Regulation of Glia-driven Neuroinflammation in Glaucoma

项目总结 最近对临床/尸检和实验样本的研究积累了支持 炎症反应与青光眼神经变性的关系。虽然炎症性方面 青光眼神经退行性变引起越来越多的关注，神经炎症是如何在 青光眼，以及我们如何防止神经退行性炎症仍是未知的。在这里，我们向大家介绍 实验数据使我们假设，NF-κB在激活神经胶质细胞驱动的 青光眼视网膜和视神经中的神经炎症以及针对该转录激活因子的 机制是提供免疫调节和避免二次损伤过程的合理策略。怀着敬意 星形胶质细胞和小胶质细胞在诱导炎症/神经毒性中的密切关系 表型，炎性介质的关键转录激活因子NF-κB对这两种胶质细胞都是关键的 青光眼神经炎的亚型及其相互作用。为了检验这一假设，我们将比较 研究针对神经胶质亚型的条件转基因株系(其中NF-κB正则途径通过以下方式失活 药物诱导星形胶质细胞或小胶质细胞中IκKβ的缺失)和有或没有眼睛的背景对照 高血压(由前房微珠/粘弹性注射引起)。我们将纵向分析神经胶质 亚型靶向转基因对不同损伤部位的不同炎症结果的影响(视网膜， 视神经头和视神经轴突)。研究结果将包括神经胶质炎症激活。 胶质细胞亚型靶向IκKβ缺失对表型和细胞类型的影响除了一组预选的 分子、新分离的星形胶质细胞和小胶质细胞样本将用定量质谱仪进行分析。 还可以获得关于转基因对NF-κB调节的炎症的影响的高通量分子信息 核因子-κB的其他转录靶点，以及与神经胶质支持功能相关的分子。由于神经胶质细胞 在青光眼中既有先天免疫反应又有获得性免疫反应，其他分析将包括T细胞的反应性 和自身抗体的产生。确定转基因是否能抑制神经胶质细胞的NF-κB(和神经炎症) 保护小鼠青光眼神经元免受免疫原性损伤，我们还将分析神经元结构(通过RGC 和轴突计数)和功能(按PERG)。我们预计，这个项目是对星形胶质细胞或小胶质细胞的补充- 具有星形胶质细胞特异性和小胶质细胞特异性反应的分子分析的靶向转基因系(在分离的 特定细胞类型的样本)将使我们能够确定NF-κB在个体贡献和相互作用中的作用 星形胶质细胞和小胶质细胞在神经炎症(视网膜、视神经头和轴突)中的关系 鉴定特定的分子组成，并评价胶质细胞NF-κB作为恢复免疫的治疗靶点的价值 在青光眼中通过免疫调节来维持体内平衡并提供神经保护。

PROJECT SUMMARY Recent studies of clinical/postmortem and experimental samples have accumulated evidence that supports a connection of inflammatory responses to neurodegeneration in glaucoma. Although inflammatory aspects of glaucomatous neurodegeneration are attracting increasing attention, how neuroinflammation is regulated in glaucoma, and how we can prevent neurodegenerative inflammation remain unknown. Here, we present experimental data leading us to hypothesize that NF-κB plays a key role in activating the glia-driven neuroinflammation in the glaucomatous retina and optic nerve, and that targeting this transcriptional activator mechanism is a logical strategy to provide immunomodulation and avoid secondary injury processes. With respect to intimate inter-relationship between astroglia and microglia in induction of the inflammatory/neurotoxic phenotype, NF-κB, the key transcriptional activator of inflammatory mediators, should be critical for both glial subtypes and their interaction in neuroinflammation in glaucoma. To test this hypothesis, we will comparatively study glial subtype-targeted conditional transgenic lines (in which the NF-κB canonical pathway is inactivated by drug-induced deletion of IκKβ in astroglia or microglia) and background controls with or without ocular hypertension (induced by anterior chamber microbead/viscoelastic injections). We will longitudinally analyze glial subtype-targeted transgenic effects on a diverse set of inflammatory outcomes at different sites of injury (retina, optic nerve head, and optic nerve axons). The studied outcomes will include the glial inflammatory activation phenotype and cell type-specific effects of glial subtype-targeted IκKβ deletion. Besides a preselected set of molecules, freshly isolated samples of astroglia and microglia will be analyzed by quantitative mass spectrometry to also gain high-throughput molecular information about transgenic effects on NF-κB-regulated inflammation pathways, other transcriptional targets of NF-κB, and molecules relevant to glial neurosupport functions. Since glia drive both innate and adaptive immune responses in glaucoma, additional analysis will include T-cell reactivity and autoantibody production. To determine whether transgenic inhibition of glial NF-κB (and neuroinflammation) protects neurons from immunogenic injury in mouse glaucoma, we will also analyze neuron structure (by RGC and axon counts) and function (by PERG). We expect that this project, complementing the astroglia- or microglia- targeted transgenic lines with molecular analysis of astroglia-specific and microglia-specific responses (in isolated cell type-specific samples) will allow us to determine the role of NF-κB in individual contribution and inter- relationship of astroglia and microglia in neuroinflammation (in the retina, optic nerve head, and axons), characterize specific molecular components, and value glial NF-κB as a treatment target to restore immune homeostasis and provide neuroprotection through immunomodulation in glaucoma.