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1877Research Article
IntroductionThe olfactory system is the only region of the
mammaliancentral nervous system (CNS) in which the olfactory
receptorneurons are unique in retaining their ability to
regeneratethroughout life, both in response to injury and as part
of normalturnover (Graziadei and Monti Graziadei, 1980; Doucette
etal., 1983). Olfactory ensheathing cells (OECs) are the glialcells
that derive from the olfactory placode and envelopolfactory axons
in the course of migration from the olfactoryepithelium to the bulb
(Ramon-Cueto and Avila, 1998). Thecells are different from the
typical glia in terms of existing inboth the peripheral nervous
system and CNS and sharing thephenotypes of both astrocytes and
Schwann cells (Ramon-Cueto and Avila, 1998; Gudino-Cabrera and
Nieto-Sampedro,2000). OECs have been reported to pioneer the
olfactory nervepathway and provide a conducive substrate for
growingprimary olfactory axons, although the specific
mechanismsremain undescribed (Tennent and Chuah, 1996).
The repair of CNS damage continues to be a majorchallenge, in
particular that of spinal cord injury. Owing to theaxonal
growth-promoting properties, OEC transplantation hasemerged as a
very promising experimental therapy to treataxonal injuries
(Franklin and Barnett, 2000; Raisman, 2001;Ramon-Cueto and
Santos-Benito, 2001). Transplanted OECshave been shown to migrate
with regenerating axons throughan unfavorable CNS environment (Li
et al., 2004), and tomingle well with astrocytes in adult brain (Li
et al., 1998;Lakatos et al., 2000; Richter et al., 2005).
Therefore, themigrating ability of OECs in the CNS was thought to
be
essential for neural regeneration and re-ensheathment
afterspinal cord injury. However, whether transplanted OECs
canmigrate over long distances in the CNS is still
controversial(Smale et al., 1996; Gudino-Cabrera et al., 2000;
Takami et al.,2002; Collazos-Castro et al., 2005; Deng et al.,
2006).
The regeneration of CNS axons following injury isdrastically
restricted by the presence of inhibitory moleculeswithin myelin
(Schwab and Bartholdi, 1996; Ng et al., 1996).Three inhibitors that
have been identified are Nogo, myelin-associated glycoprotein (MAG)
and oligodendrocyte myelinglycoprotein (OMgp) (He and Koprivica,
2004). Nogo is amember of the reticulon family and occurs in three
forms,Nogo-A, Nogo-B and Nogo-C, which are generated fromalternate
splicing (GrandPre et al., 2000). All three isoforms ofNogo share a
66-amino-acid-residue luminal/extracellulardomain (Nogo-66), which
inhibits axonal extension andfibroblast spreading (Brittis and
Flanagan, 2001; Fournieret al., 2001). The molecular cloning of
Nogo (Chen et al.,2000; GrandPre et al., 2000; Prinjha et al.,
2000) led tothe identification of a neuronal surface
glycosylphosphatidylinositol (GPI)-linked receptor that binds with
highaffinity to Nogo-66, termed the Nogo-66 receptor (NgR)(Fournier
et al., 2001). The low affinity neurotrophin receptor,p75, has been
identified as a coreceptor for NgR, andtransduces a signal upon
interaction with myelin ligands (Wanget al., 2002; Wong et al.,
2002). The downstream signalingpathway involves the activation of
small GTPases of the Rhofamily, which in turn regulate cytoskeletal
protein assemblyand mediate inhibitory effects on neurite growth
(Yamashita et
The migration of olfactory ensheathing cells (OECs) isessential
for pioneering the olfactory nerve pathwayduring development and
for promoting axonalregeneration when implanted into the injured
centralnervous system (CNS). In the present study,
recombinantNogo-66 enhanced the adhesion of OECs and inhibitedtheir
migration. Using immunocytochemistry and westernblot, we showed
that the Nogo-66 receptor (NgR) wasexpressed on OECs. When NgR was
released from the cellsurface with phosphatidylinositol-specific
phospholipase Cor neutralized by NgR antibody, the effect of
Nogo-66 onOEC adhesion and migration was markedly
attenuated.Nogo-66 was found to promote the formation of
focaladhesion in OECs and inhibited their membrane
protrusion through the activation of RhoA. Furthermore,the
co-culture migration assay demonstrated that OECmotility was
significantly restricted by Nogo-A expressedon Cos7 cell membranes
or oligodendrocytes. Moreover,treatment with anti-NgR antibody
facilitated migration ofimplanted OECs in a spinal cord hemisection
injury model.Taken together, we demonstrate, for the first time,
thatNogo, a myelin-associated inhibitor of axon regeneration inthe
CNS, enhances the adhesion and inhibits the migrationof OECs via
NgR regulation of RhoA.
