Effects of knockdown of autophagy pathway genes on C. elegans longevity are highly condition dependent

Reviewer #1 (Public review):

Summary:

Hsiung et al. investigated whether the effects of autophagy gene knockdown on the lifespan of long-lived C. elegans mutants depend on experimental conditions. The authors first compiled published data on autophagy-dependent lifespan regulation in daf-2 and wild-type backgrounds, highlighting that prior results are notably inconsistent and likely context-dependent. They then systematically tested the lifespan effects of RNAi knockdown of six autophagy genes (atg-2, atg-4.1, atg-9, atg-13, atg-18, and bec-1) in wild-type (N2), daf-2 (reduced insulin/IGF-1 signalling), and glp-1 (germlineless) animals, while varying temperature, daf-2 allele, FUDR concentration, and bacterial infection status.

The key findings are as follows. In wild-type animals, lifespan suppression by most autophagy gene knockdowns was more pronounced at 20{degree sign}C than at 25{degree sign}C, where little or no effect was observed. In daf-2 mutants, stronger lifespan suppression was seen in the weaker daf-2(e1368) allele at 20{degree sign}C, but not in the stronger daf-2(e1370) allele, and effects were largely absent at 25{degree sign}C. In glp-1 mutants, four of six gene knockdowns suppressed lifespan to a greater extent than in N2, though again in a temperature-dependent manner. FUDR at a high concentration (800 µM) abolished the life-shortening effects of most knockdowns and, in the case of atg-9 and atg-13, led to lifespan extension. Kanamycin treatment to eliminate bacterial proliferation did not fully account for the lifespan effects, suggesting that increased susceptibility to infection is not the primary mechanism. The authors also tested the programmed aging hypothesis that autophagy promotes lifespan reduction through biomass repurposing, but found no changes in vitellogenin levels upon knockdown of any of the six genes.

Altogether, among all genes tested, atg-18 knockdown produced the strongest and most consistent lifespan suppression across nearly all conditions, including both daf-2 and glp-1 backgrounds. The authors probed whether atg-18 acts through the FOXO transcription factor DAF-16 by examining dauer formation and ftn-1 expression, but found no evidence for this, suggesting a DAF-16-independent mechanism.

Strengths:

The primary strength of this work lies in its systematic and comprehensive approach to dissecting how experimental variables influence the outcome of autophagy-lifespan epistasis tests. The compilation of prior data alongside the authors' own multi-condition dataset is a genuinely useful resource for the field. The study raises a timely and important point about condition selection bias, which is relevant not only to autophagy research but to C. elegans aging studies more broadly. The finding that atg-18 behaves distinctly from other autophagy genes across all conditions is noteworthy and opens avenues for future mechanistic work.

Weaknesses:

Despite its breadth, the study has several weaknesses that limit the strength of some conclusions.

(1) Variability in control lifespan data. The N2 lifespan values under ostensibly identical conditions (e.g., GFP RNAi at 20{degree sign}C) differ substantially across experiments (compare Tables S2, S5, S6, S7, and S9). Since N2 serves as the baseline for calculating whether the effect is greater in long-lived mutants via Cox proportional hazard (CPH) analysis, this variability in controls directly affects the reliability of those comparisons.

(2) Limited biological replication. Most experiments were performed with only two biological replicates. In several cases, the two replicates yield contradictory outcomes: one showing significant lifespan suppression and the other showing no effect or even extension. The authors combine these into cumulative datasets for analysis, which, while not incorrect in principle, may obscure genuine irreproducibility. Given that the central message of the paper concerns variability and condition dependence, additional replication would have substantially strengthened confidence in the reported results.

(3) Low sample sizes in individual trials. A number of lifespan assays were conducted with only 40-50 worms per replicate, and in some cases, as few as 30. Such sample sizes are below the standard commonly used in the C. elegans aging field and are likely to contribute to the variability observed.

(4) RNAi efficacy measured only in N2 at 20{degree sign}C. The authors demonstrated that atg-2 and atg-4.1 RNAi did not significantly reduce target mRNA levels, which may explain their weaker lifespan effects. However, these same RNAi treatments significantly affected lifespan in several other conditions (e.g., daf-2(e1368) at 20{degree sign}C, glp-1 at 20{degree sign}C and 25{degree sign}C, and N2 with 15 µM FUDR). Measuring RNAi efficacy across different genetic backgrounds and conditions would be needed to properly interpret these variable results.

(5) Incomplete mechanistic exploration. The investigation of why atg-18 knockdown has uniquely strong effects was limited to DAF-16. Given published evidence that atg-18 may regulate HLH-30/TFEB, a master transcriptional regulator of autophagy and lysosomal biogenesis, testing whether atg-18 specifically affects HLH-30 nuclear localisation or activity could have provided valuable mechanistic insight and would distinguish atg-18 from the other genes tested.

Reviewer #2 (Public review):

Summary:

This study examines how genes involved in cellular recycling (autophagy) influence lifespan under different experimental conditions. The findings help clarify why previous studies have reported conflicting results about whether blocking autophagy shortens or extends lifespan. The work will be of interest to researchers studying aging and cellular stress responses, particularly those using model organisms.

Strengths:

The findings are valuable, as they help resolve inconsistencies within a specific subfield of aging research. The evidence presented is solid, as the data broadly support the primary claims of the study. In addition, the discussion is thorough and thoughtfully integrates the findings within the broader context of the field.

Weaknesses:

Additional functional validation would further strengthen the conclusions.