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The Journal of Neuroscience, July 1, 2002, 22(13):5505-5515
Localization of Nogo-A and Nogo-66 Receptor Proteins at Sites of
Axon-Myelin and Synaptic Contact
Xingxing
Wang1,
Soo-Jin
Chun4,
Helen
Treloar2,
Timothy
Vartanian4,
Charles A.
Greer2, 3, and
Stephen M.
Strittmatter1, 3
Departments of 1 Neurology and
2 Neurosurgery and 3 Section of Neurobiology,
Yale University School of Medicine, New Haven, Connecticut 06510, and 4 Harvard Institutes of Medicine, Boston, Massachusetts
02115
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ABSTRACT |
Axon regeneration in the adult CNS is limited by the presence of
inhibitory proteins. An interaction of Nogo on the oligodendrocyte surface with Nogo-66 Receptor (NgR) on axons has been suggested to play
an important role in limiting axonal growth. Here, we compare the
localization of these two proteins immunohistochemically as a test of
this hypothesis. Throughout much of the adult CNS, Nogo-A is detected
on oligodendrocyte processes surrounding myelinated axons, including
areas of axon-oligodendrocyte contact. The NgR protein is detected
selectively in neurons and is present throughout axons, indicating that
Nogo-A and its receptor are juxtaposed along the course of myelinated
fibers. NgR protein expression is restricted to postnatal neurons and
their axons. In contrast, Nogo-A is observed in myelinating
oligodendrocytes, embryonic muscle, and neurons, suggesting that Nogo-A
has additional physiologic roles unrelated to NgR binding. After spinal
cord injury, Nogo-A is upregulated to a moderate degree, whereas NgR
levels are maintained at constant levels. Taken together, these data
confirm the apposition of Nogo ligand and NgR receptor in situations of
limited axonal regeneration and support the hypothesis that this system
regulates CNS axonal plasticity and recovery from injury.
Key words:
axon regeneration; axonal growth cone; myelin; plasticity; skeletal muscle; axon repulsion
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INTRODUCTION |
Many forms of injury to the adult
nervous system spare neuronal cell bodies but sever their axonal
connections. Recovery of function is then dependent on axonal
regeneration. Although adult mammalian CNS neurons exhibit a capacity
for axonal outgrowth in permissive environments, the injured adult CNS
contains axon-inhibitory factors limiting anatomical and functional
recovery (Richardson et al., 1980 ; David and Aguayo, 1981; Benfey and
Aguayo, 1982; Fournier and Strittmatter, 2001). The CNS myelin
component is especially hostile to axonal outgrowth, and Nogo-A is a
prominent myelin-derived inhibitor of axonal outgrowth (Chen et al.,
2000; GrandPre et al., 2000; Prinjha et al., 2000). An understanding of
the cellular and subcellular distribution of the Nogo protein in
oligodendrocytes is crucial to any consideration of its pathologic role
in limiting axonal regeneration.
Nogo is expressed in three isoforms derived from alternate promoter and
splice usage (Chen et al., 2000; Fournier et al., 2000; GrandPre et
al., 2000; Prinjha et al., 2000). Nogo-A, -B, and -C proteins share a C
terminal region related to the Reticulon family of proteins. Previous
work has demonstrated that in adult rodents, Nogo-A mRNA is expressed
quite selectively in oligodendrocytes and certain neurons with little
peripheral expression (Chen et al., 2000; GrandPre et al., 2000).
Nogo-B and Nogo-C mRNA are expressed more widely with more prominent
neuronal expression and skeletal muscle expression.
The cellular distribution of Nogo protein has been only partly defined.
In transfected epithelial cells, Nogo-A and -C are concentrated in the
endoplasmic reticulum with a small percentage of the protein at the
cell surface (GrandPre et al., 2000). The surface epitope is a 66 amino
acid (aa) loop separating two hydrophobic segments near the C terminus
and is common to Nogo-A, -B, and -C. The N and C termini of the protein
are cytosolic in transfected cells. In cultured oligodendrocytes, the
66 aa loop can be detected at the cell surface as well (GrandPre et
al., 2000). The distribution of the protein in brain tissue is not well described.
The 66 aa surface loop of Nogo is inhibitory for axonal growth in
culture and acts via a Nogo-66 Receptor (NgR) expressed by neurons and
localized to their axons in vitro (GrandPre et al., 2000;
Fournier et al., 2001). Although it is clear that the NgR mRNA is
expressed selectively in adult neurons, NgR protein distribution
in vivo has not been considered. It has been hypothesized that interactions between Nogo and NgR limit both axonal plasticity and
regeneration (Thallmair et al., 1998; Brittis and Flanagan, 2001;
Fournier et al., 2001). For this hypothesis to be tenable, there must
be localized expression of NgR on the surface of axons and Nogo on the
adaxonal surface of myelin. Here, we use immunohistologic methods to
localize Nogo-A and NgR in mouse tissues. The data confirm the opposing
distributions of this ligand-receptor pair at the myelin-axon
interface and support the hypothesis that Nogo restricts axonal
sprouting in the adult nervous system.
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MATERIALS AND METHODS |
Antibodies. The polyclonal anti-NgR antibody was
raised against a GST-mouse NgR fusion protein and has been described
(Fournier et al., 2001). Anti-Nogo-A antibody was generated by
immunizing rabbits with KLH-conjugated peptide SYDSIKLEPENPPPYEEA,
corresponding to aa 623-640 of rat Nogo-A. The antibody was affinity
purified on a peptide-agarose resin before immunostaining protocols.
Immunohistochemistry. C57BL/6 mice (Charles Rivers
Laboratories, Wilmington, MA) of embryonic day 15 (E15),
postnatal day 1 (P1), P15, and adult stages were used in this study.
The animals were anesthetized intraperitoneally with 2.5% avertin and
were then perfused transcardially with 0.1 M PBS
followed by 4% paraformaldehyde. Different tissues were dissected and
postfixed in 4% paraformaldehyde in 0.1 M PBS.
We cut 40-50 µm sections on a vibrating microtome. Before staining,
free-floating sections were incubated sequentially in 0.3% Triton
X-100 in PBS (PBS-T; 30 min), 10% goat serum in PBS (30 min), and then
with primary antibodies (anti-Nogo-A, 1:8000 or anti-NgR, 1:8000; 4°C
for 24 hr). After rinsing with PBS, diluted biotinylated anti-rabbit
IgG was used as a secondary antibody (1:2000; Vector Laboratories,
Burlingame, CA). Sections were developed in diaminobenzidine (DAB kit;
Vector Laboratories), mounted, air-dried, dehydrated, and coverslipped.
Double immunofluorescence. The following monoclonal primary
antibodies were used in combination with the anti-Nogo-A or anti-NgR antibodies: anti-CNPase monoclonal antibody (clone 11-5B, 1:500; Promega, Madison, WI) and  -III tubulin monoclonal antibody (1:1000; Covance, Denver, PA). After incubating in the primary antibodies for 24 hr, sections were washed in PBS and then incubated for 1 hr at room
temperature with anti-rabbit IgG (FITC-conjugated, developed in goat,
1:200; Sigma, St. Louis, MO) and anti-mouse IgG (TRITC-conjugated,
developed in goat, 1:200; Sigma). Confocal analysis of staining was
obtained with a Zeiss LSM-5 system.
Immunoblots. Protein extracts from different tissues (20 µg) were electrophoresed through a SDS-polyacrylamide gel and
transferred to a polyvinylidene difluoride membrane. After the
transfer, the membranes were incubated overnight at 4°C with the
anti-Nogo-A antibody (1:10,000). The secondary antibody anti-rabbit IgG
(alkaline phosphatase conjugate, 1:5000; Sigma). Nogo-A-transfected
Cos-7 cells were used as a control (GrandPre et al., 2000). For NgR, membrane fractions were treated with 4% paraformaldehyde for 20 min
and washed, before immunoblotting with anti-NgR antiserum (1:3000;
Fournier et al., 2001).
Electron microscopy. Animals were perfused, and vibratome
sections stained as for routine immunohistology except that the fixative was 0.5% glutaraldehyde plus 4% paraformaldehyde. Triton X-100 was omitted from all steps, but to increase tissue penetration of
antibodies, the vibratome sections underwent a rapid freeze-thaw cycle
before staining. After the peroxidase reaction, tissue was counterstained with osmium tetroxide and embedded for thin sectioning in acrylite between plastic coverslips. Sections of 100 nm were examined on a Phillips transmission electron microscope.
