DNA methylation mechanism of intracellular zinc deficiency-induced injury in primary hippocampal neurons in the rat brain

Objective: To explore Zn2+ deficiency-induced neuronal injury in relation to DNA methylation, providing valuable data and basic information for clarifying the mechanism of Zn2+ deficiency-induced neuronal injury. Methods: Cultured hippocampal neurons were exposed to the cell membrane-permeant Zn2+ chelator N,N,N,N-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (2 mu M), and to TPEN (2 mu M) plus ZnSO4 (5 mu M) for 24 hours. We analyzed intracellular Zn2+ levels, neuronal viability, and protein/mRNA levels for DNA (cytosine-5) methyltransferase 1 (DNMT1), DNA (cytosine-5-) methyltransferase 3 alpha (DNMT3a), methyl CpG binding protein 2 (MeCP2), Brain-derived neurotrophic factor (BDNF), and growth arrest and DNA-damage-inducible, beta (GADD45b) in the treated neurons. Results: We found that exposure of hippocampal neurons to TPEN (2 mu M) for 24 hours significantly reduced intracellular Zn2+ concentration and neuronal viability. Furthermore, DNMT3a, DNMT1, BDNF, and GADD45b protein levels in TPEN-treated neurons were significantly downregulated, whereas MeCP2 levels were, as expected, upregulated. In addition, DNMT3a and DNMT1 mRNA levels in TPEN-treated neurons were downregulated, while MeCP2, GADD45b, and BDNF mRNA were largely upregulated. Addition of ZnSO4 (5 mu M) almost completely reversed the TPEN-induced alterations. Conclusion: Our data suggest that free Zn2+ deficiency-induced hippocampal neuronal injury correlates with free Zn2+ deficiency-induced changes in methylation-related protein gene expression including DNMT3a/ DNMT1/MeCP2 and GADD45b, as well as BDNF gene expression.