Reports

tubule stabilization induces such

Systemic administration of epothilone B divergent effects. Moreover, Tax-
ol cannot be used for clinical
promotes axon regeneration after spinal CNS intervention because it does
not cross the blood-brain barrier
cord injury (10).
We aimed to target microtu-
bule stabilization in the injured
Jörg Ruschel,1 Farida Hellal,1*† Kevin C. Flynn,1*‡ Sebastian CNS in a clinically feasible way
Dupraz, * David A. Elliott, Andrea Tedeschi, Margaret Bates,
1 1 1 2 and to decipher its distinct cellu-
lar actions. We used epothilones,
Christopher Sliwinski, Gary Brook, Kristina Dobrindt,
3 4,5 6
a class of FDA approved blood-
Michael Peitz,6 Oliver Brüstle,6 Michael D. Norenberg,7 Armin brain barrier permeable micro-
Blesch,3 Norbert Weidner,3 Mary Bartlett Bunge,2 John L. Bixby,2 tubule stabilizing drugs (11).
Frank Bradke1§ Mass spectrometry confirmed
1
that after intraperitoneal (i.p.)
Axonal Growth and Regeneration, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn,
2
Germany. The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, 1095 Northwest 14th injection in adult rats, epoB was
Terrace, Miami, FL33136, USA. 3Spinal Cord Injury Center, Heidelberg University Hospital, Schlierbacher Landstr. 200A, rapidly absorbed into the CNS
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.
7
Departments of Pathology, Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, i.p. injected with 0.75 mg/kg
USA.. body weight (BW) epoB at day 1
*These authors contributed equally to this work. and 15 post-injury showed in-
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
§Corresponding author. E-mail: frank.bradke@dzne.de stability (12). The dosage used
presented no obvious adverse
After central nervous system (CNS) injury, inhibitory factors in the lesion
side effects, such as reduced an-
scar 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 fi-
delayed 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 ro-
rodent 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, includ-
regeneration and improved motor function after SCI. With recent clinical
ing chondroitin sulfate proteo-
approval, 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 intrathe-
led 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 de-
ing 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 ani-
axon growth (8, 9). However, it remains elusive how micro- mals (fig. S1) indicating that neuroprotective glial sealing of

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the injury site (14) was not affected by the treatment.
Scar reduction upon epoB treatment resulted neither
from decreased cell proliferation nor from increased apop-
tosis (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 microtu-
bules (Fig. 1K) distributed throughout the cell (Fig. 1J). Simi-
larly, systemic administration of epoB after dorsal
hemisection prevented the polarization of meningeal fibro-
blasts 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 fibro-
blast 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 abun-
dant at the spinal cord lesion site (2–4, 17). In neurons ex-
pressing fluorescently-tagged microtubule plus-end binding
protein 3 (EB3-mCherry), which labels polymerizing micro-
tubules (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 Nogo-
A (Fig. 2E and movie S3). In accordance, low doses of the
microtubule destabilizing drug nocodazole abolished micro-
tubule protrusion in neurites (Fig. 2, D and F) and abrogat-
ed 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 mi-
crotubule polymerization toward the cell edges (Fig. 2G)
contrasting with the microtubule dynamics found in neu-
rons. 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 ectopi-
cally 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 ef-
fect 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 polymeriza-
tion into the distal neurite when exposed to epoB (fig. S9).
Injured axons in the rodent and human CNS form dys-
trophic retraction bulbs (Fig. 3, A to D, and table S2), a con-
sequence of microtubule depolymerization and
disorganization (Fig. 3, A and B) (22, 23). Because epoB in-
duced microtubule polymerization and axon growth in cul-
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tured neurons, we assessed its ability to promote axon re-
generation 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), re-
duced axonal dieback and increased regenerative growth
(Fig. 3, C and E). Moreover, in adult mice, systemic and
post-injury treatment with epoB, promoted axon regenera-
tion after complete dorsal column transection (Fig. 3, F and
G).
We then tested whether the treatment also promoted ax-
on 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 func-
tion 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 ad-
ministration (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). Moreo-
ver, epoB treatment increased stride length and gait regular-
ity, and reduced external rotation of the hind paws (fig. S11)
indicating improved walking balance and coordination. Ac-
cordingly, 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 func-
tional 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 inhib-
its cell division established the usage of systemic microtu-
bule stabilizing agents as a therapeutic standard for the
treatment of cancer (29). Here, at low doses, systemic ad-
ministration of the microtubule stabilizing agent epoB pro-
moted 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 obsta-
cles. This is possible due to divergent effects of pharmaco-
logical microtubule stabilization on microtubule dynamics
and, hence the polarization of neurons and meningeal fi-
broblasts. This dual effect and the efficacy after systemic
and post-injury administration, give epothilones a promis-
ing translational perspective for treatment of the injured
CNS.
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ACKNOWLEDGMENTS
Materials and methods and other supporting materials are available in the online
version of the paper. We thank L. Meyn, K. Weisheit, D. Fleischer and N. Thielen
for technical assistance and animal care and Dr. C. Hill for teaching the spinal
 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 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/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.

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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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