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• W2036693225 abstract "Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset. However, very little is known of the constituents and mechanism of formation of aggregates in ALS. We and others have shown that there is a progressive accumulation of detergent-insoluble mutant SOD1 in the spinal cord of G93A SOD1 mice. To investigate the mechanism of SOD1 aggregation, we characterized by proteome technologies SOD1 isoforms in a Triton X-100-insoluble fraction of spinal cord from G93A SOD1 mice at different stages of the disease. This showed that at symptomatic stages of the disease, part of the insoluble SOD1 is unambiguously mono- and oligoubiquitinated, in spinal cord and not in hippocampus, and that ubiquitin branches at Lys48, the major signal for proteasome degradation. At presymptomatic stages of the disease, only insoluble unmodified SOD1 is recovered. Partial ubiquitination of SOD1-rich inclusions was also confirmed by immunohistochemical and electron microscopy analysis of lumbar spinal cord sections from symptomatic G93A SOD1 mice. On the basis of these results, we propose that ubiquitination occurs only after SOD1 aggregation and that oligoubiquitination may underline alternative mechanisms in disease pathogenesis. Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset. However, very little is known of the constituents and mechanism of formation of aggregates in ALS. We and others have shown that there is a progressive accumulation of detergent-insoluble mutant SOD1 in the spinal cord of G93A SOD1 mice. To investigate the mechanism of SOD1 aggregation, we characterized by proteome technologies SOD1 isoforms in a Triton X-100-insoluble fraction of spinal cord from G93A SOD1 mice at different stages of the disease. This showed that at symptomatic stages of the disease, part of the insoluble SOD1 is unambiguously mono- and oligoubiquitinated, in spinal cord and not in hippocampus, and that ubiquitin branches at Lys48, the major signal for proteasome degradation. At presymptomatic stages of the disease, only insoluble unmodified SOD1 is recovered. Partial ubiquitination of SOD1-rich inclusions was also confirmed by immunohistochemical and electron microscopy analysis of lumbar spinal cord sections from symptomatic G93A SOD1 mice. On the basis of these results, we propose that ubiquitination occurs only after SOD1 aggregation and that oligoubiquitination may underline alternative mechanisms in disease pathogenesis. Amyotrophic lateral sclerosis (ALS) 2The abbreviations used are: ALS, amyotrophic lateral sclerosis; fALS, familial form of ALS; WT, wild type; TX, Triton X-100; 2DE, two-dimensional gel electrophoresis; IPG, immobilized-pH gradient; IEF, isoelectric focusing; WB, Western blotting; h, human; V8, endoprotease V8; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; GFAP, glial fibrillary acidic protein; ChAT, choline acetyl-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is a fatal neurodegenerative disease that specifically affects motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of ALS (fALS) (1Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. Mulder D.W. Smyth C. Laing N.G. Soriano E. Pericak-Vance M.A. Haines J. Rouleau G.A. Gusella J.S. Horvitz H.R. Brown Jr., R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar) through a gain-of-function mechanism, which remains unclear. Human SOD1 is a homodimeric antioxidant enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide and dioxygen. Each subunit contains an eight-stranded β-barrel motif, an active site that binds a catalytic copper ion and a structural zinc ion, and an intramolecular disulfide bond. This bond, a peculiarity for a cytosolic protein, is adjacent to the dimer interface and seems to be important in stabilizing the overall structure and in the function of the protein (2Lindberg M.J. Normark J. Holmgren A. Oliveberg M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15893-15898Crossref PubMed Scopus (151) Google Scholar, 3Furukawa Y. Torres A.S. O'Halloran T.V. EMBO J. 2004; 23: 2872-2881Crossref PubMed Scopus (293) Google Scholar). To date, more than 100 different SOD1 mutations have been linked to fALS. SOD1 mutations, primarily missense, are scattered throughout the gene and are believed to influence different functions of the protein (4Valentine J.S. Doucette P.A. Potter S.Z. Annu. Rev. Biochem. 