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The Journal of Neuroscience, April 1, 2003, 23(7):2833
The Wlds Mutation Delays Robust
Loss of Motor and Sensory Axons in a Genetic Model for Myelin-Related
Axonopathy
Mohtashem
Samsam1,
Weiqian
Mi2,
Carsten
Wessig1,
Jürgen
Zielasek1,
Klaus V.
Toyka1,
Michael P.
Coleman2, and
Rudolf
Martini1
1 Department of Neurology, University of
Würzburg, D-97080 Würzburg, Germany, and
2 Center for Molecular Medicine and Institute for Genetics,
University of Cologne, D-50674 Cologne, Germany
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ABSTRACT |
Mice deficient in the peripheral myelin component P0 mimic severe
forms of inherited peripheral neuropathies in humans, with defective
myelin formation and consequent axonal loss. We cross-bred these mice
with the spontaneous mutant C57BL/Wlds
typically showing protection from Wallerian degeneration because of
fusion of the ubiquitination factor E4B (Ube4b) and
nicotinamide mononucleotide adenylyltransferase (Nmnat)
genes. We found that in the double mutants, the robust myelin-related
axonal loss is reduced at 6 weeks and 3 months of age. Moreover,
retrograde labeling from plantar nerves revealed an increased survival
of motor axons. These motor axons appeared functionally active because
both the amplitude of compound muscle action potentials and muscle
strength were less reduced in the double mutants. At 6 months of age,
reduction of axonal loss was no longer detectable in the double mutants when compared with littermates carrying the P0 null mutation only, although the Wlds gene was not reduced
in its expression at this age. We conclude that myelin-related axonal
loss is a process having some features in common with Wallerian
degeneration. Introducing the Wlds
gene would be a promising approach to delaying detrimental axonal loss
in myelin disorders.
Key words:
protein zero; inherited neuropathies; Wallerian
degeneration; myelin mutant; peripheral nervous system; Schwann
cell
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Introduction |
One of the major and still
unresolved problems of myelin-related disorders, such as demyelinating
neuropathies and multiple sclerosis, is the progressive degeneration of
axons that leads to irreversible symptoms and permanent disability
(Trapp et al., 1998 ; Kornek et al., 2000; Martini, 2001). It is
presently not known how abnormal myelin or demyelination leads to
axonal destruction. In the case of inflammatory disorders, such as
multiple sclerosis and immune-mediated peripheral neuropathies, the
naked axons are particularly vulnerable because they are directly
exposed to immune cells and their secreted cytotoxic products, such as
proinflammatory cytokines, proteolytic enzymes, nitric oxide,
and others (Toyka and Hartung, 1996; Redford et al., 1997; Trapp et
al., 1999; Kornek et al., 2000; Smith et al., 2001). Moreover, axonal
loss can be caused by mutant glial cells in the absence of overt
inflammation. For instance, in proteolipid protein
(PLP)-deficient mutants and in mice overexpressing
PLP, axons degenerate in the CNS (Anderson et al., 1998;
Griffiths et al., 1998). In the peripheral nervous system,
MAG-deficient mutants and mice deficient in the myelin component P0, an
established model for severe forms of inherited neuropathies, show
substantial axonal degeneration (Carenini et al., 1997; Yin et al.,
1998; Frei et al., 1999). The molecular mechanisms underlying axonal
damage in myelin mutants are not yet understood. In the present study,
we tested the possibility that the robust myelin-related axonal loss in
P0-deficient mutants (Frei et al., 1999) follows a similar mechanism to
that of Wallerian degeneration. For this purpose, we cross-bred the
myelin mutants with the spontaneous mouse mutant
C57BL/Wlds showing protection from
Wallerian degeneration for at least 14 d after injury because of
fusion of the genes for the ubiquitination factor E4B (Ube4b) and
nicotinamide mononucleotide adenylyltransferase (Nmnat) (Conforti et
al., 2000; Mack et al., 2001). Apart from mechanical injury,
C57BL/Wlds mice have been shown recently
to be resistant to paclitaxel (Taxol)-induced sensory neuropathy (Wang
et al., 2002). We found that in distal nerve portions of 3-month-old
but not of 6-month-old dysmyelinating C57BL/Wlds mutants, myelin-related axonal
degeneration is significantly reduced. Moreover, in the younger double
mutants the rescued motor axons appeared functionally intact, as
reflected by elevated muscle response amplitudes and increased muscle
strength. Thus, introducing the Wlds
mutation into a myelin mutant significantly delays
dysmyelination-induced axonal degeneration.
