Research ArticleSpinal Cord Injury

Cbp-dependent histone acetylation mediates axon regeneration induced by environmental enrichment in rodent spinal cord injury models

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Science Translational Medicine  10 Apr 2019:
Vol. 11, Issue 487, eaaw2064
DOI: 10.1126/scitranslmed.aaw2064
  • Fig. 1 EE induces a lasting increase in the regenerative potential of sensory neurons.

    (A) Cultured mouse sciatic DRGs after exposure to EE, stained for β-III-tubulin. Scale bar, 100 μm. (B) Quantification of neurite outgrowth (mean ± SEM, unpaired Student’s t tests, ***P < 0.001; n = 4 per group). n/s, not significant. (C) Diagram illustrating the experimental design; sciatic DRGs were cultured from mice that had been placed in EE for 10 days and then returned to SH for up to 5 weeks. (D) Example images of sciatic DRGs from mice that had been in SH or EE for 10 days and then in SH for 5 weeks. Scale bar, 100 μm. (E) Quantification of neurite outgrowth in DRGs from mice that had been exposed to EE compared to SH controls (mean ± SEM, unpaired Student’s t test, **P < 0.01; n = 4 per group). (F) Sciatic nerves immunostained for superior cervical ganglion-10 Protein (SCG10) after transection and reanastomosis. Scale bar, 500 μm. (G) Quantification of regenerating axons [mean ± SEM, two-way analysis of variance (ANOVA), Holm-Sidak post hoc, ***P < 0.001; n = 6 per group]. (H) CTB-traced (red) dorsal column sensory axons after injury, 4′,6-diamidino-2-phenylindole (DAPI) (blue), lesion site (dotted line). Scale bar, 200 μm. (I) Quantification of CTB-positive regenerating axons (mean ± SEM, two-way repeated measures ANOVA, Tukey’s post hoc, **P < 0.01, ***P < 0.001, respectively; n = 5 per group). (J) Electrophysiological setup. (K) Representative compound action potentials recorded below (blue) and above (black) injury. (L) Quantification of compound action potentials above the lesion (mean ± SEM, one-way ANOVA, Fisher’s least significant difference (LSD) post hoc, ***P < 0.001; n = 4 to 7 per group).

  • Fig. 2 Proprioceptive afferent feedback is required for EE-mediated increase in DRG regenerative growth.

    (A) Schematic diagram of the experimental design; after EE and SH exposure, mice underwent sciatic nerve crush injury and CTB injection distal to the crush site, axons that regenerate across the injury site take up CTB and retrogradely transport it to the soma. (B) Representative images of colocalization between PV, substance P, or isolectin B4 (green) and CTB (red) in DRGs from EE mice that had undergone a sciatic nerve crush. Scale bar, 50 μm. (C) Quantification of the number of CTB-positive DRG neurons in mice exposed to EE compared to SH (mean ± SEM, unpaired Student’s t test, **P < 0.01; n = 6 per group). (D) Quantification of the percentage of CTB-positive neurons that colocalized with PV, substance P (SubP), or isolectin B4 after exposure to EE or SH. (E) Schematic showing Egr3 mutation resulting in degeneration of muscle spindles. (F) β-III-tubulin–stained sciatic DRGs from WT or Egr3−/− mice after exposure to SH or EE. Scale bar, 100 μm. (G) Quantification of neurite outgrowth (mean ± SEM, one-way ANOVA, Tukey’s post hoc, ***P < 0.001; n = 4 per group). (H) Example images of tdTomato (red)–positive or tdTomato–negative DRGs costained with β-III-tubulin (green) cultured from PV-cre × tdTomato mice that had been exposed to either SH or EE for 10 days. Yellow indicates colocalization between tdTomato and β-III-tubulin. Scale bar, 100 μm. (I) Quantification of neurite outgrowth (mean ± SEM, one-way ANOVA, Tukey’s post hoc, ***P < 0.001; n = 5 per group).

