The genetic secret to longevity

A handful of genes responsible for controlling the body's defense system in times of distress can also have a significant impact on health and prolong the lives of a variety of organisms. Understanding their mechanism of action may be the key to extending life expectancy in humans and also to eradicating old age diseases

You can learn quite a bit about the condition of a used car based on its year of manufacture and the number of kilometers it has covered. The wear and tear due to a lot of driving and the length of time that has passed, undoubtedly take their toll. A similar process probably also occurs when humans age, but since machines are fundamentally different from living creatures the comparison is flawed. Biological systems are able to respond to their environment and use their energy to protect themselves and repair themselves, so their deterioration process is not so drastic.

In the past, scientists believed that aging is not only a deterioration but also an active continuation of the organism's development according to its genetic program. When the individual reaches adulthood, "aging genes" begin to point him towards the grave. This idea has been dismissed, and the popular explanation today is that aging is nothing but wear and tear resulting from the science of the body's maintenance and repair systems. According to the prevailing logic, natural selection has no reason to maintain such systems after any individual has passed reproductive age.

But we and other researchers have discovered that a family of genes related to an individual's ability to survive stressful situations, such as extreme heat or lack of food or water, can preserve natural defense and repair mechanisms regardless of age. These genes improve the body's ability to survive and therefore improve the individual's chances during a crisis. And if the activity of these genes continues over time, they can also greatly improve the health of the organism and prolong its life. In fact, these genes work the opposite of the aging genes, that is, they are life-prolonging genes.

We started researching the topic about 15 years ago when we hypothesized that evolution would favor a universal control mechanism to coordinate the familiar response to stress situations. If we can identify the gene or genes that act as super-controllers of this mechanism, and therefore act as super-regulators of lifespan, we can use these natural defense mechanisms as weapons against the diseases and deterioration that are now synonymous with aging.

Many genes that have been discovered recently, bearing obscure names such as daf-2, pit-1, amp-1, clk-1 and p66Shc, affect the resistance to stressful situations and the life span of laboratory animals. This suggests that they are part of a basic mechanism that enables survival in times of distress. But both of our labs focused on a gene called SIR2, which is found in one form or another in all organisms studied so far, from yeast to humans. Adding copies of the gene extends life span in various organisms such as yeast, roundworms and fruit flies, and we are now investigating whether the same happens in larger animals, mice for example.

The SIR2 gene is one of the first life-prolonging genes discovered and one of the most characterized, so we will focus on it in this article. Its mechanism of action demonstrates how a genetically controlled survival mechanism can prolong life and improve health, and there is ample evidence that SIR2 is the central regulator of this mechanism.

Silence is golden

We discovered for the first time that SIR2 is a life-extending gene because we asked what causes yeast cells to age and whether a single gene might control aging in this simple organism. Many thought that investigating life span in yeast would teach us nothing and a half about aging in humans. Aging in yeast is measured by the number of divisions a mother cell goes through before it dies. The typical lifespan of a yeast cell is about 20 divisions.

One of us (Garnett) began scanning yeast colonies for cells with particularly long lifespans, hoping to locate genes that might be responsible for longevity. This scan yielded the discovery of a single mutation in a gene called SIR4, which encodes a protein that is in a complex with the Sir2 enzyme. The mutation in SIR4 caused the Sir2 protein to cluster in a specific region of the yeast genome that contains repetitive sequences of ribosomal DNA (rDNA). The genes in this region contain the code for the production of the ribosome proteins, which is the cellular workshop for protein production. On average, there are more than 100 rDNA repeats in the yeast genome, and the cell has trouble keeping them in a stable state. Repeated sequences tend to alternate with each other, and when the process occurs in humans it can lead to a variety of diseases, such as cancer or Huntington's disease. Our findings in yeast suggested that the aging process in the mother cell is caused by some instability in the rDNA region, and that Sir proteins help increase stability.