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Materials and Methods |
Animals and genotyping. All experiments have been
approved by the local governmental authorities of the State of Bavaria. Homozygous C57BL/Wlds mutants were
obtained from Harlan-Winkelmann (Borchen, Germany) and
cross-bred with heterozygous P0 mutants taken from our own breeding
colony. Cross-breeding of heterozygous P0 mice with other mouse mutants
has been described previously (Schmid et al., 2000; Carenini et al.,
2001). According to Mendelian law, the individuals of the
F1 generation were expected to consist
exclusively of mice heterozygous for the
Wlds mutation. All of these individuals
were genotyped for the P0 mutation by conventional PCR as described
previously (Schmid et al., 2000), and individuals heterozygously
deficient for P0 were intercrossed, leading to progenies with
homozygous, heterozygous, and no P0 deficiency. Pulsed field gel
electrophoresis was used to genotype for the 170 kb
Wlds insertion, because this
method reliably distinguishes homozygotes from heterozygotes (Mi et
al., 2002). Briefly, unfixed spleen tissue was dissociated in PBS,
filtered to make a single-cell suspension, and embedded in 0.5%
(final) low-melting point agarose in a plug mold (Bio-Rad,
Munich, Germany). After cell lysis in 0.5 M EDTA, 1% sodium-N-laurylsarcosine,
and 25 µg/ml proteinase K (48°C, 3 d), plugs containing
high-molecular-weight DNA were washed extensively in Tris-HCl, pH 7.5, and 1 mM EDTA. NotI digestion produced
restriction fragments of 220 kb (wild type), 390 kb
(Wlds) or both, which were resolved
on a Bio-Rad Chef-DR electrophoresis cell and detected by
hybridization with a single copy probe on the basis of a sequence
within the triplication (GenBank accession number AF260927).
Counting of axons and histological analysis. Mice were
transcardially perfused with sodium cacodylate buffer containing 4% paraformaldehyde (PFA) and glutaraldehyde (2% each), as described previously (Carenini et al., 2001). Plantar nerves and median nerves
were carefully removed under a dissecting microscope at the level of
the ankle joint and wrist, respectively, and postfixed in the perfusion
fixative. Tissue preparation for electron microscopy was performed as
described previously (Carenini et al., 2001).
The number of myelinated axons was counted on ultrathin sections,
because demyelinated axons cannot be reliably quantified by light
microscopy. We used a BioVision slow scan camera attached to a
Zeiss (Thornwood, NY) EM 10B and the corresponding
software analySIS 3.0 Doku. To exclude bias, in all experiments axon
counting was performed without knowledge of the
C57BL/Wlds genotype. Statistical analysis
was performed using a two-tailed t test, and p
values of <0.05 were considered significant.
Spinal motoneuron labeling and counting. Mice were
anesthetized with of a mixture of Ketanest (8 mg/ml) and Rompun
(0.08%) in 0.9% NaCl (10 µl/gm body weight) and were placed on an
operation hot plate. The plantar nerve of one side was exposed at the
level of ankle joint, transected, and immediately introduced to 3% (in 0.1 M PBS) hydroxystilbamidine methanesulfonate
(Fluorogold; Molecular Probes, Eugene, OR) in a 6-mm-long
silicone tube for 1 hr. The tube was then removed, and the skin was
sutured. The animals recovered for 5 d after Fluorogold exposure
and then were perfused transcardially with a series of solutions: 0.9%
NaCl (2-3 min), followed by 4% PFA in 0.1 M
Na-acetate buffer, pH 6.5 (15 min), followed by 4% PFA in 0.1 M Na-borate buffer, pH 9.5 (15 min), followed by
10% sucrose in 0.1 M Sörensen's buffer
(15 min). The spinal cord (L2-S1) was dissected and frozen in
Tissue-Tek freezing medium. Thirty-micrometer-thick cryosections
were mounted and analyzed under a fluorescent microscope. Motoneurons
labeled with Fluorogold were easily distinguishable in the ventral horn
and were counted. Typically, the motor neurons supplying the plantar
nerve occupied a column that was covering ~120 serial sections (30 µm thick). To be sure that all of the corresponding motor neuron
fragments had been considered, at least 80-100 sections rostral and
caudal to the last labeled motoneuron fragments were investigated, so that a total number of ~300 sections was scored for each animal. There was no contralateral motoneuron labeling, nor any labeling of
terminals or neurons in the dorsal horn. To correct for multiple counting of the cells, the total number of motoneurons was recalculated using the method of Abercrombie (1946). Statistical analysis was performed using a two-tailed t test, and p values
of <0.05 were considered significant.
Immunohistochemistry and Western blot analysis
immunohistochemistry. Mice were transcardially perfused with 4%
PFA in cacodylate buffer. Lumbar spinal cord was dissected and
processed in 25% sucrose in 0.1 M PBS overnight.
Ten micrometer cryosections of the spinal cord were treated with rabbit
polyclonal anti-Wld antibody (1:250 dilution), raised against 18 aa at
the junction between the Nmnat and Ube4b domains. The other primary
antibody was mouse anti-tubulin III (Tuj1) monoclonal antibody
(Research Diagnostics, Flanders, NJ) at 1:2500 dilution overnight.
Secondary antibodies were goat anti-rabbit Cy3 (1:200 dilution) and
goat anti-mouse Cy2 (1:100 dilution) conjugated antibodies from
Dianova (Hamburg, Germany).
Western blotting. Segments of lumbar spinal cord and
combined dorsal root ganglia (L4-L5) were placed on ice in 20 vol of 60 mM Tris-HCl, pH 6.8, 8.0 M urea, 2% (w/v) SDS, and 2% (v/v) 2-mercaptoethanol for 4 hr and then mechanically homogenized. They were
mixed with an equal volume of 2× SDS-PAGE sample buffer and subjected
to standard SDS-PAGE (10% polyacrylamide) and semidry blotting onto
nitrocellulose. Wlds antigen was detected
using anti-Wld antibody (see above), followed by horseradish
peroxidase-coupled goat anti-rabbit secondary antibody (Dianova) and enhanced chemiluminescence (Amersham
Biosciences, Arlington Heights, IL).  -Tub 2.1 monoclonal
antibody to -tubulin (Sigma, St. Louis, MO) was used as
a loading control.
Electrophysiology. Nerve conduction properties of sciatic
nerves from 3-month-old mice were determined by established
electrophysiological methods as described in detail previously
(Zielasek et al., 1996). In all experiments, the investigator was not
aware of the C57BL/Wlds genotype of the
mice. Statistical analysis was performed using a one-tailed
t test for grouped data.
Determination of muscle strength. Forelimb grip strength was
determined using a custom-made automated grip strength meter as
described previously (Masu et al., 1993). Animals were placed on a
platform and allowed to grasp a ring. Mice were pulled away until they
released the ring, and the strength was determined in Newtons by the
electronic pull strain gauge. The ratio of male to females was 1:1 in
each experimental group. In all experiments, the investigator was not
aware of the C57BL/Wlds genotype of the
mice. Statistical analysis was performed using an unpaired, two-tailed
t test.
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Results |
Wlds reduces axonal degeneration in peripheral
nerves of younger P0 mutants
The median and plantar nerves were chosen for the
determination of axonal loss in the P0-deficient knock-out
mutants (P0 /). In 4-week-old
P0/ mice, numbers of axons were only
slightly reduced, by ~7% (p < 0.05) (Fig.