  • Fig. 3 EE induces signaling pathways involved in neuronal activity, calcium mobilization, and the regenerative program of LDNs.

    (A) Heatmap of the differentially expressed (DE) genes in whole-DRGs and LDNs RNAseq (P < 0.05). Color scale represents arbitrary expression units (lowest, blue; highest, red). (B) Pie chart of genes in each functional group identified by Gene Ontology analysis of DE genes in LDNs. Functional groups are color coded. (C) Representative images of sciatic nerves transduced with AAV5-GFP (green fluorescent protein), AAV5-hM4Di-mCitrine, or AAV5-hM3Dq-mCitrine labeled with mCitrine/GFP after sciatic nerve crush. Arrowhead, lesion site. Scale bar, 500 μm. (D) Quantification of axon regeneration (mean ± SEM, two-way repeated measures ANOVA, Tukey’s post hoc, ***P < 0.001; n = 3 per group). (E) Representative time-lapse images of intracellular calcium release from whole-mount PV-genetically encoded calmodulin protein (GCaMP) DRGs before and after addition of 150 mM KCl. Scale bar, 50 μm. (F) Quantification of F/Fo (fluorescence intensity relative to baseline) ratio after 50 mM, 100 mM, and 150 mM KCl (mean ± SEM, two-way ANOVA, Sidak’s post hoc, **P < 0.01, ***P < 0.001, respectively; n = 4 per group).

  • Fig. 4 Cbp is required for EE-dependent increase in regeneration potential.

    (A) DRGs stained for H4K8ac (green), PV (red), and DAPI (blue). Scale bar, 50 μm. (B) Quantification of H4K8ac intensity (mean ± SEM, unpaired Student’s t test, ***P < 0.001; n = 6 per group). a.u., arbitrary units. (C) Example images of DRGs from mice housed in SH or EE, which were double stained for pCreb (green) and PV (red). Scale bar, 50 μm. (D) Quantification of the fluorescence intensity of pCreb in the nuclei of PV-positive DRGs (mean ± SEM, unpaired Student’s t test, ***P < 0.001; n = 4 per group). (E) Immunoblotting analysis for H4K8ac from protein extracts from whole sciatic DRGs after 10 days exposure to SH or EE (mean ± SEM, unpaired Student’s t test, **P < 0.01; n = 3 per group). H4K8ac normalized the levels of H4; glyceraldehyde phosphate dehydrogenase (Gapdh) was used as a loading control. (F) Immunoblotting analysis for pCreb from protein extracts of whole sciatic DRGs after 10 days exposure to SH or EE (mean ± SEM, unpaired Student’s t test, **P < 0.01; n = 3 per group). pCreb normalized the levels of Creb; Gapdh was used as a loading control. (G) DRGs stained for acCbp (green) and total Cbp (red). Scale bar, 50 μm. (H) Quantification of acCbp intensity (mean ± SEM, unpaired Student’s t test, ***P < 0.001; n = 11 per group). (I) Cultured DRG neurons from WT × Cbpf/f or CaMKIIa-creERT2 × Cbpf/f mice (β-III-tubulin, red; H4K8ac, green). Scale bar, 200 μm. (J) Quantification of neurite outgrowth (mean ± SEM, one-way ANOVA, Tukey’s post hoc, ***P < 0.001; n = 5 per group). (K) Quantification of H4K8ac intensity (mean ± SEM, one-way ANOVA, Tukey’s post hoc, ***P < 0.001; n = 5 per group).

  • Fig. 5 Pharmacological activation of Cbp/p300 promotes sensory axon regeneration and recovery after a dorsal hemisection SCI in mice.

