A literature review on telomerase and aging
by Wendy Ouriel
Abstract
Cell division is a normal and necessary biological process that occurs in living organisms. However, DNA becomes shortened with every cell division, which can lead to degradation. To protect the DNA, telomeres cap the ends of chromosomes, and become shortened instead of the valuable DNA. Telomeres are synthesized by the enzyme telomerase, a ribonucleotide protein whose expression serves as a biological compensatory mechanism for the shortening of telomeres. Together, telomeres and telomerase play a key role in biological aging. This review discusses some recent studies that have investigated the effects of environment and genetic factors on telomerase activity and telomere length.
Introduction: Telomeres and Telomerase
Telomeres are regions of repeated nucleotide sequences that physically cap the ends of eukaryotic chromatids. This cap serves to protect the chromosomes; without which causes susceptibility to exonucleolytic degradation. To protect against such degradation, telomeres maintain chromosome stability, and prevent depletion of genetic information (15). Telomeres are a biological necessity because the process of DNA replication naturally causes chromosomes to shorten. The presence of telomeres protects genes from truncation, by becoming shorter themselves instead of the genes (5). This is possible because telomeres are just repeated sequences of nucleotides; unlike genes they do not code for proteins. Therefore, their truncation alone will not cause any adverse effects on the organism. However, if telomeres become too short, and can no longer protect the genes, cell senescence (biological aging) will occur.
The concept of telomeres was first described in studies conducted in the 1930s and 1940s by Hermann J. Muller, who worked with Drosophila melanogaster, and Barbara McClintock, who worked with Zea mays. Muller hypothesized that chromosomes require a sort of terminal structure, what he referred to as telomeres, to maintain stability. McClintock expanded upon his work by showing that telomeres can stabilize, and possibly mend broken chromatids (15). However, subsequent studies would find that telomeres do not last forever. In 1961, Leonard Hayflick investigated the limits of cellular division using healthy human diploid fibroblast cells in vitro. His experiment found that the cells were only capable of dividing, on average, about 50 times before they started to degrade (6). This observation was termed the Hayflick Limit, and established the concept of cell senescence. This phenomenon was revolutionary in biology because it refuted earlier hypotheses that stated healthy cells are immortal (3). Furthermore, he showed that cells have a memory of how many times they had divided. When old cells were mixed in with a young population of cells, the old cells ceased to divide and entered senescence in accordance with how many times they have divided over their lifespan. This observation of cell memory implies that there is a biological mechanism in place for maintaining the youthfulness of cells (6).
The observations of Hayflick built upon the works of Muller and McClintock by showing that cells can only continue to divide up until a certain point. However, the reason for this was unknown. The association between telomere length and cell senescence was not established until 1971 when Alexey Olovnikov published a paper hypothesizing that when telomeres shorten to a critical length, the cell can no longer divide (Olovnikov). In his theory, he stated that with every round of DNA replication, there is a loss of DNA. This loss of DNA is from the chromatids. He also hypothesized that cancer cells are able to divide indefinitely due to their ability to prevent telomeres from shortening to this critical length. His theory postulated that both germ line and tumor cells are able to maintain telomeric length by expressing a special type of DNA polymerase not found in somatic cells. However, he did not elucidate further on the matter, and his work went largely unnoticed in the scientific community for many years. It was not until 1985, when Carol W. Greider and Elizabeth Blackburn discovered a special DNA polymerase that elongated the telomeres of Tetrahymena, was Olovnikov’s theory supported. This DNA polymerase, termed telomere terminal transferase by Greider and Blackburn, is known today as telomerase(5).
Telomerase is a specialized ribonucleoprotein polymerase that drives the synthesis of telomeres. It functions to replace telomeres after they have shortened during cellular division. Healthy human somatic cells express little to no telomerase, causing a loss of telomeres with every cell division, and eventual senescence of the cell (7). In cells expressing telomerase, the dividing cell can replace the lost telomeres, and can continue to do so as long as telomerase is expressed. Although high telomerase expression may sound like a positive quality in a cell, this is often not the case. Tumor cells tend to express a high level of telomerase, and are capable of maintaining telomere length after cell divisions that far surpass the Hayflick limit (7).
Research into telomerase has provided a better understanding of the aging process in humans and other organisms. It is understood that telomerase directly influences the longevity of the cell through the synthesis of telomeres. Therefore, any alterations in the expression levels of telomerase will affect the lifespan of the cell. Environmental and genetic factors are often cited as a potential cause for aging, and age-related diseases. This review will focus on some of these environmental and genetic factors that may alter the expression of telomerase, and telomere length, contributing to the aging process.
