When Rozalyn Anderson asked me to contribute an article on my reflections of the field of aging, I immediately thought of how much the field had changed since I initiated my research at Illinois State University in 1971. This was the same year that the White House Conference on Aging recommended the creation of the National Institute on Aging (NIA). Before the establishment of NIA, aging research was funded through the National Institute for Mental Health. Although President Richard Nixon vetoed the bill creating NIA in 1973, Congress in 1974 granted authority to form NIA, which would “provide leadership in aging research, training, health information dissemination, and other programs relevant to aging and older people” (taken from NIA website). Because there were no study sections for aging, aging grants were reviewed by study sections with no expertise and by reviewers with little to no interest in aging. At this time, many researchers outside of aging viewed aging as a difficult if not pointless problem to study. I saw this firsthand at a meeting NIA organized in the mid-1970s where various leaders in aging described their research to a panel of leading researchers outside of aging. I remember vividly when, Ira Wool, who was internationally recognized for his research on characterizing the ribosome, told everyone at the meeting, including NIA director and staff, that NIA should be supporting basic biological research and not aging research because aging just occurs randomly over time; it was pointless to study aging because no biological process was involved. I thought at the time, “my God how am I ever going to get funded with study section reviewers who have such a bias.” Fortunately, not all reviewers had such a bias. In the fall of 2003, 2 study sections were initiated that were designed to review grants that focused on aging: Cellular Mechanisms in Aging and Development (CMAD) and Aging Systems and Geriatrics (ASG). Two years later, these study sections were officially chartered as members of the NIH study sections.
The major problem that confronted researchers studying aging in the 1970s was that our research was primarily descriptive. Most of us were studying the effect of aging on some biological process rather than directly testing mechanisms. While it was important to define how aging was affecting various pathways and molecular and physiological processes because so little was known at this time, this research was not mechanistic and, therefore, not highly reviewed. The lack of interventions that retarded aging that could be used to test mechanisms was the primary problem that faced investigators studying aging in the 1970s and 1980s. In this article, I have highlighted those intervention studies, which I feel played a key role catalyzing the dramatic growth of biological research in aging and changed our perception of how aging occurs at the molecular level.
The Era of Caloric Restriction
In 1970, the only intervention known to increase life span was caloric restriction (CR), which had been discovered by McCay in 1935 (Figure 1A) (1). Although numerous studies over the following 3 decades showed that CR increased the life span of rats and mice (2,3), there were concerns even in the aging community in the 1970s as to whether CR increased life span by retarding aging. During the 1970s and 1980s, research lead by Edward Masoro at the University of Texas Health Science Center with rats and Roy Walford at the University of California at Los Angeles with mice demonstrated conclusively to the research community that CR had a major impact on aging, for example, it prevented/delayed the incidence of most age-related diseases and pathologies in rodents and improved a wide variety of physiological processes that declined with age. It is impossible to overstate how important the research by Masoro and Walford was to the field of aging because their research demonstrated conclusively for the first time that aging could be delayed in a mammal resulting in a more youthful phenotype. Because of their research, CR became the gold standard used for retarding aging. In addition, this research opened the door to the first studies to test potential mechanisms that could play a role in aging by studying the pathways attenuated by CR.
The effect of some of the major aging interventions on the life spans of rodents and invertebrates. Panel A: The life span of male rats fed a restricted diet or ad libitum. Data taken from McCay et al (1). Panel B: The life span of the age-1 (hx546) mutant of nematodes. Data taken from Friedman and Johnson (4). Panel C: The life span of yeast expressing the RAS gene. Data taken from Chen et al (5). Panel D: The life span of male Ames dwarf (df/df) mice. Data taken from Bartke et al (6). Panels E and F: The life spans of male and female mice fed rapamycin. Data taken from Harrison et al (7).
The research of Masoro and Walford sparked the interest of many new investigators to study CR in rodents, such as myself, Rick Weindruch, Don Ingram, George Roth, Jim Nelson, Dave Harrison, Brian Merry, Roger McCarter, Walter Ward, B. P. Yu, and Gab Fernandes, to name a few. The 1983 Gordon Conference on the Biology of Aging brought together the leaders in CR for the first time (Figure 2). Research in the 1980s and 1990s demonstrated that CR prevented/attenuated changes that occurred in almost every pathway or process that was altered by age. Therefore, the hope that CR would help identify a specific pathway or mechanism responsible for aging was not achieved.
