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P1‐133: Aberrant Micro‐Membrane Localization of γ ‐Secretase Components by Changes in Cellular Cholesterol Level Alters E‐ and/or γ ‐Cleavage of App and Alcadeinα
Author(s) -
Hu Anqi,
Piao Yi,
Hata Saori,
Suzuki Toshiharu
Publication year - 2016
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2016.06.882
Subject(s) - lipid raft , cleavage (geology) , cholesterol , presenilin , chemistry , microbiology and biotechnology , in vitro , amyloid precursor protein secretase , cell , membrane , hek 293 cells , biochemistry , amyloid precursor protein , biology , gene , medicine , alzheimer's disease , paleontology , disease , fracture (geology)
Background: Attenuated g-secretase activity, which is independent on mutations of presenilin (PS) genes, is a possible primary cause of the generation of neurotoxic Ab42 in sporadic AD, but its molecular mechanisms remain unclear. Cleavages of substrate by g-secretase occur in membrane microdomains called lipid rafts, which are enriched in cholesterol. In this study, we investigated whether changes in the cellular lipid composition result in altered cleavage of substrates by g-secretase. Methods: Using two g-secretase substrates, APP and Alcadeina (Alca), we first explored the substrate cleavages in cells which were changed cholesterol levels by treatment with cholesterol-depleted and -saturated MbCD. HEK293 cells expressing APP CTFb (C99)-FLAG and AlcaDC-FLAG or AlcaCTF were treated with MbCD. The g-cleaved products, Ab from APP CTFb and p3-Alca from AlcaCTF in medium, and the ε-cleaved product, AICD-FLAG from APP CTFb and AlcaICD-FLAG from Alca-DC-FLAG in cell, were quantified with sELISA and IP-TOF/MS. Second, the cell membrane was fractionated into DRM and non-DRM to perform in vitro gsecretase assay to examine εand g-cleavages of substrates. Results: In APP, gand ε-cleavages didn’t show substantial changes in response to altered cholesterol level of cells, while those of Alca remarkably changed, indicating that the g-secretase activity alters in cell depending on cholesterol levels. This idea was confirmed by in vitro g-secretase assay. APP g-cleavage was altered in DRM of cells with up-regulated cholesterol level, and ε-cleavage also changed in both DRM and non-DRM. These changes of g-secretase activity are likely to correlate with altered microlocalization of active g-secretase components in DRM and non-DRM. Conclusions: Altered cellular cholesterol level influences the microlocalization of active g-secretase components in membrane. This may be a cause to alter g-secretase activity, which contributes the increased production of neurotoxic Ab42. The mechanism is considered as a possible cause of sporadic AD without causative genes’ mutation.