Malene Hansen et al. (2005) International Worm Meeting "Genome-wide identification of longevity genes using RNAi"

Ao-Lin Hsu, Cynthia Kenyon, Andrew Dillin, Malene Hansen

[International Worm Meeting, 2005]

Lifespan in C. elegans is influenced by several genetic pathways and processes. The DAF-2 insulin/IGF-1 pathway, the reproductive system, food intake, and mitochondrial activity (1, 2, 3) have all been shown to regulate aging: lifespan extensions of 15 to 600% occur in animals defect in one or more of these processes (1, 4, 5). The FOXO-family transcription factor DAF-16 is required for the lifespan extension observed in daf-2 mutants, as well as in animals lacking a germline (1). However, daf-16 is not required for the lifespan extension induced by perturbations of mitochondrial activities (2, 3) or by caloric restriction (5, 6). Most of our knowledge about the regulation of aging comes from mutants originally isolated because of other phenotypes. To ask whether our current view of aging has been affected by selection bias, and to deepen our understanding of known pathways, we screened a C. elegans RNAi feeding library (7) for clones that extend lifespan. In addition to known lifespan genes, we identified 23 new genes whose wild-type function is required to prevent lifespan extension (8). Reducing the function of these loci extended mean lifespan 15-80%. The 23 new longevity genes affect signal transduction, the stress response, gene expression, and metabolism. Using phenotypic and epistasis analysis, we have assigned many of these genes to known longevity pathways. Our most important findings are (i) that caloric restriction extends C. elegans lifespan by down-regulating expression of several genes, including a gene required for methylation of macromolecules, (ii) that integrin signaling is likely to play a general, evolutionarily-conserved role in lifespan regulation, and (iii) that specific lipophilic hormones may influence lifespan in a DAF-16/FOXO-dependent fashion. Surprisingly, of the new genes that have conserved sequence domains, only one could not be associated with a known longevity pathway. Thus, our current view of the genetics of aging has probably not been distorted substantially by selection bias. We expect the further study of these genes to provide valuable information about the mechanisms of aging. (1) Tatar et al., Science, 2003 (2) Dillin et al., Science, 2002 (3) Lee et al., Nature Genetics, 2002 (4) Arantes-Oliveira et al., Science, 2003 (5) Houthoofd et al., Exp Geron, 2003 (6) Lakowski and Hekimi, PNAS, 1998 (7) Kamath et al., Nature, 2003 (8) Hansen et al., manuscript (*Hansen and Hsu contributed equally to this work)

Malene Hansen et al. (2006) European Worm Meeting "Ribosomal Proteins are New Longevity Determinants in C. elegans"

Cynthia Kenyon., Malene Hansen

[European Worm Meeting, 2006]

Malene Hansen and Cynthia Kenyon. Multiple longevity pathways and genes have been identified in C. elegans. One example is the conserved DAF-2/insulin/IGF-1 pathway, which modulates lifespan through the downstream transcription factor DAF-16. Other processes, including dietary restriction, affect lifespan in daf-16 independent ways. The molecular mechanism by which dietary restriction works is largely unknown; however, experiments from other model organisms suggest the involvement of the nutrient sensor TOR (Kapahi et al., 2004, Kaeberlein et al., 2005).. Through an RNAi screen, we found that RNAi clones for ribosomal proteins (RPs) significantly extend lifespan in a daf-16 independent fashion. Knockdown of RPs belonging to both the small and the large ribosomal subunits increased lifespan, but only when RPs were inhibited during adulthood. Collectively, these observations reveal a longevity function for ribosomal proteins in adult C. elegans. Interestingly, deletion mutants of several ribosomal proteins in yeast have recently been reported to increase lifespan (Kaeberlein et al., 2005), indicating that the function of RP in lifespan modulation is conserved.. One major regulator of ribosomal biogenesis and translation is the kinase TOR, which, like RPs, influences C. elegans lifespan in a daf-16 independent fashion (Vellai et al., 2003). We have therefore begun to investigate a potential mechanistic overlap among RPs, TOR, and some of its putative effectors (including S6 kinase and several translation initiation factors).. We expected that this group of molecules collectively involved in translation would behave similarly to influence longevity. We were therefore surprised to find significant differences in how these molecules affect lifespan. Most importantly, our genetic analysis suggests that TOR affects lifespan by a dietary restriction-like mechanism in C. elegans, whereas RPs may function by an as yet unidentified mechanism. TOR''s role in dietary restriction might therefore involve TOR-regulated processes other than translation per se. We are currently investigating this hypothesis in more detail.

