A false trail in the fight against aging?

Senescent cells

Yuri Deigin, Geektimes

Correlation does not mean causality. This phrase is drummed into the head of all students of statistics from the first classes. At the same time, correlation, of course, hints at this very causality – often the two correlating parameters, if not a direct causal relationship, then at least a common external cause. One of my favorite examples: ice cream sales correlate well with the number of drowned people, but both variables do not depend on each other, but are caused by a third factor – the weather.

In the fight against aging, new and new hypotheses of some Great Therapy are constantly emerging and are quickly coming into fashion, which will help to win a decisive victory in this struggle. Not so long ago it was Her Majesty Telomerase, but a couple of years ago she was removed from the throne of senolytics – a means of combating senescent cells. These are zombie cells that not only no longer perform their functions and at the same time refuse to die, but also poison everything around them, releasing a cocktail of pro-inflammatory substances called senescence-associated secretory phenotype, SASP.

However, as has often happened, correlation may again try to show its cunning and lead us on the wrong track: the fact that there are more senescent cells in the body as we age does not mean that they are the cause of it. And it looks very likely that they may turn out to be such a false trail. The latest research by Andrey Gudkov, professor, Doctor of Biological Sciences, founder and scientific director of the biotech company Cleveland BioLabs, and in general one of the most successful Russian biologists abroad – the results that he presented in January 2017 at the Scripps Conference on the Biology of Aging, make me more and more confident in this. In general, for me this is very interesting, I would even say revolutionary, data. Here is his full video presentation, take a look, you won't regret it. (Or don't watch the hour–long lecture in English: below its main provisions are retold and illustrated with slides - VM).

Experimental observations

What did Andrey say that was so revolutionary? And here's what:

In mice irradiated at 10 weeks of life with a "lethal" radiation dose of 11 Gy (apparently, this dose is fatal if the donor bone marrow is not immediately transplanted to mice after irradiation, which was done in Gudkov's experiments), no senescent cells are detected even 30 weeks after irradiation. Which is very strange, because in vitro (in cell cultures) irradiation is a guaranteed way to send a significant part of the cells to a senescent state (here, in the photo below, dark clusters are senescent cells; note how many of them are when irradiating a cell culture with a dose of 11 Gy):

Moreover, the fragility index in irradiated mice a year after radiation was better than in control mice who did not receive radiation, and the irradiated mice did not live dramatically less than the control ones (the average pancreas of 90 weeks versus 120 control, and the maximum pancreas of irradiated mice was 120 weeks versus 135 weeks in another experiment):

At the same time, not only no senescent cells are detected in mice, but the cytokine profile of the inflammatory response does not differ from control mice, which cannot be said about unirradiated old mice, whose inflammatory response is much higher (the same inflammatory). That is, the immune system after irradiation works perfectly at the same level as in non-irradiated peers. Here, however, it is important to remember that the bone marrow of irradiated mice is donated, and that it is the source of hematopoietic stem cells, the precursors of the vast majority of immune cells (tissue macrophages and other tissue immune residents are an exception). But in any case, it is very strange to see that radiation does not affect either the inflammatory response or the number of senescent cells in any way. The slide below shows 40-week irradiated (3rd row) mice, compared with 40-week (1st row) and 98-week (2nd row) non-irradiated mice. It is clearly seen that senescent cells (blue spots) are present only in non-irradiated 98-week-old mice. The last column on the right is the cytokine profile of the immune response:

By the way, the transcriptome of irradiated and non-irradiated 40-week-old mice is almost identical, unlike the transcriptome of 98-week-old mice:

And cancer resistance in irradiated mice is generally higher. In the induced cancer model, when mice were injected with melanoma cells (B16 melanoma cells), irradiated mice lived longer than non-irradiated ones, even though they had much more lung metastases:

And cancer tumors in irradiated mice grew much more slowly (red curve versus green), and the average survival rate was 3.5 times higher than non-irradiated ones (that is, even better than the equal average survival rate in the previous experiment):

Andrey Gudkov 's hypothesis

What is Andrey Gudkov's hypothesis on this? What, in his opinion, explains all these mysterious observations summarized on the slide below?

The hypothesis is as follows: as DNA damage occurs, most cells (not immediately killed by apoptosis), in which these damage to the body could not be repaired immediately, do not immediately turn into senescent (as in the incorrect, according to Andrey, the hypothesis generally accepted today from the slide above), but freezes in a certain state, named by him DSPC (Dormant Senescent-Prone Cells, or "hidden pro-senescent cells").

That is, these cells continue to live and function, and become senescent only if they need to divide, but then the (innate) immune system comes into play, in which the function of trapping and killing senescent cells is perfectly debugged – macrophages and immunoglobulins M (IgM) are responsible for this in it. Schematically , Andrey 's new hypothesis looks like this:

However, Andrey believes that all of the above is true only for mesenchymal cells, and epithelial cells after irradiation follow the uppermost path on the slide above, that is, DNA breakdowns in them are immediately repaired. In confirmation that mesenchymal cells are full of breakdowns, Andrey cites data that irradiated mice have an order of magnitude more double-stranded DNA breaks in them:

Whether Andrey checked epithelial cells for double-stranded breaks to test his hypothesis that breakdowns in them are repaired immediately, I do not know. But based on experimental data on mesenchymal cells, Andrey believes that after irradiation, almost 100% of them become such "pro-senescent" (DSPC), as he writes on this slide:

And it is precisely the fact that during division they become senescent and are killed by the immune system, which, according to Andrey, explains the best resistance of irradiated mice to cancer – the tumor cannot grow quickly, since new blood vessels designed to supply it with blood grow much slower in such mice, since the precursor cells of these vessels all are pro-senescent.

