Fig. 5: Neuronal reduction of F-actin polymerization slows aging via autophagy.

a Immunostaining of brains at 63x magnification from young (10-day-old) and aged (45-day-old) elavGS > UAS-Atg1-RNAi,UAS-Fhos-RNAi flies with or without RU486-mediated transgene expression from day 5 onward, showing F-actin-rich rods (red channel, phalloidin). Scale bar is 5 µm. Accompanying diagrams indicate brain regions where imaging was conducted. b Quantification of F-actin-rich rods in brains as shown in a. Young n = 6, aged uninduced n = 8, aged induced n = 10 flies, as indicated. ***p (young vs. aged uninduced) = 0.0001, ***p (aged uninduced vs. aged induced) < 0.0001, one-way ANOVA/Tukey’s multiple comparisons test. c Survival curve of elavGS > UAS-Atg1-RNAi,UAS-Fhos-RNAi flies with or without RU486-mediated transgene expression from day 5 onward. ns = non-significant, log-rank test. n = 180 uninduced and 180 induced biologically independent animals. d Intestinal integrity during aging of elavGS > UAS-Atg1-RNAi,UAS-Fhos-RNAi flies with or without RU486-mediated transgene expression from day 5 onward. n = 7 vials of 30 biologically independent animals per vial on day 10. ns = non-significant; two-way ANOVA/ Šídák’s multiple comparisons test. e Immunostaining of brains at 63x magnification from young (10-day-old) and aged (45-day-old) elavGS > UAS-Atg1-RNAi,UAS-Fhos-RNAi flies with or without RU486-mediated transgene induction from day 5 onward, showing mitochondrial morphology (green channel, anti-ATP5a) and nuclear DNA (blue channel, stained with To-Pro-3). Scale bar is 5 µm. f Quantification of mitochondrial area in brain as shown in e. Young n = 5, aged uninduced n = 7, aged induced n = 6 biologically independent animals, as indicated. *p = 0.0277, **p = 0.0021, ns = non-significant; one-way ANOVA/Tukey’s multiple comparisons test. g Staining of brains at 63x magnification from young (10-day-old) and aged (45-day-old) elavGS > UAS-Atg1-RNAi,UAS-Fhos-RNAi flies with or without RU486-mediated transgene induction from day 5 onward, showing TMRE fluorescence. Scale bar is 5 µm. h Quantification of mitochondrial membrane potential measured by TMRE staining as shown in g. Young n = 10, aged uninduced n = 12, aged induced n = 12 biologically independent animals, as indicated. *p (young vs. aged uninduced) = 0.0296, *p (young vs. aged induced) = 0.0318, ns = non-significant; one-way ANOVA/Tukey’s multiple comparisons test. RU486 or vehicle was provided in the media at a concentration of 50 ug/ml in the indicated treatment groups. Data are presented as scatter plots overlaying mean values +/− SEM.