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. 2021 Sep 22;10:e69795. doi: 10.7554/eLife.69795

Figure 2. A high throughput in vivo imaging-based chemical screen uncovers the neuroprotective effects of inhibiting the renin-angiotensin (RAAS) pathway.

(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).

Figure 2—source data 1. Secondary hit validation for RAAS compounds.
Figure 2—source data 2. Mass spectrometry data and adult behavior.

Figure 2.

Figure 2—figure supplement 1. Overview of the classically known renin angiotensin pathway and the inhibitors identified from our high throughput screen.

Figure 2—figure supplement 1.

Olmesartan, captopril, and aliskiren are antihypertensive medications working on the Renin-Angiotensin Signaling (RAAS) pathway by blocking the binding of angiotensin II to the receptor, inhibiting the conversion of angiotensin I to angiotensin II, and directly inhibiting the renin enzyme respectively. Ten other RAAS inhibitors also in the 1403-compound bioactive screen are imidapril, enalaprilat, quinapril, ramipril, moexipril, enalapril, which are angiotensin converting enzyme (ACE) inhibitors, and valsartan, telmisartan, azilsartan, and eprosartan, which are angiotensin receptor blockers (ARBs).
Figure 2—figure supplement 2. Manual validation and dose response studies of RAAS inhibitors.

Figure 2—figure supplement 2.

(A) Blind counting of ventral forebrain DA. Neurons in transgenic Tg[fuguth:gal4-uas:GFP; uas-NTRmCherry] 6dpf larvae after 24 hrs of MTZ (9 mM) and RAAS compound (10 μM) treatment (n = 6–8; *p < 0.05, ***p < 0.001, unpaired t-test compared to negative control). (B–D) Dose response studies of RAAS pathway inhibitors. Tg[fuguth:gal4-uas:GFP; uas-NTRmCherry] larvae were imaged 5dpf (Before MTZ treatment) and 6dpf (After MTZ treatment). The Y-axis is the ratio of DA neuron intensity after vs. before MTZ treatment. The MTZ concentration for all dose response studies is 10 mM. (n = 12–24; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test compared to negative control). (E) Quantification of ventral forebrain DA neurons in adult zebrafish under different treatment conditions (E). (F) Representative images showing ventral forebrain DA neurons in adult zebrafish under different treatment conditions. Note the preservation of DA neurons in in MTZ+ olmesartan condition. These DA neurons appeared, however, more scattered in the brain compared to control.
Figure 2—figure supplement 2—source data 1. Dose response quantification for captopril, aliskiren, olmesartan.
Figure 2—figure supplement 3. Agtr1a and Agtr1b morpholino phenotypes and western blot validation.

Figure 2—figure supplement 3.

(A) Translational blocking morpholino (MO) designs for agtr1a and agtr1b. The mRNA target shows the site within the agtr1a and agtr1b transcripts that is targeted by the translational blocking MO. (B) Western blot image showing successful knockdown of the agtr1 protein in the agtr1a + 1 b morpholino-injected samples compared to control (β-Actin). (C) Representative confocal images of DA neurons in different treatment conditions.
Figure 2—figure supplement 3—source data 1. AGTR1 western blot labeled.
Figure 2—figure supplement 3—source data 2. Beta actin western blot labeled.
Figure 2—figure supplement 3—source data 3. AGTR1 western blot raw.
Figure 2—figure supplement 3—source data 4. Beta actin western blot raw.
Figure 2—figure supplement 4. Olmesartan shows neuroprotective effects post neuronal injury in the NTR-MTZ DA neuron ablation model.

Figure 2—figure supplement 4.

(A) Timeline of chemical treatment for MTZ and olmesartan for experiment 1 (8 hr MTZ pretreatment) and experiment 2 (24hr MTZ pretreatment). After pretreating with MTZ for 8 or 24 hr, 10 μΜolmesartan was added and imaged after 16 hr (at 24 and 40 hr, respectively). For both experiments 1 and 2, MTZ concentration was 4.5 mM. (B) DA neuron intensity is significantly greater in the olmesartan-treated samples compared to MTZ alone for both 8 hr (left) and 24 hr MTZ pre-treatment groups (right) (n = 10–12; p < 0.05, unpaired t test).