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The Journal of Neuroscience, May 1, 1998, 18(9):3241-3250
Massive Mitochondrial Degeneration in Motor Neurons Triggers the
Onset of Amyotrophic Lateral Sclerosis in Mice Expressing a Mutant
SOD1
Jiming
Kong1 and
Zuoshang
Xu2
Departments of 1 Pharmacology and Molecular Toxicology
and 2 Cell Biology, University of Massachusetts Medical
School, Worcester Foundation Campus, Shrewsbury, Massachusetts 01545
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ABSTRACT |
Amyotrophic lateral sclerosis (ALS) involves motor neuron
degeneration, skeletal muscle atrophy, paralysis, and death. Mutations in Cu,Zn superoxide dismutase (SOD1) are one cause of the disease. Mice
transgenic for mutated SOD1 develop symptoms and pathology similar to
those in human ALS. To understand the disease mechanism, we developed a
simple behavioral assay for disease progression in mice. Using this
assay, we defined four stages of the disease in mice expressing G93A
mutant SOD1. By studying mice with defined disease stages, we tied
several pathological features into a coherent sequence of events
leading to motor neuron death. We show that onset of the disease
involves a sharp decline of muscle strength and a transient explosive
increase in vacuoles derived from degenerating mitochondria, but little
motor neuron death. Most motor neurons do not die until the terminal
stage, ~9 weeks after disease onset. These results indicate that
mutant SOD1 toxicity is mediated by damage to mitochondria in motor
neurons, and this damage triggers the functional decline of motor
neurons and the clinical onset of ALS. The absence of massive motor
neuron death at the early stages of the disease indicates that the
majority of motor neurons could be rescued after clinical
diagnosis.
Key words:
ALS; mitochondria; SOD1; paralysis; motor neuron; degeneration; spinal cord
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INTRODUCTION |
Amyotrophic lateral sclerosis (ALS)
involves motor neuron loss leading to progressive skeletal muscle
atrophy and death (Mulder et al., 1986 ; Munsat, 1989). Despite a long
history of clinical and pathological studies, the pathological
progression for ALS has not been clearly defined. This is largely
attributable to difficulties in correlating clinical symptoms and motor
neuron loss in human patients. At least two possible models could
explain the progressive nature of clinical symptoms. The first is that at the onset of muscle weakness the patient has already gone through an
extended period of gradual motor neuron death. The emergence of
symptoms reflects an exhausted functional compensation by the remaining
motor neurons. Therefore, a large fraction of motor neurons have
already been lost, and the clinical course reflects the loss of the
remaining motor neurons. The second possibility is that at the onset of
muscle weakness (or clinical disease) there is little or no motor
neuron death, but there is a decline of motor neuron function. As the
degenerative process proceeds, motor neurons gradually die, eventually
leading to paralysis and death of the patient. The distinction between
these two models has important implications for prognosis and therapy.
New animal models developed recently for ALS have made it possible to
distinguish between these two models.
ALS occurs in sporadic (SALS) and familial (FALS) forms (Mulder et al.,
1986; Munsat, 1989). Mutations in the SOD1 gene are one genetic cause
for FALS (Rosen et al., 1993; Brown, 1995). Several mouse models that
express the mutated genes develop motor neuron degeneration similar to
that in humans (Gurney et al., 1994; Ripps et al., 1995; Wong et al.,
1995; Bruijn et al., 1997). The initial characterization of these mouse
lines has proven that a dominant gain of an "adverse property" by
the mutated enzymes causes motor neuron degeneration (for review, see
Bruijn and Cleveland, 1996; Tu et al., 1997). In addition, these
analyses confirmed numerous pathological features that have been
observed in humans (Hirano, 1991; Chou, 1992), including axonal
spheroids (Tu et al., 1996), increase of ubiquitin (Wong et al., 1995;
Bruijn et al., 1997), Lewy body-like inclusions (Wong et al., 1995;
Bruijn et al., 1997), fragmentation of Golgi apparatus (Mourelatos et al., 1996), and selective loss of motor neurons (Gurney et al., 1994;
Ripps et al., 1995; Wong et al., 1995; Bruijn et al., 1997). The most
prominent new feature is the large number of membrane-bound vacuoles in
G93A and G37R lines (Dal Canto and Gurney, 1995; Wong et al., 1995).
