Cell Biology of Astrocyte-Ganglion Cell Interactions in the Retina and Optic Nerve

项目摘要 青光眼导致视网膜神经节细胞的进行性丧失，其机制尚不完全清楚。在 目前，降低眼内压（IOP）是唯一的治疗方法， 以不依赖于IOP的方式预防神经节细胞变性将是受欢迎的。有证据表明 神经节细胞变性的第一个迹象发生在视神经头中，在视神经头处，神经节细胞轴突离开视神经。 通过巩膜上的一个孔将地球仪植入眼球以形成视神经。在这个区域，轴突是无髓鞘的， 与星形胶质细胞直接接触。视神经星形胶质细胞对损伤（如IOP升高）的反应， 它们的形态和基因表达模式的变化。我们发现，至少在早期阶段， 疾病时，星形胶质细胞的反应是一种保护性反应，阻止它会导致神经节细胞的更坏结果。 细胞和视觉功能。这一结果表明，星形胶质细胞，或星形胶质细胞衍生的因子，可以利用 用于神经保护性青光眼治疗，可以添加到已经使用的降眼压药物中。 在我们寻找调节保护性反应的星形胶质细胞衍生因子时，我们鉴定了分泌型 磷蛋白1（SPP 1，也称为骨桥蛋白）。这种蛋白质在正常组织中仅以低水平表达。 视神经，但它在迄今为止研究的所有啮齿动物青光眼模型中均被稳健上调。此外，SPP 1是 在稀疏的视网膜神经节细胞群中组成型表达。使用SPP 1敲除小鼠，我们 显示SPP 1缺乏导致视神经中星形胶质细胞和轴突损伤的形态学迹象 即使在没有IOP升高的情况下也是如此。在青光眼微珠阻塞模型中，SPP 1缺陷 与野生型对照组相比，小鼠失去更多的神经节细胞，视觉功能更差。最重要的是，病毒- 视网膜中SPP 1介导的过表达高度保护神经节细胞功能， 神经节细胞损失而不影响IOP。 基于这些发现，我们认为SPP 1可能是一种有希望的神经保护疗法的候选者。 青光眼然而，目前我们还不清楚SPP 1在反应性星形胶质细胞或星形胶质细胞中的表达是否与细胞凋亡有关。 需要视网膜神经节细胞或两者来实现最佳保护。为了解决这个问题，我们有 设计了一种转基因小鼠品系，该品系允许星形胶质细胞、视网膜神经胶质细胞和视网膜神经胶质细胞中SPP 1的细胞类型特异性缺失。 神经节细胞（或其他细胞类型）。小鼠也会表达不同的荧光标记蛋白 在SPP 1缺失之前和之后的靶细胞中。我们将使用这个新工具来解决我们的假设， 视神经中的星形胶质细胞来源的SPP 1和视网膜中的神经节细胞来源的SPP 1都是必需的， 青光眼中的最佳神经节细胞保护（目的1和2）。在我们的目标3中，我们将测试 SPP 1过表达对视网膜神经节细胞功能具有长期保护作用， 可能由慢性SPP 1过表达引起的不良反应。

Project Summary Glaucoma leads to a progressive loss of retinal ganglion cells by mechanisms that are not fully understood. At present, lowering the intraocular pressure (IOP) is the only treatment, and new therapeutic approaches that prevent ganglion cell degeneration in a manner independent of IOP would be welcome. There is evidence that the first signs of ganglion cell degeneration occur in the optic nerve head where the ganglion cell axons exit the globe through a hole in the sclera to form the optic nerve. In this region, the axons are unmyelinated and come into direct contact with astrocytes. Optic nerve astrocytes react to injury – such as an increase in IOP - with changes in their morphology and gene expression pattern. We showed that, at least in the early stages of the disease, astrocyte reactivity is a protective response and preventing it leads to a worse outcome for ganglion cells and visual function. This result suggests that astrocytes, or astrocyte-derived factors, can be harnessed for a neuroprotective glaucoma therapy that could be added to IOP-lowering drugs that are already in use. In our search for astrocyte-derived factors that mediate the protective response, we identified secreted phosphoprotein 1 (SPP1, also called osteopontin). This protein is expressed only at low levels in the normal optic nerve, but it is robustly up-regulated in all rodent glaucoma models studied so far. In addition, SPP1 is constitutively expressed in a sparse population of retinal ganglion cells. Using an SPP1 knock out mouse, we showed that SPP1 deficiency leads to morphological signs of astrocyte and axon damage in the optic nerve head even in the absence of elevated IOP. In the microbead occlusion model of glaucoma, SPP1 deficient mice lose more ganglion cells and have worse visual function than wild-type controls. Most importantly, virus- mediated overexpression of SPP1 in the retina is highly protective of ganglion cell function and prevents ganglion cell loss without affecting IOP. Based on these findings, we believe that SPP1 may be a promising candidate for a neuroprotective therapy in glaucoma. However, at present we do not know whether the SPP1 expression in reactive astrocytes or in retinal ganglion cells or both are needed to achieve optimal protection. To address this question, we have designed a transgenic mouse strain that will allow for cell-type specific deletion of SPP1 in astrocytes, retinal ganglion cells (or other cell types) separately. The mice will also express different fluorescent marker proteins in targeted cells before and after the deletion of SPP1. We will use this new tool to address our hypothesis that astrocyte-derived SPP1 in the optic nerve and ganglion cell-derived SPP1 in the retina are both necessary for optimal ganglion cell protection in glaucoma (Aims 1 and 2). In our translational Aim 3, we will test whether overexpression of SPP1 is protective of retinal ganglion cell function in the long term and assess ocular tissues for adverse effects that may result from chronic SPP1 overexpression.