Aging Hours

Reset, slow down, reverse?

Margarita Pertseva, "Biomolecule"

The human body is like a clock. The arrows are constantly running forward, we are getting old. The device of the clock mechanism of this watch is very complex. But, despite this, scientists managed to understand at least some of the principles of its work.

For example, today we have already learned how to slow down the aging process of cells and return old cells to their "infant state", although we have to pay for this with the loss of cellular specialization. Is it possible to turn the clock back, that is, to "rejuvenate" the cells, preserving their functions? What about rejuvenating the whole body? Recent experiments provide answers to these questions.

What is aging?

Before understanding the rejuvenation process, let's first try to understand what aging is. Usually, aging is understood as a process in which important body functions, including the ability to reproduce and regenerate, are gradually disrupted and lost. Regarding the causes of aging, various hypotheses have been put forward and are being put forward, which are divided into two fundamentally different semantic groups.

Scientists, adherents of the first group, argue that the aging process is caused by some (unknown) program laid down by evolution, and this program can be slowed down or broken. Adherents of the second group object: there is no special program, but over time, damage and breakdowns accumulate in all structures of the body, which leads to aging.

Regardless of the correctness of one or the other, the aging processes occurring at all levels (from molecular to organismal) are the same for everyone. So, over time, mutations occur in DNA, gene expression begins to be less regulated, aggregates of damaged proteins and lipids accumulate in cells. In addition, cell division occurs less frequently, and cells perform their functions less efficiently, which, in turn, leads to a slowdown in organ regeneration, a decrease in muscle mass, a weakening of immunity, a decrease in mental abilities, etc.

The changes listed above are just a small fraction of all the not too pleasant processes that occur in an aging body. The list of these physiological problems suggests sad reflections that "the beautiful is far away", despite pleas, will be cruel. And you look longingly at the thick volumes of molecular biology, realizing how much you have aged during their reading. But, dear reader, do not rush to despair! Hundreds of scientists around the world, who have already successfully passed this path, are now working to regain the lost years. And I must say, they have achieved some success in their business.

"Zeroing" and slowing down the clock

One of the important successes of scientists was to increase the life expectancy of their laboratory pets – the roundworm Caernorhabditis elegans, the Drosophila melanogaster fly and the laboratory mouse Mus musculus with the help of induced mutations in certain genes or the selection of a calorie-limiting diet [2].

Researchers have been experimentally finding genes that affect life expectancy for a long time and trying to understand the mechanism of their action. To date, several dozen such genes have already been found (both in model organisms and in humans). For example, the mutations made in the following signaling pathways prolong the lifespan of laboratory animals:

• IGF (signaling pathway of insulin/insulin-like growth factor). A cascade of reactions triggering the inhibition of translation factors. Probably, these translation factors regulate the expression of genes that increase life expectancy.
• A protein translation pathway involving mTOR (mammalian target of rapamycin). mTOR regulates cell growth, motility, proliferation and survival, as well as transcription and protein synthesis. Inhibition of mTOR prolongs life.
• A pathway involving AMPK (5' AMP-activated protein kinase). AMPK is an enzyme that plays a role in maintaining the energy homeostasis of the cell.

It is significant that the use of such techniques prolonged not only the life, but also the youth of the subjects. It turns out that we are able to experimentally slow down the "aging clock". But is it possible to stop the clock or, moreover, to reverse its hands? It should be noted that in nature, the "zeroing" of these clocks occurs every time after the moment of fertilization. The actual age of a human egg is equal to the age of a woman.

The sperm, of course, is younger in age, but he also managed to go through a number of cell divisions. In the cell formed as a result of fertilization, the "age trace" of the parents is completely absent! The mechanisms of "zeroing out" are still unclear. But it is still clear that this process takes place under the influence of certain substances in the cytoplasm of the egg. For the survival of the species, the process described above is extremely important.

The process of "zeroing out" was used in early cloning experiments by John Gurdon. He extracted the nucleus from the egg of a spur frog and instead placed the nucleus of a muscle (or intestinal) cell of a tadpole (Fig. 1). This nucleus was reprogrammed (like the fused nuclei of the egg and sperm after fertilization) under the influence of the same factors in the cytoplasm of the egg that initiate "zeroing". Such a hybrid cell eventually developed into a normal organism, with no visible signs of premature aging. This experiment refuted the hypothesis that the process of maturation and differentiation of cells is accompanied by the loss of genetic material. In addition, he proved that the age of the donor nucleus can be "reset" [2].

Figure 1. The scheme of already classic experiments: cloning a frog by John Gurdon
and obtaining IPSC by the Shinya Yamanaki method. A drawing from the website of a Russian Reporter.

