A high content drug screen identifies ursolic acid as an inhibitor of amyloid beta protein interactions with its receptor CD36

Abstract

A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid β (Aβ) in the brain. Aβ binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of Aβ binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of Aβ binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled Aβ to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, Aβ concentration, time required for Aβ binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of Aβ binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of Aβ to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 μm, with a maximal inhibition of 64%. Ursolic acid also blocked binding of Aβ to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of Aβ to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block Aβ-CD36 interactions.

FIGURE 1.

CD36 expressed on CHO cells is a receptor for Aβ 1–42. Stably transfected CHO-CD36 and control vector-transfected CHO cells were incubated with 1 μm Hilyte-Fluor 488 Aβ 1–42 (HF488 Aβ 1–42) for 2 h, and uptake was measured by flow cytometry (A) and visualized by fluorescence microscopy (B). Only CHO-CD36 cells showed cells association with HF488 Aβ 1–42 by flow cytometry and showed evidence of intracellular Aβ by fluorescent microscopy. HF488 Aβ 1–42 is shown in green, nuclei were stained with DAPI (blue). Original magnification, ×40. Data represent mean ± S.E. (error bars); n = 3 p < 0.003).

FIGURE 2.

Time course for binding/uptake of HF488 Aβ 1–42 to CHO and CHO-CD36 cells. CHO and CHO-CD36 cells were incubated with 1 μm HF488 Aβ 1–42. At various time points the cells were washed, fixed, and analyzed by an In-Cell 1000 microscope. The data show that a 2-h incubation provides the best signal-to-background measurement. Data represent mean ± S.E. (error bars). n = 4, p < 0.04.

FIGURE 3.

Characterization of binding/uptake of CHO-CD36 cells to Aβ. A, CHO CD36 cells bind soluble and fibrillar Aβ. CHO and CHO-CD36 were incubated with either 1 μm soluble or fibrillar HF488 Aβ for 2 h, and cell-associated Aβ was measured by In-Cell, n = , p < 3 × 10⁻⁷ (for soluble Aβ) and n = 15, p < 0.01 for fibrillar Aβ. B, CHO-CD36 cells bind Aβ in a dose-dependent manner. CHO and CHO-CD36 were incubated with various concentrations of HF488 Aβ for 2 h, and the cells were analyzed by In-Cell. n = 6, p < 3 × 10⁻⁷ for 1 μm HF488 Aβ. C, CHO-CD36 cells bind HF488 Aβ in the presence of DMSO concentrations up to 1%. CHO and CHO-CD36 were incubated with 1 μm HF488 Aβ for 2 h with increasing concentrations of DMSO. Data represent mean ± S.E. (error bars). n = 15. 0% DMSO, p < 0.001; 0.1% DMSO, p < 0.0009; 0.25% DMSO, p < 0.0024; 0.5% DMSO, p < 0.0003; 1% DMSO, p < 0.01; and 2% DMSO, p < 0.05.

FIGURE 4.

Binding/uptake of CHO-CD36 cells to Aβ is miniaturized to a 384-well plate. CHO-CD36 cells were seeded in columns 1–23 and CHO cells in column 24 of a 384-well plate. All columns were incubated with 0.1% DMSO and 1 μm HF488 Aβ for 2 h. Cells were analyzed by In-Cell, and each column was averaged. Data represent mean ± S.E. (error bars), n = 4. p < 0.00001.

FIGURE 5.

Ursolic acid does not affect expression of CD36 on CD36-CHO or primary microglia. CD36-CHO and primary microglia were incubated with either ursolic acid (50 μm) or a volume equivalent of DMSO. A, surface antibody staining for CD36 revealed no difference between ursolic acid or DMSO treatment (black, unstained; red, isotype control; blue, DMSO; yellow, ursolic acid). B, qPCR for CD36 after either ursolic acid or DMSO treatment also showed no difference in CD36 expression.

FIGURE 6.

Ursolic acid competitively blocks binding of HF488 Aβ 1–42 and 1–40 species to CHO-CD36 cells. A, CHO-CD36 cells were incubated with 1 μm HF488 Aβ 1–42 for 2 h in the presence of increasing concentrations of ursolic acid DMSO concentrations remain constant in each triplicate. B, cell-associated Aβ was assessed by flow cytometry for HF488 Aβ 1–40 and HF488 Aβ 1–42. C, native Western blotting for Aβ and densitometry analysis shows the species present in the Aβ preparation are mostly 4-mers and 7-mers. Denaturing Western blotting for Aβ shows that HF488 Aβ is ∼4.5kDa. For comparison, molecular mass markers were run on the same gels, their corresponding sizes are shown in kDa). Data for A and B represent the mean ± S.E. (error bars). n = 3. p < 0.05.

FIGURE 7.

Ursolic acid partially blocks Aβ binding and Aβ-induced activation of microglia to produce ROS. A and B, freshly isolated primary microglia (A) or N9 microglia (B) incubated with 1 μm HF488 Aβ for 2 h in the presence of 50 μm ursolic acid or volume equivalent of DMSO. Cell-associated Aβ was measured by flow cytometry (p < 0.05). C, N9 microglia preincubated with ursolic acid for 30 min and then stimulated with 1 μm amyloid-β for an additional 30 min. ROS production was measured using the DHR 123 assay. Data represent mean ± S.E. (error bars), each data point is the mean of three separate experiments each done in triplicate. n = 3 (20 μm, p < 0.034; 50 μm, p < 0.0005; 100 μm, p < 0.0005).

FIGURE 8.

Ursolic acid does not affect internalization of HF488 Aβ by N9 microglia. N9 microglia were incubated with 1 μm HF488 Aβ for 2 h in the presence of 50 μm ursolic acid or volume equivalent of DMSO followed by trypan blue quenching of external fluorescence. Internalized HF488 Aβ was measured by flow cytometry. Data represent mean ± S.E. (error bars). Each data point is the mean of three separate experiments each done in triplicate. n = 3.