Elucidation of the Molecular Mechanisms of Optineurin in Glaucomatous Degeneration

Optineurin 在青光眼变性中的分子机制的阐明

基本信息

批准号：
9982675
负责人：
Hannah Webber
金额：
$ 6.02万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-18 至 2021-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9982675
关键词：
Actins Affinity Amino Acids Amyloid beta-Protein Precursor Autophagocytosis Autophagosome Axon Axonal Transport Binding Biological Assay Blindness Cell physiology Cells Confocal Microscopy Custom Cytoskeleton Electroretinography Eye Functional disorder Gamma synuclein Genes Glaucoma Goals Health Hour Immunoblotting Immunoprecipitation Inherited Injections Knock-out Lead Leupeptins LoxP-flanked allele Mediating Methods Microtubules Mitochondria Modeling Molecular Molecular Biology Monitor Morphology Motor Mus Mutation Nerve Degeneration Optic Nerve Optical Coherence Tomography Pathology Patients Pattern Play Process Proteins Proteomics Retina Retinal Ganglion Cells Role Savings Stains Symptoms TANK-binding kinase 1 Testing Therapeutic Thick Time Tissues Vision Western Blotting adeno-associated viral vector axonal degeneration cell type gain of function high intraocular pressure in vivo induced pluripotent stem cell intravitreal injection loss of function mutant neuronal cell body overexpression promoter recruit retinal ganglion cell degeneration retinal nerve fiber layer trafficking

项目摘要

ABSTRACT Elucidating the Molecular Mechanism of Optineurin in Glaucomatous Neurodegeneration Normal tension glaucoma (NTG) is characterized as retinal ganglion cell (RGC) degeneration in the absence of high intraocular pressure, and currently has no sight-saving therapies. Mutations in the optineurin (OPTN) gene have been associated with familial and sporadic NTG. OPTN acts as an adaptor to recruit ubiquitinated cargo to autophagosomes for clearance. OPTN also has binding domains for motor proteins and is known to play a pivotal role in cellular trafficking along the cytoskeleton. We hypothesize that a lack of cellular trafficking by glaucoma-associated mutants of OPTN in RGCs leads to a loss-of-function of OPTN to promote NTG. No studies have ever isolated the role of OPTN in RGCs in vivo. Using the mSncg promoter in AAV2-mediated RGC targeting, we will overexpress OPTN-WT or OPTN-E50K in mouse RGCs. We will also use AAV2-mSncg-Cre in floxed OPTN mice as a model of loss-of-function of OPTN. We will use in vivo retinal function assays and post- sacrifice tissue quantification and staining to define the levels of RGC degeneration and autophagy function. This will determine whether a gain- or loss-of-function of OPTN is associated with RGC degeneration. We have shown that OPTN truncation in RGCs leads to RGC degeneration at 8 weeks by in vivo assays and RGC soma and axon quantification, providing evidence for the need to study OPTN in RGCs. We have also found that after overexpression of OPTN-WT and OPTN-E50K specifically in RGCs, only OPTN-WT is expressed in the retinal nerve fiber layer, suggesting that OPTN-E50K can no longer function as an axon transport-related protein. RGCs have long projection axons through the optic nerve and must have well-functioning cellular trafficking capacity to regulate RGC health. Using immunostaining, proteomics, and axon targeting, we will determine the role of OPTN in RGC axons and the pathology that an E50K mutation incurs. In summary, our studies will reveal the role of OPTN in RGCs and uncover molecular mechanisms of OPTN-E50K-induced RGC degeneration.

抽象的阐明青光眼神经退行性中果蛋白的分子机制正常张力青光眼（NTG）的特征是在没有的情况下是视网膜神经节细胞（RGC）变性眼内压力高，目前没有避免视力的疗法。 Opineurin（Optn）基因的突变与家族和零星的NTG有关。 Optn充当招募泛素化货物的适配器自噬小体可清理。 OPTN还具有用于运动蛋白的结合域，众所周知可以发挥关键作用在细胞骨架沿细胞运输中的作用。我们假设缺乏细胞贩运 RGC中OPTN的青光眼相关突变体导致OPTN的功能丧失以促进NTG。不研究曾经分离出OPTN在体内RGC中的作用。在AAV2介导的RGC中使用MSNCG启动子针对性，我们将在鼠标RGC中过度表达OPTN-WT或OPTN-E50K。我们还将在 Floxed Optn小鼠作为OPTN功能丧失的模型。我们将使用体内视网膜功能分析和后牺牲组织定量并染色以定义RGC变性和自噬功能的水平。这将确定OPTN的功能或功能丧失是否与RGC变性有关。我们有表明RGC中的OPTN截断会通过体内测定和RGC SOMA在8周时导致RGC变性和轴突定量，提供了需要在RGC中研究OPTN的证据。我们还发现之后专门在RGC中的Optn-WT和Optn-E50K的过表达，仅在视网膜中表达Optn-WT 神经纤维层，表明OPTN-E50K无法再充当轴突传输相关蛋白。 RGCS 通过视神经具有很长的投影轴突，并且必须具有良好的细胞运输能力规范RGC健康。使用免疫染色，蛋白质组学和轴突靶向，我们将确定OPTN的作用在RGC轴突和E50K突变会导致的病理中。总而言之，我们的研究将揭示 RGC中的OPTN和OPTN-E50K诱导的RGC变性的分子机制。