Aggregate Formation in Cu,Zn Superoxide Dismutase-related
Proteins*
Marjatta
Son
,
C. Dyan
Cloyd
,
Jeffrey D.
Rothstein§,
Bhagya
Rajendran
, and
Jeffrey L.
Elliott
¶
From the
Department of Neurology, University of
Texas, Southwestern Medical Center, Dallas, Texas 75390 and the
§ Department of Neurology, Johns Hopkins University,
Baltimore, Maryland 21287
Received for publication, November 18, 2002, and in revised form, January 13, 2003
 |
ABSTRACT |
Aggregation of Cu,Zn superoxide dismutase (SOD1)
protein is a pathologic hallmark of familial amyotrophic lateral
sclerosis linked to mutations in the SOD1 gene,
although the structural motifs within mutant SOD1 that are responsible
for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and
extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence
identity with SOD1, particularly in the regions of metal binding, but
play no significant role in mutant SOD1-induced disease. We
hypothesized that it would be possible to form CCS- or SOD3-positive
aggregates by making these molecules resemble mutant SOD1 via the
introduction of point mutations in codons homologous to a disease
causing G85R SOD1 mutation. Using an in vitro assay system,
we found that expression of wild type human CCS or a modified
intracellular wild type SOD3 does not result in significant aggregate
formation. In contrast, expression of G168R CCS or G146R SOD3 produced
aggregates as evidenced by the presence of high molecular weight
protein complexes on Western gels or inclusion bodies on
immunofluorescence. CCS- and SOD3-positive inclusions appear to be
ubiquitinated and localized to aggresomes. These results suggest that
proteins sharing structural similarities to mutant SOD1 are also at
risk for aggregate formation.
 |
INTRODUCTION |
Mutations in Cu,Zn superoxide dismutase
(SOD1)1 cause one form of
familial amyotrophic lateral sclerosis via a toxic gain of function
(1-4). Disease-causing mutations span the entire SOD1 molecule and
encompass virtually every structural and functional domain within the
protein (5). Although the precise mechanisms underlying mutant SOD1
toxicity remain unclear, abnormal SOD1 protein folding that leads to
aggregate formation is a pathologic hallmark of the disease and may be
related to pathogenesis via inhibition of proteasome function (6-8).
SOD1 aggregates can be visualized readily either as
proteinaceous inclusions (immunofluorescence) or as high molecular
weight complexes (Western blots) in the spinal cords of both autopsied
familial amyotrophic lateral sclerosis patients and transgenic mice
expressing mutant SOD1, which serve as an animal model of the human
disease (8-13). These SOD1-positive inclusions and complexes also may
be generated in culture even in non-neuronal cells after transfection
with mutant SOD1 and proteasome inhibition (12, 13). Because
overexpression of wild type SOD1 in either culture or transgenic mice
does not produce aggregates, the formation of aggregates may be related
to structural motifs found in mutant SOD1. If this hypothesis is
correct, then such aggregate-inducing motifs, if known, could be
mimicked in proteins resembling SOD1 with the prediction that these
proteins subsequently would form aggregates.
SOD1 is not the only superoxide dismutase requiring copper and zinc
binding for normal activity. The copper chaperone for SOD1 (CCS), a
274-amino acid protein, has three structural domains and, like SOD1, is
expressed ubiquitously in the central nervous system (14-16). Wild
type CCS normally interacts with SOD1, allowing for the transfer of
copper to SOD1, but does not play a role in mutant SOD1-induced disease
(17-18). The first (residues 1-85) and third (residues 234-274)
domains of CCS have no real sequence or structural homology with SOD1.
