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Assessing the Interaction Between Telomerase Activity and Epigenetic Age in Cell Cultures

One of the more interesting aspects of the various epigenetic clocks that have been developed in recent years is that it is still largely unknown as to what exactly it is that they are assessing in our aging biochemistry. These clocks are weighted measures of epigenetic markers, such as DNA methylation, over a comparatively small number of genes. The resulting number certainly reflects chronological age, with the best clocks having a margin of error of a few years when assessed over a group of people. There is also sound evidence for it to reflect biological age, the burden of damage and dysfunction, which varies between individuals. Some people are more burdened by aging than others, and it is thought that these clocks can assess that difference.

But what underlying processes of aging are driving the results? That isn't clear at all. It is quite possible that an epigenetic clock measures the changes resulting from only a limited subset of the full range of age-related damage and dysfunction. Because aging is a global phenomenon in which all of its aspects tend to be fairly well correlated with one another, the clock nonetheless works well as a measure of overall aging. We will only find out whether or not this is the case as rejuvenation therapies start to emerge, treatments that very selectively address one and only one of the root causes of aging. Does treatment with senolytics to reduce the number of lingering senescent cells reverse the epigenetic clock measure, for example? We'll know the answer to that question in the near future, but for now those studies are still underway or pending publication.

Researchers here perform an preliminary investigation of what happens to epigenetic age in cells in which telomerase is at work. Telomerase acts to extend cell life by extending telomeres. Telomeres are the caps at the end of chromosomes, and are reduced in length with each cell division. This is a part of the countdown mechanism that leads to the Hayflick limit, preventing normal somatic cells from replicating indefinitely. Once they reach the limit, they self-destruct, or become senescent and are then destroyed by the immune system. Stem cells can replicate indefinitely because they use telomerase, and their role is to create new somatic cells with long telomeres to replace those lost over time. This split between a few privileged cells and the vast majority of limited cells is the way in which cancer risk is kept low enough for evolutionary success in higher animals.

A faction in the research and development communities are quite enthusiastic about telomerase gene therapy as a means to extend life, based on results from animal studies over the past decade or more. This most likely produces benefits through enhanced cell activity, and particularly stem cell activity, in a context in which the evolved balance of declining cell activity with age, most likely a defense against cancer, has some wiggle room. It appears possible to produce greater regeneration in later life without greatly raised risk of cancer by pressing damaged cells into undertaking more work. As is discussed by the authors of the paper below, this may also have something to do with reduced levels of senescent cells: a damaged cell that continues operating might, on average, be less immediately harmful than a lingering senescent cell, even though one would imagine this to raise cancer risk. The degree to which these and other mechanisms might contribute to the improved health and extended life observed in mice as a result of telomerase gene therapies has not yet been rigorously determined. But what does this do to epigenetic age? Running studies in cells doesn't really tell us what happens in animals; it is more a way to get a handle on the basics that can then be used to argue one position or another.

Epigenetic ageing is distinct from senescence-mediated ageing and is not prevented by telomerase expression

Ectopic expression of hTERT, the catalytic sub-unit of telomerase, which can preserve telomere length and avert senescence of some cells. It was initially thought that the functional and physical deterioration that characterise organismal ageing are a result of insufficient replenishment of cells due to telomere-mediated restriction of cellular proliferation. Senescent cells, which accumulate increasingly in tissues in function of age, were assumed to be passive and merely a consequence of the above-described processes. This notion was short-lived when senescent cells were found to secrete molecules that are detrimental to cells and tissues.

As such, it would follow that if cells were prevented from becoming senescent in the first place, ageing could be avoided. Although there are external instigators such as stress and DNA damage that can also cause cells to become senescent, replicative senescence is particular in that it is an intrinsic feature that is part of cellular proliferation and occurs even in an ideal environment. As expression of hTERT has been repeatedly demonstrated to prevent replicative senescence of many different cell types, it is reasonable to consider ectopic expression or re-activation of endogenous hTERT expression as potential means to prevent replicative senescence, delay ageing, and improve health.

