Disulphide-reduced superoxide dismutase-1
in CNS of transgenic amyotrophic lateral
Brain (2005) Page 1 of 14
P. Andreas Jonsson,1Karin S. Graffmo,2Peter M. Andersen,3Thomas Bra ¨nnstro ¨m,2
Mikael Lindberg,4Mikael Oliveberg5and Stefan L. Marklund1
Departments of Medical Biosciences,1Clinical Chemistry and2Pathology,3Pharmacology and
Clinical Neuroscience,4Biochemistry, Umea ˚ University, Umea ˚ and5Department of Biochemistry and Biophysics,
Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden
Correspondence to: Stefan L Marklund, Department of Medical Biosciences, Clinical Chemistry,
Umea ˚ University, SE-901 85, Umea ˚, Sweden
Amyotrophic lateralsclerosis (ALS) is aneurodegenerativediseaseafflicting thevoluntary motor system.More
than 100 different mutations in the ubiquitously expressed enzyme superoxide dismutase-1 (SOD1) have been
associated with the disease. To search for the nature of the cytotoxicity of mutant SOD1s, amounts, enzymic
activities and structural properties of the protein as well as the CNS histopathology were examined in multiple
transgenic murine models. In order to generate the ALS phenotype within the short lifespan of the mouse,
mice expressing human wild-type SOD1 or either of the G93A and D90A mutant proteins showed high steady-
state protein levels. The major proportion of these SOD1s in the CNS were inactive due to insufficient Cu
charging and all contained subfractions with a reduced C57-C146 intrasubunit disulphide bond. Both G85R and
the truncated G127insTGGG mutant showed low steady-state protein levels, lacked enzyme activity and had
no C57-C146 disulphide bond. These mutants were also enriched in the CNS relative to other organs, sug-
gesting inefficient recognition and degradation of misfolded disulphide-reduced SOD1 in susceptible tissues. In
end-stage disease, despite 35-fold differences in levels of mutant SOD1s, similar amounts of detergent-resistant
inclusions developed in all strains, the latter more pronounced in those with high hSOD1 levels. Widespread
vacuolizations were seen in the strains with high levels of hSOD1 but not those with low, suggesting these
alterations to be artefacts related to high hSOD1 levels and not to the ALS-causing cytotoxicity. The findings
suggest that the motoneuron degeneration could be due to long-term exposure to misfolded aggregation-
prone disulphide-reduced SOD1, which constitutes minute subfractions of the stable mutants and larger
proportions of the unstable mutants.
Keywords: amyotrophic lateral sclerosis; superoxide dismutase; disulphide bond; misfolding; aggregates
Abbreviations: ALS = amyotrophic lateral sclerosis; CCS = copper chaperone for superoxide dismutase;
G127X = Gly127insTGGG; SOD = superoxide dismutase; ww = wet weight
Received July 5, 2005. Revised September 23, 2005. Accepted October 31, 2005
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegener-
ative syndrome characterized by adult-onset progressive loss
of motoneurons in the motor cortex, brainstem and spinal
cord. About 10% of ALS cases are familial (Haverkamp et al.,
1995) and in some of these the disease has been linked to
mutations in the CuZn-superoxide dismutase (SOD1) gene
(Rosen et al., 1993). Overall, ?5% of all cases with ALS
show SOD1 mutations (Andersen et al., 2003). To date,
114 different SOD1 mutations have been identified in ALS
patients, with all but one, D90A, showing a dominant mode
of inheritance (Andersen et al., 2003). SOD1 is composed of
two equal 153 amino acid subunits, each containing one Cu
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Brain Advance Access published December 5, 2005
by guest on June 2, 2013
and one Zn ion. The enzyme catalyses the dismutation of the
superoxide anion radical under formation of hydrogen per-
oxide; 2O2?+ 2H+! H2O2+ O2. The Cu ion carries out the
catalysis, while the Zn ion stabilizes the structure of the sub-
units (Forman and Fridovich, 1973). Most mutant SOD1s
show in patients reduction in enzyme activity in erythrocytes
(Robberecht et al., 1994; Andersen et al., 1997), other cell
types (Tsuda et al., 1994) and CNS tissue (Bowling et al.,
1993). Even so, the disease is not caused by loss of function,
since ALS patients homozygous for the D90A mutation show
full SOD activity in both erythrocytes (Andersen et al., 1995)
and in the CNS (Jonsson PA, Graffmo SG, Bra ¨nnstrom T,
Andersen PM, Marklund SL, in preparation). Moreover, mice
lacking SOD1 do not develop ALS (Reaume et al., 1996) and
the SOD activity in the CNS is elevated in several of the
multiple murine mutant SOD1 transgenic ALS models that
have been generated (Gurney et al., 1994; Wong et al., 1995).
The data suggest a cytotoxic gain of function conferred by the
higher levels than in the brain and spinal cord (Marklund,
1984; Jonsson et al., 2004). Both the nature of the cytotoxicity
and the reasons for the particular susceptibility of some parts
of the CNS remain to be explained.
The different mutant SOD1 proteins are likely to cause ALS
by essentially the same mechanism. Many transgenic murine
ALS models have been generated in which mutant SOD1s of
widely different characteristics are expressed. These provide
that could help to elucidate the form of the SOD1 mutants
that exert the noxious effects, as well as the reasons for the
particular susceptibility of areas of the CNS harbouring the
motor system. In this study, we examined biochemical and
histopathological alterations in SOD1 in transgenic mice
expressing the following mutations: D90A, G93A (Gurney
et al., 1994), G85R (Bruijn et al., 1997) and G127insTGGG
human SOD1 (hSOD1) and SOD1 knockouts served as
references. The results suggest that ALS may be caused by
cytotoxic, misfolded disulphide-reduced subfractions of the
mutant enzymes which are enriched in the spinal cord and
brain relative to other organs. The susceptibility of some areas
of the CNS may be caused by their inability to recognize and
degrade misfolded SOD1s rather than by the existence of
tissue-specific vulnerable factors.