The 1983 Gordon Research Conference on the Biology of Aging. The Conference was chaired by Edward Masoro and held at Holderness School. It was the first major national meeting to focus on caloric restriction. The investigators studying caloric restriction (left to right starting at the bottom) are: 1—Rick Weindruck, 2—Roy Walford, 3—Dave Harrison, 4—Ed Masoro, 5—George Roth, 6—Gabe Fernandes, 7—Caleb “Tuck” Finch, 8—Dike Kalu, 9—Jim Nelson, 10—Don Ingram, 11—Jim Joseph, 12—Roger McCarter, 13—John Holloszy, 14—Arlan Richardson, 15—Jerry Herlihy, 16—B. P. Yu, 17—Helen Bertrand, and 18—Walter Ward.
In the late 1980s, 2 groups initiated studies testing the long-term effect of CR in rhesus monkeys: Rick Weindruch and Joe Kemnitz at the University of Wisconsin and George Roth and Don Ingram at the NIA Intramural Research Program. These studies were conducted to a large extent because researchers at the time questioned the relevance of CR to long-lived species such as humans. Over the next 3 decades, the research generated by these 2 groups (which later included Rozalyn Anderson and Ricki Colman at the University of Wisconsin and Mark Lane and Julie Mattison at NIA) showed that CR improved the health (8) and longevity (9) of rhesus monkeys much as it did in rodents. To date, CR is the only aging intervention that has been shown to retard aging in a nonhuman primate, demonstrating that interventions that increased life span and retarded aging in rodents have the potential to also work in long-lived species such as primates.
In 2001, NIA initiated the CALERIE (Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy) study, which was the first clinical trial to study the effects of prolonged CR on healthy human participants (10). It was conducted at 3 sites: Pennington Biomedical Research Center directed by Eric Ravussin and Donald Williamson, Tufts University directed by Susan Roberts, and Washington University directed by John Holloszy and Luigi Fontana. The study demonstrated the feasibility of sustained human CR (for 2 years) and showed improvement of predictors of longevity and cardiometabolic risk factors (11,12). Thus, the effect of CR on aging appears to translate from rodents to humans, demonstrating the usefulness of rodents in studying interventions of aging.
The Era of Genetic Manipulations in Aging
In the 1980s, a major shift in aging research occurred that had a profound effect on the field, the use of invertebrate models to study aging. Drosophila (Drosophila melanogaster) had been used in aging research to a limited extent in the 1960s and 1970s; however, most investigators studying aging at the time were skeptical about the relevance of findings in Drosophila to mammals. The recognition in the late 1970s that organisms from yeast to humans employed similar molecular mechanisms to regulate most biological processes led to the wide use of invertebrates in biomedical research. The ability to genetically manipulate invertebrates combined with their short life span, made them particularly attractive for aging research. Tom Johnson and Mike Jazwinski were the first advocates for using nematodes (Caenorhabditis elegans) and yeast (Saccharomyces cerevisiae), respectively, in aging research.
The initial studies with invertebrates in the 1980s focused on determining if it was possible to select for long-lived invertebrates. Michael Rose (13) and Leo Luckinbill and Bob Arking (14) obtained long-lived Drosophila by selecting female flies for late-life reproduction. These studies demonstrated that long-life and delayed senescence were genetically regulated; however, as with CR, studies with the long-lived strains of Drosophila did not lead to any major insight into the molecular pathways underlying aging because the long-lived flies showed a wide range of changes. Michael Klass took another approach to generating long-lived nematodes; he treated nematodes with a mutagen and selected for long-lived mutants. At the time, it was believed that to increase the life span in an animal, you would have to improved fitness/function. Therefore, most investigators in aging were skeptical as to whether a loss of function mutation, as one would obtain by treating nematodes with a mutagen, would yield longevity mutants. However, Klass obtained several mutants that appeared to be long-lived (15). It was assumed that these long-lived mutants had mutations in several genes, which lead to their increased longevity. However, when Tom Johnson outcrossed some of these mutants, he discovered a long-lived mutant with a recessive mutant in one gene, which he named age-1. The mutation increased life span 40% to 65% (Figure 1B) (4). Subsequently, he showed that the age-1 mutation reduced the exponential increase in mortality rate, demonstrating that the age-1 mutants were aging more slowly (16).
