Fig. 7

Aβ production/secretion and PS1 conformation are altered by Syt1 KD. a, b ELISA measurements of the endogenous (A) secreted and (B) intracellular Aβ40 and Aβ42 in parental and Syt1 KD PC12 cells stimulated for 15 minutes with 50 mM KCl. The secreted Aβ levels determined in pmol were normalized to the total protein extracted from the cells in the same well. (A) n = 5 for parental + Syt1-V5 and n = 6 for all other conditions; (B) n = 4. 100 % equals to 212.93 pmol/g and 20.06 pmol/g for secreted Aβ40 and Aβ42, respectively. 100 % for intracellular Aβ40 and Aβ42 equals to 1.92 pmol/g and 0.575 pmol/g, respectively. c FLIM analysis of the FRET efficiency [%] reflecting PS1 conformational changes in parental and Syt1 KD PC12 cells; n = 101 cells for parental Ca²⁺(-), n = 103 for parental Ca²⁺(+), n = 84 for Syt1 KD Ca²⁺(-) and n = 86 for Syt1 KD Ca²⁺(+). Higher values correspond to “closed” conformational state of PS1. The cells were placed in Ca²⁺/Mg²⁺-free (-) or Ca²⁺/Mg²⁺-containing (+) medium, prior to treatment with 50 mM KCl. d ELISA measurements of the endogenous Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the conditioned medium from parental and Syt1 KD cells (n = 8). e FLIM analysis of endogenous APP and PS1 proximity (FRET efficiency [%]) in parental and Syt1 KD PC12 cells; n = 83 cells for parental Ca²⁺(-), n = 89 for parental Ca²⁺(+), n = 70 for Syt1 KD Ca²⁺(-) and n = 64 for Syt1 KD Ca²⁺(+). The cells were treated for 5 minutes with 50 mM KCl in Ca²⁺/Mg²⁺-free or normal medium, fixed, and immunostained for PS1 and APP. The data in all panels of the Figure are presented as mean ± SEM. The statistical significance was determined using the unpaired student t-test. *p < 0.05; ** p < 0.01; ***p < 0.001. Aβ amyloid β, PS1 presenilin 1, Syt1 synaptotagmin 1, FLIM fluorescence lifetime imaging microscopy, FRET Förster Resonance Energy Transfer, APP amyloid precursor protein