Impact of insulin signaling and proteasomal activity on physiological output of a neuronal circuit in aging Drosophila melanogaster

Abstract

The insulin family of growth factors plays an important role in development and function of the nervous system. Reduced insulin and insulin-growth-factor signaling (IIS), however, can improve symptoms of neurodegenerative diseases in laboratory model organisms and protect against age-associated decline in neuronal function. Recently, we showed that chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber escape response circuit. Here, we expand our initial findings by demonstrating that reduced functional output in the giant fiber system of aging flies can be prevented by increasing proteasomal activity within the circuit. Manipulations of IIS in neurons can also affect longevity, underscoring the relevance of the nervous system for aging.

Keywords: Aging; Drosophila; Gao junctions; Giant Fiber system; Insulin signaling; Proteasomal activity; Rab11; Rab4; Recycling.

Fig. 1

Schematic representation of the Drosophila escape response pathway. The GFS consists of two descending giant fiber (GF) interneurons that originate in the brain, and downstream neurons and muscles. In the thoracic region, the GFs form mixed (chemical and electrical) synapses with the peripherally synapsing interneuron (PSI), which in turn forms cholinergic synapses with the motoneuron (DLMn) innervating dorsal longitudinal flight muscles. The GFs also directly connect (via mixed synapses) with the TTMn, the motoneuron that innervates the tergo-trochanteral, or jump, muscle. Electrical brain stimulation (green lightning bolt) activates the giant fiber interneurons, and the two output pathways can be monitored by recording from the two muscles (depolarization spikes). Abbreviations: GFS, giant fiber system; DLM, dorsal longitudinal muscle; TTM, tergo-trochanteral muscle. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Proteasomal inhibition abolishes the effect of reduced IIS. Reduced IIS (A307-GAL4/UAS-InR^dn) prevented age-related increase in response latency observed in the control genotype (+/A307-GAL4) (bars 1-4 in each panel [Augustin et al., 2017]). Proteasome inhibitors MG132 and PSI fed to adult A307-GAL4/UAS-InR^dn flies starting one day after their emergence from pupal cases (the two rightmost bars in each panel) abolished the effect of reduced IIS. N = 6–9 per genotype/condition/age. One-way ANOVA: *p < 0.05; ***p < 0.001. Error bars represent SEM. Abbreviations: ANOVA, analysis of variance; DLM, dorsal longitudinal muscle; IIS, insulin and insulin-growth-factor signaling; n.s., not significant; PSI, peripherally synapsing interneuron; SEM, standard error of the mean; TTM, tergo-trochanteral muscle.

Fig. 3

Overexpression of a proteasome regulatory (19s) subunit prevented age-related functional decline in the GFS. (A) Response latency measured in the TTM (left) and DLM (right) branch of the circuit. Rpn11 encodes one of the “lid” subunits of the proteasome 19S particle, involved in selecting and unfolding proteins targeted for degradation by the 20S core/catalytic particle. N = 4–7 per genotype per age. The genotype-age interaction (two-way ANOVA) was significant: TTM: p value = 0.0335; DLM: p value = 0.0012. One-way ANOVA (Tukey's test): *p < 0.05; ***p < 0.001. Error bars represent SEM. (B) Representative TTM and DLM traces from old flies from (A). Red arrows indicate longer response latencies in control (left) and shorter in Rpn11-overexpressing animals (right). (C) Model for the effect of reduced lysosomal and proteasomal activity on the targeting of GJ proteins to the plasma membrane (see main text). Abbreviations: ANOVA, analysis of variance; DLM, dorsal longitudinal muscle; GFS, giant fiber system; GJ, gap junction; n.s., not significant; SEM, standard error of the mean; TTM, tergo-trochanteral muscle. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

dFoxo partially mediates the effect of reduced IIS on response latency. Deletion of Foxo did not abolish the effect of attenuated IIS in the TTM branch (left), but it reversed the action of lowered IIS in the DLM (right) branch of the giant fiber circuit. N = 4–10 per genotype per age. One-way ANOVA (Tukey's test): *p < 0.05; ***p < 0.001. Error bars represent SEM. Abbreviations: ANOVA, analysis of variance; IIS, insulin and insulin-growth-factor signaling; DLM, dorsal longitudinal muscle; n.s., not significant; SEM, standard error of the mean; TTM, tergo-trochanteral muscle.

Fig. 5

IIS negatively regulates recycling mediating Rab proteins. (A) Representative immunoblots of the levels of the indicated Rab proteins and GAPDH (loading control) from RPE-1 cells treated as indicated with insulin or insulin and IR Inhib. (B) Quantification histograms of the immunoblots from (A). Levels were normalized to respective GAPDH amounts and Rab levels in resting control cells. N = 3 independent experiments. One-way ANOVA (Tukey's test): *p < 0.05; **p < 0.01. Error bars represent SEM. Abbreviations: ANOVA, analysis of variance; IIS, insulin and insulin-growth-factor signaling; IR, insulin receptor; n.s., not significant; RPE-1, retinal pigment epithelial-1; SEM, standard error of the mean.

Fig. 6

Rapamycin does not reverse the effect of age on TTM and DLM response latency in wild-type (w^Dah) flies. (Left): TTM branch. (Right): DLM branch. N = 6–8 per condition/age. One-way ANOVA (Tukey's test): *p < 0.05; ***p < 0.001. Error bars represent SEM. Abbreviations: ANOVA, analysis of variance; DLM, dorsal longitudinal muscle; SEM, standard error of the mean; TTM, tergo-trochanteral muscle.

Fig. 7

Effect on life span of attenuated IIS in neurons. (A) GFS-specific overexpression of a dominant-negative form of the Drosophila insulin receptor had no effect on median or maximum life span. Median life span (days): A307-GAL4/+ (68); UAS-InR^dn/+ (68); A307-GAL4/UAS-InR^dn (64). (B) Reduced IIS in adult neurons extended median life span. Median life span (days): UAS-InR^dn/+, RU- (76); UAS-InR^dn/+, RU+ (78); GS ELAV-GAL4/UAS-InR^dn, RU- (76); GS ELAV-GAL4/UAS-InR^dn, RU+ (86). p-value for significance between GS ELAV-GAL4/UAS-InR^dn (RU+) and other genotypes: 0.05. Abbreviations: GFS, giant fiber system; IIS, insulin and insulin-growth-factor signaling.