title: Analysis of the subcellular localization of huntingtin with a set of rabbit polyclonal antibodies in cultured mammalian cells of neuronal origin: comparison with the distribution of huntingtin in Huntington's disease autopsy brain.
abstract: Huntington's disease (HD) is a neurodegenerative disorder with a midlife onset. The disease is caused by expansion of a CAG (glutamine) repeat within the coding region of the HD gene. The molecular mechanism by which the mutated protein causes this disease is still unclear. To study the protein we have generated a set of rabbit polyclonal antibodies raised against different segments of the N-terminal, central and C-terminal parts of the protein. The polyclonal antibodies were affinity purified and characterized in ELISA and Western blotting experiments. All antibodies can react with mouse and human proteins. The specificity of these antibodies is underscored by their recognition of huntingtin with different repeat sizes in extracts prepared from patient-derived lymphoblasts. The antibodies were used in immunofluorescence experiments to study the subcellular localization of huntingtin in mouse neuroblastoma NIE-115 cells. The results indicate that most huntingtin is present in the cytoplasm, whereas a minor fraction is present in the nucleus. On differentiation of the NIE-115 cells in vitro, the subcellular distribution of huntingtin does not change significantly. These results suggest that full-length huntingtin with a normal repeat length can be detected in the nucleus of cycling and non-cycling cultured mammalian cells of neuronal origin. However, in HD autopsy brain the huntingtin-containing neuronal intranuclear inclusions can be detected only with antibodies raised against the N-terminus of huntingtin. Thus several forms of huntingtin display the propensity for nuclear localization, possibly with different functional consequences.
authors.affiliation: MGC-Department of Human Genetics, Sylvius Laboratory, Leiden University Medical Center, The Netherlands.dorsman@mail.medfac.leidenu niv.nl
authors.forename: J C
authors.initials: JC
authors.lastname: Dorsman
authors.forename: M A
authors.initials: MA
authors.lastname: Smoor
authors.forename: M L
authors.initials: ML
authors.lastname: Maat-Schieman
authors.forename: M
authors.initials: M
authors.lastname: Bout
authors.forename: S
authors.initials: S
authors.lastname: Siesling
authors.forename: S G
authors.initials: SG
authors.lastname: van Duinen
authors.forename: J J
authors.initials: JJ
authors.lastname: Verschuuren
authors.forename: J T
authors.initials: JT
authors.lastname: den Dunnen
authors.forename: R A
authors.initials: RA
authors.lastname: Roos
authors.forename: G J
authors.initials: GJ
authors.lastname: van Ommen
mesh_terms: D000818:Animals; D000906:Antibodies; D001921:Brain / Q000473:pathology*; D002454:Cell Differentiation; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics / Q000473:pathology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000473:pathology*; D009687:Nuclear Proteins / Q000032:analysis* / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013347:Subcellular Fractions / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000906:Antibodies; C086055:HTT protein, human; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins