Converging Pathways in the Regulation of Longevity and Metabolism in Caenorhabditis Elegans: A Dissertation

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

Converging Pathways in Lifespan Regulation

The processes that determine an organism's lifespan are complex and poorly understood. Yet single gene manipulations and environmental interventions can substantially delay age-related morbidity. In this review, we focus on the two most potent modulators of longevity: insulin/insulin-like growth factor 1 (IGF-1) signaling and dietary restriction. The remarkable molecular conservation of the components associated with insulin/IGF-1 signaling and dietary restriction allow us to understand longevity from a multi-species perspective. We summarize the most recent findings on insulin/IGF-1 signaling and examine the proteins and pathways that reveal a more genetic basis for dietary restriction. Although insulin/IGF-1 signaling and dietary restriction pathways are currently viewed as being independent, we suggest that these two pathways are more intricately connected than previously appreciated. We highlight that numerous interactions between these two pathways can occur at multiple levels. Ultimately, both the insulin/IGF-1 pathway and the pathway that mediates the effects of dietary restriction have evolved to respond to the nutritional status of an organism, which in turn affects its lifespan

A Comparative Study of Fat Storage Quantitation in Nematode Caenorhabditis elegans Using Label and Label-Free Methods

The nematode Caenorhabditis elegans has been employed as a model organism to study human obesity due to the conservation of the pathways that regulate energy metabolism. To assay for fat storage in C. elegans, a number of fat-soluble dyes have been employed including BODIPY, Nile Red, Oil Red O, and Sudan Black. However, dye-labeled assays produce results that often do not correlate with fat stores in C. elegans. An alternative label-free approach to analyze fat storage in C. elegans has recently been described with coherent anti-Stokes Raman scattering (CARS) microscopy. Here, we compare the performance of CARS microscopy with standard dye-labeled techniques and biochemical quantification to analyze fat storage in wild type C. elegans and with genetic mutations in the insulin/IGF-1 signaling pathway including the genes daf-2 (insulin/IGF-1 receptor), rict-1 (rictor) and sgk-1 (serum glucocorticoid kinase). CARS imaging provides a direct measure of fat storage with unprecedented details including total fat stores as well as the size, number, and lipid-chain unsaturation of individual lipid droplets. In addition, CARS/TPEF imaging reveals a neutral lipid species that resides in both the hypodermis and the intestinal cells and an autofluorescent organelle that resides exclusively in the intestinal cells. Importantly, coherent addition of the CARS fields from the C-H abundant neutral lipid permits selective CARS imaging of the fat store, and further coupling of spontaneous Raman analysis provides unprecedented details including lipid-chain unsaturation of individual lipid droplets. We observe that although daf-2, rict-1, and sgk-1 mutants affect insulin/IGF-1 signaling, they exhibit vastly different phenotypes in terms of neutral lipid and autofluorescent species. We find that CARS imaging gives quantification similar to standard biochemical triglyceride quantification. Further, we independently confirm that feeding worms with vital dyes does not lead to the staining of fat stores, but rather the sequestration of dyes in lysosome-related organelles. In contrast, fixative staining methods provide reproducible data but are prone to errors due to the interference of autofluorescent species and the non-specific staining of cellular structures other than fat stores. Importantly, both growth conditions and developmental stage should be considered when comparing methods of C. elegans lipid storage. Taken together, we confirm that CARS microscopy provides a direct, non-invasive, and label-free means to quantitatively analyze fat storage in living C. elegans

PDP-1 Links the TGF-β and IIS Pathways to Regulate Longevity, Development, and Metabolism

The insulin/IGF-1 signaling (IIS) pathway is a conserved regulator of longevity, development, and metabolism. In Caenorhabditis elegans IIS involves activation of DAF-2 (insulin/IGF-1 receptor tyrosine kinase), AGE-1 (PI 3-kinase), and additional downstream serine/threonine kinases that ultimately phosphorylate and negatively regulate the single FOXO transcription factor homolog DAF-16. Phosphatases help to maintain cellular signaling homeostasis by counterbalancing kinase activity. However, few phosphatases have been identified that negatively regulate the IIS pathway. Here we identify and characterize pdp-1 as a novel negative modulator of the IIS pathway. We show that PDP-1 regulates multiple outputs of IIS such as longevity, fat storage, and dauer diapause. In addition, PDP-1 promotes DAF-16 nuclear localization and transcriptional activity. Interestingly, genetic epistasis analyses place PDP-1 in the DAF-7/TGF-β signaling pathway, at the level of the R-SMAD proteins DAF-14 and DAF-8. Further investigation into how a component of TGF-β signaling affects multiple outputs of IIS/DAF-16, revealed extensive crosstalk between these two well-conserved signaling pathways. We find that PDP-1 modulates the expression of several insulin genes that are likely to feed into the IIS pathway to regulate DAF-16 activity. Importantly, dysregulation of IIS and TGF-β signaling has been implicated in diseases such as Type 2 Diabetes, obesity, and cancer. Our results may provide a new perspective in understanding of the regulation of these pathways under normal conditions and in the context of disease

InAKTivation of insulin/IGF-1 signaling by dephosphorylation.

