Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384891v1?rss=1 Authors: Hanbouch, L., Schaack, B., Kasri, A., Fontaine, G., Gkanatsiou, E., Brinkmalm, G., Portelius, E., Blennow, K., Mourier, G., Gilles, N., Millan, M. J., Marquer, C., Zetterberg, H., Boussicault, L., Potier, M.-C. Abstract: Background: The brains of patients with Alzheimer disease (AD) reveal increased cellular membrane levels of cholesterol. Correspondingly, we previously showed that elevating levels of membrane cholesterol in neuronal cultures recapitulates early AD phenotypes including excessive cleavage of amyloid beta peptides from the amyloid precursor protein (APP). Here we aimed to evaluate how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of APP regulates its processing. Methods: We generated seven single and two double APP mutants at amino acid positions 22, 26, 28, 29, 33, 39 of the Abeta sequence changing the charge and/or hydrophobicity of the targeted amino acids. HEK293T cells were transfected with APP constructs and secreted Abeta peptides were measured using ELISA and mass spectrometry (MS). APP processing in normal and high cholesterol condition, and endocytosis were assessed in stably expressing APPwt and APPK28A HEK293T clones. Finally, we measured the binding of synthetic peptides derived from the Abeta sequence to cholesterol-rich exosomes purified from control HEK293T cells. Results: Most mutations triggered a reduction in the production of Abeta40 and Abeta42 peptides, whereas only juxtamembrane mutants resulted in the generation of shorter Abeta peptides. We confirmed by mass spectrometry this specific change in the profile of secreted Abeta peptides for the most characteristic APPK28A mutant. A transient increase of plasma membrane cholesterol enhanced the production of Abeta40 by APPWT, an effect absent with APPK28A. The enzymatic activity of alpha, beta and gamma-secretases remained unchanged in cells expressing APPK28A. Similarly, APPK28A subcellular localization in early endosomes did not differ to APPWT. Finally, WT but not CBS mutant Abeta derived peptides bound to cholesterol-rich exosomes. Conclusions: Taken together, these data reveal a major role of the juxtamembrane region of APP in binding to cholesterol and accordingly in the regulation of APP processing. Binding of cholesterol to K28 could staple APP to the juxtamembrane region thereby permitting access to gamma-secretase cleavage at positions 40-42. The APPK28 mutant would lie deeper in the membrane, facilitating the production of shorter Abeta peptides and unveiling this specific region as a novel target for reducing the production of toxic Abeta species. Copy rights belong to original authors. Visit the link for more info