OPENSUBJECT AREAS:
SPINAL CORD INJURY
NEURAL STEM CELLS

Received
24 September 2013
Accepted
17 April 2014
Published
15 May 2014

Correspondence andrequests for materials
should be addressed toY.T. (ytakei@pharm.
kyoto-u.ac.jp)

Ecto-domain phosphorylation promotes
functional recovery from spinal cord
injuryKenji Suehiro1, Yuka Nakamura2, Shuai Xu1, Youichi Uda1, Takafumi Matsumura1, Yoshiaki Yamaguchi3,
Hitoshi Okamura3, Toshihide Yamashita2 & Yoshinori Takei4
1

Department of Genomic Drug Discovery Science, Graduate School of Medicine, Osaka University, Osaka, Japan, 2Department of
Molecular Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan, 3Department of Systems Biology, andGraduate School of Medicine, Osaka University, Osaka, Japan, 4Department of Nanobio Drug Discovery Science, Graduate
School of Pharmaceutical Science, Kyoto University, Kyoto, Japan and Graduate School of Medicine, Osaka University, Osaka,
Japan.

Inhibition of Nogo-66 receptor (NgR) can promote recovery following spinal cord injury. The ecto-domainof NgR can be phosphorylated by protein kinase A (PKA), which blocks activation of the receptor. Here, we
found that infusion of PKA plus ATP into the damaged spinal cord can promote recovery of locomotor
function. While significant elongation of cortical-spinal axons was not detectable even in the rats showing
enhanced recovery, neuronal precursor cells were observed in the region where PKA plus ATP were directly
applied. NgR1 was expressed in neural stem/progenitor cells (NSPs) derived from the adult spinal cord. Bothan NgR1 antagonist NEP1-40 and ecto-domain phosphorylation of NgR1 promote neuronal cell productionof the NSPs, in vitro. Thus, inhibition of NgR1 in NSPs can promote neuronal cell production, which could
contribute to the enhanced recovery of locomotor function following infusion of PKA and ATP.

T

he adult mammalian central nervous system (CNS) cannot be repaired spontaneously after injury. The lackof regenerative capacity in the mammalian CNS is partly due to the myelin-associated proteins including
Nogo-A1, myelin-associated glycoprotein2,3 and oligodendrocyte myelin glycoprotein4,5. Nogo-66 receptor 1(NgR1) is a common receptor for the myelin-associated inhibitors of the regeneration (i.e., Nogo-A, myelinassociated glycoprotein and oligodendrocyte myelin glycoprotein)6, as well as chondroitin sulphate proteoglycans7. Inhibition of NgR activation with neutralising antibodies against Nogo-A8, a peptide mimicking
NgR1-binding region of Nogo-A (NEP1-40)9 or the ecto-domain part of NgR1 (NgR1(310) ecto-Fc)10, canpromote functional recovery of the spinal cord after traumatic injury. Thus, inhibition of NgR activation canpromote recovery from spinal cord injury (SCI).
NgR1, along with NgR2 and NgR3, belongs to the NgR family of receptors11. Inhibition of NgRs is known topromote neurite sprouting9,12,13 and synapse formation14–17. Triple knockout of NgR1, NgR2 and NgR3, but not
single knockout, increases both dendrite outgrowth and synapse number in the mouse hippocampus18. NgR1
participates in the postnatal maturation of the CNS19,20. These reports indicate that NgRs are involved in theregulation of synapse plasticity of the CNS neurons under the physiological conditions.
We previously reported that treatment of cells with protein kinase A (PKA) phosphorylated the ecto-domainsof NgR1, NgR2 and NgR3, and that casein kinase 2 phosphorylated NgR1 and NgR213. Phosphorylation of thesereceptors impedes the binding of the myelin-associated agonists.
Here, we found that administration of PKA and ATP promotes recovery from SCI. NgR1 was expressed inneural stem/progenitor cells (NSPs) derived from the adult spinal cord. Inhibition of NgR1 during in vitro
differentiation of the NSPs enhanced neuronal cell production. Consistent with these results, administrationof PKA and ATP phosphorylated NgR1 in the spinal cord in vivo and induced cells expressing markers forneuronal precursor cells, which is strictly inhibited without the treatment.

