Investigating Cell Specific Roles of the Complement Cascade in Glaucoma

描述（由申请人提供）：我的数据显示，在青光眼期间，在视网膜（视网膜神经节细胞和小胶质细胞）和视神经乳头（小胶质细胞）中，补体级联的编码成员的基因的上调发生得非常早。重要的是，在补体成分1 qa（C1 qa）基因突变的小鼠是深刻的保护青光眼。这种强大的保护作用表明，靶向补体诱导可以证明对许多青光眼患者非常有效。然而，在推进潜在的治疗方法之前，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的机制。 首先，我将确定补体成分在两种不同的青光眼小鼠模型中的细胞类型特异性表达; DBA/2 J，一种广泛使用的遗传模型和一种最近开发的基于珠子的诱导型小鼠模型。我将使用携带C1 qa（2Gal）报告基因以及C3、C3 R和抑制剂CRRY的小鼠，使用免疫荧光和RNA原位杂交评估C1 QA的表达。这是第一次在两种对比的青光眼动物模型中评估补体蛋白，并且是确定青光眼中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RGCs两个关键变化的早期介质，轴突损伤在板层和内丛状层的突触变化。因此，我将确定RGC或小胶质细胞中的补体表达是否先于RGC的这些早期变化。使用DBA/2J.Thy1（CFP）观察轴突损伤，使用PSD 95和RIBEYE抗体评估突触变化。我将重点介绍补体级联反应中的关键分子C1 qa。关注青光眼早期的眼睛将使我能够确定在ONH和视网膜中，补体系统的变化是否先于RGC的早期损伤。 最后，我将确定使用条件基因靶向在RGC或小胶质细胞中特异性突变C1 QA的效果。C1 QA突变小鼠可免于青光眼，初步数据显示C1 QA在RGC和小胶质细胞中表达。这些发现，沿着其他研究显示，人类青光眼中补体成分上调，强调了确定补体级联如何影响青光眼的重要性。我们将使用Cre/loxP技术来特异性地消除RGC、小胶质细胞或这两种细胞类型中的C1 QA生物合成。将使用成熟的方法评估青光眼，包括轴突损伤评估、RGC索马计数和RGC突触丢失。确定每种细胞类型的补体生物合成的意义对于设计操纵补体系统的治疗干预是重要的。 公共卫生相关性：青光眼是一种致盲性疾病，影响全球7000多万人。没有临床上批准的直接靶向视网膜神经节细胞的治疗，视网膜的输出神经元在青光眼中丢失。我们和其他人已经确定，补体途径在疾病的早期起着重要作用。我们将使用两种小鼠青光眼模型充分研究补体蛋白在青光眼中的表达。我们还将评估在特定细胞类型中有条件地消融补体途径中的关键分子的影响。这项工作将确切地确定补体途径如何影响青光眼。

DESCRIPTION (provided by applicant): 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(2Gal) 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. PUBLIC HEALTH RELEVANCE: Glaucoma is a blinding disorder affecting over 70 million people worldwide. No treatments are clinically approved that directly target retinal ganglion cells, the output neurons of the retina that are lost in glaucoma. We, and others have established that the complement pathway plays an important role early in the disease. We will fully investigate the expression of complement proteins in glaucoma using two mouse models of glaucoma. We will also assess the affect of conditionally ablating a key molecule in the complement pathway in specific cell types. This work will determine exactly how the complement pathway influences glaucoma.