Rapamycin & mTOR Signaling in the Modulation of Healthspan & Lifespan: Potential and Problems
October 4, 2010
The Union Club
New York, NY
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About the Conference
Recent success in lifespan extension of mid- and late-age male and female mice by chronic treatments with the drug rapamycin could lead to a new era of pharmacological intervention for aging and age related diseases.
This conference will highlight the new and exciting discoveries in our understanding of the major metabolic pathways associated with the target of rapamycin (TOR) signaling and how these insights relate to the regulation of aging. In addition, the role(s) that TOR signaling plays in the major diseases related to aging and how preemptive intervention may lessen their burden will be a focus. TOR and associated signaling pathways in unicellular and multicellular eukaryotes are ripe for discoveries of new and better drug therapies. The conference will therefore explore the feasibility of clinical trials for aging intervention and/or disease prevention.
The conference will spotlight recent discoveries of next generation mammalian TOR inhibitors and delivery systems and address the questions: how can they best be used as tools to better understand aging and will they be better or worse than rapamycin as potential interventional approaches for the modulations of healthspan and lifespan?
Steve Austad, PhD
University of Texas Health Science Center
Richard W. Besdine, MD
Alpert Medical School of Brown University
George M. Martin, MD
University of Washington School of Medicine
Roger McCarter, PhD
Pennsylvania State University
Z. Dave Sharp, PhD
University of Texas Health Science Center
Terrie Fox Wetle, PhD
The Ellison Medical Foundation
The Glenn Foundation for Medical Research
National Institute on Aging-National Institutes of Health
On Monday, October 4, 2010, more than 100 researchers gathered at the Manhattan’s Union Club in New York City to discuss the role of rapamycin and its target protein, the mammalian target of rapamycin (mTOR). mTOR sits at the nexus of a complex web of signaling processes that coordinates growth factor, energy and nutrients for cellular growth and metabolism. A growing body of evidence links these signals with both aging and pathways involved with increased longevity mediated by caloric restriction.
An introduction to rapamycin and mTOR
Rapamycin is a chemical produced by a soil bacterium discovered on Easter Island (Rapa Nui). Although scientists originally developed the chemical for its antifungal properties, they soon found that it could suppress the immune system. It’s currently used medically as an immunosuppressant for transplant patients and as an anti-cancer drug. Scientists discovered that rapamycin binds to another protein FKBP12, which together inhibits the activity of the kinase, mTOR. mTOR serves as a signaling hub within a cell, monitoring the status vital information as nutrients (eg. amino acids), energy, hormone signals such as insulin and growth factors such as insulin like growth factor I (IGF-I). mTOR kinases are highly conserved across species, and they act by forming two different complexes, mTORC1 and mTORC2 (mTOR complex 1 and 2). The downstream effects of mTOR modulate cellular growth (in mass) and metabolism and are posited to have a major role in caloric restriction-mediated longevity.
Rapamycin and mTOR as promoters of lifespan and healthspan
As early as a decade ago, Z. Dave Sharp of the University of Texas Health Science Center (UTHSC) in San Antonio thought that rapamycin could improve longevity and health during that extended life. But because of this natural product’s ability to suppress the immune system, he says, the idea seem far-fetched to many within the research community. Over the last 20 years, a growing body of evidence has connected smaller cell and body size with longevity. Caloric restriction can retard cell growth, but data from Michael Hall’s laboratory at the University of Basel had also shown that adding rapamycin to yeast cultures served as a molecular trick which signaled cells to ignore the food. Those results pointed to mTOR as an important mediator of nutrient, thus age-related, signaling.
But recent work by Sharp and his colleagues has demonstrated that the connection between caloric restriction and the mTOR pathway is not straightforward: mice on a calorie-restricted diet show differences in mTOR activity that vary by the time of day. In the mornings, these mice show lower activity in mTOR overall. Later in the day, calorie-restricted mice show much higher mTOR activity than their counterparts on a normal diet.
