§
* Department of Cell Biology, Department of Neurology, § The Kaplan Cancer Center, New York University Medical School,
New York 10016;
Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; ¶ Department of Neurology and Neuroscience, Division of Neurobiology, Cornell University Medical College, New York 10021;
and ** Sugen, Inc., Redwood City, California 94063
We have investigated the potential role of contactin and contactin-associated protein (Caspr) in the axonal-glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homologue of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia.
MYELINATED nerve fibers play a critical role in the
vertebrate nervous system by promoting the efficient and rapid propagation of action potentials via saltatory conduction (Huxley and Stämpfli, 1949
Each of these regions of myelinated fibers are distinct
with respect to their function in impulse conduction and
organization of voltage-gated channels. Thus, voltage-gated
sodium channels are strikingly concentrated (~1,500/µm2)
at the node of Ranvier, enabling regeneration of the action potential. Na+/K+ ATPase and Na+/Ca++ exchangers are
also enriched at the node (for a recent review see Waxman
and Ritchie, 1993 The molecules that mediate the interactions between
axons and myelinating glial cells at the paranodal junctions have been elusive. Several proteins of the glial cell
are concentrated in the paranodal loops and mediate interactions between the glial processes rather than with the
axon. Among these are E cadherin, which is likely to mediate adherens-like junctions between these processes (Fannon et al., 1995 Recent studies suggested that contactin/F3/F11 might
mediate interactions between axons and glial cells (Peles
et al., 1995 Contactin is associated with the transmembrane protein
Caspr (contactin-associated protein), which has been suggested to be a coreceptor with contactin (Peles et al., 1997b We now report that contactin and Caspr/Neurexin IV
are differentially expressed and localized during myelination. Of particular note, Caspr, which is expressed exclusively by neurons, becomes concentrated in the paranodal
junctions of mature myelinated fibers in both the CNS and
PNS, whereas contactin is substantially downregulated. In
unmyelinated fibers and in nascent myelinated fibers, Caspr has a more diffuse localization along the length of the
axon, suggesting that it redistributes from the internode to
the paranode. These results implicate Caspr as a major
component of the septate junctions that form between axons
and paranodal loops and suggest that it may mediate reciprocal signaling between axons and myelinating glial cells.
Antibodies
Monoclonal antibodies used in these studies included the anti-MAG monoclonal antibody MA513 (gift of M. Schachner, Swiss Federal Institute of
Technology, Zürich, Switzerland) and anti-MBP monoclonal antibody
SMI 94 (Sternberger Monoclonals, Baltimore, MD). Rabbit polyclonal antisera included an anti-contactin/F3 antiserum (gift of C. Goridis, Institut de
Biologie du Développement de Marseille, CNRS/INSERM/Université de
la Mediterranée, Marseille, France); anti-Caspr antiserum 60/61 generated
against a bacterial fusion protein containing the Caspr cytoplasmic domain and the corresponding preimmune serum (Peles et al., 1997b Tissue Culture Methods
Primary rat Schwann cell and dorsal root ganglion (DRG) neuron cultures
and myelinating Schwann cell/neuron cocultures were established as described previously (Einheber et al., 1993 The dissociated neuronal cultures consisted of 4,000 rat embryonic day
16 or 17 DRG neurons plated onto 12-mm glass coverslips coated with
ammoniated rat tail collagen (Biomedical Technologies, Inc., Stoughton,
MA). These cultures were maintained in standard neuronal media, which
consists of MEM (Life Technologies) supplemented with 10% FBS, 2 mM
glutamine, 0.4% glucose (Sigma Chemical Co.), and 50 ng/ml 2.5S NGF
(Bioproducts for Science, Inc., Indianapolis, IN). These cultures were
treated for 2.5 wk with 5-fluorodeoxyuridine and uridine (both at 10 Myelinating Schwann cell/neuron cocultures were prepared by seeding
purified DRG neuron cultures with 200,000 Schwann cells/coverslip in standard neuronal media. The media was replaced the next day with N2 media, which consists of 5 mg/ml insulin (Sigma Chemical Co.), 10 mg/ml
transferrin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA),
20 nM progesterone (Sigma Chemical Co.), 100 mM putrescine (Sigma
Chemical Co.), 30 nM selenium (Sigma Chemical Co.), and 2 mM glutamine in a 1:1 mixture of DME and Ham's F-12 (Life Technologies) supplemented with 2.5S NGF. The cultures were maintained in N2 media for
3 d to allow the Schwann cells to populate the neurites. To initiate basal
lamina formation and myelination, the cultures were fed the standard neuronal media supplemented with 50 µg/ml ascorbic acid (Sigma Chemical Co.).
O-2A progenitors and oligodendrocytes were prepared as described
previously (Canoll et al., 1996a Immunoblotting
Cultures of DRG neurons and Schwann cells were lysed in a solution containing 95 mM NaCl, 25 mM Tris-Cl, pH 7.4, 10 mM EDTA, 2% SDS, 1 mM
PMSF, 10 µg/ml aprotinin, and 20 µM leupeptin. O-2A and oligodendrocyte lysates were prepared as described (Canoll et al., 1996a For detergent extraction experiments, cultures grown in 35-mm dishes
were rinsed with Dulbecco's PBS (dPBS; Life Technologies) and extracted with 1% Triton X-100 in dPBS in the presence of protease inhibitors at 4°C for 30 min. The supernatants containing the Triton X-100-solubilized proteins were then collected, and the remaining insoluble material
was further extracted in gel sample buffer containing 2.5% SDS for 10 min
at room temperature. All samples were spun at 12,000 g for 30 min at
room temperature, and equal volumes of the cleared supernatants were
fractionated on a 7.5% acrylamide SDS gel and blotted to nitrocellulose.
The blot was probed with Caspr polyclonal antiserum and 125I-protein A
and scanned on a phosphorimager to quantitate the immunolabeled bands.
Light Microscopy and Immunofluorescence
Immunofluorescence microscopy was done as described previously (Einheber et al., 1993 For tissue immunocytochemistry, vibratome sections of brain (60 µm
thick) and teased sciatic nerve fibers were prepared from acrolein perfused adult Sprague Dawley rats and incubated with primary antibodies as
described previously (Einheber et al., 1996 Immunoelectron Microscopy
Tissue sections immunolabeled with Caspr polyclonal antibody were processed for silver-enhanced immunogold labeling as described (Chan et al.,
1990 For electron microscopy, the immunolabeled sections were then fixed
for 1 h in 2% osmium tetroxide, dehydrated through a graded series of
ethanols and propylene oxide, and embedded in Epon 812 between two
sheets of Aclar plastic. Ultrathin sections (50 nm) were cut from the Epon-
tissue interface, collected on copper grids, counterstained with 5% uranyl
acetate and Reynold's lead citrate, and examined with an electron microscope (model 201; Philips Electron Optics, Mahwah, NJ).
