选择性剪切调控Nf1缺失小鼠成体神经干细胞分化潜能的机制研究

Neural stem cells in the adult mouse brain cannot undergo tri-lineage (neuron, astrocyte and oligodendrocyte) differentiation in vivo, thus whether they can be considered multipotent is still under intense debate. A series of studies including my first-author Cell paper have identified tumor suppressor gene Nf1 as an important regulator of adult neural stem cells. Inactivation of Nf1 unlocks a latent oligodendrocyte lineage potential of adult neural stem cells to produce all three lineages in vivo, but the downstream targets of Nf1-regulated signaling remains unclear. Alternative splicing is an important regulatory mechanism for stem cell differentiation and neural development. However, its roles in adult neural stem cell differentiation have not been systematically investigated. In our preliminary study, we have compared the expression profile of wild-type and Nf1 mutant adult neural stem cells through RNA-seq, and identified 58 genes whose transcripts undergo differential alternative splicing. We further validated 18 genes whose alternative splicing alters neural stem cell differentiation in vitro. Based on these findings, we propose to further investigate the role of these 18 alternatively spliced genes in wild-type, Nf1 mutant, as well as neurological disease models associated with hypoxia-induced oligodendrocyte defects. Using shRNA interference, lineage-tracing, functional and behavioral analysis, we plan to determine whether and how these alternatively spliced genes impact on adult neural stem cell differentiation in vivo. This proposal could elucidate how Nf1-mediated alternative splicing regulates adult neural stem cell differentiation at the cellular and molecular level, and could potentially provide novel insights for the treatment of brain disorders associated with oligodendrocyte defects.

小鼠成体神经干细胞在体内不具有三谱系（神经元、星型胶质细胞和少突胶质细胞）分化能力，其是否具有多能性有很大争议。包括申请人一作Cell论文在内的多篇研究显示，抑癌基因Nf1是抑制成体神经干细胞少突胶质细胞分化潜能的重要基因，但其下游分子机制尚不清楚。选择性剪切是干细胞分化和神经发育的重要调控方式，但其在成体神经干细胞谱系分化中的作用有待系统研究。我们前期通过RNA-seq数据统计出在Nf1缺失成体神经干细胞中有显著性选择性剪切差异的58个基因，并在体外进行了初步验证，筛选出18个功能基因。本项目拟进一步在正常、Nf1条件敲除及缺氧性少突胶质细胞损伤小鼠模型中，通过shRNA干扰和谱系示踪等手段，研究目标基因特定的选择性剪切体在体内如何调控成体神经干细胞的分化潜能。预期将阐明Nf1介导关键基因选择性剪切调控成体神经干细胞分化潜能的细胞和分子机制，并有望对治疗少突胶质细胞相关脑疾病提供新思路。

小鼠成体神经干细胞在体内不具有三谱系（神经元、星型胶质细胞和少突胶质细胞）分化能力，其是否具有多能性有很大争议。Nf1是抑制成体神经干细胞少突胶质细胞分化潜能的重要基因，但其下游分子机制尚不清楚。选择性剪切是干细胞分化和神经发育的重要调控方式，但其在成体神经干细胞谱系分化中的作用有待系统研究。在本项目资助下，我们取得了以下主要成果：1）建立了多时间点野生型和Nf1突变神经干细胞的选择性剪接图谱和生物信息学分析新方法；2）确定了一个高表达Gas1的神经干细胞亚群，在体外细胞培养时更倾向于少突谱系分化，体内生理条件下具有少突胶质和神经元双谱系分化潜能，病理条件下可以长期持续产生少突胶质谱系细胞并修复髓鞘损伤。这些成果对理解神经干细胞的选择性剪接调控和功能异质性提供了新的线索，为治疗髓鞘损伤相关神经系统疾病提供了新思路。本项目共有3篇论文发表或接受，1篇论文在投，培养博士生2名、硕士生2名。.

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