The aim of this project is to identify aging-regulated genes at the molecular and tissue levels, to investigate molecular mechanisms of lifespan extension by longevity genes, and to identify efficient prolongevity interventions. To achieve our goals, we are utilizing three invertebrate systems, the Mexican fruit fly (mexfly), A. ludens, and the fly, D. melanogaster, and the nematode, C. elegans. Lifespan is influenced by a number of genetic and environmental factors. One of the most robust environmental manipulations of lifespan is dietary restriction (DR). DR has been shown to extend lifespan in many species, ranging from invertebrates to mammals, indicating that DR might retard aging if applied in humans. However, it would be challenging to impose long-term DR in humans. An alternative strategy would be to apply pharmaceutical or nutraceutical compounds to induce responses that would mimic DR. A few compounds have been shown to have this effect in model organisms. However, the number is still small and little is known about mechanisms by which these compounds extend lifespan. Genetic analyses of model organisms have uncovered mutations in a number of genes that can affect lifespan. Changes in gene expression in aging have been observed in a number of organisms, including worms, flies, rodents, primates and human beings. However, little is known about how different tissues age, and how longevity genes and prolongevity interventions influence aging. To address tissue-specific aging, we have systematically investigated tissue-specific factors that affect lifespan and aging processes. We have measured the expression profile of aging for seven tissues from fly, including brain, muscle and tissues in the digestive and reproductive systems, which represent different physiological functions. Hundreds of genes have been identified to show significant changes at the transcript level in aging in each tissue. Very few of these tissue-specific changes were shared among all the tissues, suggesting that different tissues age in unique ways. However, some of the aging-related changes are shared among two or more tissues, suggesting that common molecular features do exist among different tissues in aging. As an example, we have found genes involved a major metabolic pathway, TCA cycle, are down-regulated in brain, gut, muscle and testis but not fat tissue, accessory gland and malpighian tubule in aging. To study the mechanisms of lifespan extension by the longevity genes at the tissue and molecular levels, we have chosen to study the methuselah mutant fly, which has an extended lifespan. We have measured molecular changes of this mutant across age for the seven tissues described above. We have compared these changes to those in the wild type fly strain at the molecular and tissue levels. Several hundreds of genes have been identified to have tissue-specific changes between wild type and methuselah flies. This assessment will elucidate molecular and cellular mechanisms on how the methuselah gene regulates lifespan at the tissue level. Similar approaches will be applied to study mechanisms by which prolongevity interventions extend lifespan at the tissue levels in the future. Dietary supplements are widely used with the belief that they can forestall disease and increase longevity. Few systematic attempts have been made to confirm prolongevity claims made or to investigate potentially effective interventions. We are currently developing a high-throughput system by using mexfly. The main reason to use mexflies is that we have easy access to millions of mexflies available daily in the Moscafrut mass-rearing facility at Tapachula, Chiapas, Mexico. This minimizes our efforts to obtain a large number of animals for mortality analysis required for the high-throughput lifespan screen. There are several other advantages as well. The size of mexflies is relatively large relative to D. melanogaster so that it is easy to sort out by sex and to measure food intake, the latter of which is critical for assessing dosage effects of compounds. Moreover, unlike D. melanogaster, mexflies and medflies can feed on a dry food source but lay their eggs on a different medium such as organdy mesh, which facilitates investigation of effects of compounds on reproduction independent of diet. Finally, the expected lifespan of mexflies on a regular diet is approximately 2 months, which is short enough for conducting a lifespan screen and but long enough for demographic studies of aging. As an example, we have assessed the effects of supplementation of two related antioxidants, alpha-tocopherol and gamma-tocopherol, on lifespan of mexflies. We have found that these two antioxidants have marginal effects on lifespan extension, which is consistent with what we have observed in D. melanogaster. Utilization of the high-throughput system will provide reliable and statistically convincing results on the effects of aging interventions. Systematic evaluation of prolongevity interventions will not only allow identification of effective anti-aging compounds but also uncover mechanisms of lifespan extension by dietary supplementation. This approach should prove valuable to advance the objective of experimental gerontology to investigate and develop aging interventions in mammals. Our understanding of molecular mechanisms of DR comes primarily from studies of genetically amenable systems including yeast, worms, and flies, where DR has been imposed by either diluting the food source or by using genetic mutations that reduce feeding efficiency. However, a major drawback of these approaches is that there remains substantial uncertainty in determining the exact caloric intake of individuals under these DR paradigms, unlike this ability in studies of higher organisms. This led us to discover and develop an alternative dietary paradigm that can extend lifespan in C. elegans. We have found that a dietary deprivation (DD) regimen, in which the food source is completely removed from adults, can prolong adult lifespan by 45%. Since this regimen involves complete removal of the food source, the problem of controlling food intake, which has hampered interpretation of past studies, is alleviated. Using this unambiguous method, we have started investigating the genetic pathways necessary for lifespan extension by diet. Genome-wide transcript profiles of DD response are measured and compared to that of DR to reveal similarities between DD and DR paradigm. To identify which genetic pathways are required for the DD response, a genetic screen is being conducted for DD-associated genes identified from genomic studies as well as for genes known to extend lifespan. This analysis should reveal mechanisms governing longevity under different environmental especially dietary conditions. Considering the similarities between DD and DR, some of the DD mechanisms should be evolutionarily conserved, which will advance knowledge about effects of diet on aging and longevity in mammals. In summary, we have applied three different invertebrate species to address issues related to dietary regulation of lifespan by taking advantage of unique features of each system. With D. melanogaster, we are studying mechanisms by which prolongevity interventions and longevity genes extend lifespan at molecular and tissue levels. We are using mexflies to identify effective prolongevity interventions, which should provide guidance for further investigation of aging interventions in mammals. By utilizing a unique and robust dietary regimen in C. elegans, we are dissecting molecular mechanisms of dietary regulation of lifespan. Identification of the conserved features in aging and efficient prolongevity interventions are clearly critical for us