Andrey proved the pro-senescence of such cells experimentally – in cell culture, the cells of irradiated mice almost immediately turn into senescent and do not grow, unlike the cells of control mice:

What else, according to Andrey, confirms this hypothesis is that on a high-calorie diet (or high-fat, to be precise), irradiated mice die even faster. However, in other experiments it has been shown that a fatty diet in itself shortens the life of mice, and the fact that the non-irradiated control group on a fatty diet also lost about 10% of the population at the same time as the irradiated group, in my eyes, gives rise to some doubts. Therefore, I would very much like to see a complete survival curve for non-irradiated control on a fatty diet:

The immune system is a forest orderly

How did Andrey show that it is the immune system that is responsible for the control and elimination of senescent cells? Very beautiful. He placed the senescent cells in a kind of structure resembling a metal cage for shark divers, and implanted these structures into a mouse. And then I looked at what kind of sharks swim to these "cages". The sharks turned out to be mainly macrophages (with the usual entourage of other immune cells – eosinophils, etc.).

This graph shows that without protective "cells", senescent cells disappear very quickly (the population decreases by 100 times) after implantation into a mouse (green curve), and when they are placed in a protective capsule, making them inaccessible to any other cells, their number practically does not decrease (blue curve):

And here are the sharks: macrophages. Moreover, what is surprising is that these macrophages themselves begin to express the senescent marker beta–galactosidase, which was previously considered a marker of exclusively senescent cells. Why this is happening, I have not yet understood, and Andrew, in my opinion, too.

Moreover, Andrey showed in another experiment that a significant part of those cells that we previously considered senescent are macrophages, which themselves are unlikely to be senescent (that is, they do not secrete SASP – the already mentioned cocktail of pro-inflammatory factors), but are most likely scattered among the population of real senescent cells, like fighters on the the battlefield:

The main question for both me and Andrey is why these fighters cope so well with senescent cells until old age, and then they stop coping abruptly. Here our points of view differ. Andrey believes that with age, a certain resource of the immune system is exhausted, and that is why it ceases to cope with them. And in irradiated mice, this resource is exhausted faster, because there are much more senescent cells there.:

Of course, I do not agree with the hypothesis of the resource. I cannot imagine that 11 Grams of radiation, which turns 100% of mesenchymal cells into pro-senescent, begins to exhaust the resource only after a year, and even then reducing the average pancreas by only 28%, and the maximum pancreas by 18-20%.

At the same time, I am very interested in why epithelial cells, according to Andrey's hypothesis, are spared from this fate. It seems to me important to understand exactly what happened in irradiated mice with rapidly separating tissues and how this fits in with the hypothesis of pro-senescent cells. After all, there are quite a lot of rapidly renewing populations in the body: intestines, stomach, lungs, reproductive system (the blood system does not count, since it is formed mainly by bone marrow, which was transplanted to irradiated mice from intact donors):

It is also not very clear to me how the mesenchymal cells of irradiated mice managed to continue functioning and accurately synthesize the right proteins for their vital activity if they had many times more DNA breakdowns (after all, proteins are built on DNA). By the way, this is another excellent counterargument against the hypothesis of "aging from the accumulation of mutations" (after all, there is still someone who believes in this hypothesis ...). If you remember, there were an order of magnitude more double-stranded DNA breaks in irradiated mice.

In any case, the concept of a programmed reduction in the quality of repair mechanisms seems much more plausible to me, which in youth, even after a mega dose of radiation, do not allow the appearance of senescent cells, and in old age, even in control mice, cause similar amounts of them.

At the same time, a fat diet is a signal for the internal clock to accelerate aging. And calorie restriction is the opposite signal, significantly prolonging the life of ordinary mice. Yes, and sublethal doses of radiation (25-50 times higher than the background), by the way, also prolonged the life of mice by 20%. Which, in my understanding, does not go well with any resource concepts. By the way, it would be interesting to see the effect of calorie restriction on irradiated mice.

So what's up with the senolitics?

Andrey has his own senolytic, EBS3899 (from Everon Biosciences), which worked perfectly in cell cultures, but when translated into a living organism, according to Andrey, it was much less effective: the effect of increasing the SPH by 13% was observed only in male mice, and only if the senolytic was used at the 89th week of life (earlier use did not lead to an increase in the pancreas, as did its use in females):

Therefore, the main conclusion of Andrey, as I heard him, is that we need to look for tools to influence other aging mechanisms (adjustable wrenches on the slide) if we want to achieve a much greater increase in the pancreas:

And it's hard not to agree with Andrey on this.

By the way, perhaps even Ned David, the head of Unity Biotechnology, the largest startup for the development of senolytics, in which Peter Thiel and Jeff Bezos invested, agrees with him. David has already met twice with Juan Carlos Ispisua Belmonte, the author of the work I love so much, and in March 2017 they already discussed some possible next steps.

Well, we will follow the further developments with great interest.

Portal "Eternal youth" http://vechnayamolodost.ru  28.06.2017