How naked diggers age
The cells of naked diggers age differently than the cells of mice
Polina Loseva, "Elements"
1. Naked diggers (Naked mole rat, Heterocephalus glaber) are very unusual animals: compared to other rodents, they live much longer, have a complex social life structure, low body temperature and other physiological characteristics, and are also able not to feel some types of pain. Photos from the website flickr.com .
Naked diggers are often called "animals that don't age." The authors of a recent article in the journal PNAS found that individual cells of these animals are quite capable of aging. But even under stress, they do it more economically and safer for the body than the cells of other rodents.
Naked diggers (Heterocephalus glaber) live about 10 times longer than their distant relatives – mice and rats. While mice in standard laboratory conditions live up to 3 years at best, diggers can live more than 30. There are also suspicions that they practically do not grow up. Many of the signs characteristic only of newborn mice can be observed in diggers throughout or almost their entire life. This is the lack of cover, and small (compared to other diggers) sizes, and sexual immaturity (diggers mature quite late), as well as the work of some enzymes, unstable body temperature and active formation of new neurons in the brain. Together, these signs allow us to talk about neoteny, that is, a slowdown in development, in which the body is essentially a "child" for most of its life, but functions as an adult (see V. P. Skulachev et al., 2017. Neoteny, Prolongation of Youth: From Naked Mole Rats to "Naked Apes" (Humans)). Finally, it is known that diggers do not age in the usual sense of the word. At least, they very rarely die from "senile" diseases – cancer, cardiovascular diseases and neurodegeneration (see: What do naked diggers and "naked monkeys" have in common?, "Elements", 06.03.2017). But does this mean that their aging mechanisms are completely disabled?
The authors of the article under discussion in the journal PNAS checked whether it is possible to detect at least some elements of aging in naked diggers. Strictly speaking, the answer to this question depends on what is meant by "aging". If we consider the organism as a whole, then aging is often referred to as an increase in mortality with age: after some age limit, a pattern is observed: the older the organism, the more likely it is to die (there is no such pattern in infancy and reproductive age). In this sense, diggers are not susceptible to old age (see J. G. Ruby et al., 2018. Naked Mole-Rat mortality rates defy gompertzian laws by not increasing with age). But if you go down to the level of individual cells, the criteria of old age become much less obvious. Neither the mortality rate is suitable here (not every dying cell in the body is old), nor the ability to reproduce (even in a newborn organism, not all cells divide anymore), nor the presence of "diseases" (for example, cancer cells contain "errors" in DNA and do not function as usual, but when at the same time, they are actively divided and do not look like the old ones).
There is still no unambiguous definition of the old cell. Cellular senescence is a physiological state in which a cell does not divide, does not differentiate and changes its metabolism. At the moment there are two ways to detect old cells.
The first is staining for β–galactosidase (β-galactosidase). This enzyme breaks down sugars during intracellular digestion. It is usually active at neutral acidity values and works poorly in an acidic environment. But during aging, the cell begins to produce it in large quantities, so even in an acidic environment, its activity can be detected. To do this, the cells are treated with a dye precursor, which turns blue after cleavage by β-galactosidase (see G. P. Dimri et al., 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo).
The second way is to study what the cell releases into the environment. Old cells are characterized by a special set of secreted proteins – SASP (senescence-associated secretory phenotype). It includes, among other things, pro-inflammatory proteins, metalloproteinases (cleaving intercellular matter) and growth factors (stimulating the division of other cells). Through these substances, the old cell affects the environment: it forces those who are still able to divide, and attracts immune cells to remove cells that have become unusable (including itself) and elements of intercellular matter. Looking for SASP proteins is a more difficult task than staining, but the result is more accurate.
The causes of cell aging are also still ambiguous. At least four independent mechanisms can be distinguished (Fig. 2):
1) Replicative aging. Each time a cell divides, its telomeres – the end sections of chromosomes - inevitably shorten. By themselves, they do not carry genetic information, but rather serve as a ballast protecting the "substantial" part of the chromosome. But the shorter the telomere, the higher the risk of losing genetic information. Therefore, when the telomere reaches a certain length, the cell cycle stops and division no longer starts.
