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Originally published In Press as doi:10.1074/jbc.M501705200 on March 7, 2005

J. Biol. Chem., Vol. 280, Issue 18, 17725-17731, May 6, 2005
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Inhibition of Chaperone Activity Is a Shared Property of Several Cu,Zn-Superoxide Dismutase Mutants That Cause Amyotrophic Lateral Sclerosis*{boxs}

Hemachand Tummala{ddagger}, Cheolwha Jung{ddagger}, Ashutosh Tiwari§, Cynthia M. J. Higgins{ddagger}||, Lawrence J. Hayward§**, and Zuoshang Xu{ddagger}**{ddagger}{ddagger}§§

From the Departments of {ddagger}Biochemistry and Molecular Pharmacology, §Neurology, and {ddagger}{ddagger}Cell Biology and the **Neuroscience Program, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Received for publication, February 14, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron degeneration, paralysis, and death. Mutant Cu,Zn-superoxide dismutase (SOD1) causes a subset of ALS by an unidentified toxic property. Increasing evidence suggests that chaperone dysfunction plays a role in motor neuron degeneration in ALS. To investigate the relationship between mutant SOD1 expression and chaperone dysfunction, we measured chaperone function in central nervous system tissue lysates from normal mice and transgenic mice expressing human SOD1 variants. We observed a significant decrease in chaperone activity in tissues from mice expressing ALS-linked mutant SOD1 but not control mice expressing human wild type SOD1. This decrease was detected only in the spinal cord, became apparent by 60 days of age (before the onset of muscle weakness and significant motor neuron loss), and persisted throughout the late stages. In addition, this impairment of chaperone activity occurred only in cytosolic but not in mitochondrial and nuclear fractions. Furthermore, multiple recombinant human SOD1 mutants with differing biochemical and biophysical properties inhibited chaperone function in a cell-free extract of normal mouse spinal cords. Thus, mutant SOD1 proteins may impair chaperone function independent of gene expression in vivo, and this inhibition may be a shared property of ALS-linked mutant SOD1 proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amyotrophic lateral sclerosis (ALS)1 is a progressive neurodegenerative disease that causes degeneration of cortical and spinal motor neurons (1). Genetic studies in humans have identified several causes for inherited (familial) forms of ALS. The ALS1 gene at chromosome 21q22 causes dominantly inherited ALS in 20% of familial ALS cases and encodes the antioxidant enzyme SOD1 (2). ALS2 at chromosome 2q33 causes a rare form of recessively inherited familial juvenile ALS (3, 4). It encodes a protein named Alsin that contains regions homologous to RCC1, RhoGEF, VPS9, MORN, and pleckstrin homology domains, suggesting a role in signaling. The third identified ALS gene, dynactin, is located at chromosome 2q13 (5). dynactin is a crucial component in the dynein complex, and this mutation compromises the retrograde axonal transport function. The fourth identified gene is senataxin (6). Several mutations in this gene cause slowly progressive juvenile ALS. Additional genes are being identified (7–9). Animal studies suggest more gene mutations that may cause human ALS, including neurofilaments (10), dynein (11), dynamitin (12), tubulin chaperon (13, 14), and vegf (15).

The pathways by which these diverse genetic mutations cause motor neuron degeneration have not been clearly delineated. However, numerous investigations using transgenic mouse models expressing mutant SOD1 have contributed important mechanistic insights. SOD1 is a ubiquitous 32-kDa homodimeric enzyme that binds one copper and one zinc ion per subunit. Although SOD1 is predominantly located in the cytoplasm, it has also been detected within mitochondria and other subcellular organelles. Copper is required for its catalytic activity, whereas zinc stabilizes the structure of the protein (16). The known function of SOD1 is dismutation of superoxide (O–2), a byproduct of oxidative metabolism, into hydrogen peroxide (H2O2) and oxygen. Together, with the downstream enzymes catalase and glutathione peroxidase (which convert H2O2 to water and oxygen), SOD1 reduces cellular free radicals. This function is important, because mice lacking the SOD1 gene develop numerous abnormalities, including reduced fertility (17), motor axonopathy (18), increased age-associated loss of cochlear hair cells (19) and neuromuscular junction synapses (20), and enhanced susceptibility to a variety of noxious assaults on the nervous system, such as axonal injury (21), ischemia (22, 23), hemolysate exposure (24), and irradiation (25).

