PS1 Conformation and Ab Table 3. Manipulation of Pen-2 lead to altered Ab42/40 ratio as well as PS1 conformation. Table 5. Pharmacological modulation of Ab42 by fenofibrate changes conformation of G-PS1-R. Acceptor Donor lifetime (psec) Condition Acceptor Donor lifetime (psec) Condition wt/flag-Pen2 wt Pen2 flag-Pen2 wt/flag-Pen2 wt Pen2 flag-Pen2 wt Pen2 flag-Pen2 Donor GFP-NT GFP-NT GFP-NT Alexa488-NT Alexa488-NT Alexa488-NT Alexa488-NT Alexa488-NT *: p,0.05 vs wt Pen2, **: p,0.05 vs wt Pen2, ***: p,0.05 vs wt Pen2. doi:10.1371/journal.pone.0007893.t003 none RFP-loop RFP-loop none Cy3-loop Cy3-loop Cy3-CT Cy3-CT 2017622 (n = 12) 17196135 (n = 19) 14306194 (n = 22)* 2223614 (n = 6) 1852659 (n = 10) 1799689 (n = 10)** 1802661 (n = 10) 16996109 (n = 10)*** the GFP-donor was significantly shorter in the Aph1B containing cells, compared to that in fibroblasts expressing Aph1A or wild- type Aph1 (Table 4). These results further support the notion that PS1 adopts a ‘‘close’’ conformation in live cells expressing Aph1B- containing c-secretase complexes, and such PS1 conformation may be responsible for a shift towards the Ab42 cleavage site. Pharmacological Manipulations of the Ab42 Production Are Associated with a Change in G-PS1-R Conformation Several compounds, referred to as c-secretase modulators (GSMs), have been shown to selectively affect production of Ab species of different lengths by affecting the APP c-secretase cleavage site. Weggen et al. [17] reported that the group of NSAIDs shifted the c-secretase cleavage site on APP from the Ab42 position to Ab38, selectively reducing Ab42 generation. Another set of compounds, such as fenofibrate, mimicked the effect of FAD mutations by increasing Ab42 production [13,25]. Now we explore whether we can detect G-PS1-R conformational change in live cells due to GSM treatment. For this, CHO cells transfected with G-PS1-R probe were treated with either 100 mM fenofibrate or DMSO. The conditioned media was collected for Ab specific ELISA and the cells were used for the analysis of PS1 NT-loop proximity by FLIM. The Ab ELISA revealed that in cells treated with fenofibrate the level of secreted Ab40 was reduced by ,42% and the level of Ab42 was increased by ,36%. FLIM analysis showed that fenofibrate treatment leads to a significant shortening of the GFP lifetime in cells expressing G-PS1-R construct, compared to that in DMSO treated cells (Table 5), Table 4. Manipulation of Aph1 lead to altered PS1 conformation. GFP donor RFP acceptor Donor lifetime (psec) Condition wt fibroblast wt fibroblast Aph1A Aph1B GFP-NT GFP-NT GFP-NT GFP-NT *: P,0.001 vs wt, Aph1A. doi:10.1371/journal.pone.0007893.t004 none RFP-loop RFP-loop RFP-loop 2110635 (n = 26) 1867680 (n = 22) 1874658 (n = 22) 1766684 (n = 22)* DMSO/Fenofibrate GFP-NT DMSO Fenofibrate DMSO Fenofibrate DMSO/Fenofibrate Alexa488-NT Donor GFP-NT GFP-NT Alexa488-NT Alexa488-NT None RFP-loop RFP-loop none Cy3-CT Cy3-CT 201368 (n = 6) 1772663 (n = 11) 1666626* (n = 10) 2083636 (n = 7) 1823625 (n = 15) 1698658** (n = 16) *: p,0.05 vs DMSO, **: p,0.02 vs DMSO. The representative experiment is shown; total four independent experiments were performed. doi:10.1371/journal.pone.0007893.t005 indicating a decrease in the GFP-PS1-NT and RFP-PS1-loop proximity. Similarly, our previously validated endogenous PS1 NT-CT conformation FLIM assay in fixed and immunostained cells showed statistically significant shortening of the donor (Alexa 488) lifetime in cells treated with fenofibrate, compared to that in the DMSO treated cells (Table 5). These data suggest that fenofibrate alters the conformation of the PS1 molecule to favor a ‘‘close’’ PS1 NT-loop and PS1 NT-CT proximity, which correlates with a shift in the APP c-cleavage site. Detection of the PS1 Conformation in Live Cells by Spectral FRET While FLIM approach provides high spatial resolution, each FLIM image takes minutes to acquire. The high temporal resolution of Spectral FRET analysis allows detection of more rapid changes in the FRET efficiency in live cells transfected with fluorescent proteins and enables monitoring these changes on a high throughput basis. First, we established optimal settings for the spectral imaging of the G-PS1-R transfected cells, and selected spectral windows in which changes in the donor and acceptor fluorescence intensity due to FRET are the most evident (Figure 2A). A construct encoded by RFP fused to GFP in a 1:1 ratio via a short linker (R-G fusion) was used as a positive control of the energy transfer, whereas G-PS1 (no acceptor) was used as a negative control. When cells transfected with the R-G fusion construct were activated by a 488 nm laser, the emission spectrum changed from that of the G-PS1, yielding a large spike at the 598 nm as a result of the energy transfer from the GFP donor to the RFP acceptor. Thus, we determined the ratio of fluorescence intensity at the 598 nm to that at the 513 nm (emission peak of the GFP) as a readout for the FRET efficiency: higher 598/513 ratio indicates higher FRET efficiency, and closer proximity between the fluorophores. When cells transfected with the G-PS1-R probe were activated by the 488 nm laser, we observed a shift in the emission spectra, indicating the presence of FRET from GFP to RFP, and resulting in a significantly higher 598/513 ratio, compared to that of the G- PS1 control. Next, we confirmed that the spectral FRET assay can detect subtle conformational differences between the wild type and L166P mutant G-PS1-R molecules. As shown in Figure 2A, the emission spectrum curve of the L166P G-PS1-R showed a small ‘hump’ at 598 nm, indicating a higher FRET efficiency compared to that of the wild type G-PS1-R. We observed a small but statistically significant increase in the average 598/513 ratio PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7893