PS1 Conformation and Ab Figure 3. Effect of GSMs on G-PS1-R conformation as detected by spectral FRET. HEK293 cells infected with G-PS1-R-lentiviral construct were treated with either (A) vehicle (DMSO) or 100 mM fenofibrate, or (B) vehicle (EtOH) or 300 mM ibuprofen for 24 hours. After the treatment, cells were activated by the 488 nm laser and analyzed by spectral FRET. The percentage of cells exhibiting certain 598/513 ratio (Y axis) was plotted against the 598/513 ratio (X axis). (A) Note that the histogram in the fenofibrate treated cells is shifted to the right of that in DMSO treated cells. This indicates a higher percentage of cells with elevated 598/513 ratio after the fenofibrate treatment (p,0.001 vs. DMSO; DMSO: n = 675 cells, fenofibrate: n = 571cells). (B) Note that higher percentage of cells shows lower 598/513 ratio after the ibuprofen treatment (p,0.001 vs. ethanol; ethanol: n = 730 cells, ibuprofen: n = 524cells). doi:10.1371/journal.pone.0007893.g003 allosterically impact PS1 conformation in a way that leads to a change in c-secretase/APP interactions and alters the exact cleavage site of APP. Very little is currently known about the structure of the PS1/c- secretase complex due to its complexity and the difficulty of crystallizing multi-pass membrane proteins. Several studies that use serial deletions and co-immunoprecipitation [28], substituted cysteine accessibility method [13,29,30] or electron microscopy- based analyses [31] reported that PS1/c-secretase may form a ring-like pore within the hydrophobic environment of a mem- brane. However, all these studies required lysing of the cells and/ or were performed using isolated c-complexes. To test the hypothesis of PS1/c-secretase structure-function correlation in living cells and to accurately report molecular changes in PS1 in its native environment, a fluorescent probe is required that adequately responds to various experimental manipulations associated with alterations in the Ab42/40 ratio. In this report, we present and fully characterize a novel probe, GFP-PS1-RFP fusion protein, which exploits NT-loop proximity as a readout of PS1 conformation in live cells. We employ a molecular imaging methodology, such as FRET-based microscopy, to show that this probe allows monitoring, and accurately reflects consistent structural changes of the PS1 molecule in live cells, and that these changes closely correlate with changes in the Ab42/40 ratio. We verified that G-PS1-R protein can be cleaved into NTF and CTF, can traffic to the plasma membrane, which is critical for the metabolism of some substrates, and can be incorporated into the c-secretase complex to support enzymatic activity in cells lacking both presenilin 1 and presenilin 2. Moreover, using both FLIM and spectral FRET approaches we determined that G-PS1-R protein adopts ‘‘close’’ or ‘‘open’’ PS1 NT-loop proximity in response to various Ab42-raising or lowering manipulations, respectively. These manipulations include introducing familial Alzheimer’s disease L166P PS1 mutation, structural change in the Pen2 N-terminus, expressing Aph1B or Aph1A isoform, or pharmacological treatment (fenofibrate, ibuprofen). We show that data obtained using G-PS1-R probe are in agreement with our previous findings using FLIM analysis of fixed, permeabilized, and immunostained cells, that utilized either NT-CT or NT-loop proximity to monitor PS1 conformation [14,20,21]. Since antibody-based FLIM analysis is based on immunolabeling of endogenous and/or untagged PS1 molecules, these results confirm that the G-PS1-R probe is a reliable indicator of PS1 conformation in living cells. The finding that the G-PS1-R probe acquires ‘‘open’’ conformation in response to ibuprofen treatment and ‘‘close’’ NT-loop proximity in response to fenofibrate is very interesting since it has recently been shown that these agents target APP substrate [19], and not c-secretase complex itself as previously believed [32,33]. This indicates that modifications in the substrate could allosterically affect conformation of the enzyme. In line with this finding, we have previously showed that APP mutations which reduce Ab42 lead to an ‘‘open’’ [15], whereas mutations that increase Ab42 induce ‘‘close’’ [16] conformation of the PS1 molecule. Thus, altered presentation of the APP substrate to c- secretase, driven by either APP mutations or by binding of the pharmacological agents to APP could, in turn, allosterically affect PS1 conformation. Collectively, our data demonstrate that regardless of the nature (genetic or pharmacological GSMs) or precise target (i.e. PS1, Pen- 2, Aph-1 or APP substrate), the final determinant of Ab40 or Ab42 generation relies on the molecular conformation of PS1. These findings strongly support the hypothesis that Ab42/40 ratio is tightly linked to the conformation of the PS1 molecule, thus making allosteric modulation of ‘‘pathogenic’’ conformation of PS1 an attractive target of therapeutic intervention in Alzheimer’s disease. In addition, we present a ratiometric spectral FRET assay that can be used for screening drugs modulating Ab42/40 ratio on a high-throughput screen platform and/or in live mouse brain in vivo. An additional advantage of using G-PS1-R probe in this assay PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7893