Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

• Published in: Journal of neurochemistry 2002-07, Vol.82 (1), p.84-92
• Main Authors: Toneff, Thomas, Mende‐Mueller, Liane, Wu, Ying, Hwang, Shin‐Rong, Bundey, Richard, Thompson, Leslie M., Chesselet, Marie‐Francoise, Hook, Vivian
• Format: Article
• Language: English
• Subjects: Age of Onset; Aged; Biological and medical sciences; Blotting, Western; brain; Brain - metabolism; Brain Chemistry; Cell Line; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Humans; huntingtin; Huntingtin Protein; Huntington Disease - genetics; Huntington Disease - metabolism; Huntington's disease; lymphoblasts; Lymphocytes - chemistry; Lymphocytes - cytology; Lymphocytes - metabolism; Medical sciences; Middle Aged; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neurology; Nuclear Proteins - analysis; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Peptide Fragments - analysis; Peptide Fragments - metabolism; Peptide Hydrolases - metabolism; Polymerase Chain Reaction; proteolysis; Trinucleotide Repeat Expansion - genetics; triplet nucleotide repeat
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2002.00940.x

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope‐specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH2‐ and COOH‐terminal htt fragments of 20–100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63–64 kDa htt fragment detected by the NH2‐domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60–61 kDa band (detected by the NH2‐domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63–64 kDa and 60–61 kDa NH2‐domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66‐kDa, instead of the 63–64 kDa N‐domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH2‐ and COOH‐terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH2‐terminal htt fragments [detected with anti(1–17) serum] compared to controls. Cortex from HD and control brains showed similar NH2‐terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH2‐terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48‐kDa COOH‐terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH2‐terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.