Modulation of optic nerve head astrocyte reactivity in glaucoma

项目摘要 青光眼是一种神经退行性疾病，视神经乳头（ONH）是青光眼轴突损伤的主要部位 这是造成全世界7 600万人不可逆转失明的主要原因。升高的眼内 眼压（IOP）是青光眼唯一可改变的危险因素，降低IOP是唯一可用的策略， 减缓视力丧失的进程。因此，迫切需要靶向肿瘤的新的治疗策略。 ONH内轴突损伤的部位。为了开发新的ONH靶向治疗青光眼，我们必须 确定导致ONH轴突损伤的早期细胞事件。星形胶质细胞（ONH内的局部神经胶质细胞）提供 轴突的结构和代谢支持。在神经退行性疾病中，包括青光眼、星形胶质细胞 变得"反应性"并显示结构和分子变化。在几个青光眼模型中，包括我们的， 显著的ONH星形胶质细胞肌动蛋白和中间体基于细胞骨架的反应性发生在 可见轴突损伤。IOP依赖性ONH星形胶质细胞的反应性是否具有神经保护作用或帮助驱动 疾病，或者这些反应性反应的调节是否改变了轴突对升高的IOP的脆弱性， 不清楚使用啮齿动物模型的急性眼压升高，我们的初步数据表明，ONH星形胶质细胞 通过收回基于肌动蛋白的细胞延伸并减少连接蛋白43标记（星形胶质细胞间隙）来做出反应 一种连接蛋白，参与维持星形胶质细胞合胞体等电位，并依赖于肌动蛋白细胞骨架 本地化）。此外，我们表明，肌动蛋白细胞骨架稳定（使用Rho激酶抑制剂 法舒地尔）显着降低ONH星形胶质细胞细胞骨架和间隙连接反应性，并保护轴突， 模型最后，使用我们实验室开发的体内手术策略，我们表明ONH星形胶质细胞可以被 通过向ONH的局部小分子递送来调节。在本提案中，我们将确定ONH的作用 急性眼压升高后轴突变性中星形胶质细胞细胞骨架反应性，结合我们的啮齿动物模型 局部和全身递送细胞骨架的小分子调节剂。接下来，我们将研究 ONH星形胶质细胞连接蛋白43在小分子和高分子介导的IOP依赖性轴突变性中的机制作用 在我们的啮齿动物模型中抑制连接蛋白43的遗传策略。轴突和星形胶质细胞特异性 ONH组织的免疫荧光将用于确定轴突损伤和星形胶质细胞损伤的程度。 眼压升高后不同时间点ONH内的反应。在这项工作中，我们将 ONH星形胶质细胞骨架和缝隙连接在IOP依赖性轴突变性中的作用，以及 它们的调制作为轴突保护的新策略。该项目的最终长期目标是 新的星形胶质细胞特异性治疗靶点，以减轻全球范围内的青光眼视力丧失负担。

Project Summary The optic nerve head (ONH) is the primary site of axon injury in glaucoma, a neurodegenerative disease that is the leading cause of irreversible blindness affecting 76 million people worldwide. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, and lowering IOP is the only available strategy to slow the progression of vision loss. Thus, there is a critical need for novel therapeutic strategies targeting the site of axon injury within the ONH. In order to develop novel ONH-targeted treatments for glaucoma, we must determine the early cellular events that lead to ONH axon injury. Astrocytes (local glia within the ONH) provide structural and metabolic support for axons. In neurodegenerative disorders including glaucoma, astrocytes become “reactive” and display structural and molecular changes. In several glaucoma models, including ours, significant ONH astrocyte actin- and intermediate filament-based cytoskeletal reactivity occurs prior to observable axon injury. Whether IOP-dependent ONH astrocyte reactivity is neuroprotective or helping drive disease, or whether modulation of these reactive responses alters axon vulnerability to elevated IOP remain unclear. Using a rodent model of acute IOP elevation, our preliminary data demonstrate that ONH astrocytes react by retracting their actin-based cellular extensions and reducing connexin43 labeling (an astrocyte gap junction protein involved in maintaining astrocyte syncytial isopotentiality, and reliant on the actin cytoskeleton for localization). Furthermore, we show that actin cytoskeletal stabilization (using the Rho kinase inhibitor fasudil) significantly reduces ONH astrocyte cytoskeletal & gap junction reactivity and protects axons in this model. Lastly, using an in vivo surgical strategy developed in our lab, we show that ONH astrocytes can be modulated by local small molecule delivery to the ONH. In this proposal, we will determine the role of ONH astrocyte cytoskeletal reactivity in axon degeneration after acute IOP elevation, by combining our rodent model with local and systemic delivery of small molecule modulators of the cytoskeleton. Next, we will examine the mechanistic role of ONH astrocytic connexin43 in IOP-dependent axon degeneration using small molecule and genetic strategies to suppress connexin43 in our rodent models. Axon- and astrocyte-specific immunofluorescence of ONH tissue will be used to determine the extent of axon injury and astrocyte responses within the ONH at various time points after IOP elevation. In the course of this work, we will address the role of the ONH astrocyte cytoskeleton and gap junctions in IOP-dependent axon degeneration, as well as their modulation as a novel strategy for axon protection. The ultimate long-term goal of this project is to bring to light new astrocyte-specific therapeutic targets to reduce the burden of glaucomatous vision loss worldwide.