title: Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis.
abstract: Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS) with accumulation of misfolded SOD1 proteins as intracellular inclusions in spinal motor neurons. Oligomerization of SOD1 via abnormal disulfide crosslinks has been proposed as one of the misfolding pathways occurring in mutant SOD1; however, the pathological relevance of such oligomerization in the SOD1-ALS cases still remains obscure.
  
  
  
  We prepared antibodies exclusively recognizing the SOD1 oligomers cross-linked via disulfide bonds in vitro. By using those antibodies, immunohistochemical examination and ELISA were mainly performed on the tissue samples of transgenic mice expressing mutant SOD1 proteins and also of human SOD1-ALS cases.
  
  
  
  We showed the recognition specificity of our antibodies exclusively toward the disulfide-crosslinked SOD1 oligomers by ELISA using various forms of purified SOD1 proteins in conformationally distinct states in vitro. Furthermore, the epitope of those antibodies was buried and inaccessible in the natively folded structure of SOD1. The antibodies were then found to specifically detect the pathological SOD1 species in the spinal motor neurons of the SOD1-ALS patients as well as the transgenic model mice.
  
  
  
  Our findings here suggest that the SOD1 oligomerization through the disulfide-crosslinking associates with exposure of the SOD1 structural interior and is a pathological process occurring in the SOD1-ALS cases.
authors.affiliation: Laboratory for Mechanistic Chemistry of Biomolecules, Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa, 223-8522, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Tokuda
authors.affiliation: Laboratory for Mechanistic Chemistry of Biomolecules, Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa, 223-8522, Japan.
authors.forename: Itsuki
authors.initials: I
authors.lastname: Anzai
authors.affiliation: Laboratory for Mechanistic Chemistry of Biomolecules, Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa, 223-8522, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Nomura
authors.affiliation: Laboratory for Mechanistic Chemistry of Biomolecules, Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa, 223-8522, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Toichi
authors.affiliation: Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Department of Neurology, Matsumoto Medical Center, Matsumoto, 399-0021, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Ohara
authors.affiliation: Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Yamanaka
authors.affiliation: Division of Pharmacology, Faculty of Pharmacy, Keio University, Tokyo, 105-8512, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Morisaki
authors.affiliation: Division of Pharmacology, Faculty of Pharmacy, Keio University, Tokyo, 105-8512, Japan.
authors.forename: Hidemi
authors.initials: H
authors.lastname: Misawa
authors.affiliation: Laboratory for Mechanistic Chemistry of Biomolecules, Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa, 223-8522, Japan. furukawa@chem.keio.ac.jp.
authors.forename: Yoshiaki
authors.identifier: 0000-0002-1918-372X
authors.initials: Y
authors.lastname: Furukawa
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology*; D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D057165:Proteostasis Deficiencies / Q000201:enzymology; D000072105:Superoxide Dismutase-1 / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C000606290:SOD1 protein, human; D000072105:Superoxide Dismutase-1
keywords: Amyotrophic lateral sclerosis; Cu/Zn-superoxide dismutase; Disulfide bond; Protein misfolding