the wild-type clones. Overall, the average Ab1–40 levels were decreased in these cell lines (Mean of two clones in pg/mL 6 SD: WT: 224.660.1, L166P: 84.3634.2, DE9: 167.2640.7, P436Q: 82.8674.7), while the Ab1–42 levels were increased (Mean of two clones in pg/mL 6 SD: WT: 69.967.9, L166P: 310.7681.0, DE9: 94.4618.5, P436Q: 141.1698.5). Consistent with previously reported data, the Ab1–42/Ab1–40 ratios were increased in all c- MEF clones overexpressing PS1 with FAD-linked mutations (Figure 2D) (Mean of two clones pg/mL 6 SD: WT: 0.3160.03, L166P: 3.860.58, DE9: 0.660.03, P436Q: 2.060.59). FAD-linked PS1 mutations alter the activity of semi- purified c-secretase complexes We next assessed the activity of c-secretase with FAD-linked PS1 mutations in microsomal extracts of c-MEFs. Membrane protein extracts were prepared and c-secretase activity was assayed on an exogenous recombinant APP-based substrate consisting of the C-terminal 99 amino acid residues of APP, and referred to as C100-Flag (Figure 3A). The generation of the cleavage product AICD-Flag was detected by Western blot analysis and measured by densitometry. As shown in Figure 3A, the levels of AICD-Flag generated from the PS1-L166P and PS1-DE9 clones accounted for 4261% and 1366% of that in the PS1-WT clones, respectively. AICD-Flag was undetectable in the clones expressing PS1-P436Q as well as in the three dominant negative aspartate PS1 variants. Similar results were observed in MEF PS1/22/2 cells overexpressing only the PS1 variants, in the absence of the other human c-secretase components (Figure S1B). We next performed activity assays under semi-purified conditions. To do so, c-secretase complexes from the different clones were co-immunoprecipitated from microsomal extracts and their activities were assayed in vitro with the C100-His substrate. Two immunoprecipitation protocols using two affinity resins (anti-Flag M2 and anti-HA 3F10, targeting respectively Flag-PEN2 and APH1aL-HA) were compared to exclude a possible heterogeneity of c-secretase complexes potentially generated under such conditions. As shown in Figure 3B, the anti-Flag and anti-HA resins immunoprecipitated similar levels of NCT, PS1 and PEN2 and the activity of the c-secretase complexes was similar regardless of the affinity resins used (Figures 3B). In agreement with the assays performed using microsomal extracts (Figure 3A), those performed with semi- purified complexes revealed that FAD-linked mutants drastically reduced AICD generation, when compared to PS1-WT (Figure 3B). Furthermore, levels of Ab1–40 produced from PS1-L166P, PS1-DE9 and P436Q variants fell respectively to ,6%, ,1.5% and ,0.5%, when compared to these produced from PS1-WT c-secretase. Following the same tendency, Ab1–42 levels generated from PS1-L166P, PS1-DE9 and PS1-P436Q c- secretase variants were respectively estimated to ,20%, ,5% and ,3% of these produced with PS1-WT-containing complexes. The Ab1-42/Ab1–40 ratio measured for WT c-MEFs was consistent with our cell-based data (compare Figures 2D and 3C), validating our in vitro experimental conditions. Interestingly, an overall Ab1–42/Ab1–40 ratio of ,1 was estimated for all PS1 mutants, with the exception of P436Q#4 (,2). However, in the latter, the ratio may have been overestimated because the Ab1– 40 levels measured were at the detection limit. This similarity in the Ab1–42/Ab1–40 ratios generated by the different mutants has, to the best of our knowledge, never been observed in previous studies. Purified c-Secretase Complexes with PS1 Mutations Highly purified c-secretase complexes with FAD-linked PS1 mutants display a loss-of-function phenotype In order to further investigate how PS1 variants affect APP- CTF cleavage and to validate our observations performed in semi- purified conditions, we purified to homogeneity c-secretase complexes from the selected clones described in Figure 1. The purification process (described in details under Materials & Methods and depicted in Figure 4A) involved three sequential affinity purifications starting from microsomal extracts. Since all cell lines were generated on a PS knockout background, this strategy allowed us to purify exclusively and selectively c-secretase complexes made of human components. As highlighted in Figure 4B, our protocol led to the production of stable, high molecular weight c-secretase complexes (HMWCs) with an apparent molecular mass on Blue Native (BN)-PAGE of ,350 kDa, which is consistent with previous reports [26,27,37]. The silver stained particles on the BN gel confirmed the high purity of these complexes (Figure 4B, upper panel), for which the identity was established by cross-reactivity with antibodies specific to NCT and PS1 (Figure 4B, lower panels). As the main goal of this experiment was to compare the efficacy of the different c- secretase complexes to process APP-based substrates, it is important to note that the final yields of the complexes purified from the different clones were very similar (Figure 4B), thus facilitating further comparative analyses. The immunoblot analysis of the purified complexes (Figure 4C) confirmed the presence of all core components (NCT-V5, APH1aL-HA, Flag-PEN2 and PS1) and globally recapitulates the maturation processes observed in the Figure 1. Interestingly, the relative PS1-NTF/CTF to PS1-FL ratios in the purified c-secretase complexes containing the three PS1 dominant-negative or the PS1-P436Q variants were higher than those estimated in the whole cell extracts before purification (Figure 1A), indicating that endoproteolysis of PS1 was not completely abolished by these mutations. To assess how FAD- linked mutations in PS1 affect c-secretase activity in these purified complexes, we performed in vitro assays by using C100-Flag substrate (Figure 3A) and equal amounts of the different purified complexes, immediately after the purification to avoid freezing/ thawing cycles that might affect enzymatic activity. As shown in Figure 5A, c-secretase activity based on AICD-Flag production was easily detected in complexes purified from the two PS1-WT clones. This is in sharp contrast to the other purified complexes that did not generate detectable AICD under our experimental conditions (Figure 5A). Next, sandwich ELISAs directed against Ab1–40 and Ab1–42 were performed in order to further characterize the specific activity of the purified complexes. Reflecting the levels of AICD and considering the detection limits of our sandwich ELISA, the production of both Ab species by c- secretase complexes containing PS1-WT was at least 10-fold higher than in complexes with the FAD-linked or dominant- negative PS1 variants (Figure 5B). The Ab1–42/Ab1–40 ratio measured in PS1-WT c-secretase complexes (,0.22) was similar to that previously reported [26,27], thus excluding a possible qualitative alteration of c-secretase activity with this protocol. Together, our results strongly support the hypothesis that the pathogenic L166P, DE9, and P436Q mutations in PSEN1 cause a drastic loss (at least 90% as estimated by ELISA) of c-secretase activity. Discussion Several conflicting results as to how Alzheimer’s disease-linked mutations in PSEN1 affect the processing of APP by c-secretase have been reported. First, it has been previously reported that PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35133