Using Epigenetic Measures of Age to Determine that Cellular Aging is Distinct From Cellular Senescence
One of the research groups involved in developing biomarkers of aging based on characteristic epigenetic changes published a most interesting paper earlier this month, linked below, in which they use their tools to investigate cellular senescence and cell aging. Biomarkers to measure biological age, the degree to which an individual is damaged and their biology has become dysfunctional in response to that damage, are an important line of development. An effective biomarker might be used to quickly assess the overall benefits of a potential rejuvenation therapy in mammals. As an alternative to running full life span studies this would dramatically reduce the time and cost required for such research. Cheaper research and faster results are certainly good for the pace of progress where they can be achieved.
Individual cells age, accumulating metabolic waste and unrepaired damage in the case of long-lived cells, or marching towards the Hayflick limit placed on the number of divisions permitted them in the case of short-lived somatic cells, but the relationship between cellular aging and tissue aging is not a straightforward one. A living being is a a dynamic system in which the majority of cells that make up its tissues are at some point replaced, on a schedule of days or months in most cases. Only in the central nervous system and a few other places do we find individual cells lasting a very long time, even the whole lifetime, and in which the steady accumulation of damage and waste are more important factors. In most tissues the importance is the pace of turnover, the number of lingering senescent cells that behave badly and refuse to die, the level of waste in the environment outside cells, and the quality of the stem cells that are responsible for producing a supply of new somatic cells to keep tissue functional.
Cells become senescent, removing themselves from the cycle of division and replication in response to a range of circumstances: damage, a toxic local environment, short telomeres as a result of reaching the Hayflick limit, and so forth. This probably serves to reduce cancer risk, at least initially, but senescent cells generate harmful signals that degrade surrounding tissues and produce localized inflammation. As their numbers grow, this damaging behavior contributes meaningfully to degenerative aging. As illustrated by recent research linking mitochondrial dsyfunction and cellular senescence, cells are very complicated machines: senescence isn't a single uniform state, not all senescent states are similar, and nor is it the case that all of the states that can be created in the laboratory are known to occur to a significant degree in living tissues. So this is an interesting area of cellular biochemistry to explore, even as clinical development is moving ahead on the blunt and direct approach of clearing senescent cells from the body so as to remove their detrimental effects. This is absolutely the way things should be: taking the fast road to therapies that will effectively treat the causes of aging even in the absence of detailed understanding, and those researchers who can raise funds for more leisurely investigation and mapping can continue their work in the meanwhile. If only this were the case in other fields relevant to aging, but for the most part only the leisurely investigation is taking place there.
Epigenetic clock analyses of cellular senescence and ageing
One model of ageing posits that the failure of tissues to function properly is due to the depletion of stem cells. Stem cells, which are the reservoir cells of tissues, may have finite capacities of proliferation such as being limited by the lengths of their telomeres. Their eventual depletion leads to the deficit of properly functioning cells, causing phenotypic changes that constitute ageing. While this model is plausible and supported by strong circumstantial evidence, it is presently difficult to prove or refute directly, not least because the identification of specific tissue stem cells is difficult. Similarly, the association between telomere length and ageing, although widely reported, is not without inconsistencies.There is however, another model of ageing which is based on the observation that the number of senescent cells in the body increases in function of organism age. While this could be interpreted to mean that senescent cells cause ageing, it could also equally mean that senescent cells are a consequence of ageing. In this regard, it is noteworthy that there is increasing evidence to demonstrate that senescent cells are not benign. Instead they secrete bio-chemicals that are detrimental to normal functioning of neighbouring cells. The senescence-associated secretory phenotype (SASP) proteins include cytokine, chemokines and proteases and their paracrine activities are very diverse and include oncogenic characteristics that stimulate cellular proliferation and epithelial-mesenchymal transition. Importantly, SASP proteins also promote chronic inflammation, which is the origin of almost all age-related pathologies. As such, SASP proteins, through their different effects on normal and cancer cells, induce deterioration of the tissue. Recently, it was demonstrated that removal of senescent cells in mice delays ageing-associated disorders, providing very strong support for the notion that senescent cells mediate the effects of ageing. Hence it follows that to understand ageing, it is necessary to understand cellular senescence. This model of active induction of ageing (via senescent cells) does not exclude the role of stem cell depletion described above, which could indeed be a result of stem cell senescence.
