the wlds gene modestly prolongs survival in the sod1g93a fals mouse
TRANSCRIPT
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Neurobiology of Disease 19 (2005) 293–300
The WldS gene modestly prolongs survival in the SOD1G93A
fALS mouse
Lindsey R. Fischer,a Deborah G. Culver,a Albert A. Davis,a Philip Tennant,a
Minsheng Wang,a Michael Coleman,c Seneshaw Asress,a Robert Adalbert,c
Guillermo M. Alexander,d and Jonathan D. Glassa,b,TaDepartment of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USAbDepartment of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USAcThe Babraham Institute, Cambridge, UKdDepartment of Neurology, Drexel University College of Medicine, Philadelphia, PA 19102, USA
Received 3 June 2004; revised 4 January 2005; accepted 12 January 2005
Available online 19 February 2005
The bslow Wallerian degenerationQ (WldS) gene is neuroprotective in
numerous models of axonal degeneration. Axonal degeneration is an
early feature of disease progression in the SOD1G93A mouse, a widely
used model of familial amyotrophic lateral sclerosis (fALS). We crossed
the WldS mouse with the SOD1G93A mouse to investigate whether the
WldS gene could prolong survival and modify neuropathology in these
mice. SOD/WldS mice showed levels of motor axon loss similar to that
seen in SOD1G93A mice. The presence of the WldS gene, however,
modestly prolonged survival and delayed denervation at the neuro-
muscular junction. Prolonged survival was more prominent in female
mice and did not depend on whether animals were heterozygous or
homozygous for the WldS gene. We also report that SOD1G93A mice
show significant degeneration of sensory axons during the course of
disease, supporting previous data from humans demonstrating that
ALS is not purely a motor disorder.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Axonal degeneration; Amyotrophic lateral sclerosis; ALS;
SOD1; WldS; Wallerian degeneration
Introduction
The WldS mouse is a spontaneous mutant with the remarkable
phenotype of prolonged survival of injured axons (bslow Wallerian
degenerationQ) (Lunn et al., 1989). The gene for WldS is created by
the splicing of fragments of two genes, Ube4b and Nmnat1, within
0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2005.01.008
T Corresponding author. Emory Center for Neurodegenerative Disease,
Whitehead Biomedical Research Building, 615 Michael Street, 5th Floor,
Mailstop 1941007001, Atlanta, GA 30322, USA. Fax: +1 404 727 3728.
E-mail address: [email protected] (J.D. Glass).
Available online on ScienceDirect (www.sciencedirect.com).
an 85kb triplication on chromosome 4 that creates a new open
reading frame coding for a novel 42-kDa protein (Coleman et al.,
1998; Conforti et al., 2000; Mack et al., 2001). The mutation is
sufficient for providing the phenotype (Mack et al., 2001; Wang et
al., 2001a), however, the mechanism for axonal protection byWldS
remains unknown. Recent data suggest that the mechanism of
protection is related to the overexpression of the Nmnat portion of
the WldS gene (Araki et al., 2004).
The WldS mutation can be considered neuroprotective. In
models of axonal degeneration due to transection (Wallerian
degeneration), transected axons from the WldS mouse survive
for up to 4 weeks (Glass and Griffin, 1991; Glass et al., 1993),
and support action potentials (Lunn et al., 1989) and axonal
transport (Glass and Griffin, 1994; Smith and Bisby, 1993) for
at least 2 weeks. The WldS gene also protects against toxin-
induced axonal degeneration. Cultured sensory neurites from
WldS mice are resistant to vincristine-induced degeneration
(Wang et al., 2001a,b) and WldS mice are resistant to Taxol-
induced sensory neuropathy (Wang et al., 2002). Moreover,
WldS protects against genetically-based axonal degeneration. In
the pmn mouse, a model of motor neuron disease (Ferri et al.,
2003), and in the protein-zero-deficient mouse, a model of
myelin-related axonopathy (Samsam et al., 2003), the WldS gene
inhibits axon loss and attenuates disease progression. In the pmn
mouse, protection of axons by WldS also prevents motor neuron
loss (Ferri et al., 2003). WldS also modifies neuropathology in
mice with axonal dystrophy by reducing the number of axonal
spheroids (Mi et al., submitted). Axonal spheroids are a typical
feature of mouse and human ALS (Borchelt et al., 1998;
Carpenter, 1968).