Materials and methods
The DNAs used for generation of transgenic mice expressing the
G127X (Jonsson et al., 2004) and D90A (Jonsson PA, Graffmo SG,
Bra ¨nnstrom T, Nilsson P, Andersen PM, Marklund SL, in prepara-
tion) mutants of hSOD1 were constructed in an 11.6 kb
EcoR1–BamHI SOD1 genomic fragment (Levanon et al., 1985).
The G127X mice used were homozygous line 716 mice and the
D90A mice were homozygous line 134 mice. The construct for
the G85R mice (Bruijn et al., 1997) was prepared in a 12 kb
EcoR1–BamH1 DNA fragment (Epstein et al., 1987), as were the
constructs for G93A and wild-type hSOD1 (N1029) transgenic mice
(Gurney et al., 1994). Two G93A lines were used, G93AGur (G1) and
used were backcrossed 4–20 generations in C57Bl6 mice. C57Bl6
mice were used as controls. For comparison purposes, SOD1 knock-
out mice were obtained from A. Reaume (Reaume et al., 1996).
The mice were killed at preselected intervals, or when they were so
terminally ill that they could not reach the food in their cages. Brain,
spinal cord and peripheral organs were rapidly dissected out, frozen
in liquid nitrogen and stored at ?80?C. Other mice were perfusion-
fixed with 4% paraformaldehyde in 0.1 M Na phosphate
buffer, pH 7.4. The animal care and experiments were carried out
in accordance with the European Communities Council Directive
Recombinant hSOD1 variants
Recombinant hSOD1 variants were coexpressed with the copper
chaperone for superoxide dismutase (CCS) in Escherichia coli and
purified as previously described (Ahl et al., 2004). CuSO4(3 mM)
and ZnSO4(30 mM ) were added to the culture medium. The metal
contents were determined by graphite furnace atomic absorption.
Following weaning at21days, G93AGur transgenic mice were put on
a chow enriched with 400 p.p.m. Cu. Parallel litters were given
ordinary chow. The mice were killed for analysis when terminally
ill, at about 124 days of age.
The Cu contents of the spinal cords of control non-transgenic
mice were determined by inductively coupled plasma atomic
emission spectrometry using a PE Optima 3000XL apparatus
(Perkin-Elmer, Boston, MA).
Total RNA was prepared from mouse brains using the Trizol reagent
(Invitrogen, Carlsbad, CA) and the Northern blots were carried out
as previously described (Jonsson et al., 2004). The samples were
normalized against b-actin and the quantifications were carried
out at least twice.
Homogenization of tissues
phosphate, pH 7.0, in 0.15 M NaCl with EDTA-free Complete
(Roche Diagnostics, Mannheim, Germany) anti-proteolytic cocktail
added, using anUltraturrax (IKA, Staufen, Germany) for 2min. This
was followed by sonication of the homogenate using a Sonifier Cell
Disruptor (Branson, Danbury, CT) for 1 min. For the analysis of
detergent-resistant aggregates, the PBS was supplemented with 0.1%
NP40 (Roche Diagnostics, Mannheim, Germany) and in studies of
the status of the intramolecular disulphide bond, 20 mM iodoacet-
amide was added to the buffer. For quantification of total tissue
SOD1 and CCS content and activity and unless otherwise stated,
a buffer containing 50mM K phosphate, pH 7.4, 3 mM DTPA,0.3 M
KBr and Complete with EDTA was used instead.
Supplementation of homogenates with Cu
The tissues were homogenized in 25 volumes of PBS as described
above. CuSO4(1 mM) or an equal volume of PBS was added to the
Page 2 of 14Brain (2005)P. A. Jonsson et al.
by guest on June 2, 2013
homogenates. They were then incubated overnight at 4?C, followed
by analysis of SOD activity. Three mice of each strain were analysed.
Non-transgenic mice and SOD1 knockout mice were used as
Analysis of detergent-resistant aggregates
Brain and spinal cord samples from the mice were homogenized in
PBS (pH 7.0) with 0.1% of the detergent Nonidet P40 (NP40) added
(Roche Diagnostics, Mannheim, Germany). The homogenized
samples were then centrifuged at 20 000 g for 30 min at 4?C. The
supernatants were removed and the pellets were resuspended and
sonicated in double the original volume of homogenizing solution,
followed by centrifugation. This washing step was repeated five
times. Following the last wash, the pellets were resuspended and
sonicated in 1 · SDS–PAGE sample buffer. The samples were
then analysed by immunoblotting, using the anti-peptide 24–39
Polyclonal rabbit antibodies were raised against keyhole limpet
haemocyanin-coupled peptides corresponding to amino acids
4–20, 24–39 (human-specific), 43–57, 58–72, 80–96, 100–115 and
131–153 in the hSOD1 sequence. Mouse SOD1-specific antibodies
were raised against a peptide corresponding to amino acids 24–36 of
the mSOD1 sequence. Antibodies directed against CCS were raised
against a peptide corresponding to amino acids 252–270 of the
human CCS sequence. The antisera were affinity-purified in two
steps, as previously described (Jonsson et al., 2004).
Immunoblotting and quantification of SOD1
The immunoblots were carried out as previously described (Jonsson
et al., 2004). For quantification of hSOD1s by immunoblotting, the
human-specific 24–39 antibody was used and wild-type hSOD1 with
the concentration determined by quantitative amino acid analysis
(Marklund et al., 1997) was used as original standard. For quanti-
mSOD1 was assumed to have the same specific activity as the human
enzyme. In general, the quantifications by immunoblot were run at
least in duplicate for each sample.
Analysis of SOD activity
SOD activity was determined by the direct spectrophotometric
method using KO2(Marklund, 1976). One unit is defined as the
SOD activity that brings about a decay of O·2
in 3 ml buffer. One unit in the assay corresponds to 4.2 ng human
wild-type and D90A mutant SOD1 (Marklund et al., 1997).
?at a rate of 0.1 s?1
Cytochrome oxidase was analysed in spinal cord homogenates
solubilized with lauryl maltoside (Birch-Machin et al., 1994).
