Single cell transcriptomics identifies master regulators of dysfunctional pathways in SOD1 ALS motor neurons

Background: Bulk RNA-Seq has been extensively utilized to investigate the molecular changes accompanying motor neuron degeneration in Amyotrophic Lateral Sclerosis (ALS). However, due to the heterogeneity and degenerating phenotype of the neurons, it has proved difficult to assign specific changes to neuronal subtypes and identify which factors drive these changes. Consequently, we have utilized single cell transcriptomics of degenerating motor neurons derived from ALS patients to uncover key transcriptional drivers of dysfunctional pathways. Results: Single cell analysis of spinal neuronal cultures derived from ALS and isogenic iPSC allowed us to classify cells into neural subtypes including motor neurons and interneurons. Differential expression analysis between disease and control motor neurons revealed downregulation of genes involved in synaptic structure, neuromuscular junction, neuronal cytoskeleton and mitochondrial function. Interestingly, interneurons did not show similar suppression of these homeostatic functions. Single cell expression data enabled us to derive a context-specific transcriptional network relevant to ALS neurons. Master regulator analysis on this network identified core transcriptional factors driving the ALS disease signature. Specifically, we were able to correlate suppression of HOXA1 and HOXA5 to synaptic dysfunction in ALS motor neurons. Our results suggest that suppression of HOX genes may be a general phenomenon in SOD1 ALS. Conclusions: Our results demonstrate the utility of single cell transcriptomics in mapping disease-relevant gene regulatory networks driving neurodegeneration in ALS motor neurons. We propose that ALS-associated mutant SOD1 leads to inhibition of transcriptional networks driving homeostatic programs specific to motor neurons, thereby providing a possible explanation for the relative resistance of spinal interneurons to degeneration in ALS.