Domain 2 (residues 86-233) does exhibit a 50% sequence identity with
human SOD1, including three of the four histidine residues that bind
copper as well as the three histidines and one aspartic acid that bind
zinc, all arranged in a pattern similar to SOD1. Wild type CCS is
catalytically inert. However, a D200H substitution, which reconstitutes
the fourth copper binding site, is capable of restoring dismutase
activity to CCS (19). Human extracellular SOD (SOD3) encoded by a gene distinct from SOD1 is composed of 240 amino acids and
harbors an 18-amino acid-long signal peptide targeting the protein for the extracellular compartment (20). In SOD3 neither the first 60 amino acids after the signal peptide nor the carboxyl-terminal portion
(residues 212-240) bears any sequence or structural homology to SOD1.
SOD3 does share a 40% sequence identity with SOD1 in the central
portion of the molecule (residues 78-212), including all copper and
zinc binding sites. Naturally occurring mutations in CCS or SOD3 have
not been reported, nor have these proteins been linked to human disease.
Because the central domains in both CCS and SOD3 bear strong homology
to SOD1, these proteins are ideal for testing whether the aggregate
potential of mutant SOD1 can be mimicked in related proteins. In this
work, we demonstrate that both CCS and SOD3 will form aggregates if
modified to resemble mutant SOD1. By making a single amino acid
substitution in CCS or SOD3 at a position homologous to a
disease-causing site in SOD1, we can induce prominent CCS and SOD3
aggregates. These results indicate that proteins bearing structural
motifs similar to mutant SOD1 are also at risk for aggregate formation.
 |
EXPERIMENTAL PROCEDURES |
Plasmids and DNA Constructs--
Wild type and G85R cDNAs
for human SOD1 were provided by Dr. Don Cleveland (University of
California, San Diego). Wild type human CCS cDNA was a gift from
Dr. Jonathan Gitlin (Washington University, St. Louis, MO).
G168R and D200H CCS cDNAs were created via PCR primer-directed
mutagenesis using the following primers: CCS D200H, 5'-GGC CTC CCC GGC
CCA GGT CAT CTT CTC CCT CAT GAA-3'; CCS G168R, 5'-CAC CGC GGA
GAC CTG CGC AAT G-3'. Wild type cDNA for SOD3 was generated by
reverse transcription PCR from human cerebellar mRNA provided by
Dr. Lawrence Honig (University of Texas, Dallas, TX). This full-length
SOD3 cDNA was used as a template to create an intracellular (IC)
version of SOD3 lacking the first 54 bp that encode an 18-amino acid
signal peptide. Two variations of IC-SOD3 were made, including a
long form with the full-length carboxyl-terminal end and a short form
lacking the last 42 bp, which encode a polybasic heparin binding motif.
In both long and short IC-SOD3, we made a G146R mutation via PCR
primer-directed mutagenesis. The following primers were used in
generating the various SOD3 constructs: 5'-CAA GCT TGC CAT GTG GAC GGG
CGA GGA CTC G-3' and 5'-AAG CGG CCG CCA TGT GGA CGG GCG AGG ACT CGG
C-3', forward primers for IC-SOD3; 5'-ATC TAG ATC ACT CTG AGT GCT CCC GCG CCT-3' and 5'-AAA CTC GAG TCA CTC TGA GTG CTC CCG CGC CTG-3', reverse primers for the short form of SOD3; 5'-CGT CGC GGA CCG CGA AGT
TGC GG-3', the primer for SOD3 G146R. All final constructs were
confirmed by sequencing. For protein expression, all cDNAs were
cloned into pBUDCE4 (Invitrogen), a eukaryotic expression vector
containing both cytomegalovirus and elongation factor 1
promoters. Two copies of each cDNA were inserted into the vector; one copy was inserted into the cytomegalovirus site, and the second copy was inserted into the elongation factor 1
site.
Transfection and Cell Extracts--
COS1 (ATCC) cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and penicillin and streptomycin. For Western blot
assays, COS1 cells were grown to 80-100% confluency, subcultured at a
density of 2.5 × 105 cells/35-mm culture well, and
transfected with the constructs of pBUDCE4 with cDNA inserts for
SOD1, CCS, or SOD3 according to the protocol for FuGENE 6 reagent (Roche Molecular Biochemicals). COS1 cells were treated
48 h after transfection with 6.5 µM lactacystin (Calbiochem) or with Me2SO as a vehicle control for 20 h. Cells were harvested 68 h after transfection by trypsinization
followed by three PBS washes, and pellets were stored at
80 °C.
