Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis

Abstract

The mammalian brain exhibits diverse types of neural plasticity, including activity-dependent neurogenesis in the adult hippocampus. How transient activation of mature neurons leads to long-lasting modulation of adult neurogenesis is unknown. Here we identify Gadd45b as a neural activity-induced immediate early gene in mature hippocampal neurons. Mice with Gadd45b deletion exhibit specific deficits in neural activity-induced proliferation of neural progenitors and dendritic growth of newborn neurons in the adult hippocampus. Mechanistically, Gadd45b is required for activity-induced DNA demethylation of specific promoters and expression of corresponding genes critical for adult neurogenesis, including brain-derived neurotrophic factor and fibroblast growth factor. Thus, Gadd45b links neuronal circuit activity to epigenetic DNA modification and expression of secreted factors in mature neurons for extrinsic modulation of neurogenesis in the adult brain.

Fig. 1

Activity-induced neuronal Gadd45b expression. (A) Quantitative polymerase chain reaction (Q-PCR) analysis of ECT-induced expression of Gadd45a, Gadd45b, and Gadd45g in the adult dentate gyrus after a single ECT. (B) Sample images of Gadd45b in situ hybridization of the adult hippocampus after ECT. Scale bar: 0.5 mm. (C and D) Gadd45 induction in the dentate gyrus after 1 hour of spatial exploration of a novel environment. (C) A summary from Q-PCR analysis. (D) Sample confocal images of Gadd45b in situ hybridization, and 4′,6-diamidino-2-phenylindole (DAPI) and Arc immunostaining. Most of the Gadd45b-positive cells (open and closed arrowheads) were Arc-positive (closed arrowheads). Scale bar: 50 μm. (E) NMDAR-dependent induction of Gadd45b, Arc, and Homer1 in the adult dentate gyrus at 1 hour after ECT. The NMDAR antagonist CPP was injected intraperitoneally at 1 hour before ECT (10 mg per kg of body weight). Values represent mean ± SEM [n = 4 animals; *P < 0.01, analysis of variance (ANOVA)].

Fig. 2

Essential role of Gadd45b in activity-induced proliferation of adult neural progenitors. (A) Sample projected confocal images of BrdU immunostaining (red) and DAPI (blue). Scale bar: 50 μm. (B) Summary of stereological quantification of BrdU⁺ cells in the dentate gyrus. Values represent mean ± SEM (n = 4 or 5 animals as indicated; *P < 0.01, ANOVA).

Fig. 3

Essential role of Gadd45b in activity-induced dendritic development of newborn neurons in the adult brain. (A) Sample projected Z-series confocal images of GFP⁺ dentate granule cells at 14 days after viral labeling. Scale bar: 50 μm. (B) Quantification of the total dendritic length of GFP⁺ dentate granule cells. Values represent mean ± SEM (n = 23 to 45 neurons for each condition; *P < 0.01, ANOVA). (C) Analysis of dendritic complexity of the same group of cells as in (B). Values represent mean ± SEM (*P < 0.01, Student's t test).

Fig. 4

Essential role of Gadd45b in activity-induced specific DNA demethylation and gene expression in the adult dentate gyrus. (A and B) Bisulfite sequencing analysis of adult dentate gyrus tissue before or at 4 hours after ECT. (A) A schematic diagram of the genomic region subjected to analysis and a summary of methylation frequency at individual CpG sites. (B) A summary of mean DNA methylation levels of individual alleles. Values represent mean ± SEM (n = 10 to 15 reads; **P < 0.01, *P < 0.05, ^#P > 0.1, ANOVA; exact P values are given in table S2). (C) Methylation-specific PCR analysis from the dentate gyrus of WT mice after one or two ECTs (“24+4”: 4 hours after two ECTs at 24 hours apart) or 7 days after lentivirus-mediated expression of Gadd45b-GFP or GFP alone without ECT. Primers are specific for methylated (M) and unmethylated alleles (UM) of the Fgf-1B promoter, or for bisulfite sequencing without CpGs (Input). (D) Summary of the mRNA and protein expression in the dentate gyrus of adult Gadd45b WT and KO mice at 4 hours after ECT and sham controls. Values represent mean ± SEM (n = 4 animals; *P < 0.01, ANOVA).