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 Previous Article | Next Article 
The Journal of Neuroscience, August 1, 2002, 22(15):6507-6514
A Myelin Galactolipid, Sulfatide, Is Essential for Maintenance of
Ion Channels on Myelinated Axon But Not Essential for Initial
Cluster Formation
Tomoko
Ishibashi1, 2, 3,
Jeffrey L.
Dupree4,
Kazuhiro
Ikenaka1, 3,
Yukie
Hirahara5,
Koichi
Honke6,
Elior
Peles7,
Brian
Popko8,
Kinuko
Suzuki9,
Hitoo
Nishino10, and
Hiroko
Baba2
1 Department of Physiological Sciences, The Graduate
University for Advanced Studies, Okazaki 444-8585, Japan,
2 Department of Molecular Neurobiology, School of Pharmacy,
Tokyo University of Pharmacy and Life Science, Hachioji 192-0392, Japan, 3 Laboratory of Neural Information, National
Institute for Physiological Sciences, Okazaki National Research
Institutes, Okazaki 444-8585, Japan, 4 Department of
Pathology and Anatomy, Eastern Virginia Medical School, Norfolk,
Virginia 23507, 5 Research Institute, Osaka Medical Center
for Maternal and Child Health, Izumi 594-1101, Japan,
6 Department of Biochemistry, Osaka University Graduate
School of Medicine, Suita 565-0871, Japan, 7 Department of
Molecular Cell Biology, The Weizmann Institute of Science, Rehovot
76100, Israel, 8 Neuroscience Center, Department of
Biochemistry and Biophysics, Program in Molecular Biology and
Biotechnology and 9 Department of Pathology and Laboratory
Medicine, University of North Carolina, Chapel Hill, North Carolina
27599, and 10 Department of Physiology, Nagoya City
University Medical School, Nagoya 467-8601, Japan
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ABSTRACT |
Myelinated axons are divided into four distinct regions: the node
of Ranvier, paranode, juxtaparanode, and internode, each of which is
characterized by a specific set of axonal proteins. Voltage-gated
Na+ channels are clustered at high densities at the
nodes, whereas shaker-type K+ channels are
concentrated at juxtaparanodal regions. These channels are separated by
the paranodal regions, where septate-like junctions are formed between
the axon and the myelinating glial cells. Although oligodendrocytes and
myelin sheaths are believed to play an instructive role in the local
differentiation of the axon to distinct domains, the molecular
mechanisms involved are poorly understood. In the present study, we
have examined the distribution of axonal components in mice incapable
of synthesizing sulfatide by disruption of the galactosylceramide
sulfotransferase gene. These mice displayed abnormal paranodal
junctions in the CNS and PNS, whereas their compact myelin was
preserved. Immunohistochemical analysis demonstrated a decrease in
Na+ and K+ channel clusters,
altered nodal length, abnormal localization of K+
channel clusters appearing primarily in the presumptive paranodal regions, and diffuse distribution of contactin-associated
protein along the internode. Similar abnormalities have been
reported previously in mice lacking both galactocerebroside and
sulfatide. Interestingly, although no demyelination was observed, these
channel clusters decreased markedly with age. The initial timing and
the number of Na+ channel clusters formed were
normal during development. These results indicate a critical role for
sulfatide in proper localization and maintenance of ion channels
clusters, whereas they do not appear to be essential for initial
cluster formation of Na+ channels.
Key words:
sulfatide; paranodal junction; myelin; node of Ranvier; Na+ channel; K+ channel
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INTRODUCTION |
Myelinated axons are divided at the
axonal surface into four discrete functional regions, including the
internode, juxtaparanodal and paranodal regions, and the node of
Ranvier (Peles and Salzer, 2000 ; Pedraza et al., 2001). The
voltage-gated Na+ channels are
concentrated at the nodes of Ranvier, whereas shaker-type Kv1.1 and
Kv1.2 K+ channels are localized primarily
at the juxtaparanodal regions. In addition, contactin-associated
protein (Caspr) and its binding partner, contactin, form a
complex at the paranodal junctions (Peles et al., 1997; Faivre-Sarrailh
et al., 2000; Rios et al., 2000) and may physically separate these two
channels by inhibiting lateral movement of these proteins.
Oligodendrocytes and/or the myelin sheath are believed to play an
instructive role in the discrete localizations of these channels;
however, the molecular mechanisms that mediate these distributions
remain largely undefined. During development, Caspr is first uniformly
expressed on the surface of axons, and later, as myelination occurs,
its distribution becomes restricted to the paranodal regions (Einheber
et al., 1997; Rasband et al., 1999). Then, the ion channels form
clusters sequentially. These observations suggest that Caspr may be a
key axonal molecule in the formation of the paranodal junction, as well
as the decision of channel localization. Support for this hypothesis
has come from recent studies using mice lacking either myelin
glycolipids (Dupree et al., 1999; Poliak et al., 2001), Caspr (Bhat et
al., 2001), or contactin (Boyle et al., 2001), in which dissociation of
the paranodal junctions and some abnormal distributions of ion channels
are displayed.
Mice generated with a disruption of the gene encoding UDP-galactose,
ceramide galactosyltransferase (CGT), showed disruption of paranodal
axo-glial junctions (Bosio et al., 1996; Coetzee et al., 1996; for
review, see Popko, 2000). Immunohistochemical studies revealed abnormal
distributions of K+ channels and Caspr in
the myelinated axons (Dupree et al., 1999; Poliak et al., 2001).
However, because this mutant lacks two major glycolipids,
galactocerebroside (GalC) and sulfatide, it is impossible to identify
the independent function of these two lipids in the paranodal junction.
