22 February 2022

Where life begins

And how much is left before death

Natalia Leskova, PCR.news


Vadim Gladyshev is a professor of medicine at Harvard Medical School, director of the Center for Redox Medicine at Brigham and Wyman (USA). His research interests include redox biology in relation to cancer, aging and reproduction.

Selenium imprints in the genome

— Vadim, before we move on to aging, let's talk about selenium. Were you elected to the National Academy of Sciences of the USA for these works?

— Yes, I would say that our articles on selenium are probably the most famous so far.

— What did you understand about him? Why does our body need selenium?

— At first, this element was considered as harmful, toxic. But then it was unexpectedly discovered that it is contained in some proteins. They began to study these proteins and realized that he was in the active center of certain enzymes. But it was unclear how many of these proteins – three, fifty, a thousand — for example, in a person? When I founded my laboratory, we tried to understand how many of these proteins and what kind of proteins they are.

— There is a 21st amino acid in these proteins - it looks like cysteine, in which sulfur is replaced by selenium. When such a protein is synthesized, selenocysteine in the matrix RNA is encoded by the UGA stop codon. That is, this codon sometimes means not the end of the protein, but the insertion of an amino acid with selenium. What does this "sometimes" depend on?

— Even before our work, it was known that when there is a structure known as SECIS (selenocysteine insertion sequence element) in the untranslated part of the mRNA, then UGA, which, as you correctly said, is a stop codon, changes its function and begins to encode selenocysteine. For this purpose, a specialized translation elongation factor, selenocysteine tRNA and several proteins are used. Thus, the UGA codon has two functions.

— And if there is a site in the gene that corresponds to SECIS, then it is a gene of a selenium-containing protein?

— Yes, actually, we just figured out how to find the genes of selenium-containing proteins using SECIS. But the difficulty was that the primary sequence of SECIS is not conservative, it is different in different genes. Therefore, we predicted hairpins and loops in RNA and calculated the free energy of potential SECIS elements for the entire sequence of the human genome. Then we looked at their conservatism at a certain evolutionary distance. And when such potential SECIS elements were found, they analyzed the coding regions of the genes right in front of the elements, found the codons within the reading of the UGA, and looked at their conservativeness. If there was both a conservative SECIS and a conservative UGA codon, after which the reading frame continued, then it was believed that this was a candidate for the selenium-containing protein gene. As a result, we predicted that there are 25 such genes in the human genome, and then experimentally confirmed that the proteins they encode actually contain selenium. We tested this by expressing them in mammalian cells and labeling them with the selenium-75 isotope. A key role in this work was played by Grigory Kryukov, who was then my graduate student.

— Our article about this was published 19 years ago in the journal Science. And today these are the same 25 genes. Obviously, we didn't miss a single protein and didn't predict incorrectly.

Thus, selenium-containing proteins have a convenient property: their genes are, as it were, labeled with SECIS elements in the genome. It is very difficult to determine, for example, a complete set of zinc-containing human proteins. And selenium-containing — it is possible. And as soon as we find a complete set of these proteins, we can understand what they do individually and in total, and therefore why selenium is needed. It turned out that this is an absolutely necessary chemical element for humans, including because several proteins, including selenium, are vital. Removing any of them will lead to lethality.

— So selenium is as necessary for living beings as iron?

— Each element has a different importance, and living beings differ in their needs. There are elements that are used in many proteins and in all or almost all organisms, for example, iron or zinc. And there are those that are necessary only for some organisms. A person, for example, does not use nickel. At the same time, there are organisms for which it is necessary. There are some that depend on tungsten. And man does not need them — we have lost the ability to use these chemical elements. But we cannot live without selenium, while, for example, yeast does not use selenium, as well as nickel and molybdenum.

— You say that we have lost the ability to use these elements. So we once had this ability?

— Living organisms evolve, respectively, their dependence on certain elements may change. The use of selenium seems to have originated in some ancestral organism when there were no archaea, eukaryotes and prokaryotes. The ability to use selenium depends on several genes, on the order of ten. If these genes are lost in the course of evolution, then it is impossible to return the use of selenium. All higher plants, almost all fungi have lost this ability, but in humans it has been preserved. And today we know for sure that it is associated with these 25 proteins.

In the same way, we identified sets of selenium-containing protein genes in all other organisms. The mouse has 24 such genes, and drosophila flies have only three. In some fish under 40, in some organisms about sixty.

