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Layer V cortical neurons require microglial support for survival during postnatal development

Abstract

Neurons require trophic support during neural circuit formation; however, how the cellular milieu contributes to neuronal survival remains unclear. We found that layer V cortical neurons require support from microglia for survival during postnatal development. Specifically, we found that microglia accumulated close to the subcerebral and callosal projection axons in the postnatal brain. Inactivation of microglia by minocycline treatment or transient ablation of microglia in CD11b-DTR transgenic mice led to increased apoptosis, specifically in layer V subcerebral and callosal projection neurons. CX3CR1 in microglia was required for the survival of layer V neurons. Microglia consistently promoted the survival of cortical neurons in vitro. In addition, we identified microglia-derived IGF1 as a trophic factor that maintained neuronal survival. Our results highlight a neuron-glia interaction that is indispensable for network formation during a specific period in the developing brain.

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Figure 1: Microglia accumulate along the subcerebral projecting axonal tracts during the postnatal period.
Figure 2: Minocycline treatment induces apoptotic cell death in layer V neurons in the postnatal brain.
Figure 3: Transient ablation of microglia induces apoptosis in layer V.
Figure 4: Cx3cr1-deficient microglia fail to support the survival of neurons in layer V.
Figure 5: Microglia enhance the survival of cortical neurons, and this is attenuated by minocycline treatment or Cx3cr1 deletion in vitro.
Figure 6: Microglia-derived IGF1 is required for the survival of layer V neurons.
Figure 7: Increases of IGFBPs in microglia and IGF1 signaling inhibition suppress the survival of layer V neurons in minocycline-treated mice.
Figure 8: Increases of IGFBPs in Cx3cr1-deficient mice and the effect of IGFBP5 on the survival of layer V neurons.

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References

  1. Oppenheim, R.W. & Johnson, J.E. Programmed Cell Death and Neurotrophic Factors (Academic Press, 2003).

  2. Huang, E.J. & Reichardt, L.F. Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24, 677–736 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Catapano, L.A., Arnold, M.W., Perez, F.A. & Macklis, J.D. Specific neurotrophic factors support the survival of cortical projection neurons at distinct stages of development. J. Neurosci. 21, 8863–8872 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Koester, S.E. & O'Leary, D.D. Connectional distinction between callosal and subcortically projecting cortical neurons is determined prior to axon extension. Dev. Biol. 160, 1–14 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Alcamo, E.A. et al. Satb2 regulates callosal projection neuron identity in the developing cerebral cortex. Neuron 57, 364–377 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Arlotta, P. et al. Neuronal subtype–specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207–221 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Ozdinler, P.H. & Macklis, J.D. IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nat. Neurosci. 9, 1371–1381 (2006).

    Article  PubMed  Google Scholar 

  8. Dugas, J.C. et al. A novel purification method for CNS projection neurons leads to the identification of brain vascular cells as a source of trophic support for corticospinal motor neurons. J. Neurosci. 28, 8294–8305 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005).

    CAS  PubMed  Google Scholar 

  10. 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  PubMed  PubMed Central  Google Scholar 

  11. 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  PubMed  Google Scholar 

  12. Tanaka, T., Ueno, M. & Yamashita, T. Engulfment of axon debris by microglia requires p38 MAPK activity. J. Biol. Chem. 284, 21626–21636 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wake, H., Moorhouse, A.J., Jinno, S., Kohsaka, S. & Nabekura, J. Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J. Neurosci. 29, 3974–3980 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kitayama, M., Ueno, M., Itakura, T. & Yamashita, T. Activated microglia inhibit axonal growth through RGMa. PLoS ONE 6, e25234 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chan, W.Y., Kohsaka, S. & Rezaie, P. The origin and cell lineage of microglia: new concepts. Brain Res. Rev. 53, 344–354 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chen, S.K. et al. Hematopoietic origin of pathological grooming in Hoxb8 mutant mice. Cell 141, 775–785 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Alliot, F., Godin, I. & Pessac, B. Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain. Brain Res. Dev. Brain Res. 117, 145–152 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Milligan, C.E., Cunningham, T.J. & Levitt, P. Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain. J. Comp. Neurol. 314, 125–135 (1991).

    Article  CAS  PubMed  Google Scholar 

  20. Ling, E.A., Ng, Y.K., Wu, C.H. & Kaur, C. Microglia: its development and role as a neuropathology sensor. Prog. Brain Res. 132, 61–79 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Hristova, M. et al. Activation and deactivation of periventricular white matter phagocytes during postnatal mouse development. Glia 58, 11–28 (2010).

