Mutant Cu/Zn-Superoxide Dismutase Proteins Have Altered Solubility and Interact with Heat Shock/Stress Proteins in Models of Amyotrophic Lateral Sclerosis*

Gayle A. Shinder, Marie-Claude Lacourse, Sandra Minotti, and Heather D. DurhamDagger

From the Department of Neurology/Neurosurgery and Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada

Received for publication, November 29, 2000, and in revised form, January 11, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis. In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-1 protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-1 and reduced formation of aggregates. In this paper, we report that mutant SOD-1 proteins have altered solubility in cells relative to wild-type SOD-1 and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-1 was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and alpha B-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation. Mutant SOD-1·HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-1 was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amyotrophic lateral sclerosis (ALS)1 is an adult-onset neurodegenerative disease characterized by death of motor neurons in the cerebral cortex, brain stem, and spinal cord. Approximately 10% of ALS cases are familial, and about 20% of these familial cases are caused by dominantly inherited mutations in the gene encoding the enzyme Cu/Zn-superoxide dismutase (SOD-1) (1). Evidence indicates that some "toxic gain of function" is responsible for motor neuron loss rather than a decreased ability of mutant SOD-1 to carry out its primary enzymatic function, dismutation of superoxide to hydrogen peroxide. Most SOD-1 mutant proteins have significant enzymatic activity, and any impairment relative to wild-type does not correlate with clinical severity of the disease (2, 3). SOD-1 knockout mice do not develop motor neuron disease (4), whereas mice expressing mutant SOD-1 transgenes develop an ALS-like phenotype (5-7).

Over 60 different mutations have been identified and associated with ALS (reviewed in Ref. 8). The distribution of such a large number of disease-causing mutations throughout all exons of the SOD-1 gene suggests that altered protein conformation may contribute to the toxic gain of function. Additional evidence comes from x-ray crystallography showing that conserved interactions within the protein that determine the conformation of the active channel are altered in mutant SOD-1 proteins (9). How this contributes to toxicity is not clear. Improperly folded SOD-1 enzyme could allow greater access of abnormal substrates to the active site; could enhance other known enzymatic functions of SOD-1, such as catalysis of protein nitration; or could alter metal binding, all of which would ultimately lead to increased oxidative damage (reviewed in Refs. 10-12). Alternatively, proteins with altered conformation could form insoluble precipitates or participate in abnormal protein-protein interactions unless prevented from doing so by association with chaperone proteins. Thus, depletion of chaperones could result in formation of mutant SOD-1 aggregates, as well as compromising cellular chaperoning function in general (13).

Mutant SOD-1 proteins appear to be degraded by the proteasome, a pathway generally involved in proteolysis of denatured or misfolded protein (14, 15). Moreover, several mutant SOD-1 proteins have a tendency to precipitate in solution relative to wild-type (16). Proteinaceous aggregates of SOD-1 have been found in the cytoplasm of cultured primary motor neurons expressing several different SOD-1 mutants (17), in motor neurons and astrocytes of mice expressing mutant SOD-1 transgenes (7, 18), in familial ALS patients at autopsy (19, 20), and in transfected cell lines (15, 21). In cultured motor neurons, coexpression of HSP70 and mutant SOD-1 expression vectors considerably reduced formation of SOD-containing aggregates and prolonged viability (13). Mutant SOD-1-transfected NIH3T3 cell lines showed up-regulation of HSP70, HSP25, and alpha B-crystallin compared with untransfected cells or cells transfected with the wild-type SOD-1 construct (13). Heat shock proteins could protect cells from mutant SOD-1 by direct interaction, thus preventing aggregation and facilitating degradation, or indirectly by protecting cells from downstream consequences of some other toxic property.

