Specific Mutations in the Cholesterol-Binding Site of APP Alter Its Processing and Favor the Production of Shorter, Less Toxic Aβ Peptides

Abstract

Excess brain cholesterol is strongly implicated in the pathogenesis of Alzheimer’s disease (AD). Here we evaluated how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of the amyloid precursor protein (APP) regulates its processing. We generated nine point mutations in the APP gene, changing the charge and/or hydrophobicity of the amino-acids which were previously shown as part of the CBS. Most mutations triggered a reduction of amyloid-β peptides Aβ40 and Aβ42 secretion from transiently transfected HEK293T cells. Only the mutations at position 28 of Aβ in the APP sequence resulted in a concomitant significant increase in the production of shorter Aβ peptides. Mass spectrometry (MS) confirmed the predominance of Aβx-33 and Aβx-34 with the APPK28A mutant. The enzymatic activity of α-, β-, and γ-secretases remained unchanged in cells expressing all mutants. Similarly, subcellular localization of the mutants in early endosomes did not differ from the APPWT protein. A transient increase of plasma membrane cholesterol enhanced the production of Aβ40 and Aβ42 by APPWT, an effect absent in APPK28A mutant. Finally, WT but not CBS mutant Aβ derived peptides bound to cholesterol-rich exosomes. Collectively, the present data revealed a major role of juxtamembrane amino acids of the APP CBS in modulating the production of toxic Aβ species. More generally, they underpin the role of cholesterol in the pathophysiology of AD.

White matter injury, cholesterol dysmetabolism, and APP/Abeta dysmetabolism interact to produce Alzheimer’s disease (AD) neuropathology: A hypothesis and review

“…Cholesterol and APOE regulate APP cleavage (Howland et al, 1998;Mills and Reiner, 1999). Mutations in the APP cholesterol-binding site alter APP processing to form less toxic Aβ peptides (Hanbouch et al, 2022). Free cholesterol fosters Aβ self-assembly on membranes (Hashemi et al, 2022), likely along with degraded myelin basic protein as noted below (Zhan et al, 2018).…”

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content

“…As the interaction of different APP fragments with cholesterol also seems to be an important factor in its processing [ 7 , 27 , 28 , 30 , 38 , 39 , 40 ], we decided to investigate if it would be possible to detect any difference in APP clusterization and APP-induced changes in a membrane structure for different concentrations of cholesterol in the lipid bilayer. The set of corresponding topographies is displayed in Figure 3 .…”

“…As for the APP, its fragment APP 672–770 , often referred to as C99, was also studied to be a cholesterol-binding site [ 27 ], especially the loop region connecting its juxtamembrane helix and N-terminal part of transmembrane helix [ 26 , 28 , 29 ]. It was later shown that indeed different mutations in the transmembrane domain of APP significantly affect cholesterol binding [ 30 ].…”

Advances in Amyloid-β Clearance in the Brain and Periphery: Implications for Neurodegenerative Diseases

“…Certain genes and molecular pathways linked to the mechanisms of neurodegenerative diseases modify their expression with progressing skin aging, possibly due to the shared ectodermal origin of the skin and nervous system [ 313 ]. BACE1 (β-secretase 1), an enzyme that degrades amyloid-β 1-40 and amyloid-β 1-42 , generates a shorter amyloid-β 1-34 peptide [ 314 ], which has been identified in human skin and could serve as a possible marker of amyloid-β degradation [ 315 ]. Additionally, a study showed that the majority of the 125 I-labeled amyloid-β 1-40 was detected in the skin with only a minor amount in the brain, demonstrating that amyloid-β produced in the brain can be cleared in the periphery [ 232 ].…”