Figure 5. AMPK mediates excitotoxic apoptosis in a Bim-dependent manner. (A) Neocortical neurons were treated with 2.5 mM AICAR for the indicated time periods. Levels of p-JNK were assessed by Western blotting. Probing for total JNK served as a loading control. (B) Mouse neocortical neurons were transfected with a vector expressing either a control (Con) or AMPK siRNA sequence. AMPK levels were assessed 48 h after transfection by Western blotting. Probing for β-actin served as a loading control. (A and B) Results are representative of at least two independent experiments. (C) Neocortical neurons transfected with either control or AMPK siRNA cultured for 48 h and subsequently treated with 100 µM NMDA/10 µM Gly or sham conditions for 5 min and fixed after 16 h. The numbers of neurons with nuclear phospho Ser63 c-Jun (p-cJun) staining were quantified. Data represent mean ± SEM from n = 3 cultures. *, P < 0.05 compared with NMDA-treated control siRNA cultures (ANOVA post-hoc Tukey). (D) Representative images of neocortical neurons treated as described in C. Note the presence of nuclear phospho Ser63 c-Jun in control siRNA–transduced neurons but not in AMPK siRNA–transduced neurons. (E) Mouse cortical neurons were transfected with a vector expressing GFP and either an siRNA targeting AMPK or a control nontargeting siRNA and then cultured for 48 h. Neurons were subsequently treated with NMDA or sham and allowed to recover for 16–24 h before staining live with Hoechst. Pyknotic nuclei within the GFP-positive cells (250–300) were scored. Data represent mean ± SEM from n = 4 cultures. This experiment was repeated twice with similar results. *, P < 0.05 compared with sham-treated neurons; #, P < 0.05 compared with control siRNA–transfected neurons at the same time point (ANOVA post-hoc Tukey). (F) Western blot demonstrating expression of Bim after NMDA treatment in neocortical neurons transfected either with control or AMPK siRNA. Probing for β-actin served as a loading control. (G) Neocortical neurons from WT and bim−/− mice were transfected with a vector expressing EGFP and either an siRNA targeting AMPK or a control nontargeting siRNA for 48 h. After NMDA treatment, pyknotic nuclei within the GFP-positive cells were scored (250–300 cells). Data represent mean ± SEM from n = 4 cultures. This experiment was repeated twice with similar results. *, P < 0.05 compared with NMDA treated with control siRNA (ANOVA post-hoc Tukey). Bar, 10 µm.
Figure 5.

AMPK mediates excitotoxic apoptosis in a Bim-dependent manner. (A) Neocortical neurons were treated with 2.5 mM AICAR for the indicated time periods. Levels of p-JNK were assessed by Western blotting. Probing for total JNK served as a loading control. (B) Mouse neocortical neurons were transfected with a vector expressing either a control (Con) or AMPK siRNA sequence. AMPK levels were assessed 48 h after transfection by Western blotting. Probing for β-actin served as a loading control. (A and B) Results are representative of at least two independent experiments. (C) Neocortical neurons transfected with either control or AMPK siRNA cultured for 48 h and subsequently treated with 100 µM NMDA/10 µM Gly or sham conditions for 5 min and fixed after 16 h. The numbers of neurons with nuclear phospho Ser63 c-Jun (p-cJun) staining were quantified. Data represent mean ± SEM from n = 3 cultures. *, P < 0.05 compared with NMDA-treated control siRNA cultures (ANOVA post-hoc Tukey). (D) Representative images of neocortical neurons treated as described in C. Note the presence of nuclear phospho Ser63 c-Jun in control siRNA–transduced neurons but not in AMPK siRNA–transduced neurons. (E) Mouse cortical neurons were transfected with a vector expressing GFP and either an siRNA targeting AMPK or a control nontargeting siRNA and then cultured for 48 h. Neurons were subsequently treated with NMDA or sham and allowed to recover for 16–24 h before staining live with Hoechst. Pyknotic nuclei within the GFP-positive cells (250–300) were scored. Data represent mean ± SEM from n = 4 cultures. This experiment was repeated twice with similar results. *, P < 0.05 compared with sham-treated neurons; #, P < 0.05 compared with control siRNA–transfected neurons at the same time point (ANOVA post-hoc Tukey). (F) Western blot demonstrating expression of Bim after NMDA treatment in neocortical neurons transfected either with control or AMPK siRNA. Probing for β-actin served as a loading control. (G) Neocortical neurons from WT and bim−/− mice were transfected with a vector expressing EGFP and either an siRNA targeting AMPK or a control nontargeting siRNA for 48 h. After NMDA treatment, pyknotic nuclei within the GFP-positive cells were scored (250–300 cells). Data represent mean ± SEM from n = 4 cultures. This experiment was repeated twice with similar results. *, P < 0.05 compared with NMDA treated with control siRNA (ANOVA post-hoc Tukey). Bar, 10 µm.

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