Key words: Nogo, Olfactory ensheathing cells, Migration,
Adhesion,RhoA, Spinal cord injury
Summary
Nogo enhances the adhesion of olfactory ensheathingcells and
inhibits their migrationZhida Su*, Li Cao*, Yanling Zhu, Xiujie
Liu, Zhihui Huang, Aijun Huang and Cheng He‡
Department of Neurobiology, Second Military Medical University,
Shanghai 200433, China*These authors contributed equally to this
work‡Author for correspondence (e-mail: [email protected])
Accepted 28 March 2007Journal of Cell Science 120, 1877-1887
Published by The Company of Biologists
2007doi:10.1242/jcs.03448
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al., 2002; Yiu and He, 2003). The mRNA for NgR has beenfound in
abundance in OECs (Woodhall et al., 2003), and theNgR coreceptor
p75 is well known as a marker protein forOECs. However, little is
known about the function of thereceptors in OECs.
Although the neuronal growth inhibition activity of Nogohas been
well documented, other important features of Nogoare only beginning
to be understood. Nogo-A has beenreported to be an important
determinant of the developmentof experimental autoimmune
encephalomyelitis (Karnezis etal., 2004), as well as an essential
player in modulating axon-glial junction architecture and possibly
K+-channellocalization during development (Nie et al., 2003).
Nogo-B,like other members of the reticulon family, is involved
inmodulating BACE1 activity and amyloid-� peptide generation(He et
al., 2004), and might function as a pro-apoptotic protein
(Tagami et al., 2000). Moreover, Nogo-B has beenreported as a
regulator of vascular remodeling(Acevedo et al., 2004). In the
present study, wedemonstrate that Nogo enhances the adhesion
andinhibits the migration of OECs via RhoAactivation by NgR.
ResultsNogo-66 inhibits the migration of OECsTo examine whether
Nogo-66 influences the OECmigration, OEC motility was detected
usingBoyden chamber migration assays. Comparedwith laminin alone,
the fusion proteinsGST–Nogo-66 and His–Nogo-66, but not GST orHis,
significantly inhibit the migration of OECsplated on the upper side
of the membrane (Fig. 1a-e). As shown in Fig. 1f, quantitative
analysisrevealed that there was significantly less
OECtransmigration through transwell membranes pre-coated with
GST–Nogo-66 or His–Nogo-66 thanthrough those pre-coated with
laminin, GST or Hisalone.
Nogo-66 enhances the adhesion of OECsTo determine the effect of
Nogo-66 on OEC
adhesion, a protein-spot assay was used. We found that OECshad a
significantly greater affinity for attaching to spotsharbouring
fusion proteins (GST–Nogo-66 or His–Nogo-66)than those containing
GST or His alone (Fig. 2A).Quantification of the results showed
that the numbers of OECsattaching to GST–Nogo-66 and His–Nogo-66
spots aftervarious time points were different (Fig. 2B). The
enhancementof adhesion by either GST–Nogo-66 or His–Nogo-66
wastime-dependent within the first 6 hours after plating.
Inaddition, the enhancement of OEC adhesion was dependent onthe
concentration of GST–Nogo-66 or His–Nogo-66 in thespot. As shown in
Fig. 2C, the number of cells attached to thespotted GST–Nogo-66 or
His–Nogo-66, but not to the spottedGST or His, increased with the
concentration of proteins.Taken together, these results suggest
that Nogo-66 has apromoting effect on the adhesion of OECs.
Journal of Cell Science 120 (11)
Fig. 1. Nogo-66 inhibits the migration of OECs. (a-e)
Photomicrograph ofcultured OECs that have transmigrated through
transwell membranes coatedeither with laminin (e, control) or with
various proteins (100 �g/ml each, dilutedin laminin): GST (a),
GST–Nogo-66 (b), His (c) and His–Nogo-66 (d).(f) Quantitative
assessment of cells transmigrated through the transwellmembranes.
Data are presented as mean ± s.d./visual field. **P
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1879Nogo regulates OEC migration
The expression of NgR on OECsThe expression of NgR in OECs was
examined by usingimmunocytochemical staining and western blotting.
OECswere double-labeled with fluorescence-conjugated
antibodiesagainst NgR and S-100, a marker of OECs. As shown in
Fig.3A, OECs exhibited positive immunostaining for NgR
(a-d),whereas Schwann cells (SCs) showed no immunoreactivity forNgR
(e). To further confirm the expression of NgR on OECs,the lysates
of SCs, OECs and PC12 cells were checked bywestern blotting with
anti-NgR. This antibody recognized aprotein that was consistent
with the expected molecular weightfor the NgR proteins expressed by
OECs and PC12 cells (as apositive control) but not by SCs (as a
negative control) (Fig.3B).
