Purified c-Secretase Complexes with PS1 Mutations submitted to a C100-His assay according to the same protocol as in (A). Protein samples were separated by SDS-PAGE and analysed by immunostaining for c-secretase subunits ((NCT164 (NCT), MAB1563 (PS1-NTF), or UD1 (PEN2)). AICD-His cleavage products were immunostained with an anti-APP-CTF antibody (A8717). *Indicates a non-specific band corresponding to the IgG light chains. (C) Ab1–40 and Ab1–42 were quantified by sandwich ELISA and represented in pg/mL (left Y-axis) or in percentage (right Y-axis) of the mean of Ab1–40 levels generated by the two wild-type clones. Ab1–42/Ab1–40 ratios are indicated on the top of the bars. The results were confirmed in three independent experiments and a representative dataset is shown. doi:10.1371/journal.pone.0035133.g003 to decrease total Ab production in PS1/PS2 knockout cells [23]. These findings suggested that endogenous PS1 and PS2 may influence the mutant phenotype in cells or in vivo. By extension, we hypothesized that the other components of c-secretase, namely APH1, NCT and PEN2, may influence the mutant phenotype as well. Indeed, c-secretase complexes are heterogeneous in compo- sition due to the existence of two human APH1 genes, APH1a and APH1b, and two splicing isoforms of APH1a (S and L), as well as two presenilin genes, PSEN1 and PSEN2. Therefore, it is plausible that a single mutation in PSEN1 confers different catalytic properties to distinct c-secretase subtypes. This hypothesis is supported by recent investigations showing that APH1 variants can modulate Ab profiles. When compared to APH1aS or L, overexpression of APH1b in MEF knockout for all APH1 genes led to increased production of longer Ab species [24]. With this regard, it is important to note that all four mouse variants of APH1 are expressed in the MEF cell line used in previous studies [23] and employed here (cf. Figure S3). Since the phenotype of PS1 mutations have mainly been assessed in vivo or in cell-based systems, we investigated the effects of FAD-linked PS1 mutants on the processing of APP-CTFs in cell-free systems, by using semi- purified and purified enzymatic complexes isolated from MEF PS1/PS2 double knockout cells stably overexpressing differentially tagged human c-secretase components. Under these conditions, the biochemical and functional properties of c-secretase complexes bearing either FAD-linked PS1 mutants (L166P, DE9, and P436Q), dominant-negative forms of PS1 or wild-type PS1 were characterized. The activity of c-secretase with PS1-WT was similar to that reported for c-secretase purified from our CHO cells overexpressing NCT-V5, APH1aL, Flag-PEN2 and PS1-WT [26,27], that resulted in Ab1–42/Ab1–40 ratios between 0.1 and 0.3. In contrast, we found a major loss in the activity of c-secretase complexes containing either the dominant-negative PS1 variants or the FAD-linked PS1 mutants. Although a total loss of activity was expected for the dominant negative forms of c-secretase [5,6,32], the drastic loss of activity seen here with the FAD-linked PS1 mutants was unexpected. Indeed, the PS1-L166P variant has previously been reported to increase Ab1–42 levels both in vivo [46] and in vitro [20], in the presence of endogenous PS1, PS2 and APH1 components. In contrast, Bentahir and colleagues found that PS1-L166P decreased both Ab1–40 and Ab1–42 production in a PS knockout background. These results are consistent with our in vitro data, although the reduction in Ab1–42 and Ab1–40 production was more pronounced in our system. However, they differ from our cell-based data in which we observed an increase in Ab1–42 associated with a decrease in Ab1–40. Taken together, these data suggest that the overexpression of the other human components of c-secretase can influence the phenotype of FAD- linked mutations. Another possible explanation for these discrep- ancies comes from the use, in previously described cellular systems, of APP carrying the Swedish mutation (K670M/N671L) [20,23,46]. Initially, this APP variant was shown to enhance the production of all Ab species by favouring its b-secretase cleavage [47,48]. However, Munter and colleagues recently demonstrated that the APP Swedish mutation can also affect the specificity of the c-secretase cleavage [49]. In particular, these authors showed that over-expression of APP Swedish in a neuronal cell line led to a 4- fold increase in secreted Ab42, associated with only a 2-fold increase of total Ab, compared with the wild-type APP. Therefore, one cannot exclude the possibility of differential interactions between PS1-WT or PS1 variants and different APP variants, as suggested earlier [50]. As far as the PS1-DE9 mutant is concerned, previous cell-based studies have reported a decrease in Ab40 levels associated with an increase in Ab42 levels [51,52], or no changes in Ab40 levels despite increased Ab42 levels [53], or a decrease in both Ab species [23]. In the present study, Ab1–40, Ab1–42, as well as AICD levels generated from purified c-secretase complexes with PS1-DE9 were close to the limit of detection, resulting in a loss of at least 95% of the c-secretase activity compared to wild-type PS1. Similarly, we found a near to complete loss of c-secretase activity for complexes with the aggressive PS1-P436Q mutant (mean onset: 28.3 years) [54]. Consistent with a recent study [35], and in contrast to PS1-L166P, the maturation of the full-length PS1- P436Q protein into PS1-NTF and PS1-CTF was impaired. Interestingly, the same study also reported that P436Q variant showed a decreased c-secretase activity and Ab40 and Ab42 secretion in PS1/PS2 double knockout cells (,75% or ,50% of the control for Ab40 or Ab42, respectively), but to a lesser extent than what we measured in this study (more than 97% for both Ab species). Overall, our data show that, under our experimental conditions, the FAD-linked PS1 mutants cause a drastic loss of activity (at least 90%) in the highly purified and homogeneous as well as semi- purified c-secretase complexes. This loss-of-function is asymmet- rical as Ab1–42 levels were relatively less affected than Ab1–40 levels. Interestingly, the ratio between the two Ab species was similar for the variants tested here (,1), suggesting a common modulatory mechanism. Our findings further support a model in which the subtype of c-secretase containing APH1aL and FAD- linked PS1 mutants generates a higher Ab42/Ab40 ratio compared to PS1-WT, as previously observed in vivo [15,38]. Taken together, the overall reduction in Ab levels strongly suggest that other regulatory mechanisms or cellular components may exist and account for the strong amyloid pathology observed in AD patients carrying these FAD-linked PSEN1 mutations. In agreement with this hypothesis, we report that the phenotype of FAD-linked PSEN1 mutations is different whether we consider the cell-based or the cell-free conditions. Indeed, even if Ab42/Ab40 ratios were globally increased in both experimental conditions, Ab production was strikingly different. One possible explanation would be that specific subtypes of c- secretase complexes are less affected than others by PSEN1 mutations. For instance, it remains unknown whether c-secretase complexes carrying APH1b or APH1aS are similarly affected by PSEN1 mutations. In support to that regard, Winkler and colleagues recently purified human c-secretase with PS1-L166P and observed that such complexes, containing both APH1a and AHP1b, led to increased Ab1–42 production associated with decreased Ab1–40 production, as compared to the PS1-WT complexes. Another possible explanation would be that purified c- secretase complexes lack binding partners modulating the PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35133