GWAS for Lifespan and Decline in Climbing Ability in Flies upon Dietary Restriction Reveal decima as a Mediator of Insulin-like Peptide Production

Abstract

Dietary restriction (DR) is the most robust means to extend lifespan and delay age-related diseases across species. An underlying assumption in the aging field is that DR enhances both lifespan and physical activity through similar mechanisms, but this has not been rigorously tested in different genetic backgrounds. Furthermore, nutrient response genes responsible for lifespan extension or age-related decline in functionality remain underexplored in natural populations. To address this, we measured nutrient-dependent changes in lifespan and age-related decline in climbing ability in the Drosophila Genetic Reference Panel fly strains. On average, DR extended lifespan and delayed decline in climbing ability, but there was a lack of correlation between these traits across individual strains, suggesting that distinct genetic factors modulate these traits independently and that genotype determines response to diet. Only 50% of strains showed positive response to DR for both lifespan and climbing ability, 14% showed a negative response for one trait but not both, and 35% showed no change in one or both traits. Through GWAS, we uncovered a number of genes previously not known to be diet responsive nor to influence lifespan or climbing ability. We validated decima as a gene that alters lifespan and daedalus as one that influences age-related decline in climbing ability. We found that decima influences insulin-like peptide transcription in the GABA receptor neurons downstream of short neuropeptide F precursor (sNPF) signaling. Modulating these genes produced independent effects on lifespan and physical activity decline, which suggests that these age-related traits can be regulated through distinct mechanisms.

Keywords: Drosophila melanogaster; GWAS; aging; dietary restriction; genetic variation; insulin-like peptides; lifespan; physical ability.

Figure 1.. Genotype influences variation in lifespan, climbing ability, and response to DR across the DGRP lines.

(A) Median lifespan of 161 DGRP lines in ascending order under AL diet (red). Adjacent lines in blue represent the same strain raised under DR diet. (B) Comparison of median lifespan under AL of each strain with its DR counterpart. Same data as in A, displayed as a scatterplot. Grey bar represents best-fit trendline. (C) Comparison of median lifespan values across biological replicates of 52 DGRP lines under AL (red) and DR (blue). (D) The age (in days) at which fewer than 20% of the surviving population can climb in the allotted time. Data are arranged in ascending order by the phenotype under AL diet (red) with adjacent lines representing the same strain under DR (blue). (E) Comparison of each strain’s climbing data between AL and DR diets. Grey line represents best-fit trendline. (F) Comparison of biological replicates for 25 tested DGRP lines for the day at which less than 20% of surviving flies are able to climb under AL (red) and DR (blue). N = 50-200 flies per strain per diet. Data used found in Data S1 and Data S2. See also Figure S1.

Figure 2.. Genotype and diet differentially influence lifespan and climbing decline.

(A-D) Comparison of each tested strain’s day below 50% of maximal climbing proportion with median lifespan, on (A-B) AL or (C-D) DR. Each bar in A and C represents a DGRP strain, ordered by median lifespan on each diet. Colored bars represent climbing half-life and grey bars represent median lifespan. (B and D) Scatter plots depicting climbing ability compared to median lifespan on the (B) AL diet or (D) DR. Each dot represents a single DGRP strain. (E) Comparison of DR responsiveness with regards to median lifespan (grey bars) and time above 50% initial climbing ability (purple bars). (F) Scatter plot depicting response to DR of each tested DGRP line with regards to median lifespan and amount of time above 50% initial climbing ability. Each dot represents a single DGRP strain. N = 50-200 flies per strain per diet. Data used found in Data S1 and Data S2. See also Figure S2.

Figure 3.. daedalus modulates DR-specific climbing ability.