Pellets were thawed in homogenization buffer consisting of 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 0.5% Triton
X-100 (Sigma), 0.5% IGEPAL CA-630 (Sigma), and 1% protease inhibitor
mixture (P 8340, Sigma) and homogenized. Samples were taken from the
homogenates, and the rest was centrifuged at 15,000 × g for 2 min. Pellets were washed once with the buffer,
centrifuged again, and sonicated with a Fisher sonic dismembrator at
10% power with short bursts. Homogenates and sonicated pellet
fractions were kept at
80 °C. Protein concentrations were
determined by BCA protein assay (Pierce).
Western Blotting--
Extracts were boiled for 5 min in 250 mM Tris-HCl, pH 6.8, containing 2.5%
-mercaptoethanol,
1% SDS, 10% glycerol, and dye. The extracts then were resolved on
4-20% Tris-glycine SDS-PAGE and transferred onto a polyvinylidene
difluoride membrane according to the manufacturer's instructions
(XCell SureLock system, Invitrogen). After transfer,
the membranes were blocked for overnight at 4 °C in blocking buffer
(PBS containing 0.2% I-Block (Applied Biosystems) and 0.1% Tween
20 (Sigma)). Membranes were incubated with primary antibody in blocking
buffer for 2-3 h at room temperature, washed four times with
blocking buffer, incubated with alkaline phosphatase-conjugated secondary antibody, diluted 1:5000 in blocking buffer (Applied Biosystems) for 2 h at room temperature, and washed again. The immunoreactive signals were detected using the CDP-Star
chemiluminescent detection system (Applied Biosystems).
Immunofluorescence--
COS1 cells were seeded at a density of
3.5 × 104 cells/chamber in 4-cell Lab-Tek II chamber
slides (Nalge Nunc International). Cells were treated 48 h
after transfection with 6.5 µM lactacystin or with
Me2SO as a vehicle control for 20 h. 68 h after
transfection, cells were washed three times in PBS, fixed with 100%
methanol for 15 min at
20 °C, washed again in PBS, and incubated
for 3 to 4 h at room temperature in blocking buffer
containing PBS with 5% normal serum from the same species as the
secondary antibody (Jackson Immunoresearch Laboratories, Inc.), 1%
bovine serum albumin (fraction V, Sigma), 0.1% gelatin (fish skin
gelatin, Sigma), and 0.05% sodium azide. Slides were incubated with
primary antibodies in blocking buffer overnight at 4 °C, washed with
PBS four times, incubated with cyanine-conjugated secondary antibodies
(goat anti-rabbit or anti-mouse IgG labeled with Cy3 or Cy2, or
donkey anti-sheep IgG labeled with Cy3 or Cy2, Jackson Immunoresearch
Laboratories, Inc.) in blocking buffer for 3 to 4 h, again washed
with PBS four times, and mounted with Gel/Mount (Biomeda Corp.). Slides
were viewed with a Nikon fluorescent microscope under a fluorescein isothiocyanate, rhodamine, or dual excitation cube.
Antibodies--
For Western blots, the following primary
antibodies were used: sheep anti-human SOD1 (diluted 1:1000,
Calbiochem), rabbit anti-CCS (diluted 1:4000, from Dr. Jeffrey
Rothstein), rabbit anti-SOD3 (diluted 1:5000, gift from Dr. James
Crapo), and mouse anti-
-tubulin (clone B-5-1-2, monoclonal, diluted
1:3000, Sigma). For immunofluorescence, rabbit anti-CCS and anti-SOD3
antibodies were used as primary antibodies as in the Western blots, and
both were diluted 1:333. Also used as primary antibodies for
immunofluorescence were the following mouse antibodies: anti-vimentin
(clone V9, monoclonal, diluted 1:600, Dako), anti-HSP 70 (monoclonal,
diluted 1:300, Stressgen), anti-
-tubulin (clone GTU-88, monoclonal,
diluted 1:300, Sigma), and anti-ubiquitin (clone FK2 against
mono- and polyubiquitinated proteins, monoclonal, diluted 1:200, Biotrend).
