The goal of geroscience, or research into old age, is to promote health during old age, and thus, to increase lifespan. In the body, the groups of biochemical reactions, or ‘pathways’, that allow an organism to sense nutrients, and regulate growth and stress, play major roles in ensuring healthy aging. Indeed, organisms that do not produce a working version of the insulin/IGF-1 receptor, a protein involved in one such pathway, show increased lifespan. In the worm Caenorhabditis elegans, mutations in the insulin/IGF-1 receptor can even double their lifespan. However, it is unclear whether this increase can be achieved once the organism has reached old age.

To answer this question, Venz et al. genetically engineered the nematode worm C. elegans so that they could trigger the rapid degradation of the insulin/IGF-1 receptor either in the entire organism or in a specific tissue. Venz et al. started by aging several C. elegans worms for three weeks, until about 75% had died. At this point, they triggered the degradation of the insulin/IGF-1 receptor in some of the remaining worms, keeping the rest untreated as a control for the experiment.

The results showed that the untreated worms died within a few days, while worms in which the insulin/IGF-1 receptor had been degraded lived for almost one more month. This demonstrates that it is possible to double the lifespan of an organism at the very end of life.

Venz et al.’s findings suggest that it is possible to make interventions to extend an organism’s lifespan near the end of life that are as effective as if they were performed when the organism was younger. This sparks new questions regarding the quality of this lifespan extension: do the worms become younger with the intervention, or is aging simply slowed down?

The goal of aging research or geroscience is to identify interventions that promote health during old age (Kennedy et al., 2014; López-Otín et al., 2013; Partridge et al., 2018). Nutrient-sensing pathways that regulate growth and stress resistance play major roles as conserved assurance pathways for healthy aging (Kenyon, 2010; López-Otín et al., 2013). One of the first longevity pathways discovered was the insulin/insulin-like growth factor (IGF)-1 signaling pathway (reviewed in Kenyon, 2010). Reducing insulin/IGF-1 signaling (IIS) increases lifespan across species (Kenyon, 2010). Mice heterozygous for the IGF-1 receptor, or with depleted insulin receptor in adipose tissue, are stress-resistant and long-lived (Blüher et al., 2003; Holzenberger et al., 2003), for example, and several single-nucleotide polymorphisms in the IIS pathway have been associated with human longevity (Kenyon, 2010). Moreover, gene variants in the IGF-1 receptor have been associated and functionally linked with long lifespans in human centenarians (Suh et al., 2008). This suggests that a comprehensive understanding of this pathway in experimental, genetically tractable organisms has promising translational value for promoting health in elderly humans. However, whether or not reducing IIS during end-of-life stages can still promote health and longevity in any organism is unknown. Therefore, we turned to the model organism Caenorhabditis elegans to investigate whether reducing IIS during old age was sufficient to increase lifespan.

The groundbreaking discovery that a single mutation in daf-2, which is the orthologue of both the insulin and IGF-1 receptors (Kimura et al., 1997), or mutations in ‘downstream’ genes in the IIS pathway, could double the lifespan of an organism was made in the nematode C. elegans (Friedman and Johnson, 1988; Kenyon et al., 1993). Since its discovery, over 1000 papers on daf-2 have been published, making it one of the most studied genes in this model organism (Source: PubMed). Genetic and genomics approaches have revealed that the DAF-2 insulin/IGF-1 receptor signaling regulates growth, development, metabolism, inter-tissue signaling, immunity, stress defense, and protein homeostasis, including extracellular matrix remodeling (Ewald et al., 2015; Gems et al., 1998; Kimura et al., 1997; Murphy and Hu, 2013; Wolkow et al., 2000). Much of our knowledge of the effects of daf-2 on aging has come from the study of reduction-of-function alleles of daf-2. Several alleles of daf-2 have been isolated that are temperature-sensitive with respect to an alternative developmental trajectory. For instance, most daf-2 mutants develop into adults at 15°C and 20°C but enter the dauer stage at 25°C (Gems et al., 1998), which is a facultative and alternative larval endurance stage in which C. elegans spends most of its life cycle in the wild (Hu, 2007). Under favorable conditions, C. elegans develops through four larval stages (L1–L4). By contrast, when the animals are deprived of food and experience an overcrowded environment and/or thermal stress (above 27°C), the developing larvae molt into an alternative pre-dauer (L2d) stage. If conditions do not improve, C. elegans enter the dauer diapause instead of the L3 stage (Golden and Riddle, 1984; Hu, 2007; Karp, 2018).