Oligodendrocyte differentiation. Oligodendrocyte precursor
cells were isolated from P2 rat brain and differentiated in
vitro (Vartanian et al., 1997). Staining with stage-dependent
antigens followed previous protocols (Vartanian et al., 1997).
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RESULTS |
To provide immunohistologic analysis of Nogo-A, an
affinity-purified anti-peptide antibody was generated. By immunoblot,
this preparation specifically detects recombinant human Nogo-A and mouse brain Nogo-A of 215 kDa (Fig. 1).
In adult mouse samples, Nogo-A protein level is high in adult brain and
spinal cord, lower in dorsal root ganglia, and undetectable in
heart, liver, lung, and kidney. Skeletal muscle expresses Nogo-A during
the E15 and P1 developmental stages, but no muscle expression is
detectable in P15 or adult samples. These findings are consistent with
previous mRNA studies of Nogo-A (Chen et al., 2000; GrandPre et al.,
2000). Anti-NgR immunoblots of aldehyde-treated tissues demonstrate a prominent band of 80 kDa in adult mouse brain and transfected COS
cells, but not in adult mouse liver or untransfected COS cells (Fig.
1B).
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Figure 1.
Immunoblot of Nogo-A and NgR expression in mouse
tissues. A, Samples (20 µg) from the indicated tissues
from mice of the indicated ages were analyzed by anti-Nogo-A
immunoblot. D, Dorsal root ganglion; B,
brain; S, spinal cord; M, skeletal
muscle; H, heart; K, kidney;
LU, lung; L, liver. The migration of
recombinant Nogo-A is shown at right with molecular
weight standards of 233, 135, and 112 kDa. B, Immunoblot
for NgR in adult mouse brain (B), adult liver
(L), NgR-expressing COS cells (COS, +), or
control COS cells (COS,  ). Molecular weight standards are indicated
at the right, in kilodaltons.
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The distributions of Nogo-A and NgR were examined in adult mouse spinal
cord sections (Fig. 2). Immunostaining
for both proteins is detected in white matter, and this staining is
blocked by inclusion of purified antigen in the incubation (Fig.
2E,F). Nogo-A-positive cells detected in the
white matter have the appearance of oligodendrocytes, and their
processes are well stained (Fig. 2A,C). Transverse
sections of the cord (Fig. 2A) reveal Nogo-A at the
outer circumference of myelin sheaths but also suggest that Nogo-A is
present at the inner adaxonal circumference of myelin. Nogo-A protein
is also observed at lower levels within axonal profiles. NgR protein is not found in oligodendrocytes or the outer myelin sheath but is detected in axons surrounded by myelin (Fig. 2B,D).
In longitudinal sections (Fig. 2C,D) and in teased fiber
preparations (data not shown), the Nogo-A and NgR protein distributions
extend along the myelinated fibers. Neither protein appears to be
confined selectively to internodal or paranodal regions.
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Figure 2.
Localization of Nogo-A and NgR in adult
mouse spinal cord. Adult mouse thoracic spinal cord sections were
sectioned in the transverse or sagittal plane and stained with
anti-Nogo-A or anti-NgR antiserum, as indicated. Note the
oligodendrocyte staining (arrowhead) for Nogo-A and the
staining along axonal profiles for both proteins
(arrows). In some cases, 50 µg/ml of purified antigen
peptide or protein was included together with primary antibody (Antigen
blockade). Scale bar, 100 µm.
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The differential distribution of the proteins is even more obvious in
transverse sections of ventral lumbar roots where axonal Nogo-A is less
prominent. The presence of Nogo-A at both the outer and the inner
adaxonal sheath of myelin is apparent (Fig.
3). NgR protein is clearly localized to
the axon but not the myelin.
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Figure 3.
Nogo-NgR distribution in ventral spinal
roots. Cross sections of lumbar ventral roots stained with the
anti-Nogo-A and anti-NgR antibodies. Note the axonal staining for NgR
(arrows in right panel) and the
myelin staining for Nogo-A, detectable at the outer
(arrowheads in left panel) and
adaxonal (arrows in left panel)
surface of the myelin. Scale bar, 100 µm.