Tom Johnson’s discovery had an enormous impact on the field because it demonstrated that one gene could influence aging, which provided the first model that could be used to directly identify a pathway(s) involved in aging. Because of Tom’s research, Cynthia Kenyon became interested in using nematodes to study the genetics of aging (17). Her group screened for long-lived nematode mutants and in 1993 reported another long-lived mutant, daf-2 (18). Subsequently, Gary Ruvkin’s group showed that the age-1 gene encoded a phosphatidylinositol 3-kinase (19), and daf-2 encoded the nematode homologue of the human insulin and IGF-1 receptors (20), which activates downstream PI3 kinase pathways. Thus, the insulin/IGF-1 pathway was the first pathway to be shown to play a role in aging in any organism.
Shortly after Tom Johnson had reported that mutations in the age-1 gene increased the life span of nematodes, Mike Jazwinski reported that overexpressing the RAS gene increased the replicative life span of yeast (Figure 1C) (5). Overexpressing RAS appeared to increase the longevity of yeast by maintaining homeostasis through metabolic control and resistance to stress (21). Interestingly, some aspects of metabolic control in yeast induced by RAS resembled the metabolic consequences seen in nematodes and Drosophila selected for extended longevity and CR in rodents. Subsequently, Lenny Guarante’s group identified long-lived yeast with mutations in the gene encoding SIR4 (22). Based on the reports in nematodes and yeast, investigators began looking for genes or mutations that lead to increased life span in Drosophila. The initial studies with Drosophila showing that the overexpression of EF-1α (23) or catalase and Cu/Zn-superoxide dismutase (24) increased the life span of Drosophila suffered from poor controls and were not replicated. In 1998, Seymour Benzer’s group reported that mutations in the mth gene, which is homologous to G protein-coupled receptors, increased the life span of Drosophila (25). Two years later, Steve Helfand’s group discovered that mutations in the Indy gene almost doubled the life span of Drosophila (6). The Indy gene coded for a cotransporter gene that is closely related to the mammalian sodium dicarboxylate cotransporter, and it was argued that these mutants create a metabolic state that mimics CR.
By the early 1990s, it was apparent that life span could be extended dramatically in nematodes and yeast by a single gene. The major question at this time was whether single gene mutations could increase the life span of mammals or was this unique to invertebrates. In 1996, Andrzej Bartke’s group made the seminal discovery that Ames dwarf mice (df/df) live significantly longer that their normal siblings (Figure 1D) (26). The Ames dwarf mice have a mutation in the Prop-1 gene, which results in a failure of somatotropes and lacotropes to differentiate, leading to mice deficient in growth hormone, prolactin, and thyroid stimulation hormone. This discovery was even more surprising because the aging community believed that dwarf mice were a model of rapid aging. Dwarf mice initially had been reported to be short-lived (27) and growth hormone production was shown to decreased with age (28). In 2001, Dave Harrison’s group reported that Snell dwarf mice, which have a mutation in the Pit-1 gene, also showed an increase in life span (29). Subsequently, it has been shown that mutations in growth hormone receptor, growth hormone-releasing hormone, and growth hormone-releasing hormone receptor increased the life span of mice (for review, see (30)). These data indicated that the growth hormone/IGF-1 pathway plays an important role in mammalian aging.
The research in the 1990s demonstrating that mutations lead to increased life span prompted investigators to study how prevalent longevity gene mutations were in invertebrates. Gary Ruvkin’s group used a systematic RNAi screen to identify gene inactivation mutations that increase life span. In 2003, they reported that RNAi inactivation of ~2% of the of 5690 genes analyzed lead to a significant increase in life span of 5% to 30%; 15% of these mutants were specific for mitochondrial function (31). The laboratories of Matt Kaeberlein and Brian Kennedy also found that ~2% of the 564 single-gene deletion strains of yeast tested showed an increase in replicative life span, with one of these genes encoding components of the TOR (target of rapamycin) signaling pathway (32). Based on their data, they predicted that 100 to 120 “aging genes” were potentially possible in yeast (7). Currently, more than 800 genes have been reported to increase the life span of nematodes and over 50 genes have been reported to increase the life span of mice (33).
