Tracking changes in functionality and morphology of repopulated microglia in young and old mice

Microglia (MG) are myeloid cells of the central nervous system supporting its homeostasis and instigating neuroinflammation in pathologies. Single-cell RNA sequencing (scRNA-seq) revealed the functional heterogeneity of MG in mice brains. Inhibition of colony-stimulating factor 1 receptor (CSF1R) signaling with inhibitors deplete microglia which rapidly repopulate. Functionalities of repopulated microglia are poorly known. We combined scRNA-seq, bulk RNA-seq, immunofluorescence and confocal imaging to study functionalities and morphology of repopulated microglia. CSRF1R inhibitor (BLZ-945) depleted MG in 21 days and their numbers were restored 7 days later as evidenced by TMEM119 staining and flow cytometry. ScRNA-seq and computational analyses demonstrate that repopulated MG originate from preexisting MG progenitors and reconstitute functional clusters but upregulate inflammatory genes. Percentages of proliferating, immature MG displaying inflammatory gene expression increase in aging mice. Morphometric analysis of MG cell body and branching shows distinct morphology of repopulated MG, particularly in old mouse brains. We demonstrate that with aging some repopulated MG fail to reach the homeostatic phenotype. These differences microglia may contribute to the deterioration of microglia protective functions with age.