Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NR1

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. were until then the then cultured vitro The experiments were performed from vitro 1 to day- in vitro 16. These analyses were performed on day- in vitro 16 neurons differentiated using undifferentiated ES cells and after five days of differentiation of embryoid bodies into neurons. Dnmt1 transcript levels were determined by quantitative RT-PCR.

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