Establishing a human cellular model of retinal ganglion cell compartmentalization in neurodegeneration and neuroinflammation

摘要 视网膜神经节细胞(RGC)是视网膜的投射神经元，起着连接 眼睛和大脑。在这一角色中，它们允许将视觉信息传输到丘脑目标，具有 损伤在受伤或疾病中对这一通路的破坏，导致视力丧失或失明。胶质细胞，尤其是星形胶质细胞 和小胶质细胞，与视神经内的视网膜节细胞相邻，在那里它们维持内环境平衡状态。 以确保RGC的健康和功能正常。相反，神经炎性疾病在以下情况下发生 星形胶质细胞和小胶质细胞被诱导进入反应性状态，导致视网膜节细胞变性。 神经炎症与多种神经退行性疾病有关，但 青光眼的神经炎症是独一无二的，因为视神经中胶质细胞的高度局部性反应。 头部作为RGC轴突离开眼睛，与青光眼中沿RGC轴突的初始损伤位置相关。而当 动物模型已经证明了神经胶质细胞在神经元发育和退化中的重要性 动物模型和人类患者之间存在差异，包括RGC的低保守性以及 在神经胶质细胞中存在大量的功能差异。因此，这些细胞相互作用的人体模型的开发 将进一步扩大我们对胶质细胞如何为视网膜节细胞提供支持以及胶质细胞如何反应的理解 在导致青光眼视网膜神经节细胞变性的神经炎性条件下。人多能干细胞 细胞(HPSCs)可以作为研究视网膜发育和疾病的强大的体外模型， 先前的研究证实了体外模拟RGC神经变性的能力。然而，这些研究 没有关注视网膜节细胞的分区性质，也没有关注反应性胶质细胞如何不成比例地影响视网膜节细胞 神经炎性条件下视网膜节细胞的轴突。因此，为了解决现有基于hPSC的 青光眼模型和更好地概括视网膜节细胞和胶质细胞之间的相互作用，目前的应用 利用健壮且可重现的体外模型重建神经胶质细胞与人RGC的空间相互作用 轴突与青光眼中观察到的神经退行性变表型有关。神经胶质细胞与RGC的相互作用 将分析静态舱和反应舱，以及反应舱的功能后果 将对RGC轴突上的胶质细胞进行表型、转录和功能评估，以确定 反应性胶质细胞调节RGC神经退行性变。成功地追求以下目标将对 一个强大的微流控平台用于分析RGC轴突，包括神经胶质细胞的神经炎症效应 并将为进一步阐明人类视网膜节细胞的基本神经退行性机制提供机会， 以及开发延缓或逆转神经变性的新治疗方法。

SUMMARY Retinal ganglion cells (RGCs) are the projection neurons of the retina that serve as the connection between the eye and the brain. In this role, they allow for the transmission of visual information to thalamic targets, with damage to this pathway in injury or disease leading to vision loss or blindness. Glial cells, particularly astrocytes and microglia, are found adjacent to RGCs within the optic nerve, where they maintain homeostatic conditions for RGCs to ensure proper health and functionality. Conversely, neuroinflammatory conditions occur when astrocytes and microglia are induced to adopt a reactive state, leading to the degeneration of RGCs. Neuroinflammation has been associated with a variety of neurodegenerative diseases, but the pathology of neuroinflammation in glaucoma is unique due to the highly localized nature of glial reactivity in the optic nerve head as RGC axons exit the eye, correlated with the initial site of injury along RGC axons in glaucoma. While animal models have demonstrated the importance of glia in neuronal development and degeneration, important differences exist between animal models and human patients, including low conservation of RGCs as well as numerous functional differences in glia. As such, the development of a human model of these cellular interactions would further expand our understanding of how glia provide support for RGCs, as well as how glia respond during neuroinflammatory conditions leading to the degeneration of RGCs in glaucoma. Human pluripotent stem cells (hPSCs) can serve as powerful in vitro models for the study of retinal development and disease, with previous studies demonstrating the ability to model RGC neurodegeneration in vitro. However, these studies have not focused upon the compartmentalized nature of RGCs, nor how reactive glia disproportionally affect the axons of RGCs in neuroinflammatory conditions. Thus, to address the shortcomings of existing hPSC-based models of glaucoma and to better recapitulate interactions between RGCs and glia, the current application leverages a robust and reproducible in vitro model to recreate the spatial interactions of glia upon human RGC axons relevant to the neurodegenerative phenotypes observed in glaucoma. Interactions between glia and RGC compartments will be analyzed in quiescent and reactive states, and the functional consequences of reactive glia upon RGC axons will be assessed phenotypically, transcriptionally, and functionally to identify the extent to which reactive glia modulate RGC neurodegeneration. The successful pursuit of the following aims will leverage a powerful microfluidic platform for the analysis of RGC axons to include the neuroinflammatory effects of glia and will provide opportunities to further elucidate fundamental neurodegenerative mechanisms in human RGCs, as well as to develop novel therapeutic approaches to slow or reverse neurodegeneration.