Northern Analysis
Total RNA was prepared from Schwann cells, O-2A progenitors, and oligodendroglia (consisting of a mixed population of O4+/O1 Caspr and Contactin Are Distinctly Expressed by
Neurons and Myelinating Glia
We first determined the expression patterns of contactin/
F3/F11 (contactin) and Caspr using well-characterized antibodies that recognize the extracellular and the cytoplasmic
domains of these proteins, respectively. Purified cultures
of sensory neurons, Schwann cells, oligodendrocyte progenitors, and differentiated progenitors (consisting of both
O4+/O1
Myelination Regulates the Expression and Distribution
of Caspr
We next examined contactin and Caspr expression in established cocultures of Schwann cells and DRG neurons
(Fig. 2). These cultures contained significant numbers of
myelinated fibers as visualized by staining for myelin basic
protein (MBP), which is a component of compact myelin
(Fig. 2, B and D). Contactin (Fig. 2 A) is significantly downregulated in the cocultures in both ensheathed and myelinated fibers compared with isolated primary neurons. Contactin continues to be expressed at relatively high levels on
the occasional unensheathed neurite that persists in such
cultures (Fig. 2 A, asterisks). These results indicate that direct Schwann cell contact is responsible for the downregulation of contactin expression.
Caspr expression is also significantly reduced in the cocultures (Fig. 2 C). Of particular note, there is a dramatic
change in its distribution, with very high levels present at
the ends of the myelin sheaths. This concentration of Caspr
is located just beyond the compact myelin sheath (as visualized by MBP staining) in the region of the paranodes. In
the case of isolated myelin segments, Caspr is present at
either end of the myelin segment (Fig. 2, C and D, arrowheads). Where two myelin segments approach each other
to form a node, Caspr staining appears as a doublet. Two
representative nodes are indicated by the arrows in Fig.
2, C and D, and a higher power view of the node in the
center of the field is shown in the inset. Of note, the Caspr
staining is found within the gap of MBP staining but does
not appear to extend into the node itself.
Caspr Is Downregulated with Myelination and Is
Associated with a Detergent Insoluble Fraction
To assess more accurately the relative levels of expression
of these proteins in the cultures, we performed Western
blotting using 125I-labeled protein A as a reporter (Fig. 3).
We compared culture lysates (50 µg) of neurons, Schwann
cells, and Schwann cell/neuron cocultures after 1 or 3 wk
in myelinating conditions; lysates (25 µg) of oligodendrocyte progenitors and differentiated O4+ oligodendroglia
were also analyzed (Fig. 3 A). Caspr migrated with an expected molecular mass of ~190 kD (Fig. 3 A, upper panels); in several experiments we also observed a minor band
of ~50 kD that may be a proteolytic fragment (data not
shown). Consistent with immunostaining results, DRG
neurons expressed Caspr at robust levels, whereas it was
undetectable in Schwann cell and oligodendrocyte lysates.
Caspr expression was greatly reduced in the cocultures compared with the neurons (more than 10-fold in several
experiments). A progressive reduction in Caspr levels was
also noticeable between 1 and 3 wk of coculture.
In parallel, we examined whether Caspr might be part of
a cytoskeleton-enriched, detergent-insoluble complex. We
extracted neuron cultures and myelinating cocultures (at
4 wk) with 1% Triton X-100 (Fig. 3 A, lanes T), solubilized
the remaining material with SDS (Fig. 3 A, lanes S), and
analyzed both fractions by Western blotting (Fig. 3 A, top
right). Only ~25% of the total Caspr in the neuron cultures was extracted by Triton (Fig. 3 A, compare lane DRG:T
to DRG:S). In the myelinating cocultures, less than 10%
was extracted by Triton ( Fig. 3 A, compare lane 3 wk:T to 3 wk:S), suggesting that even more of the Caspr is part of a
detergent-insoluble complex. In related studies, we have
found that Caspr staining persists in the cocultures after
Triton extraction, whereas MBP and MAG are completely
removed (data not shown). Likewise, extraction of brain
membrane fractions with a variety of nonionic detergents (e.g., Triton, Brij, digitonin, octylglucoside) released less than half of the total Caspr in each case (data not shown).
These results indicate that Caspr is associated with a detergent-insoluble, cytoskeleton fraction in vitro and in vivo.
We also examined contactin expression by Western blotting. Contactin is expressed by neurons and oligodendrocytes, but not by Schwann cells, and is similarly progressively downregulated in the cocultures (Fig. 3 A, bottom).
Of note, contactin is detected as a doublet on neurons and
as a single band on oligodendrocytes. We had previously
found that the upper band of this doublet is removed by
phosphatidylinositol phospholipase C, whereas the lower band is phosphatidylinositol phospholipase C-resistant
and is likely a preform of contactin (Rosen et al., 1992 A Northern blot for contactin (Fig. 3 C) confirms its
high level expression in oligodendroglia; a major band of
~6.4 kb and minor bands between 3-4 kb were present,
consistent with previous reports (Gennarini et al., 1989 Redistribution of Caspr during Myelination
To investigate further the mechanisms by which Caspr becomes concentrated at the paranodes, we have analyzed its
distribution at different times of myelination in the coculture system (Fig. 4). Schwann cells were seeded onto preestablished networks of DRG neurites and maintained for
several days in a defined media in which Schwann cells proliferate but do not ensheathe or myelinate nerve fibers.
Ascorbic acid was then added (day 0) to initiate ensheathment and myelination. Cultures were fixed at various times thereafter and double stained for Caspr and MAG, a myelin-specific protein that is expressed at the onset of myelination (Owens and Bunge, 1989
New myelin sheaths begin to form on days 3 to 4 in the
cocultures and can be detected by their expression of MAG
(Fig. 4 B, asterisks). Caspr is initially diffusely expressed
along the entire neurite at this time, although at somewhat
reduced levels compared with pure populations of neurons
(Fig. 4 A). Interestingly, in some instances there appeared
to be a slight increase in Caspr expression under some of
the forming segments of myelin (data not shown). By 6-7 d,
myelin sheaths have begun to compact, as indicated by the
redistribution of MAG into the periaxonal glial membrane, Schmidt-Lanterman incisures and the paranodal
loops. At the same time, Caspr began to accumulate into
the paranodes of a few myelinated segments. (Examples of
nascent paranodes are indicated by the arrowheads in Fig.
