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Fig. 6 ACLY inhibition rescues α-Syn-induced pathology in SNCA zebrafish model (A) Failure of nuclear translocation of p300 by A53T α-Syn expression. Representative images of antibody staining for α-Syn, p300, and DAPI were performed on cryosections across the spinal cord of transgenic Dendra-α-SYN_WT and A53T fish with mosaic expression of the transgene at 2 d.p.f. n = 14 fish per group, two-tailed unpaired t test. ∗p < 0.05 vs. Dendra-α-SYN _WT. Scale bar, 5 μm. (B) Enhanced mTORC1 activity in fish expressing human A53T α-Syn mutation. Representative images and quantification of S6K1 and p-S6K1 western blots to measure mTORC1 activity in fish with pan-neuronal expression of Dendra-α-SYN_WT and A53T at 2 d.p.f. n = 12 independent clutches, two-tailed unpaired t test. ∗p < 0.05 vs. Dendra-α-SYN_WT. (C) Inhibition of autophagic flux in fish carrying the A53T mutation. Representative images and quantification of western blots for the levels of LC3-II in fish with pan-neuronal expression of Dendra-α-SYN_WT or A53T at 2 d.p.f. in basal conditions and after treatment with 10 mM NH4Cl for 4 h. Graph represents the ratios of LC3-II vs. actin normalized to a single negative sibling control run in all blots. The same sample from a negative sibling was run in parallel on every gel and used for normalization of the data. n = 9 independent clutches, one-way ANOVA followed by Sidak’s multiple test comparison. ∗p < 0.05 vs. Dendra-α-SYN_WT; #p < 0.05 vs. Dendra-α-SYN_WT treated with NH4Cl. (D) Decreased phosphorylated α-Syn after treatment with ACLY inhibitors. Representative images and quantification of western blots showing the levels of phosphorylated α-Syn at Ser129 in fish with pan-neuronal expression of Dendra-α-SYN_ A53T treated with 0.1% DMSO, 0.5 or 1 μM SB and 50 or 100 μM HC from 1 to 3 d.p.f. n = 5 independent clutches, two-tailed one-sample t test. ∗∗p < 0.01 vs. DMSO. (E) Inhibition of mTORC1 activity after treatment with ACLY inhibitors. Representative images and quantification of S6 and p-S6 western blots to evaluate the effect of 0.5 μM SB-204990 and 50 μM HC on the mTORC1 activity in fish with pan-neuronal expression of Dendra-α-SYN_A53T treated from 1 to 2 d.p.f. n = 8 independent clutches, two-tailed one-sample t test. ∗p < 0.05 and ∗∗∗p < 0.01 vs. DMSO. (F) In vivo clearance kinetics of Dendra-α-SYN_A53T in neurons of fish treated with 0.1% DMSO (black lines), 0.5 μM SB (green line), or 50 μM HC (blue line) imaged for 48 h at 24 h intervals. Graphs represent the reduction in the percentage (%) of initial red intensity over time. n = 62 neurons/group, Sidak’s multiple comparisons two-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. DMSO. (G) Clearance of insoluble α-Syn fraction by ACLY inhibition. Representative images and quantification of western blots for the Sarkosyl-soluble and -insoluble fractions of α-Syn in 3 d.p.f. fish with pan-neuronal expression of Dendra-α-SYN_A53T treated either with 0.1% DMSO, 0.5 μM SB, and 50 μM HC. HE, high exposure. n = 5 independent experiments, 40 fish/replicate. Two-tailed one-sample t test. ∗p < 0.05 and ∗∗p < 0.01 vs. DMSO. (H) Rescue of neuronal cell death in Dendra-α-SYN_A53T zebrafish following treatment with ACLY inhibitors. Representative images show terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in transverse cryosections across zebrafish larvae brains with pan-neuronal expression of Dendra-α-SYN_A53T treated with 0.5 μM SB and 50 μM HC (1 to 2 d.p.f.) along with their A53T-negative sibling controls. The dashed rectangle in the DAPI image highlights the brain region shown at higher magnification for comparison among the groups. Apoptotic nuclei appear as bright dots. Neuronal cell death was assessed by quantifying apoptotic nuclei in the brain (white arrows), excluding other tissues such as eyes or cartilage (yellow arrows). Scale bar: 125 μm. Data are presented as the number of apoptotic neuronal nuclei per fish, quantified across five consecutive sections per fish (n = 20 fish per group). Statistical analysis was performed using a two-tailed unpaired t test. ∗∗∗∗p ≤ 0.0001 vs. A53T-negative sibling fish; ####p ≤ 0.0001 vs. A53T-positive fish treated with DMSO. Data are presented as mean values ± SD unless otherwise specified.