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Updated: Dec 22, 2025
Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Published on: December 3, 2019
RNA-seq and network analysis reveal unique glial gene expression signatures during prion infection
James A Carroll1, Brent Race2, Katie Williams2
1Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South Fourth Street, Hamilton, MT, 59840, USA. carrollja2@niaid.nih.gov.
Background:
Prion diseases and prion-like disorders, including Alzheimer's disease and Parkinson's disease, are characterized by gliosis and accumulation of misfolded aggregated host proteins. Ablating microglia in prion-infected brain by treatment with the colony-stimulating factor-1 receptor (CSF-1R) inhibitor, PLX5622, increased accumulation of misfolded prion protein and decreased survival time.
Methods:
To better understand the role of glia during neurodegeneration, we used RNA-seq technology, network analysis, and hierarchical cluster analysis to compare gene expression in brains of prion-infected versus mock-inoculated mice. Comparisons were also made between PLX5622-treated prion-infected mice and untreated prion-infected mice to assess mechanisms involved in disease acceleration in the absence of microglia.
Results:
RNA-seq and network analysis suggested that microglia responded to prion infection through activation of integrin CD11c/18 and did not adopt the expression signature associated with other neurodegenerative disease models. Instead, microglia acquired an alternative molecular signature late in the disease process. Furthermore, astrocytes expressed a signature pattern of genes which appeared to be specific for prion diseases. Comparisons were also made with prion-infected mice treated with PLX5622 to assess the impact of microglia ablation on astrocyte gene expression during prion infection. In the presence of microglia, a unique mix of transcripts associated with A1- and A2-reactive astrocytes was increased in brains of prion-infected mice. After ablation of microglia, this reactive astrocyte expression pattern was enhanced. Thus, after prion infection, microglia appeared to decrease the overall A1/A2-astrocyte responses which might contribute to increased survival after infection.
Conclusions:
RNA-seq analysis indicated dysregulation of over 300 biological processes within the CNS during prion disease. Distinctive microglia- and astrocyte-associated expression signatures were identified during prion infection. Furthermore, astrogliosis and the unique astrocyte-associated expression signature were independent of microglial influences. Astrogliosis and the unique astrocyte-associated gene expression pattern were increased when microglia were ablated. Our findings emphasize the potential existence of alternative pathways for activating the A1/A2 paradigm in astrocytes during neurodegenerative disease.
Insights
Microglia ablation in prion disease models accelerated disease and reduced survival by enhancing reactive astrocyte responses. These findings suggest microglia normally limit astrocyte activation, potentially offering new therapeutic targets for neurodegeneration.
Area of Science:
- Neuroscience
- Immunology
- Genomics
Background:
- Prion diseases and related disorders like Alzheimer's and Parkinson's involve gliosis and misfolded protein aggregation.
- Microglia ablation using CSF-1R inhibitor PLX5622 in prion-infected mice increased prion protein and decreased survival.
Purpose of the Study:
- To investigate the role of glia, specifically microglia and astrocytes, in prion disease pathogenesis.
- To compare gene expression profiles in prion-infected mice with and without microglia to understand disease mechanisms.
Main Methods:
- RNA-sequencing (RNA-seq) to analyze gene expression in mouse brains.
- Network analysis and hierarchical clustering to identify gene expression patterns.
- Comparison of gene expression between prion-infected, mock-inoculated, and PLX5622-treated prion-infected mice.
Main Results:
- Microglia in prion infection showed unique activation signatures, distinct from other neurodegenerative models.
- Astrocytes exhibited a prion disease-specific gene expression pattern, independent of microglial influence.
- Ablating microglia amplified reactive astrocyte gene expression (A1/A2 paradigm), correlating with increased disease severity.
Conclusions:
- Microglia normally modulate reactive astrocyte responses in prion disease, potentially promoting survival.
- Astrogliosis and specific astrocyte gene signatures can be independent of microglia.
- Alternative pathways for astrocyte activation (A1/A2 paradigm) may exist in neurodegenerative diseases.
