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Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord.

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Disease Mechanisms Revealed by
Transcription Profiling in SOD1-G93A
Transgenic Mouse Spinal Cord
Mary K. Olsen, BA, Steven L. Roberds, PhD, Brenda R. Ellerbrock, BA, Timothy J. Fleck, BA,
Denise K. McKinley, BA, and Mark E. Gurney, PhD
Mutations of copper,zinc-superoxide dismutase (cu,zn SOD) are found in patients with a familial form of amyotrophic
lateral sclerosis. When expressed in transgenic mice, mutant human cu,zn SOD causes progressive loss of motor neurons
with consequent paralysis and death. Expression profiling of gene expression in SOD1-G93A transgenic mouse spinal
cords indicates extensive glial activation coincident with the onset of paralysis at 3 months of age. This is followed by
activation of genes involved in metal ion regulation (metallothionein-I, metallothionein-III, ferritin-H, and ferritin-L) at
4 months of age just prior to end-stage disease, perhaps as an adaptive response to the mitochondrial destruction caused
by the mutant protein. Induction of ferritin–H and –L gene expression may also limit iron catalyzed hydroxyl radical
formation and consequent oxidative damage to lipids, proteins, and nucleic acids. Thus, glial activation and adaptive
responses to metal ion dysregulation are features of disease in this transgenic model of familial amyotrophic lateral sclerosis.
Ann Neurol 2001;50:730 –740
An important clue to the pathogenesis of amyotrophic
lateral sclerosis (ALS) was provided by the discovery of
mutations in the SOD1 gene encoding copper, zincsuperoxide dismutase (cu,zn SOD) in patients with a
familial form of ALS.1 ALS causes the progressive loss
of motor neurons from cortex, brainstem, and spinal
cord with consequent paralysis and death. From 5 to
8% of all cases of ALS show familial inheritance, and
of those roughly 20% are caused by SOD1 mutations.
The disease has an autosomal dominant mode of inheritance, indicating that one copy of the mutant gene
is sufficient to cause disease. The SOD1 mutations are
predominantly missense mutations that substitute one
amino acid for another; 92 different mutations at 67
different sites have been found to date (eg, OMIM no.
105400). These are spread throughout the length of
the protein sequence. Mutant human cu,zn SOD
causes motor neuron disease when expressed at high
levels in transgenic mice, indicating that disease is
caused by the presence of the altered protein.2 Expression of wild type human cu,zn SOD at the same level
does not cause disease.
Several different strains of mutant cu,zn SOD transgenic mice have been created.2–5 These differ in the
SOD1 mutation analyzed, the species of the SOD1
gene into which the mutation was placed, and the level
of expression of the mutant protein; all of which affects
the timing and severity of motor neuron disease.
Within the spinal cord, ventral horn motor neurons
express the highest levels of mutant human cu,zn
SOD, perhaps explaining the selective vulnerability of
this neuronal population.6
The SOD1-G93A transgenic mice [TgN(SOD1G93A)Gur1] used in this study are healthy until 3
months of age, at which point they develop motor
symptoms such as shaking in the limbs when suspended.7 Over the next month the mice grow weaker,
their stride shortens, and eventually they become paralyzed as a result of the loss of motor neurons from the
spinal cord ventral horn. Complete paralysis occurs between 4 and 5 months of age. The sequence and timing of behavioral and pathological changes in the
SOD1-G93A mice are surprisingly stereotyped. Extensive pathology develops in the spinal cord well before
the display of overt symptoms of motor neuron disease.
As early as 1 month of age, subtle vacuolar changes are
seen in motor axons where they enter the ventral roots,
and high molecular weight aggregates of cu,zn SOD
can be discerned by biochemical assay.7,8 By 2 months
of age, vacuolar changes have extended to the motor
neuron cell body. Fragmentation of the Golgi apparatus is seen, along with widespread vacuolar changes in
the mitochondria caused by the swelling of the mitochondrial intermembrane space.4,9,10 Glial activation is
From the Genomics Research Unit, Pharmacia Corporation,
Kalamazoo, MI.