2) Stress-induced aging (SIPS, stress-induced premature senescence). In the process of cellular respiration, molecules with a missing or extra electron – free radicals - are formed in the mitochondria. This is a natural process that the cell usually regulates with the help of antioxidants – substances that neutralize radicals. But occasionally they manage to escape from the mitochondria into the cytoplasm or even get into the cell nucleus, where they damage macromolecules, including proteins and DNA. The older the cell, the more such damage accumulates in it. At the same time, DNA repair proteins come into play. When there are enough error signals in the DNA, repair proteins stop the cell cycle. This is the free radical theory of aging. Under the influence of stress factors (starvation, radiation, toxins), the number of radicals increases, oxidative stress develops. If it is strong enough, the cell may age prematurely.
3) Oncogene-induced aging. It is believed that cell aging is not only a side effect of accumulating damage, but also a protective mechanism. When an ordinary cell turns into a cancerous one, the process can still be stopped at the initial stages. As soon as the genes associated with tumors (for example, ras) begin to work actively, an aging program is launched in parallel. And, if there is no mutation in the genes that stop the cell cycle, then this saves the body from a new tumor. Therefore, premature aging can be caused in cells by activating ras or other oncogenes in them (see M. Serrano et al., 1997. Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a).
4) Programmed aging (developmental senescence). At some stages of embryonic development, part of the cell mass dies, for example, for the formation of cavities or tissue rearrangement. At the same time, the "doomed" cells stop dividing and start an aging program. This process does not depend on either telomere length or stress level. It is believed that it evolutionarily preceded stress-induced aging (see D. Muñoz-Espín et al., 2013. Programmed cell senescence during mammalian embryonic development).
Fig. 2. The need to develop, DNA damage and oncogenes cause programmed, stress-induced and oncogen-induced aging, respectively, in both naked diggers and mice. An image from the article under discussion in PNAS.
Until recently, it was only known that naked diggers are not susceptible to replicative aging. Like other small rodents, telomerase, an enzyme that completes telomeres, works in their cells. In humans, telomerase is active only in embryonic and stem cells, in mature cells its gene is turned off. However, even here the diggers distinguished themselves. Sequencing of their genome showed that there are significant differences in the genes encoding their telomerase and related proteins from similar genes of other rodents. This suggests that the telomerase of diggers has been subjected to selection and now probably works more efficiently than in their distant relatives (see the genome of the naked digger – the key to the secret of longevity?, "Elements", 11.11.2011). As for other mechanisms of cellular aging, nothing was known about them. It can only be assumed that diggers are less likely to encounter oxidative stress, since they live in narrow underground passages and are less exposed to oxygen and sunlight (the first contributes to the accumulation of radicals in the cell, and the second contains ultraviolet rays that damage DNA).
The authors of the article under discussion carefully checked naked diggers for the presence of preserved aging mechanisms. The first thing they were interested in was programmed aging, as an older mechanism. To detect it, they stained newborn naked diggers and histological preparations of their organs for β-galactosidase (Fig. 3). It was found not only in the areas characteristic of mice – bone marrow and skull, but also in the skin and hair follicles. The authors believe that this fact may explain how the diggers managed to get rid of the hair cover – with the help of aging and non-dividing cells in the follicles.
Fig. 3. Results of β-galactosidase staining. A – fingers and skin of a naked digger. B – a slice of the finger and enlarged images of the nail bed area. C – section and enlarged images of the skin (left) and hair follicle. All images show areas or individual cells colored blue – this is a sign of old cells. An image from the article under discussion in PNAS.
Then the authors tried to induce oncogen-induced aging in the culture of fibroblasts from embryonic tissues and skin of diggers. They activated the expression of the ras gene in the cells, waited 12 days, and then again carried out staining for β-galactosidase (Fig. 4). Judging by the number of blue cells in the photos, this aging mechanism is also possible in diggers.
Fig. 4. Results of β-galactosidase staining in fibroblast culture. A – micrographs. The upper row of images is control, the lower one is cells after 12 days of ras oncogene operation. B is the percentage of positively colored cells. White columns – before exposure, black – after. From left to right: mouse embryonic fibroblasts (MEF), mouse cutaneous fibroblasts (MSF), digger embryonic fibroblasts (NEF), digger cutaneous fibroblasts (NSF). An image from the article under discussion in PNAS.
Stress-induced aging was next in line. The authors of the article chose gamma radiation as a stress factor. They irradiated the cells with small (10 Gy]) and stronger (20 Gy) doses, and then stained them again for β-galactosidase and measured the level of apoptosis (Fig. 5). It turned out that the cells of the naked digger are also susceptible to this aging. However, a dose of 10 Gy was not enough to cause a significant reaction: it took 20 Gy for the response of the digger cells to become comparable to the response of mouse cells at the same dose of radiation. At the same time, it is interesting that apoptosis (programmed cell death) was not triggered in the digger cells, while for mouse cells it was a characteristic response to severe stress.