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To verify the cellular origin and relative juxtaposition of Nogo-A and
NgR, sections were double labeled for these proteins and either an
oligodendrocyte marker, CNPase, or an axonal marker, -III tubulin
(Fig. 4). Clearly, the Nogo-A-positive
cells in spinal cord white matter are CNPase-positive, confirming the
oligodendrocyte origin of Nogo-A. In double stains for -III tubulin
and Nogo-A, the presence of low level Nogo-A in axons surrounded by
higher level Nogo-A in oligodendrocyte processes is verified. The NgR staining matches precisely with the -III tubulin staining,
confirming its neuronal origin.
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Figure 4.
Cellular localization of
Nogo-A and NgR. Transverse sections of the adult spinal cord were
processed for anti-Nogo-A/anti-CNPase (top panels),
anti-Nogo-A/anti--III tubulin (middle panels), or
anti-NgR/anti--III tubulin staining (bottom panels)
and examined by confocal microscopy. Note the
colocalization of Nogo-A with CNPase and NgR with -III tubulin.
Strong oligodendrocyte Nogo-A immunoreactivity surrounds axonal
staining. Scale bars, 100 µm.
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The juxtaposition of oligodendrocyte Nogo-A with axonal NgR was
investigated by two additional methods, higher magnification analysis
of confocal images and ultrastructural analysis. The Nogo-A/-III
tubulin double-stained images exhibit a rim of Nogo-A staining that
extends circumferentially beyond the axonal -III tubulin staining
(Fig. 5). When the -III tubulin stain
is digitally subtracted from the Nogo-A stain, this rim of staining is
obvious. In contrast, the NgR immunoreactivity precisely matches that
for -III tubulin.
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Figure 5.
Relationship of Nogo-A and NgR immunoreactivity to
axons. Sections stained with anti-Nogo-A or anti-NgR
(green) plus anti--III tubulin
(red) as in Figure 4 are presented as higher
magnification merged images (merged) or with the -III
tubulin signal subtracted from the Nogo-A or NgR signal
(subtracted). For Nogo-A, but not for NgR, note the rim
of staining at the inner (red arrow) and outer
(blue arrow) circumference of the myelin. Scale bar, 100 µm.
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Ultrastructural examination of NgR localization confirms these
impressions (Fig. 6). NgR is present in
myelinated axonal profiles in cortex (Fig. 6A) as
well as spinal cord (Fig. 6B,C). The HRP reaction
product is not limited to the plasmalemma, but is spread throughout the
axon where it often labels heavily microtubules and intracellular
organelles. Although many unlabeled axons are also present, it is not
clear if these are indicative of axons that lack NgR or if this is a
reflection of the difficulty of examining NgR labeling at the
ultrastructural level. In a few cases, small unmyelinated axons also
express NgR (Fig. 6B). Overall, this analysis of
Nogo-A and NgR in spinal cord supports the conclusion that the two
proteins are situated in an orientation where they may interact at the
axon-myelin interface.
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Figure 6.
Ultrastructural localization of NgR to axons. In
A, labeled axons in cortex, apparent because of
the accumulation of reaction product within the axon, are
shown (open arrows). Myelin membranes are dense in these
pictures because of the osmium treatment, and this density is present
in samples not stained with anti-NgR. It is the electron-dense reaction
product within the axons that is NgR-selective. Unlabeled axons are
also within the field of view (asterisks). In
B and C, both labeled
(arrows) and unlabeled (asterisks) are
seen. Scale bars: A, B, 5 µm;
C, 6 µm.
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Various regions of the CNS were examined to determine if different
regions might use the Nogo-A/NgR system to different degrees. The
distribution of both proteins is quite widespread in the adult mouse
nervous system (Table 1). In the
forebrain (Fig. 7), multiple layers of
the cerebral cortex express the proteins. However, as in the spinal
cord, the cellular distribution is different. NgR is detectable in many
neuronal cell bodies and the neuropil, whereas Nogo-A is observed in
oligodendrocytes and neuropil. Similarly, the hippocampus exhibits NgR
in pyramidal cells and in dentate gyrus granule cells. Nogo-A is
present only in oligodendrocyte cell bodies and in the neuropil.
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Figure 7.
Nogo-A and NgR in the adult forebrain.
