Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Distinct molecular pathways mediate glial activation and engulfment of axonal debris after axotomy

Nature Neuroscience volume 15, pages 979–987 (2012)Cite this article

Subjects

Abstract

Glial cells efficiently recognize and clear cellular debris after nervous system injury to maintain brain homeostasis, but pathways governing glial responses to neural injury remain poorly defined. We identify the Drosophila melanogaster guanine nucleotide exchange factor complex Crk/Mbc/dCed-12 and the small GTPase Rac1 as modulators of glial clearance of axonal debris. We found that Crk/Mbc/dCed-12 and Rac1 functioned in a non-redundant fashion with the Draper transmembrane receptor pathway: loss of either pathway fully suppressed clearance of axonal debris. Draper signaling was required early during glial responses, promoting glial activation, which included increased Draper and dCed-6 expression and extension of glial membranes to degenerating axons. In contrast, the Crk/Mbc/dCed-12 complex functioned at later phases, promoting glial phagocytosis of axonal debris. Our work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris and termination of responses to injury are genetically separable events mediated by distinct signaling pathways.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Drosophila Crk, dCed-12 and Rac1 are required for glial clearance of severed axons from the CNS.
Figure 2: mbc, dced-12 and rac1 mutants exhibit dominant genetic interactions in ORN axon clearance assays.
Figure 3: Constitutively active Rac1 rescues both Draper signaling and GEF pathway engulfment defects.
Figure 4: Crk and dCed-12 are not required for activation of glia after axotomy.
Figure 5: Axons remain viable engulfment targets capable of activating glia for more than 1 week after axotomy.
Figure 6: Axotomy-induced activation of phagolysosomes in engulfing glia.
Figure 7: Glial phagolysosomes are decorated with Draper and preferentially accumulate around degenerating axons.
Figure 8: Crk and dCed-12 are required for internalization of axonal debris and phagolysosome formation.

Similar content being viewed by others

References

  1. Cuadros, M.A. & Navascues, J. The origin and differentiation of microglial cells during development. Prog. Neurobiol. 56, 173–189 (1998).

    Article  CAS  Google Scholar 

  2. Marín-Teva, J.L., Cuadros, M.A., Calvente, R., Almendros, A. & Navascues, J. Naturally occurring cell death and migration of microglial precursors in the quail retina during normal development. J. Comp. Neurol. 412, 255–275 (1999).

    Article  Google Scholar 

  3. Parnaik, R., Raff, M.C. & Scholes, J. Differences between the clearance of apoptotic cells by professional and non-professional phagocytes. Curr. Biol. 10, 857–860 (2000).

    Article  CAS  Google Scholar 

  4. Block, M.L., Zecca, L. & Hong, J.S. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57–69 (2007).

    Article  CAS  Google Scholar 

  5. Sofroniew, M.V. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 32, 638–647 (2009).

    Article  CAS  Google Scholar 

  6. Savill, J., Dransfield, I., Gregory, C. & Haslett, C. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2, 965–975 (2002).

    Article  CAS  Google Scholar 

  7. Neumann, H., Kotter, M.R. & Franklin, R.J. Debris clearance by microglia: an essential link between degeneration and regeneration. Brain 132, 288–295 (2009).

    Article  CAS  Google Scholar 

  8. Lalancette-Hébert, M., Gowing, G., Simard, A., Weng, Y.C. & Kriz, J. Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain. J. Neurosci. 27, 2596–2605 (2007).

    Article  Google Scholar 

  9. Fuentes-Medel, Y. et al. Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris. PLoS Biol. 7, e1000184 (2009).

    Article  Google Scholar 

  10. Logan, M.A. & Freeman, M.R. The scoop on the fly brain: glial engulfment functions in Drosophila. Neuron Glia Biol. 3, 63–74 (2007).

    Article  Google Scholar 

  11. Luo, L. & O'Leary, D.D. Axon retraction and degeneration in development and disease. Annu. Rev. Neurosci. 28, 127–156 (2005).

    Article  CAS  Google Scholar 

  12. Kinchen, J.M. & Ravichandran, K.S. Journey to the grave: signaling events regulating removal of apoptotic cells. J. Cell Sci. 120, 2143–2149 (2007).

    Article  CAS  Google Scholar 

  13. Reddien, P.W. & Horvitz, H.R. The engulfment process of programmed cell death in Caenorhabditis elegans. Annu. Rev. Cell Dev. Biol. 20, 193–221 (2004).

    Article  CAS  Google Scholar 

  14. Hasegawa, H. et al. DOCK180, a major CRK-binding protein, alters cell morphology upon translocation to the cell membrane. Mol. Cell. Biol. 16, 1770–1776 (1996).

    Article  CAS  Google Scholar 

  15. Zhou, Z., Caron, E., Hartwieg, E., Hall, A. & Horvitz, H.R. The C. elegans PH domain protein CED-12 regulates cytoskeletal reorganization via a Rho/Rac GTPase signaling pathway. Dev. Cell 1, 477–489 (2001).

    Article  CAS  Google Scholar 

  16. Brugnera, E. et al. Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. Nat. Cell Biol. 4, 574–582 (2002).

    Article  CAS  Google Scholar 

  17. Gumienny, T.L. et al. CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration. Cell 107, 27–41 (2001).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Yu, X., Lu, N. & Zhou, Z. Phagocytic receptor CED-1 initiates a signaling pathway for degrading engulfed apoptotic cells. PLoS Biol. 6, e61 (2008).

    Article  Google Scholar 

  20. Liu, Q.A. & Hengartner, M.O. Candidate adaptor protein CED-6 promotes the engulfment of apoptotic cells in C. elegans. Cell 93, 961–972 (1998).

