Overcome aging. Part II.

Olga Volkova, "Biomolecule"A person lives a long time.

It's getting longer every decade. But he has medicine. An elephant lives the same amount, and a whale lives much longer – without using the health care system. But they're big... Recently, a person was lucky enough to get to know a very small and very strange centenarian. He lives in a stuffy dungeon, does not maintain a constant body temperature, forms castes – like an ant. Blind as a mole, naked as a man. That's probably all the analogies. Further – continuous uniqueness. He does not feel burns, does not know what cancer, atherosclerosis and neurodegeneration are, does not show classic signs of aging, is experiencing a 30-year milestone... being a small rodent. What are the secrets of a naked digger and his furry long-lived relatives that a person will have to work with in order to prolong his own healthy life?

The phenomenon of negligible aging and the prospects for studying animals demonstrating it were discussed earlier: "Senile vagaries of nature: why people stop aging, and mice do not have time to live" [1] and "Overcome aging. Part I. Who got the evolutionary jackpot?" [2]. Perhaps the most interesting objects of biogerontological research include mammals that age extremely slowly or almost do not age in the human understanding of this process. But if it is unrealistic to experiment with large organisms living for more than 100 years, then small, rapidly reproducing and forming large populations in natural conditions and captivity animals would be a real gift for scientists.

And it seems that suitable candidates have already been found: some burrowing ("underground") rodents belonging to the families Spalacidae (blind) and Bathyergidae (Damar digger and naked digger, although the latter is on the verge of separation into the family Heterocephalidae [3]) are famous for their long life for their weight category. These animals (Fig. 1) only occasionally leave the stuffy and cramped systems of underground passages and chambers, that is, they experience a chronic lack of oxygen and put up with an excess of ammonia, carbon dioxide and the product of its interaction with water – carbonic acid. The conditions can be called extreme, but relatively stable and advantageous from the point of view of safety: encounters with predators are extremely rare. Evolution took into account the insignificance of the influence of enemies and negative "terrestrial" eco-factors on the world of these rodents, selecting signs that contribute to a long healthy life in conditions of reduced comfort: slow metabolism, resistance to stress, cancer, neurodegeneration.

Figure 1. Three underground "heroes". From left to right: the champion in life expectancy is the naked digger (Heterocephalus glaber, maximum life expectancy (MPJ) exceeds 32 years), the blind man (Spalax judaei, MPJ 21 years) and the Damar digger (Fukomys damarensis, MPJ in captivity 20 years). Photos from websites lpzoo.org , paratiritis-news.com , houstonzoo.org correspondingly.

Nature has experimented especially strongly on the naked digger (see the inset "Dossier on the naked digger"), an inhabitant of the East African semi-deserts and the owner of an exceptional plateau for mammals on the mortality curve (Fig. 2) [1]. And the person decided to benefit from this by studying the living conditions of the rodent and associating them with its unique properties.

And where to start, if not by reading his genome? However, biologists acted wisely: someone sequenced and compared the results with other animals, someone plunged into the study of the unique "stability" of the digger. And some teams focused on similar properties of another burrowing rodent – the blind man – and were not disappointed either.

This is a natural consequence of inbreeding, which, however, does not reduce the viability of the offspring. Therefore, it is likely that morphological differences between individuals are formed by epigenetic mechanisms, but what kind is not yet known. The genome of the naked digger was sequenced for the first time in 2011 [9, 10]. It turned out that despite the general high similarity with the DNA of mice and rats (even in the order of the genes), it also has a number of features. The following are those that may be related to the longevity of this rodent and therefore require intensive study (see the inset "The genome of the naked digger").

Theoretically, any, if not all, genetic features associated with the work of mitochondria and telomere modeling, with the exchange and utilization of macromolecules, the balance of metabolic processes and the rate of cell division can contribute to the longevity of the naked digger. And they can prolong life, including through resistance to cancer and neurodegeneration.

Thus, it was possible to identify common genetic features of long-lived animals and unique adaptations of Heterocephalus glaber (listed in the dossier) [6]. Some genomic findings were confirmed in the study of proteins and cellular physiology of diggers.

So, what distinguishes African long-lived rodents?

It is likely that the general foundation of longevity is the mechanisms of resistance of digger cells to all kinds of stress factors – from ROS and pathological proteins to mycotoxins and xenobiotics. This is clearly manifested, for example, in the exceptional immunity to cancer and the lifelong stable maintenance of the brain functions of these animals. The peculiarities of glucose metabolism can also affect their life expectancy. As for his Majesty the Naked Digger, the word "stability" accompanies most mentions of him: stable expression of most genes, stable proteome, stable level of oxidized biomolecules, stable functioning of mitochondria and antioxidants, stable body composition, stable metabolic rate, stable functioning of organs. It is likely that this property is also characteristic of his "comrades" in the dungeon.

For example, in some lines of mice and rats, cancer mortality reaches 70-90%. At the same time, no cases of the development of this pathology in naked diggers have been reported. Their fibroblasts do not age replicatively (telomerase genes are expressed in these rodents all their lives), but they divide extremely slowly in culture. There is an inverse correlation between the rate of division of replicatively non-aging fibroblasts in vitro and life expectancy [24]. Therefore, the main questions that the naked digger must "answer" are: what mechanisms so reliably control his cell cycle, and whether they only provide insensitivity to cancer? Other long-lived rodents, apparently, will also have interesting answers.

Anti-cancer protection consists of many interrelated links: it involves strict regulation of the passage of control points of the cell cycle, and repair of DNA damage, and replicative aging, and apoptosis, and even adjusting the intensity of ubiquitin-dependent protein utilization. Interspecific differences in any of these processes explain the difference in the predisposition of animals to cancer and (partially) in their average life expectancy (Fig. 5) [24]. Mice and rats have served biomedical science faithfully for decades [25], but they are far from the best object for studying cancer resistance, since they are much more susceptible to the development of cancer than humans, not to mention diggers. This means that some preventive mechanisms do not work for them, and researchers miss important antiproliferative factors. It is known, in particular, that in humans both the most important pathways are involved in the control of cell division – pRb and p53, and in mice and rats the role of the first is minor, that is, there is practically no safety net – and this despite the fact that telomerase is active in their somatic cells. It has been experimentally established that for malignancy of mouse fibroblasts, it is enough to disrupt two cellular systems, and human ones – as many as six. Accordingly, the mouse model is not very suitable for predicting human processes, and the lack of suitable animals has so far decently limited the pace of cancer research. If classical model rodents are indispensable for practicing therapy, then it is long-lived animals whose cancer protection mechanisms work like clockwork throughout their lives that can give the missing knowledge on prevention [26].

Moreover, hypoxia is pulsating – then more oxygen, then less, or even not at all. It is not surprising that in the organisms of such rodents, in addition to high vascularization, effective respiration and active hematopoiesis, various cellular safety mechanisms have been developed, some of which are somehow related to carcinogenesis.

A distant relative of the naked digger, the blind man (Spalax, blind mole–rat) is an underground rodent common in the Middle East (Fig. 1). It also differs in longevity (20 years is not the limit) and cancer resistance: spontaneous tumors have not been observed in these animals for 50 years, and to induced cancer they are extremely stable.

Dr. Vera Gorbunova and her colleagues from the University of Rochester (New York) and other institutes in the USA and Israel, in search of an explanation for such resistance, analyzed in vitro the growth of fibroblasts of two types of blind – Spalax judaei and Spalax golani. The cell populations actively doubled only 7-20 times, then "slowed down" and began to secrete β-interferon (IFN-β), which caused massive cellular necrosis – the population was self-purified at the threshold of oncogenesis. Researchers have never observed such a one-time "end" when growing fibroblasts of 20 other rodent species. This phenomenon was not associated with either cultivation conditions or telomere shortening (the cells of the blind do not age replicatively). But the participation of cell cycle control proteins p53 and pRb in the necrotic response was confirmed [27].

Interestingly, most of the cells died from necrosis (a seemingly inaccurate process), while IFN-β caused apoptosis in mouse fibroblasts. The authors of the work attribute this to an adaptive point mutation of the p53 protein gene in blind mice, which makes it possible to disable cell division in conditions of hypoxia characteristic of underground tunnels, creating conditions for repair, but not to trigger apoptosis. It is possible that necrosis is even a more effective preventive procedure, because it allows you to clean up a suspicious place better, destroying the "unreliable" environment that has undergone tumor signaling. The same group of scientists showed that blind man's fibroblasts and even their culture medium caused the death of various tumor cell lines, including human ones [26].

Even more interesting is that the behavior of fibroblast cultures of the blind man and the naked digger is very different: the prevention of over-multiplication of fibroblasts of the latter is achieved due to hypersensitivity to population densification (see below). The cells of the naked digger prefer to respond to stress with traditional apoptosis – like the cells of mice and humans. It turns out that even related long-lived organisms can achieve the same goal in different ways (although in the cases described, the strategies still converge on the pRb path).

A whole spectrum of changes in the structure of genes or their expression caused by adaptation to hypoxia was found in the blind man. Moreover, many of these genes are directly related to carcinogenesis. The mentioned mutation of the p53 tumor suppressor gene, for example, is quite common for human malignant tumors, but, alas, it leads to a different result... The gene of the main vascular endothelial growth factor VEGF in Spalax is expressed to the maximum, and constitutively – as in tumors. In cancer patients, a high level of this factor in the blood is associated with an unfavorable prognosis: angiogenesis provoked by it provides conditions for distant metastasis [28, 29]. For a blind person, it simply facilitates the supply of oxygen to tissues. On the other hand, analysis of the Spalax transcriptome revealed many working genes associated with resistance to both cancer and hypoxia [27]. In particular, almost three dozen unique amino acid substitutions in the transcription factor Nrf2 (in mammals it is very conservative), combined with a high basal level of its synthesis, can contribute to the excellent resistance of blind cells to carcinogens and other stress factors.

The extracellular matrix is not a simple broth, but a framework for maintaining tissue architecture and a barrier protecting cells from invasion of damaging agents. The matrix is based on a three–dimensional network of collagen and elastin fibers, the space between which is filled with "jelly" containing mainly hyaluronic acid, glycoproteins and proteoglycans. The matrix plays an important role in the processes of angiogenesis, inflammation, regeneration, as well as adhesion, growth and metastasis of tumors. It is not surprising that the components of the matrix of long-lived rodents have attracted the special attention of biologists.

Carbohydrate chains of matrix proteoglycans are very often represented by heparan sulfate. Heparan sulfate proteoglycans, interacting with other components of the matrix, form a kind of depot for growth factors and cytokines. Accordingly, the destruction of the heparan sulfate framework by heparanase (HPSE) is accompanied by the release of these molecules and low molecular weight fragments of heparan sulfate itself. The latter, apparently, increase the production of interleukins and other pro-inflammatory cytokines by leukocytes and spleen cells [30]. Well, growth factors can signal to cells that it's time to divide. An excess of such signaling can provoke tumor growth and its metastasis through a new vascular network (one of the factors is angiogenic VEGF). And although normally the goals of heparanase are good – to accelerate tissue healing and vascular growth – in the case of oncopathology, it works to the detriment: there is a lot of heparan sulfate in the basement membrane of blood vessels, which serves as an obstacle to the penetration of cells that have broken away from the primary tumor into other tissues. The increased and worsening prognosis of the production of this enzyme by many tumors has long been noted.

Heparanase is now considered as a promising target not only for anticancer [31, 32] and anti-inflammatory, but even antidiabetic therapy. The fact is that white blood cells secrete this enzyme to facilitate migration from the bloodstream into tissues. And type I diabetes (autoimmune) It is associated with T-lymphocytic attacks on insulin-producing cells of the pancreas. Heparan sulfate protects these cells from migrants and free radicals, while heparanase destroys this protection. It has also been shown that it can penetrate directly into the nucleus of T-lymphocytes and activate genes associated with T-cell differentiation [33].

In the blind man (Spalax judaei), heparanase is synthesized much more intensively than in humans, and its pre-mRNA is spliced in several alternative ways. The standard version of the enzyme cuts heparan sulfate "with appetite" – just like the human version (the homology of their genes is high – 85%). However, in a blind man under conditions of pulse hypoxia, alternative proteins are also formed, including those almost devoid of heparanase activity (variants 7 and 36). Despite this, the first of them does not have an anti-cancer effect (is this not related to the described ability of heparanase to directly regulate transcription?), but the second... The introduction of a construct producing variant 36 into melanoma cells reduced the yield of heparan sulfate fragments by 80%: the non-working rodent protein dominated the active melanocytic one. Apparently, the blind man needs alternative options to modulate the work of a standard enzyme when conditions change. In vivo experiments, variant 36 significantly reduced the metastatic activity of melanoma and significantly slowed the growth of glioma in mice [34]. Thus, alternative splicing of blind man's heparanase can be included in the arsenal of strategies for inhibiting its human counterpart.

More recently, the attention of biologists was attracted by another extracellular substance – α-2-macroglobulin (A2M) of blood plasma, an inhibitor of all classes of proteinases. The fact is that in the liver of a naked digger, its gene is "read" 140 times more often than in a mouse, and in humans this protein is less [35]. Probably, at the dawn of the evolution of diggers, the underground lifestyle contributed to the consolidation of a high level of A2M in them as the main defender against bacterial proteinases. In rodents, A2M is the main protein of the acute phase (C-reactive protein is considered the main one here). Human A2M reversibly binds cytokines and growth factors, ensuring their homeostasis in tissues, and also inactivates various, including tumor, proteinases. Interaction with proteinases opens a macroglobulin binding site with the LRP1 receptor (CD91), contact with which triggers rapid purification of blood and tissues from A2M-proteinase complexes (by receptor-dependent endocytosis).

