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• W2091697287 abstract "Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed “Rosenthal fibers,” contain the protein chaperones αB-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of αB-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease. Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed “Rosenthal fibers,” contain the protein chaperones αB-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of αB-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease. Alexander disease (MIM 203450) is a rare and often fatal neurological disorder, first described by W. S. Alexander.1Alexander WS Progressive fibrinoid degeneration of fibrillary astrocytes associated with mental retardation in a hydrocephalic infant.Brain. 1949; 72: 373-381Crossref PubMed Scopus (239) Google Scholar On the basis of age at onset, the disorder has been divided into three subtypes: infantile, juvenile, and adult.2Russo Jr, LS Aron A Anderson PJ Alexander’s disease: a report and reappraisal.Neurology. 1976; 26: 607-614Crossref PubMed Google Scholar The infantile form, with onset between birth and age ∼2 years, is the most common type and is characterized by extensive loss of white matter.3Neal JW Cave EM Singhrao SK Cole G Wallace SJ Alexander’s disease in infancy and childhood: a report of two cases.Acta Neuropathol (Berl). 1992; 84: 322-327Crossref PubMed Scopus (26) Google Scholar, 4Deprez M D’Hooghe M Misson JP de Leval L Ceuterick C Reznik M Martin JJ D’Hooge M Infantile and juvenile presentations of Alexander’s disease: a report of two cases.Acta Neurol Scand. 1999; 99: 158-165Crossref PubMed Scopus (22) Google Scholar, 5Rodriguez D Gauthier F Bertini E Bugiani M Brenner M N’Guyen S Goizet C Gelot A Surtees R Pedespan J-M Hernandorena X Troncoso M Uziel G Messing A Ponsot G Pham-Dinh D Dautigny A Boespflug-Tanguy O Infantile Alexander disease: spectrum of GFAP mutations and genotype-phenotype correlation.Am J Hum Genet. 2001; 69: 1134-1140Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar A striking neuropathological feature of all forms of Alexander disease is the presence of Rosenthal fibers, unique cytoplasmic inclusions within astrocytes that contain the major astrocytic intermediate filament (IF) protein glial fibrillary acidic protein (GFAP) and the chaperones αB-crystallin and HSP27.6Tomokane N Iwaki T Tateishi J Iwaki A Goldman JE Rosenthal fibers share epitopes with alpha B-crystallin, glial fibrillary acidic protein, and ubiquitin, but not with vimentin: immunoelectron microscopy with colloidal gold.Am J Pathol. 1991; 138: 875-885PubMed Google Scholar, 7Johnson AB Bettica A On-grid immunogold labeling of glial intermediate filaments in epoxy-embedded tissue.Am J Anat. 1989; 185: 335-341Crossref PubMed Scopus (42) Google Scholar, 8Head MW Corbin E Goldman JE Overexpression and abnormal modification of the stress proteins alpha B-crystallin and HSP27 in Alexander disease.Am J Pathol. 1993; 143: 1743-1753PubMed Google Scholar Although the GFAP within Rosenthal fibers appears disorganized, astrocytes in Alexander disease also possess GFAP filaments with conventional 10-nm morphology. Recently, missense point mutations in GFAP have been identified as a genetic basis for Alexander disease.9Brenner M Johnson AB Boespflug-Tanguy O Rodriguez D Goldman JE Messing A Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease.Nat Genet. 2001; 27: 117-120Crossref PubMed Scopus (0) Google Scholar To date, all known mutations have been heterozygous, indicating that the mutant form of the protein is dominant over the wild type. This is consistent with the finding of autosomal dominant mutations in 26 other IF genes that are linked to human disease,10Omary MB Coulombe PA McLean WH Intermediate filament proteins and their associated diseases.N Engl J Med. 2004; 351: 2087-2100Crossref PubMed Scopus (372) Google Scholar, 11McLean WH Smith FJ Cassidy AJ Insights into genotype-phenotype correlation in pachyonychia congenita from the human intermediate filament mutation database.J Investig Dermatol Symp Proc. 2005; 10: 31-36Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar summarized in the online Intermediate Filament Disease Mutation Database. The list of known mutations in GFAP now includes 32 nucleotide changes that affect 24 aa spread throughout the entire sequence12Li R Johnson AB Salomons G Goldman JE Naidu S Quinlan R Cree B Ruyle SZ Banwell B D’Hooghe M Siebert JR Rolf CM Cox H Reddy A Gutierrez-Solana LG Collins A Weller RO Messing A van der Knaap MS Brenner M Glial fibrillary acidic protein mutations in infantile, juvenile, and adult forms of Alexander disease.Ann Neurol. 