Excitability and Excitotoxicity in Type-I Cochlear Afferents: Synapse Structure and Function

项目总结： 这个项目研究了耳蜗型AMPA型谷氨酸受体(AMPAs)，这是 听力，它的过度激活会导致兴奋性突触丧失和听力障碍。每个耳蜗骨 传入突触表达数百到数千个这样的AMPAR，它们都是钙离子通透性的 亚型(CP-AMPAR，缺少亚基GluA2)和不透钙亚型(CI-AMPAR，包含 亚基GluA2)。AMPAR复合体的成孔GluA亚基和辅助亚基的组合， 受跨突触黏附因子的影响，确定其生理特性和药理作用 敏感度。耳蜗AMPAR复合体具有使其在神经系统中独一无二的特性，对于 例如，GluA1的缺失。然而，耳蜗AMPAR亚基的确切组成尚不清楚。 这一建议使用了小鼠遗传学、体内和体外耳蜗电生理学、蛋白质组学和 超微结构分子解剖学研究亚基组成、药理敏感性和 耳蜗肌AMPAR复合体的功能意义。我们将测定辅助亚单位的影响 TARP--2(Stargazin)对耳蜗神经功能、突触传递和GluA亚单位表达的影响。我们将测试 GluA3KO小鼠突触形成的假说是AMPAR钙通透性增加所致 很复杂。我们将确定神经连接素1和3如何影响AMPAR亚单位的表达和听神经纤维 生理学。我们将确定GluA3、TARP-2、NLGN1和NLGN3对突触内的影响 AMPAR亚基的分布。重组表达不同组合的GluA成孔蛋白 和辅助亚单位在HEK细胞(有或没有GluA3，有或没有TARP-2)，我们将挑战我们的 通过比较药物敏感性的变化来了解耳蜗性AMPAR复合体 在天然耳蜗突触(GluA3WTvs GluA3KO，TARP-2WTvs TARP-2KO)中观察到了这些变化。 这一基础知识的获得将为设计以耳蜗肌AMPAR为靶点的小分子提供依据。使用 慢性全身给药工具化合物(CP-AMPAR阻滞剂IEM-1925)，我们将测量 突触适应和对噪声诱导的突触作用的抵抗。对于急性全身给药，我们会问是否 如果只在噪声暴露期间而不是在噪声暴露之前使用IEM-1925，如果IEM-1925- 1925+抗氧化剂联合疗法可以保护耳蜗功能不受更强烈的噪音暴露的影响。这个 这项研究的长期目标是开发针对内耳CP-AMPAR的系统性药物 同时允许通过CI-AMPAR保持听力功能，同时避免不需要的CNS侧 效果。这一合作项目的成功完成将决定准确的亚基组成 耳蜗型AMPAR复合体及其对药理敏感性的影响。

Project Summary: This project investigates the cochlear AMPA-type glutamate receptors (AMPARs) that are necessary for hearing, overactivation of which leads to excitotoxic synapse loss and hearing disorders. Each cochlear afferent synapse expresses many hundreds to a few thousand of these AMPARs, of both the Ca2+-permeable subtype (CP-AMPARs, lacking subunit GluA2) and the Ca2+-impermeable subtype (CI-AMPARs, containing subunit GluA2). The combination of pore-forming GluA subunits and auxiliary subunits of the AMPAR complex, influenced by transsynaptic adhesion factors, determine its physiological properties and pharmacological sensitivities. The cochlear AMPAR complex has properties that make it unique in the nervous system, for example, the absence of GluA1. However, the precise complement of cochlear AMPAR subunits in not known. This proposal uses mouse genetics, in vivo and ex vivo cochlear electrophysiology, proteomics, and ultrastructural molecular anatomy to investigate the subunit composition, pharmacological sensitivity, and functional significance of the cochlear AMPAR complex. We will determine the influence of auxiliary subunit TARP-2 (Stargazin) on cochlear function, synaptic transmission, and GluA subunit expression. We will test the hypothesis that synaptopathy in GluA3KO mice results from an increase in Ca2+-permeability of the AMPAR complex. We will determine how Neuroligin1 and 3 affect AMPAR subunit expression and auditory nerve fiber physiology. We will determine the influence of GluA3, TARP-2, Nlgn1, and Nlgn3 on the intrasynaptic distribution of AMPAR subunits. With recombinant expression of different combinations of GluA pore-forming and auxiliary subunits in HEK cells (with or without GluA3, with or without TARP-2), we will challenge our understanding of the cochlear AMPAR complex by comparing changes in pharmacological sensitivity with those changes observed for the native cochlear synapses (GluA3WT vs GluA3KO, TARP-2WT vs TARP-2KO). The gain of this basic knowledge will inform design of small molecules to target cochlear AMPARs. With chronic systemic administration of the tool compound (CP-AMPAR blocker IEM-1925), we will measure synaptic adaptation and resistance to noise-induced synaptopathy. With acute systemic dosing, we will ask if noise trauma can be prevented if IEM-1925 is given only during, not before, the noise exposure and if IEM- 1925 + antioxidant combination therapy can protect cochlear function from more intense noise exposures. The long-term goal of this line of investigation is to develop systemic drugs to target CP-AMPARs of the inner ear while allowing hearing function to be maintained through CI-AMPARs, and while avoiding unwanted CNS side effects. The successful completion of this collaborative project will determine the precise subunit composition of the cochlear AMPAR complex and its influence on pharmacological sensitivity.