At present, the causes of cellular senescence in vivo are not known for certain but in vitro, cells can become senescent through (i) telomere shortening via exhaustive replication (replicative senescence), (ii) over-expression of oncogene or (iii) DNA damage. While it is easy to perceive replicative senescence (RS) as part of a bona fide mechanism of ageing, it is more challenging to consider oncogene-induced senescence (OIS) as a significant contributor to natural ageing. Instead OIS has been proposed to function as a tumour suppressor mechanism. The only obvious common factor between RS and OIS is the co-opting of the DNA damage signalling mechanism to usher cells into arrest.
Recently, we developed a multivariate estimator of chronological age, referred to as epigenetic clock, based on methylation levels. The following features of this clock demonstrates that its age estimates capture several aspects of biological age: (a) it can accurately measure the age of cells regardless of tissue types including brain, liver, kidney, breast and lung (b) its accuracy is substantially higher than that of other molecular markers such as telomere length (c) it is able to predict mortality independent of health, life-style or genetic factors (d) its measurements correlate with cognitive and physical fitness amongst the elderly and (e) it is able to detect accelerated ageing induced by various factors including obesity, Down syndrome and HIV infection. Here, we apply this epigenetic clock to study the relationship between ageing and senescence of isogenic cells induced by exhaustive replication, ectopic oncogene over-expression or radiation-induced DNA damage.
We show that induction of replicative senescence (RS) and oncogene-induced senescence (OIS) are accompanied by ageing of the cell. However, senescence induced by DNA damage is not, even though RS and OIS activate the cellular DNA damage response pathway. Collectively, these two sets of observation make an effective case for the uncoupling of senescence from cellular ageing. This however, appears at first sight to be inconsistent with the fact that senescent cells contribute to the physical manifestation of organism ageing, as demonstrated elegantly by studies in which removal of senescent cells slowed down ageing. In the light of our observations however, it is proposed that cellular senescence is a state that cells are forced into as a result of external pressures such as DNA damage, ectopic oncogene expression and exhaustive proliferation of cells to replenish those eliminated by external/environmental factors. These senescent cells, in sufficient numbers, will undoubtedly cause the deterioration of tissues, which is interpreted as organism ageing. However, at the cellular level, ageing, as measured by the epigenetic clock, is distinct from senescence. It is an intrinsic mechanism that exists from the birth of the cell and continues. This implies that if cells are not shunted into senescence by the external pressures described above, they would still continue to age. This is consistent with the fact that mice with naturally long telomeres still age and eventually die even though their telomere lengths are far longer than the critical limit, and they age prematurely when their telomeres are forcibly shortened, due to replicative senescence. Hence senescence is a route by which cells exit prematurely from the natural course of cellular ageing.
Finally, it is necessary to address specifically the role of telomeres as it is easy to confound them with cellular ageing because at first view, they appear to share similar features. Since critical telomere length is attained after many rounds of proliferation, which takes a long time and hence occurs later in life, it is easy to mistake this for a functional link with age even though telomere length has only a modest correlation with chronological age, while cellular ageing as measured by the epigenetic clock has a far higher degree of association with biological ageing. The fact that maintenance of telomere length by telomerase did not prevent cellular ageing defines the singular role of telomeres as that of a means by which cells restrict their proliferation to a certain number; which was the function originally ascribed to it. Cellular ageing on the other hand proceeds regardless of telomere length.
