Epigenetics and the aging clock
Josh Mitteldorf, Methylation Aging Clock: An Update
For links, see the original article.
DNA methylation is the most well–studied mechanism of epigenetic regulation (activation and inactivation of genes). Methylation profiles are stable, except in cases of their active change. They can persist for decades and even be passed down from generation to generation.
For four years, biostatistics specialist Steve Horvath from the University of California at Los Angeles has identified a complex of 353 methylation zones that correlate most well with the chronological age of a person. Genes localized in these regions are activated and inactivated at certain stages of life. Computer analysis of DNA samples (from blood, skin or even urine) allows you to determine the age of a person with an accuracy of about 2 years.
There are two reasons why Horvath's proposed watch is of some importance. Firstly, they are the best of the existing methods for determining the biological aging of a person and provide objective results for evaluating the effectiveness of anti-aging interventions.
Suppose we have a promising new drug and we want to find out if it makes a person younger. Before the Horvath watch appeared, it had to be given to thousands of people and waited for decades to see that fewer of them would die than people who did not take the drug. Horvath's watch significantly reduces the waiting period. The drug can be given to a small number of people and their age can be estimated by methylation before and after the intervention. To get a fairly accurate idea of the effectiveness of the drug, only a few dozen people can take it for two years.
Secondly, there is evidence and theoretical justification for the idea that the methylation sites identified by Horvath are not just markers of aging, but trigger its mechanisms. This means that if we find a way to get inside the cell nucleus and change the methylation profiles on chromosomes, we will be able to influence the root cause of aging.
(Before we get too excited: "gene therapy" was invented about 20 years ago, but is still under development; we need "epigenetic therapy", which still does not exist, but is technically possible using genetically modified viruses and CRISPR technology.)
Below is a recording of two speeches by Horvath, held at the offices of the US National Institutes of Health in 2016 in Maryland and last month in Los Angeles.
In 2012-2013, 3 articles were published describing the idea that the underlying cause of aging in humans and other higher animals is an epigenetic program. Genes are activated and inactivated at different stages of life, which triggers the processes of growth, development and aging, forming a continuous sequence.
(In the fourth article, under the authorship of the Favorable, a similar idea was expressed. It is devoted to the role of the only transcription factor – a regulator of the expression of the gene encoding the mammalian rapamycin target protein, mTOR, and dismisses the conclusion that, from the point of view of natural selection, aging is the preferred outcome.)
This powerful hypothesis simultaneously provides explanations for evolutionary and metabolic issues. It contains recommendations for anti–aging research - despite the fact that epigenetics has turned out to be such a complex field that practical modification of the sequence of changes in gene expression requires a lot of fundamental work.
Unbeknownst to experts who have been working on such theoretical treatises for a long time, Steve Horvath has already been working on the calibration and evaluation of the effectiveness of the aging clock and published the basic results by the end of 2013.
One important property of Horvath's watches is that they allow predicting with higher accuracy than chronological age which of the people will develop age-related diseases and who will die before their peers.
Even though an algorithm is taken as the basis for the operation of the watch, which brings the age at the output as close as possible to the chronological age, the result obtained is guaranteed to provide more information than the chronological age. "When developing the watch, chronological age was used as an approximate indicator of biological age." People whose "methylation age" exceeds their chronological age are more likely to have a deteriorating health condition and die faster than people who have this indicator less than their chronological age.
Horvath posted his methodology and computer program in the public domain. Taking Horvath's watch as a basis, one of the California companies last year began offering a commercial age determination test by DNA methylation. To pass this test, you must send a blood or urine sample to Zymo Research.
Candidates for the Aging Clock
Horvath recounts that he came through elimination processes, starting with four candidates for the aging clock:
- telomere length;
- gene expression profile;
- proteomic data;
- DNA methylation.
This indicator was learned to evaluate quickly and cheaply more than 10 years ago, but its correlation with chronological age (and mortality) is not strong enough to be used as a biological clock.
Gene expression profile
Which genes are transcribed into human RNA at a certain time? This is determined by isolating RNA, and the data obtained are highly specific to a particular tissue. In other words, they vary depending on which part of the body is being studied.
After transcription, the genes are translated into proteins. Some of these proteins remain inside the cell, while others circulate throughout the body. The inexpensive CHIP technology makes it possible to estimate the levels of different proteins with sufficient accuracy.
This indicator is measured more simply than (2) and (3). Methylation is one of the many mechanisms for controlling gene expression that provides the most persistent effects. Horvath found that certain sets of DNA methylation regions are characteristic of a particular age, regardless of which human organ the material for analysis was obtained from.
What is DNA methylation?
Many genes are adjacent to so–called promoters - sections of DNA that store temporary information about whether a gene is activated or inactivated. The promoter sites contain a repeating sequence of nucleotide bases C-G-C-G-C-G-C. It is called a CpG island (where "p" means only that C is connected to G on the same chain, and not on a complementary one, where C is always paired with G.)
"C" means "cytosine", and the cytosine molecule can be modified by attaching an additional methyl group (CH3-) to obtain 5-methyl-cytosine.