In this study, we have demonstrated that mutant SOD-1 coimmunoprecipitates with HSP70, HSP40, and alpha B-crystallin. Furthermore, a portion of mutant SOD-1 was detergent-insoluble in three different experimental models of familial ALS: NIH3T3 cell lines stably or transiently transfected with mutant SOD-1 constructs, spinal cord cultures derived from G93A transgenic embryonic spinal cords, and lumbar spinal cord from adult G93A transgenic mice.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies and Plasmids-- The following primary antibodies were used: Rabbit anti-SOD-1 antibodies (SOD-100 diluted 1:500 and SOD-101 diluted 1:250, Stressgen Biotechnologies Corp., Victoria, British Columbia, Canada) (SOD-100 reacts with both human and mouse SOD-1 but favors human SOD-1; SOD-101 also reacts with both human and mouse SOD-1 but favors mouse SOD-1); rabbit anti-alpha B-crystallin (SPA-223) (1:250) and rabbit anti-HSP40 (SPA-400) (1:1000) antibodies from Stressgen Biotechnologies Corp.; mouse monoclonal HSP70 antibody (W27 catalog number SC-24, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1:250) (may cross-react with HSC70); mouse monoclonal actin antibody, clone C4 (691001) (1:1000) and rabbit polyclonal tubulin antibody (650951) (1:500) from ICN Biomedicals Inc. (Irvine, CA). Secondary antibodies used were goat anti-rabbit horseradish peroxidase (P0399, Dako Corp., Mississauga, ON) (1:3000) and sheep anti-mouse horseradish peroxidase (515035062, Jackson ImmunoResearch Laboratories, Willow Grove, PA) (1:10,000). cDNA encoding human HSP70 was a kind gift from D. Mosser and was subcloned into pcDNA3.

NIH3T3 Cell Lines-- NIH3T3 cell lines stably expressing human SOD-1 gene constructs (wild-type or with G93A or G41S mutations) have been described previously (13). Cultures were maintained under standard conditions in minimum essential medium supplemented with 3.7 g of NaHCO3, 5 g of dextrose, and 10% fetal bovine serum. Stable cell lines were supplemented with G418 (Life Technologies, Inc.) (250 µg/ml for G41S and G93A lines and 700 µg/ml for the wild-type line). For transient transfection, NIH3T3 cells were grown to 80-100% confluency, subcultured at a density of 2 × 105 cells/35-mm culture well, and transfected with the G93A-SOD-1/pcDNA3 construct according to the standard protocol for LipofectAMINETM reagent (Life Technologies, Inc.). Protein extracts were prepared 43 h after transfection (see below).

Transgenic Mice-- Two lines of mice transgenic for G93A mutant human SOD-1, B6SJL-TgN(SOD1-G93A)1Gur and B6SJL-TgN(SOD1-G93A)1Gurdl, as well as one line of mice transgenic for wild-type human SOD-1, B6SJL-TgN(SOD-1)2Gur, were maintained in our animal facility (breeding pairs were purchased from The Jackson Laboratory, Bar Harbor, ME). All experiments were approved by the McGill University Animal Care Committee and followed the guidelines of the Canadian Council on Animal Care. B6SJL-TgN(SOD1-G93A)1Gur (fast line) mice develop paralysis in the limbs and die at 4-5 months of age. B6SJL-TgN(SOD1-G93A)1Gurdl (slow line) mice develop paralysis in the limbs and die at 6-7 months of age.

Single Embryo Spinal Cord Cultures-- Cultures were prepared from 13-day-old mouse embryos as previously described (22) except that spinal cords from transgenic mouse embryos were dissociated and plated individually at a density of 650,000/35-mm culture well. Embryos were genotyped for the presence of human SOD-1 transgenes using the polymerase chain reaction method provided by The Jackson Laboratory.

Preparation of Detergent-soluble and Detergent-insoluble Protein Extracts-- NIH3T3 cell lines and spinal cord cultures were lysed on ice in phosphate-buffered saline, pH 8.0, containing 0.5% Nonidet P-40, 0.2% digitonin, and 0.23 mM phenylmethylsulfonyl fluoride (lysis buffer) and centrifuged to obtain detergent-soluble supernatant and detergent-insoluble pellet fractions. Pellets were washed three times in lysis buffer and then solubilized in 1× sample buffer (55 mM Tris, pH 6.8, 10% glycerol, 1% SDS, 5% beta -mercaptoethanol, and 0.04% bromophenol blue). The lumbar region of mouse spinal cord was homogenized in 500 µl of lysis buffer and centrifuged at 15,000 × g for 15 min at 4 °C. After collection of supernatants, pellets were washed five times with lysis buffer and then resuspended in 100 µl of 1× sample buffer. Protein concentrations of supernatants were determined by Bio-Rad DC protein assay.

Western Blotting-- After electrophoresis, proteins were transferred to nitrocellulose (Bio-Rad Trans-Blot® transfer medium). Membranes were blocked overnight at 4 °C in TBST-milk (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20, 5% skim milk powder). Incubations in primary and secondary antibodies were for 1 h at room temperature. Labeled proteins were detected using the Renaissance® Western blot chemiluminescence reagent (PerkinElmer Life Sciences).

Immunoprecipitation-- NIH3T3 cell lines were lysed in 700 µl of lysis buffer containing 9% Me2SO and then incubated for 1 h on ice and for 10 min at room temperature. Following the addition of 70 µl of 50 mM glycine, cells were centrifuged at 15,000 × g, and supernatants were collected. For total cell extracts, the centrifugation step was eliminated. For immunoprecipitation, 3 µl of SOD-1 antibody (SOD-100), 12.5 µl of HSP70 antibody, 5 µl of HSP40 antibody, or 5 µl of alpha B-crystallin antibody was added to 400 µg of protein and incubated at 4 °C overnight on a rotating platform. Immune complexes were captured on 40 µl of protein G-Sepharose, 4 Fast Flow (Amersham Pharmacia Biotech) by incubation on a rotating platform at 4 °C for 3 h; the beads were washed with lysis buffer, resuspended in 30 µl of 1× sample buffer, and boiled for 5 min. The beads were pelleted, half of the supernatant was electrophoresed on a 12.5% SDS-polyacrylamide gel, and immunoprecipitated proteins were detected by Western blotting.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Altered Solubility of Mutant Human SOD-1 in Transfected NIH3T3 Cells-- The tendency of mutant SOD-1 proteins to form aggregates in cultured cells and spinal cord tissue suggested that they might distribute differently in subcellular compartments compared with wild-type enzyme. NIH3T3 cell lines stably expressing human wild-type, G41S mutant, or G93A mutant SOD-1, established for previous studies (13), were used to compare the solubility of wild-type and mutant SOD-1 in nonionic detergent. Detergent-soluble supernatant and detergent-insoluble pellet fractions were prepared from these cell lines, as well as from untransfected NIH3T3 cells, and subjected to SDS-polyacrylamide gel electrophoresis. The endogenous murine, wild-type human, and mutant human SOD-1 proteins were detected by Western blotting. A blot probed with SOD-1 antibody (SOD-100, which has a higher affinity for human SOD-1 relative to mouse) is shown in Fig. 1. Endogenous murine SOD-1 and wild-type human SOD-1 were detected only in the supernatant, not in the pellet fraction. In contrast, G41S and G93A mutant human SOD-1 were present in both fractions. Larger amounts of pellet fraction were loaded on the gel relative to supernatant to demonstrate absence of wild-type and presence of mutant SOD-1 in the pellet. Using NIH Image software to compare relative band densities on Western blots and correcting for differential loading of sample, it was estimated that about 1% of total cellular mutant SOD-1 was detergent-insoluble in this experiment. This estimate was consistent with the relative band intensities observed when equal percentages of the total volume of supernatant and pellet fractions were loaded on the gel (data not shown).


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Fig. 1.   In stable transfectants of NIH3T3 cells, a proportion of mutant human SOD-1, but not wild-type human or mouse SOD-1, is insoluble in nonionic detergent. Cultures of untransfected cells (NIH3T3) and lines expressing wild-type-human SOD-1 (WT-SOD-1), G41S mutant SOD-1, or G93A mutant SOD-1 were lysed in buffer containing nonionic detergent. Shown are supernatant (S) and pellet (P) fractions after electrophoresis on a 12.5% SDS-polyacrylamide gel and Western blotting with the anti-SOD-1 antibody SOD-100, which binds preferentially to human SOD-1 (hSOD-1) but also labels the endogenous mouse SOD-1 (mSOD-1). Both mutant human SOD-1 proteins were detected in the pellet as well as the supernatant fraction.

Because Ratovitski et al. (3) reported that several different mutant SOD-1 proteins expressed in COS cells by transient transfection were essentially soluble in nonionic detergent, the experiment was repeated using NIH3T3 cells transiently transfected with the G93A mutant SOD-1 construct. As shown in Fig. 2A, mutant human SOD-1, but not murine SOD-1, was detected in the detergent-insoluble pellet by Western blotting with antibody SOD100 and was estimated to correspond to about 10% of total cellular mutant SOD-1. The absence of mouse SOD-1 in the pellet fraction was confirmed using a SOD-1 antibody with greater specificity for mouse SOD-1 (SOD101) (Fig. 2B). Reprobing the blots shown in Fig. 1 with antibody SOD101 also failed to detect murine SOD-1 in detergent-insoluble fractions of any cell line (not shown).


