Unveiling the role of ApoE in microglial development and function.

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.