Telomerase can reverse the aging process... sort of

Biologists are, at long last, beginning to understand the molecular processes responsible for aging in complex (multicellular) organisms – and to investigate ways to counteract these processes. We discussed one line of research in this recent article about a particular sirtuin (SIRT3) that helps relieve oxidative stress that can lead to DNA damage, which generally leads, in turn, to cell senescence or death.

While oxidative stress is certainly a significant factor in aging, possibly the most significant, there are others. One of these is the limitation on a cell's ability to undergo cell division in order to produce new cells of the same type. This is especially important in tissues that regularly need to regenerate, such as skin and intestinal tissue. Everyone now knows about telomeres, whose main function is to constitute protective end caps on chromosomes. The limitation on number of cell divisions happens since about 100 base pairs are lost from telomeres during each cell division. When telomeres eventually become too short signals that are similar to those associated with other kinds of DNA damage shut down a cell's ability to divide further. This mechanism indirectly helps mitigate the risks of DNA damage that are present every time a cell divides – an inherently tricky process.

However, this limitation on cell division isn't acceptable during embryonic development, when an organism's cell count is doubling most rapidly. So evolution has provided an enzyme – telomerase – that can rebuild telomeres, but is most active only during embryonic development. Except, of course, in cells that have become cancerous, where the ability to divide without limit is the name of the game. We discussed telomeres and telomerase in some detail a little over a year ago in this article, so you can go there for more.

Because of the risk of cancer, it seems imprudent to reactivate telomerase for the long term within an organism, especially in long-lived animals such as humans. (In animals like mice, which live fast and die young, it's a different matter. Telomerase may remain somewhat active in mice during adulthood. (Mentioned here.)) But what if it were possible to reactivate telomerase for a relatively short period of time (compared to the whole lifespan)... might that provide an opportunity to rebuild telomeres to some extent? Even better, might that reverse, at least to some extent, the ravages of aging?

We now have some research that seems to provide a fairly unambiguous affirmative answer... in a rather special case: Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.

But didn't we just say that mice may retain telomerase activity throughout their lives? Yes, however it's a relatively simple matter to "knock out" the main telomerase gene in mice (Tert). When that's done the resulting strain of knock-out mice – after several generations – have shortened lifespans and a general phenotype of age-related debilities, as one would expect. (The first few generations apparently still have sufficiently long teleomeres.)

Unfortunately, that's not a good enough model, since without a Tert gene, the organism has no way to manufacture telomerase. Simply giving the knock-out mice repeated infusions of telomerase is not a good way to ensure uniform distribution of the enzyme to all of the organism's cells. What to do? The experimenters came up with a rather clever solution. Normally the way that telomerase is activated in cells is by means of an "estrogen receptor" (ER), to which a form of the hormone estrogen (17β-estradiol to be precise) can bind and enable transcription of Tert. This ER can be tweaked so that estrogen binds to it only in the presence of another chemical, 4-hydroxytamoxifen (4-OHT).

A special form of the Tert gene that includes this special ER can be "knocked-in" to the mouse germline. It then turns out that 4-OHT can be efficaciously supplied to a TERT-ER mouse (in the form of a time-release subcutaneous pellet) to turn telomerase expression on and off at the experimenter's will. With that technology in place, the researchers were then able to perform a series of experiments demonstrating, in these special mice, that a month-long burst of telomerase could actually reverse a number of the ill effects of telomerase deprivation.

The first step was to show that without 4-OHT the TERT-ER mice (after a few generations) had many of the same problems, in the same degree, as later generations of knock-out mice that lacked Tert entirely. The TERT-ER mice (all of which were male) showed no signs of telomerase activity. Tissues in highly proliferative organs such as testes, spleen, and intestines showed notable atrophy. Lifespan of TERT-ER mice was about half that of normal ("wild type") mice.

The first test to investigate the effects of telomerase reactivation by means of 4-OHT was done in vitro. Fibroblast cells from TERT-ER mice were cultured and found to be essentially senescent and not undergoing cell cycles. But when the cells were placed in media containing 4-OHT, teleomerase was reactivated, telomeres lengthened, and cell proliferation resumed.

Some TERT-ER mice were then given a 4-week treatment of 4-OHT (subcutaneous pellets). At the end of that treatment there was a marked reversal of the degeneration that has occurred in testes, spleen, liver, and intestinal tissues, as well as resumption of sperm production. Survival time of these treated mice also increased. At the same time, 4-OHT had no effects on control mice that weren't lacking in telomerase and didn't have tissue degeneration.

Noteworthy results were obtained from tests to assess nervous system condition. Proliferation of neural progenitor cells was found to resume in TERT-ER mice treated with 4-OHT. Normal numbers of mature oligodendrocytes reappeared. Lastly, high-level neurological functions were restored, as indicated by resumption of nearly normal olfactory sensitivity.

An interesting conclusion that can be drawn from the neurological results is that neural progenitor cells probably survive loss of telomeres, so that they can rebuild neural cell populations if telomeres are repaired.

The really interesting question, of course, is the extent to which these results may apply, in some form, to humans. Unfortunately, there are a number of reasons to be skeptical. For one thing, telomere shortening is only one factor, and quite possibly not the main one, in human aging. Aging can be thought of as a complex disease, like cancer, with many contributing factors. The consequences of telomere truncation are only one factor.

Further, murine biology has signficant differences from human biology. Mice are less complex organisms, with rather short lifespans. Mice seem to retain some degree of telomerase activity throughout their lives, so they are not as well adapted to going for long periods without it.

It is noteworthy that evidence was not found that TERT-ER mice treated with 4-OHT became more susceptible to cancer. Still, mice don't live very long, and they are adapted to maintain active telomerase. Humans are different. If telomerase is artificially kept active for years in humans, incipient tumorigenicity could be accelerated.

Lastly, it's not necessarily easy to raise human telomerase activity levels in the first place. Although some telomerase-activating factors are known, they have not been tested extensively in humans for long periods of time, so their safety and efficacy profile is not known.

These research results are quite interesting – but they only indicate the need for much more investigation.

Jaskelioff, M., Muller, F., Paik, J., Thomas, E., Jiang, S., Adams, A., Sahin, E., Kost-Alimova, M., Protopopov, A., Cadiñanos, J., Horner, J., Maratos-Flier, E., & DePinho, R. (2010). Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice Nature, 469 (7328), 102-106 DOI: 10.1038/nature09603

Further reading: (* = especially recommended)

* Telomerase reverses ageing process (11/28/10)

* The Curious Case of the Backwardly Aging Mouse (11/29/10)

* Partial reversal of aging achieved in mice (11/29/10)

Harvard scientists reverse the ageing process in mice – now for humans (11/28/10)

Gene reactivation reverses aging-related brain deficits in mice (11/30/10)

Age-Reversing Drugs on the Horizon? Not So Fast (11/29/10)

Telomere Tweaks Reverse Aging in Mice (11/29/10)

Alzheimers and aging advances uncovered (11/29/10)

An enzyme leads the dance of immortality and death (11/29/10)

Scientists Find Way to Partially Reverse Aging in Mice (11/29/10)

Testing the Fountain of Youth in the lab

It's been more than 10 years since it was noticed that certain enzymes – the sirtuins – had life-extending properties in organisms like yeast, and later nematodes, fruit flies, and mice. The excitement spread to other compounds, such as resveratrol, that seemed to activate or assist sirtuins. Hopes were high that such things might offer the known longevity benefits of calorie restriction in a pill form. Ever since then the gold rush has been on to figure out how these things work – and if possible, to be the first to market with the Fountain of Youth in a bottle.

We've discussed sirtuins here a number of times before – here's a list of some of those discussions. If you need to brush up on the background, those would be good places to start.

The initial sirtuin that seemed to be most important for the longevity of yeast was SIR2. The gene for SIR2 is highly conserved in evolution – so it's probably kind of important. Homologs of SIR2 have been found in many sorts of higher organisms (nematodes, fruit flies, etc.). In mammals, including humans, there is a whole family of sirtuins, having at least 7 members, named SIRTx for x=1 to 7. ("SIRT" and "sirtuin" refer to SIR-two, where SIR was an acronym for "silent information regulator".)

SIR2 is primarily a histone deacetylase (HDAC), that is, an enzyme that removes acetyl groups from histone proteins (and often other types of proteins as well). Histones are the building block proteins that make up nucleosomes, around which DNA is spooled in chromosomes. Normally, DNA is tightly bound to the histones, which prevents the genes in the tightly bound portion of DNA from being transcribed into RNA in order to make proteins. In other words, the genes bound to a histone are effectively silenced. In order for a gene to be expressed, the histone closest to the portion of DNA containing the gene has to have an acetyl group attached at an appropriate location. Enzymes ("acetyltransferases") attach acetyl groups (in the process called acetylation) to histones in order to allow gene expression. Consequently, deacetylase enzymes, such as several sirtuins, are able to silence genes by removing acetyl groups from histones.

SIRT1 is the most intensively studied mammalian sirtuin. Like SIR2, it is primarily a histone deacetylase that is active in a cell nucleus to silence a wide variety of genes. Since SIRT1 can silence a large number of genes, it affects many cellular processes. However, there is one additional complication. SIR2 and SIRT1 only have their deacetylation ability in the presence of a small molecule called NAD: nicotinamide adenine dinucleotide, and only when NAD has a net positive charge, due to the loss of one electron during the process of metabolism in which cells generate needed energy. NAD⁺ denotes this form of NAD. (The neutral form of NAD is denoted by NADH.) Because of the role of NAD⁺, SIR2 is said to be a "NAD⁺-dependent" histone deacetylase.

All this is important, because research over the past 10+ years has shown that the lifespan-extending properties of calorie restriction, especially in simple organisms like yeast and nematodes, seem to be related, at least sometimes, with the deacetylation properties of SIR2 in the presence of NAD⁺. When an organism is in a calorie restricted environment, metabolism slows down, and less NAD⁺ gets used up. As a result, there is more NAD⁺ around. So SIR2 is more effective. So genes are silenced that would otherwise be expressed. Silencing these genes seem to help an organism live longer when nourishment is not ample – so that it can survive until the buffet table is restocked.

In an organism on a normal (not calorie restricted) diet, up-regulating SIR2 or otherwise enhancing its gene-silencing abilities seems to compensate for decreased amounts of NAD⁺, and thereby achieves for the organism some of the anti-aging benefits of a calorie-restricted diet without having to go hungry.

The problem is that the expression of so many different genes can be affected by SIR2 deacetylation that it's difficult to identify which genes among these are actually useful for promoting longevity or retarding aging – especially in organisms more complex than yeast or nematodes.

Now, however, research has come out involving a much less studied mammalian sirtuin, SIRT3 – Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction. (I recommend viewing this link, since the illustration on the page will be helpful in understanding what follows here.) In spite of caveats I'll mention toward the end, this is a very significant and well-done piece of research.

A number of properties of SIRT3 had already been observed prior to this latest research. It is, like SIRT1, also a NAD⁺-dependent deacetylase enzyme. But unlike SIRT1, its main activity is found in cell mitochondria instead of in the nucleus. Consequently, SIRT3 deacetylates mitochondrial proteins instead of histones.

Of particular interest, this SIRT3 activity was known to be associated with calorie restriction (CR), because of overexpression in CR conditions and presumably also because of the NAD⁺-dependence. For example, studies in mice have shown that CR increases SIRT3 expression in liver mitochondria. Further, in knockout mice without SIRT3 mitochondrial fatty acid oxidation problems are found. Under CR SIRT3 is also overexpressed in mouse heart cells and may protect these cells from oxidative stress-induced cell death. (However, in this case it's possible that the effect resulted from HDAC activity in the cell nucleus.) So SIRT3 seems to be associated with anti-oxidant activity. There is, additionally, mechanistic evidence that SIRT3 inhibits mitochondria-related carcinogenesis. For instance, knockout mice without SIRT3 are susceptible to breast tumors.

The latest research presents strong evidence that under calorie restriction SIRT3 is involved in suppressing oxidative damage. The evidence is based on studies of oxidative stress-induced cochlear cell death responsible for age-related hearing loss (AHL) in mice. AHL is a pretty typical example of health problems associated with aging – one that affects humans as well as mice. The research not only shows an association between SIRT3 and protection from oxidative damage, but goes deep into the apparent mechanism involved. A variety of different in vitro and in vivo experiments with knockout mice provide the evidence.

To begin with, at the highest level, the researchers found that SIRT3 is required along with CR to inhibit age-related cochlear cell death and hearing loss. The knockout mice used in this, and other in vivo experiments, had both copies of the SIRT3 gene knocked out. The rate of progression of AHL was first measured in wild type (WT) mice as controls. CR was found to delay or mitigate AHL in the controls – but not in the knockout mice. This implies SIRT3 is necessary for CR to inhibit the progression of AHL – there's no benefit of CR for this condition without SIRT3. Further, when the cochlear cells of the experimental mice were examined, it was found that CR retarded cell death in the control animals but not in the mice without SIRT3.

So the key process to be concerned with is progressive cell death related to aging. The next experiments showed that the cell death was the result of oxidative damage. A lot of other studies have shown that CR inhibits oxidative damage to DNA, proteins, and lipids in many types of mammalian tissues. In the present research this was confirmed by examination of DNA in cochlear, brain, and liver tissues of control mice. But CR did not inhibit oxidative damage in the same tissues of the knockout mice. So SIRT3 appears to be necessary for the inhibition of oxidative damage to DNA, which presumably was responsible for accelerated cell death.

The next issue needing to be addressed is the mechanism by which CR inhibits oxidative damage. It is known that a small molecule, glutathione, is the major small molecule antioxidant in cells. Glutathione can exist in two oxidation states – reduced (GSH) or oxidized (GSSG). A high ratio of GSH to GSSG protects other molecules in the cell from oxidative damage, and GSH predominates in the healthy mitochondria of young mice. Conversely, a low ratio of GSH to GSSG is a marker for oxidative stress and/or aging. In the present research, the GSH:GSSG ratio was tested in control and knockout mice under CR conditions, at the age of 5 months. In the mitochondria of inner ear cells, as well as in brain and liver cells, it was found that the GSH:GSSG ratio increased as a result of CR in control mice, but not in knockout mice. Once again the presence of SIRT3 was shown to be necessary for an effect.

Obviously, the next thing to look at is how the GSH:GSSG ratio is controlled. The enzyme glutathione reductase (GSR) is known to be responsible for converting GSSG to GSH. So what happens is that reactive oxygen species (ROS) get soaked up in converting GSH to GSSG, and GSR reverses this to convert GSSG back to GSH.

However, in order to work GSR requires another molecule, nicotinamide adenine dinucleotide phosphate (NADPH) to do its job. NADPH is nothing but NAD, which we encountered in connection with the HDAC function of SIRT1, with a phosphate group attached. Like NAD, NADPH also exists in an oxidized form, NADPH⁺. This latter molecule predominates in mitochondria, and needs to be converted back to NADPH for use by GSR. (All this activity is really just shuffling electrons from one place to another. The pairs of molecules that mediate the activity are called "redox couples".)

So, what is it that converts NADPH⁺ to the plain old NADPH that we need? Well, that task is handled by yet another mitochondrial enzyme, isocitrate dehydrogenase 2 (Idh2). Don't despair – this is the last step! There is just one wrinkle. Idh2 is normally found in an acetylated form, in which case it is inactive. It needs to be deacetylated in order to become active and convert NADPH⁺ to NADPH. And that is precisely where the deacetylation function of SIRT3 comes into play. The researchers hypothesized that SIRT3 was needed in order to activate Idh2.

In order to test the hypothesis, they first measured acetylation of Idh2 in the control mice, with both normal and CR diets. With a normal diet, acetylation of Idh2 was substantial, but with CR there was an 8-fold decrease of acetylation. So it only remains to find the reason for that. In knockout mice, with no SIRT3, acetylation of Idh2 was "robust" with both normal and CR diets. That's a pretty good indication that SIRT3 was required for the effect. As a further indication, SIRT3 levels in the control mice were 3 times as high with a CR diet compared to a normal diet.

So SIRT3 is necessary for deacetylation of Idh2 under CR conditions, but there's still the possibility that it isn't sufficient by itself. It's possible that CR has other effects that facilitate deacetylation – CR may cause expression or activation of other enzymes that are needed. It's also possible that CR has other effects that increase NADPH independently of Idh2.

What if NADPH levels were tested directly? It was found that in the control mice NADPH did increase in all tissue types tested when a CR diet replaced a normal one, but this effect was not found in the knockout mice.

Efforts were made to use biochemical experiments (in vitro) to determine whether SIRT3 alone is responsible for deacetylating Idh2 under CR conditions. For example, another sirtuin, SIRT5, is also a deacetylase that occurs in mitochondria. Could it be helping deacetylate Idh2? The biochemical experiments indicated this was not the case.

