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The Journal of Neuroscience, June 15, 2002, 22(12):4825-4832
Accumulation of SOD1 Mutants in Postnatal Motoneurons Does Not
Cause Motoneuron Pathology or Motoneuron Disease
Maria Maddalena
Lino,
Corinna
Schneider, and
Pico
Caroni
Friedrich Miescher Institute for Biomedical Research, Novartis
Research Foundation, CH-4058 Basel, Switzerland
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ABSTRACT |
Transgenic mice expressing high levels of familial amyotrophic
lateral sclerosis (FALS)-associated mutant superoxide dismutase 1 (SOD1) under the control of a human SOD1 minigene (hMg) accumulate mutant protein ubiquitously and develop motoneuron disease. However, restricted expression of SOD1 mutants in neurons apparently does not
cause motor impairments in mice. Here, we investigated the possible
pathogenic roles of mutant SOD1 accumulation in motoneurons. First, we
used a Thy1 expression cassette to drive high constitutive expression
of transgene in postnatal mouse neurons, including upper and lower
motoneurons. Second, we expressed human (h) SOD1(G93A) and hSOD1(G85R)
as transgenes (i.e., two SOD1 mutants with aggressive pathogenic
properties in inducing FALS). Third, in addition to clinical signs of
disease, we monitored early signs of disease onset and pathogenesis,
including muscle innervation, astrogliosis in the spinal cord, and
accumulation of ubiquitinated deposits in motoneurons and astrocytes.
We report that high-level expression and accumulation of the mutant
proteins in neurons failed to produce any detectable sign of pathology
or disease in these transgenic mice. Crossing hMg-SOD1(G93A) mice
(Gurney et al., 1994 ) with Thy1-SOD1(G93A) mice produced
double-transgenic mice with spinal cord SOD1(G93A) levels that were
approximately twofold higher than in the hMg-SOD1(G93A) single
transgenics but did not affect the onset or progression of pathology or
motoneuron disease. The accumulation of mutant SOD1 in postnatal
motoneurons is thus not sufficient and probably also not critical to
induce or accelerate motoneuron disease in FALS mice. The pathogenic
process in FALS may involve non-neuronal cells, and selective
vulnerability of motoneurons to this process may lead to motoneuron
pathology and disease.
Key words:
amyotrophic lateral sclerosis; pathophysiological
process; motoneuron; neurodegeneration; neuronal pathology; mutant
SOD1
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INTRODUCTION |
Amyotrophic lateral sclerosis (ALS)
is a late-onset neurodegenerative disease involving upper and lower
motoneurons. Approximately 15% of the total number of ALS cases are
familial (FALS), and of these, ~20% are attributable to point
mutations in cytosolic Cu/Zn superoxide dismutase (SOD1) (Deng et al.,
1993; Pardo et al., 1995). Three FALS-associated SOD1 mutants (G93A,
G85R, and G37R) were introduced into mice using a human SOD1 minigene
(hMg) approach, leading to ubiquitous high-level expression of the
human transgene and late-onset ALS-like motoneuron disease in these mice (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995; Bruijn et al., 1997; Tu et al., 1997).
Because they consistently develop motoneuron disease with predictable
onset times and progression rates, FALS mice have provided powerful
model systems to identify early pathophysiological mechanisms associated with ALS. These studies have revealed that mitochondrial pathology (Kong and Xu, 1998), fragmentation of the Golgi apparatus (Mourelatos et al., 1996), and a decrease in the rate of fast axonal
transport (Williamson and Cleveland, 1999) are detected in spinal
motoneurons long before the onset of detectable clinical signs of
disease. In addition, a selective and progressive loss of muscle
innervation is an early, sensitive, and reliable indicator of advancing
neuromuscular pathology in diseases affecting motoneurons in the mouse
(Frey et al., 2000).
Despite the availability of appropriate genetic models, the etiology of
FALS has remained difficult to define, and the cellular targets
critical to the disease process are still not known (Price et al.,
1996; Julien, 2001). Motoneuron disease in FALS mice is attributable to
a dose-dependent toxic gain-of-function mechanism specifically
associated with the accumulation of mutant SOD1 (Wong et al., 1995). In
the hMg-transgenic mice, mutant SOD1 accumulated at high levels in many
and possibly all tissues and cell types. However, because pathology and
dysfunction affected primarily motoneurons, it has seemed reasonable to
assume that accumulation of mutant protein in motoneurons is the main
cause of disease. This view was challenged by a recent report that
transgenic expression of SOD1(G37R) restricted to mouse neurons failed
to produce clinical signs of motoneuron disease. Although transgene
expression decreased in the adult in that study, and although the G37R
mutant requires particularly high expression levels to produce disease
in mice, these results suggested that FALS may not be a cell-autonomous disease of motoneurons.
Here, we investigated the possible pathogenic roles of mutant
SOD1 accumulation in motoneurons. We used a mouse Thy1.2 expression cassette (Caroni, 1997) to drive high constitutive expression of human
(h) SOD1(G93A) or hSOD1(G85R) in postnatal mouse neurons, including
upper and lower motoneurons, and monitored clinical signs of disease as
well as early signs of disease onset and pathogenesis. We report that
high-level expression and accumulation of the mutant proteins in
neurons failed to produce any detectable sign of motoneuron pathology
or disease in these transgenic mice. In addition, double-transgenic hMg-SOD1(G93A)/Thy1-SOD1(G93A) mice with spinal cord SOD1(G93A) levels that were approximately twofold higher than in the
hMg-SOD1(G93A) single-transgenic mice were undistinguishable from the
hMg-SOD1(G93A) single-transgenic mice with respect to the onset and
progression of motoneuron pathology and disease. The accumulation of
mutant SOD1 in postnatal motoneurons is thus not sufficient and
possibly also not critical to cause ALS-type pathology in motoneurons. A pathogenic process involving the accumulation of SOD1 mutants in
non-neuronal cells may thus lead to motoneuron pathology and dysfunction attributable to selective vulnerability properties of these
long-projection neurons.
