Aberrant NSUN1 activity connects m⁵C-RNA modification to TDP-43 neurotoxicity in ALS/FTD

In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the nuclear RNA-binding protein TDP-43 mislocalises to the cytoplasm and forms insoluble aggregates, but the mechanisms controlling this remain unclear. We define a native TDP-43 interactome in human SH-SY5Y cells and identify proteins linked to the 5-methylcytosine (m⁵C) RNA modification as highly enriched. Using a Drosophila model of TDP-43 pathology, we show that aberrant activity of m⁵C-RNA methyltransferases Nsun1 drives TDP-43-induced m⁵C-RNA hypermethylation, whereas Nsun1 down-regulation alleviates TDP-43-induced degeneration, lifespan deficits, and cytoplasmic accumulation. In human cells, TDP-43 selectively interacts with NSUN1 isoform 3 independently of RNA. Furthermore, NSUN1 is nucleolar and TDP-43 is largely nucleoplasmic, yet they interact in both compartments, suggesting functional roles beyond their predominant localisations. In ALS/FTD postmortem frontal cortex, NSUN1 isoform 3 persists, whereas the shorter isoform is reduced, suggesting that a pool of NSUN1 capable of contributing to pathological TDP-43 interactions remains in disease. These findings suggest that TDP-43 neurotoxicity is coupled to NSUN1 activation and m⁵C-RNA methylation, revealing a potential therapeutic axis in ALS/FTD.