Drug Screening for Neuroprotectants in Novel Zebrafish Models of Retinitis Pigmentosa

项目摘要 视网膜色素变性（RP）是一种遗传性视网膜变性（IRD）疾病，其特征是原发性视杆细胞变性， 光感受器死亡，随后是视锥细胞死亡;这导致不可逆的视力丧失。细胞死亡率在 RP相对缓慢，为治疗干预提供了延长的时间窗。然而，在这方面， 能够促进光感受器存活的药物干预仍然是未满足的需求。虽然许多 已经建立了小鼠RP模型，通过进行大规模药物筛选来加速药物发现 是不切实际的。然而，斑马鱼是一个建立人类疾病模型和测试的系统， 治疗化合物。特别是，我们以前的研究表明，斑马鱼对 大规模表型药物筛选。因此，我们通过敲除RP来产生遗传斑马鱼RP模型 与荧光标记的视杆细胞光感受器相关的基因（例如rho、pde 6a和pde 6 b）， 表型监测我们确定了五种模型，它们表现出不同的视杆细胞丢失率和严重程度，这是一个关键因素。 RP的特点。此外，虽然疾病病因尚不清楚，但我们的初步数据和最近的研究表明， 表明聚ADP核糖聚合酶（PARP）信号传导，parthanatos细胞死亡的关键调节因子， 路，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 感光细胞死亡机制，并进行体内表型药物再利用筛选 来鉴定那些能保护退化感光细胞的小分子。在目标1中，我们将测量细胞 变化和视杆细胞变性。此外，我们将评估 抑制多聚PARP信号传导，PARP 1活化的霍尔标记，以确定光感受器退化是否 得到改善。这将使用PARP抑制剂和敲除PARP 1来实现。我们将评估如何 视杆变性的程度通过用新的RP进行视觉行为测定来影响视觉功能 模型在目标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模型中促进视杆细胞的存活。我们之前筛选了大约3,000种药物， 350，000只斑马鱼幼鱼具有可诱导的视杆变性，并成功识别出视网膜神经保护剂 在鱼和小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，大部分是人类批准的， 从这个项目。在目标3中，我们将在三种RP遗传小鼠模型中验证所鉴定的神经保护剂。 这种跨物种的药物发现方法将加速已鉴定药物的临床试验转化， 该方法可应用于许多退行性疾病模型。因此，该项目的完成 将提供深入了解RP发病机制的分子基础，并确定RP临床试验的化合物。

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.