Fig. 4: F-actin stabilization disables mitophagy in the aged Drosophila brain.

a Mito-QC of brains at 63x magnification from 10-day-old and 45-day-old flies. Genotypes analyzed were elavGS > UAS-mito-QC, as controls, and elavGS > UAS-mito-QC,UAS-Fhos-RNAi. RU486-mediated transgenes were induced from day 5 onwards. Images shown of merged GFP and mCherry along with punctate mCherry-only foci (from merged images where GFP has been quenched; mitolysosomes). Scale bar is 5 µm. Accompanying diagrams indicate brain region where imaging was conducted. b Quantification of mitolysosomes per 4 mm² brain area as shown in a. Young control n = 5, aged control n = 6, aged Fhos-RNAi n = 9 biologically independent animals, as indicated. *p = 0.0475, **p = 0.0089, one-way ANOVA/Tukey’s multiple comparisons test. c Immunostaining of brains at 63x magnification from young (10-day-old) and aged (45-day-old) elavGS > 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. d Quantification of mitochondrial area in brain as shown in c. Young = 6, aged uninduced n = 4, aged induced n = 6 biologically independent animals, as indicated. **p = 0.0018, ***p = 0.0004; one-way ANOVA/Tukey’s multiple comparisons test. e Staining of brains at 63x magnification from young (10-day-old) and aged (45-day-old) elavGS > UAS-Fhos-RNAi flies with or without RU486-mediated transgene induction from day 5 onward, showing TMRE fluorescence. Scale bar is 5 µm. f Quantification of mitochondrial membrane potential measured by TMRE staining as shown in e. Young n = 9, aged uninduced n = 7, aged induced n = 7 biologically independent animals, as indicated. *p (d10 vs. d45 uninduced) = 0.0335, *p (d45 uninduced vs. d45 induced) = 0.0221; one-way ANOVA/Tukey’s multiple comparisons test. (g) Immunostaining of brains from 10-day-old, 37-day-old, and 44-day-old Canton S flies given vehicle (DMSO) or 10 µM cytochalasin D as indicated from days 37-44 post eclosion, showing mitochondrial morphology (green channel, anti-ATP5a). Scale bar is 5 µm. h Quantification of mitochondrial area in brain as shown in g. Young n = 5, aged (d37) n = 5, aged (d44) DMSO n = 8, aged (d44) CytoD n = 7 biologically independent animals, as indicated. **p (young vs. aged d37) = 0.0041, *p (aged d37 vs. aged d44 CytoD) = 0.0174, **p (aged d44 DMSO vs. aged d44 CytoD) = 0.0036; unpaired two-tailed t-tests. i Staining of brains from young (10-day-old) and aged (45-day-old) Canton S flies given vehicle (DMSO), 5 µM, or 10 µM cytochalasin D as indicated from days 37-44 post eclosion, showing TMRE fluorescence. Scale bar is 5 µm. j Quantification of mitochondrial membrane potential measured by TMRE staining in brains as shown in i. Young n = 11, aged DSMO n = 7, aged CytoD 5 µM n = 9, aged CytoD 10 µM n = 13 biologically independent animals, as indicated. ***p (young vs. aged d44 DMSO) = 0.0003, *p (aged d44 DMSO vs. aged d44 5 µM CytoD) = 0.0353, ***p (aged d44 DMSO vs. aged d44 10 µM CytoD) = 0.0002; one-way ANOVA/Tukey’s multiple comparisons test. Data are presented as scatter plots overlaying mean values +/− SEM.