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Experimental Neurology 185 (2004) 232 – 240
www.elsevier.com/locate/yexnr
Amyotrophic lateral sclerosis is a distal axonopathy:
evidence in mice and man
Lindsey R. Fischer, a Deborah G. Culver, a Philip Tennant, a Albert A. Davis, a Minsheng Wang, a
Amilcar Castellano-Sanchez, b Jaffar Khan, a Meraida A. Polak, a and Jonathan D. Glass a,b,*
b
a
Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA
Received 21 August 2003; revised 12 September 2003; accepted 7 October 2003
Abstract
The SOD1 mutant mouse is the most widely used model of human amyotrophic lateral sclerosis (ALS). To determine where and when the
pathological changes of motor neuron disease begins, we performed a comprehensive spatiotemporal analysis of disease progression in
SOD1G93A mice. Quantitative pathological analysis was performed in the same mice at multiple ages at neuromuscular junctions (NMJ),
ventral roots, and spinal cord. In addition, a patient with sporadic ALS who died unexpectedly was examined at autopsy. Mice became
clinically weak at 80 days and died at 131 F 5 days. At 47 days, 40% of end-plates were denervated whereas there was no evidence of ventral
root or cell body loss. At 80 days, 60% of ventral root axons were lost but there was no loss of motor neurons. Motor neuron loss was well
underway by 100 days. Microglial and astrocytic activation around motor neurons was not identified until after the onset of distal axon
degeneration. Autopsy of the ALS patient demonstrated denervation and reinnervation changes in muscle but normal appearing motor
neurons. We conclude that in this widely studied animal model of human ALS, and in this single human case, motor neuron pathology begins
at the distal axon and proceeds in a ‘‘dying back’’ pattern.
D 2003 Elsevier Inc. All rights reserved.
Keywords: Sclerosis; Axonopathy; Denervation
Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive weakness leading to paralysis and death. Approximately 2% of cases are
due to mutations in the superoxide dismutase (SOD1) gene.
Transgenic mice carrying human SOD1 mutations develop
progressive weakness similar to patients with ALS, making
them useful models for understanding the pathogenesis of
disease and testing new therapies. A widely used transgenic
mouse model is the high-expressing SOD1G93A mutant that
develops clinical disease at 80 – 90 days and dies at about
130 days (Chiu et al., 1995). This model has proven useful
for examining the cellular pathology of motor neuron
degeneration in ALS, and has provided a tool for developing
* Corresponding author. Emory Center for Neurodegenerative Disease,
Whitehead Biomedical Research Building, 5th Floor, 615 Michael Street,
Mailstop 1941007001, Atlanta GA 30322. Fax: +1-404 727-3728.
E-mail address: [email protected] (J.D. Glass).
0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2003.10.004
preclinical data on drugs that may work to slow the course
of ALS.
There is a general acceptance that weakness and death in
the SOD1G93A mutant mouse, and in humans with ALS,
occur as a direct consequence of motor neuron death. There
are studies, however, that have demonstrated dysfunction
and/or degeneration of the neuromuscular junction (NMJ) at
times earlier than are reported for the loss of motor neurons.
Using immunocytochemical methods, Frey et al. (2000)
showed selective loss of fast-firing neuromuscular synapses
as early as day 50, and Kennel et al. (1996) reported
progressive loss of motor unit numbers by physiologic
measures beginning at day 40. Motor neurons were not
counted in either of these studies, but reports from other
investigators do not suggest a significant loss of motor
neurons in the G93A mouse until after 80 – 90 days (Chiu
et al., 1995).
These data raise the question of where motor neuron
dysfunction begins: within the motor neuron cell body,
within the motor axon, or even at the level of the neuromuscular junction. Recent reports of abnormalities in retro-
L.R. Fischer et al. / Experimental Neurology 185 (2004) 232–240
grade axonal transport leading to motor neuron death
support a peripheral localization for factors initiating the
onset of this disease (Hafezparast et al., 2003; LaMonte et
al., 2002; Puls et al., 2003). Human studies of ALS do not
provide an answer to this question, and no data are available
correlating weakness or death with loss of spinal motor
neurons.
