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。在影响斑马鱼视觉功能的插入突变的屏幕中，我们确定了斑马鱼WRB突变体，该突变体表现出光感受器的退化和听力损失。该突变破坏了WRB基因，该基因编码了一种新的170氨基酸蛋白，没有已知功能。在初步研究中，我们发现WRB突变体表现出丝带突触成分的错误定位，在光感受器突触下的停靠丝带数量减少，并显着降低了ERG响应。这些数据强烈表明WRB可能与色带突触组装和/或功能积分。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在发育过程中是否影响色带组装，WRB的损失如何影响突触后细胞的树突形态，WRB是否阻断了突触囊泡的突触囊泡释放还是膜内细胞增多。在AIM 2中，我们将使用转基因方法来识别WRB的结合伙伴，以纯化标记的WRB复合物，并通过质谱进行随后的分析。这些研究将提供有关WRB的作用的见解，WRB是一种新型蛋白质，可能是色带突触结构和功能的关键组成部分。 WRB结合伙伴的身份以及在体内系统中WRB的随后表征将开放有关突触结构的研究的新途径，并可能提供有关盲目/耳聋疾病的候选基因的见解。 公共卫生相关性：内耳的光感受器和毛细胞都通过称为色带突触的专门结构将信息传递给下游神经元。色带突触的精确组成和功能动力学知之甚少。斑马鱼WRB突变体在色带突触结构中显示出缺陷，该突变体影响没有已知功能的新基因。研究突变WRB基因和鉴定WRB蛋白的蛋白结合伴侣的细胞后果的研究将提供有关导致失明和耳聋的机制的洞察力。