Although the characteristics of cellular ageing are still not well known, the remarkable precision with which the epigenetic clock can measure it and correlate it to biological ageing remove any doubt of its existence, distinctiveness and importance. This inevitably raises the question of what is the nature of this cellular ageing, and what are its eventual physical consequences. Admittedly, the observations above do not purport to provide the answer, but they have however, cleared the path to its discovery by unshackling cellular ageing from senescence, telomeres and DNA damage response, hence inviting fresh perspectives into its possible mechanism. In summary, the results from these experiments, while apparently simple in their presentation, untangles a conceptual knot that hitherto tied senescence, DNA damage signalling, ageing and telomeres together in an incomprehensible way. Here we propose that cellular ageing, as measured by the epigenetic clock, is an intrinsic property of cells, and while independent, its speed can be affected by some factors; a feature that would undoubtedly be exploited to characterise and elucidate its mechanism.
Hi! Excellent article!
My 2 cents. Summary : Telomeres are still It.
''This is consistent with the fact that mice with naturally long telomeres still age and eventually die even though their telomere lengths are far longer than the critical limit, and they age prematurely when their telomeres are forcibly shortened, due to replicative senescence. Hence senescence is a route by which cells exit prematurely from the natural course of cellular ageing...
The fact that maintenance of telomere length by telomerase did not prevent cellular ageing defines the singular role of telomeres as that of a means by which cells restrict their proliferation to a certain number; which was the function originally ascribed to it. Cellular ageing on the other hand proceeds regardless of telomere length.''
''These senescent cells, in sufficient numbers, will undoubtedly cause the deterioration of tissues, which is interpreted as organism ageing.
However, at the cellular level, ageing, as measured by the epigenetic clock, is distinct from senescence. It is an intrinsic mechanism that exists from the birth of the cell and continues. This implies that if cells are not shunted into senescence by the external pressures described above, they would still continue to age. ''
I am guessing when they say ''they would still continue to age'', they imply replicative lifespan potential.
''DNA methylation age and the epigenetic clock
The epigenetic clock is defined as a prediction method of age based on the linear combination of the DNA methylation levels of 353 CpGs dinucleotides''.
This is rather ironic, they measure methylation in CpG (Cytosine Guanine) DNA islands and use that as epigenetic clock.
I think they are forgetting something...telomeres/sub-telomeres/centromeres are CpG rich and the tallest total telomeres ones are Hyper-Methylated.
Methylation loss - by telomere loss (becoming demethylated/oxidative stress being the first reason to accelerating telomere loss - and thus demethylation) - Is a cause of intrinsic aging. They may use semantics and call it epigenetic clock, it still doesn't change the fact that telomeres are - behind this. Telomeres are part of the nuclear chromosomes so yes I would believe very much they have a say in intrinsic human biological aging - perhaps not in cell aging.
The fact they sa their epigenetic clock is a 'biological' counter whereas telomeres somehow are not is a bit BS. Telomeres - are - biological counters - not just some correlative thing or 'chronological' marker.
I agree though that telomeres act as proliferative stoppers to avoid tumor formation at M2 point (low critical 2 Kb telomere size and avoid tumor immortalization).
The bit that senescence is different than celluar ageing is very true. And that immortal telomerased-cell in their research still 'aged' doesn't change the fact that the telomeres maintenance allowed longer replicative potential and is a marker of Low oxidative stress in the body - allowing a longer chronological - and biological life (biological life is what is important, chronological is not).
As for using the example of mouse dying with high telomeres, that's exactly an example of Senescence acceleration in these mice such as SAMP mice (senescence accelerate prone mice) who die even quicker.
As was said, senescence and apoptosis are mechanisms that can be decoupled from telomeres, but in humans the replicative potential is a very strong indicator of biological lifespan.
Also, when they said : ''This is consistent with the fact that mice with naturally long telomeres still age and eventually die even though their telomere lengths are far longer than the critical limit,
***and they age prematurely when their telomeres are forcibly shortened, due to replicative senescence***''.
That is the most important point, in humans, we don't age as fast as mice or become dysfunctional like them. Mice accumulate so much damage fast - that is triggers the inducible senescence - despite high telomeres - not in humans.
In humans, it,s all about replicative aging, long lifespan, low oxidative stress. Diseases of humans are the inducible senescence. Regular intrinsic aging - via telomere loss - and damage accrual (AGEs, mtDNA lesion
8-oxodG, y-H2AX, protein carbonyls, lipid peroxidation chains, lipofuscin, etc) are far more important and limit the human lifespan.