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MATERIALS AND METHODS |
Transgenic mice. Transgenic mice expressing
FALS-associated hSOD1 mutants, specifically in adult neurons, were
generated using the mouse Thy1.2 expression cassette, as described
previously (Caroni, 1997). The G93A or G85R mutations were engineered
into wild-type human SOD1 cDNA using a PCR/oligonucleotide
primer-directed mutagenesis kit (Stratagene, La Jolla, CA). All cDNAs
used in this study were confirmed twice by DNA sequencing. Mutated SOD1 cDNAs were cloned upstream of an internal ribosomal entry site (IRES)-enhanced green fluorescent protein (EGFP) sequence to
generate bicistronic transcripts expressing SOD1 mutants and GFP. These constructs were subsequently cloned into the mouse Thy1.2 expression cassette. Transgenic mice were generated and screened according to
conventional methods. hMg-SOD1(G93A) mice (Gurney et al., 1994; Frey et
al., 2000) were obtained from The Jackson Laboratory (Bar Harbor,
ME) (catalog number JR2726). Thy1-SOD1(G93A), Thy1-SOD1(G85R), and hMg-SOD1(G93A) mice were all bred in the C57BL/6 background.
Transgene detection, histology, and immunocytochemistry. To
produce total protein extract fractions, neural tissues [brain, lumbar
spinal cord, lumbar ventral roots (L3-L5)] were homogenized in 120 mM NaCl, 50 mM Tris, pH
7.5, 1% Triton X-100, 0.1% SDS, and 5 mM EDTA,
plus protease inhibitors (aprotinin and PMSF). To determine SOD1
contents, equal amounts of total protein were separated on 15%
polyacrylamide gels and transferred onto nitrocellulose filters.
Endogenous mouse and mutant human SOD1 were subsequently detected using
a polyclonal antibody (Stressgen, Victoria, Canada) that binds
to both human (apparent molecular mass, 22 kDa) and mouse
(apparent molecular mass, 19 kDa) SOD1. Bound antibody was detected
with peroxidase-labeled secondary antibodies, followed by a
chemiluminescence reaction (Amersham Biosciences, Arlington Heights,
IL) and x-ray film exposure. For quantitative analysis, films were
analyzed with NIH Image software. Superoxide dismutase activity was
assayed on nondenaturing polyacrylamide gels as described previously
(Beauchamp and Fridovich, 1971). Briefly, lumbar spinal cord or brain
was homogenized in buffer containing 20 mM
Tris-Cl, pH 7.2, 1 mM EDTA, and 1% Triton X-100,
and then 20 µg of protein supernatant (centrifuged at 10,000 × g for 5 min) was loaded on each gel slot. As a
positive control and to determine the migration position of the human
enzyme, human erythrocyte SOD1 was used as a marker.
Transgene transcripts were detected by in situ hybridization
using a digoxigenin-labeled cRNA EGFP probe (Baumeister et al., 1997).
For immunocytochemistry, mice were anesthetized with Rompun (Bayer AG,
Leverhusen, Germany) and perfused with PBS, followed by 4%
paraformaldehyde in PBS. Cryostat sections (12 µm thick) of spinal
cord or brain were subsequently processed for immunocytochemistry according to standard procedures. The following antibodies were used:
rabbit anti-GFAP (Dako, Carpinteria, CA), rabbit anti-ubiquitin (Dako,
Copenhagen, Denmark), rabbit anti-human SOD1 (Stressgen), and
monoclonal antibody against phosphorylated neurofilaments (SMI31;
Stenberger Monoclonals Inc., Lutherville, MD).
Neuromuscular synapses and their innervation patterns were visualized
on fresh-frozen sections using a combined silver esterase reaction, as
described previously (Frey et al., 2000). Alternatively, innervation of
muscle was analyzed using an affinity-purified antiserum against p75
(kind gift from U. Mueller, Friedrich Miescher Institute, Basel,
Switzerland) and RITC- -bungarotoxin (Molecular Probes, Eugene, OR),
as described previously (Frey et al., 2000).
To analyze peripheral nerve regeneration, the right sciatic nerve of 2- and 6-month-old mice was crushed at midthigh level, and nerve
regeneration and reinnervation of muscle were analyzed 14 d after
the crush, as described previously (Aigner et al., 1995).
Quantitative analysis of data. Mice were tested for hindlimb
muscle strength using the loaded grid protocol (Barneoud et al., 1997).
Testing was started at 6 weeks of age and repeated weekly. For the
quantitative analysis of GFAP immunoreactivity and ubiquitin deposit
accumulation, sets of lumbar spinal cords (L3-L5) to be compared were
processed for immunocytochemistry, and photographs of the labeled
sections were processed in the same way. To quantify areas of
GFAP-immunoreactive cells, the outlines of all labeled cells in the
ventral spinal cord were traced, and total labeled areas were
determined with a computer program. Fifty sections per mouse were
analyzed, and the values are given in arbitrary units (average values
per section; pooled data from two mice each). To quantify ubiquitin
deposit accumulations, all ubiquitin-positive structures in the ventral
and dorsal spinal cord were outlined, and their areas were determined.
Forty sections per mouse were analyzed. The values are in arbitrary
units per section (averages of all data from two mice).
Muscle denervation was quantified by two independent methods.
Percentage values of nerve-free, acetylcholine esterase-positive neuromuscular junctions (NMJs) were determined for adjacent segments of
medial gastrocnemius (MGC) and lateral gastrocnemius (LGC) synaptic
regions, as described previously (Frey et al., 2000). In separate
experiments, muscle sections were double-labeled for p75 and
postsynaptic acetylcholine receptor clusters, and the percentage
of p75-positive NMJs was determined for all NMJs in the medial
compartment of the LGC on five sections per mouse. Data from two mice
each were pooled.
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RESULTS |
Transgenic mice expressing high levels of FALS-associated
SOD1(G93A) or SOD1(G85R) in postnatal neurons
To generate transgenic mice expressing high levels of
FALS-associated SOD1 mutants constitutively and specifically in
neurons, we expressed hSOD1(G93A) or hSOD1(G85R) under the control of
the mouse Thy1.2 expression cassette (Caroni, 1997). Neuron-selective expression driven by this promoter construct starts during the first
postnatal week and is unusually strong (Caroni, 1997). To monitor
transgene expression patterns in the absence of cross-reactivity with
endogenous mouse SOD1, transgenic transcripts included an IRES followed
by the coding sequence for EGFP (see Materials and Methods). Transgenic
lines were selected for high-level expression in most spinal cord and
motor cortex neurons.
