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The microglial sensome revealed by direct RNA sequencing

Abstract

Microglia, the principal neuroimmune sentinels of the brain, continuously sense changes in their environment and respond to invading pathogens, toxins and cellular debris. Microglia exhibit plasticity and can assume neurotoxic or neuroprotective priming states that determine their responses to danger. We used direct RNA sequencing, without amplification or cDNA synthesis, to determine the quantitative transcriptomes of microglia of healthy adult and aged mice. We validated our findings using fluorescence dual in situ hybridization, unbiased proteomic analysis and quantitative PCR. We found that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we refer to as the sensome. With aging, sensome transcripts for endogenous ligand recognition were downregulated, whereas those involved in microbe recognition and host defense were upregulated. In addition, aging was associated with an overall increase in the expression of microglial genes involved in neuroprotection.

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Figure 1: The microglial sensome identified by direct RNA sequencing.
Figure 2: Differences between microglia and macrophages revealed by DRS.
Figure 3: Comparative expression of the microglial and macrophages genes.
Figure 4: RNAscope dual fluorescence in situ hybridization.
Figure 5: Proteomic analysis of microglia and macrophages.
Figure 6: Effects of aging on the microglial mRNA expression profile.
Figure 7: Upregulation of alternative priming genes in microglia from aged mice.
Figure 8: The microglial sensome in aging.

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Acknowledgements

We thank F. Ozsolak (Helicos) for supervising the sequencing experiments, R. Mylvaganam for performing cell sorting and J. Liao (Applied Biomics) for analysis of the proteomic data. This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NS059005) and the National Institute of Aging (AG032349) to J.E.K.

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Authors and Affiliations

Authors

Contributions

S.E.H. co-designed the experiments performed all cell isolations from peritoneum and brain, RNA extractions and quality analysis and co-wrote the manuscript. N.D.K. assisted in all cell isolations and sorting for DRS and proteomics studies. T.K.O. and M.L.B. performed bioinformatics analyses, including development of programs in MolBioLib to annotate sequence information obtained from Helicos. L.W. performed the RNAscope experiments. T.K.M. was involved in designing experiments and data analysis. J.E.K. co-designed the experiments, analyzed the data and co-wrote the manuscript.

Corresponding authors

Correspondence to Suzanne E Hickman or Joseph El Khoury.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Overview of cell characterization and experimental flow.

a-b. Resident microglia and macrophages express CD11b in situ and by flow cytometry. Brain and peritoneal sections were stained with anti-CD11b antibodies (red-brown stain) and counterstained with hematoxylin. Microglia and macrophages were stained with Alexa 647-labeled anti-CD11b and Alexa 488-labeled anti-CD45 antibodies. The gates drawn show the populations of microglia and macrophages sorted for DRS. c. Microglia express high CD11b and low-to-intermediate CD45, while macrophages expressed higher CD11b and CD45 than microglia. The CD11b axis voltage is set lower for macrophages to allow them to be seen on the dot plot. d. Microglia isolated by flow were processed for DRS. Of the 21025 transcripts measured, we used gene ontology (GO) analysis and identified 1299 potential sensome genes. Of these, we selected the top 100 transcripts with the highest enrichment of microglia/brain and termed this gene collection as the microglial “Sensome”. e. Three dimensional image of a mouse microglia with the summary of the GO analysis of the Sensome showing the various classes of genes identified.

Supplementary Figure 2 Comparative expression of scavenger receptor genes in microglia (black) and macrophages (red).

Data were determined by DRS and are expressed as mRNA copies per million mapped reads (CMMR). a. Scara and Scarb families of scavenger receptors. Macrophages from normal mice express significantly higher mRNA levels of MSR1, Marco, and Cd36 and Scarb1 than microglia. b. Lrp (Low-density lipoprotein-related receptor protein) and Scarf families. There are significant differences between microglia and macrophages only in expression of Lrp12 and Scarf1 and Scarf2 scavenger receptor family members. Highest expression is seen with Lrp1 and Lrp10. c. Other scavenger receptors. Microglia and macrophages express similar levels of other scavenger receptors with highest expression seen for Cd68, Cd14, Cd47 and Cxcl16. *p values are <0.00001.

Supplementary Figure 3 Molecular signature of microglia

Microglia signature genes compared to brain and macrophages. Data expressed as mRNA CMMR (copies per million mapped reads) as determined by DRS (left y-axis). 3a presents only transcripts expressed at >1000 CMMR, 3b presents trasncripts expressed between 100 to 1000 CMMR, and 3c shows transcripts expressed between 10 to 100 CMMR. In all graphs, the blue line (right y-axis) represents Log2 fold enrichment of microglia over whole brain, indicating a similar level of enrichment for all transcripts shown, regardless of the level of expression.

Supplementary Figure 4 Comparison of expression of proteins identified by 2D DIGE with mRNA levels in microglia and macrophages

a-b. Expression levels of 2D DIGE-identified proteins in peritoneal macrophages relative to microglia levels (microglia levels set to 1.0). c-d. Expression of mRNA levels corresponding to identified proteins. Expression of mRNA and protein levels follow the same trend.

Supplementary Figure 5 NRG1 and Stat3 are examples of neuroprotective pathways upregulated in microglia from old mice.

Heatmaps depicting expression levels of transcripts of the NRG1 (a-c), and Stat3 (d) pathways in microglia from old and young animals. Enrichment plots for these pathways relative to the whole transcriptome are shown in figure 6.

Supplementary Figure 6 Oxidative phosphorylation is an example of a potential neurotoxic pathway downregulated in microglia from old mice.

Heatmaps depicting expression levels of transcripts of oxidative phosphorylation pathways (a,b) in microglia from old and young animals. Enrichment plots for this pathway relative to the whole transcriptome is shown in figure 6.

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Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 6081 kb)

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Hickman, S., Kingery, N., Ohsumi, T. et al. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci 16, 1896–1905 (2013). https://doi.org/10.1038/nn.3554

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