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

Kohichi Kawahara, Hiroshi Hirata, Kengo Ohbuchi, Kentaro Nishi, Akira Maeda, Akihiko Kuniyasu, Daisuke Yamada, Takehiko Maeda, Akihiko Tsuji, Makoto Sawada, Hitoshi Nakayama

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 Lys170. 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.

Original languageEnglish
Pages (from-to)1938-1961
Number of pages24
JournalGLIA
Volume64
Issue number11
DOIs
Publication statusPublished - Nov 1 2016
Externally publishedYes

Fingerprint

Furin
Microglia
Melanoma
Glycoproteins
Peptide Hydrolases
Monoclonal Antibodies
Brain
IgA receptor
Corpus Callosum
Lateral Ventricles
Cross Reactions
Peritoneal Macrophages
Prosencephalon

Keywords

  • development
  • GPNMB/osteoactivin
  • microglial heterogeneity
  • retinal pigment epithelium

ASJC Scopus subject areas

  • Neurology
  • Cellular and Molecular Neuroscience

Cite this

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. / Kawahara, Kohichi; Hirata, Hiroshi; Ohbuchi, Kengo; Nishi, Kentaro; Maeda, Akira; Kuniyasu, Akihiko; Yamada, Daisuke; Maeda, Takehiko; Tsuji, Akihiko; Sawada, Makoto; Nakayama, Hitoshi.

In: GLIA, Vol. 64, No. 11, 01.11.2016, p. 1938-1961.

Research output: Contribution to journalArticle

Kawahara, K, Hirata, H, Ohbuchi, K, Nishi, K, Maeda, A, Kuniyasu, A, Yamada, D, Maeda, T, Tsuji, A, Sawada, M & Nakayama, H 2016, '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', GLIA, vol. 64, no. 11, pp. 1938-1961. https://doi.org/10.1002/glia.23034
Kawahara, Kohichi ; Hirata, Hiroshi ; Ohbuchi, Kengo ; Nishi, Kentaro ; Maeda, Akira ; Kuniyasu, Akihiko ; Yamada, Daisuke ; Maeda, Takehiko ; Tsuji, Akihiko ; Sawada, Makoto ; Nakayama, Hitoshi. / 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. In: GLIA. 2016 ; Vol. 64, No. 11. pp. 1938-1961.
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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 Lys170. 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.",
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AU - Kuniyasu, Akihiko

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