Sci. Aging Knowl. Environ., 12 February 2003
Vol. 2003, Issue 6, p. re1
[DOI: 10.1126/sageke.2003.6.re1]


Subfield History: Use of Model Organisms in the Search for Human Aging Genes

Huber R. Warner

The author is in the Biology of Aging Program at the National Institute on Aging, Bethesda, MD 20892, USA. E-mail: warnerh{at};2003/6/re1

Key Words: National Institute on Aging • longevity • aging • longevity genes • nematode • fruit fly • mouse • human

Abstract: The National Institute on Aging (NIA) started a program in 1993 to identify genes involved in the regulation of longevity in a variety of species, including yeast, nematodes, fruit flies, and mice. The initial success of this program has attracted the interest of many investigators working with these organisms. Of primary interest are single-gene mutants that have identified genes and processes involved in longevity regulation across species. These processes include the insulin-like signaling pathway, stress resistance, and most recently, chromosome and nuclear architecture. Mutations in genes that regulate these processes indirectly are also being identified in this program. The ultimate goal of this program is to extend these results to humans to identify the major biological risk factors for age-related decline of function in human physiological systems.

Introduction Back to Top

After joining the National Institute on Aging (NIA) extramural staff in 1984, I soon became aware of Tom Johnson's early work on the age-1 mutant of Caenorhabditis elegans (see Johnson Subfield History), and the work of Leo Luckinbill and Robert Arking selecting for delayed senescence in Drosophila melanogaster. Being new to the field of gerontology, I was slow to recognize the potential implications of either of these research efforts. Even as Tom began to publish further characterization of the age-1 mutant during the period 1987-1988, I remained skeptical of the generality of the finding that a single mutation could cause an increase in longevity and maximum life-span. This scenario seemed quite unlikely to me, and possibly a result of something anomalous.

Publication: D. B. Friedman, Thomas E. Johnson, A mutation in the age-1 gene in Caenorhadditis elegans lengthens life and reduces hermaphrodite fertility. Genetics 118, 75-86 (1988).

Publication: Leo Luckinbill, Robert Arking, M. Clare, Selection for delayed senescence in Drosophila melanogaster. Evolution 38, 996-1003 (1984).

My attitude about this subject began to change when I attended a meeting at The Jackson Laboratory in September 1988 that was organized by David Harrison. At this meeting, Michael Rose presented his work on evolutionary genetics of aging in Drosophila. After discussing this topic with Michael at the meeting, I became convinced that genes that regulate longevity in animal systems could indeed be identified, and that this should be a high priority for our Genetics Program at the NIA, which was under my general supervision at the time.

When Anna McCormick joined the NIA in 1989 as director of our Genetics Program, I discussed this opportunity with her. She enthusiastically accepted the challenge to develop an initiative on what she called longevity assurance genes, or LAGs. She organized two workshops, the first held in September 1989 at the National Academy of Sciences Beckman Conference Center in Irvine, CA, and the second in June 1991 at the same site, and she encouraged the Glenn Foundation for Medical Research (GFMR) to cosponsor these two workshops. The discussion at these workshops centered around the development of "experimental strategies for the identification of candidate mammalian longevity assurance genes and evaluation of their role in determining longevity." However, in these meetings it was recognized early on that studies in invertebrate systems such as yeast, fruit flies, and nematodes could hold the key for the eventual identification of mammalian longevity genes. As a result of these discussions, McCormick developed a Request for Applications (RFA) soliciting applications to "identify candidate longevity assurance genes in several animal models of aging." This RFA was issued in mid-1992, and grants were made in September 1993 to 11 investigators at a total cost of about $3 million per year for 5 years. The funds awarded exceeded the amount we had originally set aside to fund this initiative. Applications funded included studies that used yeast, fruit flies, nematodes, mice, and human cells as model systems, and collaborations among investigations working in different systems were encouraged. Other features of the program included annual meetings of investigators whose work was funded by this initiative, but other researchers who were not specifically funded through this program were also included in these meetings to encourage the development of an interactive network of investigators. This strategy served to quickly broaden the initiative, which turned out to be crucial to its ultimate success. Mark Collins and Paul Glenn of the GFMR provided partial support for these annual meetings during the decade of the 1990s.