A major limitation in using daf-2 mutants is that several of them show L1 larval and pre-dauer stage (L2d) arrest (Gems et al., 1998). Furthermore, the daf-2 alleles have been categorized into two mutant classes depending on the penetrance of dauer-like phenotypes during adulthood, such as reduced brood size, small body size, and germline shrinkage, as observed in the daf-2 class II mutants (Arantes-Oliveira et al., 2003; Ewald et al., 2018; Ewald et al., 2015; Gems et al., 1998; Hess et al., 2019; Patel et al., 2008; Podshivalova and Kerr, 2017). RNA interference of daf-2 can be applied, which increases lifespan without dauer formation during development and circumvents induction of daf-2 class II mutant phenotypes during adulthood (Dillin et al., 2002; Ewald et al., 2018; Ewald et al., 2015; Kennedy et al., 2004). However, the increase in lifespan by RNAi of daf-2 is only partial compared to strong alleles such as daf-2(e1370) (Ewald et al., 2015). Furthermore, adult-specific RNAi knockdown of daf-2 quickly loses its potential to increase lifespan and does not extend lifespan when started after day 6 of adulthood (Dillin et al., 2002), that is, after the reproductive period of C. elegans. Whether this is due to age-related functional decline of RNAi machinery or residual DAF-2 protein levels, or whether the late-life depletion of daf-2 simply does not extend lifespan remains unclear. As such, using an alternative method to reduce DAF-2 levels beyond RNAi or daf-2 mutation may allow us to more clearly uncouple the pleiotropic effects of reduced IIS during development from those that drive daf-2-mediated longevity during late adulthood.

To this end, we used an auxin-inducible degradation (AID) system to induce the depletion of the degron-tagged DAF-2 protein with temporal precision (Zhang et al., 2015). The Arabidopsis thaliana IAA17 degron is a 68-amino acid motif that is specifically recognized by the transport inhibitor response 1 (TIR1) protein only in the presence of the plant hormone auxin (indole-3-acetic acid; Dharmasiri et al., 2005). Although cytoplasmic, nuclear, and membrane-binding proteins tagged with degron have been recently shown to be targeted and degraded in C. elegans (Beer et al., 2019; Zhang et al., 2015), to our knowledge, the AID system has not been used previously to degrade transmembrane proteins, such as the DAF-2 insulin/IGF-1 receptor. We find that using AID effectively degrades DAF-2 protein and promotes dauer formation when applied early in development. Dauer-like phenotypes are present in adults when AID of DAF-2 is applied late in development. Some of these adulthood dauer traits are induced by the loss of daf-2 in neurons, but others seem to be caused by the systemic loss of daf-2. More importantly, the post-developmental, conditional degradation of DAF-2 protein extends lifespan without introducing dauer-like phenotypes. Remarkably, we demonstrate that when more than half of the population has died at day 25 of adulthood, AID of DAF-2 in these remaining aged animals is sufficient to promote longevity. Our work suggests that therapeutics applied at even extremely late stages of life are capable of increasing longevity and healthspan in animals.

For longevity interventions to be efficient without causing undesired side effects, the time point of treatment must be chosen carefully. This is especially important for pathways such as the insulin/IGF-1 pathway that is essential for growth and development (Kenyon, 2010). Although the importance of DAF-2 in regulating lifespan is well established, the consequences of late-in-life inhibition remained unknown.

Here, we demonstrate that late-life degradation of DAF-2 extends lifespan. In this work, we have effectively engineered a degron tag into the endogenous daf-2 locus using CRISPR, representing the first report of auxin-induced degradation (AID) of a transmembrane receptor in vivo. DAF-2 receptor levels were strongly reduced via AID. Consistent with reduced insulin signaling, AID-mediated degradation of DAF-2 facilitated dauer formation, longevity, stress resistance, caused growth-related phenotypes during early adulthood, and finally increased longevity during the post-reproductive geriatric stages of life.