Cell age For cells , you can distinguish:

• actual (or chronological) age is the lifetime of a cell
• the replicative age is the number of divisions through which the cell has passed, and
• biological (epigenetic) age – it is measured using 353 epigenetic tags on DNA. It usually coincides with the chronological age (with 96% accuracy). But it has been shown that breast tissue is biologically older than the rest of the body by 2-3 years [3, 4].

Nobel Prize laureate Shinya Yamanaki's experiments on the production of induced pluripotent stem cells (iPSCs) are widely known. These cells were obtained from fibroblasts of an adult organism using only four transcription factors, which was a breakthrough in the field of stem cell production [2, 5] (Fig. 1). A new era in the field of individual regenerative medicine was predicted. However, alas. For a number of reasons, iPSCs cannot be widely used yet [5-7]:

1. iPSCs are capable of forming tumors due to their high proliferative activity,
2. when passing through the cycle "adult cell – dedifferentiation – IPSC – differentiation – adult cell", some genetic and epigenetic disorders sometimes manifest themselves,
3. this cycle takes about three weeks, and at the same time the percentage of successfully reprogrammed cells is small (the maximum efficiency is about 0.01%) [8].

Yamanaki's experiment also confirmed the hypothesis that the aging clock of adult differentiated cells can be "reset".

In all the above experiments, rejuvenation proceeds only in conjunction with dedifferentiation (that is, with the loss of cellular specialization). But is it possible to reverse the aging process while preserving specific cellular functions?

Rejuvenation without dedifferentiation

Different groups of scientists conducted experiments that proved the possibility of cell rejuvenation without losing its specificity. Moreover, by now three main ways have already been described to make the cell "remember youth" [2]:

1. creating a "young environment",
2. effects on certain genes and
3. pharmacological effects on cells.

The first method: creating a "young environment"

To understand whether the cells and tissues of an old mouse are able to rejuvenate under the influence of external biological factors, heterochronic parabiosis (GP) was used – a method in which two mice – young and old – are sewn together sideways, like Siamese twins (Fig.2, from the website ipscell.com ). At the same time, a common circulatory system and a pool of blood are created in mice. The Conboy couple (Irina Conboy and Michael Conboy) demonstrated back in 2005 that with GP, youth returns to the muscle cells and liver of an old mouse. They acquire the phenotype of young cells, and the molecular markers of aging disappear [2, 9]. Tissue-specific muscle stem cells also restore their potential.

Conboy reports that even very old stem cells do not lose their ability to repair and maintain tissue, they just need to be provided with a young environment [10].

In 2014, Harvard biologists, using GP, identified growth and differentiation factor 11 (growh differentiation factor 11, or GDF 11). Science magazine called this work the first demonstration of the rejuvenation factor, since GDF11 has all the necessary characteristics: it is synthesized in the body, reverses aging in most tissues and its level decreases with by age [11].

Biologists from Stanford also investigated the GP model. They focused on the effects that occur when young blood is exposed to the brain. It turned out that neurogenesis and the level of stem cell proliferation increased in certain areas of the mouse brain, that is, new neurons began to appear, which normally appear only in young individuals [11, 12]. Interestingly, in all experiments with GP, young mice age.

The mechanisms of the effects described above are not completely clear to date. Scientists suggest that both stem cells and various growth factors, cytokines, etc. are involved in these processes.

The second way: exposure to certain genes

When studying the molecular signs of aging, as a rule, it is not entirely clear which changes in the body are actually aging, and which are its consequence. To make such a distinction, researchers usually use genetic manipulation of biochemical pathways. An example of this is the signaling pathway of the NF–kB factor (a protein complex that controls DNA transcription). It is involved in the cellular response to stress, free radicals, bacteria, viruses, etc. When comparing old and young tissues in mice and humans, scientists have found that the expression level of genes regulated by the NF-kB signaling pathway increases in aged tissues. The establishment of this fact gave rise to the hypothesis that the NF-kB signaling pathway is necessary to maintain the age phenotype. To test this hypothesis, scientists created transgenic mice in whose skin the NF-kB signaling pathway could be inhibited at the request of experimenters at a certain moment. When the mice aged and signs of skin aging such as skin thinning became noticeable, the NF-kB inhibitor gene was turned on. This led to a noticeable rejuvenation of skin cells. At the same time, markers of cellular replicative aging, such as p16 (inhibits cell division), disappeared, and stem cells regained their proliferative activity and restored the lost skin layers [2].

The third method: pharmacological effect on cells

It is possible to restore youth at the cellular and molecular level through pharmacological intervention. The mTOR enzyme recognizes the level of nutrients in the cell, and also regulates protein synthesis and energy utilization. An mTOR inhibitor, rapamycin (a drug used as an immunosuppressant), has been shown to increase life expectancy in mice. In the hematopoietic system, aging is associated with an increase in mTOR activity in stem cells and progenitor cells. Rapamycin administered to old mice not only limited the age-related increase in mTOR, but also increased the proliferation of stem cells [2].