In fact, we discovered a surprising type of instability in rDNA. After several divisions, the mother cells produce additional copies of rDNA in the form of rings that break out of the genome. These rDNA rings, which are outside the chromosomes are called ERC (extrachromosomal rDNA circles) and are replicated side by side with the chromosomes before cell division, but remain in the nucleus of the mother cell. That is, the mother cell accumulates an increasing number of rings that eventually lead it to its death. Perhaps the reason is that the replication of ERC molecules requires so many resources that the mother cell is no longer able to replicate its own genome.

In contrast, when a copy of the SIR2 gene is added to yeast cells, the rDNA rings are not formed and the cell life is extended by 30%. This finding can indeed explain how SIR2 acts as a life-extending gene in yeast, but to our surprise we soon discovered that additional copies of SIR2 also extend the life of the roundworm by 50%. We were surprised not only by this similarity between creatures so far apart from an evolutionary point of view, but also by the fact that the adult worm's body contains only non-dividing cells. In other words, the aging mechanism that operates in yeast, which depends on DNA replication, is not suitable for worms. We therefore wanted to understand what exactly the SIR2 gene does.

As we discovered, the gene encodes an enzyme whose activity was not known until then. Inside the cell the DNA is wrapped around a clutch of packaging proteins called histones. These proteins carry chemical tags, such as acetyl groups, which determine how tightly the DNA is packed. When acetyl groups are removed from the histones, the packing is tighter and the DNA is not accessible to the enzymes responsible for separating the rDNA rings from the chromosome. DNA in such a tight state is called dead DNA, because genes found in such areas are not accessible and therefore are not activated.

At that time it was already known that Sir proteins are involved in gene silencing, and indeed the acronym SIR means silent information regulator. Sir2 is one of several enzymes that remove the acetyl tags from histones, but as we discovered, it is unique because its enzymatic activity is completely dependent on the existence of the small and common molecule NAD. NAD molecules have long been recognized as necessary in many metabolic reactions in cells. The relationship between Sir2 and NAD is exciting because it links the activity of Sir2 to metabolism and thus, perhaps, also links it to the dependence found between a low-calorie diet and the slowing of aging.

The calorie connection

The most well-known treatment for extending life span is to limit an animal's caloric intake. This was discovered more than 70 years ago, and it is still the only treatment that has proven its effectiveness. Food consumption is usually reduced by 40-30% relative to the normal consumption of the same breed. Animals such as mice, rats, dogs and maybe even great monkeys (primates) who maintain such a diet, not only live longer but are also healthier throughout their lives. This delays the development of most diseases, including cancer, diabetes and even degenerative diseases of the nervous system. It seems as if the organism is alert and ready for survival. The only visible price is the loss of fertility in some creatures.

For many decades, scientists have strived to understand the mechanisms underlying the calorie restriction phenomenon and to develop drugs that would provide similar health benefits (see: Seriously Seeking the Cure for Aging, Mark A. Lane, Donald K. Ingren, and George S. Roth, Scientific American Israel, Vol. 3, February-March 2003). For a long time, the phenomenon was attributed to a slowdown in the rate of metabolism, that is, to a slowdown in the process of creating energy in the cell from molecules of raw materials. This slowdown causes a decrease in the amount of toxic byproducts of the process in response to a reduction in the amount of food.

But this view seems to be incorrect. Limiting calorie intake does not slow down the rate of metabolism in mammals, yeast and roundworms. In fact, the metabolism is accelerated and even changed. Therefore, we believe that caloric restriction is a state of biological stress, like a lack of food in nature, which causes the body to defend itself to increase its chances of survival. In mammals, restricting calorie intake causes changes in the mechanisms of defense, repair and energy production in the cells, and also changes in the activation of a programmed cell death mechanism, known as apoptosis. We wanted to know what role Sir2 plays in these changes, so we first examined its role during caloric restriction in simple organisms.

In yeast, we found that food restriction affects two pathways that increase the enzymatic activity of Sir2 in cells. In one pathway, calorie restriction activates a gene known as PNC1, which produces an enzyme that removes the substance nicotinamide from the cells. This substance, whose small molecule is similar to vitamin B3, normally inhibits the activity of Sir2. PNC1 is also activated under other mild stress conditions that extend the life of the yeast, such as raising the temperature or excess salt. This corresponds to the theory that calorie restriction is a stressful situation that activates a survival mechanism.

