Elucidation of the Molecular Mechanisms of Optineurin in Glaucomatous Degeneration

摘要 视神经磷酸酶在青光眼神经退行性变中的分子机制 正常眼压性青光眼（NTG）的特征在于视网膜神经节细胞（RGC）变性， 高眼内压，目前还没有挽救视力的治疗方法。视神经磷酸酶（OPTN）基因突变 与家族性和散发性NTG有关。OPTN作为一种适配器，招募泛素化的货物， 自噬体进行清除。OPTN还具有马达蛋白的结合结构域，并且已知在马达蛋白的形成中起关键作用。 在沿细胞骨架沿着运输细胞中起作用。我们假设，缺乏细胞贩运， RGC中OPTN的肉瘤相关突变体导致OPTN促进NTG的功能丧失。没有 研究已经分离出OPTN在体内RGC中的作用。在AAV 2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来确定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 RGC索马体细胞和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的必要性提供证据。我们还发现， OPTN-WT和OPTN-E50K特异性地在RGC中过表达，只有OPTN-WT在视网膜中表达。 神经纤维层，表明OPTN-E50 K不再作为轴突转运相关蛋白发挥作用。RGCs 具有通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， 规管研资局的健康。利用免疫染色，蛋白质组学和轴突靶向，我们将确定OPTN的作用， 在RGC轴突和病理的E50K突变引起的。总之，我们的研究将揭示 OPTN在RGC中的作用，并揭示OPTN-E50K诱导RGC变性的分子机制。

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.