Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the ß-amyloid peptide (Aß) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aß production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and ß C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aß production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

Conformational changes induced by the A21G Flemish mutation in the amyloid precursor protein lead to increased Aß production.

Proteolysis of the ß C-terminal fragment (ß-CTF) of the amyloid precursor protein generates the Aß peptides associated with Alzheimer's disease. Familial mutations in the ß-CTF, such as the A21G Flemish mutation, can increase Aß secretion. We establish how the Flemish mutation alters the structure of C55, the first 55 residues of the ß-CTF, using FTIR and solid-state NMR spectroscopy. We show that the A21G mutation reduces ß sheet structure of C55 from Leu17 to Ala21, an inhibitory region near the site of the mutation, and increases α-helical structure from Gly25 to Gly29, in a region near the membrane surface and thought to interact with cholesterol. Cholesterol also increases Aß peptide secretion, and we show that the incorporation of cholesterol into model membranes enhances the structural changes induced by the Flemish mutant, suggesting a common link between familial mutations and the cellular environment.