Environmental Effects on Telomerase Activity and Telomere Length
Certain environmental stressors, such as alcohol consumption, have been attributed to an increased rate of aging, mental decline, and cancer (9). Although environmental stressors may contribute to the aging process in more ways than one, a 2013 study by Romano et al. investigated the effect of such stressors on telomere length homeostasis (Romano). Saccharomyces cerevisiae, a strain of yeast, was exposed to environmental stressors such as alcohol (isopropanol, ethanol, and methanol), caffeine, high temperatures (37 C), acetic acid, hydroxyurea, and oxidative stress (hydrogen peroxide). The yeast were grown in culture for 100-400 generations, and their telomere length measured via telomeric Southern blot analysis. The results of the Southern blot revealed that exposure to alcohol (isopropanol, ethanol, and methanol), and acetic acid lengthened telomeres, while caffeine, hydroxyurea, and high temperatures shortened telomeres. The effect of the environmental stressors was also observed to be dosage and time dependent. Yeast exposed to the stressor for longer periods of time, and/or at higher concentrations had a greater change in telomere length compared to yeast exposed for fewer generations or at a lower dosage.
Interestingly, oxidative stress was found to have no effect on telomere length. This challenges past research that has suggested oxidative stress to be a potential disruptor to telomere length maintenance (8, 11,13). However, these past studies used human cells exposed to hydrogen peroxide in vitro. The difference in telomere length could be attributed to the fact that the human cells were given the additional stressor of being cultured in a dish. The stress of being in a foreign environment may be enough to alter telomere length, and should be investigated in future research.
Romano et al. sought to further investigate the cause for this change in telomere length, by examining key genes involved in telomere length maintenance (TLM). It was hypothesized that the environmental factors that altered telomere length did so by altering the expression of these TLM genes. Using microarray analysis of yeast exposed to caffeine, and ethanol, it was found that genes involved in telomere length maintenance were affected. Among these genes, Rap1, a gene that encodes for an essential protein that binds to telomeric repeats, was affected by the presence of alcohol. Rif1 and Rif2, negative regulators of telomerase, interact with Rap1 to maintain telomere length homeostasis. In the presence of ethanol, Rap1 was reduced in yeast mutant for Rif1 and Rif2, but not when it was introduced with a tetracycline promoter. Telomere lengthening occurred when Rap1 was exposed to the mutant strains, but was not observed when under tetracycline promoter, suggesting that Rap1 is involved in maintaining telomere length, but is inhibited in the presence of ethanol. Southern blot analysis also revealed that a deletion of Rif1 lowers the response to ethanol, but a Rif2 deletion will enhance it. To further support that ethanol inhibits Rif1 function, ChIP analysis was performed on yeast exposed to ethanol, and compared to a control exposed to no environmental stressor. The analysis looked for the association of Rif1 and Rif2 protein to the telomeres, and found that in the presence of alcohol, there was a 54% reduction in the amount of Rif1 protein found at the telomeres, compared to a 33% reduction in Rif2. Deletion of Rif1 and Rap1 also reduced the telomeric response to caffeine, but had a shortening effect on telomeres.
The Romano et al. study is of particular importance because it demonstrates that environmental factors can alter telomere length. Humans consume alcohol and caffeine, and in turn this may have an effect on the aging process. The main genes identified that were affected by environmental stress in yeast are genes that are present in the human genome. Therefore, the impact of such stressors on human health should be further explored.
Humans may contribute to their own aging by consuming alcohol and caffeine; but we may also be able to slow the aging process by what we eat. The Mediterranean diet (MD) is a diet that consists mainly of fruits, vegetables, lean meats, and monounsaturated fatty acids. It is a diet that has been hailed for its low morbidity and mortality for cardiovascular diseases, and higher longevity among consumers. According to population surveys, these benefits are not just observed in Mediterranean countries, but in non-Mediterranean countries where the diet is consumed. This suggests that it is the diet itself, and not a consequence of genetics, that is the key factor in the observed benefits (2). An in vitro study compared three diets, the Mediterranean diet, a saturated fatty acid diet, and a low-fat high carbohydrate diet in elderly subjects to investigate the biological component of healthy eating on longevity. The subjects consumed one of these three diets for four weeks, and researchers measured the amount of intracellular reactive oxidative species (ROS), cellular apoptosis, and telomere length in the subject’s umbilical endothelial cells. It was found that those consuming the Mediterranean diets had lower intracellular ROS production, cellular apoptosis, and percentage of telomere degradation. The authors suggested that the Mediterranean diet protects cells from oxidative stress, but did not explore the matter further (10). However, Boccardi et al., builds upon this finding by hypothesizing that the Mediterranean diet may boost longevity and ward off cell senescence by boosting telomerase activity.
