fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans

Reviewer #1 (Public Review):
Summary:
This interesting and well written article by Tuckowski et al. summarizes work connecting the flavin-containing monooxygenase FMO-4 with increased lifespan through a mechanism involving calcium signaling in the nematode Caenorhabditis elegans.

The authors have previously studied another fmo in worms, FMO-2, prompting them to look at additional members of this family of proteins. They show that fmo-4 is up in dietary restricted worms and necessary for the increased lifespan of these animals as well as of rsks-1 (s6 kinase) knockdown animals. They then show that overexpression of fmo-4 is sufficient to significantly increase lifespan, as well as healthspan and paraquat resistance. Further, they demonstrate that overexpression of fmo-4 solely in the hypodermis of the animal recapitulates the entire effect of fmo-4 OE.

In terms of interactions between fmo-2 and fmo-4 they show that fmo-4 is necessary for the previously reported effects of fmo-2 on lifespan, while the effects of fmo-4 do not depend on fmo-2.

Next the authors use RNASeq to compare fmo-4 OE animals to wild type. Their analyses suggested the possibility that FMO-4 was modulating calcium signaling, and through additional experiments specifically identified the calcium signaling genes crt-1, itr-1, and mcu-1 as important fmo-4 interactors
in this context. As previously published work has shown that loss of the worm transcription factor atf-6 can extend lifespan through crt-1, itr-1 and mcu-1, the authors asked about interactions between fmo-4 and atf-6. They showed that fmo-4 is necessary for both lifespan extension and increased paraquat resistance upon RNAi knockdown of atf-6.

Overall this clearly written manuscript summarizes interesting and novel findings of great interest in the biology of aging and suggests promising avenues for future work in this area.

Strengths:
This paper contains a large number of careful, well executed and analysed experiments in support of its existing conclusions, and which also point toward significant future directions for this work. In addition it is clear and very well written.

Weaknesses:
Within the scope of the current work there are no major weaknesses. That said, the authors themselves note pressing questions beyond the scope of this study that remain unanswered. For instance, the mechanistic nature of the interactions between FMO-4 and the other players in this story, for example in terms of direct protein-protein interactions, is not at all understood yet. Further, powerful tools such as GCaMP expressing animals will enable a much more detailed understanding of what exactly is happening to calcium levels, and where and when it is happening, in these animals.

Reviewer #2 (Public Review):

Summary:
Members of a conserved family of flavin-containing monooxygenases (FMOs) are necessary and at least partly sufficient for lifespan extension induced by diet restriction and hypoxia. Of 5 FMOs in C. elegans, fmo-2 has received the majority of attention, but this study identifies that fmo-4 is also an important, positive modulator of lifespan. Based on differential requirements of fmo-2 and fmo-4 in stress resistance and lifespan extension paradigms, the authors conclude that fmo-4 acts through mechanisms that are overlapping, but distinct from fmo-2. Ultimately, the authors place fmo-2 genetically within a pathway involving atf-6, calreticulin, the IP3 receptor, and mitochondrial calcium uniporter, which was previously shown to link ER calcium homeostasis to mitochondrial homeostasis and longevity. Because the known enzymatic activity of FMOs involves oxygenating xenobiotic and endogenous metabolites, these findings highlight a potential new link between redox/metabolic homeostasis and ER-mitochondrial calcium signaling, while revealing that different FMO family members regulate stress resistance and lifespan through distinct mechanisms.

Strengths:
The authors have used genetics to discover an interesting and unanticipated new link between conserved FMOs and ER calcium pathways known to regulate lifespan.

The genetic epistasis patterns for lifespan and stress resistance phenotypes are generally clean and compelling.

Weaknesses:
The effects of carbachol and EDTA on intracellular calcium levels are inferred, especially in the tissues where fmo-4 is acting. Validating that these agents and fmo-4 itself have an impact on calcium in relevant subcellular compartments is important to support conclusions on how fmo-4 regulates and responds to calcium.

Experiments are generally reliant on RNAi. While in most cases experiments reveal positive results, indicating RNAi efficacy, key conclusions could be strengthened with the incorporation of mutants.

While FMO-4 is clearly placed in the ER calcium pathway genetically, a putative molecular mechanism by which FMO-4 would alter ER calcium remains unclear. Notably, Tuckowski et al. highlight this gap in the discussion as well.

Reviewer #3 (Public Review):

Summary:
The authors assessed the potential involvement of fmo-4 in a diverse set of longevity interventions, showing that this gene is required for DR and S6 kinase knockdown related lifespan extension. Using comprehensive epistasis experiments they find this gene to be a required downstream player in the longevity and stress resistance provided by fmo-2 overexpression. They further showed that fmo-4 ubiquitous overexpression is sufficient to provide longevity and paraquat (mitochondrial) stress resistance, and that overexpression specifically in the hypodermis is sufficient to recapitulate most of these effects.

Interestingly, they find that fmo-4 overexpression sensitizes worms to thapsigargin during development, an effect that they link with a potential dysregulation in calcium signalling. They go on to show that fmo-4 expression is sensitive to drugs that both increase or decrease calcium levels, and these drugs differentially affect lifespan of fmo-4 mutants compared to wild-type worms. Similarly, knockdown of genes involved in calcium binding and signalling also differentially affect lifespan and paraquat resistance of fmo-4 mutants.

Finally, they suggest that atf-6 limits the expression of fmo-4, and that fmo-4 is also acting downstream of benefits produced by atf-6 knockdown.

Strengths:
• comprehensive lifespans experiments: clear placement of fmo-4 within established longevity interventions.
• clear distinction in functions and epistatic interactions between fmo-2 and fmo-4 which lays a strong foundation for a longevity pathway regulated by this enzyme family.

Weaknesses:
• no obvious transcriptomic evidence supporting a link between fmo-4 and calcium signalling: either for knockout worms or fmo-4 overexpressing strains.
• no direct measures of alterations in calcium flux, signalling or binding that strongly support a connection with fmo-4.
• no measures of mitochondrial morphology or activity that strongly support a connection with fmo-4.
• lack of a complete model that places fmo-4 function downstream of DR and mTOR signalling (first Results section), fmo-2 (second Results section) and at the same time explains connection with calcium signalling.