After formalin perfusion, fixation and paraffin embedding, tissue
pieces from mice of different ages were sectioned. Immunohisto-
chemical staining was carried out with the Ventana immunohisto-
chemistry system using the anti-SOD1 peptide antibodies and
anti-GFAP (Dako, Denmark).
Overexpressed mutant hSOD1s have
different molecular characteristics, but
similar noxious effects
The mutants of hSOD1 expressed in the different mouse
strains cover a wide range of molecular, thermodynamic
and functional properties. D90A is fully active both in
human erythrocytes (Andersen et al., 1995) and in the CNS
(Jonsson PA, Graffmo SG, Bra ¨nnstrom T, Nilsson P,
Andersen PM, Marklund SL, in preparation), suggesting
high in vivo stability. G93A is intermediately stable, whereas
G85R is more rapidly degradedin cell cultures(Borchelt et al.,
the b-barrel (Jonsson et al., 2004). As a consequence, it is
unlikely that G127X would adopt any native structure under
physiological conditions. Since the noxious effects of mutant
SOD1s show strong gene-dosage effects (Jonsson et al., 2004),
we determined the levels of gene expression in all the strains
used in this comparison study (Table 1). hSOD1 was most
highly expressed in the G93AGur mice (here set to 100%) and
these mice also showed the shortest survival time in our
laboratory (124 days). The other transgenic mouse strains
showed mRNA expression levels around half of that of
G93AGur: D90A, 51%; G93AGurdl, 50%; G85R, 43% and
G127X, 63%. Their mean survival lengths were 407, 253,
345 and 250 days, respectively. Thus, despite their distinctly
different thermodynamic and structural properties, the mut-
ant hSOD1s have relatively similar cytotoxic effects on the
spinal cord. The level of hSOD1 mRNA in the brain of the
wild-type hSOD1 transgenic mice was 60% of that found in
the G93AGur mice.
Markedly different steady-state levels of
hSOD1 protein in the transgenic mice
different levels of hSOD1 protein in the spinal cord and brain
(Fig. 1A and B). After 100 days of age, there were only small
Table 1 Expression of human SOD1 mRNA in
Transgenic strainPercentage of G93AGur 6 SD
60 6 4%
51 6 8%
50 6 10%
43 6 6%
63 6 8%
mRNA was analysed by Northern blot using a purified PCR
fragment, covering exons 1–3 of the hSOD1 sequence, as a
probe. Samples were normalized against b-actin. The values are
the means of three individual mice and are expressed as
percentage of the values for G93A mice, which were run in
parallel. Each mouse was quantified at least twice.
Disulphide-reduced mutant SOD1 enriched in the CNSBrain (2005)Page 3 of 14
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Fig. 1 Analysis of SOD1 protein and activity and CCS in murine tissue homogenates. (A and B) Time courses of levels of SOD1 protein in
spinal cord and brain, respectively, of control and transgenic mice. In transgenic mice the levels of hSOD1 were analysed by quantitative
immunoblot. In non-transgenic control mice, the mSOD1 activities were analysed and protein levels were calculated using the specific
activity of hSOD1. Details are given in Materials and methods. For non-transgenic mice and the D90A, G127X and wild-type hSOD1
transgenic mice, the data represent means of analysis of three different mice and for the others, analysis of pools from three mice.
(C) Western immunoblot of mSOD1 in spinal cord from 100-day-old non-transgenic and transgenic mice. (D) mSOD1 in different
organs from 100-day-old non-transgenic mice and hSOD1 in organs from 100-day-old transgenic mice. (E) hSOD1 levels in tissues of
100-day-old transgenic mice. Protein levels were determined by immunoblot, or calculated from SOD1 activities and the specific activity of
hSOD1. The figure also shows such data following overnight incubation at 4?C with 1 mM CuSO4. (F) Murine CCS levels in spinal cords
from hSOD1 transgenic mice, non-transgenic control mice and SOD1 null mice.
Page 4 of 14Brain (2005)P. A. Jonsson et al.
by guest on June 2, 2013
increases with time. This suggests that the levels found rep-
resent steady-states rather than accumulation of the hSOD1s.
Accumulations of mutant hSOD1 were discernible in the final
phase of the disease only in the spinal cords of G85R mice and
G127X mice (Fig. 1A), as has been described previously
(Bruijn et al., 1997; Jonsson et al., 2004). No such accumu-
lations were seen in the corresponding brains (Fig. 1B).
At 100 days, the levels of hSOD1 protein in spinal cords
from G93AGurdl, G93AGur, D90A and wild-type hSOD1
transgenic mice were 8-, 17-, 20- and 24-fold higher, respect-
ively, than the levels of mSOD1 in control mice (Fig. 1A). The
G85R and G127X mice were distinctly different, with levels of
mutant hSOD1 that were only 90% and 45% of the mSOD1
level. Except for G93AGur, the hSOD1s are synthesized at
similar rates in the mice according to the levels of mRNA
(Table 1). The widely different steady-state contents thus
reflect widely different in vivo stabilities and degradation
rates of the various hSOD1s.
The level of endogenous mSOD1 is
not influenced by transgenic
overexpression of hSOD1s
The high degree of hSOD1 expression in the transgenic mod-
els could conceivably influence the turnover of mSOD1. In
G127X, G85R, G93AGur, G93AGurdland wild-type hSOD1
transgenic mice, however, there was no significant difference
in mSOD1 content in spinal cord (Fig. 1C), brain, liver and
kidney (not shown). The exception to the rule was the (strain
134) D90A mice, in which the mSOD1 levels were 50–60% of
the controls in spinal cord, brain, liver and kidney. To explore
the reasons for this result, another D90A transgenic strain,
154, with a mean survival length of 480 days, was also
examined (Jonsson PA, Graffmo SG, Bra ¨nnstrom T,
Nilsson P, Andersen PM, Marklund SL, in preparation). In
spinal cord (Fig. 1C) or in other organs. This suggests that the
location of the transgene insertion in the 134 strain causes
reduced transcription of mSOD1.
