Reduced Insulin Signaling Targeted to Serotonergic Neurons but Not Other Neuronal Subtypes Extends Lifespan in Drosophila melanogaster

Abstract

Reduced Insulin/IGF-like signaling (IIS) plays an evolutionarily conserved role in improving longevity and some measures of health-span in model organisms. Recent studies, however, have found a disconnection between lifespan extension and behavioral health-span. We have previously shown that reduction of IIS in Drosophila neurons extends female lifespan but does not improve negative geotaxis senescence and has a detrimental effect on exploratory walking senescence in both sexes. We hypothesize that individual neuronal subtypes respond differently to IIS changes, thus the behavioral outcomes of pan-neuronal IIS reduction are the balance of positive, negative and neutral functional effects. In order to further understand how reduced IIS in neurons independently modulates lifespan and locomotor behavioral senescence we expressed a dominant negative Insulin receptor transgene selectively in individual neuronal subtypes and measured the effects on lifespan and two measures of locomotor senescence, negative geotaxis and exploratory walking. IIS reduction in cholinergic, GABAergic, dopaminergic, glutamatergic, and octopaminergic neurons was found to have either no affect or a detrimental effect on lifespan and locomotor senescence. However, reduction of IIS selectively in serotonergic neurons resulted in extension of lifespan in females with no effect on locomotor senescence. These data indicate that individual neuronal subtypes respond differently to IIS changes in the modulation of lifespan and locomotor senescence, and identify a specific role for the insulin receptor in serotonergic neurons in the modulation of lifespan.

Keywords: Drosophila; ageing; behavioral senescence; insulin/IGF-like signaling; serotonergic neurons.

FIGURE 1

Survival of male and female flies with neuron specific (GAL4/UAS-InR^DN) reductions in IIS. (A) Survival of TrhGAL/UAS-InR^DN once mated female flies compared to TrhGAL/+ and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: TrhGAL/UAS-InR^DN = 64 days, N = 125; TrhGAL/ + = 57 days, N = 112; and UAS-InR^DN/+ = 61 days, N = 113. TrhGAL/UAS-InR^DN females showed an increased survival compared to both controls by log rank tests (P < 0.0001). (B) Survival of TrhGAL/UAS-InR^DN male flies compared to TrhGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: TrhGAL/UAS-InR^DN = 51 days, N = 91; TrhGAL/ + = 54 days, N = 87; d UAS-InR^DN/ + = 54 days, N = 87. (C) Survival of ThGAL/UAS-InR^DN once mated female flies compared to ThGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: ThGAL/UAS-InR^DN = 49 days, N = 150; ThGAL/ + = 73.5 days, N = 148; and UAS-InR^DN/ + = 73.5 days, N = 144. ThGAL/UAS-InR^DN females showed a decreased survival compared to both controls by log rank tests (P < 0.001). (D) Survival of ThGAL/UAS-InR^DN male flies compared to ThGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: ThGAL/UAS-InR^DN = 34 days, N = 149; ThGAL/ + = 73.1 days, N = 153; and UAS-InR^DN/ + = 74.5 days, N = 153. ThGAL/UAS-InR^DN males showed a decreased survival compared to both controls by log rank tests (P = ??). (E) Survival of VglutGAL/UAS-InR^DN once mated female flies compared to VglutGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: VglutGAL/UAS-InR^DN = 54 days, N = 100; VglutGAL/ + = 56.5 days, N = 100; and UAS-InR^DN/ + = 61 days, N = 100. VglutGAL/UAS-InR^DN females showed a decreased survival compared to both controls by log rank tests (P = 0.0008 to VglutGAL4/ + and P < 0.0001 to UAS-InR^DN/ +). (F) Survival of VglutGAL/UAS-InR^DN male flies compared to VglutGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: VglutGAL/UAS-InR^DN = 45 days, N = 100; VglutGAL/ + = 61 days, N = 100; and UAS-InR^DN/ + = 59 days, N = 100. VglutGAL/UAS-InR^DN males showed a decreased survival compared to both controls by log rank tests (P < 0.0001). (G) Survival of Gad1GAL/UAS-InR^DN once mated female flies compared to Gad1GAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: Gad1GAL/UAS-InR^DN = 56 days, N = 148; Gad1GAL/ + = 59 days, N = 149; and UAS-InR^DN/ + = 54 days, N = 154. (H) Survival of Gad1GAL/UAS-InR^DN male flies compared to Gad1GAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: Gad1GAL/UAS-InR^DN = 54 days, N = 148; Gad1GAL/ + = 54 days, N = 148; and UAS-InR^DN/ + = 54 days, N = 161. (I) Survival of ChATGAL/UAS-InR^DN once mated female flies compared to ChATGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: ChATGAL/UAS-InR^DN = 41.5 days, N = 100; ChATGAL/ + = 41.5 days, N = 102; and UAS-InR^DN/ + = 61 days, N = 99. ChATGAL/UAS-InR^DN and ChATGAL/ + females showed a decreased survival compared to the UAS-InR^DN/ + control females by log rank tests (P < 0.0001). (J) Survival of ChATGAL/UAS-InR^DN male flies compared to ChATGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: ChATGAL/UAS-InR^DN = 33.5 days, N = 100; ChATGAL/ + = 46 days, N = 100; and UAS-InR^DN/ + = 55 days, N = 100. ChATGAL/UAS-InR^DN males showed a decreased survival compared to both controls by log rank tests (P < 0.0001). (K) Survival of TdcGAL/UAS-InR^DN once mated female flies compared to TdcGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: TdcGAL/UAS-InR^DN = 60 days, N = 153; TdcGAL/ + = 60 days, N = 153; and UAS-InR^DN/ + = 60 days, N = 125. (L) Survival of TdcGAL/UAS-InR^DN male flies compared to TdcGAL/ + and UAS-InR^DN/ + controls. Median lifespans and sample sizes were: TdcGAL/UAS-InR^DN = 54 days, N = 71; TdcGAL/ + = 51 days, N = 84; and UAS-InR^DN/ + = 51 days, N = 40.

