RAN translated C9ORF72 arginine rich poly-dipeptides alter gene transcription in ALS/FTD cell model

Amyotrophic Lateral Sclerosis (ALS) and frontotemporal dementia (FTD) are associated with several mutated proteins such as: mutant SOD1, TDP-43, FUS, VCP, OPTN and C9ORF72. Expanded (G4C2)n stretch of C9ORF72 give rise to poly di-peptide repeats (DPRs) that are produced by repeat-associated non-ATG (RAN) translation, a mechanism originally identified for CAG triplet repeat sequences. DPRs misfold and aggregate into cytoplasm or nuclei of motor neuron as it has been already demonstrated in polyQ containing proteins. DPRs alter the protein quality control system which maintains protein homeostasis by re-folding (by chaperone) or by degradation (by autophagy or proteasome) and it clears misfolded proteins to counteract proteotoxicity. Chaperone assisted selective autophagy (CASA) is involved in misfolded protein degradation and is mediated by the HSPB8-BAG3-HSP70 complex. We previously demonstrated that DPRs aggregation and toxicity are prevented by autophagy facilitation through HSPB8 overexpression. We developed a novel inducible human neuronal model to identify aberrant mechanisms altered by RAN-DPR and PolyQ peptides. We first evaluated DPRs and polyQ stability and induced toxicity. RTqPCR show that DPRs mRNA are less expressed than polyQ. This is also recapitulated in toxicity assay where only polyQ cells shows marked cell death. We performed differential genetic profiling of neuronal transcriptional response to DPRs and polyQ, followed by bioinformatics analyses. We found a selective alteration of specific transcripts in cells expressing the two most highly aggregation prone DPRs: polyGR and Poly PR. Gene set enrichment analysis showed specific pathways modulated by polyGR and/or polyPR expression. Notably, PCSK1N related to ALS and FTD and TOMM5 related to mitophagy and protein metabolism are influenced by polyGR and/or polyPR expression. Collectively, these data show that aggregating prone DPRs overexpression alters gene expression in our cell model.