Despite the importance of normal SOD1 function, mice lacking SOD1 are, nonetheless, viable and do not develop an ALS-like phenotype. Studies (26, 27) of transgenic mice expressing mutant SOD1 convincingly demonstrate that SOD1 mutants cause motor neuron degeneration by acquiring a toxic property, rather than by losing superoxide dismutase activity. What this toxic property is has not been resolved, although at least three hypotheses have been put forward. Beckman et al. (28), Crow et al. (29), and Estevez et al. (30) proposed that SOD1 mutants have an enhanced activity to catalyze nitration of tyrosine by producing peroxynitrite (ONOO–), which is generated by the reaction of superoxide with nitric oxide (28–30). An alternative hypothesis is that the mutant SOD1 has an enhanced peroxidase activity. Two groups demonstrated that this peroxidase activity was elevated in the mutant compared with normal SOD1 in vitro (31–33). The third hypothesis suggests that the toxicity may stem from the propensity of SOD1 mutants to aggregate (34, 35). It is possible that such aggregates may be detrimental as in other neurodegenerative diseases by contributing to oxidative stress, sequestration, and depletion of important cellular proteins, and/or disruption of protein chaperone and proteasome function (36–38). The validity of all three hypotheses is being debated and requires further investigation (39–42).

Much work has been done to characterize the downstream events related to mutant SOD1 toxicity. Mutant SOD1 is associated with mitochondria and may contribute to mitochondrial dysfunction and degeneration (43, 44). Mutant SOD1 may cause excitotoxicity in motor neurons by inducing mitochondrial dysfunction and impairing glial glutamate transporter function (45, 46). Additionally, mutant SOD1 causes disorganization of the cytoskeleton and disrupts axonal transport (47, 48), compromises proteasome activity (37, 49), (which in turn causes accumulation and aggregation of mutant SOD1 (35, 50)), and induces inflammation (51–54) (which may accelerate the disease progression (55–57)). These downstream effects of mutant SOD1 presumably trigger the final programmed cell death in motor neurons (58).

Genetic studies in simple organisms, such as Drosophila and Caenorhabditis elegans, demonstrate that chaperones are suppressors of neurodegenerative disorders, including Parkinson and Huntington diseases (59–61). Increasing evidence suggests that chaperone dysfunction plays a role in the pathogenesis of ALS. Experiments on cultured neuronal cells show that motor neurons have a high threshold for induction of chaperones in response to stress (62). Mutations in small heat shock proteins, such as HSP27 and HSP22, cause axonal degeneration in motor neurons (63, 64). Overexpression of chaperones can suppress mutant SOD1 aggregation, protect neuronal function, and enhance motor neuron survival in culture (38, 65). Pharmacological stimulation of chaperone expression extends survival of mice expressing mutant SOD1 G93A (66). Mutant SOD1 may directly interact with different chaperones (67, 68), perhaps leading to chaperone dysfunction in mutant SOD1 transgenic mice (38).

In this study, we investigated the temporal and spatial occurrence of chaperone dysfunction in mice expressing G93A and G85R mutant SOD1. We found that chaperone dysfunction developed early, before the onset of muscle weakness in mutant SOD1 transgenic mice. This dysfunction was restricted to the spinal cord, where motor neurons degenerate. In subcellular fractions of the spinal cord, chaperone dysfunction was detected only in the cytosol but not in the nucleus and mitochondria. In addition, multiple SOD1 mutants that possess very different biochemical and biophysical properties inhibited chaperone function in a cell-free extract of wild type mouse spinal cords. Thus, mutant SOD1 can inhibit chaperone function independent of changes in gene expression, and inhibition of chaperone function may be a general property of mutant SOD1 proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Mice—The low expresser line of human SOD1 mutant G93A transgenic mice (C57BL/6J-TgN(SOD1-G93A)1Gurdl) and the human wild type SOD1 [TgN(SOD1)2GUR] were purchased from The Jackson Laboratory. Mice transgenic for the human SOD1 mutant G85R were generously provided by Dr. Cleveland (69). The G85R line was also bred to homozygosity (in some experiments, the G85R homozygous mice were not used, because this line stopped breeding in the late period of these experiments. The three mutant transgenic lines display different clinical courses. Both the G93A and G85R homozygote mice have an average disease onset at ~160 days, paralysis at ~240 days, and death at ~260 days. The G85R heterozygotes have an average disease onset at ~270 days, paralysis at ~330 days, and death at ~350 days. Transgenic positives were identified using PCR according to Gurney (70). All mice were bred at the University of Massachusetts Medical School animal facility. All animal procedures are approved by University of Massachusetts Medical School Institutional Animal Care and Use Committee.

