自噬通过抑制Notch信号通路参与毛细胞再生机制

Notch inhibition is a key signal for the initiation of hair cell regeneration. However, it is still largely unclear how it is regulated, which hampers the development of research in hair cell regeneration. It is well known that autophagy is a degradation pathway to maintain homeostasis. However, it is recently reported that autophagy can be a cell fate-determiner by directly degrading Notch receptor. In our pilot studies, a zebrafish model of hair cell injury and regeneration induced by impulse noise has been established. The hair cells of the saccule and lagena regenerated vigorously 1-2 day after impulse noise exposure. Accordingly, RNA-seq analysis of the saccular and lagenar epithelia shows that autophagy related proteins are up-regulated significantly while the members of Notch pathway suppressed at the same time point. These data strongly support the idea that autophagy may act as an important role in Notch inhibition. For this purpose, we will use the zerbafish model of injury and regeneration of hair cells and the iPSC-derived hair cell model in this proposal. Up-regulation and down-regulation of autophagy using genetic methods or drugs will be performed, and then the members of Notch pathway will be analyzed. The regeneration level of hair cells in inner ear of zebrafish and the differentiation level of iPSCs will be evaluated. Meanwhile, candidate genes will be revealed using zebrafish and iPSC models to explore the autophagy-Notch pathway-hair cell regeneration axis and to understand how autophagy regulates hair cell generation via Notch inhibition. The study will be beneficial for both the theory and technology of hair cell regeneration.

早期Notch信号抑制是毛细胞再生的关键启动环节，但其调控机制依然不详，限制了毛细胞再生研究的深入发展。新近研究显示，自噬不仅能维持细胞稳态，而且能独立于经典途径直接降解Notch分子，决定着细胞分化的方向。我们利用声损伤诱发的斑马鱼内耳毛细胞再生模型，在伤后1-2天观察到显著的毛细胞再生，之后持续存在但逐步下降；听上皮转录组分析显示，伤后1天即观察到显著的自噬活性上调及Notch信号通路活性下调，之后两者逐步恢复正常，强烈提示自噬在毛细胞再生早期Notch信号抑制中扮演重要角色。为此,本研究将使用上述斑马鱼动物模型及iPS细胞向毛细胞分化的细胞模型，通过遗传和药物干预的方法调控自噬活性，检测Notch信号通路分子表达及毛细胞再生水平的变化；并筛选和研究与该调控轴相关的候选基因，阐明自噬通过早期Notch信号抑制参与毛细胞再生的分子机制。本项目将有助于完善毛细胞再生理论与再生技术。

毛细胞再生过程中，早期Notch信号抑制是关键启动环节，但其调控机制依然不详。新近研究显示，自噬不仅能维持细胞稳态，而且能独立于经典途径直接降解Notch分子，决定着细胞分化的方向。我们利用声损伤诱发的斑马鱼内耳毛细胞再生模型，在伤后1-2天观察到显著的毛细胞再生，之后持续存在但逐步下降；听觉上皮转录组分析显示，伤后1天即观察到显著的自噬活性上调及Notch信号通路活性下调，之后两者逐步恢复正常，提示自噬在毛细胞再生早期Notch信号抑制中扮演重要角色。为了对深入明确这一调控机制,本研究使用斑马鱼动物模型、iPS细胞向毛细胞分化的细胞模型及条件基因敲除小鼠进行了损伤-自噬-毛细胞再生的相关研究。本课题研究了毛细胞损伤后Fgf-Mapk/Erk信号激活自噬系统，导致早期 Notch信号抑制，进而促进毛细胞再生的作用机制；建立了系统的斑马鱼外周和中枢听觉损伤及毛细胞、中枢神经细胞再生研究模型，为进一步再生研究奠定基础；制备Atg2a基因敲除斑马鱼、Atg2a条件基因敲除小鼠和人Atg2a基因iPS细胞系，为自噬和听力关系研究提供了多种研究模型。本课题研究提示，自噬参与毛细胞再生为该领域的研究提供了新的思路。