These vacuoles emerge before the end stage of the disease and appear to
be derived from dilated mitochondria (Dal Canto and Gurney, 1995; Wong
et al., 1995) and endoplasmic reticulum (Dal Canto and Gurney,
1995).
In these early studies, pathological changes were correlated with the
age of animals but not with disease stage. Because of the highly
heterogeneous onset and duration of the disease in individual animals
(see below), the subjects of study at any age were a mixture of
individuals at different disease stages. Consequently, pathological
changes seen in animals of the same age were not necessarily the ones
that develop at the same disease stage. Similarly, pathological
features that appear in younger animals do not necessarily precede
those seen in older animals in disease progression. Transient changes,
which correspond to specific disease stages, are particularly difficult
to recognize. In short, pathological studies without definition of the
disease stage cannot delineate the sequence of events leading to motor
neuron death and thus do not use the full potential of these transgenic
animal models.
To effectively study these models, we have conducted the first
experiments to analyze pathological changes in the context of clinical
progression. We first developed a simple, objective assay to measure
muscle strength in mice and used this assay to follow the clinical
progression in a transgenic line that expresses the G93A SOD1 mutation
(Gurney et al., 1994). We then quantitatively measured several
pathological features in the mice with defined disease stages. We show
that the disease progression goes through four stages, each with
specific and time-dependent features. Of particular interest are the
early stages in which mitochondrial abnormalities are the most
prominent feature and neuronal function declines sharply, whereas
neuronal death is minimal. Thus, mitochondrial degeneration is an
important early event in triggering the decline of motor neuron
function and, consequently, clinical disease. The minor motor neuron
loss for a substantial period after the onset of clinical disease
offers a period of time after diagnosis to rescue the majority of motor
neurons.
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MATERIALS AND METHODS |
Human SOD1 transgenic mice. Mice transgenic for the
mutated human SOD1 G93A (TgN[SOD1-G93A]1Gur) and wild-type human SOD1 (TgN[SOD1]2Gur) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in the University of Massachusetts Medical School
animal facility. Both lines were originally made and characterized by
Gurney et al. (1994). The survival of the purchased lines was prolonged
in comparison with the original line because of a reduced number of
transgene copies
(lena.jax.org/resources/documents/imr/SODletter.html). The
transgenic mice were identified using the PCR method described by
Gurney et al. (1994).
Muscle strength test. The test was conducted using a device
designed in this laboratory. Mice were allowed to grab onto a vertical
wire (2 mm in diameter) with a small loop at the lower end. A vertical
wire allows mice to use both fore- and hindlimbs to grab onto the wire.
Although in the first few tests some mice used forelimbs predominantly,
they usually learned to use all four limbs after a few trials. This
results in a significant improvement during the first three trials,
after which the performances stabilize. Thus, both the fore- and
hindlimbs contribute to the measured muscle strength in this assay.
The wire was maintained in a vertically oriented circular motion (the
circle radius was 10 cm) at 24 rpm. Early tests indicated that
maintaining the wire in motion gave much more consistent measurements
than a stationary wire. The time that the mouse was able to hang onto
the wire was recorded with a timer. Because most mice fell within 5 min, we cut off testing at 300 sec to test more animals in a limited
time period. Mice were usually tested once a week starting when they
were 90 d old, and testing continued until they could no longer
hang onto the wire.
Morphological analysis. Animal fixation, tissue dissection,
and microscopic analysis were performed as described previously (Xu et
al., 1993). In brief, mice were anesthetized and perfused with a
solution of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.6. Tissues were kept in the same solution for further fixation. The L4 and L5 spinal nerve roots and
lumbar spinal cords (a 2 mm segment centered at the L5 root entry
level) were dissected out and post-fixed with 2% osmium tetroxide in
100 mM cacodylate buffer, pH 7.6. After dehydration in
graded alcohol, the tissue blocks were embedded in Epon. Sections (1 µm) were stained with toluidine blue and examined with light microscopy.
For quantification of ventral roots, complete microscopic pictures of
L4 and L5 ventral roots were taken from 1 µm transverse sections and
digitized using a Nikon LS-1000 scanner. The area and equivalent radius
of all axons in a ventral root larger than 1.5 µm in diameter were
measured using MetaMorph software (Universal Imaging Corporation, West
Chester, PA). Degenerating axons were counted directly under light
microscope.
To quantify motor neurons in spinal cord, serial transverse sections 8 µm thick were cut from Epon-embedded L5 lumbar spinal cord. Every
fifth section (five sections total from each animal) was collected and
stained with toluidine blue. All neurons with nuclei and located in the
lateral motor column (as defined in Fig.
1) were drawn using a Nikon drawing tube.
The drawings were scanned into computer using an HP flat-bed scanner.
Several parameters, including the number and size of motor neurons and
their nuclear size, were measured from these drawings using MetaMorph
software.
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Figure 1.
The definition of L5 lateral ventral horn
where neuronal numbers were quantified was derived as follows.
Perpendicular to the midline M, line N
passing the central canal was drawn. Parallel to line N
and across point B was line L. Point
B was the first point on the contour of the ventral gray
matter (tracing from the midline) at which the contour made a >180°
turn. Connecting point C (the cross-point of line
N and the contour of gray matter) and point
D (at one-third the distance from point B
to A), line P was drawn. All neurons
within the area encompassed by line P and the contour of
ventral horn (hatched area) were drawn and
measured.
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To measure vacuoles, two ventral horns were photographed from a 1 µm
transverse section of the spinal cord. The picture frame contained an
area 280 × 433 µm in size and covered about three-quarters of
the ventral horn. These images were digitized using a Nikon LS-1000
scanner. Measurements were performed using MetaMorph image analysis
software. Vacuoles were quantified using the threshold function.
Nonvacuolar structures such as nuclei and large blood vessels were
manually eliminated from the measurement. Capillary vessels were
usually indistinguishable from the vacuoles under the light microscope
and therefore were included in the measurement. However, this
background was estimated by making the same measurements in wild-type
animals.
For electron microscopy, thin sections of ventral horn were cut from
the Epon tissue blocks, stained with uranyl acetate and lead citrate,
and visualized using a Philips EM-400 transmission electron
microscope.
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RESULTS |
Mouse ALS undergoes four functional declining stages
To follow disease progression in G93A mice, we developed a simple
and objective assay to measure muscle strength. In this assay, the time
that a mouse was able to hang onto a wire was measured as an indication
of muscle strength (see Materials and Methods). We first tested mice in
different age groups, which revealed an age-dependent decline of
hanging time for G93A mice, but not for wild-type mice (WT) and wild
type human SOD1 (WS) transgenic mice (Fig.
2A).
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Figure 2.
Four stages of muscle strength change in
G93A mice. The time that mice were capable of hanging onto a wire was
measured as an indication of muscle strength. A,
Age-dependent decline of muscle strength in G93A mice. Groups of mice
at different ages were tested. The number of mice in each age group
ranged from 3 to 20. G93A, Mutant SOD1 mice;
WT, nontransgenic (wild-type) mice; WS,
wild-type SOD1 transgenic mice. Error bars indicate SEM.
B, Highly variable onset of muscle weakness among
different G93A mice. Each trace represents measurements from one
animal. For clarity, only measurements around the RD stage in 20 animals are shown. C, Synchronized plot of muscle
strength decline in individual G93A mice. Zero week represents the time
point just before the decline begins. For clarity, only 10 traces are
shown. D, Average time course of muscle strength decline
in G93A mice (n = 12). PMW, Pre-muscle
weakness stage; RD, rapid declining stage;
SD, slow declining stage; Para.,
paralysis stage. Error bars represent SEM.
