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The Journal of Neuroscience, March 1, 1998, 18(5):1642-1649
Myelin Galactolipids Are Essential for Proper Node of Ranvier
Formation in the CNS
Jeffrey L.
Dupree1,
Timothy
Coetzee1,
Andrew
Blight2,
Kinuko
Suzuki1, 3, and
Brian
Popko1, 4, 5
1 Neuroscience Center, 2 Department of
Surgery, Division of Neurosurgery, 3 Department of
Pathology and Laboratory Medicine, 4 Department of
Biochemistry and Biophysics, and 5 Program in Molecular
Biology and Biotechnology, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599
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ABSTRACT |
The vertebrate myelin sheath is greatly enriched in the
galactolipids galactocerebroside (GalC) and sulfatide. Mice with a disruption in the gene that encodes the biosynthetic enzyme
UDP-galactose:ceramide galactosyl transferase (CGT) are incapable of
synthesizing these lipids yet form myelin sheaths that exhibit major
and minor dense lines with spacing comparable to controls. These CGT
mutant mice exhibit a severe tremor that is accompanied by hindlimb
paralysis. Furthermore, electrophysiological studies reveal nerve
conduction deficits in the spinal cord of these mutants. Here, using
electron microscopic techniques, we demonstrate ultrastructural myelin abnormalities in the CNS that are consistent with the
electrophysiological deficits. These abnormalities include altered
nodal lengths, an abundance of heminodes, an absence of transverse
bands, and the presence of reversed lateral loops. In contrast to the
CNS, no ultrastructural abnormalities and only modest
electrophysiological deficits were observed in the peripheral nervous
system. Taken together, the data presented here indicate that GalC and
sulfatide are essential in proper CNS node and paranode formation and
that these lipids are important in ensuring proper axo-oligodendrocyte interactions.
Key words:
node of Ranvier; galactocerebroside; sulfatide; myelin; UDP galactose:ceramide galactosyl transferase; axo-glial
interaction
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INTRODUCTION |
Processes originating from oligodendrocytes in the
CNS and Schwann cells in the peripheral nervous system (PNS) align
along and spiral around axons forming myelin segments known as
internodes, which are divided by regularly spaced unmyelinated regions,
the nodes of Ranvier (Morell et al., 1994 ). The paranodal region is the
portion of the myelin segment adjacent to the node where the lamellae
open along the major dense line, forming cytoplasmic pockets known as
lateral loops. These loops turn toward and terminate on the axon with
the outermost lateral loop terminating closest to the node.
Furthermore, regularly arrayed densities traverse the periaxonal space
between the lateral loops and the axolemma. These structures are known
as transverse bands (TBs) and are common features of the
axo-oligodendrocytic junctional complex (Peters et al., 1976). Working
together, these myelin structures play an integral role in dramatically
enhancing conduction velocity (Salzer, 1997).
Our understanding of myelin has been dramatically facilitated by the
availability of mutant models in which proteins of the myelin sheath
are affected (Griffiths, 1996). More recently, mouse models have been
generated that target myelin lipids, which constitute >70% of the dry
weight of myelin. Bosio et al. (1996) and Coetzee et al. (1996) have
generated mice with a disruption in the gene that encodes
UDP-galactose:ceramide galactosyl transferase (CGT), the enzyme that
catalyzes an essential step in the biosynthetic pathway of the two
major myelin galactolipids galactocerebroside (GalC) (Morell and Radin,
1969) and its sulfated derivative sulfatide (Fleischer and Zambrano,
1974).
Although slightly thinner in the white matter tracts of the spinal
cord, myelin in young CGT-deficient mice appears normal (Coetzee et
al., 1996). The mutant animals, however, have a profound tremor as
early as 2 weeks of age, and electrophysiological analysis reveal
conduction deficits. By 6 weeks of age the sheaths exhibit extensive
regional vacuolar degeneration in the ventral columns of the spinal
cord, and the animals begin to display hindlimb weakness. Treatment of
spinal cords from young animals, before demyelination, with
4-aminopyridine (4-AP), a blocker of potassium channels located in the
paranodal region, partially restores the amplitude and duration of the
compound action potential in the GalC-deficient mice (Coetzee et al.,
1996). Ordinarily, 4-AP has no effect on action potentials of
myelinated fibers, because these potassium channels, which are occluded
by the myelin sheath, are not accessible to the inhibitor. Thus, the
findings suggest that these channels are abnormally exposed to the
extracellular milieu, perhaps because of paranodal abnormalities.