NgR is a protein anchored to the membrane via a GPIlinkage that
can be released by PI-PLC (Fournier et al., 2001).As shown in Fig.
3C, the immunostaining of NgR was greatlyreduced after treatment of
OECs with PI-PLC. The results werefurther confirmed by western
blotting. After treatment of cellswith PI-PLC, NgR was released
from a membrane-boundfraction to a supernatant fraction.
Correspondingly, the residualamount of NgR remaining on the surface
of OECs wassignificantly decreased. Without PI-PLC
treatment,immunoreactivity for NgR was undetectable in the
supernatantfraction and had no change in the membrane fraction
(Fig. 3D).
NgR mediates the effect of Nogo-66 on the adhesionand migration
of OECsAfter confirming that endogenous NgR was expressed onOECs,
we next tested if Nogo-66 was bringing about its effecton OECs
through the NgR. Firstly, we investigated whethertreatment of
PI-PLC could attenuate the effect of Nogo-66mediating the
enhancement of cell adhesion and inhibition ofcell migration. As
shown in Fig. 4, the effect of Nogo-66 onthe migration (Fig. 4A)
and adhesion (Fig. 4B) of OECs was
greatly attenuated by PI-PLC treatment in a dose-dependentmanner
(Fig. 4C). Secondly, pre-incubation of OECs with anti-NgR antibody
which had been reported to be a functionblocking antibody
(Domeniconi et al., 2002), but not anirrelevant IgG, depressed the
ability of Nogo-66 to inhibit themotility (Fig. 4A) and enhance the
adhesion of OECs (Fig. 4B).Importantly, the characteristic of
migration (Fig. 4A) andadhesion (Fig. 4B) of SCs in which
endogenous NgR isundetectable was not changed by GST–Nogo-66 or
His–Nogo-66. These results strongly suggested that the enhancement
ofOECs substratum adherence and inhibition of OECs
migrationmediated by Nogo-66 were dependent on the NgR expressedon
the plasma membrane of these cells.
RhoA is involved in the regulation of Nogo-66 onadhesion and
migration of OECsRecent evidence suggests that activation of RhoA
is crucial formediating the inhibitory effect of Nogo on neurite
growth (Heand Koprivica, 2004). In addition, the Rho family
GTPasesparticipates in regulation of the actin cytoskeleton and
variouscell adhesion events (Van Aelst and D’Souza-Schorey,
1997;Hall, 1998). Therefore, we next tested whether RhoA
wasinvolved in the intracellular signaling of Nogo-66 on OECs.
Inthe adhesion assay, OECs were pretreated with 10 �M Y-27632, a
selective inhibitor of RhoA-associated kinase. Asshown in Fig.
5A,B, pretreating OECs with Y-27632 markedlyreduced the number of
adherent cells on GST–Nogo-66 andHis–Nogo-66. In the migration
assay, inhibition of OECmotility by Nogo-66 was greatly attenuated
after pre-treatmentof cells with Y-27632 (Fig. 5C). To further
examine whetherNogo activates RhoA in OECs, the pull-down assays
with theRho binding domain of rhotekin were performed using
OECsseeded on Nogo-66 substrates. As shown in Fig. 5D, theamount of
GTP-bound RhoA was significantly increased inOECs plated on
GST–Nogo-66 or His–Nogo-66 substrates. All
Fig. 3. NgR is expressed on plasmamembrane of cultured OECs.(A)
OECs were double-stained withantibodies against S-100 and NgR
aftertreatment with Triton X-100 (a-c).OECs (d) and Schwann cells
(e) weresurface stained (without treatment withTriton X-100) with
anti-NgR antibody.(B) Cell lysates from SCs (negativecontrol), OECs
and PC12 (positivecontrol) cells were subjected toimmunoblotting
with anti-NgR (upperpanel) and anti-GAPDH (lower panel).The
arrowhead points to NgR (80kDa). (C) Immunostaining for NgR
onplasma membrane of OECs before (–)and after (+) treatment with
PI-PLC(0.1 U/ml) at 37°C for 2 hours.(D) The supernatant (S) and
membranefractions (P) of OECs treated (+) oruntreated (–) with
PI-PLC (0.1 U/ml)at 37°C for 40 minutes were subjectedto
immunoblotting with anti-NgR(upper panel) and anti-GAPDH
(lowerpanel). Bars, 50 �m.
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these results suggest that RhoA activation is critical
formediating the effect of Nogo-66 on OECs.
