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Copper-binding-site-null SOD1 causes ALS in
transgenic mice: aggregates of non-native
SOD1 delineate a common feature
Jiou Wang
1
, Hilda Slunt
1
, Victoria Gonzales
1
, David Fromholt
1
, Michael Coonfield
1
,
Neal G. Copeland
3
, Nancy A. Jenkins
3
and David R. Borchelt
1,2,
*
1
Department of Pathology and
2
Department of Neuroscience, The Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA and
3
Mouse Cancer Genetics Program, NCI-Frederick Cancer Research
and Development Center, Frederick, MD 21702, USA
Received June 3, 2003; Revised August 19, 2003; Accepted August 29, 2003
Cu/Zn superoxide dismutase (SOD1), a crucial cellular antioxidant, can in certain settings mediate toxic
chemistry through its Cu cofactor. Whether this latter property explains why mutations in SOD1 cause FALS
has been debated. Here, we demonstrate motor neuron disease in transgenic mice expressing a SOD1 variant
that mutates the four histidine residues that coordinately bind Cu. In-depth analyses of this new mouse
model, previously characterized models and FALS human tissues revealed that the accumulation of
detergent-insoluble forms of SOD1 is a common feature of the disease. These insoluble species include full-
length SOD1 proteins, peptide fragments, stable oligomers and ubiquitinated entities. Moreover, chaperones
Hsp25 and aB-crystallin specifically co-fractionated with insoluble SOD1. In cultured cells, all 11 of the FALS
variants tested produced insoluble forms of mutant SOD1. Importantly, expression of recombinant peptide
fragments of wild-type SOD1 in cultured cells also produced insoluble species, suggesting that SOD1
possesses elements with an intrinsic propensity to aggregate. Thus, modifications to the protein, such as
FALS mutations, fragmentation and possibly covalent modification, may simply act to augment a natural, but
potentially toxic, propensity to aggregate.
INTRODUCTION
Mutations in Cu/Zn SOD1 have been linked to familial
amyotrophic lateral sclerosis (FALS) (1), a degenerative disease
characterized by loss of lower and upper motor neurons in the
motor cortex, brain stem and spinal cord (2). Over 70 different
missense substitutions at more than 50 residues of the 153 amino
acid protein have been described in individuals and kindreds
affected by SOD1-linked FALS (www.alsod.org). A small
number of mutations lead to early translation termination in
the last of the five exons. Although intensely studied, the nature
of the common abnormality by which all these mutations in
SOD1 cause FALS has not been definitively identified. Most
investigators accept the notion that the SOD1 mutations cause
the disease through a gain-of-property mechanism (3–7), but just
what this toxic property is has not be resolved.
One hypothesis has focused on the metal binding properties
of Cu/Zn SOD1. The ‘Cu hypothesis’ holds that mutations
diminish the normal shielding of the Cu cofactor in the
enzyme, enhancing entry of less favored substrates including
H
2
O
2
and ONOO. Reactions between the Cu cofactor and
H
2
O
2
can produce potentially toxic radical species (8–11)
whereas reaction with ONOO can catalyze the covalent
nitration of tyrosine residues (12–14). However, whether these
reactions are central to the toxicity of mutant SOD1 has been
debated (15,16).
Another hypothesis suggests that aggregation of SOD1 may
selectively injure motor neurons. Abnormal inclusions have
been observed in human ALS spinal cords from both familial
and sporadic cases (17–19), in spinal cords from transgenic mice
expressing FALS variants (7,19), and in cytoplasm of cultured
COS cells, or motor neurons, expressing FALS-SOD1 variants
(20,21). Detergent-insoluble forms of mutant SOD1 have been
detected in cultured cells and mice expressing FALS-SOD1
variants (22–24). The principal criticisms of this hypothesis are
that relatively few mutants have been examined in model
*To whom correspondence should be addressed at: Department of Pathology, The Johns Hopkins University School of Medicine, 720 Rutland Ave.,
Room 558, Baltimore, MD 21205, USA. Tel: þ1 4105025174; Fax: þ1 4109559777; Email: drbor@jhmi.edu
Human Molecular Genetics, 2003, Vol. 12, No. 21 2753–2764
DOI: 10.1093/hmg/ddg312
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systems, and in some of the transgenic mouse models, SOD1
positive inclusions in affected spinal cords are relatively rare
(19,24).
To test the Cu hypothesis, we have focused on disease
associated mutations that occur in the Cu-binding site of
SOD1. There are four histidines that coordinately bind Cu
[histidines 46, 48, 63, and 120 (25)], and disease causing
mutations have been described at histidines 46 and 48 (H46R
and H48Q). Experimental mutants of SOD1 that combine these
two mutations can induce motor neuron disease in transgenic
mice (26). To eliminate the Cu binding site, we combined the
two disease-causing mutations at histidine 46 and 48 with two
experimental mutations at histidines 63 and 120 (H63G and
H120G), yielding a protein (SOD1-Quad) that was surprisingly
stable but completely inactive. When we expressed this variant
in mice, via a mutated version of the human genomic fragment
of the sod1 gene (27), we observed motor neuron disease
similar in clinical and pathological appearance to other mouse
models of SOD1-linked FALS. We interpret this outcome as
evidence that motor-neuron specific toxicity does not require a
normally configured active site to produce a protein toxic to
motor neurons. In analyzing diseased tissues from mice
expressing SOD1-Quad, we found evidence of aggregated
forms of mutant protein by both size exclusion filter assay, and
differential detergent extraction and centrifugation. We utilized
the latter approach to compare aggregating SOD1 species in
this new model to four other previously characterized models,
demonstrating that SOD1-Quad acquires characteristics similar
to natural FALS-SOD1 mutants. We also developed cell culture
assays to examine 11 different FALS variants, finding that
misfolding to produce detergent insolubility is a common
feature. We suggest that misfolded and aggregating species of
SOD1 are strong candidates for the toxic species in the disease.
