Figure 4. AMPK is activated during excitotoxic apoptosis and is sufficient to induce bim expression. (A) Neocortical neurons were treated with 100 µM NMDA/10 µM Gly or sham conditions for 5 min and allowed to recover for the indicated time periods. ATP levels were assessed and expressed relative to sham-treated controls. Data represent mean ± SEM from n = 6 cultures. This experiment was repeated twice with similar results. *, P < 0.05 compared with sham-treated cultures (ANOVA post-hoc Tukey). (B) Chromatograms of adenine nucleotides on reverse-phase column from sham (left) and 100 µM glutamate/10 µM Gly for 10 min (right). (C) Western blotting of phospho Thr172 AMPK levels in neocortical neurons at the indicated time periods after NMDA excitation. Total AMPK served as a loading control. Results are representative of at least two independent experiments. (D) Western blotting of phospho Thr172 AMPK levels in CGNs at the indicated time periods after glutamate excitation. Probing for β-actin served as loading control. Results are representative of at least two independent experiments. The vertical black line indicates that intervening lanes have been spliced out. (E) Neocortical neurons were treated with 2.5 mM AICAR for the indicated time periods. The levels of phospho Thr72 AMPK (p-AMPK), AMPK, Bim, and β-actin (loading control) were assessed by Western blotting. Results are representative of at least two independent experiments. (F) CGNs transfected with a vector containing a 0.8-kB fragment of the bim promoter and either a vector expressing CA-AMPK or control empty vector (control). Luminescence activity was normalized to the activity of the cotransfected TK-Renilla luciferase. Mean ± SEM is shown. *, P < 0.05 compared with control vector (t test). (G) Neocortical neurons from WT and bim−/− mice were cotransfected with GFP and either a vector expressing CA-AMPK or a control empty vector, and the levels of pyknotic nuclei in 150–200 neurons per culture were assessed 24 h after transfection. Data represent mean ± SEM from n = 4 cultures per treatment. This experiment was repeated twice with similar results. *, P < 0.05 compared with CA-AMPK–transfected WT neurons (ANOVA post-hoc Tukey).
Figure 4.

AMPK is activated during excitotoxic apoptosis and is sufficient to induce bim expression. (A) Neocortical neurons were treated with 100 µM NMDA/10 µM Gly or sham conditions for 5 min and allowed to recover for the indicated time periods. ATP levels were assessed and expressed relative to sham-treated controls. Data represent mean ± SEM from n = 6 cultures. This experiment was repeated twice with similar results. *, P < 0.05 compared with sham-treated cultures (ANOVA post-hoc Tukey). (B) Chromatograms of adenine nucleotides on reverse-phase column from sham (left) and 100 µM glutamate/10 µM Gly for 10 min (right). (C) Western blotting of phospho Thr172 AMPK levels in neocortical neurons at the indicated time periods after NMDA excitation. Total AMPK served as a loading control. Results are representative of at least two independent experiments. (D) Western blotting of phospho Thr172 AMPK levels in CGNs at the indicated time periods after glutamate excitation. Probing for β-actin served as loading control. Results are representative of at least two independent experiments. The vertical black line indicates that intervening lanes have been spliced out. (E) Neocortical neurons were treated with 2.5 mM AICAR for the indicated time periods. The levels of phospho Thr72 AMPK (p-AMPK), AMPK, Bim, and β-actin (loading control) were assessed by Western blotting. Results are representative of at least two independent experiments. (F) CGNs transfected with a vector containing a 0.8-kB fragment of the bim promoter and either a vector expressing CA-AMPK or control empty vector (control). Luminescence activity was normalized to the activity of the cotransfected TK-Renilla luciferase. Mean ± SEM is shown. *, P < 0.05 compared with control vector (t test). (G) Neocortical neurons from WT and bim−/− mice were cotransfected with GFP and either a vector expressing CA-AMPK or a control empty vector, and the levels of pyknotic nuclei in 150–200 neurons per culture were assessed 24 h after transfection. Data represent mean ± SEM from n = 4 cultures per treatment. This experiment was repeated twice with similar results. *, P < 0.05 compared with CA-AMPK–transfected WT neurons (ANOVA post-hoc Tukey).

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