Results
Effects of PKA and ATP on the damage from SCI. As described schematically in Fig. 1a, we performed a dorsal
hemisection on the spinal cords of Wistar rats at the T9 vertebral level. Hind limb locomotor function of the
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Figure 1 | Treatment with PKA plus ATP diminishes damage from traumatic SCI. (a) The depth of injury and location of sections used in (c) areillustrated schematically. The dorsal corticospinal tract (dCST) and the dorsolateral corticospinal tract (dlCST) were severed. (b) The BBB scores ofvehicle-treated, PKA-treated, ATP-treated and PKA1ATP-treated SCI rats were assessed at the indicated days after SCI. The points on the graph indicate
the average BBB score from six independent rats, and the error bars indicate the standard deviation (S.D.) (*p , 0.05, **p , 0.01, ***p , 0.001 vs.
vehicle-treated rats, Student’s t-test). (c) The BDA-labelled dCST was visualised. Images are taken from transverse sections at either 5 mm caudal or
rostral to the lesion, as shown in (a). The bar indicates 25 mm. (d) The number of BDA-positive axons at T8 or T10 was normalised to the number of BDApositive axons at C1 (intact region of the spinal cord). The average and the S.D. from three independent animals are shown. No significant differences
between the vehicle-treated rats and the PKA/ATP-treated rats were observed (*p , 0.05, **p , 0.01, Student’s t-test).

injured rats, both with and without administration of PKA plus ATP,was completely blocked after the induction of SCI (Fig. 1b). From 5days after SCI, locomotor function gradually improved. By the Day
56 after SCI, the Basso-Beattie-Bresnahan (BBB) score of the injured
rats treated with PBS vehicle, PKA alone or ATP alone was less than10, demonstrating limited spontaneous recovery of locomotor
function (Fig. 1b). However, the BBB score of injured rats cotreated with PKA and ATP was significantly augmented relative to
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the controls at every experimental time point after the Day 10, and it
reached to 17 on the Day 56 (Fig. 1b). The hind limbs of the injured
rats co-treated with PKA and ATP supported the body weight of theanimals (Supplemental Movies). These observations demonstratethat co-treatment with PKA and ATP encourages functional
locomotor recovery following traumatic injury to the spinal cord.
To examine elongation of corticospinal axons beyond the lesion
epicentre, an anterograde neuronal tracer biotinylated dextran2

