Investigating cerebellar microglia diversity through development and injury

BBSRC strategic theme: Understanding the rules of life

As 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.