Molecular mechanisms of auditory nAChR synapse assembly

摘要： 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。最近，毛细胞 成熟哺乳动物内耳可诱导再生和部分功能恢复。然而，几乎没有 已知的分子机制指导功能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定义这些机制并识别分子 在耳聋的内耳中组装突触和恢复听力所必需的。这位多人调查员 合作项目利用了米歇尔·雅各布(Michele Jacob)的互补专业知识(分子机制 神经元突触组装)、耶霍阿什·拉斐尔(感觉毛细胞再生)和基思·邓肯(离子 感觉毛细胞的通道功能)。我们将集中于传出的橄榄耳蜗胆碱能传入。 大脑连接到感觉毛细胞。OC胆碱能活动调节脑电活动的敏感性和频率选择性 听证。含9/10烟碱型乙酰胆碱受体(NAChRs)介导毛发突触传递 细胞。此外，正常的活动需要9/10-nAChRs的功能偶联和紧密定位 电导钙激活钾通道(SK2)。内毛也需要SK2的早期表达 传入初级听觉突触前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。指导突触定位的机制？9/10- NAChR和SK2通道未定义。我们预测了OC突触的分子组织 我们对神经元3-nAChR突触关键成分的鉴定和Shared的初步发现 毛细胞成分？9/10-nAChR突触。在Aim1中，我们将定义核心突触后复合体 鸟类OC突触的支架和细胞骨架调节蛋白。AIM2将定义特定的适配器 将9/10-nAChRs和SK2通道连接到突触后复杂成分的蛋白质。Aim3将测试In 接头蛋白在指导9/10-nAChR和SK2突触定位和功能中的活体作用 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。我们将使用功能损失和功能获得策略，开发自发再生和 反转鸡感觉毛细胞的遗传、分子、形态、生化和功能优势。 我们的发现将为这两种发育过程中尼古丁突触的分子组织提供新的见解 和再生的毛细胞。我们将为9/10-nAChRs和SK2通道寻找新的结合伙伴。 此外，这些研究将提供对体内分子相互作用的第一次鉴定，这些相互作用对于 耳聋内耳的突触组装和听力恢复。

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.