(A) A flow chart outlining the screening pipeline. 5dpf transgenic larvae expressing Tg[fuguth:gal4-uas:GFP;uas:NTRmCherry] were arranged in glass bottom 96-well plates and treated with MTZ (4.5 mM, 48 hrs) along with each of the 1403 bioactive compounds (n = 3 per screening compound). The dual flashlight plot of Brain Health Score (BHS) and Strictly Standardized Mean Difference (SSMD) score was used to quantify the neuroprotective effects of all compounds in the screen. (B) Wilcoxon rank sum test was performed to compare data of all 1403 compounds with those representing RAAS inhibitors (n = 13) in the screened compound set, revealing a significantly higher SSMD score distribution in the RAAS inhibitor group (p = 0.012, Wilcoxon rank sum test). (C) Secondary hit validation. To obtain more precise data, before and after treatment imaging was carried out for each larva embedded in agarose and a treatment regimen with 9 mM MTZ for 24 hr was used. Compounds including the RAAS inhibitors and the N-acetyl cysteine (NAC) control compound were tested at 10 µM with increased sample size (n = 40 per group; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test). (D) Confocal images of ventral forebrain DA neurons. Positive control (0.2 % DMSO), negative control (9 mM MTZ), and 9 mM MTZ +10 µM olmesartan following 24 hrs of treatment. (E) Schematic of the chronic drug treatment and behavior test for adult zebrafish. (F) Quantification of total distance traveled across 5 min recording in the home tank for adult zebrafish treated with 0.2 % DMSO (positive control), 5 mM MTZ (negative control), 5 mM MTZ +10 mM levodopa, and 5 mM MTZ +10 µM olmesartan (with daily change of drug solutions after behavioral recording). Distance recordings were conducted for baseline, 3, 6, 9, 12, and 14 days. ANOVA and post-hoc Tukey test showed significant difference in 12 and 14 days for the MTZ versus MTZ+ olmesartan-treated groups. [n = 6 (three males, three females) for MTZ and MTZ+ Olm, n = 4 (two males, two females) for DMSO control and levodopa; p < 0.01, one-way ANOVA post-hoc Tukey’s test]. (G) Mass spectrometry data of adult zebrafish homogenized brain versus body samples after 14 days of chronic treatment with Olmesartan (n = 6, three males and three females). (H) Quantification of relative fluorescent intensity of DA neurons at 6 dpf in positive control (0.2% DMSO), negative control (9 mM MTZ, 24 hr from 5 dpf to 6 dpf), agtr1a morphant +9 mM MTZ, agtr1b morphant +9 mM MTZ, agtr1a/agtr1b double morphant +9 mM MTZ, and 10 µM olmesartan +9 mM MTZ (n = 10–12; *p < 0.05, ***p < 0.001, unpaired t test).
