Yana Slesarenko

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How to choose the optimal combination of genes?

There are various strategies for choosing the optimal combination of genes to increase lifespan, including the evolutionary approach, transcriptome analysis, and “hallmarks of aging”.

Genetics

Evolutionary approach

Different types of animals and plants have different life spans.
But if you look closely, aging is not an indispensable attribute of any living systems. What kind of aging can we talk about in populations of unicellular organisms that reproduce by symmetrical division? And, if there is perhaps no aging in such populations, why should the aggregates of cells that gave rise to the evolution of multicellular organisms senesce?

Then the question arises: why, in the course of natural selection, the phenomenon of aging not only did not eliminate, although it obviously reduced the fitness of an aging organism, but, moreover, evolved?

Undoubtedly, internal and external factors contribute to an increase in mortality with age. With the development of knowledge in the field of physiology and molecular genetics, attention was paid to various kinds of damage to DNA, cells and tissues. The mechanisms that promote aging are themselves stochastic. However, lifespans within species are the same, with different species having very different lifespans.

Apparently, the species life expectancy has a genetic basis. So another problem that evolutionary theories of aging have to explain is the difference in lifespan between species.

Speaking about the evolutionary approach, it is worth referring to the theories of aging.
One of them was proposed by Peter Medawar back in 1952. According to his theory, aging is explained by the accumulation of harmful mutations. That is, genes with harmful mutations that appear in adulthood do not meet with the resistance of natural selection, so the changes accumulate further, leading to aging. At the same time, harmful mutations are severely screened out by natural selection from an early age. And all because it negatively affects survival and reproductive fitness. For example, children with genetic diseases (phenylketonuria, spinal muscular atrophy, progeria, and others) often do not reach reproductive age and do not have time to pass on their genes further.

At the same time, mutations that are beneficial at an early age (associated with the same reproductive ability, for example) are supported by natural selection, in contrast to genes that are beneficial in old age (reducing the risk of cancer or neurodegenerative diseases)1. As a result, in the course of evolution, the accumulation of harmful mutations in older individuals became possible. This becomes more noticeable and obvious in favorable living conditions, when we all stop dying from external factors (from the paws of predators, for example).

Another theory proposes the idea of ​​trade-offs. Williams suggested in 1957 that genes that are beneficial to fitness at an early age but detrimental to fitness later in life will be selected due to the diminishing power of natural selection over time. This theory is now known as the antagonistic pleiotropic theory of aging. So it turns out that aging evolved as a by-product of natural selection to have a beneficial effect on early reproductive ability. Williams’ proposed pleiotropic genes were supposed to explain the aging process. Such genes persist in the population because they have a positive effect on reproduction at a young age, although they do not give anything good at a later age (up to which, in any case, an insignificant part of the population survives)2.

And here is an example, the TP53 gene, which increases the chances of successful reproduction, it stops the reproduction of cells with DNA damage, which are read as potential cancer cells (the famous “guardian of the genome”). At the same time, its increased activity can suppress the division of stem cells, so the body ceases to renew and replace worn-out tissues during aging3

Williams’ arguments were developed in the disposable soma theory. Here the compromise between reproduction and somatic repair and maintenance is already emphasized. This theory of Kirkwood (1977) suggests that an increase in the life expectancy of individuals is possible if the rest of the population lives in favorable, safe conditions. Then, having saved the species from the eternal struggle with predators and natural selection, it is possible to significantly increase the time of existence of each of its representatives4.

Of course, if we talk about the genetic aspects of aging, then it is also important to mention the theory of somatic mutations and DNA damage (DNA damage is everywhere in the biological world and is the main cause of aging)5, the theory of telomeric aging (telomeres are such repetitive sequences that protect the ends of our chromosomes ), to restore telomeres, the enzyme telomerase works for us. As we age, it becomes less able to repair telomeres and our cells begin to break down6, mitochondrial theory7 and others.

