Drug Screening for Neuroprotectants in Novel Zebrafish Models of Retinitis Pigmentosa

项目总结 摘要视网膜色素变性是一种遗传性视网膜变性疾病，以原发视杆细胞为特征。 感光细胞死亡后视锥细胞死亡；这会导致不可逆转的视力丧失。细胞死亡的比率 RP相对较慢，为治疗干预提供了一个较长的时间窗口。然而， 能够促进光感受器存活的药物干预仍然是一个未得到满足的需求。虽然很多人 已经建立了小鼠RP模型，通过进行大规模的药物筛选来加速药物发现 在老鼠身上是不切实际的。然而，斑马鱼是一个用于模拟人类疾病和测试的既定系统 治疗性化合物。特别是，我们之前的研究表明，斑马鱼对 大规模表型药物筛选。因此，我们通过敲除RP产生了遗传的斑马鱼RP模型 相关基因(如Rho、pde6a和pde6b)与荧光标记的杆状光感受器 表型监测。我们确定了五个模型，这些模型显示了不同的视杆细胞损失率和严重程度，这是一个关键 RP的功能。此外，虽然疾病的病因尚不清楚，但我们的初步数据和最近的研究 提示多聚腺苷二磷酸核糖聚合酶(PARP)信号转导通路是甲状旁腺细胞死亡的关键调节因子 途径，起着重要的作用。因此，我们建议利用新的斑马鱼RP模型来研究 光感受器细胞死亡机制(S)，并进行体内表型药物再利用筛选 以识别保护退化的光感受器的小分子。在目标1中，我们将测量细胞 在新的RP模型中的变化和杆状感光细胞变性。此外，我们还将评估 抑制多聚PARP信号，PARP1激活的霍尔标志，以确定光感受器是否退化 已经得到了改善。这将通过使用PARP抑制剂和敲除PARP1来实现。我们将评估如何 视杆变性的程度通过使用新的RP进行视觉行为分析来影响视觉功能 模特们。在目标2中，我们将使用我们开发的表型药物筛选平台来鉴定化合物 在我们的RP模型中促进杆状感光细胞存活。我们之前在超过3,000种药物中进行了筛选 35万尾斑马鱼幼体视杆细胞可诱导变性及视网膜神经保护剂的成功鉴定 在鱼和老鼠模型中都显示出有效性。在这里，我们将筛选约200种热门药物，其中大部分是人类批准的， 从那个项目中。在目标3中，我们将在三个RP遗传小鼠模型中验证所识别的神经保护剂。 这种跨物种的药物发现方法将加快已确定的药物用于临床试验的转换，并 这一方法可以应用于许多退行性疾病模型。因此，这个项目的完成 将深入了解RP发病的分子基础，并为RP临床试验鉴定化合物。

PROJECT SUMMARY Retinitis pigmentosa (RP), an inherited retinal degeneration (IRD) disease, is characterized by primary rod photoreceptor death followed by cone cell death; this results in irreversible vision loss. The rate of cell death in RP is relatively slow, providing a protracted time window for therapeutic intervention. However, pharmacological intervention capable of promoting photoreceptor survival remains an unmet need. While many mouse RP models have been established, accelerating drug discovery by conducting large-scale drug screens in mice is impractical. Zebrafish, however, are an established system for modeling human disease and testing therapeutic compounds. In particular, our previous studies have shown zebrafish are especially valuable for large-scale phenotypic drug screens. Thus, we generated genetic zebrafish RP models by knocking out RP associated genes (e.g. rho, pde6a, and pde6b) with fluorescently labeled rod photoreceptors for easy phenotype monitoring. We identified five models which exhibit various rates and severity of rod cell loss, a key feature of RP. In addition, while the disease etiology is unclear, our preliminary data and recent studies suggest that poly-ADP ribose polymerase (PARP) signaling, a key regulator of the parthanatos cell death pathway, plays a significant role. Therefore, we propose to utilize new zebrafish RP models to investigate photoreceptor cell death mechanism(s), and perform an in vivo phenotypic drug re-purposing screen to identify small molecules that preserve degenerating photoreceptors. In Aim 1, we will measure cellular changes and rod photoreceptor degeneration in the new RP models. In addition, we will evaluate the effect of suppressing poly-PARP signaling, a hall mark of PARP1 activation, to determine if photoreceptor degeneration is ameliorated. This will be accomplished using PARP inhibitors and knocking out PARP1. We will assess how the extent of rod degeneration affects visual function by conducting visual behavior assays with the new RP models. In Aim 2, we will use a phenotypic drug screening platform we developed to identify compounds promoting rod photoreceptors survival in our RP models. We previously screened ~3,000 drugs in over 350,000 zebrafish larvae with inducible rod degeneration and successfully identified retinal neuroprotectants showing efficacy in both fish and mouse models. Here, we will screen ~200 hit drugs, mostly human-approved, from that project. In Aim 3, we will validate the identified neuroprotectants in three RP genetic mouse models. This cross-species drug discover approach will accelerate the translation of identified drug for clinical trial, an approach which can applied to a number of degenerative disease models. Therefore, completion of this project will provide insight into the molecular basis of RP pathogenesis and identify compounds for RP clinical trials.