Our goal is to use three model organisms, the budding yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the mouse, as a bridge toward understanding the molecular basis of human aging. Everyone ages; yet, little is known about the mechanisms controlling this ubiquitous process. Our lack of knowledge is derived in large part from the intractability of aging research. Mammals have long life spans, and it is not clear which phenotypes in cell culture are most related to organismal aging. Simple eukaryotic models that have short life spans, such as yeast, worms, and flies, can serve as a bridge to understanding the molecular details of mammalian aging. Although it remains to be seen how well aging in model systems will mimic aging in humans, at least some aspects of the aging process are highly conserved. For example, mutant alleles in a few orthologous gene pairs and one environmental intervention (calorie restriction) are known to increase life span in both yeast and C. elegans, and in the case of calorie restriction, in flies and mammals as well. Given the limited number of genes examined thus far, it seems clear that many more conserved longevity determinants remain to be identified.
Most attempts to identify "public" mechanisms of aging, those that are shared among evolutionarily distinct organisms, have focused on specific genes and preconceived models. While valuable insights have been gained from these studies, we believe that a multi-organism, genomic approach will allow for an unbiased analysis of the conserved genes and pathways that determine aging and longevity. Toward this end, we have developed technologies to allow high-throughput identification of genes that determine life span in the budding yeast. For each yeast gene identified, a search for homologs in C. elegans will be carried out and the role of each homolog in determination of worm life span examined. Any gene that regulates aging similarly in both yeast and worms will be a suitable candidate for further study in the mouse mammalian model system.
Two paradigms of aging have been developed in yeast (Figure 1). Replicative age is defined by the number of mitotic cycles that a cell can undergo, and has been proposed as a model for stem cell aging. Chronological age is the length of time that a cell retains viability in a non-dividing state, similar to post-mitotic cells in higher organisms. We are currently carrying out parallel quantitative analyses of both replicative and chronological life span for more than 4,500 single-gene deletion strains contained in the yeast ORF deletion collection. Characterization of genes identified from these genome-wide screens will be a primary focus of the consortium over the next few years.
Measurement of yeast replicative life span requires micromanipulation of daughter cells away from mother cells following each mitotic cycle. The time-consuming nature of this assay has precluded large-scale analyses of replicative aging. Dr. Kaeberlein and Dr. Kennedy have developed a method to allow semi-quantitative measurement of replicative life span based on the aging properties of a small number of cells. To date, we have determined the replicative life span phenotypes for approximately 20% (~1000 strains) of the ORF deletion collection. Completion of this analysis is estimated to take approximately 1-2 years. Dr. Kaeberlein, in collaboration with Trey Powers, a graduate student in the Fields lab, has also developed technology that allows the simultaneous determination of chronological life span for several thousand strains in a highly quantitative manner. We have used this technology to carry out an initial screen the ORF deletion collection for genes that affect chronological aging. The initial results from both of these screens will be published in the near future and have uncovered an unexpectedly high degree of evolutionary conservation between the cellular response to nutrients and the aging of both mitotic and post-mitotic cells.
The genome-wide longevity screens described above will provide a means to directly compare aging in yeast to aging in higher eukaryotes. Worm, fly, and mammalian orthologs will be sought for each yeast gene found to affect life span. In some cases, life span phenotypes for worms treated with RNAi against the orthologous gene will be available from published studies in C. elegans. In cases where the life span data are unavailable or inconclusive, we will carry out experiments to determine the life span phenotype resulting from decreased expression of candidate genes by RNAi.
Genes that increase life span in C. elegans when knocked-down will be characterized genetically by epistasis analysis with known aging genes. For example, long-lived RNAi strains will be tested in a Daf-16 mutant as well as a Daf-2 mutant background, in order to place them into or out of the well-characterized insulin/IGF-1-like pathway. Each long-lived mutant will also be combined with two different models of calorie restriction (eat-1 and reduced bacterial feeding) in order to determine whether the gene product is involved in mediating the calorie restriction effect on aging.
We anticipate that at least two dozen genes will pass the criteria of conservation in yeast and C. elegans, as described above. For each of these genes, a mouse models will be made using a knock-in strategy using a cre-excised flox-stopper sequence in the endogenous gene. The use of drug-inducible and/or tissue specific cre recombinase will enable the disruption of expression of the candidate gene at specific developmental times and/or in specific cell lineages.
Effects of genetic knockouts on the aging process in mice will be studied at three different levels: 1) effects on mean and maximal span, 2) attenuation of age-related declines in biological functions, and 3) reduced incidence and/or delayed onset of age-related pathologies. Follow this link for further information on each.
This portion of the project is also supported by the resources of the U of W Nathan Shock Center of Excellence in the Basic Biology of Aging Transgenic Animal Resource Core, funded by the National Institute of Aging. Under the direction of Warren Ladiges, this resource has been responsible for generation of a large number of aging-related transgenic, knockout and allele replacement mouse models (Treuting P, Hopkins H, Ware C, Rabinovitch P, Ladiges W. Generation of genetically altered mouse models for aging studies. Experimental Mol Path. Feb;72(1):49-55, 2002). Thus, while not "high throughput" in the terms often used for yeast, the ability to systematically test in mammals dozens of yeast and worm-validated knockouts in mouse models represents a new scale for mammalian gene discovery in the biology of aging.