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Fig. 2.   In transient transfectants of NIH3T3 cells, a proportion of mutant SOD-1, but not endogenous mouse SOD-1, is insoluble in nonionic detergent. Two days after transfection with the G93A SOD-1 construct, cultures were lysed in buffer containing nonionic detergent. Shown are supernatant (S) and pellet (P) fractions of two separate transfectants (#1 and #2) after electrophoresis on a 12.5% SDS-polyacrylamide gel and Western blotting with anti-SOD-1 antibody: SOD-100, which binds preferentially to human SOD-1 (hSOD-1) but also binds to the endogenous mouse SOD-1 (mSOD-1) (A) and SOD-101, which binds preferentially to mouse SOD-1 (B). A fraction of mutant human SOD-1, but not the endogenous enzyme, was detected in the pellet fraction.

Altered Solubility of Mutant Human SOD-1 in Spinal Cord Cultures and Spinal Cord Tissue-- Whereas cell lines are convenient models for biochemical analysis, the properties of mutant protein in these cells do not necessarily reflect properties in primary tissue affected by the disease. We examined the solubility of G93A mutant SOD-1 in primary spinal cord cultures prepared from G93A and wild-type human SOD-1 transgenic mouse embryos (Fig. 3) and in lumbar spinal cord isolated from adult, presymptomatic (214-day-old) transgenic mice (Fig. 4). These experiments confirmed our findings in transfected NIH3T3 cells in that both detergent-soluble and detergent-insoluble forms of G93A SOD-1 were detected. In extracts of primary cultures, but not lumbar spinal cord, a small amount of transgenic wild-type SOD-1 was detected in the detergent-insoluble pellet fraction, but this amount was considerably less than the amount of mutant protein detected. Endogenous mouse SOD-1 was not detected in the detergent-insoluble pellet.


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Fig. 3.   A proportion of mutant human SOD-1 from spinal cord cultures prepared from G93A SOD-1 transgenic mouse embryos is detergent-insoluble. Spinal cord cultures were prepared from G93A SOD-1 (fast line) or wild-type (WT) human SOD-1 transgenic mouse embryos. After 3-4 weeks in vitro, cultures were lysed with buffer containing nonionic detergent. Distribution of SOD-1 in the supernatant (S) and pellet (P) after electrophoresis on 12.5% SDS-polyacrylamide gels and Western blotting with SOD-100 antibody. G41S SOD-1/NIH3T3: supernatant fractions from the NIH3T3 line stably expressing G41S mutant human SOD-1 were run as a control to identify human (h) and mouse (m) SOD-1. To compare protein loading, blots were reprobed with antibody to tubulin, a protein that distributes to both detergent-soluble and detergent-insoluble fractions. Shown are samples from three different cultures for each transgenic mouse line.


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Fig. 4.   A proportion of mutant human SOD-1 isolated from lumbar spinal cord of G93A SOD-1 transgenic mice is detergent-insoluble. Lumbar spinal cords from 190 day-old wild-type (WT) and G93A SOD-1 (slow line) transgenic mice were homogenized in lysis buffer containing nonionic detergent. Shown is the distribution of human (h) and mouse (m) SOD-1 in the supernatant (S) and pellet (P) fractions after electrophoresis on 12.5% SDS-polyacrylamide gels and Western blotting with SOD-100 antibody. To compare protein loading, blots were reprobed with antibody to tubulin, a protein that distributes to both detergent-soluble and detergent-insoluble fractions.