In a previous study, we demonstrated that the progression of
disease in the SOD1G93A mutant mouse, a widely-used model for
familial amyotrophic lateral sclerosis (fALS), is a distal axonop-
athy (Fischer et al., 2004). Degeneration of motor axons progressed
L.R. Fischer et al. / Neurobiology of Disease 19 (2005) 293–300294
from distal to proximal, with a significant proportion of neuro-
muscular junctions (NMJ) showing denervation prior to any
noticeable abnormalities in ventral roots or lumbar motor neurons.
We hypothesized that protection against axonal degeneration in this
model of ALS would positively alter the clinical characteristics of
disease, as was demonstrated in the pmn mouse model of motor
neuron disease (Ferri et al., 2003). We found that the presence of
WldS delayed denervation at the NMJ and modestly prolonged
survival in fALS mice. Increased lifespan was most prominent in
females.
Materials and methods
Animal breeding
All animal protocols were approved by the Emory University
Institutional Animal Care and Use Committee. Animals were
housed in microisolator cages on a 12-h light/dark cycle and given
free access to food and water. Breeding pairs of SOD1G93A high-
expressing mice (B6SJLGUR1) were originally obtained from
Jackson Laboratories (Bar Harbor, ME). WldS mice are maintained
in a colony at Emory University. These animals were crossed to
yield littermates of 3 genotypes for comparison (Fig. 1): SOD/WT,
SOD/WldS (heterozygous for the WldS gene), and SOD/WldS/
WldS (homozygous for the WldS gene). Control animals were 80-
day old C57BL/6J (n = 5) and littermate WldS mice not carrying
the mutant SOD1 gene (n = 5). The day of death for fALS mice
was defined as when they could not right themselves 30 s after
being placed on their backs. Kaplan–Meier analysis was
performed to compare survival using Prism 4 software (GraphPad,
San Diego, CA).
Rotarod testing
Mice were tested weekly beginning at age 50 days for their
ability to maintain balance on a Rotarod apparatus (Columbus
Instruments, OH). Two protocols were used: constant velocity at
15 rpm and an accelerating paradigm of 1.4 rpm + 4 rpm/min.
Animals were tested three times during each session, and the best
performance (maximum 600 s) was recorded.
Genotyping
Identification of SOD1 mutant mice was by standard PCR
analysis on tail snip DNA (Fischer et al., 2004). Designation of
WldS heterozygotes and homozygotes was determined by pub-
lished methods (Mi et al., 2002). Transgene copy number was
estimated using real-time quantitative PCR to determine the
difference in threshold cycle (number of PCR cycles when DNA
is amplified above a baseline) between the transgene (human
SOD1) and a reference gene (mouse interleukin-2), as previously
reported (Alexander et al., 2004).
Fig. 1. Breeding strategy to obtain necessary genotyp
Estimation of hSOD1 protein levels
Brain and spinal cord tissues from end-stage SOD1 mice were
homogenized in SDS buffer and prepared for Western blotting by
standard methods (Glass et al., 2002). Thirty micrograms of protein
was loaded into each well and membranes were sequentially reacted
with antibodies to human Cu/Zn SOD1 (hSOD1, polyclonal sheep
from Calbiochem, 1:1000 dilution) and beta-tubulin (monoclonal
IgG from Sigma, 1:5000). Bands were visualized using appropriate
fluorescent secondary antibodies (Alexa fluor 680 from Calbio-
chem). Band intensities were quantified on a LiCor Odyssey fluore-
scence imager. Protein levels of hSOD1 were normalized to tubulin.
