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Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass
Sameera Zia1, Brady P Hammond1, Martin Zirngibl1
1Neuroscience and Mental Health Institute, University of Alberta, Edmonton, Canada.
Background:
Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood.
Methods:
To understand how microglia respond during demyelination, we fed mice cuprizone-a potent demyelinating agent-and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response.
Results:
The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination.
Conclusions:
Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.
Insights
Microglia play a key role in white matter diseases like demyelination. This study reveals that microglia actively clear dead cells, adopting a specific state linked to phagocytosis during this process.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are critical immune cells in the central nervous system, regulating responses to injury and disease.
- In white matter diseases, microglia can contribute to demyelination, but their precise role and response mechanisms remain unclear.
Purpose of the Study:
- To investigate microglial dynamics and subpopulations during cuprizone-induced demyelination.
- To elucidate the role of microglia in clearing dead oligodendrocytes.
- To understand the factors contributing to the distinct microglial state observed during demyelination.
Main Methods:
- Mice were fed cuprizone, a demyelinating agent, and genetically fate-mapped microglia dynamics were assessed.
- Single-cell RNA sequencing identified microglial subpopulations during demyelination.
- Microglial ablation and acridine orange staining were used to study oligodendrocyte clearance.
- Primary microglial cultures were treated to model cuprizone-induced demyelination.
Main Results:
- Cuprizone induced a significant microglial response by week 4.
- Single-cell RNA sequencing identified distinct cuprizone-associated microglia (CAM) clusters with signatures of cytokine regulation, ROS production, and phagocytosis.
- Microglia preferentially phagocytose lytic cell debris.
- In vitro, phagocytosis of lytic carcasses induced a CAM-like state in microglia.
Conclusions:
- Microglia exhibit diverse roles in demyelination, with a transcriptomic state similar to other neurodegenerative conditions.
- Phagocytosis of cellular debris is a likely driver of this common microglial state in neurodegeneration.