Unsurprisingly, both normal and knockout mice were found to be leaner when fed a CR diet. Is it possible that lower body mass, especially resulting from less fat tissue, had some role in the protection from oxidative damage resulting from a CR diet? Perhaps, but other factors like that certainly weren't sufficient, as it was pretty clear that SIRT3 (absent in the knockout mice) was necessary, at least as far as AHL is concerned. It's still possible that SIRT3 isn't necessary for anti-aging effects of CR in tissue types that weren't tested (i. e. other than inner ear, brain, and liver tissue), or in mammals other than mice. The case is pretty solid for AHL in mice, but obviously there are many other age-related conditions and other species that should be investigated.

I should apologize for all the biochemical details presented here, but at least they should give you a good indication of just how complicated the effects of CR on aging and longevity can be – and probably are. There's a whole lot of work yet to be done before a reliable anti-aging pill can be developed for humans. Enthusiastic claims that this research "could lead to" therapies to slow down aging in general are basically BS. Yeah, these findings will help, but a heck of a lot more will be needed as well.

(As an example of just how badly misleading journalists who write about this stuff can be, consider this report, which begins with the claim: "In a remarkable demonstration of the ability of calorie restriction to blunt the effects of aging, scientists at the University of Wisconsin-Madison have succeeded in delaying age-related hearing loss in mice." Although the research showed that calorie restriction can do this, it did not produce any new way to do it. Instead, it shows how CR probably works by showing how CR doesn't work if SIRT3 is absent.)

So what's the bottom line here? It's pretty clear from this and many other studies that oxidative damage in cells is a cause of cell death and therefore of various health problems associated with aging. Undoubtedly there are a number of other factors that contribute to aging-related problems, such as cell death due to other causes and weakening or disregulation of the immune system. And even in the case of oxidative damage, there are many ways it can come about, and also many ways it might be inhibited. If you think of aging as a complex disease, like cancer – a point of view that has its detractors – then there are bound to be many causes and contributing factors. And also many ways to inhibit or arrest the process. The example considered here is just one of many.

Someya, S., Yu, W., Hallows, W., Xu, J., Vann, J., Leeuwenburgh, C., Tanokura, M., Denu, J., & Prolla, T. (2010). Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction Cell, 143 (5), 802-812 DOI: 10.1016/j.cell.2010.10.002

Further reading:

Scientists ferret out a key pathway for aging (11/18/10)

Calorie restriction delays age-related hearing loss, UW study finds (11/18/10)

Scientists ID key protein that links dietary restriction with healthy hearing, aging (12/16/10)

Calorie Restrictions Slow Aging by Enzyme Pathway (11/19/10)

Rapamycin and lifespan extension

Will a pill containing the immunosuppressant drug rapamycin someday extend human lifespan a few years? In spite of the hopeful research announcements that appeared a few days ago, I wouldn't recommend getting one's hopes up just yet.

This is a topic I've discussed before: Calorie restriction, TOR signaling, and aging. And for related stuff on mTOR: here.

The executive summary is that inhibition of mTOR signaling has been shown to extend lifespan in yeast, roundworms, and fruit flies. Mice can now be added to this list, in experiments that included rapamycin in their diet.

Here's the press release:

Easter Island Compound Extends Lifespan Of Old Mice: 28 To 38 Percent Longer Life (7/8/09)

On July 8, in the journal Nature, The University of Texas Health Science Center at San Antonio and two collaborating centers reported that the Easter Island compound – called "rapamycin" after the island's Polynesian name, Rapa Nui – extended the expected lifespan of middle-aged mice by 28 percent to 38 percent. In human terms, this would be greater than the predicted increase in extra years of life if cancer and heart disease were both cured and prevented.

Although rapamycin and some related compounds have been investigated as anti-cancer therapies, the hypothesized lifespan-extending benefits are thought to be related to the by now well-documented benefits of calorie restricted diets. (For very recent news on that front, see here, for example.)

Aging researchers currently acknowledge only two life-extending interventions in mammals: calorie restriction and genetic manipulation. Rapamycin appears to partially shut down the same molecular pathway as restricting food intake or reducing growth factors.

It does so through a cellular protein called mTOR (mammalian target of rapamycin), which controls many processes in cell metabolism and responses to stress.

A decade ago, Dr. [Dave] Sharp proposed to his colleagues that mTOR might be involved in calorie restriction. "It seemed like an off-the-wall idea at that time," Dr. Richardson said.

Experiments were performed in parallel at three separate research centers and consisted of feeding hundreds of mice, starting at an age of 20 months, a diet containing a special formulation of rapamycin designed to evade breakdown in the digestive system. It was found that the age at which 90% of mice had died rose from 1,078 days to 1,179 days in male mice, compared to controls, and from 1,094 days to 1,245 days in females. The total lifespan extension, on average, was therefore 9.4% in males and 13.8% in females.

Note that some accounts of the research claim lifespan extensions of 28% to 38%, but this is misleading, since those figures represent the extension of the "old age" period of mouse life beginning at 20 months. They do not mean that the mice lived up to almost 40% longer in total. (Some pretty shoddy reporting going on here....) And there was no particular evidence to indicate that extensions of such size would have occurred if the special diet began at an earlier age. However, in experiments still going on, there is evidence for some extension when addition of rapamycin to the diet begins for mice 270 days old.

Of course, even an extension of human lifespan in the 10% range – 7 or 8 years – would be quite an accomplishment, provided quality of life in the final years remained about where it is today. (Which is a big if.)

But there are various reasons to suspect that even a 10% extension in humans is rather optimistic. Some reasons:

1. Rapamycin is an immunosuppressant, currently used therapeutically to prevent organ transplant rejection. The experimental mice were maintained under conditions that carefully protected them from infection – conditions that would not be realistic for humans.
   
2. Although mice and humans are both mammals, their genetics are not all that similar. The complete sequence of the mouse genome was recently announced (see here), and it turns out that about 20% of mouse genes are different from human analogs, or not found in humans at all. (It's been 90 million years since the last common ancestor of mice and humans.)
   
3. Rapamycin is known to inhibit an important protein kinase called mTOR (mammalian target of rapamycin). mTOR plays a key role in regulating cell growth, proliferation, and survival, so it's not all that surprising that rapamycin might affect cell biology relevant to aging and longevity. This same property of rapamycin makes it interesting as an anti-cancer agent. Rapamycin and similar compounds that inhibit mTOR have in fact been found to have anti-cancer properties in animal models. Several analogs of rapamycin have been investigated as anti-cancer therapies, and one has even been approved for human use (Torisel). But even in the anti-cancer setting, mTOR inhibitors haven't yet been slam-dunk successes.
   
4. It is not clear that rapamycin in these experiments was working the same way as calorie restriction. None of the rapamycin-fed mice lost body weight, and calorie restriction usually works best when started relatively early in life.
   
5. Experimental mice that received rapamycin got a dose of 2.24 mg per kg of body weight. That's quite a lot – about 30 to 60 times (per kg) what would be given to a 60 kg human for immunosuppression.
   

The unfortunate truth is that cell signaling pathways that affect cell growth, proliferation, and survival are rather complicated, and any interventions in such pathways are very likely to not have the expected effects and/or to have various unexpected side-effects. Here's a diagram of just some of the important pathways mTOR is involved in. Imagine that were an electrical circuit and you made ad hoc changes to important components of the circuit.... Perhaps you can see how trying to affect mTOR in order either to control cancer or enhance longevity might be a dicey proposition.

In spite of all the reservations, there are still promising signs for the role of mTOR inhibition in lifespan extension. The mechanism of action need not be the same as calorie restriction, even though that hasn't been ruled out either. For example, TOR is known from yeast and nematode studies to promote protein production in ribosomes and to inhibit protein degradation via autophagy. Invertebrate studies have shown that reversal of these TOR effects can increase lifespan. And TOR signaling is also known to influence cell growth, cell-cycle progression, mitochondrial metabolism, and insulin-analog signaling.

Remember what we said about the diversity of effects of mTOR signaling? That's definitely a sword that can cut both ways – it's powerful, but hard to predict and control. We need to understand a lot more of the biological details – otherwise we're just swinging the sword in the dark.