FIGURE 2

Effect of constitutive IIS reduction in specific neuronal subtypes on negative geotaxis senescence. (A) Negative geotaxis performance index over the lifespan of TrhGAL4/UAS-InR^DN once mated female flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (B) Negative geotaxis performance index over the lifespan of TrhGAL4/UAS-InR^DN male flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (C) Negative geotaxis performance index over the lifespan of ThGAL4/UAS-InR^DN once mated female flies compared to ThGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (D) Negative geotaxis performance index over the lifespan of ThGAL4/UAS-InR^DN male flies compared to ThGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (E) Negative geotaxis performance index over the lifespan of VglutGAL4/UAS-InR^DN once mated female flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (F) Negative geotaxis performance index over the lifespan of VglutGAL4/UAS-InR^DN male flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (G) Negative geotaxis performance index over the lifespan of GAD1GAL4/UAS-InR^DN once mated female flies compared to GAD1GAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (H) Negative geotaxis performance index over the lifespan of GAD1GAL4/UAS-InR^DN male flies compared to GAD1GAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (I) Negative geotaxis performance index over the lifespan of ChATGAL4/UAS-InR^DN once mated female flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. At the age of 22 days ChATGAL4/UAS-InR^DN showed decreased negative geotaxis response compared to the controls (P = 0.0001 to ChATGAL4/ + and P = 0.0003 to UAS-InR^DN/ +) (J) Negative geotaxis performance index over the lifespan of ChATGAL4/UAS-InR^DN male flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. At the age of 12, 32, and 39 days ChATGAL4/UAS-InR^DN showed decreased negative geotaxis response compared to the controls (12 days: P = 0.0169 to ChATGAL4/ + and P = 0.0160 to UAS-InR^DN/ +; 32 days: P = 0.0133 to ChATGAL4/ + and P = 0.0164 to UAS-InR^DN/ +; 39 days: P = 0.0281 to ChATGAL4/ + and P = 0.0134 to UAS-InR^DN/ +) (K) Negative geotaxis performance index over the lifespan of TdcGAL4/UAS-InR^DN once mated female flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype. (L) Negative geotaxis performance index over the lifespan of TdcGAL4/UAS-InR^DN male flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls, N = 3 (groups of 10 flies) for each genotype.

FIGURE 3

The effect of neuronal subtype specific IIS reduction on locomotion during exploratory walking senescence in female flies. Exploratory walking senescence parameters for a cohort of once-mated female flies of the indicated genotypes run in parallel with the survival experiments shown in Figure 1. Data are shown as mean value for each parameter ± SEM, N = 16 for the indicated genotype. Data were analyzed by two-way ANOVA (age and genotype) and age was found to be a significant effect (p < 0.05) for all genotypes. (A) Mean total distance walked vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (B) Mean duration of walking vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (C) Mean speed of walking vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (D) Mean total distance walked vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (E) Mean duration of walking vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (F) Mean speed of walking vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (G) Mean total distance walked vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (H) Mean duration of walking vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (I) Mean speed of walking vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (J) Mean total distance walked vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (K) Mean duration of walking vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (L) Mean speed of walking vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (M) Mean total distance walked vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (N) Mean duration of walking vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (O) Mean speed of walking vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (P) Mean total distance walked vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (Q) Mean duration of walking vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (R) Mean speed of walking vs. age of TdcGAL4/UAS-InR^DN flies compared to tdcGAL4/ + and UAS-InR^DN/ + controls.

FIGURE 4

The effect of neuronal subtype specific IIS reduction on decision making during exploratory walking senescence in female flies. Exploratory walking senescence parameters for a cohort of female flies of the indicated genotypes run in parallel with the survival experiments shown in Figure 1. Data are shown as mean value for each parameter ± SEM, N = 16 for the indicated genotype. Data were analyzed by two-way ANOVA (age and genotype) and age was found to be a significant effect (p < 0.05) for all genotypes. (A) Mean duration flies spent in the central zone vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (B) Mean latency to first rotation vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (C) Mean rotation frequency vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (D) Mean duration flies spent in the central zone vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (E) Mean latency to first rotation vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (F) Mean rotation frequency vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (G) Mean duration flies spent in the central zone vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (H) Mean latency to first rotation vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (I) Mean rotation frequency vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (J) Mean duration flies spent in the central zone vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (K) Mean latency to first rotation vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (L) Mean rotation frequency vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (M) Mean duration flies spent in the central zone vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (N) Mean latency to first rotation vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (O) Mean rotation frequency vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (P) Mean duration flies spent in the central zone vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (Q) Mean latency to first rotation vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (R) Mean rotation frequency vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls.

FIGURE 5

The effect of neuronal subtype specific IIS reduction on locomotion during exploratory walking senescence in male flies. Exploratory walking senescence parameters for a cohort of male flies of the indicated genotypes run in parallel with the survival experiments shown in Figure 1. Data are shown as mean value for each parameter ± SEM, N = 16 for the indicated genotype. Data were analyzed by two-way ANOVA (age and genotype) and age was found to be a significant effect (p < 0.05) for all genotypes. (A) Mean total distance walked vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (B) Mean duration of walking vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (C) Mean speed of walking vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (D) Mean total distance walked vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (E) Mean duration of walking vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (F) Mean speed of walking vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (G) Mean total distance walked vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (H) Mean duration of walking vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (I) Mean speed of walking vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (J) Mean total distance walked vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (K) Mean duration of walking vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (L) Mean speed of walking vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (M) Mean total distance walked vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. At the age of 13 days ChATGAL4/UAS-InR^DN showed decreased walking duration compared to the controls (P = 0.0011 to ChATGAL4/ + and P = 0.0001 to UAS-InR^DN/ +). (N) Mean duration of walking vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. At the age of 13 days ChATGAL4/UAS-InR^DN showed decreased walking duration compared to the controls (P = 0.0011 to ChATGAL4/ + and P = 0.0001 to UAS-InR^DN/ +). (O) Mean speed of walking vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. At the age of 13 days ChATGAL4/UAS-InR^DN showed decreased speed of walking compared to the controls (P = 0.0045 to ChATGAL4/ + and P < 0.0001 to UAS-InR^DN/ +). (P) Mean total distance walked vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (Q) Mean duration of walking vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (R) Mean speed of walking vs. age of TdcGAL4/UAS-InR^DN flies compared to tdcGAL4/ + and UAS-InR^DN/ + controls.

FIGURE 6

The effect of neuronal subtype specific IIS reduction on decision making during exploratory walking senescence in male flies. Exploratory walking senescence parameters for a cohort of male flies of the indicated genotypes run in parallel with the survival experiments shown in Figure 1. Data are shown as mean value for each parameter ± SEM, N = 16 for the indicated genotype. Data were analyzed by two-way ANOVA (age and genotype) and age was found to be a significant effect (p < 0.05) for all genotypes. (A) Mean duration flies spent in the central zone vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (B) Mean latency to first rotation vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (C) Mean rotation frequency vs. age of TrhGAL4/UAS-InR^DN flies compared to TrhGAL4/ + and UAS-InR^DN/ + controls. (D) Mean duration flies spent in the central zone vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (E) Mean latency to first rotation vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (F) Mean rotation frequency vs. age of ThGAL4/UAS-InR^DN flies compared to ThGAL4/ + and UAS-InR^DN/ + controls. (G) Mean duration flies spent in the central zone vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (H) Mean latency to first rotation vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (I) Mean rotation frequency vs. age of VglutGAL4/UAS-InR^DN flies compared to VglutGAL4/ + and UAS-InR^DN/ + controls. (J) Mean duration flies spent in the central zone vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (K) Mean latency to first rotation vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (L) Mean rotation frequency vs. age of Gad1GAL4/UAS-InR^DN flies compared to Gad1GAL4/ + and UAS-InR^DN/ + controls. (M) Mean duration flies spent in the central zone vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (N) Mean latency to first rotation vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. (O) Mean rotation frequency vs. age of ChATGAL4/UAS-InR^DN flies compared to ChATGAL4/ + and UAS-InR^DN/ + controls. At the age of 13 days ChATGAL4/UAS-InR^DN showed decreased rotation frequency compared to the controls (P = 0.0193 to ChATGAL4/ + and P < 0.0001 to UAS-InR^DN/ +). (P) Mean duration flies spent in the central zone vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (Q) Mean latency to first rotation vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls. (R) Mean rotation frequency vs. age of TdcGAL4/UAS-InR^DN flies compared to TdcGAL4/ + and UAS-InR^DN/ + controls.