Larimore Jennifer L, Chapleau Christopher A, Kudo Shinichi, Theibert Anne, Percy Alan K, Pozzo-Miller Lucas
Department of Neurobiology, Evelyn McKnight Brain Institute, Civitan International Research Center, The University of Alabama at Birmingham, 35294-2182, USA.
Neurobiol Dis. 2009 May;34(2):199-211. doi: 10.1016/j.nbd.2008.12.011. Epub 2009 Jan 3.
The expression of the methylated DNA-binding protein MeCP2 increases during neuronal development, which suggests that this epigenetic factor is crucial for neuronal terminal differentiation. We evaluated dendritic and axonal development in embryonic day-18 hippocampal neurons in culture by measuring total length and counting branch point numbers at 4 days in vitro, well before synapse formation. Pyramidal neurons transfected with a plasmid encoding a small hairpin RNA (shRNA) to knockdown endogenous Mecp2 had shorter dendrites than control untransfected neurons, without detectable changes in axonal morphology. On the other hand, overexpression of wildtype (wt) human MECP2 increased dendritic branching, in addition to axonal branching and length. Consistent with reduced neuronal growth and complexity in Rett syndrome (RTT) brains, overexpression of human MECP2 carrying missense mutations common in RTT individuals (R106W or T158M) reduced dendritic and axonal length. One of the targets of MeCP2 transcriptional control is the Bdnf gene. Indeed, endogenous Mecp2 knockdown increased the intracellular levels of BDNF protein compared to untransfected neurons, suggesting that MeCP2 represses Bdnf transcription. Surprisingly, overexpression of wt MECP2 also increased BDNF levels, while overexpression of RTT-associated MECP2 mutants failed to affect BDNF levels. The extracellular BDNF scavenger TrkB-Fc prevented dendritic overgrowth in wt MECP2-overexpressing neurons, while overexpression of the Bdnf gene reverted the dendritic atrophy caused by Mecp2-knockdown. However, this effect was only partial, since Bdnf increased dendritic length only to control levels in mutant MECP2-overexpressing neurons, but not as much as in Bdnf-transfected cells. Our results demonstrate that MeCP2 plays varied roles in dendritic and axonal development during neuronal terminal differentiation, and that some of these effects are mediated by autocrine actions of BDNF.
甲基化DNA结合蛋白MeCP2的表达在神经元发育过程中增加，这表明这种表观遗传因子对神经元终末分化至关重要。我们通过在体外培养4天时测量胚胎第18天海马神经元的总长度并计算分支点数，评估了树突和轴突的发育情况，此时突触形成尚未开始。用编码小发夹RNA（shRNA）的质粒转染以敲低内源性Mecp2的锥体神经元，其树突比未转染的对照神经元短，轴突形态未检测到变化。另一方面，野生型（wt）人MECP2的过表达除了增加轴突分支和长度外，还增加了树突分支。与雷特综合征（RTT）大脑中神经元生长减少和复杂性降低一致，携带RTT个体常见错义突变（R106W或T158M）的人MECP2的过表达减少了树突和轴突长度。MeCP2转录调控的靶标之一是Bdnf基因。实际上，与未转染的神经元相比，内源性Mecp2敲低增加了BDNF蛋白的细胞内水平，表明MeCP2抑制Bdnf转录。令人惊讶的是，wt MECP2的过表达也增加了BDNF水平，而RTT相关MECP2突变体的过表达未能影响BDNF水平。细胞外BDNF清除剂TrkB-Fc可防止wt MECP2过表达神经元中的树突过度生长，而Bdnf基因的过表达可逆转由Mecp2敲低引起的树突萎缩。然而，这种作用只是部分的，因为Bdnf仅将树突长度增加到突变MECP2过表达神经元中的对照水平，但不如在Bdnf转染细胞中增加得多。我们的结果表明，MeCP2在神经元终末分化过程中的树突和轴突发育中发挥多种作用，其中一些作用是由BDNF的自分泌作用介导的。
