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NeuroMolecular Medicine
Copyright © 2004 Humana Press Inc.
All rights of any nature whatsoever reserved.
ISSN1535-1084/04/05:193–204/$25.00
ORIGINAL ARTICLE
The Neuroprotective Factor Wlds Does Not Attenuate Mutant
SOD1-Mediated Motor Neuron Disease
Christine Vande Velde,1 Michael L. Garcia,1 Xinghua Yin,2
Bruce D. Trapp,2 and Don W. Cleveland*,1
1
Ludwig Institute for Cancer Research and Departments of Medicine and Neuroscience, University
of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093; 2The Lerner Research Institute,
The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44105
Received August 18, 2004; Revised September 15, 2004; Accepted September 20, 2004
Abstract
Selective degeneration and death of motor neurons in SOD1 mutant-mediated amyotrophic
lateral sclerosis (ALS) is accompanied by axonal disorganization and reduced slow axonal transport in the three most frequently used mouse models of mutant SOD1-mediated ALS. To test
whether suppression of axonal degeneration (frequently known as Wallerian degeneration)
could slow disease development, we took advantage of a spontaneous mouse mutant Wlds
(Wallerian degeneration slow) in which the programmed axonal degenerative process that is
normally activated after axonal injury is significantly delayed. Despite its effectiveness in delaying axonal loss in other neurodegenerative models, the presence of Wlds did not slow disease
onset, ameliorate mutant motor neuron death, axonal degeneration, or preserve synaptic attachments in mice that develop disease from ALS-linked SOD1 mutants SOD1G37R or SOD1G85R.
However, presynaptic endings in both the presence and absence of Wlds showed high accumulations of mitochondria and synaptic vesicles, implicating errors of retrograde transport as
a consequence of SOD1-mutant damage to axons.
Index Entries: SOD1; Wallerian degeneration; motor neuron; amyotrophic lateral sclerosis
(ALS); axon.
Introduction
Amyotrophic lateral sclerosis (ALS) or Lou
Gehrig’s disease is a fatal neurodegenerative dis-
ease characterized by the selective loss of motor
neurons in the spinal cord, brainstem, and motor
cortex, resulting in progressive muscle weakness,
atrophy, and inevitable paralysis (Cleveland and
*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]
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Rothstein, 2001). Although the majority of ALS cases
are of unknown etiology (referred to as sporadic
ALS), a small proportion of familial cases have been
attributed to dominant missense mutations in the
ubiquitously expressed enzyme copper/zinc superoxide dismutase (SOD1) (Rosen et al., 1993). Despite
significant efforts to identify the toxic property of
mutant SOD1, the mechanism by which it selectively kills motor neurons remains unknown. However, it is well documented that large caliber
neurofilament-rich axons are susceptible in sporadic
ALS (Kawamura et al., 1981) and in mutant SOD1mediated disease in rodents (Bruijn et al., 1997).
Also, axonal disorganization due to misaccumulation of neurofilaments is a hallmark of disease in
both sporadic (Hirano et al., 1984) and familial
SOD1-mediated disease (Hirano et al., 1984; Rouleau
et al., 1996; Shibata et al., 1996; Kokubo et al., 1999).
Furthermore, defects in slow axonal transport are
an early feature in two well-studied mutant SOD1
models, SOD1G37R and SOD1G85R (Williamson and
Cleveland, 1999). Most intriguingly, a distal to proximal axonal degeneration has recently been
described in SOD1G93A mice (Fischer et al., 2004),
albeit whether this derives from axonal or perikaryal
action of mutant SOD1 is unknown.
Originally described by Waller (1850) as a passive process occurring in response to injury, the systematic disassembly and removal of an injured
axon, referred to as Wallerian degeneration, is now
considered to be an active programmed mechanism
intrinsic to neurons and distinct from the apoptotic
process that occurs in neuronal cell bodies (Lunn
et al., 1989; Glass et al., 1993; Coleman and Perry,
2002). The discovery of the spontaneous mouse
mutant Wallerian degeneration slow (Wlds) demonstrated that despite an otherwise normal phenotype, the distal portion of an injured nerve in Wlds
animals fails to degenerate within the normal
24–48 h, but rather has significantly delayed degeneration, with axons in a distal nerve segment persisting for 2–3 wk and capable of action potential
conduction (Perry et al., 1990; Glass et al., 1993).