Genetic Regulation of Nematode Longevity Back to Top

Productivity within the network was meager at first, but a breakthrough came when Cynthia Kenyon and Pamela Larsen independently reported that several of the daf mutations of C. elegans, particularly daf-2, resulted in greatly increased longevity as compared to that observed in the wild type.

Publication: Cynthia Kenyon, C. Chang, E. Genesch, A. Rudner, R. Tabtiang, A C. elegans mutant that lives twice as long as wild type. Nature 366, 461-464 (1993).

Publication: Pamela L. Larsen, Patrice S. Albert, Donald L. Riddle, Genes that regulate both development and longevity in Caenorhabditis elegans. Genetics 139, 1567-1583 (1995).

These results provided additional proof of the principle that single-gene mutations can increase longevity, and laid the groundwork for the isolation and characterization of such genes. This next phase began when Gary Ruvkun, who was not originally funded as part of the LAG initiative, showed in 1996 that age-1 and daf-23 mutations occur in the same gene, now called age-1, and that this gene codes for a phosphatidylinositol-3-OH kinase-like protein (PI 3-kinase). This result was quickly followed by Ruvkun's demonstration in 1997 that the daf-2 gene codes for an insulin receptor-like protein. This finding placed the DAF-2 and AGE-1 proteins in the same biological pathway, because PI 3-kinase is known to be activated as a result of binding of a ligand to the insulin receptor; this pathway is referred to as the insulin-signaling pathway.

Publication: Jason Morris, Heidi Tissenbaum, Gary Ruvkun, A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382, 536-539 (1996).

Publication: Koutarou D. Kimura, Heidi A. Tissenbaum, Yanxia Liu, Gary Ruvkun, daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942-946 (1997).

Continuing research on C. elegans has revealed several other classes of mutations that appear to be informative about aging mechanisms. One class, characterized by Lakowski and Hekimi (1998), includes the eat mutations (some of which result in life-span extension), which cause defects in pharyngeal function. Thus, these mutations might mimic the effects of caloric restriction (CR), which is known to increase life-span in many organisms, including S. cerevisiae, C. elegans, D. melanogaster, and mice and might also be delaying aging in primates. Another is the clk-1 mutation, which results in an inability to synthesize coenzyme Q9. However, the clk-1 mutant is long-lived when fed Escherichia coli, which supplies coenzyme Q8 instead of Q9 [Lakowski and Hekimi (1996)]. This work has been followed up by Larsen and Clarke, who showed that removal of coenzyme Q8 from the diet also extends the life-span of nonmutant nematodes. These authors hypothesize that withdrawal of coenzyme Q8 leads to decreased production of reactive oxygen species. A third class includes the old mutations that occur in receptor tyrosine kinase genes and thus interrupt signal transduction, as shown by Murakami and Johnson. Overexpression of the old genes increases both life expectancy and resistance to stress. Finally, the sir2.1 gene codes for an NAD+-dependent histone deacetylase, and Tissenbaum and Guarente demonstrated that overexpression of this gene increases life expectancy, presumably by changing patterns of gene expression.

Fig. 1 summarizes the C. elegans single-gene mutations that increase longevity. For additional details, the reader is referred to Tom Johnson's Subfield History, which describes the use of C. elegans to study aging.

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Fig. 1. Mutations that extend longevity in nematodes, fruit flies, and mice. References: Friedman and Johnson, 1988, Morris et al., 1996, Kenyon et al., 1993, Larsen et al., 1995, Kimura et al., 1997, Lakowski and Hekimi, 1996, Lakowski and Hekimi, 1998, Lin et al., 1998, Rogina et al., 2000, Tatar et al., 2001, Clancy et al., 2001, Miller, 1999, Brown-Borg et al., 1996, Flurkey et al., 2002, Coschigano et al., 2000, and Migliaccio et al., 1999.


Genetic Regulation of Fruit Fly Longevity Back to Top

The first success in the search for longevity-associated genes in fruit flies occurred as the result of open-ended genetic screens to find mutations that extend life-span. Seymour Benzer and colleagues mutagenized fruit flies by P-element insertion and screened them for increased longevity. The first mutation described was named methuselah (mth), and mth flies live about 35% longer at 25°C than do wild-type flies. The mth mutation causes partial loss of function, and mth flies are also more resistant to paraquat (a compound that causes oxidative stress), high temperature, and starvation. The mth gene has been cloned and sequenced and shown to code for a protein with seven hydrophobic regions suggestive of transmembrane domains, and homology to guanosine triphosphate-binding regulatory protein-coupled receptors. Thus, the authors speculate that fruit flies "use signal transduction pathways to modulate both stress response and life-span."

Publication: Yi-Jyun Lin, Laurent Seroude, Seymour Benzer, Extended life-span and stress resistance in the Drosophila mutant methuselah. Science 282, 943-946 (1998).

Rogina et al. (2000) used a similar approach and isolated five independent mutations in a gene they called Indy (for I'm not dead yet). The sequence of this gene indicates that the encoded product is closely related to a mammalian sodium dicarboxylic acid cotransporter, which is the membrane protein that transports dicarboxylic acids across membranes. The various Indy mutations increase mean longevity by about 90% at 25°C, and these authors hypothesize that mutations might lead to low levels of dicarboxylic acid intermediates in the mitochondria, thus creating a metabolic state resembling that produced by CR.

Publication: Blanka Rogina, Robert A. Reenan, Steven P. Nilsen, Stephen L. Helfand, Extended life-span conferred by cotransporter gene mutations in Drosophila. Science 290, 2137-2140 (2000).

Motivated by the idea that oxidative stress is a risk factor for aging (see "The Two Faces of Oxygen" and Praticò Review), other fruit fly researchers studied the effects of overexpressing candidate antioxidant genes. Parkes et al. found that selective overexpression of human superoxide dismutase 1 (the Cu/Zn cytoplasmic form) in motor neurons increases the life expectancy of flies, and Sun and Tower reported that overexpression of the fruit fly SOD gene (SOD1 ) in all cells correlates positively with life expectancy. Overproduction of another antioxidant enzyme, methionine sulfoxide reductase A (predominately in the nervous system), also increases fruit fly life expectancy (Ruan et al., 2002). This enzyme directly converts methionine sulfoxide residues in proteins, which represent a form of oxidative damage, back to methionine, using thioredoxin as the reducing agent. Earl Stadtman has suggested that methionine side chains provide an antioxidant shield for proteins that can be easily maintained via the action of methionine sulfoxide reductase A, obviating the need to degrade and resynthesize damaged proteins. These transgenic overexpressors of methionine sulfoxide reductase A are resistant to paraquat and remain physically and reproductively active much longer.

Publication: Hongyu Ruan, Xiang D. Tang, M.-L. Chen, M.-L. A. Joiner, G. Sun, N. Brot, H. Weissbach, S.H. Heinemann, L. Iverson, C.-F. Wu, T. Hoshi, High-quality life extension by the enzyme peptide methionine sulfoxide reductase. Proc. Natl. Acad. Sci. U.S.A. 99, 2748-2753 (2002).

The discovery that the insulin signaling pathway is involved in longevity regulation in nematodes was noticed by the fruit fly gerontologists, and they sought to determine whether similar life-extending mutations in the insulin signaling pathway could be identified in fruit flies. Creation of mutations in a Drosophila insulin-like receptor substrate protein (chico) (Clancy et al., 2001), and in the insulin-like receptor (Inr) itself (Tatar et al., 2001), resulted in increased life expectancy in females but a much smaller increase in male flies. Growth of the chico mutant at various concentrations of food indicates that CR also extends the life expectancy of these flies (Clancy et al., 2002), but not at very low food concentrations (see "The Road More Traveled"). These authors suggested that the chico mutation and CR increase life expectancy by at least partially overlapping mechanisms.

Publication: David J. Clancy, David Gems, Lawrence G. Harshman, Sean Oldham, Hugo Stocker, Ernst Hafen, Sally J. Leevers, Linda Partridge, Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292, 104-106 (2001).

Publication: Marc Tatar, A. Kopelman, D. Epstein, M.-P. Tu, C.-M. Yin, R.S. Garofalo, A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine functon. Science 292, 107-110 (2001).

Publication: David J. Clancy, David Gems, Ernst Hafen, Sally J. Leevers, Linda Partridge, Dietary restriction in long-lived dwarf flies. Science 296, 319 (2002).

Single-gene mutations in fruit flies that increase longevity are summarized in Fig. 1, and they support the concept that common pathways and mechanisms might be involved in the regulation of longevity among diverse species. This conclusion narrows the playing field in the search for genes and provides powerful insights about likely candidate genes and pathways in humans.

Genetic Regulation of Mouse Longevity Back to Top

Because mice live much longer than nematodes or fruit flies, and survival analysis is considerably more expensive, no genetic screens searching for mutations that increase life expectancy have been reported or, to my knowledge, have even been attempted. Nevertheless, seven long-lived mouse mutants have been identified using other approaches. Foremost among these are the four dwarf mouse strains that either cannot produce growth hormone or cannot respond to it (see Bartke Viewpoint). These dwarf mice include the Snell and Ames dwarf mice that are defective in pituitary development (because of Pit1dw and Prop1df mutations, respectively), the growth hormone receptor (GHR ) gene knockout mouse (ghr-KO, or Laron mouse), and a strain (Little), which contains a mutation in the growth hormone-releasing hormone receptor (ghrhr). Because one of the roles of growth hormone is to increase the concentration of circulating insulin-like growth factor-1 (IGF-1), these four mutants suggest that alterations in pathways controlled by insulin-like signals may modulate aging in both nematodes and mice. None of these dwarf mice was generated as part of the NIA LAG initiative, but their further characterization is funded by the NIA. Of particular interest is the observation by Bartke et al. (2001a) that CR further increases the longevity of the Prop1df mice, indicating that the pathways responsible for increasing longevity in the Ames dwarf mice and by CR are not identical (Bartke et al., 2001a). Because of the shapes of the survival curves, these authors suggest that "caloric restriction decelerates aging whereas the Prop1df mutation delays it." This interpretation comes from the observation that CR slows the rate of death, whereas the df mutation delays death without changing the slope of the survival curve. The phenotypes of these four mutant mice have been summarized by Bartke et al. (2001b).

Publication: Andrzej Bartke, J. Chris Wright, Julie A. Mattison, D. K. Ingram, R. A. Miller, G. S. Roth, Longevity: Extending the lifespan of long-lived mice. Nature 414, 412 (2001a).

Publication: Andrzej Bartke, Karen Coschigano, John Kopchick, Varadaraj Chandrashekar, Julie Mattison, Beth Kinney, Steven Hauck, Genes that prolong life: Relationships of growth hormone and growth to aging and life span. J. Gerontol. 56A, B340-B349 (2001b).

A mouse with a mutation in the IGF-1 receptor gene (IGF-1R) has also been recently described by Holzenberger et al., and although female heterozygotes live about 30% longer than their wild-type counterparts, they are only slightly smaller. Thus, dwarfism can be uncoupled from extended longevity in this mouse mutant.

The mouse p66shc gene is involved in stress response, and Migliaccio et al. (1999) showed that a targeted mutation in this gene increases resistance to oxidative stressors such as paraquat and also increases life expectancy. The mutation apparently induces the overexpression of catalase (CAT) and reduces apoptosis in response to oxidative stress. This work is described in more detail in "One for All".

Publication: Enrica Migliaccio, Marco Giorgio, Simonetta Mele, G. Pelicci, P. Reboldi, P. P. Pandolfi, L. Lanfrancone, P. G. Pelicci, The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402, 309-313 (1999).

The seventh long-lived mouse mutant apparently genetically recapitulates the CR paradigm, perhaps by suppressing appetite (Miskin and Masos, 1997).

Publication: R. Miskin, T. Masos, Transgenic mice overexpressing urokinase-type plasminogen activator in the brain exhibit reduced food consumption, body weight and size, and increased longevity. J. Gerontol. 52, B118-B124 (1997).

An entirely different approach was taken by NIA grantee Richard Miller. His strategy was to start with a heterogeneous mouse population produced in a four-way cross among grandparental strains BALB/cJ, C57BL/6J, C3H/HeJ, and DBA/2J (Jackson et al., 2002). The individual mice were genotyped to determine which markers they obtained from each grandparent, killed when they had clearly become moribund, and then autopsied. When the first 20% to die were excluded, three genetic loci were found that predict life expectancy; two of these are associated with longevity only in male mice. Actual genes at these loci have not yet been implicated in longevity regulation. Also yet to be determined are whether these genetic differences influence the pattern of progression of age-sensitive traits. Miller et al. (2002) have also shown that body weight at 2 months of age is a predictor of life-span in this genetically heterogeneous mouse population.

