©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Superoxide Dismutase 1 Subunits with Mutations Linked to Familial Amyotrophic Lateral Sclerosis Do Not Affect Wild-type Subunit Function (*)

(Received for publication, September 27, 1994)

David R. Borchelt (1)(§)(¶) Michael Guarnieri (1) Philip C. Wong (1) Michael K. Lee (2)(**) Hilda S. Slunt (1) Zuo-Shang Xu (2)(§§) Sangram S. Sisodia (1) Donald L. Price (1)(§) (3) (4)(¶¶) Don W. Cleveland (2) (3)(A)

From the  (1)Departments of Pathology, (2)Biological Chemistry, (3)Neuroscience, and (4)Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Familial amyotrophic lateral sclerosis (FALS) (^1)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 HO(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) (^2)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.


EXPERIMENTAL PROCEDURES

Human sod-1 Genomic DNA Mutagenesis

The FALS-linked G41D mutation was introduced, by polymerase chain reaction strategies, into a plasmid clone of the human genomic sod-1 gene (pHGSOD-SVneo), which contains the entire human sod-1 gene within a 14-kilobase DNA fragment flanked by EcoRI and BamHI restriction sites(17) . Briefly, a 2-kilobase PstI-XbaI restriction endonuclease fragment of human sod-1 DNA, which encompasses all of exon 2, was excised and subcloned into pBluescript (Stratagene Inc, La Jolla, CA). The G41D mutation was then introduced using a three-primer polymerase chain reaction involving a mutated synthetic oligonucleotide that spans codon 41 and two primers that are complementary to sequences in the plasmid vector. The amplified product was digested with PstI and XbaI and inserted into pBluescript. Plasmids carrying mutated sequences were initially identified by restriction enzyme digestion (the G41D mutation destroys a StuI restriction endonuclease site), with additional confirmation by nucleotide sequence analysis of the entire exon 2 and flanking mRNA splicing signals. The complete gene was reassembled by ligating an NdeI-XbaI subfragment of the mutant plasmid with EcoRI-NdeI and XbaI-EcoRI fragments of the parent pHGSOD-SVneo plasmid in a three-way ligation. The integrity of the reassembled plasmids was verified by restriction endonuclease digestion.

The wild-type and mutant HuSOD1 cDNA expression plasmids used in this study have been described previously(14) .

Expression of HuSOD1 in Cultured Cells

Two cultured cell lines were used for transient and stable gene transfection studies. In COS-1 cells, previously described pEF-BOS expression plasmids encoding wild-type and mutant human SOD1 cDNAs were used(14) . Transient transfection was performed using methods described by Chen and Okayama(18) . In experiments to coexpress wild-type and mutant HuSOD1, expression plasmids were mixed at a ratio of 1:5 (wild-type to mutant). As a control, wild-type HuSOD1 expression plasmid was mixed (1:5) with a pEF-BOS expression plasmid that codes for a deletion mutant of human APP695 comprising the NH(2)-terminal 22 codons and the COOH-terminal 104 codons of the human (Alzheimer) amyloid precursor protein. Experimental manipulations, such as metabolic radiolabeling or analysis of SOD1 activity, on these transiently transfected COS-1 cells were carried out 48 h after transfection.

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) .

Assessment of SOD1 Activity

To assess the level of SOD1 free radical scavenging activity, extracts of cells were prepared by freeze-thaw lysis in 0.125 M Tris-Cl, pH 6.8, with 20% glycerol, 0.025% bromphenol blue, and 0.1% Nonidet P-40. After centrifugation at 10,000 times g for 5 min, the supernatant was separated by electrophoresis on 7.5% polyacrylamide gels, and SOD1 activities were determined as described previously(14, 20) .

