Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways (original) (raw)

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Acknowledgements

We thank D. Vollrath for discussions and MERTK antibody. We also thank H. M. Lee for helping us set up electrophysiology experiments as well as for discussions. Part of the data was acquired at Stanford Neuroscience Microscopy Service (NMS), supported by NIH NS069375. W.-S.C. was supported in part by a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation (DRG 2020-09). L.E.C. was supported in part by an EMBO ALTF fellowship. This work was supported by grants from the NIH (5 R21NS072556, B.A.B.) and a Brain Disorder Award from the Mcknight Foundation to B.A.B. We thank V. and S. Coates for their generous support.

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Author notes

  1. Laura E. Clarke and Gordon X. Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurobiology, Stanford University, School of Medicine, Stanford, 94305, California, USA
    Won-Suk Chung, Laura E. Clarke, Chandrani Chakraborty, Julia Joung & Ben A. Barres
  2. Department of Molecular and Cellular Physiology, Stanford University, School of Medicine, Stanford, 94305, California, USA
    Gordon X. Wang & Stephen J. Smith
  3. Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, 48105, Michigan, USA
    Benjamin K. Stafford
  4. Santa Cruz Institute of Particle Physic and Department of Physics, University of California, Santa Cruz, 95064, California, USA
    Alexander Sher
  5. Institute of Molecular and Cell Biology, A *Star, 61 Biopolis Drive, Proteos Building, 138673 Singapore ,
    Lynette C. Foo
  6. Children’s Hospital, Harvard Medical School, 300 Longwood Avenue, CLS12250, Boston, Massachusetts 02115, USA ,
    Andrew Thompson & Chinfei Chen

Authors

  1. Won-Suk Chung
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  2. Laura E. Clarke
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  3. Gordon X. Wang
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  4. Benjamin K. Stafford
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  5. Alexander Sher
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  6. Chandrani Chakraborty
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  7. Julia Joung
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  8. Lynette C. Foo
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  9. Andrew Thompson
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  10. Chinfei Chen
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  11. Stephen J. Smith
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  12. Ben A. Barres
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Contributions

W.-S.C. and B.A.B. designed the experiments and wrote the paper. W.-S.C. performed experiments and analysed data. L.E.C. performed and analysed electrophysiology recordings from LGN neurons with support from A.T. and C. Chen. G.X.W. performed and analysed array tomography experiments. B.K.S. performed and analysed spontaneous retinal wave recording with support from A.S. C. Chakraborty, J.J., L.C.F. and S.J.S. provided technical support.

Corresponding author

Correspondence toWon-Suk Chung.

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Competing interests

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Extended data figures and tables

Extended Data Figure 1 MERTK protein is localized to multiple cell types.

a, b, Confocal P5 dLGN images showing MERTK (red) protein expression in endothelial cells (arrows) stained with BSL (blue) as well as in astrocytic processes (asterisks) labelled by _Aldh1l1_-EGFP (green). c, d, Confocal P5 dLGN images showing MERTK (red) protein expression in microglia (arrows) stained with IBA1 (blue) as well as in astrocytes (asterisks) labelled by _Aldh1l1_-EGFP (green). Scale bar: 10 μm.

Extended Data Figure 2 MEGF10 and MERTK are continuously localized to cortical astrocytes throughout life.

a–f, MEGF10 (red; a, c, e) and MERTK (red; b, d, f) are localized to cortical astrocytes (arrows) labelled by _Aldh1l1_-EGFP (green) in the P5 (a, b), P30 (c, d) and 1-year-old (e, f) mouse cortex. Whereas MEGF10 is specifically localized to astrocytes, MERTK is also localized to microglia (arrowheads) as well as endothelial cells (asterisks). Scale bar: 20 μm.