Era of Pharmacological and Pharmaceutical Interventions in Aging—The Aging Intervention Testing Program
It is impossible to overstate how important the ability to genetically manipulate invertebrates and mice was to the advancement of our understanding of potential mechanisms responsible for aging. However, translating these discoveries to pharmacological/pharmaceutical interventions that potentially could be taken to humans was limited at the beginning of the 21st century. The establishment of the NIA-funded Intervention Testing Program (ITP) in 2004 was a major step toward identifying pharmacological/pharmaceutical interventions in aging. Don Ingram came up with the concept of a program to test longevity-increasing interventions in mice, and in 1997, he proposed such a program to the Board of Scientific Councilors of the Intramural NIA Program. Working with Huber Warner, who was the Director of the Division of Aging Biology at NIA, they organized a workshop, which was held in Texas in 1999, to discuss the concept of such a program. As part of the workshop, I remember that all of the participants were very positive about need for a program that would rigorously test the ability of compounds to increase the life span of mice. At the time, there were a number of “antiaging” compounds being promoted in the news media, for example, DHEA (dehydro-3-epiandrosterone) and melatonin, for which the life-span data were weak. While there was strong support for the program, the general consensus was that it was not realistic to argue that the primary goal of the program was to identify compounds that increase life span because the research community generally felt it was unlikely that any one compound would have a significant impact on aging/life span. Rather, it was felt that it would be better to argue that such a program was needed to test rigorously whether compounds had a significant impact on life span by an independent group of investigators who had no involvement in the compounds being studied. The workshop recommended that the life span should be tested at 3 sites in both male and female UM-HET3 mice, a line of genetically heterogeneous mice that had been recently developed by Rich Miller. As a result of the workshop, Huber Warner presented the concept of the ITP to the annual NIA retreat in 1999: “at first, Dr. Ingram and I could convince few if any of our NIA colleagues that this was a worthwhile enterprise. Nevertheless, we tried again the following year, once more with only limited success, but on third try, Dr. Hodes gave us the green light...” (34). In 2003, a request for applications (RFA) was issued, and 3 sites were funded in 2004: the Jackson Laboratory led by Dave Harrison, the University of Michigan, led by Rich Miller, and the University of Texas Health Science Center at San Antonio, led by Randy Strong. Since 2004, the ITP has been testing the ability of compounds to increase the life span of mice as a measure of a compound’s ability to retard aging. These compounds were proposed to the ITP by investigators studying aging.
A major breakthrough occurred in 2009, when the ITP reported that rapamycin significantly increased the life span of both male and female mice (Figure 1E and F) (35). Dave Sharp had proposed testing rapamycin to the ITP in 2004. He was a very creative molecular biologist studying cancer at the University of Texas Health Science Center in San Antonio and was part of a program project I directed in 1998–2003 that used transgenic and knockout mouse models to study CR. Dave became interested how reducing calories could delayed aging at the molecular level. Around 2000, he came up with the idea that TOR might be the key to CR because it was a nutrient sensor. Based on data from Michael Hall’s group (36), which showed rapamycin mimicked the starvation phenotype in yeast by inhibiting TOR, Dave hypothesized that the reducing TOR signaling by feeding mice rapamycin would lead to increased life span. He was not able to obtain funding for this study because it was viewed as very risky and because there were no data on treating mice long term with rapamycin. The brief report in 2003 by Vellai et al (37) showing that TOR deficiency doubled the life span of nematodes provided Dave with the support he needed to convince the ITP to test rapamycin.