1A) compared with
age-matched wild-type mice, although features indicative of a severe
dysmyelinating neuropathy were already evident at this age. At 6 weeks
of age, axonal loss was more clearly detectable, with a reduction of
~24% of fibers in plantar nerves (Fig. 1A,B). At 3 months of age in P0/ mice these nerves
showed a profound axonal loss when compared with age-matched wild-type
mice (surviving axons numbered 60% of normal in the median and 59% of
normal in the plantar nerve) (Fig. 1A,C,D),
corroborating previous findings in the nerves of the toes (Frei et al.,
1999).
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Figure 1.
A, Schematic representation of
numbers of axons in plantar nerves. Plantar nerves of
P0/ mice show a progressive axonal degeneration.
At 1 month, ~7% of axons are lost compared with age-matched wild
types. At 3 months, degeneration occurs with a higher rate leading to
40% axonal loss. Axonal degeneration progresses with age, with a lower
rate leading to ~60% axonal loss in 1-year-old
P0/ mice. B, Schematic
representation of axon numbers in plantar nerves of 1.5-month-old
single and double mutants.
P0//C57BL/Wlds
double mutants show a reduced axonal degeneration compared with
age-matched P0//wild type
(WT) (p < 0.05).
C, D, Schematic representation of numbers of axons in
plantar (C) and median (D)
nerves of 3-month-old P0+/+ and
P0/ mice with and without the
Wlds mutation. Note that the
Wlds mutation has no influence on axon
numbers in P0+/+ genotypes. However, in
P0/ mice, loss of axons is significantly reduced
in mice carrying the Wlds mutation. In
C, values from P0/ mice
heterozygous for the Wlds mutation are
also given, showing an intermediate axonal loss because of a
dosage-dependent rescue effect of the
Wlds mutation. Error bars are SDs.
***p < 0.0001; **p < 0.002;
*p < 0.05. n, Number of
individuals of the respective genotype investigated.
hom, Homozygous; het, heterozygous.
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We then cross-bred the P0 knock-out mice with
C57BL/Wlds mice. In this mutant, Wallerian
degeneration after nerve injury is greatly delayed (Lunn et al., 1989).
We aimed to investigate whether the Wlds
mutation could rescue the myelin-related loss of axons seen in P0/ mice at the age of 6 weeks and 3 months. The median and plantar nerves of
C57BL/Wlds mice were morphologically
indistinguishable from nerves of wild-type mice (data not shown) and
contained a similar number of axons (Fig. 1C,D).
P0/ mutants with the homozygous
Wlds mutation showed the same
dysmyelinating neuropathy as single mutant
P0/ mice (Fig.
2). However, at 6 weeks of age, in
plantar nerves, there was a significant reduction in the degree of
axonal degeneration in the double mutants, in that only 15%
degenerated instead of 25% (Fig. 1B). Even more
striking was the rescue effect of the Wlds
mutation at 3 months, when axonal loss in P0 single mutants had substantially increased compared with 6-week-old P0 mutants (Fig. 1C). At 3 months of age, ~40% degenerated in the
P0/ single mutants, whereas only 27%
degenerated in the homozygous double mutants. In other words, ~30%
of those axons that degenerated in the
P0/ single mutants were rescued (Fig.
1C). A similar rescue effect by the
Wlds mutation was detected in the median
nerve (Fig. 1D). Here, again, 40% degenerated in the
P0/ single mutants, whereas ~30%
degenerated in the homozygous double mutants. Because protection from
Wallerian degeneration by the Wlds gene is
dosage dependent (Mack et al., 2001), we investigated whether
P0/ mice with a heterozygous
Wlds mutation showed a rescue of plantar
axons. Although the relative increase in axon numbers was less
conspicuous but still significant, the intermediate value observed in
P0//C57BL/Wlds
heterozygotes further supports a protective role for the
Wlds mutation (Fig. 1C).
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Figure 2.
Electron micrographs of plantar nerves from
P0/ single mutants (A) and
P0/ mice with the homozygous
Wlds mutation
(B). Note that P0/ mutants
with the homozygous Wlds mutation
(B) show the same dysmyelinating neuropathy as
single mutant P0/ mice
(A). Arrows point to
myelin-competent axons devoid of myelin. Scale bar in B
(for A and B), 2 µm.