    Article  CAS  Google Scholar 

  21. Freeman, M.R., Delrow, J., Kim, J., Johnson, E. & Doe, C.Q. Unwrapping glial biology. Gcm target genes regulating glial development, diversification, and function. Neuron 38, 567–580 (2003).

    Article  CAS  Google Scholar 

  22. Awasaki, T. et al. Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis. Neuron 50, 855–867 (2006).

    Article  CAS  Google Scholar 

  23. Hoopfer, E.D. et al. Wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning. Neuron 50, 883–895 (2006).

    Article  CAS  Google Scholar 

  24. Williams, D.W., Kondo, S., Krzyzanowska, A., Hiromi, Y. & Truman, J.W. Local caspase activity directs engulfment of dendrites during pruning. Nat. Neurosci. 9, 1234–1236 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Doherty, J., Logan, M.A., Tasdemir, O.E. & Freeman, M.R. Ensheathing glia function as phagocytes in the adult Drosophila brain. J. Neurosci. 29, 4768–4781 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. Reddien, P.W. & Horvitz, H.R. CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nat. Cell Biol. 2, 131–136 (2000).

    Article  CAS  Google Scholar 

  32. Wu, Y.C., Tsai, M.C., Cheng, L.C., Chou, C.J. & Weng, N.Y. C. elegans CED-12 acts in the conserved crkII/DOCK180/Rac pathway to control cell migration and cell corpse engulfment. Dev. Cell 1, 491–502 (2001).

    Article  CAS  Google Scholar 

  33. Logan, M.A. et al. Negative regulation of glial engulfment activity by Draper terminates glial responses to axon injury. Nat. Neurosci. 15, 722–730 (2012).

    Article  CAS  Google Scholar 

  34. Park, D. et al. BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 450, 430–434 (2007).

    Article  CAS  Google Scholar 

  35. Cabello, J. et al. The Wnt pathway controls cell death engulfment, spindle orientation, and migration through CED-10/Rac. PLoS Biol. 8, e1000297 (2010).

    Article  Google Scholar 

  36. Bechmann, I. & Nitsch, R. Astrocytes and microglial cells incorporate degenerating fibers following entorhinal lesion: a light, confocal, and electron microscopical study using a phagocytosis-dependent labeling technique. Glia 20, 145–154 (1997).

    Article  CAS  Google Scholar 

  37. Kapadia, S.E. & LaMotte, C.C. Deafferentation-induced alterations in the rat dorsal horn: I. Comparison of peripheral nerve injury vs. rhizotomy effects on presynaptic, postsynaptic, and glial processes. J. Comp. Neurol. 266, 183–197 (1987).

    Article  CAS  Google Scholar 

  38. LaMotte, C.C. & Kapadia, S.E. Deafferentation-induced alterations in the rat dorsal horn: II. Effects of selective poisoning by pronase of the central processes of a peripheral nerve. J. Comp. Neurol. 266, 198–208 (1987).

    Article  CAS  Google Scholar 

  39. Petersen, M.A. & Dailey, M.E. Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices. Glia 46, 195–206 (2004).

    Article  Google Scholar 

  40. Aldskogius, H. & Kozlova, E.N. Central neuron-glial and glial-glial interactions following axon injury. Prog. Neurobiol. 55, 1–26 (1998).

    Article  CAS  Google Scholar 

  41. Leiserson, W.M., Harkins, E.W. & Keshishian, H. Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment. Neuron 28, 793–806 (2000).

    Article  CAS  Google Scholar 

  42. Ito, K., Urban, J. & Technau, G.M. Distribution, classification and development of Drosophila glial cells in the late embryonic and early larval ventral nerve cord. Rouxs Arch. Dev. Biol. 209, 289–307 (1995).

    Google Scholar 

  43. Ng, J. et al. Rac GTPases control axon growth, guidance and branching. Nature 416, 442–447 (2002).

    Article  CAS  Google Scholar 

  44. Han, C., Jan, L.Y. & Jan, Y.N. Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila. Proc. Natl. Acad. Sci. USA 108, 9673–9678 (2011).

    Article  CAS  Google Scholar 

  45. Ishimaru, S., Ueda, R., Hinohara, Y., Ohtani, M. & Hanafusa, H. PVR plays a critical role via JNK activation in thorax closure during Drosophila metamorphosis. EMBO J. 23, 3984–3994 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Rørth (Institute of Molecular and Cell Biology, Singapore), N. Franc, S. Waddell (University of Massachusetts Medical School, Worcester), T. Awasaki (Janelia Farm), H. Hing (University of Illinois, Urbana), Y.-N. Jan (University of California, San Francisco) and Y. Nakanishi (Kanazawa University) for fly strains and antibodies. We thank L. Neukomm and A.N. Fox for critical reading of the manuscript and the entire Freeman laboratory for discussions. This work was supported by US National Institutes of Health grant RO1 NS053538 to M.R.F., and M.R.F. is an Early Career Scientist with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Jennifer S Ziegenfuss, Johnna Doherty & Marc R Freeman

  2. Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Marc R Freeman

Authors
  1. Jennifer S Ziegenfuss
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Johnna Doherty
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marc R Freeman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.S.Z. and M.R.F. designed the experiments; J.S.Z. conducted the majority of the experiments; J.D. performed a subset of the Rac1 studies; M.R.F. and J.S.Z. wrote the manuscript.

Corresponding author

Correspondence to Marc R Freeman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 (PDF 1428 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ziegenfuss, J., Doherty, J. & Freeman, M. Distinct molecular pathways mediate glial activation and engulfment of axonal debris after axotomy. Nat Neurosci 15, 979–987 (2012). https://doi.org/10.1038/nn.3135

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3135

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing