Figure 1

The Ab42/40 ratio-modulating APP mutations induce changes in APP positioning relative to the membrane . A) ELISA detection of the human Aβ40 and Aβ42 in conditioned media of the cells transiently transfected with human APP-RFP constructs with designated mutations. The amount of each Aβ species was normalized to that obtained from the cells expressing wild-type APP-RFP (Aβ₄₀ ≈70 pMol/L, Aβ₄₂ ≈1 pMol/L). Three independent experiments were performed. (mean ± SD; *p < 0.001 vs. wild-type APP-RFP, ANOVA, n: number of wells in the representative experiment shown). B) FLIM analysis of the proximity between APP-CT RFP and myrGFP labeled membrane. The pseudo-colour images show distribution of the EGFP and myrGFP donor fluorophore lifetimes in the presence (bottom) or absence (top) of the RFP acceptor fluorophore fused to the wild-type APP-CT. Only cells transfected with myrGFP as a donor fluorophore showed lifetime shortening (red and yellow pixels) in the presence of APP-RFP, with the shortest lifetime at the cell periphery. Scale bar: 10 μm. Colorimetric scale shows GFP fluorophore lifetime in picoseconds. C) Quantitative FLIM analysis of the GFP lifetimes in APP/APLP2 dKO cells expressing wild-type and mutant APP-RFP constructs. In cells transfected with the EGFP as a donor fluorophore (grey bars), the donor lifetime did not change significantly in the presence of either wild-type or V717I APP-RFP. In cells transfected with myrGFP as a donor (black bars), the donor lifetime was significantly shortened in the presence of RFP acceptor at the APP CT. FAD-linked APP mutations (V717I and I716F) significantly shortened, whereas V717K APP mutation significantly increased the lifetime of myrGFP donor, compared to that of the wild-type APP (mean ± SD; *p < 0.001, **p < 0.01, ANOVA). Data from one of the three independent experiments is shown; n = cell number. D) CHO cells were transfected with APP-GFP to serve as a donor fluorophore (green) in the FLIM assay. Plasma membrane was stained with CM-DiI to serve as an acceptor fluorophore (red). Merged image shows that APP signal is outlined by red membrane. Scale bar; 10 μm. E) The graph shows average lifetime of GFP donor fluorophore in CHO cells from (D). The lifetime of GFP donor was shortened in cells with CM-DiI membrane staining. In the presence of I716F and V717I mutations, the GFP lifetime was significantly shorter than that in wild-type APP-GFP transfected cells (mean ± SD; *p < 0.01, ANOVA). Three independent experiments were performed (n: total cell number).