A Potential Zebrafish Model of Glaucoma by Genetic Ablation and Modification of t

描述（由申请人提供）：青光眼是一组异质性疾病，其特征为视神经病变、视网膜神经节细胞脱落和视野丧失。据估计，它影响着7000万人，是全球第二大致盲原因。青光眼发生的确切机制尚不清楚。目前的治疗方法远远不能令人满意。我们研究的目的是在细胞和分子水平上了解青光眼的病理生理学，并利用这些知识开发未来的治疗方法，以预防和治疗这种重要的致盲疾病。本申请探讨了使用遗传和分子工具在斑马鱼中开发青光眼遗传模型的可能性。斑马鱼（Danio rerio）已成为研究人类疾病的主要模式动物，因为它允许进行强大的正向和反向遗传分析，以快速识别遗传位点并研究其在疾病过程中的作用。斑马鱼和人类在维持眼内压（IOP）方面发挥关键作用的重要眼组织的解剖学、生理学和遗传学保守性强烈表明，斑马鱼是研究青光眼的有前途的模型动物。斑马鱼眼的环状韧带（AL）是一种特殊的结构，在解剖学上位于哺乳动物小梁网（TM）所在的眼睛中。由于TM是调节IOP的主要组织，这是青光眼的关键危险因素，如果通过基因消融或AL细胞的修饰改变其AL的正常结构，则斑马鱼可能发展青光眼或青光眼样症状。为了验证这一假设，将通过在鱼的基因组中插入三种类型的基因来产生转基因鱼：毒性基因（白喉毒素A，DTA）、E.大肠杆菌硝基还原酶（NTR）基因，其蛋白质将无害的化合物（前药）转化为细胞毒性物质，以及突变的肌球蛋白基因，其是青光眼的病因，并且已知改变TM细胞。这些基因在AL中的表达将导致细胞死亡或聚集，这可能导致房水流出受阻和IOP升高。组织特异性细胞消融和修饰由AL特异性启动子和诱导型cre/loxP系统控制。将使用组织学、生理学、免疫组织化学和分子方法来评估转基因鱼在眼睛的整体结构、AL的形态、IOP的变化、视网膜神经节细胞的细胞数量和活力以及视神经的外观方面发生的情况。本研究中开发的转基因斑马鱼可以模拟人类青光眼，并为进一步研究青光眼的病因学和开发新药提供了一个非常宝贵的工具，作为一个负担得起的和高通量的系统。 斑马鱼（Danio rerio）是一种小型淡水鱼，也是研究人类疾病的流行模型动物，通过选择性去除或改变对调节正常眼内压（IOP）至关重要的细胞进行遗传修饰，IOP是眼内流体压力的测量值，有助于保持眼睛的形状。在青光眼患者中，IOP通常升高，导致不可逆的视力丧失。本研究中产生的鱼可能具有升高的IOP并发展成青光眼，为青光眼药物和治疗开发提供了负担得起的高通量系统。

DESCRIPTION (provided by applicant): Glaucoma is a group of heterogeneous disorders characterized by optic neuropathies, dropout of retinal ganglion cells, and visual field loss. It affects an estimated 70 million people and is the second leading cause of blindness worldwide. The exact mechanisms through which glaucoma occurs are not clearly known. Current treatments are far from satisfactory. The objective of our studies is to understand the physiopathology of glaucoma at the cellular and molecular levels and use this knowledge to develop future therapies to prevent and treat this significant blinding disease. This application explores the possibility of developing a genetic model of glaucoma in zebrafish using genetic and molecular tools. The zebrafish (Danio rerio) has become a leading model animal for the studies of human diseases because it allows powerful forward and reverse genetic analysis to be performed to quickly identify genetic loci and study their roles in the disease process. Anatomical, physiological, and genetic conservation of the important eye tissues that play key roles in maintaining intraocular pressure (IOP) in zebrafish and humans strongly suggests that the fish is a promising model animal for studying glaucoma. The annular ligament (AL) in the zebrafish eye is a specialized structure that is anatomically localized in the eye where the mammalian trabecular meshwork (TM) resides. Because TM is the primary tissue in regulation IOP, a key risk factor for glaucoma, zebrafish may develop glaucoma or glaucoma-like symptoms if its normal structure of the AL is altered by genetic ablation or modification of the AL cells. To test this hypothesis, transgenic fish will be produced by inserting three types of genes into the fish genome: a toxic gene (diphtheria toxin A, DTA), the E. coli nitroreductase (NTR) gene whose protein converts a harmless chemical compound (prodrug) into cytotoxic species, and the mutant myocilin gene that is causative for glaucoma and is known to alter the TM cells. Expression of these genes in the AL will lead to cell death or aggregation, which may result in obstruction of the aqueous humor outflow and elevated IOP. Tissue-specific cell ablation and modification is controlled by the AL-specific promoter and an inducible cre/loxP system. Histological, physiological, immunohistochemical, and molecular methods will be used to evaluate what happens to the transgenic fish in terms of overall structure of the eye, morphology in the AL, changes in IOP, cell number and viability in retinal ganglion cells, and appearance of optic nerve. The transgenic zebrafish developed in this proposal may mimic human glaucoma and provide an invaluable tool for further studying the etiology of glaucoma and for developing new drug as an affordable and high throughput system. The zebrafish (Danio rerio), a small fresh water fish and a popular model animal for the studies of human diseases, is genetically modified by selective removal or alteration of cells important to regulate normal intraocular pressure (IOP), a measurement of the fluid pressure inside the eye that helps maintain the shape of the eye. In glaucoma patients, IOP is often elevated, leading to irreversible vision loss. The fish generated in this study may have a raised IOP and develop glaucoma, providing an affordable and high throughput system for glaucoma drug and therapy development.