Preparation of Tissue Homogenates and Subcellular Organelle—Mice were anesthetized with halothane and perfused with phosphate-buffered saline to remove blood. After decapitation, the forebrain, cerebellum, and spinal cord were collected. For preparation of the homogenates, the tissues were homogenized with a polytron homogenizer in a buffer containing 50 mM HEPES, pH 7.4, 100 mM KCl, 5% glycerol, 1 mM MgCl2, 0.1% IGEPAL CA-630 and the protease inhibitor mixture (Sigma, P8340) for 2 min on ice. The homogenates were aliquoted and stored at –80 until use. For preparation of subcellular fractions, the issues were minced in TES (10 mM Tris-HCl, 0.5 mM EDTA, and 0.25 M sucrose, pH 7.4) with protease inhibitors and homogenized using Dounce homogenizers, 20 strokes each with pestle A (clearance 0.12 mm) and pestle B (clearance 0.06 mm). Unbroken cells and tissue debris were removed by centrifuging at 800 x g for 10 min. The nuclear fraction was centrifuged down at 1000 x g for 20 min. The nuclear pellet was washed three times with TES and analyzed under microscope after staining with propidium iodide.

The mitochondrial fraction was separated as described by Rajapakse et al. (71). In brief, after removing the nuclear fraction, the homogenates were mixed to achieve a 12% final concentration of Percoll. This mixture was layered over 24 and 40% Percoll density gradient. Fraction 2 containing mitochondria were separated after centrifuging at 30,000 x g for 6 min. The mitochondrial fraction was washed twice with and stored in mitochondrial isolation buffer (400 mM mannitol, 10 mM KH2PO4, 5 mM sodium succinate, 50 mM Tris-HCl, pH 7.2, with 5 mg/ml–1 BSA). The integrity of the mitochondria was assessed by recording the mitochondrial potential and monitoring its response to the addition of succinate using fluorescent dye JC1 (1.5 µM). To obtain the cytosolic fraction, the homogenate was centrifuged at 1,000 x g for 20 min to remove nuclei and 16,000 x g for 15 min to remove mitochondria. The resulting supernatant was centrifuged at 100,000 x g for 1 h, and the supernatant was considered as a cytosolic fraction (72). The enrichment of each subcellular fraction was evaluated by Western blot using antibodies against markers TOM20, LaminA/C (Santa Cruz Biotechnology), and calmodulin (Abcam, Inc.).

Chaperone Activity Assay—Assays were performed as described by Hook and Harding (73), based on the ability of chaperones present in the protein extracts to prevent the heat denaturation of a substrate protein catalase. In brief, tissues or isolated organelle were lysed in 50 mM HEPES, pH 7.4, 100 mM KCl, 5% glycerol, 1 mM Mg Cl2, and 0.1% IGEPAL CA-630. Protein concentrations were determined using a bicinchoninic acid protein assay (Pierce). Forty µg/ml of the extract was mixed with catalase (200 µg/ml) in phosphate-buffered saline. The reaction mixture was heated at 55 °C, and at different time points, aggregation of the denatured protein was followed by measuring light scattering at 360 nm with a spectrophotometer. The relative initial aggregation velocity was estimated using the plots (supplemental Fig. S1, A and B) based on the Michaelis-Menten equation (74). The reduction in the velocity of catalase aggregation was used as a measure of chaperone activity (supplemental Fig. S1, C and D).

