Translating ribosome affinity purification from C9orf72-associated amyotrophic lateral sclerosis patient-derived iPSC motor neurons

The C9orf72 hexanucleotide repeat expansion [GGGGCC]n is the most common cause of familial amyotrophic lateral sclerosis. The endogenous role of the C9orf72 protein has not been fully characterised, nor how the presence of the repeat expansion affects downstream mechanisms. Numerous cellular and molecular pathologies have been characterised in patient-derived induced pluripotent stem cell (iPSC) motor neuron cultures. This includes the accumulation of nuclear RNA foci and cytoplasmic dipeptide repeat proteins, altered calcium homeostasis, accumulation of apoptotic factors, nucleocytoplasmic defects and reduced excitability. Targeted antisense oligonucleotide knockdown of mutant C9orf72 mRNA transcripts mitigates some of these pathological phenotypes, though whether gene expression is restored to normal is unknown. Transcriptomic analysis of iPSC-motor neurons is challenging due to multiple sources of experimental variance, such as the presence of up to 30% of cells in culture being of unknown identity. The aim of this project is to establish a translating ribosome affinity purification (TRAP) method to isolate an enriched population of RNA from human iPSC-motor neurons. This will help achieve a more accurate analysis of the translatome profile of this model. Differential gene expression analysis between control and C9orf72 ALS motor neurons will be performed to identify patient motor neuron-specific changes in gene expression.