Figure 5

Ca²⁺ overload triggers conformational change of PS1. (A) Increase in the intracellular calcium levels in neurons treated with 50 mmol/L KCl is monitored by Oregon Green BAPTA-1/AM (images) or by Indo-1 ratiometric probe (graph). The pseudo-colored images show intracellular calcium levels pre- and post-treatment. The red pixels correspond to higher calcium levels. The graph presents quantification of the intracellular calcium levels. n = 310 (KCl−) n = 251 (KCl+), n = total number of neurons analyzed from three independent experiments mean ± SEM, two-tailed unpaired Student t test. The scale bar corresponds to 20 µm. (B) FLIM analysis of the PS1 conformation pre- and post-KCl treatment in neurons. Increase in the FRET efficiency, reflective of pathogenic “closed” PS1 conformation, is detected after 15 min of KCl treatment. n = 84 (KCl−) n = 85 (KCl+), n = total number of neurons analyzed from 3 independent experiments, mean ± SEM, two-tailed unpaired Student t test. The data are normalized to the values recorded in the neurons in KCl− conditions. (C) Increase in the intracellular calcium levels in neurons treated with 5 µmol/L A2317 calcium ionophore monitored by Oregon Green BAPTA-1/AM. The pseudo-colored images show intracellular calcium levels pre- and post-treatment. The red pixels correspond to higher calcium levels. The scale bar corresponds to 20 µm. (D) Spectral FRET analysis of the PS1 conformation pre- and post-A2317 calcium ionophore treatment in primary neurons. Increase in FRET efficiency (R/G ratio) reflective of pathogenic “closed” PS1 conformation is detected in A2317-treated neurons. n = 180 (vehicle) n = 185 (A2317), n = total number of neurons analyzed from three independent experiments, mean ± SEM, two-tailed unpaired Student t test. Aβ — amyloid β; PS1 — presenilin 1; FRET — Förster resonance energy transfer.