More recently, mice incapable of synthesizing sulfatide but not GalC
through disruption of galactosylceramide sulfotransferase
(CST) gene have been generated (Honke et al., 2002).
These mice are normal at the time of birth, develop neurological deficits after 6 weeks of life, and survive to >1 year of age. Thus
the CST-deficient mouse is a useful tool for studying the function of
sulfatide in myelin independent of that of GalC.
In the present study, we address the following two questions: First,
does sulfatide have a unique function on the organization of axonal
domain formation? Second, are the axonal channel clusters stable under
the condition of such incomplete axo-glial junctions? To clarify these
questions, the distributions of paranodal proteins as well as ion
channels on the axons were examined in CST-deficient mice of different
ages. Our findings suggest that sulfatide is not necessary for initial
cluster formation but is an essential myelin component for the proper
localization of axonal proteins as well as the maintenance of these
proteins around the nodes.
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MATERIALS AND METHODS |
Antibodies. The polyclonal antibody against
Na+ channel was generated against a highly
conserved 18 aa segment (TEEQKKYYNAMKKLGSKK) between homologous domains
III and IV of the channel  -subunit. A mouse monoclonal antibody,
K58/35, against Na+ channel (anti-pan
Na+ channel antibody) was purchased from
Sigma (St. Louis, MO). The polyclonal and monoclonal antibodies against
K+ channel - and  -subunits (Trimmer,
1991) and a mouse monoclonal antibody, 28/43, against amino acids
77-299 of human postsynaptic density (PSD)-95 were kindly provided by
Dr. J. S. Trimmer (State University of New York, Stony Brook, NY)
and were used at a dilution of 1:100. The polyclonal anti-Caspr
antibody was used at a dilution of 1:2500 (Peles et al., 1997). A mouse
monoclonal antibody against myelin basic protein (MBP) was purchased
from Boehringer Mannheim (Mannheim, Germany). A rat monoclonal antibody
(AA3) against the C-terminal portion of myelin proteolipid protein
(PLP) (Yamamura et al., 1991) was kindly provided by Dr. M. Lees
(E. K. Shriver Center, Waltham, MA).
CST-deficient mice. The CST-deficient mice were created by
gene targeting (Honke et al., 2002). The targeting construct was designed to replace the exon portions that encode for the transmembrane domain and the 5'-PAPS-binding motif in the CST gene
with a neomycin-resistance cassette. Genotypes of pups from intercross
between heterozygous mice were determined by the PCR method using a
specific primer set. The mouse line was maintained in the animal
facility of the Osaka Medical Center for Maternal and Child Health
(Osaka, Japan).
Immunohistochemistry. Optic nerves and cervical (C3-C5)
spinal cords from homozygotes of CST-deficient mice as well as
wild-type controls were fixed by transcardinal perfusion with 4%
paraformaldehyde in 0.1 M phosphate buffer (PB),
pH 7.4, and the tissue was cryoprotected with 30% sucrose in PBS, pH
7.4, for 24 hr at 4°C. After embedding in optimal cutting
temperature (OCT)-mounting medium (Miles, Elkhart, IN), the
blocks were cut in 7-µm-thick sections. The sections were collected
on 3-aminopropyltriethoxysilane (Sigma)-rubbed glass slides and allowed
to air dry. After washing in PBS to remove OCT, the sections were
permeabilized for 2 hr in 0.1 M PB, containing 0.3% Triton X-100 and 10% goat serum, pH 7.4 (PBTGS). For
double-labeling experiments, sections were incubated overnight at 4°C
with primary antibodies diluted to appropriate concentration in PBTGS.
Then the sections were thoroughly rinsed in PBS, followed by
application of fluorescently labeled secondary antibodies for 40 min at
room temperature (RT). Secondary antibodies used in this study were FITC-labeled goat anti-rabbit IgG (1:400; Cappel Laboratories, Aurora,
OH) and tetramethylrhodamine isothiocyanate-labeled goat anti-mouse IgG (1:400; Jackson ImmunoResearch, West Grove, PA). Finally, labeled cryosections were rinsed consecutively in PBTGS, 0.1 M PB, and 0.05 M PB for 5 min each and mounted in Vectashield (Vector Laboratories, Burlingame,
CA). Images were captured with a laser-scanning microscope LSM510 (Carl
Zeiss, Oberkochen, Germany). Digitized images were transferred to a
laboratory computer for later analysis using Image Pro (Media
Cybernetics, Silver Spring, MD). In quantification of ion
channel clusters and cluster lengths of
Na+ channel, a focal region of
immunofluorescence was considered to represent a cluster (aggregate) of
Na+ or K+
channels if it clearly stood out from background label, and the fluorescence intensity was  150 using LSM510 software (Carl Zeiss). Based on the criteria mentioned above, the cluster numbers of ion
channels were counted, and the length of each
Na+ channel cluster was measured by
digital linear gauges using LSM510 software. The clusters with lower
intensities (<150) were also counted. Gauge outputs were transferred
to a laboratory computer, and at least two sections of nerve from each
animal were analyzed.
Electron microscopic analysis. CST-deficient and littermate
wild-type mice (14, 29, and 36 weeks of age) were intracardially perfused through the left ventricle with a fixation solution consisting of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Millonigs buffer, pH 7.3. The perfusions were
followed by a 2 week incubation in the same fixative solution at 4°C.
Tissue samples from spinal cord and optic nerve were collected and
processed for transmission electron microscopic analysis using standard
techniques. Briefly, the optic nerves were sectioned 0.75 cm anterior
to the optic chiasm and the spinal cord images were obtained from the
ventral columns from level C3. The tissues were postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and then analyzed using a Leo 910 transmission electron microscope (Leo
Electron Microscopy Ltd., Cambridge, UK).