— And yet, why would a person need selenium-containing proteins? I understand that if it were not for your research on selenium, then the topic of aging would not have appeared. Is that true?

— And so, and not so. There is an element of randomness in science. You study one thing, and you find something else. It's interesting, you're moving in that direction. This has been happening all my life. Indeed, we have studied and continue to study selenium. But it turned out that the main function of selenium-containing proteins is redox regulation. So we found ourselves in the field of redox biology, which is closely related to aging.

Age is a moving quantity

— You have papers about biological age clocks, or molecular clocks. What is it?

— I would call them the aging clock. This is a tool that helps to determine the age of the body. This has been thought about for a long time, but until recently, scientists could not pinpoint it. They could determine approximately by looking at a person. Let's say we see that a person is about 60 years old. But then it turns out that a person is 80, he just looks good, or a person in 40 looks 60, because he drinks, smokes and works in the mine.

To determine age, scientists tried to use markers of oxidative damage, measured some functions or telomere length. But it turned out that all this is very inaccurate. And about ten years ago, Steve Horvath from the University of California in Los Angeles came up with the so-called epigenetic aging clock, and they turned out to be quite accurate.

— In Steve Horvath's watch, the age is estimated by the methylation pattern, and how does it work? There are different epigenetic clocks — how do they differ?

— Such clocks work based on age-dependent changes in DNA methylation patterns. Methylation patterns are obtained, for example, using microchips or bisulfite sequencing. Specific sites (cytosines) that contribute to the model are found by machine learning methods. Such models can be trained for chronological age, phenotypic changes, future mortality, and so on. You can also train models on individual tissues, on multiple tissues, on individual mammalian species, and even on multiple species. Therefore, epigenetic clocks are obtained differently, with different application windows and different accuracy.

After that, there was, one might say, a revolution in our region. This field of science has greatly expanded, and epigenetic clocks have been used for a variety of measurements. Our contribution here is that we came up with a clock on mice. Mice are the main model organism in the study of aging. It is also a mammal, like a human, but it lives less, and therefore it is very convenient to test different interventions and mechanisms on mice. After that, we also made many different other watches.

"And for a human?"

— And for a human. This biomarker, methylation, is able to determine not the passport, but the biological, real age. Although, as I said, the watch can be trained to determine the chronological age. And you can train these clocks to predict how long a person has left to live.

— Almost like Woland…

— Naturally, the prediction will be with a certain degree of probability, since no one is immune from random events. Or you can train your watch for the age of occurrence of aging-related diseases.

Our contribution here is that we were the first to come up with a clock that works on individual cells, and this is important because the "unit of aging" is precisely the cell. In general, the unit of life is a cell, and it is the cells that age.

Before that, we took a tissue containing millions of cells, and the average age is determined. In fact, the age of a tissue consists of the age of individual cells. And we have learned to determine the age of individual cells, which is much more important. We published an article just a month ago on this topic.

— Can you already give a specific person a forecast about his condition, about how long he will live? And is it possible to take these data into account in order to live longer?

— Yes, our laboratory uses these watches. There are hundreds of other laboratories that are also engaged in such research. At this stage, the accuracy of predicting the biological age for a particular person is not very good. Scientists rather work with populations, determine general patterns. Now an incredible number of articles are being published on this topic. A certain picture emerges from all this: indeed, biological age is a parameter that can be influenced.

If a person gets sick, his age may increase. Or he will rest — and "get younger", get healthier. Thus, age is a moving quantity. Of course, if a person smokes or drinks a lot, then his biological age is higher than the chronological age. But it is interesting that if a person quits smoking, he gradually returns almost to where he would be if he did not smoke. A lot of such associations can be found. For example, if a person eats red meat, he ages faster. If he is a vegetarian, he ages more slowly.

Ground Zero

— In one of your articles, you introduce the concepts of "Ground Zero" - Ground Zero. What is it?

— We introduced this concept to define the beginning of aging. Aging ends when the body dies. But when does aging begin?