4 C.) Of additional note, Caspr expression frequently appeared attenuated at the center of corresponding internodes.
By 11 d, there is a striking accumulation of Caspr into
multiple paranodes (Fig. 4, E and F). Concentrations of
Caspr were invariably associated with well-myelinated segments, particularly those associated with MAG-positive
Schmidt-Lanterman incisures, and accumulated into both
paranodes of a myelinated segment at approximately the
same time. Expression in the internode was reduced in
those segments containing concentrations of Caspr at the
paranodes compared with other segments that were still in
the process of myelination. Two examples are indicated by
arrows in Fig. 4, E and F, which mark paranodal accumulations. It may be seen that the mature myelin internodes located above the arrows express less Caspr than the two nascent myelin internodes located below the arrows. Taken
together, these results indicate that Caspr accumulation in
the paranodal region is a late event that occurs with myelin compaction and maturation and is likely to reflect a redistribution from the internode.
Caspr Is Concentrated in Paranodes in the CNS
and PNS
To determine whether Caspr is localized at the paranodes
of myelinated fibers in the PNS and CNS in vivo, whole
mounts of teased rat sciatic nerve fibers and sections through
various regions of the rat brain were immunolabeled with
anti-Caspr antibody and examined by light microscopy. Intense paranodal staining for Caspr was observed in sciatic
nerve fibers (Fig. 5 A), and virtually all of the fiber tracts
in the brain, including the large myelinated axons of the
facial nerve (Fig. 5 B) and the small, thinly myelinated axons of the corpus callosum (Fig. 6 A). Minimal staining, if any at all, was evident in the internodes. Paranodal staining was not observed with the corresponding control preimmune serum (data not shown). Although the protein
appeared to be absent from the outer surfaces of the paranodal loops, it could not be determined at the light microscopic level whether Caspr immunoreactivity was associated
with the inner surfaces of the paranodal loops adjacent to
the axon, the axonal membrane, or with both. More precise localization of the protein was resolved by immunoelectron microscopy (see below).
In addition to staining of the fiber tracts, low to moderate levels of diffuse Caspr immunoreactivity were present
throughout the neuropil of the brain. An example of such
diffuse staining in the lateral septal nucleus is shown in
Fig. 6 A (SN). Considerable punctate staining, most likely
representing paranodal regions of small myelinated fibers
but possibly representing other neuronal structures, was
found throughout the neuropil. In general, neuronal cell
bodies in most regions of the brain examined were either unlabeled or only lightly labeled by the Caspr antibody.
The distribution of contactin immunoreactivity in brain
sections was also examined by light microscopy (Fig. 6 B).
In contrast to Caspr, and consistent with the in vitro studies described above, contactin was not concentrated at
paranodal regions of myelinated fibers in these sections.
However, relatively strong contactin staining of cells, possibly interfascicular oligodendrocytes, was observed in fiber tracts such as the corpus callosum (Fig. 6 B inset, arrows). Light to moderate contactin immunostaining was
also prominent in many neurons distributed throughout
the brain, such as those of the cerebral cortex (Fig. 6 B).
Caspr Is Localized to the Septate-like Junctions of
the Paranodes
To determine more precisely the localization of Caspr
within the paranode and to distinguish whether it was associated with neuronal and/or glial cell membranes, the
distribution of the protein in the corpus callosum and the
facial nerve (within the pons) was examined by immunoelectron microscopy. The results of these studies confirmed the light microscopic observations that Caspr is
primarily concentrated at the paranodal regions of myelinated fibers. Furthermore, they showed that the protein is
a component of the axonal membrane and not that of the
paranodal loops of the glial cell. Representative nodal regions of myelinated fibers in the corpus callosum showing
the silver-enhanced immunogold particles denoting the distribution of Caspr are shown in Fig. 7, A-C. Similar patterns of paranodal labeling were observed in sections of
the facial nerve (data not shown). Most of the immunolabeling at nodal regions was restricted to the inner surface
of the axonal membrane containing the septate-like junctions. An example of the characteristic septae of these junctions are shown at high magnification in the inset of Fig. 7 A.
This staining of the inner membrane surface is expected
given the reactivity of the antibody with cytoplasmic determinants on Caspr. In favorable planes of section, such
as that shown in Fig. 7 B, strikingly intense immunolabeling was observed along the presumptive inner surface of
the paranodal axonal membrane. Importantly, virtually no
labeling was observed in the nodal gap itself and only occasional particles were detected in the axonal membrane
or cytoplasm in the internodes of myelinated fibers. Taken
together, these results indicate that Caspr is likely to be a
component of the septate-like junctions at the paranodal region.
In addition to its concentration at the paranodal region
of myelinated fibers, rather intense Caspr immunogold labeling was also observed around the inner surfaces of axonal membranes of some small-diameter nerve fibers that
appeared to be unmyelinated. Examples of such fibers
from the corpus callosum cut in cross section are shown in
Fig. 7 D (u). These labeled profiles are unlikely to represent cross sections through the paranodal regions of myelinated fibers because they appear to lack the increased number of microtubules in the axon or surrounding myelin
lamellae characteristic of this region (Peters et al., 1991 We have shown that contactin and Caspr, which are both
diffusely expressed on unensheathed nerve fibers, have very
different fates during myelination. Contactin is nearly undetectable on myelinated fibers, whereas Caspr becomes
highly concentrated in the paranodal region. These studies
indicate that, although contactin and Caspr interact laterally (i.e., exhibit cis interactions) when they are in the same
membrane in other settings (Peles et al., 1997b Contactin Expression during Myelination
In characterizing the expression of contactin, we have confirmed a recent report that oligodendrocytes express this
protein in culture (Koch et al., 1997 In contrast, Schwann cells do not express contactin based
on immunofluorescence, Western, and Northern analyses.
These results differ from those of a recent report demonstrating expression of F11, the avian homologue of contactin/F3, in chick Schwann cells in vitro and in sciatic nerve
(Willbold et al., 1997 Caspr Is the First Marker Specific for
Paranodal Junctions
The structure of the paranodal junctions that form between axons and the paranodal loops of oligodendrocytes
and myelinating Schwann cells were described over thirty
years ago (Bargmann and Lindner, 1964 Sequential Reorganization of the Axolemma
during Myelination
These studies emphasize that the organization of the axon
into three distinct longitudinal domains, i.e., the node, the
paranodal region, and the internode, occurs sequentially
during myelination. Previous studies have demonstrated
that initial events in the formation of the node, characterized by clustering of intramembranous particles and sodium channels, occurs at the onset of myelination before
compact myelin formation (for review see Black et al., 1995 The dramatic changes in the distribution of Caspr also
indicate that myelination is critical to this sequential reorganization. Caspr is uniformly expressed on the surface of
unensheathed DRG neurites. In the cocultures, Caspr remains diffusely expressed along the axon until later stages
of myelination (see Fig. 4); it is persistently expressed on
unmyelinated axons in the CNS in the adult (see Fig. 7 D).
