Abstract Background The brains of patients with Alzheimer’s 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 β (Aβ) 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 Aβ sequence changing the charge and/or hydrophobicity of the targeted amino acids. HEK293T cells were transfected with APP constructs and secreted Aβ peptides were measured using ELISA and mass spectrometry (MS). APP processing in normal and high cholesterol condition, and endocytosis were assessed in stably expressing APP wt and APP K28A HEK293T clones. Finally, we measured the binding of synthetic peptides derived from the Aβ sequence to cholesterol-rich exosomes purified from control HEK293T cells. Results Most mutations triggered a reduction in the production of Aβ40 and Aβ42 peptides, whereas only juxtamembrane mutants resulted in the generation of shorter Aβ peptides. We confirmed by mass spectrometry this specific change in the profile of secreted Aβ peptides for the most characteristic APP K28A mutant. A transient increase of plasma membrane cholesterol enhanced the production of Aβ40 by APP WT , an effect absent with APP K28A . The enzymatic activity of α-, β- and γ-secretases remained unchanged in cells expressing APP K28A . Similarly, APP K28A subcellular localization in early endosomes did not differ to APP WT . Finally, WT but not CBS mutant Aβ 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 γ-secretase cleavage at positions 40-42. The APPK28 mutant would lie deeper in the membrane, facilitating the production of shorter Aβ peptides and unveiling this specific region as a novel target for reducing the production of toxic Aβ species.

Mutations in the juxtamembrane segment of the cholesterol-binding site of APP alter its processing and promotes production of shorter, less toxic Aβ peptides