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

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, here we compared males and female mice at a highly symptomatic timepoint in the R62 HD mouse model against the non-transgenic control by total RNA-seq. we microdisected two brain regions, the striatum and cortex, which are areas of interest in Huntington's disease. To investigate the role of TDP-43 on the mouse transcriptome, here we compared male C57Bl/6J striatums with and without TDP-43 ASO by total RNA-seq To investigate the role of mHTT on human neurons enriched for medium spiny neurons that are derived from induced pluripotent stem cells. Further we investigated how TDP-43 knockdown relates to the effect of mHTT by studying the transcriptomic changes via total RNA-seq.亨廷顿舞蹈症（Huntington's Disease, HD）是一种由HTT基因（HTT gene）第一外显子区域发生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，HD中RNA加工紊乱的具体分子机制仍未阐明。本研究鉴定出RNA结合蛋白TDP-43（RNA-binding TDP-43）以及N6-甲基腺嘌呤（N6-methyladenosine, m6A）写入蛋白METTL3（writer protein METTL3），为多种HD模型中外显子跳跃（exon skipping）事件的上游调控因子。研究证实，HD模型小鼠与人类患者大脑中存在TDP-43的核定位（nuclear localization）异常以及磷酸化TDP-43（phosphorylated TDP-43）的胞质聚集现象；TDP-43与HTT核聚集样小体发生共定位（co-localizing），该结构区别于突变HTT包涵体（mutant HTT inclusions）与此前报道的TDP-43病理特征。同时，HD相关差异表达基因（differentially expressed genes）与异常剪接基因（aberrantly spliced genes）的编码RNA上，TDP-43的结合水平显著下降。最后，HD R6/2模型小鼠纹状体（striatum）中异常表达的RNA上，m6A修饰水平出现降低，其中包含位于TDP-43结合位点邻近的成簇修饰位点。本研究结果表明，TDP-43功能丧失伴随m6A修饰异常，是HD可变剪接异常的潜在分子机制，并凸显了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HTT外显子1对小鼠转录组的调控作用，本研究选取R6/2 HD小鼠模型中症状显著时期的雌雄小鼠，通过全转录组测序（total RNA-seq）对比其与非转基因对照小鼠的转录组差异；实验中显微切割了HD研究中备受关注的两个脑区——纹状体与皮层。为探究TDP-43对小鼠转录组的调控功能，本研究通过全转录组测序对比了转染与未转染TDP-43反义寡核苷酸（Antisense Oligonucleotide, ASO）的雄性C57Bl/6J小鼠纹状体的转录组差异。此外，为探究突变HTT在诱导多能干细胞（induced pluripotent stem cells, iPSC）来源的中等棘状神经元（medium spiny neurons）中的作用，本研究通过全转录组测序分析转录组变化，并进一步研究了TDP-43敲低与突变HTT效应之间的关联。