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amine (BDA) was injected into the sensorimotor cortex at the Day 42
after SCI. In the absence of SCI, injected BDA stained the corticospinal tract (CST) at the Day 56, as shown in Figure 1c (intact).
However, when a lesion was created on the spinal cord at the T9
vertebra, BDA-positive axons were decreased significantly in thesections prepared from 5 mm caudal to the lesion (Fig. 1c, SCI,
vehicle). The number of BDA-positive axons was also decreased in
the CST 5 mm rostral to the lesion, suggesting the degeneration ofaxons after SCI. Co-treatment with PKA and ATP failed to increase
the number of neuronal fibres stained with BDA, either rostral or
caudal to the lesion (Fig. 1c and d). These results indicate that both
SCI treated with vehicle and with PKA and ATP received comparable
damage on the spinal cord, and that the treatment with PKA andATP does not promote axonal outgrowth from the sensorimotor
cortex.
Effects of co-treatment with PKA and ATP on neural stem cells.
To investigate further the effects of the treatment with PKA and ATP,the spinal cord was dissected on the Day 7 of SCI, when the effects ofco-treatment with PKA and ATP began to become evident (Fig. 1b).
It was reported that NSPs migrate into the peri-injury site after injury
of the CNS21–24. While NSPs can differentiate into both neurons andglial cells in vitro, they preferentially produce glial cells in the periinjury site in vivo. However, we observed cells expressing markers forneuronal precursor cells, neurogenin 2 (Fig. 2c) and doublecortin(Fig. 2d), only in the tissues co-treated with PKA and ATP. The
proportion of cells expressing doublecortin and neurogenin 2 wereincreased approximately 20-fold and 10-fold, respectively, relative toinjured spinal cords exposed to PBS vehicle (Fig. 2b). The location of
the cells expressing markers for neuronal precursor cells was limitedin the tissues directly under the end-point of the tube from theinfusion pump, where PKA plus ATP were directly applied. These
results suggest that exposure to PKA plus ATP increases the numberof neuronal precursor cells, although the affected region is limited.
The number of cells expressing nestin, a common marker of NSPs,
was amplified in the dorsal funiculus after SCI, as was the number ofcells expressing Ki67, a marker of dividing cells (Fig. 2e and f). In
addition, the number of cells expressing Sox9, a transcription factor
expressed in ependymal cell-like and astrocyte-like NSPs, wasincreased in the dorsal funiculus (Fig. 2e and g). These results indicate that treatment with PKA plus ATP did not affect migration of
NSPs into the peri-injury site of SCI.
Western blotting analysis with an anti-NgR1 antibody detectedsimilar levels of NgR1 expression in vehicle-treated spinal cords vs.
PKA/ATP-treated spinal cords (Fig. 2h). This demonstrates that
overall NgR1 expression was not changed by concomitant exposure
to PKA and ATP. Meanwhile, Western blotting with an antibody
against phospho-serine281 NgR1 revealed a strong signal in PKA/
ATP-treated spinal cords. Thus, administration of PKA plus ATP
results in the in vivo phosphorylation of ecto-domains of membraneproteins, including NgR1 at serine281, which can inhibit agonistbinding to NgR113.
Expression of NgR1 in NSPs derived from the adult spinal cord.
To ask the relationship between ecto-domain phosphorylation andincreased number of neuronal progenitor cells, NSPs were preparedfrom the adult spinal cord by using a neurosphere culture method
and were then maintained as a monolayer culture. The majority ofthe obtained cells expressed Sox2, nestin and Sox9, but neither glial
fibrillary acidic protein (GFAP) nor oligodendrocyte transcription
factor 2 (Olig2) (Fig. 3a and b). Several types of cells, oligodendrocyte
precursors, astrocytes and ependymal cells, have been reported to be
NSPs in the spinal cord21,22,25,26. Sox2, as well as nestin, is a common
marker for those NSPs. GFAP is a marker for astrocytes. Olig2 isexpressed in oligodendrocyte lineage cells and some types ofneuronal precursor cells27–29. Therefore, expression profile of theobtained cells is similar to that of ependymal cells.
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RT-PCR indicated expression of NgR1 in the NSPs obtained fromthe adult spinal cord (Fig. 3c). Immuno-staining indicated that coexpression of Sox9 and NgR1. More than 90% of NgR1-positive cells
expressed Sox9, whereas only less than 5% of NgR1-positive cells
expressed GFAP (Fig. 3d and e). Thus, majority of cells expressing
NgR1 was Sox91GFAP2-cells. These results indicate that NgR1 isexpressed in ependymal cell-like NSPs derived from the adult spinal
cord.
Effects of ecto-domain phosphorylation on the neuronal cell production of adult NSPs. To ask the effects of PKA-mediated ectodomain phosphorylation on neuronal cell production, the NSPs weredifferentiated by withdrawal of EGF and FGF2 (i.e., no growth
factors for the first 2 days of culture, followed by the addition ofbrain-derived neurotrophic factor (BDNF) from the Day 3 onward).
After induction of differentiation, NgR1 expression was increased,
and treatment with PKA and ATP had no effects on the increase in
NgR expression (Fig. 4a). Furthermore, Nogo-A expression was also
increased after induction of differentiation (Fig. 4b). When the cells
were treated with PKA and ATP, transient increase in Nogo-A
expression was observed on the Day 4 of differentiation.
The percentage of GFAP-positive cells increased from about 5% toapproximately 30% after 5 days in culture under differentiation conditions (Fig. 4c–e). On the Day 10 of differentiation, the proportionof GFAP-positive cells increased to approximately 40%. Both PKA
plus ATP and NEP1-40 tend to decrease the proportion of GFAPpositive cells on the Day 10 of differentiation, although it was not
significant.
Proportion of Olig2-positive cells was not changed by 10 days
differentiation, and it was less than 5%. Nonetheless, treatment with
PKA plus ATP increased the proportion to approximately 50% at the
Day 5 of differentiation. NEP1-40 also increased the proportion of
Olig2-positive cells. No Olig2-positive cells were detected at the Day
10 of differentiation.
The proportion of beta III tubulin-positive neurons made up lessthan 1% of the total cell population even on the Day 5 of differentiation, and it was increased to approximately 50% on the Day 10.
Treatment with PKA plus ATP increased the proportion of beta III
tubulin-positive neurons to more than 75%. NEP1-40 also increased
the percentage of beta III tubulin-positive neurons.
Treatment with PKA plus ATP had no effects on the change of thetotal cell number in the course of differentiation (Fig. 4f). Taken
together, these results indicate that PKA-mediated ecto-domain
phosphorylation can increase production of neuronal cells from
NSPs derived from adult spinal cord, via transient increase in
Olig2-positive cells.
Ecto-domain phosphorylation of NgR1 is required for the increased
production of neurons. Next, we examined the contribution of NgR1
phosphorylation to the increased neuronal cell production. Wild type
NgR1 and the non-phosphorylatable S281A NgR1 mutant13 wereseparately overexpressed in the NSPs. Expression levels andtransfection efficiencies of wild type and the mutant NgR1 werecomparable (Fig. 5a and b). Treatment with PKA plus ATP
increased percentage of beta III tubulin-positive cells differentiatedfrom NSPs overexpressing wild type NgR1 (Fig. 5c and d), as theresults indicated in Figure 4. However, the treatment had no effects
on the percentage of beta III tubulin-positive cells differentiated from
NSPs overexpressing the mutant NgR1 (Fig. 5c and d). These results
indicate that phosphorylation of NgR1 is essential for promotion ofneuronal cell production by co-treatment with PKA and ATP.

