DEVELOPMENT OF IN VITRO MODELS TO INVESTIGATE THE PATHOGENESIS OF HUNTINGTON'S DISEASE AND SCREEN FOR THERAPEUTIC AGENTS

Background Huntington’s Disease (HD) is an inherited neurodegenerative disorder caused by the expansion of a CAG repeat in the HTT gene. We have recently shown that incomplete splicing of HTT mRNA, in both HD patients and mouse models, produces a HTTexon1 and Httexon1 transcript respectively, that is translated into the highly pathogenic exon 1 HTT protein. Aims The aim of this study was to investigate the role of this Httexon1 transcript in the pathogenesis of the disease, and to characterize primary cells from HD mouse models that could be used to screen for agents designed to lower the levels of the HTT transcripts. Methods/techniques We have established mouse embryonic fibroblasts (MEFs) from the zQ175 mouse model as well as deriving mouse cortical neuronal cultures. We have used our novel multiplex Quantigene assay, to measure the levels of all mouse Httexon1 and full-length Htt transcripts in these cells, and RNAscope to localize these transcripts. We have developed a TR-FRET assay that is specific for the exon 1 HTT protein. We have used immunoprecipitation western blot and TR-FRET to detect full-length and exon 1 HTT. Results/outcome We show that our Quantigene assay generates comparable data to the much more time-consuming quantitative PCRs for the zQ175 MEFs. We show that most of the mutant transcript in the zQ175 MEFs is incompletely spliced, and that these cells can be used for compound screening by multiplex Quantigene assays. The exon 1 HTT protein can be measured in these cells. Primary neurons from zQ175 mice show a higher level of incompletely spliced Htt than has been detected in brain tissue. The localization of the Htt transcripts in primary neurons will be discussed. Conclusions We conclude that we have good in vitro models and a variety of techniques to investigate the role of the Httexon1 transcripts in the pathogenesis of HD and screen for therapeutic agents. This work is supported by the CHDI foundation.