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)的特征是视网膜神经节细胞(RGC)变性。 高眼压，目前还没有挽救视力的疗法。视神经磷酸酶(OPTN)基因突变 与家族性和散发性NTG有关。OPTN充当适配器，招募泛素化的货物到 自动吞噬体待清除。OPTN也有与马达蛋白结合的结构域，并已知在 在沿细胞骨架的细胞运输中的作用。我们假设，由于缺乏细胞贩运， 视网膜节细胞中与青光眼相关的OPTN突变导致OPTN失去促进NTG的功能。不是 曾经有研究分离出OPTN在活体视网膜节细胞中的作用。MSncg启动子在AAV2介导的RGC中的应用 靶向，我们将在小鼠视网膜神经节细胞中过表达OPTN-WT或OPTN-E50K。我们还将在中使用AAV2-mSncg-CRE OPTN小鼠作为OPTN功能丧失模型。我们将使用在体视网膜功能检测和术后检查 处死组织进行定量和染色，以确定RGC退变和自噬功能的水平。 这将决定OPTN的功能增强或功能丧失是否与RGC变性有关。我们有 体内实验和RGC胞体分析表明，OPTN在RGCs中的截短导致RGC在8周时变性 和轴突的定量，为研究视网膜节细胞的OPTN提供了证据。我们还发现，在 OPTN-WT和OPTN-E50K在视网膜节细胞中的表达 神经纤维层，提示OPTN-E50K不再作为轴突运输相关蛋白发挥作用。RGCs 有通过视神经的长投射轴突，必须具有良好的细胞运输能力 规范研资局的健康。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E50K突变引起的病理中。总而言之，我们的研究将揭示 OPTN在RGC中的表达，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.