STAT1-Galectin-3通过促进活化的视乳头星形胶质细胞吞噬作用调控青光眼视神经损伤的机制研究

Glaucoma is the leading cause of irreversible blindness in the world. Astrocyte activation plays an important role in glaucomatous neuropathy. However, the molecular mechanism of activated astrocyte and its function are not clear. In the early study, we found that astrocytes in Myelination transition zone(MTZ) activated and form new longitudinal processes in the optic nerve head of mouse glaucoma model, and confirmed that these newly formed longitudinal processes engulfed the degenerating mitochondria from the nearby axon of the optic nerve by retrograde mitochondrial tracing. We also found galectin-3, which is a phagocytosis-related gene, is highly expressed in MTZ astrocytes in glaucomatous mouse. Therefore, we hypothesized that activated MTZ astrocytes help the nearby axon to clear the degenerating mitochondrial. This project will use single cell separation technique, primary culture of optic nerve head astrocytes and GFP+ transgenic mice to establish a glaucoma model. This project will demonstrate that STAT1-Galectin-3 regulates the phagocytic function of activated astrocytes in MTZ by engulfing degenerated mitochondria from the nearby axons in vivo and in vitro and explore the molecular mechanism of up-regulation of galentin-3, and finally to find out the effect of phagocytosis of astrocytes on the glaucomatous optic nerve injury. This project will describe a new phenotype and molecular mechanism of activation of MTZ astrocytes in glaucoma, providing a potential therapeutic strategy for optic nerve protection.

青光眼是全球第一位不可逆性致盲眼病，星形胶质细胞活化在青光眼性视神经损伤中具有重要作用，但其活化的分子机制和功能尚未阐明。我们前期首次报道了在青光眼小鼠中视乳头髓鞘移行区（MTZ）星形胶质细胞活化并生成触手样突起，并证实触手样突起可吞噬视神经轴突内退化线粒体。进一步发现在青光眼小鼠MTZ星形胶质细胞内吞噬相关基因galectin-3高表达。因此，提出活化的MTZ星形胶质细胞协助轴突跨细胞代谢线粒体。本项目将用单细胞分离技术、视乳头星形胶质细胞原代培养及GFP+转基因小鼠建立青光眼模型。在体内外证实STAT1-Galectin-3通过促进MTZ活化的星形胶质细胞吞噬轴突内退化的线粒体调控视神经损伤修复；明确galentin-3表达上调的分子机制；探究星形胶质细胞吞噬功能改变对视神经损伤的影响。本项目将阐述青光眼中视乳头星形胶质细胞活化的一种新表型及其分子机制，为视神经保护提供潜在治疗策略。

星形胶质细胞是分布在视神经乳头处主要的胶质细胞类型之一，它对维持神经细胞的生理功能极为重要。其生理功能包括：提供组织构架、维持细胞外微环境、调节突触功能、释放神经递质及调节血流等。大量研究表明，在青光眼或视神经损伤发生时，星形胶质细胞对机械压力引起的视神经损伤产生强烈应答，发生活化，如细胞体特征性肥大和突起结构紊乱；活化特异性标记物的上调（胶质纤维酸性蛋白（Glialfibrillary acidic protein，GFAP）、Vimentin等）；伴随细胞功能改变（细胞增殖、迁移以及释放细胞外基质等）。星形胶质细胞可能存在神经毒性（A1型星形胶质细胞）及神经保护（A2型星形胶质细胞）两种状态。星形胶质细胞活化与视神经损伤密切相关，如可通过旁分泌作用，限制神经炎症反应，协调神经元稳态，但亦可能会抑制视神经纤维轴突再生。.本项目主要研究内容及结果：1）利用小鼠慢性高眼压动物模型，明确视神经视乳头区域活化的视乳头星形胶质细胞功能的异质性，高眼压诱导的视神经损伤伴随着损伤区域的星形胶质细胞活化及吞噬能力增强。2）通过逆向示踪法证实活化的星形胶质细胞可协助损伤的轴突进行跨细胞线粒体代谢。通过单细胞分离技术进行视乳头星形胶质细胞单细胞种类的定量PCR，显示高眼压小鼠模型中，视乳头星形胶质细胞活化标记以及吞噬相关的基因（Gulp1和Lgals3）表达上调。同时证明Lgals3基因产物Galectin-3仅在星形胶质细胞中表达上调。3）建立改良视网膜视神经体外组织培养技术。联合线粒体逆行示踪技术，可在更加接近在体条件下，研究视乳头星形胶质细胞活化吞噬作用。4）利用腺病毒敲低和过表达STAT1\Galectin-3观察其在外植体培养模型中对星形胶质细胞的活化吞噬的影响。转录因子STAT1调控的Galectin-3可促进星形胶质细胞的活化和跨细胞吞噬RGCs轴突的线粒体。.基于上述结果，本研究发现高眼压发生时，可能通过STAT1/ Galectin-3通路促进视乳头区域星形胶质细胞活化以及吞噬作用增强，从而协助损伤的视神经轴突进行线粒体跨细胞代谢。为探寻新的视神经保护机制提供理论依据。