Publication: Anne U. Jackson, Andrzej T. Galecki, David T. Burke, Richard A. Miller, Mouse loci associated with life span exhibit sex-specific and epistatic effects. J. Gerontol. 57A, B9-B15 (2002).

Publication: Richard Miller, James Harper, Andrzej Galecki, David Burke, Big mice die young: early body weight predicts longevity in genetically heterogeneous mice. Aging Cell 1, 22-29 (2002).

The relative success of the LAG initiative during the period 1993-1998 encouraged Anna McCormick and the NIA to issue a second RFA in 1998 to continue the program. A greatly expanded program was funded in 1999 and continues to contribute to our understanding of the genetic regulation of longevity in animal model systems.

Translation of Results to Humans Back to Top

The ultimate goal of studies using animal models is to identify genes and processes that regulate longevity and functional decline in humans. The single-gene mutations that increase longevity in C. elegans, D. melanogaster, and mice are summarized in Fig. 1. The similarities among species shown in this figure suggest that special attention should be given to comparable genes in humans. Related research in model organisms has produced a rich harvest of additional genes and processes of possible relevance in regulation of longevity. Pharmacological examples include the demonstration by Melov et al. that EUK-134, a catalase-SOD mimetic, extends longevity in nematodes, and the demonstration by Kang et al. that 4-phenyl butyrate, an inhibitor of histone deacetylase, extends longevity in fruit flies. Therefore, of particular interest should be genes that code for proteins involved in either repair or prevention of damage caused by stresses such as heat, oxygen free radicals, and other genotoxic compounds, as well as genes that regulate gene expression.

Mutations in several human genes lead to segmental progeroid syndromes (reviewed by Martin and Oshima), in which at least some aging phenotype(s) occur relatively early in life. The most relevant syndrome appears to be Werner syndrome (WS), and although the gene (WRN) responsible for this condition has been isolated and characterized as encoding a DNA helicase, the mechanistic basis for the disease is still poorly understood (see Fry Review and "Of Hyperaging and Methuselah Genes"). What is clear is that the syndrome is associated with defects in one of more of the following processes: DNA replication, DNA repair, transcription, or recombination. Work on animal models of WS is included in the LAG initiative.

Another area of research in the genetics of human longevity involves identifying single nucleotide polymorphisms (SNPs) in genes that affect the development of one or more aging phenotypes. It seems likely that individual differences in aging patterns among individuals in a population may be at least partly due to these subtle genetic differences found in our genes. Identification of these SNPs is only marginally included in NIA's LAG initiative so far. However, it is clear that as human candidate aging genes are identified through the LAG initiative, and as SNPs in these genes are discovered, the groundwork will be laid for identifying which of these SNPs may be associated with the development of aging phenotypes. The best documented example of this so far is the apoE gene, which was discovered through the approach known as demographic selection (Schächter et al. and Perls et al.). The apoE alleles present in an individual greatly affect the risk of developing Alzheimer's disease (see McGeer Review, Honig Case Study, and "Detangling Alzheimer's Disease").

Finally, two genes possibly related to longevity have recently been identified in human populations. An unknown gene on chromosome 4 linked to exceptional longevity in humans has been reported by Puca et al., and Arking et al. reported an association between SNPs in the KLOTHO locus and age-related phenotypes in several human populations. The klotho gene in mice has previously been associated with premature death in mice by Kuro-o et al. (Klotho mice). Future research to identify human genes with possible effects on longevity will be informed by results with animal models such as those discussed here.

February 12, 2003 Citation: H. R. Warner, Subfield History: Use of Model Organisms in the Search for Human Aging Genes. Sci. SAGE KE 2003 (6), re1 (2003).

Developing a Research Agenda in Biogerontology: Basic Mechanisms.
H. R. Warner (2005)
Sci. Aging Knowl. Environ. 2005, pe33
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