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Microglia phagocytose myelin sheaths to modify developmental myelination

Nature Neuroscience volume 23, pages 1055–1066 (2020)Cite this article

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Abstract

During development, oligodendrocytes contact and wrap neuronal axons with myelin. Similarly to neurons and synapses, excess myelin sheaths are produced and selectively eliminated, but how elimination occurs is unknown. Microglia, the resident immune cells of the central nervous system, engulf surplus neurons and synapses. To determine whether microglia also prune myelin sheaths, we used zebrafish to visualize and manipulate interactions between microglia, oligodendrocytes, and neurons during development. We found that microglia closely associate with oligodendrocytes and specifically phagocytose myelin sheaths. By using a combination of optical, genetic, chemogenetic, and behavioral approaches, we reveal that neuronal activity bidirectionally balances microglial association with neuronal cell bodies and myelin phagocytosis in the optic tectum. Furthermore, multiple strategies to deplete microglia resulted in oligodendrocytes maintaining excessive and ectopic myelin. Our work reveals a neuronal activity-regulated role for microglia in modifying developmental myelin targeting by oligodendrocytes.

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Fig. 1: Microglia dynamically engage with myelin sheaths in the spinal cord.
Fig. 2: Microglia phagocytose myelin sheaths during developmental myelination.
Fig. 3: Microglia phagocytose myelin sheaths with minimal phagocytosis of oligodendrocyte somas.
Fig. 4: Neuronal activity regulates microglia–neuron interactions.
Fig. 5: Neuronal activity regulates myelin phagocytosis by microglia.
Fig. 6: Microglial ablation during myelination increases myelin sheath number.

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Data, code and materials availability

All data are available in the manuscript, the Source Data files or the Supplementary Information. Fiji scripts are available in the Supplementary Information. All plasmids and transgenic zebrafish are available by request.

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Acknowledgements

We thank M. Preston (InVivo Biosystems) and J. Hines (Winona State University) for providing pME-NTR and p5E-myrf plasmids, respectively, and W. Macklin, E. Hughes and S. Bromley-Coolidge for comments on the manuscript. This work was supported by US National Institutes of Health (NIH) grant no. R01 NS095679, a gift from the Gates Frontiers Fund to B.A. and a National Science Foundation Graduate Research Fellowship (no. DGE-1553798) to A.N.H. The University of Colorado Anschutz Medical Campus Zebrafish Core Facility was supported by NIH grant no. P30 NS048154.

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Authors and Affiliations

  1. Neuroscience Graduate Program, University of Colorado, Aurora, CO, USA

    Alexandria N. Hughes

  2. Department of Pediatrics, Section of Developmental Biology, University of Colorado, Aurora, CO, USA

    Bruce Appel

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  1. Alexandria N. Hughes
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Contributions

A.N.H. and B.A. conceptualized the project. A.N.H. designed and performed the experiments and collected and analyzed the data. A.N.H. wrote, and B.A. edited, the manuscript.

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Correspondence to Bruce Appel.

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Peer review information Nature Neuroscience thanks Marie-Ève Tremblay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Calcium events in microglial processes contacting myelin sheaths compared to processes not contacting sheaths and comparison with event proximity to the soma.

a, b, Max dF/F (a) and event area (b) for large events (dF/F >0.1, area > 3 µm2) at non-sheath-contacting and sheath-contacting processes of microglia at 4 d.p.f. in Tg(mpeg1.1:GCaMP6s-CAAX; sox10:mRFP) larvae. Wilcoxon rank-sum test, n=41 events from 9 microglia in 9 fish. c, d, To distinguish if distance from cell soma is responsible for sheath-contacting vs non-contacting event differences, we split 645 events that occurred in 31 cells (31 fish) into distal and proximal halves and assessed event dF/F (c) and duration (d); ns by Wilcoxon rank-sum test.

Extended Data Fig. 2 Anti-Mbpa detects myelin within microglia.

a, Coronal section of Tg(mpeg1.1:mVenus-CAAX) 6 d.p.f. larval spinal cord stained with anti-Mbpa to detect myelin. Note anti-Mbpa inclusions (magenta) within microglia (yellow) in orthogonal views (right panels) taken at the locations marked (i), (ii), (iii). b, Coronal section of Tg(mbpa:mCherry-CAAX) 6 d.p.f. tectal commissure stained with anti-Mbpa (cyan) to detect myelin. Oligodendrocyte membrane (mCherry-CAAX) colocalizes with anti-Mbpa (cyan), both in the intact commissure and as a limited amount of extracellular debris (inset). Scale bars, 10 µm.

Extended Data Fig. 3 Acute treatment of larvae with bafilomycin A1 does not affect oligodendrocyte myelination.

a, Representative individual oligodendrocytes in transient-transgenic myrf:mScarlet-CAAX expressing larvae treated with bafilomycin A1 or DMSO. Scale bar, 10 µm. b, c, Quantification of sheath length and number of oligodendrocytes in each treatment group. n=12 larvae/12 fish (DMSO), 8 larvae/8 fish (bafilomycin A1). Data in (b) and (c) analyzed by Wilcoxon rank-sum test.

Extended Data Fig. 4 Glutamate uncaging evokes Ca2+ transients most reliably in neurons located < 20 µm away from the uncaging site.

a, Ratio of Ca2+ transient-experiencing neurons to those without transients binned in 10 µm bins from the uncaging site. b, Distance of neurons with Ca2+ transients (defined as dF/F>0.5) or no transients from the uncaging point in larvae treated with DMSO or MNI-glutamate, analyzed by Wilcoxon rank-sum test (n=fish/neurons, n=5/85 DMSO, 9/70 MNI-glutamate).