Thus, it is possible to separate the processes of aging and differentiation. It remains only to figure out how the mechanisms of "zeroing" cells and their rejuvenation differ.

Meet Epigenetics

The main mechanisms of reprogramming of the cell nucleus and its dedifferentiation are objects studied by epigenetics [13]. This science studies the mechanisms that change the expression of genes, but do not affect the DNA sequence.

Example. The cells of our body have the same genetic material, but they express different genes, which leads to cellular specialization (liver cells, bones, neurons). It is important that epigenetic mechanisms not only "guide" differentiation, but also constantly support the specialization of the formed cell. Differentiated cells divide repeatedly and are constantly exposed to destabilizing external factors, but retain their functions [2]. Despite the epigenetic stability, the experiments described above demonstrate that the status of cells is plastic and reversible. Studying the dynamics of reprogramming of mouse cells (fibroblasts) in IPSC, scientists found out that on the fourth or seventh day, the epigenome destabilization occurs in the cells.

Figure 3. Illustration of the hypothesis explaining the differences between the processes of rejuvenation and reprogramming of cells. During rejuvenation, cells are exposed to short-term effects of reprogramming factors, so they lose only those signs that are less stable (age). With prolonged exposure to these factors, cells lose even the stable signs of the cell (specialization).

But the occurrence of destabilization does not guarantee that the cells will pass into iPSCs. If the reprogramming factors are eliminated at this very time, the cells turn back into fibroblasts [6]. Based on this, a curious hypothesis has emerged (not yet confirmed) explaining the difference between the mechanisms of dedifferentiation and rejuvenation (Fig. 3). During rejuvenation, cells are exposed to destabilizing factors for a short time. Therefore, age-related epigenetic labels (as less stable) are erased, and epigenetic labels responsible for cell specialization are preserved. Therefore, the cells return to their original state. When "zeroing" cells are exposed to prolonged exposure to reprogramming factors. Therefore, all epigenetic marks that existed before in the cell are erased. Specialization and the age of the cell are lost, and it turns into a stem cell. A third way is also possible: the transition from a state of epigenetic instability immediately into a different type of cell. There are already ways to convert fibroblasts into cardiomyocytes or into neurons using only a few transcription factors [14].

Conclusion

Experiments on cell rejuvenation, as well as the identification of the rejuvenation factor GDF11, are certainly impressive. It may seem that the elixir of youth is about to be invented. But, unfortunately, it's not that simple. The discoveries made to date are only the very first steps. Scientists are far from fully understanding the mechanisms of aging and how it can be controlled/regulated.

We should also not forget that the experiments were conducted on mice. Whether the same factor GDF 11 will act on a person is unknown. And finally, there is a possibility that the mobilization of stem cells in old people initiates the occurrence of malignant tumors or other side effects. Therefore, if you are offered rejuvenation for the sake of drinking "young blood tinctures" or "stem cell cocktail", do not rush. Youth clinics are unlikely to appear in the next ten years. But if you want to be young and strong right now, act the old-fashioned way: go in for sports, run, eat vegetables and fruits. The body will not remain in debt to you.

Literature

1. biomolecule: "Is aging a payment for suppressing cancerous tumors?";
2. Rando T.A., Chang H.Y. (2012). Aging, Rejuvenation, and Epigenetic reprogramming: Resetting the Aging Clock. Cell 148, 46–57;
3. Gibbs W.W. (2014). Biomarkers and ageing: The clock-watcher. Nature 508, 168–170;
4. Biomolecule: "Epigenetic clock: how old is your methylome?";
5. biomolecule: "There was a simple cell, it became a stem cell";
6. Manukyan M., Singh P.B. (2012). Epigenetic Rejuvenation. Genes Cells 17, 337–343;
7. biomolecule: "French researchers managed to rejuvenate the cells of centenarian people";
8. biomolecule: "A snowball of problems with pluripotence";
9. Conboy I.M., Conboy M.J. (2005). Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433, 760–764;
10. Conboy I.M., Rando T.A. (2005). Aging, stem cells and tissue regeneration: lessons from muscle. Cell Cycle 4, 407–410;
11. Bouchard J., Villeda S.A. (2014). Young Blood’s Anti-Aging Powers in Mice: Perspectives on New Paper. IPScell.com;
12. Bouchard J., Villeda S.A. (2014). Aging and Brain Rejuvenation as Systemic Events. J. Neurochem. doi: 10.1111/jnc.12969;
13. Biomolecule: "Development and epigenetics, or the story of the Minotaur";
14. Biomolecule: "Nobel Prize in Physiology or Medicine (2012): induced stem cells".

Portal "Eternal youth" http://vechnayamolodost.ru 12.11.2014