Truncation of mutant huntingtin in knock-in mice via CRISPR-Cas9 uncovers exon1 huntingtin as a key pathogenic form

It remains unknown how polyglutamine expansion in widely expressed proteins can cause selective neurodegeneration. In Huntington's disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Considerable efforts have been devoted to investigating how HTT is cleaved and whether blocking its cleavage is therapeutically beneficial. However, using CRISPR-Cas9 to truncate full-length mutant Htt in HD140Q knock-in (KI) mice, we found that exon1 Htt is stably present in the brain, regardless of truncation sites in full-length Htt. This N-terminal Htt led to similar HD phenotypes and age-dependent Htt accumulation in striatum in different KI mice. Exon1 Htt is constantly generated but its selective accumulation in the striatum is caused by the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function accounts for the selective neuropathology in HD and highlight therapeutic importance in regulating this function. We performed RNAseq analysis using striatal tissues from WT, KI-96, KI-571 and KI-FL mice, which are different lines of HD KI mice. KI-FL expresses full-length mutant Huntingtin, KI-571 expresses an N-terminal Huntingtin fragment, KI-96 expresses an N-terminal Huntingtin fragment shorter than KI-571. Please note that the processed data columns T1_1 to T1_4 (in the processed_count_matrix.txt) should be disregarded.广泛表达的蛋白中多聚谷氨酰胺扩增如何引发选择性神经退行性变，这一问题至今尚未明确。 在亨廷顿病（Huntington's disease, HD）中，蛋白水解加工会产生具有毒性的N端亨廷顿蛋白（HTT）片段，这些片段可优先杀伤纹状体神经元。 学界已投入大量精力研究HTT的切割机制，以及阻断其切割是否具有治疗益处。 然而，本研究在HD140Q敲入（KI）小鼠中利用CRISPR-Cas9系统截短全长突变型亨廷顿蛋白时，发现无论在全长亨廷顿蛋白的哪个位点进行截短，脑中均稳定存在外显子1来源的亨廷顿蛋白片段。 该N端亨廷顿蛋白片段会在不同KI小鼠模型中引发相似的HD表型，并导致纹状体中亨廷顿蛋白随年龄积累。 外显子1来源的亨廷顿蛋白片段持续产生，但其在纹状体中的选择性积累，是由纹状体富集的分子伴侣抑制蛋白HspBP1的年龄依赖性表达所介导的。 本研究结果表明，组织特异性的分子伴侣功能是HD发生选择性神经病理改变的原因，并凸显了调控该功能的治疗价值。 我们对野生型（WT）、KI-96、KI-571及KI-FL HD敲入小鼠的纹状体组织进行了RNA测序（RNAseq）分析，上述品系均为HD KI小鼠模型。其中，KI-FL可表达全长突变型亨廷顿蛋白，KI-571表达N端亨廷顿蛋白片段，KI-96所表达的N端亨廷顿蛋白片段比KI-571更短。 请注意：processed_count_matrix.txt文件中的T1_1至T1_4处理数据列应予以忽略。