title: The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain.
abstract: To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961.
authors.affiliation: Department of Molecular Cell Function, Faculty of Life Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto, 862-0973, Japan. kkawa@nupals.ac.jp.|Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata, 956-8603, Japan. kkawa@nupals.ac.jp.
authors.forename: Kohichi
authors.identifier: 0000-0003-3195-6649
authors.initials: K
authors.lastname: Kawahara
authors.affiliation: Department of Molecular Cell Function, Faculty of Life Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto, 862-0973, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hirata
authors.affiliation: Department of Molecular Cell Function, Faculty of Life Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto, 862-0973, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Ohbuchi
authors.affiliation: Department of Molecular Cell Function, Faculty of Life Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto, 862-0973, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nishi
authors.affiliation: Department of Molecular Cell Function, Faculty of Life Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto, 862-0973, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Maeda
authors.affiliation: Department of Molecular Cell Pharmacology, Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto, 860-0082, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kuniyasu
authors.affiliation: Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata, 956-8603, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamada
authors.affiliation: Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata, 956-8603, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Maeda
authors.affiliation: Department of Biological Science and Technology, the University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima, 770-8506, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tsuji
authors.affiliation: Department of Brain Functions, Research Institute of Environmental Medicine, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Sawada
authors.affiliation: Department of Molecular Cell Function, Faculty of Life Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto, 862-0973, Japan. jin@gpo.kumamoto-u.ac.jp.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakayama
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D001921:Brain / Q000166:cytology*; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D053331:Ectodysplasins / Q000378:metabolism; D004622:Embryo, Mammalian; D005123:Eye / Q000196:embryology / Q000254:growth & development / Q000378:metabolism; D005260:Female; D045683:Furin / Q000235:genetics / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D018664:Interleukin-12 / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology*; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D015703:Antigens, CD; D002135:Calcium-Binding Proteins; D053331:Ectodysplasins; D045683:Furin; D018664:Interleukin-12; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D011509:Proteoglycans; D000099216:Chondroitin Sulfate Proteoglycan 4; C100745:Aif1 protein, rat; C503043:Gpnmb protein, rat
keywords: GPNMB/osteoactivin; development; microglial heterogeneity; retinal pigment epithelium