Neuropathology
SOD/WT and SOD/WldS animals were deeply anesthetized and
killed by cardiac transection at 80, 100, and 120 days and at age of
death. Tissues were harvested and fixed appropriately for evalua-
tion of end-plates/neuromuscular junctions (NMJ) and nerve roots.
A cohort of SOD/WldS/WldS animals was observed until age of
death for survival data only.
End-plate/NMJ
Medial gastrocnemius, soleus, and tibialis anterior muscles
were dissected, pinned in mild stretch, and fixed by immersion for
20 min in 4% paraformaldehyde/PBS (pH 7.4). After rinsing in
PBS, muscles were cryoprotected in 20% sucrose/PBS (overnight
at 48C) and flash-frozen in supercooled isopentane. Muscles were
sectioned at 40 AM and placed on glass slides for staining. Sections
were stained first with rhodomine bungarotoxin (1:40, Molecular
Probes) for 30 min. After rinsing and postfixing in ice-cold
methanol, sections were labeled with monoclonal antibodies to
neurofilament (NF160 1:200, Chemicon) and SV2 (1:30, Iowa
Developmental Hybridoma Bank). Secondary antibody was FITC-
labeled goat anti-mouse (1:100, Jackson Immunoresearch).
Stained sections were examined under a fluorescence micro-
scope. End-plates were scored as binnervatedQ if there was
complete overlap with the axon terminal or bdenervatedQ if the
end-plate was not associated with an axon. Some neuromuscular
junctions were associated with a pre-terminal axon only or showed
partial overlap between end-plate and terminal. These were labeled
as bintermediate.Q Each muscle was sectioned exhaustively so that
all neuromuscular junctions could be evaluated. Mean counts for
each group were compared by ANOVA using InStat software
(GraphPad, San Diego, CA).
Nerve roots
Nerve roots were exposed and immersion-fixed in 5% buffered
glutaraldehyde (pH 7.4) at 48C for 48 h. Ventral and dorsal L4
roots were dissected out and stored in 0.1 M phosphate buffer at
48C. Tissue was treated with 1% osmium tetroxide for 90 min,
es. SOD (SOD1G93A mutant), WT (wild type).
Fig. 2. Rotarod analysis displayed graphically for acceleration and constant velocity protocols. Values are mean times until falling calculated for all surviving
animals at each time point. Female animals are analyzed separately.
L.R. Fischer et al. / Neurobiology of Disease 19 (2005) 293–300 295
dehydrated through graded alcohols, and embedded in Epon plastic
(EM Sciences, Cincinnati, OH). Cross-sections (720 nm) were
stained with toluidine blue, rinsed, and coverslipped.
Nerve root sections were imaged at 100� magnification using a
Leitz Dialux 22 microscope (Leica Microsystems, Germany) and
individual frames were captured using an attached live-feed video
camera (DAGE-MTI, Michigan City, IN). Multiple overlapping
images were captured so that all axons were counted. Measure-
ments of axon numbers and calibers were made with Image Pro
software (Media Cybernetics, Silver Spring, MD) running on a
Gateway personal computer (Gateway, San Diego, CA). Axon
interiors were manually marked as solid objects and the mean
diameter of each object was measured. The data were exported to a
Microsoft Excel (Microsoft Corporation, Redmond, WA) spread-
Table 1
Survival (in days) by sex and genotype
Breeding 1
Male Female Total
SOD/WT
Mean 130.6 132 131.1
SD 2.8 4.4 3.7
Sample size 16 14 30
SOD/WldS
Mean 137.6 145.4 141.9
SD 7.9 8.2 8.9
Sample size 28 34 62
sheet for analysis. Means for total number of axons, as well as
small-diameter (0–3.99 AM) and large-diameter (z4.0 AM) axons
were compared by ANOVA with post-hoc comparison.