Four Thy1-SOD1(G93A) lines (lines 1, 11, 13, and 16) and two
Thy1-SOD1(G85R) lines (lines 5 and 6) were retained. Figure
1 shows lumbar spinal cord and motor
cortex in situ hybridization data for Thy1-SOD1(G93A) line
13 (Fig. 1A,B) and Thy1-SOD1(G85R) line 6 (Fig.
1D,E) mice. Prominent transgenic transcript
accumulation was detected in large ventral horn neurons (Fig.
1A,D) and in layer 5 motor cortex projection neurons
(Fig. 1B,E), starting around postnatal day 5 (P5)
(data not shown). The other transgenic lines selected for additional
analysis (lines 1, 11, 16, and 5) exhibited comparable expression
patterns (data not shown). The transgene hSOD1(G93A) was metabolically
active (Fig. 1C). To monitor the accumulation levels of
mutant SOD1 protein in the transgenic mice, we performed an immunoblot
analysis of lumbar spinal cord (Fig. 1C,F), ventral
roots 3-5 (Fig. 1C), and brain homogenates (data not
shown). Transgene accumulation levels for the Thy1-SOD1(G93A) lines
were compared with those of high-expression hMg-SOD1(G93A) mice (Gurney
et al., 1994), which die of motoneuron disease at ~136 d. As shown in
Figure 1C, all Thy1-SOD1(G93A) lines selected for this study
expressed transgene levels that were nearly as high as those detected
in the reference line hMg-SOD1(G93A). Densitometric analysis of the
immunoblots revealed total SOD1(G93A) lumbar spinal cord levels that
ranged between 58 and 85% of the values in the reference line.
SOD1(G85R) is known to have a low metabolic stability, and transgene
accumulation levels in the corresponding Thy1 lines were substantially
lower than those in the Thy1-SOD1(G93A) lines (Fig.
1F). However, when the transgene signal is compared
with that of endogenous mouse SOD1, relative transgene accumulation levels are equal to if not higher than those reported for the highest
expressing hMg-SOD1(G85R) lines.
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Figure 1.
Establishment of transgenic mouse lines expressing
high levels of mutant SOD1(G93A) or SOD1(G85R) in postnatal neurons,
including spinal and upper motoneurons. A-C,
Thy1-SOD1(G93A) transgenic lines. A, Expression of
transgene in lumbar spinal cord (s.cord) neurons
[Thy1-SOD1(G93A) line 13, 1 month; EGFP mRNA; arrows
point to transgene-positive large ventral horn neurons]. In control
experiments, when the EGFP probe was applied to sections from
nontransgenic mice, it yielded no detectable signals at up to fivefold
longer color development times (data not shown). B,
Expression of transgene in motor cortex neurons [Thy1-SOD1(G93A) line
13, 1 month; EGFP mRNA; arrow points to
transgene-positive layer 5 neurons]. C, Top
three rows, Immunoblots of human (h) and
mouse (m) SOD1 in 1 month lumbar spinal cord
homogenates (top two rows) and 1 month L3-L5
ventral roots from wild-type (wt), hMg-SOD1(G93A)
high-expression line (hMg) (Gurney et al., 1994), and
Thy1-SOD1(G93A) transgenic lines 1, 11, 13, and 16. Bottom
row, SOD activity in 1 month spinal cord homogenates from
wild-type (left) and Thy1-SOD1(G93A) 13 mice
(right, brain homogenate). Note that on this
nondenaturing gel, human SOD1 migrated faster than endogenous mouse
SOD1. D-F, Thy1-SOD1(G85R) transgenic lines. D,
E, Expression of transgene in lumbar spinal cord
(D) and motor cortex layer 5 (E) neurons (1 month mouse, details as in
A and B). F, Immunoblot of
human and mouse SOD1 in lumbar spinal cord homogenates (1 month) from
Thy1-SOD1(G85R) transgenic lines 5 and 6. G-I, EGFP
fluorescence in peripheral nerves from a wild-type
(G) and Thy1-SOD1(G93A) line 13 mouse (H,
I; 14 months). G, Level of peroneal nerve (as in
I). H, Level of the tibial nerve,
where three muscular branches arise that run along the external sural
(arrow), internal sural, and posterior tibial vessels,
respectively. I, Level of common peroneal nerve, where
it divides into superficial (arrow) and deep peroneal
branches. J, Hindlimb muscle strength as a function of
age in wild-type, Thy1-SOD1(G93A) line 13, Thy1-SOD1(G85R) line 6, and
high-expressing hMg-SOD1(G93A) mice. The values are averages from three
mice each.
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To verify that the transgene was expressed in neurons innervating
hindlimb muscles in our mice, we monitored the accumulation of EGFP in
ventral roots and in distal sections of peripheral nerves. Figure
1H,I shows that in the transgenic lines, peripheral nerves exhibited strong fluorescent GFP signals, whereas no
fluorescence was detected in corresponding nontransgenic littermates
(Fig. 1G). Closer examination of ventral roots, peripheral
nerves, and intramuscular nerves suggested that in these mice, the
transgenes were expressed in most and possibly all motoneurons.
Absence of motoneuron disease and FALS-associated pathology in
Thy1-SOD1(G93A) and Thy1-SOD1(G85R) mice
The Thy1-transgenic lines were bred and followed longitudinally
for survival and signs of motor dysfunction. None of the lines exhibited any obvious behavioral phenotype, and survival curves were
undistinguishable from those of wild-type mice. Hindlimb muscle
strength in the transgenic mice was assayed with a loaded grid
protocol. Mice from the reference line hMg-SOD1(G93A) lost most of
their hindlimb muscle strength between P70 and P110 (Fig. 1J). In addition, presumably because they were
smaller than their nontransgenic littermates, hMg-SOD1(G93A) mice
exhibited reduced strength as early as P50. In contrast, all
Thy1-SOD1(G93A) and Thy1-SOD1(G85R) mice exhibited hindlimb muscle
strength values undistinguishable from those of wild-type littermates
at any age tested (Fig. 1J). For the data shown in
Figure 1J, muscle strength values were monitored
systematically between P50 and P500. In addition, we analyzed a smaller
sample of Thy1-SOD1(G93A) line 13 mice between 18 and 20 months of age,
and these were again undistinguishable from wild-type littermates (data
not shown).