2005; 74: 563-593Crossref PubMed Scopus (614) Google Scholar). However, at least some mutant enzymes share common properties in vitro. In comparison with wild type (WT) SOD1, mutants display general structural instability (5Lindberg M.J. Tibell L. Oliveberg M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16607-16612Crossref PubMed Scopus (176) Google Scholar), the disulfide bond being more susceptible to reduction (6Tiwari A. Hayward L.J. J. Biol. Chem. 2003; 278: 5984-5992Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), with an enhanced propensity to form aggregates (7Stathopulos P.B. Rumfeldt J.A. Scholz G.A. Irani R.A. Frey H.E. Hallewell R.A. Lepock J.R. Meiering E.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7021-7026Crossref PubMed Scopus (225) Google Scholar). In vivo, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from fALS patients, mutant SOD1 animals, and cellular models (8Wood J.D. Beaujeux T.P. Shaw P.J. Neuropathol. Appl. Neurobiol. 2003; 29: 529-545Crossref PubMed Scopus (122) Google Scholar). In fALS mice, SOD1 aggregates localize in neurons and astrocytes (9Stieber A. Gonatas J.O. Gonatas N.K. J. Neurol. Sci. 2000; 173: 53-62Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), as perikaryal deposits and as macromolecular complexes associated with various mitochondrial compartments (10Manfredi G. Xu Z. Mitochondrion. 2005; 5: 77-87Crossref PubMed Scopus (174) Google Scholar), and inside the endoplasmic reticulum (11Kikuchi H. Almer G. Yamashita S. Guegan C. Nagai M. Xu Z. Sosunov A.A. McKhann 2nd, G.M. Przedborski S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6025-6030Crossref PubMed Scopus (264) Google Scholar). In fALS mice SOD1 insoluble protein complexes are formed before the onset of motor dysfunction (12Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (513) Google Scholar) and are found exclusively in tissues affected by the disease (13Wang J. Xu G. Borchelt D.R. Neurobiol. Dis. 2002; 9: 139-148Crossref PubMed Scopus (176) Google Scholar). All of these observations indicate that mutant SOD1 aggregation may play a role in the pathogenesis and that aggregation may be a toxic property acquired by SOD1. What we know about the aggregate protein constituents comes principally from immunohistochemistry studies on post-mortem tissues of ALS patients or spinal cords of mutant SOD1 mice (8Wood J.D. Beaujeux T.P. Shaw P.J. Neuropathol. Appl. Neurobiol. 2003; 29: 529-545Crossref PubMed Scopus (122) Google Scholar). In sporadic and fALS patients the most widely seen inclusions (in almost all cases) immunostain for ubiquitin. In mutant SOD1 mice protein inclusions mainly immunoreactive for SOD1 and ubiquitin have been detected (14Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Crossref PubMed Scopus (1001) Google Scholar, 15Watanabe M. Dykes-Hoberg M. Culotta V.C. Price D.L. Wong P.C. Rothstein J.D. Neurobiol. Dis. 2001; 8: 933-941Crossref PubMed Scopus (346) Google Scholar, 16Cheroni C. Peviani M. Cascio P. De Biasi S. Monti C. Bendotti C. Neurobiol. Dis. 2005; 18: 509-522Crossref PubMed Scopus (87) Google Scholar). However, a clear co-localization between SOD1 and ubiquitin signals was never shown. Ubiquitinated inclusions have been found in other neurodegenerative diseases (17Ross C.A. Poirier M.A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2504) Google Scholar) and might represent the overwhelmed cellular defense against misfolded and/or abnormally modified proteins, which are normally linked to polyubiquitin chains and then degraded by the proteasome system. Mutant SOD1 is catabolized by the proteasome in vitro, in transfected cells, and in mice spinal cord tissues (18Urushitani M. Kurisu J. Tsukita K. Takahashi R. J. Neurochem. 2002; 83: 1030-1042Crossref PubMed Scopus (228) Google Scholar, 19Puttaparthi K. Wojcik C. Rajendran B. DeMartino G.N. Elliott J.L. J. Neurochem. 