Harrison, D., Strong, R., Sharp, Z., Nelson, J., Astle, C., Flurkey, K., Nadon, N., Wilkinson, J., Frenkel, K., Carter, C., Pahor, M., Javors, M., Fernandez, E., & Miller, R. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice Nature DOI: 10.1038/nature08221

Further reading:

Tests raise life extension hopes (7/8/09) – BBC news story

Immune drug boosts lifespan (7/8/09) – TheScientist.com

Fountain of Youth on Easter Island? (7/8/09) – ScienceNOW

Cancer Drug Delays Aging in Mice (7/8/09) – Wired.com

A pill for longer life? (7/8/09) – Nature.com

Ageing: A midlife longevity drug? (7/8/09) – Nature.com PDF

Rapamycin extends life in mice, raising hopes of life-prolonging drug for humans (7/9/09) – The Times (UK)

What Does Life-Extending Drug Mean for Humans? (7/9/09) – Time

New clues in search for elixir of youth (7/9/09) – New Scientist

Antibiotic Delayed Aging in Experiments With Mice (7/8/09) – New York Times

First Drug Shown to Extend Life Span in Mammals (7/8/09) – Technology Review

Longevity pill on the horizon? (7/10/09) – press release

Rapamycin: “An anti-aging drug today”? (3/6/07) – Ouroboros blog post

Tags: rapamycin, biogerontology, aging, longevity

Not good news for antioxidant therapies

Forget The Antioxidants? Researchers Cast Doubt On Role Of Free Radicals In Aging (2/17/09)

For more than 40 years, the prevailing explanation of why we get old has been tied to what is called oxidative stress. This theory postulates that when molecules like free radicals, oxygen ions and peroxides build up in cells, they overwhelm the cells' ability to repair the damage they cause, and the cells age.

An industry of "alternative" antioxidant therapies -- such as Vitamin E or CoQ10 supplements in megadose format -- has sprung up as the result of this theory. However, clinical trials have not shown that these treatments have statistically significant effects.

And now researchers at McGill University, in a study published in the February issue of the journal PLoS Genetics, are calling the entire oxidative stress theory into question. Their results show that some organisms actually live longer when their ability to clean themselves of this toxic molecule buildup is partially disabled. Collectively, these molecules are known as reactive oxygen species, or ROS for short.

Author summary of the open-access paper:

Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends Lifespan in Caenorhabditis elegans

In this paper, we examine the oxidative stress theory of aging using C. elegans as a model system. This theory proposes that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS). To test this theory, we examined the effect of deleting each of the five individual superoxide dismutase (SOD) genes on lifespan and sensitivity to oxidative stress. Since SOD acts to detoxify ROS, the oxidative stress theory predicts that deletion of sod genes should increase oxidative stress and decrease lifespan. However, in contrast to yeast, flies, and mice, where loss of either cytoplasmic or mitochondrial SOD results in decreased lifespan, we find that none of the sod deletion mutants in C. elegans exhibits a shortened lifespan despite increased sensitivity to oxidative stress. Surprisingly, we find that sod-2 mutant worms have extended lifespan and even worms with the primary cytoplasmic, mitochondrial, and extracellular sod genes deleted can live longer than wild-type worms. By examining genetic interactions with other genes known to extend lifespan and by comparing the phenotype of worms lacking sod-2 to that of known long-lived mitochondrial mutants such as clk-1 or isp-1, we provide evidence that the loss of sod-2 extends lifespan through alteration of mitochondrial function.

IGF-1, calorie restriction, exercise, and longevity

Loyal readers here (both of you) may recall that back here I mentioned the hormone IGF-1 and promised to deal with it more throughly. The occasion was that IGF-1 is a growth factor, like BDNF.

Basically, a growth factor is a protein for signaling between cells. Growth factors typically bind to specific receptors on a cell's surface, in order to promote cell survival, growth, or proliferation.

The following recent news item now gives me an excuse to make good on my promise:

Hormone May Hold Key To Helping Elderly Men Live Longer (5/27/08)

Elderly men with higher activity of the hormone IGF-1--or insulin-growth factor 1--appear to have greater life expectancy and reduced cardiovascular risk, according to a new study.

IGF-1 is a hormone similar in molecular structure to insulin. It is released from the liver and plays an important role in childhood growth and continues to have anabolic effects in adults. ...

Subjects with the lowest IGF-1 function had a significantly higher mortality rate than subjects with the highest IGF-1 bioactivity. These results were especially significant in individuals who have a high risk to die from cardiovascular complications.

So, does that mean we need to find ways to increase our body's IGF-1 production in order to extend lifespan? Well, not necessarily. It's more complicated than that, as we'll see shortly.

Any hint of longevity enhancement, of course, is something worth paying attention to, but in the case of IGF-1, there's a lot more to the story. It's actually kind of a big deal for several additional reasons.

To begin with, the full name of the hormone is insulin-like growth factor. It is so-named because, as the news item mentions, its molecular structure is similar to that of insulin.

But that's just the beginning of the similarity. Both IGF-1 and insulin affect metabolism. In fact, IGF-1 can bind to the same receptor that insulin does, although a lot less strongly. That, and the not coincidental structural similarity to insulin suggest that perhaps, sometime far back in evolution, the same gene may have coded for some ancestor of both insulin and IGF-1.

If you take into account a striking fact about the IGF-1 receptor, this hypothesis of a common origin for insulin and IGF-1 becomes even more intriguing. The fact is that the (gene for the) IGF-1 receptor is a homologue of the daf-2 gene of the nematode Caenorhabditis elegans (as is the gene for the insulin receptor also). In fact, DAF-2 (the protein product of daf-2) is the only insulin-like receptor in nematodes, so biologists now regard daf-2 as the ancestor of the mammalian receptors for IGF-1 and insulin.

I first mentioned this relationship back here, and went into more detail here, in connection with understanding the effect of sirtuin proteins on aging and longevity of C. elegans.

But the "coincidences" don't stop there. The important function of a receptor is the effect it has, when activated, upon signaling downstream inside the cell. All of the receptors we're talking about here are of the sort called tyrosine kinase receptors. Let's unbundle that term. Tyrosine is one of the 20 amino acids that make up proteins. A kinase is a type of protein enzyme whose function is to attach phosphate groups to specific kinds of amino acids in other proteins. This process is called phosphorylation. When another protein of the right sort is phosphorylated, it becomes able to act as a tyrosine kinase itself, and go on to affect yet other proteins.

This whole process is called signal transduction. The process begins (in the case here) with a receptor tyrosine kinase, which is a cell surface receptor protein that is also a tyrosine kinase – for example DAF-2, and the receptors for IGF-1 and insulin. There may be a number of intermediate steps, but the eventual result is the phosphorylation of a transcription factor, which enters the cell nucleus and facilitates the transcription of certain genes in order to produce new proteins.

In C. elegans, DAF-16 is the transcription factor that is activated by signaling mediated by DAF-2. We discussed DAF-16 in the aforementioned posts here and here. DAF-16 belongs to a family of transcription factors called forkhead box proteins. We have discussed these before too, or rather the subclass called FoxO transcription factors.

We're getting pretty far into the technical weeds here, so if you want more details on this stuff, refer to the earlier posts.

To make the long story short, the effects of the external signaling hormones like insulin and IGF-1 ultimately result from proteins coded for by the genes expressed because of the appropriate transcription factors that were activated by the signaling cascade. There are probably many proteins involved, and sorting them all out, figuring out how they collectively affect longevity, is very much an ongoing project.

The story is interesting to understand because longevity is one of its main themes. In addition to the news item already mentioned, there's more recent news with the same theme. Here are summaries of some of these research announcements:

When It Comes To Living Longer, It's Better To Go Hungry Than Go Running, Mouse Study Suggests (5/14/08)

It is once again verified that a low-calorie diet can extend the lifespan of rodents. This benefit is beyond what can be achieved with a higher-calorie diet offset by exercise. However, rats that consumed the most calories, and has less longevity, also had the highest levels of IGF-1. Rats that consumed the fewest calories had the best longevity and the lowest levels of IGF-1. Exercise could only partially counteract the higher IGF-1 levels and reduced longevity of rats on a high-calorie diet. In this study, IGF-1 levels were inversely correlated with longevity. This is a "live-fast, die-young" scenario, which is especially typical of rodents, but not necessarily of humans.

More on this study: here

Shorter Women May Have Very Long Lives: Gene Mutation Found (3/4/08)

This study focused attention on the (adult) daugheters of especially long-lived Ashkenazi Jews. A control group consisted of daughters of the same age as the others, but whose families had no history of unusual longevity. The finding was that female children of long-lived individuals (aged 95-110) were on average 2.5 cm shorter than female controls. It was also found that both the centenarians and their daughters were much more likely than the controls to have mutations in the genes for their IGF-1 receptors. However, the daughters also had blood plasma levels of IGF-1 that were 35% higher than the levels in the control group. The interpretation is that the higher IGF-1 levels were due to an attempt to compensate for disruption of IGF-1 signaling due to irregularities of the receptor proteins. This would be consistent with a number of animal studies in which reduced IGF-1 signaling correlates with increased longevity.

