In astrocytes, 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]. brain sample from the AxD patient harboring R239H GFAP (AxD#1) by the SMI-21 antibody revealed two proteolytic fragments (lane 1), which were of similar sized as the corresponding fragments generated in HeLa Solifenacin cells transiently transfected with R239H GFAP (lane 2, p30 and p26 indicated by arrows). (B) Purified recombinant R239H GFAP was either untreated (lane 1) or treated with 2.5 U of active caspase 3 (lane 2) for 1 h at 37C. The reaction products were analyzed by immunoblotting using the polyclonal anti-panGFAP antibodies. Note that GFAP cleaved by active caspase 3 generated two prominent proteolytic fragments, p30 and p20 (B, lane 2, arrows).(PDF) pone.0180694.s003.pdf (113K) GUID:?8F95294E-C24F-4054-A9C5-90CC5E36294F Data Availability StatementAll relevant data are within the paper and its Supporting Information files. 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. TSPAN3 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 Solifenacin 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. Introduction Intermediate filaments (IFs) are a highly dynamic cytoskeletal component that provides a structural scaffold and a signaling platform for the organization of the cytoplasm. In humans, at least 70 different IF proteins have been identified [1], many of which are expressed in cell type specific patterns. In astrocytes, GFAP, together with lesser amounts of vimentin [2], nestin [3], and synemin [4], are the major IF proteins that constitute the glial filaments. Solifenacin Alexander disease (AxD) is a primary astrocyte disease Solifenacin caused by autosomal dominant mutations in the gene encoding GFAP [5]. Clinically, 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. Milder forms of AxD with variable ages of onset also exist [6]. The Type II form differs markedly in clinical presentation, and patients pathology predominantly in the cerebellum, brainstem and cervical spinal cord. Pathologically, AxD is characterized by the presence of ubiquintinated protein inclusions known as Rosenthal fibers (RFs) that are found almost exclusively in astrocytes throughout the central nervous system. In addition to GFAP, RFs have been reported to contain other IF proteins, including vimentin [7, 8], synemin [9], plectin [10], as well as the small stress proteins B-crystallin and HSP27 [11, 12]. How RFs alter the biochemistry, morphology, and function of astrocytes are not clear. Mouse models created through both transgenic [13, 14] and knock-in [15] approaches showed that simply elevating the level of wild-type GFAP or expressing a mutant form of this protein leads to the formation of RFs. Astrocytes cultured from AxD mice exhibit several biochemical and functional changes, including spontaneous formation of GFAP inclusions, increased caspase activity, decreased cell viability [16] and changes in cell morphology [17]. Cellular models and in vitro studies have provided additional evidence to show that GFAP accumulation increased levels of ubiquitinated GFAP species [18], inhibited proteasome activity [18, 19], and stimulated autophagy [20, 21]. Analyses of GFAP levels and post-translational modifications by antibody-based immunoassays are therefore essential to better understand the pathological sequence of events leading to disease-linked aggregation of glial filaments that occur in the context of this disease. To achieve this goal, well-characterized anti-GFAP antibodies are needed. Although a large number of antibodies specific to GFAP have been widely available [22C29], few if any of them have defined epitopes. Here we determine the epitopes recognized by a panel of anti-GFAP antibodies and evaluate their use in the analysis of GFAP, with a special focus on biochemically.