The second pathway activated in yeast in response to calorie restriction is respiration, an energy-producing process in which NAD accumulates as a byproduct simultaneously with a decrease in the amount of its chemical partner, NADH. It turns out that not only does NAD activate Sir2, but that NADH inhibits the enzyme, in such a way that changing the ratio between NAD and NADH in the cells greatly affects the activity of Sir2.

After we understood how a life-extending stress condition increases the activity of Sir2, the question was asked, is Sir2 necessary for life extension? The answer is probably a resounding "yes". One way to test whether Sir2 is essential for this process is to remove the gene and see if caloric restriction still extends life. It was found that in complex organisms such as fruit flies, SIR2 is indeed essential for life extension due to calorie restriction. And since the body of the adult fly contains many tissues that correspond to different organs in mammals, we believe that SIR2 would also be essential in mammals for calorie restriction to have an effect.

However, extreme dieting is not a reasonable option for people who want to enjoy the health benefits of calorie restriction. It will therefore be necessary to find drugs that can affect the activity of Sir2 and its like (collectively called Sirtuins). Such compounds, capable of activating sirtuins are called STACs, and one such, resveratrol, seems particularly interesting. Resveratrol is a small molecule found in red wine and produced by a variety of plants during stressful situations. At least 18 other compounds produced in plants in response to stress conditions affect sirtuins, suggesting that plants use such molecules to control the activity of their own Sir2 enzymes.

If you give resveratrol to yeast, worms or flies, or if you limit their calorie intake, their lifespan is extended by about 30%, but only in the presence of the SIR2 gene. Furthermore, a fly that overproduces Sir2 lives longer than a normal fly, and its life cannot be extended with resveratrol or caloric restriction. The simplest explanation is that caloric restriction and resveratrol - both extend the life of the fly by activating Sir2.

Flies fed resveratrol live longer even if they eat ad libitum, and they also do not suffer from the decline in fertility often caused by calorie restriction. This is good news for those of us hoping to treat human disease with molecules that affect Sir2 enzymes. But first we need to better understand the role of Sir2 in mammals.

the conductor of the orchestra

The mammalian version of the SIR2 gene in yeast is called SIRT1 (SIR1 homolog number 2). It codes for the production of the protein, Sirt1, whose enzymatic activity is the same as Sir2 but it removes acetyl groups from a wider variety of proteins, both in the cell nucleus and in the cytoplasm. Some of the proteins that Sirt1 acts on have been identified and found to control vital processes such as apoptosis, cell defense and metabolism. It seems, then, that the ability to extend life of the genes from the SIR2 family has been preserved in mammals. But not surprisingly for larger and more complex creatures, the pathways involving sirtuins are also more complex.

For example, increasing Sirt1 activity in mice and rats allows some of the animals' cells to survive stressful situations that under normal circumstances would cause programmed cell suicide. Sirt1 does this by regulating the activity of several other key cellular proteins, such as p53, FoxO and Ku70, which are involved in setting the threshold for apoptosis or in controlling repair mechanisms in the cell. That is, Sirt1 increases the repair mechanisms in the cell and also extends the life of the cell to allow them to function.

During life, cell loss due to apoptosis may be an important component of the aging process, especially in non-regenerating tissues such as the heart and brain. Therefore, slowing cell death processes may be one way in which sirtuins improve health and extend life. An impressive example of Sirt1's ability to promote survival in mammalian cells can be seen in the Wallerian mutant mouse strain. In these mice, a single gene was duplicated, and this mutation makes their nerve cells particularly resistant to stressful situations. This condition protects the mice from stroke, from toxicity due to chemical treatment and from neurodegenerative diseases of the nervous system.

In 2004, Jeffrey D. Milbrandt of Washington University in St. Louis and his research colleagues showed that the mutation in Wallerian mice increases the activity of the enzyme that produces NAD, and that excess NAD appears to protect neurons by activating Sirt1. Furthermore, Millbrandt's group discovered that STAC compounds such as resveratrol protected neurons of normal mice in a manner similar to the Wallerian mutation.

A more recent study conducted by Christian Neri of the French National Institute of Health and Medical Research showed that resveratrol and another STAC compound, fisetin, prevented the death of nerve cells due to Huntington's disease in two different laboratory animals (worms and mice). In both cases, sirtuin activity was required for the STAC compounds to have an effect.

The nature of the protective effect of sirtuins in individual cells is becoming clearer. But if indeed these genes are responsible for the benefits of calorie restriction, the question arises as to how nutrition can regulate their activity and therefore the rate of aging throughout the body. In a recent study by Per Pwigserber from the Johns Hopkins University School of Medicine and colleagues, the researchers saw that NAD levels increase in liver cells under fasting conditions, which causes an increase in Sirt1 activity. Among the proteins that Sirt1 affects is a protein that controls transcription, PGC-1α which, in turn, causes changes in the metabolism (metabolism) of glucose in the cell. That is, the study showed that Sirt1 serves as both a sensor of nutrient availability and a controller of the liver's response.

Similar results led to the belief that Sirt1 is a central metabolic controller in the liver, muscle and fat cells, because it senses the fluctuations in the nutritional level through changes in the ratio between NAD and NADH in the cells and in turn causes drastic changes in the pattern of gene expression in these tissues. This reasoning could explain how Sirt1 links many of the genes and pathways that influence longevity and are described on page 54.

However, it should be remembered that Sirt1 may affect the body's activity through several mechanisms. Another interesting hypothesis is that mammals sense the level of food availability through the amount of energy they have stored in the body in the form of fat. Fat cells secrete hormones that signal to other tissues in the body, but the content of the signals depends on the level of fat stores. It is possible that by reducing fat stores, calorie restriction causes the formation of a hormonal signal pattern that transmits "deficiency", a condition that activates the cell's defense systems. This hypothesis is supported by the fact that mice genetically engineered to remain extremely thin, regardless of the amount of food they eat, tend to live longer.

This possibility led us to ask whether Sirt1 also regulates fat stores in response to diet. Indeed, the activity of Sirt1 in fat cells increases due to food restriction, which causes the fat stores to exit the cells into the bloodstream to be converted into energy in other tissues. We hypothesize that Sirt1 senses the level of nutrition, then dictates the level of fat stores and thus the hormonal pattern produced by fat cells. This effect on adipose tissue and the signals it produces will, in turn, determine the rate of aging in the entire body and therefore place Sirt1 in a key position as a longevity controller induced by caloric restriction in mammals. This will also link aging and metabolic diseases, such as adult diabetes, associated with excess fat. Drugs that affect the Sirt1 pathway in fat cells may, therefore, delay not only aging but also the onset of various diseases.

Another important process affected by Sirt1 is inflammation, which is involved in several diseases such as cancer, arthritis, asthma, heart disease and neurodegenerative diseases. A recent study by Martin W. Mew and his colleagues at the University of Virginia showed that Sirt1 inhibits NF-κB, a protein coupling that triggers the inflammatory response. Resveratrol, the compound that activates Sirt1, has a similar effect. This finding is particularly encouraging because in the field of drug development there is a keen search for a molecule that will inhibit NF-κB, and because another known effect of calorie restriction is the suppression of hyper-inflammation.

If SIR2 is indeed the master controller of the aging regulatory system activated by stress conditions, it may be working as a kind of conductor in an orchestra that includes a hormonal communication network, intracellular control proteins and other longevity-related genes. One of the interesting discoveries in recent years is that Sirt1 regulates insulin production and insulin-like growth factor 1 (IGF-1) production, and that these two powerful signaling agents in turn regulate Sirt1 production as part of a complex feedback mechanism. The relationship between Sirt1, IGF-1 and insulin is interesting because it explains how Sirt1 activity in one tissue can affect other cells in the body. Furthermore, it is known that the level of insulin and IGF-1 in the blood affects the life span of a variety of creatures such as worms, flies, mice and possibly even humans.