Discussion
The adult CNS shows only poor recovery from traumatic injury, andinhibition of NgR activation has beneficial effects on the recov3

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Figure 2 | Extracellular treatment with PKA plus ATP increases the number of neuronal precursor cells in the peri-injury site. Rats with SCI weretreated with either vehicle or PKA plus ATP for 1 week. (a) Location of sections used in (c, d, f, g) is illustrated schematically. The sections were preparedfrom the spinal cord directly under the end-point of the tube from the infusion pump. (b–g) Transverse sections prepared from the spinal cord directly
under the end-point of the tube from the infusion pump were used for immunofluorescence with indicated antibodies (c, d, f, g). Representative images ofthe dorsal funiculus were shown. The bars indicate 25 mm. In (b) and (e), signals from indicated antibodies were quantified by using ImageJ 1.48k(available at http://rsb.info.nih.gov/ij/). The average of the three independent animals was plotted and error bars indicate the S.D. (*p , 0.05, Student’s ttest). (h) Western blotting analysis of proteins expressed in the spinal nerves. Cell extracts were prepared from the spinal cord at 1 week after SCI, and40 mg of protein was analysed for each condition. Experiment was repeated for 3 times and intensities of signals were quantifyd by using Image J 1.48k.
The average was plotted and error bars indicate the S.D. (*p , 0.05, Student’s t-test).
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Figure 3 | NSPs derived from the adult spinal cord express NgR1. (a and d) After three rounds of neurosphere formation, NSPs were maintained as amonolayer culture. Cells were seeded onto plastic coverslips coated with polyornithine and laminin. After 2 days in culture, cells were fixed and used for
immunofluorescence analysis with the indicated antibodies. The bars indicate 50 mm. (b) Cells expressing the indicated proteins were manually counted.
DAPI-positive cells were also counted to obtain the total cell number. Each experiment was repeated four times, and more than 200 cells per sample were
examined. Each bar and error bar represents the average cell number and the S.D., respectively. (c) Total RNA was prepared from cultured NSPs and usedas the template for RT-PCR. Complementary DNA prepared from the adult rat cortex was used as a positive control. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was employed as an internal standard. (e) Sox9-expressing cells and GFAP-positive cells were counted in NgR1-positive cells,
respectively. They were indicated as percentage against the number of examined NgR1-positive cells. The results were obtained from 3 independent clones
of NSPs and their average was shown. Experiments were repeated 3 times. Error bars indicate the S.D.

