Determining Molecular and Cellular Mechanisms of Glaucoma

确定青光眼的分子和细胞机制

基本信息

批准号：
9003054
负责人：
Kayarat Saidas Nair
金额：
$ 39.63万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
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/9003054
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 Proteins Proteome Proteomics Retina Retinal Retinal Ganglion Cells Risk Factors Role Serine Protease Signal Transduction Site Specificity Staging Structure System Technology Testing Tissues Trabecular meshwork structure Western Blotting cDNA Library conditional mutant fluid flow gain of function high intraocular pressure in vivo killings lens mouse model mutant new therapeutic target novel postnatal programs therapy design

项目摘要

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）（青光眼的一个严重亚型）的分子和细胞机制。在ACG中，由于各种解剖学和生理学因素的结合，虹膜被向前推，导致眼部引流结构的物理阻塞。这导致房水排出效率低下，从而导致高眼压（IOP）和青光眼。 ACG 的潜在机制很大程度上尚不清楚。我最近描述了一只突变小鼠的特征，它概括了人类原发性 ACG 的特征，包括眼睛尺寸适度减小、晶状体相对较大和角度较窄。因果突变发生在编码新型丝氨酸蛋白酶 Prss56 的基因中。重要的是，同一基因的突变会导致后节缩小（后小眼症）的人类出现 ACG。我将利用这个小鼠模型来解决 ACG 的底层机制。我有三个目标：目标 1：预计小鼠和人类 PRS556 中的已知突变不会破坏这种蛋白酶的催化活性。因此，尚不清楚突变体 PRSS56 介导的 ACG 是否是通过其无法蛋白水解裂解内源底物或通过获得新的或增强的活性来控制的。为了解决这个问题，我将生成具有 Prss56 条件等位基因的小鼠，该小鼠可以使用 cre/loxP 系统选择性失活，从而产生催化失活的蛋白酶。我将 ablte Prss56 并确定它们对 ACG 相关表型的影响，包括其对眼轴长度、角度配置和 IOP 的影响。目标 2：2a。我将测试视网膜对介导突变体 PRSS56 的影响的贡献。视网膜是介导突变型 Prss56 诱导的 ACG 的有力候选者。众所周知，来自视网膜的信号在确定眼轴长度方面发挥着重要作用。因此，异常的视网膜PRSS56可导致眼睛尺寸减小（ACG的重要组成部分）。眼睛大小的改变与巩膜成分的变化有关，巩膜成分的变化会阻碍经巩膜的液体流动，从而进一步加剧 ACG。我将仅在视网膜细胞中有条件地消融 Prss56，并评估它们对 ACG 相关表型的影响。 2b.我使用 Prss56 突变小鼠的研究表明，仅出生后眼睛尺寸的发育减小不足以引起房角闭合和高眼压。成人眼组织的改变也必定参与疾病进展。为了确定 ACG 中特定阶段变化的作用，我将使用诱导型 Cre 从不同年龄的眼睛中选择性地消除 Prss56，并评估 ACG 相关表型。目标 3：PRSS56 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 PRSS56 底物。首先，使用开放阅读框 (ORF) - 噬菌体展示阵列来识别被 PRSS56 切割的目标。其次，采用称为底物末端胺同位素标记 (TAILS) 的蛋白质组策略来识别 PRSS56 底物。我将使用分子方法验证这些相互作用的体内特异性。