You might recall that researchers recently demonstrated that increased levels of TRF1, a component of the shelterin protein complex, could modestly extend healthy (but not overall) life span in mice. The effect is likely mediated through raised levels of stem cell activity in older individuals, somewhat turning back the usual trend towards declining tissue maintenance. The paper I'll point out today makes an good companion piece, in that it examines the shelterin component POT1, finding that increased levels of this protein also help to maintain stem cell activity. Both POT1 and TRF1 decline with advancing age, and the argument made by some researchers is that shelterin activity is one of the more relevant mechanisms in stem cell aging. That, of course, says comparatively little about where this fits in the chain of cause and effect. If there is less POT1 and TRF1, what caused that? I'm inclined to think that changes in protein expression, and the epigenetic alterations needed to increase or decrease production of proteins from their genetic blueprints, are reactions to more fundamental cell and tissue damage.
What is shelterin and what does it do? This complex is involved in defending telomeres, the repeated DNA sequences found at the end of chromosomes, from various DNA repair and other processes that would cheerfully and destructively cut them short at any moment. Telomere length is an important part of the mechanisms that permit or restrict cell replication: a little of their length is lost with each cell division, and when too short a cell either becomes senescent or self-destructs. The vast majority of cells in the body are restricted in the number of divisions they can carry out, on a countdown to destruction, and this is the foundation of all of the methods used by complex organisms such as mammals to suppress cancer to a sufficient degree to get by. Only a small number of cells, the germline and the stem cells responsible for tissue maintenance, use telomerase to lengthen their telomeres and thus replicate indefinitely. Keeping only a small number of cells privileged in this way greatly reduces the risk of one of them becoming damaged in a way that causes it to run rampant, the seed for a cancer. Too little shelterin and stem cells start to fail in their self-renewal, becoming inactive, senescent, or destroying themselves, because they progressively fail to maintain their long telomeres. More shelterin produces the opposite effect, making stem cell populations better maintained and more active in older individuals.
Stem cells have evolved to decline with age. The current consensus is that this, like a very large number of line items in cellular biochemistry, involves resistance to cancer. Evolutionary pressures lead to a species that attains a certain life span, but how exactly that life span is achieved by cell biochemistry may vary. Our species, long-lived in comparison to our nearest primate cousins, appears to have achieved a large enough resistance to cancer to obtain those additional years at the cost of a slow decline into frailty and organ failure. It doesn't have to be that way - one can look at elephants, for example, who achieved sufficient resistance to cancer to live as long as they do via much more efficient cancer suppression mechanisms. In this context, each species' biochemistry ends up where it does through the forces of natural selection favoring a certain life span, interacting with the happenstance of moving from point A to point B in the biochemistry of cells through evolutionary time. Changes in the availability of shelterin over a lifetime are just one small part of this picture.
Appropriate regulation of haematopoietic stem cell (HSC) self-renewal is critical for the maintenance of life long hematopoiesis. However, long-term repeated cell divisions induce the accumulation of DNA damage, which, along with replication stress, significantly compromises HSC function. This sensitivity to stress-induced DNA-damage is a primary obstacle to establishing robust protocols for the ex vivo expansion of functional HSCs. Telomeres are particularly sensitive to such damage because they are fragile sites in the genome. As HSCs lose telomeric DNA with each cell division, which ultimately limits their replicative potential, HSCs therefore require a protective mechanism to prevent DNA damage response (DDR) at telomeres in order to maintain their function.
The shelterin complex - which contains six subunit proteins, TRF1, TRF2, POT1, TIN2, TPP1, and RAP1 - has a crucial role in the regulation of telomere length and loop structure, as well as in the protection of telomeres from DDR signaling pathways such as ATR. Protection of telomeres 1 (POT1) binds to telomeric single-stranded DNA (ssDNA) and thereby prevents ATR signaling. Human shelterin contains a single POT1 protein, whereas the mouse genome has two POT1 orthologs, Pot1a and Pot1b, which have different functions at telomeres. Pot1a is required for the repression of DDR at telomeres. In contrast, Pot1b is involved in the maintenance of telomere terminus structure. It has recently been shown that shelterin components TRF1, Pot1b, and Tpp1 critically regulate HSC activity and survival. However, due to embryonic lethality in Pot1a knockout mice, the role of Pot1a in maintaining HSC function is still unclear and it is not known if POT1/Pot1a has a non-telomeric role in HSC regulation and maintenance.
Here, we found that Pot1a regulates HSC activity by inhibiting ATR-dependent telomeric DNA damage, and thereby protecting cells from associated apoptosis. These results indicate that the formation of the shelterin complex at the telomeric region is important to Pot1a mediated maintenance of HSC activity. However, in addition to this telomeric role we have also identified a novel non-telomeric role, preventing the production of reactive oxygen species (ROS). Due to these protective functions, we find that treatment with exogenous Pot1a maintains HSC self-renewal and function ex vivo and improves the activity of aged HSCs. This new non-telomeric role is particularly interesting since reduction of ROS is thought to be crucial in inhibiting global DNA damage in HSC in culture.
In addition to its role in protecting against stress we also found that Pot1a has a central role in regulating stem cell activity during aging. We observed that expression of Pot1a is lost during aging, and this loss results in the accumulation of DNA damage, alterations in metabolism, and an increase in ROS production, which in turn compromises aged HSC function. However, we observed that this decline is reversible: remarkably ex vivo treatment of aged HSCs with recombinant POT1a is able to re-activate aged HSCs. Since Pot1a overexpression inhibited the expression of Mtor and Rptor in aged HSCs, the regulation of mTOR signaling by Pot1a may participate in this re-activation of aged HSC function.
Although the precise mechanisms by which this functional improvement occurs have yet to be fully determined, our results indicate that exogenous Pot1a can both prevent telomeric and non-telomeric DNA damage and inhibit ROS production, thereby inducing a more potent immature phenotype in aged HSCs upon ex vivo culture. It will be interesting to clarify how these mechanisms are related to one another and determine, for example, whether telomere insufficiencies precede metabolic changes and ROS production or vice versa.