RESULTS
Motor neuron disease and SOD1 aggregates in
H46R/H48Q/H63G/H120G mice
Previous characterizations of SOD1 reactivity with H
2
O
2
and
ONOO have dealt with enzymes where Cu is loaded into its
normal active site (8–11,13,14,28). There are four histidine
residues that coordinately bind Cu, residues 46, 48, 63 and 120
(25). Mutations at histidines 46 and 48 have each been reported
in FALS patients (www.also.org), and mice expressing an
SOD1 with a double mutation at histidines 46 and 48 (H46R/
H48Q) develop motor neuron disease (26). Enzymes harboring
H46R and H48Q mutations alone, or in combination, lack
detectable superoxide scavenging activity (26). Hence, partial
destruction of the Cu-binding site in SOD1 renders the enzyme
inactive, while retaining the capacity to cause motor neuron
disease.
To completely destroy the Cu-binding site, we combined the
H46R/H48Q double FALS mutation with additional mutations
at histidines 63 and 120 (H46R/H48Q/H63G/H120G; the Quad
mutant). Two lines (87 and 125) were identified that developed
typical motor neuron disease, which progressed to hindlimb
paralysis by 8–12 months of age (Fig. 1A and B). The levels of
SOD1-Quad expression that were required to induce disease at
these ages were similar to that of other FALS-SOD1 variants
(Fig. 1D). In a native gel assay of SOD1 activity in spinal cords
of non-transgenic mice (NTg), mice expressing high levels of
wild-type human SOD1 (WT), and mice expressing SOD1-
Quad, no bands corresponding to human SOD1 were noted in
tissue extracts from the SOD1-Quad mice (Fig. 1C). Moreover,
the band of activity corresponding to endogenous mouse SOD1
homodimers was neither diminished in its intensity nor altered
in its migration in the SOD1-Quad mice, suggesting the SOD1-
Quad protein may not dimmerize with the mouse enzyme.
Pathological examination of symptomatic SOD1-Quad mice
demonstrated marked motor neuron loss (by silver impregna-
tion) and dramatically increased glial fibrillary acidic protein
(GFAP) immunostaining (Supplementary Material Fig. S1).
Spinal cords and brain stems from affected mice also showed
conspicuous inclusion body pathology, which was revealed by
immunostaining with ubiquitin antibodies or thioflavin-S
staining (Fig. 2 and Supplementary Material Fig. S2). These
pathologies were absent in cerebral cortex, cerebellum, or other
parts of the forebrain. As controls, age-matched wild-type
SOD1 transgenics and non-transgenic littermates remained
healthy and free of abnormal inclusions. Although immunos-
taining with SOD1 antibodies failed to detect obvious
inclusions (data not shown), by size exclusion filter assay
aggregated species of SOD1-Quad were abundant in homo-
genates of spinal cord and brain stem tissues from paralyzed
SOD1-Quad mice (Fig. 2E). These structures were detected in
cerebellum and cerebral cortex, but at much lower levels.
Figure 1. Transgenic mice expressing H46R/H48Q/H63G/H120G (Quad)
mutant SOD1 develop motor neuron disease. (A) Hindlimb paralysis in
SOD1-Quad mice (line 125). (B) SOD1-Quad mice (lines 125 and 87) deve-
loped complete hind limb paralysis at the age of 9–12 months, and 8–11
months, respectively. (C) Mice expressing SOD1-Quad do not produce excess
superoxide scavenging activity. Only activity associated with endogenous
SOD1 equal to that in nontransgenic (NTg) control mice is detected; 100 mg
of protein from spinal cord homogenates of 2-month-old mice were tested for
SOD1 activity in the native gel assays. (D) Disease progression is correlated
with protein expression levels in different lines of SOD1-Quad mice; the higher
the expression level (line 87>125>121), the earlier the onset of paralysis. A
similar dose dependency for disease onset in mice expressing other SOD1
mutants including, the H46R/H48Q mutant (e.g. line 139>58), has been noted
previously (26). Protein levels were determined by western blotting of spinal
cord homogenates from 2-month-old mice.
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Overexpressing WT SOD1 in mice did not induce formation of
these structures [Fig. 2E and Wang et al. (24)].
SOD1 monomers, fragments, and SDS-resistant
oligomers are components of detergent-insoluble
complexes in FALS mice and human patients
The appearance of motor neuron disease and aggregated
species of mutant SOD1 in the SOD1-Quad mice suggested to
us that aggregation of the mutant protein may be the key to
disease. To characterize further the SOD1 species accumulating
in the symptomatic mice, we used differential detergent
extraction and high speed centrifugation to enrich for forms
of the mutant protein in non-native structure (see Materials and
Methods). Proteins insoluble in non-ionic detergent (P2) and
SDS (P3) were then solubilized by boiling in the SDS sample
buffer before being analyzed by western blotting with two
polyclonal antibodies: hSOD1 antibody, a peptide antiserum
binding to amino acids 24–36 (not conserved between mouse
and human SOD1); and m/hSOD1 antibody, a peptide
antiserum recognizing amino acids 124–136 (completely
conserved between mouse and human protein; Fig. 3A).
Spinal cord extracts from five different strains of FALS mice
were examined; G37R (line 29), G85R (line 164), G93A (line
1—high expression), H46R/H48Q (line 139), and SOD1-Quad
(line 125). As compared with either non-transgenic mice, or
mice expressing high levels of wild-type human SOD1 (line
76), spinal cords from the paralyzed mutant mice contained
large amounts of detergent-insoluble SOD1 species (Fig. 3A).
However, the majority of both wild-type and mutant SOD1,
from all animals, was soluble in non-ionic detergent [Fig. 3B;
the soluble (S1) fraction contained 600 mg total protein of
which only 1 mg was analyzed by SDS-PAGE; the P2 fraction
contained 50 mg of total protein of which 3 mg was analyzed
by immunoblot—see legend to Fig. 3].
