FIGURE 1.

RO-02, a novel non-acidic GSM, lowers Aβ₄₀ and Aβ₄₂ generation with high potency. A, structure of RO-02. B, dose-response analysis of γ-secretase activity modulation by RO-02 in HEK293/sw cells by Aβ sandwich immunoassay. Data are represented as mean ± S.E. (n = 3). Note that for most data points, error bars are too small to be displayed. C, RO-02 dose-response analysis of NICD and AICD formation in HEK293 cells stably co-expressing F-NEXT and C99-6myc as assessed by immunoblot analysis. Quantitation of NICD and AICD formation is shown in the right panel. Data are represented as mean ± S.E. (RO-02, n = 3–5; DAPT (1 μm), n = 2). veh., vehicle. D–F, RO-02 modulates the activity of all six γ-secretase complexes. D, immunoblot analysis of expression of γ-secretase complex components in HEK293/sw cells stably expressing the individual combinations of PS and APH-1 with calnexin as loading control. NCT, nicastrin; m., mature; im., immature; S, short splice variant; L, long splice variant. Profile of secreted Aβ species (E) and Aβ₄₂ response to RO-02 (F) of the six γ-secretase complexes as determined by Aβ sandwich immunoassay. Data in E and F are represented as mean ± S.E. (n = 3). Asterisks indicate significant differences between corresponding PS1- and PS2-containing complexes for their relative Aβ₄₂ generation in the presence of RO-02 (*, p < 0.05; **, p < 0.01; ***, p < 0.001, two-tailed unpaired Student's t test). G, RO-02 dose-response analysis of Aβ₄₀ and Aβ₄₂ generation in cultured rat primary cortical neurons by Aβ ELISA. Data are represented as mean ± S.E. (n = 2). H, RO-02 dose-response analysis in a cell-free assay using purified γ-secretase by immunoblot analysis of Aβ species separated by Tris-Bicine urea SDS-PAGE and total Aβ and AICD. I, mass spectrometry analysis of AICD species generated in the presence or absence (vehicle) of RO-02 (1 μm).