Expression and Purification of SOD1—Human SOD1 or ALS-related mutant SOD1 enzymes (A4V, H46R, G85R, G93A, and D125H) containing biologically incorporated metal ions were isolated from a baculoviral expression system (75). Protein concentrations were estimated by absorbance measurement using a dimeric molar extinction coefficient at 280 nm of 10,800 M–1 cm–1 (76). The purity, molecular mass, and metal ion contents for copper and zinc were determined as described previously (75). The metal content of purified SOD1 were (in equivalents/dimer): wild type (WT), 0.74 copper, 1.45 zinc; A4V, 0.6 copper, 2.2 zinc; H46R 0.02 copper, 0.07 zinc; G85R 0.02 copper, 0.04 zinc; G93A 0.73 copper, 1.97 zonc; and D125H 0.09 copper, 0.36 zinc.

Western Blot—Protein concentration of the cytosol prepared was determined using bicinchoninic acid reagent (Pierce). Electrophoresis was carried out using the buffer system of Laemmli (90) on 10–15% polyacrylamide gels. The proteins were transferred onto polyvinylidene difluoride membranes and incubated for 3 h with primary and 1 h with horse radish peroxidase secondary antibodies at room temperature. The blots were developed using the enhanced chemiluminescence method (Pierce). Antibodies against all heat shock proteins (HSP90, SPA846; HSP70, SPA810; HSP60, SPA805; HSP40, SPA450; HSP25, SPA801, {alpha} crystallin, SPA224) were purchased from Stressgen Bioreagents. The antibody against SOD1 is purchased from Biodesign (Saco, ME).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chaperone Activity in the Lysates of Spinal Cord, but Not of Forebrain and Cerebellum Is Inhibited in Mice Expressing Mutant SOD1—Early work showed that chaperone activity was decreased in spinal cord in symptomatic SOD1 G93A mice (38). Whether this decrease occurs specifically in the spinal cord in the central nervous system has not been determined. Additionally, the temporal relationship of this decrease to age and disease stages has not been investigated. Different mutants are known to differ widely in terms of copper content, superoxide dismutase activity, and stability (75, 77, 78); therefore, it is important to determine whether other SOD1 mutants besides G93A share a similar inhibitory effect on the chaperone activity. We therefore measured the chaperone activity in tissue homogenates from different central nervous system regions in two mutant transgenic lines; one expresses SOD1 G93A, and the other expresses SOD1 G85R. We chose these two mutant lines, because these two mutant proteins have contrasting properties. SOD1 G93A contains similar levels of copper compared with SOD1WT, possesses nearly normal levels of the dismutase activity, and shows nearly normal stability. In contrast, SOD1 G85R contains very low copper content, has no detectable dismutase activity, and exhibits low stability (75, 77, 78). Our question was whether these two very different mutants influence chaperone activity in a similar manner.

In spinal cord lysates, chaperone activity, as measured by reduction in initial velocity of heat-induced catalase aggregation (see "Materials and Methods"), showed little difference between mice expressing wild type human SOD1 and non-transgenic mice at all ages (Fig. 1, top panel). In contrast, chaperone activity progressively decreased in G85R and G93A SOD1 mutant mice as they aged (Fig. 1, top panel). In other central nervous system regions, including the forebrain and cerebellum, this progressive decrease was not observed (Fig. 1, middle and lower panels). Because both regions are minimally affected or unaffected pathologically while the spinal cord is the site of severe pathology, the region-specific decrease in chaperone activity correlates with the region-specific pathology.



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  		FIG. 1.
Chaperone activity is significantly decreased in spinal cord of mutant SOD1 transgenic mice compared with the wild type SOD1 transgenic mice. Top graph, two factor analysis of variance between G93A and WS, p = 0.0006; between G85R and WS, p = 0.00002; between G85R+/+ and WS, p = 0.0004). This decrease is not observed in the forebrain and cerebellum (middle and lower graphs; none of the comparisons similar to the spinal cord reached the significance level at p = 0.05), two areas with mild or no pathology compared with the spinal cord. Non-Tg, non-transgenic mice; SOD1WT, wild type human SOD1 transgenic mice; G93A+/–, SOD1 G93A heterozygous transgenic mice; G85R+/– and G85R+/+ are SOD1 G85R heterozygous and homozygous mice, respectively. The control mice (Non-Tg and WS), used for comparison with the paralysis group, are 295–320 days old. All measurements are normalized as described under "Materials and Methods" and supplemental Fig. S1. Each bar represents measurements from three animals. Error bars are standard error.

