调控MeCP2促进小鼠神经干细胞向成熟多巴胺神经元分化的研究

Cell replacement therapy is highly promising for its potential application in the treatment of Parkinson’s disease (PD). The most significant question to answer is how to make cells for transplantation both phenotypically dopaminergic and functionally neuronal. Epigenetic mechanisms have been shown to underlie the transcriptional regulation of dopamine (DA)-related genes in midbrain DA neuron development. Our previous studies (Stem Cells, 2011; Development, 2014; Stem Cells, 2015) demonstrated that MeCP2, an epigenetic regulator related with DNA methylation, was involved in stimuli-induced DA neuron differentiation and responsible for the inhibition of DA phenotype-specific gene expression in the early stage of neural stem cell (NSC) differentiation. Meanwhile, MeCP2 is highly expressed in mature neurons, indicating its key roles in neuronal functionality. We propose here that a spatio-temporally conditional regulation of MeCP2 might facilitate mouse NSCs differentiating towards large-scale mature DA neurons. The protocol will be concisely optimized in regard of the level and period of MeCP2 expression during the early and late stage of differentiation. What’s more, we will also study the underlying epigenetic mechanism of MeCP2's regulation in those stages of DA neuron differentiation. This study will reveal new roles of MeCP2 in DA neuron differentiation and maintenance, further our understanding of PD and MeCP2-related neurological disorders, and offer better strategy for the cell replacement therapy in hope of PD treatment.

细胞替代治疗是当今极具前景的治疗帕金森病手段。用于移植的多巴胺（DA）神经元必须是高得率的DA表型和功能成熟的神经元。研究如何获得能满足这两条件的DA神经元具有重要科学意义及临床价值。我们新近多项研究（Stem Cells 2011，2015， Development 2014）表明，表观遗传调控机制在神经干细胞（NSC）向DA神经元分化中起关键作用。MeCP2在NSC分化前期是抑制DA表型相关基因表达，而其在分化后期成熟神经元中呈高表达，并参与神经元的各种功能，可见其在NSC分化前期和神经元分化后期的作用不同。本研究拟通过条件性调控来影响MeCP2在分化前、后期表达，促进小鼠NSC向DA神经元分化，以获得高得率DA表型且有成熟神经元功能的DA细胞。我们采用的是不同以往的调控DA神经元分化新思路，研究结果将为深入理解DA神经元发育机制及开展DA神经元细胞替代疗法提供新的理论基础和技术支持。

诱导神经干细胞向中脑多巴胺神经元分化是干细胞疗法治疗帕金森病的关键步骤，技术难点集中在如何获得高得率、具有成熟神经功能的中脑多巴胺神经元。识别鉴定影响这一过程的关键分子并阐明其作用机制，对推进这一技术的发展具有重要的科学意义及应用价值。本研究结果显示，在时间和表达量维度上维持MeCP2在神经干细胞中的正常表达对其多巴胺表型特化具有重要的影响，而在中脑多巴胺神经元分化后改变MeCP2表达水平则不再能影响多巴胺表型的正常表达，证实MeCP2是调控中脑多巴胺神经元发育分化的重要因子，具有神经干细胞特异的生物学功能。共过表达实验证实，MeCP2通过干扰中脑多巴胺神经元表型获得的关键转录因子Nurr1，而抑制神经干细胞的多巴胺表型特化。通过一系列表观遗传学相关实验，本研究更进一步显示，MeCP2在不同阶段的差异作用与多巴胺表型基因在神经干细胞与中脑多巴胺神经元这两个不同发育阶段中的DNA甲基化程度差异密切相关，提示多巴胺表型基因DNA甲基化或是调控中脑多巴胺神经元分化的关键靶点。通过CRISPR-dCas9技术靶向多巴胺表型基因的DNA甲基化位点，能够改变MeCP2在不同阶段对多巴胺表型基因表达的影响，这一结果不仅验证了以上假设，更提示DNA甲基化或为中脑多巴胺神经元功能失常的风险因素。另一方面，本研究更探索了Rett综合症相关的多种MeCP2蛋白突变体及MeCP2转录抑制复合物其他成分CoREST、Sin3A等在以上研究对象中发挥的作用，为深入开展后续系列研究打下了较为充实的实验和理论基础。总而言之，本研究首次较为系统地阐明了MeCP2在中脑多巴胺神经元发育分化中的作用并揭示了其分子机制，为理解多巴胺神经元相关疾病如帕金森病的发病机制、寻求通过干细胞及表观遗传手段改善治疗此类疾病的疗法提供了创新思路。