 |
RESULTS |
Generation of Mutant CCS and Mutant SOD3--
The alignment of
amino acid sequences for human wild type SOD1, CCS, and SOD3 is shown
in Fig. 1. A glycine to arginine change at codon 85 of SOD1 is capable of producing motor neuron disease both
in humans and in transgenic mice characterized by prominent SOD1-positive aggregates (10). This glycine residue is conserved in
both wild type CCS (codon 168) and wild type SOD3 (codon 146). To test
whether CCS and SOD3 can mimic mutant SOD1 and form aggregates, we made
glycine to arginine mutations at this conserved codon (G168R in CCS and
G146R in SOD3). We also made CCS bearing a D200H mutation either alone
or in association with the G168R change. This D200H mutation restores
the fourth copper binding site of CCS and establishes catalytic
activity for the molecule. Because SOD3 is a secreted protein, we
generated wild type and G146R SOD3 without the signal peptide to create
an intracellular SOD3. The carboxyl terminus of SOD3 contains a
polybasic heparin binding domain that may be cleaved or maintained
depending on tissue type (21). For these experiments, we generated
intracellular wild type and G146R SOD3 with and without these polybasic
residues to give long and short forms of SOD3.

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Fig. 1.
Alignment of human SOD1, CCS, and SOD3
sequences. The amino acid residues shared by either SOD1 and CCS
or SOD1 and SOD3 are dark gray; the residues shared by all
three are light gray. An asterisk marks
copper-binding amino acids, a number sign marks zinc-binding
amino acids, a carat marks the Gly-85 codon in SOD1, a
bracket indicates the SOD3 leader sequence, and an
arrowhead indicates the site where the carboxyl-terminal
heparin-binding residues were removed in SOD3.
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Aggregate Formation in G168R CCS--
To assess potential
aggregate formation with CCS, we modified an in vitro method
used previously to study SOD1 aggregates (12). In this assay, COS1
cells transfected with G85R SOD1 but not with wild type SOD1 form high
molecular weight SOD1-positive complexes on Western gels. These SOD1
complexes are concentrated in pellet fractions of total cell lysate and
can be accentuated by proteasome inhibition (Fig.
2). We therefore transfected COS1 cells
with human wild type, D200H, G168R, and G168R/D200H CCS. Cells
transfected with these four CCS constructs expressed similar levels of
CCS protein when examined in Western blots on total cell homogenates
using
-tubulin as a control (Fig.
3A). CCS antibody clearly
recognized the various mutant forms of CCS in addition to wild type
CCS. Base-line COS1 cell expression of CCS was extremely low and barely
detectable in Western blots. No clear CCS-positive aggregates were
observed in these total cell lysates even after treatment with the
proteasome inhibitor lactacystin.

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Fig. 2.
SOD1-positive complexes in cells expressing
human G85R SOD1. Pellet fractions of COS1 cell extracts
transfected with WT or G85R human SOD1 and treated with lactacystin (+)
or vehicle ( ) were loaded on a Western gel (30 µg of protein).
Blots were probed with anti-human SOD1 antibody for detection
of complexes and with anti- -tubulin antibody after stripping for
assessment of protein loading. Transfected, pelletized cells were
homogenized and centrifuged at 15,000 × g for 2 min.
High molecular weight complexes were enriched in the pellet fractions,
which represented 10-16% of the total protein. Spinal cord
(SC) homogenate from a weak 7.5-month-old G93A SOD1
transgenic mouse was used as a control for complex formation.
Expression of G85R SOD1 but not wild type SOD1 results in complex
formation.
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Fig. 3.
Cells expressing G168R or G168R/D200H CCS
form CCS-positive complexes. COS1 cells were transfected either
with human WT (lane a), D200H (lane b), G168R
(lane c), or G168R/D200H CCS (lane d) or with
vector alone (Con), and the cells were treated with
lactacystin (+) or vehicle ( ). Transfected, pelletized cells were
homogenized, and aliquots were taken for homogenate samples; the rest
were centrifuged at 15,000 × g for 2 min. High
molecular weight complexes were enriched in the pellet fractions, which
represented 10-16% of the total protein. A, 35 µg of
total cell homogenates from transfected COS1 cells were loaded on
Western gels and probed with anti-CCS antibody (hCCS) and
then with anti- -tubulin antibody after stripping. Similar levels of
human CCS protein were expressed for the differing CCS constructs.
B, 30 µg of pellet fractions of cell extracts were loaded
on Western gels and probed with anti-CCS antibody (hCCS) and
then with anti- -tubulin antibody. Five independent experiments were
performed with similar results; G168R and G168R/D200H mutations in CCS
lead to CCS-positive complexes, whereas WT or D200H mutations do not.
C, 30 µg of pellet fractions of lactacystin-treated cell
extracts were loaded on Western gels. Blots were probed with anti-human
SOD1 antibody. Cells transfected with CCS mutations do not form
SOD1-positive complexes.
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Because SOD1-positive complexes are concentrated in the pellet fraction
rather than in total cell homogenates, we performed Western blots on
the pellet fraction from CCS-transfected cells. Visible CCS-positive
high molecular weight protein complexes were observed in the pellet
fraction of COS1 cells transfected with G168R or G168R/D200H CCS but
not with wild type or D200H CCS. After treatment with the proteasome
inhibitor lactacystin, cells transfected with G168R or G168R/D200H CCS
generated even more prominent CCS-positive high molecular weight
protein complexes, whereas cells transfected with wild type and D200H
CCS did not form these complexes (Fig. 3B). Thus, complex
generation is not related to the overexpression of CCS protein but
rather to the expression of CCS bearing the G168R mutation. The
observation that D200H expression alone did not induce complexes
indicates that a single mutation in CCS by itself does not
automatically induce complex formation.
Because of the known interaction between CCS and SOD1, we next
investigated whether cells with CCS-positive complexes also developed
SOD1-positive complexes. Western blot analysis did not confirm the
presence of SOD1-positive complexes in CCS-transfected cells (Fig.
3C).
We used immunofluorescence to assay possible inclusion body formation
with the various CCS constructs. Expression of G168R and G168R/D200H
CCS yielded prominent CCS-positive inclusions (Fig.
4A). The quantitation of
CCS-positive inclusions in COS1 cells transfected with wild type or
mutant CCS is shown in Fig. 4B. In non-lactacystin-treated
cultures, few cells transfected with G168R or G168R/D200H CCS formed
CCS-positive inclusions. Virtually no inclusions were noted in cells
expressing wild type or D200H CCS. After lactacystin treatment, close
to 45% of transfected cells expressing either G168R or G168R/D200H CCS
formed CCS-positive inclusions. Again, cells transfected with wild type
or D200H CCS generally did not form CCS-positive inclusions. The
quantitation of SOD1-positive inclusions in COS1 cells transfected with
wild type or G85R SOD1 is shown in Fig. 4C. Expression of
G85R SOD1 produced SOD1 inclusions, whereas WT SOD1 did not.
These results indicate that CCS bearing G168R mutations will
form CCS inclusions under conditions similar to those in which
disease-causing SOD1 mutations have been found to cause SOD1-positive
inclusions.

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Fig. 4.
CCS-positive inclusions are formed in
cells expressing G168R-containing CCS mutants. COS1
cells were transfected with WT, D200H, G168R, or G168R/D200H
CCS and treated with lactacystin. After fixation, transfected cells
were identified with immunofluorescence by staining with rabbit
anti-CCS antibody followed by Cy3-labeled secondary anti-rabbit
antibody (red). A, CCS-positive inclusions form
in cells transfected with G168R or G168R/D200H CCS but not with WT or
D200H CCS. B, quantitation of CCS-positive inclusions in
cells transfected with WT, D200H, G168R, or G168R/D200H CCS. Five
separate experiments were performed, and in every experiment, at least
200 cells that were CCS-transfected and CCS-positive were counted in
each group. Data show the percentage of CCS-transfected cells with
CCS-positive inclusions in the presence (+) or absence ( ) of
lactacystin. C, quantitation of SOD1-positive inclusions in
cells transfected with human WT or G85R SOD1. Data show the percentage
of SOD1-transfected cells with SOD1-positive inclusions in the presence
(+) or absence ( ) of lactacystin.
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Characterization of CCS-positive Inclusions--
To determine
whether CCS inclusions were present in aggresomes, we used antibodies
against
-tubulin and vimentin.
-Tubulin colocalized with
CCS-positive inclusions, indicating that the inclusions were found in
the organizing center of the microtubules (Fig.
5A). Many CCS-positive
inclusions also were found within vimentin-positive cages, thereby
confirming localization within aggresomes (Fig. 5B,
arrowhead). However, occasional CCS-positive inclusions were
located clearly outside of this vimentin-positive cage (Fig.
5B, arrow). CCS-positive inclusions in
G168R/D220H-transfected cells were smaller after a shorter treatment
with lactacystin (7 h), but their localization was similar (data not
shown). It is therefore possible that CCS-positive inclusions not
localized in aggresomes may be in transit either to or from
aggresomes.

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Fig. 5.
Characterization of CCS-positive
inclusions. COS1 cells were transfected with G168R/D200H CCS and
treated with lactacystin. A, after fixation, cells were
labeled with rabbit anti-CCS antibody and stained with secondary Cy3
anti-rabbit antibody (red) and with mouse anti- -tubulin
(lane A), anti-vimentin (lane B), anti-ubiquitin
(lane C), or anti-HSP 70 antibody (lanes D and
E), all of which were stained with secondary Cy2 anti-mouse
antibody (green). Several, but not all, of the CCS-positive
inclusions appear to be in aggresomes. CCS-positive inclusions
colocalize with ubiquitin but not always with HSP 70. Magnification,
×600 (lanes A-D) or ×200 (lane
E).
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Further dual localization studies confirmed that CCS-positive
inclusions appear to be ubiquitin-positive (Fig. 5C).
Protein chaperones such as HSP 70 have been suggested to be important in modulating mutant SOD1 protein folding and have been associated with
SOD1-positive inclusions in mutant SOD1 transgenic mice (7). Treatment
with lactacystin causes a nonspecific increase in HSP 70 expression
with the formation of HSP 70-positive inclusions (data not shown). CCS
inclusions were positive for HSP 70 in some cells (Fig. 5, D
and E, marked with
) but not in others (Fig. 5,
D and E, marked with >), suggesting that CCS
inclusions are not always dependent on HSP 70 expression.
G146R IC-SOD3 Expression Causes Aggregate Formation--
To assess
possible aggregate formation with G146R IC-SOD3 expression, we used the
same COS1 cell assay system. COS1 cells were transfected with
intracellular wild type and G146R SOD3 (both short and long
forms) and then treated with proteasome inhibitors. Western blots
confirmed expression of IC-SOD3 within COS1 cells (Fig.
6A). However, we were not able
to distinguish SOD3 complexes clearly in the pellet fractions on
Western blots either in the presence or absence of lactacystin.

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Fig. 6.