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To determine how muscle weakness develops in individual animals, we
tested G93A and wild-type animals once a week beginning at 90 d of
age and continued the tests until the G93A mice could no longer hold
onto the wire. The results revealed several interesting aspects of
muscle strength change. First, both the onset and the duration of
muscle strength decline were highly variable among different
individuals, ranging from age 113 to 188 d for the former and 35 to 94 d for the latter (Fig. 2B, Table
1). Second, the time course of muscle
strength change apparently went through four different stages (Fig.
2C,D): a pre-muscle weakness stage (PMW), during which
muscle strength was maintained at the normal level; a rapid declining
stage (RD), during which the hanging time declined sharply (usually by
>50% within a period of 2 weeks); a slow declining stage (SD), which
followed the RD stage and lasted for 4-11 weeks; and a paralysis stage
(Para), during which paralysis of limbs began and the mouse could no
longer hold onto the wire (Fig. 2C,D). The sharp decline in
muscle strength during the RD stage suggests that there may be a
synchronized pathological change at the onset of the disease.
Pathological progression in motor axons at different stages
To elucidate the sequence of events leading to motor neuron death,
and in particular to see whether there is a synchronized loss of motor
neurons at the onset of the disease, we examined the motor axons in
ventral roots (Fig. 3). We found few
pathological changes in the PMW stage (Fig. 3C) compared
with the WT (Fig. 3A) or WS (Fig. 3B) mice.
Surprisingly, the sharp muscle strength decline during the RD stage was
not correlated with a loss of a large number of motor axons (Fig.
3D). Instead, for a long period during the SD stage (up to
50 d after the onset of RD), a significant fraction of motor axons
remained (Fig. 3E-G). It was not until the paralysis stage
that the vast majority of motor axons became degenerated (Fig.
3H).
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Figure 3.
Axonal degeneration in L5 ventral roots at
different stages of ALS in G93A mice. Plastic sections (1 µm) were
stained with toluidine blue. A, WT; B,
WS; (C-H), G93A at the following stages:
(C) PMW, (D) RD,
(E-G) 20, 33, and 50 d after the onset of
RD, respectively; H, paralysis.
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To quantify the changes in motor axons, we measured the axon size and
number in the L4 and L5 ventral roots. As the disease developed, there
was a progressive loss of large motor axons after the onset of muscle
weakness (Fig. 4). Two features of this
loss have emerged from this analysis. First, two processes, atrophy and
degeneration, contributed to the loss of large axons during the early
stages. This was particularly evident when the axons were divided into
two groups, one larger and the other smaller than 4.5 µm in diameter.
Beginning from the RD stage, there was a significant loss of large
motor axons (Fig. 5). Meanwhile, the number of small axons was increased up to 20 d after the onset of
muscle weakness, before it reverted back to the original number (Fig.
5). These results indicate that large motor axons undergo atrophy
before degeneration.
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Figure 4.
Axon size distribution in L4 and L5 ventral roots.
The distribution of axons in the two roots was similar, and therefore
the measurements were pooled together in one plot. Three animals were
measured for each plot. The three PMW animals (C)
were between 120 and 160 d old and therefore were near the RD
stage. A-H, Same as in Figure 2.
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Figure 5.
Motor axons undergo atrophy before degeneration.
A, Total number of axons in L4 and L5 ventral roots at
different disease stages. B, Number of axons larger than
4.5 µm in diameter at different disease stages. C,
Number of axons smaller than 4.5 µm in diameter at different disease
stages. D, Number of degenerating axons in L4 and L5
ventral roots at different disease stages. Asterisks
indicate significance level in comparison with the WT using two-tailed
Student's t test. One asterisk
represents p < 0.05; two asterisks
represent p < 0.01. n = 3 for
all stages.
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Second, taking into account the substantial fraction of atrophic large
axons, the actual fraction of degenerated large axons during the SD
phase was relatively low (Table 2). As
long as paralysis had not begun, even at 50 d after the onset of
muscle weakness, the fraction of degenerated large axons was <50%
(Figs. 3-5, Table 2), which implies that more than half of the large
motor neurons were still alive. The largest single loss of motor axons occurred at the paralysis stage (Fig. 5, Table 2), when the majority of
large motor neurons were lost permanently. These data indicate that the
vast majority of motor neurons are still alive when muscle weakness
develops, and the initial muscle weakness reflects motor neuron
dysfunction rather than loss (see below). Thus, there is a period of
time to rescue motor neurons after muscle weakness begins.