2003; 87: 851-860Crossref PubMed Scopus (58) Google Scholar, 20Di Noto L. Whitson L.J. Cao X. Hart P.J. Levine R.L. J. Biol. Chem. 2005; 280: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). One possible explanation for the protein inclusions is that mutant SOD1 accumulates as a consequence of a low level or reduced activity of the proteasome. We and other groups have investigated the proteasome degradation pathways in connection with ALS, but it is still inexplicably contentious whether the constitutive proteasome activity is inhibited or not and whether the induction of immunoproteasome subunits eventually compensates for proteasome dysfunction (16Cheroni C. Peviani M. Cascio P. De Biasi S. Monti C. Bendotti C. Neurobiol. Dis. 2005; 18: 509-522Crossref PubMed Scopus (87) Google Scholar, 21Kabashi E. Agar J.N. Taylor D.M. Minotti S. Durham H.D. J. Neurochem. 2004; 89: 1325-1335Crossref PubMed Scopus (122) Google Scholar, 22Puttaparthi K. Elliott J.L. Exp. Neurol. 2005; 196: 441-451Crossref PubMed Scopus (50) Google Scholar). In line with previous observations (12Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (513) Google Scholar, 23Shinder G.A. Lacourse M.C. Minotti S. Durham H.D. J. Biol. Chem. 2001; 276: 12791-12796Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), we have shown that in the spinal cords of G93A SOD1 mice there is progressive accumulation of high molecular mass and detergent-insoluble mutant SOD1 (16Cheroni C. Peviani M. Cascio P. De Biasi S. Monti C. Bendotti C. Neurobiol. Dis. 2005; 18: 509-522Crossref PubMed Scopus (87) Google Scholar). We have now thoroughly characterized SOD1 isoforms in a detergent-insoluble fraction from spinal cord of G93A SOD1 mice at different stages of the disease, with a view to elucidating the mechanism of SOD1 aggregation and the possible link to the disease pathogenesis. We unambiguously established that SOD1 accumulates in spinal cord detergent-insoluble aggregates of symptomatic G93A SOD1 mice in part as mono- and oligoubiquitinated forms. Transgenic Mouse Models—Transgenic mice originally obtained from Jackson Laboratories and expressing a high copy number of mutant human (h) SOD1 with a Gly93 → Ala substitution or wild type (WT) hSOD1 mice were bred and maintained on a C57BL/6 mice strain at the Consorzio Mario Negri Sud, S. Maria Imbaro (CH), Italy. Transgenic mice are identified by PCR (1Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. Mulder D.W. Smyth C. Laing N.G. Soriano E. Pericak-Vance M.A. Haines J. Rouleau G.A. Gusella J.S. Horvitz H.R. Brown Jr., R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar). The mice were housed at 21 ± 1 °C with relative humidity 55 ± 10% and 12 h of light. Food (standard pellets) and water were supplied ad libitum. In this study, female G93A SOD1 mice were killed at 7, 12, 17, and 26 weeks of age, corresponding to the early presymptomatic, presymptomatic, early symptomatic, and end stages of the disease. Female WT SOD1 or nontransgenic mice were used as controls. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. No. 116, G.U. Suppl. 40, Feb. 18, 1992, Circolare No. 8, G.U., 14 luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJ L 358,1, Dec.12, 1987; NIH Guide for the Care and use of Laboratory Animals, United States National Research Council, 1996). Extraction of Detergent-insoluble Proteins—The tissues were processed as previously described (16Cheroni C. Peviani M. Cascio P. De Biasi S. Monti C. Bendotti C. Neurobiol. Dis. 2005; 18: 509-522Crossref PubMed Scopus (87) Google Scholar), with some modifications. Briefly, they were homogenized in ice-cold homogenization buffer, pH 7.6, containing 15 mm Tris-HCl, 1 mm dithiothreitol, 0.25 m sucrose, 1 mm MgCl2, 2.5 mm EDTA, 1 mm EGTA, 0.25 m sodium orthovanadate, 2 mm sodium pyrophosphate, 5 μm MG132 proteasome inhibitor (Sigma), 1 tablet of Complete™/10 ml of buffer, Mini Protease Inhibitor Mixture (Roche Applied Science), and in some experiments 5 mg/ml of iodoacetamide, to ensure inhibition of ubiquitin-cleaving isopeptidases (24Matsui S. Sandberg A.A. Negoro S. Seon B.K. Goldstein G. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1535-1539Crossref PubMed Scopus (64) Google Scholar). The samples were centrifuged at 10,000 × g at 4 °C for 15 min. The supernatant was centrifuged at 100,000 × g for 1 h to obtain the cytosolic fraction. The pellet was suspended in ice-cold homogenization buffer with 2% of Triton X-100 (TX) and 150 mm KCl added, sonicated three times for 10 s, and shaken for 1 h at 4 °C. The samples were then centrifuged twice at 10,000 × g at 4 °C for 10 min to obtain TX-resistant pellets. The proteins were quantified by the Bradford assay. The pellets were frozen at -20 °C until further analyses. Cytosolic fractions were analyzed in some experiments after stable modification of Cys residues: (i) reduction in the presence of 10 mm dithiothreitol and alkylation with 5 mg/ml iodoacetamide or (ii) oxidation with performic acid (25Persson C. Kappert K. Engstrom U. Ostman A. Sjoblom T. Methods. 2005; 35: 37-43Crossref PubMed Scopus (36) Google Scholar). Two-dimensional Gel Electrophoresis (2DE)—The samples were dissolved in 7 m urea, 2 m thiourea, 4% (w/v) CHAPS, 0.5% (v/v) immobilized pH gradient (IPG) buffer (Amersham Biosciences), and 12 μl/ml DeStreak™ Reagent (Amersham Biosciences), which form stable disulfide bonds and prevent unspecific Cys residue oxidation during isoelectric focusing (IEF) (26Olsson I. Larsson K. Palmgren R. Bjellqvist B. Proteomics. 2002; 2: 1630-1632Crossref PubMed Scopus (99) Google Scholar). The samples were loaded by in-gel rehydration (1 h at 0 V, 270 Vhr at 30 V) on pH 4-7 linear or pH 3-10 nonlinear 7-cm IPG strips (Amersham Biosciences). IEF was done on an IPGphor (Amersham Biosciences) according to the following schedule: 200 Vhr at 200 V, 925 Vhr of a linear gradient up to 3500 V, 10,500 Vhr at 3500 V, 14,375 Vhr of a linear gradient up to 8000 V, and 48,000 Vhr at 8000 V. The strips were then re-equilibrated in NuPAGE LDS sample buffer (Invitrogen), and second dimension was run on precast, 4-12% polyacrylamide gradient gel, NuPAGE® Bis-Tris (Invitrogen). The gels were stained with SYPRO® Ruby protein gel stain (Molecular Probes). Western Blotting (WB)—The proteins were transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore). Detection of ubiquitinated proteins required pretreatment of the membrane to expose the ubiquitin epitope and favor antibody recognition. After blotting, the membranes were treated with 6 m guanidine-HCl, 20 mm Tris-HCl, pH 7.5, 1 mm phenylmethylsulfonyl fluoride, and 5 mm dithiothreitol for 30 min at room temperature. For the reaction with primary antibodies, the membranes were incubated for 1 h at room temperature with a blocking buffer (5% milk in Tris-buffered saline containing 0.1% Tween 20) and probed overnight at 4 °C with rabbit polyclonal antibody anti-hSOD1 (Upstate) diluted 1:2000 in blocking buffer or with rabbit polyclonal antibody anti-ubiquitin (DakoCytomation) diluted 1:800 in blocking buffer. The membrane was then washed and incubated for 1 h at room temperature with goat anti-rabbit peroxidase-conjugated secondary antibody diluted 1:5000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The blots were developed by the ECL Plus protein detection system (Amersham Biosciences). Immunoreactivity was normalized to the actual amount of proteins loaded on the membrane as detected after Coomassie Blue staining. In double immunostaining experiments the membranes were stripped by Restore™ Western blot stripping buffer (Pierce). Image Analysis—SYPRO Ruby-stained two-dimensional gel images were captured by the laser scanner Molecular Imager® FX (Bio-Rad) using a 532-nm laser as excitation source, and 530-nm long pass as emission filter. Two-dimensional WB images were captured by an Expression 1680 Pro scanner (Epson) at 16 bit and 300 d.p.i. resolution. Densitometry and image analysis were done by the Progenesis PG240 v2006 software (Nonlinear Dynamics). Gel/blot matching was done by using the specific warping algorithm of the software in the manual mode, placing seeding points on recognizable, intense immunopositive spots. Mass Spectrometry—Protein