Coimmunoprecipitation of Mutant SOD-1 and HSP70-- In response to different types of stresses, cells up-regulate production of various heat shock/stress proteins. When NIH3T3 cells were transfected with G41S or G93A mutant human SOD-1, many of the cells died; however, those cells that were able to survive and were selected as stable transfectants had elevated levels of heat shock proteins as compared with wild-type-transfected and untransfected cells (13). Furthermore, coexpression of HSP70 and mutant SOD-1 expression vectors protected primary cultured motor neurons from toxicity of several different SOD-1 mutants, reducing formation of SOD-containing aggregates and prolonging viability (13). To determine whether heat shock proteins protect cells from mutant SOD-1 by direct protein-protein interaction, coimmunoprecipitation experiments were performed. Because NIH3T3 cell lines expressing mutant SOD-1 have elevated endogenous levels of heat shock proteins (13), this model was best suited for this purpose. When total cell extracts were immunoprecipitated with antibody to SOD-1 (SOD100), HSP70 was coimmunoprecipitated from both G93A and G41S expressing cell lines but not from untransfected NIH3T3 or lines expressing wild-type human SOD-1 (Fig. 5A). Immunoprecipitation of total cell extracts with antibody to HSP70 (W27) pulled down G93A and G41S mutant SOD-1 but not wild-type human or endogenous murine SOD-1 (Fig. 5B). The same results were obtained when the cross-linking agent, dithiobis(succinimidyl propionate), was used to stabilize HSP-SOD interactions (data not shown). HSP70·mutant SOD-1 complexes were distributed mainly in the detergent-insoluble fraction. Regardless of whether anti-SOD-1 or anti-HSP70 antibodies were used for immunoprecipitation, the amount of HSP70 or mutant SOD-1, respectively, coprecipitated from detergent-soluble supernatants was minimal compared with the amount coprecipitated from total cell extracts. This was not due to absence of HSP70 in supernatants of cell lines expressing mutant SOD-1; HSP70 was immunoprecipitated by W27 anti-HSP70 antibody from supernatant fractions of both mutant cell lines (Fig. 5C) and was detected in both detergent-soluble supernatant and detergent-insoluble pellet fractions of nonimmunoprecipitated samples (Fig. 5D). Similarly, G41S and G93A mutant SOD-1 were localized to both supernatant and pellet fractions (Fig. 1) and were immunoprecipitated from supernatants by the SOD100 antibody (not shown).


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Fig. 5.   A-C, SOD-1 mutants, but not wild-type human or mouse SOD-1, interact with the stress protein HSP70. Total cell extracts or detergent-soluble extracts (supernatant) of NIH3T3 cell lines were immunoprecipitated (IP) with either SOD-1 antibody (SOD-100) (A) or HSP70 antibody (W27) (B and C). Proteins were separated on 12.5% SDS-polyacrylamide gels, and Western blots were probed for the presence of HSP70 (A and C) and SOD-1 (B). Ig indicates the immunoglobulin light chain of immunoprecipitating antibody. Also shown is nonimmunoprecipitated (not IP'd) G41S detergent-soluble extract to show the location of human (h) and mouse (m) SOD-1 and HSP70. D, HSP70 is up-regulated in NIH3T3 cell lines stably expressing human SOD-1 and is partially detergent-insoluble in the presence of mutant SOD-1. Cultures of untransfected cells (NIH3T3), a cell line expressing wild-type human SOD-1 (WT) and lines expressing G41S or G93A mutant human SOD-1 were lysed in buffer containing nonionic detergent. Shown are supernatant (S) and pellet (P) fractions after electrophoresis on a 12.5% SDS-polyacrylamide gel and Western blotting with antibody to HSP70 (W27). Blots were reprobed with antibody to actin.

The data shown in Fig. 5D also confirm our previous findings that HSP70 is up-regulated in cell lines expressing mutant SOD-1. Although no association of HSP70 with wild-type human SOD-1 was detected (Fig. 5, A and B), this could have been due to the relatively low level of HSP70 in this cell line. To test this hypothesis, cells stably expressing wild-type human SOD-1 were transiently transfected with HSP70. Despite high level expression of HSP70, verified by Western blotting of total cell lysates with antibody to HSP70, neither wild-type human nor murine SOD-1 coimmunoprecipitated with HSP70 (data not shown).

Another important observation from Fig. 5D is that HSP70 was not detected in pellet fractions of untransfected or wild-type human SOD-1 expressing NIH3T3 cultures. Thus, HSP70 and wild-type SOD-1 proteins are largely detergent-soluble proteins; however, in G41S and G93A-expressing cell lines, a proportion of both HSP70 and mutant SOD-1 are detergent-insoluble. How much of the detergent-insoluble forms represents complexes of HSP70 and mutant SOD-1 cannot be determined from these experiments.