 
Because in these experiments relative chaperone activities were measured, the results shown in Fig. 1 regarding the spinal cord may be interpreted in two ways: 1) either the chaperone activities in the non-transgenic and SOD1WT transgenic mice rise during aging (wherein the activities in mutant transgenic mice remain flat) or 2) the chaperone activities in the non-transgenic and SOD1WT transgenic mice remain constant, but the activities in mutant transgenic mice decrease with age. To determine which of these two possibilities was true, we measured chaperone activities in non-transgenic animals at different ages. Chaperone activities in the spinal cord remained constant in non-transgenic mice (supplemental Fig. S2). Thus, mutant (but not wild type) SOD1 expression in mice causes the decrease in chaperone activity specifically in the spinal cord.

The Inhibition of Chaperone Activity in Spinal Cord Is Due to Neither a Low ATP Level nor Low Levels of Several Common Chaperones—Some chaperone functions depend on ATP (79). Because structural and functional mitochondrial abnormalities have been observed in the mutant SOD1 transgenic mice (43, 80, 81), the decreased chaperone activity might be caused by lowered levels of ATP in the spinal cord. To test this possibility, exogenous ATP was added to the chaperone assay. Neither ATP nor ADP supplementation altered the decrease of chaperone activity in the mutant SOD1 transgenic mice (Fig. 2A), indicating that the lowered chaperone activity in the mutant spinal cord homogenates are not due to low ATP levels.



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  		FIG. 2.
The decreased chaperone activity is not because of a lack of ATP or decreased levels of several chaperones in the spinal cord. A, supplementation of ATP did not reverse the decrease in chaperone activity in mutant SOD1 transgenic mice (~160 days old; n = 3). Two factor analysis of variance comparison of the control with either ATP or ADP addition did not reach the significance level (p = 0.38 and 0.63, respectively). SC represents spinal cord. Other symbols and the normalization are the same as described in the legend to Fig. 1. B, levels of several common heat shock proteins are unchanged or increased in the mutant SOD1 transgenic animals (~160 days old). Blots for HSP90, HSP70, and HSP40 G85R+/+ were not done because of the loss of the homozygous line.

 
Lowered chaperone activity might also be caused by decreased chaperone protein levels. To test this possibility, we carried out protein blots for several widely expressed chaperones using specific antibodies. We did not detect decreases in the levels of these chaperones (Fig. 2B). HSP25 levels were increased, probably reflecting the increased expression in astrocytes as previously reported (62, 82, 89). There was also a slight increase in the levels of HSP90 in the mutant SOD1 mice (Fig. 2B). Immunohistochemical staining of HSP90, however, showed a wide distribution of HSP90 in the neuropil that did not reveal a specific pattern (data not shown). These results demonstrate that the decreased chaperone activity in the spinal cord is not because of decreased levels of these chaperones.

Cytosolic Chaperone Activity Is Inhibited in the Spinal Cord of Mutant SOD1 Transgenic Mice—Because mitochondria are affected in mutant SOD1 transgenic mice and mutant SOD1 is present in mitochondria (43, 44, 81), the reduced chaperone activity might be caused by inhibition of chaperone activity in mitochondria. To test this, we made subcellular fractions that enrich nuclei, mitochondria, and cytosol (Fig. 3A). Chaperone activities were not decreased in mitochondrial and nuclear fractions, but were decreased in the cytosol fraction (Fig. 3B). The extent of the decrease in chaperone activity in the cytosol was similar to the decrease observed in the unfractionated spinal cord homogenate (Fig. 1A), indicating that loss of chaperone activity in the cytosol represents the majority, if not all, of the chaperone activity loss in the spinal cord.



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  		FIG. 3.
Cytosolic (but not mitochondrial and nuclear) chaperone activity is decreased in the spinal cord of mutant SOD1 transgenic mice. A, Western blot of markers for different subcellular fractions demonstrating enrichment of different organelles. Lane 1 is whole spinal cord homogenate; lanes 2, 3, and 4 are nuclear, mitochondrial, and cytosolic fractions, respectively. TOM-20 (a subunit in transporter of mitochondrial outer membrane) is a mitochondrial marker. Lamins are markers for the nucleus. Calmodulin is a cytosolic marker. B, chaperone activity is reduced in cytosolic (but not in mitochondrial and nuclear) fractions (n = 3). This reduction was only observed in the spinal cords of mutant SOD1 transgenic mice (Student's t test, p = 0.05).

 
Inhibition of Chaperone Activity Is a Gained Property of Mutant SOD1 Protein—The results shown above demonstrate that expression of mutant but not wild type SOD1 inhibits chaperone activity in vivo. One possible mechanism for this inhibition was that mutant SOD1 alters gene expression. For example, it could reduce expression of one or more chaperones or induce expression of genes that inhibit chaperone function or increase chaperone degradation. Alternatively, mutant SOD1 could inhibit chaperones independent of alterations of gene expression by directly or indirectly interacting with chaperones. To differentiate between these two possibilities, we added purified recombinant mutant SOD1 to the spinal cord cytosol prepared from wild type mice. With prolonged incubation at room temperature, we observed inhibition of chaperone activity by two purified SOD1 mutants G93A and G85R (Fig. 4A), and the inhibition was even greater by 24 h of incubation. Recombinant wild type SOD1 also inhibited chaperone activity but to a much lesser degree compared with the mutants (Fig. 4A). This mutant-induced inhibition was also dose-dependent (Fig. 4B), whereas incubation with even 6% (w/w) of WT SOD1 had minimal effect.



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  		FIG. 4.
SOD1 mutants (but not the wild type), when exogenously added and incubated, inhibit chaperone activity in the cytosolic fraction of the spinal cords from non-transgenic mice. A, BSA or SOD1 variants (added to 4% of the spinal cord cytosolic protein) were incubated at room temperature with the spinal cord cytosol from non-transgenic mice for the length of times indicated before measurement of chaperone activity (n = 3). Mutant SOD1 significantly inhibited the chaperone activity compared with the wild type SOD1 (two factor analysis of variance between G93A and WS, p = 0.006; between G85R and WS, p = 0.007). This inhibition is also time-dependent (p = 0.007 for G93A and 0.02 for G85R). B, inhibition of chaperone activity correlates with the concentration of SOD1 mutants. BSA, SOD1WT, G93A, and G85R were mixed with the spinal cord extracts from non-transgenic mice at the percentage of the total protein indicated and incubated for 8 h before the measurement of chaperone activity. The chaperone activities were normalized to the cytosol that was incubated with BSA (n = 3). Mutant SOD1 significantly inhibited the chaperone activity compared with the wild type SOD1 (two factor analysis of variance between G93A and WS, p = 3 x 10–7; between G85R and WS, p = 2 x 10–5). This inhibition is also concentration-dependent (p = 2 x 10–7 for G93A and 4 x 10–5 for G85R). C, chaperone activity in the spinal cord was also inhibited by other SOD1 mutants. BSA or SOD1 proteins were added to spinal cord cytosol from non-tg mice to 4% of the total protein. Chaperone activities were measured at 0 and 24 h after incubation (n = 4). Significant differences exist after 24 h of incubation between WS and each of the mutants, A4V, H46R, or D125H (Student's t tests, p = 0.03, 0.006, and 0.003, respectively).

 
To determine whether purified SOD1 variants other than G93A and G85R could inhibit chaperone activity, we tested three additional mutants, A4V, H46R, and D125H. The A4V substitution occurs near the dimeric interface of the protein; this enzyme incorporates less copper compared with wild type SOD1 during expression in insect cells (75) and exhibits destabilization of the dimeric contacts (83). The H46R mutant is similar to G85R in that it contains little copper and zinc and is inactive; H46R SOD1 also contains unstructured zinc-binding and electrostatic loops (84) that potentially interact with chaperones. D125H contains low levels of copper and zinc and has low levels of SOD1 activity. Its thermostability profile differs from all the other mutants (75, 78). Despite these different properties of these mutants, they all showed enhanced inhibition of the chaperone activity (Fig. 4C). These results suggest that inhibition of chaperone activity is a gained property of the ALS-associated SOD1 mutants.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate an impairment of chaperone activity in transgenic mice that express either G93A or G85R SOD1 mutants. This impairment is specifically observed in the spinal cord (but not in the forebrain and cerebellum) and does not occur in tissue lysates from control mice. There are several possible mechanisms for this tissue- and mutant-specific inhibition. First, the decreased chaperone activity may merely reflect the degeneration and loss of motor neurons in affected regions. This is unlikely, because this decrease was observed at ~3 months or more before the first detection of muscle weakness and the initiation of significant motor neuron degeneration (Fig. 1) (85). Second, the spinal cord may express more mutant SOD1 than the forebrain and cerebellum. Our Western blots of SOD1 indicate that this is the case for both G93A and G85R (supplemental Fig. S3) (69). Therefore, the selective decrease of chaperone activity in the spinal cord could be related to the burden of mutant SOD1. This possibility, however, does not convincingly explain the regional specificity, because in both forebrain and cerebellum, the mutant SOD1 is expressed, yet there is no decrease in the chaperone activity (Fig. 1). Third, chaperones in the cerebellum or forebrain may be resistant to mutant SOD1 inhibition, whereas chaperones in the spinal cord may not. This appears unlikely, at least for the cerebellum, because prolonged incubation of purified mutant SOD1 with the cerebellar cytosol produced similar inhibition as observed in the spinal cord cytosol (supplemental Fig. S4). Fourth, forebrain and cerebellum may have compensatory mechanisms that counter the inhibitory effect of mutant SOD1. Our data appear consistent with this possibility.

