Microglia-specific localisation of a novel calcium binding protein, Iba1

doi:10.1016/S0169-328X(98)00040-0

Molecular Brain Research

Volume 57, Issue 1, 1 June 1998, Pages 1-9

https://doi.org/10.1016/S0169-328X(98)00040-0 Get rights and content

Abstract

Recently it has been shown that mRNA of Iba1 (ionized calcium binding adaptor molecule 1), which was a novel calcium binding protein cDNA-cloned by our group, is specifically expressed in microglia in cultures of rat brain cells [Imai et al. Biophys. Biochem. Res. Commun., 224 (1996) 855–862]. In the present study, immunocytochemical and immunohistochemical examinations demonstrated that Iba1 protein is expressed in microglia alone both in cultured brain cells and in the brain, respectively. In a mixed cell culture of embryonic rat brain, immunocytochemically positive for Iba1 protein were the microglia but it was not detectable in neurons, astroglia, or oligodendroglia. Immunohistochemical staining of adult rat brain sections showed Iba1 protein to be specifically localised in ramified microglia. In addition, immunohistochemical staining and immunoblot analysis of activated microglia in the facial nucleus after facial nerve axotomy shows that expression of Iba1 protein was upregulated and peaked at 7 days. These results indicated that localisation of Iba1 protein is restricted to microglia both in vitro and in vivo, and that Iba1 protein plays a role in regulating the function of microglia, especially in the activated microglia.

Introduction

Calcium ions are known to be one of the most important signal mediators in all cells including central nervous system (CNS) cells. Calcium ions exert their signaling activity through association with various calcium binding proteins, many of which are classified into a large protein family, the EF hand protein family [25]. We have previously reported a cDNA cloning of a novel EF hand protein, Iba1 (ionized calcium binding adaptor molecule 1) [12].

Iba1 protein is a small 17-kDa protein consisting of 147 amino acids. It contains two EF hand motifs in the central third of the molecule and this portion shows homology with other EF hand proteins, but none of the other amino- or carboxy-terminal regions have any homology with any known proteins. The genomic copy of the Iba1 gene was mapped to within the major histocompatibility complex (MHC) class III region between the BAT2 and TNF alpha genes. Using a specific antibody, Iba1 protein was clearly shown to be expressed in monocytic cell lines. Iba1 mRNA has been found to be highly and specifically expressed in microglia among the cultured brain cells. This suggested that Iba1 protein functions as an adapter molecule that mediates calcium signals in the monocytic lineage, including microglia. In the present study, patterns of expression of Iba1 protein in CNS cells were investigated in vitro and vivo, mainly by immunocytochemical and immunohistochemical techniques using mixed brain cell cultures and adult rat brain sections.

Facial nerve axotomy is a well-established experimental system by which microglia are locally activated to show morphological change, proliferation and migration to attach and surround the axotomized motoneurons [32]. We have furthermore examined Iba1 expression of the activated microglia detected in the axotomized facial nucleus, and also discussed the role of Iba1 protein in the activation of microglia.

Section snippets

Immunoblot analysis of primary cultured cells

Primary cultures of neurons, microglia, astroglia, and meningeal fibroblasts were prepared from rat brain according to the method described previously 12, 19, 28, 33. After seeding, neurons were cultured in poly-l-lysine-coated culture dishes for 1 day in minimum essential medium (MEM) containing 10% fetal calf serum (FCS). Microglia were cultured for 1 day in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS and astroglia and meningeal fibroblasts were cultured for 7 days in same type

Microglia-specific expression of Iba1 protein in cultured brain cells

In our previous reports 12, 21, we have reported cDNA cloning of iba1. RNA blot analysis demonstrated that Iba1 mRNA is expressed in cultured microglia but not in neurons or astroglia. To confirm the cell-type localisation of Iba1 protein in the brain, microglia, neurons, astroglia, and meningeal fibroblasts were prepared and immunoblotted with the anti-Iba1 antibody. A 17-kDa band corresponding to Iba1 protein was clearly detected in cultured microglia, but not in neurons, astroglia or

Discussion

In the present study we have shown that Iba1 is specifically expressed in microglia on the protein level, but not in neurons, astroglia, or oligodendroglia, both in vitro and in vivo. Immunocytochemical staining of cultured mixed brain cells showed that the cells that stained with the anti-Iba1 antibody also stained with the anti-ED-1 antibody (Fig. 2G,H), which is one of the well-established antibody reacts with microglia [3]. In addition, immunohistochemical staining of normal rat brain

Acknowledgements

This study was supported by Grants from the Japanese Ministry of Health and Welfare, the Science and Technology Agency, Japan, and by a Grant-in-Aid for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science, Sports and Culture.

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Research reportMicroglia-specific localisation of a novel calcium binding protein, Iba1

Abstract

Introduction

Section snippets

Immunoblot analysis of primary cultured cells

Microglia-specific expression of Iba1 protein in cultured brain cells

Discussion

Acknowledgements

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding

Anal. Biochem.

El tercer elemento de los centros nerviosos: I. La microglia en estados normal; II. Intervencio de la microglia en los processos patologicas; III. Naturaleza probable de la microglia

Bol. Soc. Espanola Biol.

The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognised by monoclonal antibodies ED1, ED2 and ED3

Immunology

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Acta Neuropathol.

Characterisation of two new monoclonal antibodies directed against rat microglia

J. Comp. Neurol.

Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells

J. Neurosci. Res.

Identity of ED2-positive perivascular cells in rat brain

J. Neurosci. Res.

Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy

Neurosci. Lett.

Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain

Acta Neuropathol.

Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo

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A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage

Biochem. Biophys. Res. Commun.

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

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The T4 gene encodes the AIDS virus receptor and is expression in the immune system and the brain

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Research report
Microglia-specific localisation of a novel calcium binding protein, Iba1