Western blot analysis. Preparation of mouse total brain and
immunoblotting were performed as described previously (Peles et al.,
1995). Myelin was purified and extracted as described previously (Huber
et al., 1994; Kim et al., 1995) based on the procedures of Norton and
Poduslo (1973). Protein concentrations of the lysates were determined
by Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The
proteins (5-50 µg) were analyzed by either 5, 7.5, or 15% SDS-PAGE
and electroblotted to Hybond-P transfer membrane (Amersham Pharmacia,
Buckinghamshire, UK). The blots were blocked for 1 hr in 5% nonfat
milk in 10 mM Tris-HCl, 100 mM NaCl, and 0.1% Tween 20, pH 7.5 (blocking
buffer), at RT and probed with antibodies specific for anti-Caspr
(1:3000), anti-mouse monoclonal Na+
channel antibody, K58/35 (1:1000), anti-MBP (1:3000), and anti-PLP (1:1000) diluted in blocking buffer for 1 hr at RT. They were washed
and exposed to goat anti-rabbit, mouse, and rat IgG conjugated by
horseradish peroxidase diluted in blocking buffer for 30 min at RT. The
conjugates were detected using the ECL system (Amersham Pharmacia),
according to the manufacturer's instructions.
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RESULTS |
CST-deficient mice exhibit a histological phenotype similar to
CGT-deficient mice
The CST-deficient mice, which lacked sulfatide but not GalC,
exhibited paranodal abnormalities that were similar to CGT-deficient mice by electron microscopic study (Honke et al., 2002). To understand the function of myelin sulfatide on the characteristic domain formation
of the axons, we first analyzed the distribution of the voltage-gated
Na+ and K+
channels on the CNS (optic nerve and spinal cord) sections from the
CST-deficient mice. In the control CNS axons, the
Na+ channels were concentrated at the
nodes of Ranvier, and the K+ channels were
localized in the juxtaparanodal regions, which were adjacent to the
paranodal regions as described previously (Fig.
1a). In contrast, the
localization and form of both channels were altered on the CNS axons
from the CST-deficient mice (Fig. 1b-e). In mutant mice,
although Na+ channels were concentrated in
the small regions that were presumptive nodes of Ranvier, the lengths
of the Na+ channel clusters were
occasionally longer than in the wild-type mice (Fig. 1, compare
a and b). We measured the nodal length in the
optic nerves, which was indicated by the
Na+ channel immunoreactivity. In
16-week-old littermate controls, the nodal lengths were nearly constant
(0.79 ± 0.05 µm; >100 nodes were examined from two mice). In
contrast, in the optic nerve from the CST-deficient mice, the lengths
were significantly longer in comparison with those of the control mice
(1.25 ± 0.05 µm; >75 nodes were examined in each of two mice;
p < 0.001 by t test). In addition, the
binary appearance of Na+ channel clusters
was frequently observed in the mutant mice (Fig. 1d). In the
optic nerves from the 10-week-old mutant mice, 11% (25 of 246 clusters
calibrating the use of two animals) of the Na+ channel clusters were binary shaped,
and 12% (31 of 245 clusters calibrating the use of two animals) were
binary shaped in the 14-week-old mice. Such binary forms were never
observed in wild-type mice of the same ages.
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Figure 1.
Alteration of ion channel clusters in
CST-deficient mice. Double immunostaining of Na+ and
K+ channels was performed in the CNS and PNS axons.
a, In the spinal cord of wild-type mice,
Na+ channels (green) were
restricted to the nodes of Ranvier, whereas the K+
channels (red) were concentrated in the juxtaparanodes.
b-e, In contrast to the spinal cord of CST-deficient
mice, the K+ channels were aberrantly localized to
the paranodes (b, c, d). The Na+
channels formed clusters in the nodes; however, the domains were longer
(b) in comparison with wild type
(a). d, The binary form of
Na+ channel clusters is frequently observed.
e, Heminode formation that labeled just one side of the
juxtaparanode or paranode with the Kv1.1 antibody was also observed.
f, In the spinal root of wild-type mouse,
Na+ channels (green) were
restricted to the nodes of Ranvier, whereas the K+
channels (red) were concentrated in the juxtaparanodes.
g, In contrast, in the spinal root of this mutant mouse,
the K+ channel clusters were aberrantly localized to
the paranode. Scale bar, 5 µm.
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Conversely, K+ channel distribution was
significantly more affected in the CST-deficient mice. Although the
K+ channel antibody occasionally stained
the paranodal regions in the wild-type mice, the intensity of the
paranodal staining was significantly lower than that of the main
juxtaparanodal clusters (Fig. 1a). In the mutant mice, in
contrast, the K+ channel clusters
frequently accumulated in the region adjacent to the
Na+ channel cluster, in presumptive
paranodal regions, and in the juxtaparanodal regions (Fig.
1b-d). As shown in Figure 1c, the intensity of
the K+ channel antibody staining in some
of the presumptive paranodes was even brighter than the staining in the
juxtaparanodal regions. Heminodal formation that labeled just one side
of the juxtaparanode or presumptive paranode with the antibody against
Kv1.1 K+ channel subunit was also
observed in the mutant mice (Fig. 1e). The staining patterns
using the antibodies against other K+
channel subunits, Kv1.2 and Kv2, revealed the same results (data not
shown). These results were similar to the findings in the CGT-deficient
mice in the CNS (Dupree et al., 1999).
We then examined whether the localizations of the ion channels in the
PNS were affected to a similar extent as those in the CNS. The
characteristic channel distribution was observed in the wild-type
spinal root, as shown in Figure 1f, which was the same as in
the spinal cord (Fig. 1a). However, the
K+ channels accumulated markedly in the
presumptive paranodal as well as the juxtaparanodal regions (Fig.
