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Review
. 2020 Apr;68(4):740-755.
doi: 10.1002/glia.23767. Epub 2019 Dec 17.

Transcriptional profiling of microglia; current state of the art and future perspectives

Emma Gerrits  1 , Yang Heng  1 , Erik W G M Boddeke  1 , Bart J L Eggen  1
Affiliations
Review

Transcriptional profiling of microglia; current state of the art and future perspectives

Emma Gerrits et al. Glia. 2020 Apr.

Abstract

Microglia are the tissue macrophages of the central nervous system (CNS) and the first to respond to CNS dysfunction and disease. Gene expression profiling of microglia during development, under homeostatic conditions, and in the diseased CNS provided insight in microglia functions and changes thereof. Single-cell sequencing studies further contributed to our understanding of microglia heterogeneity in relation to age, sex, and CNS disease. Recently, single nucleus gene expression profiling was performed on (frozen) CNS tissue. Transcriptomic profiling of CNS tissues by (single) nucleus RNA-sequencing has the advantage that it can be applied to archived and well-stratified frozen specimens. Here, we give an overview of the significant advances recently made in microglia transcriptional profiling. In addition, we present matched cellular and nuclear microglia RNA-seq datasets we generated from mouse and human CNS tissue to compare cellular versus nuclear transcriptomes from fresh and frozen samples. We demonstrate that microglia can be similarly profiled with cell and nucleus profiling, and importantly also with nuclei isolated from frozen tissue. Nuclear microglia transcriptomes are a reliable proxy for cellular transcriptomes. Importantly, lipopolysaccharide-induced changes in gene expression were conserved in the nuclear transcriptome. In addition, heterogeneity in microglia observed in fresh samples was similarly detected in frozen nuclei of the same donor. Together, these results show that microglia nuclear RNAs obtained from frozen CNS tissue are a reliable proxy for microglia gene expression and cellular heterogeneity and may prove an effective strategy to study of the role of microglia in neuropathology.

Keywords: human; microglia; mouse; single-cell RNA-sequencing; single-nucleus RNA-sequencing; transcriptomes.

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Figures

Figure 1
Figure 1
Microglia nuclear transcriptomes are a reliable proxy for cellular gene expression profiles in mice. (a) Experimental design. Mice received an ip injection with PBS or LPS (1 mg/kg; three mice per group) and after 3 hr, the animals were terminated. Microglia were isolated by FACS as CD11bposCD45intLy6Cneg. From a part of the isolated microglia, nuclei were sorted as DAPIposCD45neg CD11bneg events. After RNA isolation, the cellular and nuclear RNA was expression profiled using 3′ Quantseq (Lexogen). (b) Principal component analysis (PCA) of the transcriptomes across different groups. (c) Heatmap depicting LPS‐responsive genes (297 genes) in cells and nuclei (n = 3 mice). The colors indicate row‐z‐scores. Both rows and columns were ordered by unsupervised clustering. (d) Four way plot depicting genes significantly differentially expressed (logFC >1 and FDR < 0.05) between cells and nuclei from PBS or LPS‐injected mice. The X‐axis depicts the logFC in cells, the y‐axis the logFC in nuclei. Genes indicated in cyan have a logFC <1 in the PBS/LPS nuclei comparison. (e) GO analysis of LPS‐induced genes in cells and nuclei. The top eight most significant GO terms, associated with LPS‐upregulated genes in cells and nuclei are shown. The size of the circle indicates the number of genes associated with the respective GO term. (f) Heatmap of the 22 genes differentially expressed between cells/nuclei and PBS/LPS conditions. DE, differentially expressed
Figure 2
Figure 2
Single cell and nucleus RNA sequencing profiles of mouse microglia are highly similar. (a) UMAP plot with five clusters identified in the merged single microglia cell and nucleus transcriptomes from PBS‐ and LPS‐treated mice. Cells and nuclei from three mice were pooled and loaded on 10× chips. (b) UMAP plot where colors indicate the different experimental samples: microglia and nuclei from PBS‐ and LPS‐treated animals. (c) The distribution of clusters across the indicated experimental groups. (d) UMAP plot depicting expression (log‐transformed UMI counts per 10,000 transcripts) of canonical microglia gene C1qa, homeostatic genes P2ry12, Cx3cr1, and Mef2c, and LPS responsive genes Nfkbia, Cxcl10, and Gpr84. (e) A four way plot depicting genes significantly differentially expressed between cells and nuclei from PBS or LPS‐injected mice (average logFC <0.25 and adjusted p value <.01). The X‐axis depicts the logFC in cells, the y‐axis the logFC in nuclei. Genes indicated in cyan color have a logFC <0.25 in the in the PBS/LPS nuclei comparison. (f) Violin plots depicting distributions of normalized relative expression levels of cell‐enriched and (g) nucleus‐enriched genes
Figure 3
Figure 3
Single cell and nucleus RNA sequencing of human CNS tissues indicates that both fresh and frozen nuclear transcriptomes closely approximate and reflect microglia gene expression heterogeneity. (a) PCA plot of fresh‐tissue derived cells and nuclei, and nuclei isolated from adjacent frozen tissue samples. (b) UMAP plot depicting five clusters identified in the merged single cell and nucleus transcriptomes of microglia from two human donors. (c) UMAP plot where colors indicate the different experimental samples, fresh tissue‐derived microglia cells and fresh and frozen tissue‐derived nuclei. (d) UMAP plot depicting expression (log‐transformed UMI counts per 10,000 transcripts) of canonical microglial genes C1AQ, P2RY12, and CD74. (e) The proportion of clusters across the indicated experimental samples
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