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. 2019 Jul 3;39(27):5269–5283. doi: 10.1523/JNEUROSCI.1833-18.2019

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Copyright © 2019 Mazaud et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 6. — Effect of Gat, Gs2 and dEAAT1 levels on behavioral and lifespan deficits caused by repo knock-down. All rescue experiments in this figure and in the related Figures 7 and 8, were performed at 25°C and genotypes used were w¹¹¹⁸; UAS-repo^IR 1/+; tubGal80^ts, repoGal4/tubGal80 (negative control) // w¹¹¹⁸; UAS-repo^IR 1/UAS-lacZ; tubGal80^ts, repoGal4/UAS-eGFP // w¹¹¹⁸; UAS-repo^IR 1/UAS-Gs2; tubGal80^ts, repoGal4/UAS-dEAAT1 // w¹¹¹⁸; UAS-repo^IR 1/UAS-Gs2,UAS-Gat; tubGal80^ts, repoGal4/+ // w¹¹¹⁸; UAS-repo^IR 1/UASeGFP; tubGal80^ts, repoGal4/UAS-repo // w¹¹¹⁸; UAS-repo^IR 1/UAS-Gs2; tubGal80^ts, repoGal4/UAS-repo // w¹¹¹⁸; UAS-repo^IR 1/UAS-Gat; tubGal80^ts, repoGal4/UAS-repo // w¹¹¹⁸; UAS-repo^IR 1/UAS-dEAAT1; tubGal80^ts, repoGal4/UAS-repo. A, Evolution over time of the negative geotaxis response. Ten to 15 flies were assessed three times at different time points at 25°C. Coexpression of UAS-Gs2 and UAS-Gat delays the decrease in climbing ability over time compared with the positive control, whereas coexpression of UAS-Gs2 and UAS-dEAAT1 transiently aggravates the phenotype. Blue asterisks indicate significance compared with the positive control (two-way ANOVA, Tukey's multiple-comparisons test p < 0.0001 and p = 0.0011, respectively). B, Fly lifespan was assessed with control flies carrying the tubGal80 transgene (not temperature sensitive) to block the Gal4 activity. The positive control flies expressing control proteins in a repo-KD background display a significantly shorter lifespan compared with the negative control. Coexpression of UAS-Gs2 and UAS-dEAAT1 further shortens the lifespans of repo-KD flies, whereas coexpression of UAS-Gs2 and UAS-Gat displays a trend toward rescue but no significant difference. Black asterisks indicate significance compared with the negative control; blue asterisks indicate significance against the positive control (log–rank Mantel–Cox test p = 0.0207, p = 0.0001 and p = 0.0134, respectively). C, Western blot showing the downregulation of Repo protein in rescued-repo-KD compared with control flies. Actin is used as a loading control. D, Quantification of Repo protein expression. Mann–Whitney, n = 6, p = 0.0022. E, UPL qPCR assay for Gat (as performed in Fig. 5A). Values are normalized using eIF4A as control. Unpaired t test, two-tailed, n = 3 p = 0.024. F, Evolution over time of the negative geotaxis response as in A. Coexpression of UAS-dEAAT1 and UAS-repo accelerates the decrease in climbing ability over time. Black asterisks indicate significance compared with the negative control (two-way ANOVA, Tukey's multiple-comparisons test, p = 0.0068, p = 0.0001, and p = 0.0003, respectively). G, Fly lifespan was assessed as in B. Similar to the climbing assay, the coexpression of UAS-dEAAT1 beside UAS-repo does not rescue and rather aggravates the lifespan phenotype. Asterisks indicate significance compared with UAS-repo alone (log–rank Mantel–Cox test p = 0.000010383935). H, Evolution over time of the negative geotaxis response as in A. The flies coexpressing UAS-Gat and UAS-repo display a significantly improved climbing abilities compared with UAS-repo alone or UAS-repo and UAS-Gs2. Black asterisks indicate significance in genotype/time interactions compared with UAS-repo alone in two-way ANOVA (Tukey's multiple-comparisons test p < 0.0001, p = 0.0009, p < 0.0001, and p < 0.0001, respectively). Colored asterisks indicate significance between the different genotypes and UAS-repo alone at a specific time point (main genotype factor in two-way ANOVA, Tukey's multiple-comparisons test p = 0.0408, p = 0.0001 and p = 0.0207, respectively). I, Conditions of the lifespan similar to G, but with the addition repo-KD flies as a positive control genotype (w¹¹¹⁸; UAS-repo^IR 1/+; tubGal80^ts, repoGal4/+). The coexpression of UAS-Gs2 does not affect the lifespan, whereas UAS-Gat mildly but significantly increases the lifespan of 70% of the flies. Surprisingly, ∼30% of the flies in this condition die within 10 d, whereas 25–30% of the population have a strong increase in lifespan. Asterisks indicate significance compared with UAS-repo alone (log–rank Mantel–Cox test p = 0.000000018838).