The neuroprotective factor in these mice arises from
a genomic triplication generating a fusion protein
in which 70 amino-terminal residues of the ubiquitination factor E4b (UbE4b) is fused to the entire
reading frame of the nicotinamide mononucleotide
adenylyl transferase (Nmnat) (Conforti et al.,
2000; Mack et al., 2001). Recently, Wld s-mediated
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Vande Velde et al.
axonal protection has been attributed to increased
nuclear nicotinamide adenine dinucleotide (NAD)
biosynthesis and downstream activation of the
effector SIRT1, a protein deacetylase involved in
gene regulation (Araki et al., 2004). The neuroprotective genetic program that SIRT1 presumably regulates remains undetermined.
Neuroprotective effects of Wld s have been
reported for experimental models of vincristineinduced peripheral neuropathy (Wang et al., 2001a)
and paclitaxel-induced sensory neuropathy (Wang
et al., 2002). Furthermore, introduction of the dominant Wlds mutation has produced significant positive effects in two neuropathy models, including the
pmn/pmn mouse model of peripheral neuropathy
(Ferri et al., 2003) and the P0-deficient mouse model
of myelin-related axonopathy (Samsam et al., 2003).
The role of axonal degeneration in the context of
mutant SOD1-mediated disease remains undetermined. To directly test if maintenance of axonal
structure and function impacts motor neuron disease, we introduced the dominant Wlds mutation
into two mouse models of mutant-SOD1 mediated
motor neuron disease.
Materials and Methods
Mice
C57BL/Wlds mice were obtained from HarlanOlac (Bicester, UK). C57BL/6 mice carrying mutant
human SOD1 (SOD1G37R and SOD1G85R) transgenes
have been previously described (Wong et al., 1995;
Bruijn et al., 1997). Wlds heterozygotes were mated
with both SOD1G37R and SOD1G85R heterozygote animals. All animals were genotyped for Wlds (Mi et al.,
2002) and SOD1 mutants (Wong et al., 1995; Bruijn
et al., 1997) as previously described. Littermates were
maintained as controls. The entire cohort of animals,
which included approximately equivalent numbers
of males and females, were weighed and monitored
at regular intervals and disease onset was scored as
the point at which there was a 10% decrease in body
weight. Animals were scored as end-stage when they
could no longer right themselves within 10 s.
Immunoblot Analysis
Whole brains and spinal cords were processed
for immunoblot analysis as previously described
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(Samsam et al., 2003). Polyclonal anti-Wlds antibody has been previously described (Conforti et
al., 2000) and was kindly provided by Dr. Michael
Coleman (University of Cologne, Germany). Polyclonal anti-SOD1 peptide antibody that recognizes
human SOD1 has been previously described
(Bruijn et al., 1997). Monoclonal anti-α-tubulin
(DM1A) antibody was purchased from Sigma (St.
Louis, MO).
Morphological Analysis
Lumbar spinal cords and L5 ventral roots from
transcardially perfused mice were processed as
previously described (Bruijn et al., 1997). Thick
sections (1 µm) for light microscopy were stained
with cresyl violet or toluidine blue. Axon number
and caliber distribution were determined from
cross-sections, blindly with respect to genotype,
as previously described (Rao et al., 2002). Floating 40 µm sections were collected from gluteus
muscles of animals perfused with 4% paraformaldehyde. Post-synaptic membranes were visualized
with Alexa Fluor 488-conjugated bungarotoxin
(Molecular Probes, Eugene, OR) and presynaptic
axons were immunostained with antibodies to
phosphorylated neurofilaments (SMI31; Sternberger Monoclonal Inc., Lutherville, MI) and
synaptophysin (Zymed, San Francisco, CA) (Yin
et al., 2004). Secondary antibodies were Cy5labeled donkey anti-mouse and goat anti-rabbit
(Jackson Immunochemicals, West Grove, PA). Sections were mounted in ProLong Antifade reagent
(Molecular Probes) and imaged on a Bio-Rad confocal microscope. For ultrastructural studies,
diaphragm and soleus muscles dissected from animals perfused with 2.5% glutaraldehyde, 4%
paraformaldehyde were processed and embedded
in Epon resin. NMJs were identified in 1 µm sections and then sectioned for electron microscopy,
as described (Yin et al., 2004).
