Impact of endogenous metabolic biases on ototoxic oxidative damage

描述（由申请人提供）：本项目旨在确定耳蜗内毛细胞和外毛细胞（IHC， OHC）之间的基本代谢差异，并确定这些差异如何介导细胞对耳毒性物质的反应。活性氧（ROS）是正常代谢的副产物，当线粒体代谢紊乱时，它可以上升到致命的水平。大量研究表明，ROS的产生和破坏作用是多种听力损失（HL）病理的共同特征，包括氨基糖苷（AG）诱导的耳毒性、噪声诱导的HL （NIHL）和年龄相关性HL （ARHL）。虽然内耳ROS引起HL的观点毋庸置疑，但ROS产生的确切机制仍存在争议。内源性NADH（代谢中间体）双光子共聚焦成像技术的最新进展，首次实现了活体耳蜗制剂中线粒体代谢实时变化的成像。以庆大霉素（GM）为代表的AG/耳毒性药物，研究ihc和OHCs的即时代谢反应（NADH荧光变化）和随后ROS的产生。这一建议明确了ROS的产生是如何从转基因直接诱导的线粒体代谢抑制中产生的。在转基因暴露期间特异性阻断线粒体ROS生成的一系列实验中，本研究首次评估了之前提出的ROS生成生物活性铁- ag复合物在介导ag诱导的耳蜗hc中ROS生成中的作用。本文提出的创新模型描述了在IHC代谢相对不受限制的情况下，转基因诱导的OHC特异性抑制NADH产生、琥珀酸脱氢酶活性和电子传递链中的电子流，如何导致IHC和OHC ROS产生的差异。每个特定目标将测试由于线粒体代谢的直接调节而提出的转基因诱导耳毒性模型的有效性。这些研究的结果将彻底改变我们对AG耳毒性的认识：1)区分即时和长期影响，2)确定转基因诱导的ROS产生是否来自线粒体功能障碍，而不是生物活性铁-AG复合物，3)确定IHC和OHC线粒体代谢的根本差异是否决定了对一系列耳毒性药物的不同反应。通过确定ROS产生的确切位点，该项目促进了针对关键ROS产生位点的新的、优化的HL预防和治疗策略的产生。位点特异性减少代谢ROS产生和随后HC损失的药物将被确定为关键的药物中间体，可以减少临床转基因治疗过程中经历的耳毒性。

DESCRIPTION (provided by applicant): This project aims to identify the fundamental metabolic differences between cochlear inner and outer hair cells (IHC, OHC) and to determine how these differences mediate cellular responses to ototoxic agents. Reactive oxygen species (ROS), normal byproducts of metabolism, can rise to lethal levels when mitochondrial metabolism is perturbed. Numerous studies have shown that the production and destructive actions of ROS are common features of multiple hearing loss (HL) pathologies, including aminoglycoside (AG)-induced ototoxicity, noise-induced HL (NIHL), and age-related HL (ARHL). Although there is little doubt that inner ear ROS cause HL, the exact mechanism(s) responsible for ROS production is/are controversial. Recent advances in two-photon confocal imaging of endogenous NADH (metabolic intermediate) allowed the first imaging of real-time changes in mitochondrial metabolism in live cochlear preparations. Using gentamicin (GM) as a representative AG/ototoxic agent, the immediate metabolic responses (NADH fluorescence change) and subsequent production of ROS in IHCs and OHCs will be examined. This proposal pinpoints how ROS production results from a direct GM-induced inhibition of mitochondrial metabolism. In a series of experiments that specifically block mitochondrial ROS production during GM exposure, this proposal provides the first assessment of the previously proposed role of ROS-producing bioactive iron-AG complexes in mediating AG-induced ROS production in cochlear HCs. The innovative model proposed herein describes how differential IHC and OHC ROS production can occur as a consequence of a rapid GM-induced OHC-specific inhibition of NADH production, succinate dehydrogenase activity, and electron flow through the electron transport chain while IHC metabolism remains relatively unfettered. Each Specific Aim will test the validity of the proposed model of GM-induced ototoxicity due to direct modulations of mitochondrial metabolism. Results from these studies will revolutionize our knowledge of AG ototoxicity by 1) differentiating between immediate and long-term effects, 2) determining whether GM-induced ROS production results from mitochondrial dysfunction rather than bioactive iron-AG complexes, and 3) determining whether fundamental differences in IHC and OHC mitochondrial metabolism dictate differential responses to a host of ototoxic agents. By determining the exact site(s) of ROS production, this project spurs the production of new, optimized HL prevention and treatment strategies that specifically target key ROS production site(s). Agents that site- specifically reduce metabolic ROS production and subsequent HC loss will be identified as key pharmaceutical intermediates poised to reduce the ototoxicity experienced during clinical GM treatment.