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Wlds leads to improved conduction properties and
reduced motor axon loss in P0 mutants
Next, we investigated whether the axons that had been rescued by
the Wlds mutation and looked
morphologically normal are functionally intact and active in the
3-month-old double mutants. In an initial series of experiments, we
investigated the nerve-conduction properties with particular emphasis
on the amplitude of compound muscle action potentials of the small foot
muscles as an indicator of functional integrity of axons and synapses.
In 3-month-old P0 wild-type and homozygous
C57BL/Wlds mice, a biphasic muscle
response with a normal amplitude of ~12 mV was recorded on electrical
stimulation of the distal sciatic nerve. In
P0/ single mutants of the same age, we
found a polyphasic response with a strikingly reduced amplitude of the
main component (~1.5 mV), whereas the partially protected
double homozygous
(P0//C57BL/Wlds-homozygous)
mice primarily showed a biphasic response. Although there was still a
clear decline in the amplitude compared with the wild-type mice, the
double homozygous mice showed more than twice the amplitude found in
the myelin single mutants (Fig.
3A,B), and this difference was
statistically significant (p < 0.05). The
amplitudes of P0/ mice with a
heterozygous Wlds mutation still showed a
trend toward higher values compared with P0/ single mutants (Fig.
3A).
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Figure 3.
A, Schematic representation of
amplitudes of compound action potentials from small foot muscles of
3-month-old P0+/+ and P0/
mice with and without the Wlds
mutation on distal stimulation of sciatic nerves. Note that the
Wlds mutation has no influence on
amplitudes in P0+/+ genotypes. Amplitudes are
markedly reduced in the absence of P0
(P0/), but reduction is less pronounced
in the myelin mutants carrying the homozygous
Wlds mutation. Amplitudes from
P0/ mice heterozygous for the
Wlds mutation show a slight trend
toward higher values compared with P0/ single
mutants. ***p < 0.0001 and *p < 0.05. n, Number of individuals of the
respective genotype investigated. B, Original recordings
of compound action potentials from small foot muscles of
P0+/+ and P0/ mice with and
without the Wlds mutation on distal
stimulation of sciatic nerves. Note the compact shape of muscle
response in P0+/+ mice independent of the
Wlds mutation.
P0/ mice without the
Wlds mutation show predominantly
dispersed responses, as shown here for two examples with relatively low
and high amplitudes. Responses from
P0//C57BL/Wlds
mice are usually more compact, as presented here for a typical case
(lower recording). Note the different scales of time and amplitudes.
CMAP, Compound muscle action potential;
WT, wild type; hom, homozygous;
het, heterozygous.
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Because the increased amplitudes of compound muscle action potentials
might be indicative of improved motor axon survival, we aimed to
confirm the putative preservation of motor axons by an independent
technique. Retrograde transport of Fluorogold and counting of the
corresponding spinal motoneurons is an established technique for
scoring intact motor axons in a peripheral nerve (Brushart, 1993).
Using this technique, a significantly lower number of retrogradely
labeled motoneurons was counted in P0/
mice (136 ± 21) than in age-matched wild-type mice (177 ± 26) (Fig. 4). Most interestingly, the
loss of ~20% of retrogradely labeled motoneurons as seen in the
single mutants could no longer be detected in the double mutants
(171 ± 18), demonstrating again the protective role of the
Wlds gene (Fig. 4).
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Figure 4.
Schematic representation of retrogradely labeled
spinal motoneurons of 3-month-old mice using Fluorogold.
P0//WT mice show a significantly lower number of
back-labeled spinal (lumbosacral) motoneurons than
P0+/+/WT mice (*p < 0.05). In 3-month-old
P0//C57BL/Wlds
double mutants, however, the number of labeled motoneurons is as high
as in P0+/+ mice, indicating a significant
preservation of P0/ motor axons on
Wlds expression. n,
Number of individuals of the respective genotype investigated.
WT, Wild type; hom, homozygous;
het, heterozygous.