As the myelin sheath matures, Caspr levels dramatically
increase in the paranodal region, accumulating in the paranodes of the most mature myelin sheaths first (see Figs. 2
and 4), consistent with morphologic studies of paranodal junction development (Tao-Cheng and Rosenbluth, 1982 Potential Role of Paranodal Junctions in Channel
Distribution and Signaling
It has been proposed that invertebrate septate junctions
serve several functions, including a role analogous to that
of tight junctions in providing a transcellular barrier to the
passage of solute molecules and in promoting the generation of apical and basal lateral compartments (Lane,
1991 With this report, we have shown that paranodal junctions not only resemble invertebrate septate junctions in
their morphology but also share homologous proteins. The
molecular characterization of septate junctions has progressed rapidly in recent years, largely from analysis of
Drosophila mutants. Invertebrate septate junctions have
been found to be associated with actin filaments and associated proteins (Lane and Flores, 1988 While it is not yet known whether Caspr is similarly
linked to any band 4.1 superfamily members, current evidence supports its association with the axon cytoskeleton.
The spacing between the septae at the paranodes is highly
regular (25-30 nm and see Fig. 7 A, inset) (Peters et al.,
1991 The structure of the cytoplasmic region of Caspr suggests
it may also regulate intracellular signaling pathways in axons. Notably, this segment contains a proline-rich sequence
with at least one canonical SH3 domain-binding site (Peles
et al., 1997b An important question is the identity of the glial ligand(s)
for Caspr and whether Caspr regulates glial function.
Freeze fracture studies have demonstrated that there are
glial particles within the paranodal loops that are juxtaposed over axolemmal particles in the paranodal junctions
(Wiley and Ellisman, 1980 Caspr is likely to have other functions beyond its role in
paranodal junctions. We have observed diffuse, high level
expression in gray matter (see Fig. 7) not restricted to
paranodes. This may reflect its putative role as a signaling
coreceptor with contactin (Peles et al., 1997b Caspr and Other Neurexins Are Novel Candidates to
Mediate Axon-Glia Interactions
The neurexins comprise a large family of neuronal surface
proteins that are candidate mediators of synaptic interactions and, potentially, target recognition (Ushkaryov et al.,
1992 In summary, we have shown that the expression and distribution of Caspr and contactin are differentially regulated
during myelination. Of particular interest is the localization
of Caspr to the septate-like junctions of the paranode. This
is the first component of these vertebrate junctions to be
identified and this, together with its homology to Drosophila Neurexin IV, suggests that it is a major transmembrane component required for their integrity. These findings
have important implications for the identity of other proteins that are associated with Caspr in these junctions and for
the potential role of Caspr in reciprocal axon-glia signaling.
).
This mode of conduction requires the organization of myelinated fibers into longitudinal domains that are anatomically and functionally distinct. Three domains
the internode, the paranodal region, and the node of Ranvier
form as the result of, and can be distinguished by, specific
interactions between axons and myelinating glial cells, i.e.,
Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system (Peters et al., 1991
; Salzer, 1997
). In the internode, the most stereotypic
portion of myelinated fibers, the axon is separated from
the inner glial membrane by a regular space of 12 nm or
more and is surrounded by a compact myelin sheath. In
the paranodal region, the compact myelin lamellae open
up into a series of cytoplasmic (paranodal) loops that spiral around and physically invaginate the axon. These glial
loops are closely apposed to the axon, being separated by a gap of only 2.5-3 nm, and form a series of septate-like
junctions with the axon (shown schematically in Fig. 8). In
electron micrographs of longitudinal sections through the
paranodal region, these junctions appear as a series of ladder-like densities that arise from the outer leaflet of the
axolemma and contact the glial membranes. At the nodes,
which represent gaps between the myelin sheaths, the
axon is relatively exposed to the extracellular environment. In larger myelinated fibers, Schwann cell microvilli and astrocytic processes are also closely associated with the nodal
axolemma in the peripheral and central nervous system
(PNS and CNS)1, respectively.
Fig. 8.
Schematic structure of the nodal region and the location of Caspr. The longitudinal organization of a myelinated axon
at the node of Ranvier is shown. Myelinated axons contain three
distinct domains: the internode, where the axon is surrounded by
a compact myelin sheath; the paranodal region, where the axon is
invaginated by and forms septate-like junctions with paranodal
loops of the glial cell; and the node itself. The location of Caspr in
the axon membrane is illustrated.
[View Larger Version of this Image (42K GIF file)]
). Delayed rectifier potassium channels are enriched in the juxtaparanodal regions, where they may
contribute to repolarization and ionic homeostasis (Wang
et al., 1993
; Mi et al., 1995
). By contrast, the internode,
which exhibits a reduced capacitance, has significantly lower
concentrations of these voltage-gated channels. The mechanisms that regulate the distinct distributions of voltage-gated channels along the axon are not well understood. Paracrine and juxtacrine signals from glial cells appear to
initiate clustering of sodium channels and other nodal
components (Kaplan et al., 1997
; for review see Salzer,
1997
). The paranodal junctions may also contribute by
providing a physical barrier that prevents lateral diffusion
of ion channels and thereby separates these distinct domains (Rosenbluth, 1976
; Gumbiner and Louvard, 1985
).
), and connexin 32, which is a component of
the gap junctions that form between these glial processes
(Scherer et al., 1995
). The myelin-associated glycoprotein
(MAG), which is enriched in the inner glial membrane and
appears to promote adhesion to the axon in the internode,
is enriched in paranodal loops in the PNS but not the CNS
(for review see Quarles et al., 1992
). Its function in the
paranodal loops of Schwann cells is presently unclear. Several Ig-related cell adhesion molecules (CAMs) on the
axon, including specific isoforms of neurofascin and NrCAM, are concentrated at the node of Ranvier, rather
than in the paranodes, and may have a role in sodium channel localization (Davis et al., 1996
).
; Koch et al., 1997
). Contactin/F3/F11 is a GPI-anchored neural Ig CAM that promotes nerve fiber outgrowth. It was recently shown to be expressed by Schwann
cells in the chick (Willbold et al., 1997
) and by rat oligodendrocytes (Koch et al., 1997
), although characterization
of its expression by these cells in vivo, including its distribution along myelinated fibers, is still incomplete.
