title: Alexander disease causing mutations in the C-terminal domain of GFAP are deleterious both to assembly and network formation with the potential to both activate caspase 3 and decrease cell viability.
abstract: Alexander disease is a primary genetic disorder of astrocyte caused by dominant mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). While most of the disease-causing mutations described to date have been found in the conserved α-helical rod domain, some mutations are found in the C-terminal non-α-helical tail domain. Here, we compare five different mutations (N386I, S393I, S398F, S398Y and D417M14X) located in the C-terminal domain of GFAP on filament assembly properties in vitro and in transiently transfected cultured cells. All the mutations disrupted in vitro filament assembly. The mutations also affected the solubility and promoted filament aggregation of GFAP in transiently transfected MCF7, SW13 and U343MG cells. This correlated with the activation of the p38 stress-activated protein kinase and an increased association with the small heat shock protein (sHSP) chaperone, αB-crystallin. Of the mutants studied, D417M14X GFAP caused the most significant effects both upon filament assembly in vitro and in transiently transfected cells. This mutant also caused extensive filament aggregation coinciding with the sequestration of αB-crystallin and HSP27 as well as inhibition of the proteosome and activation of p38 kinase. Associated with these changes were an activation of caspase 3 and a significant decrease in astrocyte viability. We conclude that some mutations in the C-terminus of GFAP correlate with caspase 3 cleavage and the loss of cell viability, suggesting that these could be contributory factors in the development of Alexander disease.
authors.affiliation: Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Yi-Song
authors.initials: YS
authors.lastname: Chen
authors.forename: Suh-Ciuan
authors.initials: SC
authors.lastname: Lim
authors.forename: Mei-Hsuan
authors.initials: MH
authors.lastname: Chen
authors.forename: Roy A
authors.initials: RA
authors.lastname: Quinlan
authors.forename: Ming-Der
authors.initials: MD
authors.lastname: Perng
mesh_terms: D038261:Alexander Disease / Q000209:etiology / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001254:Astrocytoma / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics*; D002498:Centrifugation; D019938:Cyclin D1 / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D016368:Frameshift Mutation / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D018832:Molecular Chaperones; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D020125:Mutation, Missense / Q000502:physiology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; C530104:CCND1 protein, human; D000939:Epitopes; D005904:Glial Fibrillary Acidic Protein; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D025801:Ubiquitin; D014746:Vimentin; D038203:alpha-Crystallin B Chain; D019938:Cyclin D1; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; D046988:Proteasome Endopeptidase Complex