Sciatic nerve
To confirm that the WldS phenotype remains active in
SOD1G93A animals, we investigated Wallerian degeneration in
transected sciatic nerves from SOD/WT and SOD/WldS mice.
Sciatic nerves from 50-day-old mice were transected and the distal
stumps prepared for light microscopy 5 days post-transection
(according to the protocol for nerve roots). Nerve cross-sections
were imaged at 100� magnification and axons observed for signs
of Wallerian degeneration.
Breeding 2
Male Female Total
SOD/WldS
Mean 135 145 140
SD 5.5 5.1 7.3
Sample size 4 4 8
SOD/WldS/WldS
Mean 138.4 147 140.3
SD 3.6 2.8 5
Sample size 7 2 9
L.R. Fischer et al. / Neurobiology of Disease 19 (2005) 293–300296
Results
Analysis of disease onset
Animals were tested on a Rotarod apparatus weekly as a
measure of onset of disease. We saw no significant differences
between SOD/WT and SOD/WldS mice in terms of their stability
on the Rotarod, and thus conclude that disease onset was not
affected (Fig. 2). However, visual inspection of the graphs shows
an apparent delay in the initial decline in slope in the SOD/WldS
animals as compared to SOD/WT using the constant velocity
protocol. This difference was not statistically significant. We also
recognize that the Rotarod measure of disease onset is rather coarse
and is unlikely to identify small differences in onset when
comparing populations.
Survival
On average, SOD/WT animals survived 131.1 F 3.7 days (n =
30), while SOD/WldS animals survived 141.9 F 8.9 days (n = 62,
P b 0.0001, unpaired t test). SOD/WldS/WldS animals survived
140.33 F 5 days (n = 9, P b 0.0001 versus SOD/WT, unpaired t
test). Thus, the presence of the WldS gene prolonged survival for
approximately 10 days, and survival did not depend on whether
animals were heterozygous or homozygous for the WldS gene
(Table 1 and Fig. 3). However, survival in mice with the WldS
gene was sex-dependent. SOD/WldS females survived 145.4 F 8.2
days, while SOD/WldS males survived 137.6 F 7.9 days (P =
0.0004, unpaired t test). Similarly, SOD/WldS/WldS females
survived 145 F 5.1 days, while SOD/WldS/WldS males survived
only 135 F 5.5 days (P = 0.037, unpaired t test). Survival in
SOD/WT mice was not sex-dependent.
To confirm that increased survival was not due to a reduction of
transgene copy number (Alexander et al., 2004), we estimated
hSOD1 copy number using real-time PCR. DNA was analyzed
from four animals in each group (12 total) spanning the range of
lifespans within each group. Ages were as follows: SOD/WT 121,
127, 132, and 135 days; SOD/WldS heterozygotes 133, 143, 145,
and 149 days; SOD/WldS homozygotes 129, 138, 141, and 150
days. The threshold cycle for all of the specimens was higher than
7.00, a value that is found in mice with at least 24 copies of the
transgene (Alexander et al., 2004). This number is consistent with
that determined for the high-expressing SOD1G93A mice (Gurney
et al., 1994).
We also asked whether increased survival of SOD/WldS
might be due to an unexpected reduction of hSOD1 protein
expression in these animals. Direct comparisons of protein levels
in brain and spinal cord, normalized to expression of tubulin,
showed no differences, indicating that WldS does not increase
survival in this model by inhibiting hSOD1 protein expression
(Fig. 4). It must be noted that the mice used for these
experiments were not the same ones used for the survival
studies.
Fig. 3. Kaplan–Meier survival analysis. (A) SOD/WT versus SOD/WldS
(from breeding 1). (B) Male survival comparison. (C) Female survival
comparison. (D) Comparison of survival of WldS heterozygotes vs.
homozygotes. (E) Comparison of survival of male vs. female WldS
homozygotes. P values are noted on the graphs. Note that WldS
homozygotes show no increased survival when compared to heterozygotes.