By having telomeres stopping the maximum lifespan, wether the cell continues aging or not, humans can't go above the 120s years old.
Telomeres are prime targets, mitochondrial complex ROS prodution and lysosome lipofuscin-ROS production create damage of mitochondrial DNA, which limits energy production, creates nuclear damage and DDR (DNA-damage response as mentioned in paper, DDR is telomere aging/response mechanism to Repair it quick via Nucleotide-Repair Enzyme Systems (such as Werner Helicase (failing in Werner progeria)), as seen with accelerating telomeres loss with 'premature aging progeria' old-like 'young' people'syndromes.
The fact that immortalized human Werner syndrome fibroblasts - accumulate no lipofuscin for at least 250 PDs, shows that replicative lifespan potential - via telomere loss- is the main driver of intrinsic aging in humans.
Whether the epigenetic clock of cell has 'aged' despite the cell being immortalized by telomerase, doesn't make any difference - it could be epigenetic clocked age '300 years' as long as kept on proliferating (like in cancers). When the cell stops dividing, it's where problems arrive (such as M2 crisis telomere). Small telomeres stop the cell from dividing to block tumor immortalization.
As such the are - tied - together epigenetic clock - and telomeres. A human of 120 years is biologically, chronologically and epigenetically/clock - and telomerically - older than a 20 year old (so far, biorejuvenation therapy could change that - only if started at 20 years old could you be 120 chronologically). So basically makes not that much difference. Other than showing that, in humans, not mice, losing your telomeres is a problem because Hayflick/replicative potential, relevant in humans (at least in long-lived post-mitotic somatic telomerase-devoid cells like neurons, CNS cells among others) irrelevant in mice, is, a problem.
I agree.
Liz Parrish used gene therapy to induce telomerase expression in her body because research in mice has shown rejuvenation of tissues and an increase in median lifespan in middle-age (24%) and old mice (13%). Whether this effect is due to longer telomeres or some other benefit of telomerase is unknown. Thanks to her self-experimentation we may soon know more about the effect in a human. Aging isn’t just one thing so telomerase won’t cause complete restoration to a young state but telomerase therapy might help some of us reach “escape velocity” by adding a decade or two.
Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494070/
@Anonymous
Hi Anonymous !
Me too, I'm very eager of Liz Parrish's telomerase self-therapy results.
Exactly, telomerase will not reverse certain accumulated irreversible residues from oxidative damage process (such as ecm crosslinks or lysosomal lipofuscin).
But will definitely increase telomere size (only the smallest ones) as seen with astragalus (activates telomerase); I am guessing the therapy effect will be close to astragalus's effect - because they both act on the same enzyme (Telomerase) and activate it in somatic cells (where it is absent). Albeit, the therapy may be much stronger in effect, that remains to be seen (I highly doubt it since astragalus activates maximal telomerase (something like 2 to 5 fold activation of hTERT/hTERC activity and catalytic subunit mRNA expression, and that's about the max I read in other studies activating hTERT by genetic manipulation).
Out out chromosomal urgency for genome stability and telomere stability, short unstable telomeres are the target ones of telomerase; short telomeres are highly unstable (prone to fusions, joining (chromosome fusions, (SCE, sister chromatid exchange), mutational immortalization or activating senescence, long ones are already stable (elongated enough) and so telomerase does (with reason) nothing on them; like a negative feedback mechanism where telomerase stops elongating telomeres - once long enough (too long telomeres would create chromosomal abberations) and does not elongate or touch - already longest telomeres.
Since telomerase is shunted away from nucleus to mitochondria during certain high oxidative stress states (to mitigate mitochondrial DNA damage/mtROS production), I believe the therapy will be mostly therapeutical (improve health and allow her perhaps a decade extra in good health, but never increase the human maximum lifespan) as her longest telomeres - will still shorten during her remaining life (while she will have an 'averaging' effect on the shortest telomeres to increase 'overall' the shortest ones - to approach the size of her tallest ones - an 'averaging effect'. Exactly, what happened with astragalus/cycloastragenol/TA-65).
