Artificially stimulating retrotransposon activity increases mortality and accelerates a subset of aging phenotypes in Drosophila
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Evaluation Summary:
This manuscript examines the role of transposable element (TE) expression and the transcription factor FOXO on aging of Drosophila melanogaster. Increased TE expression in aged organisms compared to their younger counterparts has been observed in several animals, including Drosophila. Here, the authors show that artificially inducing transcription of a specific TE can reduce fly lifespan and exacerbate some aging phenotypes-paraquat resistance and rhythmicity. The authors also argue that the detrimental effects of increased TE expression can be rescued by FOXO expression, although this is less convincing.
(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. The reviewers remained anonymous to the authors.)
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Abstract
Transposable elements (TEs) are mobile sequences of DNA that can become transcriptionally active as an animal ages. Whether TE activity is simply a by-product of heterochromatin breakdown or can contribute toward the aging process is not known. Here, we place the TE gypsy under the control of the UAS GAL4 system to model TE activation during aging. We find that increased TE activity shortens the life span of male Drosophila melanogaster . The effect is only apparent in middle-aged animals. The increase in mortality is not seen in young animals. An intact reverse transcriptase is necessary for the decrease in life span, implicating a DNA-mediated process in the effect. The decline in life span in the active gypsy flies is accompanied by the acceleration of a subset of aging phenotypes. TE activity increases sensitivity to oxidative stress and promotes a decline in circadian rhythmicity. The overexpression of the Forkhead-box O family (FOXO) stress response transcription factor can partially rescue the detrimental effects of increased TE activity on life span. Our results provide evidence that active TEs can behave as effectors in the aging process and suggest a potential novel role for dFOXO in its promotion of longevity in D. melanogaster .
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Author Response
Reviewer #2 (Public Review):
This work will be of potential interest to biologists studying aging. While transposable elements have been reported to have higher expression as organisms age, it was previously unclear if their expression can exacerbate aging phenotypes or if they are a byproduct of aging. The authors present evidence in this manuscript that artificially increasing transposable element expression during the whole Drosophila life cycle can worsen aging phenotypes.
Strengths
The authors provide direct evidence that expression of their gypsy construct across the whole life of animals decreases fly lifespan (Figure 4), and that this outcome is dependent on reverse transcriptase (Figure 6).
Monitoring TE mobilization can be difficult in general and is often expensive when using a sequencing approach. …
Author Response
Reviewer #2 (Public Review):
This work will be of potential interest to biologists studying aging. While transposable elements have been reported to have higher expression as organisms age, it was previously unclear if their expression can exacerbate aging phenotypes or if they are a byproduct of aging. The authors present evidence in this manuscript that artificially increasing transposable element expression during the whole Drosophila life cycle can worsen aging phenotypes.
Strengths
The authors provide direct evidence that expression of their gypsy construct across the whole life of animals decreases fly lifespan (Figure 4), and that this outcome is dependent on reverse transcriptase (Figure 6).
Monitoring TE mobilization can be difficult in general and is often expensive when using a sequencing approach. The authors accurately monitor gypsy mobilization from their ectopic copy through qPCR and sequencing.
Weaknesses
Experiment design, data interpretation, and story structure:
The current model proposes that TE increases activity in aged animals and potentially contributes to the aging process. However, this paper artificially drives gypsy activation throughout the whole fly life cycle. Under this design, TE may already bring deleterious effects from early developmental stages or young age, thus ultimately shortening their life cycle. To truly test the function of TE during the aging process, the authors need to temporally control gypsy expression and only express their construct in aged animals.
Figure 1: I am not sure I got any convincing messages from this figure. First, flies at 30 days of age should not be considered as old. Second, the authors try to claim that TE expression increased with aged FOXO mutants. However, there is no data to show the comparison between aged wild-type and FOXO mutants (panel e is young wt vs young FOXO null). Meanwhile, Figure 1 has nothing to do with Gypsy. How could this figure fit into the story?
It is clear that we did not do a good job explaining this section. First, we did not mean to imply that the 30-day flies are old. They are simply older than the 5-day flies. The 30-day timepoint was chosen to match previous experiments and data sets in the literature. It was also chosen to minimize any survivor bias that could occur by doing the assay in very old flies. We have clarified this in the text and figures.