1g), and the stained area occasionally overlapped with those
of Na+ channels in the mutant spinal root.
These changes were quite similar to those in the mutant CNS (Fig.
1b-d). Thus, sulfatide seems to be a myelin component that
is necessary for the proper domain formation of PNS as well as CNS axons.
Because disruption of the paranodal axo-glial junction was frequently
observed by electron microscopic studies in CST-deficient mice (Honke
et al., 2002), we then analyzed the distribution of the axonal adhesion
molecule Caspr. This protein is normally concentrated in the paranodal
regions in the optic nerve as well as in the spinal root. Hence, double
labeling with Caspr and K+ channel
antibodies revealed the paired patterns of these proteins next to the
unlabeled node in the middle (Fig.
2a,d, respectively). On the
contrary, this type of paired staining disappeared in the CST-deficient
mice, and Caspr appeared to be diffusely distributed along the axons,
as shown in Figure 2b,c (optic nerve) and Figure 2e (spinal root). In 6-week-old mice, although the small
numbers of Caspr clusters were observed adjacent to some of the
K+ channel clusters, most of these channel
clusters were not adjacent to Caspr (Fig. 2b). In
10-week-old mice, Caspr clusters were hardly recognized, and a diffuse
staining pattern was observed throughout the axons in the CST-deficient
mice (Fig. 2c). This protein was diffusely distributed
throughout the axolemma in the spinal root of the 6-week-old mutant
mice as well (Fig. 2e). Western blot analysis of brain
revealed no differences in the protein levels of Caspr between the
CST-deficient and wild-type mice (Fig. 2f), indicating that the diminished immunoreactivity both in the CNS and in
the PNS axons was, in fact, a result of dispersed distribution of this
protein in the entire internode. These localization changes of axonal
proteins were also observed in the CGT-deficient mice. Together, these
results indicate that the lack of sulfatide is responsible for the
abnormal distribution of axonal proteins in both CGT- and CST-deficient
mice.
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Figure 2.
CST-deficient mice revealed severely disrupted
Caspr clustering both in the CNS and in the PNS. a, d,
In 6-week-old wild-type mice, Caspr (green) was
highly concentrated in the paranodal regions of the optic nerve
(a) and in the paranodal region of the spinal
root (d). b (6 weeks),
c (10 weeks), e (6 weeks), In contrast,
CST-deficient mice exhibited a more diffuse labeling pattern. The
number of K+ channel clusters is markedly decreased
in the mutant optic nerve as well as in the spinal root.
f, Western blot analysis of brain homogenate revealed no
differences in the amount of Caspr protein between wild-type (+/+) and
mutant ( /) mice in the CNS axons at 18 weeks of age. Scale bar, 10 µm.
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The ion channel clusters were markedly reduced with aging in the
CST-deficient mice
Recently, several mutants with disruptions of paranodal junctions
have been reported, such as CGT-deficient mice (Dupree et al.,
1998a,b), Caspr-deficient mice (Bhat et al., 2001), and
contactin-deficient mice (Boyle et al., 2001). All of these mice
exhibited basically the same abnormal distributions of axonal proteins
around the node as we have presently described in the CST-deficient
mice. However, the long-term effects of paranodal disclosure on the localization of axonal proteins still remained uncertain. To determine whether these abnormal paranodes eventually affect the ion channel localizations, we counted the numbers of channel clusters that showed
the same fluorescence intensities as those in wild type in the optic
nerves from postnatal day 17 (P17) to 22-week-old mice. These numbers
were represented per field of view (FOV) (1 FOV = 73.1 × 73.1 µm2). An average of the numbers in
12 FOVs was calculated from littermates of the same age (Fig.
3). In the wild-type mice, the numbers of the K+ (Fig. 3g) and
Na+ (Fig. 3h) channel clusters
rapidly increased until 6 weeks or 4 weeks of age, respectively. These
numbers were maintained without any significant changes throughout all
of the ages examined (Fig. 3a-c, graph with
diamond shapes in g and h). In
contrast, although the numbers of both channel clusters were already
significantly lower in the optic nerves of 4-week-old CST-deficient
mice in comparison with those of wild type, these numbers kept
decreasing constantly with age (Fig. 3d-f, graph
with squares in g and h). Ultimately,
these clusters disappeared dramatically in the 22-week-old optic nerve,
and only 8% of the K+ channel or 12% of
the Na+ channel clusters were detected in
comparison with those of the wild type (Fig. 3g,h). Moreover
the remaining channel clusters at this age exhibited irregular forms,
as shown in Figure 3f (arrow). In addition, the
changes in the nodal lengths demonstrated by the
Na+ channel immunoreactivities were
examined in the optic nerves from mice of different ages (Fig.
3i). Each column shows the length of
Na+ channel clusters in 4- to 22-week-old
mice. The nodal lengths in the littermate controls (Fig. 3i,
white columns) were nearly constant through 6-22 weeks of
age (0.79 ± 0.05 µm; >95 nodes were examined in two mice of
each age). In the CST-deficient mice, the lengths from >150 nodes in
two mice of each age (Fig. 3i, black columns)
were significantly longer in all of the ages in comparison with the
littermate controls except for 4-week-old animals
(p < 0.05 or p < 0.001 by
t test).
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Figure 3.
