Presenilin transmembrane domain 8 conserved AXXXAXXXG motifs are required for the activity of the γ-secretase complex.

Understanding the molecular mechanisms controlling the physiological and pathological activity of γ-secretase represents a challenging task in Alzheimer disease research. The assembly and proteolytic activity of this enzyme require the correct interaction of the 19 transmembrane domains (TMDs) present in its four subunits, including presenilin (PS1 or PS2), the γ-secretase catalytic core. GXXXG and GXXXG-like motifs are critical for TMDs interactions as well as for protein folding and assembly. The GXXXG motifs on γ-secretase subunits (e.g. APH-1) or on γ-secretase substrates (e.g. APP) are known to be involved in γ-secretase assembly and in Aß peptide production, respectively. We identified on PS1 and PS2 TMD8 two highly conserved AXXXAXXXG motifs. The presence of a mutation causing an inherited form of Alzheimer disease (familial Alzheimer disease) in the PS1 motif suggested their involvement in the physiopathological configuration of the γ-secretase complex. In this study, we targeted the role of these motifs on TMD8 of PSs, focusing on their role in PS assembly and catalytic activity. Each motif was mutated, and the impact on complex assembly, activity, and substrate docking was monitored. Different amino acid substitutions on the same motif resulted in opposite effects on γ-secretase activity, without affecting the assembly or significantly impairing the maturation of the complex. Our data suggest that AXXXAXXXG motifs in PS TMD8 are key determinants for the conformation of the mature γ-secretase complex, participating in the switch between the physiological and pathological functional conformations of the γ-secretase.

Gamma-secretase inhibitor activity of a Pterocarpus erinaceus extract.

BACKGROUND: Accumulation of ß-amyloid peptides (Aß) 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 Aß 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 Aß release. Our aim here was to characterize the mechanisms by which this extract reduces Aß levels. METHODS: We tested P. erinaceus extract at non-toxic concentrations on cells expressing the human amyloid precursor protein (APP695) or its amyloidogenic ß-cleaved C-terminal fragment (C99), as well as on neuronal cell lines. P. erinaceus extract was found to inhibit Aß 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.

Bioprofiling of Salicaceae bud extracts through high-performance thin-layer chromatography hyphenated to biochemical, microbiological and chemical detections.

The buds of poplars (Populus L.) and willows (Salix L.), both from the same family (Salicaceae Mirbel), are increasingly used in gemmotherapy and importantly contribute to the production of the physiologically active propolis by European bee Apis mellifera L. In order to study their phenolic profiles, polar extracts of buds from P. nigra L. were compared to those of P. alba L. and S. alba L. through high-performance thin-layer chromatography (HPTLC). Five chemotypical patterns were distinguished after derivatisation with the Natural Product reagent and confirmed by principal component analysis. The HPTLC analysis was directly hyphenated to various microbiological and biochemical assays as well as spectrometric techniques, directly linking to active molecules in the chromatograms. At a glance, polyvalent compounds were evident when all derivatisation and activity assays, to which HPTLC was hyphenated at ease, were combined together. In Populus buds, at least three antimicrobial compound zones were detected using Aliivibrio fischeri and Bacillus subtilis bioassays, and one phyto-œstrogen with the planar yeast œstrogen screen. In all samples, several inhibitors of acetyl- and butyrylcholinesterase and rabbit liver esterase were detected. Hyphenation to high resolution mass spectrometry supported the assignment of bioactive compounds, as shown for chrysin as selective cholinesterase inhibitor as well as caffeic acid and galangin as antimicrobials in P. nigra and P. alba. This fast and cost-efficient method can be appropriately extended and applied to the botanical origin determination and quality control of bud extracts and propolis samples.

Characterization of Pterocarpus erinaceus kino extract and its gamma-secretase inhibitory properties.

ETHNOPHARMACOLOGICAL RELEVANCE: The aqueous decoction of Pterocarpus erinaceus has been traditionally used in Benin against memory troubles. AIM OF THE STUDY: New strategies are needed against Alzheimer׳s disease (AD), for, to date, AD treatment is symptomatic and consists in drugs treating the cognitive decline. An interesting target is the ß-amyloid peptide (Aß), whose accumulation and progressive deposition into amyloid plaques are key events in AD aetiology. Identifying new and more selective γ-secretase inhibitors or modulators (none of the existing has proven so far to be selective or fully efficient) appears in this respect of particular interest. We studied the activity and mechanisms of action of Pterocarpus erinaceus kino aqueous extract, after the removal of catechic tannins (KAST). METHODS AND RESULTS: We tested KAST at non-toxic concentrations on cells expressing the human Amyloid Precursor Protein (APP695), as well as on primary neurons. Pterocarpus erinaceus extract was found to inhibit Aß release in both models. We further showed that KAST inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that KAST is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit the cleavage of Notch, another γ-secretase substrate responsible for major detrimental side effects observed with γ-secretase inhibitors. Epicatechin was further identified in KAST by HPLC-MS. CONCLUSION: Pterocarpus erinaceus kino extract appears therefore as a new γ-secretase inhibitor selective towards APP processing.

In vitro screening on ß-amyloid peptide production of plants used in traditional medicine for cognitive disorders.

AIM OF THE STUDY: The aim of the study was to investigate the activity on ß-amyloid peptide production of crude extracts of 9 plant species traditionally used in Benin or in Madagascar for the treatment of cognitive disorders, in order to select candidates for Alzheimer's disease treatment. MATERIALS AND METHODS: For each species, hexane, dichloromethane, ethyl-acetate and water extracts were tested, at non-toxic concentrations, on CHO cells overexpressing the human neuronal ß-amyloid peptide precursor (APP695) to measure variations of APP processing (by Western-blotting) and, for the most active, of Aß-amyloid production (by ECLIA). RESULTS: We observed, at non-toxic concentrations, a significant increase in CTF/APP ratio with Oldenlandia affinis cyclotide-enriched fraction, Prosopis africana EtOAc extract, Pterocarpus erinaceus aqueous extract and Trichilia emetica hexane extract. We also showed that the Pterocarpus erinaceus extract significantly decreased Aß production, displaying effects similar to those of DAPT (γ-secretase inhibitor) on APP processing, but may act on another inhibition site. CONCLUSION: These active extracts are worth further studies to isolate the compounds responsible for the observed activities, to analyze their mode of action and determine their clinical potentials.