Mice that received telomerase therapy and had increased lifespan, barely benefitted from longer telomeres, only their smallest ones increased and even then, mice have huge telomeres and have more telomerase than humans, by nature (hence their long telomeres). Thus, telomerase would not have acted as an elongator but as reducer of oxidative ROS production in their mitochondrias and nucleus. Telomerase has a dual role of elongator and oxidative stress reducer (by modulating ROS production). What I want really want to know, has her cancer/tumor proliferation markers increased that much by the therapy or not at all (astragalus increases hTERT in non-cancerous healthy somatic cells but is capable of inhibiting cancer proliferation precisely in cancerous cells - at the same time - making up the safer strategy than this therapy which we don't know it's effects in cancerous cells (it has must more likeliness of increase telomerase in cancers than astragalus does, because astragalus modulates genetically the cancer cells to die (it activates hTERT, only, in the right cells, and deprives it, in cancer cells. That is exactly, what the OncoSENS therapy should do, not block telomerase in non-malignant somatic cells, only in cancerous ones who highjack telomerase for their metastatic invasion).
This is rather ironic, they measure methylation in CpG (Cytosine Guanine) DNA islands and use that as epigenetic clock.
I think they are forgetting something...telomeres/sub-telomeres/centromeres are CpG rich and the tallest total telomeres ones are Hyper-Methylated.
Methylation loss - by telomere loss (becoming demethylated/oxidative stress being the first reason to accelerating telomere loss - and thus demethylation) - Is a cause of intrinsic aging. They may use semantics and call it epigenetic clock, it still doesn't change the fact that telomeres are - behind this. Telomeres are part of the nuclear chromosomes so yes I would believe very much they have a say in intrinsic human biological aging - perhaps not in cell aging.
Assuming I interpreted your assertions correctly, then perhaps the 353 identified CpG sites are a surrogate marker of telomere loss? If that is the case, then that interpretation may not explain the epigenetic changes taking place in the cells immortalized by transduction with the hTert homologue. That said, there did not appear to be any data showing that telomores were in fact elongated relative to non-transduced cells.
If there were telomere elongation taking place and the same epigenetic changes were evident suggesting the aging process still occurring, then would that have any affect on your intepretation?
@Aaron
Hi Aaron !
It wouldn't. Cells that are about to immortalize cannot continue dividing after they reach M2 crisis (the second and final barrier, before spontaneous immortalization, that a few cells reach after a couple more population doublings, after escaping M1 crisis (the first barrier is replicative senescence). M1 is anywhere from 5 to 3 kb (kilobytes/kilobase pairs) telomere length, M2 is at 2 kb (replicative senescence happens again like at M1, if the cell re-escapes replicative senescence at M2, it immortalizes at that point; creating a cancerous cell that uses hTERT or ALT-recombination to divide infinitely.
That there is no telomere elongation, above current telomeres' length, is not the important point; it is that they maintain telomeres - from shortening - further -/below current length. Telomerase acts on the smallest telomeres to lengthen them/average them to the tallest ones.
In immortal cells, hTERT is highjacked illegitly and is mutated form of hTERT (dysfunctional/unstable), cancerous cells transform this hTERT to never shut off, to lengthen their telomeres - as they shorten - to yield a net unshortening telomere size of 2kb - that never goes below that or above that (constant, stable, unmoving 'frozen/immortalized' size). They judiciously 'pace' telomerase activity/rate or ALT-recombination rate to remain in the 2 kb region (a perfect area for chromosomal instability and just enough inflammation, a middle-ground/cancer
'sweet spot' to promote mutational cancerous transformation) and stay immortal.
These epigenetic changes may manifest themselves below telomeres in the chromosomes and, thus, would partly explain the discrepancy between telomeres and epigenetic clock.
For humans, it is not most relevant, because our intrinsic aging is telomeres bound and replicative senescence bound (thus M1 M2 bound). Like that example of a 115 years old supercentenarian woman whose blood leukocytes cells had an average of 3 kb telomeres length. That is M1 very very soon. After that, death, she had 10 years or less left. Large scale replicative senescence is incompatible with human survival.