In vivo dopamine neuron imaging-based high throughput screening in larval zebrafish identifies potential neuroprotective compounds. (A) Overview of the high throughput screening assay. 3 dpf larvae are transferred to 96 well plates with 10 μM screening compounds. DMSO (positive control) or 4.5 mM MTZ (negative control) was added 3 hours later and the treatment lasted for 24 hr, followed by imaging with brightfield and TexRed channels on InCell 6,000. Images were analyzed with the Cellprofiler pipeline. (B) Schematic of the image processing pipeline using the custom generated MATLAB “fishplatebrowser” and Cellprofiler. The brightfield and TexRed images were used to automatically detect the eye and diencephalic region of the brain and to quantify DA neurons. (C) Dual flashlight plot generated from custom made GUI “HitDataBrowser” with MATLAB. Compounds can be selected and exported with SSMD, BHS, and corresponding sample number. (D) Compounds in the top right quadrant with high BHS and SSMD scores based on manual selection. Details of the compounds are shown in Table 1. PTU: 1-phenyl 2-thiourea MTZ: Metronidazole DMSO: dimethyl sulfoxide.

FIGURE 2. Curation and pathway analysis of the screening dataset identify novel mechanisms of neuroprotection. (A) Schematic showing the data processing and analysis pipeline. The example output of the annotations are shown on the right side with the corresponding numbers of each step. Hit calling was based on three criteria, including manual selection with good BHS and SSMD score, Wilcoxon rank sum test, and Reactome pathway analysis. (B) A list of significant pathways from the Reactome pathway analysis sorted from highest to lowest significance (Padj <0.01). (C) Significant pathways from the non-topology-based pathway analysis of the screening dataset. BHS of the chemicals in the same pathway were compared against BHS of all compounds in the dataset. (n = 5 to 13; Padj <0.05, Wilcoxon rank sum test). ADRA2A: Alpha-2A adrenergic receptor, PIK3: Phosphoinositide 3-kinase, COX1: Cytochrome c oxidase subunit I, OPRM1: Mu type opioid receptor, CHRNA1: Cholinergic Receptor Nicotinic Alpha 1 Subunit, RAAS: Renin angiotensin system, MAPK: Mitogen-activated protein kinase, PCP/CE: Planar cell polarity and convergent extension, DPP4: Dipeptidyl peptidase-4, TP53: Tumor protein P53.

Establishment of a secondary hit validation assay and validation of candidate hit compounds. (A) Schematic of the secondary hit validation assay using agarose embedding and automated imaging. At 5 dpf, larvae were embedded in 1.2% agarose and imaged under brightfield and DsRed channels. The larvae were treated with 0.2% DMSO or 9 mM MTZ with or without hit compounds. At 6 dpf, larvae were again imaged with the same x,y,z coordinates on the microscope. Image shown is an example of a 0.2% DMSO control. (B) Comparison of 40 and 50 μL 1.2% low melting point agarose for embedding. Samples embedded with 40 μL agarose showed significant difference between DMSO control and 9 mM MTZ (n = 8; p < 0.05, unpaired t test), whereas those with 50 μL agarose did not, due to increased distance between the objective and the samples. (C) Evaluation of Z′-factor for the secondary hit validation assay. The 0.2% DMSO control and 24 h of 9 mM MTZ treatment showed a significant difference in DA neuron intensity with a z’factor of 0.58. (D) Secondary hit validation of compounds with the embedding assay. Samples were treated with 10 μM of each candidate compound and 9 mM MTZ for 24 h. Etodolac, nepafenac, NAC, aloperine, Protionamide, olmesartan, and captopril showed significantly greater “BHS After treatment” to “BHS before treatment” ratio compared to the negative control (9 mM MTZ) (n = 22 to 30; one-way ANOVA F = 12.33, p = 0.003, post-hoc Fishers LSD *p < 0.05, **p < 0.01, ***p < 0.001). MTZ: metronidazole, DMSO: dimethyl sulfoxide, NAC: N-Acetyl Cysteine, NMDA: N-methyl-d-aspartate, MMF: Mycophenolate mofetil, SGC: SGC-CBP30.

Manual screening and combination screening of hit candidates based on secondary assay. (A) Manual screening of the significant compounds identified from the secondary hit validation assay. All samples were manually quantified in a blinded manner after 24 h treatment with candidate compounds and MTZ as described above. (n = 7 to 8; one-way ANOVA F = 16.72, p < 0.001, post-hoc Fishers LSD *p < 0.05, **p < 0.01). (B) Heatmap matrix showing the BHS for testing hit compounds in combination. All candidate compounds were 10 μM in concentration. The combination of etodolac-nepafenac, etodolac-protionamide, and etodolac-aloperine showed greater BHS compared to the administration of either alone. 0.2% DMSO for positive control and 9 mM MTZ for negative control. (n = 12 to 16; *p < 0.05, **p < 0.01, unpaired t test).

Validation of candidate compounds in a chemically induced Gaucher disease model. (A) High throughput imaging of DA neurons with the InCell 6,000 platform for the positive control, CBE, and the candidate compounds. The bottom left image shows the DA neuron isolation process in the custom Cellprofiler pipeline used for image analysis. (B) Hit validation of candidate compounds with 48 h treatment of 500 μM CBE. At 5 dpf, larvae were treated with 0.2% DMSO (positive control), 500 μM CBE (negative control), and the CBE+ 10 μM candidate compounds for 48 h. At 7 dpf, the larvae were imaged with a confocal microscope. The 500 μM CBE showed significant reduction in DA neurons compared to the 0.2% DMSO control (N = 12; p = 0.0012, unpaired t-test). Nepafenac, olmesartan, and aloperine showed significant neuroprotection when co-treated with CBE (N = 10 to 12; one-way ANOVA F = 6.205, p < 0.001, post-hoc Fishers LSD **p < 0.01, ***p < 0.001, unpaired t-test). CBE: Conduritol B epoxide.