Characterization of Hit Compounds Identified from High-throughput Screening for their Effect on Blood-brain Barrier Integrity and Amyloid-β Clearance: In Vitro and In Vivo Studies

Abstract

In Alzheimer's disease (AD) the blood-brain barrier (BBB) is compromised, thus therapeutic targeting of the BBB to enhance its integrity and function could be a unique approach to treat, slow or hold the progression of AD. Recently, we have developed an in vitro high-throughput screening assay to screen for compounds that increase the integrity of a cell-based BBB model. Results from primary screen identified multiple hit compounds that enhanced the monolayer integrity. Herein, further characterization of selected hit compounds, namely 8-bromoguanosine cyclic monophosphate, JW74, 1,10-phenanthroline monohydrate, SB216763 and α-tocopherol was performed. Compounds were subjected to concentration-dependent studies to determine their EC50 and potency to enhance the cell-based model integrity by the Lucifer Yellow permeability and amyloid-beta (Aβ) transport across the monolayer. The compounds demonstrated different EC50s to enhance the monolayer integrity ranging from 0.4 to 12.8 µM, and different effect on enhancing Aβ transport with highest transport observed for α-tocopherol (2.2-fold increase). Such effects were associated with increased levels of tight junction proteins such as claudin-5 and/or ZO-1, and Aβ major transport proteins LRP1 and P-glycoprotein. In vivo studies for α-tocopherol were performed in AD mouse model; consistent with the in vitro results α-tocopherol significantly increased BBB integrity measured by IgG extravasation, and reduced brain Aβ levels. In conclusion, findings support our developed cell-based BBB model as a functional predictive in vivo tool to select hit compounds, and suggest that enhancing BBB tightness and function has the potential to reduce Aβ pathology associated with AD.

Keywords: IgG extravasation; amyloid-β clearance; blood–brain barrier; brain endothelial cells; high-throughput screening; α-tocopherol.

Figure 1

Hit compounds secondary screen. 10-Point concentration-response curves of hit compounds showed significant enhancement in the barrier function of the bEnd3 cells-based BBB model. The compounds were tested in the concentration range 0.078–20 μM to calculate compounds EC50s. Data represent mean±SD, n = 6 for each concentration.

Figure 2

Representative western blot and densitometry analysis of ZO-1 (a), claudin-5 (b) and β-actin, as loading control, proteins levels in bEnd3 cells treated for 24 h with 10 μM of JW74, Phen, SB2, BrG and Tph. Data are presented as mean ± SD of 3 independent experiments. ***P<0.001 compared to control group, ns=not significant.

Figure 3

(a) Effect of bEnd3 cells treatment for 24 h with 10 μM of JW74, Phen, SB2, BrG and Tph on the transport of ¹²⁵I-Aβ₄₀, added to the lower chamber of the inserts, across bEnd3 monolayer. ¹²⁵I-Aβ₄₀ transport is determined as transport quotient (Aβ-TQ_B→A). (b) Effect of increasing concentration of Tph on the transport of ¹²⁵I-Aβ₄₀, and (c) permeability of ¹⁴C-inulin. Data are presented as mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ns=not significant.

Figure 4

Representative western blot and densitometry analyses of (a) P-gp and (b) LRP1 proteins levels in bEnd3 cells treated for 24 h with 10 μM of JW74, Phen, SB2, BrG and Tph. Data are presented as mean ± SD of 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ns=not significant.

Figure 5

Effect of Tph treatment (10 mg/kg/day for one month) on BBB tightness in 5XFAD mice. The figure shows representative brain sections stained with anti-IgG antibody to detect IgG extravasation (green) and collagen antibody (red) to detect microvessels, and their optical density quantitation in cortex (a) and hippocampus (b) regions. Data are presented as mean ± SEM of five mice in each group. ***P<0.001 compared to control group. Scale bar, 50 μm.

Figure 6

Effect of Tph treatment (10 mg/kg/day for one month) on tight junction proteins and Aβ transport proteins in microvessels isolated from 5XFAD mice brain. (a) Representative blots and densitometry analysis of claudin-5 and ZO-1. (b) Representative blots and densitometry analysis of P-gp and LRP1. Data are presented as mean ± SEM of five mice in each group. **P<0.01, ***P<0.001.

Figure 7

Effect of Tph treatment (10 mg/kg/day for one month) on total Aβ brain levels in 5XFAD mice. Representative brain sections stained with 6E10 antibody against Aβ to detect total Aβ load (green) and collagen antibody (red) to detect microvessels, and their optical density quantitation in cortex (a) and hippocampus (b) regions demonstrate significant reduction in Aβ load. Data are presented as mean ± SEM of five mice in each group. ***P<0.001 compared to control group. Scale bar, 50 μm.