Fig. 4

Activity-driven PS1-Syt1 interaction stabilizes open PS1 conformation and modulates Aβ production/secretion. a The conformation of endogenous PS1 in primary neurons pre-treated with PS1-LNT or scramble peptide at basal (KCl(-)) and high calcium (upon 15-min 50 mM KCl stimulation) conditions was determined by FLIM assay, monitoring proximity between PS1 N-terminus and PS1 loop domain. Cells were immunostained with anti-PS1 NT and anti-PS1 loop antibodies, followed by fluorescently conjugated secondary antibodies. The pseudo-colored images present the donor fluorophore lifetime in picoseconds. The orange-red pixels indicate PS1 in “closed” conformation; scale bar 10 μm. The adjacent graph presents normalized FRET efficiency between PS1-NT and PS1 cytosolic loop. The KCl stimulation shifts PS1 to “closed” conformation in both scramble and PS1-LNT pre-treated neurons but the shift is greater in the PS1-LNT conditions. The data are presented as mean ± SEM, n = 64 for scramble (KCl(-)), n = 57 for PS1-LNT (KCl(-)), n = 59 for scramble (KCl(+)) and n = 65 for PS1-LNT (KCl(+)), n = number of neurons analyzed in 3 independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t-test, **p < 0.01. The fluorescence lifetimes and corresponding FRET efficiency values are shown in the Additional file 1. b The graph presents relative Aβ40, Aβ42 levels and Aβ42/40 ratio in conditioned medium collected from the neurons pre-treated with PS1-LNT and depolarized with KCl to induce calcium influx. The values, determined by ELISA, were normalized to the scramble treated cells. The data are presented as mean ± SEM, n = 19-26. Statistical significance was determined using two-tailed unpaired Student’s t-test, *p < 0.05, **p < 0.01. c The graph presents relative Aβ40 and Aβ42 levels in cell lysates prepared from the neurons used in (b). The values were normalized to the scramble treated cells. The data are presented as mean ± SEM, n = 7-12. Statistical significance was determined using two-tailed unpaired Student’s t-test, *p < 0.05, **p < 0.01