It is well established that DAF-2/insulin/IGF-1 receptor signaling connects nutrient levels to growth and development (Murphy and Hu, 2013). This is attributed to insulin-like peptides binding DAF-2 and activating a downstream phosphorylation kinase cascade that alters metabolism (Murphy and Hu, 2013). Surprisingly, we find that DAF-2 receptor abundance is linked to nutrient availability and perhaps dietary content. Starving C. elegans leads to decreased DAF-2 receptor abundance, consistent with previous in situ antibody staining (Kimura et al., 2011), whereas mimicking a high-energy diet by adding glucose to the bacterial food source increases DAF-2 receptor abundance. This suggests an additional layer of regulating IIS by connecting food cues to adapt metabolism via DAF-2 receptor levels, potentially as an internal representation of the environment.

Environmental conditions are carefully monitored by developing C. elegans. First, larval stage (L1) wild-type C. elegans that are food-deprived, exposed to elevated temperatures (>27°C), and/or are crowded, enter into a pre-dauer L2d stage, where they continue monitoring their environment before committing and molting into dauers (Hu, 2007; Karp, 2018). However, previous temperature-shifting experiments (from 15°C to 25°C) with daf-2 mutants have indicated the existence of a ‘dauer decision time window’ between the L1 and L2 stage (Swanson and Riddle, 1981). By using AID to manipulate DAF-2 levels directly, we found that AID degradation of DAF-2 during a narrow time period in the mid-L1 stage is sufficient to induce dauer formation, suggesting that the decision to enter dauer relies upon a threshold of DAF-2 protein levels. This decision is uncoupled from temperature or food abundance. Thus, absolute DAF-2 protein abundance appears to be a key factor in the animal’s decision to enter into the dauer state during early development.

Although IIS is reduced in daf-2(e1370 or e1368) mutants at lower temperatures (Ewald et al., 2018; Gems et al., 1998), dauer larvae are formed only when these daf-2 mutants are exposed to higher temperatures. The observation that mutant DAF-2 protein (Kimura et al., 2011) but not wild-type DAF-2 protein (Figure 5) is lost at elevated temperatures suggests a model where DAF-2 mutant protein becomes unstable with increasing temperatures and might be subsequently targeted for degradation. This hypothesis might explain why strong class II daf-2 mutants, such as daf-2(e979) and daf-2(e1391), which have much lower DAF-2 protein levels at 15°C and 25°C compared to other daf-2 mutants (Tawo et al., 2017), exhibit higher propensities toward dauer formation at any temperature (Gems et al., 1998). Thus, the severity of classical daf-2 mutant alleles in regard to dauer formation and adult dauer traits might be linked to DAF-2 receptor abundance.

At higher temperatures, adult daf-2 class II mutants exhibit significant penetrance of undesired phenotypes (Ewald et al., 2018). In these animals, a clear remodeling of the body and internal organs, including constriction of the pharynx and shrinkage of the germline, remains present, along with an altered neuronal morphology and electrical synapse connectome that drives behavioral changes such as quiescence, diminished foraging behavior, and altered egg-laying programs (Arantes-Oliveira et al., 2003; Ewald et al., 2015; Gems et al., 1998; Hess et al., 2019; Bhattacharya et al., 2019; Patel et al., 2008; Podshivalova and Kerr, 2017). We showed that L4-specific AID of the DAF-2::degron results in a non-conditional reduction of body size and brood size, whereas egg retention and germline shrinkage only occur at higher temperatures. This indicates that the non-conditional daf-2 class II traits are not a residual effect of a dauer-like developmental program but instead are side effects caused by reduced functions of DAF-2 during later stages of development.

