JF: Conceptualization, Formal analysis, Data curation, Writing - original draft
JV: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing - review & editing
Various genetic, molecular and environmental factors influence the lifespan of an organism, which includes the highly conserved insulin signaling pathway. In
Survivorship curves upon expression of miR-100, mIR-190, miR-8, miR-184, mIR-312, miR-375 and miR-285 sponges in adult IPCs (A-B). An increase in lifespan was observed when miR-100 sponge was expressed in the adult IPCs (A). An increase in survival in early lifespan was observed when miR-312 sponge and miR-285 sponge were expressed in the adult IPCs (B). Percentage flies crossing 5 cm in 3 secs upon expression of miR-100, miR-190, miR-8, miR-184, miR-312, miR-375 and miR-285 sponges in adult IPCs on day 24 (C ). An increase in climbing ability on day 24 was seen upon expression of miR-312 sponge and miR-375 sponge in the adult IPCs (C ). n ≥ 56. Log-rank test (A-B), Fleming-Harrington test (A-B) and a student's t-test were performed to test statistical significance (*,
The lifespan of organisms is controlled by both genetic and environmental factors. Nutrition is one of the key environmental factors that regulates the lifespan of an organism (Kwang et al. 2008). Among the molecular pathways, insulin signaling (Bartke 2008; Schuh et al. 2011; Tatar et al. 2001), TOR signaling (Kapahi et al. 2004) and JNK (c-Jun N-terminal Kinase) signaling (Wang, Bohmann, and Jasper 2005) have been reported to affect lifespan. Various other factors like oxidative stress (Liochev 2013), metabolic status, telomere length (Armanios et al. 2009; Vera et al. 2012), and DNA damage (Flurkey, Currer, and Harrison 2007) have previously been implicated to regulate lifespan in mammals.
The insulin/insulin-like growth factor (IGF) pathway links nutritional status to growth, metabolism and lifespan. In
Earlier studies have demonstrated an increase in lifespan in flies that are defective for insulin signaling (Clancy et al. 2001; Tatar et al. 2001). Ablation of the IPCs was also found to increase the lifespan (Broughton et al. 2005; Min et al. 2008). A significant increase in longevity was observed in
microRNAs are short non-coding RNAs that post-transcriptionally regulate the levels of various target mRNAs that they bind to via sequence complementarity (Ambros 2001). microRNAs are known to either regulate insulin levels directly or regulate insulin signaling in the responsive tissues. miR-8 in
Quite a few microRNAs have been discovered to regulate the levels of insulin-like peptides directly. miR-14 has previously been discovered to regulate the insulin-like peptides and thereby the metabolism via its target
Recently various studies have been carried out to estimate the levels of all microRNAs in the fly brain/head to discover any role that microRNAs could play in aging. The level of miR-34 was found to go up in the head of flies with age and thus regulate lifespan via its target
Adult specific overexpression of miR-184 and let-7 led to significant changes to fat metabolism and life span in Drosophila (Gendron and Pletcher. 2017). Both let-7 and miR-125 were found to regulate
Here, we carried out a mini-genetic screen to identify microRNAs which when downregulated in the IPCs play a role in regulating lifespan. We analyzed the effects of downregulation of 7 conserved microRNAs which are known to be expressed in the fly head. The screen was carried out by expressing microRNA-sponges (Fulga et al. 2015) specifically in the Insulin-producing cells in the adult male
A significant increase in lifespan was observed when miR-100 sponge was expressed in the IPCs of adult male flies (Figure 1.A). In a prior study, miR-100 levels were found to increase with age (Chawla et al. 2016b). Therefore it would be interesting to verify any role that the microRNA may play in regulating DILPs from the IPCs in adult flies. To check for any early or late changes in death, we also carried out a Fleming-Harrington weighted log rank-test Test wherein, flies started dying later when miR-312 sponge (p=0.0294) and miR-285 (p=0.0459) sponge was expressed in the IPCs of adult male flies, without affecting the overall lifespan (Figure 1.B). Expression of miR-184 sponge in the IPCs led to a similar but not statistically significant increase in survival during early lifespan (p=0.0567). None of the other microRNA sponges tested showed any effects on lifespan. The climbing ability of flies in response to expression of miR-375 (p=0.00653) and miR-312 sponges (p=0.00656) in the IPCs was found to be significantly higher than that of the control (Figure 1.C). Expression of miR-100 sponge in the adult IPCs also improved the climbing capacity of older flies (though this was not statistically significant) (Figure 1.C). In a screen carried out previously, overexpression of miR-312 and miR-285 in the IPCs led to an increase in wing size (Suh et al. 2015). Further experiments are needed to verify the role that miR-100, miR-375, miR-285 and miR-312 play in the IPCs in neuronal function and aging.
Survival assay
Larvae were raised on standard media at 18 O C, flies were allowed to mate at 29 O C for two days. Batches of 10-15 male flies per vial were provided fresh food every alternate day with dead flies being recorded every day. A Log rank test and a weighted-log rank test with a Fleming-Harrington test were performed using the OASIS 2 software.
Climbing assay
Climbing assay was carried out using long vials. Flies were transferred to assay vials and acclimatised for 10 minutes, the vial was tapped thrice in an arrhythmic fashion, and the number of flies that cross 5 cms in 3 seconds were noted. This assay was carried out thrice with a gap of about 3 minutes between each trial. A student's t-test was carried out to calculate if there were any significant changes if any.
Statistical analysis
Prism 8 was used to plot all the data. OASIS 2 (Han et al. 2016) was used for the statistical analysis for the lifespan experiments. A log-rank test was carried out to check for differences in lifespan while a Fleming-Harrington test was carried out to check for differences in early or late survival. The percentage of flies crossed 5 cms in 3 secs was calculated. The percentages were compared with the control using a student's t-test.
Fly stocks
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miR-190-sp |
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61397 |
miR-100-sp |
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61391 |
miR-8-sp |
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61374 |
miR-184-sp |
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61396 |
miR-312-sp |
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61429 |
miR-375-sp |
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61436 |
miR-285-sp |
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61417 |
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Funding was provided by IISER TVM Institutional Support (to JV); an Extra-Mural Grant (EMR/2016/004978) from Science and Engineering Research Board, Department of Science and Technology, Government of India (to JV); Ramanujan Fellowship (SR/S2/RJN-140/2011) from Science and Engineering Research Board, Department of Science and Technology, Government of India (to JV) and Junior/Senior Research Fellowship from Council of Scientific and Industrial Research, Government of India (to JF)