(Received for publication, September 27, 1994)
From the
Mutations in superoxide dismutase 1 (SOD1) have been linked to familial amyotrophic lateral sclerosis, a dominantly inherited motor neuron disorder of midlife. Because SOD1 is a homodimeric enzyme, dimerization of mutant and wild-type SOD1 subunits could dominantly alter the activity, stability, or localization of wild-type SOD1 subunits. To explore these possibilities, we used transient and stable gene transfection to express high levels of either of two mutant human SOD1 subunits in the presence of limited levels of wild-type mouse and/or human SOD1 subunits. Although both mutant subunits displayed diminished half-lives and free radical scavenging activities, their presence caused no change in the half-life or activity of wild-type SOD1 subunits. Our data indicate that mutant subunits do not dominantly affect the function of wild-type SOD1 subunits. These findings, together with observations that many mutant SOD1 subunits retain significant stability and activity, suggest that motor neuron damage in familial amyotrophic lateral sclerosis is caused by the acquisition of injurious properties by mutant SOD1 subunits.
Familial amyotrophic lateral sclerosis (FALS) ()is an
autosomal dominant disorder (1, 2) , in which missense
mutations in the enzyme Cu/Zn superoxide dismutase 1 (SOD1) underlie
disease in a subset of
cases(3, 4, 5, 6, 7, 8) .
SOD1 is a 153-amino acid enzyme, containing copper and zinc cofactors,
which catalyzes the conversion of O
into O
and
H
O
(9, 10, 11) .
Although some FALS mutations either fully or partially diminish the
free radical scavenging activity of the mutant enzymatic
subunits(3, 12, 13) , other mutants retain
high levels of activity(4, 7, 14) . All
mutations examined to date diminish the stability of SOD1
polypeptides(14) .
The interactions between the subunits of
purified homodimeric SOD1 are very stable in
vitro(11) ; if this is true in vivo, then
dimerization of mutant subunits with wild-type subunits could
negatively affect the function of wild-type subunits to diminish
overall SOD1 activity, providing a basis for the dominant inheritance
of FALS(3) . However, a recent study reported that one line of
transgenic mice expressing an FALS-linked human (Hu) SOD1 mutant
develop motor neuron disease, despite elevated levels of total
SOD1 activity(15) . Moreover, we have observed motor neuron
degeneration in transgenic mice that express a HuSOD1 mutant
(HuSOD1-G37R) ()which retains high activity and significant
half-life(14) . Since expression of wild-type HuSOD1 at high
levels does not cause motor neuron disease, the simplest interpretation
of these data is that the mechanism by which mutant subunits cause
motor neuron death involves an acquisition of injurious properties.
However, it is important to note that disease was absent in transgenic
mice expressing the codon 4 (Ala
Val) FALS variant of HuSOD1,
despite robust transgene expression(15) . As the Ala
Val
mutation diminishes both the half-life and specific activity of the
mutant enzyme subunit(14) , it is possible that where mutations
do not substantially diminish enzyme activity or longevity, the
mechanism of motor neuron degeneration involves a gain of injurious
property by the mutant enzyme, whereas when enzyme function is severely
compromised, it is a loss of free radical scavenging activity which
underlies disease. Indeed, the chronic inhibition of SOD1 in rat spinal
cord organotypic cultures causes apoptotic death of
neurons(16) . If loss of SOD1 activity underlies disease in
those SOD1-linked FALS cases in which the activity or half-life of the
mutant enzyme is severely compromised, then it remains possible that
dimerization of mutant subunits with wild-type subunits causes a
diminution in the activity and/or stability of wild-type subunits and
that this phenomenon is an important factor in disease.
To test whether mutant SOD1 subunits can affect the activity or stability of wild-type subunits, we used transient and stable transfection of primate and murine cells to express high levels of mutant HuSOD1 subunits in the presence of wild-type mouse (Mo) or Hu SOD1 subunits to favor the dimerization of newly synthesized wild-type subunits with newly synthesized mutant subunits. For these studies, we selected two mutants (G41D and G85R) that possess little or no activity and markedly diminished polypeptide half-life(14) . Because SOD1 dimers are very stable molecules in vitro(11, 17) , we hypothesized that newly synthesized SOD1 subunits might dimerize shortly after synthesis and remain associated until degradation of the dimer. However, our data indicate that, in one case (HuSOD1-G85R), mutant SOD1 subunits do not form stable dimers with wild-type SOD1 subunits. More importantly, in the other case (HuSOD1-G41D), mutant and wild-type subunits readily dissociated and exchanged in vivo. These data indicate that even severely compromised SOD1 mutants lack the ability to influence the activity or longevity of wild-type SOD1 subunits.