    (A) Cultured DRG neurons treated with control (CSP) or Cbp/p300 pharmacological activator (CSP-TTK21) (β-III-tubulin, red; H4K8ac, green). Scale bar, 50 μm. (B) Quantification of neurite outgrowth (mean ± SEM, unpaired Student’s t test, **P < 0.01; n = 4 per group), H4K8ac intensity (mean ± SEM, unpaired Student’s t test, **P < 0.01; n = 11 per group), and neurite branching (mean ± SEM, unpaired Student’s t test, *P < 0.05; n = 4 per group). (C) Schematic view of a T9 dorsal column axotomy that lesions the ascending sensory axons in the dorsal columns. (D) CTB (red) was injected into the sciatic nerve 5 weeks after SCI. (E) CTB-traced (red) dorsal column axons after SCI, Gfap (green), DAPI (blue), and lesion site (dashed line). Scale bar, 200 μm. (F) Quantification of CTB-positive regenerating axons (mean ± SEM, two-way repeated measures ANOVA, Holm’s, Sidak post hoc, ***P < 0.001, *P < 0.05, respectively; n = 5 per group). (G) Quantification of slips (mean ± SEM, two-way repeated measures ANOVA, Fisher’s LSD post hoc, *P < 0.05; n = 10 per group). (H) Quantification of the time required to first contact an adhesive pad placed on the hindpaws (mean ± SEM, two-way repeated measures ANOVA, Fisher’s LSD post hoc, ***P < 0.001, **P < 0.01, *P < 0.05, respectively; n = 10 per group). (I) Representative image from a control CSP-treated mouse showing CTB-positive regenerating axons rostral to the lesion and colocalization with the presynaptic marker vGlut1. Lesion site is marked by the dashed line and asterisk. Scale bar, 100 μm (scale bar for insets, 10 μm). (J) Representative image from a CSP-TTK21–treated mouse showing colocalization of regenerating CTB–positive axons (red) rostral to the SCI site (marked by the dashed line and asterisk) with the presynaptic marker vGlut1 (green) to identify prospective nascent synapses (marked by arrows). Scale bar, 100 μm. Higher magnification images of insets show colocalization of CTB-positive axons (red) and vGlut1 (green). Scale bar, 10 μm. (K) Quantification of CTB and vGlut1 colocalization rostral to the lesion site (mean ± SEM, unpaired Student’s t test, ***P < 0.001; n = 6 per group). (L) Representative compound action potentials recorded below (gray) and above (black) the lesion. (M) Quantification of compound action potentials above the lesion (mean ± SEM, unpaired Student’s t test, *P < 0.05; n = 8 to 10 per group). (N) vGlut1-positive boutons (white) from group Ia afferents in proximity to hindlimb motoneurons (red, CTB) below the injury (L1 to L4). Scale bar, 25 μm. (O) Quantification of vGlut1-positive boutons opposed to motoneurons (MN) (mean ± SEM, unpaired Student’s t test, **P < 0.01; n = 8 per group).

  • Fig. 6 Pharmacological Cbp/p300 activation enhances sprouting of both descending motor and ascending sensory axons leading to functional recovery after contusion SCI in rats.

    (A) Image showing joints used for reconstruction of hindlimb movements. MTP, metatarsophalangeal. (B) Representative hindlimb kinematics after treatment with CSP or CSP-TTK21. Black, orange, and gray correspond to stance, drag, and swing phases of gait, respectively. (C) PC analysis of gait parameters averaged for each group at weeks 1, 4, and 8 and quantification of average scores on PC1, which quantifies the locomotor performance of rats treated with CSP or CSP-TTK21 (mean ± SEM, two-way ANOVA, Fisher’s LSD post hoc, *P < 0.05; n = 10 per group). (D) Bar plots of drag duration and step height (mean ± SEM, unpaired Student’s t test, *P < 0.05, **P < 0.01; n = 10 per group). (E) Schematics showing strategy for tracing vGi axons, T9 contusion, and the L4 ventral horn analyzed for vGi and 5-hydroxytryptamine (5HT) sprouting. (F) Representative images of descending vGi axons (red) sprouting around motoneurons [choline acetyltransferase (ChAT), cyan] in the lumbar ventral horn after treatment with CSP or CSP-TTK21. Scale bar, 50 μm. (G) Quantification of vGi intensity in the ventral horn (mean ± SEM, unpaired Student’s t test, ***P < 0.001; n = 8 per group). (H) Representative images of descending 5HT axons (magenta) sprouting around motoneurons (ChAT, cyan) in the lumbar ventral horn after treatment with CSP or CSP-TTK21. Scale bar, 50 μm. (I) Quantification of 5HT intensity in the ventral horn (mean ± SEM, unpaired Student’s t test, ***P < 0.001; n = 9 per group). (J) Representative sagittal sections showing sprouting of descending vGi (red) and 5HT (green) axons around motoneurons (ChAT, white) below in the injury in the lumbar ventral horn after treatment with CSP or CSP-TTK21. Scale bar, 50 μm. (K) vGlut1-positive boutons (yellow) from group Ia afferents in proximity to motoneurons (ChAT, cyan) below the injury (L1 to L4). Scale bar, 25 μm. (L) Quantification of vGlut1-positive boutons opposed to motoneurons (mean ± SEM, unpaired Student’s t test, ***P < 0.001; n = 9 per group). (M) Quantification of the H wave amplitude after treatment with CSP or CSP-TTK21 (mean ± SEM, one-way ANOVA, Tukey’s post hoc, *P < 0.05; n = 5 to 10 per group).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/487/eaaw2064/DC1

    Fig. S1. Exposure to EE enhances neurite outgrowth on inhibitory myelin substrate to a similar extent as a conditioning SNA injury.

    Fig. S2. Inhibiting transcription with actinomycin-D blocks the EE-mediated increase in DRG neurite outgrowth.

    Fig. S3. Exposure to EE enhances axon elongation rather than branching.

    Fig. S4. The full EE increases DRG neurite outgrowth compared to the running wheel alone.

    Fig. S5. Exposure to EE enhances muscle reinnervation by proprioceptive DRG neurons.

    Fig. S6. EE promotes axon regeneration and the formation of putative synapses.

    Fig. S7. Neurotrophin and cytokine levels in the DRG and blood serum are not affected by EE.

    Fig. S8. RNAseq and proteomic datasets demonstrate that EE strongly modulates pathways involved in neuronal activity, calcium signaling, gene expression, and cytoskeletal changes.

    Fig. S9. Efficient transduction and DREADD expression in PV-positive DRGs.

    Fig. S10. The EE-mediated increase in DRG neurite outgrowth is mediated by neuronal activity.

    Fig. S11. H3K27ac is increased in PV-positive DRGs after exposure to EE, but the levels of H3K4me2, H3K9ac, and H3K4me3 do not change compared to SH.

    Fig. S12. Levels of H4K8ac but not of acCbp or pCreb remain elevated in PV-positive DRGs for 5 weeks after exposure to EE.

    Fig. S13. Increasing neuronal activity augments the level of H4K8ac and acCbp in DRG neurons.

    Fig. S14. The CaMKIIa promoter is active in DRG neurons and drives strong expression of tdTomato after tamoxifen treatment.

    Fig. S15. CSP-TTK21 treatment significantly enhances the time to remove adhesive tape placed on the hindpaw.

    Fig. S16. CSP-TTK21 treatment promotes sprouting of afferent fibers below the level of injury.

    Fig. S17. CSP-TTK21 treatment enhances H4K8ac in the DRG.

    Fig. S18. CSP-TTK21 treatment does not affect the glial scar after a thoracic dorsal spinal cord hemisection in mice.

    Fig. S19. List compiling the 78 parameters used for quantifying gait features.

    Fig. S20. CSP-TTK21 reduced the number of slips during locomotion along a horizontal ladder.

    Fig. S21. CSP-TTK21 treatment enhances levels of H4K8ac in the raphe nucleus and reticular formation.