To determine whether Thy1-SOD1(G93A) and Thy1-SOD1(G85R) transgenic
mice exhibited any sign of muscle denervation, an early hallmark of
disease in FALS mice, we analyzed triceps sural innervation patterns using a combined silver esterase method. This histological method reveals nerves in black (silver reaction precipitate) and NMJs
in blue (synaptic acetylcholine esterase reaction product). As shown in
Figure 2A, extensive
denervation of lateral segments of the LGC was detected as early as P50
in the reference line hMg-SOD1(G93A). In contrast, no denervation was
detected in Thy1-SOD1 mutant mice (Fig. 2B shows LGC
data for a 14-month-old line 13 mouse). A detailed quantitative
analysis revealed that none of the Thy1-SOD1 transgenic lines exhibited
any significant denervation of LGC, MGC, or soleus between 1 and 15 months of age (Fig. 2C).
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Figure 2.
Absence of muscle denervation in Thy1-SOD1(G93A)
and Thy1-SOD1(G85R) transgenic mice. A, B, Combined
silver esterase reaction of LGC [medial end of medial compartment
(mLGC), as indicated by the red bar in
D] from an hMg-SOD1(G93A) mouse at P50
(A) and a Thy1-SOD1(G93A) line 13 mouse at P480.
Insets on the right show a detail from
the same region. Synaptic regions are in blue
(acetylcholine esterase reaction product), and nerves are in
black. Some of the nuclei on the section yield a
black background color. Asterisks,
Innervated NMJs; arrows, denervated NMJs.
C, Double-labeling immunocytochemistry for p75 and
-bungarotoxin in LGC (medial compartment). The Thy1 transgenic mouse
was from the Thy1-SOD1(G93A) 13 line. Arrows point to
some of the -bungarotoxin-positive NMJs. The weak p75 signal on the
sections from wild-type and Thy1 transgenic mice was associated with
sensory nerves, whereas the strong signal in the hMg mice was
attributable to Schwann cells that had lost contact with motor nerve.
The small size of the acetylcholine receptor cluster signals in the
hMg mouse is attributable to chronic denervation of mLGC in this
mouse. Scale bars: A, B, 120 µm; inset
in A and B, 50 µm; C, 60 µm. D, Quantitative analysis of denervation
(denerv.) along the synaptic band of MGC and LGC muscle
(see Materials and Methods for details). Each curve represents average
values from two mice. E, Quantitative analysis of mLGC
denervation (p75-positive NMJs; see Materials and Methods for
details). W, Wild type; Mg, minigene,
hMg-SOD1(G93A). Average values from two mice each are shown.
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Schwann cell p75 immunoreactivity is slightly upregulated at paralyzed
NMJs and strongly upregulated at denervation. To further investigate
the possibility that a few individual NMJs may exhibit signs of
denervation in the Thy1-SOD1 mice, we performed a detailed analysis of
p75 immunoreactivity at the NMJ and in peripheral nerves. As expected,
prominent upregulation of p75 was detected in the LGC of hMg-SOD1(G93A)
mice (Fig. 2D). In contrast, and as in wild-type
controls (Fig. 2F,G), Thy1-SOD1(G93A) (Fig.
2E,G) and Thy1-SOD1(G85R) mice (Fig. 2G)
exhibited no signs of muscle denervation at any of the times analyzed
in this study.
To determine whether, despite the absence of FALS-related
functional deficits, Thy1-SOD1(G93A) or Thy1-SOD1(G85R) mice may exhibit signs of FALS-associated pathology in the spinal cord, we
monitored the distribution and appearance of ubiquitin-rich deposits in
these mice. As expected, hMg-SOD1(G93A) reference mice
exhibited a profusion of ubiquitin-rich deposits in ventral spinal cord
neurons (Fig. 3A; P120 mouse).
In contrast, no ubiquitin deposits were detected in Thy1-SOD1(G93A)
mice (Fig. 3A; P430 mouse) or in Thy1-SOD1(G85R) mice (data
not shown). In reference line hMg-SOD1(G93A) mice, ubiquitin deposit
accumulation was evident from P80 on, and the density of the deposits
increased with age. In contrast, we failed to detect ubiquitin-rich
spinal cord deposits in Thy1-SOD1(G93A) and Thy1-SOD1(G85R) mice
between P30 and P520. In additional experiments, we also did not detect
abnormal accumulations of SOD1 or phosphorylated neurofilament
immunoreactivity in the Thy1-SOD1 mutant mice, whereas these signs of
pathology were prominent in the hMg-SOD1(G93A) mice (data not shown).
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Figure 3.
Absence of ubiquitinated deposits and astrocytosis
in Thy1-SOD1(G93A) mice. The data are from Thy1-SOD1(G93A) line 13 mice, but similar results were obtained with the other Thy1-SOD1(G93A)
lines and with Thy1-SOD1(G85R) lines. A, Ubiquitin
immunoreactivity in lumbar spinal cord (ventral horn). The
arrows point to ubiquitin-positive inclusions; the
inset shows a large neuron with inclusions.
B, GFAP-positive astrocytes (arrows) in
lumbar spinal cord (ventral horn). Scale bar: A, 40 µm; B, 100 µm.
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Reactive astrocytosis, as revealed by the appearance of hypertrophic
astrocytes with high expression levels of GFAP, is a prominent early
marker of dysfunction in FALS mice. As expected, reference line
hMg-SOD1(G93A) mice exhibited extensive astrocytosis in the spinal cord
(Fig. 3B). In contrast, spinal cord GFAP immunoreactivity in
Thy1-SOD1(G93A) (Fig. 3B) and Thy1-SOD1(G85R) mice (data not shown) was indistinguishable from that detected in wild-type
littermates (Fig. 3B; for a quantitative analysis, see Fig.
4).
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Figure 4.