In order to better understand the progression of disease in
the SOD1G93A mutant mouse, we undertook a systematic
pathological study of these animals at multiple time points
along the course of disease. In the same animals, we
quantified the numbers of spinal motor neurons, axons in
the nerve roots, and the degree of denervation at neuromuscular junctions, providing a sequential view of motor
neuron pathology in these animals. Our findings demonstrate that before any loss of motor neurons, there is a severe
loss of ventral root motor axons and significant denervation
at corresponding neuromuscular junctions. Progression of
abnormalities was from distal to proximal, indicating a
‘‘dying back’’ pathophysiology. In addition, we had the
opportunity to study at autopsy a patient with sporadic ALS
who died early and unexpectedly. His pathology also
suggested a pattern of disease similar to our findings in
the mouse.
Materials and methods
Animal breeding and behavioral analysis
All animal protocols were approved by the Emory
University Institutional Animal Care and Use Committee.
Animals were housed in microisolator cages on a 12h light – dark cycle and given free access to food and water.
Breeding pairs of G93A high-expressing mice were
obtained from Jackson Laboratories (Bar Harbor, ME),
and identification of mutant mice was by standard PCR
analysis on tail snip DNA.
Beginning at age 50 days, SOD-1 mutant mice (12 males
and 12 females) were tested weekly for their ability to
maintain balance on a Rotarod apparatus (Columbus Instruments, Ohio). Two protocols were used, constant velocity at
15 rpm and an accelerating paradigm of 1.4 + 4 rpm/min.
Animals were tested three times during each session, and the
best performance was recorded. Times were recorded to a
maximum of 300 s. Weight was also recorded weekly. Age
of death was defined as the time when animals could not
right themselves within 30 s of being placed on their backs.
Survival was 131 F 5 days; there were no differences in
survival between males and females.
Neuropathology
A separate cohort of transgenic SOD-1 mutants and nontransgenic littermates (controls) were deeply anesthetized
and killed by cardiac transection at 28 days (three mutants,
233
three controls), 47 days (three mutants, three controls), 80
days (four mutants, three controls), 100 days (five mutants,
three controls), and 120 days (seven mutants, three controls). A group of six SOD-1 mutants was also analyzed at
the time of natural death (131 F 5 days). Tissues were
harvested and fixed appropriately for evaluation of endplates– neuromuscular junctions (NMJ), ventral roots, and
lumbar motor neurons.
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 4jC) 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
microscope. End-plates (red) were scored as ‘‘innervated’’ if
there was complete overlap with the axon terminal (green),
or ‘‘denervated’’ if the end-plate was not associated with an
axon. Some neuromuscular junctions were associated with a
preterminal axon only, or showed partial overlap between
end-plate and terminal. These were labeled as ‘‘intermediate’’. 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).
Ventral roots
Nerve roots were exposed and immersion-fixed in 5%
buffered glutaraldehyde (pH 7.4) at 4jC for 48 h. Ventral
roots from L-4 were dissected out and stored in 0.1 M
phosphate buffer at 4jC. Tissue was treated with 1%
osmium tetroxide for 90 min, 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. Measurements 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 inte-
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L.R. Fischer et al. / Experimental Neurology 185 (2004) 232–240
Lumbar motor neurons
Fig. 1. Clinical analysis of motor neuron disease in SOD1G93A mice.
Animal weight reported as % baseline and Rotarod performance expressed
as % survival (note: ‘‘survival’’ here refers to the ability of the animal to
remain on the Rotarod for the specified time).
riors were visually identified and manually marked as solid
objects. The mean diameter of each object was measured.
The data were exported to a Microsoft Excel (Microsoft
Corporation, Redmond, WA) spreadsheet for analysis. Axon
caliber distributions for whole populations and specific
subsets were analyzed using histogram plots. Means were
compared by ANOVA with Dunnett’s post hoc comparison.
The spinal cord was fixed in situ with 4% buffered
paraformaldehyde for 48 h. The entire lumbar enlargement
was then dissected, embedded in paraffin wax, and exhaustively cross-sectioned at 8 AM, six sections to a slide (123 F
12 slides or 5.90 F 0.58 mm of tissue per specimen). Tissue
was deparaffinized in xylene, stained with cresyl violet,
differentiated, and coverslipped.
