Abstract
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.
Footnotes
The authors declare no competing financial interests.
This work was supported in part by the Swedish Research Council [K2013-62X-21398-04-4, K2011-61P-20945-01-3, K2014-62X-20404-08-5 to D.K. and A.P.], Region of Skane ALF grants to DK and AP, Royal Physiographic Society of Lund to BB, National Institutes of Health/National Institute of Neurological Disorders and Stroke [R01 NS079651 to I.S.S], the Agence Nationale pour la Recherche, the JPND program on ModelPolyQ, Neuratris (Grant Q5 ‘‘Investment for the future’’ ANR-11-INBS-0011), the Laboratoire d’Excellence Revive (Investissement d’Avenir; ANR-10-LABX-73), the French National Health Institute (INSERM), and the AFM-Téléthon (Association Française contre les Myopathies) to A.L.P. J.B is supported by a fellowship the Laboratoire d’Excellence Revive (Investissement d’Avenir; ANR-10-LABX-73). Human brain tissue samples were received from the Sydney Brain Bank, which is supported by Neuroscience Research Australia and the University of New South Wales, and from the Victorian Brain Bank Network, supported by the Mental Health Research Institute, Alfred Hospital, Victorian Forensic Institute of Medicine, The University of Melbourne, Helen Macpherson Smith Trust, Parkinson’s Victoria and Perpetual Philanthropic Service. G.M.H. is a National Health and Medical Research Council of Australia Senior Principal Research Fellow (#1079679).
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