Therefore, we examined nodal and paranodal structures in the
CGT-deficient animals. Whereas the ultrastructure of the PNS was
unaffected, we found several nodal and paranodal defects in the CNS, in
the absence of apparent oligodendrocyte pathology, that correlate with
the physiological abnormalities. Taken together, our findings indicate
that myelin galactolipids are essential in establishing
axo-oligodendrocytic interactions that ensure proper nodal and
paranodal formation.
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MATERIALS AND METHODS |
Histological analysis. Thirty- and 45-d-old
CGT-deficient mice were perfused intracardially through the left
ventricle with an ice-cold solution of 4% paraformaldehyde and 2.5%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The
perfusions were followed by a 24 hr immersion fixation at 4°C in the
same solution. Tissue samples from both midbrain and hindbrain,
cervical (C3) spinal cord, and sciatic nerve, 0.5 cm distal to the
sciatic notch, were harvested, embedded in both cross and longitudinal
orientations, and processed for electron microscopic analysis.
The prevalence of abnormal nodes of Ranvier in the ventral columns of
the spinal cord was quantitatively assessed. The number of nodes was
determined per field, defined as a single square from a 200 mesh grid
(Electron Microscopy Sciences, Ft. Washington, PA, catalog #G200Cu
Gilder). Counts were restricted to fields that were completely filled
with longitudinally oriented processes. Because older animals exhibit
extensive myelin vacuolar degeneration, only 30-d-old animals were
quantitatively analyzed, therefore ensuring that the presence of the
abnormal structures was not attributable to the processes of
demyelination and remyelination. For the quantitative studies, four
CGT-deficient mice and three littermate controls were used, and
typically 8-10 fields per animal were counted. Values are presented as
mean ± SE, and all counts were statistically evaluated by
t tests.
Expression of myelin genes. Total RNA was isolated from the
brains of CGT mutant and wild-type animals using Trizol according to
the manufacturer's specifications (Life Technologies, Grand Island,
NY). RNAs were separated on 0.8% formaldehyde-agarose gels,
transferred to nylon, and probed as described previously (Coetzee et
al., 1996). Total protein extracts were isolated by homogenization of
brains in 1% SDS for 10 sec using a Polytron homogenizer. The protein
extract was then boiled for 10 min and insoluble material removed by
centrifugation. Myelin was extracted as described previously (Coetzee
et al., 1996). After mild delipidation, myelin proteins were
resuspended in 1% SDS. Total brain and myelin proteins were separated
on 12.5% polyacrylamide gels, transferred to nitrocellulose, and
probed with antibodies to myelin basic protein (MBP) (SMI-99;
Sternberger Monoclonals, Inc., Baltimore, MD) or proteolipid protein
(PLP) (kindly provided by Dr. Joyce Benjamins, Wayne State University,
Detroit, MI) (Benjamins et al., 1994). Antibodies were detected by
chemiluminescence following the instructions of the manufacturer
(Boehringer Mannheim, Indianapolis, IN).
Lipid analysis. Thin-layer chromatography was used to
determine myelin lipid content in the PNS. Total lipids were extracted and pooled from 10 sciatic nerves from 30-d-old animals. Lipids were
extracted and analyzed as described previously (Coetzee et al.,
1996).
Electrophysiological analysis. Conduction properties of
myelinated axons were examined in acutely isolated sciatic nerves from
60-d-old CGT-deficient and wild-type mice. Recordings were made with
two different sucrose gap recording arrangements. The first, identical
to that described by Coetzee et al. (1996), was used to examine
compound potential amplitude and the response to 4-AP, for direct
comparison with the findings in spinal cord axons in the earlier study.
