Molecular mechanisms of auditory nAChR synapse assembly

描述(申请人提供)：感觉神经性听力损失影响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。最近，成熟的哺乳动物内耳被诱导毛细胞再生和部分功能恢复。然而，对于在正常发育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定义这些机制，并确定耳聋内耳中突触组装和听力恢复所需的分子。这个由多名研究人员合作的项目利用了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(感觉毛细胞的离子通道功能)的互补专业知识。我们将重点介绍从大脑到感觉毛细胞的传出橄榄耳蜗胆碱能传入。OC胆碱能活动调节听觉的敏感性和频率选择性。19/10的烟碱型乙酰胆碱受体(NAChRs)介导毛细胞的突触传递。此外，正常的活动需要19/10-nAChRs与小电导钙激活钾通道(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胞吐活动所必需的，在初级听神经元的传入突触前输入信号向大脑发出声音接收信号。指导19/10-nAChRs和SK2通道突触定位的机制尚未确定。我们根据我们在神经性13-nAChR突触的关键成分和毛细胞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突触的骨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-nAChRs和SK2通道与突触后复杂成分联系起来的特定适配器蛋白。AIM3将在体内测试适配蛋白在指导19/10-nAChR和SK2突触定位和功能耦合方面的作用，这是正常听力所必需的。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，开发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中和再生毛细胞中尼古丁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，这种相互作用对耳聋内耳的突触组装和听力恢复至关重要。 与公共卫生相关：数百万人遭受永久性听力损失，因为衰老、过度接触噪音和感染都会导致感觉毛细胞退化。我们计划的研究将提供第一次在体内鉴定分子相互作用，这是耳聋内耳的突触组装和听力恢复所必需的。

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.