Alzheimer’s Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified c-Secretase Complexes Matthias Cacquevel, Lore` ne Aeschbach, Jemila Houacine, Patrick C. Fraering* E´cole Polytechnique Fe´de´rale de Lausanne, Brain Mind Institute, Laboratory of Molecular and Cellular Biology of Alzheimer’s Disease, Lausanne, Switzerland Abstract 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 c-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Ab) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Ab production by enhancing the Ab1–42/Ab1–40 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 Ab1–42 production, decreasing Ab1–40 production, or both. Because it could be explained by the heterogeneity in the composition of c-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, DE9, 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 c-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified c-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of c-secretase activity phenotype, in terms of Ab1–40, Ab1–42 and APP intracellular domain productions in vitro. Conclusion/Significance: Our data support the view that PS1 mutations lead to a strong c-secretase loss-of-function phenotype and an increased Ab1–42/Ab1–40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer’s disease. Citation: Cacquevel M, Aeschbach L, Houacine J, Fraering PC (2012) Alzheimer’s Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified c-Secretase Complexes. PLoS ONE 7(4): e35133. doi:10.1371/journal.pone.0035133 Editor: Stefano L. Sensi, University G. D’Annunzio, Italy Received February 20, 2012; Accepted March 13, 2012; Published April 18, 2012 Copyright: 2012 Cacquevel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Swiss National Foundation (to and LA and PCF; grant 31003A_134938/1) and the National Centre for Competence in Research (‘‘Neuronal Plasticity and Repair’’ (to JH and PCF)), and the Strauss foundation (to PCF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: patrick.fraering@epfl.ch Introduction Since their discovery in 1995 and their association with early onset familial Alzheimer’s disease (FAD) [1,2], the presenilin genes PSEN1 and PSEN2 have been widely studied, and the complexity of their biological role is becoming increasingly evident. PSEN1 and PSEN2 encode transmembrane proteins PS1 and PS2, respectively, that constitute the catalytic core of c-secretase, the founding member of an emerging class of unconventional, Intramembrane-Cleaving Proteases (I-CLiPs) [3]. Active c-secre- tase is a multiprotein complex composed of PS1 or PS2 together with nicastrin (NCT), the anterior pharynx-defective protein 1 (APH1), and the presenilin enhancer 2 (PEN2). Experimental evidence such as the binding of transition-state analogue c- secretase inhibitors to PS1 [4], as well as the abolishment of c- secretase activity when PS1 lacks the aspartate residues critical for proteolysis [4,5,6], have confirmed that presenilins harbour the active site of the enzymatic complex. PS1 and PS2 play fundamental roles in cell signalling as part of the c-secretase complex. The latter cleaves numerous type-I membrane proteins in their transmembrane domain [7], releasing their corresponding intracellular domains, which are capable of influencing gene expression. For some substrates like NOTCH1 or ERBB4, the c-secretase cleavage is essential for their biological function, whereas for other substrates like DCC or NEUREXIN- 3b [8], the possible role of the cleavage in signalling events has not been formally established (see [9] for a review). The amyloid precursor protein (APP) is processed by the successive actions of b- secretase (BACE1) and c-secretase, generating amyloid-beta peptides (Ab) of different lengths, ranging from 37 to 46 amino acids [10]. Cleavage of the APP C-terminal fragments (APP-CTFs) by c-secretase also releases the APP intracellular domain (AICD), which has been recently involved in the regulation of brain ApoE expression, a major genetic determinant of AD, and in cholesterol metabolism [11]. In addition, PS1 has been shown to interact with a growing list of proteins that modulate c-secretase activity [9,12,13,14]. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35133