Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis.
Amyloid peptide (Aβ) is generated by sequential cleavage of the amyloid precursor protein (APP) by β-secretase (Bace1) and γ-secretase. Aβ production increases after plasma membrane cholesterol loading through unknown mechanisms. To determine how APP-Bace1 proximity affects this phenomenon, we developed a fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET) technique for visualization of these molecules either by epifluorescence or at the plasma membrane only using total internal reflection fluorescence. Further, we used fluorescence correlation spectroscopy to determine the lipid rafts partition of APP-yellow fluorescent protein (YFP) and Bace1-green fluorescent protein (GFP) molecules at the plasma membrane of neurons. We show that less than 10 min after cholesterol exposure, Bace1-GFP/APP-mCherry proximity increases selectively at the membrane and APP relocalizes to raft domains, preceded by rapid endocytosis. After longer cholesterol exposures, APP and Bace1 are found in proximity intracellularly. We demonstrate that cholesterol loading does not increase Aβ production by having a direct impact on Bace1 catalytic activity but rather by altering the accessibility of Bace1 to its substrate, APP. This change in accessibility is mediated by clustering in lipid rafts, followed by rapid endocytosis.
Centre de Recherche de l'Institut du Cerveau et de la Moelle, UPMC, INSERM UMR-S975, Centre National de la Recherche Scientifique (CNRS) UMR7225, Hôpital de la Pitié-Salpêtrière, Paris, France.
Catherine
C
Marquer
Viviane
V
Devauges
Jack-Christophe
JC
Cossec
Géraldine
G
Liot
Sandrine
S
Lécart
Frédéric
F
Saudou
Charles
C
Duyckaerts
Sandrine
S
Lévêque-Fort
Marie-Claude
MC
Potier
D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000378:metabolism
C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D002784:Cholesterol; D053829:Amyloid Precursor Protein Secretases