Investigating Cell Specific Roles of the Complement Cascade in Glaucoma

描述(申请人提供)：我的数据显示，编码补体级联成员的基因在青光眼期间很早就发生上调，在视网膜(视网膜神经节细胞和小胶质细胞)和视神经头(小胶质细胞)中都是如此。重要的是，携带补体成分1qA(C1qA)基因突变的小鼠可以有效地预防青光眼。这种强大的保护作用表明，靶向补体诱导可能对许多青光眼患者非常有效。然而，在开发潜在的治疗方法之前，有必要在多种动物模型中充分了解补体生物合成影响青光眼的机制。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：广泛使用的遗传性模型DBA/2J和新近发展的基于珠粒的诱导性小鼠模型。我将使用携带C1qA(2Gal)报告以及C3、C3R和抑制物CRRY的小鼠，使用免疫荧光和RNA原位杂交来评估C1QA的表达。这是首次在两种不同的青光眼动物模型中评估补体蛋白，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神经节细胞两个关键变化的早期中介，这两个变化是板层的轴突损伤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。轴突损伤将使用DBA/2J.Thy1(CFP)可视化，突触变化将使用PSD95和Ribeye抗体进行评估。我将重点介绍补体级联中的关键分子C1qa。关注早期青光眼的眼睛将使我能够确定补体系统的变化是否先于ONH和视网膜的RGC早期损伤。最后，我将使用条件性基因打靶来确定突变C1QA在视网膜节细胞或小胶质细胞中的作用。C1QA突变小鼠可以预防青光眼，初步数据显示，C1QA在视网膜节细胞和小胶质细胞中都有表达。这些发现，以及其他显示人类青光眼补体成分上调的研究，突显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技术来特异性地阻断RGC、小胶质细胞或这两种细胞中C1QA的生物合成。青光眼将使用成熟的方法进行评估，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重要性对于设计操纵补体系统的治疗干预措施很重要。 与公共卫生相关：青光眼是一种致盲疾病，全世界有超过7000万人患有青光眼。临床上没有批准直接针对视网膜神经节细胞的治疗方法，视网膜神经节细胞是在青光眼中丢失的视网膜输出神经元。我们和其他人已经证实，补体途径在疾病的早期发挥着重要作用。我们将使用两种青光眼小鼠模型来全面研究补体蛋白在青光眼中的表达。我们还将评估有条件地去除补体途径中的一个关键分子对特定细胞类型的影响。这项工作将准确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.