Focal adhesions are at the termini of stress fibre bundles
thatserve in longer-term anchorage (Burridge et al., 1998), andRhoA
is a central player in the formation of focal adhesionsand actin
stress fibre bundles (Nobes and Hall, 1995; Allen etal., 1997). To
determine further whether the effect of Nogo-66on OECs is
associated with RhoA activity, OECs and Y-27632-treated OECs were
double-stained for paxillin and F-actin aftercells were plated on
GST, GST–Nogo-66, His, or His–Nogo-66. In the OECs on GST and His,
there was little staining forthe focal adhesion protein paxillin
(Fig. 5Eb,n). These cellsrevealed multiple membrane protrusions
where there werethick bundles of actin filaments (Fig. 5Ec,o). In
OECs platedon GST–Nogo-66 or His–Nogo-66, filamentous actin
extendedalong the base of the cell and organized in fine bundles
parallelto the cell cortex at the cell periphery (Fig. 5Eg,s). In
thesecells, additionally, paxillin was distributed in dense
basalplaques at the peripheral focal adhesions (Fig.
5Ef,r),coinciding with the insertion points of actin filaments
(Fig.5Eh,t). Quantitative analysis revealed that the number
ofpaxillin punctae of OECs on GST–Nogo-66 or His–Nogo-66substrates
was significantly increased (Fig. 5F). When OECswere pretreated
with Y-27632 and plated on GST–Nogo-66(Fig. 5Ei-l) or His–Nogo-66
(Fig. 5Eu-x), they exhibited asimilar staining pattern and membrane
protrusion to theuntreated OECs plated on GST or His. The number of
paxillinpunctae was significantly decreased in pretreated OECs
platedon Nogo-66 fusion proteins (Fig. 5F). These
resultsdemonstrated that Nogo-66, via activation of RhoA,
promotedthe formation of OEC focal adhesions and inhibited
theirmembrane protrusion.
Nogo-A is involved in the augmentation of OECadhesion and
restriction of OEC migration byoligodendrocytesAs shown in Fig. 6A,
cultured oligodendrocytes (OLs)exhibited positive immunostaining
for Nogo-A and OL-specific markers O1 and MBP. Nogo-A was primarily
localizedto the cell body and major processes. To test whether
endogenous Nogo-A expressed by OLs influenced OECadhesion, OECs
were labeled with Di-I (3,3�-dioctadecyloxacarbocyanine
perchlorate) and applied to anadhesion assay. As shown in Fig. 6B,
OECs had a significantlygreater affinity for remaining attached to
an OL monolayerthan to laminin, indicating that OLs may express or
secretesome molecule that enhances the adhesion of OECs. To
testwhether OLs expressing endogenous Nogo-A also inhibit
themigration of OECs, a standard inverted coverslip migrationassay
was carried out. When inverted on GST–Nogo-66,His–Nogo-66 and OL
monolayers, few OECs migrated awayfrom the coverslip fragments, in
terms of both the distancescovered by OECs (Fig. 6C,D) and the
number of cellsemerging from the fragment (Fig. 6C,E). By contrast,
OECsmigrated further on laminin, GST and His controls. Theseresults
suggest that OECs migrate poorly on OL monolayersand the enhanced
adhesion to OL monolayers may contributeto the decreased number of
OECs migrating over OLsmonolayer.
The ability of OL monolayers to enhance OEC adhesion andrestrict
their migration could, in principle, be due to secretedmolecules,
factors associated with the extracellular matrix,
orcell-membrane-associated molecules. As shown in Fig. 7A,B,both
oligodendroglial matrix and oligodendrocyte-conditionedserum-free
medium have no inhibitory effect on OECmigration, which indicates
that the poor migration of OECs onOL monolayers may be mediated by
cell-membrane-associatedmolecules. This is consistent with the fact
that Nogo-A is amembrane-associated protein. To further confirm the
functionof Nogo-A expressed on cell membrane, a
transienttransfection system using Cos-7 cells was used (Fig. 7C).
Asshown in Fig. 7D-F, compared with that of mock-transfectedCos-7
cells, OECs adhere well to the monolayer of Cos-7 cellstransfected
with Nogo-A and exhibit a poor capacity tomigrate. To test whether
the effect of OL monolayers on OECmigration is NgR-dependent we
used an inverted coverslipmigration assay: OECs were pretreated
with PI-PLC to releaseNgR from the cell surface or with anti-NgR to
block thebinding to Nogo-A. When pretreated with PI-PLC or NgR,OECs
migrate well over OL monolayers (Fig. 7G,H), which
Journal of Cell Science 120 (11)
Fig. 4. The effects of Nogo-66 on themigration and adhesion of
OECs aremediated by NgR. (A) OECs were pre-treated with or without
PI-PLC (0.1U/ml), NgR antibody (1:100) or goatIgG (1:100) for 2
hours and applied toBoyden chamber assays. The trans-wellmembranes
were coated with laminin,GST, GST–Nogo-66, His or His–Nogo-66 (100
�g/ml each). SCs were served
as a negative control. (B) After pre-treatment with or without
either PI-PLC, NgRantibody or goat IgG, OECs or SCs were plated for
6 hours on spots of GST,GST–Nogo-66, His or His–Nogo-66.
