End-of-life targeted auxin-mediated degradation of DAF-2 Insulin/IGF-1 receptor promotes longevity free from growth-related pathologies

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Preferably, lifespan-extending therapies should work when applied late in life without causing undesired pathologies. However, identifying lifespan-extending interventions that are effective late in life and which avoid undesired secondary pathologies remains elusive. Reducing Insulin/IGF-1 signaling (IIS) increases lifespan across species, but the effects of reduced IIS interventions in extreme geriatric ages remains unknown. Using the nematode C. elegans , we engineered the conditional depletion of the DAF-2/insulin/IGF-1 transmembrane receptor using an auxin-inducible degradation (AID) system that allows for the temporal and spatial reduction in DAF-2 protein levels at time points after which interventions such as RNAi may lose efficacy. Using this system, we found that AID-mediated depletion of DAF-2 protein efficiently extends animal lifespan. Depletion of DAF-2 during early adulthood resulted in multiple adverse phenotypes, including growth retardation, germline shrinkage, egg-retention, and reducing offspring. By contrast, however, AID-mediated depletion of DAF-2 specifically in the intestine resulted in an extension of lifespan without these deleterious effects. Importantly, AID-mediated depletion of DAF-2 protein in animals past their median lifespan allowed for an extension of lifespan without affecting growth or behavioral capacity. Thus, both late-in-life targeting and tissue-specific targeting of IIS minimize the deleterious effects typically seen with interventions that reduced IIS, suggesting potential therapeutic methods by which longevity and healthspan can be increased in even geriatric populations.

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  1. Evaluation Summary:

    This paper reports careful work using auxin-mediated degradation to manipulate the DAF-2 insulin/IGF-1 receptor in specific tissues and at different ages of the nematode C. elegans. Since its initial discovery as a gene that could dramatically alter lifespan in this organism, daf-2 has been extensively studied. The authors make excellent and thorough use of their novel reagents to successfully add important new findings to our understanding of this broadly conserved aging pathway, including a finer dissection of the spatial and temporal requirements for DAF-2 in multiple processes, such as the decision window for entering dauer arrest and that altering the levels of this protein very late in life can still have dramatic effects on lifespan. Overall the data are convincing, but the presentation and clarity of the text could be improved.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    This paper by R. Venz et al. describes careful work involving the use of auxin-mediated degradation of the DAF-2 insulin/IGF-1 receptor in the nematode Caenorhabditis elegans. Since its initial discovery as a gene that could dramatically alter lifespan in this organism, daf-2 has been very extensively studied. Nevertheless, the authors make excellent and thorough use of their novel reagents to successfully add important new findings to our understanding of this broadly conserved aging pathway.

    One of the most impactful findings described here is that initiating auxin-mediated degradation of DAF-2 at a time late in life, when ~50% of the worms have already died, can still lead to dramatic increases in lifespan, and does not appear to lead to any other "side" effects of lowered DAF-2 activity. As the authors point out, this is informative in terms of what might be possible in intervening in human aging. The authors are also similarly able to restrict the effects only to those involving lifespan by limiting auxin-mediated degradation of DAF-2 only to specific tissues.

    Interestingly, in addition to the extensive results focused on the lifespan-related phenotypes mediated by DAF-2, the authors are able to draw a useful new conclusion about regulation of dauer diapause by DAF-2, namely that "AID-degradation of DAF-2 during a narrow time period in the DAF-2 degron mid-L1 stage is sufficient to induce dauer formation, suggesting that the decision to enter dauer relies upon an all-or-nothing threshold of DAF-2 protein levels."

    Finally, it is worth noting that successful auxin-induced degradation of transmembrane proteins has not been previously published in C. elegans, and the authors' use of this technique here broadens future application of degron-linked degradation for this class of proteins in this model.

    Overall, these findings are of broad interest to the field. The work is thorough, and the manuscript is extremely clearly written overall, with only a few small exceptions as noted in the Specific Comments. The main and supplementary figures are also very clear, as are their captions, and these add greatly to the overall understanding of the authors' conclusions. I do not have any additional experiments to suggest that would obviously be within the scope of the current study.