There are low amounts of SOD1 in
the CNS compared to other tissues
and inversion in mice carrying
In control mice, the spinal cord and brain contain low levels
of mSOD1 as compared to other tissues (Fig. 1D). Liver and
kidney contain 8- and 3-fold more, respectively, whereas only
skeletal muscle contains less. Similar relationships regarding
SOD1 content between tissues have been found in humans
(Marklund, 1984). In the transgenic mice with high levels of
hSOD1, however, the relative differences in hSOD1 content
between brain and spinal cord, kidney and liver are clearly
the low-level mutants G85R and G127X are higher in brain
and spinal cord than in peripheral organs.
Insufficient Cu-charging of highly
expressed hSODs in the CNS
of transgenic mice
The truncated G127X protein lacks SOD activity (Jonsson
et al., 2004). We have also found, as reported previously
(Bruijn et al., 1997), that the G85R hSOD1 protein appears
to lack activity in transgenic mice. The SOD1 activity in spinal
cords of G85R transgenic mice was no different from that in
control mice [11200 6 1060 (SD) U/g wet weight (ww) as
opposed to 11020 6 1170 (SD) U/g ww; both n = 11]. We
analysed theactivity of an E.coli-produced recombinant G85R
hSOD1preparationthat was11% Cu-chargedandcompletely
charged with Zn. Related to the Cu-content, the SOD activity
of this preparation was 93% of that of a recombinant wild-
type hSOD1 preparation. Thus, Cu-charged G85R hSOD1
shows full SOD activity. The lack of activity in the transgenic
Cu in competition with other ligands in vivo in mice. The
G93AGur mice showed very high SOD1 activities, but the
levels in brains and spinal cords—calculated from the
SOD1 activity measurements and the specific activity of
Cu-charged hSOD1 (Marklund et al., 1997)—were only 71
and 37%, respectively, of the protein levels measured in
quantitative immunoblots (Fig. 1E). No such discrepancies
were seen for kidney and liver. Likewise, in spinal cords from
G93AGurdl, D90A and wild-type hSOD1 transgenic mice, the
overexpressed hSOD1s proteins appeared to be only 65, 21
and 21% active (Fig. 1E).
In order to find an explanation for the discrepancies, three
G93A brain and spinal cord extracts were incubated with
1 mM CuSO4overnight. This increased the SOD1 activities
of the brain extracts from 121700 6 14200 to 183800 6
14900 (SD) U/g ww and of the spinal cord extracts from
These SOD activities almost corresponded to the total SOD1
protein levels (Fig. 1E). Likewise, Cu-incubations of spinal
cord extracts from the other high-level hSOD1 transgenic
mice resulted in large increases in the SOD activities
(Fig. 1E). The increases recorded were not due to unspecific
effects of Cu in the SOD assay; 1 mM Cu reduced the total
SOD activity of a spinal cord extract of a SOD1 null mouse
from 800 to 190 U/g ww. The treatment thus appeared to
partially inactivate the SOD2 activity. The SOD1 activities of
spinal cord extracts from three control mice rose by 7%, from
11200 6 1400 to 12000 6 600 (SD) U/g ww. Finally, Cu-
incubation increased the activity of three G85R spinal cord
extracts from 13100 6 3300 to 17700 6 5200 (SD) U/g ww.
Subtracting the increase seen in the non-transgenic control
mice, the G85R-related 28% increase corresponds to a 30%
activity of the G85R protein.
The Cu content of spinal cords from non-transgenic con-
trol mice was 3.5 6 1.05 (SD, n = 5) mg/g ww. To Cu-charge
all hSOD1 in the spinal cords of 100-day-old wild-type
Thus, to charge the hSOD1s in the bodies of transgenic mice,
Disulphide-reduced mutant SOD1 enriched in the CNS Brain (2005) Page 5 of 14
by guest on June 2, 2013
much Cu is required. To determine whether the incomplete
Cu-charging of hSOD1 could be caused by an insufficiency in
dietary availability of Cu, G93AGur mice were kept on diets
containing 400 p.p.m. Cu from weaning until terminally ill.
There were no significant differences in SOD1 activities in
spinal cords between these mice [127600 6 18500 (SD) U/g
ww, n = 5) and mice on a normal diet (121300 6 9300 (SD)
U/g ww, n = 4]. Supplementation with Cu did not influence
the survival time of the mice.
Cytochrome oxidase, like SOD1, is charged via chaperones
that derive Cu mainly from Ctr-1 in the plasma membrane
(Valentine and Gralla, 1997). The activity of cytochrome oxi-
dase is a measure of the Cu availability in tissues. The cyto-
chrome oxidase activities in spinal cord extracts from the
transgenic mouse strains (3–9 mice of each strain) were
not significantly altered; all were within 615% of the levels
in controls (data not shown). This result provides evidence
that the deficient Cu charging of SOD1 is not caused by a
general insufficiency in the Cu uptake of the tissue.
(Culotta et al., 1997), the levels of murine CCS were analysed
by immunoblotting of spinal cord extracts from 100-day-old
mice (Fig. 1F). CCS was found to vary with the steady-state
levels of SOD1. In mSOD1 knockout mice, the CCS levels
were below half of thosefound in control mice. In G85R mice,
the CCS levels were doubled and they were 25% higher in
G127X mice. Finally, in the high-level mice, the G93AGurdl,
G93AGur, D90A or wild-type hSOD1 transgenics, the CCS
levels were 3–5-fold higher than in non-transgenic control
mice. These increases are far less than the 8–24-fold increases
in hSOD1 protein, suggesting that CCS may be limiting with
regard to Cu-charging in the transgenic mice. This idea is
supported by the fact that the largest proportion of active
SOD1 (65%) is found in the strain with least hSOD1 protein,
Significant fractions of both human and
murine SOD1 carry a reduced intrasubunit
disulphide bond in transgenic mice
Human SOD1 contains four cysteines (C6, C57, C111 and
C146). Two of these, C57 and C146, form an intrasubunit
disulphide bond which links the flexible Zn-binding loop to
b-strand 8 in the central b-barrel of the SOD1 subunit.