From defensive to forward

For tens of thousands of years, humanity has tried to slow down aging without success, so there will be those who find it difficult to believe that the aging process can be controlled with the help of a handful of genes. However, we know that aging in mammals can be delayed with a simple dietary change, namely calorie restriction, and we have shown that sirtuins control many molecular pathways that are affected by calorie restriction. Without actually understanding the exact, and perhaps even many, causes of aging, we were able to delay aging in a variety of animals by influencing a small number of proteins that, in turn, affected the health of the organism.

We also know that the genes belonging to the SIR2 family evolved a long time ago, because today they can be found in a variety of organisms from yeast, the Leishmania parasite and roundworms to flies and humans. Sirtuins dictate lifespan in all these creatures except humans, where this has not yet been tested. This fact alone convinces us that human sirtuins are probably the key to health and longevity in humans as well.

The research laboratories of both of us are currently conducting well-controlled studies in mice that will soon teach us whether the SIRT1 gene controls health and lifespan in mammals. But many decades will pass before we know for sure how sirtuins affect longevity in humans. Therefore, those of us who hope to swallow a bullet and live to be 130, were born a little too early. Despite this, we may get to see drugs that affect the sirtuin enzymes for the treatment of diseases such as Alzheimer's, cancer, diabetes and heart disease. In fact, several such drugs for the treatment of diabetes, herpes and neurodegenerative diseases are currently in clinical trials.

In the longer term, we anticipate that deciphering the secrets of longevity genes will allow society to move beyond the treatment of old age diseases and prevent their development in the first place. It may be difficult to imagine what life will look like when ninety-year-olds feel young and healthy relative to their age, and some may wonder if it is even worthwhile to extend the human lifespan. But at the beginning of the 20th century life expectancy was around 45 years. It has risen for about 75 years thanks to the discovery of antibiotics and the improvement of public health that allow people to recover from infectious diseases or avoid them altogether. Society has adapted to this dramatic change in life expectancy, and there aren't many people who would want to go back to life without an introduction. There is no doubt that future generations who will be accustomed to living beyond 100 years will look upon our current attempts to improve health as relics of a bygone era.

Overview/Delay the End

Genes that control the organism's ability to survive in times of distress cause changes in the body that make it, for a time, particularly vulnerable to survival. This response to stressful situations that is activated over time extends life span and inhibits the development of diseases in a variety of creatures.

Sirtuins are a family of genes that are candidates for being master controllers of this survival mechanism.

If we understand how they cause health and longevity, we can develop cures for disease and ultimately live longer and healthier lives.

SIR2 and stress states in yeast

Moderate stress conditions extend the life of the yeast by about 30% by increasing the activity of the Sir2 enzyme. Stressors can increase Sir2 activity through two separate pathways (discussed below), which ultimately result in inhibitory silencing of Sir2. Therefore, an excess of activated Sir2 proteins eliminates genomic instability that would otherwise cause the death of the yeast cell after about 20 cell divisions.

Food shortages and other stress conditions, such as low nitrogen levels or excess salt or heat, activate the PNC1 gene in yeast. The gene encodes a cellular protein that reduces the level of nicotinamide, which is a substance that inhibits Sir2.
Calorie restriction also causes the mitochondria in the cell to switch from a state of fermentation to an energy-producing state known as respiration. In respiration, NADH is converted to NAD. NADH is an inhibitor of Sir2, while NAD activates the enzyme.

Sir2 removes acetyl tags from the histone proteins that pack the DNA, thereby causing tighter packing. The enzyme removes the acetyl groups from a certain region of DNA that tends to produce rings of excess genetic material that detach from the genome when the cell replicates its DNA in preparation for cell division.

Yeast cells reproduce by division. Each cell is divided into a mother cell and a daughter cell. After several divisions, the mother cell begins to accumulate excess DNA rings. After about 20 divisions, the cell is damaged by the accumulation of DNA and dies.

Increased Sir2 activity protects yeast cells from forming excess rings by causing the vulnerable region of the genome to pack more tightly. This allows the yeast to stay young and keep dividing longer.

Genetic pathways that extend life span

Scientists who study longevity have identified a variety of genes that can affect lifespan in different creatures. Like SIR2 and its genetic relatives (the sirtuins), some of the genes identified extend life when the number of copies of the gene increases, or when the activity of the protein encoded by the gene increases. However, many genes have a negative effect on life expectancy, so a decrease in their activity causes longevity.