Because SOD1 mutants have been found in mitochondria and cause mitochondrial damage (43, 44, 81, 86), we investigated the possibilities that mutant SOD1 inhibits chaperone activity by lowering ATP or by inhibiting chaperones in mitochondria. Supplementing ATP in our chaperone activity assay did not reverse the inhibition in the spinal cord extracts from mutant SOD1 transgenic mice, indicating the decrease was not caused by a lack of ATP (Fig. 2). Measurement of chaperone activities in subcellular fractions showed that the chaperone activity was not decreased in mitochondrial and nuclear fractions but was decreased in the cytosol (Fig. 3). The absence of inhibition of chaperone activity in mitochondria does not rule out the possibility that dysfunction of cytosolic chaperones may cause mitochondrial dysfunction and degeneration, because mitochondrial protein import heavily depends on the functions of cytosolic chaperones (87).

SOD1 is constitutively expressed. The long term exposure of the spinal cord cells to mutant SOD1 might cause alterations in gene expression, resulting in a down-regulation of chaperone proteins levels or induction of chaperone inhibitors. Our results do not agree with this possibility. First, Western blots of several common chaperones showed an increase, rather than a decrease, of some chaperones in the spinal cord extract (Fig. 2B). Although this did not rule out that other chaperones might be down-regulated, it did not support that the down-regulation of chaperone levels caused the decrease in chaperone activity. Second, incubation of purified SOD1 mutants with post-nuclear cytosol inhibited chaperone activity (Fig. 4). This suggests that inhibition of chaperone activity in the spinal cord is mediated through protein-protein interactions, but not through alterations of gene expression.

In these in vitro chaperone activity assays, there is a possibility that SOD1 mutants are unfolding during heating, thus competing with catalase for chaperones. This is unlikely, because addition of SOD1 mutants to the spinal cord cytosol immediately before the chaperone activity assay does not produce detectable inhibition. Only after a prolonged incubation could the inhibition of chaperone activity be detected (Fig. 4, A and C). This indicates that SOD1 mutants have to co-exist with the chaperone for a considerable period of time before they can inhibit the chaperone activity. How these mutants inhibit chaperone activity will require further investigation, but direct binding between the SOD1 mutants and chaperones have been demonstrated (67, 68).

Our findings suggest that the inhibition of chaperone activity is likely a general property of mutant SOD1, because two mutants, G93A and G85R, can inhibit chaperone activity in spinal cord lysates (Fig. 1). These two mutants possess very different biochemical and biophysical properties. G93A is a wild type-like mutant in that it is relatively stable, contains normal amounts of metal ions, has normal levels of superoxide dismutase activity, and causes mitochondrial vacuolation in vivo (43, 75, 77, 78). G85R, however, is a metal binding region mutant. It is much less stable, contains almost no metal ions, has undetectable levels of superoxide dismutase activity, and does not cause mitochondrial vacuolation (34, 75, 77, 78). Despite the differences, our results indicate that these two mutants share the inhibitory activity to the chaperone activity both in vivo and in vitro, thus suggesting that inhibition of chaperone activity may be a shared property in all mutants. Further support for this notion is that three other mutants, A4V, H46R, and D125H, showed similar inhibition (Fig. 4C).