Reductional cleavage of the disulphide bond substantially
weakens thedimericinteraction andleadstomonomerization
of the subunits in the absence of metals (Arnesano et al.,
2004). The integrity of the disulphide bond is also critical
for the monomeric state. In its absence, the proportion of
denatured and potentially aggregation-prone species is
To study the status of the disulphide bond, fresh tissues
from transgenic mice were homogenized in buffer containing
20 mM iodoacetamide to react with and block free thiol
groups in proteins and other compounds. When such homo-
genates were subjected to SDS-PAGE and immunoblot
analysis in the absence of reductant in the sample buffer,
two bands were seen (Fig. 2A). These bands appeared to differ
by 2–3 kDa in molecular weight. The upper band is assigned
to SOD1 with the disulphide bond reduced, since all
SOD1 protein appears at that position if the sample is
(Fig. 2A). Analogous patterns were seen in homogenates trea-
ted with another thiol-blocker, N-ethylmaleimide (not
shown). The separation in the gel could thus arise from
the linearized disulphide-reduced SOD1 being more retarded
in the matrix than the corresponding species restricted by the
disulphide bond. To further confirm the assignment of the
E.coli (Ahl et al., 2004) were used as reference materials
(Fig. 2B). Consistently, the wild-type recombinant hSOD1
showed mobility similar to that of the disulphide-oxidized
band inaG93AGurspinal cord homogenate. Also, thecontrol
mutant hSOD1 with all four cysteines mutated to alanines
(CallA)had mobilitysimilar tothat of the disulphide-reduced
band in the brain homogenate. The hSOD1 with only Cys6
and Cys111 mutated to alanines behaved as if it were
disulphide-oxidized. In reducing gels, the SOD1s in the
G93A spinal cord and the reference samples showed identical
mobilities (Fig. 2B). Similar mobility differences have been
reported for in vivo disulphide-reduced and oxidized forms of
a prokaryotic SOD1 (Battistoni et al., 1999) and in vitro
reduced and oxidized recombinant hSOD1s (Furukawa and
The proportion of disulphide-reduced SOD1 in a sample
not treated with reductant can be determined by comparison
with a standard curve created from dilutions of reduced tissue
extract (Fig. 2A). From this comparison, it becomes obvious
that the disulphide-oxidized subunit has a lower antigenic
reactivity than the reduced subunit in the blot. The propor-
tion of disulphide-reduced hSOD1 in the G93A spinal cord
extract in Fig. 2A was ?6%. It is important that the thiol
blocker iodoacetamide is present in the homogenates to pre-
vent artificial oxidative formation of the disulphide bond. In
its absence the disulphide-reduced band becomes weaker with
a half-life of 2 h in homogenates kept at room temperature
Disulphide-reduced hSOD1 was analysed in spinal cord,
brain, kidney and liver in the different transgenic mouse
strains and was detected in all cases (Table 2). Throughout,
the proportions were nearly equal in brain and spinal cord
and lower in kidney and liver. The proportion of disulphide-
reduced hSOD1 did not change significantly with the age of
the mice (Fig. 2C). No disulphide-oxidized band could be
discerned in the G85R (Fig. 2D) and G127X mice. To ascer-
tain that disulphide-oxidized and disulphide-reduced G85R
hSOD1 can be differentiated from each other in the blotting
assay, G85R in a spinal cord extract was compared with a
recombinant G85R preparation produced in E.coli (Fig. 2E).
The recombinant preparation showed a higher mobility
in the non-reducing gel and was apparently all in
disulphide-oxidized form. Upon reduction of the samples
Page 6 of 14 Brain (2005)P. A. Jonsson et al.
by guest on June 2, 2013
by mercaptoethanol, both showed the same mobility as the
G85R from spinal cord in the non-reducing gel. From the
standard curves, the recoveries of reduced G85R and G127X
(which, owing to the C-terminal truncation, cannot be
disulphide oxidized) were both around 50%. This can be
lar weight in the non-reduced sample, which disappear in the
presence of mercaptoethanol (Fig. 2D). These bands most
Fig. 2 Analysis of disulphide-reduced SOD1 by immunoblot. (A) Western immunoblot of spinal cord extract from a 100-day-old G93AGur
mouse. In the left lane, reductant was omitted from the sample buffer. In the other lanes, the extract was diluted as indicated and sample
buffer with 5% of the reductant mercaptoethanol (ME) was used. (B) Comparison of a G93AGur spinal cord extract with recombinant (r-)
hSOD1s; wild-type hSOD1, a mutant with all cysteines changed to alanines (r-CallA) and a mutant with Cys6 and Cys111 changed to
alanines (r-C6A/C111A). Mercaptoethanol was omitted from the sample buffer in the lanes to the left and was present in the lanes to
the right. (C) Time course of occurrence of disulphide-reduced human and murine SOD1 in spinal cords from G93AGur mice. The ages in
days are indicated. Term: mouse in terminal stage of disease (124 days). Reductant was omitted from the sample buffer in all lanes.
(D) Western immunoblot of a spinal cord homogenate from a 100-day-old G85R mouse. Lanes are as described under (A). (E) Comparison
of G85R hSOD1 in a spinal cord extract with recombinant G85R which had Cys6 and Cys111 mutated to Ala. Mercaptoethanol was
omitted from the sample buffer in the lanes to the left and was present in the lanes to the right. (F) Disulphide-reduced and oxidized
mSOD1 in spinal cords from 100-day-old control and transgenic mice. Reductant was omitted from the sample buffer in all lanes.
Disulphide-reduced mutant SOD1 enriched in the CNSBrain (2005) Page 7 of 14
by guest on June 2, 2013
probably represent hSOD1 disulphide-coupled to other
SOD1 molecules or other proteins, which suggests that (mis-
folded disulphide-reduced) hSOD1 easily engages in such
reactions. They could also indicate increased oxidant stress
in the tissue (Cumming et al., 2004). The proportions of the
higher-molecular-weight bands were lower in the transgenic
mutant mice with high levels of hSOD1 protein (not shown).
The disulphide-oxidation state of the mSOD1 was also
examined, using the mSOD1-specific antibody (Fig. 2F).