If you ask this question to different scientists who work in the field of aging, they give completely different answers. Someone will say that aging begins when an egg is fertilized. A new organism arises, life begins — and aging begins. Another scientist will say that aging begins at the moment of birth. And an evolutionary biologist can say that aging begins when development ends. He considers the so-called power of natural selection, and as soon as a person reaches the period of puberty and can leave offspring, the power of selection falls. The importance of continuing the life of this organism is becoming less. For example, at the age of 20, this importance is greater, because a person has not yet left offspring, and at the age of 50, a person usually has adult children, and therefore whether a person will live or not is not so important from an evolutionary point of view. Because of this, harmful changes that occur with age are not particularly susceptible to negative selection.

But when we started using the aging clock, it turned out that this question can be answered quantitatively. We began to look: is aging already going on at the age of 20? Apparently, it's coming. At 15? Goes. At five years old? Goes.

— At what point is it not coming yet?

— From our point of view, aging begins in the initial or middle phase of embryogenesis. When an egg is fertilized, a zygote occurs. It turned out that the age of the zygote is not zero, and during early embryogenesis this age falls and reaches a minimum value, after which, in fact, aging begins.

— Is this moment Ground Zero?

- yes. This seems to be the beginning of aging. We determined that it corresponds to gastrulation in the middle of embryogenesis.

— How did you come to the idea that the fertilized egg is not zero and the gastrula is "younger"? From what considerations did it follow that it should be so? After all, the experimental confirmation with the epigenetic clock was later, did I understand correctly?

— The very idea of Ground Zero came to mind when two years ago I listened to a report by a colleague from Germany about the phylotypic period in plants — the stage of embryogenesis, when the embryos of different species are as similar as possible to each other. It usually occurs in the middle of embryogenesis, and not at the very beginning: the differences decrease, and then increase again. I suddenly realized that this or a similar period could be the lowest point of biological age. In fact, this is a good example of why conferences are needed. When we listen to the reports of other scientists, we apply their ideas and observations to our work. And sometimes a "eureka moment" happens.

I read a lot of literature about development, and the further I read, the more convinced I became that the model made sense. That's why I wrote a theoretical article about it. To understand something better, I need to write about it, and I did so. And then we began to test this model experimentally. And it turned out that the biological age is really minimal in the middle of embryogenesis. It is an indescribable feeling when you realize that you have found something new, especially when you managed to predict it yourself.

The only thing that was not quite accurate in the model was that initially it seemed to me that the biological age should begin to decrease soon after fertilization, but it turned out that at an early stage of embryogenesis it changes little and only then drops sharply.

The clock is going backwards

— In the same article, you list different approaches to the fight against aging — there are six of them?

— Well, it's like counting. They can be classified in different ways. I would say there are two main ways: we can try to slow down aging if, for example, we eat less, exercise less, give up bad habits. And you can rejuvenate the body — try to transfer it from the biological age at which it is located to a younger state.

Until recently, scientists believed that this was impossible, that age could only grow. But everything changed the discovery of the Japanese scientist Shinya Yamanaki, who was able to transfer the cells of an adult organism into an embryonic state. He studied the so-called process of dedifferentiation, when the cell returns to the embryonic state, and then it turned out that the cells actually become young. This means that it is possible to rejuvenate cells, which means, perhaps, the whole body.

— And the fact that the minimum age of the embryo does not coincide with the beginning of embryogenesis also confirms this?

—That's right. There is a zygote with a non-zero age, and then the age drops. So, there is a process of natural rejuvenation, and it occurs at the very beginning of life.

— But embryonic rejuvenation, as you say, does not apply to the genomic sequence, does not correct those mutations that already exist in the genome. It turns out that this method is not omnipotent?

— He is omnipotent in the sense that the age of the organism is "reset". The germ lines of the mother and father age, because the organisms age, and then fertilization occurs, a zygote with a non-zero age occurs, and then the rejuvenation process takes place. But, of course, the mutations that were in the germ lines of the mother and father cannot be reset, they are irreversible.

Genome rejuvenation occurs at the population level: those combinations of genes that include too many harmful mutations are rejected at the embryonic stage. An embryo with such mutations does not survive. There is evidence in the literature that only 1/3 of all successful fertilization processes reaches the period of puberty. Moreover, the main mortality occurs, apparently, in the earliest pregnancy. Thus, these harmful mutations leave the population and are not passed on to the next generation.

— And then you experimentally proved the Ground Zero hypothesis. Please tell us how you did it.

— We used the epigenetic aging clock and measured the biological age of the organism in the early stages of embryogenesis. This was mainly done on mice, because there is more data on them. But first we took cells from early embryogenesis — embryonic stem cells and induced pluripotent stem cells, which were obtained by the Yamanaki method. We looked to see if they were aging in culture.