Although the predominant SOD1 species solubilized after
boiling in SDS consisted of full-length SOD1 monomers (open
arrow), there were distinct immunoreactive peptide fragments
(solid arrow) and high molecular weight (solid arrowhead)
species in these insoluble fractions (Fig. 3A). Notably, the G85R
variant always migrates, in SDS–PAGE, slightly faster than
wild-type SOD1 or other FALS variants (4). This characteristic
was also evident in 28 and 38 kDa species detected in the G85R
mice. Interestingly, detergent-insoluble, SOD1-immunoreactive
species in the spinal cords of the paralyzed mice displayed
patterns that were not entirely identical when examined by
immunoblot with the two different antibodies. The hSOD1
antibody recognized, with variable abundance, a set of 7 kDa
fragments that were particularly abundant in the H46R/H48Q
mice. Additionally, there were several species that appeared to be
slightly smaller than the full-length protein; these fragments
were not recognized by the m/hSOD1 antibody, suggesting that
these fragments lack C-terminal residues. By contrast, the m/
hSOD1 antibody specifically recognized several fragments in the
range of 9–15 kDa; these fragments were particularly abundant
in spinal cords from the G85R and H46R/H48Q mice. Both
antibodies recognized a pair of prominent SOD1 species
(marked by asterisks), which have relative molecular masses
of 28 and 38 kDa. Note that the hSOD1 antibody detected, at
lower abundance, additional species just below these 28 and
38 kDa species and since these were not recognized by the
m/hSOD1 antibody we infer that these molecules are truncated
at the C-terminus.
Next, we examined spinal cord tissues from an FALS patient
known to harbor an SOD1-A4V mutation, together with two non-
SOD1 FALS patients, 12 sporadic ALS patients, and three
controls. Only tissue homogenates from the patient harboring
the A4V mutation showed enhanced levels of detergent-insoluble
SOD1 monomers, fragments, and high molecular weight species
(Fig. 3C). By contrast, little or no insolubleSOD1 was detected in
spinal cord tissues from patients with sporadic and non-SOD1
familial ALS (Supplementary Material Fig. S3).
To determine whether covalent ubiquitination of any of these
SOD1 species might be contributing to the complexity of the
SOD1-immunoreactive profiles seen in immunoblots probed
Figure 2. Fibrillar inclusions and SOD1-containing aggregates in symptomatic
SOD1-Quad mice. (Aand B) Immunostains of spinal cords with antibodies to
ubiquitin revealed inclusions (open arrow) and degenerating cell bodies/neuro-
pil (solid arrow) in SOD1-Quad transgenic mice (A) as compared with NTg lit-
termate controls (B). (Cand D) Sagittal sections through the juncture of brain
stem and spinal cord stained with thioflavin-S revealed long fibrils in sympto-
matic SOD1-Quad mice (C) as compared with NTg controls (D). Scale bars:
100 mm. (E) Size exclusion filter trapping and immunodetection (described in
Materials and Methods and 24) demonstrated high molecular weight forms
of SOD1-Quad in spinal cord and brain stem of symptomatic mice. The same
amount of protein from transgenic mice overexpressing wild-type SOD1 or
NTg mice was filtered and immunoblotted as controls.
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with SOD1 antibodies, we probed immunoblots of the SDS
insoluble pellet (P3) with monoclonal antibodies to ubiquitin.
In all five of the SOD1 FALS models we examined, we found
that the insoluble pellets contain ubiquitinated entities includ-
ing two species with apparent molecular mass of mono and di-
ubiquitinated mutant SOD1 (Fig. 4A). To confirm whether
ubiquitinated SOD1 was indeed present in these fractions, we
dissociated the P2 pellet fraction by boiling in 2% SDS, and
then diluted the solubilized protein to 0.2% SDS and immuno-
precipitated SOD1 protein with the m/hSOD1 antibody.
Immunoprecipitated proteins were then subjected to immuno-
blot with ubiquitin antibody (Fig. 4B), noting the detection of
two prominent proteins with relative molecular masses of 28
and 38 kDa (Fig. 4B), which are roughly the predicted sizes for
mono- and di-ubiquitinated SOD1 proteins.
FALS mutations promote non-native folding to
form detergent-insoluble complexes
As noted above, and from our previous work (24), high-
molecular weight, detergent-insoluble, mutant SOD1 is most
abundant in tissues that develop visible pathology: brain stem
and spinal cord. In this setting, it is impossible to determine
whether the formation of these altered species of SOD1 is an
inherent property of the protein or consequence of the disease
process. To address this issue, we studied the detergent
solubility of different SOD1 mutants expressed at high levels
in cultured cells. Wild-type SOD1 and 11 FALS mutant cDNAs
[A4V, G37R, G41D, H46R, H48Q, H46R/H48Q, G85R, G93C,
I113T, Quad, and FS126 (frame shift 126–stop 131)] were
inserted into the pEF-BOS expression vector (4) before transient
transfection of human HEK-293 cells. Northern analysis of the
transfected cultures showed similar SOD1 mRNA levels (data
not shown) for each variant. As expected, the accumulated
steady-state levels of the different mutants varied according to
the intrinsic half-life of the individual proteins (Fig. 5A) (4,29).
Twenty-four hours after transfection, cells were harvested and
differentially extracted as described in Materials and Methods.
The non-ionic detergent-insoluble pellets (P2) and supernatant
(S1) were separated, and SOD1 levels were quantified by
western blotting with the hSOD1 antibody and
125
I-labeled
secondary antibodies, followed by Phosphorimager detection
and quantification (Fig. 5A and B). All of the cells transfected
with FALS variants contained forms of mutant SOD1 that were
insoluble in non-ionic detergents (Fig. 5A) and SDS (data not
shown). Notably, however, wild-type human SOD1 was barely
detectable in the insoluble fractions, even though the wild-type
protein accumulated to the highest steady-state level in the
soluble fraction. Comparing the relative amounts of SOD1 in
the pellet and supernatant fractions, we found that each mutant
has a statistically higher aggregation potential than wild-type
SOD1 (Fig. 5B). Interestingly, the A4V, G41D, G85R and
FS126 variants, all short-lived (4,29), produced more detergent
insoluble species than any of the other mutants. The FS126
variant, the only truncated protein, appeared to be the most
aggregation prone. These results strongly suggest that FALS
variants are prone to adopting non-native conformations that
lead to detergent-insoluble structures, and that this property is
intrinsic to the mutants and does not require extrinsic events
such as may occur in degenerating and diseased tissues.
