Fas Ligand Cleavage regulates ocular homeostasis and glaucoma

Fas 配体裂解调节眼稳态和青光眼

基本信息

批准号：
10867990
负责人：
MEREDITH GREGORY-KSANDER
金额：
$ 4.36万
依托单位：
SCHEPENS EYE RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10867990
关键词：
Acceleration Address Apoptosis Astrocytes Binding Blindness Cause of Death Cell Death Cells Cessation of life Chronic Disease Data Defect Development Effector Cell Embryo Eye Eye diseases Funding Gene Expression Gene Targeting Glaucoma Goals Homeostasis Immune Infiltration Inflammation Inflammatory Injections Integral Membrane Protein Ligands Link LoxP-flanked allele Mediating Membrane Metalloproteases Modeling Monitor Mus Mutation Neuroglia Onset of illness Pathogenesis Pathway interactions Patients Production Proteins Research Retina Retinal Ganglion Cells Signal Pathway Site TIMP1 gene Tumor Necrosis Factor Ligand Superfamily Member 6 Vascular Endothelial Cell cell type chemokine constitutive expression improved insight intravitreal injection membrane activity neurotoxic new therapeutic target novel prevent programs receptor recruit sortase therapeutic target tool vector

项目摘要

Project Summary (from original funded application) The type II transmembrane protein Fas ligand (FasL) was first identified as a death receptor ligand that induced Fas+ target cells to undergo apoptosis. As such, its constitutive expression in the eye has historically been linked to the phenomenon of immune privilege and its ability to kill activated eye-infiltrating Fas+ effector cells, or eye- infiltrating Fas+ vascular endothelial cells. However, this notion is confounded by the fact that many non- hematopoietic cell types in the eye, including retinal ganglion cells (RGCs), constitutively express Fas. In fact, FasL-mediated destruction of RGCs is a key factor in glaucoma pathogenesis, either by direct killing of RGCs and/or by inducing the production of proinflammatory chemokines by Fas+ glial cells (eg. astrocytes), that recruit proinflammatory cells to the retina and thereby causing neurotoxic inflammation. This apparent conundrum can be explained if one accepts our hypothesis that constitutive metalloproteinase-mediated cleavage of membraned-bound FasL (mFasL), releases a soluble fragment (sFasL) that opposes the neurotoxic activity of mFasL. This premise is supported by preliminary data showing: (a) mice with a gene-targeted mutation of FasL that eliminates this FasL cleavage site (mFasLmice) develop accelerated glaucoma in spontaneous and inducible glaucoma models; (b) in healthy eyes, retinal FasL is constitutively cleaved, but in glaucomatous eyes, retinal FasL is membrane-bound; and (c) intravitreal injection of an AAV2-sFasL vector prior to disease onset can prevent the development of glaucoma, while injection of AAV2-sFasL after disease onset can reverse functional defects. Together, these data point to FasL as an important therapeutic target for patients with glaucoma. However, a number of key questions remain unanswered and will be addressed by the proposed 3 specific aims: (Aim 1) When and how is FasL cleavage suppressed during the development and progression of glaucoma and how do ADAM10 and TIMP1 in regulate FasL cleavage ?; (Aim 2) To what extent does the direct engagement of Fas, expressed by astrocytes and/or RGCs, contribute to the development of glaucoma?; and (Aim 3) Can sFasL directly engage Fas to elicit a protective gene expression program? Our research strategy will involve both accepted and novel experimental tools, including (a) sortase-tagged-FasL mice (provide by Dr. Ploegh), that will greatly facilitate our ability to monitor mFasL vs sFasL protein levels in the eye, (b) Fas-floxed mice crossed to RGC- and astrocyte/müller-specific Cre-deleter lines, that will allow us to identify the importance of these cells in the development of glaucoma; (c) allophenic (tetraparental) chimeric mice made by fusing Fas+ and Fasneg embryos, that will allow us to distinguish direct and indirect effects of FasL engagement in the context of glaucoma, and (d) AAV2-sFasL vectors that will allow us to determine if sFasL functions independently of mFasL. The mechanistic insights gained from the proposed studies are likely to reveal improved strategies for the effective manipulation of Fas/FasL interactions in patients afflicted with glaucoma and other ocular disorders.

项目摘要（来自原始资助申请） II型跨膜蛋白Fas配体（FasL）首先被鉴定为死亡受体配体，可诱导死亡 Fas+ 靶细胞发生凋亡，因此，其在眼中的组成型表达历来被联系在一起。免疫豁免现象及其杀死激活的眼部浸润性 Fas+ 效应细胞或眼- 浸润 Fas+ 血管内皮细胞然而，这一概念因许多非 - 血管内皮细胞的存在而被混淆。眼睛中的造血细胞类型，包括视网膜神经节细胞 (RGC)，组成型表达 Fas。 FasL 介导的 RGC 破坏是青光眼发病机制的关键因素，无论是通过直接杀死 RGC 和/或通过诱导 Fas+ 神经胶质细胞（例如星形胶质细胞）产生促炎趋化因子，促炎细胞进入视网膜并引起神经毒性炎症。如果有人接受我们的假设，即组成型金属蛋白酶介导的裂解膜结合的 FasL (mFasL)，释放可溶性片段 (sFasL)，对抗 FasL 的神经毒性活性 mFasL 的前提得到了初步数据的支持，该数据显示：(a) 具有 FasL 基因靶向突变的小鼠消除 FasL 裂解位点 (mFasLmice) 会加速自发性青光眼的发展诱导性青光眼模型；(b) 在健康眼睛中，视网膜 FasL 是组成性裂解的，但在青光眼眼睛中，视网膜 FasL 是膜结合的；(c) 在疾病发作前玻璃体内注射 AAV2-sFasL 载体可以预防青光眼的发展，而发病后注射 AAV2-sFasL 可以逆转总之，这些数据表明 FasL 是患有功能缺陷的患者的重要治疗靶点。然而，一些关键问题仍未得到解答，将由提议的 3 解决。具体目标：（目标 1）FasL 裂解在细胞发育和进展过程中何时以及如何受到抑制青光眼以及 ADAM10 和 TIMP1 如何调节 FasL 裂解？（目标 2）直接作用程度如何？由星形胶质细胞和/或 RGC 表达的 Fas 的参与会导致青光眼的发生？（目标 3）sFasL 能否直接与 Fas 结合来引发保护性基因表达程序？将涉及公认的和新颖的实验工具，包括（a）分选酶标记的 FasL 小鼠（由 Dr. Ploegh），这将极大地促进我们监测眼中 mFasL 与 sFasL 蛋白水平的能力，（b）Fas-floxed 小鼠与 RGC 和星形胶质细胞/穆勒特异性 Cre 删除系杂交，这将使我们能够确定重要性这些细胞在青光眼发展中的作用；（c）通过融合 Fas+ 制成的异体（四亲）嵌合小鼠和 Fasneg 胚胎，这将使我们能够区分 FasL 参与的直接和间接影响青光眼，以及 (d) AAV2-sFasL 载体，使我们能够确定 sFasL 的功能是否独立于 mFasL 从拟议的研究中获得的机制见解可能会揭示改进的策略有效控制青光眼和其他眼部疾病患者的 Fas/FasL 相互作用。