Results and Discussion
Wlds Does Not Ameliorate Mutant
SOD1-Mediated Disease
To determine whether inhibiting axonal degeneration ameliorates ALS pathogenesis, SOD1G37R
and SOD1G85R mice were generated that also express
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Fig. 1. Generation of SOD1 G37R ;Wld s and
SOD1G85R;Wlds mice. Whole brain (for Wlds) and spinal
cord (for mutant SOD1) homogenates were prepared
from mice at disease onset and analyzed by
immunoblot as indicated. Tubulin content is shown as
a loading control.
the dominant neuroprotective factor Wlds, arising
from the spontaneous mutant Wlds. Since SOD1G37R
and SOD1G85R are enzymatically active and inactive, respectively, utilizing both models permitted
examination of the neuroprotective effects of Wlds
in disease arising from SOD1 mutants of divergent
biochemical characteristics. Examination of mutant
SOD1 protein levels in spinal cord extracts prepared from animals at disease onset demonstrated
that the presence of Wlds did not affect mutant
SOD1 protein levels (Fig. 1). In addition, relative
to non-mutant SOD1 littermates Wlds protein levels
remained constant in both SOD1G37R and SOD1G85R
animals (Fig. 1) through disease onset at 5 and 12
mo, respectively, consistent with and extending
previously published reports demonstrating undiminished Wlds protein levels during aging (Gillingwater et al., 2002; Samsam et al., 2003). Despite the
continuous presence of Wld s, survival of either
mutant line was not significantly different from
that in littermate SOD1 mutant mice without the
Wlds mutation. Specifically, disease end-stage (Fig.
2C,D) was determined as 175 ± 12 d and 175 ± 13
d in SOD1 G37R and SOD1 G37R;Wld s mice, respectively; and 350 ± 52 d and 363 ± 33 d in SOD1G85R
and SOD1G85R;Wlds mice, respectively. Disease onset
was similarly unchanged (Fig. 2A,B; summarized
in Table 1). For both mutant SOD1 lines, no genderspecific differences in survival or disease onset were
observed in the presence or absence of Wlds.
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Vande Velde et al.
Fig. 2. Onset and survival of mutant SOD1-mediated motor neuron disease is not altered by the presence of
Wlds. Disease onset, as determined by 10% weight loss, in (A) SOD1G37R;Wlds (n = 23) and SOD1G37R mice (n =
16); and (B) SOD1G85R;Wlds (n = 19) and SOD1G85R mice (n = 10). Kaplan–Meier survival curves of (C) SOD1G37R;Wlds
(n = 24) and SOD1G37R mice (n = 19); and (D) SOD1G85R;Wlds (n = 16) and SOD1G85R mice (n = 8).
Wlds Does Not Prevent Mutant
SOD1-Mediated Motor Neuron Loss
and Axonal Loss/Degeneration
Wld s has been demonstrated to actively delay
Wallerian degeneration of transected axons in
mice ranging from 1 to 16 mo (Crawford et al.,
1995). Also, Wld s has previously been shown to
functionally delay and/or prevent axonal degeneration in other models of neurodegeneration
(Wang et al., 2001a,b; Ferri et al., 2003; Samsam
et al., 2003). Therefore, despite its inability to slow
disease onset or extend survival, we investigated
whether Wld s rescued the obvious Wallerian
degeneration and/or axonal loss that is characteristic of diseased mutant SOD1 mice. Examination of L5 ventral roots of SOD1 mutant mice
with or without the Wld s gene revealed, how-
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Table 1
Onset of Disease and Survival of Mutant SOD1 Mice
in the Presence or Absence of Wlds
Onset (d)
Survival (d)
SOD1G37R;Wlds
SOD1G37R
p-Value
160 ± 15
153 ± 16
0.75
175 ± 13
175 ± 12
0.98
SOD1G85R;Wlds
SOD1G85R
p-Value
354 ± 38
349 ± 59
0.94
363 ± 33
350 ± 52
0.84
Data were analyzed using unpaired Student’s t-test
and expressed as the mean ± standard error.
ever, that degeneration at end-stage disease was
indistinguishable (Fig. 3A). Counting of surviving motor axons in these L5 ventral roots revealed
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Fig. 3. Wlds does not preserve axonal number or morphology in mutant SOD1 mice. (A) Representative micrographs of the L5 ventral root of end-stage animals. (B, C) Axon counts and distribution of calibers of end stage
animals (n = 3 to 5 per genotype).
that the Wld s gene product did not confer any
significant protection against axonal degeneration (Fig. 3B) nor did it prevent the preferential
loss of large caliber axons, producing essentially
indistinguishable size distributions of axons
remaining at end stage (Fig. 3C). Unexpectedly,
by end-stage disease rather than offering protection against axonal loss, in both SOD1 G37R and
SOD1 G85R mice, Wld s expression led to slightly
fewer axons. Wld s also offered no protection for
motor neuron cell bodies (Fig. 4) and did not ameliorate the minor sensory axon loss (data not
shown) that has been previously reported in
SOD1 G37R mice (Wong et al., 1995) or SOD1 G85R
mice (Williamson et al., 1998).