Assessment of SOD1 Polypeptide Half-life

The half-lives of SOD1 polypeptides were measured by pulse-chase metabolic radiolabeling with [S]cysteine, followed by immunoprecipitation of labeled SOD1, polyacrylamide gel electrophoresis separation, and PhosphorImaging. In transiently transfected COS-1 cells, a single mixture of wild-type or mutant SOD1 encoding DNA was transfected into a 60-mm dish of COS-1 cells using the reagent DOTAP (Boehringer Mannheim) or the methods of Chen and Okayama(18) . Twenty-four h after transfection, the cells were split into four replicate 2-cm dishes. After an additional 24 h, the cells were labeled, and SOD1 polypeptides were immunoprecipitated with either of two previously described polyclonal antisera raised against intact erythrocyte HuSOD1 polypeptide, or a synthetic HuSOD1 peptide (amino acids 125-137; DDLGKGGNEESTK), which is completely identical in MoSOD1 and HuSOD1(14) . Both antisera gave identical results. The SOD1(125-137) peptide antiserum recognized both HuSOD1 and MoSOD1, with only weak reactivity for COS-1 SOD1, whereas the antiserum to erythrocyte HuSOD1 recognized all three species of SOD1. The immunoreactivity of these antisera was fully competed by excess peptide or whole protein, respectively (not shown). Immunoprecipitated polypeptides were separated by SDS-polyacrylamide gel electrophoresis (21) and visualized by autoradiography. Radiolabeled SOD1 polypeptide bands were quantified with a Molecular Dynamics (Mountain View, CA) PhosphorImager. To estimate half-lives of the wild-type HuSOD1 and each mutant, the percent of radiolabeled SOD1 polypeptides remaining in the cells after a given period of chase was plotted on a log scale and fit to a curve with the assumption that degradation follows first-order kinetics (Cricket Graph III, Computer Associates International, Inc.).

In Vitro Assay of SOD1 Subunit Exchange

Naive COS-1 cells were lifted from culture dishes by treatment with 5 mM EDTA in phosphate-buffered saline. The cells were pelleted by centrifugation at 3,000 times g for 5 min then resuspended in 5-10 volumes (packed cells) of deionized water. The cells were lysed by two cycles of freeze-thaw. After centrifugation at 3,000 times g for 10 min, the supernatant was adjusted to 10 mM NaPO(4), 3 mM KCl, 2 mM KPO(4), and 140 mM NaCl. The supernatant was divided into 30-µl portions, and 200 ng of purified human erythrocyte SOD1 (Sigma) was added. After varied periods of time at 37 °C, the reactions were chilled to 4 °C and loaded onto SOD1 assay gels.


RESULTS

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. box, 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 geq 48 h (Fig. 3, lanes 2-5). In contrast, the half-life of the HuG41D mutant was leq 7 h, but the half-life of the MoSOD1 remained geq 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. box, HuSOD1-wt; circle, 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.




DISCUSSION

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.^2

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.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants AG 05146, NS 20471, NS 27036, and NS 22849 and by the American Health Assistance Foundation and the Amyotrophic Lateral Sclerosis Association (made possible by a gift from Curt and Shonda Schilling). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipients of Leadership and Excellence in Alzheimer's Disease Award AG 07914.

To whom correspondence should be addressed: Dept. of Pathology, Johns Hopkins Medical Institutions, 720 Rutland Ave., 558 Ross Bldg., Baltimore, MD 21205-2196. Tel.: 410-955-5632; Fax: 410-955-9777.

**
Supported by a National Institutes of Health postdoctoral fellowship.

§§
Supported by a Muscular Dystrophy Association postdoctoral fellowship.

¶¶
Recipient of Javits Neuroscience Investigator Award NS 10580.

A
Recipient of Javits Neuroscience Investigator Award NS 27036.

(^1)
The abbreviations used are: FALS, familial amyotrophic lateral sclerosis; SOD1, Cu/Zn superoxide dismutase 1; Hu, human; Mo, mouse.

(^2)
P. C. Wong, C. A. Pardo, D. R. Borchelt, M. K. Lee, N. G. Copeland, N. A. Jenkins, S. S. Sisodia, D. W. Cleveland, and D. L. Price, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Shigekazu Nagata for graciously providing the pEF-BOS expression plasmid and Dr. Yoram Groner for providing the pHGSOD-SVneo plasmid.


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