Investigating Cell Specific Roles of the Complement Cascade in Glaucoma

项目摘要/摘要 我的数据显示，编码补体级联成员的基因上调发生得很早 在青光眼期间，视网膜(视网膜神经节细胞和小胶质细胞)和视神经头(在 小胶质细胞)。重要的是，携带补体成分1qA(C1qa)基因突变的小鼠 彻底保护您免受青光眼的伤害。这种强大的保护表明，靶向是对 对于许多青光眼患者来说，诱导可能被证明是非常有效的。然而，在推进之前， 对于潜在的治疗方法，有必要充分了解补体 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个不同细胞类型中的特异性表达 小鼠青光眼模型；DBA/2J，一种广泛使用的遗传性模型和最近开发的基于珠子的 诱导型小鼠模型。我将使用携带C1qA(？Gal)报告的小鼠来评估C1QA的表达 免疫荧光和RNA原位杂交检测C3、C3R和抑制物CRRY。这是 首次在两种不同的青光眼和青光眼动物模型中评估了补体蛋白 是确定青光眼补体级联的细胞特异性机制的必要步骤。 其次，我假设补体是视网膜节细胞轴突两个关键变化的早期中介。 板层受损，内网状层突触改变。因此，我将确定是否 RGCs或小胶质细胞的补体表达先于RGCs的这些早期变化。轴突损伤将是 使用DBA/2J.Thy1(CFP)和突触变化将使用PSD95抗体进行评估 还有里贝眼。我将重点介绍补体级联中的关键分子C1qa。聚焦在眼睛上 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 ONH和视网膜中的RGCs受损。 最后，我将确定突变C1QA对视网膜神经节细胞或小胶质细胞的影响。 条件性基因打靶。初步数据显示，C1QA突变小鼠可预防青光眼 C1QA在视网膜神经节细胞和小胶质细胞中均有表达。这些发现以及其他研究表明 人类青光眼补体成分上调，强调确定如何 补体级联会影响青光眼。我们将使用CRE/loxP技术来专门消融C1QA 在视网膜节细胞和/或小胶质细胞中的生物合成。青光眼的评估将使用公认的 方法包括轴突损伤评估、RGC胞体计数和RGC突触丢失。确定 每种细胞类型的补体生物合成的意义对于设计治疗方案很重要 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.