The cell contains molecular agents whose function is to selectively attach methyl groups to certain DNA sites and remove them from other sites. The essence of this is that methylated cytosine is a label meaning "not to transcribe the adjacent gene". Removal of methyl groups is a signal for the resumption of transcription.
Methyltransferase enzymes are localized in certain regions of the genome and perform the function of activating and inactivating genes. Methylation may be transient. There is evidence of the existence of circadian methylation cycles. It can also be quite long-term. Methylation profiles can persist for decades. They can be copied during cell division and passed on to the descendants of the organism as part of their epigenetic inheritance. However, inherited methylation regions are an exception, since most of the genome is programmed anew shortly after birth. During the formation of eggs and spermatozoa, methylation profiles are formed that ensure their pluripotency.
How the methylation clock works
Using a standard statistical algorithm, Horvath identified 363 CpG regions that were most strongly correlated with chronological age, regardless of which part of the body the cells were isolated from. The same algorithm provided multiplication of 353 numbers by the values of the methylation levels of each of the regions, and then added all the values. The resulting number is not a direct measure of age, at the last stage of the assessment, a table (an empirically constructed curve) is used to determine the age corresponding to the number.
This curve is a rough representation of the function before its transformation into an indicator of age. It should be noted that the methylation profile changes at a high rate during the first five years of life, which gradually decreases during the growth phase and levels out to a stable decrease after about 18 years.
Despite the fact that Horvath's watch was developed regardless of which tissue DNA was isolated from, certain variations are possible. The most severe deviations are characteristic of the breast, aging faster than the rest of the body, and the brain, which ages much slower. Blood and bone tissue are characterized by slightly accelerated aging; spermatozoa and eggs are at "zero age" regardless of the age of the person. Placentas of women of any age also have zero age.
Similarly, induced pluripotent stem cells (obtained using 4 Yamanaki factors) have zero age. At the same time, a similar effect can turn differentiated cells of one type into cells of another type, for example, skin cells into neurons. This has no effect on epigenetic age.
Liver cells tend to be older than the rest of the body in overweight people and younger in underweight people. Apparently, this pattern does not apply to other tissues. For example, the age of methylation of fat cells of obese people does not exceed the corresponding indicator for the whole body. And, perhaps unexpectedly, a decrease in body weight does not normalize the age of liver cell methylation (at least, this did not happen during the 9-month follow-up period in one of the studies devoted to this issue.)
A number of studies have revealed correlations between the age of methylation and the risk of developing various diseases, as well as mortality. During such work, the analysis is corrected for all environmental factors, including smoking, obesity, physical activity, harmful production factors, and so on. In the complex, these influences are called "external factors". The results obtained showed that exposure to such factors increases the age of methylation and, regardless of this, the age of methylation correlates with internal (genetic) factors that affect life expectancy. According to Horvath's estimates, the genotype is responsible for 40% of the age variability in methylation, which provides a discrepancy with chronological age. The age of methylation of men is slightly higher than this indicator for women. This is obvious by the age of two. Delayed menopause corresponds to a younger age for methylation. The level of cognitive function has an inverse correlation with age by brain methylation.
Speaking to Horvath at the same conference, Jim Watson stated that there are many dietary supplements and medications that can slow down Horvath's clock. He devotes his speech to metformin, which, according to Watson, affects epigenetics through a mechanism completely unrelated to lowering blood sugar levels (the reason why metformin is prescribed to tens of millions of people with diabetes).
There is a very interesting hint: a small number of children never develop or grow and continue to look like babies up to 20 and possibly more years. These children have a normal methylation age. Whatever is blocking their growth is not a change in the methylation process of their DNA. Does this mean that there are other epigenetic mechanisms, more powerful than methylation, regulating growth and development? Or do children with this syndrome have normal epigenetic development, but something below the expression of genes blocks their growth? On the contrary, Hutchinson-Guilford progeria is caused by a defect in the LMNA gene that causes premature aging and death occurring earlier than adulthood. According to Horvath's watch, children with this disease have a normal methylation age.
Radiation, as well as smoking and oxidation under the influence of environmental factors, accelerate the aging of the body. This does not depend on the age of methylation, which is not affected by radioactive radiation. Neither smoking nor exposure to radiation affect epigenetic age. HIV also accelerates the aging process and does not affect the age of methylation.
Indicators of methylation age and telomeric age correlate with chronological age, but they predict mortality and morbidity regardless of chronological age. At the same time, these two indicators do not correlate with each other. In other words, the data provided by the methylation clock and telomere length measurements complement each other, and their combined use provides a higher efficiency in predicting age-related extinction that will occur in the future than their use separately.
The diet has a weak effect on the age of methylation. Diets with very high carbohydrate and very low protein content are noticeably worse. In addition to this evidence in favor of the fact that the "golden mean" are two approaches, namely the protein-depleted Ornish diet and the type of diets, which include the "Zone" diet and the Atkins diet. These evidences are not strong enough to be unambiguously convincing, but they indicate the possible effectiveness of these approaches.
