DILP-producing median neurosecretory cells in the Drosophila brain mediate the response of lifespan to nutrition

Abstract

Dietary restriction extends lifespan in diverse organisms, but the gene regulatory mechanisms and tissues mediating the increased survival are still unclear. Studies in worms and flies have revealed a number of candidate mechanisms, including the target of rapamycin and insulin/IGF-like signalling (IIS) pathways and suggested a specific role for the nervous system in mediating the response. A pair of sensory neurons in Caenorhabditis elegans has been found to specifically mediate DR lifespan extension, but a neuronal focus in the Drosophila nervous system has not yet been identified. We have previously shown that reducing IIS via the partial ablation of median neurosecretory cells in the Drosophila adult brain, which produce three of the seven fly insulin-like peptides, extends lifespan. Here, we show that these cells are required to mediate the response of lifespan to full feeding in a yeast dilution DR regime and that they appear to do so by mechanisms that involve both altered IIS and other endocrine effects. We also present evidence of an interaction between these mNSCs, nutrition and sleep, further emphasising the functional homology between the DILP-producing neurosecretory cells in the Drosophila brain and the hypothalamus of mammals in their roles as integration sites of many inputs for the control of lifespan and behaviour.

Figure 1. The effect of DR by yeast dilution on lifespan and fecundity of DILP-producing mNSC ablated females and controls

(A and C) Median lifespans of d2GAL/UAS-rpr females and their controls (d2GAL/+ and UAS− rpr/+) across a yeast dilution series in two independent experiments. (A) Experiment 1. d2GAL/UAS-rpr on: 0.1×Y N = 111; 0.5×Y N = 74; 1.0×Y N = 71; 1.5×Y N = 72; 2.0×Y N = 74. d2GAL/+ on: 0.1×Y N = 108; 0.5×Y N = 87; 1.0×Y N = 76; 1.5×Y N = 96; 2.0×Y N = 89. UAS-rpr/+ on: 0.1×Y N = 81; 0.5×Y N = 61; 1.0×Y N = 65; 1.5×Y N = 66; 2.0×Y N = 66. (C) Experiment 2. d2GAL/UAS-rpr on: 0.1×Y N = 94; 0.5×Y N = 105; 1.0×Y N = 97; 1.5×Y N = 93; 2.0×Y N = 86. d2GAL/+ on: 0.1×Y N = 96; 0.5×Y N = 122; 1.0×Y N = 112; 1.5×Y N = 109; 2.0×Y N = 111. UAS-rpr/+ on: 0.1×Y N = 113; 0.5×Y N = 102; 1.0×Y N = 111; 1.5×Y N = 80; 2.0×Y N = 119. Survival curves for each genotype were compared between the yeast dilution giving the peak DR lifespan (0.5 or 1.0×Y) and 2.0×Yeast food using nonparametric log rank tests and p values calculated (median lifespans, the percentage DR induced increase in lifespan above 2.0×Y food and statistical analyses are given in Table 1). (B and D). Fecundity of d2GAL/UAS-rpr females and their controls (d2GAL/+ and UAS-rpr/+) from the survival experiments in (A) and (C): (B) Fecundity in experiment 1; (D) Fecundity in experiment 2. Data were analysed by ANOVA with genotype and food dilution as the main effects. At each food dilution, planned comparisons of mean eggs laid by genotype were performed by Tukey HSD or Students t as appropriate and * indicates significant difference to controls, p<0.05. Mean number of eggs laid/female, the % increase in fecundity between the peak food for lifespan and 2.0×Y food, and statistical analyses by food dilution are given in Table 2.

Figure 2. Feeding rates of mNSC ablated and control flies on Sugar/Yeast food dilutions

Flies were reared at standard density on 1.0×Y food. After a 48 hour mating period on 1.0×Y food, females of the indicated genotype were transferred to 0.1×Y/0.1×S, 0.1×Y/0.5×S, 0.5×Y, 1.0×Y or 2.0×Y foods prior to analysis. (A) Undisturbed feeding behavior. 6 day old flies of each genotype were transferred to fresh food of the appropriate yeast dilution the evening before the assay. Feeding was measured during a 60 minute period the next morning by observation of proboscis extension behaviour. The proportion of flies feeding during this period is presented as a proportion of feeding events/possible feeding events ±SEM. (B) Calibration of feeding observations by direct quantification of food consumption. 7 day old flies were transferred to blue dye containing fresh food of the appropriate yeast dilution. Observations of proboscis extension behavior were performed over a 30 minute period, followed by quantification of food consumed by colour spectrophotometry. Data are presented as mean μg of food per mg of fly, ±SEM. The calibration analysis is shown in Supplementary Figure 1. For both experiments, N=10 vials of 5 flies per genotype, per food.

