Astrocyte scar formation aids central nervous system axon regeneration

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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.

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Figure 1: Preventing astrocytic scar formation does not lead to spontaneous regrowth of CST, AST or 5HT axons after SCI.
Figure 2: No spontaneous regrowth of CST, AST or 5HT axons after ablating chronic astrocyte scars.
Figure 3: CSPG production by non-astrocyte cells in SCI lesions after ablation or attenuation of astrocytic scars.
Figure 4: Genomic dissection of astrocytes and non-astrocyte cells in SCI lesions.
Figure 5: Robust regrowth of AST axons can be stimulated after wild-type SCI and is significantly attenuated by preventing astrocytic scar formation.

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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

Change history

  • 13 April 2016

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

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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

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.

Correspondence to Michael V. Sofroniew.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 SCI model schematic, locomotor behavioural effects, and AAV vector targeting specificity and effects.

a, Schematic of severe lateral crush SCI at thoracic level T10 that generates a large lesion core (LC) of non-neural tissue surrounded by an astrocytic scar (AS) and completely transects descending and ascending axons. b, Open field hindlimb locomotor score at various times after SCI assessed using a 5-point scale where 5 is normal and 0 is no movement of any kind17. No significant differences were observed among any of the experimental groups at any time point. n = 6 mice at all time points P > 0.5 (ANOVA with Newman–Keuls post hoc analysis). WT, wild type. c, Horizontal sections through a severe SCI lesion of a representative tdTomato (tdT) reporter mouse51 injected with an AAV vector with a minimal Gfap promoter regulating Cre (AAV2/5-GfaABC1D-Cre) into the lesion at two weeks after SCI and perfused at three weeks. tdTomato labelling demonstrates that this AAV2/5-GfaABC1D-Cre efficiently and specifically targets GFAP-positive astrocytes. In this mouse, the amount AAV2/5-GfaABC1D-Cre injected was intentionally titrated on the basis of previous trial and error to target primarily the astrocytic scar border in an approximately 500 μm zone immediately abutting the SCI lesion core. High-magnification analysis of individual fluorescence channels stained for tdTomato plus various cell markers shows the specificity of Cre activity targeting to cells expressing the astrocyte marker, GFAP, but not to cells expressing either the neuronal marker, NeuN, or the mature oligodendrocyte marker, GSTπ. AAV2/5-GfaABC1D-Cre was prepared using a previously described and well-characterized cloning strategy53,54,55. d, Open field hindlimb locomotor scores at various times after SCI. There was no difference in scores of control mice and loxP-STOP-loxP-DTR (diphtheria toxin receptor) mice that received AAV2/5-GfaABC1D-Cre before injections of diphtheria toxin (DT). Five weeks after DT injections, loxP-DTR mice that received AAV2/5- GfaABC1D-Cre exhibited a slightly, but significantly, lower locomotor score. Hindlimb locomotion was assessed using a 5-point scale where 5 is normal and 0 is no movement of any kind17. n = 6 mice per group; *P < 0.05 versus wild-type (ANOVA with Newman–Keuls). e, GFAP immunohistochemistry of a sagittal section after ablation of a chronic astrocytic scar plus adjacent astrocytes. DT was administered to a transgenic mouse expressing DTR targeted selectively to astrocytes around a severe SCI. In this case, the amount of AAV2/5-GfaABC1D-Cre injected was titrated to target not only primarily the astrocytic scar border but also adjacent astrocytes spread over approximately 2 mm on either side of the centre (Cn) of the SCI lesion core (LC). Note the profound degeneration of neural tissue resulting from the selective ablation of the chronic astrocytic scar plus adjacent astrocytes after SCI. Source data

Extended Data Figure 2 Single-channel CSPG and GFAP immunofluorescence and stained area quantification.