Malene Hansen et al. (2007) International Worm Meeting "Autophagy is required for dietary restriction to extend lifespan in C. elegans."

Cynthia Kenyon, Malene Hansen

[International Worm Meeting, 2007]

Malene Hansen et al. (2003) International Worm Meeting "Genome-wide identification of longevity genes in C. elegans using systematic RNAi"

Ao-Lin Hsu, Malene Hansen, Ravi Kamath, Andrew Fraser, Andrew Dillin, Cynthia Kenyon, Julie Ahringer

[International Worm Meeting, 2003]

Lifespan in C. elegans is determined by multiple genetic pathways and processes. The DAF-2 insulin/IGF-1 pathway, the reproductive system, food intake, and mitochondrial activity (1, 2, 3) have been shown to regulate aging: lifespan extensions of 15 to 500% occur in animals defect in one or more of these processes (1). The forkhead transcription factor DAF-16 is required for the lifespan extension observed in daf-2 mutants, as well as in animals defective in germline signaling (1). However, daf-16 is not required for the lifespan extension induced by pertubations of mitochondrial activities (2, 3) or in calorically restricted animals (6). Identification of additional genes is needed to clarify how these pathways and processes differ in lifespan regulation. To identify such new genes that regulate aging, we carried out a genome-wide RNA interference (RNAi) screen. Specifically, we looked for genes that, when inhibited by RNAi, lead to a long-lived phenotype. Our RNAi feeding library contains 87% of the predicted C. elegans open reading frames. In addition to known lifespan genes, we identified 31 new genes whose wild-type function is required to prevent lifespan extension. Reducing the function of these loci extended lifespan anywhere between 15-80%. The lifespan extension observed with 10 of these clones was dependent on daf-16, suggesting that they might encode new components in the DAF-2 insulin/IGF-1 signaling pathway, or, potentially, daf-2-independent, daf-16-dependent genes. We are currently characterizing these genes further. In contrast, the lifespan extension observed with the remaining 21 genes, including components of the mitochondrial respiratory chain (see 2), was not dependent on daf-16. We are continuing the epistasis analysis of these genes to determine their involvement, if any, in other known aging pathways. In summary, our genome-wide RNAi screen has identified over 30 new genes involved in regulation of lifespan in C. elegans. We expect the study of these genes to provide a wealth of information about the aging process, and possibly to define new aging pathways in C. elegans. (1) Guarente and Kenyon, Cell, 2000 (2) Dillin et al., Science, 2002 (3) Lee et al., Nature Gen, 2002 (4) Lin et al., Science, 1997(5) Ogg et al., Nature, 1997(6) Lakowski and Hekimi, PNAS, 1998

Malene Hansen et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Autophagy Genes Affect in the Extension of Lifespan by Dietary Restriction in C. elegans"

Abha Chandra, Cynthia Kenyon, Malene Hansen, Laura Mitic, Brian Onken, Monica Driscoll

