Molecular mechanisms of auditory nAChR synapse assembly

感音神经性听力损失影响着3000万美国人。衰老、过度噪声、感染和耳毒性抗生素都会导致感觉毛细胞变性和永久性听力损失。最近，在成熟的哺乳动物内耳中诱导毛细胞再生和部分功能恢复。然而，很少有人知道的分子机制，指导功能性突触组装在正常发育或再生的毛细胞。我们计划的研究将在体内确定这些机制，并确定在受损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借鉴了Michele Jacob（神经元突触组装的分子机制），Yehoash Raphael（感觉毛细胞再生）和基思邓肯（感觉毛细胞中的离子通道功能）的互补专业知识。我们将集中在传出橄榄耳蜗（OC）胆碱能输入从大脑到感觉毛细胞。OC胆碱能活性调节听觉的灵敏度和频率选择性。含19/10的烟碱乙酰胆碱受体（nAChRs）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（SK 2）的功能偶联和紧密定位。早期SK2表达也是内毛细胞功能成熟和在传入突触前输入到初级听觉神经元的正常胞吐活动所需的，所述初级听觉神经元向大脑发出声音接收信号。指导19/10-nAChR和SK 2通道的突触定位的机制尚未确定。我们预测的OC突触的分子组织的基础上，我们确定的关键组件在神经元13-nAChR突触和毛细胞19/10-nAChR突触的共享组件的初步研究结果。在目标1中，我们将确定在鸟类OC突触的支架和细胞骨架调节蛋白的核心突触后复合物。Aim 2将定义将19/10-nAChR和SK 2通道连接到突触后复合物组分的特定衔接蛋白。Aim 3将测试衔接蛋白在指导19/10-nAChR和SK 2突触定位和功能耦合中的体内作用，这些功能对正常听力至关重要。我们将在鸟类发育和再生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通道的新型结合伴侣。此外，这些研究将首次在体内鉴定分子相互作用，这对受损内耳的突触组装和听力恢复至关重要。 公共卫生相关性：数以百万计的人遭受永久性听力损失，因为老化，噪音过度和感染都导致感觉毛细胞退化。我们计划的研究将提供第一个识别的分子相互作用，在体内，这是需要突触组装和听力恢复在受损的内耳。

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.