Mechanisms of naturally-occurring astrocyte death during retinal development

视网膜发育过程中星形胶质细胞自然死亡的机制

• 批准号: 10583310
• 负责人: Jeremy N Kay
• 金额: $ 40.39万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10583310
• 关键词: Acute; Affect; Apoptosis; Astrocytes; Automobile Driving; Biological Assay; Blood Vessels; Candidate Disease Gene; Cell Death; Cell surface; Cells; Central Nervous System; Cessation of life; Characteristics; Cues; Data; Defect; Development; Disease; Eating; Excision; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Goals; Human; Knock-out; Knowledge; Learning; Link; Mediating; Mediator; Membrane Lipids; Metabolic; Metabolic stress; Microglia; Mitochondria; Molecular; Mus; Mutant Strains Mice; Nerve Fibers; Nervous System; Neurodegenerative Disorders; Outcome; Output; Pathologic; Pattern; Pattern Formation; Phagocytes; Phagocytosis; Phosphatidylserines; Population; Population Sizes; Positioning Attribute; Public Health; Publishing; Research; Retina; Retinal Degeneration; Retinopathy of Prematurity; Role; Shapes; Signal Transduction; Stress; System; Testing; Therapeutic; Vascular Diseases; Vision; Work; angiogenesis; cell type; conditional mutant; density; genetic manipulation; in vivo; in vivo evaluation; insight; mitochondrial metabolism; neonatal mice; nervous system development; neuron loss; novel; photoreceptor degeneration; prevent; programs; recruit; respiratory; response; retina circulation disorder; retinal angiogenesis; selective expression; single-cell RNA sequencing; stressor; transcription factor; uptake

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.

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。视网膜星形细胞 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;这种机制对pat至关重要， 将星形胶质细胞转化为控制血管生成的模板。尽管如此重要， 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，我们必须知道细胞和分子- 杀死星形胶质细胞的小胶质细胞的典型特征，但这也仍然未知。这里的目标是 以确定执行发育性星形胶质细胞死亡的小胶质细胞和实现这一功能的机制。 中心假设是，发育短暂的小胶质细胞群体，称为神经纤维层- 相关的小胶质细胞（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， 是的。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成为可能 星形胶质细胞和血管网络是如何达到它们的成熟模式的--在正常的和病理性的退化中， - 谢谢此外，由于非凋亡性“吞噬细胞死亡”也可发生在神经退行性疾病中， 对死亡机制的了解可能与疾病病理学有着广泛的相关性。具体目标是：1） 识别由星形胶质细胞“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“吃我”信号， 神经胶质。单细胞RNA测序确定了一个小胶质细胞群，NFL-M，这可能是星形胶质细胞- 反应亚群这一工作假设将通过操纵星形胶质细胞密度和PtdSer expo来检验。 当然，如果假设是正确的，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很高的 线粒体输出NFL-M显示强烈线粒体代谢的转录特征，包括ac- 激活Nrf 2信号传导，这是对代谢应激的关键反应。这些数据表明NFL-M是高度吞噬的 对星形胶质细胞;这一假设将在体内测试使用遗传操作，防止小胶质细胞 假设NFL-M状态或激活Nrf 2。3)鉴定星形胶质细胞相关的 调节其功能的小胶质细胞。如果星形胶质细胞被转录上独特的小胶质细胞群杀死- 因此，使它们独特的基因应该支持这种功能。lgals 3，一个在噬菌体中具有已知作用的基因- 细胞分裂，由NFL-M选择性表达。初步数据显示， 在视网膜变性中，支持星形胶质细胞去除也需要它的工作假设。 这将使用Lgals 3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.这将是有意义的，因为它将为确定吞噬细胞死亡的潜在机制开辟道路。 以及这种新的死亡机制对神经系统发育和疾病的贡献。