    Fig. S22. CSP-TTK21 treatment does not affect the glial scar after a thoracic contusion SCI in rats.

    Movie S1. EE-mediated calcium mobilization in proprioceptive DRG neurons.

    Movie S2. SH-mediated calcium mobilization in proprioceptive DRG neurons.

    Movie S3. Treatment with CSP-TTK21 enhances hindlimb function and over-ground locomotion.

    Data file S1. Differentially expressed genes and proteins after EE compared to SH.

    Data file S2. Functional classification of EE-dependent gene expression changes.

    Data file S3. Raw data.

    References (5558)

  • The PDF file includes:

    • Fig. S1. Exposure to EE enhances neurite outgrowth on inhibitory myelin substrate to a similar extent as a conditioning SNA injury.
    • Fig. S2. Inhibiting transcription with actinomycin-D blocks the EE-mediated increase in DRG neurite outgrowth.
    • Fig. S3. Exposure to EE enhances axon elongation rather than branching.
    • Fig. S4. The full EE increases DRG neurite outgrowth compared to the running wheel alone.
    • Fig. S5. Exposure to EE enhances muscle reinnervation by proprioceptive DRG neurons.
    • Fig. S6. EE promotes axon regeneration and the formation of putative synapses.
    • Fig. S7. Neurotrophin and cytokine levels in the DRG and blood serum are not affected by EE.
    • Fig. S8. RNA-seq and proteomic datasets demonstrate that EE strongly modulates pathways involved in neuronal activity, calcium signaling, gene expression, and cytoskeletal changes.
    • Fig. S9. Efficient transduction and DREADD expression in PV-positive DRGs.
    • Fig. S10. The EE-mediated increase in DRG neurite outgrowth is mediated by neuronal activity.
    • Fig. S11. H3K27ac is increased in PV-positive DRGs after exposure to EE, but the levels of H3K4me2, H3K9ac, and H3K4me3 do not change compared to SH.
    • Fig. S12. Levels of H4K8ac but not of acCbp or pCreb remain elevated in PV-positive DRGs for 5 weeks after exposure to EE.
    • Fig. S13. Increasing neuronal activity augments the level of H4K8ac and acCbp in DRG neurons.
    • Fig. S14. The CaMKIIa promoter is active in DRG neurons and drives strong expression of tdTomato after tamoxifen treatment.
    • Fig. S15. CSP-TTK21 treatment significantly enhances the time to remove adhesive tape placed on the hindpaw.
    • Fig. S16. CSP-TTK21 treatment promotes sprouting of afferent fibers below the level of injury.
    • Fig. S17. CSP-TTK21 treatment enhances H4K8ac in the DRG.
    • Fig. S18. CSP-TTK21 treatment does not affect the glial scar after a thoracic dorsal spinal cord hemisection in mice.
    • Fig. S19. List compiling the 78 parameters used for quantifying gait features.
    • Fig. S20. CSP-TTK21 reduced the number of slips during locomotion along a horizontal ladder.
    • Fig. S21. CSP-TTK21 treatment enhances levels of H4K8ac in the raphe nucleus and reticular formation.
    • Fig. S22. CSP-TTK21 treatment does not affect the glial scar after a thoracic contusion SCI in rats.
    • Legends for movies S1 to S3
    • Legends for data files S1 to S3
    • References (5558)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). EE-mediated calcium mobilization in proprioceptive DRG neurons.
    • Movie S2 (.mp4 format). SH-mediated calcium mobilization in proprioceptive DRG neurons.
    • Movie S3 (.mp4 format). Treatment with CSP-TTK21 enhances hindlimb function and over-ground locomotion.
    • Data file S1 (Microsoft Excel format). Differentially expressed genes and proteins after EE compared to SH.
    • Data file S2 (Microsoft Excel format). Functional classification of EE-dependent gene expression changes.
    • Data file S3 (Microsoft Excel format). Raw data.

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