ery8,9,10. Effects of NgR activation are extensively studied with differentiated neurons30. However, NgR expression appears not to be limited in the differentiated neurons. Wang, F. and Zhu, Y. reported
expression of NgR1 in nestin-positive cells derived from the spinal
cord of postnatal day 1 rats31. They indicated that an agonist for
NgR1 Nogo-p4 can inhibit neurite outgrowth of neuronal cells differentiated from the nestin-positive cells. Wang, B. et al. reported
that addition of another NgR1 agonist Nogo66 decreases in neuronalcell production of nestin-positive cells derived from the telencepharon of neonatal rats32. Since both papers employed nestin as
the only marker for NSPs, types of NSPs expressing NgR1 were
unknown. Moreover, since both papers used cells derived from theimmature CNS, it was unknown whether NgR1 expression is maintained in NSPs in the adult CNS or not.
This paper indicated expression of NgR1 in ependymal cell-like
NSPs derived from the mature spinal cords. Moreover, Nogo-A
expression was detected in the differentiating NSPs, and both
NEP1-40 and ecto-domain phosphorylation of NgR1 promoted
neuronal cell production from the adult NSPs without ectopic addition of NgR1 agonists. These results indicated that NgR1 has a role inthe cell fate decision of NSPs, at least under the in vitro conditions.
Thus, signalling through NgR1 appears to be able to affect the cells
involved in the neural cell lineage from very early stage of neuronaldifferentiation to terminally differentiated neurons.
During the in vitro differentiation of the adult NSPs, transient
increase in the population of Olig2-positive cells was observed on
the Day 5 of differentiation of both cells treated with PKA plus ATP
and with NEP1-40. Nogo-A expression is also transiently increased
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on the differentiation Day 4 of cells treated with PKA plus ATP. Sinceboth Olig2 and Nogo-A are expressed in the oligodendrocyte lineage
cells, it suggests a possibility that inhibition of NgR1 activation could
induce differentiation of NSPs into oligodendrocytes, but the cells
could not survive in the in vitro conditions. Since oligodendrocytes
can promote survival of neuronal cells33,34, the transient increase inoligodendrocyte population could contribute to the augmented proportion of neuronal cells observed in the differentiation Day 10. Our
results indicated that inhibition of NgR1 in adult NSCs can promote
neuronal cell production. However, it is still uncertain whether theincreased neuronal cell production is due to promotion of neuronalcell differentiation or survival.
While administration of PKA plus ATP had no effects on the
migration of NSPs following SCI, the treatment phosphorylated
NgR1 and increased the number of neuronal precursor cells, whichis consistent with the in vitro experiments. Although ecto-domain
phosphorylation of NgR1 was essential for the augmentation of neuronal cell production in vitro, our results do not exclude the possible
contribution of ecto-domain phosphorylation of other proteins, invivo, in addition to the ecto-domain phosphorylation of NgR1. Theseobservations suggest that ecto-domain phosphorylation by PKA canallow production of neuronal cells in the adult spinal cord, which isstrictly inhibited in vivo.
Neuronal progenitor cells in the peri-injury site can be beneficial
for regeneration of the injured spinal cord, as indicated by cell transplantation studies35–37. While some mechanisms have been proposed
for the enhanced recovery, including production of new neurons38,39
and secretion of growth factors and neurotrophic factors40–43, the5www.nature.com/scientificreports