Published online Nov 1, 2001; DOI 10.1002/ana.1252
Received Apr 3, 2001, and in revised form Jul 5. Accepted for publication Jul 31, 2001.
730
© 2001 Wiley-Liss, Inc.
Address correspondence to Dr Gurney, deCODE Genetics, Lynghals 1, IS-110 Reykjavik, Iceland. E-mail: mark@deCODE.is
Table 1. Disease Progression in SOD1-G93A Transgenic Mice
Age (days)
Symptoms
Spinal Cord Pathology
30
60
Healthy
Healthy
90
Shaking in limbs when suspended,
disinhibition of spinal reflexes
Weakness, shortening of stride,
decrease in nightly running
Slight vacuolar changes in motor axons entering the ventral root
Ventral horn motor neurons show fragmentation of the golgi
apparatus, extensive vacuolar changes involving both endoplasmic reticulum and mitochondria
Evidence of motor neuron loss, denervation and reinervation of
motor endplates, demyelination of descending spinal tracts
Motor neuron loss, astrocytic and microglial cell activation, ubiquitinated inclusions visible in ventral horn
120
prominent by 3 months of age, as is formation of ubiquitinated, cytoplasmic inclusion bodies11; some or
most of which contain aggregated SOD.11–14
DNA chip technology was used to compare and
contrast alterations in gene expression over the course
of disease in transgenic SOD1-G93A mutant mice.
Gene expression monitoring was based on hybridization to high-density arrays of synthetic oligonucleotides
(Affymetrix, Santa Clara, CA). Such array-based methods allow the simultaneous monitoring of large numbers of probe sequences for expression in tissues and
for alteration of their expression because of disease.
Monitoring of gene expression in whole spinal cord
tissue allowed us to monitor changes in bulk cellular
activation and overall changes in cellular composition
as well as homeostatic responses to neural degeneration, myelin fragmentation, and lipid release. Extensive
microglial and astrocytic activation concident with the
onset of paralysis at 3 months of age is reflected in
striking transcriptional alterations in genes involved in
inflammation, scavenging, and resorption. As disease
severity worsens, this is followed by the activation of
genes involved in metal ion regulation.
Materials and Methods
The colonies of TgN(SOD1-G93A)1Gur and TgN(SOD1)2Gur transgenic mice, obtained from the Jackson Laboratory, were housed and bred as described previously,2,15 in
accordance with Institutional Animal Care and Use Committee guidelines. For each time point and genotype, three to
five mouse spinal cords were obtained by “blow out” and
pooled. Genotyping of mice was by polymerase chain reaction (PCR). C57BL/6J-Apoetml UNC mice were obtained
from the Jackson Laboratory. The combined silver–cholinesterase stain for motor axons at the neuromuscular junction
has been described elsewhere.2
Total RNA was purified from mouse spinal cord using
Tri-Reagent (Life Technologies, Carlsbad, CA), and
poly(A)⫹ RNA was isolated with a Qiagen kit (Venlo, The
Netherlands). Samples for hybridization were prepared by reverse transcription of cDNA followed by in vitro transcription of RNA in the presence of biotinylated nucleotides. Affymetrix murine 6500 oligonucleotide microarrays were
processed as recommended by the manufacturer using a
GeneChip Fluidics Station 400. Labeled samples prepared
from each pool of mouse spinal cords were hybridized with a
single murine 6500 GeneChip. The probed arrays were
scanned with a Gene-Array Scanner (Hewlett Packard, Palo
Alto, CA). The scanned images were analyzed using Affymetrix GeneChip v 3.1 software. Images were scaled globally
to compensate for minor variations in fluorescence, and to
bring the mean average difference intensity (ADI) for all of the
genes on each array to 2,500 intensity units (IU). Each gene
discussed in the text is cross-referenced by its GenBank Accession number (eg, W41883 for thymosin B4). OMIM accession numbers refer to entries in Online Inheritance in Man
(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db⫽OMIM).