More on this study: here, here, here

Interestingly enough, IGF-1 had already been recognized to have an effect on body size – in mice and dogs. The dog research is described here:

One gene between tiny dogs and giant ones? (10/13/06)

Nate Sutter, a geneticist at the National Human Genome Research Institute in Bethesda, Maryland, wanted to know the reason why big dogs, such as Irish wolfhounds, can grow up to 50 times larger than other members of their own species, such as chihuahuas. So he started out looking at large and small dogs of one breed — the Portuguese water dog. ...

The team found that one of the few differences in these Portuguese water dogs occurred in a gene called 'insulin-like growth factor 1', or Igf-1 .

This is one of many genes already known to influence the size of mice: when Igf-1 is knocked out, the animals grow up to be mini-mice.

(The article is subscription-only, but you can find another reference to it here.)

The researchers went on to do further analysis of the IGF-1 gene in many different dog breeds of all sizes, and also in foxes and wolves. They found that almost all of the small breeds had the same variant of the IGF-1 gene as the small Portuguese water dogs had, while almost none of the large breeds had that variant. The team concluded that the IGF-1 variant in small breeds is responsible for the difference because it reduces production of the growth factor.

This should also explain what dog people have always known – that small breed dogs generally live longer than large ones.

Here's a later report of the same research:

What Makes Little Dogs Small? Researchers Identify Gene Involved In Dog Size (4/5/07)

In their study, researchers explored the genetic basis for size variation among dogs by comparing the DNA of various small dog breeds, including Chihuahuas, Toy Fox Terriers and Pomeranians, to an array of larger dog breeds, including Irish Wolfhounds, Saint Bernards and Great Danes. Their investigation found that variation in one gene - IGF-1, which codes for a protein hormone called insulin-like growth factor 1 - is very strongly associated with small stature across all dog breeds studied.

Further reading:

Scientists Explore Queen Bee Longevity (5/8/07) – press release describing research on various factors, including IGF-1 signaling, in queen bee longevity

Mechanisms of lifespan regulation by IGF-I (2/25/08) – blog post that considers some of the paradoxical effects of IGF-1 that may be beneficial in some ways but also shorten lifespan

Not so fast, daf-2: IGF-I is all kinds of good for you (1/23/08) – another blog post on the paradoxical effects of IGF-1

IGF-1 attenuates cardiac aging (11/15/06) – blog post about research on cardioprotective properties of IGF-1

It’s not easy being wee: Does IGF-1 deficiency slow down the brain? (8/30/06) – one more blog post on paradoxical effects of IGF-1

A Single IGF1 Allele Is a Major Determinant of Small Size in Dogs – 4/6/07 research article in Science (sub. rqd.)

Tags: IGF-1, calorie restriction, longevity, aging

Calorie restriction, TOR signaling, and aging

Now that I've given some pointers to information about how TOR signaling is involved with metabolism (see here), it seems like an opportune time to mention a recent research announcement in this general area.

How Dietary Restriction Slows Down Aging (4/17/08)

University of Washington scientists have uncovered details about the mechanisms through which dietary restriction slows the aging process. Working in yeast cells, the researchers have linked ribosomes, the protein-making factories in living cells, and Gcn4, a specialized protein that aids in the expression of genetic information, to the pathways related to dietary response and aging.

Here's the key background:

Previous research has shown that the lifespan-extending properties of dietary restriction are mediated in part by reduced signaling through TOR, an enzyme involved in many vital operations in a cell. When an organism has less TOR signaling in response to dietary restriction, one side effect is that the organism also decreases the rate at which it makes new proteins, a process called translation.

The researchers investigated various strains of yeast cells that had low rates of protein production, but increased lifespan. They found that a common characteristic of such cells was mutations to one part of the cell's ribosomes, the complex of RNA and certain proteins which manufactures all new proteins in the cell. The result of these ribosome changes was a decrease in the production of most proteins, except for one, called Gcn4, a transcription factor, whose production increased. The effect seems to depend on the same pathway affected by reduced TOR signaling. Gcn4 is associated with control of amino acid synthesis, and is activated when a cell is starved for amino acids.

To make the link between Gcn4 and longevity, the scientists then asked whether preventing the increase of Gcn4 would block life span extension. In every case, cells lacking Gcn4 did not respond as strongly as Gcn4-positive cells.

"The increased production of Gcn4 in long-lived yeast strains, combined with the requirement of Gcn4 for full life-span extension, makes a compelling case for Gcn4 as an important downstream factor in this longevity pathway," Kaeberlein said.

One might speculate that increased Gcn4 production somehow helps the cell cope with lack of nutrients, and one effect is that the cell takes steps to conserve its resources and slow the rate of aging.

Since reduction of TOR signaling is one way to bring about this effect, TOR inhibitors might help slow aging and increase lifespan, at least in yeast. However, since TOR affects so many other cell functions, the chance for harmful side effects of reduced TOR signaling is high.

"The role of TOR and translation in aging is known to be conserved across many different species, so it's plausible that this function of Gcn4 is conserved as well," Kennedy said. Future research will be aimed at testing this hypothesis.

"Clearly TOR signaling is one component, and perhaps the major component, of the beneficial health effects associated with dietary restriction," said Kaeberlein. "The difficulty with TOR as a therapeutic target, however, is the potential for negative side effects. As we learn more of the mechanistic details behind how TOR regulates aging, we will hopefully be able to identify even better targets for treating age-associated diseases in people."

Tags: calorie restriction, TOR signaling

Wnt signaling

We've discussed Wnt signaling a couple of times before, here, and here.

Wnt refers to a family of proteins now numbering perhaps 20 or more, which have been found in a wide range of multicellular animals, from fruit flies, to fish, to mice and humans. Wnt proteins carry messages between cells, and are especially important in embryogenesis. They are known to play a large role in the control of stem cells and regeneration of body parts (in species where this occurs). In mammals, including humans, Wnt signaling, when it malfunctions, also seems to be involved in many types of cancer, degenerative diseases of aging, and other aging-related problems such as insulin resistance. It may be possible to ameliorate a number of these disease conditions once we have a better understanding of the details of Wnt signaling.

The "Wnt signaling pathway" refers to a sequence of proteins that, in the presence of earlier members of the pathway, change in behavior to affect proteins later in the pathway. The pathway begin when a Wnt protein (secreted by a nearby cell) binds to a cell surface protein, such as the whimsically-named Frizzled. Various other proteins in the pathway then interact, and eventually result in the build-up of a protein called β-catenin, which enters the cell nucleus, where it combines with various transcription factors to affect gene expression.

The name "Wnt" originates from the realization that two genes discovered earlier were homologous – the "wingless" gene in fruit flies (which, when mutated, yields flies without wings), and the Int genes found in mouse tumors.

Although Wnt genes and proteins have now been studied for nearly 20 years, the pace of discovery continues to increase. This is because of the large number of very interesting processes heavily influenced by Wnt signaling – from proliferation and differentiation of stem cells to embryonic development, regeneration of body parts, cancer, and degenerative diseases of aging.

The following summaries of research reports from just the past half year or so will give a buffet-style sample of Wnt-related investigations.

Carbohydrate Regulates Stem Cell Potency (2/1/08)

Embryonic stem cells are characterized by an ability to continually self-renew, but also to give rise to any adult cell type. Stem cell renewal is driven by several external signaling proteins and growth factors, including Wnt, FGF (fibroblast growth factor), and BMP (bone morphogenetic protein). In particular, Wnt signaling stimulates β-catenin to produce the transcription factor Nanog, which maintains pluripotency. However, the ability of these proteins to attach to stem cell surface proteins in order to induce a response seems to depend on the presence of a carbohydrate molecule called heparan sulfate (HS). Stem cells were found to reproduce less frequently but differentiate more frequently in proportion to experimental inhibition of HS production.

Beta-catenin Gradient Linked To Process Of Somite Formation (12/27/07)

In a developing vertebrate embryo somites are masses of a type of tissue (mesoderm) that will eventually develop into such adult tissue types as skeletal muscle and vertebrae. This research on mouse embryos demonstrates the importance of β-catenin as the principal mediator of the Wnt-signaling pathway, in the process of somite formation. In particular, there is a gradient in levels of β-catenin found in cells of the presomitic mesoderm (PSM), and this gradient is critical in regulating mesoderm maturation. This leads to the development of the characteristic vertebral column in embryos of vertebrate animals.