Coronal sections of adult mouse brain were examined for anti-Nogo-A and
anti-NgR staining. Note the small oligodendrocyte cell bodies
(arrows) and neuropil staining for Nogo-A, and the
multiple neuronal cell bodies (arrows) and neuropil
staining for NgR. The abbreviations are: II and
IV, layers II and IV of the cortex; CA1
and CA3, pyramidal cell layers of the hippocampus;
DG, dentate gyrus of the hippocampus. Scale bars,
100 µm.
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Analysis of cortex at the utrastructural level reveals NgR at both pre-
and post-synaptic sites (Fig. 8). NgR is
evident presynaptically at asymmetrical synapses where the DAB reaction
product distributes diffusely among cytoplasmic elements, including
synaptic vesicles and mitochondria (Fig. 8A,B). In
the NgR-immunoreactive postsynaptic profiles of asymmetric synapses,
reaction product is evident at the postsynaptic density as well as on
microtubules and mitochondria (Fig. 8C,D). No synapses with
both presynaptic and postsynaptic NgR are observed. Localization of NgR
to synaptic junctions broadens the potential roles of this molecule to
include modulation of synaptic structural plasticity.
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Figure 8.
NgR localizes to both presynaptic and postsynaptic
profiles in cortex. In A and B, labeled
presynaptic terminals (white t) are seen making
asymmetric synapses (arrows) onto unlabeled processes
(black asterisks). In C and
D, unlabeled presynaptic terminals (black
t) are seen making asymmetric synapses (arrows)
onto labeled processes (white asterisk). In all
examples, small accumulations of spherical vesicles are observed in the
presynaptic terminal while a thick postsynaptic specialization
characterizes the postsynaptic element. Scale bar (shown in
C): A, 0.5 µm; B, 1.3 µm; C, 0.75 µm; D, 1.0 µm.
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In the cerebellum, Nogo-A immunoreactivity is again prominent in
oligodendrocyte cell bodies scattered in the deep white matter and the
granule cell layer, with some neuropil staining (Fig. 9). In contrast, the NgR is expressed by
multiple neurons, including Purkinje cells and granule cells, and is
found throughout the neuropil.
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Figure 9.
Presence of Nogo and NgR protein in the hindbrain
Sagittal sections of the cerebellum and medulla oblongata were analyzed
with anti-Nogo-A and anti-NgR antibodies. Oligodendrocyte cell bodies
(arrowheads) and myelinated fibers
(arrows) contain Nogo-A, whereas NgR immunoreactivity is
present in numerous neuronal cell bodies, including Purkinje cells and
myelinated fibers (arrows). m, Molecular
layer; p, Purkinje cell layer; g, granule
cell layer; wm, deep white matter layer. Scale
bars, 100 µm.
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Longitudinal sections through white matter tracts in the medulla
oblongata demonstrate the same pattern seen in longitudinal sections of
the spinal cord (Fig. 9). Axonal staining for NgR is prominent, whereas
Nogo-A is visualized both in oligodendrocyte cell bodies and in
myelinated fiber profiles.
The developmental regulation of Nogo-A and NgR was examined
in mice from E15 through adulthood (Fig.
10). At E15 and P1, the forebrain
exhibits strong staining for Nogo-A. The pattern is reticular, with few
stained cell bodies. Because NgR is not yet expressed, the role of this
early Nogo-A expression may relate to a Reticulon-like function rather
than to a cell surface interaction with axonal NgR. By P15, the adult
Nogo-A pattern of strongly stained oligodendrocyte cell bodies becomes
apparent. Developing brain samples show little or no NgR
immunoreactivity in comparison to P15 and adult samples. This is
consistent with our previous findings of late developmental expression
of NgR in chick (Fournier et al., 2001) and with the late onset of
responsiveness to axonal inhibition by CNS myelin (Cai et al.,
2001).
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Figure 10.
Developmental pattern of Nogo-A and NgR
expression in the forebrain. Sections of the indicated tissues from
mice of the indicated ages were stained for Nogo-A or NgR protein. Note
the shift of Nogo-A immunoreactivity from a diffuse reticular pattern
at E15 to a predominantly oligodendrocyte pattern in the adult. NgR is
present only at low levels before birth but is widely expressed in
neurons postnatally. Scale bars, 100 µm.