).
Caspr is a type I integral membrane protein with a molecular mass of 190 kD that is highly expressed in the CNS; it
is also present at reduced levels in several extraneuronal
tissues (Peles et al., 1997b
). It copurifies with contactin
when the carbonic anhydrase domain of the receptor protein tyrosine phosphatase
is used as an affinity ligand.
Contactin and Caspr also coimmunoprecipitate, suggesting
that they are constitutively complexed. The extracellular domain of Caspr contains a series of laminin G-like and
EGF-like domains characteristic of the neurexins; accordingly, Caspr is a member of the neurexin superfamily. The
cytoplasmic segment of Caspr contains potential binding
sites for SH3 domain-containing proteins and band 4.1 proteins but lacks the carboxy-terminal motif found in
other neurexins that binds to PDZ domains. Of interest, the Drosophila homologue of Caspr, Neurexin IV (Baumgartner et al., 1996
), which has a similar extracellular domain
organization, and a 30% amino acid identity (Peles et al.,
1997a
; Littleton et al., 1997
), is expressed by glial cells and
is a component of their septate junctions that compose the
blood-nerve barrier (Baumgartner et al., 1996
).
Materials and Methods
).
) with minor modifications. Neonatal rat Schwann cells were maintained in standard media consisting of
DME (Whittaker Bioproducts, Inc., Walkersville, MD), 10% FBS (HyClone Laboratories, Inc., Logan, UT), and 2 mM glutamine (Life Technologies, Gaithersburg, MD) and were amplified in standard media supplemented with 5 ng/ml GGF2 (Cambridge Neurosciences, Cambridge,
MA) and 4 µM forskolin (Sigma Chemical Co., St. Louis, MO). To examine Caspr and contactin expression by immunocytochemistry, 50,000 Schwann cells were plated onto poly-L-lysine-coated 12-mm coverslips
and kept in standard media for a minimum of 3 d before fixation.
5 M)
(Sigma Chemical Co.), which were added to the standard neuronal media
in alternate feedings to eliminate nonneuronal cells.
). To examine Caspr and contactin expression on oligodendrocytes by immunocytochemistry, 50,000 O-2A cells
were plated onto poly-L-lysine-coated Lab-Tek chamber slide wells (Fisher
Scientific, Pittsburgh, PA) and kept in DM+ media for 3 d before fixation.
). Sciatic
nerve lysates were prepared from nerves immersed in liquid nitrogen after
their dissection (Einheber et al., 1993
). Protein concentrations of the lysates were determined by the Micro BCA method (Pierce Chemical Co.,
Rockford, IL). Lysates were subjected to SDS gel electrophoresis and
blotted onto nitrocellulose. Blots were first probed with primary antibody
against Caspr followed by 125I-labeled protein A (Amersham Corp., Arlington Heights, IL). Appropriate regions of the blots were cut out and reincubated with antibodies to contactin/F3 and 125I-protein A. The blots
were then scanned and quantitated on a Molecular Dynamics (Sunnyvale,
CA) phosphorimager.
) with minor modifications. Cultures grown on coverslips
were washed with dPBS (Life Technologies), fixed with 4% paraformaldehyde in dPBS for 15 min, washed in dPBS, and then permeabilized with
100% methanol at
20°C for 15 min. The coverslips were then washed
with dPBS and blocked for 30 min with Leibovitz's L-15 media (Life Technologies) supplemented with 10% heat-inactivated FBS. The coverslips
were incubated for 1 h at room temperature with primary antibodies diluted in the blocking solution, washed three times with blocking solution,
and incubated for 1 h at room temperature with species-specific affinity-purified rhodamine-conjugated donkey anti-rabbit IgG or fluorescein-conjugated donkey anti-mouse IgG (Chemicon International, Inc., Temecula, CA) diluted 1:100 in the blocking solution. The coverslips were washed five times, mounted in Citifluor (Ted Pella, Inc., Redding, CA) on
glass slides, and examined by epifluorescence microscopy.
). Primary antibodies bound to
the tissue were detected using the avidin-biotin complex (ABC) peroxidase method and the chromagen 3,3
diaminobenzidine (Aldrich Chemical
Co., Milwaukee, WI) (Einheber et al., 1996
).
). Briefly, immunolabeled tissue was incubated for 2 h in 1:50 dilution
of 1-nm colloidal gold-conjugated goat anti-rabbit IgG (Amersham
Corp.), postfixed in 2% glutaraldehyde in PBS for 10 min, and reacted
with silver solution using the intenSEM kit (Amersham Corp.).
and O1+
cells), rat brain, and postnatal sciatic nerves by CsCl2 gradient centrifugation (Chirgwin et al., 1979
). Equal samples (10 µg) of total RNA were
electrophoresed in 1% agarose, 2.2 M formaldehyde gels, transferred to
nylon membranes (Duralon; Stratagene, La Jolla, CA) in 6× SSC, and
UV cross-linked. Blots were prehybridized, hybridized, and washed using
standard techniques; the final stringency of the wash was 0.2× SSC at
65°C for 30 min. cDNAs used as probes included an ~500-bp fragment to
rat Caspr and an ~1.5-kb fragment to human contactin as described (Peles
et al., 1997b
). 32P-labeled cDNA probes with specific activities of 2-5 × 109 cpm/mg were prepared by primer extension with random hexamers using the Prim-a-gene kit (Promega Corp., Madison, WI) according to the
manufacturer's instructions.
Results
prooligodendrocytes and O1+ oligodendrocytes)
were prepared, and expression was analyzed by immunofluorescence (Fig. 1). Contactin is expressed robustly by
neurons (Fig. 1 A), by oligodendrocytes (Fig. 1 E), and by their progenitors but not by Schwann cells (Fig. 1 C). By
contrast, Caspr expression is restricted to neurons and their
processes (Fig. 1 B); no staining of Schwann cells (Fig. 1 D)
or of cells in the oligodendrocyte lineage (Fig. 1 F) was observed. Both Caspr and contactin are diffusely distributed
along the entire surface of neurons and their processes.
Fig. 1.
Expression of contactin and Caspr by neurons,
Schwann cells and oligodendrocytes. Primary cultures of sensory
neurons (A and B), Schwann cells (C and D), and oligodendrocytes (E and F) were stained with antibodies to contactin (A, C,
and E) and Caspr (B, D, and F). Bar, 50 µm.
[View Larger Version of this Image (156K GIF file)]
Fig. 2.
