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Systemic administration of epothilone B promotes axon regeneration after spinal cord injury

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Science  17 Apr 2015:
Vol. 348, Issue 6232, pp. 347-352
DOI: 10.1126/science.aaa2958
  • Fig. 1 EpoB reduces inhibitory fibrotic scarring after SCI by abrogating meningeal fibroblast polarization and migration.

    (A) Mass spectrometric analysis of CNS tissue and blood after a single i.p. injection of epoB; n = 4 rats per time point. (B) Immunoblots (IB) of indicated proteins in lesion extracts; n = 3 rats. (C) Human spinal cord after injury (asterisk); laminin immunolabeling. (D) Immunolabeling for laminin, glial fibrillary acidic protein (GFAP), or chondroitin sulfates (CS-56) after rat spinal cord hemisection. (E) Laminin-immunopositive (+) area at the lesion; n = 7 to 8 rats per group. (F) Glycosaminoglycan amounts in spinal cord lesion extracts; n = 8 rats per group. (G) Rat meningeal fibroblasts (RMFs) in wound-healing assays. (H) Percentage of the area shown in (G) occupied with RMFs after 48 hours, n = 3 experiments. (I and J) Immunolabeling of tyrosinated (TyrTub) and detyrosinated tubulin (DetyrTub, arrowheads). (K) IB of indicated proteins in RMFs 24 hours after treatment. (L) Immunolabeling for fibronectin, detyrosinated and tyrosinated tubulin [4′,6-diamidino-2-phenylindole (DAPI); nuclear staining] in the rat meninges at the lesion. Bottom panel, magnification of fibroblasts (arrowheads) in top panel. dpi, days postinjury. Scale bars, 50 μm. Schemes in (D) and (L) indicate lesion and displayed region (red box). Values are plotted as means + SEM. *P < 0.05, ***P < 0.001 by Student’s t test.

  • Fig. 2 EpoB promotes microtubule protrusion and axon elongation in neurons while dampening microtubule dynamics in scar-forming fibroblasts.

    (A) Beta-3 tubulin (Tuj-1) immunolabeling of neurons on inhibitory substrates (CSPGs, chondroitin sulfate proteoglycans; Sema 3A, Semaphorin 3A). (B) Neurite length of cortical neurons after 48 hours under indicated conditions; n = 3 to 4 experiments. (C) EB3-mCherry time-lapse projections in Nogo-A exposed neuron before and after epoB treatment (asterisks, stable landmarks). Bottom panels, high magnification of boxed areas in top panels. (D) EB3-mCherry fluorescence intensity in neurites under indicated conditions; n = 9 to 16 neurons (from three experiments). (E) Neurite growth on Nogo-A. Black arrowhead, time of indicated treatment. n = 12 to 15 neurons (from three experiments). (F and G) EB3-mCherry time-lapse projections of nocodazole-treated neuron (F) and epoB-treated meningeal fibroblast (G). Bottom panels, high magnification of boxed areas in top panels. (H) EB3-mCherry time-lapse projections before and after epoB treatment in cultured meningeal fibroblasts with (arrowhead) or without Tau-expression. Bottom panels, magnification of boxed areas in top panels. (I) EB3-mCherry fluorescence intensity in fibroblast periphery under indicated conditions; n = 20 cells per condition (from four experiments). Scale bars, 25 μm. Values are plotted as means [+ SEM in (B) and (E)]. *P < 0.05, **P < 0.01 by Student’s t test. n.s., not significant.

  • Fig. 3 EpoB reduces dystrophy and promotes regeneration of injured spinal cord axons.

    (A) Electron microscope images of human SCI. (Top) Undamaged axon containing microtubules (black arrowheads). (Bottom) Retraction bulb (indicated by white arrowheads) without microtubules. (Middle) Magnification of boxed area in bottom panel. Scale bars, 500 nm. (B) Beta-3 tubulin (Tuj-1) immunolabeling of retraction bulbs in chronic human SCI. Scale bar, 10 μm. (C) Lesioned GFP-positive spinal cord axons in mice forming retraction bulbs (yellow arrowheads), dying back (red arrowheads), or regenerating (green arrowheads). Boxed area in top panels, displayed region in panels below. Scale bars, 100 μm. (D and E) Percentage of injured axons forming retraction bulbs (D) and distance between injured axons and injury site (E); n = 8 mice per group. Values are plotted as means + SEM. (F) Microruby-traced mouse dorsal column axons after injury (white arrowheads), laminin and GFAP immunolabeling (dashed line, lesion border). Scale bar, 100 μm. (G) Average distance between caudal lesion margin and injured axons in individual animals (circles) and group means (vertical bars) ± SEM. *P < 0.05, **P < 0.01 by Student’s t test.

