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

Abstract

BackgroundBulk 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. ResultsSingle cell analysis of spinal neuronal cultures derived from SOD1 E100G ALS and isogenic iPSCs 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, neuronal cytoskeleton, mitochondrial function and autophagy. 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 based on this network identified core transcriptional factors driving the ALS MN gene dysregulation. Specifically, we identified activation of SMAD2, a downstream mediator of the TGF-{beta} signalling pathway as a potential driving factor of ALS motor neuron degeneration. Our phenotypic analysis further confirmed that an activated TGF-{beta} signalling is major driver of motor neuron loss in SOD1 ALS. Importantly, expression analysis of TGF{beta} target genes and computational analysis of publicly available datasets indicates that activation of TGF{beta} signalling may be a common mechanism shared between SOD1, FUS and sporadic ALS. ConclusionsOur results demonstrate the utility of single cell transcriptomics in mapping disease-relevant gene regulatory networks driving neurodegeneration in ALS motor neurons. We find that ALS-associated mutant SOD1 targets transcriptional networks that perturb motor neuron homeostasis.