The second technique used two sets of bipolar silver-silver chloride
wire hook electrodes to stimulate the nerve at two points, 5.5 mm
apart. The difference in conduction delay of the compound action
potential from stimulation at these two points was then used to
calculate conduction speed in the usual manner. These two techniques
provided stable recording and stimulating conditions over the course of
the 1-2 hr experiments.
The animals were anesthetized with an intraperitoneal injection of
ketamine HCl (60 mg/kg), xylazine (10 mg/kg), and acepromazine maleate
(0.6 mg/kg). They were then decapitated, and the sciatic nerves on both
sides were removed over the length from the knee to the exit from the
spine. The isolated nerves were incubated in oxygenated Krebs'
solution (in mM, NaCl 124, KCl 2, KH2PO4 1.2, MgSO4 1.3, CaCl 1.2, dextrose 10, NaHCO3 26, and sodium ascorbate 6, equilibrated with 95% oxygen and 5% carbon dioxide). After a period
of at least 1 hr, the nerve was removed to the recording chamber and
mounted in the sucrose gap. The main part of the nerve was superfused
continuously with oxygenated Krebs' solution at a rate of 2 ml/min.
4-AP (Regis Technologies, Morton Grove, IL) was applied to the nerve in
a concentration of 0.1 mM, dissolved in Krebs' solution,
with pH adjusted to 7.3, as necessary. The temperature of the chamber
was controlled with a Peltier unit, and recordings were made at 37°C.
Data were recorded using a Neurodata Instruments bridge amplifier,
digitized, and recorded on videotape for subsequent analysis using
Labview software on a Macintosh computer.
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RESULTS |
CGT-deficient mice form compact myelin sheaths that exhibit normal
periodicity up to 1 month of age, followed by the onset of vacuolar
degeneration of the CNS sheaths by 1.5 months of age. With this in
mind, 30- and 45-d-old animals were analyzed, and similar
ultrastructural abnormalities were observed at both time points. The
major difference between the two time points was that demyelination was
not yet detected by 30 d of age. Therefore, the majority of the
data presented is from 30-d-old mice, thus increasing the likelihood
that all structural anomalies in the CGT-deficient animals were formed
during the initial process of myelination and not by demyelination or
during remyelination.
Abnormal node and paranode formation in the CNS
Because the electrophysiological deficits in the spinal cord of
the CGT / mice were suggestive of nodal and paranodal abnormalities (Coetzee et al., 1996), we used electron microscopic techniques to
analyze longitudinal sections of these regions. Our results revealed a
significantly higher incidence of aberrantly formed nodal and paranodal
structures. In the CGT-deficient animals, 3.10 ± 0.04 abnormal
nodes and paranodes per field were observed compared with 0.29 ± 0.03 per field in the littermate controls (p < 0.0001). Typically, nodes were longer in the mutant mice than in the
controls (Fig. 1, compare
A,B), and heminodes, paranodes juxtaposed to extended unmyelinated
axonal regions, were present (Fig. 1C). Adjacent myelin
sheaths with overlapping paranodal regions that completely occluded the
formation of the node were also occasionally observed (Fig.
1D). Moreover, in the mutant mice, the periaxonal
space between the lateral loops and the axolemma lacked TBs (Fig.
2A,B). Furthermore,
paranodal regions were frequently configured in a reversed orientation
with the lateral loops facing away from the axon and with the outermost
lamella terminating farthest from the node (Fig. 2C,D).
Finally, astrocytic processes, which were identified by the presence of
bundles of 10 nm filaments, presumptive glial fibrillary acidic
protein, and glycogen particles, were observed in the periaxonal space
of <5% of the myelinated axons (Fig.
3).
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Figure 1.
