27 August 2018

Candidate for senolitics: details

Senolytics prolonged the mice's life and improved its quality

Polina Loseva, "Elements"

One of the causes of aging of the body is the accumulation of old, or senescent, cells. A recent study showed that transplanting these cells to young mice accelerates the aging of the body. On the contrary, selective destruction of senescent cells with the help of senolytic drugs improves the health of mice and prolongs their life. Moreover, clinical trials of senolytics for the treatment of senile diseases in humans are already beginning.

During the life of an organism, its cells gradually age. However, this means not so much the age of the cells (which may be different depending on the tissue), as the change in their activity. They lose the ability to divide, perform their functions worse in the body and instead stimulate inflammation and tissue restructuring. Such cells are called "senescent" in order not to confuse the aging of the body as a whole with the aging of individual cells. At the moment, there is no clear explanation why ordinary cells turn into senescent. Probably, this happens as a result of the action of many factors: during the divisions, chromosomes are shortened, mutations in DNA and damage in proteins accumulate. Then, according to the theory of hyperfunctionation, if the damaged cell continues to receive signals that it needs to divide and grow, which it can no longer do, it becomes senescent (M. V. Blagosklonny, 2012. Answering the ultimate question "What is the Proximal Cause of Aging?").

There are several ways to detect senescent cells in the body. For example, it is often used to stain beta-galactosidase associated with aging, an enzyme whose activity changes in senescent cells. Another option is to measure the activity of genes that block cell death: after all, senescent cells, like cancer cells, carry a lot of damage and, in order not to die, must be resistant to apoptosis (programmed cell death). Finally, the third option is to pay attention to the substances that these cells secrete outside. It turned out that this is a fixed set of molecules specific to senescent cells – it is called SASP (Senescence-associated secretory phenotype). It includes, among others, protease proteins that break down the intercellular substance and trigger its restructuring, growth factors for surrounding cells and pro-inflammatory signaling molecules (cytokines) that attract immune cells.

The effect of SASP on other cells in the body is ambiguous. In small doses, it is rather favorable and stimulates stem cells to divide and renew the cellular composition of tissues (see B. Ritschka et al., 2017. The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration). But when a lot of SASP proteins accumulate, they act depressingly on neighboring cells, and "infect them with old age" – neighbors also become senescent (similar processes are described when extracts of old organisms act on young cells.

The authors of the article under discussion tested the effect of senescent cells on the aging of mice. To do this, they took fat stem cells from one mouse (since such cells take root well during transplantation), caused aging in them (in two ways – with the help of radiation or with the help of toxic substances that disrupt DNA synthesis) and transplanted another mouse. To track the fate of the transplanted cells, a transgenic animal producing luciferase, a bioluminescent enzyme, was used as a donor mouse. Figure 1 shows how luciferase was used to track the location of the transplanted cells in the mouse body; they remained there for about 40 days.


Fig. 1. Senescent adipose tissue cells transplanted into the body of a female mouse. A transgenic mouse carrying the enzyme luciferase became a donor of fat senescent cells, so the transplanted cells glow. Maximum luminescence is indicated in red, minimum luminescence is indicated in blue. It can be seen that the cells were in the area of fat deposits, that is, they reached their destination. An image from additional materials to the discussed article in Nature Medicine.

The health of mice was assessed by a variety of parameters: maximum running speed, muscle strength (when grabbing objects), physical endurance (the time during which a mouse can run on a treadmill and hang on a wire), intensity of daily activity, food intake and body weight. Not all of these parameters changed during transplantation of senescent cells compared to transplantation of healthy cells or injections of a pure solution for injection (Fig. 2). But some showed a significant decrease (for example, running speed or grip strength). This allows the authors to say that senescent cells really worsen the health of the organism they enter. At the same time, 200,000 cells are not enough to cause serious deterioration, and starting from 500,000 cells, the changes become significant. Recalculating this by the total number of cells in the mouse body, the authors concluded that one senescent cell per 7-15 thousand cells in the body (0.01–0.03%) or one of 350 cells in adipose tissue (0.28%) is enough to cause visible deterioration in health (such as a decrease in running speed or grip strength).


Fig. 2. Health indicators of mice one month after transplantation of senescent cells. d – the maximum running speed (as a percentage of the original); e – how much the mouse was able to hang on the wire (seconds multiplied by the weight of the mouse in grams); f – grip strength (in Newtons); g – daily activity (number of movements). Mice were injected: blue color – phosphate buffer (control solution), black – 1,000,000 healthy cells, pink – 200,000 senescent cells, orange – 500,000 senescent cells, red – 1,000,000 senescent cells. Significant differences (those that cannot be explained by the spread of data) are indicated by brackets and asterisks. Image from the discussed article in Nature Medicine.

