Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

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Abstract

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

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Figure 1: Localization of MEGF10 and MERTK to astrocytes and their phagocytic roles in purified astrocytes.
Figure 2: Astrocytes mediate synapse elimination in the developing dLGN.
Figure 3: Astrocytes mediate developmental synapse pruning and remodelling through MEGF10 and MERTK pathways.
Figure 4: dLGN neurons are abnormally innervated by multiple weak inputs in Megf10−/−; Mertk−/− mice.
Figure 5: Neural activity promotes astrocyte-mediated synapse elimination through MEGF10 and MERTK.
Figure 6: Astrocytes in the adult cortex continuously engulf synapses.

References

  1. 1

    Barres, B. A. The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60, 430–440 (2008)

    CAS  Article  Google Scholar 

  2. 2

    Cahoy, J. D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008)

    CAS  Article  Google Scholar 

  3. 3

    Ziegenfuss, J. S. et al. Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling. Nature 453, 935–939 (2008)

    CAS  Article  ADS  Google Scholar 

  4. 4

    MacDonald, J. M. et al. The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 50, 869–881 (2006)

    CAS  Article  Google Scholar 

  5. 5

    Zhou, Z., Hartwieg, E. & Horvitz, H. R. CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans . Cell 104, 43–56 (2001)

    CAS  Article  Google Scholar 

  6. 6

    Hamon, Y. et al. Cooperation between engulfment receptors: the case of ABCA1 and MEGF10. PLoS ONE 1, e120 (2006)

    Article  ADS  Google Scholar 

  7. 7

    Wu, H. H. et al. Glial precursors clear sensory neuron corpses during development via Jedi-1, an engulfment receptor. Nature Neurosci. 12, 1534–1541 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Kinchen, J. M. et al. Two pathways converge at CED-10 to mediate actin rearrangement and corpse removal in C. elegans . Nature 434, 93–99 (2005)

    CAS  Article  ADS  Google Scholar 

  9. 9

    Prasad, D. et al. TAM receptor function in the retinal pigment epithelium. Mol. Cell. Neurosci. 33, 96–108 (2006)

    CAS  Article  Google Scholar 

  10. 10

    Duncan, J. L. et al. An RCS-like retinal dystrophy phenotype in mer knockout mice. Invest. Ophthalmol. Vis. Sci. 44, 826–838 (2003)

    Article  Google Scholar 

  11. 11

    Finnemann, S. C. Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors. EMBO J. 22, 4143–4154 (2003)

    CAS  Article  Google Scholar 

  12. 12

    Wu, Y., Singh, S., Georgescu, M. M. & Birge, R. B. A role for Mer tyrosine kinase in αvβ5 integrin-mediated phagocytosis of apoptotic cells. J. Cell Sci. 118, 539–553 (2005)

    CAS  Article  Google Scholar 

  13. 13

    Tung, T. T. et al. Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper. J. Biochem. 153, 483–491 (2013)

    CAS  Article  Google Scholar 

  14. 14

    Hochreiter-Hufford, A. & Ravichandran, K. S. Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb. Perspect. Biol. 5, (2013)

  15. 15

    Foo, L. C. et al. Development of a method for the purification and culture of rodent astrocytes. Neuron 71, 799–811 (2011)

    CAS  Article  Google Scholar 

  16. 16

    Shatz, C. J. & Sretavan, D. W. Interactions between retinal ganglion cells during the development of the mammalian visual system. Annu. Rev. Neurosci. 9, 171–207 (1986)

    CAS  Article  Google Scholar 

  17. 17

    Penn, A. A., Riquelme, P. A., Feller, M. B. & Shatz, C. J. Competition in retinogeniculate patterning driven by spontaneous activity. Science 279, 2108–2112 (1998)

    CAS  Article  ADS  Google Scholar 

  18. 18

    Hooks, B. M. & Chen, C. Distinct roles for spontaneous and visual activity in remodeling of the retinogeniculate synapse. Neuron 52, 281–291 (2006)

    CAS  Article  Google Scholar 

  19. 19

    Cuervo, A. M. & Dice, J. F. A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273, 501–503 (1996)