No disulphide-reduced band could be discerned in control
mice, or in G85R and G127X transgenic mice. On the other
hand, in the G93AGur, D90A and wild-type hSOD1 trans-
genic mice, the proportions of disulphide-reduced mSOD1
appeared to be similar to the proportions of reduced hSOD1
in the same extracts. The time courses of disulphide-reduced
mutanthuman and endogenousmurine SOD1in spinal cords
were similar in G93A transgenic mice (Fig. 2C).
Detergent-resistant hSOD1 aggregates increase with time
in the spinal cords of all transgenic mouse strains.
Spinal cord homogenates were extracted five times with the
detergent NP40 and the final pellets were analysed by
immunoblotting (Fig. 3A–E). Pelleted hSOD1 was seen at
all times, but with a distinct accumulation at the terminal
phase of thedisease. Inall cases,bandsat 30–40 kDa wereseen
in the final pellets, together with heavy widespread smearing.
Similar findings have been reported previously in some mod-
els (Johnston et al., 2000; Shinder et al., 2001; Wang et al.,
2002; Jonsson et al., 2004). In comparison, the brains of
transgenic mice with terminal disease contained much less
detergent-resistant hSOD1 aggregates (Fig. 3A–C). Strikingly,
a similar increase in detergent-resistant aggregates was seen in
spinal cord extracts from wild-type hSOD1 transgenic mice,
although with a slower time course.
Immunoblots of detergent-resistant hSOD1 material from
equal amounts of terminal spinal cord extracts are shown in
Fig. 3E. Despite the very large differences in steady-state levels
of the enzyme (Fig. 1A), the total amounts of hSOD1 in
aggregates in terminal spinal cords were relatively similar
The time course of histopathological alterations in spinal
cords was examined in transgenic mice. The mice formed
tissue reaction. The mouse strains with low levels of hSOD1,
G85R and G127X, could be grouped together in terms of
histopathological changes, as could the mice with high levels,
i.e. D90A, G93AGur and G93AGurdl. The wild-type hSOD1
transgenic mice were similar to the latter group, but with less
advanced changes and delayed occurrence. The motoneuron
loss did not differ to any obvious degree between the different
ALS models studied. In all strains of mice, an appreciable
number of cells persisted even at terminal stages.
In all mouse strains, there was more background SOD1
seemed to increase with time and it was, as expected, stronger
in the mice with high levels of hSOD1, than in mice with low
levels (Fig. 4A, B, D, E, G, H, J, K, M, N, P and Q). With time,
this staining condensed to larger more homogenous inclu-
sions, with a terminal surge in occurrence. These inclusions
were clearly more abundant in the mice with high levels of
hSOD1 (Fig. 4E, H and K). This condensation of hSOD1
affects motoneurons to different degrees and at later time
points one can in one section see motoneurons with almost
normal appearance, while other motoneurons contain many
inclusions. There were also small dense granular SOD1-
immunoreactive inclusions in motoneurons and the neuropil
(Fig. 4B, D, E, G, H, J, K, N and Q). The neuropil inclusions
may exist in the astrocytic and dendritic compartments, or
they may represent phagocytosed remnants of degenerate
cells. These small granular inclusions were the alteration
that differed least between the transgenic ALS models and
they thus form a common denominator.
levelstrains (Fig.4OandR).Inthehigh-leveltransgenic mice,
there were also marked vacuolizations in ventral funiculi and
roots and in most of the vacuoles there was a rim of SOD1-
positivity. No such alterations were seen in the low-level
Likewise, there were distinct progressive vacuolizations
with rim staining in the neuropil of the high-level transgenic
mice (Fig. 4B, E, H and K), but not in G85R and G127X mice
(Fig. 4N and Q). The similarities suggest that the neuropil
alterations mainly represent pathology in axons and axon
collaterals. The absence of such changes in the low-level trans-
genic mice suggests that the vacuolization axonal pathology is
an artefact associated with extreme levels of hSOD1 protein in
some of the models and it is possibly not relevant to ALS.
Smaller vacuoles could also be discerned in motoneuron
Table 2 Proportion of disulphide-reduced hSOD1 in
different transgenic mouse strains
The percentage of disulphide-reduced hSOD1 in tissues from
100-day-old mice of different transgenic strains was determined
as outlined inFig. 2A. Mean values for three mice in the G93AGur
group and for two mice in the others, are given. For G85R and
G127X, the amount of disulphide-reduced hSOD1 appeared to
be around 50 according to the standard curve. This discrepancy
appears to be largely accounted for by the presence of multiple
bands of high molecular weight in the non-reduced lane (Fig. 2D).
These are not seen in the reduced lanes in the figure. The
proportions of disulphide-reduced subunits are presented
Page 8 of 14Brain (2005)P. A. Jonsson et al.
by guest on June 2, 2013
but not in G85R and G127X mice (Fig. 4N and Q). These
probably represent swollen mitochondria (Wong et al., 1995;
Jaarsma et al., 2001) and as with the axonal changes, are
apparently related to the occurrence of extreme levels of
In all models, there was a time-dependent increase in the
number and staining intensity of glial fibres as judged from
GFAP immunocytochemistry (not shown). There was no sig-
nificant difference between the different strains of mice
expressing mutant hSOD1s. A weaker and delayed reaction
was seen in the transgenic mice expressing wild-type hSOD1.
Estimation of SOD1 turnover
The levels of hSOD1 in the spinal cords of wild-type hSOD1
transgenic mice were 24-fold higher than the endogenous
levels of mSOD1 in control mice (Fig. 1A). No significant
changes in the background levels of mSOD1 were observed
(Fig. 1C). There was thus no overload of the systems that
degrade mSOD1 as a result of high-level expression of
hSOD1. The murine and human wild-type SOD1s should
have similar rates of turnover in the spinal cords of transgenic
mice. If this is the case, the rate of synthesis of wild-type
hSOD1 is about 24-fold higher than that of the endogenous
mSOD1. Comparison of the relative mRNA levels (Table 1)
suggests that the rates of synthesis of hSOD1 in the transgenic
strains vary between 17 (G85R) and 40 times (G93AGur) the
background synthesis rate of mSOD1. Thus, very high syn-
thesis rates of mutant SOD1s appear to be necessary for
expression of ALS-like phenotypes within the short lifespan
of mice. There is a strong inversely proportional gene dosage
effect. In two lines of G127X mice, the survival times were
almost twice as long in hemizygous mice as in homozygous
Fig. 3 Time courses of hSOD1 in detergent-resistant pellets from spinal cord extracts of transgenic mice. (A) D90A, (B) G127X and
(C) G93A transgenic mice at different ages and in terminal-stage disease. The last lane is from a brain pellet from the terminal mouse.