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Because the early embryonic brain demonstrates widespread
Nogo-A expression, myelin-forming cells of the adult brain may express the protein from the earliest precursor stage through that of fully
mature oligodendrocytes. Alternatively, Nogo-A expression may be
modulated by the differentiation process. The onset of Nogo-A
expression during oligodendrocyte differentiation was assessed in
cultured oligodendrocytes from P2 rat forebrain (Fig.
11). Nogo-A is detected at the
oligodendrocyte-type II astrocyte (A2B5-positive) precursor stage,
persists through the O4 and O1 stages, and maintains high levels in
fully differentiated MBP-positive oligodendrocytes. Thus, Nogo-A
protein is present at all stages of oligodendrocyte differentiation.
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Figure 11.
Nogo-A expression during oligodendrocyte
differentiation. Oligodendrocyte precursors isolated from rat forebrain
were differentiated in vitro and stained for the
stage-dependent antigens recognized by A2B5, O4, O1, and anti-MBP
antibodies (green). Nogo-A immunoreactivity is
observed in double-stained cells from each stage of oligodendrocyte
differentiation (red). The merged images include nuclear
staining (blue) by propidium iodide.
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Mouse embryos were also examined for peripheral Nogo-A expression (Fig.
12). Sections of the developing E15
embryo reveal peripheral nerve Nogo-A that colocalizes with
neurofilament. Obvious skeletal muscle Nogo-A is present at this stage,
but not in the adult. NgR expression is nil at these stages (data not
shown).
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Figure 12.
Peripheral expression of Nogo-A in E15
mouse embryo. A, B, Parasagittal sections
of the head of the E15 mouse embryo were stained for Nogo-A expression.
Note the strong skeletal muscle expression of Nogo-A in
A and the nerve fiber expression in B.
Anterior is to the left, dorsal is up.
C-E, Nogo-A (C, E, green) and
neurofilament (D, E, red) are colocalized in a section
similar to that in B. The presence of Nogo-A in
neurofilament-positive structures is clear. F, Higher
magnification of A reveals a Nogo-A-positive nerve fiber
innervating a Nogo-A-positive muscle. G, Adult skeletal
muscle expresses little or no Nogo-A protein as compared with the high
level E15 expression in F. Scale bars, 100 µm.
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Because the Nogo-NgR interaction at the axon-myelin
interface may limit axonal regeneration after trauma, it is critical to consider whether basal expression is altered after injury. Complete spinal cord transections were made at the T6 level, and protein expression was examined at various times thereafter. The expression of
NgR at the spinal cord injury site and in the cerebral cortex did not
change significantly (data not shown). In contrast, there was a
moderate increase in Nogo-A staining around the injury site (Fig.
13). This was prominent at 1 week after
injury and declined to baseline levels by 1 month after injury. The
increased expression of Nogo-A was not present within the scarred area
itself but in the region 5 mm caudal and rostral to the injury. In
fact, the glial scar exhibits decreased Nogo-A immunoreactivity. At the cellular level, numerous small cells in the perilesional area were
positive for Nogo-A.
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Figure 13.
Nogo-A in transected adult spinal cord.
Parasagittal sections of the thoracic spinal cord were processed for
Nogo-A (A) immunohistology 1 week after complete
spinal cord transection (asterisk). Note the moderate
increase of Nogo-A in the region immediately surrounding the lesion
site and the low level within the glial scar. Higher magnification of
the perilesional site stained for Nogo-A at 1 week
(B), 2 weeks (C), or 1 month after injury (D). Scale bars, 100 µm.
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DISCUSSION |
The distribution of NgR and Nogo-A supports the hypothesis that
these two proteins interact at contact sites between axons and myelin.
A majority of Nogo-A is expressed by oligodendrocytes in the adult CNS,
and a proportion of the protein localizes to the adaxonal membrane. The
NgR protein is found throughout axons in the adult and maturing CNS.
Expression of NgR is minimal before myelination. The juxtaposed
expression of this ligand-receptor pair is detected in the uninjured
brain and spinal cord, and there is a modest increase in Nogo-A
expression at sites of trauma.
