Molecular mechanisms of auditory nAChR synapse assembly

摘要： 感音神经性听力损失影响着3000万美国人。衰老、噪声过度、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，毛细胞 在成熟的哺乳动物内耳中诱导再生和部分功能恢复。不过小 在正常发育或发育过程中， 再生的毛细胞我们计划的研究将在体内定义这些机制并识别分子 这是受损内耳中突触组装和听力恢复所必需的。这位多方调查员 合作项目借鉴了米歇尔雅各布（分子机制的补充专业知识， 神经元突触组装）、Yehoash Raphael（感觉毛细胞再生）和基思邓肯（离子 感觉毛细胞中的通道功能）。我们将集中研究来自小脑顶核的传出橄榄耳蜗（OC）胆碱能输入。 大脑到感觉毛细胞。OC胆碱能活性调节的敏感性和频率选择性 听证会含9/10的烟碱乙酰胆碱受体（nAChRs）介导毛发中的突触传递 细胞此外，正常的活动需要9/10-nAChR与小NAChR的功能偶联和紧密定位。 电导钙激活钾通道（SK2）。内毛也需要早期SK2表达 初级听觉传入突触前输入的细胞功能成熟和正常胞吐活动 向大脑发出声音接收信号的神经元。指导9/10突触定位的机制- nAChR和SK2通道未定义。我们预测OC突触的分子组织是基于 我们鉴定了神经元3-nAChR突触的关键成分， 毛细胞9/10-nAChR突触的组成部分。在Aim 1中，我们将定义核心突触后复合体， 支架和细胞骨架调节蛋白在鸟类OC突触。Aim 2将定义特定的适配器 连接9/10-nAChR和SK 2通道与突触后复合物组分的蛋白质。Aim 3将测试 衔接蛋白在指导9/10-nAChR和SK 2突触定位和功能中的体内作用 这是正常听力所必需的。我们将在鸟类发育和再生的毛发中测试该模型 细胞我们将使用功能丧失和功能获得策略，并利用自发再生， 逆转鸡感觉毛细胞的遗传、分子、形态、生化和功能优势。 我们的研究结果将为发育中的两种细胞中烟碱突触的分子组织提供新的见解。 和再生的毛细胞我们将确定新的9/10-nAChR和SK2通道的结合伙伴。 此外，这些研究将首次在体内鉴定分子相互作用，这对 突触组装和听力恢复在受损的内耳。

Abstract: 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. ¿9/10-containing nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in hair cells. Further, normal activity requires the functional coupling and close positioning of ¿9/10-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 ¿9/10- nAChRs and SK2 channels are undefined. We predict the molecular organization of the OC synapse based on our identification of key components at neuronal ¿3-nAChR synapses and preliminary findings of shared components at hair cell ¿9/10-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 ¿9/10-nAChRs and SK2 channels to postsynaptic complex components. Aim3 will test the in vivo roles of the adapter proteins in directing ¿9/10-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 ¿9/10-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.