The wild-type and mutant HuSOD1 cDNA expression plasmids used in this study have been described previously(14) .
Stably transfected lines
of mouse neuroblastoma N2a cells were derived by selection with the
drug G418, following transfection (18) with the wild-type and
mutant (G41D) pHGSOD-SVneo plasmids described above. The pHGSOD-SVneo
plasmid contains a 3-kilobase fragment that contains a transcription
unit for the gene that confers resistance to the neomycin analog
G418(19) . Colonies were screened for expression of wild-type
and mutant HuSOD1 by metabolic radiolabeling for 20 min with
[S]cysteine and immunoprecipitation with SOD1
antibodies as described(14) .
To determine whether mutant subunits can affect the activity or stability of wild-type SOD1 subunits, we focused on two previously characterized mutants (G41D and G85R), which display diminished specific activity (50 and 100% reduction) and polypeptide half-life (60 and 70% reduction)(14) . Each mutation substitutes a charged amino acid for uncharged glycine, altering the rate of electrophoretic migration of the dimeric holoenzyme on SOD1 assay gels(14) . Transient expression of wild-type and mutant (G37R and G41D) HuSOD1 in COS-1 cells yielded novel free radical scavenging activities in such assay gels (Fig. 1, lanes 2 and 3, filled and open arrowheads, HuSOD1 homodimer and human/monkey heterodimer, respectively); no activity could be detected for HuSOD1-G85R (lane 4), as noted in a previous study(14) . To demonstrate that cotransfection of two unlinked plasmids yielded coexpression in each transfected cell, plasmids encoding wild-type and G37R HuSOD1 were mixed, transfected, and SOD1 activity assessed by the gel assay (Fig. 1, lane 5). Previous studies of lymphoblasts from heterozygous FALS (G37R) individuals demonstrated the ability of HuSOD1-G37R subunits to heterodimerize with wild-type subunits(14) . The appearance of activities (lane 5, open arrow) that migrate between homodimeric wild-type HuSOD1 (filled arrow) and homodimeric HuSOD1-G37R (filled arrowhead) in these assay gels clearly establishes that mutant and wild-type expression plasmids enter cells simultaneously and are coexpressed, yielding heterodimeric wild-type/G37R HuSOD1(14) . Activities with the mobility of mutant and wild-type human SOD1 heterodimers were also detected after cotransfection of wild-type and G41D HuSOD1 expression plasmids (lane 6, open arrow). Heterodimers of human and monkey SOD1 (open arrowheads) were detected in all transfections.
Figure 1: SOD1 activity assay gel of transiently transfected COS-1 cells. COS-1 cells (60-mm dish) were transiently transfected with wild-type or mutant SOD1 expression plasmids as described under ``Experimental Procedures.'' After 48 h, the cells were lysed, and SOD1 activities were assayed by activity gel assay (20) . Lane 1 contains a lysate of cells transfected with vector only. Lanes 2-4 contain lysates of cells transfected with 10 µg of HuSOD1-G37R, HuSOD1-G41D, and HuSOD1-G85R expression plasmids, respectively. Lanes 5-7 contain lysates of cells transfected with mixtures of wild-type (wt, 2 µg) and mutant (10 µg) expression plasmid DNA (lane 5, G37R + wild-type; lane 6, G41D + wild-type; lane 7, G85R + wild-type). Lane 8 contains a lysate of cells transfected with a mixture of wild-type HuSOD1 (2 µg) and pEF-BOS.humanAPP592-695 (10 µg) expression plasmids. Heterodimeric mutant/wild-type HuSOD1 species (open arrows) were identified on the basis of electrophoretic mobility between homodimeric species of the contributing subunits(17, 19) ; filled arrows, homodimeric wild-type HuSOD1; filled arrowheads, homodimeric mutant HuSOD1; open arrowheads, heterodimeric human/monkey SOD1.