Nodal and paranodal abnormalities in the
CGT-deficient mice. Myelinated processes from the cervical region of
the spinal cord of 30-d-old CGT-deficient mice demonstrating
ultrastructurally normal (A) and abnormal nodes
of Ranvier (B-D). B, An
aberrantly formed node with lateral loops of the outermost lamellae
forming farther from the node than the more medial lamellae, and these loops are facing away from the axon. Furthermore, the length of the
nodes (bracketed) in the CGT-deficient mice is typically
longer than in the age-matched wild types (bracketed in
A). C, The most commonly observed
aberrant nodal structure was the heminode, a myelinated segment
adjacent to a nonmyelinated region of the axon. In addition, heminodes
are the extreme example of node elongation. D, Less
frequently observed was the overlapping of paranodal regions. Two
myelin sheaths (1, 2) failed to form a node of Ranvier,
because the regions of lateral loop formation overlap, thus excluding the nodal region. Scale bar, 1.0 µm.
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Figure 2.
High magnification of aberrant paranodal
structures in the CGT mutant animals. A, A paranodal
region from a 30-d-old littermate control displays a periaxonal space
with regularly arrayed densities known as transverse bands
(arrowheads). These structures are prominent features of
the junctional complex normally formed between the axolemma and the
myelin sheath. B, Transverse bands were not found in
age-matched CGT-deficient mice. Lateral loops in both the spinal cord
(C) and brain (D)
frequently are reversed and face away from the axon. Furthermore, the
outer most lamella terminates farther from the node. Ax,
Axon; G, glial fibrillary acid protein. Scale bar, 0.1 µm.
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Figure 3.
Astrocytic intrusion between the myelin sheath and
the axon. Myelinated process from the spinal cord of a CGT mutant
animal demonstrating the intrusion of an astrocytic process, as
identified by the presence of glycogen particles
(arrowheads) and bundles of filaments
(stars), between the myelin sheath and the axolemma. Scale bar, 1.0 µm.
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Assessment of oligodendrocyte function
Because abnormal myelin structures have been attributed to
oligodendrocytic pathology (Dentinger et al., 1982; Duncan et al., 1983), we used a combination of ultrastructural and biochemical parameters to assess the function of these cells. The oligodendrocytes in the CGT-deficient mice appeared ultrastructurally normal with respect to membrane integrity and organelle structure and produced myelin sheaths that exhibit major and minor dense line spacing comparable to age-matched littermate controls (Coetzee et al., 1996;
data not shown). In addition, we found that expression of MBP and PLP
mRNAs were unaffected in the mutant mice relative to the littermate
controls at 30 and 45 d of age (Fig.
4A). Moreover, the
levels of MBP and PLP protein were not significantly different between
the mutants and the controls at these ages in either the total brain
extracts or in myelin fractions (Fig. 4B), indicating that normal numbers of myelinating oligodendrocytes are present in the
mutant animals at both time points.
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Figure 4.
Northern and Western blot analyses of myelin
proteins. A, Message levels of MBP and PLP are similar
between CGT-deficient mice and littermate controls at 30 and 45 d
of age. B, Analysis of both total brain and isolated
myelin revealed no difference in the levels of MBP and PLP at 30 and
45 d of age.
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Analysis of PNS structure and function
The alterations in both the structure and function of the myelin
in CNS prompted the investigation of PNS myelin. Thin-layer chromatography of the sciatic nerve demonstrated no difference in the
pattern of the myelin lipid profiles between the PNS and that reported
for the CNS (Coetzee et al., 1996). PNS myelin from the CGT-deficient
mice lacked both normal fatty acid (NFA)- and hydroxy fatty acid
(HFA)-containing GalC and sulfatide and revealed bands with mobility
patterns consistent with HFA-glucocerebroside and HFA-sphingomyelin
(Fig. 5).
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Figure 5.
Lipid analysis of the PNS. Both NFA- and
HFA-containing GalC and sulfatide were absent in the sciatic nerves of
the CGT/ mouse. Bands with mobility patterns consistent with
HFA-glucocerebroside and sphingomyelin were prominent in the mutant
but not in the wild-type. Chol, Cholesterol;
HFA-GalC, hydroxy fatty acid galactocerebroside; NFA-GalC, normal fatty acid galactocerebroside;
HFA-GlcC, hydroxyglucocerebroside; PE,
phosphatidyl ethanolamine; PC, phosphatidyl choline;
SPM, sphingomyelin; HFA-SPM, hydroxy
fatty acid sphingomyelin.
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Given the CNS nodal and paranodal abnormalities, we examined these
regions in the PNS of the CGT/ mice. Sciatic nerves from 30-, 45-, and 90-d-old animals did not exhibit vacuolar degeneration (Fig.
6A,B). In addition,
heminodes were not present; lateral loops were not reversed; and node
length was not altered (Fig. 6C,D).
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Figure 6.
Structural analysis of the PNS. Sciatic nerve
myelin sheaths revealed no differences between CGT+/+ (A,
C) and CGT/ (B, D) mice with respect to
thickness (A, B) or node formation (C, D). Scale bar, 1.0 µm.
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Electrophysiological parameters indicated some alteration in the
function of the PNS in the CGT-deficient mice (Table
1). As depicted in Figure
7 and Table 1, CGT/ animals
demonstrated ~50% reduction in compound action potential amplitude
and 30-40% reduction in conduction velocity. Furthermore, a slight
increase in the responsiveness to blockade of the fast potassium
channels with 4-AP (Fig. 7D) was observed; however, this
effect was not statistically significant as assessed by ANOVA. None of
these effects was as dramatic as those seen in the CNS (Coetzee et al., 1996).
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Figure 7.
Electrophysiological analysis of the PNS.
A, Diagram of the recording apparatus used for
measurement of conduction velocity. The isolated nerve was placed in a
Plexigal chamber and superfused with oxygenated Krebs' solution, and
one end was isolated by a flowing sucrose gap in a separate chamber
slowly perfused with isotonic KCl solution (120 mM).
Silver-silver electrodes were used to record the potential across the
gap and to stimulate the nerve at two points, 5.5 mm apart in the main
chamber. An example of compound potentials recorded in response to
stimulation of the nerve at proximal and distal electrode pairs is
illustrated in B. The conduction time for the length of
nerve between the stimulating electrodes was derived from the time
between the compound potential peaks in each recording.
C, Three recordings are superimposed showing the effect
of superfusing a nerve from a CGT+/+ mouse with Krebs' solution
containing 100 µM 4-AP. There was a slight increase in
amplitude of the response and the depolarizing afterpotential. The
compound action potential increase reversed more readily with 10 min of
washing in normal Krebs' solution. D, Recordings,
similar to those in C, show a slightly more pronounced
effect of 4-AP on the amplitude of the compound potential and
depolarizing afterpotential in a nerve from a CGT/ mouse.
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DISCUSSION |
We have examined the structure of the paranode and the node of
Ranvier in mice incapable of synthesizing the myelin galactolipids GalC
and sulfatide. Whereas nodal structure was unaffected in the PNS,
several abnormalities were observed in the CNS. Nodal length was
frequently increased; TBs were absent; and paranodal orientation was
reversed. In addition, heminodes were abundant. One explanation for the
abundance of heminodes is the occurrence of segmental demyelination.
Whereas this possibility cannot be disregarded, cross sectional
examination of the spinal cords from 30-d-old mice revealed minimal
signs of demyelination. Although the mechanism for proper formation of
nodal and paranodal structures is unknown, the data indicate that GalC
and sulfatide are essential in this process and abnormalities in these
regions are the result of disruptions in axo-oligodendrocytic
interactions. These CNS myelin anomalies, however, occurred in the
absence of detectable alterations in oligodendrocyte form or function.
In contrast, despite the absence of the myelin galactolipids, the PNS
appeared structurally normal, and the electrophysiological studies
revealed only modest deficits.
Because the proper formation of a node of Ranvier is likely dependent
on communications between the axon and myelinating cells, one
explanation for the abnormal formation of nodal and paranodal structures is the disruption of these cell-cell interactions. Such a
disruption could lead to misalignment of the oligodendrocytic processes
along the axon resulting in increased nodal lengths and the formation
of heminodes. Furthermore, the presence of reversed lateral loops and
the improper order of lateral loop termination also suggest a loss of
cell-cell communication. Finally, the presence of myelin sheaths that
appear to overlap and occlude node formation is further evidence of
oligodendrocytic process misalignment during myelinogenesis. Taken
together, these data indicate that the abnormal nodal formations are
the result of a disruption in axo-oligodendrocytic interactions that
are dependent on the presence of functional myelin galactolipids.
