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

I 型耳蜗传入神经的兴奋性和兴奋性毒性：突触结构和功能

• 批准号: 10589830
• 负责人: Mark Allen Rutherford
• 金额: $ 63.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-10 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589830
• 关键词: Acids; Acoustic Nerve; Acute; Address; Adhesions; Affect; Anatomy; Antioxidants; Basic Science; Cells; Chronic; Cochlea; Collaborations; Combined Modality Therapy; Complement; Complex; Data; Discipline; Dose; Drug Delivery Systems; Ear; Electrophysiology (science); Excitatory Amino Acid Antagonists; FDA approved; Family; Functional disorder; Funding; Genetic; Glutamate Receptor; Glutamates; Goals; Hearing; Hearing problem; Heterogeneity; Histology; International; Intervention; Investigation; Knockout Mice; Knowledge; Labyrinth; Measures; Mediating; Molecular; Morphology; Mus; Nerve Fibers; Nervous System; Neurotransmitter Receptor; Noise; Permeability; Pharmaceutical Preparations; Pharmacology; Physiological; Physiology; Polyamines; Prevention; Property; Proteins; Proteomics; Recombinants; Resistance; Role; Structure; Synapses; Synaptic Transmission; Testing; antagonist; channel blockers; cochlear development; cochlear synaptopathy; design; excitotoxicity; in vivo; local drug delivery; mouse genetics; neuroligin 1; neuroligin 3; neurophysiology; noise exposure; noise trauma; patch clamp; pharmacologic; postsynaptic; prevent; prevent hearing loss; receptor; response; side effect; small molecule; sound; stargazin; tool; translational potential; transmission process

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.

项目概要： 本项目研究耳蜗AMPA型谷氨酸受体（AMPAR）， 听觉，其过度激活导致兴奋毒性突触丧失和听觉障碍。每个耳蜗 传入突触表达数百到数千种AMPAR，包括Ca 2+渗透性的 亚型（CP-AMPAR，缺乏亚基GluA 2）和Ca 2+不渗透亚型（CI-AMPAR，含有 亚基GluA 2）。成孔GluA亚基和AMPAR复合物的辅助亚基的组合， 受跨突触粘附因子的影响，决定其生理特性和药理作用 敏感性耳蜗AMPAR复合物具有使其在神经系统中独特的性质， 例如，缺乏GluA 1。然而，耳蜗AMPAR亚基的精确互补尚不清楚。 该提案使用小鼠遗传学、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，药理学敏感性， 耳蜗AMPAR复合体的功能意义。我们将确定辅助亚基的影响 TARP-102（Stargazin）对耳蜗功能、突触传递和GluA亚基表达的影响。我们将测试 GluA 3 KO小鼠的突触病是由AMPAR的Ca 2+渗透性增加引起的假设 复杂.我们将确定Neuroligin 1和3如何影响AMPAR亚基表达和听觉神经纤维 physiology.我们将确定GluA 3、TARP-102、Nlgn 1和Nlgn 3对突触内突触的影响。 AMPAR亚基的分布。通过重组表达不同组合的GluA孔形成 和辅助亚基（有或没有GluA 3，有或没有TARP-β 2），我们将挑战我们的 通过比较药物敏感性的变化， 对于天然耳蜗突触观察到的那些变化（GluA 3 WT对GluA 3 KO，TARP-12 WT对TARP-12 KO）。 这些基本知识的获得将为靶向耳蜗AMPAR的小分子设计提供信息。与 工具化合物（CP-AMPAR阻断剂IEM-1925）的慢性全身给药，我们将测量 突触适应和对噪声诱导的突触病的抵抗。对于急性全身给药，我们会问， 如果仅在噪声暴露期间而不是之前给予IEM-1925， 1925 +抗氧化剂联合治疗可以保护耳蜗功能免受更强烈的噪音暴露。的 该研究的长期目标是开发靶向内耳CP-AMPAR的全身药物 同时允许通过CI-AMPAR维持听力功能，同时避免不需要的CNS侧 方面的影响.这个合作项目的成功完成将决定精确的亚基组成 耳蜗AMPAR复合体的作用及其对药理学敏感性的影响。