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Wlds leads to improved muscle strength in
P0 mutants
Next, we addressed the question of whether, as a consequence of
improved motor axon survival, the Wlds
mutation would also lead to improved muscle strength in
P0/ mutants by performing quantitative
grip strength analysis. Wild-type mice at 3 months of age showed a grip
strength of ~1.3 N, a very similar value to that obtained in
age-matched homozygous C57BL/Wlds mice
(Fig. 5). As expected,
P0/ mice were weaker, with only 70%
of the strength of mice with normal myelination. Three-month-old
P0/ double mutants with the homozygous
Wlds mutation displayed significantly more
strength than the single mutant P0/
mice (Fig. 5), reflecting the milder axonal loss in median nerves of
the double mutants (Fig. 1D).
P0/ mice with a heterozygous
Wlds mutation still showed a mild yet
nonsignificant trend toward higher values compared with
P0/ single mutants (Fig. 5). Thus,
cross-breeding of C57BL/Wlds mice with
P0/ mice not only reduced axonal loss
but also improved electrophysiological properties and physical strength
compared with P0/ single mutants.
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Figure 5.
Schematic representation of muscle strength (grip
test of forelimb) of P0+/+ and
P0/ mice (3 months of age) with and without the
Wlds mutation. The
Wlds mutation has no influence on
muscle strength in P0+/+ genotypes. Muscle strength
is strongly reduced in P0//WT mice, but
reduction is less pronounced in
P0//Wlds-homozygous mutants.
Muscle strength of P0/ mice carrying a
heterozygous Wlds mutation show a
small, nonsignificant trend toward higher values compared with
P0/ single mutants. ***p < 0.0005; *p <0.05. n, Number of
individuals of the respective genotype investigated. WT,
Wild type; hom, homozygous; het,
heterozygous.
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Wlds does not lead to reduced axon loss
in 6-month-old P0/ mutants
In a subsequent step, we investigated whether the rescue effect of
the Wlds mutation is still detectable in
P0/ mice of >3 months of age. By
investigating axon numbers in plantar nerves of 6-month-old
P0//C57BL/Wlds
homozygous animals,
P0//C57BL/Wlds
heterozygotes, and P0/ mice, we no
longer observed a decrease in axon loss in the presence of the
Wlds mutation (Fig.
6A). Correspondingly,
neither the amplitude of compound muscle action potentials nor the grip
strength was significantly elevated in
P0/ mice carrying the homozygous or
heterozygous Wlds mutation (Fig.
6B,C). These results indicate that the
Wlds mutation delays, but does not
completely abolish, the myelin-related axonal loss in
P0/ mice.
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Figure 6.
Schematic presentation of numbers of axons in
plantar nerve (A), amplitudes of compound muscle
action potentials (B), and muscle strength
(C) of 6-month-old P0+/+,
P0/, and
P0//C57BL/Wlds
mice. A, In 6-month-old
P0//C57BL/Wlds
double mutants, axonal loss is no longer reduced compared with
P0//WT mice. B, The amplitudes of
compound muscle action potentials of small foot muscles of
P0//C57BL/Wlds
double mutants are not significantly different from those of
P0//WT mutants. C, The muscle
strength (grip test) of 6-month-old
P0//C57BL/Wlds
double mutants is not significantly higher than that of
P0//WT mutants. n, Number of
individuals of the respective genotype investigated.
CMAP, Compound muscle action potential;
WT, wild type; hom, homozygous;
het, heterozygous.
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Wlds protein expression is not decreasing
with age
Next, we examined whether loss of axonal protection in older
double mutants is caused by any decrease in
Wlds expression.
Wlds protein expression has been reported
to be unaltered in brain homogenates with aging (Gillingwater et al.,
2002), but it has not been studied in motor or sensory neurons, nor has
it been studied in the context of the
P0/ mutation. Using a highly specific
polyclonal antibody against amino acids 71-88 of the
Wlds protein (T. G. A. Mack and M. P. Coleman, unpublished observations), we showed the nuclear
localization of the Wlds protein in the
spinal motoneurons of 6-week-old and 6-month-old Wlds mutants (Fig.