(D) Corresponding time course of extracts from wild-type hSOD1 transgenic mice. The brain pellet was from the 600-day-old mouse.
(E) Comparison of terminal-disease spinal cord detergent-resistant pellets from hSOD1 transgenic mice and a pellet from a 600-day-old
wild-type hSOD1 transgenic mouse. The figures under the lanes show the mean values for amounts of hSOD1 in aggregates from
terminal-disease spinal cords (n = 3).
Disulphide-reduced mutant SOD1 enriched in the CNS Brain (2005)Page 9 of 14
by guest on June 2, 2013
mice for the insertions (Jonsson et al., 2004). The G93AGurdl
mice, which showed mRNA levels that were 50% of those of
G93AGur mice, survived 253 days as compared to 124 days.
Broadly, a 25-fold increased rate of mutant hSOD1 synthesis
appears to cause disease within a year in the murine models.
By simple inference from the data of this study, one can
predict that humans heterozygous for SOD1 mutations
(50% of the control synthesis rate), would develop terminal
Fig. 4 Time courses of changes in spinal cord ventral horns from different hSOD1 transgenic mouse strains analysed for hSOD1 by
immunohistochemistry. Micrographs of ventral horn or ventral roots of sections stained with the anti-hSOD1 antibody 4–20. The sections
were taken from wild-type hSOD1 Tg (A–C), D90A (D–F), G93AGurdl(G–I), G93AGur (J–L), G85R (M–O) and G127X (P–R) transgenic
mice at 100 and 600 days of age or at terminal disease as indicated. The scale bar represents 60 mm.
Page 10 of 14Brain (2005) P. A. Jonsson et al.
by guest on June 2, 2013
disease at 25/0.5 · 1 year = 50 years of age, as is actually the
case (Andersen et al., 2003).
Cytotoxic and cytocidal effects of mutant SOD1s have been
higher than the background endogenous SOD1 levels in the
cells and the noxious effects are usually observed within days.
The susceptible areas of the CNS of mice are thus much more
resistant to mutant SOD1s than cultured cells, suggesting that
the noxious mechanisms could differ between the in vivo
situation and the in vitro cell culture studies.
A limitation of the present study is that whole tissue was
analysed, while primarily motoneurons are injured in the
transgenic models. The cell type(s) in which mutant SOD1s
exert their noxious effects, however, is/are unknown. Specific
expression of mutant SOD1s in astrocytes (Gong et al., 2000),
neurons in general (Pramatarova et al., 2001) and motoneur-
as in co-cultures of glial and neuronal cells (Ferri et al., 2004)
have indicated that the simultaneous presence of mutant
hSOD1 in several cell types causes a tissue reaction that
results in motoneuron injury. These latter studies support
the validity of the present approach using tissue analysis.
Accumulation of hSOD1 aggregates is a
hallmark of the terminal phase of ALS
associated with mutant SOD1s
In the spinal cords of terminally ill mice, all the mutant
hSOD1s developed detergent-resistant aggregates of multiple
molecular forms, with heavy smearing (Fig. 3A–C and E). An
almost identical picture was seen in spinal cord ventral horns,
but not elsewhere in the CNS, in a patient carrying the G127X
mutation (Jonsson et al., 2004). Numerous studies have
indicated early-commencing, long-term noxious effects of
mutant SOD1s in mice (for references see Jonsson et al.,
2004), which suggests that the terminal aggregates appear
too late to be major participants in the pathogenesis. Import-
antly, however, they constitute a least common denominator
for all the transgenic models (Fig. 3A–C, E and 4) (Johnston
et al., 2000; Shinder et al., 2001; Wang et al., 2002; Jonsson
et al., 2004) and for patients carrying SOD1 mutations
(Jonsson et al., 2004). The aggregates can possibly be regarded
as terminal markers of the long-term assault from cytotoxic
Is the oxidative formation of the C57-C146
disulphide bond linked to Cu-charging?
SOD1 species with a reduced disulphide bond existed in all
transgenic strains and G85R hSOD1 appeared completely
reduced. The endogenous mSOD1 in the transgenic mice
was found to mimic the disulphide bond reduction of the
G93A, D90A and wild-type, but not the G127X and G85R
hSOD1s (Fig. 2C and F). This suggests that the incomplete
oxidation of the disulphide bond is related to insufficient
Cu-charging in the high-level mouse strains, which should
also pertain to the mSOD1. The mechanisms by which struc-
tural disulphide bonds can be formed in the strongly reducing
cytosol are not understood (Rietsch and Beckwith, 1998). The
present data indicate that formation and maintenance of the
disulphide bond in SOD1 are at least partially linked to the
Cu-charging and possibly the CCS itself, as has also been
suggested by studies in yeast (Brown et al., 2004; Furukawa
et al., 2004). However, since the major part of the inactive
SOD1 is disulphide-oxidized, the oxidation cannot be com-
pulsorily linked to the Cu-charging.
Model for ALS-causing toxicity of SOD1
The 114 mutant SOD1s associated with ALS most likely cause
the disease by the same mechanism. The compositions of
hSODs in spinal cords were found to be complex and to vary
a great deal among the various transgenic strains (Fig. 5). A
consideration of the data suggests that the damage is caused
by minute amounts of a common cytotoxic hSOD1 species.