Other studies had demonstrated that rapamycin treatment could extend lifespan in yeast, fruit flies and in the worm, C. elegans, so Sharp and his colleagues designed a multi-site study to see whether chronic rapamycin treatment mimicked the effects of caloric restriction in mice. In a study published in Nature in 2009, Sharp and his colleagues at the Intervention Testing Program (ITP) demonstrated for the first time that a specially prepared formulation of rapamycin developed by Dr. Randy Strong of the ITP also extends lifespan in mammals. Rapamycin showed similar effects whether first administered to older mice (20 months) or to young mice (9 months). Chronic rapamycin treatment increased the longevity of both groups overall, and the oldest mice in each group lived almost 20 percent longer. Almost all of the rapamycin-treated mice eventually died of cancers.
Follow-up studies have confirmed these original findings and researchers have also been examining the healthspan of mice: looking at whether mice treated with chronic rapamycin show health traits and behavior consistent with younger animals. A number of videos demonstrated that male and female mice at 38-months of age, equivalent to a human centenarian, are far more active and have shinier coats than their untreated counterparts of a similar age. Dosing studies indicate that 14 ppm, the dose used in previous studies, appears to be optimal when compared with 4ppm and 40 ppm.
In collaboration with Carolina Livi of UTHSC, Sharp and his colleagues have looked at the effect of doses of rapamycin on gene expression in mouse fat and liver tissues. Approximately 40 genes in fat and 50 in the liver are regulated by rapamycin. Rapamycin regulates three genes that occur in both tissue types, which makes them potential biomarker candidates for treatment with the drug.
Although the effects of rapamycin treatment and caloric restriction overlap, they also have distinct differences, Sharp says. Both strategies delay age-related pathology. Caloric restriction can both increase and decrease mTOR signaling. Unlike caloric restriction, rapamycin treatments are effective even when started late in life. Rapamycin treatment does not produce leaner and lighter mice, and treatment with the drug decreases the body’s ability to regulate glucose.
mTOR’s connection to growth
Michael Hall of the University of Basel gave an extensive overview of mTOR signaling. mTOR is a highly conserved protein kinase whose function is to control cell growth in response to nutrients. The complexity and diversity of mTOR signaling is mediated by two structurally and functionally distinct mTOR complexes termed mTORC1 and mTORC2. mTORC1 and mTORC2 control a large number of cellular processes that collectively determine cell growth and aging, including transcription, protein synthesis, ribosome biogenesis, nutrient uptake, and autophagy. mTORC1 phosphorylates 4E-BP and S6K, and is sensitive to rapamycin. mTORC2 phosphorylates Akt and other targets, and is insensitive to rapamycin. Hall presented unpublished results from his laboratory on the regulation of mTORC2.
The diabetes drug metformin and mTOR signaling
George Thomas of the University of Cincinnati described how the diabetes drug metformin interacts with the mTOR signaling pathway. Previously, researchers had thought that this drug regulated mTOR signaling through the enzyme 5’-adenosine monophosphate-activated kinase (AMPK), which is regulated by glucose signaling. However, Thomas and his colleagues found that metformin works differently than previously thought—it regulates mTORC1 but independently of AMPK. Unlike rapamycin and other small molecules which can erode insulin sensitivity, metformin improves sensitivity to insulin. Therefore this drug could have wider applications as an anti-aging compound.
Thomas and his colleagues are also working on studies in fruit flies to better understand the role of ribosomal S6 kinase (S6K), an enzyme regulated by mTOR and important for cell growth and aging.
Linking mTOR to protein translation
Brian Kennedy of the Buck Institute for Age Research in Novato, California, discussed data that ties dietary restriction, mTOR signaling and aging to translation, the production of proteins within cells. mTOR signaling represents a particularly interesting mechanism for controlling translation, he says, because researchers already know that mTOR activates S6K which sends signals to a number of ribosomal proteins and translation initiation factors.
One of the promising areas for this type of research, Kennedy says, is the emergence of the “anti-aging” compounds, rapamycin and resveratrol. If researchers can extend this list of compounds, this small molecule approach could both improve understanding of the complex network of pathways involved in the aging process and provide potential treatments.
Kennedy and his colleagues have been looking at connections between aging and translation in several organisms. They initially thought that just turning down the translation of ribosomal proteins might conserve energy within a cell and therefore increase lifespan. But as they looked at the knockdown of genes involved in increased longevity, those genes primarily produced proteins in a single subunit of the ribosome, the 60s subunit, rather than being distributed among both ribosomal subunits. Instead, the results connected lifespan extension with altered translation of mRNA from specific genes. In most cases, long-lived yeast mutants translate mRNA less efficiently, Kennedy says. But a few genes, such as that of the yeast transcriptional activator GCN4, are translated more efficiently. This induction of GCN4 translation occurs normally under conditions of dietary restriction and reduced 60S subunit biogenesis copies this effect. Kennedy presented data indicating that enhanced GCN4 translation under these conditions is important for extending yeast lifespan.