7E,F), but no signal
was observed in the absence of the Wlds
mutation (Fig. 7A,B). Furthermore, we observed no overt
difference in the intensity of Wlds
immunoreaction in spinal motoneurons from young versus aged
Wlds mice (Fig. 7C,D). In
contrast, there was a clear difference in Wlds protein expression between homozygous
and heterozygous Wlds mutants, proving
that with the immunohistochemical method applied we can detect a
decrease in protein expression of ~50% (Fig.
7D,F). Similarly, we could find no evidence of a
decrease in Wlds protein expression in
spinal cord or dorsal root ganglia between 6-week-old and 6-month-old
double mutants
(P0//C57BL/Wlds-homozygous)
by Western blot analysis (Fig. 8).
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Figure 7.
Wlds protein immunoreactivity
is not decreased in the spinal motoneurons of young versus old
C57BL/Wlds single or
P0//C57BL/Wlds
double mutants. A primary antibody against Wlds
stains nuclei of the spinal motoneurons of
C57BL/Wlds mice (C,
red). -Tubulin III outlines the cell body and
processes (green). The nuclear
Wlds signal is missing in P0+/+
or P0/ mice that do not express this chimeric
gene (A, B). There is no detectable
decrease in Wlds immunoreactivity in young versus
old C57BL/Wlds single mutant mice
(C, D). Reduced expression of
Wlds protein is detectable in
Wlds-heterozygous versus
Wlds-homozygous mutants
(D, F). Scale bar, 20 µm.
WT, Wild type; hom, homozygous;
het, heterozygous.
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Figure 8.
Western blot analysis of young versus old
P0//C57BL/Wlds
double mutants. Lumbar spinal cord (SPC) and lumbar
(L4-L5) dorsal root ganglia (DRG) homogenates of
6-week-old versus 6-month-old
P0//C57BL/Wlds
double mutants show a similar Wlds expression
(Wlds). Transgenic (Tg
Wlds)
C57BL/Wlds and wild-type
lanes show that the observed band is specific for
Wlds mice. -Tubulin (-Tub)
expression in spinal cord and dorsal root ganglia homogenates are shown
as controls. hom, Homozygous.
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Discussion |
Here, we demonstrate that, in 6-week-old and 3-month-old
P0/ mice, a unique neuroprotective
gene, Wlds, can partially rescue axons
prone to undergoing myelin-related degeneration and in doing so
alleviate the symptoms of mice suffering from inherited dysmyelination.
We also present the first evidence that the
Wlds mutation can protect axons in
vivo from the effects of chronic neurological disease, in addition
to its well documented delay in axonal degeneration on acute injury
(Gillingwater and Ribchester, 2001). The effect of the
Wlds mutation on the disorder is robust,
because it rescues ~30% of those axons that would degenerate in the
3-month-old P0/ single mutants. It is
of note that motor axons have been rescued by
Wlds, as reflected by an increase in
muscle strength, by elevated amplitudes of the muscle responses, and by
an increased number of retrogradely labeled spinal motoneurons.
Additionally, a reduced temporal dispersion of the muscle responses was
found, possibly reflecting improved conduction velocities of some
slowly conducting fibers in the double mutants. It is tempting to
speculate that in the P0/ single
mutants, these particularly slowly conducting fibers comprise those
prone to or being in the stage of degeneration, thus leading to reduced
myelin function because of impaired axon-glia signaling.
We were able to show that in the double mutants especially motor
axons are well preserved, because in 3-month-old
P0//Wlds
mice, the number of back-labeled motoneuron cell bodies was as high as
in P0+/+ mice. Comparing this robust
rescue effect on motor axons with the compound muscle action
potential amplitudes and grip test strength of the double
mutants, the physiological and functional rescue effect has to be
considered lower than one would expect from the high number of
preserved motor axons. The most probable explanation for this
discrepancy is the recent finding that presynaptic motor nerve
terminals are less well preserved by the
Wlds mutation than the motor axon
(Gillingwater et al., 2002).
