Tracking changes in functionality and morphology of repopulated microglia in young and old mice
Zuzanna M Luczak-Sobotkowska1, Patrycja Rosa1,2, Maria Banqueri Lopez1
1Laboratory of Molecular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland.
Background:
Microglia (MG) are myeloid cells of the central nervous system that support homeostasis and instigate neuroinflammation in pathologies. Single-cell RNA sequencing (scRNA-seq) revealed the functional heterogeneity of MG in mouse brains. Microglia are self-renewing cells and inhibition of colony-stimulating factor 1 receptor (CSF1R) signaling depletes microglia which rapidly repopulate. The functions of repopulated microglia are poorly known.
Methods:
We combined scRNA-seq, bulk RNA-seq, immunofluorescence, and confocal imaging to study the functionalities and morphology of repopulated microglia.
Results:
A CSRF1R inhibitor (BLZ-945) depleted microglia within 21 days and a number of microglia was fully restored within 7 days, as confirmed by TMEM119 staining and flow cytometry. ScRNA-seq and computational analyses demonstrate that repopulated microglia originated from preexisting progenitors and reconstituted functional clusters but upregulated inflammatory genes. Percentages of proliferating, immature microglia displaying inflammatory gene expression increased in aging mice. Morphometric analysis of MG cell body and branching revealed a distinct morphology of repopulated MG, particularly in brains of old mice. We demonstrate that with aging some repopulated MG fail to reach the homeostatic phenotype. These differences may contribute to the deterioration of MG protective functions with age.
Insights
Repopulated microglia in aging mice show distinct inflammatory gene expression and morphology, potentially impairing their protective functions. This study investigates their altered characteristics after depletion and regrowth.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia (MG) are crucial myeloid cells in the central nervous system, maintaining homeostasis and driving neuroinflammation.
- Single-cell RNA sequencing (scRNA-seq) has highlighted the diverse functions of microglia in mouse brains.
- Microglia are self-renewing; inhibiting colony-stimulating factor 1 receptor (CSF1R) depletes them, followed by rapid repopulation, but their functions post-repopulation are unclear.
Purpose of the Study:
- To investigate the functionalities and morphology of repopulated microglia.
- To understand how aging impacts repopulated microglia phenotypes and functions.
Main Methods:
- Combined scRNA-seq, bulk RNA-seq, immunofluorescence, and confocal imaging.
- Utilized a CSF1R inhibitor (BLZ-945) for microglia depletion and assessed repopulation.
- Performed morphometric analysis to evaluate cell body and branching characteristics.
Main Results:
- CSF1R inhibition led to microglia depletion and subsequent restoration within days, confirmed by TMEM119 staining and flow cytometry.
- ScRNA-seq revealed repopulated microglia originated from progenitors, reconstituted functional clusters, but upregulated inflammatory genes.
- Aging mice showed increased percentages of proliferating, immature microglia with inflammatory gene expression and distinct morphologies, failing to reach homeostatic phenotypes.
Conclusions:
- Repopulated microglia exhibit altered inflammatory profiles and morphologies, especially in aging brains.
- Aging impairs the ability of some repopulated microglia to achieve a homeostatic state.
- These age-related changes in repopulated microglia may contribute to diminished protective functions.
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