The ion channel clusters were reduced with age in
CST-deficient mice. The optic nerves from 4-week-old (a,
d), 10-week-old (b, e), and 22-week-old
(c, f) animals were double stained by
Na+ (green) and
K+ (red) channel antibodies. In
wild-type mice (a-c), the numbers of
Na+ and K+ channel clusters were
maintained without any changes throughout all ages. In the mutants
(d-f), both types of ion channel clusters were
reduced, and irregular forms (f) became
prominent with age. Scale bar, 10 µm. g, h, Numbers of
K+ channel (g) and
Na+ channel (h) clusters per
field of view in the optic nerves of wild-type ( ) and mutant mice
( ) plotted at different ages. An average of 12 FOVs was analyzed at
each age. Single and double asterisks
indicate p < 0.01 and p < 0.05, respectively. Wherever statistics are used, results are given as
mean ± SD. i, The nodal lengths in the optic
nerves of wild-type mice (white bar) and mutant mice
(black bar), which were revealed by
Na+ channel immunoreactivity. Error bars indicate
SD. Single and double asterisks indicate
p < 0.001 and p < 0.05, respectively. 4w, Four weeks; 6w, six
weeks; 10w, 10 weeks; 14w, 14 weeks;
16w, 16 weeks; 22w, 22 weeks.
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Because not only the numbers of Na+
channel clusters but also the nodal lengths indicated by the
Na+ channel immunoreactivities were
changed significantly after 6 weeks of age, we examined how these
changes progressed with age. The optic nerves from 6 to 36 weeks of age
were immunostained with anti-Na+ channel
subunit antibody, and all of the confocal images were taken under
the same conditions (detector gain and amplifier offset levels).
Fluorescence intensities of almost all of the clusters in wild-type
mice (Fig. 4a) were >150
using LSM510 software (see details in Materials and Methods). In
contrast, various shapes of Na+ channel
clusters with low-fluorescence intensities became more and more
prominent in the older CST-deficient mice (Fig. 4b-e). Figure 4g-i shows representative clusters with abnormal
shapes (Fig. 4g,h, asterisks) and low intensities
(Fig. 4h,i, arrowheads) compared with those in
wild type (Fig. 4f). Therefore, we classified the
clusters into two groups depending on their fluorescence intensities and counted these two groups of clusters independently in each age in
the optic nerves from 6 to 36 weeks of age per FOV (1 FOV = 48.7 × 48.7 µm2) (Table
1). Even then, the total cluster numbers
were significantly less in old mutant mice. In addition, the clusters
with long shapes, as shown in Figure 4i, were increased with
age, and the relatively homogeneous staining along the axons was
observed especially at 36 weeks of age (Fig. 4e), suggesting
that Na+ channels became distributed more
diffusely.
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Figure 4.
The changes of shapes and fluorescence intensities
of Na+ channel clusters with age in CST-deficient
mice. The optic nerves from wild-type mice (a)
and 6-week-old (b), 16-week-old
(c), 22-week-old (d), and
36-week-old (e) mutant mice were stained by
Na+ channel antibodies. In the mutant, the numbers
of Na+ channel clusters decreased significantly with
age, and the clusters of longer shapes with lower intensities became
more and more prominent in older mutant animals (compare
d or e with a).
f, Typical Na+ channel clusters in
wild-type mice whose intensity levels are >150 using LSM510 software
(see Materials and Methods). g-i, The representative
clusters with abnormal shapes (g, h, *) and
intensities (h, i, arrowheads) observed
in the mutants. Scale bars: a-e, 7 µm; f,
g, 2 µm. j, An immunoblot of wild-type (+/+)
and mutant (/) mice spinal cords at 36 weeks of age probed with
anti-mouse Na+ channel antibody. The bands at ~260
kDa represent Na+ channel subunit. There were no
differences in the amount of Na+ channel protein
between wild-type and mutant mice.
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The similar changes of Na+ channel
clusters were also observed in the spinal cord (data not shown).
Western blot analysis of cervical spinal cord revealed no differences
in the protein levels of Na+ channels
between the CST-deficient and wild-type mice (Fig. 4j). These findings suggested that sulfatide is necessary not only for the
proper localization of channels but also for the maintenance of these
channel clusters around the node. Furthermore, the CST-deficient mice
showed a pronounced tremor and progressive ataxia from 6 weeks of age
(Honke et al., 2002), when the loss of channel clusters became
prominent. Thus, such maintenance of channel clusters may be critical
for normal conduction.
The CST-deficient mice do not exhibit apparent signs
of demyelination
Because the immunohistological abnormalities during aging in the
CST-deficient mice were suggestive of demyelination, we examined the
myelin membrane by immunostaining with the antibodies against two major
myelin proteins, MBP and PLP/DM20. No differences were observed in the
staining pattern of MBP between the mutant and control optic nerves at
22 weeks of age (Fig. 5a,b),
which is a different result from that observed in the demyelinating
mutant mice generated by overexpression of PLP gene (Fig.
5c) (Kagawa et al., 1994). The PLP/DM20 staining of the
optic nerves also revealed no significant changes between the mutant
and wild type (data not shown). Furthermore, each protein level in the
myelin fraction of CNS was examined by Western blot analysis. As shown in Figure 5d,e, the intensities of the specific bands of MBP
(d) and PLP/DM20 (e) were unaltered between the
mutants and the controls. Thus, even in 22-week-old mice, no evidence
of demyelination was detectable by immunohistochemical as well as
Western blot analysis. These results were confirmed by electron
microscopic study. Cross-sectional analysis of optic nerves from
29-week-old wild-type (Fig. 5f) and CST-deficient
(Fig. 5g) mice revealed no difference in myelin stability.
In addition, common indications of demyelination, such as myeloid
figures and microglial activation, were not observed in the
sulfatide-deficient mice at any of the ages analyzed. Spinal cord
analysis revealed similar findings (data not shown). Together, these
results demonstrated that the disappearance of the channel clusters
during aging in this mutant was not attributable to demyelination but
probably related to paranodal disruptions, although the axons were
covered with normal-appearing compact myelin.