    Article  PubMed  Google Scholar 

  22. Streit, W.J. Microglia and macrophages in the developing CNS. Neurotoxicology 22, 619–624 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Dittgen, T. et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl. Acad. Sci. USA 101, 18206–18211 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yrjänheikki, J., Keinanen, R., Pellikka, M., Hokfelt, T. & Koistinaho, J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc. Natl. Acad. Sci. USA 95, 15769–15774 (1998).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Tikka, T., Fiebich, B.L., Goldsteins, G., Keinanen, R. & Koistinaho, J. Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J. Neurosci. 21, 2580–2588 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Duffield, J.S. et al. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J. Clin. Invest. 115, 56–65 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Akazawa, H. et al. Diphtheria toxin–induced autophagic cardiomyocyte death plays a pathogenic role in mouse model of heart failure. J. Biol. Chem. 279, 41095–41103 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Valverde, F., Lopez-Mascaraque, L., Santacana, M. & De Carlos, J.A. Persistence of early-generated neurons in the rodent subplate: assessment of cell death in neocortex during the early postnatal period. J. Neurosci. 15, 5014–5024 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ransohoff, R.M. Chemokines and chemokine receptors: standing at the crossroads of immunobiology and neurobiology. Immunity 31, 711–721 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cardona, A.E. et al. Control of microglial neurotoxicity by the fractalkine receptor. Nat. Neurosci. 9, 917–924 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Jung, S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell Biol. 20, 4106–4114 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Brewer, G.J. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674–683 (1995).

    Article  CAS  PubMed  Google Scholar 

  33. Pietrzkowski, Z., Wernicke, D., Porcu, P., Jameson, B.A. & Baserga, R. Inhibition of cellular proliferation by peptide analogues of insulin-like growth factor 1. Cancer Res. 52, 6447–6451 (1992).

    CAS  PubMed  Google Scholar 

  34. Firth, S.M. & Baxter, R.C. Cellular actions of the insulin-like growth factor binding proteins. Endocr. Rev. 23, 824–854 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Frade, J.M. & Barde, Y.A. Microglia-derived nerve growth factor causes cell death in the developing retina. Neuron 20, 35–41 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. Marín-Teva, J.L. et al. Microglia promote the death of developing Purkinje cells. Neuron 41, 535–547 (2004).

    Article  PubMed  Google Scholar 

  37. Antony, J.M., Paquin, A., Nutt, S.L., Kaplan, D.R. & Miller, F.D. Endogenous microglia regulate development of embryonic cortical precursor cells. J. Neurosci. Res. 89, 286–298 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  39. Gianino, S. et al. Postnatal growth of corticospinal axons in the spinal cord of developing mice. Brain Res. Dev. Brain Res. 112, 189–204 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Canty, A.J. & Murphy, M. Molecular mechanisms of axon guidance in the developing corticospinal tract. Prog. Neurobiol. 85, 214–235 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Wang, C.L. et al. Activity-dependent development of callosal projections in the somatosensory cortex. J. Neurosci. 27, 11334–11342 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. O'Leary, D.D. & Terashima, T. Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and “waiting periods”. Neuron 1, 901–910 (1988).

    Article  CAS  PubMed  Google Scholar 

  43. Beck, K.D., Powell-Braxton, L., Widmer, H.R., Valverde, J. & Hefti, F. Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hippocampal granule and striatal parvalbumin-containing neurons. Neuron 14, 717–730 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Chesik, D., Glazenburg, K., Wilczak, N., Geeraedts, F. & De Keyser, J. Insulin-like growth factor binding protein-1–6 expression in activated microglia. Neuroreport 15, 1033–1037 (2004).

    Article  CAS  PubMed  Google Scholar 

  45. Murase, S. & Hayashi, Y. Expression pattern and neurotrophic role of the c-fms proto-oncogene M-CSF receptor in rodent Purkinje cells. J. Neurosci. 18, 10481–10492 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Del Río, P. et al. GDNF-induced osteopontin from Muller glial cells promotes photoreceptor survival in the Pde6brd1 mouse model of retinal degeneration. Glia 59, 821–832 (2011).

    Article  PubMed  Google Scholar 

  47. Haynes, S.E. et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat. Neurosci. 9, 1512–1519 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  49. Ueno, M. & Yamashita, T. Strategies for regenerating injured axons after spinal cord injury - insights from brain development. Biologics 2, 253–264 (2008).

    PubMed  PubMed Central  Google Scholar 

  50. Ueno, M., Hayano, Y., Nakagawa, H. & Yamashita, T. Intraspinal rewiring of the corticospinal tract requires target-derived BDNF and compensates lost function after brain injury. Brain 135, 1253–1267 (2012).

    Article  PubMed  Google Scholar 

  51. Cailhier, J.F. et al. Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation. J. Immunol. 174, 2336–2342 (2005).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank H. Ozawa for experimental technical support, M.K. Yamada (University of Tokyo), P. Osten (Cold Spring Harbor Laboratory) and H. Miyoshi (RIKEN) for their kind gift of the lentivirus plasmid and helpful suggestions, and Y. Yoshida (Cincinnati Children's Hospital Medical Center) for critical comments on the manuscript. This work was supported by a grant for Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST) to T.Y. and a Grant-in-Aid for Young Scientists (B) from Japan Society for the Promotion of Science to M.U.

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M.U. and T.Y. conceived the project, designed the experiments and wrote the paper. M.U. and Y.F. performed the experiments and analyzed the data. T.T. contributed to the culture experiments, and Y.N. contributed to the culture experiments and in situ hybridization. J.K. and M.I. contributed to the experiments using Cx3cr1-deficient mice. T.Y. coordinated and directed the project.

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Correspondence to Masaki Ueno or Toshihide Yamashita.

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Ueno, M., Fujita, Y., Tanaka, T. et al. Layer V cortical neurons require microglial support for survival during postnatal development. Nat Neurosci 16, 543–551 (2013). https://doi.org/10.1038/nn.3358

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