Journal Article

A03 Alternative processing of human HTT MRNA in YAC128 mice: implications for Huntington’s disease therapeutics

Gomez-Paredes, Casandra, Osborne, Georgina F, Phillips, Jemima M, Fienko, Sandra, Landles, Christian, Kordasiewicz, Holly B, McAteer, Sean, Bates, Gillian P, Milton, Rebecca, Jones, Samuel T, Sathasivam, Kirupa

2022

Overview

BackgroundYAC128 transgenic mice carry human HTT with an expanded CAG repeat. This model is particularly useful for evaluating therapies targeting the human HTT gene and/or protein.AimsTo better understand the molecular phenotype of YAC128 mice at the RNA and protein level.MethodsA QuantiGene assay was designed to gain insights into incomplete splicing in the context of human HTT, and to evaluate the lowering efficiency of therapeutic compounds. RNAscope was implemented to visualize HTT transcripts and immunohistochemistry to characterise HTT aggregation spatiotemporally. HTRF measured the levels of the total HTT protein and the pathogenic exon 1 HTT. Mouse embryonic fibroblasts (MEFs) from YAC128 mice were used for screening agents targeting human HTT transcript.ResultsMicroscopic analysis revealed that the full-length HTT mRNA (FL-HTT) was retained in RNA nuclear clusters together with the incompletely spliced HTT1a transcript. These clusters were not observed in zQ175 HD mouse model where, instead, FL-Htt and Htt1a mRNAs were detected as mostly cytoplasmic molecules. Immunohistochemistry showed a progressive appearance of aggregated HTT in nuclei in the cortex, striatum, hippocampus and cerebellum. HTRF indicated that the level of exon 1 HTT was highest in the cerebellum. Soluble mutant exon 1 HTT decreased with age, with concomitant increase in aggregated HTT. In YAC128 MEFs, HTT1a was detected and ASOs targeting HTT were efficient in lowering HTT levels in this model system.ConclusionsHuman HTTundergoes incomplete splicing in brains of YAC128 mice. The RNA clusters detected may have direct therapeutic implications, with pathogenic or protective consequences

• Publisher: BMJ Publishing Group Ltd,BMJ Publishing Group LTD
• Subject: A: Pathogenic mechanisms / aggregation / huntingtin / Huntingtons disease / Proteins / RNA / RNA clusters / Splicing