Impact of endogenous metabolic biases on ototoxic oxidative damage

描述(申请人提供)：该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调节细胞对耳毒性药物的反应。当线粒体代谢受到干扰时，正常的代谢副产物--活性氧物种(ROS)会上升到致命的水平。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，包括氨基糖苷类耳毒性、噪声性HL(NIHL)和年龄相关性HL(ARHL)。虽然内耳ROS导致HL几乎是毫无疑问的，但ROS产生的确切机制(S)是有争议的。内源性NADH(代谢中间体)的双光子共聚焦成像的最新进展使人们能够首次实时成像活体耳蜗剂中线粒体代谢的变化。以庆大霉素(GM)为代表的AG/耳毒性药物，将检测IHC和OHC的即刻代谢反应(NADH荧光变化)和随后ROS的产生。这一建议明确了ROS的产生是如何由GM诱导的对线粒体代谢的直接抑制所导致的。在一系列专门阻断GM暴露期间线粒体ROS产生的实验中，这项提议首次评估了先前提出的产生ROS的生物活性铁-银复合体在介导AG诱导的耳蜗癌ROS产生中的作用。本文提出的创新模型描述了在IHC代谢相对自由的情况下，由于GM诱导的OHC特异性抑制NADH产生、琥珀酸脱氢酶活性和电子流经电子传递链，IHC和OHC ROS的差异产生是如何发生的。每一个特定的目标都将检验所提出的转基因耳毒性模型的有效性，该模型是由于直接调节线粒体代谢而引起的。这些研究的结果将通过1)区分即时和长期影响，2)确定GM诱导的ROS产生是否源于线粒体功能障碍而不是生物活性铁-AG复合体，以及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，从而彻底改变了我们对AG耳毒性的认识。通过确定ROS生产的确切地点(S)，该项目刺激针对关键ROS生产地点(S)的新的、优化的HL预防和治疗策略的生产。定点减少代谢ROS产生和随后的HC损失的药物将被确定为关键的药物中间体，有望减少在临床GM治疗期间经历的耳毒性。

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.