Quantification assays for total and polyglutamine-expanded huntingtin proteins

• Published in: PloS one 2014-05, Vol.9 (5), p.e96854-e96854
• Main Authors: Macdonald, Douglas, Tessari, Michela A, Boogaard, Ivette, Smith, Melanie, Pulli, Kristiina, Szynol, Agnieszka, Albertus, Faywell, Lamers, Marieke B A C, Dijkstra, Sipke, Kordt, Daniel, Reindl, Wolfgang, Herrmann, Frank, McAllister, George, Fischer, David F, Munoz-Sanjuan, Ignacio
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animal models; Animals; Antibodies - immunology; Assaying; Biological Assay - methods; Biology and Life Sciences; Brain; Brain - metabolism; Cell Line; Clinical trials; Electrochemiluminescence; Female; Humans; Huntingtin; Huntingtin Protein; Huntington's disease; Huntingtons disease; Immunoglobulins; Luminescent Measurements; Male; Medical research; Medicine and Health Sciences; Mice; Molecular Sequence Data; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - immunology; Nerve Tissue Proteins - metabolism; Nuclear Proteins - chemistry; Nuclear Proteins - immunology; Nuclear Proteins - metabolism; Pathology; Peptides - metabolism; Polyglutamine; Proteins; Rats; Research and Analysis Methods; RNA-mediated interference; Solubility; Tissues; Trinucleotide repeat diseases; Trinucleotide repeats
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0096854

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.