RT Journal Article
SR Electronic
T1 Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay
JF eneuro
JO eNeuro
FD Society for Neuroscience
SP ENEURO.0234-18.2018
DO 10.1523/ENEURO.0234-18.2018
VO 5
IS 4
A1 Barbara Baldo
A1 Muhammad Umar Sajjad
A1 Rachel Y. Cheong
A1 Julie Bigarreau
A1 Ravi Vijayvargia
A1 Catriona McLean
A1 Anselme L. Perrier
A1 Ihn Sik Seong
A1 Glenda Halliday
A1 Åsa Petersén
A1 Deniz Kirik
YR 2018
UL http://www.eneuro.org/content/5/4/ENEURO.0234-18.2018.abstract
AB The neurodegenerative Huntington’s disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pm and optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cells in vitro and in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.