Virus-mediated Dnmt1 and Dnmt3a deletion disrupts excitatory synaptogenesis and synaptic function in primary cultured hippocampal neurons.
Virus-mediated Dnmt1 and Dnmt3a deletion disrupts excitatory synaptogenesis and synaptic function in primary cultured hippocampal neurons.
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DOI：
10.1016/j.bbrc.2020.03.094
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发表时间：
2020-03
影响因子：
3.1
通讯作者：
Wei-yang Sun;Q. Kong;Meng Zhang;Xue Mi;Xiaomin Sun;Ming Yu;Tengbo Yu;Yu Zhou
中科院分区：
文献类型：
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作者：
Wei-yang Sun;Q. Kong;Meng Zhang;Xue Mi;Xiaomin Sun;Ming Yu;Tengbo Yu;Yu Zhou
Dnmt 1、Dnmt 3a和Dnmt 3是编码DNA甲基转移酶（DNA methyltransferases，Dnmts）的主要基因，它们催化DNA甲基化，并在不改变DNA序列的情况下调节基因表达。我们的前期研究发现，Dnmt 1和Dnmt 3基因双敲除可导致前脑兴奋性神经元突触可塑性受损，并导致海马区依赖性学习记忆障碍，但其机制尚不清楚。本研究通过转染Cre表达病毒，选择性地下调Dnmt 1，3a 2 flox/2a 3小鼠原代培养海马神经元中Dnmt 1和Dnmt 3的表达，研究Dnmt 1和Dnmt 3介导的DNA甲基化对突触发生和突触功能的影响。结果发现，病毒介导的Dnmt 1和Dnmt 3去除后，体外培养15天（DIV 15）的海马神经元胞体大小相似，但树突变长，分支数目减少，兴奋性突触形成密度降低。支持，培养的神经元与Dnmt 1和Dnmt 3a缺陷显示减少的频率和幅度的微型兴奋性突触后电流（mEPSC），这表明前和突触后功能障碍的参与。此外，我们用Rhod-3AM进行的Ca ~（2+）图像研究显示，Dnmt 1和Dnmt 3缺失后谷氨酸诱发的胞浆[Ca ~（2+）]升高受到抑制。总之，我们的研究结果提供了新的证据，Dnmt 1和Dnmt 3在海马神经元的正常表达是必要的兴奋性突触的发生和突触功能。
Dnmt1,Dnmt3aandDnmt3bare main genes encoding DNA methyltransferases (Dnmts) which catalyze DNA methylation and regulate gene expression without changing DNA sequence. Our previous study disclosed that double knockout ofDnmt1andDnmt3ain forebrain excitatory neurons impaired synaptic plasticity and led to hippocampus-dependent learning and memory deficits, however the underlying synaptic mechanisms remain uncertain. In this study, we selectively knocked down the expression ofDnmt1andDnmt3ain primary cultured hippocampal neurons derived from embryonicDnmt1,3a2flox/2floxmice by transfection with Cre-expressing virus, to study the effect of Dnmts and mediated DNA methylation on synaptogenesis and synaptic function. We found that the hippocampal neurons at 15 daysin vitro(DIV15) exhibited similar size of cell body, but longer dendrites with reduced number of branches and lower density of excitatory synapses formation after virus-mediatedDnmt1andDnmt3adeletion. Supportively, cultured neurons with Dnmt1 and Dnmt3a deficiency displayed reduced frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), indicating that both pre- and post-synaptic dysfunction are involved. In addition, our Ca2+-image study with Rhod-3AM revealed suppression of glutamate-evoked elevation of cytoplasmic [Ca2+] afterDnmt1 and Dnmt3adeletion. Altogether our findings provide new evidence that normal expression ofDnmt1andDnmt3ain hippocampal neurons are essential for excitatory synaptogenesis and synaptic function.