Intrigued by the appearance of detergent insoluble fragments
of SOD1 in the mouse and human tissues (Fig. 3), we used
the cell culture transfection system to ask whether these
molecules may also be inherently prone to adopt non-native
Figure 3. Accumulation of detergent-insoluble SOD1 monomers, fragments, and high molecular weight species in spinal cords of affected mice and human
patients. (A) Mutant SOD1 from the spinal cords of affected mice fractionated in the detergent-insoluble pellet fraction (P3). Spinal cords were differentially
extracted and sedimented in non-ionic detergent and SDS as described in Materials and Methods. P3 is the final pellet after sequential extraction in non-ionic
detergent and SDS. Fractions were separated by tricine-peptide SDS–PAGE. Western blotting was done with the hSOD1 antibody, which binds to residues 24–
36 specific to human SOD1, or the m/hSOD1 antibody, which recognizes residues 124–136 common to both the human and mouse protein. Bound primary anti-
bodies were detected by protein A-horseradish-peroxidase and chemiluminescence. In mice expressing wild-type human SOD1 and in normal NTg controls, all
SOD1 was fully soluble in detergent. By contrast, insoluble species of SOD1 were abundant in symptomatic mutant mice. SOD1 monomers (open arrow), frag-
ments (solid arrow), and high molecular weight species (solid arrow head) are indicated. Apparent dimers (asterisk) and trimers (double asterisk) are noted. (B) The
majority of SOD1 in both mutant and control mice is fully soluble in detergent (S1). For every 600 mg of total protein in the supernatant S1 fraction, roughly 200 mg
of protein fractionated in P2 and 50 mg of protein fractionated in P3. One microgram of S1 protein was loaded in (B), compared with 3 mg of P3 protein loaded in
(A). With the same blotting condition and antibody titration, the exposure time for (A) is three times that for (B), which was <5s. The immunoblot in (B) was
probed with the m/hSOD1 antiserum. (C) The spinal cord of a patient with the A4V mutation contains SOD1 monomers, fragments and oligomers that are inso-
luble in non-ionic detergent (P2). A representative age-matched non-ALS control case was examined in parallel. Immunoblots were probed with hSOD1 orm/
hSOD1 antiserum as described in (A).
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conformations. We examined the aggregation potential of
several experimental variants of wild-type SOD1 truncated at
the N- and C-terminus to approximate the sizes of fragments
observed in mouse and human tissues. Although the steady-
state levels of each recombinant fragment were much lower
than wild-type protein, each fragment produced detergent
insoluble species (Fig. 6). These results support the notion that
structural elements in SOD1 have an unusually high potential
to aggregate, and that in the wild-type protein these elements
must be effectively ordered in the native structure to avoid
aggregation.
Up-regulation of Hsp25 and aB-crystallin in
symptomatic SOD1 mutant mice
Because molecular chaperones are crucial participants in
protein folding, degradation and aggregation, we used a panel
of antibodies against heat shock proteins to determine whether
any of the known chaperones were up-regulated in the spinal
cords of FALS mice. Using non-ionic detergent extraction
protocols described above, we also asked whether any of these
chaperones were also associated with the detergent-insoluble
SOD1 complexes (Fig. 7). Although we found no appreciable
differences between control and symptomatic mice in analyzing
the levels and solubility of Hsp40, Hsp60, Hsp70 and Hsp90,
we observed a dramatic up-regulation in the levels of Hsp25. In
affected mice from five different SOD1 mouse models, the
level of Hsp25 was elevated in both the soluble and insoluble
fractions. In the soluble fraction, Hsp 25 appeared as a doublet
band, whereas in the pellet fraction only a single species of
HSP25 was detected (Fig. 7A). Another small chaperone
protein, aB-crystallin, was found to be significantly up-
regulated in the mutant mice, with the majority of the induced
component being localized to the detergent-insoluble fraction.
Because these two proteins are known to form highly organized
structures (30), we do not know whether they are integral
components of SOD1 aggregates. However, both chaperones
co-fractionated with SOD1 aggregates in the SDS insoluble
fraction P3 (data not shown).
Immunocytochemical examination of paralyzed mice loca-
lized Hsp25 to both neuronal and glial cells and aB-crystallin
to oligodendrocytes. In neither case, were neuropil inclusions
(recognized by ubiquitin antibodies) specifically marked (data
not shown). Hence, we are uncertain as to whether these
chaperones are directly binding protein contained in large
visible inclusions, or protein organized in smaller structures not
visible by light microscopy.
DISCUSSION
Our demonstration of pathologic and behavioral symptoms of
motor neuron disease in strains of mice expressing SOD1-Quad
proves that disease can be induced by mutant forms of SOD1
lacking the histidine residues that coordinately bind Cu in the
active site. The pathologic features of disease in the SOD1-
Quad mice were identical to that of other FALS-SOD1 mice;
these features include ubiquitin-immunoreactive inclusions,
thioflavin-S positive inclusions, robust astrogliosis, and loss of
motor neurons. As we have previously demonstrated in other
FALS-SOD1 mouse models (24), diseased tissues contained
high molecular weight forms of SOD1-Quad that could be
trapped by size exclusion filter trap. Similarly, behavioral
phenotypes were identical; initial symptom of hindlimb
weakness progressing to hindlimb paralysis before involvement
of forelimbs. Thus by all criteria we have used to characterize
FALS-SOD1 mice, the SOD1-Quad mice appear to have
authentic motor neuron disease.
Have we definitively disproved the Cu hypothesis?
