Determining Molecular and Cellular Mechanisms of Glaucoma

确定青光眼的分子和细胞机制

• 批准号: 8788029
• 负责人: Kayarat Saidas Nair
• 金额: $ 38.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8788029
• 关键词: Address; Adult; Affect; Age; Alleles; Amines; Angle-Closure Glaucoma; Animal Model; Aqueous Humor; Blindness; Cell physiology; Cells; Cleaved cell; Co-Immunoprecipitations; Code; Cre-LoxP; Development; Disease; Disease Progression; Drainage procedure; Ethylnitrosourea; Etiology; Eye; Functional disorder; Genes; Genetic; Glaucoma; Goals; Health; Human; Induced Mutation; Iris; Label; Lead; Length; Link; Location; Mediating; Medical emergency; Microphthalmos; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Names; Neurodegenerative Disorders; Obstruction; Open Reading Frames; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Phage Display; Phenotype; Physiologic Intraocular Pressure; Physiological; Play; Primary Angle Closure Glaucoma; Process; Property; Protease Gene; Proteins; Proteome; Proteomics; Retina; Retinal; Retinal Ganglion Cells; Risk Factors; Role; Signal Transduction; Site; Specificity; Staging; Structure; System; Technology; Testing; Tissues; Trabecular meshwork structure; Western Blotting; cDNA Library; fluid flow; gain of function; high intraocular pressure; human MADHIP protein; in vivo; killings; lens; mouse model; mutant; new therapeutic target; postnatal; programs; therapy design

DESCRIPTION (provided by applicant): My goal is to identify the molecular and cellular mechanisms of Angle-Closure Glaucoma (ACG), a severe subset of glaucoma. In ACG, due to a combination of various anatomical and physiological factors, the iris is pushed forward causing physical blockage of the ocular drainage structure. This results in inefficient aqueous humor exits, thereby causing high intraocular pressure (IOP) and glaucoma. The mechanisms underlying ACG are largely unidentified. I have recently characterized a mutant mouse that recapitulates features of human Primary ACG including modestly decreased ocular size, a relatively larger lens and a narrow angle. The causal mutation is in a gene coding for a novel serine protease Prss56. Importantly, mutations in the same gene contribute to ACG in humans with reduced posterior segment (posterior microphthalmia). I will exploit this mouse model to resolve the mechanisms underlying ACG. I have three aims: Aim 1: The known mutations in mouse and human PRS556 are not predicted to disrupt the catalytic activity of this protease. Hence, it is unclear if the mutant PRSS56-mediated ACG is controlled by its inability to proteolytically cleave endogenous substrate or by gaining a new or enhanced activity. To address this, I will generate mice with a Prss56 conditional allele that can be selectively inactivated using the cre/loxP system to give rise to a catalytically inactive protease. I will ablte Prss56 and determine their impact on ACG relevant phenotypes, including its effect on ocular axial length, angle configuration and IOP. Aim 2: 2a. I will test the contribution of the retina i mediating the effect of the mutant PRSS56. The retina is a strong candidate in mediating mutant Prss56-induced ACG. Signals from the retina are known to play an important role in determining ocular axial length. Therefore, abnormal retinal PRSS56 can induce reduced ocular size (an important component of ACG). Alteration in ocular size has been linked to changes in scleral composition, which can further exacerbate ACG by impeding transcleral fluid flow. I will conditionally ablate Prss56 only in the retinal cells and assess their effect on ACG relevant phenotypes. 2b. My studies using Prss56 mutant mice suggest that postnatal developmental decrease in ocular size alone is insufficient to cause angle closure and high IOP. Alterations in adult ocular tissues must also participate in disease progression. To determine a role of stage-specific changes in ACG, I will use an inducible Cre to ablate Prss56 selectively from eyes at different ages and assess ACG related phenotypes. Aim 3: Identification of PRSS56 protease substrates is critical in understanding the molecular pathways contributing to ACG. I will employ two state-of-the-art approaches to identify PRSS56 substrates. First, use an open-reading frame (ORF)- phage display array to identify targets that are cleaved by PRSS56. Second, employ a proteome-wide strategy named terminal amine isotopic labeling of substrates (TAILS) to identify PRSS56 substrates. I will validate the in vivo specificity of these interactions using molecular approaches.

描述（由申请人提供）：我的目标是确定闭角型青光眼（ACG）的分子和细胞机制，ACG是一种严重的青光眼亚型。在ACG中，由于各种解剖学和生理学因素的组合，虹膜被向前推动，导致眼部引流结构的物理阻塞。这导致无效的房水排出，从而引起高眼内压（IOP）和青光眼。ACG的潜在机制在很大程度上尚未确定。我最近对一只突变小鼠进行了表征，该小鼠重现了人类原发性ACG的特征，包括眼球尺寸适度减小、相对较大的透镜和狭窄的角度。致病突变是在编码新型丝氨酸蛋白酶Prss 56的基因中。重要的是，同一基因中的突变有助于后节减少（后小眼）的人类ACG。我将利用这个小鼠模型来解决ACG的潜在机制。我有三个目标：目标1：在小鼠和人类PRS 556中的已知突变预计不会破坏这种蛋白酶的催化活性。因此，目前尚不清楚突变体PRSS 56介导的ACG是否受其不能蛋白水解切割内源性底物或获得新的或增强的活性的控制。为了解决这个问题，我将产生一个Prss 56条件等位基因的小鼠，可以选择性地使用cre/loxP系统失活，产生一个催化失活的蛋白酶。我将切除Prss 56并确定它们对ACG相关表型的影响，包括其对眼轴长度、眼角构型和IOP的影响。 目标2：2a。我将测试视网膜介导突变体PRSS 56的作用的贡献。视网膜是介导突变Prss 56诱导的ACG的强有力的候选者。已知来自视网膜的信号在确定眼轴长度中起重要作用。因此，异常的视网膜PRSS 56可以诱导眼睛尺寸减小（ACG的重要组成部分）。眼球大小的改变与巩膜成分的变化有关，巩膜成分的变化可通过阻碍经巩膜流体流动而进一步加重ACG。我将仅在视网膜细胞中有条件地消融Prss 56，并评估其对ACG相关表型的影响。 2b.我使用Prss 56突变小鼠的研究表明，出生后发育性眼大小减少单独不足以引起房角闭合和高IOP。成人眼组织的改变也必须参与疾病进展。为了确定ACG中阶段特异性变化的作用，我将使用诱导型Cre从不同年龄的眼睛中选择性地消融Prss 56并评估ACG相关表型。 目的3：PRSS 56蛋白酶底物的鉴定对于理解ACG的分子途径至关重要。我将采用两种最先进的方法来识别PRSS 56底物。首先，使用开放阅读框（ORF）-噬菌体展示阵列来鉴定被PRSS 56切割的靶标。第二，采用蛋白质组范围内的策略命名为底物的末端胺同位素标记（TAILS），以确定PRSS 56底物。我将使用分子方法验证这些相互作用的体内特异性。