Differences in neomycin and gentamicin toxicity in the zebrafish lateral line

描述（由申请人提供）：尽管氨基糖苷类抗生素具有众所周知的耳毒性副作用，但仍在使用。我们实验室的初步数据表明，不同的氨基糖苷类药物可能通过部分重叠的细胞死亡途径发挥作用，但不同的抗生素也可能激活独特的途径。这项研究将检查新霉素与庆大霉素诱导斑马鱼侧线毛细胞死亡的不同分子途径。具体来说，该提案重点关注 Bcl-2 蛋白家族，该蛋白家族包含促凋亡和抗凋亡成员。该提案测试了这样的假设：Bcl-2 和 Bax 在斑马鱼侧线中专门涉及新霉素毒性，但不涉及庆大霉素毒性。本研究的具体目的是 1) 表征正常斑马鱼毛细胞中 Bcl-2 相关蛋白的分布，并将这种分布与新霉素或庆大霉素处理的毛细胞进行比较。 2) 从遗传和药理学角度操纵 Bcl-2 家族成员，以研究它们对毛细胞死亡的精确贡献。目标 1 将使用免疫细胞化学定位不同治疗组中的蛋白质来实现。对于目标 2，反义寡核苷酸（吗啉代）将用于低表达实验，并且将创建转基因用于过表达研究。最后，这项研究将 3) 筛选两个已批准药物和小药物分子库，以寻找特异性调节庆大霉素诱导的毛细胞死亡的化合物。这种庆大霉素特异性筛选将补充我们之前使用新霉素的筛选，并应鉴定出差异调节氨基糖苷类耳毒性的新分子靶点。这些目标代表了氨基糖苷类引起的听力损失治疗干预的细胞凋亡途径中的推定点，以及使用其他耳毒性刺激（例如噪音）的未来研究的目标。简单描述：拟议的研究使用斑马鱼侧线系统作为整体动物模型来研究感觉毛细胞的死亡，感觉毛细胞负责所有脊椎动物的听力和平衡。这项研究着眼于抗生素引起的毛细胞死亡的特定分子，并将研究预防这种死亡的方法，可能为预防听力损失提供新的治疗靶点。

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics are still used despite their well-known ototoxic side-effects. Preliminary data in our lab suggests that different aminoglycosides may act via partially overlapping cell death pathways but that different antibiotics may also activate unique pathways. This research will examine molecular pathways that are differentially involved in neomycin vs. gentamicin-induced hair cell death in the zebrafish lateral line. Specifically, this proposal focuses on the Bcl-2 family of proteins, which contains both pro- and anti- apoptotic members. This proposal tests the hypothesis that Bcl-2 and Bax are specifically involved in neomycin toxicity but not gentamicin toxicity in the zebrafish lateral line. The specific aims of this study are 1) to characterize the distribution of Bcl-2 related proteins in normal zebrafish hair cells and to compare this distribution to hair cells treated with neomycin or gentamicin. 2) To genetically and pharmacologically manipulate Bcl-2 family members in order to study their precise contributions to hair cell death. Aim 1 will be accomplished using immunocytochemistry to localize proteins in different treatment groups. For aim 2, antisense oligonucleotides (morpholinos) will be used for underexpression experiments and transgenics will be created for overexpression studies. Finally, this research will 3) screen two libraries of approved drugs and small drug-like molecules for compounds that specifically modulate gentamicin-induced hair cell death. This gentamicin-specific screen will complement our previous screen using neomycin and should identify new molecular targets that differentially regulate aminoglycoside ototoxicity. These targets represent putative points in apoptotic pathways for therapeutic intervention of aminoglycoside-induced hearing loss and targets of future studies using other ototoxic stimuli such as noise. Lay description: The proposed research uses the zebrafish lateral line system as a whole-animal model to study death of sensory hair cells, the cells responsible for hearing and balance in all vertebrates. This research looks at specific molecules underlying hair cell death in response to antibiotics and will examine ways to prevent this death, potentially providing new therapeutic targets for prevention of hearing loss.