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

描述（由申请人提供）：毛细胞是内耳的主要感觉受体，负责检测声音和平衡。听力损失可归因于许多因素；这包括通过很大程度上不清楚的机制引起耳毒性的药物。我们的实验室正在研究这些药物在毛细胞死亡中的致病作用，希望找到保护策略。由于研究耳毒素诱导的哺乳动物毛细胞死亡具有挑战性，因此我们采用斑马鱼系统作为研究此类事件的工具。除了内耳的毛细胞外，斑马鱼在其表面的感觉系统中也有毛细胞，称为侧线。虽然它们的外部位置使斑马鱼能够感知环境线索，但我们作为研究人员可以很容易地观察和成像细胞内的事件，几乎没有扰动。由于斑马鱼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毛细胞相似，因此是研究影响毛细胞的信号传导和细胞死亡途径的理想对象。我们已经开始认真研究这些途径，并开始关注Ca2+信号在毛细胞死亡中的作用。尽管Ca2+在许多细胞内通路（包括促凋亡通路）中发挥着众所周知的作用，但在暴露于耳毒性物质后，Ca2+在毛细胞中的作用尚未得到广泛的研究。我建议利用斑马鱼侧线系统的许多好处来研究细胞内Ca2+在毛细胞死亡中的作用。我利用之前在我们实验室进行的剂量-反应表征，用耳毒素剂量治疗斑马鱼，杀死大约50%的侧线毛细胞，并量化死亡和存活毛细胞之间Ca2+水平的差异。我的初步分析已经确定，在暴露于氨基糖苷新霉素或抗癌治疗顺铂的垂死毛细胞中，细胞内Ca2+水平明显更高。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统一的细胞群体表现出不同的细胞死亡反应。我将利用针对毛细胞内各种亚细胞区室的基因编码Ca2+指示器来实时可视化这些事件。这项工作的大部分预计将确定毛细胞死亡途径中的其他步骤。这些研究应该为耳毒素作用的方法以及在耳毒素暴露过程中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Ca2+亚细胞流动的药理学药物，并且了解这些耳毒素如何影响细胞内Ca2+，为未来设计保护人类毛细胞免于死亡的治疗提供了希望。

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.