Our findings imply that myelin-related degeneration of axons has some
features in common with injury-induced Wallerian degeneration. So far,
the principle mechanisms that underlie Wallerian degeneration are only
incompletely understood. It has been shown previously that
Ca2+ influx and activation of the
Ca2+-dependent protease calpain are
involved (George et al., 1995; Wang et al., 2000). Wallerian
degeneration does not seem to follow caspase-3-mediated apoptosis (Finn
et al., 2000), although it would seem likely that an active mechanism
of some kind is involved, because it appears to be a regulated process.
The recent characterization of the mutation underlying delayed
Wallerian degeneration in C57BL/Wlds mice
revealed an 85 kb triplication on chromosome 4 (Coleman et al., 1998)
creating the chimeric gene Ube4b/Nmnat (Conforti et al.,
2000) that, when transmitted to transgenic mice, confers protection
from Wallerian degeneration (Mack et al., 2001). Thus, characterization
of the mechanism by which this gene protects the axon should give
valuable insights into mechanisms underlying both injury-induced and
dysmyelination-related degeneration of axons.
The rescue effect of the Wlds mutation in
P0/ mice is transient. Whereas in
3-month-old mice ~30% of axons prone to degeneration could be
rescued, at 6 months the rescue effect was no longer detectable. This
might be related to the fact that in
C57BL/Wlds mice the rescue of motor nerve
terminals and the ability of peripheral nerves to transmit compound
action potentials is reduced considerably between the 2 and 4 months of
age (Perry et al., 1992; Gillingwater et al., 2002). However, this age
dependency on axon rescue does not correlate with preservation of axon
structure (Crawford et al., 1995; Gillingwater et al., 2002). We also
demonstrate that the loss of axons in 6-month-old
P0//Wlds double
homozygotes cannot be explained by any decrease in
Wlds protein expression with age. Thus,
the most likely explanation is that Wlds
protected axons in these experiments for only a limited period. It is
now important to find ways to extend this period of protection, perhaps, for example, by expressing Wlds
protein at still higher levels.
Because the Wlds mutation significantly
prohibits myelin-related axonal degeneration, our study may be relevant
for the clinical aspects of axon degeneration in inherited
dysmyelinating peripheral neuropathies of the Charcot-Marie-Tooth type
in humans. The major medical problem of patients suffering from these
relatively frequent disorders (Skre, 1974) is progressive and disabling
weakness and muscle atrophy on the basis of distal motor axonal loss
(Berciano et al., 2000; Martini, 2001). Similarly, in other relatively
frequent myelin disorders, such as multiple sclerosis, axonal loss
seems to be the major reason for the clinical impact of the disease (Trapp et al., 1998; Kornek et al., 2000; Kuhlmann et al., 2002). Thus,
additional studies will be necessary to investigate the reasons for the
functional decline of the Wlds protein on
the myelin-related axonopathy in adult animal models of incurable human
nerve disorders.
| |
FOOTNOTES |
Received Oct. 30, 2002; revised Jan. 13, 2003; accepted Jan. 16, 2003.
This work was supported by grants from the German Research Council
(SFB-581, to R.M. and K.V.T.), Gemeinnützige Hertie-Stiftung (GHS-191/00/01, to R.M.), Federal Ministry of Education and Research (FKZ: 01-KS-9502, to M.P.C.), and Center for Molecular Medicine Cologne, University of Cologne (ZMMK, to M.P.C. and W.M.); and local
research funds from the University of Würzburg. We are grateful
to Heinrich Blazyca and Carolin Kiesel for excellent technical
assistance, Dr. Robert Adalbert for genotyping one group of double
mutants, Helga Brünner for animal care, and Drs. Jochen Ulzheimer, Martin Berghoff, and in particular Igor Kobsar for help with
data processing and statistics.
Correspondence should be addressed to Dr. Rudolf Martini,
Department of Neurology, Developmental Neurobiology, University of
Würzburg, Josef-Schneider-Strasse 11, D-97080 Würzburg,
Germany. E-mail:
rudolf.martini{at}mail.uni-wuerzburg.de.
| |
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TOP
ABSTRACT
Introduction