2005; 57: 310-326Crossref PubMed Scopus (187) Google Scholar (see also the Alexander Disease Web site). The mutations usually arise spontaneously during spermatogenesis,13Li R Johnson AB Salomons GS van der Knaap MS Rodriguez D Boespflug-Tanguy O Gorospe JR Goldman JE Messing A Brenner M Propensity for paternal inheritance of de novo mutations in Alexander disease.Hum Genet. 2006; 119: 137-144Crossref PubMed Scopus (35) Google Scholar with familial cases being quite rare because of the high morbidity associated with the disease. The mutation studied in this report, R416W, is one of the four mutations reported in familial cases and is also found in sporadic cases.12Li R Johnson AB Salomons G Goldman JE Naidu S Quinlan R Cree B Ruyle SZ Banwell B D’Hooghe M Siebert JR Rolf CM Cox H Reddy A Gutierrez-Solana LG Collins A Weller RO Messing A van der Knaap MS Brenner M Glial fibrillary acidic protein mutations in infantile, juvenile, and adult forms of Alexander disease.Ann Neurol. 2005; 57: 310-326Crossref PubMed Scopus (187) Google Scholar Like other IF family members, GFAP has a characteristic domain structure comprising a central α-helical rod domain flanked by non–α-helical N-terminal “head” and C-terminal “tail” domains.14Herrmann H Aebi U Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics.Curr Opin Cell Biol. 2000; 12: 79-90Crossref PubMed Scopus (407) Google Scholar The rod domain contains characteristic heptad repeats of hydrophobic residues, which are the underlying basis for the coiled-coil dimer in the filament, and the highly conserved LNDR and TYRKLEGGE motifs that are present at the start and the end of this central rod domain.10Omary MB Coulombe PA McLean WH Intermediate filament proteins and their associated diseases.N Engl J Med. 2004; 351: 2087-2100Crossref PubMed Scopus (372) Google Scholar Both of these motifs are highly conserved throughout the whole IF family,15Herrmann H Aebi U Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular scaffolds.Annu Rev Biochem. 2004; 73: 749-789Crossref PubMed Scopus (514) Google Scholar and those mutations in Alexander disease and other genetic IF protein disorders found within them usually correlate with the severest forms of the diseases.10Omary MB Coulombe PA McLean WH Intermediate filament proteins and their associated diseases.N Engl J Med. 2004; 351: 2087-2100Crossref PubMed Scopus (372) Google Scholar, 11McLean WH Smith FJ Cassidy AJ Insights into genotype-phenotype correlation in pachyonychia congenita from the human intermediate filament mutation database.J Investig Dermatol Symp Proc. 2005; 10: 31-36Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar The crystallization of regions containing these two motifs from vimentin, a closely related type III IF protein16Strelkov SV Herrmann H Geisler N Wedig T Zimbelmann R Aebi U Burkhard P Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly.EMBO J. 2002; 21: 1255-1266Crossref PubMed Scopus (218) Google Scholar, 17Strelkov SV Herrmann H Aebi U Molecular architecture of intermediate filaments.Bioessays. 2003; 25: 243-251Crossref PubMed Scopus (199) Google Scholar that coassembles with GFAP, has provided the atomic structure of these particular highly conserved motifs. Our knowledge of the important higher order interactions within the filament, however, is still limited to low-resolution studies18Wu KC Bryan JT Morasso MI Jang SI Lee JH Yang JM Marekov LN Parry DA Steinert PM Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments.Mol Biol Cell. 2000; 11: 3539-3558Crossref PubMed Scopus (75) Google Scholar, 19Yamada S Wirtz D Coulombe PA Pairwise assembly determines the intrinsic potential for self-organization and mechanical properties of keratin filaments.Mol Biol Cell. 2002; 13: 382-391Crossref PubMed Scopus (76) Google Scholar, 20Bernot KM Lee CH Coulombe PA A small surface hydrophobic stripe in the coiled-coil domain of type I keratins mediates tetramer stability.J Cell Biol. 2005; 168: 965-974Crossref PubMed Scopus (29) Google Scholar; therefore, the full structural impact of most of these rod mutations