Quantitative assessment was obtained bydetermining the percentage
of the cell numbers within the spots compared with outsidethe spots
(vehicle, 100%). (C) Dose-dependency of the effect of PI-PLC. After
beingexposed to PI-PLC at the indicated concentration for 2 hours,
cells were harvested andused in an adhesion assay. Values were
reported as mean ± s.d./visual field. **P
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1881Nogo regulates OEC migration
indicates that the inhibitory effect of OL monolayers on
themigration of OECs is NgR-dependent.
The migration of implanted OECs is facilitated byneutralizing
NgR in vivoTo investigate whether the migration of implanted OECs
wasaffected by Nogo, an in vivo migration assay was performedas
previously reported (Cao et al., 2006). OECs prelabeled withDi-I
were injected into the injured spinal cord. Ten days
afterinjection, OECs were found to migrate longitudinally
andlaterally from the injection sites. However, in the presence
ofanti-NgR antibody, OECs migrated further in the verticaldirection
(parallel with the long axis of spinal cord) whencompared with OEC
migration using normal saline (N.S.) or
the irrelevant IgG, at the rostral (Fig. 8) as well as the
caudalinjecton site. There was no difference in migration
distancebetween OECs injected at rostral and caudal sites (data
notshown). Quantitative analysis revealed that treatment of
OECswith anti-NgR increased the maximum migration distancealong the
vertical direction, and there was no significantdifference in the
maximum migration distance along thehorizontal direction (Fig. 8B).
These results indicated thatneutralizing NgR with anti-NgR antibody
facilitated OECmigration through white matter tracts in vivo.
DiscussionCell migration is an essential process in
embryonicdevelopment, growth, wound repair and inflammation, as
well
Fig. 5. RhoA activation is critical formediating the effect of
Nogo-66 onOECs. (A,B) Y-27632 treatmentsignificantly attenuates
theenhancement of Nogo-66 on adhesionof OECs. OECs (a,c,e,g,i) and
Y-27632-treated OECs (b,d,f,h,j) were plated ondishes coated with
laminin (a,b), GST(c,d), GST–Nogo-66 (e,f), His (g,h) orHis–Nogo-66
(i,j). After incubation for30 minutes with gentle shaking,adherent
cells were photographed andcounted. The values were normalizedwith
respect to the values obtained forlaminin. (C) Y-27632
treatmentsignificantly attenuates the inhibition ofNogo-66 on
migration of OECs. OECswere pre-treated with or without Y-27632 and
applied to Boyden chamberassays. The trans-well membranes
werecoated with laminin, GST, GST–Nogo-66, His or His–Nogo-66 (100
�g/mleach). Values were reported as mean ±s.d./visual field. *P
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1882
as tumor cell dissemination. During development, OECs derivefrom
the olfactory placode and migrate along olfactory nervetracts,
modulating their growth and guidance (Tisay and Key,1999). The
ability of OECs to assist in the growth of olfactoryaxons in the
olfactory system has led them to becomecompelling candidates for
transplant-mediated repair of CNSlesions. Several studies have now
confirmed the use of OECsin spinal cord injuries, which include the
promotion of axonalregeneration following spinal cord injuries and
the replacementof myelin in demyelinating diseases. The anatomical
evidenceof regeneration (Franklin et al., 1996; Li et al., 1997; Li
et al.,1998; Imaizumi et al., 2000; Nash et al., 2002) and
functionalimprovements (Li et al., 1998; Ramon-Cueto et al.,
1998;Ramon-Cueto et al., 2000; Lu et al., 2001; Lu et al.,
2002;López-Vales et al., 2006) have been noted in a variety of
spinalcord repair models, including complete
transection,hemisection, tract lesion, contusion and
demyelination.However, there are some negative reports of
OECtransplantation after spinal cord injury (Resnick et al.,
2003;Barnett and Riddell, 2004), and it is still debatable
whethertransplanted OECs can migrate long distances in the
injuredCNS. Smale et al. reported that no significant cell
migrationwas detected when OECs from fetal rat olfactory bulb
wereimplanted into the damaged adult rat brain (Smale et al.,
1996).The recent study by Takami et al. showed that grafted
OECssimply disappeared from the lesion cavity with no evidencethat
they had migrated away into the surrounding neuropil(Takami et al.,
2002). Deng and colleagues reported that both
rat and human OECs showed similar migration after injectioninto
the thoracic spinal cord. Importantly, both rat and humanOECs
migrated for shorter distances, in both rostral and
caudaldirections, in the injured cord of animals with a
concomitantcontralateral hemisection (Deng et al., 2006). Data from
otherstudies also demonstrated that adult rat or human OECsmigrated
over shorter distances after they were transplantedinto the
injuried CNS (Gudino-Cabrera et al., 2000; Collazos-Castro et al.,
2005). However, the underlying specificmechanisms for the poor
motility of OECs in the damagedCNS remain unknown. In the present
study, we first show thatboth Nogo-66 and Nogo-A enhance the
adhesion of OECs andinhibit their migration. These studies may, at
least partially,explain why OECs fail to migrate long distances in
the injuredCNS.