What is the role of chaperone dysfunction in motor neuron degeneration? At present the answer is not known. Given the indispensable roles of chaperones in many vital cellular functions, including protein synthesis, folding, transport, translocation across different cellular compartments, and upon preventing protein aggregation during stress, this chaperone dysfunction is likely to contribute significantly to the cause for motor neuron degeneration (88). It is puzzling, therefore, that there is not an obvious correlation between the extent of decrease of chaperone activity and the life expectancy in the mutant SOD1 transgenic mice. Although the chaperone activity is similarly inhibited, G93A (heterozygous) and G85R (homozygous) mice develop disease faster than G85R singly transgenic mice. Three non-mutually exclusive possibilities might explain this: 1) the variability of the chaperone activity assay is too large to reveal small differences, 2) the chaperone activity in the spinal cord might have been maximally inhibited, and 3) survival may depend on both the extent of chaperone inhibition and other parallel pathways, including protein aggregation, mitochondrial dysfunction, oxidative stress, and excitotoxicity. Although these SOD1 mutants might similarly inhibit chaperone activity, their other properties differ. For example, G93A causes mitochondrial vacuolation, but G85R does not, and this could contribute to the earlier onset of the disease in G93A mice than in G85R mice.

Although the role of chaperone dysfunction in ALS remains to be more precisely defined, our observations, including the early onset of the decrease of chaperone activity (which precedes the onset of clinical symptoms by more than three months), the region specificity of this inhibition and the ability of SOD1 mutants to inhibit chaperone function strongly suggest that chaperone dysfunction is an upstream event in the mutant SOD1-induced motor neuron degeneration pathway, and this event contributes significantly to the pathogenesis of ALS. Several observations in the literature support this view. Motor neurons have a high threshold for induction of chaperones under stress (62). Mutations in two small heat shock proteins, HSP27 and HSP22, cause degeneration of motor axons in humans (63, 64). Interestingly, HSP25, a mouse homologue of HSP27, appears to be selectively decreased in motor neurons in mutant SOD1 transgenic mice (89). Overexpression of chaperone HSP70 protects cultured motor neurons from mutant SOD1 toxicity (38). Pharmacological stimulation of chaperone expression slows ALS progression in mutant SOD1 transgenic mice (66). Taken together, understanding of the role of chaperone dysfunction and the prevention of this dysfunction may constitute an additional avenue for developing therapies for ALS. Further exploration of therapeutic approaches will be accelerated by identification of specific chaperone(s) inhibited by mutant SOD1 and clarification of the mechanism(s) by which such inhibition occurs.


   FOOTNOTES
 
* This work was supported by NINDS, National Institutes of Health Grant RO1 NS41739, the Amyotrophic Lateral Sclerosis (ALS) Association, and The Robert Packard Center for ALS Research at Johns Hopkins (to Z. X.), and NINDS, National Institutes of Health Grant R01 NS44170, the ALS Association, and the Muscular Dystrophy Association (to L. J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

Supported by an ALS Association fellowship. Back

|| Supported by a National Institutes of Health post-doctoral fellowship. Back

§§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605. Tel.: 508-856-3309; Fax: 508-856-2003; E-mail: zuoshang.xu{at}umassmed.edu.

1 The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu,Zn-superoxide dismutase; HSP, heat shock protein; BSA, bovine serum albumin; WT, wild type; WS, wild type human SOD1. Back


   ACKNOWLEDGMENTS
 
We thank Ellen Trang for maintaining transgenic mice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Aberrantly Increased Hydrophobicity Shared by Mutants of Cu,Zn-Superoxide Dismutase in Familial Amyotrophic Lateral Sclerosis
J. Biol. Chem., August 19, 2005; 280(33): 29771 - 29779.
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J. Wang, G. Xu, H. Li, V. Gonzales, D. Fromholt, C. Karch, N. G. Copeland, N. A. Jenkins, and D. R. Borchelt
Somatodendritic accumulation of misfolded SOD1-L126Z in motor neurons mediates degeneration: {alpha}B-crystallin modulates aggregation
Hum. Mol. Genet., August 15, 2005; 14(16): 2335 - 2347.
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