BACE-1 inhibition prevents the γ-secretase inhibitor evoked Aβ rise in human neuroblastoma SH-SY5Y cells

Abstract

BackgroundAccumulation of amyloid β-peptide (Aβ) in the plaques is one of the major pathological features in Alzheimer's disease (AD). Sequential cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE-1) and γ-secretase results in the formation of Aβ peptides. Preventing Aβ formation is believed to attenuate AD progression and BACE-1 and γ-secretase are thus attractive targets for AD drug development.MethodsCombining BACE-1 and γ-secretase inhibition on Aβ secretion from human neuroblastoma SH-SY5Y cells was evaluated in this study. Secreted Aβ40 and Aβ42 levels were measured from SH-SY5Y cells stably transfected with APPwt or APPswe genes. A selective BACE inhibitor and the γ-secretase inhibitor LY450139 (semagacestat) were used to inhibit respective secretase.ResultsLY450139 increased Aβ40 and Aβ42 secretion from SH-SY5Y APPwt cells at low concentrations (by 60% at 3 nM) followed by subsequent inhibition at higher concentrations (IC50 90 nM). Washout studies showed that the Aβ increase evoked by 3 nM LY450139 was not due to enhanced cleavage following substrate accumulation but rather to activation of Aβ formation. By contrast, LY450139 inhibited Aβ formation from SH-SY5Y APPswe in a monophasic manner (IC50 18 nM). The BACE inhibitor per se inhibited Aβ secretion from both SH-SY5Y APPwt and SH-SY5Y APPswe cells with IC50s ranging between 7 - 18 nM and also prevented the increased Aβ secretion evoked by 3 nM LY450139. Combining the BACE inhibitor with higher inhibitory concentrations of LY450139 failed to demonstrate any clear additive or synergistic effects.ConclusionBACE-1 inhibition attenuates the Aβ increase evoked by LY450139 while not providing any obvious synergistic effects on LY450139-mediated inhibition.

Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons

“…The α-secretase ADAM-10 is known to be associated with exosomes [40,57] and further studies should test if CTF-η inserted in exosomes released by dendrites can give rise to toxic Aη in the synaptic vicinity. Aβ peptides were enriched in exosomes compared to cells, and as expected from previous work [58][59][60], the level of amyloid peptides tended to be higher in APPswe than in APPwt-expressing N2a cells. Accordingly, there was nearly five times more Aβ40 in exosomes from APPswe-than in those of APPwtexpressing N2a (Fig.…”

Interplay between α-, β-, and γ-Secretases Determines Biphasic Amyloid-β Protein Level in the Presence of a γ-Secretase Inhibitor

“…That γ-secretase inhibition can lead to an increase in the production rate of its Aβ product has been reported in several in vitro and in vivo systems (12, 13, 21–23, 27). However, no mechanism has been proposed to explain the origin of this paradoxical behavior by only inhibiting this enzyme.…”

“…Here, we demonstrate that the model can quantitatively recapitulate in vitro data. It has been reported that dose-response curves of Aβ 1–40 /Aβ 1–42 against GSIs have two different behaviors depending on the cell type where the compounds are tested (13, 17, 21, 29). For example, it has been shown that in HEK cell lines stably transfected with wild-type APP (HEK APPwt), a biphasic response in the levels of Aβ occurs on treatment with increasing concentrations of GSI.…”

“…Semagacestat in vitro dose-response data from Jamsa et al (21) were used to fit the in vitro model, whereas the pharmacokinetic model was fitted using the concentration of the drug in plasma available from the trial data (supplemental Section 4 supplemental Fig. S2 and supplemental Tables S2 and S3).…”

“…As we expect, the addition of inhibitors of γ-secretase in cell-free assays produces a monotonic decrease in the production of Aβ (13, 16–20). However, in vitro concentration-response curves for a wide range of inhibitors show two types of behaviors consistently depending on the cell line used (13, 21). In some cell lines, the Aβ production decreases with inhibitor concentration as for the cell-free assay.…”