Reports tubule stabilization induces such divergent effects. Moreover, Taxol cannot be used for clinical CNS intervention because it does not cross the blood-brain barrier (10). We aimed to target microtubule stabilization in the injured CNS in a clinically feasible way Jörg Ruschel,1 Farida Hellal,1*† Kevin C. Flynn,1*‡ Sebastian 1 1 1 2 and to decipher its distinct celluDupraz, * David A. Elliott, Andrea Tedeschi, Margaret Bates, lar actions. We used epothilones, 3 4,5 6 Christopher Sliwinski, Gary Brook, Kristina Dobrint, Michael a class of FDA approved bloodPeitz,6 Oliver Brüstle,6 Michael D. Norenberg,7 Armin Blesch,3 brain barrier permeable microNorbert Weidner,3 Mary Bartlett Bunge,2 John L. Bixby,2 Frank tubule stabilizing drugs (11). Mass spectrometry confirmed Bradke1§ that after intraperitoneal (i.p.) 1 Axonal Growth and Regeneration, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn, injection in adult rats, epoB was 2 Germany. The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, 1095 Northwest 14th rapidly absorbed into the CNS Terrace, Miami, FL33136, USA. 3Spinal Cord Injury Center, Heidelberg University Hospital, Schlierbacher Landstr. 200A, 69118 Heidelberg, Germany. 4Institute for Neuropathology, RWTH Aachen University, Steinbergweg 20, 52074, Aachen, and remained at comparable Germany. 5Jülich-Aachen Research Alliance–Translational Brain Medicine. 6Institute of Reconstructive Neurobiology, levels for 6 days (Fig. 1A). Rats Life&Brain Center, University of Bonn and Hertie Foundation, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany. i.p. injected with 0.75 mg/kg 7 Departments of Pathology, Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, body weight (BW) epoB at day 1 USA.. and 15 post-injury showed in*These authors contributed equally to this work. creased levels of detyrosinated †Present address: Institute for Stroke and Vascular Dementia Research, University of Munich Medical Center, Max and acetylated tubulin in lesion Lebsche Platz 30, 81377 Munich, Germany. site extracts 4 weeks after spinal ‡Present address: Department of Molecular Medicine, Max Planck Institute of Biochemistry, Am Klopferspitz 18, cord dorsal hemisection (Fig. 1B) 82152 Martinsried, Germany. indicating increased microtubule stability (12). The dosage used §Corresponding author. E-mail: [email protected] presented no obvious adverse After central nervous system (CNS) injury, inhibitory factors in the lesion side effects, such as reduced anscar and poor axon growth potential prevent axon regeneration. imal weight or decreased white Microtubule stabilization reduces scarring and promotes axon growth. blood cell counts (fig. S1). However, the cellular mechanisms of this dual effect remain unclear. Here, Fibrotic scar tissue rich in fidelayed systemic administration of a blood-brain barrier permeable bronectin and laminin forms at microtubule stabilizing drug, epothilone B (epoB), decreased scarring after the lesion site after SCI in rorodent spinal cord injury (SCI) by abrogating polarization and directed dents (8) and humans (Fig. 1C; migration of scar-forming fibroblasts. Conversely, epothilone B reactivated table S1). This scar tissue poses a neuronal polarization by inducing concerted microtubule polymerization key impediment to regenerating into the axon tip, which propelled axon growth through an inhibitory axons, because it contains axon environment. Together, these drug elicited effects promoted axon growth inhibitory factors, includregeneration and improved motor function after SCI. With recent clinical ing chondroitin sulfate proteoapproval, epothilones hold promise for clinical use after CNS injury. glycans (CSPGs) (1, 8). Adult rats systemically treated post-injury An ideal treatment to induce axon regeneration in the in- with 0.75 mg/kg BW epoB showed a significant reduction of jured CNS should reduce scarring (1) and growth inhibitory fibronectin (Fig. 1B) and of laminin-positive fibrotic scar factors at the lesion site (2–4), reactivate the axon growth tissue even 4 weeks after dorsal hemisection (Fig. 1D and E). potential (5) and be administrable as a medication after in- We found a comparable decrease of fibrotic scarring when jury. Recently, a number of combinatorial approaches have epoB was locally delivered to the injury site via an intratheled to axon regeneration (6, 7). These approaches, however, cal catheter (fig. S2) (8). Reduction of fibrotic scar tissue by involve multiple drugs, enzymes and interventions render- systemic epoB administration was associated with a deing clinical translation difficult. Moderate microtubule sta- crease of CSPGs (Fig. 1, D and F), including neurocan (Fig. bilization by the anti-cancer drug Taxol promotes axon 1B) and NG2 (13), at the injury site (fig. S3). Astrogliosis and regeneration by reducing fibrotic scarring and increasing lesion area were similar between treated and control aniaxon growth (8, 9). However, it remains elusive how micro- mals (fig. S1) indicating that neuroprotective glial sealing of / sciencemag.org/content/early/recent / 12 March 2015 / Page 1 / 10.1126/science.aaa2958