spots were located and excised with an EXQuest™ spot cutter (Bio-Rad). The spots were processed and gel-digested alternatively with trypsin or endoprotease V8 (V8) (Sigma), essentially as previously described (27Casoni F. Basso M. Massignan T. Gianazza E. Cheroni C. Salmona M. Bendotti C. Bonetto V. J. Biol. Chem. 2005; 280: 16295-16304Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Peptide mass fingerprinting was analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) on a ReflexIII™ (Bruker Daltonics) instrument using α-cyano-4-hydroxycinnamic acid as matrix. The mass spectra were internally calibrated with trypsin or V8 autolysis fragments, routinely obtaining accuracy better than 30 ppm. For the detection of additional post-translational modifications of SOD1, liquid chromatography-MS/MS analysis was done on a reverse phase microbore liquid chromatography Suveyor system coupled to an ion trap mass spectrometer LCQ Deca XPPlus (Thermo Finningan), as described (28Pastorelli R. Carpi D. Campagna R. Airoldi L. Pohjanvirta R. Viluksela M. Hakansson H. Boutros P.C. Moffat I.D. Okey A.B. Fanelli R. Mol. Cell Proteomics. 2006; 5: 882-894Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Immunohistochemical Confocal Analysis—Three WT SOD1 mice and five G93A SOD1 mice at a late symptomatic stage of disease were analyzed in each experiment. The mice were anesthetized and transcardially perfused, and the spinal cords were removed, as previously described (27Casoni F. Basso M. Massignan T. Gianazza E. Cheroni C. Salmona M. Bendotti C. Bonetto V. J. Biol. Chem. 2005; 280: 16295-16304Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Immunolabeling was done on lumbar spinal cord sections (30-μm-thick floating cryosections). In each experiment, some of the sections were processed without primary antibody to verify the specificity of the staining. The sections were blocked in phosphate-buffered saline containing 5% normal goat serum and 0.05% TX for 60 min at room temperature and then incubated with anti-ubiquitin antibody (DakoCytomation) diluted 1:750. The sections were incubated with the anti-rabbit biotinylated antiserum (Vector Laboratories, diluted 1:500), and the immune reaction was revealed by the tyramide signal amplification kit (Cy5; PerkinElmer Life Sciences), as described (29Tortarolo M. Veglianese P. Calvaresi N. Botturi A. Rossi C. Giorgini A. Migheli A. Bendotti C. Mol. Cell Neurosci. 2003; 23: 180-192Crossref PubMed Scopus (143) Google Scholar). After the ubiquitin staining, a second staining was done with the anti-hSOD1 antibody (Upstate Biotechnology, Inc.) diluted 1:2000. The reaction was revealed by incubation with anti-rabbit antibody conjugated to Alexa 488 (1:500 dilution; Molecular Probes). No direct reaction was detected between this secondary fluorescent antibody and the anti-ubiquitin primary antibody using the dilution of antibodies above described. For the co-localization with glial fibrillary acidic protein (GFAP), the sections were first labeled with anti-ubiquitin and anti-hSOD1 antibodies, incubated with the anti-GFAP antibody (1:2500 dilution; Chemicon) and then with anti-mouse Alexa 546 antibody (1:500 dilution; Molecular Probes). For labeling with the motor neuron marker choline acetyl-transferase (ChAT), the sections were probed with the anti-ChAT antibody (1:200 dilution, Chemicon) and then with anti-mouse Alexa 546 antibody (1:500 dilution, Molecular Probes). Fluorescence-labeled sections were mounted with Fluorsave (Calbiochem) and analyzed under an Olympus Fluoview laser scanning confocal microscope. Wavelengths of 488 nm (Laser Ar-Kr), 546 nm (Laser He-Ne green), and 647 nm (Laser He-Ne red) were used to excite Alexa 488 and 546 and Cy5 fluorophore, respectively. Emission radiations (510-550 nm for Alexa 488 and 670 nm for Cy5) were collected on separate detectors. To eliminate the risk of cross-talk between channels, the sections were scanned in a sequential mode. Post-embedding Immunogold Electron Microscopy—For electron microscopy, G93A SOD1 mice at symptomatic stage (n = 3) and nontransgenic littermates (n = 3) were transcardially perfused with 4% paraformaldehyde under anesthesia, and blocks of their lumbar spinal cord were embedded in hydrophilic resin (LR white embedding medium, Sigma). Thin sections of the ventral horn cut with an ultramicrotome were collected on Formvar-coated nickel grids and processed with a standard postembedding immunogold staining protocol (30Bendotti C. Atzori C. Piva R. Tortarolo M. Strong M.J. De Biasi S. Migheli A. J. Neuropathol. Exp. Neurol. 