Neuronal Activity–Induced Gadd45b Promotes Epigenetic DNA Demethylation and Adult Neurogenesis
Dengke K. Ma1,2*,
Junjie U. Guo1,2,
Richard A. Flavell4,
Guo-li Ming1,2,3, and
1 Institute for Cell Engineering, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA.
2 The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA.
3 Department of Neurology, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA.
4 Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520, USA.
5 Department of Immunology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261, USA.
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)].
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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).
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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).
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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).
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