The Role of Wrb in Vertebrate Ribbon Synapse Formation

描述（由申请人提供）：脊椎动物光感受器和感觉毛细胞的突触通过称为突触带的特殊结构释放神经递质。在筛选影响斑马鱼视觉功能的插入突变时，我们鉴定出斑马鱼wrb突变体，该突变体表现出光感受器变性和听力丧失。这种突变破坏了wrb基因，该基因编码一种新的170个氨基酸的蛋白质，没有已知的功能。在初步研究中，我们发现wrb突变体表现出带状突触成分定位错误，光感受器突触上的停泊带数量减少，ERG反应显著降低。这些数据有力地表明Wrb可能完整地参与了带状突触的组装和/或功能。为了验证这一假设，我们将通过组织学和分子方法的结合来研究Wrb在斑马鱼突触功能中的作用。在Aim 1中，我们将使用转基因方法确定Wrb的亚细胞定位，我们将确定Wrb是否影响发育过程中的带状组装，Wrb的缺失如何影响突触后细胞的树突形态，以及Wrb是否阻断突触囊泡释放或膜内吞噬。在目标2中，我们将使用转基因方法纯化标记的Wrb复合物并随后通过质谱分析来鉴定Wrb的结合伙伴。这些研究将为Wrb的作用提供新的见解，Wrb是一种新的蛋白质，可能是带状突触结构和功能的关键组成部分。Wrb结合伙伴的身份和Wrb在体内系统的后续表征将为突触结构的研究开辟新的途径，并可能为盲/耳聋疾病的候选基因提供见解。

DESCRIPTION (provided by applicant): The synapses of vertebrate photoreceptors and sensory hair cells release neurotransmitter through specialized structures known as synaptic ribbons. In a screen for insertional mutations affecting visual function in zebrafish, we identifie the zebrafish wrb mutant, which exhibited photoreceptor degeneration and hearing loss. The mutation disrupts the wrb gene, which encodes a novel 170 amino acid protein with no known function. In preliminary studies, we found that wrb mutants exhibited mislocalization of ribbon synapse components, reduced numbers of docked ribbons at photoreceptor synapses, and significantly reduced ERG responses. These data strongly suggest that Wrb could be integrally involved in ribbon synapse assembly and/or function. To validate this hypothesis, we will study the role of Wrb in zebrafish synaptic function through a combination of histological and molecular approaches. In Aim 1, we will identify the subcellular localization of Wrb using transgenic approaches, we will determine if Wrb affects ribbon assembly during development, how loss of Wrb affects dendritic morphology of postsynaptic cells, and whether Wrb blocks synaptic vesicle release or membrane endocytosis. In Aim 2, we will identify binding partners of Wrb using a transgenic approach to purify tagged Wrb complexes and subsequent analysis by mass spectroscopy. These studies will provide insights into the role of Wrb, a novel protein that may be a critical component to ribbon synapse structure and function. The identity of Wrb binding partners and the subsequent characterization of Wrb in an in vivo system will open new avenues of research on synaptic architecture and possibly provide insight into candidate genes for blindness/deafness disorders. PUBLIC HEALTH RELEVANCE: Both photoreceptors and hair cells of the inner ear transmit information to downstream neurons through specialized structures termed ribbon synapses. The precise composition and functional dynamics of ribbon synapses are poorly understood. The zebrafish wrb mutant shows defects in ribbon synapse structure and this mutant affects a novel gene with no known function. Investigations into the cellular consequences of mutating the wrb gene and identifying protein binding partners of the Wrb protein will provide insight into the mechanisms that lead to blindness and deafness.