A new genetic strategy for targeting microglia in development and disease

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Abstract

As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include border-associated meningeal, perivascular and choroid plexus macrophages. Using immunofluorescent labeling, flow cytometry and Cre-dependent ribosomal immunoprecipitations, we describe P2ry12-CreER, a new tool for the genetic targeting of microglia. We use this new tool to track microglia during embryonic development and in the context of ischemic injury and neuroinflammation. Because of the specificity and robustness of microglial recombination with P2ry12-CreER, we believe that this new mouse line will be particularly useful for future studies of microglial function in development and disease.

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The Microglial Sensome Revealed by Direct RNA Sequencing

Suzanne E Hickman, Nathan D. Kingery, Toshiro K Ohsumi … (2013)

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 by fluorescent dual in-situ hybridization, unbiased proteomic analysis and quantitative PCR. We report here that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we term the “sensome”. With aging, sensome transcripts for endogenous ligand recognition are downregulated, whereas those involved in microbe recognition and host defense are upregulated. In addition, aging is associated with an overall increase in expression of microglial genes involved in neuroprotection.

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The P2Y12 receptor regulates microglial activation by extracellular nucleotides.

Sharon E Haynes, Gunther Hollopeter, Guang Yang … (2006)

Microglia are primary immune sentinels of the CNS. Following injury, these cells migrate or extend processes toward sites of tissue damage. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia express several purinoceptors, including a G(i)-coupled subtype that has been implicated in ATP- and ADP-mediated migration in vitro. Here we show that microglia from mice lacking G(i)-coupled P2Y(12) receptors exhibit normal baseline motility but are unable to polarize, migrate or extend processes toward nucleotides in vitro or in vivo. Microglia in P2ry(12)(-/-) mice show significantly diminished directional branch extension toward sites of cortical damage in the living mouse. Moreover, P2Y(12) expression is robust in the 'resting' state, but dramatically reduced after microglial activation. These results imply that P2Y(12) is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury.

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Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.

Elisenda Sanz, Linghai Yang, Thomas Su … (2009)

Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22(HA), which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.

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Author and article information

Contributors

Gabriel L McKinsey:

ORCID: https://orcid.org/0000-0002-5503-2830

Thomas D Arnold

Isaac M Chiu: Role: Reviewing Editor

Satyajit Rath: Role: Senior Editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic, pub): 23 June 2020

Publication date Collection: 2020

Volume: 9

Electronic Location Identifier: e54590

Affiliations

[1 ]Department of Pediatrics, University of California San Francisco San FranciscoUnited States

[2 ]Cardiovascular Research Institute, University of California San Francisco San FranciscoUnited States

Harvard Medical School United States

Indian Institute of Science Education and Research (IISER) India

Harvard Medical School United States

Boston Children's Hospital United States

Author information

Gabriel L McKinsey https://orcid.org/0000-0002-5503-2830

Nicolas Santander https://orcid.org/0000-0001-8919-833X

Article

Publisher ID: 54590

DOI: 10.7554/eLife.54590

PMC ID: 7375817

PubMed ID: 32573436

SO-VID: f305a5e4-fdda-4a9b-8fcc-5fc9ad0e50e7

License:

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

History

Date received : 19 December 2019

Date accepted : 23 June 2020

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: K08NS96192

Award Recipient : Thomas D Arnold

Funded by: FundRef http://dx.doi.org/10.13039/100000968, American Heart Association;

Award ID: 20POST35120371

Award Recipient : Nicolas Santander

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Author impact statement P2ry12-CreER robustly and specifically labels microglia in fate-mapping and ribosomal profiling experiments, revealing new markers for myeloid subpopulations in the central nervous system.

ScienceOpen disciplines: Life sciences

Keywords: microglia,fate mapping,ribosomal profiling,neuroinflammation,stroke,multiple sclerosis,mouse

Data availability:

ScienceOpen disciplines: Life sciences

Keywords: microglia, fate mapping, ribosomal profiling, neuroinflammation, stroke, multiple sclerosis, mouse

A new genetic strategy for targeting microglia in development and disease

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Karger: Neurology and Neuroscience

Most cited references 26

The Microglial Sensome Revealed by Direct RNA Sequencing

The P2Y12 receptor regulates microglial activation by extracellular nucleotides.

Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.

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