It is also known that the epigenetic clock does not work in malignant tumors.
Improvement of the original clock
Initially, the watch was optimized to track chronological age, but it randomly provides much more information. At the second stage of the work, Horvath set himself the goal of learning how to accurately track biological age. He used blood samples preserved since the 1990s and compared them with medical histories and death certificates in order to identify the methylation regions most strongly correlated with age-associated clinical outcomes. As a result, he invented a phenotypic clock called DNAm PhenoAge. They use 523 methylation regions to predict:
- mortality from any cause;
- mortality from cardiovascular diseases;
- development of lung diseases;
- development of cancer;
- development of diabetes mellitus;
- the extinction of physical strength;
- extinction of cognitive function.
The epigenetic clock is adapted to work effectively with the most accessible cells – skin and blood. A sufficient number of epithelial cells for the DNAm test can be painlessly scraped off the mucous membrane of the oral cavity (the inner surface of the cheeks).
Relationship with parabiosis and plasma transfusions
Several research groups have started experimenting with plasma transfusions of young donors as a possible method of rejuvenation of the body. Horvath describes promising observations: sometimes elderly people develop a variant of leukemia that requires transfusions of blood and bone marrow (containing stem cells that give rise to new blood cells) from a donor. According to the data obtained, after such therapy, the patient's blood demonstrates the age of the donor's methylation, and not the recipient himself.
Epigenetic aging and telomere aging are interconnected according to the principle of "swing"
In different people, the methylation age is higher or lower than the chronological age by an average of 2 years. 40% of this variability is due to heredity. Some common gene variants may cause the epigenetic clock to go faster or slower. The most significant genetic variants link telomeric aging with age by methylation. The faster a person's epigenetic clock goes, the longer his telomeres are. The slower the epigenetic clock, the shorter the telomeres.
This is mentioned in the genetic theory of aging, known as the theory of antagonistic pleiotropy. In 1957, George Williams suggested that aging-causing genes should have both positive and detrimental effects. This would serve as an explanation for the fact that natural selection allowed aging to appear, despite the fact that it reduces fitness. According to Williams, "nature had no choice but to accept the genes that cause aging, since there was no other way to get the positive influence of these same genes" (which, according to his assumption, contributes to increased fertility).
In the author's understanding, the theory of antagonistic pleiotropy does not describe the situation of "forced selection". Rather, aging is an important factor in preserving the health of society and Mother Nature is faced with a dilemma: how to preserve aging despite the natural selection directed against it at the individual level. Aging is so important to society that the goal of evolution was to preserve it despite the short-term temptation for natural selection to promote the survival of those who live longer and have more chances to leave descendants. According to the author's hypothesis, evolution gave rise to pleiotropy to solve this problem. The telomerase/epigenetic clock is an example of this. There is no physical need for the existence of a relationship between telomeric and epigenetic aging, however, in the course of evolution, a relationship has formed between them on the principle of a swing, which complicates the elimination of the aging process as a result of the accumulation of certain mutations.
This also applies to the latest refutation of the existence of a relationship between telomerase and cancer. At first glance, it was doubtful that genetic variants that increase the length of telomeres may be associated with an increased risk of developing certain types of cancer. We have a clue: genetic variants that increase the length of telomeres simultaneously accelerate the epigenetic aging program. A particular example of cancer most closely associated with elevated telomerase levels is melanoma, a malignant tumor that is less sensitive to age than other types of cancer. Melanoma usually develops at an earlier age than other skin tumors. Therefore, it can be assumed that for genetic variants providing longer telomeres, other pleiotropic relationships with mechanisms specifically associated with the development of melanoma will also be found.
The data available to date indicate that programmed methylation is an important, but not the only trigger of the aging process. Smoking affects life expectancy, but does not change the age of methylation. A decrease in body weight has a positive effect on life expectancy, but does not affect the age of methylation in any way. The most interesting fact is that there are children who are not developing or prematurely aging due to genetic defects, but, at the same time, have a normally progressive age for methylation.
Why does radiation age the body without shifting the methylation clock? It is possible that the accumulation of damage is part of the aging phenotype, although I would like to believe that the body will retain the ability to repair these damages even in the later stages of life if it is reprogrammed for this. Why does AIDS speed up the aging clock? Perhaps the immune system is the central signaling mechanism of the aging process.
Thus, aging is "methylation plus". Plus what? Not just "methylation plus damage": despite the fact that we can shorten our lives with radiation or smoking, we cannot lengthen it by avoiding toxins. "Methylation plus other epigenetic programs" is the first thing that comes to mind. "Methylation plus mitochondrial status" is the second thing that comes to mind. Methylation occurs inside the nucleus, and the cytoplasm of the cell, apparently, retains information independently and is even able to reprogram the state of the nucleus, as evidenced by the results of experiments on parabiosis. There is also evidence for the existence of a combination of "methylation plus telomere shortening".
Evgenia Ryabtseva, portal "Eternal Youth" http://vechnayamolodost.ru