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.

中文翻译：

---

人类 HTT mRNA 的替代加工对亨廷顿病治疗的影响。


亨廷顿病是由亨廷顿基因 (HTT) 外显子 1 中的 CAG 重复扩增引起，该重复扩增被翻译为亨廷顿蛋白 (HTT) 中的聚谷氨酰胺片段。我们之前表明，携带扩展的 CAG 重复序列的 HTT mRNA 不完全剪接生成 HTT1a，这是一种仅外显子 1 的转录物，它被翻译产生高度聚集倾向和致病性的外显子 1 HTT 蛋白。这种情况发生在所有亨廷顿病敲入小鼠模型中，并且可以在患者细胞系和死后大脑中检测到。为了将这些发现扩展到表达人类 HTT 的模型系统，我们利用了 YAC128 小鼠，该小鼠是转基因酵母人工染色体，携带人类 HTT 和扩展的 CAG 重复序列。我们发现 HTT1a 转录本可以在 YAC128 小鼠的整个大脑中检测到。我们使用 RNAscope 在单分子水平上可视化 HTT 转录本，发现全长 HTT 和 HTT1a 一起保留在大型核 RNA 簇中，并且在细胞质中作为单个转录本存在。均质时间分辨荧光分析表明 HTT1a 转录物已被翻译产生外显子 1 HTT 蛋白。 YAC128 小鼠中外显子 1 HTT 的水平与 HTT 聚集相关，支持外显子 1 HTT 启动聚集过程的假设。降低亨廷顿蛋白的策略是亨廷顿病治疗开发的一个主要焦点。这些方法通常仅针对全长 HTT，预计不会降低致病性外显子 1 HTT 水平。我们已经建立了 YAC128 小鼠胚胎成纤维细胞系，并表明，与我们的 QuantiGene 多重检测一起，这些为针对 HTT 转录物的药物提供了有效的筛选工具。 目前的靶向策略对核 RNA 簇的影响尚不清楚，这些结构可能具有致病作用，或者可以通过将 HTT1a 保留在细胞核中并阻止其翻译来起到保护作用。鉴于最近停止的反义寡核苷酸试验，开发针对 HTT1a 的药物至关重要，并且了解 HTT 降低策略对所有 HTT 转录本及其各种 HTT 蛋白亚型的亚细胞水​​平的影响。