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Dietary restriction (DR) extends lifespan in multiple organisms, at least in part, by down-regulation of the TOR pathway (1,2). In C. elegans, both DR and TOR inhibition reduce protein translation (1) and extend lifespan via a DAF-16/FOXO independent mechanism (3-5). Even though inhibition of protein translation extends lifespan (1, 6-8), it remains unclear if this is the sole process by which DR promotes longevity or whether this is also an independent avenue of regulation. Besides protein translation, TOR also regulates autophagy. This cellular process of cytoplasmic degradation is generally induced following nutritional deprivation (9). To better understand the molecular mechanism underlying DR, we have addressed the role of autophagy in this process in C. elegans (10). We find that DR and TOR inhibition trigger autophagy in this organism, and that inhibiting genes required for autophagy during adulthood prevents DR and TOR inhibition from extending lifespan. The longevity response to DR in C. elegans is not a passive consequence of reduced energy levels, because it is known to require transcription, in part through the PHA-4 transcription factor (11). Interestingly, we find that the autophagic response to DR also requires PHA-4 activity, indicating that autophagy in relation to lifespan, too, is a transcriptionally-regulated response to food limitation. Finally, in spite of the rejuvenating effect that autophagy is predicted to have on cells, we find that autophagy does not appear to be sufficient to extend lifespan. Long-lived daf-2 insulin/IFG-1 receptor mutants require both the transcription factor DAF-16/FOXO (12) and the autophagy for longer life. Taken together with our finding that autophagy takes place in the absence of DAF-16, we conclude that autophagy is necessary but not sufficient for lifespan extension in the context of insulin/IGF-1 signaling. Perhaps, by recycling cellular components, autophagy provides raw material for new protein construction, and transcription factors such as DAF-16 program the cells to recycle these components into cell-protective longevity proteins.

Gelino, Sara et al. (2013) International Worm Meeting "Role of Autophagy in Long-lived C. elegans Subjected to Dietary Restriction."

Chang, Jessica, Hansen, Malene, Gelino, Sara

[International Worm Meeting, 2013]

Multiple conserved pathways and processes can modulate lifespan, including dietary restriction (DR). While the underlying mechanism for how DR promotes longevity remains poorly understood, we and others have recently shown a direct role for the cellular recycling process autophagy, in lifespan extension induced by DR. Specifically, autophagy is induced in response to DR, and autophagy genes are required for DR to extend lifespan in C. elegans (Hansen et al., 2008). However, these studies did not address how autophagy may prolong organismal lifespan in response to DR at the cellular or molecular level. To address the cellular role of autophagy in DR, we are investigating autophagy in different tissues of dietary-restricted C. elegans. First, we are using different markers to systematically monitor the autophagy process in all major tissues. Second, we are utilizing tissue-specific RNA interference models to inactivate autophagy and functionally evaluate the role of autophagy in select tissues. Knowledge of where in the animal that autophagy is occurring and may have a rejuvenating effect will shed light on the underlying mechanism by which autophagy promotes longevity. Our preliminary studies suggest that DR induces autophagy in multiple tissues and depletion of autophagy genes in single tissues, including the intestine, is sufficient to abrogate DR-mediated lifespan extension. These data indicate that DR broadly induces autophagy in multiple tissues of C. elegans, and several tissues engage autophagy in a lifespan-promoting fashion in response to DR. Future work will investigate if autophagy induced by DR is similarly engaged in all tissues. We propose that the autophagic turnover of tissue-specific material, the nature of which is still to be identified, may prolong the youthfulness of a cell, tissue, and organism.

Kumsta, Caroline et al. (2011) International Worm Meeting "Translational Control of Tumor Formation in C. elegans."

Hansen, Malene, Kumsta, Caroline

[International Worm Meeting, 2011]

The major risk factor for cancer development is increased age. With age, the likelihood of tumor formation and cancer incidence increases in many organisms, including humans. Recently, C. elegans was employed as a model system for tumor formation, and mutations that increase lifespan, e.g., in the insulin/IGF-1 receptor (InR/daf-2), decrease tumor growth in this model, suggesting that aging and tumor progression are linked mechanistically. This link creates the opportunity to identify longevity modulators that have effects on tumor formation in C. elegans. Similarly to inhibition of InR/daf-2, the reduction of components of the mRNA translation initiation complex was recently found to increase longevity in C. elegans. Conversely, increased function of translation initiation factors has been observed in many human cancers, yet the precise mechanisms by which these factors affect tumor formation are unclear. This study aims to address the hypothesis that reduced translation initiation could constitute a tumor suppressor mechanism in C. elegans. Specifically, we investigate the oncogenic function of the translation initiation machinery on the C. elegans tumor model. These tumors arise due to increased Notch/GLP-1 signaling in the C. elegans germline, which leads to the massive overproliferation of germ cells and their subsequent break out of the gonad, causing the animals to die prematurely. Notably, we have found that the reduction of specific components of the translation initiation machinery rescues tumor growth and the premature death in C. elegans. These findings indeed suggest that impairment of the translation initiation complex has tumor-suppressive effects in C. elegans. We have now begun to investigate the possible underlying mechanisms by which the translation initiation complex affects the formation of a tumorous germline in C. elegans. This study, which is rapidly carried out in C. elegans due to its tractable genetics and short lifespan, has the potential to identify novel tumor-suppressor and oncogenes that might influence tumor formation also in mammalian systems.

She, Xingyu et al. (2015) International Worm Meeting "Applying a Spatial-Temporal Controlled Gene Expression System to Aging Research."

She, Xingyu, Hansen, Malene

[International Worm Meeting, 2015]

C. elegans has been an effective model organism for identifying genes with important physiological functions, including in aging. However, to fully dissect gene function an effective spatial-temporal controlled gene expression system is needed. In C. elegans, several protocols have been put forward for this purpose, including driving temporal gene expression using heat shock inducible promoters (Bacaj et al. 2007), and achieving tissue-specific gene expression using site-specific recombination methods (Cre-loxP, FLP-FRT). However, none of these systems have been used for simultaneous temporal- and spatial control and it is uncertain how applicable they would be for aging studies (see Xu & Kim, 2011). While several chemicals (e.g., tetracycline, RU486) are utilized to induce gene expression in other systems, such protocols are not available in C. elegans. However, the Shen lab recently adapted the Q system to C. elegans, a system in which inducible gene expression is obtained by the chemical quinic acid (Wei et al. 2013). This binary system, originally discovered in the fungus Neurospora crassa, is comprised of the transcription factor QF and its repressor QS. In the presence of quinic acid, the QS repressor is released from binding with QF, allowing QF to activate the expression of transgenes. Temporal control is achieved through the timing of quinic acid supplementation, and spatial control by use of tissue-specific promoters that drive the expression of QS and QF (Wei et al. 2013). We are interested in applying the Q system for our research on genes with roles in aging. Towards this goal, we are focusing on three main aspects. First, we have tested the effects of quinic acid and the Q system on C. elegans lifespan and stress resistance and detected no apparent alterations on longevity or fitness, suggesting that the Q system is suitable for aging studies. Second, we are driving the elements of the Q system with tissue-specific promoters to test inducibility in major tissue types of adult animals. Preliminary results indicate that the Q system can be used to induce expression in selected tissues, including neurons and muscle. Third, we are creating a library of Gateway-compatible plasmids that contain the various elements of the Q system and tissue-specific promoters to expedite the generation of Q system transgenic strains for future gene-specific projects. We will present our current progress towards applying the Q system as a powerful tool for spatial- and temporal analysis of gene function in adult C. elegans. This work will also be discussed at the "Spatial and temporal analysis of gene function in C. elegans" workshop.

Gelino, Sara et al. (2015) International Worm Meeting "Autophagy in the Intestine Acts to Influence Healthspan and Longevity During Dietary Restriction."

