Rotation 1: Age-dependent regenerative mechanisms in the brain

由于中枢神经系统在损伤或神经元丢失时没有固有的修复能力，最近的研究集中在内源性干细胞群体替代丢失神经元的能力上。小脑是研究再生的绝佳模型；最近的研究强调了出生时损伤后新生儿小脑中巢蛋白表达祖细胞（NEPs）的再生能力（颗粒细胞祖细胞（gcp）的丧失），尽管这种能力在成年后就会丧失。鉴于祖细胞不能孤立地发挥作用，并且需要来自周围环境的广泛信号来控制其行为，免疫系统可能有助于调节NEPs在发育和损伤期间的行为。小胶质细胞是大脑内的巨噬细胞，是一个异质性的群体，在整个发育过程中表现出功能多样性，并在神经祖细胞的维持和生长调节中发挥重要作用。单细胞技术的最新发展使得小胶质细胞的分子多样性亚群得以识别，这些亚群在发育和衰老过程中具有特定的功能。然而，大多数关于小胶质细胞分子异质性的现有研究并没有专门针对小脑，这表明需要进一步研究小胶质细胞- nep在发育和稳态过程中的串扰。这个项目的主要目的之一是探讨小胶质细胞的异质性损伤在新生儿和成人小脑。我假设出生后小脑中的小胶质细胞向NEPs发送促再生信号，促进其谱系可塑性。为了验证这一假设，我将进行单细胞RNA测序（scRNA-seq）实验，以鉴定小鼠小脑中分子上不同的小胶质细胞亚型，以及基因表达在损伤条件下如何变化。scRNA-seq的发现将使用多种技术进行验证，包括组织学、流式细胞术和体外培养实验。我对先前发表的全脑小胶质细胞scRNA-seq数据集进行了初步分析，发现了一个在新生儿阶段富集的小胶质细胞亚群，以及参与神经祖细胞调节和维持的高表达基因。Spp1（或骨桥蛋白）是该簇中最富集的基因，是一种分泌的糖蛋白，先前已发现它在神经干细胞的存活、增殖和迁移中发挥作用。我发现SPP1在小鼠新生小脑中强烈表达，而在成年小鼠小脑中未检测到表达。SPP1的一个特殊受体，细胞表面受体CD44，在新生小脑的再生NEPs中高度表达。该项目的第二个目标是功能性测试SPP1-CD44通路和细胞因子信号传导在促进新生儿小脑NEPs再生中的作用。解决NEP在发育和适应性重编程过程中的行为机制，以及NEP如何受到免疫系统的影响，对神经发育障碍和损伤的潜在治疗具有长期影响。

BBSRC strategic theme: Understanding the rules of lifeAs the central nervous system has no inherent capacity for repair upon injury or neuronal loss, recent research has focused on the capacity of endogenous populations of stem cells to replace lost neurons. The cerebellum is an excellent model for studying regeneration; recent research has highlighted the regenerative capacity of nestin-expressing progenitors (NEPs) in the neonatal cerebellum following injury at birth (loss of granule cell progenitors, GCPs), though this ability is lost by adulthood. Given that progenitor cells do not function in isolation, and require extensive signalling from their surrounding environment to govern their behaviour, it is likely that the immune system contributes to regulating the behaviour of NEPs during development and upon injury. Microglia, the resident macrophages of the brain, are a heterogenous population that exhibit functional diversity throughout development, and have been found to play an important role in the regulation of neural progenitor cell maintenance and growth. Recent developments in single-cell technologies have allowed the identification of molecularly diverse subpopulations of microglia that have specific functions throughout development and aging. However, the majority of the existing research on the molecular heterogeneity of microglia is not focused on the cerebellum specifically, highlighting a clear need for further investigation into microglia-NEP crosstalk during development and homeostasis. One of the main aims of this project is to explore microglia heterogeneity upon injury in the neonatal and adult cerebellum. I hypothesise that microglia in the postnatal cerebellum send pro-regenerative signals to NEPs that facilitate their lineage plasticity. To test this hypothesis, I will perform single-cell RNA sequencing (scRNA-seq) experiments to identify molecularly distinct microglia subtypes in the mouse cerebellum, and how gene expression changes in injury conditions. Findings from scRNA-seq will be validated using a variety of techniques, including histology, flow cytometry and in vitro culture experiments. My preliminary analysis of previously published microglia scRNA-seq datasets on whole-brains identified a subpopulation of microglia that are enriched in the neonatal stage, and highly express genes involved in the regulation and maintenance of neural progenitors cells. Spp1 (or osteopontin), the top enriched gene within this cluster, is a secreted glycoprotein that has been found previously to play roles in the survival, proliferation and migration of neural stem cells. I identified that SPP1 was strongly expressed in the mouse neonatal cerebellum, whilst expression was undetectable in the adult mouse cerebellum. One particular receptor for SPP1, the cell surface receptor CD44, is highly expressed in regenerative NEPs in the neonatal cerebellum. The second aim of this project is to functionally test the SPP1-CD44 pathway and the role of cytokine signalling in facilitating the regenerative of NEPs in the neonatal cerebellum. Resolving the mechanisms of NEP behaviours during development and adaptive reprogramming, and how NEPs are impacted by the immune system, have long term impact for potential therapies against neurodevelopmental disorders and injury.