Longevity is regulated by genetic pathways and influenced by environmental factors. This project's goals are to (a) elucidate mechanisms by which genes affect longevity and (b) identify environmental and physiological factors that regulate aging. These studies focus on aging in the nematode, Caenorhabditis elegans, because C. elegans is one of the premier organisms for studying the genetic control of longevity. The current popularity of C. elegans as a focus for studies of aging comes from its short lifespan (2-3 weeks), ease of cultivation and genetic amenability.? ? Insulin-like signaling pathways are important regulators of longevity in several species, from worms to flies to mice, and could also affect human longevity. In the nematode, Caenorhabditis elegans, lifespan can be tripled by mutations that disrupt signaling downstream of the insulin receptor-like (IR) protein, DAF-2. In addition to regulating lifespan, DAF-2/IR signaling regulates larval development and also stress resistance and metabolism in adults. The phenotypic effects of disrupting C. elegans insulin signaling come from the consequential activation of a FOXO transcription factor, DAF-16, which is a shared output of insulin-like pathways in many animals. Thus, FOXO transcription factors may be evolutionarily conserved regulators of longevity. ? ? Current work aims to define how FOXO transcription factors affect longevity and to identify FOXO-interacting pathways. Signaling by DAF-2/IR and its major effector, AGE-1/PI3K, could promote wildtype lifespan non-cell autonomously from a variety of cells throughout the body. We have recently confirmed and extended the previous findings and shown that insulin signaling in cells throughout the nervous system, or in other tissues, have the capability of promoting normal lifespan. We also identified a new phenotype of insulin pathway mutants, which is resistance to a cellular fasting response. Interestingly, regulation of this fasting response was primarily mediated by insulin signaling within intestinal cells. Furthermore, restricted insulin signaling to some non-intestinal cells could promote wildtype lifespan but did not rescue the mutant fasting phenotype. Thus, insulin signaling may regulate functional outputs through both non-cell autonomous (longevity) and cell-autonomous (fasting response) pathways. ? ? We have also investigated whether DAF-16 functions cell-autonomously or non-cell autonomously in regulating longevity and fasting responses. Although DAF-16 could regulate the fasting response cell-autonomously, DAF-16 did not display the same non-cell autonomous effect on longevity as DAF-2/IR and AGE-1/PI3K. Therefore, we tentatively conclude that DAF-16 functions primarily cell-autonomously. This suggests that the insulin signaling pathway couples to DAF-16 through both cell-autonomous and non-cell autonomous pathways. DAF-16, in turn, regulates target genes that cell-autonomously promote longevity of the whole organism. We are currently testing this model by examining the effects of tissue-restricted insulin signaling on the expression of DAF-16 target genes using a microarray-based analysis of gene expression. We are also studying the regulation of known DAF-16 target genes to identify additional pathways that may collaborate with insulin signaling to regulate longevity. ? ? Recently, we have identified several mutations that affect lifespan by modifying insulin pathway signaling. We have cloned a collection of 4 mutant genes that modulate signaling downstream of DAF-2/IR. These are all mutations that promote normal levels of insulin signaling in animals lacking AGE-1/PI3K. All 4 mutations were new alleles of known components of the DAF-2/IR effector pathway. Two were loss of function mutations in daf-16. The remaining two alleles were gain-of-function mutations that activated signaling by PI3K-dependent serine/threonine kinases, AKT-1 and PDK-1. Given that the mutant AKT-1 and PDK-1 enzymes can promote insulin signaling in the absence of AGE-1/PI3K, we speculate that the mutations activate basal enzyme activity in the absence of AGE-1-generated phospholipids. ? ? We are working to rapidly identify compounds with prolongevity activity in C. elegans. Lead compounds that clear this screen can be further studied for their effects on aging in mammals, which requires more time-consuming and costly procedures. Current work is focused on two classes of compounds, blueberry polyphenols and the electron spin-trap nitroxide compound, tempol. Blueberries contain a high abundance of polyphenolic compounds. In vitro, these compounds possess high antioxidant activity. We have found that treatment with blueberry polyphenols can significantly prolong C. elegans lifespan and delay the accumulation of aging-related damage. The bioactive compounds co-fractionate with a proanthocyanidin-enriched fraction, but not with other antioxidant polyphenols. Genetic analysis revealed that treatment with blueberry polyphenols did not lead to induction of stress resistance pathways. Our current model is that blueberry polyphenols protected cells from intrinsic stress that leads to cellular decline during aging. C. elegans lifespan was also significantly prolonged by treatment with the electron spin-trap nitroxide compound, tempol. At higher doses, tempol inhibited feeding and movement and caused larval lethality. Tempol did not appear to act as an antioxidant in vivo, as tempol treatment did not rescue lethality in mutants with increased oxidative stress. We hypothesize that tempol might interfere with normal mitochondrial function, since previous studies had shown that reductions of mitochondrial respiration can extend lifespan. ? ? We are studying normal aging in C. elegans to identify how aging causes functional and structural declines in tissues, particularly muscles. Current work investigates the basis for aging-related declines of the pharynx muscles. The pharynx is the worm's feeding organ and is composed of 20 muscle cells. We have found that aging primarily affects the ability of pharynx muscles to respond to neuronal stimulation, and probably does not significantly affect neuronal function. In addition, we have found that muscle contraction is probably a major contributor to functional declines in this organ during aging. Both genetic and longitudinal approaches are being used to define specific factors responsible for pharynx functional decline during aging. The results of these studies may provide new avenues for treating muscle deterioration in human aging.
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