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. 2021 Jan 8;24(2):102043. doi: 10.1016/j.isci.2021.102043

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© 2021 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

14-3-3ε prevents stress-induced depressive phenotype via rescue of Bad-mediated apoptosis in VLO

(A) Schematic of experiment. Mice were injected with virus that expressed 14-3-3ε (AAV9-CaMKIIa-bGlobin-14-3-3ε-eGFP-3FLAG; n = 32) or the GFP control virus (AAV9-CaMKIIa-bGlobin-eGFP-3FLAG; n = 30) into bilateral VLO. Then, these mice were allowed to recover for 2 weeks to allow for adequate gene expression before 8 weeks of CUMS exposure. At the end of CUMS regime, the behavioral tests were carried out to assess the depressive phenotype and molecular changes in VLO.

(B) Left: eGFP-labeled cells was observed in the VLO 80 days after injection of AAV-CaMKIIa-14-3-3ε-eGFP. Scale bar = 1 mm. Right: immunoblot showing the expression level of 14-3-3ε in VLO was significantly increased by the 14-3-3ε overexpressing virus (n = 8 per group; unpaired t test, t ₁₄ = 4.871, p < 0.01).

(C) CUMS mice with 14-3-3ε overexpression in VLO exhibited increased sucrose preference in SPT (one-way ANOVA, followed by Tukey's post hoc, 14-3-3ε versus GFP, t ₈₀ = 3.893, p < 0.05) and open arm time in EPM (one-way ANOVA, Tukey's post hoc, 14-3-3ε versus GFP, t ₈₀ = 3.698, p < 0.05), and significantly decreased immobility time in the FST (one-way ANOVA, Tukey's post hoc, 14-3-3ε versus GFP, t ₈₀ = 3.942, p < 0.05).

(D) 14-3-3ε overexpression within VLO significantly changed the constituent ratio of depressive phenotypes (Fisher's exact test, p < 0.05) and effectively reduced the number of Dep (D-3) mice. No-depressive mice (noDep) were defined as those with D-score 0, 1 and 2.

(E) 14-3-3ε overexpression mice have obvious body weight gains than GFP mice (one-way ANOVA, Tukey's post hoc, 14-3-3ε versus GFP, t ₈₀ = 8.836, p < 0.0001).

(F) TrkB and phospho-Akt^Thr308 were decreased in both GFP and 14-3-3ε overexpressing mice compared with the non-stressed Ctrl mice. Phosphorylated Akt was expressed as phosphorylated Akt to total Akt. Protein expression levels were analyzed with one-way ANOVA, followed by Tukey's post hoc test.

(G) Co-immunoprecipitation revealed that 14-3-3ε specifically bound to Bad. The GFP group has similar ratio of 14-3-3ε to Bad with that in the non-stressed Ctrl mice. The ratio of 14-3-3ε to Bad is significantly higher in the 14-3-3ε overexpression group than that in the GFP group (n = 6–8 per group; one-way ANOVA, Tukey's post hoc, 14-3-3ε versus GFP, t ₁₇ = 5.786, p < 0.01).

(H) The expression of the apoptotic markers such as Bax, Cyt c, and cleaved caspase-3 were increased in the GFP mice and decreased by 14-3-3ε overexpression in VLO of CUMS mice. Protein levels were analyzed with one-way ANOVA, followed by Tukey's post hoc test. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001 compared between the two indicated groups.