Abstract
Alexander disease (AxD) is a neurodegenerative disease caused by heterozygous mutations in the GFAP gene, which encodes the major intermediate filament protein of astrocytes. This disease is characterized by the accumulation of cytoplasmic protein aggregates, known as Rosenthal fibers. Antibodies specific to GFAP could provide invaluable tools to facilitate studies of the normal biology of GFAP and to elucidate the pathologic role of this IF protein in disease. While a large number of antibodies to GFAP are available, few if any of them have defined epitopes. Here we described the characterization of a panel of commonly used anti-GFAP antibodies, which recognized epitopes at regions extending across the rod domain of GFAP. We show that all of the antibodies are useful for immunoblotting and immunostaining, and identify a subset that preferentially recognized human GFAP. Using these antibodies, we demonstrate the presence of biochemically modified forms of GFAP in brains of human AxD patients and mouse AxD models. These data suggest that this panel of anti-GFAP antibodies will be useful for studies of animal and cell-based models of AxD and related diseases in which cytoskeletal defects associated with GFAP modifications occur.
Highlights
Intermediate filaments (IFs) are a highly dynamic cytoskeletal component that provides a structural scaffold and a signaling platform for the organization of the cytoplasm
To determine the epitopes recognized by a panel of pre-existing monoclonal anti-GFAP antibodies, we developed a series of vectors for expression in HeLa cells, a human cervix epitheloid carcinoma cell line that has no endogenous GFAP
Immunoblotting showed that the SMI-23 antibody (Fig 1A) did not react with the amino acids 1–312 of GFAP, it did bind to GFAP residues 1–340, suggesting that the epitope for this antibody lies between amino acid residues 312 and 340 of GFAP
Summary
Intermediate filaments (IFs) are a highly dynamic cytoskeletal component that provides a structural scaffold and a signaling platform for the organization of the cytoplasm. At least 70 different IF proteins have been identified [1], many of which are expressed in cell type specific patterns. GFAP, together with lesser amounts of vimentin [2], nestin [3], and synemin [4], are the major IF proteins that constitute the glial filaments. Alexander disease (AxD) is a primary astrocyte disease caused by autosomal dominant mutations in the gene encoding GFAP [5]. AxD is a fatal leukoencephalopathy that often affects infants and young children. This early-onset Type I form is characterized by symptoms including psychomotor retardation, seizures, and megalencephaly.
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