Signal transduction pathways are tightly regulated by phosphorylation-dephosphorylation cycles and yet the mammalian genome contains far more genes that encode for protein kinases than protein phosphatases. Therefore, to target specific substrates, many phosphatases associate with distinct regulatory subunits and thereby modulate multiple cellular processes. One such example is the C. elegans PP2A regulatory subunit PPTR-1 that negatively regulates the insulin/insulin-like growth factor signaling pathway to modulate longevity, dauer diapause, fat metabolism and stress resistance. PPTR-1, as well as its mammalian homolog B56beta, specifically target the PP2A enzyme to AKT and mediate the dephosphorylation of this important kinase at a conserved threonine residue. In C. elegans, the major consequence of this modulation is activation of the FOXO transcription factor homolog DAF-16, which in turn regulates transcription of its many target genes involved in longevity and stress resistance. Understanding the function of B56 subunits may have important consequences in diseases such as Type 2 diabetes and cancer where the balance of Akt phosphorylation is deregulated

A new DAF-16 isoform regulates longevity

The insulin/IGF-1 signalling (IIS) pathway has diverse roles from metabolism to longevity. In Caenorhabditis elegans, the single forkhead box O (FOXO) homologue, DAF-16, functions as the major target of the IIS pathway. One of two isoforms, DAF-16a, is known to regulate longevity, stress response and dauer diapause. However, it remains unclear how DAF-16 achieves its specificity in regulating these various biological processes. Here we identify a new isoform, DAF-16d/f, as an important isoform regulating longevity. We show that DAF-16 isoforms functionally cooperate to modulate IIS-mediated processes through differential tissue enrichment, preferential modulation by upstream kinases, and regulating distinct and overlapping target genes. Promoter-swapping experiments show both the promoter and the coding region of DAF-16 are important for its function. Importantly, in mammals, four FOXO genes have overlapping and different functions, and in C. elegans, a single FOXO/DAF-16 uses distinct isoforms to fine-tune the IIS-mediated processes in the context of a whole organism

A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation.

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research

TOR Signaling and Rapamycin Influence Longevity by Regulating SKN-1/Nrf and DAF-16/FoxO

The TOR kinase, which is present in the functionally distinct complexes TORC1 and TORC2, is essential for growth but associated with disease and aging. Elucidation of how TOR influences life span will identify mechanisms of fundamental importance in aging and TOR functions. Here we show that when TORC1 is inhibited genetically in C. elegans, SKN-1/Nrf, and DAF-16/FoxO activate protective genes, and increase stress resistance and longevity. SKN-1 also upregulates TORC1 pathway gene expression in a feedback loop. Rapamycin triggers a similar protective response in C. elegans and mice, but increases worm life span dependent upon SKN-1 and not DAF-16, apparently by interfering with TORC2 along with TORC1. TORC1, TORC2, and insulin/IGF-1-like signaling regulate SKN-1 activity through different mechanisms. We conclude that modulation of SKN-1/Nrf and DAF-16/FoxO may be generally important in the effects of TOR signaling in vivo and that these transcription factors mediate an opposing relationship between growth signals and longevity.National Institutes of Health (U.S.) (Grant CA129105)Ellison Medical FoundationAmerican Federation for Aging ResearchStarr FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT. Frontier Research ProgramNational Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (DRC Grant)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award) (F32 Postdoctoral Fellowship)American Diabetes Association (Fellowship

Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence

In Caenorhabditis elegans, ablation of germline stem cells (GSCs) extends lifespan, but also increases fat accumulation and alters lipid metabolism, raising the intriguing question of how these effects might be related. Here, we show that a lack of GSCs results in a broad transcriptional reprogramming in which the conserved detoxification regulator SKN-1/Nrf increases stress resistance, proteasome activity, and longevity. SKN-1 also activates diverse lipid metabolism genes and reduces fat storage, thereby alleviating the increased fat accumulation caused by GSC absence. Surprisingly, SKN-1 is activated by signals from this fat, which appears to derive from unconsumed yolk that was produced for reproduction. We conclude that SKN-1 plays a direct role in maintaining lipid homeostasis in which it is activated by lipids. This SKN-1 function may explain the importance of mammalian Nrf proteins in fatty liver disease and suggest that particular endogenous or dietary lipids might promote health through SKN-1/Nrf. DOI: http://dx.doi.org/10.7554/eLife.07836.00

DAF-16/Forkhead Box O Transcription Factor: Many Paths to a Single Fork(head) in the Road

The Caenorhabditis elegans Forkhead box O transcription factor (FOXO) homolog DAF-16 functions as a central mediator of multiple biological processes such as longevity, development, fat storage, stress resistance, and reproduction. In C. elegans, similar to other systems, DAF-16 functions as the downstream target of a conserved, well-characterized insulin/insulin-like growth factor (IGF)-1 signaling pathway. This cascade is comprised of an insulin/IGF-1 receptor, which signals through a conserved PI 3-kinase/AKT pathway that ultimately downregulates DAF-16/FOXO activity. Importantly, studies have shown that multiple pathways intersect with the insulin/IGF-1 signaling pathway and impinge on DAF-16 for their regulation. Therefore, in C. elegans, the single FOXO family member, DAF-16, integrates signals from several pathways and then regulates its many downstream target genes. Antioxid. Redox Signal. 14, 623–634