The above proposition would be valid if senescent cells were indeed the only cause of ageing. Relatively recently, an apparently distinct form of ageing, called epigenetic ageing was described. This discovery stems from observations that the methylation states of some specific cytosines that precede guanines (CpGs) in the human genome changed rather reliably and strictly with age. This allowed supervised machine learning methods to be applied to DNA methylation data to generate an DNA methylation-based age estimator, which in the majority of the human population is similar with chronological age. Epigenetic age is not merely an alternative means of determining chronological age but is to some degree a measure of biological age or health; a proposition that is further supported by the impressive demonstration that acceleration of epigenetic ageing is associated with increased risk of all-cause mortality.

We recently developed a new epigenetic age estimator, referred to as skin and blood clock that is more accurate in estimating age of different cell types including fibroblasts, keratinocytes, buccal cells, blood cells, saliva and endothelial cells. Studies employing skin and blood clock and the pan-tissue epigenetic age clock revealed a startling consistency of epigenetic age across diverse tissues from the same individual, even though cellular proliferation rates and frequencies of these tissues are not the same. This suggests that the ticking of the epigenetic clock is not a reflection of proliferation frequency, which is in stark contrast to telomere length, which enumerates cellular division. It would therefore appear that the process of epigenetic ageing is distinct from that which is driven by telomere-mediated senescence.

To understand their relationship or interaction, if one indeed exists, we set out to test the impact of hTERT on epigenetic ageing. To this end we employed wild type hTERT that can prevent telomere attrition and its mutants that cannot, with some still able to nevertheless prolong cellular lifespan. Expressing these hTERT constructs in primary cells from numerous donors, ages and cell types, we observe that while hTERT expression can indeed prevent cellular senescence, it does not prevent cells from undergoing epigenetic ageing and that extension of cellular lifespan is sufficient to support continued epigenetic ageing of the cell. These simple observations provide a very important piece to the puzzle of the ageing process because it reveals the distinctiveness of epigenetic ageing from replicative senescence-mediated ageing. They provide further empirical support to the epidemiological observation that hTERT variant that is associated with longer telomeres are also associated with greater epigenetic ageing.

Comments

Totally in agreement with my previous comments, this further work by Horvath clearly shows greater methylation changes occur when cells live for longer. It's a simple as a reflection of the replacement rate from stem cells, which presumably being less metabolically active, have a younger epigenetic age.

So any supplement, lifestyle change or medical treatment that decreases somatic cell turnover will increase average epigenetic age. Whilst increasing turnover will reduce it, at the cost of greater telomere loss.

Posted by: Mark at October 21st, 2018 3:17 AM

Would not mtDNA damage also account for these results?

Posted by: Abelard Lindsey at October 21st, 2018 12:32 PM

I'm not sure the experiment supports the conclusion about epigenetic aging. TERT can restore replication ability to stem cells in the body. But it will not repair those cells that are already damaged. If aging is largely due to the toxic environment created by those damaged cells, is it not reasonable to assume that TERT by itself is not going to fix the problem and, thus, extend healthy lifespan?

What would senolytics and TERT done together accomplish? That is the appropriate follow-on experiment.

What is meant by epigenetic aging? Is this the breakdown of appropriate gene expression/supression, which is controlled by Methylization? Or is it something else?

I think the researchers doing this experiment are advocates of "programmed" aging theories.

Posted by: Abelard Lindsey at October 21st, 2018 1:02 PM

Moreover, is it not possible that the toxic environment created by scenescent cells may be the actual cause of replicative scenescence (aka Hayflick limit) of the functional stem cells themselves? It may also be possible that this toxic envrionment may, in and of itself, be the cause of telomere shortening.

Posted by: Abelard Lindsey at October 21st, 2018 1:05 PM

Basically most cells senescese because of ROS, so yes - it is down to the toxic environment generated by cells themselves - but as these cells die the remaining cells have to divide to replace them, so Telomeres shorten, gene expression starts shifting, cells cycling slows down, and the whole situation gets worse. So yes, you could say telomeres shorten because of mtROS, or atleast partly.

It's an open question what senolytics will do long term - will those cells that should have died but haven't being removed improve the environment such that other cells (including stem cells) do better? Or are they hanging on for good reason, and removing them will hasten overall decline (due to other cells having to divide more)? Evidence so far points to improvement, atleast in mice.

Who knows what the Horvath clock really shows - I doubt the methylation changes he records are harmful in themselves (and they don't seem to alter gene expression as far we can tell), but they probably are indicative of the problems that might eventually happen if somatic cells had no limit on their replicative lifespan.

Posted by: Mark at October 21st, 2018 2:52 PM

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