A systematic and unbiased sampling procedure was
designed using stereologic principles to insure that every
motor neuron in the lumbar spinal cord had an equal
probability of being counted (Coggeshall and Lekan,
1996). A pilot counting study was done using an 80-day
animal to determine the approximate number of sections that
would need to be analyzed to count at least 100 –200 motor
neurons for each spinal cord specimen. This sample size has
been repeatedly shown to produce coefficients of error
within reasonable limits for a biological system (West et
al., 1991). Based on this pilot study, every 10th slide (a 480Am interval, amounting to 10 –14 slides counted per animal)
was chosen for counting, with the first slide chosen by
random selection. One ventral horn of one tissue section
was analyzed per slide. Sections were imaged in overlapping frames (20 objective) using a Hamamatsu digital
camera. Each Nissl-stained neuron in the ventral horn with a
Fig. 2. NMJ immunocytochemistry. Motor endplates are identified with a-bungarotoxin (red); axons are identified with neurofilament + SV2 (green). Images
depict (A) innervated endplates (yellow indicates overlap), (B) intermediate endplates, (C) denervated endplates, (D) quantitative analysis. Data are shown as %
of endplates classified as innervated, denervated, or intermediate. Numbers in parenthesis indicate number of endplates quantified per time point.
L.R. Fischer et al. / Experimental Neurology 185 (2004) 232–240
distinct nucleolus and a darkly stained cytoplasm was
manually traced using Image Pro software. Area and mean
diameter were recorded for each neuron. a-Motor neurons
were defined as having cross-sectional area z250 Am2
(Drachman et al., 2002).
Data were analyzed as both mean number of a-motor
neurons per section and as total number of a-motor neurons
per lumbar spinal cord, estimated using the fractionator
method (Coggeshall and Lekan, 1996). This method takes
into account the fractions used to select the sample to be
counted, and then estimates the total population by multiplying back by those fractions. Means were compared by
ANOVA with Tukey –Kramer post hoc comparison.
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Human neuropathology
Autopsy was performed approximately 24 h after death.
The entire brain and spinal cord was examined. Microscopic
sections were prepared from motor cortex, and from spinal
cord and nerve roots from cervical, thoracic, lumbar, and
sacral levels. Spinal cord sections were stained with H and
E, luxol fast blue for myelin, and with GFAP and HAM-56
for astrocytes and microglia, respectively (Glass et al.,
1995).
Results
Spinal cord immunocytochemistry
SOD1 clinical evaluation
Spinal cord sections adjacent to those used for counting
were stained using standard techniques for astrocytes (GFAP
1:500, DAKO) and microglia (CD11b/Mac1 1:25, Serotec).
Sections were developed using the Vectastain ABC kit
(Vector Labs, Burlingame, CA), reacted with diaminobenzidine (DAB), and counterstained with hematoxylin.
The first indication of clinical disease was decreased
performance on the accelerating Rotarod at day 78.
Performance on the constant Rotarod began to decline
at day 85. Weight was not a sensitive measure of disease
onset, and did not deviate from baseline until after 120 days
(Fig. 1).
Fig. 3. (A) Light microscope images of L4 ventral roots from SOD1G93A mice at the time points indicated. (B) Number of ventral root axons reported as %
control. Number of animals examined at each time point in parentheses. (C) Small versus large axons from 80 days to death. Note relative increase in
proportion of small caliber axons, indicating regeneration. Data are means F SEM. Numbers in parentheses indicate number of animals examined (** indicates
P < 0.01 vs. control).
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L.R. Fischer et al. / Experimental Neurology 185 (2004) 232–240
Neuropathology
Initial evaluation of end-plates and ventral roots in 80day-old presymptomatic animals showed significant abnormalities in both, prompting us to look for the initial
neuropathologic changes at earlier time points. At 28 days,
virtually 100% of end-plates were innervated, and there was
no evidence of axonal degeneration in the ventral root.
Denervation of end-plates was significant by day 47
(40%) and continued to progress up to the time of death
(Fig. 2). Neuromuscular junctions showing terminal axons
but no end-plate overlap (‘‘intermediate’’) represented approximately 10% of the population between days 47 and
120, and dropped to minimal numbers at the time of death.
Pathological changes in the ventral root were not seen
until 80 days (Fig. 3). By day 80, the density of intact motor
axons was markedly reduced, with the larger caliber axons
preferentially affected. Actively degenerating fibers were
recognized at all time points after 80 days, constituting
14.71 F 6.01% of total axons at day 80, 11.25 F 1.35% at
day 100, 7.29 F 1.61% at day 120, and 6.95 F 1.60% at
death. The total number of ventral root fibers, however, did
not decrease after 80 days. In fact, there was a statistically
significant increase in axon numbers at 120 days as compared to 80 and 100 days (Fig. 3B). There was also a
marked increase in the proportion of small caliber axons
(Fig. 3C), suggesting regeneration. Regenerating ‘‘clusters’’
of axons, however, were not seen.