has not yet been detailed. One of the other common mutations outside the central rod domain of GFAP that causes Alexander disease is R416W. This mutation occurs in the tail domain within the RDG motif, which is conserved among all GFAP proteins from multiple species, as well as the related type III IF proteins vimentin and desmin. Here, we describe the effects of this mutation on GFAP assembly and use this R416W GFAP mutant to identify the early events in the development of Alexander disease. Total RNA was extracted from human astrocytoma U373MG cells with the RNeasy kit (Qiagen). The complete human GFAP cDNA was amplified by RT-PCR, with the SuperScript RT-PCR system (Invitrogen), with use of oligonucleotides 5′-CATATGGAGAGGAGACGCAT-3′ and 5′-TCACATCACATCCTTGTGCT-3′ as forward and reverse primers, respectively. The amplified PCR product was cloned into the pGEM-T Easy vector (Promega), to generate pGEM-T Easy-WTGFAP, and the entire sequence was confirmed against the GenBank database entry for GFAP (accession number J04569). The R416W mutation was introduced by QuickChange site-directed mutagenesis (Stratagene) with use of the pGEM-T Easy-WTGFAP vector as a template. The following mutagenic oligonucleotides that contained the desired C→T mutation at np 1246 were synthesized: 5′-GAAGACCGTGGAGATGTGGGATGGAGAGGTCAT-3′ and 5′-ATGACCTCTCCATCCCACATCTCCACGGTCTTC-3′. The amplified PCR product containing the R416W mutation was cloned into the pGEM-T Easy vector, and the mutation sequence was confirmed by DNA sequencing. For expression in cultured mammalian cells, both the wild-type and R416W GFAP cDNA in the pGEM-T Easy vector were subcloned into the pcDNA3.1(−) vector (Invitrogen) with use of the XbaI and EcoRI restriction sites. The GFAP mutant R239C was also subcloned into pCDNA3.1 from the pRSVi vector21Hsiao VC Tian R Long H Der Perng M Brenner M Quinlan RA Goldman JE Alexander-disease mutation of GFAP causes filament disorganization and decreased solubility of GFAP.J Cell Sci. 2005; 118: 2057-2065Crossref PubMed Scopus (60) Google Scholar with use of the restriction enzyme HindIII. For expression in bacteria, wild-type and R416W GFAP cDNA samples were subcloned into the pET23b vector (Novagen) with use of the NdeI and EcoRI restriction sites. For bacterial expression of proteins, pET 23b vector containing either wild-type or mutant GFAP cDNA samples were transformed into Escherichia coli strain BL21(DE3) pLysS (Novagen). After transformation, cultures were grown in Luria Bertani medium supplemented with appropriate antibiotics to OD600 of 0.5–0.6, and protein expression was induced by the addition of 0.5 mM isopropyl-1-thio-β-d-galactopyranoside for 3 h. Overexpressed GFAP formed inclusion bodies, which were prepared as described elsewhere.22Ralton JE Lu X Hutcheson AM Quinlan RA Identification of two N-terminal non-alpha-helical domain motifs important in the assembly of glial fibrillary acidic protein.J Cell Sci. 1994; 107: 1935-1948Crossref PubMed Google Scholar The final pellets, consisting predominantly of GFAP, were solubilized in extraction buffer (8 M urea, 20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol [DTT], and 1 mM phenylmethylsulfonyl fluoride [PMSF]) at room temperature for 3 h, and any insoluble material was removed by centrifugation, at 100,000 g, in a benchtop Optima MAX Ultracentrifuge with use of an MLA-80 rotor (Beckman Coulter). GFAP was further purified by ion-exchange chromatography with use of a Merck-Hitachi Biochromatography system equipped with a Fractogel-EMD TMAE 650S column (Merck) pre-equilibrated in the column buffer (6 M urea, 10 mM Tris-HCl [pH 8.0], 5 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). GFAP was eluted from the column with a linear gradient of 0–0.5 M NaCl in the same buffer over 1 h at a flow rate of 1 ml/min. The GFAP-enriched fractions were pooled, concentrated, and applied to a Fractogel EMD COO- 650S column (Merck) and were pre-equilibrated with column buffer (6 M urea, 20 mM sodium formate [pH 4.0], 5 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After washing with buffer B, GFAP was eluted with a linear gradient of 0–0.5 M NaCl in the same buffer. Column fractions were analyzed by SDS-PAGE, and those containing purified GFAP were collected and stored at −80°C. Protein concentrations were determined by bicinchonic acid assay (BCA reagent [Perbio