We focused initially on the G85R subunit, reasoning
that if the inactive G85R subunit could stably combine with wild-type
SOD1, then the combination of the G85R charge variant subunit with
active wild-type subunits should create a novel species of activity
(like that observed for G37R and wild-type subunits in lane 5, open arrow). Alternatively, the mutant subunit could diminish
(or eliminate) the activity of the wild-type subunit when present in
heterodimers. Neither of these outcomes was observed as the only active
enzyme we detected in cotransfections of G85R and wild-type (lane
7) comigrated with the wild-type enzyme generated in control
transfections (lane 8). Moreover, the level of wild-type
HuSOD1 homodimer activity was undiminished (compare lanes 7 and 8) even when five times more G85R expression plasmid,
relative to wild-type expression plasmid, was used in the transfection
mixture. Metabolic radiolabeling and immunoprecipitation studies
confirmed that the synthetic rate of G85R subunits in these experiments
exceeded that of wild-type subunits by 3-4-fold (Fig. 2).
After cotransfection with wild-type SOD1 and G85R HuSOD1, the cells
were metabolically radiolabeled with [S]cysteine
for 20 min and then incubated in unlabeled medium for varied periods
followed by lysis and immunoprecipitation with SOD1 antiserum. As the
G85R SOD1 subunits display accelerated electrophoretic migration on
SDS-polyacrylamide gel electrophoresis relative to wild-type
polypeptides (14; Fig. 2, compare lanes 1 and 5) the synthetic rate of HuSOD1-G85R wild-type subunits could
easily be determined. PhosphorImaging showed that the synthesis of
mutant subunits exceeded the rate of wild-type subunits by
3-4-fold (Fig. 2, lane 9). Further analysis of
labeled SOD1 remaining in cells after incubation in unlabeled medium
demonstrated that the degradation of the G85R and wild-type
polypeptides followed independent kinetics (lanes 9-12),
with each displaying a half-life indistinguishable from that of each
individual polypeptide when expressed alone (lanes 1-8).
Data from two determinations are displayed graphically below the
figure.
Figure 2:
Degradation of SOD1 subunits in
transiently transfected COS-1 cells. COS-1 cells were transiently
transfected with a mixture of wild-type (wt) HuSOD1 (2 µg)
and HuSOD1-G85R (10 µg) expression plasmids, as well as each
plasmid separately, then metabolically radiolabeled for 20 min as
described under ``Experimental Procedures.'' After varied
periods of chase, SOD1 polypeptides were extracted and
immunoprecipitated with SOD1 peptide antiserum as described under
``Experimental Procedures.'' Lanes 1-4 contain
wild-type HuSOD1 polypeptides immunopurified from cells after 0, 12,
24, and 48 h of chase, respectively. Lanes 5-8 contain
HuSOD1-G85R polypeptides immunopurified from cells after 0, 12, 24, and
48 h of chase, respectively. Lanes 9-12 contain SOD1
polypeptides from cells transfected with a mixture of wild-type and
G85R expression plasmids after 0, 12, 24, and 48 h of chase,
respectively. The amount of radioactive SOD1 polypeptide in these gels
was quantified on a Molecular Dynamics PhosphorImager. To normalize
values from various experiments, each value for each time point was
converted into a percentage of the maximum value (in most cases this
maximum value was the value at time 0 h of chase, but in some cases the
12-h chase value was slightly higher). The normalized values were
plotted on a log scale and fit to a curve that assumes
exponential decay, using Cricket Graph III software. The graphs for
wild-type alone and HuSOD1-G85R alone represent data collected from
three determinations; the graphs for wild-type + HuSOD1-G85R
represent data from two determinations.
, HuSOD1-wt;
,
HuSOD1-G85R.
