Title: Inhibition of Chaperone Activity Is a Shared Property of Several Cu,Zn-Superoxide Dismutase Mutants That Cause Amyotrophic Lateral Sclerosis
Abstract: 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. 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. Amyotrophic lateral sclerosis (ALS) 1The 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. is a progressive neurodegenerative disease that causes degeneration of cortical and spinal motor neurons (1Rowland L.P. Shneider N.A. N. Engl. J. Med. 2001; 344: 1688-1700Crossref PubMed Scopus (1597) Google Scholar). 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 (2Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. Rahmani Z. Krizus A. 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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. 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 (69Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. 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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 × g for 10 min. The nuclear fraction was centrifuged down at 1000 × 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. (71Rajapakse N. Shimizu K. Payne M. Busija D. Brain Research Protocols. 2001; 8: 176-183Crossref PubMed Scopus (61) Google Scholar). 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 × 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 × g for 20 min to remove nuclei and 16,000 × g for 15 min to remove mitochondria. The resulting supernatant was centrifuged at 100,000 × g for 1 h, and the supernatant was considered as a cytosolic fraction (72Hemachand T. Gopalakrishnan B. Salunke D.M. Totey S.M. Shaha C. J. Cell Sci. 2002; 115: 2053-2065Crossref PubMed Google Scholar). 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 (73Hook D.W. Harding J.J. Eur. J. Biochem. 1997; 247: 380-385Crossref PubMed Scopus (64) Google Scholar), 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 (74Montgomery R. Swenson C.A. Quantitative Problems in the Biochemical Sciences. 2nd Ed. W. H. Freeman and Company, New York1976: 222-265Google Scholar). 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 (75Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Protein concentrations were estimated by absorbance measurement using a dimeric molar extinction coefficient at 280 nm of 10,800 m–1 cm–1 (76Goto J.J. Gralla E.B. Valentine J.S. Cabelli D.E. J. Biol. Chem. 1998; 273: 30104-30109Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The purity, molecular mass, and metal ion contents for copper and zinc were determined as described previously (75Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). 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 (90Laemmli U.K. Nature. 1970; 15: 680-685Crossref Scopus (207218) Google Scholar) 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, α crystallin, SPA224) were purchased from Stressgen Bioreagents. The antibody against SOD1 is purchased from Biodesign (Saco, ME). 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 (38Bruening W.