Fas Ligand Cleavage regulates ocular homeostasis and glaucoma

II型跨膜蛋白Fas配体(Fasl)首次被鉴定为一种死亡受体配体，可诱导 Fas+靶细胞发生凋亡。因此，它在眼睛里的构成表达在历史上是有联系的 对免疫豁免现象及其对活化的眼浸润性Fas+效应细胞或眼浸润性细胞的杀伤作用 Fas+血管内皮细胞。然而，这一概念被以下事实混淆了：许多非造血组织 眼内的细胞类型，包括视网膜神经节细胞(RGC)，结构性地表达Fas。事实上, FasL介导的视网膜节细胞破坏是青光眼发病机制中的一个关键因素，无论是通过直接杀伤视网膜节细胞 和/或通过诱导Fas+胶质细胞产生促炎趋化因子(例如，星形胶质细胞)，即招募 促炎症细胞进入视网膜，从而引起神经毒性炎症。这一明显的难题可能 如果人们接受我们的假设，即结构性金属蛋白酶介导的切割 膜结合FasL(MFasL)，释放一种可溶性片段(SFasL)，对抗 MFasL.这一前提得到了初步数据的支持，初步数据显示：(A)具有Fasl基因靶向突变的小鼠 这消除了这个Fasl裂解位点(mFasl小鼠)在自发性和自发性青光眼中发展为加速青光眼 诱导性青光眼模型；在健康眼中，视网膜Fasl呈结构性分裂，但在青光眼中， 视网膜FasL是膜结合的；和(C)发病前玻璃体内注射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。综上所述，这些数据表明FasL是治疗慢性阻塞性肺疾病患者的重要靶点 青光眼。然而，一些关键问题仍未得到回答，将由拟议的3 具体目标：(目标1)Fasl裂解在肿瘤的发生和发展过程中何时和如何被抑制 青光眼与ADAM10和TIMP1如何调节FasL裂解？(目标2)ADAM10和TIMP1在多大程度上指导 星形胶质细胞和/或视网膜节细胞表达的Fas参与青光眼的发展？ (目的3)sFasL能否直接与Fas结合以诱导保护性基因表达程序？我们的研究策略 将涉及公认的和新的实验工具，包括：(A)标记了Sortase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/穆勒特异的前缺失品系杂交，这将使我们能够识别其重要性 这些细胞在青光眼发展中的作用；(C)通过融合Fas+构建的四亲性嵌合小鼠 和FASN·9胚胎，这将使我们能够区分FasL参与在上下文中的直接和间接影响 和{d}个AAV2-sFasL载体，这将使我们能够确定sFasL是否独立于 MFasL.从拟议的研究中获得的机械论见解可能会揭示出改进的战略 青光眼等眼病患者Fas/Fasl相互作用的有效操作

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.