Pathological progression in ventral horn motor neurons at
different stages
To investigate the sequence of events in the vicinity of motor
neuron cell bodies, we analyzed changes in the ventral horn spinal cord
at different disease stages. Consistent with changes in motor axons,
there were considerable numbers of neurons remaining in the ventral
horn after muscle weakness began (Fig.
6D-G). Quantification of neuronal number and size in the lateral ventral horn showed a
selective loss of large neurons (Fig.
7A). However, this was not
evident until 33 d after the onset of muscle weakness (Fig. 7A). At the paralysis stage, most of large motor neurons
were lost, whereas the number of small neurons was unaffected (Figs. 6H, 7A).
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Figure 6.
Changes in ventral horn at different disease
stages. Plastic sections (1 µm) were stained with toluidine blue.
A-H, Same as in Figure 2.
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Figure 7.
Changes in spinal cord at different disease
stages. A, Changes in the number of neurons in the
ventral horn. All numbers were averages from two animals and were
normalized to the average number of neurons in each wild-type mouse
(145 neurons). Filled bars, Total number of neurons;
open bars, number of neurons with diameters <25 µm;
shaded bars, number of neurons with diameters >25 µm.
Note that the division of large and small neuronal groups at 25 µm
diameter is arbitrary because there was not a clear division of large
and small neuron peaks in the size distribution (data not shown).
Changes in vacuole number (B) and vacuole size
(C) at different disease stages. Two ventral
horns were measured in each animal. The small number of vacuoles in the
WT mice are capillary blood vessels that could not be distinguished
from the real vacuoles under the light microscope.
Asterisks indicate significance level in comparison with
the WT, using two-tailed Student's t test. One
asterisk represents p < 0.05; two
asterisks represent p < 0.01. n = 3 for all stages.
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The most prominent change in the early stages of the disease was
vacuolation. At the PMW stage, there was a slight increase of vacuoles
(Figs. 6C, 7B) compared with WT (Figs.
6A, 7B) and WS (Figs.
6B, 7B) mice. However, vacuoles were
greatly increased at the onset of the disease (RD stage) (Figs.
6D, 7B). Although conspicuous throughout
the SD stage, the number of vacuoles gradually declined toward the end
stage of the disease (Figs. 6E-H, 7B). But the size of vacuoles increased slightly from the RD stage until
33 d after the onset of muscle weakness, and then declined as the
disease progressed toward the paralysis stage (Fig. 7C). These results indicate that the massive vacuolation in motor neurons is
transient in the disease process and suggest that small vacuoles may
fuse or cluster before being lost with the motor neurons.
Mitochondrial damage precedes the onset of the disease
The light microscopic data described above showed only a minor
increase in vacuoles during the PMW stage. To determine what change(s)
triggers the onset of RD stage, we used electron microscopy (EM) to
examine the spinal cords. The most prominent feature at the PMW stage
was abnormal mitochondria, which were present in abundance in dendrites
and axons but scarce in the cell bodies. Shown in Figure
8 are several examples of mitochondrial
changes, including those with dilated and disorganized cristae (Fig.
8A,B), leakage of the outer membrane (Fig.
8A, arrow), broken outer membrane (Fig.
8C, arrows), and early vacuoles that still carry
remnants of mitochondria (Fig. 8D,E, arrows). The
abnormal mitochondria often appeared swollen compared with the normal
ones in adjacent synaptic terminals (Fig. 8C,
arrowheads). Densely accumulated neurofilaments can be seen
in axons in close proximity with vacuoles (Fig. 8D)
and also at many other proximal axon sites without vacuoles (data not
shown), suggesting that slow axonal transport begins to fail before the
onset of the RD stage. At the RD stage, abundant vacuoles in dendrites
as well as in axons were seen, some of which were no longer carrying
the remnants of mitochondria (Fig. 8F). Only rarely
were vacuoles present in cell bodies.