Sciatic nerve transection
To determine whether SOD/WldS mice retained the slow
Wallerian degeneration phenotype, sciatic nerves were transected
Fig. 6. Quantitative analysis of innervation at the NMJ of SOD/WT versus
SOD/WldS mice. Note the protection in SOD/WldS mice at 80 and 100
days. All data are means F SEM. * indicates P b 0.05 (unpaired t test).
Fig. 4. Western blots measuring hSOD1 protein expression in brain and
spinal cords of SOD/WT and SOD/WldS mice. Quantitation of band
intensity is normalized to beta-tubulin.
L.R. Fischer et al. / Neurobiology of Disease 19 (2005) 293–300 297
and compared to uncut nerves after 5 days (Fig. 5). While
transected axons from SOD/WT mice had undergone extensive
degeneration, transected axons from SOD/WldS mice were
morphologically similar to axons from SOD/WT uncut nerve. This
demonstrates that the WldS phenotype is retained in the SOD/WldS
mice.
Axonal pathology
Innervation of NMJs and axon numbers in nerve roots were
quantified in SOD/WT and SOD/WldS animals to determine
whether differences in survival could be linked to morphology.
SOD/WldS animals had a higher percentage of innervated motor
end-plates at 80 days, 100 days, and at the time of death (Fig. 6).
The differences reached statistical significance only at day 80,
where SOD/WldS animals had 32.9% more innervated end-plates
than SOD/WT animals (P b 0.05, unpaired t test). Very few
significant differences were noted between SOD/WldS and SOD/
WT animals in the number of total, small-diameter, or large-
diameter ventral root axons (Table 2). Likewise, no differences in
the numbers of motor axons were noted between male and female
SOD/WldS animals. Insufficient numbers of animals were available
to compare male and female NMJ innervation.
Sensory neuropathy in SOD/WT and SOD/WldS mice
Quantitative evaluation of dorsal roots in SOD/WT and SOD/
WldS mice revealed a significant loss of large- and small-diameter
axons at all time points (Figs. 7 and 8). By day 80 (onset of motor
weakness), SOD/WT animals had lost approximately 53% of total
dorsal root axons (P b 0.01 versus 80-day wild-type controls,
Fig. 5. Photomicrographs of sciatic nerve cross-sections. (A) SOD/WT (uncut). (B
Note in panel B the loss of axons and presence of myelin ovoids and phagocytes ty
confirms the WldS phenotype. All animals were 50 days old (scale bar = 25 AM)
Dunnett’s test), and SOD/WldS animals had lost 44% of dorsal
root axons (P b 0.01 versus 80-day WldS controls, Dunnett’s test).
This loss appeared to be nonprogressive, as the number of axons
remained fairly constant from 80 days through death. The only
significant change was an increase in the number of large-
diameter axons (z4.0 AM) between day 80 and day 120 for both
SOD/WT and SOD/WldS animals (P b 0.05, Tukey–Kramer).
Direct comparisons of sensory axon numbers in SOD/WT and
SOD/WldS showed no significant differences for either males or
females.
Discussion
Prolonged survival of SOD/WldS mice
The WldS gene modestly prolonged survival in SOD1G93A
mice, showing greater protection in female mice versus male mice.
In the present study, survival of WldS heterozygotes did not
significantly differ from that of WldS homozygotes. This may not
be surprising since a similar lack of dose effect was demonstrated
in crossing the WldS with the pmn mouse (Ferri et al., 2003).
However, in studies of Wallerian degeneration and in crosses with
) SOD/WT 5 days post-transection. (C) SOD/WldS 5 days post-transection.
pical of Wallerian degeneration at 5 days. The presence of axons in panel C
.
Table 2
Morphometry of ventral root axons
Control 80 days 100 days 120 days Death
Total ventral root axons
SOD/WT 1071.6 F 34 312.25 F 26 336.6 F 24 443 F 14 393.5 F 18
SOD/WldS 986 F 30 393 F 30 383.4 F 49 412.71 F 26 346.8 F 8
P value 0.095 0.098 0.42 0.35 0.11
Small ventral root axons (b4 lM)
SOD/WT 489.8 F 28 228 F 14 255.6 F 26 340.17 F 22 318.2 F 13
SOD/WldS 455.4 F 24 275.83 F 12 295.2 F 40 318.14 F 20 295.4 F 8
P value 0.37 0.033 0.43 0.47 0.28
Large ventral root axons (z4 lM)
SOD/WT 581.8 F 22 84.25 F 20 80.8 F 9 102.83 F 10 75.3 F 7
SOD/WldS 530.6 F 11 117.17 F 20 88.2 F 16 94.57 F 11 51.4 F 2
P value 0.071 0.31 0.60 0.70 0.03
L.R. Fischer et al. / Neurobiology of Disease 19 (2005) 293–300298
the protein-zero demyelinating mutant, the WldS gene acted in a
dose-dependent manner in protection against axonal degeneration
(Mack et al., 2001; Samsam et al., 2003).
There were no differences between groups in the numbers of
surviving motor axons. This lack of axonal protection was
unexpected given the remarkable positive effects shown in other
neurodegenerative models including the pmn mouse (Ferri et al.,
Fig. 7. Photomicrographs of L4 dorsal roots from 120-day old mice. (A)
Wild-type; (B) SOD/WT; (C) SOD/WldS. There is marked degeneration of
axons in animals with the SOD1G93A mutation (scale bars = 50 AM).
2003), the protein-zero knockout mouse (Samsam et al., 2003), and
in Taxol neuropathy (Wang et al., 2002). The WldS phenotype was
not lost in the SOD1/WldS animals, as was demonstrated by the
observed delay in Wallerian degeneration of transected sciatic
nerves. We could, however, demonstrate protection against axonal
degeneration at the NMJ in SOD1/WldS mice. This protection was
most robust at the 80-day time point where the differences in the
number of innervated end-plates reached statistical significance.
Later time points showed no differential effects at the NMJ, which
Fig. 8. Quantitative analysis of axonal degeneration in dorsal roots. (A)
SOD/WT; (B) SOD/WldS. Note significant loss of large and small fibers in
both groups by day 80. The slight increase in axons at later time points may
represent attempts at regeneration. All data are means F SEM. ** indicates
P b 0.01 versus control for all axon size groups (ANOVA).
L.R. Fischer et al. / Neurobiology of Disease 19 (2005) 293–300 299
is consistent with data showing that protection at the synaptic
terminal decreases with age, despite the continued expression of
the WldS protein (Gillingwater et al., 2002). Thus, while neuro-
muscular synapses are still partially protected by WldS at 80 days,
this early protection may be blostQ at the age when animals
typically become symptomatic with motor neuron disease. It is
therefore difficult to explain why the SOD/WldS animals survived
longer than their SOD/WT littermates since at late stages of disease
there were no differences either in motor axon numbers or NMJ
innervation. Even so, the effect of WldS at the NMJ in younger
animals implies that, if this protection could be maintained to an
older age, one might expect an increased positive effect on
lifespan.
The prolonged survival of SOD/WldS mice could not be
explained by reduction of the number of copies of the mutant
transgene as has been shown previously in this model (Alexander
et al., 2004). We also did not see a reduction in hSOD1 protein
expression in SOD1/WldS as compared to SOD1/WT mice. It must
be noted that these protein determinations were not performed in
the same animals used for survival analysis and neuropathology.
However, if WldS had the effect of reducing hSOD1 protein
expression, we likely would have seen this in these other animals
from the same colony.
Issues of possible effects of background strain were also
considered. The WldS mouse is a spontaneous mutation in the
C57BL/6 strain. The transgenic SOD1G93A strain used for these
studies was built on a B6/SJL background. The cross breeding with
WldS mice certainly introduced the C57BL/6 background, an
intervention that may prolong the survival of fALS mice (Greg
Cox, Jackson Laboratories, personal communication). Several
observations argue against this having a major effect in our
studies. First, the SOD1 mutant mice used for comparison were the
non-WldS littermates. These mice coming from the same parents
should have similar genetic backgrounds. Second, the accentuated
survival noted in female WldS animals would not be expected if
this were only an issue of background effects. Third, we have
continued this breeding strategy now for over 8 generations and
have not seen prolongation of life in non-WldS SOD1 mutants. Of
course, we cannot exclude the possibility that the WldS gene is
closely linked to another influential gene in the C57BL/6
background and that they co-segregate.
There is a growing body of evidence suggesting that motor
neuron pathology in the SOD1G93A mouse may be a dying-back
process that begins at the NMJ (Fischer et al., 2004; Frey et al.,
2000; Kennel et al., 1996). According to this hypothesis, age-
dependent protection at the synaptic terminal may explain the
relatively modest effects of the WldS gene on survival in
SOD1G93A mice compared to other mouse models. WldS gene
expression in pmn mice extended survival, on average, from 40
days to 62 days, and delayed disease onset by over 1 week (Ferri et
al., 2003). However, the pmn mutation, which affects a tubulin
chaperone gene, is primarily an axonal insult (Martin et al., 2002).
Likewise, the effects of peripheral myelin protein-zero deficiency
are seen at the level of the axon rather than at the synaptic terminal,
so protection may not be subject to the same age-dependent
constraints (Samsam et al., 2003).
The reason behind sex-dependence of SOD/WldS survival is
unknown. The majority of data on the interaction of androgens and
motor neurons demonstrate a supportive effect of androgens both
in cell growth and response to injury (Brooks et al., 1998;
Gonzalez Deniselle et al., 2001). Low serum levels of testosterone
have also been reported in some ALS patients (Militello et al.,
2002). Our data showing prolonged survival in female mice do not
easily conform to models where androgens are protective in ALS,
although the influence of sex on the expression of the WldS
phenotype in this model may eventually provide clues to the
mechanisms of either mutant SOD1 toxicity or WldS protection.
No sex-related differences in the survival of injured axons have
been observed in WldS mice (independent unpublished data from
Glass and Coleman laboratories).
Sensory neuropathy
This is the first quantitative assessment of sensory pathology in
the SOD1G93A mutant mouse. The loss of 53% of dorsal root axons
by day 80 indicates that mutant SOD1-mediated neuropathology is
not limited to the motor system in these animals. As with motor
fibers, the addition of WldS did not provide protection against
degeneration of sensory fibers. The loss of sensory axons appeared
to be nonprogressive, with a trend towards increased numbers of
axons with age. This finding is reminiscent of our published data
from motor axons (Fischer et al., 2004) and may suggest
regenerative efforts in the sensory system. Further study is needed
to determine the physiological consequences of this sensory axon
loss.
The presence of sensory pathology in the SOD1G93A mouse is
consistent with the sensory pathology seen in human ALS.
Morphological studies have indicated a loss of approximately
30% of large myelinated fibers in the sural nerve (Bradley et al.,
1983; Heads et al., 1991), a 27% decrease in large-diameter L5
dorsal root axons, and a 54% reduction in large, L5 dorsal root
ganglion cell bodies (Kawamura et al., 1981). Functionally, the
sural nerve axonal transport rate is reduced by 44% (Bradley et al.,
1983), and sensory conduction velocity is slower in ALS patients
versus control subjects (Shefner et al., 1991; Theys et al., 1999).
Sensory nerve action potential (SNAP) amplitude remains within
the normal range but has been shown to deteriorate with disease
progression (Gregory et al., 1993).
Acknowledgments
We thank Dr. Terry Heimann-Patterson for assistance with
SOD1 genotyping, Raphael James, Karen Carney, and Daniela
Grumme for technical assistance. Funded by a grant from the
Packard Center for ALS Research.
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