Science]) with use of BSA as standard. Purified GFAP was diluted to 0.3 mg/ml in 6 M urea in a buffer of 10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 mM EGTA, and 1 mM DTT and was dialyzed stepwise against 3 M urea in the same buffer for 4–6 h at room temperature and then against the same buffer without urea overnight at 4°C. Filament assembly was completed by dialyzing against assembly buffer (20 mM imidazole-HCl [pH 6.8], 100 mM NaCl, and 1 mM DTT) for 12–16 h at room temperature. The efficiency of in vitro assembly was assessed by sedimentation assay as described elsewhere.23Nicholl ID Quinlan RA Chaperone activity of alpha-crystallins modulates intermediate filament assembly.EMBO J. 1994; 13: 945-953Crossref PubMed Scopus (390) Google Scholar In brief, the assembly mixture was layered onto a 0.85-M sucrose cushion in assembly buffer and was centrifuged at 80,000 g for 30 min. To investigate the effect of mutations on filament-filament interactions, assembled filaments were subjected to low-speed centrifugation at 3,000 g for 10 min in a bench-top centrifuge (Eppendorf). The supernatant and pellet fractions were analyzed by SDS-PAGE24Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202762) Google Scholar and were visualized by Coomassie blue staining. In some instances, the proportion of GFAP distributed between pellet and supernatant fractions was measured using an image analyzer (LAS-1000plus [FujiFilm]). Coomassie blue signals for individual bands were quantified using the Image Gauge software (v. 4.0) (FujiFilm). GFAP was diluted in assembly buffer to 100 μg/ml and was negatively stained with 1% (w/v) uranyl acetate (Agar Scientific). Samples on carbon-coated copper grids were examined with a Phillips 400T transmission electron microscope, with use of an accelerating voltage of 80 kV. Images were acquired at a magnification of 17,000× on Kodak 4489 film and then were digitized at 1,200×1,200–pixel resolution before being processed further in Adobe Photoshop 7 (Adobe Systems). Human breast cancer epithelial MCF7 cells were obtained from the European Collection of Cell Cultures (Sigma). Human adrenal cortex carcinoma SW/cl.1 and SW13/cl.2 cells were kindly provided by Dr. Robert Evans (University of Colorado Health Sciences Center, Denver). The human astrocytoma cell line U343MG was a gift from Dr. Rutka (Toronto), and the cells were grown in αMEM (Invitrogen). These cells express vimentin and GFAP as well as HSP27 and αB-crystallin. Primary mouse astrocytes from wild-type and vimentin/GFAP double-knock littermates were a generous gift of Dr. Milos Pekny (Göteborg, Sweden). They were prepared and grown as described elsewhere.25Ding M Eliasson C Betsholtz C Hamberger A Pekny M Altered taurine release following hypotonic stress in astrocytes from mice deficient for GFAP and vimentin.Brain Res Mol Brain Res. 1998; 62: 77-81Crossref PubMed Scopus (72) Google Scholar Unless otherwise stated, cells were grown in Dulbecco's modified Eagle medium supplemented with 10% (v/v) fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma) and were maintained at 37°C in a humidified incubator of 95% (v/v) air and 5% (v/v) CO2. For transient transfection studies, cells grown on 13-mm coverslips at a density of 40%–50% confluency were transfected with pcDNA3.1(−) expressing either wild-type or R416W GFAP, with use of GeneJuice transfection reagent (Novagen) according to the manufacturer's protocol. In some experiments, the R239C GFAP21Hsiao VC Tian R Long H Der Perng M Brenner M Quinlan RA Goldman JE Alexander-disease mutation of GFAP causes filament disorganization and decreased solubility of GFAP.J Cell Sci. 2005; 118: 2057-2065Crossref PubMed Scopus (60) Google Scholar and myc-tagged ubiquitin (His6-myc-Ubiquitin)26Ward CL Omura S Kopito RR Degradation of CFTR by the ubiquitin-proteasome pathway.Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1088) Google Scholar were used. Cells were analyzed by double-label immunofluorescence microscopy 48 h after transfection. Mouse monoclonal antibodies were made that specifically recognized human but not wild-type R416W GFAP. The immunogen was a peptide dodecamer centered on the mutation site, KTVEMWDGEVIK (Genemed Synthesis), which was linked to keyhole limpet hemocyanin. Monoclonal antibodies were produced by the UAB Epitope Recognition Core, which also performed an initial ELISA screen against purified recombinant wild-type and R416W mutant GFAP. The corresponding wild-type peptide, KTVEMRDGEVIK, failed to produce monoclonal antibodies specific to the wild-type sequence. Immunocytochemistry of cultured cells was performed on coverslips washed twice with PBS, and the cells were fixed in either ice-cold methanol/acetone (1:1 [v/v]) for 20 min or in 4% (w/v) paraformaldehyde/PBS for 10 min. In the case of paraformaldehyde fixation, cells were subsequently permeabilized with 0.5% NP-40 in PBS for 10 min. After being washed twice with PBS containing 0.02% (w/v) sodium azide and 0.02% (w/v) BSA (PBS/BSA/azide), cells were blocked with 10% (v/v) goat serum in PBS/BSA/azide for 20 min and then were incubated with primary antibodies at room temperature for 1 h. The primary antibodies used in this study were mouse monoclonal anti-GFAP (G-A-5, 1:500 [Sigma]), rabbit polyclonal GFAP antibodies (Z0334, 1:500 [Dako]), monoclonal anti-human GFAP (SMI-21, 1:500 [Sternberger Monoclonals]), monoclonal anti-keratin 18 (LE41 [kindly provided by Prof. Birgit Lane, University of Dundee, Dundee, United Kingdom), monoclonal anti-R416W GFAP (19.2 and 1A3, described below, 1:500), rabbit polyclonal anti-GFAP (clone 3270, 1:200),27Perng MD Cairns L van den IJsse P Prescott A Hutcheson AM Quinlan RA Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin.J Cell Sci. 1999; 112: 2099-2112Crossref PubMed Google Scholar polyclonal anti-vimentin (clone 3052, 1:200),28Sandilands A Prescott AR Carter JM Hutcheson AM Quinlan RA Richards J FitzGerald PG Vimentin and CP49/filensin form distinct networks in the lens which are independently modulated during lens fibre cell differentiation.J Cell Sci. 1995; 108: 1397-1406PubMed Google Scholar and the myc-epitope monoclonal antibody (Clone 9E11, 1:10).29Evan GI Lewis GK Ramsay G Bishop JM Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.Mol Cell Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2136) Google Scholar After cells were washed with PBS/BSA/azide, the primary antibodies were detected using Alexa 488 (1:400 [Molecular Probes]) or Alexa 594 (1:600 [Molecular Probes]) conjugated secondary antibodies. All antibodies were diluted in PBS/BSA/azide buffer. The glass coverslips were mounted on slides with the fluorescent protecting agent Citifluor (Citifluor Labs) and were observed with a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss). Optical sections were set to ∼1.0 μm. Images were processed and prepared for figures with Adobe Photoshop 7 (Adobe Systems). Quantitation of the GFAP filament phenotypes was by visual assessment of the cells and by scoring cells for the presence or absence of GFAP-containing aggregates. Approximately 100–150 transfected cells were assessed, and each experiment was repeated at least three times. Immunohistochemistry from normal human and Alexander disease brain sections was performed on 6 μm–thick paraffin sections kindly provided by Drs. Jim Goldman and Goumei Tang (Columbia Medical School, New York). Internal review board approval was obtained from Columbia Medical School for these studies. Archival material for the infantile R416W Alexander disease case used in this study was described elsewhere.30Iwaki T Wisniewski T Iwaki A Corbin E Tomokane N Tateishi J Goldman JE Accumulation of alpha B-crystallin in central nervous system glia and neurons in pathologic conditions.Am J Pathol. 1992; 140: 345-356PubMed Google Scholar Primary antibodies were rabbit anti-cow GFAP (Z0334, 1:5,000 [Dako]), and mouse anti-R416W GFAP (19.2, described below, 1:2,000). Secondary antibody for the peroxidase method was peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) (1:2,000), with staining visualized using 3,3′-diaminobenzidine tetrachloride (DAB, metal-enhanced Substrate Kit [Pierce]). Secondary fluorescent antibodies are described above. Some sections were counterstained with Hoechst 33258 (Sigma) to reveal nuclei. MCF7 cells grown on 10-cm2 petri dishes (Greiner Bio-One) were transfected with either wild-type or R416W GFAP for 48 h. Cells were then fixed directly in 80 mM cacodylate buffer (pH 7.2) containing 1.25% (v/v) glutaraldehyde and 1% (w/v) paraformaldehyde for 30 min at room temperature. Cells were scraped off the dish by a rubber policeman, were pelleted by low-speed centrifugation, and were washed three times with cacodylate