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: 10444754
• 负责人: Mark Allen Rutherford
• 金额: $ 69.54万
• 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/10444754
• 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 structure; Neurotransmitter Receptor; Noise; Permeability; Pharmaceutical Preparations; Pharmacology; Physiological; Physiology; Polyamines; Prevention; Property; Proteins; Proteomics; Recombinants; Resistance; Role; Structure; Synapses; Synaptic Transmission; Testing; Time; 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; 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.

项目摘要： 该项目研究了人工耳蜗AMPA型谷氨酸受体（AMPARS） 听力，过度激活会导致兴奋性突触丧失和听力障碍。每个人工耳蜗 传入的突触表达式数百至数千个AMPAR，包括Ca2+可渗透 亚型（CP-Ampars，缺乏亚基GLUA2）和Ca2+可耐受性亚型（CI-AMPAR，包含 亚基glua2）。 AMPAR复合物的孔子组合亚基和辅助亚基的组合， 受经透射性粘附因子的影响，确定其物理特性和药物 灵敏度。人工耳蜗AMPAR建筑具有使其在神经系统中独特的特性，因为 例如，没有Glua1。但是，未知的人工耳蜗AMPAR亚基的精确完成。 该建议使用小鼠遗传学，体内和体内耳蜗电生理学，蛋白质组学和 超微结构分子解剖结构，以研究亚基组成，药物敏感性和 耳蜗AMPAR复合物的功能意义。我们将确定辅助亚基的影响 TARP-2（Stargazin）关于人工耳蜗功能，突触传播和GLUA亚基表达。我们将测试 GLUA3KO小鼠突触性疾病的假设是由于AMPAR的Ca2+渗透率提高而引起的 复杂的。我们将确定Neuroligin1和3如何影响AMPAR亚基表达和听觉神经纤维 生理。我们将确定GLUA3，TARP-2，NLGN1和NLGN3对introsynaptic的影响 AMPAR亚基的分布。与Glua孔形成不同组合的重组表达 HEK细胞中的辅助亚基（有或没有GLUA3，有或没有TARP-2），我们将挑战我们的 通过将药物敏感性的变化与 天然耳蜗突触观察到的这些变化（Glua3wt vs Glua3ko，TARP-2WT与TARP-2KO）。 这种基本知识的增益将为小分子的设计提供针对人工耳蜗的设计。和 工具化合物的慢性系统给药（CP-Ampar Blocker IEM-1925），我们将测量 突触适应和对噪声引起的突触病的抗性。使用急性全身剂量，我们会问 如果IEM-1925仅在噪声暴露期间，以及IEM- 1925年 +抗氧化剂组合疗法可以保护人工耳蜗免受更强烈的噪声暴露。这 这一调查线的长期目标是开发全身药物以靶向内耳的CP-pampar 同时允许通过CI-Ampars保持听力功能，并避免使用不必要的CNS侧 效果。该协作项目的成功完成将确定精度亚基组成 人工耳蜗AMPAR复合物及其对药物灵敏度的影响。