Interrogating a white matter degeneration-specific astrocyte reactivity state and its role in governing repair-associated microglia specification and function.

项目摘要 从退化的中枢神经系统（CNS）白色物质中清除细胞碎片是低效的。 碎片积累，特别是来自髓鞘的碎片积累，沉淀适应不良的神经炎症，从而进一步促进疾病 发展并阻止修复。慢性白色物质变性，产生细胞碎片， 先天性免疫炎症是多种疾病的标志，包括创伤性和缺血性CNS损伤， 多发性硬化症（MS）。旨在增强碎片清除以促进修复的疗法的发展是 因此具有广泛的翻译相关性。旨在揭示中央监管机制的研究 因此，在白色物质损伤后促进碎片清除的炎症反应的基础是 谨慎和必要的。这项研究的首要目标是剖析星形胶质细胞的分子通路- 控制对白色变性的促恢复CNS先天免疫应答的小胶质细胞相互作用。 本研究将探讨一种新发现的细胞间通讯机制， 白色变性反应性星形胶质细胞的独特亚群调节小胶质细胞的特化， 清除炎性髓鞘碎片和组织修复所需的功能。在目标1中，我们将使用 转基因、细胞类型特异性功能丧失系统、单核RNA测序（snRNA-Seq）和小鼠 急性和慢性白色变性模型，以评估这种独特的星形胶质细胞的参与， 介导的信号通路在调节髓鞘碎片所需的损伤反应性小胶质细胞特化中的作用 清除和髓鞘再生。在目标2中，我们将剖析的直接影响和受体依赖性机制， 一种新的星形胶质细胞衍生分子线索对小胶质细胞分子谱、运动性和髓鞘能力的作用 使用小鼠原代和人iPSC衍生的小胶质细胞的培养物的碎片吞噬作用。在目标3中，我们 确定白色物质的分子和功能不同状态下的细胞内在调节途径 使用细胞配对snRNA-Seq和转座酶可降解单核测定的星形胶质细胞反应性 在慢性白色变性的小鼠模型中，使用测序（snATAC-Seq）的染色质随时间的变化。 一个综合分析管道将用于生成星形胶质细胞反应性的基因调控程序， 包括染色质结构的动态变化和转录调节因子的特定组合 (TR)，它们共同控制遗传可及性和转录组谱中的不同反应性变化。选择 白色物质星形胶质细胞反应性的分子不同状态的TR将通过以下方法在体内进行功能性询问： 功能丧失研究。总之，这项研究将加强对星形胶质细胞-小胶质细胞如何 相互作用塑造了能够修复白色物质的CNS先天免疫应答。这些数据还可以提供信息， 利用恢复性先天免疫反应的新治疗途径，涉及星形胶质细胞和 小胶质细胞，以预防或减轻适应不良的白色物质炎症，以及改善结果， 促进CNS损伤后和疾病中的恢复。

Project Summary Clearance of cellular debris from the degenerating central nervous system (CNS) white matter is inefficient. Debris accumulation, especially from myelin, precipitates maladaptive neuroinflammation which furthers disease progression and prevents repair. Chronic white matter degeneration that produces cellular debris and protracted innate immune inflammation is a hallmark of diverse disorders, including traumatic and ischemic CNS injury and Multiple Sclerosis (MS). The development of therapies aimed enhancing debris clearance to promote repair is thereby of broad translational relevance. Research aimed at uncovering central regulatory mechanisms underlying the inflammatory response that facilitates debris clearance after white matter insult is therefore prudent and necessary. The overarching objective of this research is to dissect molecular pathways of astrocyte- microglia interaction that govern pro-restorative CNS innate immune responses to white matter degeneration. The present research will investigate a newly identified mechanism of intercellular communication, through which a distinct subpopulation of white matter degeneration-reactive astrocytes regulate microglia specification and function required for the removal of inflammatory myelin debris and tissue repair. In Aim 1, we will use a transgenic, cell type-specific loss-of-function system, single-nucleus RNA-Sequencing (snRNA-Seq) and mouse models of acute and chronic white matter degeneration to assess the involvement of this unique astrocyte- mediated signaling pathway in regulating damage-responsive microglia specification required for myelin debris clearance and remyelination. In Aim 2, we will dissect the direct effects and receptor dependent mechanism of action for a novel astrocyte-derived molecular cue on microglia molecular profile, motility, and capacity for myelin debris phagocytosis using cultures of mouse primary and human iPSC-derived microglia. In Aim 3, we will determine cell-intrinsic regulatory pathways underlying molecularly and functionally distinct states of white matter astrocyte reactivity using cell-paired snRNA-Seq and single-nucleus Assay for Transposase-Accessible Chromatin using Sequencing (snATAC-Seq), over time, in a mouse model of chronic white matter degeneration. An integrative analysis pipeline will be used to generate gene regulatory programs of astrocyte reactivity, including dynamic changes in chromatin structure and context-specific combinations of transcriptional regulators (TR), which together govern genetic accessibility and distinct reactive changes in transcriptomic profile. Select TR of molecularly distinct states of white matter astrocyte reactivity will be functionally interrogated in vivo by loss-of-function studies. Together, this research will enhance the understanding of how astrocyte-microglia interactions shape CNS innate immune responses that enable white matter repair. These data may also inform new therapeutic avenues for harnessing restorative innate immune responses, involving astrocytes and microglia, to prevent or attenuate maladaptive white matter inflammation, as well as improve outcome and promote recovery after CNS injury and in disease.