Alternative processing of humanHTTmRNA with implications for Huntington’s disease therapeutics

Abstract

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 retained 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 produce 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 approaches 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 antisense 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.

Early detection of exon 1 huntingtin aggregation in zQ175 brains by molecular and histological approaches

“…We have previously shown that in R6/2 mice with 90 CAGs and YAC128 mice, HTT aggregation in the nucleus remained relatively diffuse throughout disease progression. 36,51 We have also previously demonstrated that, in HTTexon1 and knock-in mouse models of Huntington's disease, the steady-state levels of soluble HTT isoforms are cytoplasmic and that HTT fragments remain in the nucleus because they have aggregated into high-molecular weight complexes. 36,42,52 Here, we have shown that HTT aggregation in zQ175 striatal nuclei was present as early as 6 weeks of age, well before transcriptional dysregulation has been reported 49 and consistent with transcriptional dysregulation being caused by aggregated HTT.…”

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

“…Another interesting issue to examine in the future is alternative transcript variants of both genes, ATXN3 and HTT . In this research, we wanted to focus on the main variants of each transcript, but analyses on, e.g., HTT intron1 variant [ 14 , 42 ] or ATXN3 transcripts which does not contain exon 11 (which lead to the translation of ataxin3-a long and ataxin3-a short proteins) [ 26 ], might be interesting in terms of revealing new facts regarding the pathogenesis of both diseases.…”

“…The exact role of mutant transcripts in pathogenic mechanisms in polyQ diseases is being unraveled [10][11][12][13]. RNA-related studies have demonstrated so far: incomplete splicing of HTT mRNA [14], abnormal interactions of RNAs containing expanded CAG repeats with proteins [15,16], and RNA foci formation [17,18], and potential importance of alternative polyadenylation of various polyQ transcripts [19]. Furthermore, mutant polyQ disease transcripts have been shown to be promising therapeutic targets in strategies that aim to downregulate mutant gene expression [2,9,20].…”

Mutant huntingtin messenger RNA forms neuronal nuclear clusters in rodent and human brains

“…While preparing this manuscript, another recently published study from the Bates lab recapitulates the data in the present study, further solidifying the potential importance of RNA clusters in HD. 88 Additional work is necessary to delineate and link the molecular mechanisms involved in mHTT mRNA clustering to HD pathology. However, this work provides clear evidence identifying mutant mRNA nuclear aggregation as one of the biomolecular signatures of HD, thus adding HD to a growing list of repeat-associated, neurodegenerative disorders with the demonstrated abnormalities in RNA processing and localization.…”