Article | Published:

Astrocyte scar formation aids central nervous system axon regeneration

Nature volume 532, pages 195200 (14 April 2016) | Download Citation

This article has been updated

Abstract

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 13 April 2016

    Figure 5b and c were corrected to remove duplication of the ‘LC’ label in the bottom panels.

Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

Raw and normalized genomic data have been deposited in the NCBI Gene Expression Omnibus and are accessible through accession number GSE76097 and via a searchable, open-access website https://astrocyte.rnaseq.sofroniewlab.neurobio.ucla.edu

References

  1. 1.

    Degeneration and Regeneration of the Nervous System (Oxford Univ. Press, 1928)

  2. 2.

    et al. Sustained axon regeneration induced by co-deletion of PTEN and SOCS3. Nature 480, 372–375 (2011)

  3. 3.

    , , & Neuronal intrinsic mechanisms of axon regeneration. Annu. Rev. Neurosci. 34, 131–152 (2011)

  4. 4.

    , & Axons from CNS neurons regenerate into PNS grafts. Nature 284, 264–265 (1980)

  5. 5.

    & Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats. Science 214, 931–933 (1981)

  6. 6.

    Functions of Nogo proteins and their receptors in the nervous system. Nature Rev. Neurosci. 11, 799–811 (2010)

  7. 7.

    & Can regenerating axons recapitulate developmental guidance during recovery from spinal cord injury ? Nature Rev. Neurosci. 7, 603–616 (2006)

  8. 8.

    & Collagen matrix in spinal cord injury. J. Neurotrauma 23, 422–435 (2006)

  9. 9.

    & Regeneration beyond the glial scar. Nature Rev. Neurosci. 5, 146–156 (2004)

  10. 10.

    , & Inhibition of formation of a glial barrier as a means of permitting a peripheral nerve to grow into the brain. J. Comp. Neurol. 96, 359–369 (1952)

  11. 11.

    Regeneration of axons in the vertebrate central nervous system. Physiol. Rev. 36, 427–440 (1956)

  12. 12.

    & Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway. Science 237, 642–645 (1987)

  13. 13.

    & Reactive gliosis and the multicellular response to CNS damage and disease. Neuron 81, 229–248 (2014)

  14. 14.

    Astrocyte barriers to neurotoxic inflammation. Nature Rev. Neurosci. 16, 249–263 (2015)

  15. 15.

    et al. Leukocyte infiltration, neuronal degeneration and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron 23, 297–308 (1999)

  16. 16.

    et al. Reactive astrocytes protect tissue and preserve function after spinal cord injury. J. Neurosci. 24, 2143–2155 (2004)

  17. 17.

    et al. STAT3 is a critical regulator of astrogliosis and scar formation after spinal cord injury. J. Neurosci. 28, 7231–7243 (2008)

  18. 18.

    et al. Glial scar borders are formed by newly proliferated, elongated astrocytes that interact to corral inflammatory and fibrotic cells via STAT3-dependent mechanisms after spinal cord injury. J. Neurosci. 33, 12870–12886 (2013)

  19. 19.

    et al. Combined genetic attenuation of myelin and semaphorin-mediated growth inhibition is insufficient to promote serotonergic axon regeneration. J. Neurosci. 30, 10899–10904 (2010)

  20. 20.

    et al. The unusual response of serotonergic neurons after CNS injury: lack of axonal dieback and enhanced sprouting within the inhibitory environment of the glial scar. J. Neurosci. 31, 5605–5616 (2011)

  21. 21.

    et al. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nature Methods 2, 419–426 (2005)

  22. 22.

    & Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody. Cell 38, 811–822 (1984)

  23. 23.

    & Biosynthesis and function of chondroitin sulfate. Biochim. Biophys. Acta 1830, 4719–4733 (2013)

  24. 24.

    & Where no synapses go: gatekeepers of circuit remodeling and synaptic strength. Trends Neurosci. 36, 363–373 (2013)

  25. 25.

