Unveiling the role of ApoE in microglial development and function

由小胶质细胞领导的大脑固有免疫系统对中枢神经系统的功能和功能障碍的贡献现在已经得到了很好的证实。小胶质细胞的功能对维持正常的大脑发育、神经连接和体内平衡至关重要。然而，我们对小胶质细胞作用的理解大多来自啮齿动物模型的研究，而人类小胶质细胞的起源、维持和作用的图景尚未定义：我们需要进化到实验系统来研究人类小胶质细胞。在这里，我们提出了一个新的，嵌合的，器官型培养的发展，其中研究人类小胶质细胞在复杂的生理环境的背景下。具体来说，我们将应用这个新的平台来研究ApoE的作用，ApoE是一个最近被描述为小胶质细胞功能和功能障碍的关键基因。我们假设ApoE（2/3/4）的不同等位基因变异会影响小胶质细胞的发育和功能，直接影响大脑的生理机能。为了解决这个假设，学生将解决以下目标：1。研究ApoE变异在小胶质细胞群体发育中的作用，以及由小胶质细胞2指示的关键脑发育标志。建立用于人小胶质细胞研究的嵌合器官型切片培养平台分析ApoE变异对人类小胶质细胞调节大脑稳态功能的影响学生将通过使用IHC，流式细胞术和纯化小胶质细胞的RNAseq相结合，通过比较ApoE4和ApoE3靶向替代小鼠中发育中的小胶质细胞群体的动态和特征来解决目标1。这项研究将与主要脑细胞群的连通性和发育的研究相匹配。目标2将需要开发一种新的器官型系统，将人类ipsc衍生的小胶质细胞整合到小鼠海马切片中，从而允许在复杂生理系统的背景下研究人类小胶质细胞。该模型将结合CRISPR-CAS诱导人类小胶质细胞中不同ApoE变异，研究它们对小胶质细胞谱（单细胞RNAseq）的影响以及对脑生理的影响。这个项目不仅将为博士生提供一个坚实的训练平台，而且还将为小胶质细胞的关键作用提供有价值的见解，直接转化为我们对大脑功能和功能障碍的理解。

The contribution of the brain's resident innate immune system, led by microglia, to the function and dysfunction of the central nervous system is now well established. Microglia function is critical to maintain normal brain development, wiring and homeostasis. However, most of our understanding of the roles of microglia arise from the study of rodent models, while the picture of the origin, maintenance and role of human microglia is not yet defined: we need to evolve to experimental systems that investigate human microglia in context. Here, we propose the development of a novel, chimeric, organotypic culture, in which to study human microglia in the context of a complex physiological environment. Specifically, we will apply this novel platform to the investigation of the role of ApoE, a gene recently described as key for the function and dysfunction of microglia. We hypothesise that different allelic variants of ApoE (2/3/4) will affect the development and functions of microglia, having a direct impact on brain's physiology. To address this hypothesis, the student will tackle the following objectives: 1. Study the role of ApoE variants on the development of the microglial population, as well key brain developmental hallmarks instructed by microglia 2. Develop a chimeric organotypic slice culture platform for the study of human microglia in context 3. Analyse the impact of ApoE variants on the function of human microglia in regulating homeostasis of the brain The student will address objective 1 by comparing the dynamics and profile of the developing microglial population in ApoE4 vs ApoE3 targeted replacement mice, using a combination of IHC, flow cytometry and RNAseq of purified microglia. This study will be matched to study of connectivity and development of the main brain cellular populations. Objective 2 will entail the development of novel organotypic system integrating human iPSC-derived microglia into mouse hippocampal slices, allowing the study of human microglia in context of a complex physiological system. This model will be combined with CRISPR-CAS induction of the different ApoE variants in the human microglia, to study their effect on microglia profile (single-cell RNAseq) and impact on brain physiology. This project will not only provide a solid training platform for a PhD student, but also valuable insights to key roles of microglia, with direct translation into our understanding of brain function and dysfunction.