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：
9211347
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 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9211347
关键词：
Address Adult Affect Age Alleles Amination Amines Anatomy Angle-Closure Glaucoma Animal Model Aqueous Humor Blindness Cell physiology Cells Cleaved cell Co-Immunoprecipitations Code Development Disease Disease Progression Drainage procedure Ethylnitrosourea Etiology Eye Functional disorder Genes Genetic Glaucoma Goals Human Induced Mutation Iris Isotope Labeling 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 Structure 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 public health relevance recombinase-mediated cassette exchange 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）：我的目标是识别角度闭合青光眼（ACG）的分子和细胞机制，这是严重的青光眼子集。在ACG中，由于各种解剖学和生理因素的组合，将虹膜推动，从而导致眼部排水结构的物理阻塞。这导致效率低下的幽默出口，从而导致高眼内压（IOP）和青光眼。 ACG基本的机制在很大程度上是未识别的。我最近表征了一种突变小鼠，该突变小鼠概括了人类原发性ACG的特征，包括较小的眼大小，相对较大的镜头和狭窄的角度。因果突变是编码新型丝氨酸蛋白酶PRSS56的基因中。重要的是，同一基因中的突变在后段降低（后微感染）的人类中有助于ACG。我将利用该小鼠模型来解决ACG的基础机制。我有三个目标：目标1：小鼠和人PRS556中已知的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是否通过其无法蛋白水解裂解内源性底物或获得新的或增强的活性来控制。为了解决这个问题，我将使用PRSS56有条件的等位基因生成小鼠，可以使用CRE/LOXP系统选择性地灭活，从而产生催化性无活性蛋白酶。我将ABLTE PRSS56确定它们对ACG相关表型的影响，包括其对眼部轴向长度，角构型和IOP的影响。目标2：2a。我将测试视网膜I介导突变prss56的效果的贡献。视网膜是介导突变体PRSS56诱导的ACG的强大候选者。众所周知，来自视网膜的信号在确定眼部轴向长度方面起着重要作用。因此，视网膜PRSS56异常可诱导眼大小（ACG的重要组成部分）。眼大小的改变与巩膜组成的变化有关，这可以通过阻碍经逆流动流动来进一步加剧ACG。我只有在视网膜细胞中有条件地烧掉PRSS56，并评估其对ACG相关表型的影响。 2b。我使用PRSS56突变小鼠的研究表明，单独的眼部大小的产后发育减少不足以导致角度闭合和高IOP。成年眼组织的改变还必须参与疾病进展。为了确定ACG阶段特异性变化的作用，我将使用诱导的CRE从不同年龄的眼睛中选择性地烧蚀PRSS56并评估与ACG相关的表型。 AIM 3：识别PRSS56蛋白酶底物对于理解有助于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，使用开放阅读框架（ORF） - 噬菌体显示数组来识别PRSS56裂解的目标。其次，采用一种蛋白质组的策略，称为终端胺同位素的底物（TAILS）来识别PRSS56底物。我将使用分子方法来验证这些相互作用的体内特异性。