Investigating Cell Specific Roles of the Complement Cascade in Glaucoma

项目总结/摘要 我的数据显示，编码补体级联反应成员的基因的上调发生在非常早的时候， 在青光眼期间，在视网膜（视网膜神经节细胞和小胶质细胞）和视神经头（视网膜神经节细胞和小胶质细胞）中， 小胶质细胞）。重要的是，在补体成分1 qa（C1 qa）基因中具有突变的小鼠， 大大保护了青光眼。这种强大的保护作用表明， 诱导可以证明对许多青光眼患者非常有效。然而，在推进 对于潜在的治疗，有必要充分了解补充的机制， 生物合成在多种动物模型中影响青光眼。 首先，我将确定补体成分在两种不同细胞中的细胞类型特异性表达， 小鼠青光眼模型; DBA/2 J，一种广泛使用的遗传模型和最近开发的基于珠粒的 诱导型小鼠模型。我将使用携带C1 qa（半乳糖）报告基因的小鼠评估C1 QA的表达， 用免疫荧光和RNA原位杂交技术检测C3、C3 R和CRRY抑制剂的表达。这是 首次在两种对比的青光眼动物模型中评估补体蛋白， 是确定青光眼中补体级联反应的细胞特异性机制的必要步骤。 其次，我假设补体是RGCs两个关键变化的早期介导者，轴突 板层损伤和内丛状层突触改变。因此，我将决定， RGC或小胶质细胞中的补体表达先于RGC的这些早期变化。轴突损伤将是 使用DBA/2J.Thy1（CFP）可视化，并使用PSD 95抗体评估突触变化 和Ribeye。我将重点介绍补体级联反应中的关键分子C1 qa。专注于眼睛， 青光眼的早期阶段将使我能够确定补体系统的变化是否先于早期青光眼。 对ONH和视网膜中的RGC的损害。 最后，我将确定在RGC或小胶质细胞中特异性突变C1 QA的效果， 条件基因打靶C1 QA突变小鼠免受青光眼的影响，初步数据显示 C1 QA在RGC和小胶质细胞中都有表达。这些发现，沿着其他研究表明， 人类青光眼中补体成分的上调，强调了确定如何 补体级联影响青光眼。我们将使用Cre/loxP技术专门消融C1 QA 在RGC、小神经胶质细胞或两种细胞类型中的生物合成。青光眼将使用公认的 方法包括轴突损伤评估、RGC索马计数和RGC突触丢失。确定 每种细胞类型补体生物合成的重要性对于设计治疗 操纵补体系统的干预措施。

Project Summary/Abstract My data shows that upregulation of genes encoding members of the complement cascade occurs very early during glaucoma, in both the retina (in retinal ganglion cells and microglia) and optic nerve head (in microglia). Importantly, mice with a mutation in the gene for complement component 1qa (C1qa) are profoundly protected from glaucoma. This strong protection suggests that targeting complement induction could prove highly efficacious for many glaucoma patients. However, before advancing towards potential treatments it is necessary to fully understand the mechanisms by which complement biosynthesis impacts glaucoma in multiple animal models. Firstly, I will determine the cell-type specific expression of complement components in two different mouse models of glaucoma; DBA/2J, a widely used inherited model and a recently developed bead-based inducible mouse model. I will assess the expression of C1QA using mice carrying a C1qa(¿Gal) reporter as well as C3, C3R and the inhibitor CRRY using immunofluorescence and RNA in situ hybridization. This is the first time complement proteins have been assessed in two contrasting animal models of glaucoma and is a necessary step in determining the cell-specific mechanisms of the complement cascade in glaucoma. Secondly, I hypothesize that complement is an early mediator of two key changes in RGCs, axon damage at the lamina and synapse changes in the inner plexiform layer. Therefore, I will determine if complement expression in RGCs or microglia precede these early changes to RGCs. Axon damage will be visualized using DBA/2J.Thy1(CFP) and synapse changes will be assessed using antibodies for PSD95 and RIBEYE. I will focus on C1qa, a key molecule in the complement cascade. Focusing on eyes with early stages of glaucoma will allow me to determine if complement system changes precede early damage to RGCs in the ONH and retina. Finally, I will determine the effect of mutating C1QA specifically in either RGCs or microglia using conditional gene targeting. C1QA mutant mice are protected from glaucoma and preliminary data shows that C1QA is expressed in both RGCs and microglia. These findings, along with other studies showing upregulation of complement components in human glaucoma, highlight the importance of determining how the complement cascade influences glaucoma. We will use Cre/loxP technology to specifically ablate C1QA biosynthesis in RGCs, microglia or both cell types. Glaucoma will be assessed using well-established methods including axon damage assessment, RGC soma counts and RGC synapse loss. Determining the significance of complement biosynthesis by each cell type is important for designing therapeutic interventions that manipulate the complement system.