Differences in neomycin and gentamicin toxicity in the zebrafish lateral line

说明(申请人提供)：氨基糖苷类抗生素尽管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。这项研究将检测在新霉素和庆大霉素诱导的斑马鱼侧线毛细胞死亡中存在差异的分子途径。具体地说，这项提案关注的是包含促凋亡和抗凋亡成员的Bcl-2蛋白家族。这一建议验证了这样一种假设，即在斑马鱼的侧线中，Bcl-2和Bax特异性地参与了新霉素的毒性，而不是庆大霉素的毒性。本研究的具体目的是：1)研究正常斑马鱼毛细胞中Bcl2相关蛋白的分布，并与新霉素或庆大霉素处理的毛细胞进行比较。2)从遗传学和药理学的角度对Bcl2家族成员进行调控，以研究其在毛细胞死亡中的确切作用。目标1将使用免疫细胞化学来定位不同治疗组中的蛋白质。对于目标2，反义寡核苷酸(吗啉)将用于低表达实验，转基因将用于过度表达研究。最后，这项研究将筛选两个经批准的药物和小类药物分子库，以寻找专门调节庆大霉素诱导的毛细胞死亡的化合物。这一庆大霉素特异性筛查将补充我们之前使用新霉素的筛查，并应识别不同调节氨基糖苷类耳毒性的新分子靶点。这些靶点代表了氨基糖苷类药物所致听力损失治疗干预的细胞凋亡途径中的假设点，也是未来使用其他耳毒性刺激如噪音进行研究的靶点。描述：这项拟议的研究使用斑马鱼侧线系统作为一个整体动物模型来研究感觉毛细胞的死亡，感觉毛细胞是所有脊椎动物中负责听力和平衡的细胞。这项研究着眼于抗生素导致毛细胞死亡的特定分子，并将研究防止这种死亡的方法，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.