The Boccardi et al. study investigated the impact of MD on telomerase activity in elderly subjects. The study consisted of 217 people, 115 men and 102 women, with an average age of 78 years. The subjects varied in their diet, some had a high adherence to the Mediterranean diet (vegetables, legumes, fruits, cereal, fish and ratio of monounsaturated fats to saturated fats, little to no alcohol), while others did not (diet heavy in meat, dairy, high alcohol consumption). Their white blood cells were collected and analyzed by telomeric Southern blot. It was found that those with the highest adherence to MD had the longest telomeres. Telomere length was still longer for those on MD even after adjusting for age, and gender, suggesting that the diet plays a beneficial role in the aging process. Telomerase activity was also highest for those with the greatest adherence to MD, even after controlling for age and gender. The findings of this study suggest that the foods found in MD may protect the cells from oxidative stress, and other causes of cell senescence. It also shows that a poor diet can be detrimental to ones health, as those who ate “poorly” were found to have the lowest telomeres activity and shortest telomere length. A good future study would be to examine the potential genes that may be affected by oxidative stress, and telomerase, and how foods found in MD may alter their expression.
Social Environment Can Have An Effect on Telomeres
Social stress is another environmental factor that can have an effect on telomerase expression and telomere length. In a recent 2014 study by Aydinonat et al., researchers examined the telomere length of captive African grey parrots. The telomere length of single parrots was compared to parrots living with a companion to investigate the effects of social isolation on telomere length. Parrots are social animals by nature; they form lifelong partnerships, and are rarely isolated in nature. However, when held in socially isolated environments in captivity, they exhibit aggressive behavior, self-mutilate, and suffer recurring illness1. Humans under psychological stress exhibit similar behavior. People with high amounts of stress may be more susceptible to certain diseases due to oxidative damage. The authors cite previous studies that suggest constant exposure to stressful conditions may impede upon the body’s anti-oxidative defense mechanism, notably in the telomeres. As mentioned prior, some studies have shown that telomerase activity is affected in human cells exposed to oxidative stress. These studies found that telomerase can be diminished, leading to senescence, or enhanced, leading to cancer. Therefore, studying the effects of psychological stress on the telomeres of the African grey parrot may help to better understand its implications in human health. The authors hypothesized that due to the psychological stress imposed upon parrots living in isolation, their telomere length will be shorter compared to parrots living with a companion.
The study was conducted from 2011 to 2013, and involved 45 captive African grey parrots. There were 21 females and 24 males, and their ages ranged from 0.75-45 years. DNA samples were taken from the parrots, and their relative telomere length measured using quantitative PCR methods. It was found that telomere length decreases with age regardless of the housing situation, but the parrots housed alone had shorter telomeres than parrots of the same age that were housed with a companion. There was also no measured correlation between sex of the parrot, and telomere length.
The researchers wanted to not just compare telomere length between isolated and partnered parrots, but they sought to investigate whether social isolation may accelerate telomere degradation. Using statistical analysis (general additive model) of the relative telomere length data, it was found that telomeres shortened at a faster rate for isolated parrots. For example, the average relative telomere length for an isolated parrot at age 10 was about 0.9, and declined to an average of 0.3 for isolated parrots at age 35. However, average relative telomere length for partnered parrots at age 10 was 1.3, and only declined to an average relative telomere length of 1.0. Therefore, not only was telomere length shorter for isolated parrots, telomere degradation occurred at a faster rate.
This study was novel because it was the first to investigate the effects of social stress on telomere length. Social stress was found to drastically reduce the length of telomeres in isolated parrots, which may explain their heightened susceptibility to illness. The findings of this study greatly support the notion that stress can negatively impact ones health, and may do so at the DNA level. The effects of social stress on human health, especially its role in telomerase activity and telomere maintenance, should therefore be explored further. Further research into the implications of stress, aging, and disease progression in the context of telomerase activity may be key to developing effective treatment. For future studies, it is suggested that the authors perform microarray analysis on the DNA of isolated and partnered parrots. This will provide information regarding the genes affected by social stress, including which genes may be silenced or overexpressed. This information will allow us to better understand the genes affected by psychological stress, and how they affect telomerase activity and telomere length.
Paternal Genetic Factors May Influence Telomerase and Telomere Length In Offspring
Parents that have children at an older age may be more likely to pass on a genetic mutation than if they were younger. Autism, and schizophrenia are two disorders that may be caused by genetic mutation passed down from an elderly parent to their offspring (4). However, there may be some genetic benefit to having an older parent, especially in the case of older fathers. Somatic cells have little to no telomerase activity, however the telomerase activity in the germ cells is very high, specifically in sperm cells. The telomeres, as a result, have been shown to increase in length with age, which is the exact opposite of what somatic cells are able to do. This observation explains why men are capable of producing offspring for their entire life, and doing so in older age may be of benefit to their children. By having children at an older age, the father is passing on sperm cells with longer telomeres for his offspring to inherit. New data also suggests that his grandchildren will also inherit the longer telomere length.
