Figure 7.

Stigmasterol reduces Aβ generation via pleiotropic mechanisms. A, Effect of stigmasterol on β-secretase BACE1. Dark-gray bars, Stigmasterol reduces β-secretase activity directly in purified membranes of SH-SY5Y wt cells and of mouse brains. Light-gray bars, Stigmasterol has no effect on BACE1 expression. Sterol concentration used and analysis of secretase activities and BACE1 expression as described for Figure 3A. B, Influence of stigmasterol on γ-secretase. Dark-gray bars, Stigmasterol does not affect γ-secretase activity directly in purified membranes of SH-SY5Y wt cells and of mouse brains. Light-gray bars, left, Stigmasterol significantly decreases gene expression of all γ-secretase components, PS1, PS2, APH1A, APH1B, PEN2, and NCSTN, resulting in a reduced PS1 protein level. Sterol concentration, analysis of gene transcription, and PS1 protein level as described for Figure 3B. Light gray bar, right, Influence of stigmasterol on secreted Aβ levels in SH-SY5Y cells stably expressing SPC99. SPC99-expressing SH-SY5Y cells were incubated with 10 μm stigmasterol or solvent control. Secreted Aβ levels were immunoprecipitated with antibody W02, and WB analysis was performed with antibody W02. C, Biotinylation of cell surface localized proteins in presence of stigmasterol. SH-SY5Y wt cells were incubated with 10 μm stigmasterol or solvent control, scraped off, and biotinylation was performed as described in Material and Methods. Biotinylated proteins were immunoprecipitated using streptavidin agarose. WB analysis of biotinylated APP was performed with antibody W02, WB analysis of biotinylated BACE1 with antibody B0806. Cell-lysates were analyzed for total protein level of EEA1 of stigmasterol-treated cells or control cells. WB analysis was performed with antibody ab2900. D, Stigmasterol reduces BACE1 internalization. Stigmasterol-treated cells (10 μm) and control cells were fractionated using OptiPrep density gradient centrifugation. Collected fractions were loaded and separated on 10–20% Tris-Tricine gels. WB analysis was performed with anti-EEA1 antibody. EEA1-positive fractions were pooled and analyzed for BACE1 protein level as described for Figure 3A. β-CTF was detected using antibody W02 for WB analysis. EEA1-positive fractions were also analyzed for β-secretase activity. E, Stigmasterol affects cholesterol level, γ-secretase activity, and PS1 protein level in lipid rafts. Stigmasterol-treated cells (10 μm) and control cells were subjected to buoyant sucrose density centrifugation and 16 fractions were collected. Fractions were loaded, separated on 10–20% Tris-Tricine gels and subjected to WB analysis. For the detection of raft-positive gradient fractions, the antibody anti-flotillin 610821 was used, for the detection of nonraft fractions the antibody anti-cadherin ab6528. Determination of cholesterol level, γ-secretase activity, and PS1 protein level in raft and nonraft membrane microdomains. For the determination of the cholesterol content, SH-SY5Y wt cells were pulsed with ¹⁴C-cholesterol and afterward incubated with 10 μm stigmasterol as described in Materials and Methods. Cholesterol levels were determined by liquid scintillation counting in a Tri-Carb2800TR, γ-secretase activity via the fluorescent γ-secretase assay as described in Material and Methods. PS1 protein level was detected using antibody sc7860. A–E, Representative WBs for protein analysis experiments are shown. Illustration and statistical significance as described for Figure 2; n ≥ 4. F, Stigmasterol distribution to raft and nonraft microdomains. Pooled raft/nonraft samples were subjected to derivatization and mass spectrometry as described in Materials and Methods to determine stigmasterol content in raft and nonraft membrane microdomains.