a, Individual fluorescence channels of CS56 and GFAP immunohistochemistry from horizontal sections of uninjured mice and at two weeks after severe SCI shown in Fig. 3b. Sections are taken from wild-type (WT) mice and mice with transgenic ablation (TK+GCV) or attenuation (STAT3-CKO) of astrocytic scar formation. b, Example of black and white thresholding of single channels of immunofluorescence staining for image analysis to quantify (using NIH Image J software) the amount of CSPG- or GFAP-stained area in different tissue compartments in SCI lesions. Boxes denote areas quantified to obtain values for lesion core (LC) and grey (g) or white (w) matter in astrocytic scar (AS) or equivalent regions in uninjured tissue. Graphs show percentage of areas (means ± s.e.m.) stained for CSPG or GFAP determined using ImageJ. n = 4 (wild type mice); n = 6 (TK+GCV and STAT3-CKO mice); #P < 0.05 versus uninjured white matter; *P < 0.05 versus uninjured grey matter in same experimental group (ANOVA with Newman–Keuls); ^P < 0.05 versus equivalent anatomical region in wild-type (ANOVA with Newman–Keuls). Source data

Extended Data Figure 3 Specificity of haemagglutinin targeting to astrocytes and enrichment of haemagglutinin immunoprecipitation for astrocyte-specific RNA transcripts.

a, Individual fluorescence channels of immunohistochemistry for transgenically targeted haemagglutinin (HA) plus various cell markers showing the specificity of HA targeting to cells expressing the astrocyte marker, GFAP, and not to cells expressing either the neuronal marker, NeuN, or the mature oligodendrocyte marker, GSTπ, in uninjured grey and white matter and in astrocytic scars at 2 weeks after SCI. b, CNS-cell-type-specific gene transcript enrichment of ribosome-associated mRNA (ramRNA) isolated from wild-type (WT) uninjured spinal cord by HA immunoprecipitation (HA-IP). Differential expression analysis by RNA-seq indicates significant enrichment (red) for astrocyte-specific gene transcripts, and de-enrichment (green) for gene transcripts enriched in other CNS cell types, FDR < 0.1. A log2 scale is used so that positive and negative differences are directly comparable. The mean numerical enrichment of three quintessential astrocyte genes, Gfap, Aldh1l1 and Aqp4, is 25-fold greater in HA samples than in flow-through samples. c, Gene transcript enrichment of HA-IP ramRNA relative to P7 mouse primary cortical astrocytes103. Of the 200 most highly expressed genes previously described103 for post-natal mouse cortical astrocytes, 71.5% (red line) are at least fourfold enriched (blue line) in HA-IP ramRNA isolated from uninjured spinal cord relative to flow through RNA from non-astrocyte cells. d, Pearson correlation plots of total normalized RNA-seq reads from individual biological replicates for each treatment condition. Correlation colouring indicates little (white) to high (red) similarity. n = 4 mice each for uninjured controls and wild-type SCI (SCI-WT); n = 3 mice for STAT3-CKO SCI (SCI-STAT3). FDR < 0.1 for differential expression and enrichment analysis. Raw and normalized data have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE76097 and via a searchable, open-access website https://astrocyte.rnaseq.sofroniewlab.neurobio.ucla.edu.

Extended Data Figure 4 Comparison of genomic data from astrocytes and non-astrocyte cells from WT and STAT3-CKO mice after SCI.

a, Heat maps depicting all significantly differentially expressed genes (DEG), as determined by RNA-seq, for wild-type (WT) and STAT3-CKO astrocytes and non-astrocytes from independent biological replicates two weeks after SCI relative to uninjured wild-type control. Red upregulated, green downregulated. b, Total numbers and Venn diagrams of significant DEGs in wild-type and STAT3-CKO astrocytes and non-astrocytes two weeks after SCI relative to uninjured control. Red and green numerical values indicate significantly upregulated and downregulated genes, respectively. c, Comparison of altered gene expression in our SCI-reactive astrocytes and previously reported forebrain stroke-reactive astrocytes27. Of the 200 most highly elevated genes in forebrain astrocytes one week following stroke27, 58.5% (red line) are also significantly elevated in astrocytes after SCI, relative to uninjured. d, Comparison of expression by wild-type SCI and STAT3-CKO SCI reactive astrocytes of a selected cross-section of genes that are highly regulated after SCI by wild-type reactive astrocytes. Many of the regulated genes exhibit changes that are expected and implicated in wild-type reactive astrogliosis mechanisms and roles, and some of the changes appear to be newly identified in this context. Note that many of the genes are not regulated or exhibit attenuated changes in STAT3-CKO SCI astrocytes. n = 4 mice each for uninjured and wild-type SCI; n = 3 mice for STAT3-CKO SCI (SCI-STAT3). FDR < 0.1 for differential expression and enrichment analysis.