Vacuolation of large motor neuron cell bodies, indicating
active degeneration, was first observed at day 80 and
continued through death (Fig. 4). The numbers of large
motor neurons did not decrease, however, until the 100-day
time point. Quantitative analysis of a-motor neurons was
performed both as mean number of a-motor neurons per
section, and as an estimate of total neuron number using the
fractionator method. Both methods yielded a similar outcome, showing a loss of approximately 40% of neurons with
area z250 Am2 between days 80 and 100, when compared
to littermate controls (Fig. 5).
There was an apparent increase in the number of a-motor
neurons with age in the control groups. Post hoc analysis
demonstrated that the mean cross-sectional area of motor
neurons increased from 181.82 Am2 at day 28 to 254.67 Am2
at day 120. Since size-exclusion (z250 Am2) was used to
define a-motor neurons, this explains the increase with age
in the number of neurons in this category. The total number
of neurons counted in the control groups, however, did not
change over time (data not shown).
In order to indirectly examine the ‘‘health’’ of lumbar
motor neurons at time points before frank degeneration, we
stained the spinal cords with markers for activated astrocytes and microglia (Hall et al., 1998). Reactive astrocytosis at a level different from littermate controls was barely
evident at 47 days and increased in intensity to the time of
death (Fig. 6). Microglial activation was less prominent
than astrocytosis at all stages and did not precede patho-
Fig. 4. (A) Macroscopic view of lumbar spinal cord. Boxed area is region defined as ventral horn for motor neuron counting. (B) Representative images from
the ventral horn of SOD1G93A animals at the ages indicated. Twenty-eight-day micrograph depicts outlining of neuron as seen with Image Pro analysis
software.
L.R. Fischer et al. / Experimental Neurology 185 (2004) 232–240
237
logic changes at the NMJ or the ventral roots (data not
shown).
Case report
The patient was a 58-year-old man with a 6-month
history of weakness and wasting. Physical examination
demonstrated diffuse fasciculations, muscle atrophy, normal
Fig. 5. (A) Number of a-motor neurons per section. Numbers in parentheses
indicate number of animals examined. (B) Total number of a-motor neurons
in lumbar spinal cord, estimated using the fractionator method. Data are
means F SEM (** indicates P < 0.01, *** indicates P < 0.001 vs. control).
Fig. 6. GFAP staining for astrocytes in SOD1G93A animals 28 days through
death. Staining is first distinguishable from control spinal cord at 47 days.
Fig. 7. Neuropathology from human ALS case. (A) H and E stained section
showing grouped atrophy and angulated fibers (insert), indicating acute and
chronic denervation – reinnervation. (B) ATPase (pH 4.2) showing fibertype grouping. (C) Lumbar spinal cord showing a normal complement of
motor neurons. Morphology of motor neurons better seen in insert.
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L.R. Fischer et al. / Experimental Neurology 185 (2004) 232–240
to brisk reflexes, and normal sensation. Forced vital capacity
was 2.7 l (57% of predicted). Electromyographic examination showed changes consistent with acute and chronic
denervation in upper and lower extremities, and in thoracic
paraspinal muscles. Two weeks after this examination, he
died unexpectedly during a minor surgical procedure.
Neuropathological analysis (Fig. 7) demonstrated
grouped atrophy and fiber-type grouping in skeletal muscles
from the legs and thorax. Ventral roots at all levels showed
postmortem autolytic changes but little axonal degeneration,
and all levels of the spinal cord appeared normal. Astrocytic
and microglial activations were not prominent at any level.
Corticospinal tract degeneration was not noted, and motor
cortex appeared normal.
Discussion
We examined the cell body, axon, and NMJ at multiple
time points to illustrate the spatiotemporal progression of
motor neuron pathology in the high-expressing SOD1G93A
mutant mouse and related these findings to the clinical onset
of weakness in these animals. As in previous studies, we first
recognized signs of clinical disease at about 80 days, but there
were significant pathological changes at much earlier time
points. Quantitative analysis demonstrated denervation at the
NMJ by day 47, followed by severe loss of motor axons from
the ventral root between days 47 and 80, and loss of a-motor
neuron cell bodies from the lumbar spinal cord after day 80.
