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The Journal of Neuroscience, January 15, 2000, 20(2):660-665
Restricted Expression of G86R Cu/Zn Superoxide Dismutase in
Astrocytes Results in Astrocytosis But Does Not Cause Motoneuron
Degeneration
Yun H.
Gong1,
Alexander
S.
Parsadanian2,
Albina
Andreeva1,
William D.
Snider3, and
Jeffrey L.
Elliott1
1 Department of Neurology, University of Texas,
Southwestern Medical Center, Dallas, Texas 75235, 2 Department of Neurology, Washington University School of
Medicine, St. Louis, Missouri 63110, and 3 University of
North Carolina Neuroscience Center, School of Medicine, University of
North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
Evidence garnered from both human autopsy studies and genetic
animal models has suggested a potential role for astrocytes in the
pathogenesis of amyotrophic lateral sclerosis (ALS). Currently, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) represent the only known cause of motoneuron loss in the disease, producing 21q linked familial ALS (FALS). To determine whether astrocytic dysfunction has a primary role in familial ALS, we have
generated multiple lines of transgenic mice expressing G86R mutant SOD1
restricted to astrocytes. In GFAP-m SOD1 mice, astrocytes exhibit
significant hypertrophy and increased GFAP reactivity as the animals
mature. However, GFAP-mutant SOD1 transgenic mice develop normally and
do not experience spontaneous motor deficits with increasing age.
Histological examination of spinal cord in aged GFAP-mSOD1 mice reveals
normal motoneuron and microglial morphology. These results indicate
that 21q linked FALS is not a primary disorder of astrocytes, and that
expression of mutant SOD1 restricted to astrocytes is not sufficient to
cause motoneuron degeneration in vivo. Expression of
mutant SOD1 in other cell types, most likely neurons, is critical for
the initiation of disease.
Key words:
amyotrophic lateral sclerosis; glutamate; mouse; transgenic; glia; gliosis
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INTRODUCTION |
Amyotrophic lateral sclerosis (ALS)
is characterized by the progressive loss of motoneurons, leading to
profound weakness and death of affected individuals. Because it is
motoneurons that invariably die in ALS, most attention has focused on
these cells as the primary site where pathophysiological injury is
initiated. However, evidence garnered from human autopsy studies and
genetic animal models has suggested a potential role for astrocytes in the pathogenesis of ALS. Abnormalities in astrocytic function particularly related to glutamate transport have been well documented in ALS patients. Astrocytes from the brains of ALS patients manifest selective decreases in expression of full-length and functional EAAT-2
protein, which serves as the principle glutamate transporter in the CNS
(Rothstein et al., 1994 , 1995, 1996). Recent investigations have shown
that aberrant processing of EAAT-2 mRNA transcripts occurs in ALS
astrocytes and yields alternatively spliced forms of the protein that
act as dominant negative inhibitors of normal EAAT-2 expression
function (Lin et al., 1998). Such decreases in functional astroglial
EAAT-2 expression in vivo potentially explain the
significant reduction in glutamate transport function observed in
synaptosomes from ALS brains and, as well, the overall elevation
in CSF glutamate levels observed in ALS patients (Rothstein et al.,
1992). Together these findings have led to the formulation of a
hypothesis concerning ALS pathogenesis in which astrocyte dysfunction
is critical (Bai and Lipton, 1998). However, partly because the
pathogenesis of sporadic ALS is unknown, no model system exists to test
whether observed alterations in astrocytes contribute significantly to
the disease process.
Although the etiology of sporadic ALS is unknown, mutations in the gene
encoding Cu/Zn superoxide dismutase (SOD1) have been found to cause one
form of familial ALS (FALS) (Rosen et al., 1993). The process by which
mutant (m) SOD1 leads to motoneuron degeneration remains unclear, but
studies involving the use of transgenic animals clearly point to a
toxic gain of function model for the abnormal protein. Mice with
targeted deletions of both SOD1 alleles do not develop spontaneous
motoneuron loss (Reaume et al., 1996). In contrast, transgenic mice
overexpressing mSOD1 develop spontaneous motoneuron degeneration and
represent an excellent animal model of the disease (Gurney et al.,
1994; Ripps et al., 1995; Wong et al., 1995; Tu et al., 1996).
