will kick what they call the antioxidant defense system (ADS) and heat shock proteins (hsps) into action and will wipe some "aging" off the body, thus leading to lower mortality.  This activation is semi-locked by an epigenetic mechanism, thus leading to a clustering of people with lower mortality separate from the main cluster.  They hypothesis that testing young people to see whose ADS get upregulated easiest will tell you which people will leave longer.

One big issue with the paper for me is their evidence that upregulated ADS and hsps lead to longer lifespan. Their data is from fruit flies, but they mention mice, in which just upregulating CuZnSOD (superoxide dismutase) doesn't lead to longer lifespan.  In mice, they go by the suggestion that since calorie-restricted mice have upregulated CuZnSOD and catalase, that these two are important reasons of the lifespan extension. I suspect the situation in humans to be even fuzzier.

Another bit of the paper I found interesting is at the beginning where they list possible theories for the decelerated/decreased mortality effect: one, that this is just part of the aging process; second, that this is a predicted effect of the reliability theory I'm fond of by the Gavrilovs (I find these two explanations to be compatible), and third, that the population is genetically heterogenous, so each subsection would have its own Gompertz gradient. 

Role of Cell Senescence in Human Aging

Summary: Cell senescence is the problem, but we won't talk about cancer.

Interestingness: 7

Paper by Michael Fossel in the Journal of Anti-Aging Medicine, Volume 3, Issue 1, Spring 2000.

(((

This paper is mostly a defense of the senescence model of aging which consists of saying that cell senescence is the main reason for aging. The paper consists of clarifications on possible misinterpretations of the theory. The model, it says, only pertains to human aging. It says that in critical tissue, enough cells senesce for it to have organism-wide effects, either by their inability to replicate, or by their changed gene expression patterns.

The clarifications are presented by examples. One main example is that heart attacks and strokes are compatible with the theory. Damage to the endothelial cells, the surface layer, cause neighbouring endothelial cells to replicate. At some point they senesce, at which point the holes on the surface aren't fixed any more and the plasma has direct access to the subendothelial layer, triggering the rest of the effects. A bit of supporting evidence is that the places on the blood vessels at which atherosclerosis is usually formed are the same places at which telomere length of endothelial cells is shortest.

The paper has doubts about the ability of the model to explain Alzheimer's but it suggests that since astrocytes divide, measuring telomere length in astrocytes and comparing to Alzheimer's propensity would be a good test.

There is an interventionist undertone to the paper, which is why I think it is interesting. It wants to shove human telomerase (hTERT) in tissue (leukocyte stem cells, the skin of Hutchinson Gilford Syndrome patients, arterial endothelial cells) and see if that fixes them or affects longevity. It mentions unpublished experiments, at least unpublished at that time, about using young cells vs old cells vs old cells with telomerase, in forming skin layers on a naked mouse. The old cells formed skin that looked like old human skin, while the young and telomerased cells formed normal, "grossly, microscopically and genetically", looking skin.

I am a bit surprised that the paper doesn't mention cancer at all. Senescence seems like a method of cancer control so I'd expect something to be said about it. The theory is vague and broad enough to be compatible with lots of other theories of aging, but I think it is not compatible with the mitochondrial free radical theory of aging. The one presented here seems easier to test.

)))

The notion that cell senescence might, ultimately, be central to human aging has been attractive but unsubstantiated for the past four decades. Recent genetics and cell biology work has strongly supported this position. The model has been criticized, largely because few understand what the model actually says about aging. The cell senescence model (often mislabeled the "telomere theory of aging") suggests that changes in gene expression within senescent cells underlie most common age-related pathology, for example those occurring in the coronary arteries in atherosclerosis. It does not suggest that most somatic cells senesce, but rather that those cells which do senesce (e.g., endothelial cells, chondrocytes, fibroblasts, keratinocytes, microglia, hepatocytes, etc) are common denominator of human aging and age-related disease as well as the most efficient point for therapeutic intervention. The cell senescence model of human aging remains elegant and consistent with all known data on human aging and disease; an appropriate criticism is that it remains yet unproven.

The Telomere Shortening Signal May Be Explained by a Fountain Mechanism Modulating the Expression of Eukaryotic Genes

Summary: Speculation on the mechanism involved in telomere-shortening bringing about cell senescence

Interestingness: 4

Paper by AM Olovnikov in the Journal of Anti-Aging Medicine, Volume 2, Issue 1, Spring 1999.

(((
This dude hypothesised that telomere shortening was the trigger for cell senescence and the existence of telomerase back in the 70s. He now gets to publish whatever he wants about telomeres like the hypothesis in this paper. By the present time (2010) biologists probably know if the theory has any merit but I don't, so to me it's still interesting. The idea is more about basic cell biology than about aging. Its only link with aging is that it explains cell senescence via telomere shortening.

