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Pomezia, Italy

Reid S.J.,University of Auckland | Patassini S.,University of Auckland | Handley R.R.,University of Auckland | Rudiger S.R.,South Australian Research And Development Institute | And 11 more authors.
Journal of Huntington's disease

Huntington's disease is a neurodegenerative disorder, typically with clinical manifestations in adult years, caused by an expanded polyglutamine-coding repeat in HTT. There are no treatments that delay or prevent the onset or progression of this devastating disease. In order to study its pre-symptomatic molecular progression and provide a large mammalian model for determining natural history of the disease and for therapeutic testing, we generated and previously reported on lines of transgenic sheep carrying a full length human HTT cDNA transgene, with expression driven by a minimal HTT promoter. We report here further characterization of our preferred line, OVT73. This line reliably expresses the expanded human huntingtin protein at modest, but readily detectable levels throughout the brain, including the striatum and cortex. Transmission of the 73 unit glutamine coding repeat was relatively stable over three generations. At the first time-point of a longitudinal study, animals sacrificed at 6 months (7 transgenic, 7 control) showed reduced striatum GABAA α1 receptor, and globus pallidus leu-enkephalin immunoreactivity. Two of three 18 month old animals sacrificed revealed cortical neuropil aggregates. Furthermore, neuronal intranuclear inclusions were identified in the piriform cortex of a single 36 month old animal in addition to cortical neuropil aggregates. Taken together, these data indicate that the OVT73 transgenic sheep line will progressively reveal early HD pathology and allow therapeutic testing over a period of time relevant to human patients. Source

Fodale V.,IRBM Promidis | Kegulian N.C.,University of Southern California | Verani M.,IRBM Promidis | Cariulo C.,IRBM Promidis | And 16 more authors.

Background: In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. Methodology/Principal Findings: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTTstructure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein. Conclusions/Significance: The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease. © 2014 Fodale et al. Source

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