Telomerase can reverse the aging process... sort of

Biologists are, at long last, beginning to understand the molecular processes responsible for aging in complex (multicellular) organisms – and to investigate ways to counteract these processes. We discussed one line of research in this recent article about a particular sirtuin (SIRT3) that helps relieve oxidative stress that can lead to DNA damage, which generally leads, in turn, to cell senescence or death.

While oxidative stress is certainly a significant factor in aging, possibly the most significant, there are others. One of these is the limitation on a cell's ability to undergo cell division in order to produce new cells of the same type. This is especially important in tissues that regularly need to regenerate, such as skin and intestinal tissue. Everyone now knows about telomeres, whose main function is to constitute protective end caps on chromosomes. The limitation on number of cell divisions happens since about 100 base pairs are lost from telomeres during each cell division. When telomeres eventually become too short signals that are similar to those associated with other kinds of DNA damage shut down a cell's ability to divide further. This mechanism indirectly helps mitigate the risks of DNA damage that are present every time a cell divides – an inherently tricky process.

However, this limitation on cell division isn't acceptable during embryonic development, when an organism's cell count is doubling most rapidly. So evolution has provided an enzyme – telomerase – that can rebuild telomeres, but is most active only during embryonic development. Except, of course, in cells that have become cancerous, where the ability to divide without limit is the name of the game. We discussed telomeres and telomerase in some detail a little over a year ago in this article, so you can go there for more.

Because of the risk of cancer, it seems imprudent to reactivate telomerase for the long term within an organism, especially in long-lived animals such as humans. (In animals like mice, which live fast and die young, it's a different matter. Telomerase may remain somewhat active in mice during adulthood. (Mentioned here.)) But what if it were possible to reactivate telomerase for a relatively short period of time (compared to the whole lifespan)... might that provide an opportunity to rebuild telomeres to some extent? Even better, might that reverse, at least to some extent, the ravages of aging?

We now have some research that seems to provide a fairly unambiguous affirmative answer... in a rather special case: Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.

But didn't we just say that mice may retain telomerase activity throughout their lives? Yes, however it's a relatively simple matter to "knock out" the main telomerase gene in mice (Tert). When that's done the resulting strain of knock-out mice – after several generations – have shortened lifespans and a general phenotype of age-related debilities, as one would expect. (The first few generations apparently still have sufficiently long teleomeres.)

Unfortunately, that's not a good enough model, since without a Tert gene, the organism has no way to manufacture telomerase. Simply giving the knock-out mice repeated infusions of telomerase is not a good way to ensure uniform distribution of the enzyme to all of the organism's cells. What to do? The experimenters came up with a rather clever solution. Normally the way that telomerase is activated in cells is by means of an "estrogen receptor" (ER), to which a form of the hormone estrogen (17β-estradiol to be precise) can bind and enable transcription of Tert. This ER can be tweaked so that estrogen binds to it only in the presence of another chemical, 4-hydroxytamoxifen (4-OHT).

A special form of the Tert gene that includes this special ER can be "knocked-in" to the mouse germline. It then turns out that 4-OHT can be efficaciously supplied to a TERT-ER mouse (in the form of a time-release subcutaneous pellet) to turn telomerase expression on and off at the experimenter's will. With that technology in place, the researchers were then able to perform a series of experiments demonstrating, in these special mice, that a month-long burst of telomerase could actually reverse a number of the ill effects of telomerase deprivation.

The first step was to show that without 4-OHT the TERT-ER mice (after a few generations) had many of the same problems, in the same degree, as later generations of knock-out mice that lacked Tert entirely. The TERT-ER mice (all of which were male) showed no signs of telomerase activity. Tissues in highly proliferative organs such as testes, spleen, and intestines showed notable atrophy. Lifespan of TERT-ER mice was about half that of normal ("wild type") mice.

The first test to investigate the effects of telomerase reactivation by means of 4-OHT was done in vitro. Fibroblast cells from TERT-ER mice were cultured and found to be essentially senescent and not undergoing cell cycles. But when the cells were placed in media containing 4-OHT, teleomerase was reactivated, telomeres lengthened, and cell proliferation resumed.

Some TERT-ER mice were then given a 4-week treatment of 4-OHT (subcutaneous pellets). At the end of that treatment there was a marked reversal of the degeneration that has occurred in testes, spleen, liver, and intestinal tissues, as well as resumption of sperm production. Survival time of these treated mice also increased. At the same time, 4-OHT had no effects on control mice that weren't lacking in telomerase and didn't have tissue degeneration.

Noteworthy results were obtained from tests to assess nervous system condition. Proliferation of neural progenitor cells was found to resume in TERT-ER mice treated with 4-OHT. Normal numbers of mature oligodendrocytes reappeared. Lastly, high-level neurological functions were restored, as indicated by resumption of nearly normal olfactory sensitivity.

An interesting conclusion that can be drawn from the neurological results is that neural progenitor cells probably survive loss of telomeres, so that they can rebuild neural cell populations if telomeres are repaired.

The really interesting question, of course, is the extent to which these results may apply, in some form, to humans. Unfortunately, there are a number of reasons to be skeptical. For one thing, telomere shortening is only one factor, and quite possibly not the main one, in human aging. Aging can be thought of as a complex disease, like cancer, with many contributing factors. The consequences of telomere truncation are only one factor.

Further, murine biology has signficant differences from human biology. Mice are less complex organisms, with rather short lifespans. Mice seem to retain some degree of telomerase activity throughout their lives, so they are not as well adapted to going for long periods without it.

It is noteworthy that evidence was not found that TERT-ER mice treated with 4-OHT became more susceptible to cancer. Still, mice don't live very long, and they are adapted to maintain active telomerase. Humans are different. If telomerase is artificially kept active for years in humans, incipient tumorigenicity could be accelerated.

Lastly, it's not necessarily easy to raise human telomerase activity levels in the first place. Although some telomerase-activating factors are known, they have not been tested extensively in humans for long periods of time, so their safety and efficacy profile is not known.

These research results are quite interesting – but they only indicate the need for much more investigation.

Jaskelioff, M., Muller, F., Paik, J., Thomas, E., Jiang, S., Adams, A., Sahin, E., Kost-Alimova, M., Protopopov, A., Cadiñanos, J., Horner, J., Maratos-Flier, E., & DePinho, R. (2010). Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice Nature, 469 (7328), 102-106 DOI: 10.1038/nature09603

Further reading: (* = especially recommended)

* Telomerase reverses ageing process (11/28/10)

* The Curious Case of the Backwardly Aging Mouse (11/29/10)

* Partial reversal of aging achieved in mice (11/29/10)

Harvard scientists reverse the ageing process in mice – now for humans (11/28/10)

Gene reactivation reverses aging-related brain deficits in mice (11/30/10)

Age-Reversing Drugs on the Horizon? Not So Fast (11/29/10)

Telomere Tweaks Reverse Aging in Mice (11/29/10)

Alzheimers and aging advances uncovered (11/29/10)

An enzyme leads the dance of immortality and death (11/29/10)

Scientists Find Way to Partially Reverse Aging in Mice (11/29/10)

Telomerase and Wnt signaling

Now that research into telomeres and telomerase has (finally) garnered a Nobel Prize, it's a good time to write about recent research on the subject.

Seminal work on telomeres by Elizabeth Blackburn, one of the Nobel winners, was published way back in 1978, and active studies have been going on ever since. So perhaps it's not surprising that the rate of new findings is not so rapid as occurs in newer areas – such as stem cells.

But fascinating new results on telomeres and telomerase do still appear, and one of them connects with more recent research areas – such as Wnt signaling and... stem cells.

Here's the press release:

Discovery pinpoints new connection between cancer cells, stem cells (7/1/09)

A molecule called telomerase, best known for enabling unlimited cell division of stem cells and cancer cells, has a surprising additional role in the expression of genes in an important stem cell regulatory pathway, say researchers at the Stanford University School of Medicine. The unexpected finding may lead to new anticancer therapies and a greater understanding of how adult and embryonic stem cells divide and specialize.

Don't bother getting excited about the "new anticancer therapies" bit. That's just boilerplate that about 77.3% of all press releases dealing with cell biology contain, presumably to impress the rubes. If you need something to get excited about, you might recall that telomerase is also being investigated intensively in connection with issues of aging and longevity, independently from cancer. However, while it's possible that something of medical significance may come from this research, that's probably way down the road.

Before discussing the new research, let's review some of the background on telomeres, telomerase, and Wnt signaling.

To begin with, a telomere is a series of short repeated segments of DNA found at the ends of chromosomes in all eukaryotic cells. In cells of vertebrate animals the repeated segment is TTAGGG (where the letters represent nucleobases: T=thymine, A=adenine, G=guanine).

The total number of nucleotides in these repeated segments varies a great deal from species to species, but in humans it is (initially) about 10,000 nucleotides (or ~1700 complete segments). I say "initially", because part of the telomere is lost every time a cell divides – perhaps 50 to 200 nucleotides per division. Obviously, this means that an adult cell newly derived from a stem cell can divide at most 50-200 times before the telomere is all gone. In practice, the number is a lot less than the maximum, perhaps 40 to 60 times, which is called the Hayflick limit. Cells are programmed to stop dividing on reaching this limit, since otherwise useful DNA would be lost or damaged upon further division.

Why does this loss occur? It seems to be somewhat of an accident of the nitty-gritty details of how DNA replication occurs during cell division. I won't go into that, since it's best explained with some diagrams; you can read about it at Wikipedia. In fact, in the early days of molecular biology (around 1972), what happens at the end of chromosomes during DNA replication was rather puzzling, and the puzzle was called the "end replication problem". Now it's pretty well understood, though somewhat messy.

In any case, the loss of DNA from the telomeres during cell division is a fact, and it conveniently explains the existence of the Hayflick limit, which had been recognized since 1965. What happens when the teleomere is all used up and the limit is reached? Cells that have reached the limit don't necessarily die (though they might), but they do stop dividing, and they enter a static phase of cell life known as senescence.

If you think about it, senescence can be a problem, especially in certain tissues that need to continually replenish their cells, such as skin and the lining of the intestines, as well as hair, fingernails, etc. How is senescence circumvented in such tissues? The answer is (adult) stem cells. It turns out that stem cells are not subject to the Hayflick limit. It's not clear whether they are subject to limits at all.

Like any other cell type, stem cells also divide (by the process technically known as mitosis). But there is one difference from the way "ordinary" cells divide. Stem cells can divide asymmetrically, where one daughter cell is another stem cell, but the other daughter is a "progenitor cell", which is close to a normal, fully-differentiated adult cell of a fixed tissue type. Progenitor cells differentiate further into the final form when they divide, and they are able to divide only a limited number of times.

So how is it that stem cells are able to escape the Hayflick limit, dividing an indefinite number of times, even though they are also subject to the same loss of telomere nucleotides with each division? The answer is the enzyme telomerase, which is able to rebuild shortened teleomeres. It's fortunate for the longevity of complex organisms that telomerase exists, otherwise stem cells would not be able to divide often enough to allow tissues exposed to harsh conditions (such as skin and intestinal lining) to be replenished.

In order to explain the results of the research to be described here we need to say more about telomerase, but first let's summarize the function of telomeres. At first it may seem that all they do is compensate for the sloppy way that DNA replication works, by providing long stretches of expendable DNA at the ends of chromosomes. But there is one other notable function: teleomeres are a useful "cap" at the end of a chromosome that is recognizable to cellular mechanisms responsible for detecting DNA damage. If it weren't for the telomeres, the ends of chromosomes would be indistinguishable from DNA damage, which can result from errors in the replication process, as well as damage due to external agents such as ionizing radiation or harmful chemicals. Cells have various mechanisms for repairing many kinds of damage, but even if repair isn't possible, other mechanisms exist that recognize the damage and prevent further cell division or cause programmed cell death (apoptosis).

Not all DNA damage can be repaired or compensated for by apoptosis or cessation of cell division. Unrepaired DNA damage (however it occurs) is the main cause of cancer (though not the only one). So the existence of the Hayflick limit as a result of teleomere shortening acts as one defense against cancer. Cancer can be defined as the uncontrolled proliferation of cells as a result of DNA damage (affecting existing mechanisms that normally control proliferation) or other causes. So fixed limits on the number of times a cell can divide is one of a number of mechanisms organisms have to guard against cancer.

Before moving on, here's a quick summary of the functions served by telomeres: (1) compensate for the chromosome "end replication problem"; (2) make it possible for cells to distinguish chromosome ends from damaged chromosomes; (3) provide natural limits to the number of times ordinary cells can divide, as protection against cancer.

As noted above, the third of these functions is a problem for stem cells that do need the ability to divide an indefinite number of times. Repair of tissues exposed to harsh conditions is not the only circumstance this ability is needed. Another very important case is that of embryonic development. Multicellular, sexually-reproducing organisms start from a single cell (zygote). Yet there are close to 10¹⁴ cells in an adult human.

It is true that a single cell that underwent 47 cycles of cell division could theoretically produce that many cells. But that's really pushing the limits, since some cell types are needed in much larger numbers than others. The bottom line is that embryonic development is the other main circumstance when limits on cell division need to be overcome.

Telomerase is what makes this overcoming of telomere limits possible. And it does it in a pretty straightforward way. Telomerase is a complex molecule with three distinct parts. Two of these are proteins: Telomerase Reverse Transcriptase (TERT) and dyskernin, which are coded for by distinct genes. TERT does most of the work. The other part is a short piece of RNA, called the telomerase RNA component (TERC), which contains and is somewhat longer than the repeat unit (TTAGGG in vertebrates). Like any other reverse transcriptase enzyme (other examples of which occur in RNA viruses such as HIV), TERT simply translates a piece of RNA into DNA and inserts it into a chromosome. In the case of telomerase, the RNA is carried inside the enzyme complex itself. Telomerase does its job simply by replacing the telomere DNA that is lost from chromosomes during mitosis.

So telomerase performs the function of rebuilding telomeres, and this is essential in tissues where cells must proliferate rapidly, such as in embryonic development and tissue regeneration. But as we noted above, if the restriction on cell division is circumvented, the risk of cancer goes up. Although telomerase serves an essential purpose in specialized contexts, it also makes it possible for cells to become cancerous. Consequently, telomerase is not expressed in most adult body cells. However, telomerase is expressed in about 90% of tumor cells.

It might seem as though one approach to controlling or even destroying cancer could involve either inhibiting telomerase or developing a vaccine using telomerase to induce an immune response against telomerase-rich cancer cells. There are in fact various clinical trials exploring both techniques. But this is tricky and rather risky, because as we've observed, telomerase is needed in stem cells required for tissue regneration, at least once the cells have begun proliferating. Those cells need to be protected while they are simply doing their job – replenishing skin and intestinal linings, for example. We need to understand how such cells are controlled so that they work without leading to cancer.

Anyhow, TERT is a protein that is an essential component of telomerase, which plays an important role in cell proliferation. The new research we're finally almost ready to discuss shows that, surprisingly, TERT also plays a role in a completely different aspect of cell proliferation having nothing to do with telomeres. That's where Wnt signaling comes in.

Wnt signaling is a subject we've looked at several times before. Some of the relevant articles are here, here, here, and here. Wnt was first noticed in connection with embryonic development and tissue regeneration. This article has many examples. The name Wnt alludes to a gene called "wingless", because the gene causes fruit flies to lack wings when the gene is mutated.

Recently Wnt's importance in stem cells has also received a lot of attention. Some of our discussion of that may be found here, here, here, and here. Wnt's relevance to cancer and cancer stem cells is often touched on in most of all the articles listed.

Basically, Wnt is the name of a family of proteins that play an important role in signals promoting cell proliferation, especially in the context of embryonic development and tissue renewal (skin, intestines, hair, and immune system cells). Since Wnt proteins promote proliferation, they also play a role in cancer.

The fact, then, that new research shows teleomerase can enhance Wnt signaling is signficant as a second, entirely separate route through which it plays a role in both normal stem cell function and cancer.

Here's the research abstract:

Telomerase modulates Wnt signalling by association with target gene chromatin (7/2/09)

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/β-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/β-catenin signalling by serving as a cofactor in a β-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior–posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert^-/- mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/β-catenin signalling pathway.

That's a pretty good summary of the paper, but I imagine most people would like a bit more explanation of what's going on.