Extended Data Figure 5 Immunohistochemistry of specific CSPGs.

a, Absence of aggrecan (ACAN) production by scar-forming astrocytes. Images show individual fluorescence channels of ACAN and GFAP immunohistochemistry from horizontal sections two weeks after severe SCI in a representative wild-type (WT) mouse. Boxes denote areas of astrocytic scar (AS) or uninjured tissue (Uninj) shown at higher magnification. Note that ACAN is: (i) heavily present in the perineuronal nets that surround neurons in uninjured tissue; (ii) almost absent from astrocytic scar and lesion core (LC); and (iii) not detectably produced by newly generated scar-forming astrocytes (arrows). b, Brevican (BCAN) production by scar-forming astrocytes and non-astrocyte cells. Images show individual fluorescence channels of BCAN and GFAP immunohistochemistry from horizontal sections two weeks after severe SCI, in wild-type mice and mice with transgenic ablation (TK+GCV) or attenuation (STAT3-CKO) of astrocytic scar formation. Note that BCAN is produced both by GFAP-positive scar-forming astrocytes (arrowheads) and by non-astrocyte cells (arrows).

Extended Data Figure 6 Immunohistochemistry of specific CSPGs.

a, Neurocan (NCAN) production by scar-forming astrocytes and non-astrocyte cells. Images show individual fluorescence channels of NCAN and GFAP immunohistochemistry from horizontal sections two weeks after severe SCI, in a representative wild-type (WT) mouse. Box denotes area of lesion core (LC) and astrocytic scar (AS) shown at higher magnification. Note that NCAN is produced both by GFAP-positive scar-forming astrocytes and by non-astrocyte cells (arrows) in the lesion core. b, NG2 (CSPG4) production by newly proliferated scar-forming astrocytes. Images show individual channels and various combinations of immunofluorescence staining for NG2, GFAP, tdTomato (tdT), BrdU (proliferation marker) and DAPI showing astrocytes in a mature SCI scar. The images are representative of findings from tdTomato-reporter mice51 injected with AAV2/5-GfaABC1D-Cre vector53 into multiple sites of the uninjured spinal cord to label mature astrocytes. Three weeks after AAV2/5-GfaABC1D-Cre injection, the mice received a severe SCI and were administered BrdU from days 2–7 after SCI. The mice were perfused after two weeks after SCI. Images comparing individual fluorescence channels show that astrocytes labelled 1 and 3: (i) incorporated BrdU and thus are newly proliferated after SCI; (ii) express the tdTomato reporter; (iii) express GFAP, the prototypical marker of reactive and scar-forming astrocytes; and (iv) express NG2 both intracellularly and along their cell surfaces. In contrast, astrocyte number 2 is also BrdU-labelled and expresses both tdTomato and GFAP, but does not appear to express detectable levels of NG2. c, CSPG5 (Neuroglycan C) production by scar-forming astrocytes. Images show individual channels and various combinations of immunofluorescence staining for CSPG5 or GFAP. Note that CSPG5 is present within and along the processes of GFAP-positive scar-forming astrocytes (arrows).