Previous studies have established that Hu- and MoSOD1 form
active heterodimers(17, 19) . To examine interactions
between HuSOD1-G41D subunits and wild-type SOD1, we stably transfected
mouse neuroblastoma N2a cells with a 14-kilobase human genomic DNA
fragment, contained within the plasmid pHGSOD-SVneo (17, 19) encoding either wild-type SOD1 or a gene
mutated to encode HuSOD1-G41D. Mutant and wild-type pHGSOD-SVneo
plasmids yielded a similar number of G418-resistant colonies. Cloned
cell lines were screened by metabolic radiolabeling with
[
S]cysteine and immunoprecipitation with SOD1
antiserum to identify cultures in which the synthetic rate of human
polypeptides exceeded that of mouse polypeptides (MoSOD1 and HuSOD1
possess 3 and 4 cysteines, respectively). For wild-type HuSOD1, a clone
was identified in which the synthetic rate of human polypeptide was
three to four times greater than mouse polypeptide (Fig. 3, lane 2). For G41D HuSOD1, a colony was isolated in which the
human polypeptide was synthesized at two to three times the rate of
mouse polypeptide (Fig. 3, lane 6). In cells expressing
wild-type HuSOD1, both MoSOD1 and HuSOD1 polypeptides possessed
half-lives of
48 h (Fig. 3, lanes 2-5). In
contrast, the half-life of the HuG41D mutant was
7 h, but the
half-life of the MoSOD1 remained
48 h (Fig. 3, lanes
6-9).
Figure 3:
Degradation of SOD1 subunits in stably
transfected N2a cells. Mouse N2a cells that constitutively express
wild-type (wt) HuSOD1 or HuSOD1-G41D were derived as described
under ``Experimental Procedures.'' Duplicated confluent
cultures in six-well plates were metabolically radiolabeled for 20 min,
and SOD1 polypeptides were immunoprecipitated with peptide SOD1
antiserum that recognizes both Mo- and HuSOD1, as described under
``Experimental Procedures.'' Lane 1 contains MoSOD1
immunoprecipitated from parental N2a cultures after 48 h of chase. Lanes 2-5 contain wild-type HuSOD1 and wild-type MoSOD1
immunoprecipitated from cultures after 0, 12, 24, and 48 h of chase. Lanes 6-9 contain HuSOD1-G41D and wild-type MoSOD1
immunoprecipitated from cultures after 0, 12, 24, and 48 h of chase.
Plots of SOD1 degradation were generated as described in the legend to Fig. 2. Each graph represents data collected from three
determinations. , HuSOD1-wt;
, MoSOD1-wt;
,
HuSOD1-G41D.
To determine whether HuSOD1(G41D) and MoSOD1 subunits form heterodimers as seen in previous transfection assays with wild-type HuSOD1(17, 19) , extracts of N2a cells expressing wild-type or G41D SOD1 were analyzed by gel assay. In cells expressing the wild-type HuSOD1, nearly all MoSOD1 subunits appeared to be heterodimerized with human SOD1 subunits (Fig. 4, lane 2, open arrow)(17, 19) . In contrast, the level of homodimeric MoSOD1 in cells expressing the G41D mutant (lane 3) was comparable to that of the parental N2a cells (lane 1) despite the high rate of HuSOD1-G41D synthesis (see above). Although the short half-life of HuSOD1-G41D subunits precluded the accumulation of significant levels of activity, low levels of heterodimeric Hu-G41D/MoSOD1 (lane 4, open arrowhead) and homodimeric HuSOD1-G41D (lane 4, filled arrowhead) could be detected.
Figure 4: SOD1 activity assay gel of stably transfected N2a cultures. Lysates of N2a cultures (confluent 60-mm dish) were prepared as described under ``Experimental Procedures.'' The level of SOD1 activity in cell lysates was assessed as described(20) . Lane 1 contains a lysate from parental N2a cells. Lane 2 contains a lysate from N2a cells that constitutively express wild-type (wt) HuSOD1. Lanes 3 and 4 contain lysates from N2a cultures that constitutively express HuSOD1-G41D (lane 4 contains twice the amount of lane 3). Filled arrow, homodimeric HuSOD1. Open arrow, heterodimeric Mo/HuSOD1(17, 19) .
