AGES for Ageing: Evaluating the auxin-inducible gene expression system for use in Drosophila ageing studies

Reviewer #1 (Public review):

Summary:

The authors set out to evaluate whether AGES, a recently developed auxin/TIR1-based conditional GAL4 expression system, is a suitable tool for Drosophila ageing research. They characterise induction efficiency across sex, transgene insertion site, auxin dose and age, then test whether AGES can replicate a well-established pro-longevity manipulation (dominant-negative insulin receptor expression).

Strengths:

The study is thorough and methodical. The authors use appropriate genetic controls throughout, which is required to properly interpret AGES-based experiments. They identify an important issue, in that activation of the AGES machinery itself (independent of any UAS-transgene) shortens lifespan and alters protein levels, while high-dose auxin independently affects body mass, and even a moderate dose (5 mM) impairs stress resistance across all genotypes. These findings are important for researchers when interpreting their experiments. The tissue and age mapping of induction efficiency (brain, fat body, gut) is also useful, and the inclusion of driver-only positive controls at each age (Figure 2) establishes that da-GAL4 activity itself is stable across the ages tested, ruling out declining driver activity as an explanation for the reduced induction seen in older flies (though, as noted below, reduced auxin ingestion with age remains a very plausible contributing factor alongside declining AGES efficacy).

Weaknesses:

Longevity and stress assays were conducted only in females, which, combined with the finding that males show weaker and less consistent induction, means the study cannot speak to whether the metabolic and survival costs of auxin/AGES activation observed here also apply to, or differ in, males. The KCl vehicle control matches the potassium cation (K⁺) content of K-NAA across conditions; therefore, chloride (Cl⁻) concentration differs between control and auxin-fed media (both minor weaknesses).

Achievement of aims and impact:

The authors achieve their stated aim. Rather than validating AGES as unambiguously suitable for longevity work, they set out to characterise its behaviour and limitations in this context, which they do convincingly. The data support their overall conclusion that AGES can be used to conditionally induce transgene expression at advanced ages, but that its use in longevity/healthspan studies requires caution and rigorous control genotypes. This is a useful contribution with direct practical value: it will help other researchers make informed decisions about whether and how to deploy AGES in ageing-related work, and the cautionary findings regarding auxin/AGES toxicity are likely to be of broad relevance to the growing community of AGES users beyond the ageing field specifically.

Reviewer #2 (Public review):

McGilvary et al. evaluate the recently developed auxin-based gene expression system (AGES) for use in aging studies of Drosophila melanogaster. This system is based on the widely used Gal4/UAS system that enables cell-specific expression of UAS-transgenes under Gal4 activator control. AGES uses an auxin-inducible degron-tagged Gal80 repressor that should prevent Gal4-dependent activation unless flies are fed auxin, providing a useful approach for temporal control of transgene induction - something that would be highly useful for aging studies. The authors perform a comprehensive analysis of AGES-dependent transgene induction in male and female flies at different ages with multiple controls, demonstrating some moderate induction in female flies only - albeit with some substantial background induction even in the absence of auxin.

Overall, transgene induction appears to be both much lower with the AGES system compared to Gal4 driver controls and very leaky, with some tissue-specific differences in induction observed as well. Combined with their observations that auxin feeding has impacts on body mass, triacylglycerol and protein levels, and lifespan, these data raise some concerns regarding the interpretation of data obtained using the AGES system for aging or longevity studies in flies. This study provides well-needed validation for the recently developed AGES system and highlights critical caveats that will support future studies.

Most conclusions of the paper are well supported by data, but additional controls and textual edits would strengthen and clarify the findings. In addition, the abstract and conclusions of this study should more accurately reflect the limitations of transgene induction using this AGES system in adult flies.

Reviewer #3 (Public review):

Summary:

In this useful work, the authors characterize the auxin-based gene expression system (AGES) as a tool for studying ageing. They found that this system can be applied to ageing studies. In addition, they identified important drawbacks of the methods, including effects of insertion sites and sex on induction of the system, and that some auxin doses may have inadvertent effects on body mass and physiology. Overall, the study extends the AGES system for use in fly ageing studies and highlights some caveats. While the findings are solid pointers, more extensive characterization is needed to benchmark the extent of the caveats identified.

Strengths:

The study provides the first longitudinal evaluation of the AGES system's induction efficiency across the entire Drosophila lifespan. The authors also highlighted a number of caveats of the AGES system in ageing animals. These are all important points to be considered when using this system, and findings should be interpreted keeping these caveats in mind.

Weaknesses:

There were inconsistencies with auxin dosages between the figures.

(1) The authors used a higher dose of auxin (20mM) compared to the original AGES paper (McClure, 2022) in Fig 3. The auxin dose-dependent effects are not linear for TAG and protein levels, highlighting that the genotype-dependent effects may be highly variable and may yield quite different results in other studies.

(2) The results in Figure 4 showing the lack of induction in the brain are quite interesting; however, only 5mM auxin is tested. Characterizing dose-dependence for the variability of the induction in tissue types would be useful. At the very least, recapitulating prior results at 10mM should be done.

(3) Food intake and hydration status were not measured alongside body mass, TAG, and protein endpoints. The changes seen could be an effect of decreased feeding or fluid balance rather than metabolic reprogramming.

Author response:

We thank the editorial team and all three reviewers for their time and attention to detail in reviewing our manuscript. We are particularly grateful for comments recognising the “direct practical value” and “comprehensive analysis” in our work (Reviewer #2) as well as its “thorough and methodical” approaches (Reviewer #1).

We also appreciate the reviewers’ constructive comments to improve our manuscript, which we plan to address in a revised version. Specifically, we plan to more explicitly acknowledge some of the limitations of our study, including clearer highlighting of the experiments performed only in females (Reviewer #1); the inability of our experimental design to control for chloride concentration (Reviewers #1 and #2); the limitations of transgene induction using the AGES system in adult flies (Reviewer #2); and the rationale for using different concentrations of auxin across different experiments (Reviewer #3). We also note that Reviewers #1 and #2 have provided additional recommendations beyond the public reviews (largely relating to helpful ways to clarify our text and more explicitly acknowledge limitations), which we also plan to address.

In addition, we plan to perform additional experiments to address specific points raised by reviewers in both their public reviews and additional recommendations. In the first instance, we plan to follow Reviewer #3’s suggestion to characterise induction in the brain using 10mM auxin, as well as Reviewer #2 and #3’s suggestions to explore feeding behaviour of flies fed auxin within our experimental setup.

We look forward to submitting an improved manuscript guided by the reviews, with the aim of strengthening this “well-needed validation” study that also “highlights critical caveats that will support future studies” (Reviewer #2).