关键词: DNA methylation, Dnmt1, Dnmt3a, Dnmt3b, learning and memory, hippocampus

Abstract: Objective: DNA methylation is one of the most important epigenetic modulation, which is catalyzed primarily by three methyltransferases, DNMT1, DNMT3a and DNMT3b. The aim of our study is to investigate the modulatory effect of DNMTs on hippocampus-dependent memory formation, and to explore the underlying molecular, cellular and synaptic mechanisms. Methods:  Dnmt1, 3a and 3b were selectively deleted in the CA1 region of dorsal hippocampus by Cre/LoxP recombinase systerm, either with virus-mediated Cre expression in CA1 of Dnmtsflox/flox mice or conditional αCaMKII-Cre; Dnmtsflox/flox mice. Hippocampus-dependent and hippocampus-independent memory performance were evaluated in adult KO and control mice. RNA-seq analysis was conducted to screen differential expression genes in the hippocampus after conditional Dnmts knockout. Real-time qPCR and Western blot analysis were used to confirm the differential expression. We also analyzed the alteration in DNA methylation by Whole Genome Bisulfite Sequencing. Neuronal excitability, synaptic transmission and plasticity were measured in CA1 pyramidal neurons of hippocampal slices. Finally, we checked whether virus-mediated shRNA expression in hippocampal CA1 could ameliorate abnormal synaptic function and memory deficit observed in αCaMKII-Cre; Dnmstflox/flox mice. Results: We found that both Dnmt1flox/floxDnmt3aflox/flox mice and Dnmt3bflox/flox mice receiving AAV-Cre virus infection into CA1 region displayed recognition memory deficit to object place, but normal memory to novel object. All mice showed similar performance in fear memory tests. Also, virus infection and Dnmts deletion did not changed anxiety- or depression-like behavior. The object place recognition memory deficit was also observed in both αCaMKII-Cre; Dnmt1flox/floxDnmt3aflox/flox mice and αCaMKII-Cre; Dnmt3bflox/flox mice. The Cre expression in αCaMKII-Cre mice was verified to be dominant in hippocampal CA1. RNA-seq based gene expression and followed real-time qPCR and western blot analysis confirmed significant upregulation of certain genes after Dnmts deletion in aCaMKII-expression excitatory neurons in the hippocampus. WGBS analysis showed differentiated DNA methylation in related genes. Normal basal synaptic transmission but impaired LTP was observed in SC-CA1 path of both αCaMKII-Cre; Dnmt1flox/floxDnmt3aflox/flox mice and αCaMKII-Cre; Dnmt3bflox/flox mice. AAV-virus mediated specific shRNA expression in CA1 region of dorsal hippocampus interfered upregulation of candidate genes, rescued abnormal synaptic function, and ameliorated object place cognition impairment in both αCaMKII-Cre;Dnmt1flox/floxDnmt3aflox/flox mice and αCaMKII-Cre;Dnmt3bflox/flox mice. Virus-mediated shRNA expression in CA1 region of dorsal hippocampus did not affect recognition memory to novel object. Conclusion: In conclusion, our findings suggest that Dnmts in CA1 excitatory neurons plays an important role in regulating synaptic function and hippocampus-dependent recognition memory process by control the expression of certain target genes.

Key words: DNA methylation, Dnmt1, Dnmt3a, Dnmt3b, learning and memory, hippocampus