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 Optineurin 在青光眼神经变性中的分子机制正常眼压性青光眼 (NTG) 的特征是视网膜神经节细胞 (RGC) 在缺乏视网膜神经节细胞 (RGC) 的情况下变性。眼压高，目前没有挽救视力的疗法。 optineurin (OPTN) 基因突变与家族性和散发性 NTG 相关。 OPTN 充当适配器来招募泛素化货物自噬体用于清除。 OPTN 还具有运动蛋白的结合域，并且已知在其中发挥着关键作用沿细胞骨架的细胞运输中的作用。我们假设缺乏细胞运输 RGC 中青光眼相关的 OPTN 突变体导致 OPTN 促进 NTG 的功能丧失。不研究已经分离出 OPTN 在体内 RGC 中的作用。在 AAV2 介导的 RGC 中使用 mSncg 启动子靶向，我们将在小鼠 RGC 中过表达 OPTN-WT 或 OPTN-E50K。我们还将使用 AAV2-mSncg-Cre 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测定和后牺牲组织定量和染色以确定 RGC 变性和自噬功能的水平。这将确定 OPTN 功能的获得或丧失是否与 RGC 变性相关。我们有通过体内测定和 RGC 体细胞显示，RGC 中的 OPTN 截短导致 8 周时 RGC 变性和轴突定量，为研究 RGC 中 OPTN 的必要性提供了证据。我们还发现，之后 OPTN-WT 和 OPTN-E50K 在 RGC 中过度表达，仅 OPTN-WT 在视网膜中表达神经纤维层，表明 OPTN-E50K 不能再充当轴突运输相关蛋白。 RGC 具有穿过视神经的长投射轴突，并且必须具有功能良好的细胞运输能力调节 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 RGC 轴突和 E50K 突变引起的病理学。总之，我们的研究将揭示 RGC 中的 OPTN 并揭示 OPTN-E50K 诱导 RGC 变性的分子机制。