The lifespan of an organism is determined by a complex array of genetic, environmental and nutritional factors. Yet single gene manipulations have been shown to significantly extend lifespan in several model organisms. Of all the genes that have been studied thus far, components of the insulin/IGF-1 signaling (IIS) pathway have emerged as the most robust regulators of longevity. In addition, IIS also regulates development, energy metabolism and the response to stress in a conserved manner. In Caenorhabditis elegans, signaling through this pathway is initiated by activation of the insulin/IGF-1 receptor tyrosine kinase DAF-2, which then activates a PI3-kinase signaling pathway involving additional downstream serine/threonine kinases such as PDK-1, AKT-1, AKT-2 and SGK-1. The concerted action of these kinases results in the negative regulation of the single FOXO transcription factor homolog DAF-16. Under reduced signaling conditions, active DAF-16 is able to translocate into the nucleus and regulate the expression of hundreds of genes regulating longevity, stress resistance, metabolism and development. The PTEN phosphatase homolog DAF-18, which antagonizes IIS at the level of PI3-kinase, is a major negative regulator of the pathway. However, not much was known about additional phosphatases that negatively regulated the kinases in the pathway. Dephosphorylation is a critical regulatory mechanism by which cellular signaling homeostasis is maintained. Aberrant hyper-activation of growth factor signaling pathways, including IIS, has been implicated in several cancers. In addition, deregulation of IIS is also closely linked to Type II diabetes. Therefore, the identification phosphatases that balance kinase activity will provide a better understanding of the regulation of the IIS pathway under normal as well as disease conditions. A directed RNAi screen using dauer diapause was conducted in our lab to identify serine/threonine phosphatases that modulated IIS. My work in the Tissenbaum Lab has primarily focused on characterization of the top three candidates from this screen, the genes pptr-1, pdp-1 and fem-2. From these studies, we have also uncovered novel crosstalk between the IIS and TGF-β signaling pathways. In Chapter 2, we demonstrate that PPTR-1, a PP2A phosphatase regulatory subunit negatively regulates the IIS pathway by modulating AKT-1 dephosphorylation. PPTR-1 modulates several outputs of IIS similar to DAF-18. In addition, PPTR-1 co-localizes and physically interacts with its substrate, AKT-1. PPTR-1 modulates dephosphorylation of AKT-1 at a conserved threonine site and we show the molecular conservation of this interaction in mammalian adipocytes. Ultimately, this negative regulation by PPTR-1 results in increased DAF-16 nuclear localization and transcriptional activity. Next, in Chapter 3, we show how PDP-1 is a novel link between the IIS and TGF-β signaling pathways. Similar to DAF-18 and PPTR-1, PDP-1 regulates multiple outputs of the IIS pathway and promotes DAF-16 activity. Interestingly, PDP-1 acts at the level of DAF-8 and DAF-14, two R-SMAD proteins that function in a TGF-β pathway. Our data suggests that PDP-1 may negatively regulate TGF-β signaling to downregulate the expression of several insulin(s). Without the insulin ligands, there is less activation of the IIS pathway, and DAF-16 is more active, thereby promoting transcription of genes that act to enhance longevity and stress resistance. In Chapter 4, we investigate possible crosstalk between IIS and the TGF-β signaling pathways, as the latter was previously considered as a parallel independent pathway. From our studies on PDP-1, we knew that this phosphatase, despite acting in the TGF-β pathway, was a robust modulator of multiple outputs of IIS. Using double mutant combinations as well as RNAi we unravel complex and extensive crosstalk between the two pathways. Importantly, our results suggest that DAF-16 is likely to be the most downstream component of the two pathways. In Chapter 5, we describe genetic characterization of fem-2, and its regulation of the IIS pathway. RNAi of fem-2 results in robust suppression of dauer formation, similar to pptr-1 and pdp-1 RNAi but this phenotype is only observed in the e1370 allele of daf-2. While knockdown of pptr-1 and pdp-1 suppress dauer formation of additional alleles of daf-2, fem-2 RNAi has no effect. These results reveal a complex genetic interaction between fem-2 and the daf-2 receptor. Taken together, our results identify several novel regulators of IIS that modulate this pathway by distinct mechanisms.

Supplementation with nicotinamide riboside (NR), a form of vitamin B3 and a precursor of nicotinamide adenine dinucleotide (NAD+) extends lifespan in the nematode C. elegans and delays aging-related pathologies in mammals. During aging, levels of NAD+ decline causing metabolic dysfunction and oxidative damage. Studies in C. elegans found that when NR was administered during larval development it induced the mitochondrial unfolded protein response (UPRmt), which is frequently associated with lifespan extension. Both calorie restriction (CR) and ketogenic diets (KD) have been shown to extend lifespan, in part through increasing NAD+ and through increasing levels of the pro-longevity ketone body beta-hydroxybutyrate (BHB). In a previous study from my lab, NR increased C. elegans lifespan to a much larger extent when administered starting at the L1 larval stage as compared to when started at the L4 larval stage. Conversely, a high dose of BHB greatly increased lifespan when administered starting at the L4 stage. But this same dose decreased lifespan when it was first administered at the L1 stage. I further found that NR greatly extended lifespan when only administered during larval development and that the combination of both individual pro-longevity NR and BHB treatments significantly decreased lifespan. These chemical epistasis experiments suggest that NR and BHB function in parallel pathways to extend C. elegans lifespan through a common downstream target with hormesis playing a role. Therefore, human subjects who supplement with both NAD+ precursors and ketone esters should be aware of possible negative interactions when high doses of both are administered.