Alternative processing of human HTT mRNA with implications for Huntington’s disease therapeutics Sandra Fienko, 1 Christian Landles, 1 Kirupa Sathasivam, 1 Sean J. McAteer, 1 Rebecca E. Milton, 1 Georgina F. Osborne, 1 Edward J. Smith, 1 Samuel T. Jones, 1 Marie K. Bondulich, 1 Emily C. E. Danby, 1 Jemima Phillips, 1 Bridget A. Taxy, 1 Holly B. Kordasiewicz 2 and Gillian P. Bates 1 Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington’s disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were re- tained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to pro- duce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington’s disease. These ap- proaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted anti- sense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood. 1 Department of Neurodegenerative Disease, Huntington’s Disease Centre and UK Dementia Research Institute at UCL, Queen Square Institute of Neurology, UCL, London WC1N 3BG, UK 2 Ionis Pharmaceuticals, Carlsbad, CA 92008, USA Correspondence to: Gillian Bates Department of Neurodegenerative Disease Queen Square Institute of Neurology UCL, Queen Square House Queen Square, London WC1N 3BG, UK E-mail: gillian.bates@ucl.ac.uk Keywords: Huntington’s disease; YAC128 mice; nuclear RNA clusters; HTT1a transcript; exon 1 HTT and aggregation Abbreviations: 3′ UTR = 3′ untranslated region; ASO = antisense oligonucleotide; HTRF = homogeneous time-resolved fluorescence; MEF = mouse embryonic fibroblast; YAC = yeast artificial chromosome Received January 14, 2022. Revised May 17, 2022. Accepted June 11, 2022. Advance access publication July 6, 2022 © The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which per- mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1093/brain/awac241 BRAIN 2022: 00; 1–16 | 1 Downloaded from https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awac241/6632804 by guest on 21 October 2022