Mechanisms of naturally-occurring astrocyte death during retinal development

自然发生的细胞死亡对于发育中的神经系统的模式形成至关重要。视网膜天文- 细胞经历由小胶质细胞介导的不寻常的非凋亡形式的死亡；这个机制对于pat-至关重要 将星形胶质细胞转变为控制血管生成的模板。尽管如此重要，但该机制 哪种小胶质细胞杀死星形胶质细胞尚不清楚。要解开这些机制，我们必须了解细胞和运动 杀死星形胶质细胞的小胶质细胞的分子特征，但这也仍然未知。这里的目标是 识别执行发育性星形胶质细胞死亡的小胶质细胞以及实现该功能的机制。 中心假设是发育短暂的小胶质细胞群，称为神经纤维层 - 相关小胶质细胞（NFL-M），响应星形胶质细胞衍生的“吃我”信号并介导星形胶质细胞消除 。这项工作的基本原理是星形胶质细胞死亡机制的知识将使学习 星形胶质细胞和血管网络如何在正常和病理状态下达到成熟模式 发展。此外，由于非凋亡“吞噬细胞死亡”也可能发生在神经退行性疾病中，因此 对死亡机制的洞察可能与疾病病理学具有广泛的相关性。具体目标是：1) 识别星形胶质细胞“吃我”信号招募的视网膜小胶质细胞亚群。初步研究 表明磷脂酰丝氨酸（PtdSer）是星形胶质细胞表面的“吃我”信号，可招募吞噬细胞 神经胶质细胞。单细胞 RNA 测序鉴定出一个小胶质细胞群 NFL-M，它可能是星形胶质细胞 响应子集。这一工作假设将通过操纵星形胶质细胞密度和 PtdSer 暴露来检验 当然，如果假设正确，这应该会改变 NFL-M 的数量和/或视网膜分布。 2）识别 执行星形胶质细胞吞噬的小胶质细胞群。长时间的吞噬作用需要高 线粒体输出。 NFL-M 显示出强烈的线粒体代谢的转录特征，包括 ac- Nrf2 信号传导的激活，这是对代谢应激的关键反应。这些数据表明 NFL-M 具有高度吞噬能力 朝向星形胶质细胞；该假设将通过阻止小胶质细胞的基因操作在体内进行测试 假设 NFL-M 状态或激活 Nrf2。 3) 鉴定星形胶质细胞相关表达的基因 小胶质细胞介导其功能。如果星形胶质细胞被转录独特的小胶质细胞群杀死 化，使它们独特的基因应该支持这种功能。 Lgals3，一种在噬菌体中具有已知作用的基因 胞质作用，由 NFL-M 选择性表达。初步数据表明，它对于小胶质细胞的吞噬作用至关重要 在视网膜变性中，支持了星形胶质细胞去除也需要它的工作假设。 这将使用 Lgals3 条件突变小鼠进行测试。这项工作的完成预计将确定具体的 杀死发育中的星形胶质细胞的小胶质细胞亚群及其对视网膜星形胶质细胞和血管模式的贡献- ing。这将具有重要意义，因为它将为定义吞噬细胞死亡的机制开辟道路 以及这种新的死亡机制对神经系统发育和疾病的贡献。

Naturally-occurring cell death is essential to pattern formation in the developing nervous system. Retinal astro- cytes undergo an unusual non-apoptotic form of death mediated by microglia; this mechanism is crucial for pat- terning astrocytes into a template that controls angiogenesis. Despite this importance, the mechanism by which microglia kill astrocytes is unknown. To unravel these mechanisms one must know the cellular and mo- lecular characteristics of the microglia that kill astrocytes, but this too remains unknown. The objective here is to identify microglia that execute developmental astrocyte death and the mechanisms that enable this function. The central hypothesis is that a developmentally transient microglial population, termed nerve fiber layer- associated microglia (NFL-M), responds to astrocyte-derived “eat-me” signals and mediates astrocyte elimina- tion. The rationale for this work is that knowledge of astrocyte death mechanisms will make it possible to learn how the astrocyte and vascular networks arrive at their mature pattern – in both normal and pathological de- velopment. Further, as non-apoptotic “death by phagocyte” can occur also in neurodegenerative disease, in- sight into death mechanisms may have broad relevance for disease pathobiology. The Specific Aims are: 1) Identify retinal microglial subsets that are recruited by astrocyte “eat-me” signals. Preliminary studies show that phosphatidylserine (PtdSer) is an astrocyte cell-surface “eat-me” cue that recruits phagocytic micro- glia. Single-cell RNA-sequencing identified a microglial population, the NFL-M, that is likely to be the astrocyte- responsive subset. This working hypothesis will be tested by manipulating astrocyte density and PtdSer expo- sure, which should alter the number and/or retinal distribution of NFL-M if the hypothesis is correct. 2) Identify microglial populations that execute astrocyte engulfment. Prolonged bouts of phagocytosis require high mitochondrial output. NFL-M show transcriptional signatures of intense mitochondrial metabolism including ac- tivation of Nrf2 signaling, a key response to metabolic stress. These data suggest NFL-M are highly phagocytic towards astrocytes; this hypothesis will be tested in vivo using genetic manipulations that prevent microglia from assuming the NFL-M state or activating Nrf2. 3) Identify genes expressed by astrocyte-associated microglia that mediate their function. If astrocytes are killed by a transcriptionally unique microglial popula- tion, the genes that make them unique should support this function. Lgals3, a gene with known roles in phago- cytosis, is selectively expressed by NFL-M. Preliminary data show that it is crucial for microglial phagocytosis in retinal degeneration, supporting the working hypothesis that it will also be required for astrocyte removal. This will be tested using Lgals3 conditional mutant mice. Completion of this work is expected to define specific microglial subsets that kill developing astrocytes and their contribution to retinal astrocyte and vascular pattern- ing. This will be significant because it will open the way to defining mechanisms underlying death by phagocyte and the contributions of this novel death mechanism to nervous system development and disease.