Expression of human wild type and G146R
intracellular SOD3 in vitro. COS1 cells
were transfected with short WT IC-SOD3, long WT IC-SOD3, short G146R
IC-SOD3, long G146R, or vector alone (Con). A, 35 µg of cell homogenates were loaded on Western gels. Blots were probed
with anti-human SOD3 antibody. B, quantitation of
SOD3-positive inclusions. COS1 cells were transfected with WT IC-SOD3
or G146R IC-SOD3, either short form (S) or long form
(L), or with WT extracellular (EC) SOD3. After
fixation, transfected cells were identified with immunofluorescence by
staining with rabbit anti-human SOD3 antibody followed by Cy3-labeled
secondary anti-rabbit antibody. Five separate experiments were
performed, and in every experiment, at least 200 SOD3-transfected and
SOD3-positive cells were counted in each group. Data show the
percentages of SOD3-transfected cells with SOD3-positive inclusions in
the presence (+) or absence ( ) of lactacystin. In the absence of
lactacystin, G146R IC-SOD3 formed inclusions. After lactacystin
treatment, cells transfected with all intracellular forms of SOD3
produce inclusions. However, significantly more inclusions are formed
in cells transfected with G146R mutant forms of IC-SOD3 than with WT
forms of IC-SOD3. C, characterization of SOD3-positive
inclusions. Cells were transfected with the long form of G146R IC-SOD3
and treated with lactacystin. After fixation, cells were labeled with
anti-human SOD3 antibody and stained with secondary Cy3 anti-rabbit
antibody (red) and with either mouse anti-vimentin or
anti-ubiquitin antibody with secondary Cy2 anti-mouse antibody
(green). The overall pattern of SOD3-positive inclusions
appears to be similar to what is observed for CCS inclusions.
SOD3-positive inclusions appear to be ubiquitinated, and many appear to
localize within vimentin cages.
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We then used immunofluorescence to determine whether expression of
G146R IC-SOD3 would yield SOD3-positive inclusions. In the absence of
lactacystin treatment, less than 4% of cells transfected with wild
type intracellular SOD3 (either short or long forms) formed
SOD3-positive inclusions (Fig. 6B). In contrast, 14% of G146R SOD3 short- and 10% of G146R SOD3 long-transfected cells exhibited SOD3-positive inclusions without proteasome inhibition. These
percentages are considerably larger than those observed for COS1 cells
transfected with either G168R CCS or G85R SOD1, suggesting that the
intracellular expression of G146R IC-SOD3 may have greater aggregation potential.
We treated transfected cells with the proteasome inhibitor lactacystin
to determine whether the percentage of inclusion-forming cells could be
increased. As a control, we found that expression of wild type human
native SOD3 (extracellular), even in the presence of lactacystin, did
not yield inclusions. After lactacystin treatment, a large percentage
of cells transfected with intracellular SOD3, either wild type or
G146R, formed complexes. However, the percentage was significantly
higher in cells expressing G146R compared with wild type SOD3,
indicating that the presence of a G146R mutation contributed to a
substantial increase in the aggregation potential of the SOD3 protein.
Other changes in the protein, such as the presence or absence of the
terminal heparin binding domain, had no appreciable effect on the
inclusion formation of G146R SOD3.
We also performed colocalization studies of SOD3-positive inclusions.
SOD3-positive inclusions appear to colocalize partially with vimentin
cages (Fig. 6C) and with
-tubulin (data not shown). SOD3
inclusions also appear to be ubiquitin-positive (Fig. 6C) but not always HSP 70-positive (data not shown). These results indicate
that SOD3-positive and CCS-positive inclusions share overall similar characteristics.
