Journal: Cell Reports

Article Title: ADSL deficiency is a secondary mitochondrial disease affecting organelle homeostasis and ERK2/AKT signaling in a linear genotype-phenotype relation

doi: 10.1016/j.celrep.2025.116230

Figure Lengend Snippet: Drosophila model of ADSL deficiency (A) The table lists all GAL4 promoters employed to drive tissue-specific expression of UAS-ADSL RNAi. For each driver, we indicate the target tissue, the tested temperature, whether the knockdown resulted in lethality or caused any phenotype. (B) Western blotting analysis of ADSL and SYN proteins in Drosophila larvae expressing ADSL RNAi in muscles. Densitometry analysis of ADSL and SYN normalized against Giotto is shown (right). Values are mean ± SEM ( n ≥ 5). (C) Analysis of locomotion activity of adult flies expressing ADSL RNAi in neurons (69B-GAL4). Values are mean ± SEM ( n ≥ 22 flies from three independent experiments). (D) NMJ (muscle 6 and 7) immunofluorescence analysis of third-instar larvae on expressing d ADSL RNAi under Mef2-GAL4 driver. NMJs were co-stained with fluorescent labeled phalloidin (marker of muscle actin fibers) and anti-Discs-large (marker of subsynaptic reticulum). Discs-large signal intensity was measured and divided by NMJ total area for normalization. Around 10 NMJs for each genotype were analyzed (control n = 9 NMJs, ADSL RNAi n = 12 NMJs). All crosses for the NMJ analysis were held at 29°C. Scale bars, 20 μm. The violin plots (right) report median (dashed lines), first and third quartile (dotted lines), and density plot (outside lines). (E) Immunofluorescence analysis of third-instar-larva NMJs on muscle 6 and 7 of flies expressing dADSL RNAi under Mef2-GAL4 driver. Anti-HRP was used to highlight presynaptic membranes and representative images are presented. Few representative NMJs on muscle 12 and 13 were also added. The area of all boutons of each NMJ was measured and normalized by the length of the junction. The area of each abnormal bouton above a fixed threshold (area > 1,000 a.u.) was considered and summed. Around 20 NMJs for each genotype were analyzed (control n = 19 NMJs, ADSL RNAi n = 19 NMJs). Scale bars, 10 μm. The violin plots (bottom) report median (dashed lines), first and third quartile (dotted lines), and density plot (outside lines). (F) Immunofluorescence analysis of the mitochondrial network of Drosophila larval muscles expressing ADSL RNAi and mito-GFP reporter (green); phalloidin (red) was used to mark actin filaments. Scale bar, 10 μm. The measure of mitochondrial fragmentation is also shown (left). Values are mean ± SEM ( n ≥ 7). (G) Representative transmission electron microscopy (TEM) images of larval muscles expressing ADSL RNAi under Mef2-GAL4 driver. Mitochondria are highlighted with red arrowheads. Scale bar, 500 nm. Quantification of mitochondrial fragmentation is shown (bottom) and was assessed by analyzing multiple independent electron microscopy (EM) images per condition and defined as the percentage of damaged or fragmented mitochondria relative to the total number of mitochondria per field (from three independent experiments). Values are mean ± SEM. Statistical analyses were performed using unpaired Student’s t test or Wilcoxon matched-pairs signed rank test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001.

Article Snippet: The primary antibodies used in this study were as follows: p -AKT Thr308 (Cell Signaling Technology #9275, WB 1:1000); AKT (Cell Signaling Technology #9272, WB 1:1000); p -ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology #9101, WB 1:1000); ERK1/2 (Cell Signaling Technology #9102, WB 1:1000); ADSL (C-11) (Santa Cruz Biotechnology #sc-365623, WB 1:1000), GAPDH (6C5) (Santa Cruz Biotechnology #sc-32233, WB 1:1000), HSP90α/β (F-8) (Santa Cruz Biotechnology #sc-13119, WB 1:3000), TOM20 (F-10) (Santa Cruz Biotechnology #sc-17764, WB 1:1000, IF 1:200); UBIQUITIN (Santa Cruz Biotechnology #sc-8017, WB 1:1000); ACTIN (Sigma-Aldrich #A2066, WB 1:3000); OxPhos Human antibody cocktail (Thermo Fisher Scientific #45–8099); SYN (3C11 (anti SYNORF1) Developmental Studies Hybridoma Bank, US, WB 1:1000); Giotto ( was a gift from GL Cestra; https://www.antibodyregistry.org/AB_2892585 , WB 1:3000); HRP (Peroxidase AffiniPure Rabbit Anti-Human IgG (H + L), Jackson ImmunoResearch #309-035-003); Discs large (4F3 anti-discs large, Developmental Studies Hybridoma Bank, US, IF 1:200); LC3B (D11, XP, Cell Signaling Technology #3868, WB 1:1000); Cyclin B1 (Cell Signaling Technology #4138, WB 1:1000); OPA1 (Cell Signaling Technology #80471, WB 1:1000); p -DRP1 Ser616 (Cell Signaling Technology #3455, WB 1:1000); DRP1 (Cell Signaling Technology #8570, WB 1:1000); MFN2 (Santa Cruz Biotechnology #sc-515647, WB 1:1000); FLAG (M2, Sigma-Aldrich #F1804, WB 1:1000).

Techniques: Expressing, Knockdown, Western Blot, Muscles, Activity Assay, Immunofluorescence, Staining, Labeling, Marker, Control, Transmission Assay, Electron Microscopy