Skip to main content
. 2022 Mar 30;8(13):eabj8658. doi: 10.1126/sciadv.abj8658

Fig. 3. Neuronal C/EBPβ selectively triggers GABAnergic neuronal degeneration, related to figs. S2 to S4.

Fig. 3.

(A) C/EBPβ overexpression promotes GABAnergic neuronal apoptosis. Rat primary neurons (DIV 12) were infected with FOXO1 or C/EBPβ lentivirus for 72 hours. Neurons were fixed and permeabilized; after terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, cells were incubated with vGlut1 and GAD67 antibodies. vGlut1 marked the glutamatergic neurons, and GAD67 marked GABAnergic neurons. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Control, vector virus. Scale bar, 50 μm. OE, overexpression. (B) Quantification of TUNEL-positive neurons induced by C/EBPβ overexpression, and the percentage of glutamatergic or GABAnergic neuron also showed. (Means ± SEM; n = 6; **P < 0.01; unpaired t test with Welch’s correction). (C) Immunoblotting analysis using C/EBPβ-overexpressed neurons. C/EBPβ overexpression inhibited Akt activation and FOXO1 T24 phosphorylation, and it promoted MST1 activation and FOXO1 S212 phosphorylation. The treated neurons from (A) were used for Western blotting (WT) and detected with various indicated antibodies. MW, molecular weight. (D) The LAP/LIP isoform ratios were calculated from quantification by immunoblots of (C). Data are represented as means ± SEM; n = 4 per group. **P < 0.01; two-way ANOVA and Bonferroni’s post hoc test. (E) C/EBPβ regulates p-FOXO1 status. C/EBPβ overexpression inhibited p-FOXO T24 and promoted p-FOXO1 S212 in GABAnergic other than glutamatergic neurons, leading to p-FOXO1 S212–induced GABAnergic neuronal degeneration. Rat primary neurons (DIV 12) were infected with control virus or C/EBPβ lentivirus for 72 hours. Neurons were fixed and permeabilized, and cells were incubated with p-FOXO1, vGlut1, and GAD67 antibodies. The nuclei were stained with DAPI. Control, vector virus. Scale bar, 50 μm. (F) Quantification of p-FOXO1 T24 or p-FOXO1 S212–positive neurons induced by C/EBPβ overexpression, and the percentage of glutamatergic or GABAnergic neurons also showed. (Means ± SEM; n = 6; **P < 0.01; unpaired t test with Welch’s correction).