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DISCUSSION |
Aggregate formation of mutant proteins is characteristic of many
adult onset neurodegenerative conditions (22-25). This pattern holds
true for SOD1-related disease, in which SOD1-positive aggregates are
found in both human beings and transgenic mice that harbor a mutant
SOD1 gene. Unlike Huntington's disease, where
disease-causing polyglutamine repeats represent an aggregate-forming
motif found in proteins linked to neurodegenerative diseases, the
aggregate-forming motifs within SOD1 remain obscure. It is also
unclear whether proteins that are related to mutant SOD1 and harbor
similar structural motifs form aggregates. In this work, we demonstrate
that selective single amino acid mutations in proteins related to SOD1
will induce aggregate formation of these proteins. These results
suggest that motifs shared in mutant SOD1, CCS, and SOD3 can lead to
aggregate formation.
Although the structural properties within SOD1, CCS, and SOD3 that
predispose these proteins to form aggregates are unclear, there is
evidence to suggest that metal binding may be important. These proteins
share similar copper- and zinc-binding residues arranged in nearly
identical spatial patterns. Although much attention was focused
initially on copper within SOD1 as possibly related to disease
pathogenesis, recent work has suggested that copper binding may not be
relevant (18, 26). Our findings here provide additional evidence that
copper binding may not be critical to aggregate formation in that
mutations in the copper-binding Asp-200 residue of CCS do not
alter the ability of CCS to form aggregates.
There is stronger evidence to support an important role for zinc
binding to SOD1 for both disease pathogenesis and aggregation potential. Altering the levels of zinc-binding proteins can
significantly affect zinc levels within the central nervous system and
can change the disease course in mutant SOD1 transgenic mice (27).
Although zinc binding sites may be preserved in SOD1 mutations causing disease, studies have shown that zinc binding to mutant SOD1 is abnormal and has consequences for both the enzymatic and structural integrity of the protein (28-29). Changes in zinc binding can enhance the tendency of certain proteins, including prion protein and
-amyloid, to form insoluble complexes, and such mechanisms
may contribute to the formation of SOD1 aggregates observed in the disease (30-32). Prion protein is especially interesting in that like
SOD1, it possesses dismutase activity and binds both copper and zinc
(33-35). Mutations in prion protein are responsible primarily for
several human dementias, but cases of motor neuron involvement with amyotrophy, similar to amyotrophic lateral sclerosis, have been
reported (36).
The biologic consequences of mutant SOD1 aggregate formation are
unclear. Protein aggregates may be directly harmful to cells. Recent work has found that protein aggregates interfere with
proteasome function, thus potentially leading to a cycle of protein
processing dysfunction, increased aggregate formation, and consequent
further declines in proteasome protein clearing (6). However, the
formation of protein aggregates also might be helpful for cells and
might be used as a potential way to sequester harmful proteins (37). It
also is possible that aggregates may be of little positive or negative
consequence in disease; rather, they are only a marker of cellular
pathology (38). Whether protein aggregates contribute to mutant
SOD1-induced motor neuron disease is unclear, but certainly there
exists a good correlation between aggregate formation and the disease
state. Based on this fact, we would predict that expression of G168R
CCS or G146R intracellular SOD3 in vivo would lead to a
motor phenotype similar to that found in expression of G85R SOD1. Such
experiments will test the biological significance of aggregate
formation in motor neuron disease and are being pursued actively.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Ling Yi Chang (National Jewish
Medical and Research Center, Denver, CO) for supplying antibody to
SOD3, Dr. Jonathan Gitlin (Washington University School of Medicine,
St. Louis, MO) for wild type CCS cDNA, and Dr. Don Cleveland
(University of California, San Diego, CA) for wild type and G85R
SOD1 cDNAs.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health, the Robert Packard Center for ALS Research, and the John G. Penson Fund.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
214-648-2871; Fax: 214-648-6320; E-mail:
jeffrey.elliott@utsouthwestern.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211698200
 |
ABBREVIATIONS |
The abbreviations used are:
SOD1, Cu,Zn
superoxide dismutase;
CCS, copper chaperone(s) for SOD1;
SOD3, extracellular Cu,Zn superoxide dismutase;
IC, intracellular;
PBS, phosphate-buffered saline;
WT, wild type;
HSP 70, heat shock protein
70;
Cy, cyanine.
 |
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