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

总结 视网膜神经节细胞（RGC）是视网膜的投射神经元，其充当视网膜神经元之间的连接。 眼睛和大脑。在这个角色中，它们允许将视觉信息传输到丘脑目标， 在损伤或疾病中对该通路的损害导致视力丧失或失明。胶质细胞，特别是星形胶质细胞 和小胶质细胞，在视神经内与RGCs相邻，在视神经内它们维持稳态 以确保RGC的健康和功能。相反，神经炎性疾病发生时， 星形胶质细胞和小胶质细胞被诱导采取反应性状态，导致RGC变性。 神经炎症与多种神经退行性疾病相关，但神经炎症的病理学改变与神经退行性疾病相关。 青光眼中的神经炎症是独特的，这是由于视神经中神经胶质反应性的高度局部化性质 头部作为RGC轴突离开眼睛，与青光眼中沿沿着RGC轴突损伤的初始部位相关。而 动物模型已经证明了神经胶质细胞在神经元发育和变性中的重要性， 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 神经胶质细胞的许多功能差异。因此，这些细胞相互作用的人类模型的开发 将进一步扩大我们对神经胶质细胞如何为RGC提供支持以及神经胶质细胞如何响应的理解， 在导致青光眼中RGC变性的神经炎性病症期间。人多能干 细胞（hPSC）可以作为研究视网膜发育和疾病的强大体外模型， 先前的研究证明了体外模拟RGC神经变性的能力。但是这些研究 没有关注RGC的区室化性质，也没有关注反应性胶质细胞如何影响神经元的功能。 神经炎症条件下RGCs的轴突。因此，为了解决现有的基于hPSC的免疫治疗的缺点， 青光眼模型，并更好地概括RGC和神经胶质细胞之间的相互作用，目前的应用 利用一个强大的和可重复的体外模型，以重建人类RGC上胶质细胞的空间相互作用 与青光眼中观察到的神经变性表型相关的轴突。胶质细胞与RGC的相互作用 将在静止和反应状态下分析隔室，以及反应的功能后果。 将在表型、转录和功能上评估RGC轴突上的神经胶质，以确定 其中反应性胶质细胞调节RGC神经变性。成功实现以下目标将有助于 一个强大的微流体平台，用于分析RGC轴突，包括神经胶质细胞的神经炎症作用 并将为进一步阐明人类RGC中的基本神经退行性机制提供机会， 以及开发新的治疗方法来减缓或逆转神经变性。

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.