Extended Data Fig. 5 Microglia ablation does not change oligodendrocyte number or distribution.

a, Max projection images of hemi-spinal cords in 4 d.p.f. Tg(mbpa:TagRFP-T) larvae in each microglia ablation group and controls. Scale bar, 10 µm. b, c, Total number (b) and dorsal/ventral distribution (c) of oligodendrocytes per 3.5 hemisegments in each ablation group paired with corresponding controls, analyzed by Wilcoxon rank sum test (n=fish/oligodendrocytes, groups left to right, n=9/293, 12/442, 7/222, 8/271, 7/225, 6/209, 8/274, 6/208).

Extended Data Fig. 6 Working model of activity-regulated myelin phagocytosis by microglia.

Microglia phagocytose myelin to regulate myelin sheath number and targeting. Neuronal activity attracts microglia to contact neuronal cell bodies and reduces the amount of myelin that microglia can phagocytose, whereas reductions in neuronal activity promote myelin phagocytosis from axons.

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and legends for Supplementary Videos 1–8.

Reporting Summary

Supplementary Video 1

Microglia exhibit Ca2+ transients at sheath- and non-sheath-contacting processes. Microglia in Tg(mpeg1.1:GCaMP6s-CAAX; sox10:mRFP) 4 d.p.f. larvae. Ca2+ events (cyan) are visible at process tips, along branches and in the soma. Note moving engulfed mRFP+ material within microglia and at points of microglia contact with sheaths. Videos are representative of microglial Ca2+ events (31/31 microglia imaged exhibited transients during 10 min). Scale bar, 10 µm.

Supplementary Video 2

Microglia exhibit Ca2+ transients at sheath- and non-sheath-contacting processes. Microglia in Tg(mpeg1.1:GCaMP6s-CAAX; sox10:mRFP) 4 d.p.f. larvae. Ca2+ events (cyan) are visible at process tips, along branches and in the soma. Note moving engulfed mRFP+ material within microglia and at points of microglia contact with sheaths. Videos are representative of microglial Ca2+ events (31/31 microglia imaged exhibited transients during 10 min). Scale bar, 10 µm.

Supplementary Video 3

Microglia Ca2+ transients before phagocytosis. Microglia in Tg(mpeg1.1:GCaMP6s-CAAX; sox10:mRFP), a 4 d.p.f. larva. A Ca2+ event at the distal process is followed by beading, engulfment and intracellular movement of mRFP+ phagocytosed material. Scale bar, 10 µm.

Supplementary Video 4

Microglia Ca2+ transients before phagocytosis. Microglia in Tg(mpeg1.1:GCaMP6s-CAAX; sox10:mRFP), a 4 d.p.f. larva. A Ca2+ event in the first frame (arrowhead) is followed by breakdown of the associated sheath (rectangle); a later event (second arrowhead) is followed by sheath beading but not loss within the acquisition time. Scale bar, 10 µm.

Supplementary Video 5

Engulfment of myelin sheaths by microglia. Top, timelapse imaging (~5 min per frame, timestamped in upper left) of a microglia (yellow) interacting with an oligodendrocyte (magenta) in a Tg(mpeg1.1:mVenus-CAAX; sox10:mRFP) larva. Two myelin sheaths (magenta rectangles) are intact and visibly surrounded by microglial processes initially, but become engulfed by the microglia soon after. Bottom, same video but only the RFP channel is shown to highlight the nascent sheaths that are engulfed. Scale bar, 10 µm.

Supplementary Video 6

3D rotation of a microglia containing myelin inclusions. A microglia (yellow) in a Tg(mpeg1.1:mVenus-CAAX; mbpa:mCherry-CAAX) larva containing phagocytosed myelin (magenta inclusions). Note intact myelin sheaths next to the microglia (magenta tubes). Movie generated with 3D Viewer plugin in Fiji.

Supplementary Video 7

3D rotation of microglia, myelin and neurons in the dorsal optic tectum. A 3D rendering of a ~40-µm z-stack of dorsal optic tectum in a 6 d.p.f. Tg(mpeg1.1:mVenus-CAAX; mbpa:mCherry-CAAX) larva mosaically expressing neuroD:mTagBFP-CAAX to sparsely label neurons. Microglia (yellow) are situated between the tectal commissure (magenta) and tectal neurons (cyan). Movie generated with 3D Viewer plugin in Fiji.

Supplementary Video 8

Microglial response to glutamate uncaging in tectal cell body layer. Timelapse imaging (1 min per frame) of a microglia in a Tg(mpeg1.1:mVenus-CAAX) larva with MNI-glutamate focally uncaged in the tectal cell body layer. Closed circle after the third minute represents the time and area of 405-nm laser application to uncage glutamate; open circle for remaining frames indicates the area where glutamate was uncaged. Scale bar, 20 µm.

Source data

Source Data Fig. 1

Source data for all experiments and plots in Fig. 1.

Source Data Fig. 2

Source data for all experiments and plots in Fig. 2 and Extended Data Figs. 1 and 2.

Source Data Fig. 3

Source data for all experiments and plots in Fig. 3 and Extended Data Fig. 3.

Source Data Fig. 4

Source data for all experiments and plots in Fig. 4 and Extended Data Fig. 4.

Source Data Fig. 5

Source data for all experiments and plots in Fig. 5.

Source Data Fig. 6

Source data for all experiments and plots in Fig. 6 and Extended Data Fig. 5.

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Hughes, A.N., Appel, B. Microglia phagocytose myelin sheaths to modify developmental myelination. Nat Neurosci 23, 1055–1066 (2020). https://doi.org/10.1038/s41593-020-0654-2

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