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Systemic administration of epothilone B promotes axon regeneration after spinal cord injury
the injury site (14) was not affected by the treatment. Scar reduction upon epoB treatment resulted neither from decreased cell proliferation nor from increased apoptosis (fig. S4) but from a migratory defect of scar-forming meningeal fibroblasts (15). In wound healing assays, epoB inhibited migration of meningeal fibroblasts (Fig. 1, G and H, and movies S1 and S2) by changing their microtubular network. Control cells polarized by forming a leading edge enriched in stable detyrosinated microtubules and a trailing edge containing dynamic, tyrosinated microtubules (Fig. 1I), both hallmarks of directed cell migration (16). In contrast, epoB treated fibroblasts were round and nonpolar (Fig. 1J and fig. S5) with elevated levels of detyrosinated microtubules (Fig. 1K) distributed throughout the cell (Fig. 1J). Similarly, systemic administration of epoB after dorsal hemisection prevented the polarization of meningeal fibroblasts at the lesion site into a bipolar, migratory shape (Fig. 1L), which reduced scar formation (Fig. 1D and E). In co-cultures of meningeal fibroblasts and postnatal cortical neurons, epoB treatment (1 nM) perturbed fibroblast polarization while enhancing axon growth (fig. S5). Moreover, epoB restored axon growth when these neurons were confronted with the inhibitory molecules Nogo-A, CSPGs or Semaphorin 3A (Fig. 2, A and B), which are abundant at the spinal cord lesion site (2–4, 17). In neurons expressing fluorescently-tagged microtubule plus-end binding protein 3 (EB3-mCherry), which labels polymerizing microtubules (18), epoB induced rapid and concerted microtubule polymerization into the neurite tips (Fig. 2, C and D, and movie S3) causing axon elongation despite inhibitory NogoA (Fig. 2E and movie S3). In accordance, low doses of the microtubule destabilizing drug nocodazole abolished microtubule protrusion in neurites (Fig. 2, D and F) and abrogated the growth promoting effect of epoB (Fig. 2B). EpoB also promoted axon growth of human cortical neurons under growth permissive as well as non-permissive conditions (fig. S6). In meningeal fibroblasts, however, epoB prevented microtubule polymerization toward the cell edges (Fig. 2G) contrasting with the microtubule dynamics found in neurons. This dichotomy was due to neuron-specific expression of the microtubule associated protein Tau (fig. S7), which regulates microtubule dynamics, bundling, and binding of microtubule stabilizing agents (19, 20). In fibroblasts ectopically expressing Tau, epoB induced an accumulation of bundled microtubules (fig. S8) that polymerized toward the cell edge (Fig. 2, H and I, and movie S4), mimicking the effect observed in neurons. In turn, neurons depleted of Tau, by transfection with a plasmid encoding small hairpin (sh) RNA for tau (21), showed reduced microtubule polymerization into the distal neurite when exposed to epoB (fig. S9). Injured axons in the rodent and human CNS form dystrophic retraction bulbs (Fig. 3, A to D, and table S2), a consequence of microtubule depolymerization and disorganization (Fig. 3, A and B) (22, 23). Because epoB induced microtubule polymerization and axon growth in cul-
tured neurons, we assessed its ability to promote axon regeneration after SCI. In vivo imaging of adult transgenic mice, expressing green fluorescent protein (GFP) in spinal cord dorsal column axons (23, 24), revealed that transected axons of animals injected with 1.5 mg/kg BW epoB exhibited significantly fewer retraction bulbs (Fig. 3, C and D), reduced axonal dieback and increased regenerative growth (Fig. 3, C and E). Moreover, in adult mice, systemic and post-injury treatment with epoB, promoted axon regeneration after complete dorsal column transection (Fig. 3, F and G). We then tested whether the treatment also promoted axon regrowth of descending axons important for locomotion. In adult rats post-injury injected with 0.75 mg/kg BW epoB, we found a 3-fold increase of serotonergic fibers caudal to a dorsal hemisection (Fig. 4, A and B). Increased serotonergic innervation strongly correlates with recovery of motor function after SCI (25–27). Therefore, we asked whether the treatment improves walking of adult rats that underwent a moderate, mid-thoracic spinal cord contusion, a clinically relevant SCI model (28). After contusion injury, epoB administration (0.75 mg/kg BW) reduced fibrotic scarring at the injury site (fig. S10) and promoted serotonergic axon regrowth in the caudal spinal cord (Fig. 4, C and D). Moreover, epoB treatment increased stride length and gait regularity, and reduced external rotation of the hind paws (fig. S11) indicating improved walking balance