Gelino, Sara, Chang, Jessica, Hansen, Malene

[International Worm Meeting, 2015]

Autophagy is a cellular recycling process with key roles in development and aging. Work from several organisms including C. elegans has found that autophagy plays a beneficial role in several longevity models, including dietary restriction (DR). However, it remains unknown how autophagy may prolong organismal lifespan in response to DR at the cellular and molecular level.Autophagy is a multi-step process during which double-membrane vesicles called autophagosomes recruit cytosolic material that subsequently undergoes degradation when the autophagosomes fuse with lysosomes. To address the cellular role of autophagy in DR, we monitored autophagy reporters in multiple tissues of wild-type animals and dietary-restricted eat-2 mutants with age. Noticeably, of the tissues investigated, the intestine showed the most significant changes in autophagosome numbers in eat-2 mutants compared to wild type. Noticeably, additional assays indicated that this induction in the intestine of eat-2 mutants occurs mainly by enhanced lysosomal turnover. This analysis is the first to assess the relative contribution of the different steps of the autophagy process in adult C. elegans.To complement these studies, we also analyzed the functional role of autophagy in the intestine. Consistent with an important role for autophagy in this tissue, we found that knock-down of autophagy genes specifically in the intestine of eat-2 mutants is sufficient to shorten their lifespan. We next investigated the role of autophagy in intestinal barrier function to learn more about how autophagy is utilized in the intestine. We observed that intestinal integrity, assessed by feeding the animals blue food dye, is improved in eat-2 mutants compared to wild type. Interestingly, we observed that intestinal-specific inhibition of autophagy greatly impairs intestinal barrier function in eat-2 animals, suggesting that autophagy plays a critical role in maintaining intestinal integrity, which likely contributes to overall health of the organism.Collectively, our study indicates that DR causes changes in autophagy in multiple tissues of C. elegans, and highlights a key role for autophagy in the intestine of dietary-restricted C. elegans to ensure improved healthspan and lifespan. These cellular insights will likely be critical for targeting autophagy to improve healthspan in higher organisms.

Tan, Ee Phie et al. (2019) International Worm Meeting "Probing Mechanisms of Selective Autophagy Relevant to Aging."

Hansen, Malene, Choy, Elizabeth, Tan, Ee Phie

[International Worm Meeting, 2019]

Autophagy is an evolutionarily conserved cellular recycling process, which is critical for development, disease, and aging. Therefore, determining the molecular action of autophagy is crucial to better understand organismal health. Notably, autophagy is increasingly appreciated as a selective process by which specific types of cytoplasmic material or cargo, such as mitochondria or lipids are sequestered into double-membrane vesicles called autophagosomes, which fuse with lysosomes to facilitate degradation of the contents. Selective cargo sequestration to the autophagosome is achieved by autophagy receptors, which simultaneously bind specific cargo and Atg8/LC3/GABARAP, a protein localized to the autophagosomal membrane. While accumulating evidence indicates that dysregulation in selective types of autophagy can contribute to the development of age-related diseases, the relevant selective autophagy receptors and their role in aging remain understudied. To identify selective autophagy receptors relevant to aging, we performed a candidate RNA interference (RNAi) screen using genes previously reported to be potential autophagy receptors in other model systems. We used C. elegans expressing GFP-tagged Atg8/LGG-1 and looked for aberrant Atg8/LGG-1 expression upon gene knockdown. In this primary screen, we identified many candidate genes to increase LGG-1 levels. Currently, we are performing secondary RNAi screens of candidates from the primary screen, utilizing cargo-specific reporters to look for genes that increase cargo levels upon knockdown. In a parallel approach, we are also performing unbiased proteomic screenings to identify proteins that interacts with Atg8/LGG-1 and which could be potential autophagy receptors. Proteins of interests from these screening approaches will be analyzed further by immunoprecipitation and immunohistochemistry assays to determine if they are potential autophagy receptors, and if they are critical for the healthspan and longevity of the animal. This study will improve our molecular understanding of the regulation of the physiological relevance of selective autophagy in aging. Such insights may provide new entry points for designing targeted therapeutic interventions against age-related diseases.