Second, it is the number of transposons that show an increase in expression in the dFOXO null animals that we mean to highlight (18 for dFOXO vs only 2 for wDAH). Panel e is meant to illustrate that the transposon landscapes, even in young flies differ by genotype making a direct transposon to transposon comparison impossible. We have added text to clarify these points.
Third, we also do not mean to imply that anything here is specific for gypsy. The work going forward in the paper uses gypsy as a tool because it is one of the better understood retrotransposons, there existed a validated active clone of the transposon and it has already been implicated in aging in the fly. We took gypsy as a model retrotransposon. We have added text to clarify here.
Figure 3: While the data presented in this Figure is sound, it is unclear how this data fits into the overall narrative that transposon activity drives aging.
Figure 3 is a continuation of the characterization of our ectopic gypsy. We wanted to rule out that there is a “hotspot” of insertion that would account for any phenotypes we observe. We find no hotspot in the males we use for analysis suggesting it is the act of transposition, not a specific target gene that is important. We have added to the text to clarify the motivation for these experiments.
Figure 5: It is interesting to see the copies of gypsy are not increased after 5 days. Does gypsy still mobilize after this young age? If yes, the authors should observe increased gypsy gDNA in later time points, unless the cells having gypsy new insertions keep dying. The authors should specifically check tissues with low cell turnover (such as brain) or high cell turnover (such as gut).
Reviewer 2 makes a great observation. In fact, using primer pairs that specifically detect the ectopic gypsy, we consistently see insertion numbers go down in very old animals (figure 5a&b). With our current understanding of retrotransposition, we should not be able to see loss of insertions unless the host cells are being lost from the analysis. We favor the idea that the reviewer suggests; that the cells that have high levels of insertion are dying and disappearing from the analysis. We think this is also reflected in the bias for intergenic or intronic sequences in our insertion mapping of figure 3. In an attempt to address this question we did measure insertions in heads versus bodies. In male flies aged 14 days there was no difference in the average number of insertions (although the variability was greater in heads). This data is reported in Supplemental Figure 6a.
Figure 8: Using Ubiquitin GAL4 to drive both gypsy and FOXO expression could dilute the expression of each individual gene. Thus, it is possible the rescue effect seen by expressing FOXO in addition to gypsy may just be due to lower gypsy expression. Including qPCR data showing gypsy expression levels in Ubi>gypsy, UAS FOXO flies compared to Ubi>gypsy flies would be helpful.
We included this data in Figure 2b and 8c. Unfortunately, we did not clearly direct the reader to compare the values. Comparing Figure 2b with Figure 8c shows the RNA level of the ectopic gypsy is comparable in both cases. Perhaps even slightly higher in the UAS-FOXO case. We have added a sentence to make this clear.
It is unclear if FOXO can rescue TE-specific aging phenotypes. While it appears that FOXO overexpression rescues the decrease in lifespan caused by gypsy expression, the authors did not test if FOXO overexpression could rescue the effects of gypsy in the paraquat resistance assays or rhythmicity experiments.
We include in this revision data showing dFOXO overexpression rescues the paraquat resistance and lowers the levels of overall insertions in the animals.
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Evaluation Summary:
This manuscript examines the role of transposable element (TE) expression and the transcription factor FOXO on aging of Drosophila melanogaster. Increased TE expression in aged organisms compared to their younger counterparts has been observed in several animals, including Drosophila. Here, the authors show that artificially inducing transcription of a specific TE can reduce fly lifespan and exacerbate some aging phenotypes-paraquat resistance and rhythmicity. The authors also argue that the detrimental effects of increased TE expression can be rescued by FOXO expression, although this is less convincing.