In conducting the rapamycin study, the ITP was faced with the problem of how to administer rapamycin to the mice. All of the previous studies injected rapamycin intraperitoneally, which was not realistic to do in a life-span experiment. However, ~85% of the rapamycin was degraded in preparing the diet containing rapamycin. Therefore, Randy Strong came up with the novel concept of encapsulating rapamycin with a material developed by the Southwest Research Institute (San Antonio) that would protect rapamycin from degradation in the diet as well the stomach and would be solubilized by the basic pH in the small intestine, thereby delivering rapamycin to the animal. The ITP had generated a cohort of UM-HET3 mice so they could start feeding rapamycin at 4 months of age. However, the mice were ~19 months of age when the rapamycin-containing diet became available. There was a discussion of whether to study the effect of treating these mice with rapamycin because at this the time the existing data on CR indicated that an intervention had to be implemented relatively early in life for it to be successful. Fortunately, the ITP decided to feed the old mice rapamycin and the rest is history. The ITP not only discovered the first pharmaceutical intervention to increase life span in a mammal, but equally important, they demonstrated for the first time that an intervention could increase the life span of mice when initiated later in life. The editors of Science selected this discovery as one of the top 10 scientific breakthroughs in 2009 (Science 326, 1602–1603, 2009). This was the first time a discovery in aging was recognized by the scientific community as a major breakthrough in science. Two years after the discovery that rapamycin increased the life span of mice, a second discovery in aging was selected by Science as one of the top 10 scientific breakthroughs (Science 334, 1635, 2011). van Deursen’s group discovered that genetically removing senescent cells postponed age-related disease in mice (38). Currently, 15 studies have shown that rapamycin increases the life span of various strains of mice (33,39), demonstrating the robustness of the effect of rapamycin on life span of mice. Rapamycin also been shown to attenuate many age-related conditions, including cancer, neurodegeneration (including Alzheimer’s disease), and cardiac disease/function (39).
Translating Basic Biomedical Research to the Clinic
For the first time in human history, we were in a position to test aging interventions in humans that have been rigorously validated to increase life span and retard aging in animal models. However, several hurdles needed to be overcome before this was possible. First, a protocol for testing the ability of a drug to affect aging in humans needed to be developed because it is impractical to do a life-span study to demonstrate the efficacy of a compound in humans as is done with invertebrates and mice. The first step in developing such a protocol was initiated in 2013 when Steve Austad, Jim Kirkland, and Nir Barzilai received a R24 grant from NIA to bring together experts in the biology of aging and clinical geriatrics to discuss how to conduct a clinical trial that targets aging in humans. As a result of this grant, the first clinical, geroscience-guided trial, TAME (Targeting Aging and Metformin) was proposed. Metformin was chosen as the first drug to text because it had been safely used to treat diabetes for over 60 years with minimal side effects, it reduced the incidence of many age-related diseases, and altered several pathways known to be involved in aging (eg, insulin levels, IGF-1 signaling, and mTOR signaling) (40). With Nir Barzilai leading the effort, the TAME trail received FDA approval in 2015 and funding at the end of 2019. Ironically, the funds for clinical trial of TAME came from private sources rather than NIA/NIH. TAME is a randomized clinical trial involving 14 institutions with over 3000 participants. The trial will follow the incidence of age-related diseases and a panel of 9 blood-based biomarkers (41) in participants receiving metformin or a placebo control. In the next decade, we should know if the unique protocol developed for the TAME clinical trial can be used for testing the effect of other drugs on aging in humans.
Another problem confronting aging researchers is how to take compounds that the ITP has found to work in mice to humans. In developing the ITP, we never guessed that it would be as successful as it has been; therefore, there has been no serious discussion to date as to what steps should be used when taking a compound from mice to humans. One cannot assume that pharmacological interventions in mice will automatically translate to humans, for example, only about 1 in 10 successful mouse cancer interventions ever makes it to the clinic and none of the 300+ mouse interventions in Alzheimer’s disease has proven effective in humans. Due to the cost in time and money of human clinical trials, it is critical that that NIA develop a plan/program to test the translatability of compounds identified by the ITP in mice, for example, determine the effect of the compounds on aging in other species as well as screening for potential side effects.
Even in the absence of a such a plan, several investigators have begun testing the translatability of the ITP data. Currently, Daniel Promislow and Matt Kaeberlein at the University of Washington are studying the effect of rapamycin on aging in dogs as part of their Dog Aging Project (42), and Adam Salmon is funded to study the effect of rapamycin on aging in the common marmoset (43). Steve Austad and I have proposed using rats as a follow up to the ITP studies with mice. Rats differ from mice in a variety of physiological parameters making them more similar to humans in many aspects than mice, including end-of-life pathology and drug toxicity (44). A major advantage to using the rat is their life span is relatively short (similar to mice), for example, much shorter than that of dogs or nonhuman primate models, making them an ideal model to quickly test the antiaging effects of compounds identified by ITP.