Mutant SOD-1 Proteins Coimmunoprecipitate with HSP40 and alpha B-crystallin-- The interaction of mutant SOD-1 with stress proteins was not limited to HSP70. Both SOD-1 mutants, but neither wild-type human nor endogenous murine SOD-1, were pulled down when total cell extracts of NIH3T3 cell lines were immunoprecipitated by antibodies to HSP40 (Fig. 6A). As in experiments with HSP70, failure to detect complexes of HSP40 and wild-type human SOD-1 was not due to lack of HSP expression. Considerable HSP40 was present in both supernatant and pellet fractions of all cell lines (Fig. 6B).


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Fig. 6.   Mutant SOD-1, but not wild-type human or mouse SOD-1, coimmunoprecipitates with the stress proteins HSP40 and alpha B-crystallin. Total cell extracts of NIH3T3 cell lines were immunoprecipitated (IP) with antibody to HSP40 (A) or alpha B-crystallin (C). Proteins were separated on 12.5% SDS-polyacrylamide gels, and Western blots were probed for the presence of SOD-1 using antibody SOD-100. Ig indicates the immunoglobulin light chain of the immunoprecipitating antibody. Also included is nonimmunoprecipitated G41S extract to show the location of human (h) and mouse (m) SOD-1. B, although HSP40 is found in both supernatant and pellet fractions of all cell lines, only mutant, not wild-type SOD-1, coimmunoprecipitates with HSP40. Shown is a Western blot of supernatant (S) and pellet (P) fractions of untransfected NIH3T3 cells and lines expressing wild-type human SOD-1 (WT) or G41S mutant SOD-1 probed with HSP40 antibody.

Similar results were obtained when immunoprecipitations were carried out using antibody to alpha B-crystallin. Both G41S and G93A SOD-1 mutants, but not wild-type human or endogenous murine SOD-1, were coprecipitated (Fig. 6C).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In response to various physiological stresses, including heat shock or exposure to toxic agents, cells up-regulate families of heat shock/stress proteins. Their function is to prevent the accumulation of improperly folded proteins by binding to and promoting proper refolding of the damaged proteins or by facilitating their degradation by proteasomes (23-25). Our studies have provided evidence that heat shock proteins play a role in protecting cells from the toxicity of mutant SOD-1 proteins associated with human familial ALS. Coexpression of HSP70 prolonged viability of cultured motor neurons expressing mutant SOD-1 proteins and reduced the formation of cytoplasmic aggregates (13).2 Survival of NIH3T3 cells expressing two different SOD-1 mutants, G41S and G93A, correlated with up-regulation of heat shock proteins (13). Finally, HSP70, HSP40, and alpha B-crystallin were coimmunoprecipitated with mutant SOD-1 proteins from mutant SOD-1-transfected cell lines (this study). These data support the hypothesis that heat shock proteins protect cells, at least in part, by direct association with mutant protein.

SOD-1, being a cytoplasmic enzyme, is normally soluble in nonionic detergent. However, a small percentage of mutant human SOD-1 protein was consistently detected in the detergent-insoluble fraction of protein extracts from NIH3T3 transfectants, spinal cord cultures prepared from embryos of G93A mutant SOD-1 transgenic mice, and lumbar spinal cord of adult G93A transgenic mice. Although mutant SOD-1 proteins can form homodimers with wild-type protein of different species (26), the endogenous murine SOD-1 was not found in the detergent-insoluble pellet fraction despite overloading of gels with protein and blotting with antibody preferentially recognizing this species.

HSP70 and mutant SOD-1 were coimmunoprecipitated from total cell extracts of the two mutant SOD-1 transfected NIH3T3 cell lines, whereas only trace amounts were detected from the supernatant fractions, indicating that HSP70·mutant SOD-1 complexes were predominantly detergent-insoluble. Distribution of proteins, including heat shock proteins, to the detergent-insoluble pellet occurs under a variety of stressful conditions, including heat shock, oxidative stress, ATP depletion, calcium overload, and treatment with sulfhydryl reagents (27-29). Whether all detergent-insoluble mutant SOD-1 was complexed with HSP70 cannot be determined from the present experiments. Future studies will address whether posttranslational modification of mutant SOD-1 is required either to alter solubility or to promote binding of HSPs. That a relatively small fraction of total cellular mutant SOD-1 (estimated at 0.5-10%) was found in the pellet fraction in which the HSP·SOD-1 complexes were localized suggests the possibility that altered conformation conferred by the mutation alone may not be sufficient. Regardless, binding of heat shock proteins would be protective by preventing inappropriate protein-protein interactions and/or facilitating degradation.

Although 3T3 cells may be physiologically different from spinal cord cells affected in familial ALS, detergent-insoluble mutant SOD-1 also was detected in spinal cord cultures prepared from embryos of G93A mutant SOD-1 transgenic mice and from lumbar spinal cord of adult G93A transgenic mice. Because significant expression of HSP70 was not detected in spinal cord (13), additional studies are under way to characterize expression of heat shock proteins and their interaction with mutant SOD-1 in these models.

Although a proportion of mutant SOD-1 is detergent-insoluble in the transfected NIH3T3 cell lines, no large aggregates are visible by immunocytochemistry in these cells (13). However, in the study by Johnston et al. (15), formation of large aggregates of mutant SOD-1 was induced in transfected HEK cells by inhibiting proteasomal activity with lactacystin. The detergent insolubility of mutant SOD-1 and association with heat shock proteins observed in our study could represent an intermediate stage between soluble mutant SOD-1 and protein aggregated into inclusions. Inclusions may form when insufficient heat shock proteins are expressed to sequester mutant protein and facilitate its degradation in proteasomes. This could result from proteasomal inhibition, as in the experiment by Johnson et al. (15), or from an inadequate stress response to increase levels of heat shock proteins.

The propensity of mutant SOD-1 proteins to form aggregates differs according to cell type (17). Motor neurons are particularly susceptible, perhaps in part because they have a high threshold for activation of the stress response (30-33). For example, when spinal cord cultures are heat shocked, glial cells up-regulate HSP70, but motor neurons do not, even when the level of thermal stress is lethal.3 In cultured motor neurons expressing mutant SOD-1 proteins, formation of aggregates is tightly linked to loss of viability (22). However, this does not necessarily mean that aggregates cause toxicity. The presence of aggregates may reflect the inability of the cell to elicit a sufficient stress response to maintain general chaperoning function in the cell, thus increasing its vulnerability to a variety of other physiological and environmental stresses, which ultimately contribute to cellular dysfunction and death.

Similar arguments apply to other motor neuron diseases involving altered protein conformation. Colocalization of heat shock proteins and mutant protein within proteinaceous aggregates occurs in many neurodegenerative disorders, including those caused by expansion of CAG (polyglutamine) repeat sequences (reviewed in Ref. 34). Spinal bulbar muscular atrophy (Kennedy's disease) is a motor neuron disease resulting from CAG repeat expansion in the androgen receptor gene (35). Overexpression of heat shock proteins (HSP70, HSP40, and HDJ2/HSDJ) in HeLa or Neuro2A cell culture models of spinal bulbar muscular atrophy reduced both formation of aggregates and toxicity (36, 37). Aberrant distribution and altered ratios of the 20 S proteasomal core and PA700 proteasomal cap proteins, the two major components of 26 S proteasomes, were found in HeLa cells with aggregates of polyglutamine-expanded androgen receptor. Thus, both deficiency of chaperones and impaired proteolysis could contribute to pathogenesis (36).

The data presented here indicate that HSP70 may protect cells from mutant SOD-1 proteins at least in part by direct interaction with the mutant protein. This does not exclude other cytoprotective mechanisms for heat shock proteins, such as chaperoning other proteins damaged as a result of mutant SOD-1 expression or a general antiapoptotic effect (38-40). Additional studies are required to assess the feasibility of targeting stress protein pathways to treat motor neuron disease and other neurodegenerative disorders characterized by accumulation of aberrant proteins.

    FOOTNOTES

* This research was supported by the Amyotrophic Lateral Sclerosis Association, the Amyotrophic Lateral Sclerosis Society of Canada, the Muscular Dystrophy Associations of Canada and the United States, and the Canadian Institutes for Health Research.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.

Dagger A Killam Scholar. To whom correspondence should be addressed: Rm. 649, Montreal Neurological Institute, 3801 University St., Montreal, Quebec H3A 2B4, Canada. Tel.: 514-398-8509; Fax: 514-398-1509; E-mail: mddm@musica.mcgill.ca.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010759200

2 J. Roy, S. Minotti, D. A. Figlewicz, and H. D. Durham, unpublished observations.

3 Z. Batulan, S. Minotti, and H. D. Durham, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: ALS, amyotrophic lateral sclerosis; HSP, heat shock protein; SOD-1, Cu/Zn-superoxide dismutase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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