Specificity of presenilin-1- and presenilin-2-dependent c-secretases towards substrate processing Serena Stanga a, # ,C  eline Vrancx a, # , Bernadette Tasiaux a , Claudia Marinangeli b , Helena Karlstr € om c , Pascal Kienlen-Campard a, * a Alzheimer Research group, Institute of Neuroscience, Universit  e catholique de Louvain, Brussels, Belgium b Inserm, CHU Lille, UMR-S 1172 – JPArc – Centre de Recherche Jean-Pierre AUBERT, University of Lille, Lille, France c Center for Alzheimer Research, Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Stockholm, Sweden Received: March 24, 2017; Accepted: July 27, 2017 Abstract The two presenilin-1 (PS1) and presenilin-2 (PS2) homologs are the catalytic core of the c-secretase complex, which has a major role in cell fate decision and Alzheimer’s disease (AD) progression. Understanding the precise contribution of PS1- and PS2-dependent c-secretases to the production of b-amyloid peptide (Ab) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Ab release without affecting the processing of other c-secretase substrates. To that end, we studied PS1- and PS2-dependent sub- strate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1-PS2 double-KO noted PSdKO) or stably re-expressing human PS1 or PS2 in an endogenous PS-null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L-685,458). We found that murine PS1 c-secretase plays a predominant role in APP and Notch processing when compared to murine PS2 c-secretase. The inhi- bitors blocked more efficiently murine PS2- than murine PS1-dependent processing. Human PSs, especially human PS1, expression in a PS- null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1- than human PS2-dependent c-secretase activity. Keywords: presenilins  c-secretase complex  APP  Notch  reporter gene assay  c-secretase inhibitors  Alzheimer’s disease Introduction Presenilins 1 and 2 are homologous polytopic proteins that represent the catalytic subunits of the c-secretase complex. The assembly and activation of the c-secretase is achieved by the association of either PS1 or PS2 [1] with three other membrane proteins: nicastrin (Nct), anterior pharynx defective 1 (Aph1a or Aph1b in humans; Aph1a, Aph1b or Aph1c in rodents [2]) and presenilin enhancer 2 (Pen-2), giving rise to four and six different c-secretases in humans and rodents, respectively. The c-secretase is responsible for the cleavage of up to 90 type I integral membrane proteins [3, 4] that have previ- ously undergone an ectodomain shedding by processing at a cleavage site close to their transmembrane domain [5]. This results in the gen- eration of a membrane-anchored C-terminal fragment (CTF), which is subsequently cleaved by the c-secretase within its transmembrane domain to generate a truncated membrane stub and a soluble intra- cellular domain (ICD). The APP and the Notch receptor are the pre- dominant substrates of c-secretase (PSs) [6]. Their processing is of critical importance in AD aetiology and in cell proliferation, develop- ment and tissue homoeostasis. The onset and progression of AD relies on the release of various Ab generated by the c-secretase cleav- age of APP [7]. Ab is the main component of the senile plaques found in brains of AD patients [8]. Notch signalling is triggered by the c-secretase release of its intracellular domain (NICD) that exerts essential functions during development [9]. Much attention in AD research has focused on the c-secretase- mediated cleavage of the amyloidogenic CTF of APP (b-CTF or C99) that generates the intracellular domain of the protein (AICD) and the Ab peptide [10]. To block amyloid deposition and the formation of senile plaques, many pharmacological approaches have been designed to target the c-secretase activity, but clinical trials of c-secretase # These authors contributed equally. *Correspondence to: Pascal KIENLEN-CAMPARD E-mail: pascal.kienlen-campard@uclouvain.be ª 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. doi: 10.1111/jcmm.13364 J. Cell. Mol. Med. Vol XX, No X, 2017 pp. 1-11