The Role of Wrb in Vertebrate Ribbon Synapse Formation

Wrb 在脊椎动物带状突触形成中的作用

• 批准号: 8301306
• 负责人: Brian D Perkins
• 金额: --
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8301306
• 关键词: Affect; Afferent Neurons; Affinity Chromatography; Amino Acids; Anatomy; Antibodies; Architecture; Auditory; Binding; Biochemistry; Biological Assay; Blindness; Candidate Disease Gene; Cells; Coiled-Coil Domain; Communication; Complex; Data; Defect; Development; Disease; Docking; Ear; Electron Microscopy; Endocytosis; Exhibits; Functional disorder; Gene Expression; Genes; Hair Cells; Hearing; Human; Immunohistochemistry; In Situ Hybridization; Investigation; Knowledge; Labyrinth; Lead; Learning; Mass Spectrum Analysis; Membrane; Membrane Potentials; Modeling; Molecular; Molecular Probes; Morphology; Mutate; Mutation; Neurons; Perception; Phenotype; Photoreceptors; Property; Protein Binding; Proteins; Reagent; Research; Retina; Role; Sensory; Sensory Hair; Stimulus; Structure; Synapses; Synaptic Cleft; Synaptic Transmission; Synaptic Vesicles; System; Testing; Transgenes; Transgenic Animals; Transgenic Organisms; Transmembrane Domain; Vertebrate Photoreceptors; Vesicle; Vision; Visual; Zebrafish; base; deafness; hearing impairment; in vivo; insight; interdisciplinary approach; mutant; neurotransmitter release; novel; photoreceptor degeneration; postsynaptic; presynaptic; protein protein interaction; research study; response; ribbon synapse; synaptic function; synaptogenesis

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.

描述（由申请人提供）：脊椎动物感光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。在对影响斑马鱼视觉功能的插入突变的筛选中，我们鉴定了斑马鱼wrb突变体，该突变体表现出光感受器变性和听力损失。该突变破坏了 wrb 基因，该基因编码一种新的 170 个氨基酸的蛋白质，其功能尚不清楚。在初步研究中，我们发现 wrb 突变体表现出带状突触成分的错误定位，减少了光感受器突触处对接带的数量，并显着降低了 ERG 反应。这些数据强烈表明 Wrb 可能整体参与带状突触组装和/或功能。为了验证这一假设，我们将通过组织学和分子方法相结合来研究 Wrb 在斑马鱼突触功能中的作用。在目标 1 中，我们将使用转基因方法鉴定 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 的丢失如何影响突触后细胞的树突形态，以及 Wrb 是否阻止突触小泡释放或膜内吞作用。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 的结合伴侣。这些研究将深入了解 Wrb 的作用，Wrb 是一种新型蛋白质，可能是带状突触结构和功能的关键组成部分。 Wrb 结合伴侣的身份以及随后在体内系统中对 Wrb 的表征将为突触结构研究开辟新途径，并可能提供对失明/耳聋疾病候选基因的深入了解。 公共健康相关性：内耳的光感受器和毛细胞都通过称为带状突触的特殊结构将信息传输到下游神经元。人们对带状突触的精确组成和功能动力学知之甚少。斑马鱼wrb突变体显示出带状突触结构的缺陷，并且该突变体影响一个功能未知的新基因。对 wrb 基因突变的细胞后果的研究以及鉴定 Wrb 蛋白的蛋白结合伴侣将有助于深入了解导致失明和耳聋的机制。