Although similar ultrastructural myelin abnormalities have been
reported in the CNS of myelin-deficient rat (Rosenbluth, 1987) and
shaking pup (Griffiths et al., 1981), these models are the products of
a point mutation in the PLP gene that results in oligodendrocyte cell
death (Dentinger et al., 1982; Duncan et al., 1983), apparently because
of the accumulation of the mutant protein in the endoplasmic reticulum
(Gow et al., 1994). In contrast, our data provide no evidence for the
loss of oligodendrocytic viability in the CGT-deficient mice. The
oligodendrocytes appear ultrastructurally normal, form abundant amounts
of myelin with correct periodicity, and express normal levels of MBP
and PLP mRNAs and protein. Therefore, the loss of CGT function and not
oligodendrocyte death is the likely cause of the abnormal formation of
nodal and paranodal structures in these animals.
The absence of TBs, prominent features of the axo-oligodendrocytic
junctional complex in the periaxonal space of the paranode, in the CNS
of the CGT mutant animals strongly supports a role for the myelin
galactolipids in establishing cell-cell interactions. Rosenbluth
(1987) suggested that in the absence of TBs, the lateral loops are not
tightly opposed to the axon, exposing the paranodal axolemma to the
extracellular milieu. Therefore, the lack of formation of TB in the
CGT/ mice likely explains our previous findings with 4-AP, an
inhibitor of potassium channels positioned in the paranodal region of
the axolemma. Because these channels are normally occluded by the
myelin sheath, treatment of control animals with the channel blocker
had little or no effect on the action potential. In the CNS of the
CGT-deficient mice, however, 4-AP treatment partially restored the
compound action potential amplitudes (Coetzee et al., 1996), indicating
that the inhibitor is capable of gaining access to the channels via a
compromised axo-oligodendrocytic junction. Moreover, further evidence
for a disruption in this axo-glial junction is provided by the
intrusion of astrocytic processes into the periaxonal space between the
myelin sheath and the axolemma. In the presence of normal
axo-oligodendrocytic junctions, astrocytes are unable to penetrate the
paranodal region. In the case of the CGT-deficient mice, astrocytic
penetration into the periaxonal space of the paranodal region indicates
that the myelin galactolipids are essential in the formation of TBs, and thus in establishing proper axo-oligodendrocytic interactions.
The CNS myelin abnormalities also provide a structural correlate for
the electrophysiological deficits previously observed (Coetzee et al.,
1996). The frequency of abnormal node formation, particularly the
prevalence of heminodes, likely accounts for the conduction deficits in
the spinal cord of the CGT mutant animals. With regard to the
heminodes, long stretches of unmyelinated axons would inhibit the
efficient spreading of the depolarization down the axon, and conduction
would be compromised. Moreover, the lateral loops are frequently
reversed and not in contact with the axolemma. Such aberrant structures
are consistent with the 4-AP studies (Coetzee et al., 1996), which
indicate current leakage into the paranodal region resulting in the
reduction of depolarization efficiency. In addition, the absence of TBs
may alter saltatory conduction because these structures have been
implicated in retarding abnormal ion fluxes and maintaining proper
distribution of sodium and potassium channels (Salzer, 1997).