Additional temperature-sensitive daf-2 class II traits of egg retention and gonad shrinkage are mediated by loss of daf-2 in neurons only at higher temperatures. Why these traits only manifest at higher temperatures remains unclear. One explanation may be that DAF-2 levels are reduced in temperature-sensing neurons, which then elicits a systemic effect that drives germline shrinkage and egg retention. Neurons are refractory to RNAi, suggesting that daf-2(RNAi) effects work through other tissues than neurons to extend lifespan. Furthermore, treating class I mutants daf-2(e1368) with daf-2(RNAi) doubles their longevity without causing adult daf-2 class II traits (Arantes-Oliveira et al., 2003), suggesting that daf-2(RNAi) would lower DAF-2 receptor levels in other tissues than neurons for this additive longevity effect.

Consistent with neuronal regulation of these traits is that daf-2 RNAi applied to wild type does not result in dauers but results in dauer formation when applied to neuronal-hypersensitive RNAi C. elegans strains (Dillin et al., 2002; Ewald et al., 2015; Kennedy et al., 2004). We find that DAF-2 degradation in neurons or intestine increases lifespan. This is consistent with a previous finding by Apfeld and Kenyon that either mosaic loss of daf-2 in AB cell lineage that gives rise to neurons and other cells (epidermis, seam, pharyngeal, vulval cells) or loss of daf-2 in EMS cell lineage that gives rise to intestinal and other cells (pharyngeal and gonadal cells) results in increased lifespan (Apfeld and Kenyon, 1998). By contrast, our neuronal DAF-2 depletion did not result in a doubling of lifespan as seen by loss of daf-2 in the AB lineage (Apfeld and Kenyon, 1998), suggesting that loss of daf-2 in other tissues, in combination with neurons or intestine, is required to recapitulate full lifespan extension. Given that reducing DAF-2 in neurons results in daf-2 class II traits at higher temperatures, one might target intestinal DAF-2 for degradation to uncouple longevity from any undesired phenotypes. Yet, DAF-2 is essential for growth. We find the best time point for DAF-2 inhibition is rather late in life to bypass these undesired side effects to promote longevity.

We find that as late as day 25 of adulthood, when almost three-quarters of the population had died, AID of DAF-2 is sufficient to increase lifespan. The only other manipulation that was able to increase the lifespan so late in life was the transfer of old C. elegans to culture plates without food (Smith et al., 2008). However, C. elegans do not feed after reaching mid-life (Collins et al., 2008; Ewald et al., 2016), suggesting that it might not be the intake of calories that promotes longevity. Instead, the old C. elegans could sense the absence of food and thereby reduce DAF-2 levels to promote longevity. Along these lines, it would be interesting to determine if late-life bacterial deprivation works synergistically with DAF-2 AID or not in future studies.

In mammals, mid-life administration of IGF-1 receptor monoclonal antibodies to 78-week-old mice (a time when all mice are still alive and 6 weeks before first mice start to die) is sufficient to increase their lifespan and improve their healthspan (Mao et al., 2018). Other parallels between mammals and nematodes are the tissues from which lower insulin/IGF-1 receptor levels promote longevity. Mice carrying brain-specific heterozygous IGF-1 receptor knockout are long-lived (Kappeler et al., 2008), as are the mice with adipose-specific knockout of the insulin receptor (Blüher et al., 2003). This is reminiscent of our findings that AID of DAF-2::degron in neurons or intestine (the major fat-storage tissue in C. elegans) was sufficient to increase the lifespan. These similarities could reflect conserved functions, as daf-2 is considered the common ancestor to both insulin and IGF-1 receptors (Kimura et al., 1997).

Although it is known that neurons and the intestine are important for food perception and regulation of food intake, the effects of food perception or intake on insulin receptor and IGF-1 receptor levels are poorly understood. Starving rats for 3 days increases the abundance of insulin-bound insulin receptors (Koopmans et al., 1995), possibly to increase glucose uptake. It is unknown whether prolonged starvation would lead to lower basal insulin/IGF-1 receptor levels. However, alterations of the IGF-1 receptor levels are associated with altered lifespan. For instance, heterozygous IGF-1 receptor knockout mice, which have lower IGF-1 receptor levels, have an increased lifespan (Holzenberger et al., 2003; Kappeler et al., 2008; Xu et al., 2014). Also, overexpression of the short isoform of p53 (p44) increases IGF-1 receptor levels and shortens the lifespan of mice (Maier et al., 2004). Furthermore, the administration of recombinant human IGF-1 increases IGF-1 receptor abundance in murine embryonic cells (Maier et al., 2004). Food components themselves can affect insulin receptor levels. For instance, palmitate activates PPARα to induce miR-15b, which targets insulin receptor mRNA for degradation (Li et al., 2019). Potentially, food components could impact the murine insulin/IGF-1 receptor also via its degradation.