Figure 3. The effect of DR by yeast dilution on transcription of dilps 2, 3 and 5 in adult heads of mNSC ablated and control flies

Relative mRNA abundance of dilps 2, 3 and 5 from adult heads of flies of the indicated genotypes maintained on 0.1×Yeast, 0.5×Yeast, 2.0×Yeast and 0.1×Sugar/Yeast food dilutions were measured by quantitative RT-PCR and normalised to the abundance of actin5C: (A) Relative abundance of dilp2 in each genotype; (B) Relative abundance of dilp3; (C) Relative abundance of dilp5. Data are shown as means of 4 independent experiments (N=4) ±SEM. For each genotype, ANOVAs were performed and expression levels of dilps 2, 3 or 5 were compared between the three food dilutions using Tukey HSD. * indicates significant difference to the 2.0×Y expression level, p<0.05.

Figure 4. The effect of DR by yeast dilution on DILP protein

(A) Western blot analysis of DILP2 levels in protein extract from female heads of w^Dah flies following 6 hrs and 24 hrs of the indicated food treatment. (B-E) Immunohistochemical analysis of DILP5 and DILP3 protein in control d2GAL/+ 7 day old female brains following 48hours treatment with 0.1×Y, 0.5×Y and 2.0×Y foods. Representative images of DILP expression from the analysis of brains examined at the same confocal microscope settings are shown. (B) (a) DILP5 protein was virtually undetectable following 0.1×Y food treatment in 3/6 brains examined and was detectable at very low levels in 2/6 brains (see Supplementary Figure 1 for images of all brains examined). (b) DILP5 protein was detectable in 6/6 brains after 0.5×Y treatment (see Supplementary Figure 3). (c) DILP5 protein was detected in 6/6 brains after 2.0×Y treatment (see Supplementary Figure 3). (C) Quantification of DILP5 levels using Image J performed on samples shown in (B) and in Supplementary Figure 3. Data are shown as mean expression ±SEM, and * indicates significant difference to 2.0×Y level by Tukey HSD, p<0.05. (D) DILP3 was detectable at similar levels in all brains at all food treatments. (a) 0.1×Y, DILP3 was detected in 5/5 brains. (b) 0.5×Y, DILP3 was detected in 3/3 brains. (c) 2.0×Y, DILP3 was detected in 3/3 brains. (E) Quantification of DILP3 levels using Image J. Data are shown as mean expression ±SEM.

Figure 5. Survival, activity and sleep behavior of mNSC ablated and control flies maintained on low sugar and yeast foods

(A and B) Median lifespans of d2GAL/UAS-rpr females and their controls on 0.1×Yeast/0.1×Sugar food in two independent experiments. Survival curves by genotype were compared using nonparametric log rank tests and p values calculated. Data are presented as median lifespan and * indicates significant difference to both controls, p<0.0001. (A) Median lifespans in Experiment 1: d2GAL/UAS-rpr = 22, N = 72; d2GAL/+ = 18, N = 78; UAS-rpr/+ = 18, N = 72. (B) Median lifespans in Experiment 2: d2GAL/UAS-rpr = 27, N = 116; d2GAL/+ = 22, N = 121; UAS-rpr/+ = 22, N = 101. (C) Mean total activity levels of d2GAL/UAS-rpr and control females on 0.1×Yeast/0.5×Sugar, 0.5×Yeast/0.1×Sugar and 0.1×Yeast/0.1×Sugar foods. (D) Mean quantity of night sleep (minutes/12hours) ±SEM of d2GAL/UAS-rpr and control females on 0.1×Yeast/0.5×Sugar, 0.5×Yeast/0.1×Sugar and 0.1×Yeast/0.1×Sugar foods. N=16 for each genotype on each food. (E) Mean quantity of day sleep (minutes/12hours) ±SEM of d2GAL/UAS-rpr and control females on 0.1×Yeast/0.5×Sugar, 0.5×Yeast/0.1×Sugar and 0.1×Yeast/0.1×Sugar foods. N=16 for each genotype on each food. Data were analysed by ANOVA and planned comparisons of means performed using Tukey HSD, and * indicates significant differences (p<0.05) between indicated genotype/food treatments.