线虫MYRF-1通过内质网形态构架调控突触重塑的机制研究

During development, circuits are reorganized through the dynamic addition and removal of synapses, collectively called synaptic remodeling, which are essential steps towards the circuitry refinement and maturity. Despite its universality, much remains to be learned about the molecular mechanisms underlying synaptic remodeling. We took advantage of C. elegans as a genetic model to study synaptic remodeling in DD motor neurons. Through forward genetic screening we identified myrf-1 as the essential gene regulating DD synaptic remodeling. MYRF is highly conserved throughout the metazoan, and its molecular function is poorly defined. C. elegans MYRF-1 is localized to endoplasmic reticulum (ER), but its N’-terminal fragment can be released upon auto-cleavage and translocate into the nucleus. MYRF-1’s nuclear function is essential for synaptic remodeling. The DDs of myrf-1 mutant show defective ER morphology in processes and disrupted ER polarity in soma. Our proposed research is to establish fluorescent markers to label ER specializations and examine specific distributions of the ER sheets and tubules in remodeling DD. We will determine whether perturbations of the ER dynamics will lead to the alteration of the remodeling process. We will employ ChIP-Seq to analyze the DNA binding sites of MYRF-1, as well as FACS-RNA-Seq to identify downstream targets of MYRF-1. Our work will provide critical insights into understanding the synaptic plasticity during development and help to build a solid foundation for better treatment for relevant neurological diseases.

突触重塑是指通过突触形成和突触消除来重组现有神经回路的过程，是大脑突触连接精细化的必要步骤，在早期发育中十分普遍，但是对其分子机理所知甚少。我们借助于线虫发育中的DD神经元，建立了独到的突触重塑的分子遗传模型。我们通过筛选突变体鉴别到调控突触重塑的核心基因myrf-1，该基因在动物界高度保守，其功能缺陷导致突触重塑严重阻断。MYRF-1定位于内质网上，发生自我剪切后其N’端片段入核，参与调控突触重塑。MYRF-1蛋白功能缺陷导致内质网的形态和极性异常。本申请将以分子遗传学手段建立对神经元内质网特异形态的观察和干扰体系，探索内质网形态构架及动态变化和突触重塑之间的关联。本项目将深入开展对于myrf-1相关分子途径的研究，以ChIP-Seq结合DD特异表达谱来鉴别MYRF-1的下游转录靶点，建立调节突触重塑的分子途径。本研究探索突触发育的可塑性原理，并为了解神经发育疾病提供重要信息。

神经突触重塑是神经回路发育中的必要步骤，这个过程是由内源程序和环境信号协同控制的。线虫DD神经元在发育中出现树突和轴突功能域的反转，但其细胞形态没有明显变化，这给研究突触重塑的机制提供了一个优秀的分子遗传学模型。我们鉴别到线虫的突触重塑受保守的、跨膜的转录因子MYRF家族蛋白控制。MYRF如何调控突触重塑未知。并且，MYRF对动物发育（包括线虫、小鼠）是必需的。近年来多项研究表明MYRF的单倍体功能缺陷导致多种发育紊乱综合症。但是MYRF如何调控发育未知。我们就MYRF在发育中的调控机制展开了研究。之前在细胞系中获得的数据显示：MYRF定位于内质网，发生自动剪切，释放N-MYRF，入核，进行转录调控。我们的数据表明：MYRF定位于细胞膜上，在发育过程的特定阶段从细胞上释放，并入核。MYRF定位在细胞膜上是线虫发育所必需的。我们鉴别到富含亮氨酸的重复结构域蛋白PAN-1是细胞膜上MYRF的互作因子，PAN-1和MYRF通过胞外结构域相互作用。PAN-1的缺失能够消除MYRF在细胞膜上的定位，从而阻断了突触重塑和幼虫的发育。因此，通过与细胞膜上的协同因子的相互作用，MYRF可能将细胞外信号与转录过程连接起来以促进发育。我们的发现揭示了MYRF蛋白活性的重要机制，为了解神经发育疾病提供了重要信息。