2004; 63: 113-119Crossref PubMed Scopus (80) Google Scholar). Briefly, the grids were incubated overnight at 4 °C with either a monoclonal anti-human SOD1 antiserum (dilution 1:2000) (Medical & Biological Laboratories Co., Nagoya, Japan) or a polyclonal anti-ubiquitin antiserum (UG9510, Biomol; dilution 1:2000) for single labeling or with a mixture of both antisera for double labeling. After several rinses in buffers, the grids were incubated for 1 h at room temperature in a goat anti-mouse secondary antiserum conjugated to 15-nm gold particles to detect the monoclonal anti-human SOD1 antiserum or in a goat anti-rabbit secondary antiserum conjugated to 15-nm gold particles to detect the polyclonal anti-ubiquitin antiserum, whereas for double labeling the grids were incubated in a mixture of goat anti-mouse secondary antiserum conjugated to 10-nm gold particles to detect human SOD1 and goat anti-rabbit secondary antiserum conjugated to 20-nm gold particles to detect ubiquitin. All of the gold-conjugated secondary anti-sera were from British Biocell International (Cardiff, UK) and diluted 1:25. Control grids were processed in the same way, with omission of the primary antiserum; in double labeling experiments omission of one of the two primary antisera resulted in absence of the corresponding gold particles. All of the grids were counterstained with aqueous uranyl acetate and lead citrate and observed and photographed with a Jeol T8 electron microscope. 2DE Characterization of TX-insoluble G93A SOD1 Isolated from Spinal Cord of fALS Mice at Different Stages of the Disease—We have recently shown that in the spinal cord of G93A SOD1 mice there is a progressive accumulation of mutant SOD1 correlated with a decrease of its solubility in TX, forming evident aggregates at a late stage of the disease (16Cheroni C. Peviani M. Cascio P. De Biasi S. Monti C. Bendotti C. Neurobiol. Dis. 2005; 18: 509-522Crossref PubMed Scopus (87) Google Scholar). In this study we further analyzed, using 2DE, SOD1 in TX-insoluble extracts of spinal cord from SOD1 G93A mice, with a view to elucidating the mechanism of SOD1 aggregation. We analyzed samples from spinal cords of G93A mice at different stages of the disease, two presymptomatic stages, 7 and 12 weeks of age, an early symptomatic stage, 17 weeks of age, and a late symptomatic stage, 26 weeks of age. As controls, we analyzed samples from spinal cord of 26-week-old WT SOD1 mice and hippocampus, tissue not affected by the disease, of late symptomatic G93A SOD1 mice. Fig. 1 shows anti-hSOD1 two-dimensional WB of 2DE-separated TX-insoluble proteins from spinal cord of fALS mice at the different stages of the disease and control samples. There is a marked difference between the two-dimensional WB of the samples from presymptomatic G93A SOD1 mice (Fig. 1, A and B), where only one spot (Fig. 1A) or three (Fig. 1B) spots are visible, and the two-dimensional WB of the samples from mice at symptomatic stages of the disease (Fig. 1, C and D), where several and highly intense spots are present. In panels A and B, the spots at ∼16 kDa correspond to monomeric SOD1 isoforms. In panel C and D, two trains of spots in the 5-6 pI range are most intense, one with at least three spots at 16 kDa, corresponding to monomeric SOD1 isoforms, and one with at least four spots at an apparent molecular mass of 37 kDa, corresponding to dimeric SOD1 isoforms. Although proteins are dissolved in highly denaturing conditions (7 m urea, 2 m thiourea) and with reducing agents, some dimeric SOD1 isoforms are detected, because of resistance to complete disassembly of the multimeric SOD1 complexes abundantly present in symptomatic fALS