Astrocytic alterations occur in mSOD1 transgenic mice. Pathological
examination of transgenic mice expressing a G85R mSOD1 demonstrates
abnormal protein inclusions in astrocytes even at early clinically
presymptomatic time points when motoneuron pathology is not readily
apparent (Bruijn et al., 1997). In addition, these mice also exhibit
selective loss of the principal astrocytic glutamate transporter GLT-1
and manifest alterations in glutamate transport function, mirroring
those observed in human disease (Bruijn et al., 1997; Canton et al.,
1998). However, experiments to date have been unable to delineate
whether the astrocyte abnormalities observed in mSOD1 transgenic mice
represent primary dysfunction of this cell type or occur secondary to
neuronal injury, let alone determine whether these astrocytic changes
contribute to disease pathogenesis or represent epiphenomena.
Conventional lines of mSOD1 mice are problematic for understanding the
role of astrocytes in the disease process. Because these lines use the
endogenous SOD1 promoter to drive transgene expression, mSOD1 is
expressed ubiquitously in all cells including neurons and glia.
Consequently, these animals are unsuitable to determine which cell type
is critical in initiating the process leading to motoneuron loss. We
therefore have generated transgenic mice expressing mSOD1 under the
control of an astrocyte-specific promoter and asked whether such mice
develop spontaneous motoneuron degeneration and astrocytic pathology.
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MATERIALS AND METHODS |
Construct design and generation of GFAP-G86R transgenic
mice. A cDNA encoding full-length normal murine SOD1 was obtained using P1 mouse brain RNA, reverse-transcribed with random primers (Superscript reverse transcriptase). An aliquot of this mixture was
then used to amplify normal murine SOD1 cDNA. Primers were used
(5[prime- ACT AGT ATG GCG ATG AAA GCG GTG-3' and 5'-GGA TCC TGT TTA
CTG GGC AAT CCC-3') to amplify the full-length murine SOD1 with
Spe1 and BamHI restriction sites on the 5' and 3'
end, respectively. PCR conditions were 94°C for 4 min, 94°C for 1 min, 60°C for 1 min, and 72°C for 1.5 min for 35 cycles followed by a 7 min extension at 72°C. A band corresponding to a 465 nucleotides was obtained and cloned directly into a PCR II cloning vector (Invitrogen, San Diego, CA). After sequencing to confirm normal SOD1
sequence, this plasmid was digested with SpeI and
BamHI and then cloned into pBluescript II(p-SOD2). This
plasmid was then used to generate G86R SOD1. For this amino acid
substitution, the base at position 256 must be changed from G to C. A
reverse primer incorporating this change was designed using sequence
250 to 294, which also has BalI site at the 3' end (5'-CAC
ATT GGC CAC ACC GTC CTT TCC AGC AGT CAC ATT GCG CAG GTC-3'). This
primer and the original 5' SOD1 primer (listed above) were used to
amplify a 295 bp fragment, which was then cloned (p-BAL frag) and
sequenced to confirm the appropriate G to C change at 256. After a
Spe1and BalI digestion, this altered 295 bp SOD
fragment was ligated into PSOD2, which had also undergone
SpeI and BalI digestion to then yield a
full-length SOD1 with a G to C change at 256 (p-BAL-SOD1). This plasmid
was confirmed again by sequencing. A 2.3 kb fragment (Brenner et al.,
1994) containing the 5' flanking DNA sequence derived from the human
glial fibrillary acidic protein (GFAP) gene was available in Bluescript
SK II with flanking SpeI sites. This GFAP promoter was
excised with SpeI and ligated into the SpeI site
of p-BAL-SOD to generate p-GFAP-mSOD. An 850 bp SV40 small t-antigen
with an intron and polyadenylation sequence was cloned into PGFAP-SOD
using EcoRV and SalI sites to generate
p-GFAP-mSOD1-SV40, which was then excised with NotI and
SalI and used for pronuclear injection (strainB6/CBA).
Integration of the transgene into the mouse genome was determined by
Southern blotting and PCR on genomic DNA extracted from mouse tails.
Southern blot and PCR analysis. Ten micrograms of genomic
DNA isolated from mouse tail were digested with digested with
BamHI and KpnI, separated on a 0.8% agarose gel,
and then transferred to a positive charged nylon membrane (Boehringer
Mannheim, Indianapolis, IN) via overnight capillary transfer. A
dig-dUTP 450 bp probe generated by PCR (PCR-dig kit; Boehringer
Mannheim), corresponding to full-length SOD1 cDNA, was used for
hybridization in DIG Easy Hyb solution (Boehringer Mannheim) at 42°C.
Hybridization of probe and DNA fragment were visualized the using the
DIG-Luminescent Kit (Boehringer Mannheim) following the instructions of
the manufacturer. Films were be scanned and compared semiquantitatively
between the differing transgenic lines using NIH Image software
(version 1.61). PCR analysis (30 cycles: 94°C for 1 min, 64°C for
45 sec, and 72°C for 1 min 20 sec) was performed for routine
identification of mice carrying the transgene using primers
encompassing the 5' end of SOD1 and extending 150 bp into the SV40 sequence.
In situ hybridization. In situ hybridization
was performed following methods previously described (Deckwerth et al.,
1996; Parsadanian et al., 1998). Animals were deeply anesthetized with sodium pentobarbital and then decapitated. Spinal cord and brain were
rapidly dissected out, frozen on dry ice, and 12 µM sections were cut on a Zeiss micom
cryostat. Plasmid (p-SV40-1) containing the fragments encoding the 850 bp SV40 small t-antigen were linearized with appropriate restriction
enzymes for subsequent sense and antisense in vitro
transcription The antisense probe recognizes only transgene mRNA. cRNA
probes were transcribed in vitro using the appropriate RNA
polymerase in the presence of 50 µCi
33P[UTP] (NEN). Hybridization occurred
overnight at 55°C in the presence of 1 × 106 cpm
33P[UTP] labeled riboprobe diluted in a
hybridization mixture. The following day slides were rinsed in 4× SSC,
at 37°C, followed by a 30 min wash in RNase A (20 mg/ml) at 45°C.
Slides were then rinsed at 37°C in 2× SSC, 0.5× SSC, and 0.1× SSC.
Slides were then exposed to Biomax film (Eastman Kodak, Rochester, NY)
for 3-4 d to generate autoradiographs. Slides were dipped in Kodak NTB-2 emulsion and stored in light-tight boxes for 2 weeks, developed with Kodak developer and fixer, and counterstained with hematoxylin and eosin.
Western blotting. Animals were overdosed with sodium
pentobarbital (250 mg/kg, i.p.). Spinal cords were dissected,
homogenized (in mM: 20 Tris-HCl, pH 7.5, 2 DTT, 1 EDTA,
and 1 EGTA, with leupeptin 0.1 mg/ml), then centrifuged at 13,000 × g with pelleted debris discarded. Protein concentration
was measured using the BCA protein assay (Pierce, Rockford, IL). Five
micrograms of protein from each sample was loaded on a 14%
Tris-glycine gel (Novex). A range of known concentrations of bovine
SOD 1 (Sigma, St. Louis, MO) were loaded. Gel was gently rinsed with
transfer buffer (Tris base 12 mM, glycine 96 mM, and 20% methanol) and placed against a
polyvinylidene difluoride membrane. Transfer was performed at 25 V, 100 mA for 2 hr. Membranes were washed in PBS followed by incubation in
blocking solution [0.2% I-block (Tropix), PBS, and 0.1% Tween 20]
overnight at room temperature. Membrane was incubated with primary
antibody, a polyclonal rabbit anti-bovine SOD1 (Chemicon, Temecula, CA)
antibody, at 1:4000 dilution in blocking buffer for 1-2 hr. After
washing, membrane was incubated with a secondary anti-rabbit
antibody-alkaline phosphatase conjugate (Tropix) (1:5000 in blocking
buffer) as well as with an Avidix-AP streptavidin-alkaline phosphatase
conjugate (1:20,000) for 1 hr. After washing, the membrane underwent a
5 min incubation with CSPD chemoluminescent alkaline phosphatase
substrate (Tropix). After exposure to Kodak X-omat film, films were
scanned and then imported into NIH Image for quantitation of band density.
Stride test. This test was performed with modifications from
Gurney et al. (1994). Animals were trained to walk across a 1 m
flat board. These animals then had their hindpaws dipped with a
nontoxic ink and were allowed to walk across the 1 m board that had white construction paper over the top for recording footprints. Stride lengths were measured in millimeters. Statistics on data were be
performed using Student's t test. All procedures performed on mice were approved by an animal research committee and conform to
National Institutes of Health guidelines.
Immunochemistry and histology. After overdose with
pentobarbital, animals were perfused with 4% paraformaldehyde and had
spinal cords and brains removed. For routine histology, tissue was
embedded in plastic, sectioned (1 µm), and stained with toluidine
blue. For immunohistochemistry, paraffin-embedded sections (4 µm)
were incubated with a rabbit polyclonal GFAP antibody (Dako, Glostrup, Denmark) (1:200 dilution) and visualized using an immunoperoxidase reaction (the one kit, Sternberger Monoclonals). For studies of morphology, four GFAP-mSOD1 and three wild-type littermates were used
for each time period studied (4 and 12 months of age). Multiple sections from GFAP-mSOD1 and wild-type sibling lumbar spinal cords were
processed and stained simultaneously. Photomicrographs were taken,
scanned, and then imported into NIH Image version 1.61, where the area
of GFAP-positive astrocytes (in pixels) was obtained. At least 250 astrocytes from GFAP-mSOD1 and wild-type littermates of 4 and 12 months
of age were used per age group. Measurements were performed by a single
investigator who was unaware of the animal genotype. To assess
microglia, sections were washed in TBS followed by overnight incubation
with a tomato (Lycopersicon esculentum) lectin (Sigma)
conjugated to fluorescein (1:70 dilution). G93A conventional mSOD1 mice
were obtained from The Jackson Laboratory (Bar Harbor, ME).
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RESULTS |
Construct design and line analysis of GFAP-(m)SOD1
transgenic mice
To generate transgenic mice expressing G86R mSOD1 restricted to
astrocytes, we designed a construct that uses a 2.3 kb flanking sequence 5' to the human GFAP gene (Brenner et al., 1994). This promoter region has been shown to drive high levels of
astrocyte-specific expression in transgenic mice beginning at early
postnatal periods and extending into adulthood (Brenner et al., 1994;
Raeber et al., 1997). This promoter fragment is also capable of driving transgene upregulation in the setting of an injury, which would typically result in increased GFAP protein synthesis, such as occurs in
conventional mutant SOD1 transgenic mice. We selected the murine G86R
SOD1 mutation (G85R in humans) because earlier experiments had shown
that expression of this altered protein under the control of the
endogenous SOD1 promoter is capable of producing motoneuron disease
in vivo with both neuronal and astrocytic dysfunction (Ripps
et al., 1995; Bruijn et al., 1997). Figure 1a demonstrates the overall
construct design used for pronuclear injection and the generation of
GFAP-SOD1 transgenic mice.
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Figure 1.
a, Schematic representation of
construct used in generation of GFAP-mSOD1 mice. The C for G
substitution in the first base at codon 86 causes a glycine to arginine
change. b, Southern blot analysis of GFAP-mSOD1
transgenic lines. BamHI and KpnI
digestion of DNA yields a 729 bp fragment in transgenic mice.
Con, p-GFAP-mSOD1-SV40; WT, wild-type.
c, Western blot analysis of SOD1 in spinal cords from
adult GFAP-mSOD1 and wild-type mice using rabbit anti-bovine SOD1.
Lanes 1, 2, Wild-type (WT) mouse;
2.5 µg and 5 µg of total protein loaded, respectively. Lanes
3, 4, Line 5512-2; 2.5 µg and 5 µg of total protein
loaded. Lanes 5, 6, Line 5512-29; 5 µg and 2.5 µg
of total protein loaded. Lane 7, Bovine SOD1 control;
0.02 µg of protein loaded.
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After injection of the transgene construct, six founder mice (5512-2,
5512-4, 5512-9, 5512-19, 5512-29, and 5512-31) were identified by
initial PCR screening. Relative transgene copy number was determined by
Southern blot analysis after digestion of genomic DNA with
BamHI and HindIII (Fig. 1b). This
digestion yields a 729 bp fragment of the transgene that encompasses
the entire SOD1 cDNA and a 250 bp segment of the 3' end of the GFAP
promoter. Relative transgene copy number differed for each line varying from line 5512-9 (1x) to line 5512-29 (10x). Lines 5512-29 and 5512-2 demonstrated the highest number of transgene copies and were
used for further analysis of transgene transcription and translation.
Line 5512-19 exhibited a rearrangement of the transgene, and although
still bred, was not used for further experiments.
To assess whether transgene incorporation resulted in increased protein
production, we performed Western blot analysis on spinal cords from
lines 5512-29 and 5512-2 (Fig. 1c). Available antibodies
against SOD1 were not capable of distinguishing between normal and
mutant murine SOD1, so total SOD1 protein levels were assayed. Initial
blots performed on tissue from 6-week-old animals revealed
significantly increased levels of total SOD1 protein expression in
spinal cords from transgenic GFAP-mSOD1 transgenic mice as compared to
wild-type controls. Correlating with copy number, line 5512-29
demonstrated higher total SOD1 expression than did line 5512-2.
Increased total SOD1 expression in spinal cord was also observed in
older transgenic GFAP-mSOD1 mice up to 5 months of age (the oldest age
examined). We next used in situ hybridization to test
whether expression of the mutant SOD1 was in fact restricted to
astrocytes (Fig. 2). By generating a riboprobe complimentary to the SV40 t-antigen, we were able to distinguish the cells that were transcribing the transgene. In situ hybridization demonstrated robust transgene synthesis
in both white and gray matter portions of spinal cord from line
5512-29. High-power bright-field microscopy revealed that expression
of transgene was limited to glial cells and not neurons. Similar findings were observed in brainstem sections from 5512-29 mice. GFAP-mSOD1 mice from line 5512-2 also expressed transgene message limited to glial cells in spinal cord and brainstem, but again correlating with total copy number and results from Western blotting, line 5512-2 exhibited overall less robust transgene expression than
did 5512-29.
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Figure 2.
Expression of mSOD1 transgene is restricted to
non-neuronal cells in the spinal cord. a , b, Transverse
sections of spinal cord from 6-week-old wild-type
(a) and line 5512-29 GFAP-mSOD1
(b) mice hybridized with antisense riboprobe for
the SV40 sequence, which detects only transgene expression. Dark-field
microscopy, Magnification, 40×. c, d, Bright-field
views of wild-type (c) and GFAP-mSOD1
(d) ventral horn. Arrowheads
indicate neuronal cell bodies. Silver grains are observed only over
glial cells in GFAP-mSOD1 mice (arrows). Magnification,
400×.
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GFAP-SOD1 survival and motor function
All six GFAP-mSOD1 transgenic founders were bred and followed
longitudinally for signs of motor dysfunction as well as for survival
analysis. GFAP-mSOD1 mice exhibited normal early development, reproductive capacity, and survival curves comparable to wild-type mice
at least until 16 months of age (the oldest age yet attained). Founder
5512-2 did die suddenly at 4 months of age. However, this founder
demonstrated no signs of motor dysfunction before death, and its
transgenic offspring are alive at 1 year of age. Even up to 16 months
of age, GFAP-mSOD1 mice do not display abnormalities in gross motor
function or postural reflexes. The stride length test was used to
assess motor function quantitatively, in a method that had been used to
test motor function in conventional mutant SOD1 mice (Fig.
3). Compared to wild-type littermates,
GFAP-mSOD1 mice exhibited an identical pattern of stride length
distance beginning at 1 month of age and continuing until past 1 year
of age. Thus, at least for the first 16 months of life, GFAP-mSOD1 mice
do not appear to develop clinical signs of motor neuron
dysfunction.
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Figure 3.
Stride length in GFAP-mSOD1 transgenic female mice
from line 5512-29 and wild-type littermates. There is no significant
difference in stride length between the two groups.
n = 4 for each group.
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Neuronal and glial morphology in GFAP-mSOD1 transgenic mice
We performed histological and immunhistochemical analysis on
1-year-old GFAP-mSOD1 mice from line 5512-29 to determine whether these mice developed any pathological evidence of neuronal or glial
dysfunction. Thin plastic sections of spinal cord ventral horn
demonstrated normal motoneuron morphology without evidence of
cytoplasmic vacuoles (Fig. 4).
Imunnohistochemical analysis with antibodies directed against
phosphorylated neurofilaments or ubiquitin in GFAP-mSOD1 mouse tissue
did not reveal any neuronal inclusions in spinal cord motor neurons
that are characteristic of affected neurons in conventional mSOD1 mice
(data not shown).
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Figure 4.
Toluidine-stained plastic sections of the ventral
horn from 1-year-old wild-type (a, c) and line 5512-29
GFAP-mSOD1 mice (b, d). Arrows indicate
motoneurons. Magnification, 200× for a and
b; 600× for c and
d.
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We next asked whether glia manifested any morphological or biochemical
abnormalities. Immunostaining spinal cord from GFAP-mSOD1 demonstrated
increased GFAP reactivity as compared to wild-type age-matched
littermates (Fig. 5). In addition,
astrocytic morphology had changed from fine stellate-appearing cells in
wild-type animals to a larger and more globular appearance in
GFAP-mSOD1 mice. GFAP-immunoreactive spinal cord astrocytes were
significantly larger in GFAP-mSOD1 mice compared to age-matched
wild-type controls [mean area (pixels) ± SEM: WT, 147 ± 8;
GFAP-mSOD1, 225 ± 8; p < 0.001]. However, changes in both GFAP reactivity and astrocytic hypertrophy in GFAP-mSOD1 mice were not as extensive as those observed in endstage conventional mutant SOD1 transgenic mice with a mean area of 392 ± 29 pixels for astrocytes. Interestingly, changes in astrocytic morphology were not observed in the ventral horn of younger GFAP-mSOD1 mice. At 4 months of age, there was no difference in size between spinal cord astrocytes in wild-type (138 ± 5 pixels) and
GFAP-mSOD1 mice (143 ± 9 pixels). This finding indicates that the
astrocytosis in GFAP-mSOD1 mice is not present at younger ages but
develops as the animals mature.
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Figure 5.
Astrocyte morphology in the ventral horns of
1-year-old wild-type (A), 1-year-old GFAP-mSOD1
(B), and 8-month-old (endstage) G93A SOD1
(C) mice identified by GFAP immunoreactivity.
Magnification, 400×. D, Area of ventral horn
GFAP-reactive cells from 1-year-old WT and 5512-29 mice as well as
endstage (8 month) G93A SOD1 mice. *p < 0.0001 versus wild-type.
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To assess changes in microglia we used a lectin (L. esculentum) that binds poly N-acetyl lactosamine
residues in both activated and nonactivated microglia (Acarin et al.,
1994). Both wild-type and GFAP-mSOD1 mice exhibit rare microglia in
spinal cord in contrast with the markedly increased number of microglia
present in conventional mutant SOD1 mouse spinal cord (data not shown).
These results indicate that if mutant SOD1 expression is astrocyte
specific, than pathological changes appear restricted to this cell type and do not promote subsequent neuronal dysfunction or microglial proliferation.
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DISCUSSION |
Although previous studies have described abnormalities in
astrocytes in both autopsied human ALS tissue as well as in transgenic mouse models of FALS, they have not adequately addressed the issue of
whether primary astrocytic dysfunction is capable of causing motoneuron
degeneration in ALS. Mutant SOD1 expression in vivo provides
a model system to test whether astrocytic changes induced by mSOD1 are
critical in the pathogenesis of ALS. Here we demonstrate that mutant
SOD1 expression limited to astrocytes results in significant changes to
this cell type in transgenic mice but is not sufficient to cause
motoneuron death or motor dysfunction in vivo. Although it
is possible that with even higher levels of mSOD1 production in
astrocytes, we may have observed motoneuron loss, we feel that this
occurrence is unlikely for a number of reasons. First, in conventional
mSOD1 transgenic mice, expression of even small amounts of G85R SOD1,
representing a fraction of total SOD1 protein produced, is sufficient
to cause disease with a time course resulting in death by ~12-14
months of age (Bruijn et al., 1997). In addition, our in
situ hybridization experiments demonstrate that robust transgene
transcription in our highest expressing lines of GFAP-mSOD1 mice is
widely distributed, occurring not only in glial cells surrounding
motoneurons in the ventral horn but also in glia throughout the spinal
cord and into white matter tracts. Third, the morphological changes we
have observed in GFAP-mSOD1 astrocytes also would offer support of
transgene expression at levels capable of altering basic astrocytic properties.
Recently, Trotti et al. (1999) have demonstrated that mSOD1 selectively
inactivates the GLT-1 transporter in vitro when both proteins are expressed in Xenopus oocytes and propose that
this finding accounts for the toxic properties of mSOD1 leading to motoneuron degeneration. Their experiments and conclusion again imply a
principal and critical role for astrocytes in initiation of the disease
process. However, our results indicate that 21q linked FALS is not a
primary disease of astrocytes as might be predicted by the glutamate
hypothesis, but that expression of mutant SOD1 in another cell type is
critical for manifestations of disease. It is likely, although not
proven, that neuronal expression of mutant SOD1 is necessary for the
disease process and that mutant SOD1 would likely exert its toxic
effects directly on motoneurons. Although precise mechanisms of mutant
SOD1 toxicity within neurons are unclear, several possibilities
including enhanced generation of hydroxyl radicals or increased
peroxynitrite formation by the mutant enzyme have been suggested by a
number of investigations (Beal et al., 1997; Crow et al., 1997;
Ferrante et al., 1997; Bogdanov et al., 1998; Pedersen et al., 1998).
Our results do not exclude the possibility that astrocytes may
contribute to progression of FALS after the disease process has been
initiated in another cell type. Neurons appear capable of regulating
expression of astrocytic proteins possibly via the secretion of
diffusable factors as has been demonstrated for glutamate transporter
subtypes (Swanson et al., 1997). Primary neuronal dysfunction could
trigger inappropriate and harmful astrocytic responses that potentiate
neuronal injury or allow its propagation, including release of
cytokines or alterations in glutamate transporter expression (Campbell
et al., 1993; Chavany et al., 1998).
Although GFAP-mSOD1 mice do not develop motor dysfunction or motoneuron
pathology, astrocytes in these animals do undergo changes, including
hypertrophy and increased GFAP expression. Because such changes occur
in GFAP-mSOD1 mice in whom motoneuron pathology does not occur, this
indicates that mSOD1 expression in astrocytes alone is sufficient to
cause at least some degree of astrocytosis that is not reactive but
primary. The fact that the astrocytosis in GFAP-mSOD1 develops as the
mice mature and is not present in younger adult mice suggests that
there is a gradual accumulation of potentially noxious factors within
astrocytes leading to injury but not degeneration.
It is interesting to note that pronounced alterations in astrocytic
morphology have been observed in conventional mutant SOD1 transgenic
mice that develop weakness. Remarkably, these changes can be observed
in astrocytes located not only in close proximity to motor neuron
populations (i.e., ventral horn) but also in astrocytes at a distance
from motor pools such as in the dorsal horn or rostral pons. One
possible explanation for this phenomenon is that mutant SOD1 exerts its
toxic effects on many differing populations of neurons extending beyond
motor neuron pools and that astrocytes throughout the CNS then undergo
changes secondary to this diffuse neuronal injury (Dal Canto and
Gurney, 1994; Kliveny et al., 1999). However, our experiment would
suggest that mSOD1 expression directly within astrocytes is capable of
altering astroglial morphology. Together, these results suggest that
the astrocytosis in conventional mSOD1 mice is derived from a
combination of both secondary astrocytic reaction to neuronal
dysfunction as well as primary direct astrocytic dysfunction.
Although this study demonstrates that mutant SOD1-induced disease is
not a primary disease of astrocytes, 21q linked FALS is responsible for
only ~20% of familial cases and <5% of all ALS cases. It is
unclear whether the conclusions reached from studying a transgenic
murine model of FALS can be broadened to include the sporadic form of
disease, despite common pathological and biochemical features that have
been observed both in the animal model and human disease.
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FOOTNOTES |
Received Aug. 19, 1999; revised Oct. 20, 1999; accepted Oct. 22, 1999.
This work was supported by grants from the National Institute of
Neurological Diseases and Stroke (NS01853) to J.L.E. and Muscular
Dystrophy Association to J.L.E. and W.D.S. We thank Laura Lee Deane for
expert technical assistance and Dr. Larry Honig for valuable discussions.
Correspondence should be addressed to Dr. Jeffrey L. Elliott,
Department of Neurology, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235. E-mail: jellio{at}mednet.swmed.edu.
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ABSTRACT
INTRODUCTION
Substance via MeSH