In worms, for example, the gene that encodes cellular receptors for insulin and insulin-like growth factor 1 (IGF-1) is called daf-2. Suppressing the activity of the daf-2 gene in adult worms interferes with the activity of insulin and IGF-1 and extends the life of the organism up to 100%. Suppression of other genes related to growth, or interference with the molecular pathways they activate - also encourages longevity.

Some of the genes listed below, or their proteins, control the sirtuins or are controlled by them due to calorie restriction. That is, it is possible that they are part of a super control network that controls the aging process. The authors hypothesize that SIR2 and similar proteins orchestrate this network.

sirtuins in the cell

The Sirt1 enzyme is the most characterized sirtuin, but it is not the only one found in mammals. Genes corresponding to SIRT1 produce similar enzymes that act at a variety of sites in the cell. Sirt1 acts in both the nucleus and the cytoplasm by removing acetyl groups from other proteins, thereby altering their activity. Many of its target proteins are transcription factors that directly activate genes or activate proteins that control transcription factors. Sirt1 controls in this way a wide variety of essential cellular activities.

Scientists are just beginning to understand the roles played by other sirtuins and to determine whether they also affect longevity. For example, Sirt2 is known to affect tubulin, a component of the cytoskeleton, and may affect cell division. Sirt3 is active in the mitochondria, the organelles in the cell responsible for energy production, and appears to participate in body temperature regulation. The role of Sirt4 and Sirt5 is unknown. Mutations in the gene encoding Sirt6 are associated with premature aging.

Some target proteins of SIRT1

FoxO1, FoxO3 and FoxO4: transcription factors for genes involved in cellular defense mechanisms and glucose metabolism.
Histones H3, H4 and H1: package the DNA in the chromosomes.
Ku70: a transcription factor that accelerates DNA repair and cell survival.
MyoD: a transcription factor that promotes muscle development and tissue repair.
NCoR: a control protein that affects many genes, including those involved in fat metabolism, inflammation and the function of other control proteins such as PGC-1α.
NF-κB: a transcription factor that controls inflammation, and cell survival and growth.
P300: a control protein that adds acetyl groups to histones.
P53: a transcription factor that causes programmed cell suicide in damaged cells.
PGC-1α: a control protein of cellular respiration, and probably plays a role in muscle development.

About the authors

David A. Sinclair (Sinclair) and Lenny Garnett (Guarente) began working together to identify genes that control longevity and their mechanism of action in 1995, when Sinclair began his postdoctoral training in Garnett's laboratory at the Massachusetts Institute of Technology (MIT). Sinclair currently directs the Paul P. Glenn Laboratories for the Study of the Biological Mechanisms of Aging at Harvard Medical School, and is a researcher at the Broad Institute in Cambridge, Massachusetts. Garnett, a Novartis Endowed Professor of Biology, has been an MIT faculty member for 25 years. His laboratory was the first to discover that the SIR2 gene controls lifespan in yeast and that the enzyme it encodes is responsible for the beneficial effect on calorie restriction in this organism. Currently the two authors are studying SIRT1, the gene corresponding to SIR2 in mammals. The company founded by Sinclair, Sirtis, and the one founded by Garnet, Elixir, are developing molecules that activate sirtuins for medicinal use.

And more on the subject

Ageless Quest: One Scientist's
Search for Genes That Prolong
Youth. Lenny Guarente. Cold
Spring Harbor Laboratory Press,
2002.

The Secrets of Aging. Sophie
L. Rovner in Chemical &
Engineering News, Vol. 82,
no. 34, pages 30–35;
August 23, 2004. http://pubs.acs.org/cen/
coverstory/8234/8234aging.html

Calorie Restriction, Sirt1 and Metabolism: Understanding Longevity. Laura Bordone and Leonard Guarente in Nature Reviews Molecular and Cell Biology, Vol. 6, pages 298–305; April 2005.

Toward a Unified Theory of Caloric Restriction and Longevity Regulation. David A. Sinclair in Mechanisms of Aging and Development, Vol. 126, no. 9, pages 987–1002; September 2005.