    & Local translation and directional steering in axons. EMBO J. 26, 3729–3736 (2007)

  26. 26.

    et al. Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc. Natl Acad. Sci. USA 106, 13939–13944 (2009)

  27. 27.

    et al. Genomic analysis of reactive astrogliosis. J. Neurosci. 32, 6391–6410 (2012)

  28. 28.

    et al. Modulation of the proteoglycan receptor PTPsigma promotes recovery after spinal cord injury. Nature 518, 404–408 (2015)

  29. 29.

    Lecticans: organizers of the brain extracellular matrix. Cell. Mol. Life Sci. 57, 276–289 (2000)

  30. 30.

    & Sugar-dependentmodulation of neuronal development, regeneration, and plasticity by chondroitin sulfate proteoglycans. Exp. Neurol. 274, 115–125 (2015)

  31. 31.

    et al. Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity. Neuron 33, 689–702 (2002)

  32. 32.

    & Peripheral injury enhances central regeneration of primary sensory neurones. Nature 309, 791–793 (1984)

  33. 33.

    & Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron 23, 83–91 (1999)

  34. 34.

    et al. Robust Axonal regeneration occurs in the injured CAST/Ei mouse CNS. Neuron 86, 1215–1227 (2015)

  35. 35.

    et al. Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury. Nature Neurosci. 12, 1106–1113 (2009)

  36. 36.

    et al. Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro. Mol. Cell. Neurosci. 39, 50–62 (2008)

  37. 37.

    et al. Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles. Nature 417, 424–428 (2002)

  38. 38.

    et al. Biocompatibility of amphiphilic diblock copolypeptide hydrogels in the central nervous system. Biomaterials 30, 2881–2898 (2009)

  39. 39.

    et al. Sustained local delivery of bioactive nerve growth factor in the central nervous system via tunable diblock copolypeptide hydrogel depots. Biomaterials 33, 9105–9116 (2012)

  40. 40.

    & Concepts and methods for the study of axonal regeneration in the CNS. Neuron 74, 777–791 (2012)

  41. 41.

    & Contrasting the glial response to axon injury in the central and peripheral nervous systems. Dev. Cell 28, 7–17 (2014)

  42. 42.

    , & The extending astroglial process: development of glial cell shape, the growing tip, and interactions with neurons. J. Neurosci. 8, 3124–3134 (1988)

  43. 43.

    & Reactive astrocytes are substrates for the growth of adult CNS axons in the presence of elevated levels of nerve growth factor. Neuron 7, 1019–1030 (1991)

  44. 44.

    et al. Short hairpin RNA against PTEN enhances regenerative growth of corticospinal tract axons after spinal cord injury. J. Neurosci. 33, 15350–15361 (2013)

  45. 45.

    , , & Astroglial-derived periostin promotes axonal regeneration after spinal cord injury. J. Neurosci. 34, 2438–2443 (2014)

  46. 46.

    et al. Thermoresponsive copolypeptide hydrogel vehicles for CNS cell delivery. ACS Biomater. Sci. Eng . 1, 705–717 (2015)

  47. 47.

    et al. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury. Science 348, 347–352 (2015)

  48. 48.

    , & Axonal growth therapeutics: regeneration or sprouting or plasticity ? Trends Neurosci. 31, 215–220 (2008)

  49. 49.

    et al. Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice. Cell 93, 189–201 (1998)

  50. 50.

    et al. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J. Immunol. 161, 4652–4660 (1998)

  51. 51.

    et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neurosci. 13, 133–140 (2010)

  52. 52.

    et al. Tunable diblock copolypeptide hydrogel depots for local delivery of hydrophobic molecules in healthy and injured central nervous system. Biomaterials 35, 1989–2000 (2014)

  53. 53.

    et al. Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses. J. Gen. Physiol. 141, 633–647 (2013)

  54. 54.

    , , & Imaging intracellular Ca2 + signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators. J. Vis. Exp . 93, e51972 (2014)

  55. 55.

    et al. Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington’s disease model mice. Nature Neurosci. 17, 694–703 (2014)

  56. 56.

    et al. Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis. Nature Neurosci. 16, 571–579 (2013)

  57. 57.

    , , & *Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39, 175–191 (2007)

  58. 58.

    et al. Reversible Ponceau staining as a loading control alternative to actin in western blots. Anal. Biochem. 401, 318–320 (2010)

  59. 59.

    et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  60. 60.

    , & HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  61. 61.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)

  62. 62.

    et al. The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth. J. Cell Biol. 125, 669–680 (1994)

  63. 63.

    et al. Phosphacan and neurocan are repulsive substrata for adhesion and neurite extension of adult rat dorsal root ganglion neurons in vitro. Exp. Neurol. 182, 1–11 (2003)

  64. 64.

    , , & Deoxyribozyme-mediated knockdown of xylosyltransferase-1 mRNA promotes axon growth in the adult rat spinal cord. Brain 131, 2596–2605 (2008)

  65. 65.

    , , , & Tenascin-R as a repellent guidance molecule for developing optic axons in zebrafish. J. Neurosci. 23, 6232–6237 (2003)

  66. 66.

    Molecular mechanisms of axon guidance. Science 298, 1959–1964 (2002)

  67. 67.

    et al. Netrin-1 acts as a repulsive guidance cue for sensory axonal projections toward the spinal cord. J. Neurosci. 28, 10380–10385 (2008)

  68. 68.

    et al. Plexin A is a neuronal semaphorin receptor that controls axon guidance. Cell 95, 903–916 (1998)

  69. 69.

    , & Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron 32, 39–51 (2001)

  70. 70.

    & Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell 90, 739–751 (1997)

  71. 71.

    et al. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature 432, 179–186 (2004)

  72. 72.

    et al. Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell 87, 175–185 (1996)

  73. 73.

    et al. Draxin inhibits axonal outgrowth through the netrin receptor DCC. J. Neurosci. 31, 14018–14023 (2011)

  74. 74.

    et al. Neogenin mediates the action of repulsive guidance molecule. Nature Cell Biol. 6, 756–762 (2004)

  75. 75.

    et al. RGM is a repulsive guidance molecule for retinal axons. Nature 419, 392–395 (2002)

  76. 76.

    , & SLITRK1 binds 14-3-3 and regulates neurite outgrowth in a phosphorylation-dependent manner. Biol. Psychiatry 66, 918–925 (2009)

  77. 77.

    et al. Draxin, a repulsive guidance protein for spinal cord and forebrain commissures. Science 323, 388–393 (2009)

  78. 78.

    et al. NG2 glial cells provide a favorable substrate for growing axons. J. Neurosci. 26, 3829–3839 (2006)

  79. 79.

    , , , & Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse. BMC Neurosci . 8, 80 (2007)

  80. 80.

    , & Axon regeneration through scars and into sites of chronic spinal cord injury. Exp. Neurol. 203, 8–21 (2007)

  81. 81.

    et al. Adult NG2 + cells are permissive to neurite outgrowth and stabilize sensory axons during macrophage-induced axonal dieback after spinal cord injury. J. Neurosci. 30, 255–265 (2010)

  82. 82.

    et al. Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis. J. Biol. Chem. 281, 24970–24978 (2006)

  83. 83.

    et al. Tenascin-C contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting sites for neurons. J. Cell Biol. 132, 681–699 (1996)

  84. 84.

    et al. Alpha9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration. J. Neurosci. 29, 5546–5557 (2009)

  85. 85.

    & Syndecan promotes axon regeneration by stabilizing growth cone migration. Cell Rep . 8, 272–283 (2014)

  86. 86.

    , & The role of neuronal versus astrocyte-derived heparan sulfate proteoglycans in brain development and injury. Biochem. Soc. Trans. 42, 1263–1269 (2014)

  87. 87.

    , & BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp. Neurol. 191, 344–360 (2005)

  88. 88.

    , , & Cellular delivery of neurotrophin-3 promotes coricospinal axonal growth and partial functional recovery after spinal cord injury. J. Neurosci. 17, 5560–5572 (1997)

  89. 89.

    & Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination. J. Comp. Neurol. 467, 403–417 (2003)

  90. 90.

    et al. Leukemia inhibitory factor augments neurotrophin expression and corticospinal axon growth after adult CNS injury. J. Neurosci. 19, 3556–3566 (1999)

  91. 91.

    et al. Leukemia inhibitory factor determines the growth status of injured adult sensory neurons. J. Neurosci. 21, 7161–7170 (2001)

  92. 92.

    , & Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation. Brain 130, 3308–3320 (2007)

  93. 93.

    & IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nature Neurosci. 9, 1371–1381 (2006)

  94. 94.

    et al. Fibroblast growth factor-2 promotes axon branching of cortical neurons by influencing morphology and behavior of the primary growth cone. J. Neurosci. 21, 3932–3941 (2001)

  95. 95.

    , & TGF-α increases astrocyte invasion and promotes axonal growth into the lesion following spinal cord injury in mice. Exp. Neurol. 214, 10–24 (2008)

  96. 96.

    , , & Astrocyte-associated fibronectin is critical for axonal regeneration in adult white matter. J. Neurosci. 24, 9282–9290 (2004)

  97. 97.

    , & Perlecan antagonizes collagen IV and ADAMTS9/GON-1 in restricting the growth of presynaptic boutons. J. Neurosci. 34, 10311–10324 (2014)

  98. 98.

    , , , & Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats. Proc. Natl Acad. Sci. USA 109, 9155–9160 (2012)

  99. 99.

    et al. Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons. Neurobiol. Dis. 32, 88–95 (2008)

  100. 100.

    et al. Galectin-1 regulates initial axonal growth in peripheral nerves after axotomy. J. Neurosci. 19, 9964–9974 (1999)

  101. 101.

    & Neural cell adhesion molecules of the immunoglobulin superfamily: role in axon growth and guidance. Annu. Rev. Cell Dev. Biol. 13, 425–456 (1997)

  102. 102.

    et al. The extracellular-matrix protein matrilin 2 participates in peripheral nerve regeneration. J. Cell Sci. 122, 995–1004 (2009)

  103. 103.

    et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014)

Download references

Acknowledgements

We thank D. W. Bergles for the NG2 antibody, and the Microscopy Core Resource of the UCLA Broad Stem Cell Research Center-CIRM Laboratory. This work was supported by the US National Institutes of Health (NS057624 and NS084030 to M.V.S.; P30 NS062691 to G.C. and NS060677, MH099559A, MH104069 to B.S.K.), and the Dr. Miriam and Sheldon G. Adelson Medical Foundation (M.V.S. and T.J.D.), and Wings for Life (M.V.S.).

Author information

Author notes

    • Mark A. Anderson
    •  & Joshua E. Burda

    These authors contributed equally to this work.

    • Mark A. Anderson
    •  & Yilong Ren

    Present addresses: School of Life Sciences, Swiss Federal Institute of Technology (EPFL), SV BMI UPCourtine, Station 19, CH-1015 Lausanne, Switzerland (M.A.A); Department of Spine Surgery, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China (Y.R.).

Affiliations

  1. Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1763, USA

    • Mark A. Anderson
    • , Joshua E. Burda
    • , Yilong Ren
    • , Yan Ao
    • , Timothy M. O’Shea
    •  & Michael V. Sofroniew
  2. Departments of Psychiatry and Neurology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1761, USA

    • Riki Kawaguchi
    •  & Giovanni Coppola
  3. Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1751, USA

    • Baljit S. Khakh
  4. Departments of Bioengineering, Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California 90095-1600, USA

    • Timothy J. Deming

Authors

  1. Search for Mark A. Anderson in:

  2. Search for Joshua E. Burda in:

  3. Search for Yilong Ren in:

  4. Search for Yan Ao in:

  5. Search for Timothy M. O’Shea in:

  6. Search for Riki Kawaguchi in:

  7. Search for Giovanni Coppola in:

  8. Search for Baljit S. Khakh in:

  9. Search for Timothy J. Deming in:

  10. Search for Michael V. Sofroniew in:

Contributions

M.A.A., J.E.B., B.S.K., T.J.D. and M.V.S. designed experiments; M.A.A., J.E.B., Y.R. and Y.A. conducted experiments; M.A.A., J.E.B., Y.A., T.M.O., R.K., G.C. and M.V.S. analysed data. M.A.A., J.E.B., T.M.O., B.S.K, T.J.D. and M.V.S. prepared the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael V. Sofroniew.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Text.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature17623

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.