Results
Gene Expression Changes in SOD1-G93A Mouse
Spinal Cord
The progression of disease in SOD1-G93A transgenic
mice is outlined in Table 1. Figure 1 shows the timing
of sample collection from SOD1-G93A transgenic
mice, nontransgenic littermate controls (NonTg), and
control SOD1 transgenic mice. Both transgenes are expressed at high levels in these two lines such that the
SOD1-G93A and SOD1 transgenic mice are balanced
for human cu,zn SOD protein and elevation of total
dismutase activity toward superoxide in the mouse spinal cord.7,16
Looking at overall patterns of gene expression profiled with the Affymetrix murine 6500 chips, there
were essentially no differences in gene transcript levels
Fig 1. Times of tissue harvest (days) and comparisons made
between SOD1-G93A, NonTg and SOD1 transgenic mice.
Olsen et al: Gene Expression Profile in ALS Transgenic Mice
731
vation of scavenge pathways for proteins and lipids,
and dysregulation of metal ion homeostasis.
Fig 2. (A) Comparison of gene expression profiles in SOD1
transgenic versus nontransgenic mice (A; R 2 ⫽ 0.925), and
in mutant SOD1-G93A at end-stage disease (120 days) versus
nontransgenic mice (B; R 2 ⫽ 0.841).
between the SOD1 and NonTg spinal cords sampled
at 120 days (R2 ⫽ 0.925; Fig 2). Considerably more
dispersion was seen when comparing transcript levels
between SOD1-G93A spinal cord at 120 days and
NonTg (R2 ⫽ 0.841; see Fig 2) or SOD1 mouse spinal cord (not shown).
Changes in transcript profiles at 30 and 60 days of
age between SOD1-G93A mice and NonTg littermate
controls were negligible. Transcripts for thymosin B4
(W41883) and a 21kDa peptide under translational
control (X06407) were increased more than twofold at
30 days of age, but returned to control values at older
ages (Fig 3). Thymosin B4 gene transcription was developmentally regulated with a sharp increase at 120
days of age in all three genotypes. Transcripts for the
KIF3-associated protein (KAP3A/B), a regulator of fast
axonal transport, were previously shown to be upregulated in the SOD1-G86R transgenic model17; however,
no alteration in that transcript was seen in SOD1G93A mice.
Changes in gene expression in SOD1-G93A mice
due to disease became prominent at 90 and 120 days
of age, consistent with changes in glial activation, acti-
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Changes in Cellular Composition
and Glial Activation
Motor neurons are a minor neuronal population in spinal cord. In SOD1-G93A mice, they are depleted
roughly 50% at end-stage disease.7 Perhaps correlated
with neuron loss, a decrease in the neurofilament NF-L
(M20480) and NF-H (M35131) transcripts was seen
in SOD1-G93A mouse spinal cord at end-stage disease, whereas there was no change in NF-M (X05640)
transcripts (see Fig 3).
Changes in glial activation were the earliest expression
changes seen in the SOD1-G93A mice. Astrocytic activation is a prominent histological feature of motor neuron disease in the SOD1-G93A transgenic mice.9 This
was reflected by increased expression of glial fibrillary
acidic protein GFAP(K01347) and vimentin (M26251)
mRNA transcripts at 90 and 120 days of age. Expression
of the microglial activation marker CD63 (D16432)
also was increased at 90 and 120 days of age (Fig 4).
CD63 is expressed on mouse microglial cells and on
mouse macrophages (where activation increases expression), although its distribution in brain has not been
reported.18,19 Expression of the ␣ integrin MAC-1/
CD11B (X07640) found on macrophages and microglial cells was induced at 120 days of age in SOD1G93A mice. LFA-1 (M60778) expression was also
elevated slightly at 120 days of age. Astrocytic and microglial activation is normally accompanied by an increase in MCP-1, a chemotactic factor for microglia and
perhaps activated astrocytes,20 but expression of MCP-1
(U50712) was not altered by disease in these mice. Of
the four macrophage inflammatory protein (MIP) genes,
only MIP-1a (M73061) was present on the murine
6500 chip. Its expression increased with age, but was not
altered by disease. Expression of transcripts encoding
tumor necrosis factor-␣ (TNF␣), interleukin-1␣
(IL-1␣), interleukin-1␤ (IL-1␤), interleukin-5 (IL-5),
interleukin-10 (IL-10), and transforming growth
factor-␤3 were detected in spinal cord but were not altered by disease, whereas expression of interleukin-6
(IL-6) and epidermal growth factor were not detected in
mouse spinal cord with the Affymetrix murine 6500
chip. Increased expression of interleukin-1␤ convertase
has been reported previously in SOD1-G93A mice,21
but the transcript was not detected by hybridization to
the Affymetrix chip.
Increased expression of complement C1Q␤ has been
reported previously in sporadic ALS,22 and we extend
that result to the SOD1-G93A transgenic mice (see Fig
4). C1Q␣ and C1Q␤ transcripts were expressed normally and at low levels at 30 and 60 days of age, but
increased rapidly by 90 and 120 days of age (see Fig 4).
The complement regulators clusterin (apoJ), MCP,
Fig 3. The earliest significant change in gene expression in SOD1-G93A transgenic mouse spinal cord is an increase in transcripts
for thymosin B4 and a 21kDa polypeptide under translation control (X06407). At end-stage disease (120 days), alterations in NF
gene transcripts are observed, while glial activation as indicated by an increase in GFAP. Vimentin gene expression is detected with
onset of symptoms at 90 days of age. G93A, lines with squares; NTG, lines with triangles; SOD1, arrowhead.
DAF, and CD59 were not present on the mouse 6500
chip, whereas C3 and complement receptor mRNA expression were detected at low levels that did not vary.
Increased complement biosynthesis by microglia is reported after transient global cerebral ischemia, in Huntington’s disease, and in Alzheimer’s disease.23–25
All three forms of nitric oxide synthase are present
on the murine 6500 chip. nNOS gene expression is
elevated in SOD1-G93A mice at 120 days of age
(1,038 ADI) as compared with SOD1 and nonTg mice
(308 ADI and 431 ADI, respectively), whereas eNOS
(2,086 ADI, 2,985 ADI, and 2,948 ADI, respectively)
and iNOS (888 ADI, 1,563 ADI, and 1,407 ADI, respectively) gene transcripts are unchanged. Probes for
cyclooxygenase-1 (COX-1) and COX-2 are not present
on the chip. COX-2 is increased significantly in symptomatic SOD1-G93A mouse spinal cord.26
Apolipoprotein E
Among the most striking changes in gene expression
occurring in end stage SOD1-G93A spinal cord were
those associated with induction of scavenge pathway
genes for lipid mobilization and protein catabolism.
Apolipoprotein E (ApoE; AA036067) gene expression
was normal until 3 months of age, and then was induced more than fivefold in SOD1-G93A at 120 days
of age, just prior to end-stage disease (Fig 5). ApoD
(L39123) gene expression similarly was increased in
SOD1-G93A mice at 4 months, although to a much
lesser extent; ApoA-I (X64263) and ApoA-II (U35456)
Olsen et al: Gene Expression Profile in ALS Transgenic Mice
733
Fig 4. Markers of microglial activation increase as early as 90 days of age coincident with the onset of symptomatic disease in
SOD1-G93A transgenic mice. Lines with squares ⫽ G93A; lines with triangles ⫽ SOD1, arrowhead.
were detected at low levels but remained unchanged.
ApoE and ApoD are strongly induced in peripheral
nerve as a consequence of nerve damage; ApoA-I and
ApoA-IV accumulate to a lesser extent.27 Immunoblot
analysis of spinal cord extracts from 4-month-old
SOD1-G93A and control SOD1 and nontransgenic
mice indicated that there was an increase in ApoE protein that paralleled the increase in ApoE gene expression observed by microarray analysis (see Fig 5).
As disease develops in the SOD1-G93A mice, loss of
motor axons from peripheral nerves stimulates sprouting and reinnervation by the motor axons remaining.
Sprouting is robust until about 3 months of age, after
which the rapid loss of motor neurons from spinal cord
causes extensive denervation in muscle.7 ApoE is produced by macrophages infiltrating into damaged peripheral nerve and may accumulate to 2 to 5% of the
total extracellular protein, suggesting that it plays a
central role in the mobilization and reutilization of
lipid and cholesterol contained in degenerating myelin
fragments.28,29 However, peripheral nerve regeneration
is apparently unimpaired.30 To determine whether the
absence of ApoE might influence sprouting, gluteus
muscles were harvested from SOD1-G93A and SOD1G93A;ApoE null mice at 90 days of age and stained
with a combined silver–acetylcholinesterase technique
to reveal the fine details of motor endplate innervation
and morphology. No difference was seen in compensatory motor axon sprouting, or in the number of denervated endplates (Table 2).
To examine the role of ApoE induction on disease
severity, the SOD1-G93A locus was crossed into an
ApoE null background. We then compared the course
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of disease in SOD1-G93A and SOD1-G93A;ApoE
null mice littermates from crosses of SOD1-G93A;
ApoE ⫺/⫹ mice that were heterozygous at both loci.
Fewer SOD1-G93A;ApoE null mice were bred than
expected, suggesting either embryonic or perinatal lethality. The distribution of other genotypes in the cross
indicates that the SOD1-G93A and ApoE loci segregate independently. Interestingly, the absence of ApoE
prolonged survival of SOD1-G93A transgenic mice by
about 10 days (Fig 6, Table 3). The same prolongation
of survival was seen in a second cohort of SOD1G93A;ApoE null mice that also were bred by crossing
SOD1-G93A,ApoE⫺/⫹ mice heterozygous at both
loci. Thus, an absence of ApoE appears to confer protection in the disease model; however, the cross does
not rule out the less likely possibility that the prolongation of survival is caused by cosegregation of a second neuroprotective gene contributed by the C57Bl6
parent that is tightly linked to the mouse ApoE locus,
and that therefore is not separated by recombination.
Table 2. Denervation, Sprouting, and Reinnervation in the
Gluteus Muscles of SOD1-G93A and SOD1-G93A;ApoE
Null Mice
Genotype
SOD1-G93A
SOD1-G93A;
ApoE null
Innervated
Sprouts
Denervated
77 ⫾ 6
79 ⫾ 6
4.8 ⫾ 1.3
4.9 ⫾ 2.1
19 ⫾ 6
15 ⫾ 4
Values tabulated are mean ⫾ SEM. Approximately 100 endplates
were scored per muscle.
Fig 6. Survival analysis for the timing to end-stage disease of
SOD1-G93A (lines with triangles) and SOD1-G93A;ApoE
(KO; lines with squares) null mice. The absence of ApoE
causes a slight but significant prolongation of the timing to
end-stage disease.
Fig 5. ApoE transcripts and protein are increased in SOD1G93A transgenic mice at end-stage disease (120 days of age).
The inset shows the hybridization of Affymetrix chip elements
to the probes prepared from SOD1-G93A and nontransgenic
NonTg mouse spinal cord. The average difference intensity is
calculated as the difference in hybridization intensity of the
probe to the exact match oligonucleotides (top element in each
pair) as compared with the mismatch oligonucleotides (bottom
element in each pair). Each gene is probed by 20 pairs of
oligonucleotide elements.
Scavenge Pathways
The strong induction in ApoE and ApoD transcripts
during late-stage disease in the SOD1-G93A transgenic
mice was paralleled by induction of transcripts for a
variety of other proteins involved in lipid or glycolipid
mobilization and protein catabolism. Hexosaminidase
is a multisubunit enzyme that degrades saccharide substrates containing a terminal, ␤-linked, N-acetylated
hexosaminide such as GM2-ganglioside, particular globosides, oligosaccharides, or glycoproteins. The two
subunits of hexosaminidase A (HEXA and HEXB)
combine with a third, small glycolipid transport protein, the GM2 activator protein (GM2A), which acts
as a substrate-specific cofactor for hexosamindase catabolism of GM2-ganglioside. A deficiency of any one of
these proteins leads to storage of the ganglioside, primarily in the lysosomes of neuronal cells, and one of
the three forms of GM2-gangliosidosis whose clinical
phenotype includes juvenile or adult-onset loss of motor neuron viability (OMIM no. 272800, 26880). Both
HEXA(U05837) and HEXB(Y00964) were strongly induced in SOD1-G93A mouse spinal cord beginning at
90 days of age, coincident with the average age of clin-
ical disease onset (Fig 7). Expression of the GM2A
gene remained unchanged.
Expression of brain fatty acid binding protein (BFABP, U04827) declined with age but was induced
strongly in SOD1-G93A during late-stage disease (see
Fig 7). FABPs may act as molecular scavengers, binding oxidized fatty acids produced by oxidative stress
and perhaps induced as a response to lipid peroxidation. Perhaps the induction of B-FABP is a protective
response, as lipid peroxidation is increased in SOD1G93A mouse spinal cord at late-stage disease.31
Expression of a variety of lysosomal proteases important to protein catabolism was also elevated in SOD1G93A mice. Cathepsin D and S gene expression was
strongly induced beginning at 90 days of age, coincident with the average onset of clinical disease (Fig 8,
see Fig 7). The cathepsins are cysteinyl lysosomal proteases that play a role in the catabolism of intracellular
and extracellular proteins. Cathepsin D is present in all
cell types in the brain, whereas cathepsin S is localized
primarily to microglial cells.32
Alterations in Expression of Genes Regulating Metal
Ion Homeostasis
Metal ion homeostasis is regulated through metal uptake, intracellular storage, and utilization. Copper and
zinc are transported into the cell through selective upTable 3. Timing to End-Stage Disease for SOD1-G93A and
Littermate SOD1-G93A;ApoE Null Mice
Genotype
SOD1-G93A
SOD1-G93A; ApoE null
Days
Number
p
133 ⫾ 12
143 ⫾ 7
37
11
—
0.0113
Values tabulated are mean ⫾ SD.
Olsen et al: Gene Expression Profile in ALS Transgenic Mice
735
Fig 7. Induction of scavenge pathways for lipids, glycolipids
and proten catabolism occurs after the onset of symptomatic
disease in SOD1-G93A transgenic mice. G93A, lines with
squares; NTG, lines with triangles; SOD1, arrowhead.
take systems where they are sequestered by specific
chaperones or other low-molecular weight, metalbinding proteins called metallothioneins. Mutations in
the Wilson’s disease copper transporting ATPase
(ATP7B) and Menke’s disease copper-effluxing ATPase
(ATP7A) are associated with multisystem degeneration
caused by excess tissue accumulation of copper. The
ATP7B protein resides in the trans-Golgi network, and
transports copper into the secretory pathway where the
metal is delivered to vesicle-bound ATP7B by the copper chaperone Atox1 for incorporation into ceruloplasmin.33 A parallel pathway in the cytoplasm via the
copper chaperone for SOD (CCS) loads copper into
cu,zn SOD. ATP7B transcripts were reduced twofold
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in SOD1-G93A mice at end-stage disease (120 days of
age), suggesting downregulation of copper transport
into the secretory pathway. Components of the copper
regulatory pathway, including ATP7A, CCS, the
plasma membrane copper transporter CTR, and
ATOX1 (HAH1) were not present on the Affymetrix
chip. Expression of the ZNT-1 zinc transporter
(U17132) was not altered.
Metallothioneins have been proposed to detoxify
metals; to play a role in zinc and copper homeostasis;
to regulate synthesis, assembly, or activity of zinc metalloproteins; and to protect against formation of reactive oxygen species by free metal ions. Targeted disruption of the mouse MT-I and MT-2 genes renders mice
more sensitive to heavy metal toxicity and to oxidative
stress.34 Three metallothionein transcripts were detected in mouse spinal cord (MT-I, MT-III, and MT–
IV; Fig 9). MT-1 expression occurs widely among tissues. In brain it is expressed principally in
astrocytes,35,36 whereas MT-3 expression is restricted
largely to neurons.37 MT expression within cells is inducible by zinc through activation of the metallothionein transcription factor MTF-1. As previously reported,38 MT-I and MT-III gene expression was
increased in SOD1-G93A mice. Both MT transcripts
were induced at end-stage disease in SOD1-G93A
mice but were unchanged in SOD1 mice (Fig 10).
Low expression of MT-IV also was detected in mouse
spinal cord, although it was expressed at similar levels
in SOD1, SOD1-G93A, and nontransgenic mice.
MTF-1 transcripts were not detected in the experiment.
Iron levels in the brain are regulated through uptake
and intracellular storage. Iron is needed as a cofactor
for enzymes in the electron transport chain, for
NADPH reductase activity, for myelination of axons,
and for neurotransmitter synthesis. Yet when released
in unbound form, Fe2⫹ has the potential to catalyze
formation of the strong oxidant, hydroxyl radical, from
hydrogen peroxide via the Fenton reaction. Hydroxyl
radicals can damage proteins, lipids, sugars, and nucleic
acids. Ferritin is the major intracellular iron storage
protein,39 consisting of heavy and light subunits (FTH
and FTL). The FTH and FTL transcripts accumulated
to extraordinarily high levels in diseased SOD1-G93A
mouse spinal cord (see Figs 9 and 10). The FTH signal
reached a level of 60,000 ADI. This saturated the signal on the GeneChip, whereas the FTL signal was induced threefold to an ADI of approximately 15,500.
Accumulation of FTH and FTL transcripts occurred at
120 days of age, at end-stage disease.
Iron is transported into the cell bound to transferrin
via uptake through the transferrin receptor. Transferrin
receptor mRNA (X57349) was low or undetectable in
SOD1 or nontransgenic control mouse spinal cord, but
was similarly induced at 120 days of age in SOD1-
Fig 8. Expression profiles for genes involved in microglial activation, antioxidant defense, electron transport, and ATP synthesis in
SOD1-G93A transgenic mice and controls at end-stage disease.
G93A mice (to ⬃1,200 ADI). Transferrin probes were
not present on the murine 6500 gene chip.
Regulation of ferritin and transferrin receptor levels
occurs at the level of mRNA stability and translation
by binding of iron regulatory protein-1 (IRP-1) or
IRP-2 to an mRNA structure motif called the ironresponsive element (IRE).40 In the absence of iron, the
IRP bind with high affinity to IRE present in the 5⬘
UTR of ferritin mRNA and repress translation. The
level of IRP-1 (X61147) mRNA expression was unchanged (⬃2,300 ADI in the three types of mice);
IRP-2 gene expression was not detected with the Affymetrix probes.
Antioxidant Enzymes, the Electron Transport Chain,
and ATP Synthase
No changes in gene expression profiles were seen for
SOD1 and SOD2 gene transcripts (note that the murine 6500 chip does not detect the abundant human
SOD1 gene transcripts in the SOD1-G93A and SOD1
transgenic mice; see Fig 8). Interestingly, SOD3
(AA071956) transcripts encoding extracellular cu,zn
SOD were detected in brain and were down 90% in
SOD1-G93A spinal cord. No changes were seen in the
expression of transcripts for catalase, different forms of
glutathione peroxidase, or thioredoxin-dependent peroxide reductase. No changes were seen in the expres-
sion of transcripts encoding components of the electron transport pathway or ATP synthase were noted
(see Fig 8), despite the increase in complex I activity
reported previously.41
Discussion
Debate focuses on the pathogenic mechanism in animal models of ALS with at least two views emerging.42,43 The first is that motor neuron disease in the
mutant SOD1 transgenic mice is predominantly a disease of protein or lipid oxidation caused by the excess
generation of reactive oxygen species by mutant cu,zn
SOD. In this view, disease is caused by metal-catalyzed
free radical mechanisms. The second is that disease is
caused by the propensity of mutant cu,zn SOD to agFig 9. Differential induction of genes for metal ion regulation
in SOD1-G93A transgenic mice at end-stage disease (120
days of age).
Olsen et al: Gene Expression Profile in ALS Transgenic Mice
737
Fig 10. Time course of induction of genes important for metal ion homeostasis in SOD1-G93A transgenic mice. G93A, lines with
squares; NTG, lines with triangles; SOD1, arrowhead.
gregate into cytoplasmic inclusion bodies that impair
neuronal function. Of course, both views may be correct because damage by oxygen radicals can cause protein misfolding and consequent aggregation. cu,zn
SOD itself is more highly oxidized in SOD1-G93A
transgenic mice than in controls, perhaps contributing
to the formation of SOD containing cytoplasmic inclusions.44
The changes in gene expression seen in SOD1G93A transgenic mouse spinal cord are consistent with
induction of homeostatic mechanisms in response to
dysregulation of metal ion sequestration. The ferritin-L
and -H genes are induced, as are the MT-1 and MT-3
metallothionein genes, whereas ATP7B transcripts encoding the Wilson’s disease copper transporting
ATPase are reduced. In yeast, MT are important regulators of copper homeostasis, but as isolated from
mammals the MT primarily are loaded with zinc and
cadmium, perhaps because of the low level of free intracellular copper resulting from sequestration by copper chaperones such as CCS and ATOX1/HAH1.
MT-1 gene expression is only weakly induced by copper (it is induced more strongly by cadmium and zinc),
whereas MT-3 gene expression has not been shown to
be inducible by metals. Thus, increased MT gene transcription in SOD1-G93A spinal cord may be an adap-
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tive response to stress rather than a response to an increase in intracellular copper.
Induction of ferritin–H and –L chain gene expression in the SOD1-G93A mice is suggestive of an adaptive response to a rise in intracellular iron. What might
be the source of that iron? Mitochondrial dysfunction
is implied in the SOD1-G93A mice by the mitochondrial pathology that develops with age.4,10,45 Proteins
comprising the electron transport chain represent the
highest density of iron-binding proteins in the cell.
Complex I, for example, contains two Fe2S2 clusters
and four to five Fe4S4 clusters or up to 25mol Fe/mol
carrier protein. In the entire electron transport chain
the ratio is 42 mol Fe/mol carrier protein. Thus, mitochondrial destruction, through releasing iron into the
cytoplasm, may induce ferritin–H and –L gene expression as well as induction of ferritin mRNA translation
through derepression of IRP in the presence of iron.
Free metals (Fe2⫹ or Cu2⫹) can catalyze the formation
of the strong oxidant, hydroxyl radical, from hydrogen
peroxide via the Fenton reaction, leading to oxidation
of proteins, lipids, and nucleic acids. Tissue content of
oxygen radicals is increased in SOD1-G93A mouse spinal cord,16,46 as is the content of oxidized proteins,44
products of lipid peroxidation,31 and protein nitration.47 Thus, the increase in ferritin gene transcripts
may be a protective response intended to limit Fecatalyzed oxidative damage caused by mitochondrial
destruction.
The strong fingerprint of microglial activation seen
in the SOD1-G93A mice suggests that inhibition of
neuroinflammation might be of therapeutic benefit in
this model of ALS. COX-2 is elevated in the transgenic
model and in sporadic ALS spinal cord,26 whereas
COX-2 inhibitors rescue motor neurons in an organotypic model of glutamate toxicity.48 It would seem
worthwhile to evaluate COX-2 inhibitors in ALS, although COX-2 may not be the sole source of prostanoids in diseased or damaged spinal cord.49
Our profiling of gene expression in SOD1-G93A
mouse spinal cord using high-density oligonucleotide
arrays reveals changes in bulk cellular activation consistent with neuroinflammation, metal ion dysregulation,
and induction of scavenge pathways for protein catabolism and lipid mobilization. Clearly, it will be of interest to profile gene expression specifically in vulnerable cells, the spinal cord motor neurons, and extend
the analysis to sporadic and familial ALS tissues.
We thank Carol Himes, Alla Karnofsky, and Tom Vidmar for their
assistance and Joan Valentine, Tracey Roualt, and Tom O’Halloran
for helpful discussions.
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