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Figure 8.
Mitochondrial abnormalities at PMW
(A-E) and RD (F) stages.
A, A swollen dendritic mitochondrion with dilated
cristae (asterisk) and leaking outer membrane
(arrow). B, Swollen dendritic
mitochondria with dilated and disorganized cristae. A synaptic terminal
on the dendrite contains normal mitochondria
(arrowhead). C, A proximal dendrite
containing mitochondria with broken outer membranes
(arrows). Adjacent synaptic terminals contain normal
mitochondria (arrowheads). D, Early
vacuoles in a proximal axon. Arrows point to
mitochondrial remnants. E, Early vacuoles in a dendrite.
Arrows point to mitochondrial remnants.
F, Massive dendritic vacuolation at the RD stage.
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DISCUSSION |
Pathological observations without correlation with disease stages
are largely a description of scattered phenomena and provide only
limited information regarding the mechanism of disease progression. Because of difficulties involved in studying human cases, particularly because of the near impossibility of obtaining pathological specimens at the early stages of the disease, descriptions of end-stage disease
have dominated the study of ALS since the original identification of
the disease more than a century ago (Chou, 1992; Hirano, 1991). Transgenic mice expressing mutated SOD1 have provided an unprecedented opportunity to study the early stages of the disease. Indeed, several
new pathological features including mitochondrial vacuolation and early
astrogliosis were revealed (Dal Canto and Gurney, 1995; Wong et al.,
1995; Morrison et al., 1996; Bruijn et al., 1997).
In all studies preceding the current work, pathological features were
correlated with age of animals but not with stage of the disease. As
shown in Figure 1, the onset and duration of disease in mice mirrors
human disease (Cudkowicz et al., 1997; Juneja et al., 1997) and are
highly heterogeneous. As a result, it was difficult to determine the
sequence of events in the disease progression and the significance of
the pathological features that were observed. To solve this problem, we
used a novel approach by developing an assay to determine the disease
stages and analyzing pathological changes in mice with defined disease
stages. By this approach, a coherent picture of the functional and
pathological progression of ALS in the G93A mice begins to emerge (Fig.
9).
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Figure 9.
Sequence of pathological events leading to motor
neuron death in G93A mice. All the data points were derived from the
same set of animals. Each data point represents the average of three
animals. All the values are normalized against the highest measure
(100%) in the sequence. For the hanging test, only the last hanging
times before the animals were killed are shown. Error bars have been
shown in Figures 3 and 6 and are omitted here for clarity.
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The progression of ALS in G93A mice can be divided into four stages
based on changes in muscle strength, and each of these stages is
correlated with specific pathological features. The PMW stage (just
before the RD stage) shows little difference from the wild type under
the light microscope. However, abundant mitochondrial abnormalities can
be found by EM (Fig. 8). The onset of muscle weakness (RD stage)
correlates with a massive mitochondrial vacuolation, signifying the
beginning of a degenerative process in motor neurons that compromises
their function (Fig. 9). The SD stage correlates with axonal atrophy
and a gradual loss of motor neurons (Fig. 9). However, the
majority of motor neurons are alive until the very end stage of the
disease (Fig. 9, Table 2).
Several implications can be drawn from these results. First, abundant
mitochondrial abnormalities are the most prominent pathological feature
before the onset of muscle strength decline. In addition, the onset of
a sharp muscle strength decline is correlated with a massive
mitochondrial vacuolation. This is the first demonstration that
mitochondrial degeneration is associated with the onset of the disease
and suggests that the "gained toxicity" of the mutant SOD1 damages
mitochondria, triggering the decline of motor neuron function and the
onset of clinical disease. Second, the vacuolation is a transient
process. It correlates with the onset of muscle weakness but decreases
toward the end stage of the disease. It has been pointed out that
vacuoles were not a noted feature in human ALS (Bruijn et al., 1997).