Kennedy and his colleagues are also developing mouse models that lack some ribosomal proteins as possible models of various age-related diseases. One such model has a deletion in Rpl22, which shows a normal response to a high-fat diet but is protected against heart enlargement caused by angiotensin. Female mice with a deletion in Rpl29 are also protected in the angiotensin assay and from obesity when fed a high-fat diet.
In addition, the researchers are studying A-type lamin proteins. Mutations in these proteins are connected with genetic diseases including various forms of muscular dystrophy and Hutchison-Gilford progeria, a disease believed to be based on premature aging. A mouse knockout of this protein shows 6 to 7 week survival, reduced body weight and muscular dystrophy and cardiomyopathy. Kennedy presented evidence that these mice have enhanced mTOR signaling in the heart and proposed that rapamycin, a mTOR inhibitor, would offset the cardiac pathology.
How dietary restriction affects mTOR signaling
Pankaj Kapahi of the Buck Institute for Age Research is interested in how mTOR mediates the effects of dietary restriction. In earlier work, he and his colleagues showed that blocking mTOR signaling extended lifespan in fruit flies by a process that was similar to the effects of dietary restriction. Turning on mTOR signaling activates processes that lead a cell to grow and reproduce, but turning it off sets off signals that put an organism in survival mode that extends lifespan. mTOR integrates signals from a variety of environmental sources, Kapahi says: nutrients such as glucose and amino acids, ATP, growth factors, insulin, and stresses can all activate this pathway in a variety of organisms.
Kapahi and his colleagues are examining these molecular connections. HIF-1 is a transcription factor that helps cells respond to low oxygen conditions, and it’s also downstream of signals from mTORC1 and S6K. The researchers have demonstrated that HIF-1 critical in mediating lifespan extension through dietary restriction in the worm C. elegans. This effect is particularly important in muscle cells and may be present in other organisms, Kapahi says. Data from human patients suggests patients with elevated HIF-1 have a reduced tolerance for exercise, hinting at a connection with oxidative metabolic pathways in humans.
In fruit flies, Kapahi and his colleagues have shown another way that nutrient signals affect lifespan. Through a TOR-mediated pathway, lower levels of nutrients elevate the activity of 4E-BP, a protein that leads to differential translation of mRNA. As a result, the mitochondria rev up their production of energy within the cell, which extends lifespan in the fruit flies.
Kapahi and his colleagues are also looking at the role of mTOR pathways in inflammation.
The brain on mTOR
In his research at New York University, Eric Klann has been looking at the role of mTOR signaling in learning and memory. Certain types of long-term memory formation are thought to require a type of long-lasting synaptic plasticity called late phase long-term potentiation (L-LTP). Both long-term memory and L-LTP are dependent on the translation of new proteins. Because the mTORC1 complex plays a role in this translation initiation, it also acts within the hippocampus to help form memories. Klann and his colleagues have demonstrated these results in mice, where they developed knock-out mice that lacked the protein 4E-BP2, an mTORC1 substrate. They measured lower levels of LLTP in these mice, and in behavioral tests the mice showed diminished long-term memory consistent with a requirement for mTORC1 signaling in the hippocampus. Other mouse mutants that lacked S6K, another mTORC1 substrate, also showed lowered synaptic plasticity and altered memory.
They’ve also examined the role of FKBP12, the protein that recruits rapamycin’s binding to mTOR, by developing mice with selective deletions of that protein in particular areas of the brain. These mice show enhanced L-LTP and enhanced contextual memory. Even with a boost in some memory processes, these mice have other cognitive and behavioral deficits. These mice tend to prefer familiar objects rather than exploring a novel object, as a normal mouse would. This preference for normal objects is a hallmark of anxiety disorders such as obsessive-compulsive disorder and autism spectrum disorders. These mice also show problems in their working memory in a more complicated memory task. The researchers set up water mazes with a submerged platform that allow the mice to escape the water. When researchers move that platform to various locations, FKBP12 knockout mice will stubbornly search in the old location for the platform, while wild-type mice will search and find the new location of the platform more quickly. FKBP12 knockouts also show other autism-related behaviors such as impaired social interaction.
