Gamma-Secretase Inhibitor Activity of a <b><i>Pterocarpus erinaceus</i></b> Extract

Abstract

Background: Accumulation of β-amyloid peptides (Aβ) and its progressive deposition into amyloid plaques are key events in the aetiology of Alzheimer's disease (AD). To date, AD treatment is symptomatic and consists of drugs treating the cognitive decline. Objective: Identifying molecules specifically targeting Aβ production or aggregation represents a huge challenge in the development of specific AD treatments. Several molecules reported as γ-secretase inhibitors or modulators have been evaluated, but so far none of them have proven to be selective or fully efficient. We have previously investigated the potential interest of plant extracts and we reported that Pterocarpus erinaceus stem-bark extract was active on Aβ release. Our aim here was to characterize the mechanisms by which this extract reduces Aβ levels. Methods: We tested P. erinaceus extract at non-toxic concentrations on cells expressing the human amyloid precursor protein (APP695) or its amyloidogenic β-cleaved C-terminal fragment (C99), as well as on neuronal cell lines. P. erinaceus extract was found to inhibit Aβ release. We further showed that this extract inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that P. erinaceus extract is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit γ-secretase-dependent Notch intracellular domain release. Conclusion: P. erinaceus extract appears as a new potent γ-secretase inhibitor selective towards APP processing.

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

“…Cell medium was then collected, and A␤ was quantified according to the manufacturer's instructions. Two A␤ multiplex assays, 4G8 and 6E10, have been used to measure rodent and human A␤, respectively (54).…”

“…In Vitro ␥-Secretase Activity Assay-A cell-free in vitro ␥-secretase activity assay was applied for measuring the activity of ␥-secretase with a fluoroprobe mimicking the APP ␥-cleavage site (54, 58), performed as previously described (54). All of the steps were carried out at 4°C unless otherwise specified.…”

“…AICD Monitoring in Cell-free Assays-A cell-free assay to monitor AICD production has been applied as described previously (54). All steps were performed at 4°C unless otherwise specified.…”

“…For NICD production, MEFs were transfected 24 h after seeding with the HES1 luciferase reporter gene, Renilla luciferase vector, and Notch⌬E or with the corresponding empty (mock) vector (54). Transfected cells were collected and analyzed by Western blotting, to monitor Notch⌬E expression and processing; NICD release was measured by a luciferase assay (see below).…”

“…1, C and D). A␤ quantification was performed by ECLIA using a specific antibody recognizing human A␤ (6E10) (54). Compared with PS1-WT, the levels of A␤40 and A␤42 released from PS1-AA, PS1-AG, and PS1-DA were reduced (Fig.…”