Then the authors of the article checked the remaining, initially healthy, cells of the recipient organism for markers of aging (beta-galactosidase associated with aging and others). It turned out that 2 months after transplantation, 10-15% of senescent cells can be detected in the mouse body. At the same time, there is no luciferase in them, that is, they are not donor cells, but the recipient's own cells. This means that the "old age" of cells is really contagious and senescent cells affect their neighbors.

In order to fight senescent cells, it is necessary to come up with a strategy for their selective destruction. Substances that allow this to be done are called senolytics. The general principle of their work is as follows: like cancer cells, senescent cells are resistant to apoptosis, that is, special proteins are active in them, and these proteins can be blocked. This is how many anti-cancer drugs work, and you can also try to use them as senolytics. One of the first candidates for this role were dasatinib and quercetin – the first is already actively used in the fight against cancer, the effectiveness of the second is not so obvious. However, many scientists believe that their combination may be more successful than using each of them alone (see Yi Zhu et al., 2015. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs).

Before proceeding to animal testing, the authors of the article under discussion tested the effect of dasatinib and quercetin on cell cultures. They used adipose tissue obtained from obese patients during surgery (it is believed that obesity is accompanied by accelerated cell aging; see C. Franceschi, 2017. Healthy aging in 2016: Obesity in geroscience – is cellular senescence the culprit?). Immediately after the operation, the cells were treated with senolytics or a pure solution for two days. Two days later, the number of senescent cells (carrying the corresponding markers) and the amount of SASP in the culture medium were measured. Both parameters significantly decreased under the influence of senolytics, and the level of apoptosis in culture also increased, which means that anti–apoptotic mechanisms were blocked.

Finally, to evaluate the effect of senolytics on animals, the authors of the article used elderly mice aged 20 months (the average life expectancy of a mouse is about two years). For four months, one group (control) received a placebo, the second (experimental) – a combination of senolytics. After that, the authors again assessed their health by a variety of parameters, and the experimental group was more successful in everything (Fig. 3). And the number of SASP proteins in their body decreased.


3. Health indicators of mice that received placebo (V) or a combination of senolytics (D+Q) for two months. b – the maximum running speed (as a percentage of the original); c – how much the mouse was able to hang on the wire (seconds multiplied by the weight of the mouse in grams); d – the grip force (in newtons); e – body weight; f – how much energy the mouse spent on the treadmill (in kilojoules); g – daily activity (number of movements); h – food intake (in grams). Image from the discussed article in Nature Medicine.

After that, the authors tested the effect of senolytics on the lifespan of mice. They gave the drugs to very old mice, aged 24-27 months (which corresponds to 75-90 years in humans). These animals lived 36% longer than their placebo-treated peers (Fig. 4), and until the last weeks, the physical activity of the long-lived mice remained no lower than that of the control group.


4. Survival curves of mice treated with placebo (V, black line) and senolytics (D+Q, red line). On the left – survival in days after the first intake, on the right – survival in days of life. In contrast to the 50 percent survival rate, the average life expectancy of the groups was noted. Image from the discussed article in Nature Medicine.

This suggests that senolytics prolong a full life without compromising health (Fig. 5).


Fig. 5. Health indicators of mice that lived the longest (top 40%). The upper row is males, the lower row is females. Black is a control group, red is an experienced one. From left to right: the maximum running speed (the maximum number of turns of the track per minute at which the mouse can stay), how long the mouse was able to hang on the wire (seconds multiplied by the weight of the mouse in grams), the total lifespan (in days). Image from the discussed article in Nature Medicine

Moreover, the set of age-related diseases that could cause death did not differ in the control and experimental groups (Fig. 6).


Fig. 6. The number of diseases that could lead to death in the control (V, black) and experimental (DQ, red) groups. n.s. (not significant) – differences that do not have statistical significance. From left to right: males + females, males, females. The left columns are the number of diseases in each mouse, the right columns are the number of tumors in each mouse. Image from the discussed article in Nature Medicine.

Thus, dasatinib and quercetin performed very well in mice, reducing the amount of SASP in their body and prolonging their life. At the same time, no side effects and new life-threatening diseases were detected, and physical activity persisted until death. Of course, one should not make premature conclusions about how much such therapy could help people. The authors themselves warn of the need for preclinical studies that would check the possible toxicity of drugs for healthy cells. In addition, what works well in mice is not always as effective for humans.

At the same time, UNITY Biotechnology recently announced the start of clinical trials of senolytics. The company offers two products: these are not dasatinib and quercetin, but other drugs, but the mechanism of operation is similar – they also target anti-apoptotic proteins. In a trial clinical trial, the first senolytic will be used to treat osteoarthritis. This disease is associated with age, and it is believed that one of its mechanisms is the accumulation of senescent cells. Thus, in small steps, starting with the treatment of individual diseases, we are approaching clinical trials of drugs that slow down aging in humans.

A source: Ming Xu et al., Senolytics improve physical function and increase lifespan in old age // Nature Medicine, 2018.

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