The role of intracellular calcium in ototoxin-mediated hair cell death

描述(申请人提供)：毛细胞是内耳的主要感官感受器，负责检测声音和平衡。许多因素可归因于听力损失；其中包括通过基本上不清楚的机制引起耳毒性的药物。我们的实验室正在调查这些药物在毛细胞死亡中的致病作用，希望确定保护策略。由于研究耳毒素诱导的哺乳动物毛细胞死亡具有挑战性，我们使用斑马鱼系统作为研究此类事件的工具。除了内耳的毛细胞，斑马鱼的表面也有毛细胞，这种感觉系统被称为侧线。虽然斑马鱼的外部位置使其能够感知环境线索，但作为研究人员，我们可以很容易地观察和成像细胞内的事件，几乎没有扰动。由于侧线毛细胞在结构上和对耳毒素的反应上都与哺乳动物内耳的毛细胞相似，斑马鱼是研究影响毛细胞的信号和细胞死亡途径的理想选择。我们已经开始认真研究这种途径，并开始关注钙信号在毛细胞死亡中的作用。尽管钙离子在许多细胞内途径中扮演着众所周知的角色，包括促凋亡途径，但在暴露于耳毒性药物后的毛细胞中，钙离子尚未被广泛研究。我建议利用斑马鱼侧线系统的许多好处来研究细胞内钙离子在毛细胞死亡中的作用。我利用我们实验室以前进行的剂量-反应特性，用耳毒素剂量处理斑马鱼，这种毒素可以杀死大约50%的侧线毛细胞，并量化死亡毛细胞和存活毛细胞之间的钙水平差异。我的初步分析发现，无论是氨基糖苷新霉素还是抗癌药物顺铂，濒临死亡的毛细胞内钙离子水平都显著高于对照组。这里提出的工作旨在揭示导致这种增加的亚细胞事件，以及更好地理解为什么看似一致的细胞群体表现出不同的细胞死亡反应。我将利用基因编码的钙离子指示剂，针对毛细胞内的各种亚细胞隔间，实时可视化这些事件。预计这项工作的大部分将在毛细胞死亡的途径中定义额外的步骤。这些研究应该为耳毒素作用的方法以及在耳毒素暴露期间阻止毛细胞死亡的可能性提供新的见解。存在能够调节细胞内钙离子亚细胞流动的药物，了解这些耳毒素如何影响细胞内钙离子有望为未来旨在保护人类毛细胞免受死亡的治疗提供希望。 与公共卫生相关：内耳毛细胞死亡是人群中听力损失的主要原因。其中一个致病因素源于常见治疗药物的副作用，其中包括氨基糖苷类抗生素和铂类抗癌药物。由于这些药物的成本和/或有效性，尽管它们有耳毒性作用，但仍在继续使用。因此，这些药物的副作用仍然是一个重大的健康问题。因此，有必要更好地了解耳毒素诱导毛细胞死亡的机制。这项建议旨在实时监测毛细胞死亡途径对耳毒素的反应，并利用这些发现确定在具有耳毒性副作用的治疗剂治疗期间保护毛细胞的方法。

DESCRIPTION (provided by applicant): Hair cells are the primary sensory receptors of the inner ear, and are responsible for detecting sound as well as balance. A number of factors are attributable to hearing loss; this includes drugs that elicit ototoxicity through mechanisms that are largely unclear. Our lab is investigating the causative role of these drugs in hair cell death in hopes of identifying protective strategies. Because it is challenging to study ototoxin-induced hair cell death in mammals, we employ the zebrafish system as a tool to study such events. In addition to hair cells of the inner ear, zebrafish also possess hair cells on their surface in a sensory system termed the lateral line. While their external location enable zebrafish to sense environmental cues, we as researchers can easily observe and image intracellular events with little perturbation. Because lateral line hair cells resemble hair cells of the mammalian inner ear both structurally and in their response to ototoxins, zebrafish are ideal for the study of signaling and cell death pathways that affect hair cells. We have begun to examine such pathways in earnest, and have started to focus on the role of Ca2+ signaling in hair cell death. Despite its well-known role in many intracellular pathways, including pro-apoptotic ones, Ca2+ has not been extensively examined in hair cells following exposure to ototoxic agents. I propose to examine the role of intracellular Ca2+ in hair cell death utilizing the many benefits of the zebrafish lateral line system. I have taken advantage of previous dose-response characterizations performed in our lab to treat zebrafish with doses of ototoxin that kill approximately 50% of lateral line hair cells, and quantify differences in Ca2+ levels between hair cells that die and those that survive. My initial analysis has identified that intracellular Ca2+ levels are significantly higher in dying hair cells exposed to either the aminoglycoside neomycin or the anticancer therapeutic cisplatin. The work proposed here aims to reveal subcellular events that lead to this increase, as well as to better understand why a seemingly uniform population of cells exhibits a differential cell death response. I will utilize genetically encoded Ca2+ indicators targeted to various subcellular compartments within the hair cell to visualize these events in real-time. Much of this work is expected to define additional steps within the pathway of hair cell death. These studies should provide new insight into methods of ototoxin action as well as the potential to block hair cell death during ototoxin exposure. Pharmacological agents exist that are capable of regulating the subcellular flow of Ca2+ within the cell, and understanding how these ototoxins affect intracellular Ca2+ holds promise for future therapies designed to protect human hair cells from death. PUBLIC HEALTH RELEVANCE: Death of hair cells within the inner ear is the prominent cause of hearing loss within the population. One such causative factor stems from the side effects of common therapeutic agents, which include aminoglycoside antibiotics and platinum-based anticancer therapeutics. Because of their cost and/or effectiveness, these drugs continue to be used despite their ototoxic effects. Thus, the side effects of these drugs remain a significant health issue. There is therefore a need to better understand the mechanisms underlying ototoxin-induced hair cell death. This proposal is aimed at monitoring the response of hair cell death pathways to ototoxins in real- time, and using these findings to identify ways to protect hair cells during treatment with therapeutic agents that possess ototoxic side effects.