Fig. 1

A screen of 2007 FDA-approved compounds revealed artesunate as a lead drug candidate upregulating PICALM in cell assays and mouse brain capillaries in vivo. A A PICALM promotor-driven secreted luciferase plasmid was integrated into HEK293t cells to create a stable luciferase reporter cell line for PICALM drug screening. B Using the HEK293t luciferase reporter line, 2007 FDA-approved drugs were evaluated for PICALM upregulation. Screen was run in triplicate at 3 μM drug concentration. Each point represents luciferase signal for each drug in the screen relative to SEAP internal control and normalized to DMSO control luciferase signal in the absence of drugs. Drugs with luciferase increases over 3 SD above the mean DMSO-normalized luciferase/SEAP value for all drugs (dashed line) were considered “hits” and further evaluated (see Methods and Results). C Secondary mRNA evaluation of drug hits by RT-qPCR in human Eahy926 endothelial cells. Relative abundance of PICALM mRNA after incubation of Eahy926 endothelial cells with top drug hits at 3 μM for 24 hours, normalized by the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels and compared to DMSO vehicle. Data are mean ± SEM; n = 3 independent cultures per condition. Significance by ANOVA vs DMSO followed by Dunnett’s multiple comparisons test. D, E Relative abundance of PICALM mRNA (D) and protein levels (E) after incubation of Eahy926 endothelial cells with top drug hit artesunate (Art) at 3 μM for 24 hours. The relative abundance of PICALM mRNA was normalized by the GAPDH mRNA levels (D) or β-actin protein (E), and compared to DMSO vehicle. n = 5 replicates for D, and n = 3 replicates for E. F Relative abundance of PICALM mRNA normalized by the GAPDH levels in the presence of different concentrations of artesunate. Bars represent mean ± SEM at each concentration studied; n = 4–6 for each concentration. Individual values indicated by circles; gray line shows a sigmoid curve fit to the data for determination of the EC50 value. G Relative abundance of EGR1 mRNA after incubation of Eahy926 endothelial cells with 3 μM artesunate for 24 hours. The relative abundance of EGR1 mRNA was normalized by GAPDH mRNA levels, and compared to DMSO vehicle. n = 3 replicates. H Relative abundance of PICALM protein levels after incubation of Eahy926 endothelial cells with 3 μM Art for 24 hours after silencing EGR1 (siEGR1) or in the presence of control siRNA (siControl). n = 3 replicates. I-J PICALM protein levels in brain capillaries (I) and capillary-depleted brains (J) from Picalm^+/− mice treated i.p. with 32 mg/kg artesunate or vehicle for 1 week. n = 4 mice per group. Individual replicates in D, E, G, H, or mice in I, J indicated by circles; All data in these panels are mean ± SD. Significance in D, E, G, I and J by Student’s two-tail t-test. Significance in H, by one-way ANOVA followed by Tukey post-hoc test. Full blots for E, H-J shown in supp. Fig. 5