The distinct calcium signatures generated by NMDAR stimulation, APOE3 treatment and APOE4 treatment. A, Rat primary cortical neurons (DIV15) were treated with recombinant APOE3/APOE4 protein (15 nm) for 20 min in the presence or absence of extracellular calcium (ACSF with or without calcium) and probed for the phosphorylation of eEF2. Left, Representative immunoblots indicating levels of phospho-eEF2, eEF2, and Tuj1. Right, Graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data are represented as mean ± SEM, N = 3–4, one-way ANOVA (p = 0.0004) followed by Tukey's multiple comparison test. B–D, Rat primary cortical neurons (DIV15) were subjected to calcium imaging for 5 min. The graphs represent the time trace for the change in Flou4-AM fluorescence compared with initial fluorescence (ΔF/F0) under the following conditions. B, NMDAR stimulation (20 μm), nifedipine (50 μm) pretreatment followed by NMDAR stimulation (20 μm), and MK801 (25 μm) pretreatment followed by NMDAR stimulation (20 μm). C, APOE3 treatment (15 nm), nifedipine (50 μm) pretreatment followed by APOE3 addition (15 nm), and MK801 (25 μm) pretreatment followed by APOE3 addition (15 nm). D, APOE4 treatment (15 nm), nifedipine (50 μm) pretreatment followed by APOE4 addition (15 nm), and MK801 (25 μm) pretreatment followed by APOE4 addition (15 nm). E–H, Box plots represent the quantification of the change in Flou4-AM fluorescence (ΔF/F0) after 2 min of APOE addition. N = 4–5 experiments, each experiment has the average value of 40–50 neurons. E, Unpaired Student's t test. F, One-way ANOVA (p = 0.0058) followed by Tukey's multiple comparison test. G, One-way ANOVA (p = 0.0029) followed by Tukey's multiple comparison test. H, One-way ANOVA (p = 0.0038) followed by Tukey's multiple comparison test. I–L, Box plots represent the quantification of the change in Flou4-AM fluorescence (ΔF/F0) after 1 min of APOE addition. N = 4–5 experiments, each experiment has the average value of 40–50 neurons. I, Unpaired Student's t test. J, One-way ANOVA (p = 0.0008) followed by Tukey's multiple comparison test. K, One-way ANOVA (p = 0.0038) followed by Tukey's multiple comparison test. L, One-way ANOVA (p < 0.0001) followed by Tukey's multiple comparison test. M, Rat primary cortical neurons (DIV15) were treated with APOE4 recombinant protein (15 nm) for 24 h and subjected to calcium imaging using Fluo8-AM. The graph shows the resting cytosolic calcium measured in these neurons. N = 50–60 neurons from 3 independent experiments. Kolmogorov–Smirnov test. N, Rat primary cortical neurons (DIV15) were treated with APOE KO or APOE4 (10–15 nm) conditioned media for 24 h and subjected to calcium imaging using Fluo8-AM. The graph shows the resting cytosolic calcium measured in these neurons. N = 50–60 neurons from 3 independent experiments. Kolmogorov–Smirnov test.