Indeed, there is some evidence suggesting the regulation of insulin/IGF-1 receptor abundance by E3 ubiquitin ligases. For instance, the E3 ligase CHIP regulates insulin/IGF-1 receptor levels in C. elegans, Drosophila, and human cell cultures (Tawo et al., 2017). In mice, the muscle-specific mitsugumin 53 (MG53) E3 ligase targets the insulin receptor for degradation (Song et al., 2013). High-fat diet results in the reduction of insulin receptor levels (Li et al., 2019) via higher MG53-mediated degradation (Song et al., 2013). MG53 is upregulated under a high-fat diet in mice, and MG53-/- deficient mice are protected from high-fat diet-induced obesity, insulin resistance, and other metabolic syndrome-associated phenotypes (Song et al., 2013). Furthermore, another E3 ligase, MARCH1, is overexpressed in obese humans and targets the insulin receptor for ubiquitin-mediated degradation (Nagarajan et al., 2016). Taken together, these observations suggest that food abundance controls mammalian insulin receptor levels via E3 ligase-mediated degradation. Although in C. elegans we found the opposite changes in DAF-2 receptor levels, that is, they were reduced upon starvation and increased upon high glucose feeding, our observations suggest that nutritional cues may regulate insulin/IGF-1 receptor levels via a variety of mechanisms, including ubiquitination and proteasomal degradation, across species.

In summary, we have demonstrated that interventions at almost the end of life can increase lifespan. We have established that auxin-induced degradation is suitable for targeting transmembrane receptors for non-invasive manipulations during developmental and longevity in vivo studies. We reconciled a longstanding question by providing evidence that dauer-like traits are not a spill-over of reprogrammed physiology from developing L2d pre-dauers. Instead, the essential growth-related functions of DAF-2 are causing deficits when applied during development or growth phases. We have shown that tissue-specific interventions or global interventions beyond reproduction or growth extend lifespan without pathology or deficits. Degradation of DAF-2/insulin/IGF-1 receptor might not be an artificial intervention since DAF-2/insulin/IGF-1 receptor abundance is read-out to adapt metabolism to food abundance. Dissecting intrinsic DAF-2/insulin/IGF-1 receptor abundance in response to nutritional cues may impact our understanding of nutrient sensing in promoting health during old age.

Source Data 1 includes data for all figures, Source Data 2 shows all full western blots and Source Data 3 contains raw data for all arsenite stress assays.

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Author details

1. Richard Venz

   Eidgenössische Technische Hochschule Zürich, Department of Health Sciences and Technology, Institute of Translational Medicine, Schwerzenbach-Zürich, Switzerland

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Performed all assays not listed by other authors. Wrote the manuscript with CYE in consultation with the other authors, Validation, Visualization, Writing – original draft

   Competing interests

   No competing interests declared

2. Tina Pekec

   1. University of Basel, Faculty of Natural Sciences, Basel, Switzerland
   2. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

   Contribution

   Investigation, Methodology, Crossed the DAF-2::degron strain and the transgenes into DAF-2::degron strain, Validation

   Competing interests

   No competing interests declared

3. Iskra Katic

   Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Rafal Ciosk

   1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
   2. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego, Poland
   3. University of Oslo, Department of Biosciences, Oslo, Norway

   Contribution

   Conceptualization, Funding acquisition, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2234-6216

5. Collin Yvès Ewald

   Eidgenössische Technische Hochschule Zürich, Department of Health Sciences and Technology, Institute of Translational Medicine, Schwerzenbach-Zürich, Switzerland

   Contribution

   Performed late-in-life top-coating auxin lifespans. Wrote the manuscript with RV in consultation with the other authors, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

   For correspondence

   collin-ewald@ethz.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1166-4171

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