    CAS  Article  ADS  Google Scholar 

  20. 20

    Wang, G. & Smith, S. J. Sub-diffraction limit localization of proteins in volumetric space using Bayesian restoration of fluorescence images from ultrathin specimens. PLOS Comput. Biol. 8, e1002671 (2012)

    CAS  Article  ADS  Google Scholar 

  21. 21

    Peters, A., Palay, S. L., Webster, H. & d The Fine Structure of the Nervous System: the Neurons and Supporting Cells (Saunders, 1976)

    Google Scholar 

  22. 22

    Schafer, D. P. et al. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74, 691–705 (2012)

    CAS  Article  Google Scholar 

  23. 23

    Chen, C. & Regehr, W. G. Developmental remodeling of the retinogeniculate synapse. Neuron 28, 955–966 (2000)

    CAS  Article  Google Scholar 

  24. 24

    Shatz, C. J. & Stryker, M. P. Prenatal tetrodotoxin infusion blocks segregation of retinogeniculate afferents. Science 242, 87–89 (1988)

    CAS  Article  ADS  Google Scholar 

  25. 25

    Stellwagen, D. & Shatz, C. J. An instructive role for retinal waves in the development of retinogeniculate connectivity. Neuron 33, 357–367 (2002)

    CAS  Article  Google Scholar 

  26. 26

    Huberman, A. D., Stellwagen, D. & Chapman, B. Decoupling eye-specific segregation from lamination in the lateral geniculate nucleus. J. Neurosci. 22, 9419–9429 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Xu, T. et al. Rapid formation and selective stabilization of synapses for enduring motor memories. Nature 462, 915–919 (2009)

    CAS  Article  ADS  Google Scholar 

  28. 28

    Yang, G., Pan, F. & Gan, W. B. Stably maintained dendritic spines are associated with lifelong memories. Nature 462, 920–924 (2009)

    CAS  Article  ADS  Google Scholar 

  29. 29

    Roberts, T. F., Tschida, K. A., Klein, M. E. & Mooney, R. Rapid spine stabilization and synaptic enhancement at the onset of behavioural learning. Nature 463, 948–952 (2010)

    CAS  Article  ADS  Google Scholar 

  30. 30

    Paolicelli, R. C. et al. Synaptic pruning by microglia is necessary for normal brain development. Science 333, 1456–1458 (2011)

    CAS  Article  ADS  Google Scholar 

  31. 31

    Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178 (2007)

    CAS  Article  Google Scholar 

  32. 32

    Tasdemir-Yilmaz, O. & Freeman, M. R. Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev (in the press)

  33. 33

    Lu, Q. et al. Tyro-3 family receptors are essential regulators of mammalian spermatogenesis. Nature 398, 723–728 (1999)

    CAS  Article  ADS  Google Scholar 

  34. 34

    Dunkley, P. R., Jarvie, P. E. & Robinson, P. J. A rapid Percoll gradient procedure for preparation of synaptosomes. Nature Protocols 3, 1718–1728 (2008)

    CAS  Article  Google Scholar 

  35. 35

    Beletskii, A. et al. High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye. Biotechniques 39, 894–897 (2005)

    CAS  Article  Google Scholar 

  36. 36

    Datwani, A. et al. Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity. Neuron 64, 463–470 (2009)

    CAS  Article  Google Scholar 

  37. 37

    Jaubert-Miazza, L. et al. Structural and functional composition of the developing retinogeniculate pathway in the mouse. Vis. Neurosci. 22, 661–676 (2005)

    Article  Google Scholar 

  38. 38

    Micheva, K. D., Busse, B., Weiler, N. C., O’Rourke, N. & Smith, S. J. Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68, 639–653 (2010)

    CAS  Article  Google Scholar 

  39. 39

    Stafford, B. K., Sher, A., Litke, A. M. & Feldheim, D. A. Spatial-temporal patterns of retinal waves underlying activity-dependent refinement of retinofugal projections. Neuron 64, 200–212 (2009)

    CAS  Article  Google Scholar 

Download references

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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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 to Won-Suk Chung.

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Competing interests

The authors declare no competing financial interests.

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.

af, 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.

ad, 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 (bj).

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. bd, 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. cf, 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 (bj).

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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