Molecular mechanisms of auditory nAChR synapse assembly

描述（由申请人提供）：感觉神经性听力损失影响着3000万美国人。衰老、噪音过度暴露、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丧失。近年来，在成熟哺乳动物内耳中诱导毛细胞再生和部分功能恢复。然而，在正常发育的毛细胞或再生的毛细胞中，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确定这些机制，并确定在耳聋的内耳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。我们将重点讨论从大脑输出到感觉毛细胞的耳蜗胆碱能输入。OC胆碱能活性调节听觉的灵敏度和频率选择性。19/10-含烟碱乙酰胆碱受体（nAChRs）介导毛细胞突触传递。此外，正常的活性需要19/10-nAChRs与小电导钙活化钾通道（SK2）的功能偶联和紧密定位。早期的SK2表达也是内毛细胞功能成熟和正常的外胞活动所必需的，这些外胞活动是向初级听觉神经元传递声音接收信号的传入突触前输入。指导19/10- nAChRs和SK2通道突触定位的机制尚不明确。基于我们对神经元13-nAChR突触关键成分的鉴定和毛细胞19/10-nAChR突触共享成分的初步发现，我们预测了OC突触的分子组织。在ai1中，我们将定义鸟类OC突触中支架蛋白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-nAChRs和SK2通道连接到突触后复合物组分的特定适配器蛋白。Aim3将测试适配器蛋白在指导19/10-nAChR和SK2突触定位和功能偶联中的体内作用，这是正常听力所必需的。我们将在禽类发育和再生毛细胞中测试该模型。我们将利用功能丧失和功能获得策略，利用鸡感觉毛细胞的自发再生和反向遗传、分子、形态、生化和功能优势。我们的发现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。我们将为19/10-nAChRs和SK2通道寻找新的结合伙伴。此外，这些研究将首次在体内鉴定分子相互作用，这对耳聋的内耳突触组装和听力恢复至关重要。

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.