The results also suggest that this noxious form is disulphide-
reduced. Reduced subunits would probably adopt cytotoxic
properties more easily than their disulphide-oxidized coun-
terparts, because of their higher configurational freedom and
lower stability (Lindberg et al., 2004). The ALS-associated
C146R mutant and six C-terminal SOD1 truncation mutants
(Andersen et al., 2003), which for structural reasons perman-
ently lack the disulphide bond, support this notion; the
Even so, it is clear that all disulphide-reduced species of
hSOD1 populating the cells cannot be cytotoxic. The steady-
state levels of disulphide-reduced hSOD1 are highest in wild-
type hSOD1 transgenic mice. At the same time, wild-type
hSOD1 has proved to be less susceptible to disulphide reduc-
tion in vitro than mutants associated with ALS (Tiwari and
Hayward, 2003). This suggests that the reduced wild-type
enzyme is generally more stable in vivo than the reduced
mutants. This conclusion is also consistent with the results
of thermodynamic (Lindberg et al., 2005) and melting point
(Furukawa and O’Halloran, 2005) analyses of wild-type and
mutant hSOD1s in vitro.
An additional factor that would be expected to modulate
protein stability in vivo is the binding of Zn and Cu. Ligation
of Zn compensates for the loss of stability caused by reduction
of the disulphide bond and promotes retention of the dimeric
state(Arnesanoetal.,2004).Thus, differentabilities tocoord-
inate Zn could affect the steady-state levels of the reduced
protein, possibly favouring the wild-type protein over the
mutants. The Zn-binding affinities (Crow et al., 1997) and
also the metal ion specificities (Goto et al., 2000) are signi-
ficantly weakened in the ALS-associated hSOD1 mutants
tested. Consistent with the idea that the metal-binding ability
is indeed a critical determinant of protein turnover in vivo,
the truncated variant G127X and also G85R (which appears
to be unable to retain Cu in vivo), both show low overall
Disulphide-reduced mutant SOD1 enriched in the CNSBrain (2005)Page 11 of 14
by guest on June 2, 2013
Fig. 5 Amounts of different molecular forms of hSOD1 in spinal cords from 100-day-old mice of different transgenic lines. The columns
were calculated from the data in Fig. 1A and E and Table 2. In the high-competition situation for Cu, the disulphide-reduced subfractions
of G93A, D90A and wild-type hSOD1 were presumed to be inactive. The mSOD1 in non-transgenic control mice is presented as a
Fig. 6 Hypothetical mechanism of formation of noxious forms of hSOD1 in the spinal cord. The various mutant hSOD1 proteins fold and
mature to different extents, giving rise to multiple molecular forms after synthesis (cf. Fig. 5). A selection of the many possible variants is
depicted in the illustration. The wild-type hSOD1 and stable mutants should be degraded in larger proportions via non-selective routes
such as autophagy to lysosomes. More unstable and hence short-lived, mutants unfold to greater extents and are recognized by the quality-
control systems for proteasomal degradation. Unfolded/misfolded disulphide-reduced forms of hSOD1 show higher steady-state levels
in the spinal cord and brain than in peripheral organs, suggesting slow recognition and degradation in the CNS (Fig. 1D). These forms
and possibly also oligomeric protoaggregates, may exert cytotoxic effects which injure the motor areas of the CNS.
Page 12 of 14Brain (2005)P. A. Jonsson et al.
by guest on June 2, 2013
steady-state hSOD1 levels in tissues of transgenic mice
(Figs 1A, B, D and 5). The putative cytotoxic forms of
SOD1 may constitute large proportions of these and minute
subfractions of the high-level mutants G93A and D90A.
Following synthesis, the mutant hSOD1s form native
metal-charged dimers and many other structural variants
(Figs 5 and 6). The degradation routes of stable cytosolic
proteins in the CNS are not well understood, but as in other
organs, they may proceed partially via non-selective auto-
phagy of the cytosol and lysosomal lysis (Yoshimori, 2004).
SOD1 degradation by such non-selective pathways occurs in
the liver (Rabouille et al., 1993) and this could be an innocu-
ous high-capacity route of mutant and wild-type SOD1
turnover in the CNS. The action of such non-selective
high-capacity degradation would explain the lack of influence
of high hSOD1 expression on the turnover of mSOD1
(Fig. 1C). The more stable the SOD1 variant, the greater
the proportion that is degraded via a non-selective route.
The ALS-linked hSOD1 mutants are conformationally less
stable than the wild-type enzyme and have an increased pro-
pensity to unfold (and possibly also to misfold) in vivo. Thus,
theyarealsomore likely toberecognizedanddegradedviathe
quality control systems and the proteasome. The complete
unfolding of the hSOD1 structure occurs subsequent to dis-
sociation of the homodimer and is promoted by reductive
cleavage of the C57–C146 disulphide bond (Lindberg et al.,
2004). The enrichment of G85R and G127X hSOD1 proteins
in the CNS (Fig. 1D) suggests that the quality control and
degradation systems for reduced and misfolded hSOD1s are
less efficient in the brain and spinal cord than in other organs.
Such unfolded subfractions of mutant hSOD1s could be
responsible for the toxic effects, e.g. by interaction with essen-
tial factors such as the antiapoptotic protein Bcl-2 (Pasinelli
et al., 2004), by accumulating in mitochondrial outer mem-
branes (Liu et al., 2004), or by forming oligomeric protoag-
gregates which are commonly believed to cause cytotoxicity
(Bucciantini et al., 2002; Volles and Lansbury, 2002; Walsh
et al., 2002).
This work was supported by the Swedish Science Council, the
Swedish Brain Fund/Ha ˚llsten Fund, the Swedish Medical
Society including the Bjo ¨rklund Fund for ALS Research,
the Swedish Association of Persons with Neurological Disab-
ilities, the Foundation for Medical Research at Umea ˚ Univer-
sity and Va ¨sterbotten County Council. We thank Jo ¨rgen
Andersson, Eva Bern, Ingalis Fransson, Karin Hjertkvist,
Ann-Charlott Nilsson, Ulla-Stina Spetz, Karin Wallgren
and Agneta O¨berg for technical assistance, Dr D.W. Cleveland
for the G85R hSOD1 transgenic mice and Dr A. Reaume for
the SOD1 knockout mice.
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