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Pathological amino acid substitutions in the amyloid precursor protein (APP) and chemical ? secretase modulators affect the processing of APP by the ? secretase complex and the production of the amyloid beta peptide Aß42 the accumulation of which is considered causative of Alzheimer's disease. Here we demonstrate that mutations in the transmembrane domain of APP causing aggressive early onset familial Alzheimer's disease affect both ? and e cleavage sites by raising the Aß42/40 ratio and inhibiting the production of AICD50 99 one of the two physiological APP intracellular domains (ICDs). This is in sharp contrast to ? secretase modulators which shift Aß42 production towards the shorter Aß38 but unequivocally spare the e site and APP and Notch ICDs production. Molecular simulations suggest that familial Alzheimer's disease mutations modulate the flexibility of the APP transmembrane domain and the presentation of its ? site modifying at the same time the solvation of the e site. © 2009 2012 IEEE.

BACKGROUND: Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1-42/Aβ1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1-42 production, decreasing Aβ1-40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1-40, Aβ1-42 and APP intracellular domain productions in vitro. CONCLUSION/SIGNIFICANCE: Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1-42/Aβ1-40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease.

The toxic amyloid-β (Aβ) peptides involved in Alzheimer's disease (AD) are produced after processing of the amyloid precursor protein-C-terminal fragment APP-C99 by γ-secretase. Thus, major therapeutic efforts have been focused on inhibiting the activity of this enzyme. However, preclinical and clinical trials testing γ-secretase inhibitors revealed adverse side effects most likely attributed to impaired processing of the Notch-1 receptor, a γ-secretase substrate critically involved in cell fate decisions. Here we report an innovative approach to selectively target the γ-secretase-mediated processing of APP-C99 with monoclonal antibodies neutralizing this substrate. Generated by immunizing mice with natively folded APP-C99, these antibodies bound N- or C-terminal accessible epitopes of this substrate, and decorated extracellular amyloid deposits in AD brain tissues. In cell-based assays, the same antibodies impaired APP-C99 processing by γ-secretase, and reduced Aβ production. Furthermore, they significantly decreased brain Aβ levels in the APPPS1 mouse model of AD after intracerebroventricular injection. Together, our findings support APP-C99 substrate-targeting antibodies as new immunotherapeutic and Notch-sparing agents to lower the levels of Aβ peptides implicated in AD.