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: 10374484
• 负责人: MEREDITH GREGORY-KSANDER
• 金额: $ 61.96万
• 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/10374484
• 关键词: 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; Inflammation; Injections; Integral Membrane Protein; Length; Ligands; Link; Mediating; Mediator of activation protein; 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 Fragment; Protein Isoforms; 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; cytokine; improved; 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.

首先将II型跨膜蛋白FAS配体（FASL）鉴定为诱导的死亡受体配体 FAS+靶细胞进行凋亡。因此，其在眼睛中的本构表达历史上已经连接 免疫特权现象及其杀死活化眼睛浸润的Fas+效应细胞的能力，或渗透到眼睛 FAS+血管内皮细胞。但是，这一概念被许多非脊骨to虫的事实混淆了 眼睛中的细胞类型，包括残留神经节细胞（RGC），构成表达FAS。实际上， FASL介导的R​​GC破坏是青光眼发病机理的关键因素，要么通过直接杀死RGC 和/或通过Fas+神经胶质细胞（例如星形胶质细胞）诱导促炎性趋化因子的产生，募集 促炎细胞到视网膜，从而引起神经毒性感染。这个明显的难题可以 解释是否接受我们的假设，即构型金属蛋白酶介导的裂解的裂解 膜结合的FASL（MFASL），释放一个可溶性片段（SFASL），该片段反对反对神经毒性活性 MFASL。此前提由初步数据支持：（a）具有基因靶向FASL突变的小鼠 这消除了这种FASL裂解位点（MFASLMICE）在自发和 诱导青光眼模型； {b}在健康的眼睛中，残留的fasl组成部裂解，但在青光眼的眼睛中， 视网膜FASL是膜结合的； （c）疾病发作前玻璃体内注射AAV2-SFASL载体 可以防止青光眼的发展，而疾病发作后注射AAV2-SFASL可以逆转 功能缺陷。这些数据共同将FASL作为患者的重要治疗靶标 青光眼。但是，许多关键问题仍未解决，并将由拟议的3解决 具体目的：（目标1）在开发和进展过程中，FASL裂解何时何时以及如何抑制 青光眼以及如何调节FASL裂解中的ADAM10和TIMP1？ （目标2）直接在多大程度上 由星形胶质细胞和/或RGC表达的FAS的参与有助于青光眼的发展？和 （AIM 3）SFASL可以直接参与FAS引发受保护的基因表达程序吗？我们的研究策略 将涉及接受和新颖的实验工具，包括（a）排序酶标记的FASL小鼠（由Dr.提供。 ploegh}，这将极大地支持我们监测MFASL与SFASL蛋白水平的能力，{B} FAS-FLEXED 越过RGC和星形胶质细胞/Muller特异性ERE-DERETER线的小鼠将使我们能够确定重要性 这些细胞在青光眼发展中； （c）通过融合Fas+制成的同种（四方）嵌合小鼠 和FASN•9个胚胎，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，这将使我们能够确定SFASL是否独立于功能 MFASL。拟议的研究从提出的研究中获得的机械见解可能揭示了改进的策略 患有青光眼和其他眼部疾病的患者中FAS/FASL相互作用的有效操纵。