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Figure 5.
CST-deficient mice displayed no signs of
demyelination. The staining pattern of MBP (a, b)
exhibited no differences between wild-type (a)
and CST-deficient (b) mice in optic nerves at 22 weeks of age, whereas it is apparently different in demyelinating
mutant mice generated by overexpression of PLP gene
(Kagawa et al., 1996) at 28 weeks of age (c).
Scale bar, 10 µm. Analysis of the membrane fraction of the brain from
wild-type (+/+) and mutant (/) mice revealed no differences in the
levels of MBP (d) and PLP
(e) at 18 weeks of age. Electron microscopic
analysis showed no signs of demyelination. Cross-sectional analysis of
optic nerves from 29-week-old wild-type (f) and
CST-deficient (g) mice revealed no difference in
myelin stability. In addition, common indications of demyelination,
such as myeloid figures and microglial activation, were not observed in
the sulfatide-deficient mice at any of the ages analyzed. Spinal cord
analysis revealed similar findings (data not shown).
|
|
The presence of Caspr is not a prerequisite to
Na+ channel clustering at the nodes
Because Caspr had been proposed to play an important role in
channel clustering, we examined the localization of Caspr and Na+ channel clusters carefully during
development. At P17, during the period of rapid formation of the
Na+ channel clusters in the optic nerve
(Fig. 6a), Caspr was
distributed diffusely along the axons, and no clusters were observed in
the CST-deficient mice in comparison with the littermates (Fig.
6b). In contrast to Caspr, no significant changes were
observed in the number of Na+ channel
clusters of both mutant and wild-type mice (23 ± 4 clusters per
FOV in littermate control; 21 ± 2 clusters per FOV in the mutant
mice; in both types of mice, n = 12 from two animals
each). Therefore, the clustering of Caspr may not be a prerequisite for the proper localization of the Na+ channel
at the presumptive node.
View larger version (45K):
[in this window]
[in a new window]
|
Figure 6.
Optic nerves from CST-deficient mice exhibited
severe disruptions in Caspr cluster formation during development (P17).
Optic nerves from control littermates (a) and
CST-deficient mice (b) were double labeled to
indicate the localization of Na+ channels
(green) and Caspr (red). Caspr
clustering was hardly observed, and this protein was distributed
diffusely along the axons in CST-deficient mice
(b). In contrast, Na+ channel
cluster formation was comparable between mutants (b,
arrows) and control littermates. Scale bar, 10 µm.
|
|
The distribution of the PSD-95-related molecule is altered
similarly to the distribution change of the K+
channels
Although it has not yet been fully characterized, PSD-95, which
binds to the shaker-type K+ channel
-subunits (Kim et al., 1995), or a highly related protein colocalizes with the K+ channels at the
juxtaparanodal regions on myelinated axons (Baba et al., 1999).
Therefore, we examined whether the localization of this protein was
also altered in the CST-deficient mice. In contrast to wild-type mice,
in which PSD-95-related protein formed clusters in the juxtaparanodal
regions (Fig. 7a,
red), this protein changed its localization to the paranodal
regions adjacent to the Na+ channel
clusters in the CST-deficient mice (Fig. 7b). Double immunostaining using antibodies to PSD-95 and
K+ channel revealed the colocalization of
these proteins in the paranodal region, suggesting that these two
proteins may interact significantly even in the mutant mice (data not
shown).
View larger version (34K):
[in this window]
[in a new window]
|
Figure 7.
The distribution of PSD-95-related molecule is
altered in CST-deficient mice. Optic nerves from control littermates
(a) and CST-deficient mice
(b) were double labeled by the antibodies against
Na+ channels (green) and
PSD-95 (red). In the wild-type controls, the
PSD-95-related molecule formed clusters primarily in the juxtaparanodal
regions, where the K+ channels were usually
localized. In contrast, in the mutants, the PSD-95-related molecule was
concentrated in the paranodal regions rather than at the juxtaparanode
and was sometimes distributed diffusely throughout the internode. Scale
bar, 10 µm.
|
|
| |
DISCUSSION |
Sulfatide is an essential myelin component involved in the proper
localization of axonal proteins through the axo-glial junction
Both electrical and histochemical experiments have revealed the
importance of myelin GalC and/or sulfatide in proper paranodal formation (Dupree et al., 1998a,b, 1999). So which galactolipid is
essential for the formation of the axo-glial junction? The altered
distributions of ion channels and Caspr both in the CNS and in the PNS
of the CST-deficient mice are consistent with the observations from a
previous report of CGT-deficient mice (Dupree et al., 1999). Despite
the consistency in Na+ channel cluster
nodal location in both mutants, the lengths of the nodes indicated by
Na+ channel immunoreactivity were
significantly longer in these mutants. The
K+ channel clusters were frequently
observed in the paranodal regions rather than in the juxtaparanodal
regions. Furthermore, Caspr clustering in the paranodal regions
disappeared and became evenly distributed in the axolemma throughout
the internode. Thus, all of these characteristic observations of the
axonal proteins in CGT-deficient mice were well preserved in
CST-deficient mice, suggesting that sulfatide, but not GalC, is in fact
a primary myelin component that determines the proper localization of
axonal proteins via the paranodal axo-glial junctions.