Because of the overlap of some of behaviors of FKBP12 mice with those observed in autism spectrum disorders, Klann and his colleagues are examining the possible role of mTORC1 signaling in these syndromes. Other genes connected with mTOR signaling have been tied to autism spectrum disorders, including PTEN, TSC1 and TSC2. Fragile X Syndrome is based on a mutation of a single protein, FMRP, that is believed to suppress the translation of particular mRNAs, and many patients with this disorder also fall on the autism spectrum. Work is ongoing in the group to understand the role of mTORC1 signaling in Fragile X and related disorders.
A tale of two complexes: mTORC1 and mTORC2
Dudley Lamming , a postdoctoral researcher in David Sabatini’s group at the Whitehead Institute in Cambridge, Massachusetts, described work performed in that laboratory to uncover the differences between signaling by the two different mTOR complexes, mTORC1 and mTORC2, and the role of those complexes in longevity. mTORC1, which is rapamycin sensitive, includes the proteins mTOR(mammalian target of rapamycin), mLST8, and Raptor. mTORC2, which is relatively insensitive to rapamycin, includes mTOR, mLST8, and Rictor.
The Sabatini lab has developed a strain of mice with partially depleted levels of mTOR and mLST8 (Mice without these proteins cannot survive). Female mice depleted for these two proteins have increased longevity, Lamming says. They are not calorically restricted but show decreased mTORC1 activity, leading the group to name this strain DEnACO mice (Decreased Endogenous Activation of mTOR complex One). While positive changes in glucose homeostasis, such as low insulin levels and improved glucose tolerance and sensitivity, have been linked to longevity in many mouse models and calorie restricted mice, DEnACO mice are more similar to normal mice: they become slightly glucose intolerant with age, gain weight on a high fat diet and can become insulin-resistant in response to that diet. Data from these mice suggest that increased longevity is not exclusively connected with improved regulation of glucose, Lamming says.
Treatment with rapamycin extends lifespan, but the Sabatini lab has found that normal mice treated with rapamycin become glucose intolerant and produce more glucose in the liver. To better understand how this occurs, the Sabatini laboratory has looked at mTOR signaling in the liver. To understand mTORC1 signaling, they developed knockout mice without active Raptor in the liver tissue. These mice had normal glucose tolerance, suggesting that the effects of rapamycin in the liver did not involve mTORC1. However, they found that chronic rapamycin treatment with rapamycin suppresses the activity of mTORC2 in the liver, and when they made knockout mice without Rictor in the liver, they found them to be highly glucose intolerant, Lamming says. The initial results suggest that Rictor is regulating gluconeogenesis in the mouse liver, a process thought to be important in the start of diabetes. It also raises the possibility that mTORC2 signaling, previously thought to be unaffected by rapamycin, could be regulated by this molecule.
The Sabatini lab is also looking at small molecules other than rapamycin that can serve as novel inhibitors of the mTOR pathway. These molecules inhibit the kinase activity of mTOR in both the mTORC1 and mTORC2 complexes and will allow researchers to further probe these effects in living cells.
With the evidence that rapamycin treatment can increase longevity in mice, research into the inner workings of mTOR signaling represents a boon for the field of aging. Because of rapamycin’s activity as an immunosuppressant, researchers are still trying to understand whether this activity will undermine its potential as an anti-aging drug. A major push in the field will be to come up with new compounds or combination therapies that could modulate mTOR signaling in productive ways. Aging-related diseases are intimately connected with the normal biology of aging, the researchers say, which both complicates the research questions and makes them increasingly important to tackle.
In a discussion moderated by Felipe Sierra of the NIH’s National Institute on Aging (NIA), researchers also discussed the funding and public awareness challenges in anti-aging research. The NIA receives the smallest slice of NIH funding, Sierra says, and researchers can’t solely depend on NIH funding to support this type of work. As a result researchers will increasingly need to look for grants for other sources, such as AFAR. AFAR is also in the process of partnering with AARP to provide better public awareness of aging research and its goals of improving both lifespan and healthspan.
Stay tuned for the conference videocast of presentations!