Compared with single-transgenic hMg-SOD1(G93A)
mice, double-transgenic hMg-SOD1(G93A)/Thy1-SOD1(G93A) mice express at
least twice the amount of mutant SOD1(G93A) in postnatal neurons but
exhibit no acceleration of motoneuron pathology or disease. The
immunoblot analysis of human (h) and mouse
(m) SOD1 in spinal cord (S.CORD)
from 1 month Thy1-SOD1(G93A) line 13, hMg-SOD1(G93A), and
double-transgenic hMg-SOD1(G93A)/Thy1-SOD1(G93A) mice was as described
in Figure 1C. Force was determined as described for
Figure 1J, and the values are averages from two
mice each. Lumbar spinal cord ubiquitin deposits and areas of
GFAP-immunoreactive cells were analyzed as described in Materials and
Methods. Values are averages from 40 (ubiquitin) and 50 (GFAP) spinal
cord sections; data from two mice each were pooled. The quantitative
analysis of muscle denervation (p75: mLGC) was as described for Figure
2D,E. All transgenic mice analyzed in the figure
expressed the SOD1(G93A) mutant protein.
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To determine whether spinal motoneurons in the Thy1-SOD1 mutant mice
may be more vulnerable to stress, we monitored peripheral nerve
regeneration and muscle reinnervation after sciatic nerve crush in
pairs of 2- and 6-month-old Thy1-SOD1(G93A) line 13 mice. These
experiments also failed to reveal differences between the transgenic
mice and corresponding wild-type littermates (data not shown).
Doubling the levels of mutant SOD1(G93A) in motoneurons of
postnatal FALS mice does not affect onset time and progression of
motoneuron pathology and disease
Although overexpression of high levels of FALS-associated
SOD1 mutants in postnatal neurons was not sufficient to induce
motoneuron disease or motoneuron pathology in mice, the levels of SOD1
mutant protein in motoneurons may nevertheless affect the onset time and/or the progression of dysfunction and pathology in FALS mice. To
address this possibility, we crossed reference line hMg-SOD1(G93A) mice
with Thy1-SOD1(G93A) line 13 mice. As shown in Figure 4, double-transgenic hMg-SOD1(G93A)/Thy1-SOD1(G93A) mice accumulated mutant SOD1(G93A) protein levels in the spinal cord that were approximately twice as high as those detected in single-transgenic hMg-SOD1(G93A) littermates. Despite these elevated accumulation levels
in postnatal neurons, single- and double-transgenic mice were
undistinguishable with respect to muscle strength values between P50
and P130 (Fig. 4), the time course of denervation in LGC (Fig. 4), and
the time at which paralysis prevented locomotion in these mice. In
addition, although we did detect minor transient increases during the
intermediate phase of spinal cord ubiquitin accumulation and
astrogliosis in the transgenic mice, pathology values during the onset
time and the advanced phase of the disease were undistinguishable from
those in single-transgenic hMg-SOD1(G93A) mice (Fig. 4). Therefore,
although onset time and disease progression are highly dependent on
transgene expression levels in these ALS models, doubling the levels of
mutant SOD1(G93A) in neurons did not affect the kinetic properties of
pathology and disease. These results suggest that the accumulation of
mutant SOD1(G93A) in neurons is not a critical pathogenic factor in
this model of familial ALS.
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DISCUSSION |
We have provided evidence that overexpression of FALS-associated
SOD1 mutants in postnatal neurons of mice fails to produce muscle
denervation, motoneuron disease, or motoneuron pathology. In addition,
we have shown that duplicating the load of SOD1(G93A) in motoneurons
does not affect disease onset, disease progression, and motoneuron
pathology in reference hMg-SOD1(G93A) mice. In the following, we
discuss the implications of these findings for the role of mutant SOD1
in motoneuron pathology and disease and the pathophysiology of
motoneuron disease.
In the absence of motoneuron disease, the accumulation of mutant
SOD1 in motoneurons fails to induce motoneuron pathology
Our results provide evidence that overexpression and accumulation
of FALS-associated SOD1 mutants in postnatal neurons of transgenic mice
is not sufficient to cause motoneuron disease or to induce
neuromuscular synapse loss (i.e., an early premonitory sign of
motoneuron dysfunction in motoneuron disease). The Thy1-SOD1(G93A) lines described in this study accumulated mutant protein levels in the
spinal cord and ventral roots that were 58-85% of those detected in
hMg-SOD1(G93A) mice with the highest published expression levels
(Gurney et al., 1994). Unlike the Thy1 transgenics, those mice die at
135 d, and hMg-SOD1(G93A) line 20 mice, which accumulate ~33%
of the SOD1(G93A) levels detected in our transgenics, are completely
paralyzed at 1 year of age (Dal Canto and Gurney, 1997). hMg transgenic
mice with expression levels of SOD1(G85R) lower than those of our
Thy1-SOD1(G85R) mice die at 8-14 months, depending on expression
levels, whereas none of our mutant mice exhibited any sign of
dysfunction at 18 months. These transgene accumulation comparisons do
not take into account the fact that transgene expression is not
restricted to neurons in hMg mice, whereas it is in the Thy1
transgenics. Consequently, when comparisons with hMg lines are based on
immunoblots of spinal cord or ventral root tissue, relative levels of
mutant SOD1 accumulation in neurons of Thy1 transgenic lines are
underestimated. Therefore, had the accumulation of mutant SOD1 in
postnatal motoneurons been sufficient to induce motoneuron dysfunction
and disease, we should have detected it in our mice.
A possible explanation for our findings could be that transgene
expression may have been restricted to a fraction of the motoneurons in
any motor pool, or that transgene expression levels in motoneurons varied substantially, with only a fraction of the motoneurons accumulating sufficiently high and toxic levels of mutant SOD1. However, most if not all axons in distal sections of peripheral nerves
to hindlimb muscles exhibited bright green fluorescence, and ventral
roots were homogeneously fluorescent, suggesting that most if not all
motor neurons also accumulated high levels of mutant SOD1. In addition,
if a fraction of the spinal motoneurons had been affected by the
transgene in our mice, in situ hybridization patterns with
transgene-specific probes should have revealed a selective loss of
neurons with high transgene signals as the transgenic mice became
older. However, when lumbar spinal cord in situ
hybridization samples were compared for transgenic mice aged 1 and 8 months, we did not detect alterations in the number of
transgene-expressing neurons or in the fraction of large ventral horn
cells with the highest apparent expression levels of transgene. In
addition, a detailed analysis of peripheral and intramuscular nerves
failed to reveal any sign of denervation-related upregulation of p75 immunoreactivity in transgenic mice aged 1-16 months. Therefore, SOD1
mutant accumulation restricted to postnatal neurons is not sufficient
to induce loss of peripheral synapses or clinical signs of motoneuron
disease in FALS mice.
