Aberrant splicing in Huntingtons disease accompanies disrupted TDP-43 activity and altered m6A RNA modifications [ISO-seq]

Huntingtons disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here, we identify the RNA-binding TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Dysregulated nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 is shown to be present in HD mice and human brains with TDP-43 co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions and from previously observed TDP-43 pathologies. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD and highlights the critical nature of TDP-43 loss of function across multiple neurodegenerative diseases. To investigate the role of mHTT exon1 on the mouse transcriptome and alternative splicing, here we compared males mice at a highly symptomatic timepoint in the R62 HD mouse model against the non-transgenic control by long-read sequencing with pacbio iso-seq. we microdisected two brain regions, the striatum and cortex, which are areas of interest in Huntington's disease.亨廷顿舞蹈症（Huntington's Disease, HD）是一种由HTT基因第一外显子区域CAG重复扩增引发的神经退行性疾病，可导致基因表达异常。然而，亨廷顿舞蹈症中RNA加工紊乱的具体机制仍未阐明。本研究鉴定出RNA结合蛋白TDP-43（TAR DNA-binding protein 43）以及N6-甲基腺嘌呤（N6-methyladenosine, m6A）写入酶METTL3，是多种亨廷顿舞蹈症模型中外显子跳跃事件的上游调控因子。研究发现，亨廷顿舞蹈症模型小鼠及患者大脑中存在TDP-43的核定位异常，以及磷酸化TDP-43的胞质聚集；且TDP-43可与HTT核聚集样结构共定位，该结构不同于突变型HTT包涵体与此前报道的TDP-43病理聚集体。TDP-43与编码亨廷顿舞蹈症相关差异表达基因及异常剪接基因的RNA的结合能力显著降低。最后，亨廷顿舞蹈症R6/2模型小鼠纹状体中异常表达的RNA上的m6A修饰水平降低，其中包括紧邻TDP-43结合位点的成簇修饰位点。本研究证据表明，TDP-43功能丧失伴随m6A修饰异常，是亨廷顿舞蹈症中可变剪接紊乱的潜在机制，同时也凸显了TDP-43功能丧失在多种神经退行性疾病中的关键作用。为探究突变型HTT第一外显子（mHTT exon1）对小鼠转录组及可变剪接的调控作用，本研究针对亨廷顿舞蹈症R6/2模型小鼠的高症状期雄性个体，采用PacBio Iso-Seq长读长测序技术，与非转基因对照小鼠进行对比分析。研究人员显微解剖了该模型小鼠的两个脑区——纹状体与大脑皮层，这两个区域均为亨廷顿舞蹈症研究的重点靶区。