Transcriptome analysis

The active development of technologies for analyzing gene expression has given rise to the idea that aging can be understood by analyzing transcriptomes. Mapping changes in gene expression in aging organisms across different tissues can show how biological functions change with age.

For example, epigenome and transcriptome landscapes during aging in mice have shown widespread stimulation of inflammatory responses8. Transcriptome studies in several fly species with different lifespans have also shown the potential role of gene expression regulation in lifespan extension9. In addition, epigenetic clocks of aging have been developed based on DNA methylation markers10. So it turns out that studying changes in gene expression and epigenetic regulation can help us understand the causes of aging.

For example, in one study, scientists from South Korea11 looked at changes in gene expression profiles in the brain with aging. We know that aging is also a major risk factor for many neurodegenerative diseases, including Alzheimer’s and Parkinson’s. Such a close relationship between aging and neurodegenerative diseases suggests the existence of common mechanisms for the regulation of transcriptional genes in the brain12 13.

Although transcriptome mapping itself provides rather descriptive information, the evaluation and interpretation of this information already allows planning the subsequent analysis of the functions of candidate genes and transcripts.

In general, significant changes in gene expression in many areas of the brain are found in humans in their 60s and 70s. But the aging of synapses occurs even before the destruction of neurons. And such aging underlies the increased expression of the transcription factor REST and the reduced expression of tumor protein 73 (TP73). Another important point is that although adequate immune activation is protective, chronic immune activation increases the brain’s vulnerability to aging. In this case, the microglia genes (immune cells in the brain), the C1q component of the complement system (C1QA) and the suppression of genes that encode immunosuppressive factors (for example, CX3CR1) occur11.

Other researchers from the United States and China analyzed the transcriptomes of different cell types and showed that a decrease in antioxidant defense and cell response to stress can be considered important links in ovarian aging and age-related decline in fertility in primates. The scientists studied the transcriptomic landscape of ovarian aging in cynomolgus monkeys. Changes in gene expression have shown that oxidative damage is a significant factor in ovarian aging. In addition, analysis of human granulosa cells (they surround the ovary) revealed similar aging-associated suppression of antioxidant genes. And knocking down those genes compromised the response to oxidative stress altogether14.

“hallmarks of aging”

What we have? Aging is characterized by a loss of physiological integrity, leading to dysfunction and increased vulnerability to death. Such disorders are a major risk factor for various human pathologies, including cancer, diabetes, cardiovascular disease, and neurodegenerative diseases.

Perhaps at first glance, cancer and aging may seem to be opposite processes: cancer is the result of an increase in the fitness of some cells, while aging is characterized by a loss of fitness. However, at a deeper level, cancer and aging may have a common origin. The time-dependent accumulation of cellular damage is considered to be the main cause of aging15 16 17. At the same time, cellular damage can sometimes provide benefits to certain cells that can eventually cause cancer. Therefore, cancer and aging can be considered as two different manifestations of the same basic process – the accumulation of cellular damage. In addition, some pathologies associated with aging – atherosclerosis and inflammation – are associated with uncontrolled cell overgrowth18.

Based on this concept, a number of questions have arisen in the field of aging regarding the physiological sources of damage, the relationship between different types of damage, and ultimately the possibility of intervening in these processes to slow down aging.
If we try to classify the cellular and molecular signs of aging, we can distinguish the following12 19:

  • genome instability
  • telomere depletion
  • epigenetic changes
  • loss of proteostasis
  • nutrient dysregulation
  • mitochondrial dysfunction
  • cellular aging
  • stem cell depletion
  • change in intercellular communication.

Genome instability

The integrity and stability of DNA is constantly exposed to external physical, chemical and biological factors. Meanwhile, we also have our own internal threats (errors in DNA replication, spontaneous hydrolytic reactions and reactive oxygen species (ROS))20. As a result, genetic disorders are very diverse: point mutations, translocations, changes in chromosomes, shortening of telomeres and genome disruption caused by the integration of viruses or transposons. To minimize this damage, organisms have evolved a complex network of DNA repair mechanisms that together are able to deal with most damage21.