To investigate the association between the age of the father and grandfather and telomere length in children, biological anthropologist Christopher Kuzawa and anthropological geneticists Geoff Hayes and Dan Eisenberg performed a long-term study of 3327 Philippine women that became pregnant in 1983. Their hypothesis was that grandfathers that were older when they had children would have grandchildren with longer telomeres. After collecting blood samples from mothers and their children, telomere length was analyzed. It was found that the telomeres of children with older fathers were longer compared to children of younger fathers. Statistical analysis found that with every additional ten years of age of the father, the children had a 4% increase in telomere length. This same increase was also observed for every ten years added to the age of the grandfather. However, the researchers noted that the inheritance of longer telomeres was only passed to their son’s children, and not their daughter’s children, indicating that this phenomenon is paternal. The researchers also noted that the increase in telomere length for every year older the father is at conception is roughly equivalent to the telomere length lost during the normal aging process (4).
The findings of this study suggest that older fathers pass on an evolutionary advantage to their children, granting them higher fitness with cells that can survive the aging process longer. However, there are drawbacks to having longer telomeres, as this is also suspected to be the cause of certain types of cancer. A suggested future study would be to survey fully grown children of elderly fathers and grandfathers to determine if they actually live longer, or are susceptible to any diseases compared to children of younger fathers and grandfathers.
A Human Genetic Disorder and Telomerase Activity
Telomerase activity is associated with cell longevity and health; therefore it is logical to assume that genetic mutations that inhibit telomerase activity may lead to premature aging. One such disease, Dyskeratosis congenita (DC), is a premature aging disorder that primarily affects the skin, hair, and bone marrow. This disease can be inherited in autosomal recessive, X-linked, or autosomal dominant fashion, depending on the specific mutation. People with the disorder often display symptoms characteristic of the elderly, such as premature graying of the hair, susceptibility to cancer, osteoporosis, and shortened lifespan. Past studies have found that patients with DC have shortened telomeres, and reduced TERC, the RNA component of telomerase. These observations suggest that DC is a disorder of telomere maintenance (14).
In a study by Vulliamy et al., the researchers investigated the role of the NHP2 and GAR1, two key proteins of the telomerase and small nucleolar ribonucleoprotein (snoRNP) complexes. The researchers sought to determine the cause of the autosomal recessive form of this disorder, which may be caused by a mutation in these two proteins. To investigate, human cells were cultured in vitro, and using SiRNA-mediated knockdown, a knockdown mutation of NHP2 and GAR1 was introduced into the cells. The TERC levels were measured via quantitative PCR, and telomere length was measured using Southern blot analysis. It was found that TERC levels were lowered and telomeres shortened for the cells with the NHP2 knockdown, but not for the GAR1 knockdown. These findings indicate that an NHP2 mutation causes the lowered TERC levels and telomere length associated with DC, and therefore may be the main factor in the progression of the autosomal recessive form of the disease. It also indicates that GAR1 plays a different role in the telomerase complex compared to NHP2. These findings help expand upon our present body of knowledge on the proteins involved in maintaining telomerase activity. Further research should be done so that potential treatment may be developed for people with DC.
Conclusion, Current Working Model, and Future Direction
Telomerase activity plays a key role in biological aging by maintaining telomere length. When cells in the body divide, telomeres shorten, and can only shorten so much before the cell enters senescence. It is at this point that we see the aging process to occur. The shortening of telomeres is a natural biological process, but there are certain environmental and genetic factors that also influence telomerase activity and telomere length. The studies presented in this literature review stated that environmental factors such as caffeine, alcohol, diet, and psychological stress could all affect telomerase and telomere length, and therefore affect the aging process. Studies have also shown that genetics plays a role in telomere length as well, such as the age of ones parents, or potential mutations that can affect telomerase activity.
The findings of all of these studies are significant because it helps shape our understanding of the aging process. Through our understanding of telomerase, including what influences its activity, we can develop treatment for age-related diseases, or just have the knowledge of what helps and harms our body.
Questions that still remain in the field include, do people with longer telomeres live longer? Or are these people more likely to die from cancer? Do people who live to be over 100 years have enhanced telomerase activity, or are there other biological mechanisms involved?
For future experiments, I would like to see further genetic analysis, such as DNA microarray testing performed on the parrots in the study described above. It would be interesting to determine the genetic cause for the shortening of telomeres seen in parrots under psychological stress. From this information we can investigate how certain genes alter telomerase activity.
References
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This study is of special interest because it was the first of its kind to investigate the limits to cellular division. Before this study was published, it was widely believed that cells were immortal, and could divide indefinitely. This study established the concept of the Hayflick Limit, as described in this review.
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a. This article is of special interest because it covers a wide range of environmental stressors, and explores their effect on telomere length in yeast. The genes that were affected in the yeast are also genes that humans have. Therefore, we can learn from this study which everyday environmental stressors, such as caffeine and alcohol consumption, affect our health.
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[*] Of Special Interest