This pattern implies that motor neuron disease in the
SOD1G93A mouse is actually a ‘‘dying back’’ motor neuropathy where distal axonal degeneration occurs early during the
disease, before neuronal degeneration, and onset of symptoms. The human case, though anecdotal and non-quantified,
showed a similar pattern of disease with prominent evidence
of motor neuron degeneration only in muscle. The published
neuropathology of human ALS is restricted to postmortem
analysis of late stage disease (Bradley et al., 1983; Sobue et
al., 1983). These types of analyses cannot be expected to
identify the earliest morphological changes in the motor unit.
Our quantitative analysis of the SOD1G93A suggested
partial ‘‘recovery’’ at 120 days in the ventral roots and at
end-plates. We believe the transient increase in innervated
end-plates represents a phase of compensatory reinnervation,
as was documented previously in the SOD1 mutant mouse as
well as in humans with ALS (Bjornskov et al., 1984; Frey et
al., 2000). In the ventral roots, the relative increase in the
number of axons indicates regeneration. This finding is
accentuated by the presence of actively degenerating axons
at all stages between 80 days and death. Without regeneration, active and continuous axonal degeneration should result
in reduction of axon numbers. In addition, the shift in axon
diameters toward most small axons likely reflects the presence of regenerates.
Early pathological changes in the periphery are perhaps a
surprising finding in what has long been considered classic
motor neuron disease. However, ‘‘dying back’’ or slowly
evolving distal to proximal axonal degeneration is a common
pattern seen in a wide variety of degenerative and toxic
conditions of the central and peripheral nervous systems
(Glass, 2002). The pathophysiology of distal axonal degeneration is poorly understood, but the longest and largest
nerve fibers with the highest metabolic demand seem to be
the most susceptible to ‘‘dying back’’. Thus, it has been
suggested that distal axonal degeneration represents a sizedependent ‘‘undernourishment’’ of the most distal region of
the axon (Cavanagh, 1964; Spencer and Schaumburg, 1976).
Several animal models of motor neuron disease have been
characterized as ‘‘dying back’’ neuropathies. In the mnd
(motoneuron degeneration) and the pmn (progressive motor
neuronopathy) mouse models, morphological analysis of the
NMJ showed synaptic weakening and ‘‘dying back’’ before
clinical manifestations of disease (Frey et al., 2000). Pathological analysis of the Smn mouse model of spinal muscular
atrophy indicated NMJ abnormalities and a massive (78%)
loss of ventral root axons at a time when the spinal cord
showed only a 30% loss of motor neurons (Cifuentes-Dias et
al., 2002). Analysis of the NMJ in dogs with hereditary
canine spinal muscular atrophy (HCSMA) also revealed
presymptomatic motor terminal dysfunction (Rich et al.,
2002). Early synaptic changes were linked to deficits in
quantal release of ACh before the actual degeneration of the
motor terminal and the distal axon in this model.
There are many possible explanations for the ‘‘dying
back’’ pattern seen in the SOD1 mouse. As suspected for
sensory neuropathies, this pattern might be due to a sublethal
insult to the cell body that results in undernourishment of the
distal axon. Accumulation of insoluble complexes of mutant
SOD1 protein (Johnston et al., 2000) or chronic glutamate
toxicity (Rothstein et al., 1993) could be responsible for
insufficient maintenance of the distal axon, leading to early
denervation at the NMJ while the cell body remains structurally intact. We investigated this issue by looking for
markers of early astrocyte and microglial activation, hypothesizing that glial activation might be an indirect marker of
abnormalities at the level of the spinal cord. We found some
increased staining for GFAP at 47 days, but little evidence of
microglial activation. Other investigators showed no increase in either microglial or astrocytic markers until 80
days in this model (Hall et al., 1998), long after NMJ and
ventral roots are abnormal. Also, it is not clear that glial
activation indicates local abnormalities in the motor neuron
cell bodies. Peripheral axotomy clearly results in astrocytic
and microglial activation around motor neurons (Graeber et
al., 1988; Tetzlaff et al., 1986).