For example, the PARP1 protein is involved in DNA repair, regulation of gene expression, chromatin remodeling, and mitochondrial function. During aging, the expression of this protein increases, causing replication to occur. As a result, damage due to the work of PARP leads to cell death. PARP activation can also provoke overexpression of the p53 protein (TP53 gene product). And the more the protein is activated, the more it accelerates the aging process, leading to increased cell destruction (including stem cells)22.

So it turns out that the repair of damage in DNA is not perfect, and with age, damage only accumulates.

Telomere depletion

Telomere depletion or shortening is a specific type of DNA damage at the ends of chromosomes. Normal cell division shortens telomeres, as do other processes that damage DNA. An enzyme called telomerase, which is turned off in most adult cells, can prevent telomere shortening and even restore telomere length. But telomerase deficiency in humans is associated with the premature development of certain diseases (pulmonary fibrosis, for example)23. In general, enzyme activity decreases with age. And when telomeres reach a critical length, cells turn off their replication mechanism24 25.

Epigenetic changes

Epigenetics is the study of what mechanisms regulate the expression of the genome. Gene expression can change for various reasons. And there is ample evidence that aging is accompanied by epigenetic changes and that epigenetic perturbations can provoke progeroid syndromes (accelerated aging syndrome) in model organisms.
Epigenetic changes involve complex processes: changes in DNA methylation patterns, histone modification, and chromatin remodeling26.

a) DNA methylation is such a process of adding methyl groups to the nitrogenous bases of nucleotides (cytosine, as a rule) that does not change the nucleic acid sequence. As a result of this DNA modification, the genetic fragment is either “read” and includes the gene in work (transcription), or not, and thus controls the activity of the gene27 28.

b) DNA strands are wound around coils of proteins called histones. And histones can also be modified. Increased H4K16 histone acetylation, H4K20 trimethylation or H3K4 trimethylation, and decreased H3K9 methylation or H3K27 trimethylation are age epigenetic marks. As a result, the precise coordination of gene activity can be disrupted. But one group of molecules that influence the epigenome are the sirtuins. Sirtuins are a family of proteins that perform histone deacetylation. They have been widely studied as potential factors that slow down aging. In particular, SIRT6 is an example of an epigenetically significant enzyme whose loss of function reduces lifespan and gain of function prolongs lifespan in mice.

c) Changes in the already described histone modifications and DNA methylation determine changes in chromatin architecture such as global loss and redistribution of heterochromatin (contribute to genome destabilization), which are characteristic signs of aging29 30.

Loss of proteostasis

The main task of genes is to produce proteins, which are the heart and soul of cell biology. Proteins regulate chemical reactions and provide structure to the cell. Therefore, the maintenance of proteostasis – the balance between the synthesis, breakdown of proteins, maintaining the correct structure of proteins – is necessary31.

There are special systems that restore the structure of misfolded polypeptides or destroy them so that damaged components do not accumulate. Like many processes, proteostasis changes with age. Most worryingly, chronic expression of misfolded or aggregated proteins contributes to the development of some age-related pathologies such as Alzheimer’s disease, Parkinson’s disease and cataracts31 32.

Dysregulation of nutrient perception

The mammalian somatotropic axis includes growth hormone and its secondary mediator, insulin-like growth factor-1 (IGF-1), which is produced in response to growth hormone by many cell types. The intracellular IGF-1 and insulin signaling pathway is known as the insulin and insulin-like growth factor-1 signaling pathway. Notably, this pathway is the most conserved pathway controlling aging in evolution, and among its targets are the FOXO family transcription factors and mTOR complexes, which are also involved in aging and are conserved during evolution. Genetic polymorphisms or mutations that reduce the functions of growth hormone, IGF-1 receptor, insulin receptor, or downstream intracellular effectors (AKT, mTOR, and FOXO) are associated with longevity in both humans and model organisms. It certainly shows33 34 35.