Extended Data Figure 3 Phagocytic capacity of _Megf10_−/− or _Mertk_−/− astrocytes and microglia measured by FACS.

a–d, FACS profiles of astrocytes (a, c) and enriched microglia population (b, d) for pHrodo intensity after incubating with pHrodo-conjugated synaptosomes for 24 h in the presence of 5% serum. _Megf10_−/− (a) and _Mertk_−/− (c) astrocytes (blue lines) show clear leftward shifts in pHrodo intensity compared to wild-type astrocytes (red lines). _Megf10_−/− microglia (b, blue line) do not show any difference in the FACS profile compared to wild-type microglia (red lines in b). _Mertk_−/− microglia (d, blue line) exhibit a slight leftward shift in the FACS profile showing strong pHrodo intensity (yellow rectangle) whereas there is no difference in low pHrodo intensity (green rectangle) compared to wild-type microglia. e, _Megf10_−/− and _Mertk_−/− astrocytes show a 42% and 51% reduction in the relative engulfment ability, respectively, compared to wild-type astrocytes. f, _Mertk_−/− microglia show a 25% reduction in the relative engulfment ability compared to wild-type microglia. The relative engulfment ability was calculated by comparing the percentage of the cell population expressing strong pHrodo intensity (>3 × 104). Representative data from three independent experiments are shown. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. NS, not significant. Error bars indicate s.e.m.

Extended Data Figure 4 Astrocytes in the developing dLGN engulf pre-synaptic material.

a, b, Optical sections of the P5 dLGN using structured illumination (a) and confocal (b) microscopy through xy, xz and yz axes show that synaptophysin- (a, arrows) and VGlut2- (b, arrows) positive presynaptic material are engulfed by EGFP-expressing astrocytes (green). Scale bar: 1 μm (a); 5 μm (b).

Extended Data Figure 5 Astrocytes in the developing dLGN engulf pre- and postsynaptic material, revealed by array tomography (AT).

a, 3D-max projection AT images showing EYFP (grey)-labelled P5 dLGN astrocytes (total volume = 155 μm by 125 μm by 2.8 μm). b, Close-up view of EYFP (blue)-labelled dLGN astrocytes. c, d, Close-up view of 3D-max projection AT images showing CTB-594 labelled projections (magenta) before (c) and after (d) image processing, revealing engulfed CTB-labelled debris by astrocytes (blue). e, f, Close-up view of 3D-max projection AT images showing Bassoon (red) before (e) and after (f) image processing, revealing engulfed Bassoon-positive synaptic material by astrocytes (blue). g, h, Close-up view of 3D-max projection AT images showing PSD-95 (cyan) before (g) and after (h) image processing, revealing engulfed PSD-95-positive synaptic material by astrocytes (blue). i, j, Close-up view of 3D-max projection AT images showing GluR1 (green) before (i) and after (j) image processing, revealing engulfed GluR1-positive synaptic material by astrocytes (blue). Scale bar: 50 μm (a); 20 μm (b–j).

Extended Data Figure 6 Astrocytes clear neural debris more robustly than microglia in the developing dLGN.

a, Representative image of P6 dLGN (yellow dotted line) showing astrocytes labelled by _Aldh1l1_-EGFP (green) and microglia labelled by IBA1 staining (red). b, The number of astrocytes in dLGN is much greater than microglia at P5 (10-fold), P6 (7-fold), P7 (6-fold) and P9 (4-fold). c, The phagocytic index measured by the total amount of CTB debris per unit cell volume showed that during P3–P6, microglia engulfed more CTB-labelled debris than astrocytes per unit cell volume, whereas astrocytes and microglia cleared about the same amount of debris per unit cell volume after P6. n = 5 per group. d, The phagocytic index measured by the total amount of CTB debris per imaging field showed that astrocytes clear a significantly greater amount of CTB debris than microglia during P3–P9. n = 5 per group. *P < 0.05, ***P < 0.001, _t_-test. Error bars indicate s.e.m.

Extended Data Figure 7 MERTK is dispensable for the microglia-mediated phagocytosis in developing dLGN.

a, Comparing the phagocytic index of microglia in dLGN during P3–P6 between wild-type and _Mertk_−/− mice. Microglia showed a gradual decrease in the phagocytic index measured from P3 to P6. b–d, Relative engulfment ability between wild-type and _Mertk_−/− microglia during P3–P4 (b), P4–P5 (c) and P5–P6 (d). _Mertk_−/− microglia showed a transient increase in the phagocytic index during P4–P5. However, the phagocytic index of microglia during P3–P4 and P5–P6 was comparable between wild-type and _Mertk_−/− mice. n = 4 per group. **P < 0.01, _t_-test. NS, not significant. Error bars indicate s.e.m.