The Cu hypothesis is an umbrella term that covers hypotheses
that involve the ability of SOD1 to covalently nitrate tyrosine
residues in protein (12) or to react with H
2
O
2
to produce radical
species (8). These toxic reactions have been described for
Figure 4. High molecular weight ubiquitinated entities, including ubiquitinated SOD1, are enriched in the detergent-insoluble fractions extracted from spinal cords
of mutant SOD1 mice. (A) Spinal cord homogenates from symptomatic mutant SOD1 mice and normal control mice were detergent extracted and western blotted
with anti-ubiquitin antibodies. High molecular weight, ubiquitin-immunoreactive entities fractionated in the SDS-insoluble fractions (P3) from the spinal cords of
symptomatic mutant mice as compared with mice expressing wild-type SOD1 and NTg controls (left). The majority of monomeric ubiquitin was completely
solubilized by non-ionic detergent (S1; right). Prominent 28 and 38 kDa bands, with a slight down-shift in size from the G85R mice, were found in samples from
all mutant mice. (B) The non-ionic detergent pellets (P2) of spinal cord homogenates were adjusted to 2% SDS and boiled for 10min. Then the diluted samples
were immunoprecipitated with the m/hSOD1 antibody and western blotted with the anti-ubiquitin monoclonal antibody. The two distinct protein species at 28 and
38 kDa seen in (A) clearly contain SOD1. The relative size of the two proteins is consistent with mono- and di-ubiquitinated SOD1.
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wild-type SOD1, with evidence that FALS mutations augment
these activities. The nitration chemistries attributed to both
wild-type SOD1 (28) and FALS variants (13,14) by other
investigators were described in enzymes with intact active sites.
There is no evidence that inactive enzymes can catalyze these
reactions.
Similarly, the peroxidative activity of SOD1 was originally
described for wild-type SOD1 with Cu bound in the active site
(9,31). As was the case for the nitration reaction, mutations distal
to the active site were found to augment the peroxidative reaction
(8,10). Moreover, enzyme harboring the H48Q mutation within
the active site was found to retain the peroxidative chemistry.
Although altering one of the histidine residues responsible for
binding Cu is not sufficient to eliminate this chemistry, there is
no evidence that enzyme lacking all four of the histidine residues
necessary for the coordinate binding of Cu could perform the
peroxidative chemistry.
When one sums all of the studies that have tested the Cu-
hypothesis by experimentally lowering the level of Cu binding
in the active site [the present study, the work by Subramaniam
et al. (15) to delete CCS and diminish the loading of Cu into
mutant SOD1, and our previous study of mice expressing a
double histidine mutant (H46R/H48Q) (26)], then the weight of
evidence does not favor the hypothesis as it pertains to Cu
correctly loaded in the Cu-binding pocket. It remains to be
determined whether Cu bound to other parts of the protein (the
Zn site or elsewhere) could produce SOD1 enzymes that are
capable of generating the same type, or some other type, of
toxic chemical reaction (16).
Are aggregated forms of mutant SOD1 the
toxic species?
We have examined, in great detail, five different mouse models
of SOD1-linked FALS and found that disease is always
associated with the accumulation of high molecular weight
forms of mutant SOD1 that are retained by size exclusion
filtration [Fig. 2 and (24)]. We have also found that the tissues
that are most devastated in mice (brain stem and spinal cord)
contain non-native, detergent insoluble, species of mutant
protein. We have never observed full-length wild-type protein
to possess these properties. Further, in a cell culture assay, we
have found that of 11 different FALS mutants tested, all were
found to be inherently prone to adopt non-native, detergent-
insoluble, structures. In many other neurodegenerative disease
Figure 5. Adopting structures insoluble in detergent is an inherent property of
FALS SOD1 variants. (A) Images of representative immunoblots of detergent-
soluble and insoluble fractions from HEK 293 cells transiently transfected with
wild-type and mutant SOD1 constructs. The levels of SOD1 in the pellets (P2)
and supernatants (S1) were measured by immunoblots using the hSOD1 anti-
serum and
125
I-labeled secondary antibodies followed by Phosphorimager
detection. Although highly expressed, wild-type SOD1 was not detected in
the insoluble fractions. In contrast, all tested mutants accumulated, to varying
degrees, in insoluble fractions (P2). (B) The relative ratio of detergent-insoluble
to soluble SOD1 in pellets and supernatants was used to calculate the aggrega-
tion potential of tested variants. The data represent meansSEM of nine dis-
tinct transfection experiments. Significance levels were determined by paired
Student’s t-test. All mutants were statistically different from wild-type SOD1;
*significance of difference between wild-type and FALS variants P<0.02;
**significance of difference between A4V, G41D, and G85R variants and all
other variants except FS126 P<0.04; ***significance of difference between
the FS126 variant and all the other variants P<0.02.
Figure 6. Peptide fragments of SOD1 form detergent-insoluble structures. In
addition to an authentic FALS truncation, FS126, we generated the following
variants: 1, Frag(1–75), C-terminally truncated ending at the K75 residue in
the large loop domain; 2, Frag(38–153) and Frag(49–153), which are two
N-terminally truncated peptides that start at residue L38 and E49 located at
the ends of the third and fourth beta strands, respectively. The fragments,
together with controls, were transiently expressed by the pEF-BOS vector in
HEK 293 cells. After non-ionic detergent extraction, P2 and S1 fractions
were analyzed by western blotting with the hSOD1 antibody for N-terminal
fragments and controls (lanes 1–4) and the m/hSOD1 antibody for C-terminal
fragments and controls (lanes 5–8). The relative position of SOD1 wild-type
monomers (open arrow), peptide fragments (solid arrow), and oligomers
(solid arrowhead) are indicated. In addition to FS126, all expressed wild-type
peptide fragments were preferentially enriched in the non-ionic detergent pellet
fractions (*next to lane 4; and **next to lanes 7 and 8).
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settings, loss of detergent solubility by a given protein or peptide
is a hall mark of aggregation. In prion disease a distinguishing
feature of the disease-specific isoforms of prion protein is loss of
detergent solubility (32). Similarly, aggregated forms of the
Alzheimer’ b-amyloid peptide become insoluble in detergents
(33,34). In Parkinson’s disease, a-synuclein acquires detergent
insolubility (34,35) and in Alzheimer’s disease and Fronto-
temporal dementia with Parkinson’s the tau protein adopts
detergent-insoluble conformations (34,36,37). A similar change
in solubility occurs in protein harboring expansions of
polyglutamine tracts such as occurs in Huntington’s disease
(38). In each of these cases, the appearance of detergent
insoluble entities is associated with the pathologic accumulation
of aggregated forms of the particular protein.