mice. Dimeric SOD1 isoforms are formed through intermolecular disulfide bonds, and in fact they disappeared when the sample was drastically denatured and stably alkylated at Cys residues with iodoacetamide before IEF (data not shown). Only in symptomatic fALS mice there are other four trains of spots, focalized at slightly higher pI than monomeric SOD1, at approximately 24, 32, 40, and 48 kDa. Because these four trains differ by 8 kDa, which is a sign of ubiquitination, we did double immunostaining (Fig. 2) with antibody anti-ubiquitin and anti-hSOD1 on the same membrane to see whether there was any overlapping of signals. There was a clear overlap between the signals at 24 and 32 kDa in the anti-SOD1 and anti-ubiquitin two-dimensional WB and possibly also at 40 and 48 kDa. Monomeric SOD1 is therefore probably mono- and oligoubiquitinated in TX-insoluble spinal cord extracts of symptomatic G93A SOD1 mice. In conclusion, an increasing amount of TX-insoluble mutant SOD1 was recovered from spinal cords of fALS mice as disease progressed. Only at symptomatic stages of the disease part of the insoluble SOD1 was recovered as oligoubiquitinated. A small amount of TX-insoluble nonoligoubiquitinated SOD1 was present in hippocampi of late symptomatic fALS mice (Fig. 1E) and in spinal cords of 26-week-old WT SOD1 mice (Fig. 1F).FIGURE 2Anti-ubiquitin two-dimensional WB (A) and anti-hSOD1 two-dimensional WB (B) of TX-insoluble extracts from spinal cord of a 26-week-old G93A SOD1 mouse. After anti-ubiquitin WB, the membrane was stripped and reprobed with anti-hSOD1 antibody. The arrows indicate trains of spots corresponding to monomeric and dimeric SOD1. The asterisks highlight trains of perfectly overlapping spots, indicating that G93A SOD1 is at least mono- and biubiquitinated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) G93A SOD1 Is Present in TX-insoluble Aggregates as Mono- and K48-linked Oligoubiquitinated Isoforms—We further confirmed SOD1 ubiquitination, assessing the site of ubiquitination of SOD1 and the site of branching of the polyubiquitin chains using a combination of 2DE and MALDI MS. TX-insoluble extracts from 26-week-old G93A SOD1 mice were separated by 2DE in the 4-7 pI range. The gel image was matched and warped with the anti-ubiquitin and the anti-hSOD1 two-dimensional WB by computerized image analysis. Fig. 3 reports a representative SYPRO Ruby-stained two-dimensional gel of aggregated proteins where the spots matched with two-dimensional WB are indicated. Spots 6-14 matched concomitantly with anti-SOD1 and anti-ubiquitin immunoreactive spots. On the basis of the molecular mass in 2DE, spots 6-8 correspond to monoubiquitinated SOD1, spots 9-11 correspond to biubiquitinated SOD1, spots 12-13 correspond to triubiquitinated SOD1, and spot 14 corresponds to tetraubiquitinated SOD1. Spots 6-14 were gel-digested alternatively with trypsin or V8, and their peptide mass fingerprints were analyzed by MALDI MS to confirm and better characterize SOD1 ubiquitination. Table 1 shows the m/z of the ions that matched with SOD1 and ubiquitin tryptic and V8 fragments. All of the spots analyzed (spots 6-14) contained ubiquitin- and SOD1-derived fragments. A larger number of fragments were found in correspondence with monoubiquitinated SOD1, spots 6 and 7, with, respectively, 13 and 16 fragment ions, and diubiquitinated SOD1, spot 9, with 12 fragment ions.TABLE 1MALDI MS analysis of tryptic and V8 mass fingerprints of protein 2DE spots matching concomitantl" @default.
• W2036693225 created "2016-06-24" @default.
• W2036693225 creator A5008591366 @default.
• W2036693225 creator A5009056818 @default.
• W2036693225 creator A5020722865 @default.
• W2036693225 creator A5037015741 @default.
• W2036693225 creator A5050402377 @default.
• W2036693225 creator A5052424293 @default.
• W2036693225 creator A5055570295 @default.
• W2036693225 creator A5060876047 @default.
• W2036693225 date "2006-11-01" @default.
• W2036693225 modified "2023-10-12" @default.
• W2036693225 title "Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice" @default.
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