Determining Molecular and Cellular Mechanisms of Glaucoma

确定青光眼的分子和细胞机制

• 批准号: 8784082
• 负责人: Kayarat Saidas Nair
• 金额: $ 38.61万
• 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/8784082
• 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; Emergency Situation; Ethylnitrosourea; Etiology; Eye; Functional disorder; Genes; Genetic; Glaucoma; Goals; Health; Human; Induced Mutation; Iris; Label; Lead; Length; Link; Location; Mediating; Medical; 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(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，这是青光眼的一个严重亚型。在ACG中，由于各种解剖和生理因素的结合，虹膜被向前推，导致眼部引流结构的物理阻塞。这会导致低效的房水流出，从而导致高眼压和青光眼。ACG的发病机制在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ACG的特征，包括适度缩小的眼睛尺寸、相对较大的晶状体和狭窄的角度。这种原因突变发生在一种新型丝氨酸蛋白酶Prss 56的编码基因中。重要的是，相同基因的突变会导致后部节段减少(后部小眼球)的人发生ACG。我将利用这个小鼠模型来解析ACG背后的机制。我有三个目标：目标1：小鼠和人类PRS556中已知的突变不会破坏该酶的催化活性。因此，目前尚不清楚突变体PRSS56介导的ACG是受其不能切割内源底物还是获得新的或增强的活性所控制。为了解决这个问题，我将用Prss 56条件等位基因来产生小鼠，该等位基因可以使用cre/loxP系统选择性地灭活，从而产生一种催化失活的蛋白酶。我将启用Prss 56，并确定它们对ACG相关表型的影响，包括对眼轴长度、角度配置和眼压的影响。目标2：2a。我将测试视网膜I对突变体PRSS56的影响。视网膜是介导突变体Prss 56诱导的ACG的有力候选者。众所周知，来自视网膜的信号在决定眼轴长度方面起着重要作用。因此，视网膜PRSS56异常可导致眼球缩小(ACG的一个重要组成部分)。眼球大小的改变与巩膜成分的改变有关，巩膜成分的改变会阻碍经巩膜液体的流动，从而进一步加重ACG。我将有条件地去除视网膜细胞中的Prss 56，并评估它们对ACG相关表型的影响。2B。我用Prss 56突变小鼠进行的研究表明，仅有出生后眼睛大小的发育减少不足以导致房角关闭和高眼压。成人眼组织的改变也必须参与疾病的进展。为了确定特定阶段的变化在ACG中的作用，我将使用可诱导的CRE来选择性地消融不同年龄的眼睛的Prs56，并评估ACG的相关表型。目的3：鉴定PRSS56蛋白水解酶底物是了解ACG发生的分子途径的关键。我将使用两种最先进的方法来识别PRSS56基板。首先，使用开放阅读框架(ORF)-噬菌体展示阵列来识别被PRSS56切割的靶点。其次，使用一种名为底物(尾部)末端胺同位素标记的蛋白质组策略来鉴定PRSS56底物。我将使用分子方法验证这些相互作用在体内的特异性。