Extended Data Figure 8 Spontaneous retinal wave is intact in Megf10 −/− ; Mertk −/− mice.

a, Waves occur with the same frequency (left), propagate at the same speed (middle), and are the same size (right) in Megf10 −/− ; Mertk −/− retinas. a′, Correlation index (CI), computed for spike trains from pairs of neurons and plotted as a function of the distance between electrodes on which the neurons were recorded, shows that CI decreases as a function of distance in both wild-type and _Megf10_−/−; _Mertk_−/− retinas. Plots summarize data from multiple wild-type (black; n = 5) and _Megf10_−/−; _Mertk_−/− (red; n = 5) preparations. b, Bursts fired by ganglion cells show no difference in duration (top left), mean spike rate (top middle), or the amount of time spent firing at high frequencies (top right) in _Megf10_−/−; _Mertk_−/− retinas. The per cent of all spikes that are incorporated into bursts (bottom left), and the per cent of all bursts that occur during waves (bottom right), are also unchanged in _Megf10_−/−; _Mertk_−/− retinas. Plots summarize data from multiple wild-type (black; n = 5) and _Megf10_−/−; _Mertk_−/− (red; n = 5) preparations. c, Quantification of directionality parameters shows that the same fraction of ganglion cells demonstrates a directional bias in _Megf10_−/−; _Mertk_−/− retinas (top). In addition, the magnitude of the directional bias of all neurons in a preparation (bottom) is unchanged in _Megf10_−/−; _Mertk_−/− retinas. Plots summarize data from multiple wild-type (black; n = 5) and _Megf10_−/−; _Mertk_−/− (red; n = 5) preparations. _t_-test.

Extended Data Figure 9 Analysis of astrocytic and synaptic protein localization by array tomography (AT) in the adult cortex with EYFP-expressing astrocytes.

a, 3D-max projection AT images showing EYFP (grey)-labelled astrocytes from the 4-month-old somatosensory cortex (total volume = 155 μm by 125 μm by 2.8 μm). b, Close-up view of EYFP (grey)-labelled cortical astrocytes. c–f, Close-up views of 3D-max projection AT images showing EAA2 (c, d; green) and glutamine synthetase (e, f; magenta) staining reveal specific expression of EYFP in astrocytes (d, f; grey). g, h, Close-up view of 3D-max projection AT images showing Bassoon (red) before (g) and after (h) image processing, revealing engulfed Bassoon-positive synaptic material by astrocytes (grey). i, j, Close-up view of 3D-max projection AT images showing PSD-95 (cyan) before (i) and after (j) image processing, revealing engulfed PSD-95-positive synaptic material by astrocytes (grey). Scale bar: 50 μm (a); 20 μm (b–j).

Supplementary information

Astrocytes engulf synaptosomes in vitro

3D-rendering video showing a purified astrocyte (cyan) efficiently engulfed synaptosomes (red) after incubating them in astrocyte-conditioned media for 4 hours. (MPG 12676 kb)

Astrocytes engulf synaptic material in vivo in the developing dLGN

3D-reconstruction video using micrographs from serial block-face scanning electron microscopy revealed that astrocytic processes (cyan) from the postnatal day 5 dLGN fully engulfed presynaptic material (red arrowhead) as well as the second inclusion (black arrowhead). Astrocytic processes (cyan) containing glycogen granules (black dots) make close contacts with intact synapses (yellow rectangles) in the axonal processes (orange). Synaptic vesicles in the intact synapses and the engulfed presynaptic material were shown in red and yellow spheres, respectively. (MOV 14939 kb)

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Chung, WS., Clarke, L., Wang, G. et al. Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways.Nature 504, 394–400 (2013). https://doi.org/10.1038/nature12776

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