Summary
Over the past 50 years, basic biomedical research in aging has gone from a relatively obscure area of research to one of the hottest areas of science resulting in major discoveries in science in 2009 and 2011 (summarized in Figure3). In 1970, it was generally believed that it would be difficult to intervene in aging. However, we now know that mutations in hundreds of genes can increase the life span of invertebrates and dozens of genes increase the life span of mice. In addition, the ITP has been much more successful than originally believed possible. Currently, the ITP has identified 4 compounds that have consistently shown over a 10% increase in the life span of mice: rapamycin, 17α-estradiol, acarbose, and nordihydroguaiaretic acid. Three other compounds (aspirin, protandim, and glycine) show a modest (<10%) but significant increase in life span.
Summary of the major discoveries and developments in the biology of aging in the past 50 years.
In closing, I feel it is important to recognize 2 unsung heroes who played a major role in the dramatic advancements in biological research in aging over the past 25 years: Huber Warner and Felipe Sierra. They served as directors of the Division of Aging Biology at NIA from 1989 to 2020. Based on input from the research community, they were responsible for steering the research funded by the Division of Biology on Aging through its most productive era to date. Huber was responsible for the Division’s push to identify specific genes involved in aging (the Longevity Assurance Genes, LAG, initiative) as well as the development of the ITP, as described above. Felipe was single-handedly responsible for the development of a new field in aging: Geroscience, which is the study of mechanisms through which changes in cellular/tissue function with age contribute to the onset and progression of specific diseases. Previously, most researchers studying aging went to great lengths not to study old animals when they had major diseases, such as cancer. They wanted to study “pure” aging in the absence of disease. Conversely, Institutes studying major age-related diseases ignored the role aging played in these diseases. Felipe was able to convince other Institutes at NIH about the importance of aging in the etiology of the age-related diseases they were studying. His efforts led to the creation of the Trans-NIH GeroScience Interest Group (GSIG), which focuses on exploring the intersection between aging biology and the biology of diseases. The GSIG currently involves over 20 NIH Institutes and has led to increased funding for basic research in aging through RFAs from Institutes other than the NIA, interested in exploring the role of aging in the particular diseases each Institute is studying.
Arlan Richardson received his PhD in chemistry from Oklahoma State University and did his postdoctoral training at the University of Minnesota. In 1971, he joined the faculty at Illinois State University and began his research studying the role of gene expression in aging and caloric restriction in rats. His group was the first to show that caloric restriction altered the expression of specific genes through changes in transcription factors. In 1990, he joined the faculty at the University of Texas Health Science Center at San Antonio where he became the Founding-Director of the Barshop Institute on Longevity and Aging Studies in 1995. At San Antonio, Dr. Richardson changed his focus from rats to mice to take advantage of the ability to genetically modify mice. With Dr. Holly Van Remmen, initially a postdoctoral fellow in his laboratory and later a collaborator, he directly tested the role of oxidative stress in aging using various transgenic and knockout mouse models with alterations in the antioxidant defense system to reduce or increase oxidative stress. Data generated over 15 years with multiple genetic models demonstrated that altering the level of oxidative damage had no significant impact on the life span of mice, calling into question the role oxidative stress has in aging. In 2013, Dr. Richardson joined the University of Oklahoma Health Sciences Center where he continues to study caloric restriction. Dr. Richardson served as President of the American Aging Association and the Gerontological Society of America. In addition, he has received the Robert W. Kleemeier Award from the Gerontological Society of America, the Harman Research Award from the American Aging Association, the Irving Wright Award of Distinction in Aging Research from the American Federation for Aging Research, and the Lord Cohen Medal for Services to Gerontology from the British Society for Research on Ageing.
Acknowledgments
I want to thank Drs. Donald Ingram, Felipe Sierra, Holly Van Remmen, and Michal Jazwinski for their helpful comments when reviewing the manuscript.
Funding
In writing this manuscript, the author was supported by a Senior Career Research Award from the Department of Veterans Affairs.
Conflict of Interest
None declared.
References
