Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NMDA/NR1 subunit

Abstract

High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD(67) and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1(tet/tet) mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1(tet/tet) ES cell lines, neurons derived from Dnmt1(tet/tet) cells showed abnormal dendritic arborization and branching. The Dnmt1(tet/tet) neuronal cells also showed elevated levels of functional N-methyl d-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD(67) in neuronal networking and excitatory/inhibitory balance, respectively, we studied methylation of these genes' promoters in Dnmt1(tet/tet) ES cells and neurons. Both reelin and GAD(67) promoters were not hypermethylated in the Dnmt1(tet/tet) ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.