buffer. The cells were then postfixed with 1% (w/v) osmium tetroxide in cacodylate buffer. After several washes with distilled water, cells were subjected to a series of graded ethanol dehydration, followed by overnight incubation in 1:1 propylene oxide:epoxy resin (Durcupan [Sigma]). After two changes of 100% fresh resin, cell pellets were transferred to BEEM capsules (Agar Scientific) and were polymerized in fresh resin overnight at 60°C. Ultrathin sections were generated using a Leica Ultracut ultramicrotome and were collected on pioloform and carbon-coated nickel grids (Agar Scientific). The grid specimens were then etched with 1% periodic acid, and osmium was removed by 2% (w/v) sodium periodate before incubation with blocking solution consisting of 0.5% (w/v) fish skin gelatin (Sigma) in PBS. Subsequently, sections were incubated with polyclonal anti-GFAP antibodies (clone 3270) diluted 1:20 in blocking solution for 90 min, were washed three times in PBS, and then were incubated with protein A conjugated with 5-nm gold particles (British BioCell International) for 2 h. After several washes in distilled water, specimens were stained with saturated aqueous uranyl acetate (3% [w/v]) for 30 min, followed by staining with lead citrate for 30 min.31Reynolds ES The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.J Cell Biol. 1963; 17: 208-212Crossref PubMed Scopus (17280) Google Scholar Stained samples were subsequently examined on an FEI Tecnai 12 transmission electron microscope (FEI). Cells grown on 10-cm2 petri dishes were transfected with control vector (pcDNA3.1) or vectors containing either wild-type or R416W GFAP cDNA. At 48 h after transfection, cells were lysed using two different extraction buffers, designed to test the resistance of GFAP filaments and aggregates to extraction. In the mild extraction protocol, cells were lysed on ice for 15 min in 1 ml mild extraction buffer (MEB; 20 mM Tris-HCl [pH 7.6], 140 mM NaCl, 5 mM EDTA, 1 mM EGTA, 0.5% [v/v] NP-40 supplemented with Complete protease inhibitor cocktail [Roche Diagnostics], and 1 mM PMSF). In the more severe extraction protocol, cells were lysed in 1 ml of a harsher extraction buffer (HEB) containing deoxycholate (20 mM Tris-HCl [pH 7.6], 140 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% [v/v] NP-40, 0.5% [w/v] sodium deoxycholate supplemented with Complete protease inhibitor cocktail [Roche Diagnostics], and 1 mM PMSF). Cell lysates were collected, were homogenized in a Dounce homogenizer (Wheaton), and were centrifuged at 16,000 g for 15 min at 4°C. The pellet was resuspended in pelleting buffer (20 mM Tris-HCl [pH 8.0], 10 mM MgCl2, and 1 mM PMSF) containing 250 U/ml benzonase nuclease (Novagen) and was incubated for 30 min at room temperature. After repelleting, the final pellets were washed in PBS containing 1 mM PMSF and then were resuspended in Laemmli's sample buffer,24Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202762) Google Scholar in a volume that was equivalent to the supernatant. Supernatant and pellet fractions were then boiled for 5 min in Laemmli's sample buffer, and equal volumes were analyzed by SDS-PAGE and immunoblotting. Actin was used as a loading control for these samples. Immunoblotting was performed using the semidry blotting method, according to the manufacturer's specifications (Bio-Rad Laboratories). After the blotting, protein-transfer efficiency was assessed by Ponceau S (Sigma) staining of the nitrocellulose membrane, followed by destaining in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.4], and 150 mM NaCl). Membranes were blocked for 2 h in blocking buffer containing 5% (w/v) BSA in either TTBS (TBS containing 0.2% [v/v] Tween 20) or PBST (5% [w/v] nonfat milk and 0.1% [v/v] Tween-20 in PBS) and were incubated for 2 h with mouse monoclonal anti-GFAP (GA-5), monoclonal anti-human GFAP (SMI-21), monoclonal anti-actin (AC-40 [Sigma]), or rabbit polyclonal ant" @default.
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• W2091697287 date "2006-08-01" @default.
• W2091697287 modified "2023-10-03" @default.
• W2091697287 title "The Alexander Disease–Causing Glial Fibrillary Acidic Protein Mutant, R416W, Accumulates into Rosenthal Fibers by a Pathway That Involves Filament Aggregation and the Association of αB-Crystallin and HSP27" @default.
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