The theory tries to explain how it is that telomere shortening causes senescence. It proposes that some bits of RNA bind to and open Ca2+ and Zn2+ channels on the nuclear membrane, and that the influxes of these ions into the nucleus are critical to the transcription of some/most genes. When telomeres shorten, they would physically pull genes near the telomeres out of the areas where these ion influxes happen and therefore they would stop being transcribed, or at least their transcription patterns would be significantly altered. From what I can tell, the specific bits of RNA, which he calls fountain RNAs (fRNAs), and the importance of the ions to transcription are both speculation.

He says that the location and orientation of the chromosomes between G1 and S phase are nonrandom. The telomeres attach to the nuclear membrane, and the bits of attachment are a reinforced section of the membrane that lack the ion channels in question. As the genes near the telomeres get pulled in closer to the membrane, they would also miss out on the ions.

The fRNAs would be composed of two sections, one that would bind to a section of the genome close to the genes that are going to be induced by the ions, and the other section to the ion channels. The sections of the genome to which the fRNA binds to are called converters. The section of the fRNAs that bind to them would vary depending on which section of the genome the fRNA is meant to stimulate. The other section of the fRNA that binds to the channels, in order to open them, would be constant per channel type, Ca2+ and Zn2+ (although the choice of these two doesn't seem central to the theory, and Mg2+ is listed as another option), but the fRNA wouldn't be able to bind to the channel without having first bound to its converter. The activation of the bits of DNA that code for the fRNAs themselves, called modulators, could themselves be controlled by the ionic fluxes so all sorts of feedback loops and modulation of gene expression would exist.

I had problems distinguishing which bits of the paper were speculation and which parts are presented as evidence. From what I can tell, the following are some of the snippets given as supporting evidence for the theory:

• Ca2+ can increase both transcriptional activity and mRNA stability, increases promoters and RNA levels
• There are Ca2+ releasing channels in the inner nuclear membrane and the nuclear envelope has a store of Ca2+
• Zn2+ involvement in zinc fingers, and their involvement in everything DNA
• Explains the long spacers between genes as spacers decoupling the ionic activation between the genes

)))


Abstract follows:

We propose a possible mechanism for the telomere shortening signal. The suggested solution of this as yet unsolved enigma—how cell senescence is causally linked to telomere short-ening—is based on a "fountain theory" of modulation of eukaryotic gene expression, in which gene expression is modulated by ionic channels of the inner nuclear membrane. These Ca2+ and Zn2+ channels are opened transiently through the action of a special small nuclear RNA (the fountain RNA or fRNA) on the ionic channels as conformational changes of the fRNA and channel-forming protein occur. Specific Ca2+ and Zn2+ ion channels allow these ions to pass from the perinuclear lumen to the nucleoplasmic gene surroundings. The resultant change of ionic concentration in close vicinity to certain genes, in turn, will alter some in- properties (e.g., mRNA stability, transcript maturation, chromatin configuration, transcriptional activity, and so forth).

Such fRNA-dependent ionic "fountains," may serve as a major mechanism regulating quantitative gene (phenotypic) expression in eukaryotes. We suggest that among metal-activated transcription factors, zinc-finger nuclear proteins evolved, and they are used in the nucleus as an alternative, noncalcium, path of gene-activity modulation, by means of fRNA-dependent channels, increasing the versatility of a fountain system.

We further propose that telomeres are anchored—in a compacted state—to special reinforcing shields, which are parts of the nuclear lamina along the inner nuclear membrane. This may be particularly true between Gl and S phases of the cell cycle, when chromosomes have nonrandom allocation within a nuclear space and telomeres are compacted and serve as "spacers" between the subtelomeric chromosome and the inner nuclear membrane. Each reinforcing shield would cover a portion of the inner nuclear membrane and, in doing so, prohibit the action of fRNA-dependent ion channels, causing an ionic "dead zone" in the nuclear membrane located immediately beneath the shield. When telomeres are long (e.g., in young cells), subtelomeric genes are located at a relatively greater distance from such dead zones; when telomeres shorten and reach the critical threshold, subtelomeric genes become closer to the dead zone and are deprived of contact with active ion channels. Shortening of the telomere—and therefore of the distance of subtelomeric genes from the dead zone—alters subtelomeric gene expression, decreases the functional capabilities of the cell, and results in cell senescence.

In some species, such subtelomeric genes may encode the fRNAs themselves, in addition to structural genes. If modulator genes—coding for fRNAs—require the ion fountains for optimal expression, then other structural genes (in turn modulated by such genes) will inevitably show senescence-associated gene expression as the telomere shortens. Such an alteration of gene expression, and the consequent dysfunction in cellular homeostasis, are typical of senescing cells.

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.