Contactin and
Caspr expression in myelinating cocultures. Schwann
cells were added to cultures
of dissociated sensory neurons and allowed to repopulate the neurites. Ascorbic
acid and serum were added
to promote myelination, and
cultures were fixed after an
additional 3 wk and stained
for contactin (A) and Caspr
(C). Corresponding fields (B
and D) were stained for
MBP. Asterisks in A indicate
an unensheathed fiber that
expresses contactin at high
levels. Arrows in C and D indicate nodes of Ranvier; arrowheads indicate isolated
paranodes. The insets in C
and D show the node in the
center of the field at higher
power. Bar, 50 µm.
[View Larger Version of this Image (17K GIF file)]
Fig. 3.
Immunoblot and Northern analysis of contactin and
Caspr expression. (A) Caspr and contactin expression in cultured
cells and association of Caspr with the detergent insoluble complex. 50 µg of protein lysate prepared from neuron (DRG),
Schwann cell (SC), 1-wk-old, and 3-wk-old myelinating cocultures and 25 µg of lysate from oligodendrocyte progenitors (O-2A) and O4+ oligodendroglia (Oligo) were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with antiserum
specific for Caspr (top left) or contactin (bottom). The top right of
A is an immunoblot of samples prepared from DRGs and 3-wk-old
myelinating cultures extracted with Triton X-100 (T) and SDS (S)
probed with the Caspr antiserum. (B) Changes in Caspr and contactin expression during postnatal sciatic nerve development. 100 µg of protein lysate, prepared from sciatic nerves of rats at postnatal days 1, 3, 7, 11, 15, 21, 25, 30, 40, and adult (A) were separated by SDS-PAGE, blotted onto nitrocellulose, and probed
with either Caspr (B, top) or contactin (B, bottom) antisera. Note
that the amounts of Caspr and contactin detected in sciatic nerve
were considerably smaller than that in 100 µg of hippocampal lysate (Hc). (C) Northern blot analysis of contactin expression. A
Northern blot with 10 µg per lane of total RNA isolated from
postnatal day 1, 6, 10, 15, 34, and adult sciatic nerves, total rat
brain (CNS) and cultured O-2A progenitors, O4+ oligodendroglia, and Schwann cells (Sc) was probed with a 32P-labeled human
contactin cDNA probe. Bars indicate the position of 28S and 18S
rRNA.
[View Larger Version of this Image (65K GIF file)]
). As
shown in Fig. 3 B, Caspr and contactin expression also significantly decrease during postnatal sciatic nerve development (day 1 through adulthood). At all times, they are
present at substantially reduced levels in sciatic nerves when
compared with hippocampus (Fig. 3 B, lane Hc) used as a
CNS control.
).
No expression of contactin was detected in Schwann cells or
at varying times of sciatic nerve development. By contrast,
Caspr mRNA of the expected size (6.2 kb) was detected in
the CNS control but not in glia or sciatic nerve samples
(data not shown). These results are consistent with the
protein studies described above and indicate that Caspr
and contactin in sciatic nerve are exclusively of axonal origin.
).
Fig. 4.
Redistribution of
Caspr during myelination in
vitro. Schwann cells were
added to cultures of dissociated sensory neurons and allowed to repopulate the neurites. Ascorbic acid and
serum were added to promote myelination, and cultures were fixed after an additional 4 d (A and B), 6 d (C
and D), or 11 d (E and F).
Cultures were immunostained for Caspr (A, C, and
E) and MAG (B, D, and F).
At 4 d, a few MAG-positive,
nascent myelin segments
were present (three are indicated with asterisks in B).
Caspr expression continues to be expressed in the underlying axolemma (A, asterisks). 6 d after adding ascorbate, Caspr has begun to
concentrate in some of the
forming paranodes (C, arrowheads) associated with
maturing myelin sheaths and,
at the same time, is becoming
attenuated in the corresponding internode. By 11 d,
numerous myelin segments are present, and MAG staining in the Schmidt-Lanterman incisures is apparent
(F). Caspr is concentrated in
multiple paranodes and is
substantially reduced in the
internodes of mature myelin
segments (located above the
arrows in E), whereas it continues to be abundant in the
internodes of nascent myelin
segments (located below the arrows in E). Bar, 25 µm.
[View Larger Version of this Image (22K GIF file)]
Fig. 5.
Caspr is concentrated in the paranodal regions of myelinated fibers in
the PNS and CNS. A teased
fiber preparation of adult sciatic nerve (A) and a coronal
section through the facial
nerve in the pons (B) were
stained with an antiserum
against Caspr using the immunoperoxidase technique
and visualized by Nomarski
(A) or brightfield microscopy
(B). Low-magnification photomicrographs show that
Caspr immunoreactivity is
essentially restricted to the
paranodes in both PNS and
CNS fibers. Higher magnifications of individual nodes
are shown in the insets. Bar,
20 µm.
[View Larger Version of this Image (147K GIF file)]
Fig. 6.
Contactin/F3 is not concentrated in the paranodal region. Staining of the corpus callosum (cc) with antisera against
Caspr and contactin/F3 is shown. Caspr staining (A) is concentrated in the paranodal regions of the small myelinated fibers of
the corpus callosum (shown at higher magnification in the inset). Contactin/F3 immunoreactivity is concentrated in cells that may be interfascicular oligodendrocytes based on their morphology (arrows in the higher-magnification inset) and location. Minimal contactin/F3 immunoreactivity was apparent in the paranodal regions. Moderately intense but diffuse Caspr immunoreactivity in
the lateral septal nucleus (SN in A) and light contactin/F3 staining of cells in the cortex (Ctx in B), some of which appear to be
neurons, were also apparent. Bar, 100 µm.
[View Larger Version of this Image (152K GIF file)]
Fig. 7.
Immunoelectron
microscopic localization of
Caspr in myelinated and unmyelinated nerve fibers of
the corpus callosum. Representative longitudinal sections of nodal regions of myelinated fibers with silver
enhanced immunogold particles denoting the distribution of Caspr are shown in A-C.
Gold particles were concentrated along the inner surface
of the axonal membrane beneath the septate-like junctions located in the paranodal
region (A-C). The inset in A
shows the septate-like junctions that form between the
axon and paranodal glial
loops at higher magnification.
Extremely dense labeling was
observed where the plane of
section approached the inner surface of the axonal
membrane at the paranodes
(pn; B and at higher magnification of the same field in C).
Gold particles were occasionally found associated with the
axonal membrane or cytoplasm in internodal portions
of the myelinated fibers (B
and C) but were rarely seen
in the node (N). (D) A cross
section demonstrating labeling of small caliber axons
that are unmyelinated (u).