Certain Diseases, Birth Defects May Be Linked To Failure Of Protein Recycling System (12/20/07)

The Wnt signaling protein, like other proteins, is produced in the nuclei of certain cells, and it must be transported to the cell surface, so it can be secreted into the extracellular environment to regulate the growth of tissues during (and after) embryonic development. Another protein, called Wntless (Wls), acts as a cargo container for Wnt, and plays a key role in the transport process. Another protein, called Vps35, which makes up an important part of the "retromer complex", is responsible for moving empty Wls molecules (like freight cars) to where they are needed in the cell. But mutated Vps35 proteins can fail to perform their function, and consequently lead to the failure to transport Wnt out of the cell where it has been produced.

Grape Powder Blocks Genes Linked To Colon Cancer (11/14/07)

Previous research has found that the Wnt signaling pathway is linked to more than 85 percent of sporadic (i. e. not caused by a hereditary defect) colon cancers. Additionally, in vitro studies have shown that resveratrol is capable of blocking the Wnt pathway. The present research showed that in some colon cancer patients who consumed grape powder (which contains resveratrol and possibly other active ingredients), Wnt signaling in biopsied colon tissue was significantly reduced.

Odd protein interaction guides development of olfactory system (10/29/07)

The olfactory system of fruit flies has been shown to develop abnormally when the signaling protein Wnt5 is absent. However, if large amounts of Wnt5 but no Wnt5 receptors called "derailed" are present, development is even more abnormal. Specifically, structures called glomeruli in fruit fly antennal lobes (which are analogous to human olfactory bulbs) grow abnormally when Wnt5 is absent. But if Wnt5 is present in large amounts and there are no derailed receptors, malformed glomeruli develop in locations where they should not be.

Cilia: Small Organelles, Big Decisions (10/3/07)

Research into the development of zebra fish (a favorite of developmental biologists) has shown that organelles called cilia in the cells of developing embryos play a large role in the transduction of Wnt signaling proteins that guide the development process. By blocking the production of three proteins used by cilia, researchers were able to disrupt proper balances in the interpretation of Wnt signals, resulting in developmental defects.

New Insights into the Control of Stem Cells: Keeping the Right Balance (9/15/07)

The Wnt signaling pathway plays a crucial role in embryonic development, cell growth (proliferation), and maturation of cells into specialized cells (differentiation). It is also an important regulator of stem cells. An interaction between Wnt signaling and tyrosine kinases enables the proliferating cells to mature into specialized (differentiated) cells. Normally this interaction strikes a proper balance between proliferation and differentiation. Cancers, such as breast and colon cancer, result when the interaction gets unbalanced. In 90% of human cancers the tumor suppressor APC (adenomatous polypolis coli), one of the core components of the Wnt pathway, is deregulated. This results in excessive amounts of β-catenin, which triggers the onset of breast and colon cancer when it gets into the cell nucleus and affects gene expression.

Reactivating A Critical Gene Lost In Kidney Cancer Reduces Tumor Growth (8/15/07)

Studies of an important tumor-suppressor protein, sFRP-1 (secreted frizzled-related protein 1), in clear cell renal cell carcinoma, the most common type of kidney cancer, may reveal a means to defeat the cancer. sFRP-1 was found to control 13 tumor-promoting genes along the Wnt signaling pathway, which has been linked to a number of cancers, especially colon cancer. Several close relatives of sFRP-1 are also known to affect at least 20 Wnt-related proteins, and up-regulation of members of the sFRP-1 family may be an effective way to control cancers linked to Wnt signaling. In one experiment, increasing sFRP-1 expression in human renal cancer cells was effective, and Wnt regulated oncogenes, such as c-myc, were suppressed compared to untreated cells.

Why Aging Muscles Heal Poorly (8/9/07)

Stem cells normally found in muscle tissue are responsible for repair to muscles damaged by injury or age-related degeneration. But in aged muscle tissue, stem cells tend to produce scar-tissue cells called fibroblasts, instead of normal muscle cells (myoblasts). The overproduction of fibroblasts is a condition known as fibrosis. New research shows that it isn't the age of the muscle stem cells that is the problem, but rather the age of the cellular environment itself, including blood supply to the tissue. The malfunction appears to be a problem with Wnt signaling in the aged environment rather than with the actual stem cells. Muscle stem cells from young mice exhibited the same problems when exposed to an enviroment from older animals.

Related research found that Wnt signaling increased, with detrimental effect, due to age-related deficiency of a hormone called klotho. Klotho seems to inhibit Wnt signaling, and also has some control over insulin sensitivity. However, production of klotho seems to decline with age, possibly leading to age-related problems such as cancer, arterial disease, and insulin resistance.

Not A Relay Race, But A Team Game: New Model For Signal Transduction In Cells (6/27/07)

Details of the inner workings of the Wnt signal transduction process have remained incomplete, but are gradually coming into focus. Member of the Wnt family of proteins may dock with a variety of cell-surface proteins, including LRP6 (low density lipoprotein receptor-related protein 6) and members of the family of G protein-coupled receptors known as Frizzled. After the docking, a signaling cascade is triggered that transmits molecular messages via the cytoplasm to the nucleus. This research shows that the first step after docking involves large protein complexes formed from proteins already known to be part of the signaling pathway, such as phosphorylated LRP6, axin, and Dishevelled (Dvl).

Further reading:

The Wnt Homepage

Regeneration for Repair's Sake

The answer is blowing in the Wnt

Miller on Wnt and Klotho

A hazy shade of Wnt

Tags: Wnt, embryogenesis, stem cells, signal transduction, cancer, developmental biology, aging

FoxO transcription factors

Transcription factors are proteins that help regulate genes. This regulation may involve either enabling the expression of a gene or preventing expression. In the first case, the transcription factor is an "activator", and in the second case a "repressor".

Transcription factors perform their function by binding to a particular portion of DNA that is specific to a given gene. When bound to the appropriate DNA segment, a transcription factor affects gene expression by either facilitating (activator) or inhibiting (repressor) the operation of RNA polymerase in transcribing the affected gene into messenger RNA. Usually more than one transcription factor must be present to affect gene transcription, and additional proteins (called "cofactors") may also be required.

To make things even more interesting, transcription factors usually affect multiple genes, which may be otherwise unrelated to each other.

A particularly important family of related transcriptions factors comprises what are called "forkhead box" proteins, or Fox proteins, for short. (The name refers to a sequence of 80 to 100 amino acids that are part of the protein and bind to DNA, and which was originally discovered in fruit flies (Drosophila).)

Among the genes that Fox proteins are involved with are genes related to cell growth, proliferation, differentiation, longevity, and embryonic development. So there are Fox proteins that are important for things like cancer and stem cells – and thus it's quite useful to know about them.

An important subfamily of Fox proteins are the FoxO proteins, and we'll discuss some recent examples in this note.

To begin with, perhaps the most recent example is this:

Molecular Signal That Helps Muscle Regenerate Discovered (12/19/07)

Muscle regeneration after injury is complex and requires a coordinated interplay between many different processes. Key players in regeneration are muscle stem cells, so-called satellite cells. They divide and produce many new muscle cells to fix the damage incurred by injury. A crucial regulator of muscle function and repair is a signalling molecule called calcineurin. It is activated by injury and controls the activity of other key proteins involved in differentiation and the response to damage.

It turns out that calcineurin works by inhibiting FoxO.

Using sophisticated molecular techniques, the scientists revealed that calcineurin accomplishes its effect on muscle by inhibiting another protein called FoxO. FoxO is a transcription factor, a protein that plays a crucial role in skeletal muscle atrophy through the induction of genes involved in cell cycle repression and protein degradation. Suppressing the effects of FoxO, calcineurin ensures that proliferating cells stay alive and keep dividing to produce enough cells to repair muscle damage.

In this case, the normal function of FoxO is to inhibit cell proliferation (as a check on cancer), but this needs to be bypassed (temporarily) to enable muscle regeneration.

This result follows the discovery a few months earlier of the way a specific FoxO protein (FoxO1) cooperates with another important developmental protein (Notch) to control muscle cell differentiation:

Building Muscle Requires Foxo1 (8/25/07)

The mechanisms by which Foxo proteins regulate metabolism are relatively well characterized. However, little was known about the mechanisms by which these same proteins regulate cellular differentiation.