Nevertheless, the clinical observations were distinct between the two
mutants, and several additional histological changes were observed in
CGT-deficient mice. For example, CGT-deficient mice exhibit a severe
tremoring phenotype early on, within 14 d, and die by 90 d of
age. In contrast, the clinical phenotype of CST-deficient mice is
milder, and these mice can survive to >1 year of age (Honke et al.,
2002). Furthermore, CGT-deficient mice exhibit significant
demyelination starting at ~7 weeks of age, and the CNS axons are
almost completely devoid of myelin by 13 weeks of age (Dupree et al.,
1998a). In contrast, according to our data, destruction of compact
myelin does not occur in CST-deficient mice of the same age, and
electron microscopic studies have demonstrated that it does not occur
even by 36 weeks of age. These differences suggest that the two major
glycolipids may function independently in myelin. For instance,
sulfatide bears a negative charge and thus can bind electrostatically
to any positively charged amino acid chain
(Arg+, His+,
and Lys+) on the surface of a protein (Vos
et al., 1994). This kind of binding with galactolipid has been reported
in several myelin proteins, such as MBP (for review, see Smith, 1992),
myelin-oligodendrocyte basic proteins (Holz et al., 1996), and myelin
and lymphocyte protein (Kim et al., 1995; Schaeren-Wiemers et al.,
1995; Frank et al., 1998, 2000). Additionally, tenascin-R 160 and 180, two isoforms of an extracellular matrix molecule expressed in
oligodendrocytes during myelination, have been reported to interact
with sulfatide (Pesheva et al., 1997). Together, these results indicate
that sulfatide may function differently on lipid-protein interactions. Hence, a defect in this lipid results in the disruption of junctional formation and in the abnormal localization of axonal ion channels. What, then, is the functional role of GalC in myelin? Based on the
predominance of this glycolipid in myelin and on the significantly greater instability of myelin membranes in CGT-deficient mice in
comparison with mice lacking only sulfatide, we surmise that GalC may
be an essential myelin component to maintain structural integrity.
Axo-glial interactions regulate axonal domain formation as well as
its stability
We have demonstrated that although no apparent demyelination was
observed in CST-deficient mice, the numbers of clusters of both
K+ and Na+
channels decreased significantly with age. Furthermore, Figure 4j shows that Na+ channels were
not reduced at the protein level in this mutant, suggesting that the
decrease in the clusters was attributable to the dispersed distribution
of the channels throughout the axolemma. Our studies have provided the
first evidence indicating that incomplete paranodal formation may
eventually affect the stabilities of channel localization. The relative
longevity (1 year) of these mutant mice in comparison with other
mutant mice with disruptions of paranodal junctions has allowed us to
study whether sulfatide affects the stability of ion channel clusters.
Currently, three different mouse mutants are available for studying the
molecular composition and functional roles of paranodal regions. Two of
the mutants contain deficiencies in the axonal paranodal proteins Caspr
or contactin, and the third contains a deficiency in the myelin
glycolipids GalC and sulfatide. All mutants primarily exhibit
disruptions of paranodal axo-glial junctions, as revealed by electron
microscopic studies, and changes in axonal protein localization around
the nodes. Caspr is distributed diffusely in the entire internode in
the myelin mutants, whereas it is not found in the axons in both axonal
mutants because this protein cannot be transported without contactin
and stays in the neuronal cell bodies in the contactin knock-out mice.
In each case, the K+ channel clusters move
to the presumptive paranodal regions, just next to the
Na+ channel clusters, but the densities of
both channels in the internodes remain quite low. These changes are
quite distinct from the changes observed in hypomyelinating (Baba et
al., 1999; Rasband et al., 1999) and demyelinating mutants (Baba et
al., 1999), in which both ion channels exhibit loss of specific
localization and distribute diffusely in the axons. Thus, the presence
of the compact myelin surrounding the axon can trigger channel
clustering, although the complete axo-glial junction is necessary for
the proper location of these clusters presumably because of the
formation of a diffusion barrier between both channels, as reported
recently (Poliak et al., 2001). It is also possible that the existence
of paranodal loops of myelin in the absence of axolemma binding may be
sufficient for the formation of channel clusters around the nodes.
However, at present it is still unclear whether the instability of
axonal protein localization is specific for the CST-deficient mice or
is a feature common to all mutants with paranodal abnormalities, because some other mutations result in lethality by postnatal day 20, rendering them recalcitrant to observation with age.
Caspr clustering is not essential for the initial determination of
Na+ channel clusters during development
Based on previous reports, Caspr clustering is the initial event
during CNS axonal formation that is strictly dependent on the
maturation of the myelinated fibers, subsequent to which
Na+ channel aggregation occurs, followed
by the formation of K+ channel clusters
(Rasband et al., 1999, 2000; Shrager, 2000). Proper development may not
always necessitate the sequential occurrence of these events, because
the present study using CST-deficient mice clearly demonstrates that
Na+ channel clusters can be formed in
appropriate regions at the correct time of development even in the
absence of Caspr cluster formation in the beginning. It remains to be
determined whether direct contact of the glial process is necessary to
determine the localization of Na+ channel
clusters. Nevertheless, the presence of Caspr protein in a specific
place in the axolemma does not constitute the initial guidance step for
ion channel clustering. Rather, a direct glial signal, either via a
soluble factor (Kaplan et al., 1997) or through the mediation of actual
contact, may determine future nodal location. This would be followed by
the formation of K+ channel clusters in
the presence of compact myelin, the localization of which is contingent
on the presence or absence of the axo-glial junction.
How might sulfatide mediate interactions between myelinating glial
cell and the axon?