With age, the amount of A2M in a person decreases, respectively, the quality of "cleaning" also decreases. It is assumed that this protein plays a significant role in oncogenesis, inflammation, neurodegeneration and aging: its receptor, LRP1, has been shown to be involved in lipid metabolism, blood purification from Alzheimer's amyloid, the hormone leptin and factors stimulating cancer progression (including VEGF). The auxiliary role of A2M in the "denunciation of traitors" – the presentation of cancer antigens is also described. This protein claims to be a chaperone that prevents protein aggregation, and a "supplier" of cells with zinc – an indispensable helper of many enzymes, hormones and transcription factors (zinc deficiency is associated with the development of age-dependent diseases). Anyway, the structural differences between the macroglobulins of a naked digger and a human have already been established, the most difficult thing left is to assess their impact on life expectancy. The authors of [35] cautiously remind about the improvement of cognitive functions in the sensational experiment with infusions of "young" blood to old mice. At that time, the effect was associated with the activation of the Creb protein in the senile hippocampus [36], however, it would be interesting to establish the role of A2M in such processes...

Perhaps the anti-cancer strategy associated with high-molecular hyaluronic acid of the extracellular matrix of the naked digger is best described.

The polysaccharide hyaluronic acid (HA, hyaluronan) creates a protective cover on the cell surface associated with membrane receptors, organizing proteoglycans and other surface proteins and maintaining a water supply. The average molecular weight of HA in normal biological fluids and human tissues is high – about 1-8 MDa. The protective function of this polymer is weakened by free radicals, which it is forced to actively capture, for example, during inflammation. Therefore, in order to maintain tissue homeostasis and update the extracellular matrix, a rapid exchange of HA, an optimal balance between synthesis and degradation is vital, otherwise its low-molecular fragments accumulate. These fragments disrupt the matrix architecture and compete for binding to both high molecular weight hyaluronan receptors and alternative structures, which leads to changes in receptor clustering, cytoskeleton structure, and intracellular signaling [37]. Thus, GC can be considered as a sensor of destructive processes in the cellular microenvironment. A change in the balance of a low- and high–molecular polymer is a stimulus that sets in motion numerous mechanisms of cellular response, often only intensifying inflammation and transferring it to the category of chronic [38].

It should be noted that there is a clear link between inflammation and cancer, and recently there has been an increased interest in HA as a possible helper of macrophages and fibroblasts in the construction of a pro-tumor inflammatory environment [39]. Firstly, enveloping cancer cells with hyaluronic protects them from immune attacks. Secondly, it has been shown that the amount of HA increases both during wound healing and the progression of carcinomas (intestines, lungs, breast, prostate, bladder), but at the same time the expression of hyaluronidase genes increases, which together with the hyperproduction of free radicals by macrophages leads to the cutting of long HA chains into low-molecular fragments. According to their number in the blood serum, it is possible, for example, to distinguish metastatic breast cancer from non-metastatic.

Binding of small fragments of HA to CD44 and RHAMM/HMMR receptors activates signaling pathways that stimulate not only inflammation, but also survival, migration and invasion of tumor cells (Fig. 6) [40]. A decrease in the molecular weight of HA is observed both in the cartilages of elderly people, and in the skin after UV irradiation (B-type), and in prostate tumors [37]. But the increase in the size of HA molecules is characteristic just for our favorite diggers...

Contact inhibition is a key anti–cancer mechanism that blocks cell division when a certain density is reached (in culture, usually during the formation of a monolayer). This mechanism breaks down in the cells of malignant tumors, and they are seamlessly layered on top of each other. So the fibroblasts of a naked digger in culture stop dividing at a much lower density than the same mouse cells. This process, called early contact inhibition (RCT), proceeds under the condition that the p53 and pRb pathways or at least one of them are in good condition: if the "switch" of the pRb cell cycle is lost, then apoptosis is triggered due to the p53 insurance system (this is characteristic of both mice and humans), and if only the latter deteriorates – apoptosis occurs anyway (this is an exceptional property of a naked digger). But this situation, apparently, develops infrequently. It is worth adding here that the digger has a basal level of p53 protein synthesis in fibroblasts 50 times higher than in mice, and under stressful influences it "jumps" more actively.