It is well documented that Nogo, MAG and OMgp areprominent
components of CNS myelin inhibitory activity foradult
axon-regeneration. Following injury to the spinal cord,Nogo-A mRNA
is upregulated around the lesion and Nogo-Aprotein is strongly
expressed in injured dorsal column fibresand their sprouts that
entered the lesion site (Hunt et al., 2003).Meier et al. showed
that Nogo-A expression is upregulatedafter hippocampal denervation
or kainate-induced seizures(Meier et al., 2003). Studies of
Bandtlow et al. (Bandtlow etal., 2004) demonstrated that Nogo-A
mRNA andimmunoreactivity are markedly increased in
hippocampalneurons of patients with temporal lobe epilepsy. In the
presentstudy, Nogo-A was found highly expressed in OLs, which
is
Journal of Cell Science 120 (11)
Fig. 6. Effects of OLs on the adhesionand migration of OECs. (A)
OLs wereidentified by staining with anti-O1 andMBP(a-d).
MBP-negative cell as thearrowhead points to (c,d) may
beoligodendrocyte precursor cell. Nogo-Aexpression on OECs is
revealed byimmunostaining without permeabilization(e-f). (B) OECs
adhere well tooligodendrocyte monolayers (OLs-MON). DiI-labelled
OECs were placedover laminin or OLs-MON. The valuesare normalized
against the laminin group.(C) Photomicrographs of OEC migrationfrom
the edge of the coverslip fragment.Coverslip fragments covered with
Di-I-labbled OECs were inverted with cellsfacing downward onto
laminin, GST,GST–Nogo-66, His, His–Nogo-66 orOLs-MON. (D) Average
maximumdistance traveled from the edge of theinverted fragment by
OECs upon eachsubstrate compared with laminin (100%).Migration
index is the ratio of maximummigration distance traveled by OECs on
aparticular substrate to the maximummigration distance traveled by
thesescells on laminin. (E) Distribution of thenumber of migrated
cells occupyingsuccessive 50 �m � 1 mm areas from theedge of an
inverted fragment for eachsubstrate. **P
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1883Nogo regulates OEC migration
consistent with previous reports (Moreira et al., 1999;GrandPre
et al., 2000; Huber et al., 2002). Furthermore, the co-culture
migration assay demonstrated that motility of OECswas significantly
depressed by Nogo-A on OLs. Therefore, theelevated levels of
Nogo-A, probably on OLs, in the injuredCNS, may be responsible for
the inhibition of the migration oftransplanted OECs in the damaged
CNS.
The movement of a metazoan cell involves the turnover
ofadhesions with the substratum on which it moves (Kaverina etal.,
2002). In the present study, we found that the adhesion ofOECs was
enhanced both by recombinant Nogo-66 proteinand by Nogo-A
exogenously expressed by Cos-7 cells orendogenously by OLs.
Therefore, one possibility to account forthe influence of Nogo-66
on OEC migration was that Nogo-66affected the adhesion of OECs.
Adhesion sites form as a resultof signaling between the
extracellular matrix on the outside andthe actin cytoskeleton on
the inside, and they are associatedwith specific assemblies of
actin filaments (Kaibuchi et al.,1999; Kaverina et al., 2002).
Paxillin is a cytoskeletalcomponent localized to the long-lived
focal adhesions at theend of actin stress fibres that serve in
longer-term anchorage(Burridge et al., 1998). Protrusion of the
cell surface is an early
step in several cellular processes including cell migration.
Thedriving force for the formation and extension of
membraneprotrusions is the polymerization of actin (DeMali
andBurridge, 2003). In the present study, we observed theinfluences
of Nogo-66 on focal adhesions, actin stress fibersand membrane
protrusions. It was apparent that Nogo-66regulated actin
reassembly, promoted the formation of focaladhesions, and inhibited
membrane protrusion. Taken together,these data support the
hypothesis that Nogo-66 enhances theadhesion of OECs, which
inhibits their migration.