Although we have not been able to visualize large SOD1-
immunoreactive aggregates in the FALS mice with our SOD1
antibodies, we have noted that affected tissues contain large
amounts of thioflavin-S positive inclusions. Thioflavin-S is
thought to be relatively specific for b-sheet structures and will
recognize b-amyloid peptide aggregates and aggregated forms
of tau protein (39,40). The abundance of detergent insoluble
forms of SOD1 in the brain stem and spinal cord of mice that
show thioflavin-S positive inclusions leads us to believe that
aggregated forms of mutant SOD1 are one of the targets for this
dye in tissue sections from the FALS mice.
Features of the SOD1 FALS mouse models also implicate
aggregation in the toxic process. Studies of b-peptide and poly-
glutamine protein aggregation have demonstrated that aggrega-
tion is a slow progress where nucleation and seeding is the
slowest component of the process, which is highly influenced
by the concentration of the monomeric species (41–44). In
studies of several lines of transgenic mice expressing varied
levels of SOD1-G37R (6) and SOD1-H46R/H48Q (26), a strong
correlation between the age of onset and the expression level
of mutant proteins was observed. Moreover, in the G37R (6),
G93A (3), H46R/H48Q (26) and SOD1-Quad mice, hyper-
expression of the mutant protein was required to induce the
disease. Although sometimes criticized as a flaw of these models,
the requirement for high-level expression fits well with the notion
that aggregation is crucial; at lower levels of expression the
thermodynamics of the aggregation process would preclude any
appreciable nucleation before mice reach their natural life
expectancy. It is noteworthy that mice expressing the G85R
variant develop disease, accompanied by aggregated species of
mutant SOD1, when the steady state levels of protein are much
lower—equal to or less than endogenous mouse SOD1 (7,24).
Our demonstration that G85R variant has one of the highest
aggregation potentials appears to explain how this mutant
appears to possess a greater toxicity per unit of protein than
other FALS variants tested in mice.
In our view, there is much evidence that SOD1-FALS shares
features common to other neurodegenerative diseases where the
aggregation of specific proteins is a pathologic hallmark.
However, just what the toxic species is in these protein
aggregation diseases has been hotly debated (reviewed in 45).
Are the large aggregates the toxic species, or are there small
oligomeric structures that are the real culprits? We note that, in
every mouse model we have looked at, aggregated forms of
mutant SOD1 increase in abundance as symptoms worsen (24).
However, it remains possible that the large aggregates are
biomarkers for another species of toxic protein whose detection
is more elusive.
In our study of heat shock response in FALS mice, we noted
that Hsp25 and aB-crystallin were dramatically induced in the
spinal cords of affected mice but that antibodies to these
proteins did not decorate the large ubiquitin-positive inclusions
that are prominent in affected spinal cords of the FALS mice
(data not shown). It is possible that these chaperones
are associated with much smaller SDS-insoluble structures;
these small heat shock proteins are known to bind to proteins
Figure 7. Elevated levels of Hsp25 and aB-crystallin in the spinal cords of symptomatic mutant SOD1 mice. (A) Spinal cord homogenates from symptomatic
mutant SOD1 mice and normal control mice were extracted in non-ionic detergent before immunoblot analysis with antibodies specific to Hsp25. Substantially
elevated levels of Hsp25 were observed in spinal cords from all mutant SOD1 mice, but not in cords from mice expressing wild-type SOD1 or from NTg controls.
Hsp25 was up-regulated in both supernatant and detergent-insoluble pellet fractions. (B)aB-crystallin was also found to be significantly up-regulated in the symp-
tomatic mutant mice; however, most of the elevated aB-crystallin was located in the detergent-insoluble fractions.
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in non-native conformations (46). The cellular pattern of
staining by antibodies to Hsp25 and aB-crystallin suggested
that non-neuronal cell types, astroglia in the case of Hsp25 and
oligodendrocytes in the case of aB-crystallin, were the major
sources of these proteins. Hence, from these data we could
argue that something other than a large aggregate has induced
a response in non-neuronal cells.
One of the attractive aspects of the Cu-hypothesis was the
notion that the gained-property (or activity) by SOD1 when
carrying FALS mutations need not involve the acquisition of
some entirely new function. Because wild-type SOD1 could, at
low levels, catalyze toxic peroxidative and nitration activities
(9,10,28), the disease associated mutations need only augment
these natural secondary activities for the protein to acquire
toxicity (8,12). Here, we demonstrate, through study of defined
peptide fragments of wild-type SOD1, that there are elements
in SOD1 that are prone to induce aggregation. Hence, once
again we could argue that the FALS mutations simply augment
a natural, potentially toxic, feature of the protein.
Ubiquitin inclusion pathology in FALS mice
The accumulation of ubiquitinated protein inclusions are
hallmark pathologies of many neurodegenerative diseases
(reviewed in 47). The appearance of ubiquitinated inclusions
has been construed as evidence that there may be diminished
proteasome function. A direct test of this possibility in a cell
culture paradigm demonstrated that cells containing large
aggregates of polyglutamine protein have lower proteasome
function (48). We and others have noted that ubiquitin
immunoreactive inclusions are frequently observed in spinal
cords of paralyzed FALS mice (6,7,19,26). Our results indicate
that most of the accumulated ubiquitin-immunoreactive
material in the affected tissues of our mice fractionates in the
detergent insoluble fraction (Fig. 4A). Some of this ubiquitin is
conjugated to SOD1 protein (Fig. 4B), and thus it is possible
that the ubiquitin-immunoreactive inclusion pathology seen in
the mouse models reflects the abundance of ubiquitinated
SOD1 in aggregates, which are likely to be poorly degraded by
the cell. Whether the appearance of the large ubiquitinated
inclusions reflects a general dysfunction of the ubiquitin/
proteasome system is unclear.
Why do FALS variants of SOD1 tend to aggregate?
