Single cell RNA sequencing of spinal motor neurons and interneurons differentiated from isogenic FUS and TARDBP human induced pluripotent stem cell lines harbouring ALS-causative mutations

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease that can be inherited with genetic mutations in > 25 different genes including e.g. FUS, TARDBP, and C9ORF72. It is currently unclear how these mutations lead to motor neuron degeneration and whether different gene mutations converge on similar pathogenic pathways early on, or if each mutation leads to motor neuron death in a unique manner. To address these questions in depth, we generated a number of isogenic iPSC (induced pluripotent stem cell) lines harbouring point mutations in FUS (R495X and P525L) or TARDBP (M337V) as well as a knock-out of FUS (FUS-KO), to discern loss-versus gain-of-function mechanisms in FUS. The use of isogenic lines enables identification of subtle early disturbances in the transcriptome which may be detrimental but impossible to detect when lines of different backgrounds are used. We conducted single cell RNA sequencing of motor neurons and interneurons derived from these lines using Smart-seq2. We show that ALS-causative mutations elicit a much larger transcriptional response in motor neurons than in interneurons. A large number of transcripts were coregulated across FUS R495X and P525L motor neurons and the majority of these were part of gain-of-function of FUS. Cross-comparison with isogenic ALS-linked TARDBP M337V identified an early dysfunctions shared across ALS-causative mutations unique to motor neurons that could have major implications for motor neuron survival and that might be targeted therapeutically.