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）：脊椎动物光感受器和感觉毛细胞的突触通过称为突触带的专门结构释放神经递质。在对影响斑马鱼视觉功能的插入突变进行筛选时，我们发现了斑马鱼wrb突变体，该突变体表现出光感受器退化和听力丧失。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，功能未知。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停靠的带状物的数量，并显著降低了ERG反应。这些数据有力地表明，RIPB可以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生物学相结合的方法来研究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确定Bismb的亚细胞定位，我们将确定Bismb是否影响发育过程中的带状组装，Bismb的丢失如何影响突触后细胞的树突状形态，以及Bismb是否阻断突触囊泡释放或膜内吞作用。在目标2中，我们将使用转基因方法来纯化标记的BIPB复合物并随后通过质谱分析来鉴定BIPB的结合伴侣。这些研究将为深入了解BMPB的作用提供帮助，BMPB是一种新的蛋白质，可能是带状突触结构和功能的关键成分。的身份，BRIAB的结合伙伴和随后的表征BRIAB在体内系统将开辟新的途径的突触结构的研究，并可能提供洞察候选基因的盲/聋疾病。 公共卫生关系：内耳的光感受器和毛细胞都通过称为带状突触的专门结构将信息传递给下游神经元。带状突触的精确组成和功能动力学知之甚少。斑马鱼wrb突变体显示带状突触结构缺陷，该突变体影响一个未知功能的新基因。对wrb基因突变的细胞后果的研究以及对Wrb蛋白质结合伴侣的鉴定将为我们提供对导致失明和耳聋的机制的深入了解。