Mitochondrial dysfunction

As cells and organisms age, the efficiency of the respiratory chain decreases, which increases electron leakage and reduces ATP production. A link between mitochondrial dysfunction and aging has long been suspected, but analyzing it is still a major challenge for aging research36.

Mutations and deletions in mitochondrial DNA can also contribute to aging. mtDNA is considered a major target for aging-associated somatic mutations due to the lack of protective histones and the limited efficiency of repair mechanisms compared to those of nuclear DNA.

Cellular aging

Cellular aging is a phenomenon that is associated with the loss of the ability of cell division37 38 39.  This process was originally described by Hayflick in human fibroblasts grown in the laboratory40. We now know that the aging observed by Hayflick is caused by telomere shortening41, but there are other stimuli that trigger aging. For example, nontelomeric DNA damage and derepression of the INK4/ARF locus38.

In recent years, it has been recognized that senescent cells show dramatic changes in their secretome (a set of factors that are secreted by the cell). Their secretion is particularly rich in pro-inflammatory cytokines and matrix metalloproteinases, and has been referred to as the “ageing-associated secretory phenotype”39 42. And this pro-inflammatory secretion can contribute to aging.

Depletion of the stem cell pool

One of the most obvious characteristics of aging is the decline in tissue repair ability. For example, hematopoiesis (the formation, development and maturation of blood cells) decreases with age and the so-called immunoaging occurs43. Such depletion of stem cells (SCs) has been found in virtually every part of the body, including the mouse forebrain44, bones45 or muscle fibers46. And this is due to the accumulation of DNA damage47, with the overexpression of proteins that suppress the cell cycle48. Here, telomere shortening is also an important cause of SC depletion49 50. In general, these are just examples, and in fact, a decrease in the number of SCs occurs as a result of multiple types of damage.

Just as under-proliferation of SCs is detrimental in long-term maintenance of the body, so their excessive proliferation can be detrimental, accelerating the depletion of cell niches. And it turns out that for the long functioning of the SC, a period of rest is necessary. For example, in the case of Drosophila intestinal SC. Here, excessive proliferation leads to depletion of the SC pool and premature aging51. A similar situation is observed in p21-deficient mice. They experience premature depletion of neuronal stem cells52 53. But the induction of INK4a (involved in the regulation of the cell cycle) during aging and the decrease in serum IGF-1 may be an attempt to maintain a dormant state of cells. You can also try to inhibit FGF2 signaling (involved in the proliferation and differentiation of different cells) to reduce SC depletion during aging.

Here I would like to mention the inhibition of mTORC1 by rapamycin. This makes it possible to delay aging by improving proteostasis, increasing the functional activity of SC in the skin, blood, and intestines54 55 56.

Eventually

It can be seen that a large number of processes are involved in aging, and therefore it is necessary to decide whether we will act on one system or several at once. We cannot modify the work of all genes at once, and even modifications of those genes that are probably suitable for us can hide many unexpected things. By “turning on” or “turning off” genes, it is impossible to be immune from side effects, because we are changing the course of events in a large chain of molecular processes.

But if we don’t start testing, we’ll never know how it all works. Having an idea of ​​what strategies exist for selecting candidate genes, we will select these very genes and try to combine them to achieve our goal of slowing down aging. It is important that genes be shown to have an effect on lifespan earlier in the literature.

After that, we will evaluate whether our combination works using Drosophila Melanogaster or other animal models. And then the choice of proven combinations of genes will allow the development of combinatorial gene therapy for aging.

Gene therapy modulates the architecture of the genome through both direct (eg, gene editing) and indirect (viral or non-viral vectors) approaches. And of course the clinical implementation of such methods is a long process that will require steps to solve many problems. But having already overcome these problems, their practical application can open up new possibilities in anti-aging medicine.

Yana Slesarenko

Yana Slesarenko

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