Figure 4 | Inhibition of NgR1 enhances neuronal cell production. Differentiation of NSPs derived from the adult spinal cord was induced by depletion of
EGF and FGF-2 in the presence of the indicated reagents. (a and b) Total RNA was prepared from the differentiating NSPs on indicated differentiation
day, and its cDNA was used as the template for quantitative RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as an internal
standard. Average of three independent experiments was shown and error bars indicate the S.D. (c and d) On the Day 5 (c) and the Day 10 (d) ofdifferentiation, cells were fixed and used for immunofluorescence analysis with primary antibodies against the indicated proteins. The bars indicate
25 mm. In (e), cells expressing the indicated proteins were manually counted. DAPI-positive cells were also counted to obtain the total cell number. Eachexperiment was repeated 3 times with 3 independent clones of NSPs, and more than 200 cells per sample were examined. (*p , 0.05 vs. vehicle-treated
NSPs, Student’s t-test). The bar indicates 25 mm. (f) On the Day 0, 5 and 10 of differentiation, cells were harvested and cell number of each samples werecounted, as described in the Methods section. The cell numbers were indicated as percentage against the number of cells treated with vehicle at the Day 0.
Each experiment was repeated 3 times with 3 independent clones of NSPs.
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Figure 5 | NgR1-phosphorylation is essential for enhancement of neuronal cell production induced by treatment with PKA and ATP. Plasmids
encoding either wild type NgR1 or S281A mutant NgR1 were transfected into NSPs before the induction of differentiation. Expression of wild type andmutant NgR1 were examined by Western blotting (a) and immunofluorescence analysis (b). In (a), 40 mg of each protein sample was analysed by SDSPAGE. Actin was employed as an internal standard. The bars indicate 25 mm. (c) At the Day 10 of differentiation with or without PKA plus ATP, cells were
fixed and stained for GFAP, beta III tubulin and DAPI. The bars indicate 25 mm. In (d), cells transfected with either wild type or the S281A mutant NgR1
were treated as in (c) and manually counted. Each experiment was repeated four times, and each bar and error bar represents the average proportion and
the S.D., respectively. Differences between the proportions of beta III tubulin-positive cells in the cells transected with mutant NgR1 were not statisticallysignificant. (*p , 0.05, Student’s t-test).

detail has not been elucidated yet. It is tempting to assume that ectodomain phosphorylation of NgR1 expressed in NSPs promotes neuronal cell production, contributing to the augmentation of recovery
from SCI. If this is the case, signalling through NgR1 activated after
traumatic injury could inhibit regeneration of adult CNS not only
through inhibition of synapse plasticity of differentiated neurons but
also through inhibition of neuronal cell production of NSPs migrated
into the peri-injury site. However, neuronal progenitor cells wereobserved only in the limited area even after application of PKA plus
ATP. Further study is required for assessing the contribution of theneuronal progenitor cells induced by application of PKA plus ATP to
the augmentation of recovery from the SCI.
In summary, this study demonstrated that NgR1 is expressed in
NSPs derived from adult spinal cord, where its activation can inhibit
neuronal cell production at least in vitro. Administration of PKA plus
ATP phosphorylated NgR1 in vivo, and increased the number ofneuronal precursor cells in the limited region of the spinal cord.
Furthermore, this treatment promoted functional recovery from
SCI. Thus, our results indicated that ecto-domain phosphorylation
by PKA could be beneficial for the recovery from SCI.

purchased from Sigma. Anti-phospho NgR1 (serine281) primary rabbit antibody was
prepared by Cambridge Research Biochemicals Limited (Cambridge, UK).

Methods

Locomotor testing. Behavioural analyses were performed to assess open-field
locomotor function based on the BBB scale44. The BBB open-field test was scored ondays 1, 3, 5, 7, 10, 14, 21, 28, 35, 42, 49 and 56 after SCI to evaluate hind limb motor
function with regard to stepping and coordination. Each rat was observed for 2 minduring each session.

Antibodies. Anti-Trk A and anti-Myc tag antibodies were purchased from Cell
Signaling Inc. Anti-nestin, anti-Sox2 and anti-GFAP were purchased from Merck.
Anti-Ki67, anti-Sox9 and anti-doublecortin were purchased from abcam. Antineurogenin 2 was purchased from R&D. Anti-NgR1 and anti-beta III tubulin were

