Fas Ligand Cleavage regulates ocular homeostasis and glaucoma

Fas 配体裂解调节眼稳态和青光眼

• 批准号: 10550147
• 负责人: MEREDITH GREGORY-KSANDER
• 金额: $ 60.02万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 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/10550147
• 关键词: Acceleration; Address; Apoptosis; Apoptotic; Astrocytes; Binding; Blindness; CD95 Antigens; Cause of Death; Cell Death; Cells; Cessation of life; Chimera organism; Chronic Disease; Dangerousness; Data; Defect; Development; Disease; Effector Cell; Embryo; Environment; Equilibrium; Extracellular Domain; Eye; Eye diseases; Family; Fluorochrome; Gene Expression; Gene Targeting; Genetic Transcription; Glaucoma; Glial Fibrillary Acidic Protein; Goals; Grant; Homeostasis; Immune; Infiltration; Inflammation; Inflammatory; Injections; Integral Membrane Protein; Length; Ligands; Link; Mediating; Mediator; Membrane; Metalloproteases; Microspheres; Modeling; Monitor; Morula; Muller' s cell; Mus; Mutate; Mutation; Neuroglia; Ocular Pathology; Onset of illness; Optic Disk; Optic Nerve; Outcome; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Prevention; Process; Production; Protein Isoforms; Proteins; Regulation; Research; Retina; Retinal Ganglion Cells; Role; Signal Pathway; Signal Transduction; Site; Source; TIMP1 gene; TNF gene; Tamoxifen; Therapeutic; Time; Tissue Inhibitor of Metalloproteinase-1; Tissues; Transfection; Tumor Necrosis Factor Ligand Superfamily Member 6; Vascular Endothelial Cell; cell type; chemokine; constitutive expression; cytokine; improved; inducible Cre; insight; intravitreal injection; macroglia; membrane activity; neurotoxic; new therapeutic target; novel; overexpression; prevent; programs; receptor; recruit; sortase; systemic autoimmune disease; therapeutic target; tool; vector

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-flexed mice crossed to RGC- and astrocyte/muller-specific ere-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 Fasn•9 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.

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，它在眼睛中的组成性表达在历史上一直与 免疫豁免现象及其杀死激活的眼浸润 Fas+ 效应细胞或眼浸润细胞的能力 Fas+血管内皮细胞。然而，这一观点因许多非造血细胞的事实而令人困惑。 眼睛中的细胞类型，包括视网膜神经节细胞 (RGC)，组成型表达 Fas。实际上， 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， 促炎细胞到达视网膜，从而引起神经毒性炎症。这个明显的难题可以 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 神经毒性活性 mFasL。这一前提得到了初步数据的支持，这些数据显示：(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} 在健康的眼睛中，视网膜 Fasl 是结构性分裂的，但在青光眼的眼睛中， 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 功能缺陷。总之，这些数据表明 Fasl 是患有以下疾病的患者的重要治疗靶点： 青光眼。然而，许多关键问题仍未得到解答，将通过拟议的 3 具体目标：（目标 1）Fasl 裂解在疾病的发展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）直接影响到什么程度 星形胶质细胞和/或 RGC 表达的 Fas 的参与会导致青光眼的发展？和 （目标 3）sFasL 能否直接与 Fas 结合以引发保护性基因表达程序？我们的研究策略 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睛中 mFasL 与 sFasL 蛋白水平的能力，{b} Fas-flexed 小鼠与 RGC 和星形胶质细胞/苗勒特异性 ere 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过程中的作用； (c) 通过融合 Fas+ 制成的异体（四亲）嵌合小鼠 和 Fasn•9 胚胎，这将使我们能够区分 Fasl 参与的直接和间接影响 青光眼和 {d} AAV2-sFasL 向量将允许我们确定 sFasL 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。