New data generated by Domenico Accili and colleagues at Columbia University, New York, now indicates that Foxo1 cooperates with Notch to control muscle cell differentiation in vitro.

Overexpression of either a constitutively active form of Foxo1 or a constitutively active form of Notch was found to inhibit the in vitro differentiation of a mouse myoblast cell line.

Note that the preceding alludes to the involvement of FoxO proteins in regulation of metabolism. This comes about because they affect the insulin signaling pathway, and hence also glucose and lipid metabolism.

This function is what allows yet another well-known protein, mTOR, to play a role in "metabolic syndrome" – a group of disorders that includes insulin resistance, heart disease and high lipid levels. (mTOR is short for "mammalian target of rapamycin". It's a protein kinase that modifies other proteins by phosphorylation.) The same mechanism appears relevant also to the "Atkins diet" and the effects of calorie restriction.

Fly Genetics Reveal Key Workings Of Atkins Diet (8/8/06)

Using fruit flies bred with a newly created mutant form of the gene TOR (short for target of rapamycin), Oldham and his colleagues were able to determine how the TOR pathway interacted with other important regulators of insulin, glucose and lipid metabolism.

TOR is an ancient gene, found in nearly all animal and plant cells. The researchers discovered that their new mutant fly reduced TOR function, allowing them to observe what happens when TOR's influence is removed.

Reductions in TOR function lowered glucose and lipid levels in the body. They also blocked the function of another important insulin regulator, a factor called FOXO, which is known to be a critical mediator of insulin signals and therefore glucose and lipid metabolism.

As if all that weren't enough, FoxO proteins are also involved with cancer and stem cells:

Gene Knockouts Reveal FoxOs' Vital Functions In Cancer Defense, Health Of Stem Cells (1/25/07)

In an elegant, multiple-gene knockout experiment, a team of Boston scientists has discovered that a trio of molecules, called FoxOs, are fundamentally critical in preventing some cancers, maintaining blood vessel stability, and in keeping blood-forming stem cells healthy. ...

The researchers at Brigham and Women's found that mice engineered to lack genes for the FoxO1, FoxO3, and FoxO4 molecules had serious blood abnormalities. Without the FoxO gene-regulating molecules, the rodents' blood stem cells -- master cells that give birth to working blood cells while also renewing themselves -- divided too fast and "burned out." ...

In the companion paper, lead author Ji-Hye Paik, PhD, of Dana-Farber and colleagues from the DePinho lab report that the three FoxO molecules, known as transcription factors, normally function as tumor suppressors that override maverick cells threatening to grow too fast and form tumors. When FoxOs are eliminated, it may allow cancer to develop.

And even that's not the end of it. FoxO proteins are also involved in the increased levels of inflammation often associated with the aging process. (This phenomenon has been tagged with the neologism "inflammaging".) It has been hypothesized that inflammaging results from the effect of phosphorylated FoxO on another notorious transcription factor, NF-κB (which is heavily involved in inflammation). Some of the effects of calorie restriction may also be due to FOXO phosphorylation. Reference: Restricting inflammaging (11/12/07)

FoxO is also regulated (as is P53) by SIRT1 – so this is yet another relationship to calorie restriction. Reference: Unlocking the Secrets of Longevity Genes

Additional references (for the seriously interested):

An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans.

Ageing: When Less Is More

Tags: Fox proteins, transcription factor, longevity, aging, calorie restriction

P53, a versatile gene

P53 is well-known for its role in regulating the cell cycle so as to suspend the cycle or even lead to cell death via apoptosis in case damage to a cell's DNA is detected. This function is especially important in forestalling cancer.

And as we noted here, p53 is also involved with skin tanning.

But that's not all p53 is good for. It also plays a role in fertility, which has recently been reported by one of the co-discoverers (Arnold Levine) of p53:

Cancer Fighter May Be Fertility Helper

A protein known primarily for its role in fighting cancer also helps embryos implant in the womb, according to a study in mice. The find may explain why some women have difficulty becoming pregnant.

More information: here, here

But the list of p53's goodness doesn't stop there. It also slows aging, apart from deterring cancer, but via the same mechanism:

Anti-cancer gene p53 doubles up as anti-ageing agent

The latest research suggests that one of the genes that protects us from cancer may also help delay the ageing process.

A new study has found that a particular gene, p53 which has been previously linked to premature ageing, along with one of its cellular regulators, called Arf, may boost the body's antioxidant activity to keep cells younger longer and thereby slow down the aging process.

The regulatory chemical Arf, lets p53 know that a particular cell is in trouble and marked for elimination.

More information: here, here, here, here

But, surprisingly, at least in fruit flies, reducing p53 activity may also increase lifespan, and apparently in the same way that calorie restriction does:

Key To Longer Life (in Flies) Lies In Just 14 Brain Cells

Two years ago, Brown University researchers discovered something startling: Decrease the activity of the cancer-suppressing protein p53 and you can make fruit flies live significantly longer.

Now the same team reports an intriguing follow-up finding. The p53 protein, they found, may work its lifespan-extending magic in only 14 insulin-producing cells in the fly brain.

How was this connected with calorie restriction? Simply by noting that calorie restriction in fruit flies didn't increase longevity when p53 activity was suppressed in only 14 insulin-producing cells of the flies' brains:

Studies have shown that low-calorie diets can significantly increase the lifespan of flies, worms, mice and rats. The phenomenon is of intense interest to researchers who study aging. They want to know if caloric restriction works in people and if drugs could be made to mimic its effects.

So researchers restricted the diets of the flies and ran the same experiments. The calorie-restricted flies didn't live any longer when p53 was reduced in the insulin-producing cells. This evidence supports the notion that p53 reduction is one of the direct effects of caloric restriction.

Even more intriguing, Helfand said, is the fact that the 14 insulin-producing cells that seem to be critical for lifespan extension are the equivalent of beta cells in the human pancreas. Beta cells make and release insulin, the hormone that controls the level of glucose in the blood. The research team found that when p53 activity drops, so does insulin-responsive activity in the fat body, the major metabolic organ in the fruit fly.

The involvement of insulin in this effect is especially interesting, as insulin signaling has also been found to be involved in the mechanism by which sirtuin proteins extend longevity in nematodes and fruit flies (and perhaps other organisms).

One wonders just how p53 came to play such a prominent role in cellular processes. Some researchers think they have found the answer – endogenous retroviruses that have actually proven beneficial to the host genome:

Ancient Retroviruses Spurred Evolution Of Gene Regulatory Networks In Humans And Other Primates

Scientists have long wondered how a master regulator such as p53 gained the ability to turn on and off a broad range of other genes related to cell division, DNA repair, and programmed cell death. How did p53 build its complex and powerful empire, so to speak?

Using the tools of computational genomics, the UCSC team gathered compelling evidence that retroviruses helped out. ERVs jumped into new positions throughout the human genome and spread numerous copies of repetitive DNA sequences that allowed p53 to regulate many other genes, the team contends.

"This would have provided a mechanism to quickly establish a gene regulatory network in a very short evolutionary time frame," said Ting Wang, a post-doctoral researcher at UCSC and lead author of the paper.

It's hard to avoid a suspicion that there's a lot to the story of p53 left to be discovered.

Tags: p53, aging, longevity, calorie restriction, insulin signaling

The discovery of sirtuins, part 2

Unless you're a biologist who's already familiar with the ins and outs of research into sirtuin proteins, you might want to have a look (if you haven't already) at the previous note in this series, where I describe a lot of important background and provide various other references.

But if you're ready to forge ahead, in this note I'm going to write about a gene found in the nematode Caenorhabditis elegans. The gene is called sir2-1, and it's a homologue of the yeast SIR2 gene. (I. e. the two genes have very similar DNA sequences.)

C. elegans and some of its genes (like sir2-1 and several others affected by it, as mentioned here and here) has been studied by many investigators, because it's an easily-grown model organism for many biological processes that occur in multicellular creatures. And that's in spite of its simplicity – adults have a grand total of only 959 somatic cells.

Sydney Brenner began research into the detailed biology of C. elegans in 1974. Brenner had already earned his scientific spurs for helping decipher the 3-letter DNA code in the 1960s. But the Nobel Prize he shared in 2002 was awarded for his work with worms – which is an impressive statement about the importance of that work. Other prominent names associated with research into C. elegans include Cynthia Kenyon and Gary Ruvkun.