The adjacent cellular expression of Nogo-A and NgR in the intact brain
is consistent with the hypothesis that Nogo-A limits axonal sprouting
and plasticity as part of normal physiology. A general statement of
this theory holds that axon growth and pathfinding are robust
during development. After synaptogenesis and myelination, axonal
contacts with Nogo-A and other myelin-derived outgrowth inhibitors may
serve to stabilize major myelinated tracts. Several major lines of
evidence now support this notion: (1) NgR is expressed on mature axons
and Nogo-A is present at the adaxonal oligodendrocyte membrane (this
manuscript); (2) Nogo-66 limits axonal growth in vitro via
the NgR protein (GrandPre et al., 2000; Fournier et al., 2001); and (3)
the IN-1 antibody recognizing Nogo-A and other proteins promotes
sprouting from uninjured tracts adjacent to sites of trauma (Thallmair
et al., 1998; Z'Graggen et al., 2000; Raineteau et al., 2001). The
widespread distribution of the NgR-Nogo-A system in the adult CNS
indicate that this may apply to most, if not all, neurons.
After trauma, the anatomical correlation of Nogo-A and NgR
distribution persists. If anything, Nogo-A expression in perilesional areas is increased. Further studies will be required to define the cell
of origin for the increased Nogo-A, although proliferating NG2-positive
cells are a potential source (McTigue et al., 2001). Thus, the
anatomical studies support the notion that the NgR-Nogo-A pathway
participates in the extreme limitation of axonal regeneration observed
in the adult mammalian CNS. The data are consistent with the ability of
the IN-1 antibody to promote some degree of axonal regeneration
(Bregman et al., 1995; Merkler et al., 2001).
NgR is also detected at synapses in the adult CNS. This finding raises
the possibility that the Nogo and NgR might contribute to structural
plasticity at synapses as well as along axonal pathways. Because it is
now clear that many forms of activity-dependent synaptic plasticity are
associated with structural rearrangements (Engert and Bonhoeffer, 1999;
Yuste and Bonhoeffer, 2001), it is plausible that some of the same
mechanisms may contribute to axonal rearrangements and dendritic spine
rearrangements. The potential role of Nogo-NgR in such events has not
yet been explored in functional studies.
NgR protein is primarily expressed by neurons of the adult animal in a
distribution where it may receive a Nogo signal. In contrast, Nogo-A is
expressed in many tissues exhibiting little or no NgR expression. For
example, high levels of Nogo-A are present in developing skeletal
muscle and in embryonic neurons. The role of Nogo-A in these
locations appears unrelated to its role as a ligand for NgR. The
simplest explanation for NgR-independent expression of Nogo-A is that
the protein subserves a second function in these tissues. This second
function may be derived from the sequence homology between Reticulons
and Nogo (GrandPre et al., 2000). The nature of Reticulon function
remains elusive, but by virtue of their subcellular localization, these
proteins are thought to participate in endoplasmic reticulum regulation
(van de Velde et al., 1994; GrandPre et al., 2000). Alternatively, Nogo
may regulate cellular survival in developing muscle and nerve, because Nogo-B has recently been shown to modulate apoptosis in some cancer cells (Tagami et al., 2000; Li et al., 2001).
Taken together, the data presented here support the notion that Nogo-A
interaction with NgR limits axonal regeneration after injury. In
addition, the juxtaposed distribution of these proteins in uninjured
brain is consistent with the participation of this system
in maintaining axonal tract stability and perhaps in modulating synaptic structural plasticity. NgR expression is
consistent with a specific role in receiving Nogo signals, whereas Nogo
may have additional roles unrelated to NgR in early development of
multiple tissues, including brain and skeletal muscle.
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FOOTNOTES |
Received Feb. 7, 2002; revised April 8, 2002; accepted April 15, 2002.
This work was supported by research grants from the National Institutes
of Health (S.M.S., C.A.G.) and from the McKnight Foundation and the
Institute for the Study of Aging and Biogen, Inc. (S.M.S.). S.M.S. is
an Investigator of the Patrick and Catherine Weldon Donaghue Medical
Research Foundation.
Correspondence should be addressed to Dr. Stephen M. Strittmatter,
Department of Neurology, Yale University School of Medicine, P. O. Box 208018, 333 Cedar Street, New Haven, CT 06520. E-mail: stephen.strittmatter{at}yale.edu.
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ABSTRACT
INTRODUCTION