Woodhall et al. reported that NgR mRNA is expressed inprimary
cultures of OECs (Woodhall et al., 2003). In our study,the
expression of NgR in OECs was confirmed further
byimmunocytochemistry and western blot. With enzymaticcleavage of
NgR and anti-NgR block, we confirmed that theeffect of recombinant
Nogo-66 and endogenous Nogo-Aexpressed by OLs on OECs was
NgR-dependent. To ourknowledge, this is the first evidence that
functional NgR isexpressed on OECs, although the role of p75, the
commonmarker for OECs, as an essential co-receptor needs
furtherinvestigation. The Rho family of small GTPases have
beenreported to be central players in regulating the assembly of
the
Fig. 7. The effect of OLs on OECs ismediated by Nogo-A. (A,B)
OECmigration is not affected byoligodendrocyte-derived matrix
anddiffusible factors. Coverslip fragmentscontaining OECs or
OECs-DiI wereinverted on laminin and cultured withculture medium
(Laminin), the condition-defined medium (CM),
theoligodendrocyte-conditioned medium(OLs-CM), the
oligodendrocyte-derivedmatrix with culture medium (OLs-GM),or on a
monolayer of oligodendrocyteswith culture medium (OLs-MON).
Themigration index and distribution of thenumber of migrated cells
were examined.Migration Index (%)=Maximum distancetravelled by OECs
as a percentage of themaximum distance traveled by OECs onlaminin.
**P
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1884
actin cytoskeleton, adhesion formation and membraneprotrusion
(Hall, 1998; Ridley, 2001; DeMali and Burridge,2003), in which RhoA
signals the formation and maturation offocal adhesions associated
with actin stress fibre bundles, andRac1 and Cdc42 stimulate the
formation of protrusions inassociation with lamellipodia and
filopodia, respectively.Formation of membrane protrusions is an
early central step inthe process of cell migration. When the ratio
of activitybetween these small GTPases is no longer optimal
forprotrusion and polarization of the cell, the migration will
stop(Cox et al., 2001). In the present study, we found that
activeRhoA was significantly increased in OECs plated on
Nogo-66substrates, which suggests that Nogo binding to NgR leads
toactivation of RhoA in OECs. One of the downstream effectorsof
RhoA is Rho-kinase (ROCK), which can be inhibitedeffectively by the
compound Y-27632 (Narumiya et al., 2000).Inhibition of ROCK
stimulates membrane protrusion (Cox etal., 2001; Rottner et al.,
1999; Tsuji et al., 2002) and promotescell migration (Nobes and
Hall, 1999). Here, we observed that
the effect of Nogo-66 on the adhesion and migration of OECswas
greatly attenuated when cells were pretreated with Y-27632. In
addition, we found that the Nogo-66-inducedincrease of focal
adhesion formation, alteration of actincytoskeletal structure, and
inhibition of membrane protrusionwere significantly attenuated by
pretreatment of OECs with Y-27632. All these results supported the
hypothesis that theactivity of RhoA is involved in the regulation
of adhesion andmigration of OECs by Nogo-66. However, further study
isnecessary to clarify the detailed intracellular
signalingmechanisms.
It is a continuing challenge to move toward
therapeuticapproaches for CNS injury. Recently, there has been
greatinterest in the possibility that OECs have potential for use
inthe treatment of axonal injuries and demyelinating
disease(Franklin and Barnett, 2000; Raisman, 2001; Ramon-Cuetoand
Santos-Benito, 2001). Upgrading the growth-promotingproperties of
OECs is considered a valuable strategy forpromoting CNS repair;
however, most of these studies involveOECs secreting additional
neurotrophic factors (Cao et al.,2004; Ruitenberg et al., 2003;
Ruitenberg et al., 2005). Sincethe inhibition of axonal outgrowth
by CNS myelin is one ofthe major obstacles to functional recovery
following CNSinjury, myelin inhibitors and their receptors are
recentlyemerging as potential therapeutic targets (Lee et al.,
2003).Neutralizing Nogo-A with IN-1 antibody and NgR
antagonistswere proven to improve CNS axon regeneration and
functionalrecovery after various lesions (Fouad et al., 2004;
GrandPre etal., 2002). In the present study, we found that OEC
migrationwas facilitated in vitro and in vivo when NgR was
neutralizedwith anti-NgR antibody. It is of interest to test the
idea that,combined with OEC implantation, local administration
ofNogo-A antibody or NgR antagonists would improve thetherapeutic
properties of OECs on CNS injury.
Materials and MethodsCell culturePrimary OECs were prepared from
the olfactory bulb of adult Sprague-Dawley ratsand purified by
differential cell adhesiveness (Ramon-Cueto et al., 1998).