It turned out that even if they are kept in culture for a long time, they do not age at all. At the same time, most adult cells, when grown in culture, gradually age.

And then we began to look in more detail at the different stages of embryogenesis. And we saw that, firstly, the age of the zygote is not zero, and during early development it gradually decreases. We don't know exactly the time of this minimum age yet. Our estimate is that in a mouse it is about seven days after fertilization, in a human it is about the third week after fertilization. Now we are trying to measure more precisely when this happens.

Another interesting observation is that when embryogenesis occurs, some cells then become an embryo, while others perform an auxiliary function — for example, placental cells. And we see that rejuvenation occurs precisely in the embryo, and in those cells that carry an auxiliary function, rejuvenation is not observed.

— What mechanisms are responsible for returning to the zero level in embryogenesis? Are they somehow related to Yamanaka factors that create induced stem cells?

— We don't understand all the mechanisms yet. We know that DNA methylation decreases and then increases. It looks like the methylation marks are erased first, and then re-placed. In addition, telomeres lengthen at the beginning of embryogenesis. The same period is associated with significant cell division, so the damage that accumulated in the germ line and ended up in the embryo can be diluted. How this is related to Yamanaka factors is still unclear. It is necessary to study.

Signals of youth

— Have you already tried to somehow reduce Ground Zero in mice in order to prolong their life in the future?

— This is an idea that we also wrote about — to try to influence the rejuvenation process in embryogenesis in order to achieve Ground Zero, which would be lower than natural. So that the aging process starts from an even lower level. But we don't know how to do it yet. Moreover, we don't even know if it would have helped or not.

For example, we can imagine that at the Ground Zero stage the amount of damage is very small, and we could make it even less. But if an organism produces a certain amount of damage per unit of time, then the initial difference would be so small that in the future it would be completely unimportant.

There are some other ways of rejuvenation, many laboratories are studying them, including ours — for example, the process of parabiosis. We can stitch together young and old mice so that they have a common circulatory system, and they would exchange blood. If they live together for, say, a few months, then we see that the old mouse is getting younger. And when we separate them and look after her, she still continues to be younger than her chronological age.

— And what happens to the young mouse?

— We are currently studying this. Apparently, her age is increasing.

— I imagined a terrible picture from the future: a rich old woman buys herself a poor girl, they are sewn together, the old woman rejuvenates, and the unhappy girl becomes an old woman and dies…

— We do not use these models to apply them directly to a person. We are trying to prove that age can be rolled back. We are trying to figure out how to do this. It is not necessary to stitch a mouse with a mouse or an old lady with a girl. It is possible to find ways to activate biological programs in the body that are activated in the same way as in the case of parabiosis.

There are several hypotheses about how rejuvenation occurs with parabiosis, they are tested by different laboratories. One hypothesis is that young blood carries some signals of youth. In this case, you can simply isolate these signals, these molecules, make injections and rejuvenate.

The second hypothesis: there is a dilution of harmful damage in the old body. When he connects with the young, they are distributed into two organisms. Moreover, an old organism has access to the organs of a young organism, for example, in order to better remove harmful substances through young kidneys.

The beginning of aging, the beginning of life

— It is known that from the point of view of jurisprudence, the birth of a child is considered the beginning of human life. Until he is born, he is not yet human. And from a biological point of view?

— From my point of view, most likely, the life of the organism begins at the moment of Ground Zero. When a zygote, a fertilized egg, has arisen, cellular life is formed, with a new genome, where half is from the father, half is from the mother. But so far it is only a set of cells. I wouldn't call it an organism, because, for example, you can take one cell from this embryo, separate it, and it will become a completely different embryo. This is how twins arise — one embryo is divided into two. Or two such embryos can be combined, and you get one organism. There is no individuality here yet.

— Just a kind of conditional life.

- yes. There is no immune system, there is no identity of the body. There is simply cell growth, such a preparatory phase. Then differentiation into three lines occurs, the germ line arises, and the life of the organism begins. But these are, of course, biological terms, not legal ones.

— Probably, when this hypothesis becomes generally accepted, then the legal norms will also be revised.

— Maybe. This is a question for society — how it will decide what legal mechanisms should be. In fact, this is a very important question, because now it is accepted in the world that it is possible to do experiments on a human embryo up to 14 days after fertilization, then experiments are prohibited. Therefore, we actually have no data about human embryogenesis after 14 days.