It is known that in mice and humans, contact inhibition is mediated by membrane receptors with further induction of p27Kip1, an inhibitor of cyclin–dependent kinases (from the family of Cip/Kip inhibitors), which interferes with the phosphorylation of Rb protein in the nucleus, thereby blocking the transition of the cell from the G1 phase to the S phase, that is, prohibiting it from dividing. In the naked digger, p27 also does its job – but if the early inhibition of fission is disabled, which is provided by the p16INK4a inhibitor from another family, Ink4. It turns out that the "usual" p27-inhibition only further insures the long-lived rodent from carcinogenesis in the event of a malfunction of the p16-dependent RCT system (Fig. 7, left).

But nature was so imbued with sympathy for this animal that she gave him another "anti-cancer shield". If the oncogenes complex is somehow forced to work in the digger's fibroblasts, then it will not be able to cope with hyperproliferation through the described suppression systems - oncogenes interfere with this. However, unlike mouse cells expressing oncogenes, digger fibroblasts quickly stop dividing, showing all the signs of a so-called cellular crisis: chromatin defects, multinucleation, incomplete divisions, etc. Cells can stay in this state for quite a long time. But it is suspected that if there are a lot of them, then eventually necrosis cleans up the pathological zone [42].

EVMGK accumulates in the tissues of the digger due to the low activity of its hyaluronidases and the high processability of hyaluronan synthase 2 (HAS2) with a unique structure of the active center. Moreover, the cells of this rodent are more susceptible to "hyaluronic" signaling. If hyaluronidases are activated or HAS2 is knocked out, the fibroblast culture medium loses its increased viscosity, and the cells become capable of malignancy (we remember that low molecular weight HA promotes proliferation and inflammation). However, the physical presence of EVMGK is not enough for RCT – a full-fledged work of the signaling pathway linking GC reception with the induction of the INK4 genetic locus (encoding tumor suppressors p15INK4b, Arf and p16INK4a) is necessary. Apparently, this pathway includes the CD44 HA receptor and the cytoskeleton protein NF2 (merlin). It is assumed that the tendency to accumulate EVMGK initially developed in diggers to maintain skin elasticity, which is simply necessary in tight underground tunnels, and then this property formed the basis for anti-cancer protection and longevity of these mammals. Interestingly, EUMGC is also produced by blind cells.

Since the INK4 locus is among the most frequently mutated in cancer in humans, understanding the mechanisms of its expression is difficult to overestimate. In addition, it is involved in the development of replicative, oncogen–induced and premature stress-induced aging, and possibly age-related diseases (interestingly, the expression of the p16 gene increases with aging, but not p15, although there is one locus, and they themselves are descendants of one gene duplicated in antiquity). However, in our context, it is especially important that the loss of locus operability makes early contact inhibition in the digger impossible.

More recently, it was discovered that the INK4 locus of this rodent contains an early translation terminator and encodes an additional, fourth, hybrid product p15INK4b and p16INK4a [44]. This new protein, called pALTINK4a/b (Fig. 7, right), is found both in cultured cells and in various living tissues of the digger, but it could not be detected in either humans or mice. pALTINK4a/b synthesis by cells is induced during RCT and under stressful influences: UV and gamma irradiation, loss of adhesion to the substrate and activation of oncogenes. Hyperproduction of the "fourth" protein is more likely to block the cycle of digger and human cells than hyperproduction of p15INK4b or p16INK4a. The ratio of different products of the locus is apparently tissue-specific and varies depending on the intensity and set of stimuli.

It has recently been shown that conventional contact inhibition (along with serum starvation, hypoxia, and rapamycin) suppresses the mTOR pathway and, consequently, geroconversion – the transition of reversible arrest of the cell cycle into a deep senescent state with a pathological profile of cell secretion [45]. Therefore, it is impossible to exclude the contribution of RCT acting through an unusual INK4 locus to the prevention of not only cancer, but also aging as such in a naked digger. An additional inhibitor of cyclin-dependent kinases probably serves as an additional "layer" of protection and allows the animal to fine-tune the work of cell cycle control points, providing a balance between the lifelong ability to cell proliferation and tumor suppression. It is obvious that different stages of synthesis, metabolism and signaling of hyaluronic acid can be promising targets for therapeutic effects already in the human body.

Longevity increases the chances of developing tumors, and tumors prevent you from living for a long time. Animals with negligible or delayed aging surprisingly combine extremely long youth with extreme resistance to cancer. It is obvious that the activity of the signaling pathways of the transcription factors Nrf2 and p53 plays an important role in prolonging life. Apparently, the strictest, multilevel control of the cell cycle and the quality of intra- and extracellular biomolecules is the key to the success of many negligibly aging organisms. Perhaps something from the spectrum of "animal" responses to the risk of malignancy and other age-dependent diseases will be able to be adapted for the needs of human medicine – even if at first in the form of "crutches", pharmaceuticals, but still...

08.02.2015