An important result of our study is that the accumulation of SOD1
mutants in postnatal motoneurons not only failed to cause motoneuron
disease but also failed to induce FALS-associated pathology in
motoneurons or neighboring cells in the spinal cord. Thus, no SOD1,
neurofilament, or ubiquitin-accumulating deposits were detected in
spinal cord neurons of the Thy1 transgenic mice at any age. In
addition, we did not detect any sign of astrocytosis in the transgenic
mice. Together with the finding that loss of vulnerable neuromuscular
junctions was never detected in the transgenic mice, these results
suggest that the accumulation of high levels of FALS-associated SOD1
mutants did not significantly affect the viability of motoneurons. An
important implication of these findings is that the accumulation of
deposits, including those containing SOD1, in motoneurons is probably a
consequence of disease, not a causative factor in the pathophysiology
of FALS (for a model, see Fig. 5).
View larger version (25K):
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Figure 5.
Proposed model of motoneuron disease initiation
and progression in mutant SOD1 FALS mice. The model postulates the
existence of a pathogenic process driven by the accumulation of mutant
SOD1 in non-neuronal cells. This process may lead to a chronic local
imbalance in extracellular glutamate handling in the CNS, to which
motoneurons are selectively vulnerable. In motoneurons, this would lead
to deficits in energy metabolism, axonal transport, and neuromuscular
junction maintenance, followed by cell death. According to this model,
the timing of motoneuron disease onset and the kinetics of its
progression would be determined primarily by the pathogenic process in
non-neuronal cells. Although the accumulation of mutant SOD1 in
motoneurons may aggravate damage to motoneurons, it may not be
necessary for motoneuron disease to be induced.
|
|
Accumulation levels of SOD1 mutants in motoneurons are not a
critical determinant of motoneuron pathology or disease progression
Double-transgenic hMg-SOD1(G93A)/Thy1-SOD1(G93A) mice accumulated
mutant SOD1 levels in the spinal cord and ventral roots that were
approximately twice as high as those detected in corresponding single-transgenic hMg mice. Nevertheless, we did not detect differences in the timing of muscle denervation or in the appearance of muscle weakness or paralysis between single hMg-SOD1(G93A) and
double-transgenic hMg/Thy1 mice. In addition, the onset time and extent
of ubiquitin deposits and astrocytosis in the spinal cord were
comparable in single- and double-transgenic mice. There is substantial
experimental evidence to support the notion that transgene expression
levels in FALS mice determine the timing of disease onset and its
progression rate. Because of the expression properties of the Thy1
promoter, the excess load of mutant SOD1 in the double-transgenic mice
must have been restricted to neurons, including motoneurons. Therefore, if neurons and motoneurons were the site at which SOD1 mutant accumulation affects disease onset and progression in a dose-dependent manner, the double-transgenic mice would have to exhibit accelerated pathology and disease. Our results therefore suggest that the levels of
mutant SOD1 in postnatal neurons are not a critical determinant of
motoneuron disease initiation or progression.
Our findings are consistent with the possibility that the accumulation
of mutant SOD1 in motoneurons is not necessary to induce motoneuron
disease or motoneuron pathology. However, we cannot exclude the
possibility that a minimal level of mutant SOD1 in motoneurons is
necessary to cause motoneuron disease and/or that the critical
concentrations of mutant SOD1 in motoneurons are those that accumulate
in embryonic neurons. The latter possibility appears to us unlikely,
because it would imply that the extent of an insult to motoneurons
induced by mutant SOD1 during embryonic development would be predictive
of the onset time and progression rate of disease in the adult. We did
detect a small increase in ubiquitin deposits and astrocytosis at
intermediate times during disease progression in the double-transgenic
mice. These differences between single- and double-transgenic mice may
be attributable to some toxic effect of mutant SOD1 overexpression in
the motoneurons.
Implications for the mechanisms that lead to
motoneuron disease
The results of this study suggest that neurons, and in particular
spinal motoneurons, are not the cells in which mutant SOD1 acts
primarily to initiate and propagate motoneuron disease. Instead, motoneurons appear to be selectively vulnerable to a pathological process caused by the accumulation of FALS-associated SOD1 in a
different type of cell (Fig. 5). That process subsequently leads to
mitochondrial pathology, the accumulation of deposits, a reduction in
axonal transport, and a loss of peripheral synapses in motoneurons, ultimately causing paralysis and death.
What could be the nature of the pathological process in FALS, and in
what cells may SOD1 mutants act to initiate and propagate the disease?
There is substantial experimental evidence to support the notion that
an excess of extracellular glutamate and glutamate excitotoxicity may
be a critical factor in motoneuron disease (Rothstein, 1995; Ludolph et
al., 2000). Astrocytes play an important role in removing glutamate
from the extracellular space of the neuropil, and a deficit in
excitatory amino acid transporter-2, the glutamate transporter
on astrocytes, was detected in a mouse model of FALS (Trotti et al.,
1999). In addition, early damage to astrocytes was detected in
hMg-SOD1(G85R) mice (Bruijn et al., 1997). However, transgenic mice
overexpressing SOD1(G85R) in astrocytes exhibited some astrocytosis but
did not develop motoneuron disease (Gong et al., 2000). Damage to
astrocytes may thus be involved in the pathogenesis of motoneuron
disease (Levine et al., 1999), although astrocytes may not be the
initial site of action of mutant SOD1. A recent study provided evidence
that augmenting CXC chemokine receptor-4 signaling in microglia
can lead to excess release of tumor necrosis factor-, excess
extracellular glutamate, and motoneuron pathology (Bezzi et al., 2001).
Accordingly, one possibility is that microglia may be the cells in
which mutant SOD1 acts to initiate FALS in the transgenic mouse models.
However, regardless of whether FALS may be initiated in microglia, the
sequence of pathogenic events leading to a chronic local imbalance in
glutamate handling most likely involves multiple cellular elements.