  3. Reviewer #2 (Public Review):

    One of the remarkable examples of genetic input into lifespan and healthy aging is insulin/IGF-1 signaling (IIS). Reduced IIS promotes both lifespan and healthspan across species. This phenomenon was first discovered in C. elegans and later translated into other animals including mammals. In C. elegans, there is a single insulin/IGF receptor called DAF-2. Reduced daf-2 activity increases lifespan, but this finding is complicated by other phenotypes present when daf-2 activity is reduced during development. Performing RNAi post-developmentally solves some of these problems, but RNAi is apparently ineffective later in adult life. In this study, Venz et al. create and test a new tool to conditionally degrade DAF-2 protein. Specifically, they add an AID degron to the endogenous daf-2 locus, apparently creating the first degron within a transmembrane protein in C. elegans. When this daf-2 allele is combined with a transgene expressing the TIR1 ubiquitin ligase and in the presence of auxin, DAF-2 will be degraded. The authors use Western blots to show that about 40% of DAF-2 protein is degraded under these conditions. Phenotypic analysis shows that this degradation is sufficient to cause various daf-2 phenotypes during development and young adulthood that are reminiscent of phenotypes observed in daf-2 mutants. Therefore, the authors have established an important new tool to study IIS in worms. Next, the authors use the DAF-2 degron system to determine tissue requirements for DAF-2 and to show that depletion of DAF-2 late in life can increase lifespan without any of the earlier developmental effects.

    This study provides a valuable tool and new information regarding daf-2 function in development and aging. Overall, the study is clear and well-controlled, however there are some points that require clarification. These points may or may not require more experiments and could potentially be addressed by rewording and adding more information and explanation.

    1. An important claim made in the abstract and several times in the paper is that depleting DAF-2 late in life increases lifespan while avoiding "dauer-associated" phenotypes associated with earlier depletion of DAF-2. The increase in lifespan is shown in figure 5, but "dauer-associated" phenotypes are not addressed in worms with this treatment. The authors could add experiments directly examining these phenotypes in worms treated with auxin in mid/late adulthood. Alternatively, if the phenotypes in question could not occur at this late stage, an explanation could be added to clarify this point.

    2. Another claim that is emphasized is regarding the time when DAF-2 activity is required to promote non-dauer development. There are three aspects related to this claim that require more explanation.

    First, statements are made on Line 264 and line 464 that reducing daf-2 during the mid-L1 stage causes dauer formation irrespective of conditions later in development. However, I did not see data addressing the environment at L2d or later stages, other than the observation made earlier that dauer formation is induced at all temperatures tested.

    Second, the statement on line 465 that the abundance of DAF-2 appears to be the key or sole factor in the dauer formation decision should be revised to reflect the fact that there are several interconnected dauer formation pathways and mutations in genes in any of these pathways are sufficient to cause dauer formation. There is no indication that daf-2 is more important than daf-7 or daf-12, for example.

    Third, experiments shown in supplemental figure 3 address the precise timing when daf-2 activity is needed to promote non-dauer development. There are three different criteria used to describe the stages in question. The text describes animals as mid-L1. Supplemental figures 1A-B show hours since the onset of feeding. And finally supplemental figure 3C shows the number of germ cells per animal. However, these three criteria are never put in context relative to each other, so it is difficult to understand how they all relate to each other. Under the conditions in the experiments, when does the L1 molt occur in terms of hours post-feeding? How many germ cells are present at different stages of the L1? Is the correlation between germ cell number and developmental stage based on your own observations or previously published work?

