processing of APP-based substrates, due to purification conditions affecting physical interactions of proteins. These include TMP21 or the recently reported c-secretase activating protein (GSAP) [12,13]. Further biochemical analyses are needed to test this hypothesis. The loss of activity in c-secretase containing PS1 variants also leads to decreased generation of AICD. As AICD is involved in the transcriptional regulation of several genes, including the neprilysin [55], and the lipoprotein receptor, LRP1 [11], it is likely that mutations in PSEN1 impair the regulation of these genes in vivo. For instance, it is possible that unknown genes transcriptionally regulated in vivo by the AICD might influence Ab metabolism in return. In support to these views, Veeraraghavalu and colleagues recently demonstrated that Notch signalling was impaired in transgenic mice overexpressing PS1 mutants, albeit in the presence of endogenous PS1-WT and all APH1 isoforms [56]. They found decreased self-renewal and differentiation of neuronal precursor cells in the subventricular zone, suggesting that the loss-of-function phenotype of PSEN1 mutations can also be observed in Purified c-Secretase Complexes with PS1 Mutations heterogeneous conditions. These results are reminiscent of the previous report from Saura and colleagues showing that conditional inactivation of both presenilins in vivo induces age- dependent neurodegeneration associated with memory impair- ment [57]. Altogether, the above-described data suggest that alternative pathways of neurodegeneration related to loss of c- secretase functions are possible and relevant to AD. They further support the concept that modulating rather than inhibiting c- secretase activity would be a more appropriate therapeutic strategy for AD [58]. Supporting this view, the c-secretase inhibitor Semagacestat tested in phase III clinical trials not only failed to slow cognitive decline in patients with mild-to-moderate AD, but actually made it worse [59]. Collectively, our findings support a model in which FAD-linked mutations in PSEN1 likely induce Ab pathology by perturbing the relative ratio between Ab species and by impairing developmental and cellular signalling pathways controlled by c-secretase substrates. This deleterious dual effect might explain why FAD- linked PSEN1 mutations cause early onset Alzheimer’s disease. Figure 5. Enzymatic activity of highly purified c-secretase complexes with FAD-linked or aspartate PS1 mutants. Equal amounts of the different purified c-secretase preparations characterized in Figure 4 were tested for activity on C100-Flag, as described in Figure 3. The resulting cleavage products were separated by SDS-PAGE and detected by immunostaining with an anti-Flag antibody (M2) for C100-Flag or AICD-Flag (A), and by sandwich ELISA for Ab1–40 or Ab1–42 (B). Note that the levels of Ab produced from FAD-linked c-secretase complexes were all in the non-linear range of the ELISA standards, close to the detection limit. Whenever possible, Ab1–42/Ab1–40 ratios were quantified and indicated on the top of the bars. Two independent purifications were performed on each clone and similar results were obtained. A representative dataset is shown. doi:10.1371/journal.pone.0035133.g005 PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35133