Our results raise the possibility that vacuoles are also an integral
part of human ALS, but they exist only at the very early stages of the
disease. Therefore, the lack of vacuoles in human ALS could be
attributed to the fact that the pathological examination is performed
on patients at the terminal stage of the disease.
Third, the initial muscle weakness signifies a stage in which the
degenerative process in large motor neurons begins to compromise their
function. However, it does not represent a massive loss (or death) of
these neurons. The loss of the majority of motor neurons does not occur
until the paralysis stage, the end stage of the disease. The presence
of the majority of motor axons during the RD and SD stages indicates
that even after the muscle weakness begins, most motor neurons have not
died and thus could be rescued by effective therapeutic intervention.
Fourth, axonal atrophy has been noted in human ALS patients (Chou,
1992), but it was difficult to determine whether this decrease was
caused by the death of large axons or a shift of large axons to small
ones. Our results show that large axons become atrophic before
degeneration (Fig. 4, Table 2). This suggests that there is a failure
in axonal transport at the early stage of ALS, which is consistent with the proximal accumulation of neurofilaments observed during the PMW and
RD stages in this study (Fig. 8), as well as in another study at
unspecified disease stages (Tu et al., 1996).
Previous studies have noted the presence of vacuoles. However, the
origin of the vacuoles has been controversial (Dal Canto and Gurney,
1995; Wong et al., 1995). One study proposes that vacuoles are derived
from dilation of endoplasmic reticulum and mitochondria (Dal Canto and
Gurney, 1995), whereas the other study contends that vacuoles are
derived from degenerating mitochondria (Wong et al., 1995). By
determining the precise time of the disease onset, the current study
shows that abundant abnormal mitochondria, including those with dilated
cristae and the early vacuoles with mitochondrial remnants, are present
before the massive vacuolation at the disease onset. This observation,
in conjunction with the absence of dilated endoplasmic reticulum before
the massive vacuolation, strongly supports the contention that
mitochondria are the source of vacuoles.
Thus, the current study suggests that mitochondrial damage is at the
center of the degeneration mechanism. Whether such damage is caused by
a direct effect of toxicity from the mutated SOD1 or is mediated
through other intermediates remains to be determined. However, the
involvement of mitochondrial degeneration in the early stage of the
disease is consistent with a direct effect of mutant SOD1 toxicity and
perhaps is mediated by properties gained by the mutant enzyme in
catalyzing redox reactions (Beckman et al., 1993; Wiedau-Pazos et al.,
1996; Yim et al., 1996). Mitochondria are a large source of oxidative
free radicals, and the production of free radicals is directly
correlated with metabolic activity (Beal, 1996). It is possible that
the high metabolic activity in motor neurons, combined with the gained,
toxic oxidative properties of the mutant SOD1, causes mitochondria in
motor neurons to degenerate earlier than other neurons, triggering the
onset of ALS. Consistent with this possibility are the results of a
recent report showing that mitochondria in neuroblastoma cells
transfected with G93A mutation displayed a significant loss of membrane
potential and an increase in cytosolic calcium concentration (Carri et
al., 1997).
The assay for disease progression developed in the current work sets
the stage for further investigations of the disease mechanism in animal
models. This assay enables the study of animals with defined disease
stages, thus allowing meaningful comparisons of pathological evolution
among different SOD1 mutations. By grouping mice according to their
disease stages, inconsistencies caused by heterogeneous disease stages
can be avoided, therefore allowing a full construction of the sequence
of events (including both biochemical and morphological events) leading
to motor neuron death. The assay also provides a useful means to
evaluate the effectiveness of potential therapeutic agents.
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FOOTNOTES |
Received Nov. 7, 1997; revised Feb. 12, 1998; accepted Feb. 18, 1998.
This work is supported by grants from National Institutes of Health
(RO1NS35750-01), the ALS Association, and the Markey Charitable Trust.
We thank Vivian Tung for technical assistance and Alonzo Ross and Diane
Casey for critically reading this manuscript.
Correspondence should be addressed to Zuoshang Xu, Worcester
Foundation, 222 Maple Avenue, Shrewsbury, MA
01545.
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Abstract
Introduction