However, for the initial study of SIR2-like genes in C. elegans, we can return to the laboratory of Leonard Guarente. Soon after he and others at the lab had begun to appreciate the importance of SIR2 for longevity in yeast, Guarente suggested to a postdoc in his lab, Heidi Tissenbaum, that it might be very rewarding to figure out whether there were similar genes in the nematode that played a role in longevity. Tissenbaum was a pretty natural choice for this project, since she'd just recently done her thesis work in Gary Ruvkun's lab.

Despite the simplicity of C. elegans, following up on this suggestion was more easily said than done. Some idea of the complexity involved can be gained from the fact that there are about 20,000 genes in the worm's genome, as had only very recently been realized, since C. elegans was the first animal to have its whole genome figured out. The worm has almost 90% as many different genes as a human.

Among these 20,000 or so genes were four that were like SIR2. Which, if any, of those might have longevity-prolonging effects? Guarente and Tissenbaum set about trying to answer the question. So as not to miss any genes that might affect longevity even though unlike SIR2, they did experiments that could turn up others among the 20,000. They did this by considering different strains of C. elegans, each of which had one random section of its DNA duplicated. Since a eukaryotic organism already has two copies of each gene, this meant each strain would have 50% more copies (3 instead of 2) of the genes on the duplicated segment.

They found only one out of the 40 strains they tested that had a significantly longer life span. And the duplicated section of DNA contained only one of the 4 known SIR2-like genes – sir2-1, which was also the one closest in sequence to SIR2. Talk about "things that make you go hmmmmmm..."

To further strengthen the evidence that sir2-1 was somehow responsible for the increased life span, Tissenbaum produced a strain of C. elegans whose only extra gene was one or more extra copies of sir2-1. Lo and behold, these worms indeed lived much longer.

That's all well and good, of course. But how does sir2-1 bring about this increased life span? It certainly couldn't be much like the way SIR2 works in yeast to raise longevity. As you recall, longevity in a yeast cell is measured by how many times it is capable of budding off daughter cells. Normally, this is about 20 times. But this number can be substantially increased in a yeast strain with extra copies of SIR2.

However, the biology of C. elegans is quite different. The life span of these worms is manifested in a very different way than by how often cells are capable of dividing. In fact, the cells of an adult nematode do not divide at all – they have reached a state known as "senescence", all 959 of the somatic cells. All the difference in life span of a nematode occurs after its cells become senescent.

Initially, life span of the worms was measured simply by how long it took before the creature stopped wriggling, about 20 days. Later, more careful observation showed that aging could actually be noticed visibly (under a microscope). Old worms looked wrinkled and exhibited other visible signs of decrepitude. This is of importance, because an alternative hypothesis about how sir2-1 promoted longevity was that it somehow blocked a disease state that could kill the worm. But in fact, it was found that extra sir2-1 genes indeed slowed the rate of visible aging.

So there still remained to find an account of how sir2-1 extended life span. There were several other worm genes that were already known to affect longevity. I noted two of these (daf-2 and daf-16) here. Some of this information was already known to Guarente and Tissenbaum. In fact, the latter herself had participated in some of the relevant research while working in Ruvkun's lab. This 1997 press release describes some of that research:

Inactivation Of Key Gene Allows Worms To Develop Without Insulin (10/29/97)

The team — which also includes first author Scott Ogg, PhD, Suzanne Paradis, Shoshanna Gottlieb, PhD, Garth Patterson, PhD, Linda Lee, and Heidi Tissenbaum, PhD — discovered that insulin may control metabolism via inactivation of a second gene, daf-16. The researchers found that, although insulin normally is required to regulate metabolism in the worm C. elegans, as in humans, the animal no longer needs insulin if it also carries a mutation in daf-16. This gene encodes a DNA-binding protein that passes along insulin signals within the cell to control the production of enzymes that metabolize sugars and fats. The team proposes that in the absence of insulin, the DAF-16 protein becomes unregulated, and that its runaway activity may be the key cause of metabolic disease in diabetes. In support of this model, the research team shows that metabolic defects in worms with defective insulin signaling are "cured" by the inactivation of the daf-16 gene.

(If you're confused by the capitalization of daf-16 and DAF-16, note that the former refers to the gene, and the latter to the corresponding protein. But you're not alone, since the opposite convention is sometimes used.)

I suppose that, at this point, the suspense is killing you, or at least delivering a credible threat to curtail your life span, so I'll just summarize what has been learned over the years about daf-2, daf-16, related genes, and how sir2-1 fits into the picture.

The hormone insulin plays an important role. In mammals insulin has a signaling function that stimulates cells to take up glucose and metabolize it. However, its role in C. elegans is somewhat simpler. There it doesn't directly affect glucose metabolism, but it still acts as a signal, as a trigger of the so-called "insulin-signaling pathway". This pathway keeps the daf-16 gene turned off as long as a cell-surface receptor detects insulin.

The protein coded for by daf-16, namely DAF-16 (duh), is a transcription factor, which means it enables the expression of other genes. When this happens in an immature worm, the result is an alternative developmental path, in which the worm enters a larval state, called a "dauer" (German for "enduring"). (The name "daf" is short for "dauer formation".) A dauer will eventually, after some delay, develop into a normal adult anyhow. But evolution has provided this dauer stage in case times are lean, and a delay will allow the organism to survive a little longer, on the hope that better times will come soon.

Under normal conditions, when sufficient nutrients are available, insulin is produced. A cell surface receptor (DAF-2, coded for by daf-2) detects the insulin and initiates a signaling cascade within the cell, and this in turn keeps daf-16 inactive. This does no harm to the organism, and in fact worms get along just fine even without a daf-16 gene, assuming adequate nutrition.

However, assuming insufficient nutrients, insulin levels drop. If that happens early enough in the nematode's life, daf-16 becomes active and triggers the dauer state. But what occurs after the nematode reaches adulthood and daf-16 becomes active (due to low insulin level) is perhaps even more interesting: the worm's aging slows down, and total life span increases. So this is a second way that the worm, even after it reaches adulthood, may be able to survive when food runs low, in the hope for better times.

Why isn't this second scenario simply the normal one? Why bother with the dauer stage at all? The answer is probably that nature has found this "live fast and die young" strategy the most successful in the long run, just as with small rodents. After all, a C. elegans is pretty small – 959 cells and about 1 mm in length. It's easy prey to larger predators that can enjoy a nematode meal. On the other hand, in cases there's not enough food for the worm to "live fast", it's nice to have not one but two backup strategies.

So where does sir2-1 fit in to all of this? Well, just as with SIR2, the worm homologue produces a deacetylase enzyme that inhibits the production of other proteins. One or more of these proteins is a necessary part of the signaling cascade that insulin initiates to keep daf-16 inactive. So extra sir2-1 protein interferes with the insulin signaling and, in effect, activates daf-16, which slows down aging, and extends life span – even when adequate amounts of nutrients are available.

Pretty neat, eh? That's evolution for you – always coming up with the Rube Goldberg schemes.

OK, that's how sirtuins work in nematodes. What about mammals, like us? As you might suppose, since mammals have far more than 959 cells in their bodies, things are a lot more complicated. There are even (at least) seven different homologues of SIR2. But the fact that in worms sir2-1 messes with insulin signaling and metabolism is a clue. Those are pretty important processes in mammals too.

To be continued.


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Further reading:

daf-16: An HNF-3/forkhead Family Member That Can Function to Double the Life-Span of Caenorhabditis elegans (11/14/97)

Reproductive Signals Affect Lifespan In Roundworm C. Elegans, Offering Possible Insight Into Human Aging Process (5/27/99)

Smell, Taste May Influence Lifespan Of The Roundworm C. Elegans (12/17/99)

Long-Lived Worms (3/8/01)

University Of Colorado Researchers Identify Switch That Controls Aging In Worms (12/11/01)

Stem Cells For Eggs And Sperm Also Control Aging In Roundworm (1/18/02)

DAF-16 Target Genes That Control C. elegans Life-Span and Metabolism (4/25/03)

Scientists Find What Type Of Genes Affect Longevity (7/1/03)

Old Worms, New Aging Genes (8/2/03)

Methuselah Worm Remains Energetic for Life (10/27/03)

Signs Of Aging: Scientists Evaluate Genes Associated With Longevity (4/18/05)

For The First Time: Longevity Modulated Without Disrupting Life-sustaining Function (3/11/06)

Eat Less, Live Longer? Gene Links Calorie Restriction To Longevity (5/2/07)

Genes That Both Extend Life And Protect Against Cancer Identified (10/15/07)

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Tags: sirtuins, aging, longevity, calorie restriction, caloric restriction