and coordination. Accordingly, epoB treated animals showed a 50% reduction of foot misplacements on the horizontal ladder compared to injured controls (Fig. 4E and movies S5 and S6). These functional improvements were abrogated by pharmacological ablation of serotonergic innervation (Fig. 4, D and E, and movies S7 and S8) (25). The finding that the stabilization of microtubules inhibits cell division established the usage of systemic microtubule stabilizing agents as a therapeutic standard for the treatment of cancer (29). Here, at low doses, systemic administration of the microtubule stabilizing agent epoB promoted functional recovery after SCI. Our approach differs from other experimental regenerative paradigms (1–7) by pharmacologically focusing on a single molecular target, the microtubules, yet overcoming multiple pathological obstacles. This is possible due to divergent effects of pharmacological microtubule stabilization on microtubule dynamics and, hence the polarization of neurons and meningeal fibroblasts. This dual effect and the efficacy after systemic and post-injury administration, give epothilones a promising translational perspective for treatment of the injured CNS. REFERENCES AND NOTES 1. N. Klapka, S. Hermanns, G. Straten, C. Masanneck, S. Duis, F. P. Hamers, D. Müller, W. Zuschratter, H. W. Müller, Suppression of fibrous scarring in spinal cord injury of rat promotes long-distance regeneration of corticospinal tract axons, rescue of primary motoneurons in somatosensory cortex and significant functional recovery. Eur. J. Neurosci. 22, 3047–3058 (2005). Medline doi:10.1111/j.14609568.2005.04495.x
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cord contusion injury model. We also thank C. Laskowski, C.H. Coles, A. Kania, M. Hübener, W. Jackson and G. Tavosanis for critically reading and discussing the manuscript. We are grateful for the support from the Human Spinal Cord Tissue Bank and the electron microscopy core at the Miami Project as well as Professor B. Kakulas, University of Western Australia and Royal Perth providing anonymized post mortem sections following human spinal cord injury. This work was supported by NIH, IRP, WfL and DFG. Harald Witte, Ali Ertürk, Farida Hellal, and Frank Bradke filed a patent on the use of microtubule stabilizing compounds for the treatment of lesions of CNS axons” (European Patent No. 1858498; European patent application EP 11 00 9155.0; US patent application 11/908,118). The authors declare no competing financial interests. SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.aaa2958/DC1 Materials and Methods Figs. S1 to S11 Tables S1 and S2 Movies S1 to S8 References (30–37) 14 November 2014; accepted 25 February 2015 Published online 12 March 2015 10.1126/science.aaa2958
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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 single i.p. injection of epoB, N = 4 rats/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/group. (F) Glycosaminoglycan amounts in spinal cord lesion extracts, N = 8 rats/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 (DAPI, nuclear staining) in the rat meninges at the lesion. Bottom panel, magnification of fibroblasts (arrowheads) in top panel. dpi, days post-injury. 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.
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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 = 3-4 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 = 9-16 neurons (from 3 experiments). (E) Neurite growth on Nogo-A. Black arrowhead, time of indicated treatment. N = 12-15 neurons (from 3 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 Tauexpression. Bottom panels, magnification of boxed areas in top panels. (I) EB3-mCherry fluorescence intensity in fibroblast periphery under indicated conditions, N = 20 cells/condition (from 4 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.
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Fig. 3. EpoB reduces dystrophy and promotes regeneration of injured spinal cord axons. (A) Electron microscope images of human SCI. Top panel, undamaged axon containing microtubules (black arrowheads). Bottom panel, retraction bulb (indicated by white arrowheads) without microtubules. Middle panel, 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 panel, 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/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. / sciencemag.org/content/early/recent / 12 March 2015 / Page 7 / 10.1126/science.aaa2958
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 = 7-8 rats/group. (C) Coronal sections of the lumbar spinal cord after contusion injury. Left panel, co-immunostaining 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 (7dpi), 11-12 rats (56 and 70 dpi)/group. (E) Number of footfalls on the horizontal ladder, N = 10-11 rats/group. dpi, days post-injury. 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.
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Systemic administration of epothilone B promotes axon regeneration after spinal cord injury
the injury site (14) was not affected by the treatment. Scar reduction upon epoB treatment resulted neither from decreased cell proliferation nor from increased apoptosis (fig. S4) but from a migratory defect of scar-forming meningeal fibroblasts (15). In wound healing assays, epoB inhibited migration of meningeal fibroblasts (Fig. 1, G and H, and movies S1 and S2) by changing their microtubular network. Control cells polarized by forming a leading edge enriched in stable detyrosinated microtubules and a trailing edge containing dynamic, tyrosinated microtubules (Fig. 1I), both hallmarks of directed cell migration (16). In contrast, epoB treated fibroblasts were round and nonpolar (Fig. 1J and fig. S5) with elevated levels of detyrosinated microtubules (Fig. 1K) distributed throughout the cell (Fig. 1J). Similarly, systemic administration of epoB after dorsal hemisection prevented the polarization of meningeal fibroblasts at the lesion site into a bipolar, migratory shape (Fig. 1L), which reduced scar formation (Fig. 1D and E). In co-cultures of meningeal fibroblasts and postnatal cortical neurons, epoB treatment (1 nM) perturbed fibroblast polarization while enhancing axon growth (fig. S5). Moreover, epoB restored axon growth when these neurons were confronted with the inhibitory molecules Nogo-A, CSPGs or Semaphorin 3A (Fig. 2, A and B), which are abundant at the spinal cord lesion site (2–4, 17). In neurons expressing fluorescently-tagged microtubule plus-end binding protein 3 (EB3-mCherry), which labels polymerizing microtubules (18), epoB induced rapid and concerted microtubule polymerization into the neurite tips (Fig. 2, C and D, and movie S3) causing axon elongation despite inhibitory NogoA (Fig. 2E and movie S3). In accordance, low doses of the microtubule destabilizing drug nocodazole abolished microtubule protrusion in neurites (Fig. 2, D and F) and abrogated the growth promoting effect of epoB (Fig. 2B). EpoB also promoted axon growth of human cortical neurons under growth permissive as well as non-permissive conditions (fig. S6). In meningeal fibroblasts, however, epoB prevented microtubule polymerization toward the cell edges (Fig. 2G) contrasting with the microtubule dynamics found in neurons. This dichotomy was due to neuron-specific expression of the microtubule associated protein Tau (fig. S7), which regulates microtubule dynamics, bundling, and binding of microtubule stabilizing agents (19, 20). In fibroblasts ectopically expressing Tau, epoB induced an accumulation of bundled microtubules (fig. S8) that polymerized toward the cell edge (Fig. 2, H and I, and movie S4), mimicking the effect observed in neurons. In turn, neurons depleted of Tau, by transfection with a plasmid encoding small hairpin (sh) RNA for tau (21), showed reduced microtubule polymerization into the distal neurite when exposed to epoB (fig. S9). Injured axons in the rodent and human CNS form dystrophic retraction bulbs (Fig. 3, A to D, and table S2), a consequence of microtubule depolymerization and disorganization (Fig. 3, A and B) (22, 23). Because epoB induced microtubule polymerization and axon growth in cul-
tured neurons, we assessed its ability to promote axon regeneration after SCI. In vivo imaging of adult transgenic mice, expressing green fluorescent protein (GFP) in spinal cord dorsal column axons (23, 24), revealed that transected axons of animals injected with 1.5 mg/kg BW epoB exhibited significantly fewer retraction bulbs (Fig. 3, C and D), reduced axonal dieback and increased regenerative growth (Fig. 3, C and E). Moreover, in adult mice, systemic and post-injury treatment with epoB, promoted axon regeneration after complete dorsal column transection (Fig. 3, F and G). We then tested whether the treatment also promoted axon regrowth of descending axons important for locomotion. In adult rats post-injury injected with 0.75 mg/kg BW epoB, we found a 3-fold increase of serotonergic fibers caudal to a dorsal hemisection (Fig. 4, A and B). Increased serotonergic innervation strongly correlates with recovery of motor function after SCI (25–27). Therefore, we asked whether the treatment improves walking of adult rats that underwent a moderate, mid-thoracic spinal cord contusion, a clinically relevant SCI model (28). After contusion injury, epoB administration (0.75 mg/kg BW) reduced fibrotic scarring at the injury site (fig. S10) and promoted serotonergic axon regrowth in the caudal spinal cord (Fig. 4, C and D). Moreover, epoB treatment increased stride length and gait regularity, and reduced external rotation of the hind paws (fig. S11) indicating improved walking balance and coordination. Accordingly, epoB treated animals showed a 50% reduction of foot misplacements on the horizontal ladder compared to injured controls (Fig. 4E and movies S5 and S6). These functional improvements were abrogated by pharmacological ablation of serotonergic innervation (Fig. 4, D and E, and movies S7 and S8) (25). The finding that the stabilization of microtubules inhibits cell division established the usage of systemic microtubule stabilizing agents as a therapeutic standard for the treatment of cancer (29). Here, at low doses, systemic administration of the microtubule stabilizing agent epoB promoted functional recovery after SCI. Our approach differs from other experimental regenerative paradigms (1–7) by pharmacologically focusing on a single molecular target, the microtubules, yet overcoming multiple pathological obstacles. This is possible due to divergent effects of pharmacological microtubule stabilization on microtubule dynamics and, hence the polarization of neurons and meningeal fibroblasts. This dual effect and the efficacy after systemic and post-injury administration, give epothilones a promising translational perspective for treatment of the injured CNS. REFERENCES AND NOTES 1. N. Klapka, S. Hermanns, G. Straten, C. Masanneck, S. Duis, F. P. Hamers, D. Müller, W. Zuschratter, H. W. Müller, Suppression of fibrous scarring in spinal cord injury of rat promotes long-distance regeneration of corticospinal tract axons, rescue of primary motoneurons in somatosensory cortex and significant functional recovery. Eur. J. Neurosci. 22, 3047–3058 (2005). Medline doi:10.1111/j.14609568.2005.04495.x
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cord contusion injury model. We also thank C. Laskowski, C.H. Coles, A. Kania, M. Hübener, W. Jackson and G. Tavosanis for critically reading and discussing the manuscript. We are grateful for the support from the Human Spinal Cord Tissue Bank and the electron microscopy core at the Miami Project as well as Professor B. Kakulas, University of Western Australia and Royal Perth providing anonymized post mortem sections following human spinal cord injury. This work was supported by NIH, IRP, WfL and DFG. Harald Witte, Ali Ertürk, Farida Hellal, and Frank Bradke filed a patent on the use of microtubule stabilizing compounds for the treatment of lesions of CNS axons” (European Patent No. 1858498; European patent application EP 11 00 9155.0; US patent application 11/908,118). The authors declare no competing financial interests. SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.aaa2958/DC1 Materials and Methods Figs. S1 to S11 Tables S1 and S2 Movies S1 to S8 References (30–37) 14 November 2014; accepted 25 February 2015 Published online 12 March 2015 10.1126/science.aaa2958
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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 single i.p. injection of epoB, N = 4 rats/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/group. (F) Glycosaminoglycan amounts in spinal cord lesion extracts, N = 8 rats/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 (DAPI, nuclear staining) in the rat meninges at the lesion. Bottom panel, magnification of fibroblasts (arrowheads) in top panel. dpi, days post-injury. 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.
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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 = 3-4 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 = 9-16 neurons (from 3 experiments). (E) Neurite growth on Nogo-A. Black arrowhead, time of indicated treatment. N = 12-15 neurons (from 3 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 Tauexpression. Bottom panels, magnification of boxed areas in top panels. (I) EB3-mCherry fluorescence intensity in fibroblast periphery under indicated conditions, N = 20 cells/condition (from 4 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.
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Fig. 3. EpoB reduces dystrophy and promotes regeneration of injured spinal cord axons. (A) Electron microscope images of human SCI. Top panel, undamaged axon containing microtubules (black arrowheads). Bottom panel, retraction bulb (indicated by white arrowheads) without microtubules. Middle panel, 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 panel, 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/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. / sciencemag.org/content/early/recent / 12 March 2015 / Page 7 / 10.1126/science.aaa2958
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 = 7-8 rats/group. (C) Coronal sections of the lumbar spinal cord after contusion injury. Left panel, co-immunostaining 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 (7dpi), 11-12 rats (56 and 70 dpi)/group. (E) Number of footfalls on the horizontal ladder, N = 10-11 rats/group. dpi, days post-injury. 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.
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