(A and B) Plot of the day at which fewer than 20% of flies climb in tested DGRP lines, split by genotype at the most significant locus downstream of dls on (A) AL or (B) DR. The day at which 20% or fewer surviving flies are able to climb is represented by blue dots, black bars represent mean values across all tested strains with a given genotype and diet. Significance for diet interaction p < 4E-5, FDR = 3%. (C-E) The effect of Minos element insertion on (C) lifespan and (D) climbing ability over the course of life in a w¹¹¹⁸ genetic background, and (E) the log₂ difference median lifespan and unnormalized climbing decline between mutant and controls. AL shown in red, DR in blue. Significant differences between mutant and controls are indicated by *. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005. nc = no change, ns = not significant. p values shown in Data S3. N = 200 flies per condition for each mutant experiment. Data in (C-E) show collective results from three biological replicates. Error bars represent SD between replicates. Data used found in Data S3. See also Figure S3E-G, L, Figure S4A-B, E-F.

Figure 4.. decima modulates longevity in a diet-dependent manner.

(A and B) ALignment of all 161 DGRP lines according to genotype at a particular locus in dcma and according to the day at which ≤ 75% of flies in a strain remain alive on (A) AL or (B) DR. Strains’ median lifespans are represented by blue dots, black bars represent mean values across all tested strains with a given genotype and diet. Significance for diet interaction p < 9E-5, FDR = 8%. (C-E) The effect of neuron-specific RNAi of dcma using the v30160 transgenic line in modulating (C) lifespan and (D) climbing ability over the course of life, with (E) log₂ fold-change between RNAi and control for both median lifespan and unnormalized climbing decline values. AL shown in red, DR in blue. Significant differences between RNAi and controls are indicated by *. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, determined by unpaired t test. p values shown in Data S3. nc = no change, ns = not significant. N = 200 flies per condition for each RNAi experiment. Data in (C-E) show collective results from three biological replicates. Error bars represent SD between replicates. See also Figure S3H-K, Figure S4C-D. RU486 controls shown in Figure S5.

Figure 5.. decima knockdown inhibits development, promotes elevated triglyceride and glucose levels, and improves starvation resistance.

(A) Number of larvae reaching pupariation from dcma^RNAi driven pan-neuronally (left), in IPCs (middle), and in GABA receptor cells (right). N = 60 embryos per condition. (B) Whole-body triglyceride levels of flies with GABA-B-R2-Gal4-driven dcma^RNAi (checkered boxes) versus controls with no RNAi (solid boxes). N = 15 flies per condition. (C) Whole-body glucose levels in flies with GABA-B-R2-Gal4-driven dcma^RNAi. N = 15 flies per condition. (D) Starvation resistance of flies with GABA-B-R2-Gal4-driven dcma^RNAi (dotted line) versus control (solid line). N = 100 flies per condition. RNAi represented by checkered bars and control conditions in solid colors. Significant differences between RNAi and controls are indicated by *. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, determined by unpaired t test. p values shown in Data S3. nc = no change, ns = not significant. All experiments show collective results of three biological replicates. Error bars represent SD between replicates. See also Figure S6.

Figure 6.. decima regulates insulin-like peptide production in GABA receptor neurons through sNPF signaling.

(A-C) Levels of neuronally-expressed dilps in heads of flies with dcma^RNAi driven (A) pan-neuronally under control of RU486, (B) in dilp2-producing cells, and (C) in GABA receptor neurons. (D) Relative expression of dcma and dilp2 with pan-neuronal RNAi of sNPF (left) or GABA Receptor cell-specific sNPF-R^RNAi (right) in heads of flies relative to rp49 housekeeping gene. (E) Relative expression of dcma and dilp2 with pan-neuronal overexpression of sNPF (left) or GABA Receptor cell-specific sNPF-R overexpression (right) in heads of flies relative to rp49 housekeeping gene. AL shown in red, DR in blue. RNAi represented by checkered bars and control conditions in solid bars. N = 50 fly heads per condition. Significant differences between RNAi and controls are indicated by *. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, determined by unpaired t test. p values shown in Data S3. nc = no change, ns = not significant. N = 200 flies per condition for each RNAi experiment. All experiments show collective results of three biological replicates. Error bars represent SD between replicates. (F) Model for decima’s regulation of dilp production. dcma transcription is upregulated through dietary and sNPF signals, and in turn then regulates the transcription of Drosophila insulin-like peptides in the GABA receptor neurons/insulin-producing cells. See also Figure S7.