Aberrant splicing in Huntington's disease accompanies disrupted TDP-43 activity and altered m6A RNA modifications

Huntington's 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. Overall design: 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.