Fig. 5. Experiment on irradiation of mouse and naked digger fibroblasts with gamma radiation. A – results of β-galactosidase staining. The upper row of images is control, the middle one is irradiation with a dose of 10 Grams, the lower one is irradiation with a dose of 20 grams. B is the percentage of positively colored cells. F is the percentage of apoptotic (dying) cells in culture. White columns – control, gray – a dose of 10 G, black – 20 G. From left to right: mouse embryonic fibroblasts (MEF), mouse cutaneous fibroblasts (MSF), digger embryonic fibroblasts (NEF), digger cutaneous fibroblasts (NSF). An image from the article under discussion in PNAS.
To check whether radiation damages the DNA of a mouse and a digger in the same way, the authors of the article used the method of DNA comets. After irradiation, DNA in cells is stained, and cell membranes are destroyed. The DNA is then forced to move in an electric field. If the DNA was damaged by radiation, there were breaks in it. In this case, fragments of different lengths are formed, which form the "tail of the comet" when moving in an electric field (Fig. 6). It turned out that DNA is damaged equally in diggers and mice. This means that they differ not in the degree of resistance to stress, but in the reaction to it. Both start the aging program, but the cells of the diggers, unlike the cells of mice, do not go to programmed death.
Fig. 6. The method of DNA comets in mouse and naked digger cells. DNA is colored green. From left to right: control, irradiation with a dose of 10 G, irradiation with a dose of 20 G. From top to bottom: mouse embryonic fibroblasts (MEF), mouse cutaneous fibroblasts (MSF), digger embryonic fibroblasts (NEF), digger cutaneous fibroblasts (NSF). An image from the article under discussion in PNAS.
Finally, the authors of the article analyzed how gene expression in cell cultures changes during stress-induced aging (Fig. 7). Here 's what became clear as a result:
1) In all cell cultures, the expression of many genes changes at once. However, in the mouse, the differences between embryonic and adult fibroblasts are stronger than in the digger. This is consistent with the idea that diggers practically do not "grow up".
2) SASP genes were activated in all cultures. This can be considered the second confirmation that cell aging in naked diggers is possible.
3) The set of processes that are triggered and suppressed under stress differs between a mouse and a digger. Unlike mouse cells, digger cells do not enter apoptosis, as noted above. They also suppress the work of genes responsible for the synthesis of cellular proteins, but the genes associated with the response to stress (breakdown of substances, antioxidants, etc.) work much more actively.
4) Finally, the most surprising result is the following: while the number of genes that changed expression in response to radiation was greater in diggers than in mice, the number of processes that changed their activity was less. This probably means that the aging process in diggers is more clearly organized – it is enough to run fewer genes to regulate more processes.
Fig. 7. Gene expression after a dose of gamma irradiation of 20 Gy. Dark red columns – mouse embryonic fibroblasts, red – mouse skin fibroblasts, dark green - digger embryonic fibroblasts, green – digger skin fibroblasts. An image from the article under discussion in PNAS.
The authors of the article under discussion proved that three of the four known mechanisms of aging are possible in the cells of naked diggers. This does not mean that all of them (with the exception of programmed aging) occur normally. But this shows that, unlike previous representations, naked diggers have not lost the aging program itself and are able to launch it. Another thing is that the strategy of behavior of their cells, faced with aging, is not like the one we are used to. The cells of naked diggers reduce the overall level of metabolism, therefore, they probably secrete less pro-inflammatory substances and cause less harm to the body. At the same time, the antioxidant response is enhanced in them, but apoptosis is not triggered, so the cellular resources of the body are not depleted. Separately, it is worth noting the effectiveness of the organization of the aging process: fewer genes are activated in the cells of diggers than in mice, but they control a large number of processes. It can be assumed that such an organization allows, among other things, to maintain cell metabolism at a low level and save resources.
Active research of naked diggers in laboratories began relatively recently, so now mostly "young" individuals are available for study. The most interesting thing, apparently, will begin when they massively grow up to 30 years old, and it will be possible to study the cellular physiology of "old" diggers. This is where we find out what happens to the cells of diggers at the end of life and whether they actually have aging programs.
A source: Zhao et al., Naked mole rats can undergo developmental, oncogene-induced and DNA damage-induced cellular senescence // PNAS. 2018.
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