  • Fig. 4 EpoB promotes regrowth of raphespinal axons and improves walking after spinal cord contusion injury.

    (A) Serotonin (5HT) immunolabeling (dashed line, lesion border) and (B) number of 5HT-labeled (+) fibers caudal to a spinal dorsal hemisection; n = 7 to 8 rats per group. (C) Coronal sections of the lumbar spinal cord after contusion injury. (Left panel) Coimmunostaining of 5HT, synaptophysin (Syn), and choline acetyltransferase (ChAT). (Right panels) Magnification of each marker in boxed area (left panel) visualizing serotonergic innervation of motor neurons (arrowheads). (D) Total length of 5HT-immunopositive fibers in the ventral horn (5,7-DHT, 5,7-dihydroxytryptamine); n = 4 (uninjured), 6 (7 dpi), 11 to 12 rats (56 and 70 dpi) per group. (E) Number of footfalls on the horizontal ladder; n = 10 to 11 rats per group. dpi, days postinjury. Scale bars, 50 μm. Schemes in (A) and (C) indicate lesion and displayed region (red box). Values are plotted as means + SEM. *P < 0.05; n.s., not significant by Student’s t test.

Supplementary Materials

  • Systemic administration of epothilone B promotes axon regeneration after spinal cord injury

    Jörg Ruschel, Farida Hellal, Kevin C. Flynn, Sebastian Dupraz, David A. Elliott, Andrea Tedeschi, Margaret Bates, Christopher Sliwinski, Gary Brook, Kristina Dobrint, Michael Peitz, Oliver Brüstle, Michael D. Norenberg, Armin Blesch, Norbert Weidner, Mary Bartlett Bunge, John L. Bixby, Frank Bradke

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S11
    • Tables S1 and S2
    • Captions for movies S1 to S8
    • References

    Images, Video, and Other Other Media

    Movie S1
    Meningeal fibroblasts migrate into the cell free area in a wound healing assay.
    In vitro wound healing assay of primary meningeal fibroblasts treated with DMSO. Images were acquired at 5 min intervals. Movie plays at 40 frames per second. Counter displays hour:minute.
    Movie S2
    Epothilone B inhibits meningeal fibroblast migration into the cell free area in a wound healing assay.
    In vitro wound healing assay of primary meningeal fibroblasts treated with epothilone B. Images were acquired at 5 min intervals. Movie plays at 40 frames per second. Counter displays hour:minute.
    Movie S3
    Epothilone B promotes microtubule polymerization into neurite tips which propels neurite elongation.
    EB3-mCherry dynamics in a neuron cultured with Nogo-A, before and after epothilone B treatment. Movie plays at 7 frames per second
    Movie S4
    In Tau-expressing fibroblasts EB3 accumulates specifically at the distal tips of Taudecorated microtubule bundles.
    EB3-mCherry dynamics in a Tau-expressing meningeal fibroblast, before and after epothilone B treatment. Movie plays at 7 frames per second.
    Movie S5
    Functional recovery of walking in a representative vehicle treated rat.
    Movie of a representative vehicle treated rat performing on the horizontal ladder 56 days after spinal cord contusion injury. Movie plays at 24 frames per second.
    Movie S6
    Functional recovery of walking in a representative epothilone B treated rat.
    Movie of a representative epothilone B treated rat performing on the horizontal ladder 56 days after spinal cord contusion injury. Movie plays at 24 frames per second.
    Movie S7
    Effects of the serotonergic neurotoxin 5,7-DHT on functional recovery of walking in a representative vehicle treated rat.
    Vehicle treated rat, shown in movie S4, performing on the horizontal ladder 14 days after receiving intracerebroventricular injections of 5,7-Dihydroxytryptamine (5,7-DHT). Movie plays at 24 frames per second.
    Movie S8
    Effects of the serotonergic neurotoxin 5,7-DHT on functional recovery of walking in a representative epothilone B treated rat.
    Epothilone B treated rat, shown in movie S5, performing on the horizontal ladder 14 days after receiving intracerebroventricular injections of 5,7-Dihydroxytryptamine (5,7-DHT). Movie plays at 24 frames per second.

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