Bars, 0.5 µm.
[View Larger Version of this Image (215K GIF file)]
).
Furthermore, these profiles were often present in clusters
within the callosum and much more frequent than would
be expected for paranodes, which are quite short in comparison to the length of nerve fibers. These findings suggest that unmyelinated or ensheathed axons in the CNS
may continue to express considerable amounts of Caspr
and are consistent with the in vitro studies above, which
showed that neurons grown in the absence of Schwann cells
or those during the early stages of myelination express significant levels of this protein.
Discussion
), they have
distinct roles during myelination and are not obligately associated. These findings also implicate Caspr as a major
component of the septate-like paranodal junctions. The
expression of contactin and the localization of Caspr are considered further below.
). This expression by
oligodendrocytes and their progenitors could promote the
initial interactions of axons and oligodendrocytes, possibly
via a homophilic mechanism. Contactin also interacts with
other ligands, notably receptor protein tyrosine phosphatase
(Peles et al., 1995
), which is expressed at high levels
by radial glial cells and oligodendrocyte progenitors (Canoll
et al., 1996b
). Expression of contactin may therefore promote homotypic interactions between progenitors or heterotypic interactions with radial glia on which they appear to migrate (Zerlin et al., 1995
). Interestingly, although contactin is robustly expressed by oligodendrocytes and their
progenitors in vitro and by what appear to be interfascicular oligodendrocytes in vivo (Fig. 6 B, inset), there is little
expression in white matter tracts or compact myelin. Consistent with these findings, other studies have reported contactin expression by neurons and their processes, including
occasional axons in white matter, but not in myelin itself
(Gennarini et al., 1989
; Faivre-Sarrailh et al., 1992
). Contactin was recently detected in myelin fractions by biochemical methods (Koch et al., 1997
), potentially reflecting the presence of oligodendrocyte membranes in these
myelin fractions or, alternatively, a greater sensitivity of
the blotting techniques used.
). This may reflect differences in contactin expression between avian and mammalian species.
We have also found that contactin expression on nerve fibers dramatically declines during postnatal sciatic nerve
development, a period of rapid myelination, and during
myelination in vitro. This expression pattern resembles
that of other adhesion molecules on axons, including
NCAM and L1, that are also downregulated during the
transition from ensheathment to myelination (Martini and
Schachner, 1986
; for review see Salzer, 1995
). Contactin
appears to be persistently expressed at low levels on unensheathed nerve fibers in the cocultures (see Fig. 2), indicating that downregulation depends on direct Schwann
cell contact.
; Andres, 1965
),
but their molecular composition has remained unknown.
A major finding of this study is the identification of Caspr
as the first known marker of these junctions (shown diagrammatically in Fig. 8). Caspr seems likely to be a major
transmembrane component of these junctions as well. Previous ultrastructural studies demonstrated that septate junction components arise from the axon, extending ~3-5 nm
to contact the glial membranes of the paranodal loops (Peters et al., 1991
). Freeze fracture studies have revealed circumferential rows of intramembranous particles within the
axolemma at these junctions (Wiley and Ellisman, 1980
),
potentially corresponding to Caspr. The time course of the
focal accumulation of Caspr at these sites, i.e., at later
stages of myelination, agrees well with earlier studies demonstrating that septate junctions develop with maturation
of the myelinated fiber (Tao-Cheng and Rosenbluth, 1982
).
Of particular note, Drosophila Neurexin IV, a homologue of Caspr, was recently shown to be an essential component
of the septate junctions that form between specialized glial
cells (Baumgartner et al., 1996
); Neurexin IV mutants lack
the transverse septae characteristic of these junctions. Taken
together, these results strongly support the notion that
Caspr is an integral membrane component of the septate-like junctions at the paranodes.
;
Salzer, 1997
) and can even occur in its absence (Deerinck et al., 1997
). In contrast, paranodal junctions form during
later stages of myelination, after the node has begun to
mature, and require the formation of appropriately compacted myelin (Rosenbluth, 1983
). Consistent with the sequential development of these domains, we have found
that ankyrin G, a marker of the nodal cytoskeleton (Kordeli
and Bennett, 1991
), accumulates at the node several days before the accumulation of Caspr in the paranodes (Zanazzi,
G., V. Bennett, and J. Salzer, unpublished observations).
).
Caspr accumulation in the paranodes is accompanied by a
simultaneous decrease in the internode (see Fig. 4, C and
E). This decrease may reflect, in part, more rapid turnover
of Caspr in the internode during myelination (see Fig. 3 A),
as well as its redistribution to the paranodes. Interactions
with glial ligands and/or elements of the axon cytoskeleton
may target Caspr to the paranodal region. The association
of Caspr with a detergent insoluble fraction in neurons,
myelinating cocultures, and brain membrane fractions as
well as specific sequence motifs of its cytoplasmic domain (discussed below) are consistent with the latter possibility.
). In strong support of this notion, the blood-nerve
barrier that forms between the specialized glial cells is disrupted in a Drosophila Neurexin IV mutant (Baumgartner
et al., 1996
). The paranodal junctions of myelinated fibers
probably have a similar role in forming a partial physical
barrier. Studies in which electron-dense tracers were infused in peripheral nerves, for example, have demonstrated that these junctions provide a partial barrier to the diffusion of small ions and an absolute barrier for the passage of
large molecular weight compounds into the internode
(Hirano and Llena, 1995
). By retarding ionic movements,
these junctions are likely to promote more efficient saltatory
conduction. Paranodal junctions may also regulate the distribution of voltage-gated channels. Indirect evidence, including freeze fracture studies, suggests they physically demarcate the boundaries of the node by limiting the lateral
diffusion of Na+ channels, thereby confining them to the
node (Rosenbluth, 1983
). Thus, while the paranodal junctions may not be required for the initial clustering of Na+
channels, they may restrict the subsequent distribution of
these channels. Less is known about factors that regulate
the distribution of specific voltage-gated K+ channels
(Kv1.1 and Kv1.2), which are concentrated in the juxtaparanodal region (Wang et al., 1993
; Mi et al., 1995
). The
paranodal junctions are appropriately positioned to have a
similar role in limiting the lateral diffusion of K+ channels
but have not yet been directly implicated.