To our knowledge, the dissociation constant of SOD1 subunits is unknown; however, one study has documented the slow exchange of SOD1 subunits in physiologic buffers at 0 °C(22) . To determine the rate of SOD1 subunit exchange at physiologically relevant temperatures, lysates of COS-1 cells (prepared by freeze-thaw in water followed by phosphate and saline buffering) were mixed with purified erythrocyte HuSOD1. Within 10 min at 37 °C, heterodimeric SOD1 activities (Fig. 5, lane 4) with mobility intermediate between homodimeric HuSOD1 and homodimeric COS-1 SOD1 could be detected. The amount of heterodimeric activity increased steadily with time, up to 2 h (Fig. 5, lane 7, open arrow). These data indicate that SOD1 subunits are capable of rapid exchange.
Figure 5: SOD1 subunits exchange rapidly in physiologic buffers at 37 °C. In vitro exchange reactions were prepared as described under ``Experimental Procedures.'' Lane 1, HuSOD1 from erythrocytes. Lane 2, COS-1 lysate. Lanes 3-7, mixtures of COS-1 lysates and HuSOD1 incubated for 0, 10, 30, 60, and 120 min. Lane 8, COS-1 lysate alone incubated for 120 min at 37 °C. Open arrowhead, heterodimeric COS-1/HuSOD1.
In the present study, we demonstrate that the half-lives and free radical scavenging activities of wild-type SOD1 subunits are unaltered in settings in which the rate of mutant (FALS) SOD1 subunit synthesis substantially exceeds that of wild-type subunits. Although both mutants we examined display markedly diminished half-life with reduced specific activity (14) (and thus, when heterodimerized, are good candidates to diminish wild-type subunit activity or stability), neither was found to affect the half-life or activity of wild-type subunits. Whether mutant SOD1 subunits can affect the activity or stability of mutant subunits is largely dependent upon the stability of the dimeric interaction and the relative concentration of each subunit. Because our data indicate that SOD1 subunits exchange rapidly and that mutant subunits are relatively short lived, it becomes nearly impossible for mutant subunits to affect either the activity or stability of wild-type subunits. At equilibrium, a newly synthesized wild-type subunit will have a greater likelihood of dimerizing with a preexisting wild-type subunit (which will be in greater concentration) than a newly synthesized mutant subunit.
The present data suggest
that the mechanism underlying dominant inheritance of FALS does not
involve a diminution of wild-type SOD1 subunit half-life or free
radical scavenging activity as a result of heterodimerization with
mutant subunits. Thus for any heterozygous FALS individual with a
mutation in SOD1, the level of remaining SOD1 activity will be at least
50% of normal. Combined with the finding that some FALS SOD1 mutants
retain wild-type activity and significant stability(14) , we
conclude that it is unlikely that a partial loss of free radical
scavenging activity underlies motor neuron degeneration in FALS. The
simplest interpretation of the biochemical data described here is that
the dominant character of SOD1-linked FALS is due to a gain of some
injurious property by mutant SOD1 subunits. Motor neuron disease in
transgenic mice expressing the FALS variant HuSOD1-G93A, in which a
3-fold increase in total SOD1 activity levels was observed (15) , offers further support for this conclusion, as does
motor neuron disease in transgenic mice expressing the stable and
active G37R FALS SOD1 mutant.
The key questions that remain unresolved are the nature of the gained injurious property(ies) and the basis for the apparent selective targeting of motor neurons. These uncertainties leave open additional possibilities, including one in which wild-type SOD1 possesses activities beyond free radical catabolism in motor neurons and that it is these activities that are most severely diminished by FALS-linked mutations. If SOD1 possesses alternative functions, then it remains possible that interactions between mutant and wild-type subunits, however transient, are sufficient to alter some aspect of wild-type SOD1 function. Examples of this type of process include the URE2 gene product in yeast, where a conformationally altered form of URE2 interacts with wild-type URE2, causing conformational changes that diminish function(23) . Other examples of these phenomena include the p53 oncogene where dimerization of mutant and wild-type p53 subunits causes the wild-type subunit to acquire the nonfunctional conformation of mutant subunits(24) . Both of these mechanisms share features with the apparent mechanism of prion replication(25, 26) . However, our data indicate that mutant subunits do not alter the free radical scavenging activity of wild-type subunits. The absence of evidence for alternative SOD1 functions or activities favors the idea that rather than loss of free radical scavenging (or other) activities, mutant SOD1 subunits cause motor neuron degeneration by gain of as yet undefined deleterious properties.