Activin signaling targeted by insulin/dFOXO regulates aging and muscle proteostasis in Drosophila

Abstract

Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling.

Figure 1. ChIP-Seq to identify dFOXO direct target genes and lifespan screen for 23 selected candidates.

(A) Venn diagram to show dFOXO target genes identified in ChIP-Seq analysis. 15-day-old female insulin mutants (chico ^−/+ and IPC ablation) were used in ChIP-Seq experiments. dFOXO was enriched at promoters of 273 genes common to these genotypes. (B) Pathway analysis for 273 dFOXO targets, determined by DAVID functional classification. (C–E) Expression analysis of 23 selected dFOXO target genes indicates dFOXO acts as both activator and repressor. Asterisk indicates significant difference between chico^−/− and wildtype (p<0.05); three biological replicates per genotype. (F–H) Lifespan analysis for three dFOXO target genes (daw, Glyp and Tsp42Ef) (Log-rank test, p<0.0001). Ubiquitous GeneSwitch (GS)-Gal4 drivers, Tub-GS-Gal4 or Tub-GS-dicer2-Gal4 (with UAS-dicer2 to enhance the knockdown) were used in lifespan screen (lifetable statistics summarized in Table S1).

Figure 2. Reducing Activin signaling, but not BMP signaling prolongs lifespan in Drosophila.

(A) Schematic showing distinct TGF-β pathways in Drosophila: BMP and Activin. (B) Phylogenetic analysis of TGF-β ligands from worm, fly and mouse. Ligand sequences were retrieved from Flybase, Wormbase and Genebank, respectively. The phylogeny was constructed using MEGA 5.0. (C–F) Lifespan analysis of TGF-β pathways in Drosophila using ubiquitous GeneSwitch (GS)-Gal4 drivers, Tub-GS-Gal4 or da-GS-gal4 (Log-rank test, p<0.0001). See Table S3 for survival analysis.

Figure 3. Inactivation of genes in Activin signaling (daw, Smox and babo) in muscle, but not in fat body extended lifespan.

(A) Tissue-specific gene expression pattern of daw. (B) Tissue-specific distribution of transcription factor Smox using 7-day-old Oregon R females. (C–E) Lifespan analysis of Activin signaling using muscle-specific Gal4 driver (MHC-Gal4). Lifespan was extended by inactivating Activin genes (daw, Smox and babo) in muscle (Log-rank test, p<0.0001). (F–H) Lifespan analysis of Activin signaling using adult fat body-specific Gal4 driver (S106-GS-Gal4). Fat body-specific inactivation of Activin genes (daw and Smox) shortens lifespan (Log-rank test, p<0.0001). See Table S4 for survival analysis. (I, J) mRNA expression of daw and phosphorylation of Smox are down-regulated by chico mutation and rescued by mutation of dFOXO. Muscle and fat body were dissected from 7-day-old female wildtype, chico^−/− and chico;foxo double mutants. Band intensity was quantified using Bio-Rad Image Lab software. The average band intensity from four independent experiments is shown. Asterisk indicates significant difference between treatment and control (p<0.05).

Figure 4. Activin signaling regulates muscle aging and proteostasis.

(A–C) Decline of flight with age is delayed in daw, Smox and babo RNAi flies. 40 females were scored for each genotype at each time point. Flying ability was measured at one week, three weeks and five weeks. (D) Poly-Ubiquitin-positive protein aggregates are reduced at old age in daw, Smox and babo RNAi flies. Aggregates were visualized with Poly-Ubiquitin FK2 antibody at one week, three weeks and five weeks. Scale bar: 20 µm. (E) RNAi for daw, Smox and babo preserves the decline of lysotracker-positive organelles (lysosomes). Scale bar: 20 µm. (F) Quantification of the cumulative area of protein aggregates for Figure 4D (n = 20). (G) Quantification of the number of lysotracker-positive stain for Figure 4E (n = 10). Asterisk indicates significant difference between treatment and control (p<0.05).

Figure 5. Activin regulates muscle autophagy.

(A) Autophagosomes indicated using an Atg8a-Cherry reporter in Activin RNAi flies or babo over-expressing (babo-Act) flies. 3-day old females. (B) Quantification of autophagosomes for Figure 5A (n = 20). (C) mRNA expression of autophagy genes (Atg1, Atg5, Atg6 and Atg8a) in aging muscle, at 10 days, 25 days and 45 days. (D) Phosphorylation of Smox in muscle increases with age. The average band intensity from three independent experiments was quantified using Image Lab software. (E) Inactivation of daw in muscle up-regulates autophagy gene expression. (F) RNAi against Smox in muscle up-regulates autophagy gene expression. (G) Constitutive activation of babo (babo-Act) in muscle inhibits autophagy gene expression. Asterisk indicates significant difference between treatment and control (p<0.05).

Figure 6. Activin signaling represses autophagy via transcriptional regulation on Atg8a.

(A) Schematic of Atg8a genomic region (Smad box and ChIP-PCR target regions (P1–P3) are shown). Gray bar represents UTR and orange bar represents exon. (B) ChIP-PCR shows Smox binds to the promoter of Atg8a with binding enrichment calculated as the fold change of ChIP DNA vs. input DNA. The binding to the coding region of Actin gene (Act5C) was used as a negative control. (C) Smox binds to the promoter of Atg8a, but not Atg1 and Atg6. The primers targeting the promoter regions containing putative Smad box in Atg8a, Atg1 and Atg6 were used in ChIP-PCR. (D) The binding of Smox to Atg8a promoter is abolished by chico mutation. Asterisk indicates significant difference between treatment and control (p<0.05). (E). EMSA analysis reveals that recombinant Smox protein (MH1-DNA binding domain) binds to Smad binding element (AGAC AGAC) located in Atg8a promoter. Biotin-labeled Atg8a oligonucleotide probe (5′- CATATTAGAC AGACACCATT -3′) and its mutated forms are labeled with biotin. Halo-tagged Smox-MH1 DNA binding domain (amino acids 1–140) are expressed in E. coli and purified before used in EMSA analysis. (F) Recombinant Smox protein can also bind to mammalian SBE (GTATGTCT AGACTGAA).

Figure 7. Muscle Activin signaling regulates longevity through Atg8a and remotely controls brain insulin secretion.

(A) Lifespan analysis of muscle-specific Atg8a overexpression. (B) Genetic epistasis between daw and Atg8a in muscle (MHC-Gal4). Simultaneous expression of RNAi for daw and Atg8a blocks the longevity benefit of daw RNAi alone (while Atg8a RNAi alone does not affect survival). See Table S5, S6 for survival analysis. (C, D) Muscle-specific daw RNAi reduces circulating DILP2 level, but has no effects on dilp2 mRNA expression in the head. (E) 4ebp mRNA expression in fat body is regulated by muscle Activin signaling. 4ebp mRNA is elevated in fat body when daw is reduced in muscle, while it is repressed when muscle babo is induced. (F) Female fecundity is not affected by reducing muscle Activin signaling. Asterisk indicates significant difference between treatment and control (p<0.05).