Abstract

Mutations in the RNA/DNA-binding proteins FUS and TDP-43 cause the fatal disease amyotrophic lateral sclerosis (ALS). The precise mechanisms behind the selective motor neuron degeneration remain unclear and it is uncertain if ALS-causative mutations trigger motor neuron death through shared or distinct pathogenic pathways. To address these two questions, we performed single-cell RNA sequencing across neuron types derived from isogenic induced pluripotent stem cell lines, harbouring FUS P525L, FUS R495X, TARDBP M337V mutations or FUS knockout. The mutations elicited 5- to 15-fold greater transcriptional responses in motor neurons than interneurons. Approximately 20% of transcripts uniquely dysregulated in motor neurons were shared across FUS mutations, with half being driven by FUS gain-of-function. Among these, a majority pointed towards mitochondrial impairments, with attenuated pathways shared with the TARDBP M337V mutation. Meta-analysis demonstrated convergence on mitochondrial dysfunction with C9orf72-ALS patient-derived motor neurons. We observed impaired mitochondrial motility across ALS motor axons, even in isogenic FUS R244C motor neurons, which retain FUS in the nucleus, demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS, uncoupled from protein mislocalization. These early signs of mitochondrial dysfunction unique to motor neurons could have profound implications for their survival and represent promising therapeutic targets across multiple ALS forms.