Briefly,OECs were extracted from the olfactory nerve layer by
trypsin treatment and platedon two uncoated 25 cm2 culture flasks;
each was incubated for 36 hours at 37°C in5% CO2. The non-adhesive
cell suspension was collected and then seeded onto diskspre-coated
with poly-L-lysine (PLL, 0.1 mg/ml), and incubated with
serum-containing DMEM/F-12 supplemented with 2 �M forskolin (Sigma)
and 10 ng/mlbFGF (Sigma).
Schwann cells (SCs) were obtained from sciatic nerves of
2-day-old Sprague-Dawley rat pups and purified using a modification
of the protocol previouslydescribed (Lakatos et al., 2000; Brockes
et al., 1979). Cells were cultured on poly-L-lysine (100
�g/ml)-coated dishes and maintained in DMEM/F-12 containing 15%FBS
and supplemented with Forskolin (2 �M, Sigma) and bFGF (10 ng/ml,
Sigma).The cultures were treated with cytosine arabinoside (10–5 M,
Sigma) to reducecontamination by fibroblasts.
Oligodendrocytes (OLs) were prepared from rat cerebral cortex as
described(McCarthy and de Vellis, 1980; Fok-Seang et al., 1995). In
brief, newborn ratcortical cells were dissociated and cultured in
DMEM/F-12 containing 10% FBS.The culture medium was changed at 24
hours and twice weekly thereafter. After10-12 days, the cultures
became confluent and loosely attached macrophages wereremoved by
shaking the flasks on a rotary shaker at 260 rpm for 1 hour at
37°C.The supernatant was discarded and the cultures were then
shaken in fresh culturemedium for 18 hours at 260 rpm to remove
oligodendrocyte precursor cells fromthe cell monolayer. The
detached cells were plated onto a non-coated culture dishfor 7
minutes to remove adherent cells such as microglia and astrocytes.
Theoligodendrocyte precursor cells were then transferred to a
culture dish pre-coatedwith PLL and left to adhere overnight. From
the next day on, the medium wasexchanged for serum-free chemical
defined DMEM/F-12 supplemented with 5�g/ml insulin, 50 �g/ml
transferrin, 0.66 mg/ml BSA, 20 ng/ml progesterone, 100�mol/ml
putrescine, 40 ng/ml sodium selenite and 30 nmol/ml T3, and
theoligodendrocyte precursor cells were cultured for another 1-10
days to differentiate
Journal of Cell Science 120 (11)
Fig. 8. The migration of implanted OECs is facilitated by
neutralizingNgR in a spinal cord hemisection injury model. (A) The
animalsimplanted with DiI-labelled OECs were treated with N.S.,
anti-NgRantibody or goat IgG every day through a pipe embedded in
thesubarachnoid space. After 10 days, the length, width and area of
theregion invaded by DiI-postive cells were measured by
usingMetaMorph Imaging System analysis software. The three right
panelsare locally magnified images of the three left panels. (B)
Quantitativeassessment of cell migration in vivo. Migration Index
(%)=Maximumlength, width or area traveled by DiI-positive cells in
the presence ofanti-NgR antibody or irrelevant IgG as a percentage
of the maximumlength, width or area traveled by DiI-positive cells
in the presence ofN.S. **P
-
1885Nogo regulates OEC migration
into OLs. OLs were identified by indirect immunofluorescence
labeling usingmonoclonal anti-oligodendrocyte marker O1 and
anti-myelin basic protein (MBP).
Unless indicated otherwise, cells were pretreated with or
without PI-PLC (0-0.1U/ml), anti-NgR (1:100), IgG (1:100) or
Y-27632 (15 �M) for 2 hours before contactwith Nogo-66. For PI-PLC
treatment, cells were washed once with 0.1 M PBS (PH7.4) and then
incubated with phosphatidylinositol-specific phospholipase C
(PI-PLC,Sigma; 0.001, 0.01, 0.1 U/ml) in DMEM/F-12 at 37°C for the
times indicated. Thecells were washed three times and then
processed for the migration and adhesionassay or for western blot
analysis. To confirm cell viability, cells were stained with0.4%
Trypan Blue. Trypan-Blue-incorporating cells were
-
1886
cells prepared at different times. Statistical analysis was
performed using unpairedStudent’s t-test. All data are presented as
mean ± s.d.
We thank Dana Dodd in the Schwab lab for the gift of
plasmidencoding Nogo-A. This work was supported by the National
KeyBasic Research Program (2005CB724302, 2006CB500702), theNational
Natural Science Foundation (30400128, 30325022,30530240), the
Program for Changjiang Scholars and InnovativeResearch Team in
University (IRT0528), the Shanghai Young Scienceand Technology
Phosphor Projects (04QMX1437), and the ShanghaiMetropolitan Fund
for Research and Development (04DZ14005,04XD14004).
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