Molecular mechanisms of auditory nAChR synapse assembly

听觉 nAChR 突触组装的分子机制

基本信息

批准号：
7935254
负责人：
Michele H. Jacob
金额：
$ 45.27万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-19 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7935254
关键词：
Actinin Actins Affect Age Aging American Antibiotics Architecture Auditory Basilar Papilla Binding Biochemical Biological Assay Birds Brain Calcium-Activated Potassium Channel Cell Line Cells Complex Confocal Microscopy Coupling Cytoskeleton Development Dominant-Negative Mutation F-Actin Felis catus Frequencies Functional disorder Gene Transfer Hair Hair Cells Hearing Human Immunofluorescence Immunologic Infection Inner Hair Cells Ion Channel Label Labyrinth Lead Length Lesion Link Maps Mediating Membrane Modeling Molecular Molecular Models Muscle Natural regeneration Nerve Neurons Neurotransmitters Nicotinic Receptors Noise Peptides Phenotype Positioning Attribute Precipitation Protein Binding Proteins Recombinant Proteins Recovery Relative (related person)Research Personnel Role Scaffolding Protein Sensorineural Hearing Loss Sensory Hair Sequence Analysis Signal Transduction Specificity Supporting Cell Surface Synapses Synaptic Transmission Synaptic Vesicles Testing Vesicle Viral Vector adapter protein base calcium-activated potassium channel small-conductance cholinergic gain of function genetic regulatory protein hair cell regeneration hearing impairment human EMS1 protein in vitro testing in vivo insight loss of function molecular modeling novel positional cloning postsynaptic presynaptic public health relevance receptor restoration scaffold sound synaptogenesis

项目摘要

DESCRIPTION (provided by applicant): Sensorineural hearing loss affects 30 million Americans. Aging, noise-overexposure, infection and ototoxic antibiotics all lead to sensory hair cell degeneration and permanent hearing loss. Recently, hair cell regeneration and partial function restoration were induced in the mature mammalian inner ear. However, little is known about molecular mechanisms that direct functional synapse assembly in either normal developing or regenerated hair cells. Our planned studies will define these mechanisms in vivo and identify molecules required for synapse assembly and hearing restoration in the deafened inner ear. This multi-investigator collaborative project draws on the complementary expertise of Michele Jacob (molecular mechanisms of neuronal synapse assembly), Yehoash Raphael (sensory hair cell regeneration), and Keith Duncan (ion channel function in sensory hair cells). We will focus on efferent olivocochlear (OC) cholinergic inputs from the brain onto sensory hair cells. OC cholinergic activity regulates the sensitivity and frequency selectivity of hearing. 19/10-containing nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in hair cells. Further, normal activity requires the functional coupling and close positioning of 19/10-nAChRs to small conductance calcium activated potassium channels (SK2). Early SK2 expression is also required for inner hair cell functional maturation and normal exocytotic activity at the afferent presynaptic inputs onto primary auditory neurons that signal sound reception to the brain. Mechanisms that direct the synaptic localization of 19/10- nAChRs and SK2 channels are undefined. We predict the molecular organization of the OC synapse based on our identification of key components at neuronal 13-nAChR synapses and preliminary findings of shared components at hair cell 19/10-nAChR synapses. In Aim1, we will define the core postsynaptic complex of scaffold and cytoskeletal regulatory proteins at avian OC synapses. Aim2 will define the specific adapter proteins that link 19/10-nAChRs and SK2 channels to postsynaptic complex components. Aim3 will test the in vivo roles of the adapter proteins in directing 19/10-nAChR and SK2 synaptic localization and functional coupling that are essential for normal hearing. We will test the model in avian developing and regenerated hair cells. We will use loss-of-function and gain-of-function strategies and exploit the spontaneous regeneration and reverse genetic, molecular, morphological, biochemical and functional advantages of chick sensory hair cells. Our findings will provide new insights into the molecular organization of nicotinic synapses in both developing and regenerated hair cells. We will identify novel binding partners for 19/10-nAChRs and SK2 channels. Further, the studies will provide the first identification of molecular interactions, in vivo, that are essential for synapse assembly and hearing recovery in the deafened inner ear. PUBLIC HEALTH RELEVANCE: Millions of people suffer permanent hearing loss because aging, noise-overexposure and infection all lead to sensory hair cell degeneration. Our planned studies will provide the first identification of molecular interactions, in vivo, that are required for synapse assembly and hearing restoration in the deafened inner ear.

描述（由申请人提供）：感音神经性听力损失影响着 3000 万美国人。衰老、过度接触噪音、感染和耳毒性抗生素都会导致感觉毛细胞退化和永久性听力损失。最近，在成熟的哺乳动物内耳中诱导了毛细胞再生和部分功能恢复。然而，人们对正常发育或再生毛细胞中指导功能性突触组装的分子机制知之甚少。我们计划的研究将在体内定义这些机制，并确定失聪内耳中突触组装和听力恢复所需的分子。这个多研究者合作项目利用了 Michele Jacob（神经元突触组装的分子机制）、Yehoash Raphael（感觉毛细胞再生）和 Keith Duncan（感觉毛细胞中的离子通道功能）的互补专业知识。我们将重点关注从大脑到感觉毛细胞的传出橄榄耳蜗 (OC) 胆碱能输入。 OC 胆碱能活性调节听力的敏感性和频率选择性。含有 19/10 的烟碱乙酰胆碱受体 (nAChR) 介导毛细胞中的突触传递。此外，正常活动需要 19/10-nAChR 与小电导钙激活钾通道 (SK2) 的功能耦合和紧密定位。早期 SK2 表达也是内毛细胞功能成熟和初级听觉神经元传入突触前输入的正常胞吐活动所必需的，初级听觉神经元向大脑发出声音接收信号。指导 19/10-nAChR 和 SK2 通道突触定位的机制尚未定义。我们根据对神经元 13-nAChR 突触关键成分的识别以及毛细胞 19/10-nAChR 突触共享成分的初步发现来预测 OC 突触的分子组织。在 Aim1 中，我们将定义鸟类 OC 突触的支架和细胞骨架调节蛋白的核心突触后复合体。 Aim2 将定义将 19/10-nAChR 和 SK2 通道连接到突触后复合体组件的特定接头蛋白。 Aim3 将测试接头蛋白在指导 19/10-nAChR 和 SK2 突触定位和功能耦合方面的体内作用，这对于正常听力至关重要。我们将在鸟类发育和再生的毛细胞中测试该模型。我们将使用功能丧失和功能获得策略，并利用小鸡感觉毛细胞的自发再生和逆转遗传、分子、形态、生化和功能优势。我们的研究结果将为发育和再生毛细胞中烟碱突触的分子组织提供新的见解。我们将确定 19/10-nAChR 和 SK2 通道的新型结合伙伴。此外，这些研究将首次鉴定体内分子相互作用，这对于失聪内耳的突触组装和听力恢复至关重要。公共健康相关性：由于衰老、噪音过度暴露和感染都会导致感觉毛细胞退化，数百万人遭受永久性听力损失。我们计划的研究将首次鉴定体内分子相互作用，这是聋内耳中突触组装和听力恢复所需的。