Extended Data Figure 7 Specificity and effects of treatments to stimulate AST axon regrowth after SCI.

a, BDNF and NT3 treatment does not alter the appearance or density of astrocyte scars in wild-type (WT) or STAT3-CKO mice. Images show horizontal sections of mice at two weeks after SCI or after SCI followed by delayed injection of hydrogel only (as a control) or hydrogel releasing NT3 and BDNF. Top images show GFAP immunofluorescence; boxed area denotes size of areas taken from multiple locations in the astrocytic scar (AS) for GFAP area quantification shown in graph. n = 5 mice per group; NS, P > 0.05 (ANOVA with Newman–Keuls). Bottom images show brightfield immunohistochemistry simultaneously of GFAP+TK to stain both astrocyte cell processes (GFAP) and cell bodies (TK) in mGFAP-TK transgenic mice for quantification of astrocyte cell numbers shown in graph. For these experiments the transgene-derived TK is used as a reporter protein that efficiently labels astrocyte cell bodies and thereby improves cell quantification18 and the mice were not given GCV. n = 4 mice per group; *P < 0.05 versus uninjured (ANOVA with Newman–Keuls); NS, P > 0.05 (ANOVA with Newman–Keuls). b, AST axon regrowth through scar-forming astrocytes and CSPGs in SCI lesions. Images show individual channels and various combinations of immunofluorescence staining for CTB, GFAP and CS56 to detect total CSPGs from a wild-type mouse after SCI followed by delayed injection of a hydrogel depot releasing NT3 and BDNF, shown as multichannel image in Fig. 5e. Arrows denote robust regrowth of many AST axons along, through and past scar-forming astrocytes into and through the lesion core. Note that the stimulated axons are regrowing through CSPG containing areas in the astrocyte scar and lesion core. Boxed area is shown at higher magnification in Extended Data Fig. 8. c, Graph shows numbers of AST axons at various distances past the proximal border of the astrocytic scar under different conditions. n = 5 per group; *P < 0.001 significant difference SCI+CL+BDNF+NT3 versus all other groups (ANOVA with post-hoc Newman–Keuls). Source data

Extended Data Figure 8 AST axon regrowth through scar-forming astrocytes and CSPGs in SCI lesions.

a, b, Images show individual channels and various combinations of immunofluorescence staining for CTB, GFAP and CS56 to detect total CSPGs from a wild-type (WT) mouse after SCI followed by delayed injection of a hydrogel depot releasing NT3 and BDNF, shown as multichannel images in Fig. 5f, g. Arrows in a denote robust regrowth of many AST axons along, through and past scar-forming astrocytes into and through the lesion core; note that the stimulated axons are regrowing through CSPG containing areas in the astrocytic scar and lesion core. b, High-magnification orthogonal images of axons in three visual planes. Arrows in b denote AST regrowing axons tracking along CSPG-positive and GFAP-negative structures. Arrowheads in b denote AST axons tracking along GFAP-positive and CSPG-positive astrocyte processes, passing from one astrocyte process to another.

Extended Data Figure 9 AST axon regrowth in SCI lesions is dependent on laminin.

ag, Tract tracing of AST axons using CTB and laminin immunohistochemistry. ac, Same fields imaged for CTB alone (a1c1), or CTB plus laminin (a2c2). a, d, Intact gracile–cuneate tract (GCT). b, SCI only. c, f, SCI plus conditioning lesion (CL) plus hydrogel with growth factors. e, SCI plus conditioning lesion. g, SCI plus conditioning lesion plus hydrogel with growth factors and anti-CD29. dg, High-magnification orthogonal images of axons in three visual planes. Arrows indicate regrowing axons in direct contact with laminin. Arrowheads indicate axons not in direct contact with laminin in the intact GCT (d) or with anti-CD29 treatment (g). Note the difference in appearance of axons in the intact gracile–cuneate tract (GCT), which are independent of laminin, compared with regrowing axons in lesion core (LC), which track along laminin. h, Axon length per tissue volume (means ± s.e.m.) in intact GCT or in SCI lesions under different conditions. Intact GCT values were not included in ANOVA comparison of other 3 groups. i, Percentages (means ± s.e.m.) of AST axon length in direct contact with laminin under different conditions. n = 5 mice per group; *P < 0.001 (ANOVA with post-hoc Newman–Keuls); NS, not significant (ANOVA with post-hoc Newman–Keuls). Source data

Extended Data Table 1 Axon growth inhibitory molecules and axon growth permissive molecules

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Anderson, M., Burda, J., Ren, Y. et al. Astrocyte scar formation aids central nervous system axon regeneration. Nature 532, 195–200 (2016) doi:10.1038/nature17623

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