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Surgical procedures. All surgical procedures and postoperative care were reviewed
and approved by the Animal Care and Use Committee of Kyoto University (Kyoto,
Japan), and were carried out in accordance with the approved guidelines. Female
Wistar rats, 8 weeks of age, were anesthetised by inhalation of 2.0% isoflurane. The
skin over the thoracic vertebrae was shaved and cleaned with an iodine tincture. The
skin was incised, and the connective and muscle tissues were dissected to expose
the lower thoracic spinal cord. A laminectomy was then performed between the 8th
and 10th thoracic spinal vertebrae. The dura was cut with a needle, and a dorsolateralhemisection was made with a No. 11 surgical blade at the 9th thoracic spinal vertebral
level (T9) to a depth of 1.8 mm. To ensure that the lesion was laterally complete, thesurgical blade was passed through the dorsal part of the spinal cord several times.
PKA (New England Biolab) and ATP (Sigma) were diluted to final concentrationsof 100 mM and 2.5 U/ml, respectively, in PBS containing 2 mM MgCl2 and 100 mM
trinitrophenyl (TNP)-ATP (Sigma). TNP-ATP is an inhibitor of P2X receptors, ofwhich activation causes pain. PKA alone, ATP alone or a mixture of PKA plus ATP
was applied to the injured region by intrathecal administration via a subcutaneously
implanted iPrecio micro infusion pump (Primetech, Ibaraki, Japan). Solutions wereadministered at a flow rate of 1 ml/h for 14 days. The solution in the pump waschanged every 2 days. The pump was implanted subcutaneously at the site of thelesion, and a tube originating from the pump was inserted into the subarachnoid
cavity for local administration. The pump was initiated immediately after surgery.

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Immunofluorescence analysis. Anesthetised rats were transcardially perfused with
PBS, followed by 4% paraformaldehyde in PBS. The spinal cords were removed,
postfixed in the same fixative, and immersed overnight in PBS containing 30%
sucrose. The spinal cords were then embedded in Tissue-Tek OCT and kept frozen at
280uC until use. Serial transverse sections (30 mm thick) were cut on a cryostat andmounted on Matsunami adhesive silane-coated Superfrost/Plus slides (Matsunami,
Osaka, Japan). The sections were washed three times with PBS and blocked in PBS
supplemented with 5% bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 h atroom temperature. Immunocytochemistry was performed on the mounted sections
with the indicated antibodies. Sections were incubated with primary antibodies
overnight at 4uC, followed by the appropriate secondary antibodies for 1 h at 37uC.
For immunostaining of NSPs, cells were washed once with PBS, fixed with 4%paraformaldehyde and 0.5% Triton X100 in PBS overnight at 4uC, and treated as
described above.
BDA tracing. The rats were stabilized in a stereotaxic frame (Narishige) after deep
anaesthesia with isoflurane. The scalp was retracted and a circular craniotomy 4 mm
in diameter was made with a drill on the right side with the centre at 2 mm anteroposterior, 2 mm lateral to the bregma. An anterograde tracer BDA (10% in PBS) was
injected at a depth of 1.5 mm from the cortical surface using a 5 ml micro-syringe
tipped with a pulled glass micropipette. After injection, the skin overlying the skull
was sutured. Two weeks after BDA injection, the spinal nerves were dissected andsections were prepared as described in ‘‘Immunofluorescence’’.
For visualisation of BDA, the sections were incubated at room temperature in 5%
BSA and 0.1% Triton X-100 in PBS for 1 hour, and then with Alexa Fluor 488conjugated streptavidin for 2 hours. To quantify the BDA-labelled CST axons in the
T8 and T10 vertebra, pixels in the labelled fibres of the dorsal CST were counted foreach animal, and this value was divided by the number of pixels in the labelled dorsal
CST axons located C1.
Cell culture. For the preparation of NSPs from the adult spinal cord, a cell suspension
was prepared from the spinal cord with Nerve Cell Dissociation Solution (Sumitomo
Bakelite Co., Ltd., Tokyo, Japan). The cells were seeded in a soft collagen gel (1 3 104
cells in 35 mm culture dish) and cultured for 3 weeks by using the NeuroCult NeuralColony-Forming Cell Assay kit (Stem Cell Technologies). Independent neurospheresof .2 mm in diameter in collagen gel were harvested separately and disrupted withcollagenase and pipetting. Dissociated cells from each neurosheres were cultured
separately in serum-free medium containing fibroblast growth factor (FGF)-2 andepidermal growth factor (EGF) for secondary neurosphere formation. After 10 days,
floating neurospheres were harvested and treated with Accutase Cell Detachment
Solution to prepare a single cell suspension. This step was repeated once. The
dissociated cells derived from the neurospheres were cultured in N2B27 medium45
containing FGF-2 and EGF on dishes coated with polyornithine and laminin. The
medium was changed every day.
For differentiation of NSPs, the cells were seeded on plastic cover slip coated with
polyornithine and laminin in a 24-well plate (1 3 105 cells/well), with N2B27 media
containing FGF-2 and EGF. Medium was changed to N2B27 without FGF-2 or EGF
in the next day (day 0 of differentiation) and cells were cultured for 10 days. From theDay 3 of differentiation onward, BDNF (10 ng/ml) was added to the N2B27 medium.
For cell counting, Accutase Cell Detachment Solution was used for detachment ofcells. The cells were washed with Neurobasal medium and resuspended into 500 ml ofneurobasal medium. The cell concentration was estimated with a cell counter Scepter
2.0 (Millipore). For transfection of NgR1 plasmids13 into NSPs, NeuroMag (Oz
Biosciences) was used according to the manufacturer’s recommendations.
Western blotting analysis. One week after SCI, the rats were anesthetised byinhalation of 2.0% isoflurane. The lower thoracic spinal cord was exposed bylaminectomy. A portion of the spinal cord between 10 mm rostral and caudal to thelesion epicentre was dissected. The dissected spinal cord was washed with PBS, diced
with a surgical blade, and homogenised in a buffer containing 50 mM Tris-HCl(pH 7.8), 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 2 mM DTT, 23 PhosSTOP
phosphatase inhibitor mix (Roche) and 13 Complete Protease Inhibitor Cocktail(Roche). After centrifugation at 15,000 3 g for 15 min at 4uC, the supernatant wascollected as a cell extract. Proteins (40 mg) were analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride
membrane. The membrane was incubated with blocking buffer containing 5% skim
milk and 0.5% Triton X-100 in PBS for 1 h at room temperature, followed by theindicated primary antibody in blocking buffer overnight at 4uC and a peroxidaselabelled secondary antibody for 1 h at room temperature. Enhanced
chemiluminescence-Plus reagent (GE Healthcare) was used to visualise the boundantibodies.
NSPs shown in Fig. 5 were transfected with either wild type NgR1/pDisplay or
S281A mutant NgR1/pDisplay13 and cultured overnight in N2B27 medium containing FGF-2 and EGF. Differentiation was induced by depletion of the growth
factors. Two days after the induction of differentiation, protein fractions were prepared and used for Western blotting as described above.
RT-PCR. Total RNA was prepared with Trizol reagent (Invitrogen) from NSPs
cultured as a monolayer and used for cDNA synthesis with Superscript II(Invitrogen). Complementary DNA was prepared from 250 ng total RNA and used asa template for RT-PCR with Taq polymerase (Takara Bio Inc., Shiga, Japan). The
following sequences were employed for the amplification of NgR1: 59-

