Fig. 1

sAβ_1–42 increases cytosolic Ca²⁺ concentration by inducing mitochondria Ca²⁺ release dependent extracellular Ca²⁺ influx. a Cultured neurons were pretreated as indicated and analyzed for the level of each phospho-synapsin (S9) and phospho-CaMKIV (T196). b Representative images for Fluo-4 AM after treatment with 200 nM sAβ_1–42 for 5 min (Scale bar = 5 μm). c Neurons were treated with sAβ_1–42 for the time as indicated and phosphorylation of synapsin was measured by the western blot (1 ± 0 for 0 min, 1.56 ± 0.13 for 3 min, 1.73 ± 0.19 for 2 h, 1.77 ± 0.10 for 6 h sAβ_1–42 treatment, n = 4 independent blots). Equal amount of HEK293T cell lysates and neuron lysates incubated with phosphatase were loaded to confirm the synapsin and phospho-synapsin bands. d Fluo-4 AM intensity plots after treatment with 200 nM sAβ_1–42 or 2 μM TPP in the indicated extracellular buffer (in brackets). Averaged ΔF/F₀ was calculated by averaging the last 20 points of fluorescence profiles (2.59 ± 0.39 for sAβ_1–42 in 2 mM extracellular Ca²⁺, 0.43 ± 0.05 for sAβ_1–42 in 0 mM extracellular Ca²⁺, 0.42 ± 0.08 for sAβ_1–42 in 2 mM extracellular Ca²⁺ with TPP, 0.00 ± 0.01 for sAβ_1–42 in 0 mM extracellular Ca²⁺ with TPP, 0.04 ± 0.04 for TPP in 2 mM extracellular Ca²⁺, 0.05 ± 0.03 for TPP in 0 mM extracellular Ca²⁺ (n ≥ 5 independent experiments for each group)). e Fluo-4 AM loaded neurons were pretreated as indicated (in brackets) for 5 min and treated with 200 nM sAβ_1–42 (Averaged ΔF/F₀: 1.67 ± 0.20 for sAβ_1–42 (n = 3 independent experiments), 0.00 ± 0.03 for sAβ_1–42 with FCCP (n = 4 independent experiments)). f Neurons were treated with the indicated medium for 2 h and their phospho-synapsin (S9) and total synapsin level were detected by western blot and analyzed (Phospho-synapsin/total-synapsin: 1 ± 0 for control, 1.48 ± 0.32 for control with TPP, 2.45 ± 0.11 for sAβ_1–42, 1.66 ± 0.37 for sAβ_1–42 with TPP, n = 3 independent blots). Values are means ± standard error of mean (SEM). N.S = no significant difference, * p < 0.05, ** p < 0.01 (ANOVA and Tukey’s HSD post hoc test for (c, d, f) and Student’s t-test for (e))