Previous research had disclosed some interesting "coincidences" involving stems cells and embryonic development, in which both telomerase and Wnt signaling seemed to have similar effects, even though no obvious connection was known. For example, epidermal (skin) stem cells spend most of their time in a quiescent (non-dividing) state. The main function of Wnt proteins is to carry signals between cells that inform target cells of a need to start dividing, for example during various stages of embryonic development or to heal wounds. The curious thing is that telomerase was known to have a similar effect as Wnt signaling on some stem cells. This coincidence is enough to motivate looking for a connection.

If you are really into this sort of thing, you might want to refer to this diagram of various cell signaling pathways, including that of Wnt. One of the things it illustrates is the position of β-catenin in the Wnt pathway. β-catenin operates at the end of the pathway, where it becomes a part of a protein complex that includes transcription factors (known as TCF/LEF), and the complex causes expression of a variety of Wnt-regulated genes, which go on to enable cell proliferation.

One of the main findings of the research is that TERT interacts with another protein called BRG1 (or SMARCA4), and together these become important parts ("cofactors") of the gene-regulating protein complex. It was also determined that TERT is the only component of telomerase that is involved with Wnt signaling.

Apparently TERT is more important for some instances of Wnt signaling than for others. For example, low levels of TERT in embryos of the frog Xenopus laevis resulted in very abnormal development of the frog embryos (in terms for the anterior-posterior axis of the body). But low levels of TERT at a later stage of development caused only somewhat more subtle defects in formation of ribs in the embryo. Similar somewhat minor effects also occured with low levels of TERT in mouse embryos, and these defects were much like the effects of low levels of β-catenin.

So what, then, is so interesting about this research? It's satisfying to know the reasons for what were previously just observed coincidences, such as the fact that telomerase is not only very important for the ability of stem cells to divide freely (during embryonic development and tissue regeneration) but that it also can help stimulate stem cell division.

But what makes this research especially significant is the importance of the biological processes in which both telomerase and Wnt signaling play major roles – namely embryonic development, tissue regeneration and repair, and cancer. The latter two processes are especially important for medical reasons, although we're still a long way from being able to use this knowledge about telomerase and Wnt signaling for therapeutic purposes.

As far as cancer is concerned, we need to understand much more about how telomerase and Wnt signaling work in various types of stem cells, given that stem cells may play a big role in some types of cancer (though not in others).

But tissue regeneration and repair are also of significant medical interest, since it is the eventual inability of various types of tissues to replenish themselves in old age that is responsible for the many debilities that appear in old age. Many people have speculated that telomerase could help alleviate this problem – provided it does not also lead to facilitating the development of cancer.

Finally, we are left with the puzzle of how it is that TERT happens to play important – yet quite distinct – roles in two very separate processes that are, neverthelesss, both important for cell proliferation. We don't know the answer to this, but we can speculate. Perhaps it's because TERT has to be around anyway for stem cells during embryogenesis and tissue regeneration. That being the case, perhaps at some point in evolution, TERT also happened to help boost Wnt signaling a little. The effect of amplifying Wnt signaling in the same contexts that telomerase was needed would be advantageous and worth conserving. This kind of double duty should lead to more efficient use of cell resources.

Park, J., Venteicher, A., Hong, J., Choi, J., Jun, S., Shkreli, M., Chang, W., Meng, Z., Cheung, P., Ji, H., McLaughlin, M., Veenstra, T., Nusse, R., McCrea, P., & Artandi, S. (2009). Telomerase modulates Wnt signalling by association with target gene chromatin Nature, 460 (7251), 66-72 DOI: 10.1038/nature08137

Further reading:

Cell biology: The not-so-odd couple (7/2/09) – expository article in Nature about the telomerase-Wnt research

Nobel in medicine honors discoveries of telomeres and telomerase (10/5/09) – news article in Science News

Nobel for insights into ageing and cancer (10/5/09) – news article in New Scientist

Chromosome protection scoops Nobel (10/5/09) – news article in Nature

Three Americans Win Physiology or Medicine Nobel (10/5/09) – news article at ScienceNOW.com

Work on Telomeres Wins Nobel Prize in Physiology or Medicine for 3 U.S. Genetic Researchers (10/5/09) – news article in Scientific American

Nobel Winners Isolate Protein Behind Immortality, Cancer (10/5/09) – news article at Wired.com

Tags:

telomeres, telomerase, Wnt signaling