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（Fas 1）首先被鉴定为死亡受体配体， Fas+靶细胞进行凋亡。因此，其在眼睛中的组成性表达在历史上与 免疫赦免现象及其杀死活化的眼浸润Fas+效应细胞或眼浸润Fas+效应细胞的能力， Fas+血管内皮细胞。然而，这一概念是混淆的事实，许多非造血 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。事实上， FasL介导的RGCs的破坏是青光眼发病机制中的关键因素，无论是通过直接杀死RGCs 和/或通过诱导Fas+神经胶质细胞产生促炎趋化因子（例如，星形胶质细胞）， 促炎细胞进入视网膜，从而引起神经毒性炎症。这个明显的难题可以 如果接受我们的假设，即组成型金属蛋白酶介导的切割， 膜结合的FasL（mFasL），释放可溶性片段（sFasL），其对抗 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， 在自发性青光眼中， 诱导型青光眼模型; {B}在健康的眼睛中，视网膜FasL是组成性裂解的，但是在青光眼眼睛中， 视网膜FasL是膜结合的;和（c）在疾病发作之前玻璃体内注射AAV 2-sFasL载体 可以预防青光眼的发展，而发病后注射AAV 2-sFasL可以逆转 功能缺陷总之，这些数据表明，FasL是患有糖尿病的患者的重要治疗靶点。 青光眼然而，一些关键问题仍然没有答案，将由拟议的3 具体目的：（目的1）在肿瘤的发生和进展过程中，何时以及如何抑制FasL裂解， 青光眼及ADAM 10和TIMP 1如何调节FasL裂解？(Aim（2）在多大程度上， 星形胶质细胞和/或RGCs表达的Fas参与青光眼的发展？和 (Aim（3）sFasL能否直接与Fas结合，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-弯曲 与RGC和星形胶质细胞/穆勒特异性ere-deleter系杂交的小鼠，这将使我们能够确定 （c）通过融合Fas+ CD 4 + CD 4 + T细胞制备的同种异体（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， 和{d} AAV 2-sFasL载体，其将允许我们确定sFasL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， 在患有青光眼和其他眼部疾病的患者中有效操纵Fas/FasL相互作用。