Fig. 1
From: Cholestenoic acid, an endogenous cholesterol metabolite, is a potent γ-secretase modulator
γ-Secretase modulatory effect of CA. a Chemical structure of CA with EC50 and EC90 for decreasing Aβ42 is illustrated. b Dose response curves of CA for Aβ42, Aβ38, and total Aβ in CHO-2B7 cells are shown. The Aβ levels in the conditioned media of the cells treated with CA at nine dose points for 16 h were measured by Aβ specific sandwich ELISAs. Aβ42 (red line) levels increase with the concentration, whereas Aβ38 (green line) levels decrease with the concentration. Total Aβ (black line) levels did not show significant changes. c Aβ spectra are illustrated by mass spectrometry after CA treatment at 3 μM in CHO-2B7 cells. Control refers to the conditioned media treated with DMSO in the cells, a solvent for CA. CA treatment at 3 μM increased Aβ38 peak and decreased Aβ42 peak with no significant changes in Aβ40 peak compared to the DMSO control. Identified Aβ peptides are indicated above the peaks. d In vitro γ-secretase assays show the direct effect of CA in γ-secretase modulation analyzed by ELISAs. Cmpd E is an irreversible pan γ-secretase inhibitor, which limits γ-secretase activity at the starting time point of the assay. The total γ-secretase activity was measured after 2 h of DMSO (solvent control) and CA incubation. Compared to the control, CA at 150 μM decreased Aβ42 by 50 % (n = 2 per group, repeated 2–3 times). e Aβ spectra obtained from MALDI-TOF mass spectrometry studies show that 20 μM CA from in vitro study decrease Aβ42 peak and increase Aβ38 peak compared to the DMSO treated control group. f For AICD spectra, AICD49-99 and AICD50-99 are presented as the dominant isoforms in both DMSO control and 20 μM CA treated groups. g-i The effects of CA as a GSM are shown in primary neuron-glia culture (n = 6). Mouse endogenous Aβ (mAβ) levels were measured by sandwich ELISAs. Cmpd E (γ-secretase inhibitor) decreased overall Aβ production. GSM-1, an acidic type GSM, was used as a positive control. For the primary neuron-glia culture, GSM-1 at 1 μM decreased level of mAβ42, but increased mAβ38 level. CA at 3 μM and 10 μM presented dose-dependent effects for decreasing mAβ42 and increasing mAβ38. Results were analyzed by two-way analysis of variance (ANOVA) followed by bonferroni post-hoc testing for group differences (Fig. 1d) and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons (Fig. 1g-i). (***p < 0.001, **p < 0.01, *p < 0.5)