title: Native mutant huntingtin in human brain: evidence for prevalence of full-length monomer.
abstract: Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.
authors.affiliation: Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.
authors.forename: Ellen
authors.initials: E
authors.lastname: Sapp
authors.forename: Antonio
authors.initials: A
authors.lastname: Valencia
authors.forename: Xueyi
authors.initials: X
authors.lastname: Li
authors.forename: Neil
authors.initials: N
authors.lastname: Aronin
authors.forename: Kimberly B
authors.initials: KB
authors.lastname: Kegel
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Vonsattel
authors.forename: Anne B
authors.initials: AB
authors.lastname: Young
authors.forename: Nancy
authors.initials: N
authors.lastname: Wexler
authors.forename: Marian
authors.initials: M
authors.lastname: DiFiglia
mesh_terms: D000328:Adult; D000818:Animals; D015153:Blotting, Western; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000453:epidemiology / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015995:Prevalence; D061251:Primary Cell Culture; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D012156:Reticulocytes / Q000166:cytology; D012995:Solubility; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D014015:Tissue Banks
chemical_list: D003902:Detergents; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments