47 research outputs found
Neuronal Diseases: Small Heat Shock Proteins Calm Your Nerves
AbstractMutations in HSPB1 and HSPB8, members of the small heat shock protein family, have recently been shown to cause some distal motor neuropathies. Their function in motor neurones is now under scrutiny
Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander Disease severity
Alexander Disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD
αB-crystallin is a sensor for assembly intermediates and for the subunit topology of desmin intermediate filaments
Characterization of a panel of monoclonal antibodies recognizing specific epitopes on GFAP
<div><p>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.</p></div
Clinical and genetic details of Alexander disease patient samples analyzed by immunoblotting.
<p>Clinical and genetic details of Alexander disease patient samples analyzed by immunoblotting.</p
Stochastically determined directed movement explains the dominant small-scale mitochondrial movements within non- neuronal tissue culture cells
AbstractThe apparently stationary phase of mitochondrial motion was investigated in epithelial cells by spinning disk confocal light microscopy combined with image correlation based single particle tracking using custom software producing sub-pixel accuracy measurements (∼5nm) at 10–12Hz frame-rates. The analysis of these data suggests that the previously described stationary, or anchored phase, in mitochondrial movement actually comprise Brownian diffusion, interspersed with frequent and brief motor-driven events whose duration are stochastically determined. We have therefore discovered a new aspect of mitochondrial behavior, which we call stochastically determined, directed movement
Fine mapping of the SMI-21 antibody epitope on GFAP.
<p>(A) Purified recombinant full-length (A, lane 1) and Δ179–206 GFAP (A, lane 2) were probed with either SMI-21 or SMI-23 antibody. The SMI-21 antibody was unable to recognize the GFAP with its putative epitope being removed (A, lane 2). (B) Total lysates prepared from SW13 (Vim-) cells (B, lane 1) expressing human full-length GFAP (B, lane 2) was probed with SMI-21 antibody in the absence (top panel) or presence of peptide 1 corresponding to GFAP residues 186–194 (middle panel), or peptide 2 corresponding to GFAP residues 196–206 (bottom panel). Approximate molecular weight markers (in kDa) were shown on the left. (C-F) SW13 (Vim-) cells transfected with Δ179-206GFAP were immunostained with either SMI-21 (C) or SMI-23 (E) antibody and counterstained with the anti-panGFAP antibody (D and F) to reveal transfected cells. Note that Δ179-206GFAP was readily detected by the SMI-23 antibody (E), but not the SMI-21 antibody (C). Bar = 10 μm.</p
Epitope mapping of anti-GFAP antibodies.
<p>HeLa cells were transfected with indicated GFAP constructs for 48 hours. Total cell lysates were prepared and analyzed by immunoblotting with anti-GFAP antibodies as indicated at the bottom of each blot. Representative images showed the immunoblotting pattern for SMI-23 (A), 2.2B<sub>10</sub> (B) and SMI-21 (C) GFAP antibodies. Immunoblots were also probed with the anti-panGFAP antibody to reveal transfected proteins (D-F). The ability of GFAP antibodies to detect various GFAP proteins is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180694#pone.0180694.t003" target="_blank">Table 3</a>.</p
Characterization of polyclonal antibodies specific to mouse GFAP.
<p>(A) Purified recombinant mouse (A, lanes 1, 3 and 5, labeled M) and human (A, lanes 2, 4 and 6, labeled H) GFAPs were probed with anti-mouse GFAP (A, lanes 1 and 2), anti-human GFAP (SMI-21) (A, lanes 3 and 4) and anti-panGFAP (A, lanes 5 and 6) antibodies. Notice that the anti-mouse GFAP (A, lane 1) and SMI-21 (A, lane 4) antibodies recognized mouse and human GFAP, respectively, confirming the specificity of these antibodies. The anti-panGFAP antibody, however, recognized both mouse and human GFAPs (A, lanes 5 and 6). Approximate molecular weight markers (in kDa) were shown on the left. Primary mouse astrocytes (B and C) were transiently transfected with human wild type GFAP (D and E). At 48 hours after transfection, cells were immunostained with SMI-21 (B and D) and anti-mouse GFAP antibodies (C and E). When expressed in mouse primary astrocytes, human wild-type GFAP formed filamentous networks (D) that colocalized with the endogenous mouse GFAP (E). Bar = 10 μm.</p