Recently, Bansal et al. (1999) suggested that sulfatide and/or
GalC interact with external ligands and negatively regulate oligodendrocyte differentiation. So it is possible that sulfatide may
directly bind to an axolemmal partner and mediate some signals through
axo-glial association. However, the following hypothesis can be
proposed to explain the dramatic alteration in the paranodal regions
observed in CST-deficient mice. Namely, sulfatide may indirectly
mediate axo-glial junctions by regulating intracellular protein
trafficking in myelin-forming cells, and hence, myelin protein
trafficking may be disrupted in CST-deficient mice. In support of this
possibility, there has been evidence demonstrating that glycolipids
actively select certain proteins for subcellular transport via
detergent-insoluble, glycolipid-enriched complexes [digoxigenin
(DIG)] (Ledesma et al., 1998). In many cell types, these
lipid-dependent domains are usually enriched in glucocerebroside (Simons and Ikonen, 1997). However, the composition of myelin is
atypical in many aspects. In comparison with other cell membranes, myelin contains a high amount of lipid (75-80% of dry weight), with
galactosphingolipids in particular contributing ~30% of the weight
of total lipids (for review, see Vos et al., 1994). In addition, Kramer
et al. (1997) have demonstrated that the galactolipid DIGs, isolated
from maturing oligodendrocytes and the myelin sheath, contain the
glycosyl phosphatidylinositol-linked proteins known as neural
cell adhesion molecule 120 and contactin. The protein composition of DIGs prepared from CGT-deficient mice was changed compared with that of normal brains (Bansal et al., 1999). Recently, neurofascin 155, an ankyrin-binding member of the L1 family, was identified as the first glial molecule enriched in paranodal regions (Tait et al., 2000). Poliak et al. (2001) reported that the disruption of clustering of this protein was observed in CGT-deficient mice. We
also found that clustering of neurofascin 155 was hardly detected in
the paranodal regions in the CST-deficient mice (T. Ishibashi and H. Baba, unpublished observation). Thus, sulfatide may collect such
adhesion molecules in DIGs and transport them to form axo-glial junctions.
| |
FOOTNOTES |
Received Nov. 6, 2001; revised May 21, 2002; accepted May 21, 2002.
This work was supported by a Grant for Private Universities provided by
the Ministry of Education, Culture, Sports, Science, and Technology, by
the Promotion and Mutual Aid Corporation for Private Schools of Japan
(both to H.B), and by Grants-in-Aid 10214204 and 13480270 from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan (K.I.). E.P. is an Incumbent of the Madeleine Haas Russell Career
Development Chair. J.L.D. is supported by a grant from the National
Multiple Sclerosis Society. This work was also supported by Grants
NS27336 (B.P.) and NS24453 (K.S.) from the National Multiple Sclerosis Society.
Correspondence should be addressed to Dr. Hiroko Baba,
Department of Molecular Neurobiology, School of Pharmacy, Tokyo
University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji,
Tokyo 192-0392, Japan. E-mail: hirobaba{at}ps.toyaku.ac.jp.
| |
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[Abstract]
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K. Sinha, S. Karimi-Abdolrezaee, A. A. Velumian, and M. G. Fehlings
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[Abstract]
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D. Lingwood, G. Harauz, and J. S. Ballantyne
Regulation of Fish Gill Na+-K+-ATPase by Selective Sulfatide-enriched Raft Partitioning during Seawater Adaptation
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[Abstract]
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U. Matzner, E. Herbst, K. K. Hedayati, R. Lullmann-Rauch, C. Wessig, S. Schroder, C. Eistrup, C. Moller, J. Fogh, and V. Gieselmann
Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy
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May 1, 2005;
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[Abstract]
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X. Cheng, Y. Zhang, N. Kotani, T. Watanabe, S. Lee, X. Wang, I. Kawashima, T. Tai, N. Taniguchi, and K. Honke
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[Abstract]
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G. Corfas, M. O. Velardez, C.-P. Ko, N. Ratner, and E. Peles
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N. Schaeren-Wiemers, A. Bonnet, M. Erb, B. Erne, U. Bartsch, F. Kern, N. Mantei, D. Sherman, and U. Suter
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J. Cell Biol.,
August 30, 2004;
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[Abstract]
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D. P. Schafer, R. Bansal, K. L. Hedstrom, S. E. Pfeiffer, and M. N. Rasband
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[Abstract]
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D. Ogawa, K. Shikata, K. Honke, S. Sato, M. Matsuda, R. Nagase, A. Tone, S. Okada, H. Usui, J. Wada, et al.
Cerebroside Sulfotransferase Deficiency Ameliorates L-selectin-dependent Monocyte Infiltration in the Kidney after Ureteral Obstruction
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[Abstract]
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T. Ishibashi, L. Ding, K. Ikenaka, Y. Inoue, K. Miyado, E. Mekada, and H. Baba
Tetraspanin Protein CD9 Is a Novel Paranodal Component Regulating Paranodal Junctional Formation
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[Abstract]
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L. Gollan, D. Salomon, J. L. Salzer, and E. Peles
Caspr regulates the processing of contactin and inhibits its binding to neurofascin
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December 22, 2003;
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[Abstract]
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S. Poliak, D. Salomon, H. Elhanany, H. Sabanay, B. Kiernan, L. Pevny, C. L. Stewart, X. Xu, S.-Y. Chiu, P. Shrager, et al.
Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1
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September 15, 2003;
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[Abstract]
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J. C. Rios, M. Rubin, M. St. Martin, R. T. Downey, S. Einheber, J. Rosenbluth, S. R. Levinson, M. Bhat, and J. L. Salzer
Paranodal Interactions Regulate Expression of Sodium Channel Subtypes and Provide a Diffusion Barrier for the Node of Ranvier
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[Abstract]
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M. J. Craner, A. C. Lo, J. A. Black, and S. G. Waxman
Abnormal sodium channel distribution in optic nerve axons in a model of inflammatory demyelination
Brain,
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION