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Neuromuscular Junctions Are Not Rescued
by Wlds Protein Expression
in Mutant SOD1 Mice
ALS disease is characterized by extensive muscular atrophy in both patients and rodent models
(Cleveland and Rothstein, 2001). Denervation by
examination of neuromuscular junctions has only
recently been demonstrated in the SOD1G93A mouse
model (Fischer et al., 2004), but remains unaddressed
in other models. Furthermore, it has been proposed
that motor neuron degeneration in the SOD1G93A
mouse model is initiated at neuromuscular junctions
(NMJs) and progresses in a distal to proximal direction, yielding a “dying-back” axonopathy (Fischer et
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Fig. 4. Motor neuron loss is not attenuated in the presence of Wlds. Representative micrographs of lumbar spinal
cords of (A) C57BL/6, (B) Wlds, (C) SOD1G37R, (D) SOD1G37R;Wlds, (E) SOD1G85R, and (F) SOD1G85R;Wlds at disease end-stage.
al., 2004). Wlds expression has been reported to delay
withdrawal of neuromuscular synapses following
axotomy of young (1–2 mo) animals (Gillingwater et
al., 2003). Axotomized Wlds nerve terminals, but not
wild-type ones, are also capable of neurotransmitter
release and synaptic vesicle membrane recycling for
several days following injury (Gillingwater and
Ribchester, 2001). Wlds expression has been reported
to delay degeneration at the neuromuscular junctions in axotomized animals less than 4 mo old but
not in those more than 7 mo old (Gillingwater et al.,
2002). Since disease is initiated in SOD1G37R mice at
approx 5 mo, we sought to determine whether Wlds
could preserve synaptic terminals in symptomatic
mice. Examples of both prominent denervation (in
which presynaptic end-plates are completely absent
from postsynaptic structures [Fig. 5D]) and partial
denervation (in which axonal structures appear to
make some contact with the postsynaptic specializations [Fig. 5C]) of motor end-plates were
observed in symptomatic SOD1 G37R mice (as
expected from the animal’s clinical phenotype) and
this was not ameliorated by the expression of Wlds
(Fig. 5D,F). Consistent with data reported for
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another mouse model (SOD1G93A) (Fischer et al.,
2004), 84% of end plates examined were denervated
(either partially or completely) in symptomatic
SOD1G37R mice (compared to 75% of junctions examined in SOD1G37R;Wlds mice). Neuromuscular junctions in age-matched Wlds mice were more than 95%
innervated, as expected (Fig. 5A,B).
Axonal withdrawal from synaptic terminals in
axotomized Wlds mice is characterized by dense
packing of synaptic vesicles at the presynaptic membrane and neurofilament accumulations in the center
of the bouton (Gillingwater and Ribchester, 2001). To
determine if Wlds expression influenced the axonal
ultrastructure of degenerating neuromuscular junctions in symptomatic SOD1G37R mice, transmission
electron microscopy was used to examine diaphragm
and/or soleus muscles. Relative to normal mice (Yin
et al., 2004), all synaptic terminals examined displayed abnormal ultrastructural features. Specifically, while some nerve terminals maintained close
proximity to postsynaptic specializations and were
capped by a terminal Schwann cell in both SOD1G37R
and SOD1G37R;Wlds mice (Fig. 6A,D; arrowheads),
neighboring regions of postsynaptic membrane were
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Fig. 5. Neuromuscular junctions of symptomatic SOD1G37R mice are not preserved by expression of Wlds. Representative micrographs of gluteus muscles isolated from (A, B) Wlds, and symptomatic (disease onset) (C, D)
SOD1G37R, and (E, F) SOD1G37R;Wlds animals. Insets of enlarged boxed areas illustrate innervation/denervation.
Innervation is represented by the overlap of pre-synaptic nerve terminals (red) with bungarotoxin-labeled postsynaptic motor end plates on the muscle (green). Examples of both partial denervation (in which motor end plates
are not fully opposed by pre-synaptic terminals) and complete denervations in which endplates are devoid of any
pre-synaptic apparatus, are presented for both SOD1G37R and SOD1G37R;Wlds animals.
devoid of presynaptic terminals indicating partial
denervation (Fig. 6A,C,D; brackets). In both types of
animals, synaptic terminal degeneration was evident
by the presence of both collagen fibrils and Schwann
cell processes adjacent to these unoccupied junctional
folds (Fig. 6B,C; arrowheads). There was also an
abundance of small synaptic terminals occupying a
small part of the width of a synaptic gutter (Fig. 6D),
possibly representing regenerating or retracting terminal branches (Kawabuchi et al., 2000).
Furthermore, at disease onset in both in the presence and absence of Wlds, the remaining synaptic
NeuroMolecular Medicine
terminals in SOD1G37R mice contained an unusually
dense packing of synaptic vesicles (Fig. 6, A–D;
labeled “V”), and in some cases there was an abundance of mitochondria (Fig. 6A; labeled “m”). This
phenotype is reminiscent of axotomized Wlds nerve
terminals (Gillingwater and Ribchester, 2001).
Distal vesicle and mitochondrial accumulation is
consistent with either of two previously unrecognized deficits in these SOD1 mutant mice: inhibited
retrograde transport or increased anterograde
transport, or both. While the very high increase in
synaptic vesicles could also reflect an additional
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Fig. 6. Abnormal ultrastructure of neuromuscular junctions of symptomatic SOD1G37R mice. Representative
micrographs of diaphragm and soleus muscles of (A, B) SOD1G37R and (C, D) SOD1G37R;Wlds animals at disease
onset. A schematic (A’) of the junction in A illustrates the juxtapositioning of pre- and post-synaptic structures and
a terminal Schwann cell. In all four images, there are instances of post-synaptic specializations without opposing
nerve terminals (brackets) and Schwann cell processes in the vicinity of the neuromuscular junction (arrowheads).
All remaining synaptic boutons have an unusual packing of synaptic vesicles (V), and some had abnormal accumulations of mitochondria (m). Scale bars = 1 µm.
defect in turnover/recycling/dynamics, the concomitant mitochondrial accumulation distally offers
strong evidence that aberrant fast axonal transport
is the major contributor. In light of an earlier report
of preferential association of both dismutase active
and inactive SOD1 mutants with the cytoplasmic
face of mitochondria from spinal cord, but not unaffected tissues (Liu et al., 2004), and the apparent
association of mutant SOD1 with Bcl-2 (Pasinelli
et al., 2004), an anti-apoptotic component localized
to the outer mitochondrial membrane (Krajewski
et al., 1993), these findings raise the possibility that
mutant SOD1 association with the mitochondrial
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surface directly inhibits binding and/or activation
of components of dynein-dependent retrograde
transport, but not action of one or more anterograde
kinesins. This disruption of the normal mechanisms
for insuring proper distribution of mitochondria,
which include the balance of interactions of the outer
mitochondria surface with rapid anterograde and
retrograde microtubule motors (Nangaku et al., 1994;
Morris and Hollenbeck, 1995), is not affected by
Wlds and may reflect a primary toxicity of SOD1
mutants. Mouse models in which defective retrograde
transport is causative of motor neuron degeneration and disease have been previously reported
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Axonal Degeneration in Mutant-SOD1 Mice
(LaMonte et al., 2002; Hafezparast et al., 2003).
Finally, motor neurons, as with many neuron subtypes, rely on both inter- and intra-neuronal signaling for survival. This is especially relevant for
the non-cell autonomous toxicity to motor neurons,
which requires SOD1 mutant damage acting
within non-neuronal cells (Clement et al., 2003).
The failure of a factor that is localized to motor neurons and has previously been reported to protect
against axonal degeneration and loss in other mouse
models, to ameliorate SOD1 mutant toxicity adds
further support for this non-cell autonomous disease mechanism.
Acknowledgments
We thank Dr. M. Coleman (University of Cologne,
Germany) for generously providing anti-Wlds antibody. We also thank T.M. Miller for reading the manuscript; as well as Y. Wang, C.M. Ward, J. McClure,
and J. Folmer for excellent technical assistance. This
work was supported by a grant from the NIH to B.D.T.
and D.W.C. C.V.V. and M.L.G are supported by postdoctoral fellowships from the Paralyzed Veterans of
America Spinal Cord Research Foundation and NIH,
respectively. Salary support for D.W.C. is provided
by the Ludwig Institute for Cancer Research.
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