PS1 Conformation and Ab Figure 2. Detection of the PS1 conformation in living cells by spectral FRET. (A) HEK293T cells were transfected with designated constructs and activated by the 488 nm laser. Vertical axis shows the ratio of intensities within the 513 nm to 641 nm spectral range normalized by the intensity at 513 nm. The GFP-RFP fusion construct (red) shows large ‘hump’ centered on 598 nm, compared to G-PS1 (purple) fluorescence intensity due to FRET, i.e., energy transfer from GFP to RFP. L166P G-PS1-R (green) shows smaller but significant (p,0.001) increase in the 598/513 ratio, compared to that in the wild-type G-PS1-R expressing cells (blue). (B) HEK293Tcells were transfected with either G-PS1-R or L166P mutant G-PS1-R construct. Equal numbers of cells (n = 277) were randomly chosen from each condition and cell number was plotted against the 598/513 ratio. Note that the histogram is shifted to the right for L166P mutants, indicating an increase in the number of cells that exhibit higher 598/513 ratio, i.e., closer PS1 NT- loop proximity (p,0.001). doi:10.1371/journal.pone.0007893.g002 distribution in cells expressing the FAD mutant PS1 (Figure 2B). This result indicates that in the presence of the L166P mutation, GFP at the PS1 NT is closer to or more directly aligned with the RFP in the TM6-7 loop, giving rise to more efficient energy transfer from G to R, compared to that in the wt PS1 molecules. To visualize structural differences between the G-PS1-R molecules in different subcellular compartments of the same cell, pseudo-colored ratiometric images of the 598/513 intensity were generated by Image J program after background subtraction. Several regions of interest (ROI) were randomly selected within the cell expressing G-PS1-R (Supplementary Figure S2A) and the 598/513 ratio was calculated in each ROI. As expected, ROIs located at the cell periphery yield higher 598/513 ratio (more green-to-red pixels in the pseudo-colored Spectral FRET image), compared to the ROI adjacent to the nucleus (predominantly blue pixels, Supplementary Figure S2B). This distribution is very similar to that in HEK cells transfected with G-PS1-R and analyzed by FLIM (Supplementary Figure S2C). The pseudo- colored FLIM image shows shorter lifetime of the donor GFP at the periphery of the cells (red pixels), indicating ‘closer’ NT-loop conformation of the G-PS1-R probe at/near the cell surface. Thus, both FLIM and spectral FRET imaging in live cells revealed that G-PS1-R molecules adopt more ‘‘close’’ NT-loop conforma- tion as they move towards the cell surface, a pattern which is identical to that of previously observed endogenous PS1 [21]. Finally, we asked if the effect of GSMs treatment on PS1 conformation could be detected by spectral FRET in live cells. To test this, HEK293 cells were infected with G-PS1-R-lenti viral constructs. 24 hrs after the infection, cells were treated with either 100 mM fenofibrate (closes PS1 conformation) or 400 mM ibuprofen (opens PS1 conformation [20]) for 24 hours followed by the 598/513 ratio analysis. Control cells were treated with vehicle (DMSO and ethanol, respectively). As expected, fenofi- brate treatment resulted in a spectral shift similar to that observed for the L166P G-PS1-R construct. We detected a significant increase in the 598/513 ratio in the presence of fenofibrate (Figure 3A), which indicates that fenofibrate treatment induces the ‘‘closed’’ conformation of PS1.Conversely, ibuprofen treat- ment leads to a reduced 598/513 ratio (Figure 3B), inducing an ‘‘open’’ PS1 conformation. Thus, G-PS1-R conformation changes in response to agents that selectively modulate Ab42 production in live cells. Discussion An aggregation and deposition of amyloid b (Ab) containing plaques is a common feature of normal aging and the AD brain. A shift towards an increased proportion of the longer, highly fibrillogenic and neurotoxic Ab species (i.e., Ab42) in the brain has been associated with the neurodegeneration leading to AD. Blocking Ab production can be a therapeutic target. However, direct inhibition of c-secretase activity may cause undesirable side effects due to disruption of the normal physiological function of numerous c-secretase substrates, including APP itself [26] and Notch [27]. Instead, modulators of the site at which c2secretase cleaves APP have been developed by screening libraries of compounds, or noted by genetic manipulations [13,17,25]. However, the precise molecular and cellular mechanism under- lying the resultant changes in the Ab42/40 ratio is unknown. We propose a hypothesis of ‘‘structure-function correlation’’ implying that structural changes in the PS1/c-secretase are linked to the precision of c-cleavage of APP to yield Ab species of different lengths. Our data suggest that changes in the structure of multiple members of the c-secretase complex, or even APP itself, can PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7893