Fig. 5
From: Repeat length of C9orf72-associated glycine–alanine polypeptides affects their toxicity
Increased insulin signalling rescued GA400 toxicity. A–C Protein levels of insulin-like peptides (dILP) and the insulin receptor (InR) measured by mass spectrometry-based proteomics in the brain of female flies expressing GA100, GA200 or GA400 for 5, 15 or 40 days under the control of the elav-GS driver. Shown are z-score normalized protein levels of A dILP3, B dILP5 and C InR. GA400 expression caused a significant decrease of dILP3, dILP5 and InR levels, suggesting decreased activity of brain insulin signalling upon GA400 induction (One-Way ANOVA + Tukey’s multiple corrections test; n = 3–4 replicates of 25 fly brains; *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001). D Representative eye images of female flies expressing GA100 or GA400 (upper panel) or GA100 or GA400 in combination with constitutively active insulin receptor (InRCA) (lower panel) under the control of the GMR-Gal4 driver. E Eye size of flies normalized to the mean of the eye size of GMR-Gal4/ + control flies. InRCA expression significantly increased the eye size of control, GA100 and GA400 flies (Two-way ANOVA + Bonferroni’s Tukey’s multiple comparisons test; n = 10–13 imaged fly eyes per genotype; presence of GA: ****P < 0.0001; presence of InRCA: ****P < 0.0001; interaction between GA and InRCA: ****P < 0.0001). While no interaction was found between control and GA100 flies upon InRCA expression (Two-way ANOVA; n = 10–13 imaged fly eyes per genotype; interaction of GA and InRCA: P > 0.05), a significant interaction was observed between control and GA400 flies upon InRCA expression (Two-way ANOVA; n = 10–12 imaged fly eyes per genotype; interaction of GA and InRCA: P > 0.05), indicating that the magnitude of the InRCA effect was larger in GA400 flies. F, G Climbing performance and survival of GA400 flies co-expressing InRCA. Climbing indices are represented as box plots and the mean is indicated by + . Circles indicate individual flies. InRCA co-expression significantly improved climbing of control and GA400 expressing flies (elav-GS/ + vs InRCA and GA400 vs GA400 + InRCA; Two-Way ANOVA + Tukey’s multiple corrections test; n = 35–44 flies; age: ****P < 0.0001; genotype: ****P < 0.0001; interaction of age and genotype: ****P < 0.0001). Same elav-GS/ + and GA400 flies were used between F and Additional file 13: Fig. S13C. G Co-expression the InRCA significantly extended lifespan of GA400-expressing female flies (GA400 vs GA400 + InRCA; log-rank test; n = 150 female flies per genotype; ****P < 0.0001). The same survival curve of GA400-expressing flies is shown in G and Additional file 12: Fig. S12D. H Representative western blot of head protein extracts from female flies co-expressing GA400 and InRCA probed with anti-GA and anti-p62 antibodies. I, J Quantification of the western blots. I HMW GA and J p62 protein levels. Co-expression of InRCA significantly reduced GA400 and p62 protein levels (One-way ANOVA + Tukey’s multiple comparisons test; n = 4 replicates of 20 fly heads; ****P < 0.0001)