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，视神经头是轴突损伤的主要部位 这是影响全世界7600万人的不可逆转失明的主要原因。高位眼内 压力(IOP)是青光眼的唯一可改变的危险因素，而降低IOP是唯一可行的策略 延缓视力丧失的进程。因此，迫切需要有针对性的新的治疗策略。 ONH内轴突损伤的部位。为了开发针对青光眼的新型ONH靶向治疗方法，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内的局部胶质细胞)提供 对轴突的结构和代谢支持。在包括青光眼在内的神经退行性疾病中，星形胶质细胞 变得“反应性”，并显示出结构和分子的变化。在几个青光眼模型中，包括我们的， ONH星形胶质细胞以肌动蛋白和中间丝为基础的细胞骨架活性显著发生在 明显的轴突损伤。眼压依赖的ONH星形胶质细胞的反应是神经保护还是帮助驱动 疾病，或者这些反应的调节是否改变了轴突对高眼压的易感性仍然存在 不清楚。利用急性眼压升高的啮齿动物模型，我们的初步数据表明，ONH星形胶质细胞 通过收回其基于肌动蛋白的细胞延伸和减少连接蛋白43的标记(星形胶质细胞间隙)来做出反应 连接蛋白参与维持星形胶质细胞合体等电势，并依赖于肌动蛋白细胞骨架 用于本地化)。此外，我们还证明了肌动蛋白细胞骨架的稳定性(使用Rho激酶抑制剂 法舒地尔显著降低ONH星形胶质细胞细胞骨架-缝隙连接反应性并保护轴突 模特。最后，使用我们实验室开发的体内手术策略，我们证明了ONH星形胶质细胞可以 由局部小分子输送到ONH进行调制。在这项提案中，我们将确定ONH的角色 结合我们的啮齿动物模型，急性眼压升高后轴突变性中星形胶质细胞的细胞骨架反应 通过局部和系统地传递细胞骨架的小分子调节剂。接下来，我们将研究 ONH星形细胞连接蛋白43在眼内压依赖性轴突变性中的作用 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，我们将处理 ONH星形胶质细胞细胞骨架和缝隙连接在眼内压依赖性轴突变性中的作用 它们的调节是轴突保护的一种新策略。该项目的最终长期目标是实现 新的星形胶质细胞特异性治疗靶点，以减轻全球青光眼视力丧失的负担。

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.