— I wonder why exactly 14 days?

— This figure arose as follows. In the 80s, a committee was assembled in England to give recommendations on working with embryos. The head of this committee was Mary Warnock. They had a task: to determine the time when it is possible to work with the embryo. They defined it as 14 days. It was a random number, it could be more or less. It didn't matter then, because in those years it was impossible to grow embryos. And now it's possible. Accordingly, now there is a question of revising this rule.

Last year there was a meeting of the Society of Embryologists, where they tried to answer this question. They decided to remove the "14-day rule" and leave the decision to the discretion of the local ethics committees so that there would be an opportunity to study this issue further. At the same time, it should be borne in mind that the life of the organism, if we are right, begins just around these 14 days or a little later.

"We don't even pronounce this word — immortality"

— Another interesting thought in your article: we got aging in the course of evolution along with multicellularity. It turns out that we need it evolutionarily — could there be complex organisms without aging? So, maybe we are fighting with him in vain?

— Has evolution provided for us to use airplanes? Or that we have doctors who are able to do high-tech operations?

— Evolution "conceived" us as intelligent beings so that we could create all this.

— Exactly! So we are thinking how to stop aging. What kind of contradiction can there be if the goal is for the body to live a healthy life longer, to be more productive and make a greater contribution to society? What negative connotation is there in this?

— There can always be a negative shade. For example, rich people will start taking advantage of this opportunity, and others will be deprived of it, not because they are less productive and important to society, but because they do not have such money. It will be a society of immortal oligarchs.

— Our experience shows that this is not the case. Here comes a mobile phone, and after a while everyone has it. Yes, not immediately, but gradually it comes to everyone, everyone can enjoy these benefits.

As for immortality, there is no talk about it yet. We don't even say that word. For a person at the moment there is no known intervention that increases life expectancy. Such data is available for model organisms, and there is no reason why this would not work on humans. But if these data are confirmed, then we will be able to increase human life expectancy by ten percent, no more. And we want to achieve significant rejuvenation, radically prolong life.

But so far we are talking only about the rejuvenation of cells and organs. Here, too, questions arise: if the body is 60 years old and the liver is 20, what is the age of the individual? How do older and younger parts affect each other? Anyway, it's not clear at all how you can stop the aging process. We can roll back the age of some parts of the body back, most likely. But still the body will age. Yes, people will live longer and better, but they will die anyway. Maybe the scientists of the future will come up with some ways that we can't imagine yet.

— Do you yourself try any of the possible ways of rejuvenation, in addition to a healthy lifestyle?

— There's nothing to try yet. There are only hypotheses and fundamental observations. I don't smoke, I don't drink, I have physical activity, but I can do more. Unfortunately, you have to work a lot, so there is not enough time for wellness procedures. In general, yes, there are ways that will help not to accelerate the aging process. But how to slow it down on a person, we don't have exact data. There are many people who say they know, but unfortunately there is no real way.

— They write that COVID-19 affects cells in such a way that they age. Is this so from your point of view?

— From my point of view, not quite so. It's one thing to be infected, but it's another when the infection reaches a serious illness. A serious illness is a lot of stress. When a person gets to the hospital, on a ventilator — this moment ages. And if a person has been ill easily enough, then I would say that, most likely, not.

— So we are coming to the idea of vaccination again?

Definitely. I don't understand people who might doubt her at all. It is a mystery to me why a reasonable person does not see a 20-fold difference in mortality between vaccinated and unvaccinated people. A person deliberately lowers his chances of survival by 20 times! For me, this is wild and incomprehensible. In my environment, I do not know a single person who would not be vaccinated immediately as soon as the vaccine became available.

— How do you assess the current level of development of Russian science?

It's hard for me to say about the whole science. In my area, the condition is not very pleasing. Of course, there are great groups, we cooperate with some of them, but there are infinitely few of them to compete on an equal footing with the West or even with China. But here's what's interesting: specifically in the field of aging, there are many successful scientists from Russia or originally from Russia, and in general Russian-speaking scientists. Maybe it's because of our education and the influence of intellectuals of past generations? Or maybe it's because our people have learned to cope with great difficulties and therefore solve big problems, but they don't exchange for small ones? It would be interesting to test this hypothesis as well.

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