One simple mechanism could involve interactions between
specific domains exposed only when non-native structures are
adopted. An example of this type of interaction has been
termed domain swapping (49,50). If FALS mutations were to
heighten spontaneous unfolding of the protein, then alternative
conformations could form oligomeric species. We have noted
that the distribution of disease-causing mutations is not entirely
random. Mutations are most preponderant at residues con-
served across many phyla and occur at a much higher
frequency in b-strand domains (Wang and Borchelt, unpub-
lished data), suggesting that these mutations may destabilize
native structures. Consistent with this notion, Rodriquez et al.
(51) reported that 14 FALS mutants have decreased thermal
stability, a property augmented when Cu and Zn were removed
(52). Thermal stability was also eroded by eliminating the
single disulfide bond in the protein (53). In an unfolded state,
interactions between hydrophobic b-strands could occur
between two individual molecules rather than within individual
molecules in a domain-swapping type of interaction (49,50). In
such a scenario, highly stable oligomeric molecules could form
(Fig. 8).
It is possible that ubiquitination or fragmentation of SOD1
increases its potential to form aggregating structures.
Ubiquitinated forms of mutant SOD1 were detected in the
detergent-insoluble fraction of affected spinal cords from each
of the FALS mouse models tested. However, in our cell culture
assay, ubiquitinated forms of SOD1 were less prevalent in the
detergent insoluble fraction. Thus, how ubiquitination may
affect solubility is not clear. By contrast, it appears that
fragmentation can certainly change the solubility of the protein.
The FS126, truncation, variant was the most aggregation-prone
of all variants tested. Experimentally truncated variants of wild-
type SOD1 likewise demonstrated very high aggregation
potential. Therefore it is possible that fragmentation of mutant
SOD1 may actually potentiate aggregation.
Does aggregation potential correlate with a clinical
aspect of SOD1-linked FALS?
In studies of SOD1-linked FALS, it has been noted that disease
onset is highly variable, even within a particular kindred, but
disease severity (as indicated by duration) appears to be more
predictable by the location of the mutation (54). However, with
the exception of the A4V and D90A mutations, the number of
affected individuals with a given mutation is usually quite
small. It has been noted that patients harboring the A4V
mutation tend to have very short durations (<2 years) (55–57),
suggesting a more severe mutation. Likewise, patients harbor-
ing the FS126 mutation also show short duration (3 years)
(58). Both of these mutants show very high aggregation
potential in our cell culture assay. However, disease of short
duration has also been noted in patients with the H48Q (59)
and I113T (54–57) mutations, variants that appear to have a
lower aggregation potential in our cell culture assay. Notably,
the measured half-lives of the H48Q and I113T variants are
substantially longer than that of the A4V and FS126 variants
(4,29). We note that, in our cell culture assay, the strongest
correlate to aggregation potential was the measured half-life of
the individual variant. The FS126 truncation variant, which has
the shortest recorded half-life (<5 h) (29), displayed the highest
aggregation potential. Other short-lived variants, including the
A4V, G41D and G85R variants, along with the FS126 variant,
clearly segregated into a distinct group of proteins that were
much more prone to form detergent-insoluble structures
(Fig. 5). In previous studies, we have established that these
four variants exhibit the shortest half-lives when expressed in
cultured cells (29,60). If the accumulation of mis-folded,
aggregating, SOD1 is a key pathologic process, then disease
severity may be due to a complex interaction between the
half-life of the protein and its potential to aggregate. What is
most striking to us is that proteins with relatively short half-
lives have the greatest aggregation potential. Hence, it becomes
easier to explain how these unstable proteins could possess
toxicity that is equivalent to the longer-lived variants.
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CONCLUSIONS
We demonstrate that an experimental variant of SOD1, lacking
all crucial residues for the coordinate binding of Cu, induces an
ALS-like disease in mice that is indistinguishable from the
disease phenotypes of mice expressing natural FALS variants
of SOD1. Disease in these mice, as well as all other models of
FALS mice expressing human SOD1, is accompanied by the
accumulation of high-molecular-weight SOD1 aggregates, and
non-native, detergent-insoluble species of mutant protein. In all
of these respects, the SOD1 models of FALS bear striking
resemblance to other neurodegenerative diseases where the
accumulation of mis-folded and aggregating protein culprits
features in the pathogenesis of disease.
MATERIALS AND METHODS
Transgenic mice, human tissues and cell
culture models
The H46R/H48Q/H63G/H120G mutation was engineered into
a 12 kb fragment of human genomic DNA, encompassing the
entire SOD1 gene (27), by PCR-based oligonucleotide primer-
directed mutagenesis. Each coding exon and at least 50 bp of
intronic sequences on either side of each exon were
subsequently verified by sequence analysis before injection
into mouse embryos (C3H/HeJ C57BL/6J F2). Transgenic
founder mice were identified by PCR amplification of DNA
extracted from tail biopsies. Lines were maintained by crossing
transgenic males to non-transgenic (C57BL/6J C3/HeJ F1)
females (Jackson Laboratories, Bar Harbor, ME, USA). All the
other lines of SOD1 transgenic animals used in this study have
been previously characterized; the G93A variant [B6SJL-
TgN(SOD1-G93A)1Gur; Jackson Laboratory, Bar Harbor,
ME, USA], the G85R variant (line 164) (7), the G37 variant
(line 29) (6), the H46R/H48Q variant (lines 139 and 58) (26),
and the wild-type protein (line 76) (6). The animal use protocol
was approved by the Animal Care and Use Committee of The
Johns Hopkins Medical Institutions.
Human post-mortem spinal cord tissues from a patient
harboring the A4V variant, two familial ALS patients with
unknown causes, 12 patients with sporadic ALS, and three
control cases were obtained from Johns Hopkins Alzheimer’s
Disease Research Center (n¼12) and J. Rothstein at Johns
Hopkins University, School of Medicine (n¼6). HEK 293
cells were transfected with expression plasmids encoding
engineered human SOD1 cDNAs. All point mutations and
fragments were generated based on PCR strategies and expressed
by the pEF-BOS vector (61); many of the constructs used here
have been described previously (4). Disease mutant FS126
comprises a 2bp deletion at codon 126 resulting in a shift in
reading frame and termination five amino acids downstream
from residue 125 (58). Confluent (90%) HEK 293 cells, cultured
by standard procedures in 60 mm wells, were transfected by
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and
harvested 24h later for analyses as described below.
