Translational profiling of motor neurons with C9orf72 mutations under acute oxidative stress

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease (MND) with a median survival of 30 months from disease onset and no cure available. Mutations in the gene C9orf72-SMCR8 complex subunit (C9orf72) are the most common cause of ALS in populations of European genetic heritage. Previous studies suggest that RNA processing, global translation and stress granule (SG) dynamics are dysregulated in C9orf72-ALS, which might alter the profile of translating mRNAs (translatome) in motor neurons. To test this hypothesis, I employed translating ribosome affinity purification (TRAP) to obtain the parallel transcriptome and translatome of human induced pluripotent stem cell-derived motor neurons (iPSC-MNs) from patients with C9orf72 mutations and healthy controls. I examined their expression profiles both at baseline and after transient oxidative stress. While minimal differences were present in the transcriptome and translatome at baseline and immediately after stress, 68 differentially expressed genes (DEGs) were identified in the translatome 2 h after stress removal. Functional over-representation analysis showed that they were enriched in synaptic transmission, small GTPase-mediated signalling transduction, and neuron projection. Two of the downregulated DEGs, unc-13 homolog A (UNC13A) and purine rich element binding protein A (PURA), have been previously associated with ALS. Further characterisation of the acute stress response in iPSC-MNs revealed that transient oxidative stress exacerbated or induced some key molecular phenotypes related to C9orf72-ALS. Additionally, the mutant iPSC-MNs exhibited a gradual and persistent decrease in the signal of a cell viability assay, in contrast to the control group. No differences were found between the two groups in global translational activity or SG dynamics. Preliminary investigation into the subcellular distribution of UNC13A and PURA transcripts suggested potential dysregulation in nucleocytoplasmic redistribution and sequestration by SGs in C9orf72-ALS iPSC-MNs, although further experimentation is needed to reach a definitive conclusion.