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. 2019 Nov 26;13:526. doi: 10.3389/fncel.2019.00526

Figure 5.

Figure 5

c-Abl ablation protects against Aβ oligomers-induced cell death and preserves Piccolo clusters in apoptotic and non-apoptotic neurons. (A,B) WT and c-Abl-KO neurons were treated with 5 μM AβOs for 5 h and Hoechst stained to label apoptotic nuclei (blue; PSD95 in red) (A); and co-stained with active caspase-3 (red) antibody to confirm apoptosis; cytoskeletal protein β-tubulin is shown in green (B). Graphs show the percentage of apoptotic nuclei (C) and the percentage of active caspase-3 nuclei (D) for each condition. (E–F) WT and c-Abl-KO neurons were classified into apoptotic vs. non-apoptotic neurons and analyzed for Piccolo (E) and PSD95 (F) cluster quantification per 10 μm dendrite. Both were significantly affected in non-apoptotic neurons, especially in WT neurons. While c-Abl-KO neurons display higher number of Piccolo clusters and were significantly preserved after AβOs treatment (WT: 6.52 ± 0.18 vs. WT+AβOs: 4.36 ± 0.21 clusters/10 μm dendrite, and KO: 8.19 ± 0.19 vs. KO+AβOs: 7.1 ± 0.24 clusters/10 μm dendrite; WT: n = 38, WT+AβOs: n = 43, KO: n = 29 and KO+AβOs: n = 42 dendrites). Apoptotic neurons displayed the same tendency for Piccolo clusters (WT: 4.81 ± 0.26 vs. WT+AβOs: 3.22 ± 0.23 clusters/10 μm dendrite, and KO: 6.77 ± 0.23 vs. KO+AβOs: 5.73 ± 0.25 clusters/10 μm dendrite; WT: n = 37, WT+AβOs: n = 38, KO: n = 29 and KO+AβOs: n = 42 dendrites). (F) PSD95 clusters strongly decreased in WT compared with c-Abl-KO non-apoptotic neurons while all conditions in apoptosis display significantly less clusters than healthy conditions (Non-apoptotic: WT: 6.76 ± 0.17 vs. WT+AβOs: 4.29 ± 0.17 clusters/10 μm dendrite, and KO: 5.7 ± 0.33 vs. KO+AβOs: 4.66 ± 0.22 clusters/10 μm dendrite; WT: n = 42, WT+AβOs: n = 44, KO: n = 36 and KO+AβOs: n = 42 dendrites; Apoptotic: WT: 4.65 ± 0.22 vs. WT+AβOs: 3.5 ± 0.21 clusters/10 μm dendrite, and KO: 4.69 ± 0.34 vs. KO+AβOs: 4.11 ± 0.19 clusters/10 μm dendrite; WT: n = 41, WT+AβOs: n = 42, KO: n = 27 and KO+AβOs: n = 43 dendrites; non-apoptotic: WT: n = 9, WT+AβOs: n = 10, KO: n = 7 and KO+AβOs: n = 9 neurons; apoptotic: WT: n = 9, WT+AβOs: n = 11, KO: n = 6 and KO+AβOs: n = 9 neurons). Two-way ANOVA and Tukey’s multiple comparison test, ***p < 0.001. Scale bar = 20 μm and 10 μm for magnifications. n = 2 independent cultures, 3–4 mice embryos per condition. ns: non-significant; *p < 0.5; **p < 0.01; ***p < 0.001.