In a pathological context, 185 missense mutations in PSEN1 and 13 mutations in PSEN2 have been identified and found to be associated with FAD (www.molgen.ua.ac.be/ADMutations). It was initially suggested that such mutations lead to a toxic gain of function because they are associated with a relative increased production of longer and more hydrophobic Ab species, mainly Ab1–42, that accumulate and aggregate early in the course of the disease [15,16]. Similar phenotypes have been observed by independent groups in cell-based studies performed with different mammalian cell lines [17,18,19,20,21,22]. Despite this growing consensus, the above-mentioned studies provided conflicting results regarding the relative proportions of Ab42, Ab40, and AICD resulting from the PSEN mutations. On one hand, a gain- of-function phenotype was suggested due to the observed increase in Ab42 production, accompanied or not with reduced Ab40 production, thereby leading to an increased Ab42/Ab40 ratio. On the other hand, reduced AICD production suggested a loss-of- function phenotype [20]. Differences in the methodologies and cell types used to assess the effects of PSEN mutations on Ab and AICD productions can explain this controversy as to how PS mutations exert their effects. For example, endogenous PS has been suggested to affect such analysis, as the FAD-linked PSEN1 mutations led to a decrease in the total amount of Ab generated in PS1 and PS2 double knockout cells [23]. Next, it has been reported that c-secretase complexes are heterogeneous in composition (with two PS genes and three APH1 isoforms, six combinations of c-secretase are possible), with distinct functional properties influencing the relative amount of Ab species generated [24]. In order to investigate the biochemical and functional properties of c-secretase in cell-free systems, we and others have recently purified this enzymatic complex to homogeneity [25,26,27]. In particular, protocols for the high-grade purification of proteolytically active c-secretase constituted of NCT, APH1aL, PEN2 and wild-type PS1 [26,27] allowed the reconstitution of 3D structures at 15 A˚ and 12 A˚ resolution by EM and cryo-EM and gave new insights on the structure and activity of the enzyme [28,29]. However, these studies were exclusively focused on the wild-type PS1. Here, we report for the first time the functional characterization of highly purified and homogenous human c- secretase particles carrying different FAD-linked PS1 mutants. Our biochemical and functional findings strongly suggest that pathogenic mutations in PSEN1 cause a loss of c-secretase activity. Results Generation, selection and characterization of stable cell lines overexpressing c-secretase complexes with FAD- linked PS1 mutants In order to facilitate high-grade purification of homogenous human c-secretase complexes containing different PS1 variants, we took advantage of a previously generated mouse embryonic fibroblast (MEF) cell line that lacks the two presenilins (PS1/22/2) [30,31]. Our global strategy consisted in producing stable cell lines, on a PS1/22/2 background, that overexpress tagged versions of the three human c-secretase subunits NCT, APH1 and PEN2 together with different variants of human PS1, and to purify the different human c-secretase complexes by three sequential affinity purification steps. This strategy allowed us to exclude a possible co-purification of mouse c-secretase compo- nents. First, a parental cell line was generated by co-transducing lentiviral vectors of human NCT-V5 (hNCT-V5), human APH1aL-HA (hAPH1aL-HA) and human Flag-PEN2 (Flag- hPEN2) into MEF PS1/22/2. Lentivectors are stably integrated into the cell genome and allow the generation of cell lines Purified c-Secretase Complexes with PS1 Mutations overexpressing multiple genes in a short period of time. Next, the clone expressing the highest levels of all three recombinant proteins was selected (designated c - PS1/2) and used as the parental cell line that was further transduced with lentivectors carrying the following different human PS1 variants: PS1-WT, three dominant-negative forms of PS1 that lack the crucial, catalytic site aspartate residues (D257A, D385A, and both D257A/D385A referred to as DDAA later in the article), and three FAD-linked PS1 mutants (L166P, DE9 and P436Q, with mean disease onset at 24 years, 45.5 years and 28.3 years, respectively). For each PS1 variant, two individual clones were selected based on the expression levels of the recombinant proteins, and extensively characterized (Figure 1). To distinguish those cell lines overexpressing all c-sectease components from wild-type MEFs, they are collectively designated c-MEFs. As shown in Figure 1A, the analysis of total protein extracts confirmed that PS is required for the maturation of NCT and the stability of PEN2 [23,32]. As expected, and when compared to the parental c- PS1/2 cell line, higher levels of the mature form of NCT were observed in all PS1- overexpressing clones. In contrast to PEN2, APH1aL levels were relatively similar in all PS1 clones and the parental cell line. Indeed, and as previously reported [23,32], PEN2 levels were higher in the clones overexpressing WT PS1 and FAD-linked PS1 mutants, and to a lesser extent in the clones overexpressing the dominant negative forms of PS1. Interestingly, PEN2 migrated on the gel as an apparent double band, possibly reflecting a post- translational modification. Different patterns of PS1 expression and autoproteolysis were observed in the different clones. First, and in sharp contrast to the other clones, those expressing PS1- WT or PS1-L166P displayed higher levels of PS1-NTF and PS1- CTF fragments compared with full-length PS1 (PS1-FL; Figure 1A). As estimated by densitometry, the NTF fragments of PS1-WT and PS1-L166P account for 8762% and 7967% of total PS1 (PS1-NTF+PS1-FL), respectively. Next, the PS1-DE9 clones did not show any detectable NTF or CTF fragments, as previously described [33], while P436Q and the three dominant negative variants of PS1 were characterized by low levels of PS1-NTF and PS1-CTF fragments, suggesting that these variants were less prone to endoproteolysis. We next investigated the effects of PSEN1 mutations on the processing of endogenous APP. As expected, and as indicated by increased levels of intracellular APP-CTFs (a common character- istic among known substrates in response to c-secretase pharma- cological inhibition), the activity of c-secretase was significantly altered in the PS deficient parental c - PS1/2 cell line as well as in all clones expressing dominant negative forms of PS1 (Figure 1B). In the clones expressing PS1-WT, APP-CTFs were almost undetectable due to high c-secretase activity, while clones expressing PS1-L166P or PS1-P436Q displayed APP-CTF accumulation. In PS1-DE9 clones, a mild APP-CTF accumulation was observed (Figure 1B). Although a poor recovery of c-secretase activity for the PS1-L166P and a comparatively better perfor- mance of PS1-DE9 have been reported by others under similar experimental conditions [23], the potential of PS1-P436Q mutation to restore the processing of endogenous APP-CTFs has never been assessed [34,35]. Importantly, all observations described above for the cells overexpressing simultaneously hNCT-V5, hAPH1aL-HA, Flag-hPEN2 and the different PS1 variants are very consistent with those made in MEF PS1/22/2 cells overexpressing only the PS1 variants, in the absence of the other c-secretase components (cf. Figure S1A). These data suggest that the effect of FAD mutations in PS1 on APP-CTFs PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35133