If the pathogenic process in FALS does not involve primarily
motoneurons, why are these cells selectively vulnerable to the disease?
Motoneurons have particularly long axonal projections and appear to be
particularly sensitive to excess glutamate (Ludolph et al., 2000).
Axonal transport puts a high energy load on neurons, and mitochondrial
pathology in motoneurons has been detected early in presymptomatic FALS
mice, suggesting that a deficit in energy production may be an early
component in the sequence of events leading to motoneuron dysfunction
and loss (Kong and Xu, 1999). An energy deficit in axons can
lead to the formation of cofilin-containing aggregates because of
excess dephosphorylated, active cofilin (Minamide et al., 2000). In
addition, the accumulation of neurofilaments and neurofilament
aggregates in motoneuron axons has been linked to the pathogenesis of
human motor neuron diseases, including ALS (Lee and Cleveland, 1994;
Lee et al., 1994; Tu et al., 1997; Couillard-Despres et al., 1998;
Williamson et al., 1998). Therefore, as a consequence of glutamate
excitotoxicity, deficits in energy production and axonal transport in
motoneurons (Zhang et al., 1997; Williamson and Cleveland, 1999) may
lead to neuromuscular junction dysfunction, loss of neuromuscular
synapses (Frey et al., 2000), and muscle paralysis.
| |
FOOTNOTES |
Received Nov. 21, 2001; revised March 18, 2002; accepted March 22, 2002.
We are grateful to S. Arber for valuable comments on this manuscript.
The Friedrich Miescher Institute is a branch of the Novartis Research Foundation.
Correspondence should be addressed to Pico Caroni, Friedrich Miescher
Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. E-mail:
caroni{at}fmi.ch.
| |
REFERENCES |
-
Aigner L,
Arber S,
Kapfhammer JP,
Laux T,
Schneider C,
Botteri F,
Brenner HR,
Caroni P
(1995)
Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice.
Cell
83:269-278[Web of Science][Medline].
-
Barneoud P,
Lolivier J,
Sanger DJ,
Scatton B,
Moser P
(1997)
Quantitative motor assessment in FALS mice: a longitudinal study.
NeuroReport
8:2861-2865[Web of Science][Medline].
-
Baumeister A,
Arber S,
Caroni P
(1997)
Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube end compartments during muscle morphogenesis.
J Cell Biol
139:1231-1242[Abstract/Free Full Text].
-
Beauchamp C,
Fridovich I
(1971)
Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.
Anal Biochem
44:276-287[Web of Science][Medline].
-
Bezzi P,
Domercq M,
Brambilla L,
Galli R,
Schols D,
De Clercq E,
Vescovi A,
Bagetta G,
Kollias G,
Meldolesi J,
Volterra A
(2001)
CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity.
Nat Neurosci
4:702-710[Web of Science][Medline].
-
Bruijn LI,
Becher MW,
Lee MK,
Anderson KL,
Jenkins NA,
Copeland NG,
Sisodia SS,
Rothstein JD,
Borchelt DR,
Price DL,
Cleveland DW
(1997)
ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions.
Neuron
18:327-338[Web of Science][Medline].
-
Caroni P
(1997)
Overexpression of growth-associated proteins in the neurons of adult transgenic mice.
J Neurosci Methods
71:3-9[Web of Science][Medline].
-
Couillard-Despres S,
Zhu Q,
Wong PC,
Price DL,
Cleveland DW,
Julien JP
(1998)
Protective effect of neurofilament heavy gene overexpression in motor neuron disease induced by mutant superoxide dismutase.
Proc Natl Acad Sci USA
95:9626-9630[Abstract/Free Full Text].
-
Dal Canto MC,
Gurney ME
(1997)
A low expressor line of transgenic mice carrying a mutant human Cu,Zn superoxide dismutase (SOD1) gene develops pathological changes that most closely resemble those in human amyotrophic lateral sclerosis.
Acta Neuropathol (Berl)
93:537-550[Medline].
-
Deng HX,
Hentati A,
Tainer JA,
Iqbal Z,
Cayabyab A,
Hung WY,
Getzoff ED,
Hu P,
Herzfeldt B,
Roos RP
(1993)
Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase.
Science
261:1047-1051[Abstract/Free Full Text].
-
Frey D,
Schneider C,
Xu L,
Borg J,
Spooren W,
Caroni P
(2000)
Early and selective loss of neuromuscular synapse subtypes with low sprouting competence in motoneuron diseases.
J Neurosci
20:2534-2542[Abstract/Free Full Text].
-
Gong YH,
Parsadanian AS,
Andreeva A,
Snider WD,
Elliott JL
(2000)
Restricted expression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause motoneuron degeneration.
J Neurosci
20:660-665[Abstract/Free Full Text].
-
Gurney ME,
Pu H,
Chiu AY,
Dal Canto MC,
Polchow CY,
Alexander DD,
Caliendo J,
Hentati A,
Kwon YW,
Deng HX
(1994)
Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.
Science
264:1772-1775[Abstract/Free Full Text].
-
Julien JP
(2001)
Amyotrophic lateral sclerosis: unfolding the toxicity of the misfolded.
Cell
104:581-591[Web of Science][Medline].
-
Kong J,
Xu Z
(1998)
Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1.
J Neurosci
18:3241-3250[Abstract/Free Full Text].
-
Kong J,
Xu Z
(1999)
Peripheral axotomy slows motoneuron degeneration in a transgenic mouse line expressing mutant SOD1 G93A.
J Comp Neurol
412:373-380[Web of Science][Medline].
-
Lee MK,
Marszalek JR,
Cleveland DW
(1994)
A mutant neurofilament subunit causes massive, selective motor neuron death: implications for the pathogenesis of human motor neuron disease.
Neuron
13:975-988[Web of Science][Medline].
-
Levine JB,
Kong J,
Nadler M,
Xu Z
(1999)
Astrocytes interact intimately with degenerating motor neurons in mouse amyotrophic lateral sclerosis (ALS).
Glia
28:215-224[Web of Science][Medline].
-
Ludolph AC,
Meyer T,
Riepe MW
(2000)
The role of excitotoxicity in ALS: what is the evidence?
J Neurol
247:I7-I16.
-
Minamide LS,
Striegl AM,
Boyle JA,
Meberg PJ,
Bamburg JR
(2000)
Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function.
Nat Cell Biol
2:628-636[Web of Science][Medline].
-
Mourelatos Z,
Gonatas NK,
Stieber A,
Gurney ME,
Dal Canto MC
(1996)
The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.
Proc Natl Acad Sci USA
93:5472-5477[Abstract/Free Full Text].
-
Pardo CA,
Xu Z,
Borchelt DR,
Price DL,
Sisodia SS,
Cleveland DW
(1995)
Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons.
Proc Natl Acad Sci USA
92:954-958[Abstract/Free Full Text].
-
Price DL,
Koliatsos VE,
Wong PC,
Pardo CA,
Borchelt DR,
Lee MK,
Cleveland DW,
Griffin JW,
Hoffman PN,
Cork LC,
Sisodia SS
(1996)
Motor neuron disease and model systems: aetiologies, mechanisms and therapies.
Ciba Found Symp
196:3-13[Medline].
-
Ripps ME,
Huntley GW,
Hof PR,
Morrison JH,
Gordon JW
(1995)
Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis.
Proc Natl Acad Sci USA
92:689-693[Abstract/Free Full Text].
-
Rothstein JD
(1995)
Excitotoxic mechanisms in the pathogenesis of amyotrophic lateral sclerosis.
In: Pathogenesis and therapy of ALS (Serratrice G,
Munsat T,
eds), pp 7-20. Philadelphia: Lippincott-Raven.
-
Trotti D,
Rolfs A,
Danbolt NC,
Brown RH,
Hediger MA
(1999)
SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivate a glial glutamate transporter.
Nat Neurosci
2:848[Medline].
-
Tu PH,
Raju P,
Robinson KA,
Gurney ME,
Trojanowski JQ,
Lee VM
(1996)
Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions.
Proc Natl Acad Sci USA
93:3155-3160[Abstract/Free Full Text].
-
Tu PH,
Gurney ME,
Julien JP,
Lee VM,
Trojanowski JQ
(1997)
Oxidative stress, mutant SOD1, and neurofilament pathology in transgenic mouse models of human motor neuron disease.
Lab Invest
76:441-456[Web of Science][Medline].
-
Williamson TL,
Cleveland DW
(1999)
Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons.
Nat Neurosci
2:50-56[Web of Science][Medline].
-
Williamson TL,
Bruijn LI,
Zhu Q,
Anderson KL,
Anderson SD,
Julien JP,
Cleveland DW
(1998)
Absence of neurofilaments reduces the selective vulnerability of motor neurons and slows disease caused by a familial amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutant.
Proc Natl Acad Sci USA
95:9631-9636[Abstract/Free Full Text].
-
Wong PC,
Pardo CA,
Borchelt DR,
Lee MK,
Copeland NG,
Jenkins NA,
Sisodia SS,
Cleveland DW,
Price DL
(1995)
An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria.
Neuron
14:1105-1116[Web of Science][Medline].
-
Zhang B,
Tu P,
Abtahian F,
Trojanowski JQ,
Lee VM
(1997)
Neurofilaments and orthograde transport are reduced in ventral root axons of transgenic mice that express human SOD1 with a G93A mutation.
J Cell Biol
139:1307-1315[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22124825-08$05.00/0
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|
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|
|
|
|
|
|
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|
|
|
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|
|
|
|
|
|
|
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[Full Text]
[PDF]
|
|
|
|
|
|
|
C. W. Strey, D. Spellman, A. Stieber, J. O. Gonatas, X. Wang, J. D. Lambris, and N. K. Gonatas
Dysregulation of Stathmin, a Microtubule-Destabilizing Protein, and Up-Regulation of Hsp25, Hsp27, and the Antioxidant Peroxiredoxin 6 in a Mouse Model of Familial Amyotrophic Lateral Sclerosis
Am. J. Pathol.,
November 1, 2004;
165(5):
1701 - 1718.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Vanoni, S. Massari, M. Losa, P. Carrega, C. Perego, L. Conforti, and G. Pietrini
Increased internalisation and degradation of GLT-1 glial glutamate transporter in a cell model for familial amyotrophic lateral sclerosis (ALS)
J. Cell Sci.,
October 15, 2004;
117(22):
5417 - 5426.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Dupuis, H. Oudart, F. Rene, J.-L. G. de Aguilar, and J.-P. Loeffler
Evidence for defective energy homeostasis in amyotrophic lateral sclerosis: Benefit of a high-energy diet in a transgenic mouse model
PNAS,
July 27, 2004;
101(30):
11159 - 11164.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. P. Koushika, A. M. Schaefer, R. Vincent, J. H. Willis, B. Bowerman, and M. L. Nonet
Mutations in Caenorhabditis elegans Cytoplasmic Dynein Components Reveal Specificity of Neuronal Retrograde Cargo
J. Neurosci.,
April 21, 2004;
24(16):
3907 - 3916.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. D. Nguyen, T. D'Aigle, G. Gowing, J.-P. Julien, and S. Rivest
Exacerbation of Motor Neuron Disease by Chronic Stimulation of Innate Immunity in a Mouse Model of Amyotrophic Lateral Sclerosis
J. Neurosci.,
February 11, 2004;
24(6):
1340 - 1349.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. A. Jonsson, K. Ernhill, P. M. Andersen, D. Bergemalm, T. Brannstrom, O. Gredal, P. Nilsson, and S. L. Marklund
Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis
Brain,
January 1, 2004;
127(1):
73 - 88.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Clement, M. D. Nguyen, E. A. Roberts, M. L. Garcia, S. Boillee, M. Rule, A. P. McMahon, W. Doucette, D. Siwek, R. J. Ferrante, et al.
Wild-Type Nonneuronal Cells Extend Survival of SOD1 Mutant Motor Neurons in ALS Mice
Science,
October 3, 2003;
302(5642):
113 - 117.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Z. Ellis, J. Rabe, and K. J. Sweadner
Global Loss of Na,K-ATPase and Its Nitric Oxide-Mediated Regulation in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis
J. Neurosci.,
January 1, 2003;
23(1):
43 - 51.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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ABSTRACT
INTRODUCTION
Substance via MeSH
