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. 2016 Jul 6;36(27):7253–7267. doi: 10.1523/JNEUROSCI.0319-16.2016

Figure 4.

Figure 4.

miR-124 represses HDAC4/5 and GSK3β expression. A–D, Alignment of potential binding sites for miR-124 in the 3′ UTRs of Hdac4 (A), Hdac5 (B), Gsk3b (C), and Pde4b (D) mRNAs. All of these potential target sequences of miR-124 are conserved among human, mouse, and rat. E, Levels of mRNAs for Hdac4, Hdac5, Gsk3b, and Pde4b in the hippocampus of mice subjected to a 6-week CUMS session or the NS condition receiving either water or IMI (n = 6 mice per group). CUMS-exposed mice receiving water showed elevated Gsk3b mRNA levels. This overexpression was reversed by IMI. *p < 0.05. F, Protein levels of HDAC4, HDAC5, GSK3β, and PDE4B in the hippocampus of mice subjected to 6-week CUMS or the NS condition receiving water or continuous IMI (n = 6 mice per group). CUMS-exposed mice receiving water showed elevated GSK3β protein level, which was reversed by IMI. IMI also reduced HDAC4 and HDAC5 expressions in CUMS-exposed mice compared with water-treated CUMS-exposed mice. *p < 0.05. G–I, Luciferase reporter assays showing that miR-124 targets Hdac4 3′ UTR, Hdac5 3′ UTR, and Gsk3b 3′ UTR. Mutations (MT) in both the miR-124 seed matches (seed positions 1 and 2) in Hdac4 3′ UTR, seed match in Hdac5 3′ UTR, and seed match in Gsk3b 3′ UTR blocked the inhibitory effects of miR-124. *p < 0.05 versus mock control. J. Schematic of the experimental design to demonstrate that miR-124 overexpression also alters the expression levels of HDAC4, HDAC5, and GSK3β in CUMS-exposed mice. Mice were injected with AAV–miR-124 or AAV–GFP into the bilateral hippocampus. After 3 weeks, they were subjected to CUMS for 6 weeks and then killed for expression analyses. K, Levels Hdac4, Hdac5, and Gsk3b mRNAs in the hippocampus of stressed mice injected with AAV–miR-124. CUMS upregulated Gsk3b mRNA in AAV–GFP-injected mice, an effect reversed by AAV–miR-124-induced miR-124 overexpression. n = 6 mice per group. *p < 0.05. L, Levels of HDAC4, HDAC5, and GSK3β proteins in the hippocampus of stressed mice injected with AAV–miR-124 (n = 6 mice per group). CUMS upregulated GSK3β expression in AAV–GFP-injected mice, an effect blocked by AAV–miR-124-induced miR-124 overexpression. miR-124 overexpression also suppressed HDAC4/5 expression in CUMS-exposed mice. *p < 0.05. All data are presented as mean ± SEM.