There is a growing literature describing mechanisms of
motor neuron degeneration that primarily involve defects in
axonal structure or function. Accumulation of abnormal
neurofilaments leading to slowing of anterograde axonal
transport has been shown to be an early event in SOD1 mice
(Williamson and Cleveland, 1999; Zhang et al., 1997).
Disrupted anterograde transport may prevent components
L.R. Fischer et al. / Experimental Neurology 185 (2004) 232–240
necessary for synaptic activity or trophic support of the axon
from reaching the nerve terminal, potentially resulting in
distal axonal degeneration (Griffin and Watson, 1988). More
intriguing is recent evidence of a critical role for retrograde
axonal transport in the maintenance of motor neurons.
Transgenic mice overexpressing the dynamitin subunit of
dynactin, a protein that regulates dynein activity, develop
late-onset, progressive motor neuron disease due to the
inhibition of retrograde transport (LaMonte et al., 2002).
Furthermore, two mouse mutants generated by random
mutagenesis and showing progressive motor neuron disease
were found to have missense mutations in the cytoplasmic
dynein heavy chain (Hafezparast et al., 2003). These mutations selectively inhibited fast retrograde transport in amotor neurons, resulting in neuronal loss and perinuclear
inclusions positive for ubiquitin, SOD1, CDK5, and neurofilaments. In humans, a point mutation in the gene encoding
the p150 subunit of dynactin has been identified in a family
with a slowly progressive, autosomal dominant form of
motor neuron disease (Puls et al., 2003). Therefore, in both
mice and humans, axonal abnormalities that inhibit retrograde transport confer selective damage to motor neurons,
possibly by preventing the delivery of target-derived neurotrophic factors back to the cell body.
Neuronal and axonal degeneration are not necessarily
linked, and in some situations, may occur by separate and
independent mechanisms (Coleman and Perry, 2002). For
example, Finn et al. (2000) showed that withdrawal of NGF
from cultured neurons causes death of both the cell body and
the axon, but only the cell body dies via caspase-mediated
apoptosis. The Wlds (slow-Wallerian degeneration) mouse
also provides several examples of how axonal degeneration
(or survival) can occur in isolation of events at the neuronal
cell body. When deprived of NGF, cultured Wlds sympathetic
neurons exhibit neuronal apoptosis that is indistinguishable
from wild-type neurons, while degeneration of neurites is
selectively delayed (Deckwerth and Johnson, 1994).
Experimental findings in the pmn model clearly show that
primary axonal pathology may lead to a motor neuron
disease phenotype. Pmn is due to a mutation in a tubulinspecific chaperone protein, affecting microtubule assembly
in axons (Bommel et al., 2002; Martin et al., 2003). Crossing
of the pmn mouse with the anti-apoptotic Bcl-2 overexpressing mouse rescued motor neuron cell bodies, but did not
prevent axonal degeneration or prolong survival (Sagot et al.,
1997). Conversely, crossing the pmn mouse with the Wlds
mouse prevented axonal degeneration and resulted in extended survival from 40.5 to 62 days. The Wlds gene also
delayed both retrograde transport deficits and synaptic pathology (Ferri et al., 2003). Moreover, the prevention of
axonal degeneration in pmn by the Wlds gene resulted in
prolonged survival of motor neurons, indicating that in some
circumstances, loss of innervation may result in motor
neuron death. These are perhaps the most compelling data
supporting the importance of axonal degeneration in the
pathogenesis of motor neuron diseases, as well as the idea
239
that axonal degeneration may somehow initiate the cell death
program in the parent neuron.
Treatments to rescue motor neurons according to the cell
death (or ‘‘dying-forward’’) model of motor neuron pathology in fALS have shown only limited success in SOD1 and
other mouse models (Sagot et al., 1997) as well as in
humans (Carter et al., 2003). By separating the motor
neuron from its target muscle, axonal degeneration may be
the more important contributor to the progressive deterioration of motor function in fALS, therefore representing a
very important and neglected therapeutic target (Coleman
and Perry, 2002). Furthermore, if cell body degeneration is
relatively late compared to axonal degeneration, early intervention at the first sign of motor symptoms could potentially
prevent the subsequent irreversible loss of motor neurons.
Acknowledgments
This work was supported by NIH grant P01
NS40405(JDG) and by a grant from the Robert Packard
Center for ALS Research at Johns Hopkins. We thank Dr.
Mark Rich for helpful discussion and Raphael James, Karen
Carney, and Dayna McDermott for technical assistance.
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