; Colombo et al.,
1993
). The localization of a second protein to Drosophila
septate junctions, band 4.1-Coracle (Fehon et al., 1994
),
provides a mechanism for this linkage. Band 4.1 is a prototype of a large family of cytoskeletal proteins that includes
the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins and several phosphatases (for review see Tsukita et al., 1997
). It functions as a link between transmembrane proteins and the underlying spectrin/actin cytoskeleton (Bennett and Gilligan, 1993
). It is of interest that Drosophila
Neurexin IV and Caspr both contain cytoplasmic sequences
homologous to those in glycophorin, a red blood cell protein,
that binds to the erythrocyte band 4.1 protein. Of additional
note, in Drosophila Neurexin IV mutants, band 4.1-Coracle
is no longer restricted to the septate junctions (Baumgartner et al., 1996
). These results provide strong support for a
direct interaction of Neurexin IV with band 4.1-Coracle.
), suggesting an organized structure that might be imposed by cytoskeletal associations. Indeed, previous ultrastructural studies have demonstrated a linkage between
membrane proteins of the axon and the underlying cytoskeleton at the paranodes (Ichimura and Ellisman, 1991
),
although the nature of the proteins has not yet been determined. Finally, we have shown (Fig. 3 A) that Caspr is
poorly extracted by nonionic detergents from neurons
alone and from myelinating cocultures and brain membrane preparations. Taken together, these results indicate
that Caspr is likely to be constitutively associated with the
axon cytoskeleton, possibly through a band 4.1 superfamily member. Such a linkage could regulate its targeting to
the paranodal junctions.
). Consistent with this suggestion, fusion proteins containing the SH3 domains of the signaling proteins
PLC
, src, fyn, and the p85 subunit of PI 3-kinase bound
to the cytoplasmic domain of Caspr (Peles et al., 1997b
). It
is not yet known whether these proteins are associated
with paranodal junctions in vivo. However, this would be an
attractive possibility, as the paranodal region appears to
be a site of local axon-glia signaling. For example, the diameter of the axon in the nodal region is dramatically reduced (up to 80% in large fibers). This reduced diameter
reflects alterations in the level of phosphorylation and the
packing density of axonal cytoskeletal proteins (Waegh et
al., 1992). The rate of axonal transport is also significantly
diminished in the paranodal region (Waegh et al., 1992;
Fabricius et al., 1993
), which is consistent with local signaling. Whether proteins that are linked to Caspr mediate
these effects or not will be an important area for future investigation. An additional signaling mechanism in invertebrate septate junctions may be mediated by the PDZ binding domain of Neurexin IV, which is also shared by other
neurexins (for review see Littleton et al., 1997
). Notably,
Neurexin IV may interact with the discs-large tumor suppressor protein, a MAGUK protein that is found in septate junctions and has a role in cell signaling and differentiation (Woods and Bryant, 1991
). This interaction may be
unique to Drosophila Neurexin IV, as Caspr lacks a PDZ binding domain and several mammalian homologues of
discs-large have not been localized to the paranodes (Cho et
al., 1992
; Müller et al., 1995
; Hunt et al., 1996
).
). These glial proteins are candidate ligands for Caspr. To date, they have not been identified nor are any glial proteins known that have a complementary distribution to Caspr. Like all neurexins, the
extracellular segment of Caspr contains domains homologous to laminin G domains and EGF repeats (Littleton et
al., 1997
; Peles et al., 1997). In addition, it contains a number of unique motifs including a discoidin-related (lectin-like) domain, a fibrinogen-related sequence, and repeats
of proline, glycine, and tyrosine residues that may reflect
multiple ligands. A calcium-dependent ligand for specific
neurexin isoforms has been identified, termed neuroligin-1, but it is expressed by only a subset of neurons in the CNS
(Ichtchenko et al., 1995
). However, the adhesive integrity
of the paranodal junctions is also calcium dependent (Blank
et al., 1974
), perhaps indicative of calcium-dependent adhesion mediated by Caspr.
). In addition,
septate-like junctions have been noted in the vertebrate
central nervous system in other sites, including the pituitary (Kurono et al., 1994
) and cerebellum (Sotelo and
Llinás, 1972
; Laube et al., 1996
). It is not yet known
whether these junctions are enriched in Caspr. Finally,
Caspr is expressed at low levels in several other tissues, including the ovaries and a number of enteric viscera (Peles
et al., 1997b
), perhaps reflecting a wider distribution of
septate-like junctions or additional roles for this protein.
). A recent study suggests that other neurexins may
mediate axon-glia interactions. In particular, a pan-neurexin antibody, raised to a sequence present in neurexins I-III
but not Caspr, stained the electromotor nerve of the electric fish at the axon interface with myelinating Schwann cells
(Russell and Carlson, 1997
). Whether or not other neurexins, in addition to Caspr, also mediate axon-glia interactions in mammals will require further study. This pan-neurexin antibody also stained the perineurial cells of the
electromotor nerve, raising the possibility that neurexins
may play a more general role in the tight association of
cells in this layer. By Northern analysis, we have not detected Caspr mRNA in the sciatic nerve, suggesting it is not
expressed in the vertebrate perineurium.
Received for publication 21 August 1997 and in revised form 30 September 1997.
Address all correspondence to Dr. James L. Salzer, Department of Cell Biology, New York University Medical School, 550 First Avenue, New York, NY 10016. Tel.: (212) 263-5358. Fax: (212) 263-8139.We thank Dr. Josie Schlessinger (New York University Medical Center, New York) for his encouragement and support, Peter Canoll (New York University Medical Center, New York) and Dr. Randy McKinnon (Robert Wood Johnson Medical Center, Piscataway, NJ) for assistance with oligodendrocyte progenitor cultures and lysate preparation, Sabrina Prince (Cornell University Medical College, New York) for assistance with immunocytochemistry, Jody Culkin (New York University Medical Center, New York) for photographic assistance, and John Weider (New York University Medical Center, New York) for digital imaging assistance.
This study was supported by National Institutes of Health (NIH) grants NS26001 and NS33165 (J.L. Salzer) and HL18974, MH42834, and DA08259 (T.A. Milner). W. Ching is a Medical Scientist Trainee supported by NIH training grant 5T32 GM07308 from the National Institute of General Medical Sciences.
After this paper was submitted, the cloning and localization of paranodin, a protein identical in sequence to Caspr, was reported (Menegoz, M., P. Gaspar, M. Le Bert, T. Galvez, F. Burgaya, C. Palfrey, P. Ezan, F. Aruos, and J.-A. Girault. 1997. Neuron. 19:319-331).
CAM, cell adhesion molecule; Caspr, contactin-associated protein; CNS and PNS, central and peripheral nervous system; dPBS, Dulbecco's PBS; DRG, dorsal root ganglia; MAG, myelin-associated glycoprotein; MBP, myelin basic protein.
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