Transcriptional alterations induced by polyDPRs overexpression in neuronal cell model

Familial and sporadic Amyotrophic Lateral Sclerosis (ALS)forms are associated with several mutated proteins such as: mutant SOD1, TDP-43, FUS, VCP, OPTN and C9ORF72. RAN translation has been originally identified for CAG triplet repeat sequences, but it is activated also on (G4C2)n stretch of C9ORF72 witch give rise to RAN translated poly di-peptide repeats (DPRs). As already demonstrated in polyQ containing proteins, DPRs misfold and aggregate into cytoplasm or nuclei of motor neuron. They are able to alter the proteotoxicity response machinery. The protein quality control (PQC) system maintains protein homeostasis by re-folding (by chaperone) or by degradation (by autophagy or proteasome) of misfolded proteins to counteract proteotoxicity. Chaperone assisted selective autophagy (CASA) is involved in misfolded protein degradation and play its function by HSPB8-BAG3-HSP70 complex. We previously demonstrated that autophagy facilitation by HSPB8 overexpression prevents to DPRs aggregation and toxicity. In order to characterize aberrant mechanisms altered by RAN-DPR and PolyGln peptides, we developed a novel inducible human neuronal model to study DPRs and to compare them with polyQ toxicity. 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 proteotoxic DPRs, followed by bioinformatics analyses and we found a selective alteration of specific transcript in cells expressing the two most highly aggregation prone DPRs: polyGR and Poly PR. Unfortunately, RNAseq did not identify a specific pathway modulated by polyGR and/or polyPR expression. Notably, PCSK1N related to ALS and frontotemporal dementia and TOMM5 related to mitophagy and protein metabolism are influenced by polyGR and/or polyPR expression. Collectively, these data showed that aggregating prone DPRs overexpression despite do not show toxicity they clearly alter gene expression in our cell model.