SCIENTIFIC REPORTS | 4 : 4972 | DOI: 10.1038/srep04972

CTGGAGGGTAGCAACACCATGC-39 and 39-AGTGCAGCCACAGGATAGTGAG-59. The PCR products were separated by agarose gel electrophoresis,
stained with ethidium bromide, and visualised with a UV transilluminator. For
quantitative PCR, the following sequences were employed for the amplification of
NgR1: 59-CAGCGAATCTTCCTGCATGGC-39 and 39-GTGAAGGCAGCAGCATCGATCC-59, and for the amplification of Nogo-A: 59-GTGTTCAGCATTGTCAGTGTAACG-39 and 39-GTGGCCTTCATCTGATTTCTGG-59.
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Acknowledgments
This work was supported by the Grant-In Aid for Scientific Research (C) (JSPS KAKENHI
number 23500392) from the Japan Society for the Promotion of Science, and grants fromthe Seamless Technology Transfer Program through Target-Driven R&D (numbers
AS221Z01416G and AS232Z00373F) from the Japan Science and Technology Agency. This
work was partially supported by the Shimizu Foundation for Immunology and
Neuroscience Grant for 2013.

Author contributions
K.S., Y.N., S.X., Y.U., M.T. and Y.T. performed the experiments. Y.Y., H.O., T.Y. and Y.T.
conceived the experiments. Y.T. wrote the manuscript. All authors discussed the results andcommented on the manuscript.

Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Suehiro, K. et al. Ecto-domain phosphorylation promotes
functional recovery from spinal cord injury. Sci. Rep. 4, 4972; DOI:10.1038/srep04972(2014).
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