Although our data implicate GalC and/or sulfatide in mediating
cell-cell interactions, the mechanism by which this is accomplished is
unknown. Several studies, however, have indicated that galactolipids have functional properties compatible with cell-cell interactions. First, a number of proteins with adhesive properties bind sulfatide in vitro (Vos et al., 1994), and one of these, tenascin-R,
facilitates adhesion of oligodendrocytes through a sulfatide-mediated
mechanism (Pesheva et al., 1997). Furthermore, because some CNS neurons express tenascin-R (Fuss et al., 1993; Wintergerst et al., 1993), the
interaction of this protein with sulfatide may mediate communications between the axon and the myelinating cell. Moreover, a sulfatide antibody specifically labeled nodes of Ranvier in the rabbit (Nardelli et al., 1995), indicating that sulfatide may be important in the structure and function of this region. Furthermore, implantation of
anti-GalC-producing hybridomal cells inhibited tight junction formation
between lateral loops (Rosenbluth et al., 1995), and sulfatide has been
shown to be a prominent constituent of myelin tight junctions known as
the radial component (Karthigasan et al., 1994). In addition,
galactolipids may be critical in trafficking cell adhesion proteins to
the myelin sheath (Kramer et al., 1997). Taken together, it is
tantalizing to suggest a role for GalC/sulfatide in mediating the
linkage between the axon and the oligodendrocyte at the nodal and
paranodal regions and that a disruption in this linkage could yield the
structural abnormalities observed in the CGT-deficient mice.
In contrast to the CNS, the PNS showed no structural deficit.
Nevertheless, some functional impairment was observed. It is important
to note, however, that in wild-type animals, galactolipid content in
the PNS is lower than in the CNS (Norton and Cammer, 1984). Whether
this difference can account for the heterogeneity in the tolerance of
GalC and sulfatide elimination between the Schwann cells and the
oligodendrocytes is unclear. The reduction in amplitude and conduction
velocity in the PNS may be attributed to a combination of factors,
including an overall reduction in tissue size in the mutant (data not
shown), slight (undetectable) ultrastructural abnormalities, or changes
in the lipid composition and electrical characteristics of myelin. The
changes in physiology would be consistent with a lowered resistivity of
the myelin sheath. Furthermore, 4-AP had only a slight effect,
indicating a fairly tight paranodal junction.
Collectively, the nodal abnormalities also provide an explanation for
the vacuolar degeneration that occurs in the CGT/ mice after 6 weeks of age (Coetzee et al., 1996). A prominent feature of this
degenerative process is separation of lamellae along the intraperiod
line. Most likely this is the result of penetration of fluid between
the lateral loops. The absence of TBs and the inversion of lateral
loops provide a means for fluid seepage between the lamellae. Indeed,
the ability of 4-AP to penetrate the periaxonal space provides evidence
for leakage into the paranodal region. Abnormal entrance of fluid, as
indicated by the large vacuoles associated with the demyelinated axons,
into the periaxonal space may initiate the vacuolar degeneration in
older animals (Coetzee et al., 1996). In support of this possibility,
the PNS of the CGT mutant animals does not show nodal abnormalities or vacuolar degeneration of myelin in older animals.
In summary, we demonstrate the presence of abnormal nodal and paranodal
structures in the CNS, but not in the PNS, of mice that lack the myelin
galactolipids GalC and sulfatide. These defects include altered node
length, high incidence of heminodes, absence of TBs, and reversed
lateral loops. These findings indicate that GalC and sulfatide mediate
interactions between the axon and oligodendrocyte that are essential
for proper CNS node and paranode formation.
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FOOTNOTES |
Received Aug. 26, 1997; revised Nov. 19, 1997; accepted Dec. 5, 1997.
This work was supported by Grants NS27336 (B.P.), NS24453 (K.S.), and
NS33687 (A.B.) from the National Institutes of Health and Mental
Retardation Research Center Core Grant HD03110 from the National
Institutes of Health. J.D. was supported by Training Grant HD07201 from
the National Institutes of Health and an Advanced Postdoctoral
Fellowship from the National Multiple Sclerosis Society. T.C. is a
recipient of an Advanced Postdoctoral Fellowship from the National
Multiple Sclerosis Society. B.P. is the recipient of Research Career
Development Award NS01637 from the National Institutes of Health. We
thank Clarita Langaman and Tom Kelly for their superb technical
assistance with the electron microscopic and electrophysiological
analysis, respectively. We also thank Dr. Jack Rosenbluth for his
insightful critique of the electron micrographs and Dr. Kunihiko Suzuki
for his assistance in the interpretation of the lipid analysis.
Correspondence should be addressed to Dr. Brian Popko, Neuroscience
Center, CB 7250, University of North Carolina, Chapel Hill, NC
27599-7250.
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Abstract
Introduction