Differential detergent extraction and centrifugation
Tissues and cultured cells were homogenized in 1TEN
(10 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0, and 100 mM
NaCl); (for tissues, volume : weight ¼10 : 1; for 60 mm culture
dishes, 100 ml lysis buffer per well). Tissue homogenates or cell
lysates were mixed at 1 : 1 with buffer A [1TEN; 1% Nonidet
P40; proteinase inhibitor cocktail 1 : 100 dilution (P 8340,
Sigma, St Louis, MO, USA)]. The mix was sonicated [50%
output for 30 s with a probe sonicator (70 W; TEKMAR,
Cincinnati, OH, USA)] and then centrifuged at >100 000gfor
Figure 8. Hypothetical mechanism of mutant SOD1 aggregation by a domain-swapping mechanism. The asterisk denotes the positions of FALS mutations. Each
arrow represents a b-strand domain of SOD1. The dashed arrows are b-strands contained in portions of the protein deleted by certain FALS-associated mutations.
Human Molecular Genetics, 2003, Vol. 12, No. 21 2761
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5 min to separate supernatant S1 and pellet P1 in a Beckman
Airfuge. The P1 pellet was washed with buffer B (1TEN;
0.5% Nonidet P40) by sonication (50% for 30 s), and
centrifuged at >100 000gfor 5 min to obtain pellet P2, which
was resuspended in buffer C (1TEN; 0.5% Nonidet P40;
0.5% deoxycholic acid; 0.25% SDS). The P2 fraction was
further extracted by sonication (50% for 30 s) and centrifuga-
tion (>100 000gfor 5 min) to sediment P3, which was
resuspended by buffer D (1TEN; 0.5% Nonidet P40; 0.5%
deoxycholic acid; 2% SDS).
Western blotting and immunoprecipitation
Proteins were fractionated by SDS–PAGE on either 8–16%
Tris-glycine or 16.5% tricine–peptide (for SOD1 fragments)
Criterion gels (Bio-Rad, Hercules, CA, USA). Samples were
boiled for 5 min in Laemmli sample buffer containing 2.5%
b-mercaptoethanol. SOD1 was detected by immunoblotting
with rabbit polyclonal antibody hSOD1 (7), or rabbit polyclonal
antibody m/hSOD1 (4). A
125
I-labeled donkey-anti-rabbit
antibody (Amersham, Piscataway, NJ, USA) was used in
quantitative assessment of SOD1; between 1 and 10 mgof
protein were loaded per lane. Protein amounts were measured
by a BCA kit (Pierce, Rockford, IL, USA). The signals were
read by a Phosphorimager (Bio-Rad). A monoclonal ubiquitin
antibody (5–25) was used in western blotting for ubiquitinated
proteins (Signet, Dedham, MA, USA). Antibodies against
Hsp25 (SPA-801), aB-crystallin (SPA-222), Hsp40 (SPA-450),
Hsp60 (SPA-806), Hsp70 (SPA-812), and Hsp90 (SPA-830)
were from Stressgen (Victoria, BC, Canada).
For immunoprecipitation, the m/hSOD1 polyclonal antibody
was cross-linked to immobilized protein A by using the Seize
X Protein A immunoprecipitation kit (Pierce). Forty micro-
grams of P2 fraction protein were adjusted to 2% SDS, boiled
for 10 min, diluted to 0.2% SDS, immunoprecipitated with the
above protein A/SOD1 antibody complex, separated by SDS–
PAGE, and blotted with the monoclonal ubiquitin antibody
(5–25; Signet).
SOD1 activity gel
SOD1 activities were determined as previously described (4,62).
Spinal cords were homogenized in 1phosphate buffered saline
(PBS; volume : weight ¼10 : 1), and then centrifuged at 10 000g
for 5 min. One hundred micrograms of supernatant proteins were
electrophoresed on a 7.5% polyacrylamide native gel, where an
in-gel reaction was carried out in the final step.
Filter trap assay
The assay was carried out as previously described (24). Tissue
homogenates were centrifuged at 800gfor 5 min in a micro-
centrifuge. The supernatant was frozen at 70C until used.
Protein concentrations were determined by BCA before filtering
through acetate membranes (0.2 mm pore size; Schleicher and
Schuell, Keene, NH, USA). The samples containing 100 mgof
protein were thawed and diluted with 20 vols of 1PBS (pH
7.4) containing 1% SDS. The solution was sonicated (70 W,
50% output, 30 s) before membrane filtering using a 96-well
dot-blot apparatus (Bio-Rad). Each well was washed twice with
1PBS and the membranes were immunostained with the m/
hSOD1 antiserum, in a fashion similar to a standard immuno-
blot, before enhanced chemiluminescence detection.
Histopathology and immunocytochemistry
Mice anesthetized with ethyl ether were sacrificed by trans-
cardial perfusion with 1PBS (pH 7.4), followed by 4%
paraformaldehyde in 1PBS. Brains and spinal cords were
removed, post-fixed in the same fixative, embedded in paraffin,
and sectioned for histologic and immunologic staining. Sagittal
sections of the brain and brain stem and cross sections of spinal
cord (10 mm) were stained with hematoxylin and eosin, silver
impregnation and thioflavin-S. Deparaffinized sections were
processed for immunocytochemistry, using antibodies against
ubiquitin (DAKO, Carpenteria, CA, USA). The immune
reaction was visualized with diaminobenzidine, and sections
were counterstained with hematoxylin.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG Online.
ACKNOWLEDGEMENTS
We are very grateful to Ms Debbie Swing for her help in
transgene injections. We thank Drs Wenxue Li, Michael K. Lee,
Guilian Xu and Alena V. Savonenko for their helpful discussion
and assistance. This study was supported by the ALS
Association, the Muscular Dystrophy Association, and by the
Robert Packard Center for ALS Research at Johns Hopkins
University.
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