Programming of Resident Macrophages by the Brain Environment Following Transplantation

项目摘要 小胶质细胞是大脑中的巨噬细胞，它的一个显著特征是它们能够适应变化 在大脑环境中。小胶质细胞状态的变化发生在发育和几乎所有的疾病，往往与 有害或有益的功能。更好地理解小胶质细胞状态的调控机制 因此，改变将提高对脑部疾病的理解，并发现新的治疗靶点。几十个 疾病反应状态已被确定，但对小胶质细胞如何在它们之间转换知之甚少。 即使是小胶质细胞的“稳态”也是一种由大脑环境信号积极维持的状态，并在文化中丢失。 菜.我们是分离和操作小胶质细胞的专家，并创造了一个独特的颅内模型， 在遗传性小胶质细胞耗竭后移植小胶质细胞和其它巨噬细胞。移植后， 巨噬细胞植入大脑，并在14天内经历基因表达的巨大变化。初步 数据，我们在几个时间点收获移植的巨噬细胞，并通过单细胞RNA测序， （scRNAseq），测量小胶质细胞身份随时间的渐进获得。有了这个高度控制的 在体内模型中，我们将生成移植的小胶质细胞如何 由大脑环境编程，并使用它来识别基因，途径，调节网络 可能是负责的。在目标1A中，我们将捕获培养的小胶质细胞的环境编程， 移植，使用配对的单细胞RNA/ATACseq来识别中间状态，并预测外部 信号，转录因子，受体，通路和网络负责。在目标1B中，我们将比较 用供体和宿主性别的所有组合进行移植，以确定其在小胶质细胞身份中的作用 规范.最后，由于血液浸润巨噬细胞可以类似于小胶质细胞，但仍然是一种独特的细胞类型， aim 2将在移植后测量它们的编程，以更好地理解为什么它们不能成为 小胶质细胞总之，这些目标的完成将填补关于小胶质细胞状态调节的知识空白 和身份，创建一个新的基础数据资源，并证实R 01提案，以因果关系测试身份 监管机构在这里发现。

PROJECT SUMMARY A striking feature of microglia, the brain's resident macrophages, is their ability to adapt in response to changes in the brain environment. Microglial state changes occur in development and nearly all diseases, often linked to harmful or helpful functions. A better understanding of the regulatory mechanisms underlying microglia state change will therefore improve understanding of brain diseases, and uncover new therapeutic targets. Dozens of disease reactive states have been identified, but little is known about how microglia transition between them. Even microglial “homeostasis” is a state actively maintained by brain environmental signals, and lost in a culture dish. We are experts in the isolation and manipulation of microglia, and created a unique model for intracranial transplantation of microglia and other macrophages following genetic microglia depletion. After transplantation, macrophages engraft the brain and over 14 days undergo dramatic changes in gene expression. In preliminary data, we harvested transplanted macrophages at several timepoints, and by single cell RNA sequencing (scRNAseq), measured the progressive acquisition of microglial identity over time. With this highly controlled in vivo model, we will generate a comprehensive fingerprint of how transplanted microglia are programmed by the brain environment, and use it to identify the genes, pathways, regulatory networks likely to be responsible. In aim 1A, we will capture the environmental programming of cultured microglia after transplant, using paired single cell RNA/ATACseq to identify intermediate states, and to predict the external signals, transcription factors, receptors, pathways and networks responsible. In aim 1B, we will compare transplantation with all combinations of donor and host sex, in order to determine its role in microglia identity specification. Finally, since blood infiltrating macrophages can resemble microglia but remain a distinct cell type, aim 2 will measure their programming after transplantation, to better understand why they cannot become microglia. In summary, completion of these aims will fill knowledge gaps about the regulation of microglial state and identity, create a new foundational data resource, and substantiate an R01 proposal to causally test identity regulators uncovered here.