    1. A more minor, but very interesting claim made on line 180 concerns the effect of the environment on DAF-2 levels. Building on the results of Kimura et al., the authors show two different experiments, one in which starvation causes a dramatic reduction in DAF-2 levels (Fig 1F-1G) and a second experiment in which no effect on DAF-2 levels was seen (Fig 1H). They interpret the difference between these experiments to be the different bacterial strains that worms were fed prior to starvation. However, there appears to be many differences between these two experiments. The OP50 experiments were presumably carried out on standard NGM plates, since no additional information was provided. In contrast, the HT1115 experiments mention L4440, which is the empty vector control used for RNAi. This presumably means that worms were exposed to RNAi conditions, including IPTG and ampicillin, as well as the double-stranded RNAi coming from the empty vector. Furthermore, FUDR was mentioned for this experiment but not the OP50 experiment. Therefore, in principle any one of these conditions, or a combination, could have affected DAF-2 levels. If the authors wish to make conclusions about the bacterial strain they should perform an experiment where the bacterial strain is the only difference in experimental conditions. Alternatively, they can adjust their wording to encompass all of the differences between the experiments. In any case, the experimental details for these experiments should be added to the methods section.

    2. As alluded to above, there are several places where the information in figure legends and/or methods is incomplete. Some additional examples are given directly to authors, but in general the authors are encouraged to revisit the legends and methods section and make sure the experiments are clearly described.

  4. Reviewer #3 (Public Review):

    The authors created an auxin-inducible system to degrade the DAF-2/insulin receptor protein in specific tissues and at different ages of the worm C. elegans. Because DAF-2 has pleiotropic functions, the authors used their system to identify the specific tissues from where DAF-2 acts to regulate certain physiological processes. For example, they showed that neuronal DAF-2 regulates reproductive physiology and longevity, whereas intestinal DAF-2 regulates both longevity and oxidative stress responses. On the other hand, body wall muscle-expressed DAF-2 had little or no effect on longevity or oxidative stress, although it remains unclear how effective the auxin-mediated knockdown of DAF-2 is in body wall muscles. By using this system, they also extended worm lifespan when they reduced DAF-2 levels in post-reproductive and very old adult animals. Importantly, they show that DAF-2 functions during most of post-reproductive adulthood to regulate longevity, highlighting the potential of the insulin receptor as a therapeutic target in improving the life quality of older adults in our population.

    This auxin-inducible system allows for a finer dissection of the spatial and temporal requirements for DAF-2 in multiple processes, including the decision window for entering dauer arrest, a developmental program that the animal undergoes during harsh environments. For example, Golden and Riddle (Dev Biol 1984, vol. 102, pp 368-378) previously showed that the dauer entry decision window is around the mid- to late period of the first larval (L1) stage through temperature upshift and downshift experiments under dauer-inducing conditions. In this manuscript, the authors refined this window to mid-L1-specifically, a certain threshold of DAF-2 levels at mid-L1 is required to inhibit dauer entry.

    Another example is the necessity for DAF-2 in multiple tissues in regulating longevity. Apfeld and Kenyon (Cell 1998, vol. 95, pp. 199-210) previously showed through genetic mosaic analysis that daf-2 is necessary in the EMS and AB cell lineages to regulate worm longevity. Here, the authors show that DAF-2 is required in intestinal cells, which arise from the EMS cell lineage, and in neuronal cells, which arise from the AB cell lineage, in regulating lifespan. Interestingly, while the daf-2 EMS mosaic mutants (Apfeld and Kenyon, 1998) and the intestinal-depleted DAF-2 animals (this manuscript) exhibited a similar increase in lifespan, the daf-2 AB mosaic mutants (Apfeld and Kenyon, 1998) and the neuronal-depleted DAF-2 animals (this manuscript) did not. The daf-2 AB mosaic mutants lived twice as long as control (Apfeld and Kenyon, 1998), whereas the neuronal-depleted DAF-2 animals (this manuscript) only lived about 44% longer than control (Supplementary Table 1). This could suggest the requirement for DAF-2 in non-neuronal tissues that arise from the AB cell lineage, in regulating lifespan.

    The authors also show that the lack of germline shrinkage at 15C in DAF-2-depleted animals (DAF-2::degron; auxin-treated; Figure 3C) is still accompanied by a smaller brood size at the same temperature (Figure 3D), which might suggest that DAF-2 affects multiple aspects of the animal's reproductive physiology. This again highlights how this inducible system can uncouple the DAF-2 requirements in multiple processes. Thus, this manuscript shows how this tool, which will likely be in high demand among the C. elegans community, can significantly impact the study of DAF-2/insulin receptor signaling and its pleiotropy.