(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. The reviewers remained anonymous …
Evaluation Summary:
This manuscript examines the role of transposable element (TE) expression and the transcription factor FOXO on aging of Drosophila melanogaster. Increased TE expression in aged organisms compared to their younger counterparts has been observed in several animals, including Drosophila. Here, the authors show that artificially inducing transcription of a specific TE can reduce fly lifespan and exacerbate some aging phenotypes-paraquat resistance and rhythmicity. The authors also argue that the detrimental effects of increased TE expression can be rescued by FOXO expression, although this is less convincing.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The manuscript by Rial et al. describes an interesting interaction between the dFOXO transcription factor and the transposable element (TE) activity in aging using Drosophila. The authors find that dFOXO deletion mutants lead to elevated TE expression. They go on to use Drosophila molecular genetics to over-express the gypsy retrotransposon coding sequences and show deleterious effects on lifespan. They show that a wild-type reverse transcriptase (RT) enzyme is required for the reduced lifespan. Interestingly, the effects are only observed in "middle-age" flies. The authors also go on to show that there are defects in circadian rhythms in the flies over-expressing gypsy.
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Reviewer #2 (Public Review):
This work will be of potential interest to biologists studying aging. While transposable elements have been reported to have higher expression as organisms age, it was previously unclear if their expression can exacerbate aging phenotypes or if they are a byproduct of aging. The authors present evidence in this manuscript that artificially increasing transposable element expression during the whole Drosophila life cycle can worsen aging phenotypes.
Strengths
The authors provide direct evidence that expression of their gypsy construct across the whole life of animals decreases fly lifespan (Figure 4), and that this outcome is dependent on reverse transcriptase (Figure 6).
Monitoring TE mobilization can be difficult in general and is often expensive when using a sequencing approach. The authors accurately …
Reviewer #2 (Public Review):
This work will be of potential interest to biologists studying aging. While transposable elements have been reported to have higher expression as organisms age, it was previously unclear if their expression can exacerbate aging phenotypes or if they are a byproduct of aging. The authors present evidence in this manuscript that artificially increasing transposable element expression during the whole Drosophila life cycle can worsen aging phenotypes.
Strengths
The authors provide direct evidence that expression of their gypsy construct across the whole life of animals decreases fly lifespan (Figure 4), and that this outcome is dependent on reverse transcriptase (Figure 6).
Monitoring TE mobilization can be difficult in general and is often expensive when using a sequencing approach. The authors accurately monitor gypsy mobilization from their ectopic copy through qPCR and sequencing.
Weaknesses
Experiment design, data interpretation, and story structure:
The current model proposes that TE increases activity in aged animals and potentially contributes to the aging process. However, this paper artificially drives gypsy activation throughout the whole fly life cycle. Under this design, TE may already bring deleterious effects from early developmental stages or young age, thus ultimately shortening their life cycle. To truly test the function of TE during the aging process, the authors need to temporally control gypsy expression and only express their construct in aged animals.
Figure 1: I am not sure I got any convincing messages from this figure. First, flies at 30 days of age should not be considered as old. Second, the authors try to claim that TE expression increased with aged FOXO mutants. However, there is no data to show the comparison between aged wild-type and FOXO mutants (panel e is young wt vs young FOXO null). Meanwhile, Figure 1 has nothing to do with Gypsy. How could this figure fit into the story?
Figure 3: While the data presented in this Figure is sound, it is unclear how this data fits into the overall narrative that transposon activity drives aging.
Figure 5: It is interesting to see the copies of gypsy are not increased after 5 days. Does gypsy still mobilize after this young age? If yes, the authors should observe increased gypsy gDNA in later time points, unless the cells having gypsy new insertions keep dying. The authors should specifically check tissues with low cell turnover (such as brain) or high cell turnover (such as gut).
Figure 8: Using Ubiquitin GAL4 to drive both gypsy and FOXO expression could dilute the expression of each individual gene. Thus, it is possible the rescue effect seen by expressing FOXO in addition to gypsy may just be due to lower gypsy expression. Including qPCR data showing gypsy expression levels in Ubi>gypsy, UAS FOXO flies compared to Ubi>gypsy flies would be helpful.
It is unclear if FOXO can rescue TE-specific aging phenotypes. While it appears that FOXO overexpression rescues the decrease in lifespan caused by gypsy expression, the authors did not test if FOXO overexpression could rescue the effects of gypsy in the paraquat resistance assays or rhythmicity experiments.
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